RU2832574C2 - Neo-antigen compositions and use thereof - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to biotechnology and medicine, particularly to immunotherapeutic polypeptides containing neoepitopes, polynucleotides coding them and a method of producing antigen-specific T-cells.

EFFECT: invention provides additional anticancer therapeutic vaccines to ensure effective and sufficient processing and presentation of epitopes.

13 cl, 13 dwg, 15 tbl, 9 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No. 62/860,493, filed June 12, 2019, which is incorporated herein by reference in its entirety. This application is related to International Application No. PCT/US2020/031898, filed May 7, 2020, which is incorporated herein by reference in its entirety.

LEVEL OF TECHNOLOGY

Cancer immunotherapy is the use of the immune system to treat cancer. Immunotherapies exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor antigens, which are often proteins or other macromolecules (eg, carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting tumor antigens. Passive immunotherapy enhances existing antitumor responses and involves the use of monoclonal antibodies, lymphocytes, and cytokines. Tumor vaccines typically consist of tumor antigens and immune-stimulatory molecules (eg, adjuvants, cytokines, or Toll-like receptor (TLR) ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and kill tumor cells. Tumor neoantigens, which arise from genetic alterations (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) in malignant cells, represent the most tumor-specific class of antigens and may be patient-specific or both. Tumor neoantigens are unique to the tumor cell because the mutation and its corresponding protein are present only in the tumor. They also evade central tolerance and are therefore more likely to be immunogenic. Thus, tumor neoantigens are excellent targets for immune recognition, including humoral and cellular immunity.

To elicit a T cell response to vaccination, antigen-presenting cells (APCs) must process the epitope-containing peptide and present the epitopes on major histocompatibility complex (MHC) I or MHC II. One of the critical obstacles to the development of therapeutic and tumor-specific immunotherapies is the insufficient processing and release of minimal epitopes for antigen presentation to generate adequate immune responses. Accordingly, there is a need to develop additional cancer therapeutic vaccines to ensure efficient and sufficient epitope processing and presentation.

INCLUSION BY LINK

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

ESSENCE OF THE INVENTION

In some aspects, the invention relates to a polypeptide comprising an epitope presented by an MHC class I or MHC class II antigen-presenting cell (APC), the polypeptide having the structure of Formula (I):

Y _n -B _t -A _r -X _m -A _s -C _u -Z _p

Formula (I),

or a pharmaceutically acceptable salt thereof,

(i) where X _m is an epitope, where each X is independently an amino acid from a contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject,

and where,

(a) MHC is MHC class I and m is an integer from 8 to 12, or

(b) MHC is MHC class II, and m is an integer from 9 to 25;

(ii) where each Y is independently an amino acid, its analogue or derivative, and where:

(A) if the variable r of A _r in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t -A _r -X _m ,

(B) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m , or

(C) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 1 or greater, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t ; and

where n is an integer from 0 to 1000;

(iii) where each Z is independently an amino acid, analogue or derivative, and where:

(A) if variable s of A _s in Formula (I) is 0, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m -A _s -C _u ,

(B) if variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 0, Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m , or

(C) when variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 1 or more, Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes _Cu ; and

where p is an integer from 0 to 1000;

and besides, where

if n is 0, p is an integer between 1 and 1000; and

if p is 0, n is an integer between 1 and 1000;

(iv) where A _r is a linker and r is 0 or 1;

(v) where A _s is a linker and s is 0 or 1;

(vi) where each B is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately upstream of the nucleic acid sequence in the genome of the subject that encodes X _m ,

and where t is an integer between 0 and 1000; and

(vii) where each C is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m ,

and where u is an integer between 0 and 1000;

and besides, where

(a) the polypeptide does not consist of four different epitopes presented by MHC class I;

(b) the polypeptide comprises at least two different polypeptide molecules;

(c) the epitope comprises at least one mutated amino acid; and/or

(d) Y _n and/or Z _p are cleaved from the epitope when the polypeptide is processed by the APC.

In some embodiments, the epitope is presented by MHC class II. In some embodiments, m is an integer from 9 to 25. In some embodiments, t is 1, 2, 3, 4, or 5 or more and r is 0. In some embodiments, u is 1, 2, 3, 4, or 5 or more and s is 0. In some embodiments, t is 1 or more, r is 0, and n is from 1 to 1000. In some embodiments, u is 1 or more, s is 0, and p is from 1 to 1000. In some embodiments, t is 0. In some embodiments, u is 0. In some embodiments, t is at least 1, and B _t comprises a lysine. In some embodiments, u is at least 1, and _Cu comprises a lysine. In some embodiments, B _t is cleaved from the epitope if the polypeptide is processed by an APC. In some embodiments, _Cu is cleaved from the epitope if the polypeptide is processed by the APC. In some embodiments, n is an integer from 1 to 5 or from 7 to 1000. In some embodiments, p is an integer from 1 to 4 or from 6 to 1000.

In some embodiments, the polypeptide does not consist of four different epitopes presented by MHC class I. In some embodiments, the polypeptide does not comprise four different epitopes presented by MHC class I. In some embodiments, the polypeptide comprises at least two different polypeptide molecules. In some embodiments, the epitope comprises at least one mutant amino acid. In some embodiments, the at least one mutant amino acid is encoded by an insertion, deletion, frameshift, neoORF, or point mutation in a nucleic acid sequence in the genome of the subject. In some embodiments, Y_nand/or Z_pare cleaved from the epitope if the polypeptide is processed by the APC. In some embodiments, m of X_mis at least 8, and X_mis AA₁AA₂AA₃AA₄AA₅AA₆AA₇AA₈AA₉AA₁₀AA₁₁AA₁₂AA₁₃AA₁₄AA₁₅AA₁₆AA₁₇AA₁₈AA₁₉AA₂₀AA₂₁AA₂₂AA₂₃AA₂₄AA₂₅, where each AA is an amino acid, and where one or more of the AA₉, AA₁₀, AA₁₁, AA₁₂, AA₁₃, AA₁₄, AA₁₅, AA₁₆, AA₁₇, AA₁₈, AA₁₉, AA₂₀, AA₂₁, AA₂₂, AA₂₃, AA₂₄and AA₂₅optionally present, and further wherein at least one AA is a mutant amino acid. In some embodiments, r is 1. In some embodiments, s is 1. In some embodiments, r is 1 and s is 1. In some embodiments, r is 0. In some embodiments, s is 0. In some embodiments, r is 0 and s is 0.

In some embodiments, _Ar and/or _As are a non-polypeptide linker. In some embodiments, _Ar and/or _As are a chemical linker. In some embodiments, _Ar and/or _As comprise a non-naturally occurring amino acid. In some embodiments, _Ar and/or _As do not comprise an amino acid. In some embodiments, _Ar and/or _As do not comprise a natural amino acid. In some embodiments, _Ar and/or _As comprise a bond other than a peptide bond. In some embodiments, _Ar and/or _As comprise a disulfide bond. In some embodiments, _Ar and/or _As are different. In some embodiments, _Ar and/or _As are the same.

In some embodiments, the polypeptide comprises a hydrophilic tail. In some embodiments, Y _n -B _t -A _r and/or A _s -C _u -Z _p increases the solubility of the polypeptide compared to a corresponding peptide that does not comprise Y _n -B _t -A _r and/or A _s -Z _p . In some embodiments, each X of X _m is a naturally occurring amino acid.

In some embodiments, the epitope is released from Y _n -B _t -A _r and/or A _s -C _u -Z _p if the polypeptide is processed by the APC. In some embodiments, the polypeptide is cleaved into A _r and/or A _s . In some embodiments, the polypeptide is cleaved at a higher rate when n is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m ; and/or the polypeptide is cleaved at a higher rate, if p is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of the nucleic acid sequence in the subject's genome that encodes X _m .

In some embodiments, the polypeptide is cleaved at a higher rate when n is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains B_t-X_m, where t is at least one and r is from the variable A_rin Formula (I) is 0; and/or where the polypeptide is cleaved at a higher rate, if p is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains X_m-C_u, where u is at least one and s is from variable A_s in Formula (I) is equal to 0.

In some embodiments, the polypeptide is cleaved into _Ar at a higher rate if n is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m ; and/or the polypeptide is cleaved into _As at a higher rate if p is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m .

In some embodiments, the presentation of an epitope by an APC is enhanced if n is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m ; and/or the presentation of an epitope by an APC is enhanced if p is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m .

In some embodiments, the presentation of an epitope by an APC is enhanced if n is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises B _t -X _m , where t is at least one and r of the variable A _r in formula (I) is 0; and/or where the presentation of an epitope by an APC is enhanced if p is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises X _m -C _u , where u is at least one and s of the variable A _s in formula (I) is 0.

In some embodiments, the APC presents an epitope to an immune cell. In some embodiments, the APC presents an epitope to a phagocytic cell. In some embodiments, the APC presents an epitope to a dendritic cell, a macrophage, a mast cell, a neutrophil, or a monocyte. In some embodiments, the APC presents an epitope preferentially or specifically to an immune cell, a phagocytic cell, a dendritic cell, a macrophage, a mast cell, a neutrophil, or a monocyte.

In some embodiments, the immunogenicity is increased if n is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m ; and/or the immunogenicity is increased if p is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m .

In some embodiments, the immunogenicity is increased if n is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises B _t -X _m , where t is at least one and r of the variable A _r in Formula (I) is 0; and/or where the immunogenicity is increased if p is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises X _m -C _u , where u is at least one and s of the variable A _s in Formula (I) is 0.

In some embodiments, the antitumor activity is increased if n is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m ; and/or the antitumor activity is increased if p is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m .

In some embodiments, the antitumor activity is increased when n is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that comprises B _t -X _m , where t is at least one and r of the variable A _r in formula (I) is 0; and/or where the antitumor activity is increased when p is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that comprises X _m -C _u , where u is at least one and s of the variable A _s in formula (I) is 0.

In some embodiments, Y _n and/or Z _p comprise a sequence selected from the group consisting of poly-Lys (polyK) and poly-Arg (polyR). In some embodiments, Y _n and/or Z _p comprise a sequence selected from the group consisting of polyK-AA-AA and polyR-AA-AA, wherein each AA is an amino acid, an analog, or a derivative thereof. In some embodiments, polyK comprises poly-L-Lys. In some embodiments, polyR comprises poly-L-Arg. In some embodiments, polyK or polyR comprises at least three or four contiguous lysine (SEQ ID NO: 1) or arginine (SEQ ID NO: 2) residues, respectively. In some embodiments, A _r and/or A _s are selected from the group consisting of disulfide; p-aminobenzyloxycarbonyl (PABC); and AA-AA-PABC, wherein each AA is an amino acid, an analog, or a derivative thereof. In some embodiments, AA-AA-PABC is selected from the group consisting of Ala-Lys-PABC, Val-Cit-PABC, and Phe-Lys-PABC.

In some embodiments, A _r and/or A _s is

Formula (II).

In some embodiments, A _r and/or A _s are

Formula (III), or

Formula (IV),

Where,

R ¹ and R ² are independently H or (C ₁ -C ₆ ) alkyl; j is 1 or 2; G ¹ is H or COOH; and i is 1, 2, 3, 4, or 5.

In some embodiments, the polypeptide is ubiquitinated. In some embodiments, the polypeptide is ubiquitinated prior to cleavage. In some embodiments, the polypeptide is ubiquitinated at a lysine residue. In some embodiments, the polypeptide is not cleaved prior to processing by APC prior to internalization by APC in a subject. In some embodiments, the polypeptide is not cleaved in the blood of a subject prior to processing by APC or prior to internalization by APC. In some embodiments, the polypeptide is not cleaved by a protease in the blood. In some embodiments, the polypeptide is not cleaved by plasmin, plasma kallikrein, tissue kallikrein, thrombin, or a coagulation factor. In some embodiments, the polypeptide is stable in human plasma. In some embodiments, the polypeptide has a half-life in human plasma from 1 hour to 5 days. In some embodiments, the polypeptide is cleaved in a lysosome, endolysosome, endosome, or endoplasmic reticulum (ER). In some embodiments, the polypeptide is cleaved by an aminopeptidase. In some embodiments, the aminopeptidase is leucyl cystinyl aminopeptidase (IRAP) or endoplasmic reticulum aminopeptidase (ERAP). In some embodiments, the polypeptide is processed by a trypsin-like domain of a proteasome and/or immunoproteasome. In some embodiments, the trypsin-like domain comprises trypsin-like activity, chymotrypsin-like activity, or peptidyl glutamyl peptide hydrolase (PGPH) activity. In some embodiments, the polypeptide is cleaved by a protease. In some embodiments, the protease is a trypsin-like protease, chymotrypsin-like protease, or peptidyl glutamyl peptide hydrolase (PGPH). In some embodiments, the protease is selected from the group consisting of asparagine peptide lyase, aspartic protease, cysteine protease, glutamic protease, metalloprotease, serine protease, and threonine protease. In some embodiments, the protease is a cysteine protease selected from the group consisting of calpain, caspase, cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, and cathepsin Z.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In some embodiments, the epitope binds to HLA MHC class I. In some embodiments, the epitope binds to HLA MHC class I with a stability of 10 minutes to 24 hours. In some embodiments, the epitope binds to HLA MHC class I with an affinity of 0.1 nM to 2000 nM. In some embodiments, the epitope binds to HLA MHC class II. In some embodiments, the epitope binds to HLA MHC class II with a stability of 10 minutes to 24 hours. In some embodiments, the epitope binds to HLA MHC class II with an affinity of 0.1 nM to 2000 nM, 1 nM to 1000 nM, 10 nM to 500 nM, or less than 1000 nM. In some embodiments, n is an integer from 1 to 20 or from 5 to 12. In some embodiments, p is an integer from 1 to 20 or from 5 to 12. In some embodiments, the epitope comprises a tumor-specific epitope.

In some embodiments, the polypeptide comprises at least two polypeptides, wherein two or more of the at least two polypeptides have the same formula Y _n -B _t -A _r -X _m -A _s -C _u -Z _p . In some embodiments, the polypeptide comprises at least two polypeptide molecules. In some embodiments, X _m of two or more of the at least two polypeptides or polypeptide molecules are the same. In some embodiments, Y _n of two or more of the at least two polypeptides or polypeptide molecules are the same. In some embodiments, Z _p of two or more of the at least two polypeptides or polypeptide molecules are the same. In some embodiments, A _r and/or A _s of two or more of the at least two polypeptides or polypeptide molecules are different. In some embodiments, r=0 for the first of the at least two polypeptides or polypeptide molecules, and r=1 for the second of the at least two polypeptides or polypeptide molecules. In some embodiments, s=0 for the first of the at least two polypeptides or polypeptide molecules, and s=1 for the second of the at least two polypeptides or polypeptide molecules. In some embodiments, the polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides or polypeptide molecules.

In some embodiments, the epitope is a RAS epitope. In some embodiments, the epitope comprises a mutant RAS peptide sequence that comprises at least 8 contiguous amino acids of a mutant RAS protein comprising a G12, G13, or Q61 mutation and a G12, G13, or Q61 mutation. In some embodiments, at least 8 contiguous amino acids of a mutant RAS protein comprising a mutation at G12, G13, or Q61 comprise a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation. In some embodiments, the G12, G13, or Q61 mutation comprises a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation. In some embodiments, Y _n and/or Z _p comprise an amino acid sequence of a cytomegalovirus (CMV) protein, such as pp65, human immunodeficiency virus (HIV), or MART-1. In some embodiments, n and/or p are 1, 2, 3, or an integer greater than 3. In some embodiments, Y _n and/or Z _p comprise a lysine or polylysine. In some embodiments, Y _n and/or Z _p comprise K, KK, KKK, KKKK (SEQ ID NO: 1), or KKKKK (SEQ ID NO: 3).

In some embodiments, the epitope binds to the protein encoded by the HLA allele with an affinity of less than 10 μM, less than 1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than more than 200 nM, less than 150 nM, less than 100 nM, or less than 50 nM. In some embodiments, the epitope binds to the protein encoded by the HLA allele with a stability of greater than 24 hours, greater than 12 hours, greater than 9 hours, greater than 6 hours, greater than 5 hours, greater than 4 hours, greater than 3 hours, greater than 2 hours, greater than 1 hour, greater than 45 minutes, greater than 30 minutes, greater than 15 minutes, or greater than 10 minutes. In some embodiments, the HLA allele is selected from the group consisting of the HLA-A02:01 allele, the HLA-A03:01 allele, the HLA-A11:01 allele, the HLA-A03:02 allele, the HLA-A30:01 allele, the HLA-A31:01 allele, the HLA-A33:01 allele, the HLA-A33:03 allele, the HLA-A68:01 allele, the HLA-A74:01 allele, and/or the HLA-C08:02 allele, and any combination thereof.

In some embodiments, the epitope comprises the amino acid sequence GADGVGKSAL (SEQ ID NO: 4), GACGVGKSAL (SEQ ID NO: 5), GAVGVGKSAL (SEQ ID NO: 6), GADGVGKSA (SEQ ID NO: 7), GACGVGKSA (SEQ ID NO: 8), GAVGVGKSA (SEQ ID NO: 9), KLVVVGACGV (SEQ ID NO: 10), FLVVVGACGL (SEQ ID NO: 11), FMVVVGACGI (SEQ ID NO: 12), FLVVVGACGI (SEQ ID NO: 13), FMVVVGACGV (SEQ ID NO: 14), FLVVVGACGV (SEQ ID NO: 15), MLVVVGACGV (SEQ ID NO: 16), FMVVVGACGL (SEQ ID NO: 17), YLVVVGACGV (SEQ ID NO: 18), KMVVVGACGV (SEQ ID NO: 19), YMVVVGACGV (SEQ ID NO: 20), MMVVVGACGV (SEQ ID NO: 21), DTAGHEEY (SEQ ID NO: 22), TAGHEEYSAM (SEQ ID NO: 23), DILDTAGHE (SEQ ID NO: 24), DILDTAGH (SEQ ID NO: 25), ILDTAGHEE (SEQ ID NO: 26), ILDTAGHE (SEQ ID NO: 27), DILDTAGHEEY (SEQ ID NO: 28), DTAGHEEYS (SEQ ID NO: 29), LLDILDTAGH (SEQ ID NO: 30), DILDTAGRE (SEQ ID NO: 31), DILDTAGR (SEQ ID NO: 32) ILDTAGREE (SEQ ID NO: 33), ILDTAGRE (SEQ ID NO: 34), CLLDILDTAGR (SEQ ID NO: 35), TAGREEYSAM (SEQ ID NO: 36), REEYSAMRD (SEQ ID NO: 37), DTAGKEEYSAM (SEQ ID NO: 38), CLLDILDTAGK (SEQ ID NO: 39), DTAGKEEY (SEQ ID NO: 40), LLDILDTAGK (SEQ ID NO: 41), ILDTAGKE (SEQ ID NO: 42), ILDTAGKEE (SEQ ID NO: 43), DTAGLEEY (SEQ ID NO: 44), ILDTAGLE (SEQ ID NO: 45), DILDTAGL (SEQ ID NO: 46), ILDTAGLEE (SEQ ID NO: 47), GLEEYSAMRDQY (SEQ ID NO: 48), LLDILDTAGLE (SEQ ID NO: 49), LDILDTAGL (SEQ ID NO: 50), DILDTAGLE (SEQ ID NO: 51), DILDTAGLEEY (SEQ ID NO: 52), AGVGKSAL (SEQ ID NO: 53), GAAGVGKSAL (SEQ ID NO: 54), AAGVGKSAL (SEQ ID NO: 55), CGVGKSAL (SEQ ID NO: 56), ACGVGKSAL (SEQ ID NO: 57), DGVGKSAL (SEQ ID NO: 58), ADGVGKSAL (SEQ ID NO: 59), DGVGKSALTI (SEQ ID NO: 60), GARGVGKSA (SEQ ID NO: 62), KLVVVGARGV (SEQ ID NO: 62), VVVGARGV (SEQ ID NO: 63), SGVGKSAL (SEQ ID NO: 64), VVVGASGVGK (SEQ ID NO: 65), GASGVGKSAL (SEQ ID NO: 66), VGVGKSAL (SEQ ID NO: 67), VVVGAGCVGK (SEQ ID NO: 68), KLVVVGAGC (SEQ ID NO: 69), GDVGKSAL (SEQ ID NO: 70), DVGKSALTI (SEQ ID NO: 71), VVVGAGDVGK (SEQ ID NO: 72), TAGKEEYSAM (SEQ ID NO: 73), DTAGHEEYSAM (SEQ ID NO: 74), TAGHEEYSA (SEQ ID NO: 75), DTAGREEYSAM (SEQ ID NO: 76), TAGKEEYSA (SEQ ID NO: 77), AAGVGKSA (SEQ ID NO: 78), AGCVGKSAL (SEQ ID NO: 79), AGDVGKSAL (SEQ ID NO: 80), AGKEEYSAMR (SEQ ID NO: 81), AGVGKSALTI (SEQ ID NO: 82), ARGVGKSAL (SEQ ID NO: 83), ASGVGKSA (SEQ ID NO: 84), ASGVGKSAL (SEQ ID NO: 85), AVGVGKSA (SEQ ID NO: 86), CVGKSALTI (SEQ ID NO: 87), DILDTAGK (SEQ ID NO: 88), DILDTAGREEY (SEQ ID NO: 89), DTAGHEEYSAMR (SEQ ID NO: 90), DTAGKEEYS (SEQ ID NO: 91), DTAGKEEYSAMR (SEQ ID NO: 92), DTAGLEEYS (SEQ ID NO: 93), DTAGLEEYSA (SEQ ID NO: 94), DTAGLEEYSAMR (SEQ ID NO: 95), DTAGREEYS (SEQ ID NO: 96), DTAGREEYSAMR (SEQ ID NO: 97), GAAGVGKSA (SEQ ID NO: 98), GACGVGKSA (SEQ ID NO: 8), GACGVGKSAL (SEQ ID NO: 5), GADGVGKS (SEQ ID NO: 99), GAGDVGKSA (SEQ ID NO: 100), GAGDVGKSAL (SEQ ID NO: 101), GASGVGKSA (SEQ ID NO: 102), GCVGKSAL (SEQ ID NO: 103), GCVGKSALTI (SEQ ID NO: 104), GHEEYSAM (SEQ ID NO: 105), GKEEYSAM (SEQ ID NO: 106), GLEEYSAMR (SEQ ID NO: 107), GREEYSAM (SEQ ID NO: 108), GREEYSAMR (SEQ ID NO: 109), HEEYSAMRD (SEQ ID NO: 110), KEEYSAMRD (SEQ ID NO: 111), KLVVVGASG (SEQ ID NO: 112), LDILDTAGR (SEQ ID NO: 113), LEEYSAMRD (SEQ ID NO: 114), LVVVGARGV (SEQ ID NO: 115), LVVVGASGV (SEQ ID NO: 116), REEYSAMRDQY (SEQ ID NO: 117), RGVGKSAL (SEQ ID NO: 118), TAGLEEYSA (SEQ ID NO: 119), TEYKLVVVGAA (SEQ ID NO: 120), VGAAGVGKSA (SEQ ID NO: 121), VGADGVGK (SEQ ID NO: 122), VGASGVGKSA (SEQ ID NO: 123), VGVGKSALTI (SEQ ID NO: 124), VVVGAAGV (SEQ ID NO: 125), VVVGAVGV (SEQ ID NO: 126), YKLVVVGAC (SEQ ID NO: 127), YKLVVVGAD (SEQ ID NO: 128), YKLVVVGAR (SEQ ID NO: 129) or DILDTAGKE (SEQ ID NO: 130).

In some embodiments, Y _n comprises the amino acid sequence of IDIIMKIRNA (SEQ ID NO: 131), FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC (SEQ ID NO: 132), FFFFFFFFFFFFFFFFFFFFFFFFAAFWFW (SEQ ID NO: 133), IFFIFFIIFFFFFFFFFFFFIIIIIIIWEC (SEQ ID NO: 134), FIFFFIIFFFFFIFFFFFIFIIIIIIFWEC (SEQ ID NO: 135), TEY, TEYKLV (SEQ ID NO: 136), WQAGILAR (SEQ ID NO: 137), HSYTTAE (SEQ ID NO: 138), PLTEEKIK (SEQ ID NO: 139), GALHFKPGSR (SEQ ID NO: 140), RRANKDATAE (SEQ ID NO: 141), KAFISHEEKR (SEQ ID NO: 142), TDLSSRFSKS (SEQ ID NO: 143), FDLGGGTFDV (SEQ ID NO: 144), CLLLHYSVSK (SEQ ID NO: 145), KKKKIIMKIRNA (SEQ ID NO: 146) or MTEYKLVVV (SEQ ID NO: 147). In some embodiments, Z _p comprises the amino acid sequence of KKNKKDDI (SEQ ID NO: 148), KKNKKDDIKD (SEQ ID NO: 149), AGNDDDDDDDDDDDDDDDKKDKDDDDDD (SEQ ID NO: 150), AGNKKKKKKKNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 151), AGRDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 152), SALTI (SEQ ID NO: 153), SALTIQL (SEQ ID NO: 154), GKSALTIQL (SEQ ID NO: 155), GKSALTI (SEQ ID NO: 156), QGQNLKYQ (SEQ ID NO: 157), ILGVLLLI (SEQ ID NO: 158), EKEGKISK (SEQ ID NO: 159), AASDFIFLVT (SEQ ID NO: 160), KELKQVASPF (SEQ ID NO: 161), KKKLINEKKE (SEQ ID NO: 162), KKCDISLQFF (SEQ ID NO: 163), KSTAGDTHLG (SEQ ID NO: 164), ATFYVAVTVP (SEQ ID NO: 165), LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG (SEQ ID NO: 166) or TIQLIQNHFVDEYDPTIEDSYRKQVVIDGE (SEQ ID NO: 167).

In some embodiments, the epitope is not a RAS epitope. In some embodiments, the polypeptide is not KKKKKPKRDGYMFLKAESKIMFAT (SEQ ID NO: 168), KKKKYMFLKAESKIMFATLQRSS (SEQ ID NO: 169), KKKKKAESKIMFATLQRSSLWCL (SEQ ID NO: 170), KKKKKIMFATLQRSSLWCLCSNH (SEQ ID NO: 171), or KKKKMFATLQRSSLWCLCSNH (SEQ ID NO: 172).

In some embodiments, the epitope is a GATA3 epitope. In some embodiments, the GATA3 epitope comprises the amino acid sequence of MLTGPPARV (SEQ ID NO: 173), SMLTGPPARV (SEQ ID NO: 174), VLPEPHLAL (SEQ ID NO: 175), KPKRDGYMF (SEQ ID NO: 176), KPKRDGYMFL (SEQ ID NO: 177), ESKIMFATL (SEQ ID NO: 178), KRDGYMFL (SEQ ID NO: 179), PAVPFDLHF (SEQ ID NO: 180), AESKIMFATL (SEQ ID NO: 181), FATLQRSSL (SEQ ID NO: 182), ARVPAVPFD (SEQ ID NO: 183), IMKPKRDGY (SEQ ID NO: 184), DGYMFLKA (SEQ ID NO: 185), MFLKAESKIMF (SEQ ID NO: 186), NO: 186), LTGPPARV (SEQ ID NO: 187), ARVPAVPF (SEQ ID NO: 188), SMLTGPPAR (SEQ ID NO: 189), RVPAVPFDL (SEQ ID NO: 190) or LTGPPARVP (SEQ ID NO: 191).

In some aspects, the invention relates to a cell comprising a polypeptide described in the present invention. In some embodiments, the cell is an antigen-presenting cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is a mature antigen-presenting cell.

In some aspects, the invention relates to a method of cleaving a polypeptide comprising contacting the polypeptide described herein with an antigen-presenting cell (APC). In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo.

In some aspects, the invention relates to a method of producing a polypeptide comprising linking Y _n -A _r and/or A _s -Z _p to a sequence comprising an epitope sequence, wherein the epitope sequence is presented by MHC class I or MHC class II of an antigen-presenting cell (APC); wherein (i) each Y is independently an amino acid, an analog or a derivative thereof, and wherein Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes the epitope, and n is an integer from 0 to 1000; (ii) each Z is independently an amino acid, an analog or a derivative thereof, and wherein Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the subject's genome that encodes the epitope, and p is an integer from 0 to 1000; and (iii) A _r is a linker and A _s is a linker, where at least one of r and s is 1; and where (a) the polypeptide does not consist of four different epitopes presented by MHC class I; (b) the polypeptide comprises at least two different polypeptide molecules; (c) the epitope comprises at least one mutant amino acid; and/or (d) Y _n and/or Z _p are cleaved from the epitope if the polypeptide is processed by APC.

In some aspects, the invention relates to a method of producing a polypeptide comprising linking Y _n to B _t - X _m and/or Z _p to X _m - _Cu, where X _m is an epitope sequence presented by an MHC class I or MHC class II antigen-presenting cell (APC); and wherein (i) each B is independently an amino acid encoded by a nucleic acid sequence in the subject's genome that is immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m , and wherein t is an integer from 0 to 1000; (ii) wherein each C is independently an amino acid encoded by a nucleic acid sequence in the subject's genome that is immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m , and wherein u is an integer from 0 to 1000; (iii) each Y is independently an amino acid, an analog or a derivative thereof, and wherein Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes B _t - X _m , and wherein n is an integer from 0 to 1000; and (iv) each Z is independently an amino acid, an analog or a derivative thereof, and wherein Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m - _Cu , and wherein p is an integer from 0 to 1000; and further wherein (a) the polypeptide does not consist of four different epitopes presented by MHC class I; (b) the polypeptide comprises at least two different polypeptide molecules; (c) the epitope comprises at least one mutant amino acid; and/or (d) Y _n - B _t and/or Cu _- Z _p are cleaved from the epitope if the polypeptide is processed by the APC.

In some embodiments, if n is 0, p is an integer from 1 to 1000, and if p is 0, n is an integer from 1 to 1000. In some embodiments, each X is independently an amino acid in a peptide sequence comprising any contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject, and wherein (a) the MHC is MHC class I and m is an integer from 8 to 12, or (b) the MHC is MHC class II and m is an integer from 9 to 25.

In some aspects, the invention relates to a pharmaceutical composition comprising a polypeptide described in the present invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the immunomodulatory agent or adjuvant is selected from the group consisting of poly-ICLC, 1018 ISS, aluminum salts, amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonists, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, 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, microparticles PLGA, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam2Cys, Pam3Cys, Pam3CSK4, and Aquila QS21 stimulon. In some embodiments, the immunomodulatory agent or adjuvant comprises poly-ICLC. In some embodiments, the pharmaceutical composition is a vaccine composition. In some embodiments, the pharmaceutical composition is aqueous or liquid.

In some embodiments, the epitope is present in the pharmaceutical composition in an amount of 1 ng to 10 mg or 5 μg to 1.5 mg. In some embodiments, the pharmaceutical composition further comprises DMSO. In some embodiments, the pharmaceutically acceptable excipient comprises water. In some embodiments, the pharmaceutical composition comprises a pH modifier present at a concentration of less than 1 mM or greater than 1 mM. In some embodiments, the pH modifier is a dicarboxylate or a tricarboxylate. In some embodiments, the pH modifier is succinic acid dicarboxylate or disuccinate. In some embodiments, the pH modifier is citric acid tricarboxylate or tricitrate. In some embodiments, the pH modifier is disodium succinate. In some embodiments, the succinic acid dicarboxylate or disuccinate is present in the pharmaceutical composition at a concentration of 0.1 mM - 1 mM. In some embodiments, the succinic acid dicarboxylate or disuccinate is present in the pharmaceutical composition at a concentration of 1 mM - 5 mM. In some embodiments, the immune response to the epitope is enhanced when administered to a subject.

In some aspects, the invention relates to a method of treating a disease or condition comprising administering a therapeutically effective amount of a pharmaceutical composition described in the present invention to a subject in need thereof. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, colorectal cancer, uterine cancer, and liver cancer. In some embodiments, the administration comprises intradermal injection, intranasal spray application, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, or subcutaneous injection.

In some aspects, the invention relates to a method of prophylaxis of a subject comprising contacting a cell of the subject with a polypeptide, cell, or pharmaceutical composition described herein.

In some aspects, the invention relates to a method comprising identifying an epitope expressed by tumor cells of a subject and obtaining a polypeptide comprising the epitope, wherein the polypeptide has a structure of formula (I),

Y _n -B _t -A _r -X _m -A _s -C _u -Z _p

Formula (I),

or its pharmaceutically acceptable salt,

(i) where X _m is an epitope, where each X is independently an amino acid from a contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject,

and where

(a) MHC is MHC class I and m is an integer from 8 to 12, or

(b) MHC is MHC class II and m is an integer from 9 to 25;

(ii) where each Y is independently an amino acid, its analogue or derivative, and where:

(A) if the variable r of A _r in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t -A _r -X _m ,

(B) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m , or

(C) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 1 or greater, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t ; and

where n is an integer from 0 to 1000;

(iii) where each Z is independently an amino acid, its analogue or derivative, and where:

(A) if variable s of A _s in Formula (I) is 0, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m -A _s -C _u ,

(B) if variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 0, Z _p is not encoded by a nucleic acid sequence located immediately downstream of the nucleic acid sequence in the subject's genome that encodes X _m , or

(C) if variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 1 or more, Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes _Cu ; and

where p is an integer from 0 to 1000;

and besides,

if n is 0, p is an integer between 1 and 1000; and

if p is 0, n is an integer between 1 and 1000;

(iv) where A _r is a linker and r is 0 or 1;

(v) where A _s is a linker and s is 0 or 1;

(vi) where each B is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately upstream of the nucleic acid sequence in the genome of the subject that encodes X _m ,

and where t is an integer between 0 and 1000; and

(vii) where each C is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m ,

and where u is an integer between 0 and 1000;

and besides,

(a) the polypeptide does not consist of four different epitopes presented by MHC class I;

(b) the polypeptide comprises at least two different polypeptide molecules;

(c) the epitope comprises at least one mutated amino acid; and/or

(d) Y _n and/or Z _p are cleaved from the epitope if the polypeptide is processed by the APC.

In some embodiments, identifying comprises selecting a plurality of nucleic acid sequences from a pool of nucleic acid sequences sequenced from tumor cells of the subject that encode a plurality of candidate peptide sequences comprising one or more different mutations not present in a pool of sequenced nucleic acid sequences from non-tumor cells of the subject, wherein the pool of nucleic acid sequences sequenced from tumor cells of the subject and the pool of nucleic acid sequences sequenced from non-tumor cells of the subject are sequenced by whole genome sequencing or whole exome sequencing. In some embodiments, identifying further comprises predicting or measuring which candidate peptide sequences from the plurality of candidate peptide sequences form a complex with a protein encoded by an HLA allele of the same subject using an HLA peptide binding assay. In some embodiments, identifying further comprises selecting a plurality of selected tumor-specific peptides or one or more polynucleotides encoding the plurality of selected tumor-specific peptides from candidate peptide sequences based on an HLA binding assay of the peptide.

In some embodiments, the method further comprises administering the polypeptide to the subject. In some embodiments, administering comprises intradermal injection, intranasal spray application, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, or subcutaneous injection. In some embodiments, an immune response is elicited in the subject. In some embodiments, the epitope expressed by the tumor cells of the subject is a neoantigen, a tumor-associated antigen, a mutated tumor-associated antigen, and/or wherein the expression of the epitope is higher in the tumor cells of the subject compared to the expression of the epitope in normal cells of the subject.

The invention relates to a polypeptide containing an epitope presented by MHC class I or MHC class II of an antigen-presenting cell (APC), where the polypeptide has a structure of formula (I):

Y _n -B _t -A _r -X _m -A _s -C _u -Z _p

Formula (I),

or a pharmaceutically acceptable salt thereof, wherein X _m is an epitope, wherein each X is independently an amino acid from a contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject, and wherein (a) the MHC is MHC class I and m is an integer from 8 to 12, or (b) the MHC is MHC class II and m is an integer from 9 to 25; where each Y is independently an amino acid, an analogue or a derivative thereof, and where: if variable r of A _r in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t - _{A r} - X _m , if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m , or if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 1 or more, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t ; and further where n is an integer from 0 to 1000; where each Z is independently an amino acid, an analog, or a derivative, and where: if variable s of A _s in Formula (I) is 0, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m - _{A s} - _Cu , if variable s of A _s in Formula (I) is 1 and variable u of _Cu in Formula (I) is 0, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m , or if variable s of A _s in Formula (I) is 1 and variable u of _Cu in Formula (I) is 1 or more, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes _Cu ; and, further, p is an integer from 0 to 1000; and further, if n is 0, p is an integer from 1 to 1000; and if p is 0, n is an integer from 1 to 1000; where A _r is a linker, and r is 0 or 1; where _As is a linker, and s is 0 or 1; where each B is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m , and where t is an integer from 0 to 1000; and where each C is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m , and where u is an integer from 0 to 1000; and further, where the polypeptide does not consist of four different epitopes presented by MHC class I; the polypeptide comprises at least two different polypeptide molecules; the epitope comprises at least one mutant amino acid; and/or Y _n and/or Z _p are cleaved from the epitope if the polypeptide is processed by the APC.

In some embodiments, the epitope is MHC class II and m is an integer from 9 to 25.

In some embodiments, Y _n -B _t -A _r and/or _As -Cu _{-Z p} increases the solubility of the polypeptide compared to a corresponding peptide that does not contain Y _n -B _t -A _r and/or _As -Cu _{-Z p} . In some embodiments, the epitope is isolated from Y _n -B _t -A _r and/or As _{-Cu -Z p} if the polypeptide is processed by the APC. In some embodiments, the polypeptide is cleaved at a higher rate when n is an integer from 1 to 1000 compared to the cleavage of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m ; and/or wherein the polypeptide is cleaved at a higher rate if p is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m . In some embodiments, the presentation of the APC epitope is enhanced if n is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m ; and/or wherein the presentation of the epitope by the APC is enhanced when p is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of the nucleic acid sequence in the genome of the subject that encodes X _m . In some embodiments, the APC presents the epitope to an immune cell.

In some embodiments, the immunogenicity is increased if n is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m ; and/or where the immunogenicity is increased if p is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m .

In some embodiments, the antitumor activity is increased if n is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m ; and/or wherein the antitumor activity is increased if p is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _{m .}

In some embodiments, Y _n and/or Z _p comprise a sequence selected from the group consisting of lysine (Lys), poly-Lys (polyK), and poly-Arg (polyR). In some embodiments, polyK comprises poly-L-Lys. In some embodiments, polyR comprises poly-L-Arg. In some embodiments, polyK or polyR comprises at least two, three, or four contiguous lysine (SEQ ID NO: 1) or arginine (SEQ ID NO: 2) residues, respectively.

In some embodiments, the epitope binds to HLA MHC class II. In some embodiments, the epitope binds to HLA MHC class II with a stability of 10 minutes to 24 hours. In some embodiments, the epitope binds to HLA MHC class II with an affinity of 0.1 to 2000 nM, 1 to 1000 nM, 10 to 500 nM, or less than 1000 nM.

In some embodiments, the polypeptide is not cleaved prior to APC processing or prior to APC internalization in the subject. In some embodiments, the polypeptide is stable in human plasma. In some embodiments, the polypeptide has a half-life of 1 hour to 5 days in human plasma. In some embodiments, the subject is a human.

In some embodiments, the epitope binds to the protein encoded by the HLA allele with an affinity of less than 10 μM, less than 1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than more than 200 nM, less than 150 nM, less than 100 nM, or less than 50 nM. In some embodiments, the epitope binds to the protein encoded by the HLA allele with a stability of greater than 24 hours, greater than 12 hours, greater than 9 hours, greater than 6 hours, greater than 5 hours, greater than 4 hours, greater than 3 hours, greater than 2 hours, greater than 1 hour, greater than 45 minutes, greater than 30 minutes, greater than 15 minutes, or greater than 10 minutes. In some embodiments, the HLA allele is selected from the group consisting of HLA-A02:01 allele, HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, HLA-A74:01 allele and/or HLA-C08:02 allele and any combinations thereof. In some embodiments, the epitope comprises a tumor-specific epitope. In some embodiments, the epitope comprises at least one mutant amino acid. In some embodiments, at least one mutant amino acid is encoded by an insertion, deletion, frameshift, neoORF, or point mutation in a nucleic acid sequence in the genome of the subject.

In some embodiments, the epitope is a RAS epitope. In some embodiments, the epitope comprises a mutant RAS peptide sequence that comprises at least 8 contiguous amino acids of a mutant RAS protein comprising a G12, G13, or Q61 mutation and a G12, G13, or Q61 mutation. In some embodiments, at least 8 contiguous amino acids of a mutant RAS protein comprising a mutation at G12, G13, or Q61 comprise a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation. In some embodiments, the G12, G13, or Q61 mutation comprises a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation. In some embodiments, the PAH epitope comprises the amino acid sequence VVVGAAGVGK (SEQ ID NO: 192), VVVGAAGVG (SEQ ID NO: 193), VVVGAAGV (SEQ ID NO: 125), VVGAAGVGK (SEQ ID NO: 194), VVGAAGVG (SEQ ID NO: 195), VGAAGVGK (SEQ ID NO: 196), VVVGACGVGK (SEQ ID NO: 197), VVVGACGVG (SEQ ID NO: 198), VVVGACGV (SEQ ID NO: 199), VVGACGVGK (SEQ ID NO: 200), VVGACGVG (SEQ ID NO: 201), VGACGVGK (SEQ ID NO: 202), VVVGADGVGK (SEQ ID NO: 203), VVVGADGVG (SEQ ID NO: 204), VVVGADGV (SEQ ID NO: 205), VVGADGVGK (SEQ ID NO: 206), VVGADGVG (SEQ ID NO: 207), VGADGVGK (SEQ ID NO: 122), VVVGARGVGK (SEQ ID NO: 208), VVVGARGVG (SEQ ID NO: 209), VVVGARGV (SEQ ID NO: 63), VVGARGVGK (SEQ ID NO: 210), VVGARGVG (SEQ ID NO: 211), VGARGVGK (SEQ ID NO: 212), VVVGASGVGK (SEQ ID NO: 65), VVVGASGVG (SEQ ID NO: 213), VVVGASGV (SEQ ID NO: 214), VVGASGVGK (SEQ ID NO: 215), VVGASGVG (SEQ ID NO: 216), VGASGVGK (SEQ ID NO: 217), VVVGAVGVGK (SEQ ID NO: 218), VVVGAVGVG (SEQ ID NO: 219), VVVGAVGV (SEQ ID NO: 126), VVGAVGVGK (SEQ ID NO: 220), VVGAVGVG (SEQ ID NO: 221) or VGAVGVGK (SEQ ID NO: 222). In some embodiments, Y _n comprises the amino acid sequence of KKKK (SEQ ID NO: 1), KKKKK (SEQ ID NO: 3), KKKKKKK (SEQ ID NO: 223), KKKKKKKK (SEQ ID NO: 224), KTEY (SEQ ID NO: 225), KTEYK (SEQ ID NO: 226), KTEYKL (SEQ ID NO: 227), KTEYKLV (SEQ ID NO: 228), KTEYKLVV (SEQ ID NO: 229), KTEYKLVVV (SEQ ID NO: 230), KKTEY (SEQ ID NO: 231), KKTEYK (SEQ ID NO: 232), KKTEYKL (SEQ ID NO: 233), KKTEYKLV (SEQ ID NO: 234), KKTEYKLVV (SEQ ID NO: 235), KKTEYKLVVV (SEQ ID NO: 236), KKKTEY (SEQ ID NO: 237), KKKTEYK (SEQ ID NO: 238), KKKTEYKL (SEQ ID NO: 239), KKKTEYKLV (SEQ ID NO: 240), KKKTEYKLVV (SEQ ID NO: 241), KKKTEYKLVVV (SEQ ID NO: 242), KKKKTEY (SEQ ID NO: 243), KKKKTEYK (SEQ ID NO: 244), KKKKTEYKL (SEQ ID NO: 245), KKKKTEYKLV (SEQ ID NO: 246), KKKKTEYKLVV (SEQ ID NO: 247), KKKKTEYKLVVV (SEQ ID NO: 248), IDIIMKIRNA (SEQ ID NO: 131), FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC (SEQ ID NO: 132), FFFFFFFFFFFFFFFFFFFFFFFFAAFWFW (SEQ ID NO: 133), IFFIFFIIFFFFFFFFFFFFIIIIIIIWEC (SEQ ID NO: 134), FIFFFIIFFFIFFFFFIFIIIIIIFWEC (SEQ ID NO: 135), TEY, TEYK (SEQ ID NO: 249), TEYKL (SEQ ID NO: 250), TEYKLV (SEQ ID NO: 136), TEYKLVV (SEQ ID NO: 251), TEYKLVVV (SEQ ID NO: 252), WQAGILAR (SEQ ID NO: 137), HSYTTAE (SEQ ID NO: 138), PLTEEKIK (SEQ ID NO: 139), GALHFKPGSR (SEQ ID NO: 140), RRANKDATAE (SEQ ID NO: 141), KAFISHEEKR (SEQ ID NO: 142), TDLSSRFSKS (SEQ ID NO: 143), FDLGGGTFDV (SEQ ID NO: 144), CLLLHYSVSK (SEQ ID NO: 145), KKKKIIMKIRNA (SEQ ID NO: 146) or MTEYKLVVV (SEQ ID NO: 147). In some embodiments, Z _p comprises the amino acid sequence of K, KK, KKK, KKKK (SEQ ID NO: 1), KKKKK (SEQ ID NO: 3), KKKKKKK (SEQ ID NO: 223), KKKKKKKK (SEQ ID NO: 224), KKNKKDDI (SEQ ID NO: 148), KKNKKDDIKD (SEQ ID NO: 149), AGNDDDDDDDDDDDDDDDDDKKDKDDDDDD (SEQ ID NO: 150), AGNKKKKKKKKNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 151), AGRDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 152), SALTI (SEQ ID NO: 153), SALTIQL (SEQ ID NO: 154), GKSALTIQL (SEQ ID NO: 155), GKSALTI (SEQ ID NO: 156), SALTIK (SEQ ID NO: 253), SALTIQLK (SEQ ID NO: 254), GKSALTIQLK (SEQ ID NO: 255), GKSALTIK (SEQ ID NO: 256), SALTIKK (SEQ ID NO: 257), SALTIQLKK (SEQ ID NO: 258), GKSALTIQLKK (SEQ ID NO: 259), GKSALTIKK (SEQ ID NO: 260), SALTIKKK (SEQ ID NO: 261), SALTIQLKKK (SEQ ID NO: 262), GKSALTIQLKKK (SEQ ID NO: 263), GKSALTIKKK (SEQ ID NO: 264), SALTIKKKK (SEQ ID NO: 265), SALTIQLKKKK (SEQ ID NO: 266), GKSALTIQLKKKK (SEQ ID NO: 267), GKSALTI (SEQ ID NO: 156),KKKK (SEQ ID NO: 1), QGQNLKYQ (SEQ ID NO: 157), ILGVLLLI (SEQ ID NO: 158), EKEGKISK (SEQ ID NO: 159), AASDFIFLVT (SEQ ID NO: 160), KELKQVASPF (SEQ ID NO: 161), KKKLINEKKE (SEQ ID NO: 162), KKCDISLQFF (SEQ ID NO: 163), KSTAGDTHLG (SEQ ID NO: 164), ATFYVAVTVP (SEQ ID NO: 165), LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG (SEQ ID NO: 166) or TIQLIQNHFVDEYDPTIEDSYRKQVVIDGE (SEQ ID NO: 167). In some embodiments, the polypeptide comprises the amino acid sequence KTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 268), KTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 269), KTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 270), KTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 271), KKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 272), KKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 273), KKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 274), KKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 275), KKKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 276), KKKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 277), KKKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 278), KKKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 279), KKKKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 280), KKKKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 281), KKKKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 282), KKKKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 283) KKTEYKLVVVGAVGVGKSALTIQLKK (SEQ ID NO: 284), KKTEYKLVVVGADGVGKSALTIQLKK (SEQ ID NO: 285), KKTEYKLVVVGARGVGKSALTIQLKK (SEQ ID NO: 286), KKTEYKLVVVGACGVGKSALTIQLKK (SEQ ID NO: 287), TEYKLVVVGAVGVGKSALTIQLK (SEQ ID NO: 288), TEYKLVVVGADGVGKSALTIQLK (SEQ ID NO: 289), TEYKLVVVGARGVGKSALTIQLK (SEQ ID NO: 290), TEYKLVVVGACGVGKSALTIQLK (SEQ ID NO: 291), TEYKLVVVGAVGVGKSALTIQLKK (SEQ ID NO: 292), TEYKLVVVGADGVGKSALTIQLKK (SEQ ID NO: 293), TEYKLVVVGARGVGKSALTIQLKK (SEQ ID NO: 294), TEYKLVVVGACGVGKSALTIQLKK (SEQ ID NO: 295), TEYKLVVVGAVGVGKSALTIQLKKK (SEQ ID NO: 296), TEYKLVVVGADGVGKSALTIQLKKK (SEQ ID NO: 297), TEYKLVVVGARGVGKSALTIQLKKK (SEQ ID NO: 298), TEYKLVVVGACGVGKSALTIQLKKK (SEQ ID NO: 299), TEYKLVVVGAVGVGKSALTIQLKKKK (SEQ ID NO: 300), TEYKLVVVGADGVGKSALTIQLKKKK (SEQ ID NO: 301), TEYKLVVVGARGVGKSALTIQLKKKK (SEQ ID NO: 302), or TEYKLVVVGACGVGKSALTIQLKKKK (SEQ ID NO: 303). In some embodiments, the epitope is not a RAS epitope. In some embodiments, the polypeptide is not KKKKKPKRDGYMFLKAESKIMFAT (SEQ ID NO: 168), KKKKYMFLKAESKIMFATLQRSS (SEQ ID NO: 169), KKKKKAESKIMFATLQRSSLWCL (SEQ ID NO: 170), KKKKKIMFATLQRSSLWCLCSNH (SEQ ID NO: 171), or KKKKMFATLQRSSLWCLCSNH (SEQ ID NO: 172).

In some embodiments, Y _n and/or Z _p comprise an amino acid sequence of a protein different from the protein from which the epitope is derived. In some embodiments, Y _n and/or Z _p comprise an amino acid sequence of a CMV protein, such as pp65, HIV, or MART-1. In some embodiments, n is 11, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an integer greater than 20. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an integer greater than 20.

In some embodiments, the epitope is a TMPRSS2:ERG epitope. In some embodiments, the TMPRSS2:ERG epitope comprises the amino acid sequence ALNSEALSV (SEQ ID NO: 304).

The invention also relates to a polynucleotide comprising a sequence encoding a polypeptide described in the present invention. In some embodiments, the polynucleotide is mRNA.

The invention also relates to a pharmaceutical composition comprising a polypeptide described in the present invention or a polynucleotide described in the present invention; and a pharmaceutically acceptable excipient.

The invention also relates to a method of treating a disease or condition comprising administering a therapeutically effective amount of a pharmaceutical composition described in the present invention to a subject in need thereof. In some embodiments, the disease or condition is a cancer selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, colorectal cancer, uterine cancer, prostate cancer, liver cancer, biliary tract cancer, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia, and breast cancer. In some embodiments, the administration comprises intradermal injection, intranasal spray application, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, or subcutaneous injection.

The invention also relates to a method for producing antigen-specific T cells, comprising stimulating T cells with antigen-presenting cells containing a polypeptide described in the present invention or a polynucleotide encoding a polypeptide described in the present invention. In some embodiments, the method is performed ex vivo.

BRIEF DESCRIPTION OF DRAWINGS

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 disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments employing the principles of the disclosure, and the accompanying drawings, in which:

FIG. 1 depicts a simplified exemplary epitope processing and presentation of an X epitope on an XHLA allele by antigen-presenting cells (APCs). In a natural context, a peptide comprises an amino acid or amino acid sequence that naturally flank the epitope sequence. In a rational context, a peptide comprises at the N- and/or C-terminus of the epitope sequence an amino acid or amino acid sequence that is not encoded by the genome encoding the epitope sequence, and/or a linker.

FIG. 2 shows an approximate cleavage of a cathepsin B polypeptide containing a cathepsin B cleavable linker.

FIG. 3 shows a schematic of the experimental design for in vitro screening of polypeptides for epitope processing and presentation using T cell receptor (TCR)-transduced cells (results shown in FIGS. 4 and 5).

FIG. 4 is a graph showing the level of IL-2 (pg/ml) secreted by KRAS-specific Jurkat cells after 48 hours of co-culture with an equal number of peripheral blood mononuclear cells (PBMCs) loaded with either a peptide containing only the KRAS-G12V epitope or a peptide containing the KRAS-G12V epitope and additional amino acid sequences naturally flanking the KRAS-G12V epitope at the N- and C-terminus. SEQ ID NOS 220 and 387, respectively, are described in the figure in order of appearance.

FIG. 5 is a graph showing the level of IL-2 (pg/ml) secreted by KRAS-specific Jurkat cells after 48 hours of co-culture with an equal number of peripheral blood mononuclear cells (PBMCs) loaded with either a peptide containing only the KRAS-G12V epitope, a peptide containing the KRAS-G12V epitope and additional amino acid sequences naturally flanking the KRAS-G12V epitope at the N- and C-terminus, or a peptide containing the KRAS-G12V epitope and additional amino acid sequences rationally designed not by the naturally flanking KRAS-G12V epitope (rational context) at the N- and/or C-terminus. SEQ ID NOS 220 and 387-393, respectively, are described in the figure in order of appearance.

FIG. 6 is a flow chart of the experimental design of the immunogenicity study. Mice were immunized on days 0, 7, and 14 with different polypeptide designs, and blood was collected on days 7, 14, and 21 to assess antigen-specific CD8+ T cell responses (results are shown in FIGS. 7-9).

FIG. 7 shows graphs showing overall immune responses (7A: H-2K ^b , 7B: H-2D ^b , 7C: total). The figure footnote describes "K4" as SEQ ID NO: 1 and "K4-Val-Cit" as SEQ ID NO: 384.

FIG. 8 shows graphs demonstrating that immunization with K4 epitopes ("K4" is described as SEQ ID NO: 1) enhances immune responses to H-2K ^b -presented epitopes (8A: Alg8, 8B: Lama4). The figure footnote describes "K4" as SEQ ID NO: 1 and "K4-Val-Cit" as SEQ ID NO: 384.

FIG. 9 shows graphs demonstrating that immunization with K4 epitopes ("K4" is described as SEQ ID NO: 1) increases immune responses to H-2D ^b -presented epitopes (9A: Reps1, 9B: Adpgk, 9C: Irgq, 9D: Obsl1). The figure footnote describes "K4" as SEQ ID NO: 1 and "K4-Val-Cit" as SEQ ID NO: 384.

In FIG. 10 is a graph showing that the level of IL-2 (pg/ml) secreted by Jurkat cells after 24 h co-culture with 293T cells (5:1 ratio of Jurkats to 293T cells) loaded with a peptide containing the TMPRSS2::ERG epitope alone or transduced with a plasmid that encodes a peptide containing the TMPRSS2::ERG epitope in a natural context (i.e., the peptide further comprises an amino acid or amino acid sequence that naturally flank the epitope sequence at the N- and/or C-terminus), a plasmid that encodes a peptide containing the TMPRSS2::ERG epitope in a non-natural context (i.e., the peptide further comprises an amino acid or amino acid sequence that does not naturally flank the epitope sequence), or a plasmid that encodes an irrelevant epitope in a non-natural context (i.e., the peptide further comprises an amino acid or amino acid sequence that does not naturally flank the epitope sequence), natural context (as a control).

FIG. 11 shows a graph of IL-2 concentration (pg/mL) versus peptide concentration (nM) in FLT3L-treated PBMCs exposed to increasing amounts of the indicated RAS-G12V mutant peptides after co-culture with Jurkat cells transduced with a TCR that binds to the underlined RAS-G12V MHC-associated epitope encoded by the HLA-A11:01 allele. SEQ ID NOS 220, 390, and 387, respectively, are described in the figure in order of appearance.

FIG. 12 shows data illustrating the immunogenicity of the designated RAS-G12V mutant peptides from FIG. 11 both in vitro, using PBMC from healthy donors (top), and in vivo, using HLA-A11:01 transgenic mice immunized with the peptides (bottom).

FIG. 13A depicts exemplary schematics of mRNA constructs using shortmers (9-10 amino acids, top) and longmers (25 amino acids, bottom) used for expression in cells. The linker sequences are described in the figure as SEQ ID NOS 394, 386, 385, 395, 386, and 385, respectively, in order of appearance.

FIG. 13B shows a typical graph of multimer-specific CD8+ cells as a proportion of total CD8+ cells. The antigens used for the multimer analysis are shown.

FIG. 13C depicts exemplary flow cytometric assays for the detection of multimer-positive CD8+ T cells comparing short (9-10 amino acids) and long (25 amino acids) peptide-stimulated APCs and APCs containing RNAs encoding the same short (9-10 amino acids) and long (25 amino acids) peptides.

DETAILED DESCRIPTION

The present invention describes novel immunotherapeutic compositions comprising a tumor-specific antigen or neoepitope of an individual and their use based on the discovery of methods for enhancing the processing and presentation of the epitope to stimulate an immune response. Accordingly, the present disclosure provided in the present invention relates to peptides that can be used, for example, to stimulate an immune response to a tumor-associated antigen or neoepitope, to create an immunogenic composition or an anti-cancer vaccine for use in the treatment of cancer, a disease or a condition.

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 in the present invention and therefore may vary. Those skilled in the art will understand that there are numerous variations and modifications of the present disclosure that are within its scope.

All terms should be understood as understood by a person skilled in the art. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by a person skilled in the art to which the description pertains.

The section headings used in this disclosure are for organizational purposes only and should not be construed as limiting the subject matter described.

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

The following definitions are supplementary to definitions in the art and pertain to the present application and shall not be construed as referring to any related or unrelated instance, such as any joint patent or application. Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice of testing the present disclosure, the preferred materials and methods are described in the present invention. Accordingly, the terminology used in the present invention is for the purpose of describing particular embodiments only and is not intended to be limiting.

1. Definitions

The terminology used in the present invention is intended to describe particular instances only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically indicated otherwise. As used in the present invention, the singular forms also include the plural forms unless the context clearly dictates otherwise.

In this application, the use of "or" means "and/or" unless otherwise specified. The terms "and/or" and "any combination thereof" and their grammatical equivalents as used herein may be used interchangeably. These terms may mean that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B and/or C" or "A, B, C or any combination thereof" may mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B and C." The term "or" may be used together or separately unless the context specifically indicates separate use.

The term "about" or "approximately" may mean within an acceptable range of errors for a particular value, a determination by one of ordinary skill in the art that will depend in part on how the quantity is measured or defined, i.e., the limitations of the measurement system. For example, "about" may mean a value within 1 or more than 1 standard deviation in accordance with practice in the art. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of the value. When particular values are described in the application and claims, unless otherwise indicated, the term "about" should be used to mean within an acceptable range of errors for the particular value.

In the present specification and claims, the words "comprising" (and any form of inclusion, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of inclusion, such as "include" and "includes"), or "containing" (and any form of inclusion, such as "comprises" and "comprises") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be practiced with respect to any method or composition of the present specification, and vice versa. Furthermore, the compositions of the present specification can be used to achieve the methods of the present specification.

Reference in the description to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments of the present disclosure. To facilitate understanding of the present disclosure, a number of terms and phrases are defined below.

The nomenclature used to describe peptides or proteins follows the conventional practice of having the amino group on the left (the amino or N-terminus) and the carboxyl group on the right (the carboxy or C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope, they are numbered in the amino to carboxy direction, with the first position being the residue located at the amino terminus of the epitope, or of the peptide or protein of which it may be a part. In the formula representing selected specific embodiments of the present invention, the amino and carboxy terminus groups, although not specifically shown, are in the form they assume at physiological pH, unless otherwise indicated. In the structural formula of an amino acid, each residue is typically represented by standard three-letter or single-letter notations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for amino acid residues having D-forms is represented by a lowercase single letter or a lowercase three-letter symbol. However, if three-letter symbols or full names are used without capital letters, they may refer to L-amino acid residues. Glycine does not have an asymmetric carbon atom and is simply called "Gly" or "G". The amino acid sequences of the peptides referred to in the present invention are generally designated using a standard single-letter symbol. (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).

The term "residue" refers to an amino acid residue or an amino acid residue mimetic incorporated into a peptide or protein via an amide bond or an amide bond mimetic, or a nucleic acid (DNA or RNA) that encodes the amino acid or amino acid mimetic.

"Polypeptide", "peptide" and their grammatical equivalents as used herein refer to a polymer of amino acid residues. A "mature protein" is a protein that is full-length and that optionally contains glycosylation or other modifications typical of a protein in a given cellular environment. The polypeptides and proteins (including functional portions and functional variants thereof) disclosed herein may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, 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-tetrahydroisoquinodine-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. The present disclosure further contemplates that expression of the polypeptides described herein in an engineered cell may be associated with post-translational modifications of one or more amino acids of the polypeptide constructs. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfonation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypylation, lipoylation, and iodination.

The term "peptide" refers to a series of amino acid residues linked together, usually by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues.

"Synthetic peptide" refers to a peptide derived from a non-natural source, such as an artificial one. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptides" include "fusion proteins."

An "epitope" is a set of molecular characteristics, such as primary, secondary, and tertiary peptide structure and charge, that together form a site recognized by, for example, an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor. Alternatively, an epitope may be defined as a set of amino acid residues that are involved in recognition by a particular immunoglobulin or, in the context of T cells, those residues that are required for recognition by T cell receptor proteins, chimeric antigen receptors, and/or major histocompatibility complex (MHC) receptors. A "T cell epitope" is a peptide sequence that can be linked to MHC class I or II molecules in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by T cells such as T lymphocytes or T helper cells. Epitopes can be obtained by isolation from a natural source or they can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues, "amino acid mimetics", such as D-isomers of naturally occurring L-amino acid residues or non-naturally occurring amino acid residues such as cyclohexylalanine. In the present specification, epitopes may in some cases be referred to as peptides or peptide epitopes. It should be appreciated that proteins or peptides that comprise an epitope or analog described herein, as well as additional amino acids, are still within the scope of the present disclosure. In some embodiments, the peptide comprises a fragment of an antigen. In some embodiments, there is a limitation on the length of a peptide of the present disclosure. A limited length embodiment occurs if a protein or peptide comprising an epitope described herein comprises a region (i.e., a contiguous series of amino acid residues) that has 100% identity to the native sequence. In order to avoid the determination of the epitope when reading, for example, for entire natural molecules, there is a limitation on the length of any region that has 100% identity with the native peptide sequence. Thus, for a peptide comprising an epitope described in the present invention and a region with 100% identity with the native peptide sequence, the region with 100% identity with the native sequence typically has a length of: less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, or less than or equal to 50 amino acid residues. In some embodiments, an "epitope" described herein consists of a peptide having a region of less than 51 amino acid residues that has 100% identity to a native peptide sequence in any increment of up to 5 amino acid residues; for example 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue.

The term "derivative" and its grammatical equivalents, when used to discuss an epitope, are synonymous with the term "derived" and its grammatical equivalents. A derived epitope may be isolated from a natural source or may be synthesized according to standard protocols in the art. Synthetic epitopes may contain artificial amino acid residues "amino acid mimetics" such as D-isomers of naturally occurring L-amino acid residues or non-natural amino acid residues such as cyclohexylalanine. A derived or derived epitope may be an analog of a native epitope.

An "immunogenic" peptide or "immunogenic" epitope or "peptide epitope" is a peptide that contains an allele-specific motif such that the peptide will bind to an HLA molecule and induce 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 in the present invention are capable of binding to an appropriate HLA molecule and thereafter inducing a CTL (cytotoxic) response or an HTL (and humoral) response to the peptide.

"Neoantigen" means a class of tumor antigens that arise from tumor-specific alterations of proteins. Neoantigens include, but are not limited to, tumor antigens that arise from, for example, a protein sequence substitution, a frameshift mutation, a fusion polypeptide, an in-frame deletion, an insertion, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypeptides.

The terms "mutant peptide," "tumor-specific peptide," "neoantigen peptide," and "neoantigenic peptide," as used interchangeably with "peptide" herein, refer to a series of residues, typically L-amino acids, linked to one another, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Similarly, the term "polypeptide" is used interchangeably with "mutant polypeptide," "neoantigen polypeptide," and "neoantigenic polypeptide" herein to refer to a series of residues, e.g., L-amino acids, linked to one another, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The polypeptides or peptides may be of various lengths, either in their neutral (uncharged) form or in the form of salts, and either be without modifications such as glycosylation, side chain oxidation or phosphorylation, or contain these modifications, provided that the modification does not destroy the biological activity of the polypeptides as described in the present invention. The peptide or polypeptide used in the present invention comprises at least one flanking sequence. The term "flanking sequence" as used in the present invention refers to a fragment or region of a neoantigen peptide that is not part of a neoepitope.

"Neoepitope", "tumor-specific neoepitope", "tumor-specific epitope" or "tumor antigen" refers to an epitope or region of an antigenic determinant that is not present in a reference cell, such as a non-disease cell, such as a non-cancerous cell or a germline cell, but is found in a diseased cell, such as a cancer cell. This includes situations where the corresponding epitope is found in a normal healthy cell or a germline cell, but due to one or more mutations in the diseased cell, such as a cancer cell, the epitope sequence is altered such that a neoepitope is obtained. The term "neoepitope" as used herein refers to a region of an antigenic determinant within a peptide or neoantigenic peptide. A neoepitope may comprise at least one "anchor residue" and at least one "anchor residue flanking region". A neoepitope may further comprise a "separation region". The term "anchor residue" refers to an amino acid residue that binds to specific pockets on HLA, resulting in specificity of interactions with HLA. In some cases, the anchor residue may be in a canonical anchor position. In other cases, the anchor residue may be in a non-canonical anchor position. Neoepitopes can bind to HLA molecules through primary and secondary anchor residues projecting into pockets in the peptide-binding groove. In the peptide-binding groove, specific amino acids constitute pockets that accommodate the corresponding side chains of the anchor residues of the presented neoepitopes. Peptide binding preferences exist among different alleles of both HLA I and HLA II molecules. HLA class I molecules bind short neoepitopes whose N- and C-termini are anchored in pockets located at the ends of the neoepitope-binding groove. While most HLA class I-binding neoepitopes are approximately 9 amino acids long, longer neoepitopes can be accommodated by the prominence of their central portion, resulting in binding neoepitopes of approximately 8-12 amino acids. Neoepitopes binding to HLA class II proteins are not limited in size and can contain approximately 16 to 25 amino acids. The neoepitope-binding groove in HLA class II molecules is open at both ends, allowing binding of relatively longer peptides. Although the 9-amino acid core segment contributes most to neoepitope recognition, the flanking regions of the anchor residue are also important for the specificity of the peptide for the HLA class II allele. In some cases, the flanking region of the anchor residue is the N-terminal residue. In other cases, the flanking region of the anchor residue is the C-terminal residue. In another case, the flanking region of the anchor residue is both the N-terminal residues and the C-terminal residues. In some cases, the flanking region of the anchor residue is flanked by at least two anchor residues. The flanking region of the anchor residue flanked by anchor residues is the "spacer region".

The "major histocompatibility complex" or "MHC" is a cluster of genes that plays a role in controlling cellular interactions responsible for physiological immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see Paul, Fundamental Immunology, ^3rd 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 capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential lymphocyte epitopes (e.g. a T-cell epitope and a B-cell epitope), transporting them to the cell surface and presenting 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 genetic region whose gene products, expressed on the cell surface, are important for the binding and presentation of endogenous and/or foreign antigens and thus for the regulation of immunological processes. The major histocompatibility complex is divided into two groups of genes encoding different proteins, namely MHC class I molecules and MHC class II molecules. The cell biology and expression profiles of the two MHC classes are tailored to these distinct roles.

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

"Peptide-MHC (pMHC) stability" refers to the length of time it takes for half the amount of a particular peptide to dissociate from the cognate HLA in a biochemical assay.

Antigen-presenting cells (APCs) are cells that present peptide fragments of protein antigens in association with MHC molecules on their cell surface. Some APCs can activate antigen-specific T cells. Mature professional antigen-presenting cells internalize antigen very efficiently, either by phagocytosis or receptor-mediated endocytosis, and then display the antigen fragment bound to an MHC class II molecule on their membrane. The T cell recognizes and interacts with the antigen-MHC class II molecular complex on the membrane of the antigen-presenting cell. The antigen-presenting cell then produces an additional costimulatory signal, leading to T cell activation. Expression of costimulatory molecules is a defining feature of professional antigen-presenting cells. The major types of professional antigen-presenting cells are dendritic cells, which have the broadest spectrum of antigen presentation and are probably the most important antigen-presenting cells, macrophages, B cells, and some activated epithelial cells. "Dendritic cells (DCs)" are populations of leukocytes that present antigens captured in peripheral tissues to T cells via the MHC class II and I antigen-presenting pathways. Dendritic cells are well known to be potent inducers of immune responses, and activation of these cells is an important step in the induction of antitumor immunity. Dendritic cells are conveniently classified as "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be construed as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with high antigen uptake and processing capacity, which correlates with high expression of the Fc receptor (FcR) and mannose receptor. The mature phenotype is typically characterized by lower expression of these markers but high expression of cell surface molecules responsible for T cell activation, such as MHC class I and class II, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

The terms "polynucleotide", "nucleotide", "nucleic acid", "polynucleic acid" or "oligonucleotide" and their grammatical equivalents are used interchangeably in the present invention and refer to polymers of nucleotides of any length and include DNA and RNA, such as mRNA. Thus, these terms include double-stranded and single-stranded DNA, triplex DNA, and double-stranded and single-stranded RNA. It also includes modified, such as by methylation and/or capping, and unmodified forms of a polynucleotide. The term also includes molecules that include non-naturally occurring or synthetic nucleotides, as well as nucleotide analogs. The nucleic acid sequences and vectors described or contemplated in the present invention can be introduced into a cell, such as by transfection, transformation or transduction. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid may be in vitro transcribed mRNA. In some embodiments, the polynucleotide that is administered using the methods of the present disclosure is mRNA.

A "standard" may be used to correlate and compare the results obtained in the methods of the present description from a tumor sample. Typically, the "standard" may be derived from one or more normal samples, in particular samples not affected by cancer, obtained from a patient or one or more different individuals, such as healthy individuals, in particular individuals of the same species. The "standard" may be determined empirically by testing a sufficiently large number of normal samples.

The term "mutation" or "mutant" refers to a change or difference in a nucleic acid sequence (a nucleotide substitution, addition, insertion, or deletion) compared to a reference. A "somatic mutation" can occur in any cell of the body except germ cells (sperm and eggs), and is therefore not passed on to children. These changes may (but do not always) cause cancer or other diseases. In some embodiments, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation, such as a nucleotide substitution, that actually results in an amino acid change, such as an amino acid change in a translation product. A "frameshift" occurs when a mutation disrupts the normal phase of a gene's codon periodicity (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. It is possible for different mutations in a gene to result in the same altered reading frame. A "neoORF" can be created if an open reading frame (ORF) is altered by various mutational events in the genome, such as missense mutations, fusion transcripts, frameshifts, and/or loss of stop codons. A neoORF can encode new amino acid sequences that are not present in the normal genome.

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

A "native" or "wild-type" sequence refers to a sequence that occurs in nature. Such a sequence may contain a longer sequence in nature.

As used herein, the term "affinity" refers to a measure of the strength of binding between two members of a binding pair, such as an HLA-binding peptide and HLA class I or II. K _D is the dissociation constant in molar units. The affinity constant is the reciprocal of the dissociation constant. The affinity constant is sometimes used as a general term to describe a chemical compound. It is a direct measure of the binding energy. Affinity can be determined experimentally, such as by surface plasmon resonance (SPR) using commercially available Biacore SPR devices. Affinity can also be expressed as the inhibitory concentration 50 (IC ₅₀ ), the concentration at which 50% of the peptide is displaced. Similarly, ln(IC ₅₀ ) refers to the natural logarithm of the IC ₅₀ . K _off refers to the dissociation rate constant, for example for the dissociation of an HLA-binding peptide and HLA class I or II. In this specification, "binding data" or "binding assay" results may be expressed in units of "IC ₅₀ ". The IC ₅₀ is the concentration of the test peptide in a binding assay that exhibits 50% inhibition of binding of the labeled reference peptide. Taking into account the conditions under which the assays are performed (i.e., HLA protein restriction and labeled reference peptide concentrations), these values approximate K _D values. Binding assays are well known in the art and are described in detail, for example, in PCT publications WO 94/20127 and WO 94/03205 and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding to a standard reference peptide. For example, it can be based on its IC ₅₀ relative to the IC ₅₀ of a reference standard peptide. Binding can also be determined using other assay systems, including: 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-flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)) and measurement of MHC class I 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-linking" indicates that the peptide binds to more than one HLA molecule; a synonym is degenerate binding.

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

"Antigen processing" or "processing" and its grammatical equivalents refer to the cleavage of a polypeptide or antigen into processing products that are fragments of said polypeptide or antigen (e.g., cleavage of a polypeptide into peptides) and the association of one or more such fragments (e.g., via binding) with MHC molecules for presentation by cells, e.g., antigen-presenting cells, to specific T cells.

The term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, dogs, cats, rodents, and the like, that is subject to a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

"Cell" and its grammatical equivalents refer to a cell of human or non-human animal origin.

"T cell" includes CD4 ⁺ T cells and CD8 ⁺ T cells. The term T cell also includes both type 1 T helper T cells and type 2 T helper T cells.

As used herein, the term "vaccine" refers to a pharmaceutical preparation (pharmaceutical composition) or product that, when administered, induces an immune response, such as a cellular or humoral immune response, that recognizes and attacks a pathogen or a diseased cell, such as a cancer cell. The vaccine may be used to prevent or treat a disease. The term "individualized cancer vaccine" or "personalized cancer vaccine" refers to a specific cancer patient and means that the cancer vaccine is tailored to the needs or special circumstances of an individual cancer patient.

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. A therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow the development of a disease or disorder; slow the progression of a disease or disorder; alleviate to some extent one or more symptoms associated with the disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

The terms "treatment" or "treatment" or "treat" or "alleviate" or "alleviate" refer to both (1) therapeutic measures that cure, slow down, reduce symptoms, and/or stop the progression of a diagnosed pathological condition or disorder; and (2) prophylactic or preventive measures that prevent or slow down the development of the target pathological condition or disorder. Thus, those in need of treatment include those already suffering from the disorder; those who are susceptible to the disorder; and those in whom the disorder is to be prevented.

“Pharmaceutically acceptable” refers to a composition or component of a composition that is generally non-toxic, inert, and/or physiologically compatible.

"Pharmaceutical excipient" or "excipient" includes such material as adjuvant, carrier, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives and the like. "Pharmaceutical excipient" is an excipient that is pharmaceutically acceptable.

"Immunomodulatory agent" or its grammatical equivalent as used herein may refer to a substance that can stimulate or suppress the immune system and can help an individual's body fight a disease, such as an infection, cancer, etc. Examples of specific immunomodulatory agents that affect specific parts of the immune system include, but are not limited to, monoclonal antibodies, cytokines, and vaccines. Non-specific immunomodulatory agents affect the immune system as a whole, and non-limiting examples include Bacillus Calmette-Guerin (BCG) and levamisole.

The term "cancer" and its grammatical equivalents as used herein may refer to a hyperproliferation of cells whose unique feature - loss of normal control - results in unregulated growth, lack of differentiation, local tissue damage and metastasis. With respect to the compositions and methods of the invention, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, rectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, carcinoid tumor of the gastrointestinal tract, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, hematological malignancies, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesentery, pharyngeal cancer, prostate cancer, colon cancer, renal cancer, skin cancer, small bowel cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer and/or bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissues, such as a malignant type or a benign type.

The term "exome" refers to the portion of the genome that codes for functional proteins, or the sequence encompassing all the exons, or coding regions, of protein-coding genes in the genome. It makes up approximately 1-2% of the entire genome, depending on the species.

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

By "receptor" is meant a biological molecule or group of molecules capable of binding a ligand. A receptor can serve to transmit information in a cell, a cellular formation or an organism. A receptor comprises at least one receptor unit, for example, where each receptor unit can consist of a protein molecule. The receptor has a structure that is complementary to the structure of the ligand and can form a complex of the ligand as a binding partner. Information is transmitted, in particular, by conformational changes in the receptor after complexation of the ligand on the cell surface. In some embodiments, by receptor is meant specific proteins of MHC classes I and II capable of forming a receptor/ligand complex with a ligand, in particular a peptide or a peptide fragment of an appropriate length.

By "ligand" is meant a molecule that has a structure complementary to the structure of a receptor and is capable of forming a complex with this receptor. In some embodiments, by ligand is meant a peptide or peptide fragment that has a suitable length and suitable binding motifs in its amino acid sequence, such that the peptide or peptide fragment is capable of forming a complex with MHC class I or MHC class II proteins.

In some embodiments, "receptor/ligand complex" should also be understood to mean "receptor/peptide complex" or "receptor/peptide fragment complex", including an MHC class I or class II molecule presenting a peptide or peptide fragment.

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

The term "identical" and its grammatical equivalents as used herein, or "sequence identity" in the context of two nucleic acid sequences or amino acid sequences of polypeptides, refers to residues in the two sequences that are the same when aligned for maximum correspondence within a specified comparison window. A "comparison window" as used herein refers to a segment of at least about 20 contiguous positions, typically from about 50 to about 200, more typically from about 100 to about 150, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); using 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, among others, CLUSTAL in the PC/Gene program from 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 in Higgins and Sharp, Gene, 73:237–244 (1988) and Higgins and Sharp, CABIOS, 5:151–153 (1989); Corpet et al., Nucleic Acids Res., 16:10881–10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155–165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307–331 (1994). Alignment is also often accomplished by manual inspection and alignment. In one class of embodiments, the polypeptides of the present invention at least 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 fragment thereof, for example, according to BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to the reference nucleic acid, for example, they can have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% sequence identity to a reference nucleic acid or fragment thereof, for example, according to BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. If one molecule is said to have a certain proportion of sequence identity with a larger molecule, this means that, if the two molecules are optimally aligned, the specified proportion of residues in the smaller molecule finds a matching residue in the larger molecule according to the order in which the two molecules are optimally aligned.

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

As used in the present invention, the term "vector" means a construct that is capable of delivering and typically expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors linked to cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

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

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

As used herein, the term “substantially pure” refers to a product that is at least 50% pure (i.e., free of impurities), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

"Transfection," "transformation," or "transduction," as used herein, refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Many transfection methods are known in the art and include, for example, calcium phosphate coprecipitation of DNA (see, for example, Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; liposome-mediated cationic transfection; microprojectile bombardment with tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate coprecipitation of DNA (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after growth of infectious particles in suitable packaging cells, many of which are commercially available.

2. Improved splitting and its use

One of the critical obstacles to the development of therapeutic and tumor-specific immunotherapies is the insufficient processing and release of minimal epitopes for antigen presentation to generate adequate immune responses. Antigen processing and presentation refer to the processes that occur within a cell that result in the fragmentation or proteolysis of proteins, the association of protein fragments or peptides with major histocompatibility complex (MHC) molecules, and the expression of peptide-MHC (pMHC) molecules on the cell surface for recognition by the T cell receptor (TCR) on the T cell. Antigen presentation is mediated by MHC class I molecules and MHC class II molecules found on the surface of antigen-presenting cells (APCs) and some other cells. MHC class I and MHC class II molecules deliver short peptides to the cell surface, allowing these peptides to be recognized by cytotoxic (CD8 ⁺ ) and helper (CD4 ⁺ ) T cells, respectively. The TCR can recognize antigen only in the form of a peptide bound to an MHC molecule on the cell surface, and the antigens recognized by T cells are peptides that result from the breakdown of macromolecular structures, the unfolding of individual proteins, and their cleavage into short fragments through antigen processing.

Antigen presentation on the cell surface requires proper processing of peptides to release minimal epitopes by proteasomes, cytosolic and endoplasmic reticulum (ER) aminopeptidases, an efficient transporter associated with antigen processing (TAP), and sufficient binding to MHC class I molecules. The efficiency of epitope generation depends not only on the epitope itself but also on its flanking regions or the amino acid sequence flanking the epitope amino acid sequence. The efficiency of minimal epitope processing from a peptide containing the epitope sequence and the amino acid sequence flanking the epitope sequence is not fully understood, but is known to be influenced by several factors, including the specific amino acid residues on either side of the cleavage site in the peptide and other competing cleavage sites in the vicinity.

One way to address the issue of insufficient processing and release of the minimal epitope is to explore and design specific amino acid residues or sequences that can be added to the N- and/or C-terminus of the epitope sequence to enhance peptide cleavage and processing and epitope presentation. For example, amino acid residues or sequences from other epitopes that are known to be efficiently processed can be added to the epitope sequence. Another example is to use amino acid residues that are known to be commonly observed around epitopes (Abelin, et al., 2017, Immunity 46, 315-326). This approach may provide additional benefits including easier production (e.g., synthesis, purification, and/or formulation) or easier subsequent modification (e.g., conjugation to other molecules) of the peptides.

Another way to overcome the current barriers to efficient processing and release of minimal epitopes is to use a protease-cleavable linker to target the epitope-containing peptide for site-specific protease processing to release the epitope. For example, specific linkers that can be readily cleaved within dendritic cells (DCs) to release minimal epitope sequences could be used to enhance CD8-dependent immune responses following vaccination. Furthermore, these peptides would not have non-selective binding to MHC class I molecules on the surface of non-professional APCs and would instead pass through specific pathways (e.g., endocytosis) for proper processing and presentation to T cells. Another example of ensuring sufficient epitope processing and presentation is to combine two strategies, i.e., specific amino acid residues and specific linkers.

The invention relates to a polypeptide comprising an epitope sequence encoded by the genome of a subject, an amino acid or an amino acid sequence that may or may not be encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence that encodes the epitope sequence in the genome of the subject, an amino acid or an amino acid sequence and/or a linker. Addition of an amino acid, an amino acid sequence and/or a linker to an epitope sequence can enhance epitope processing and presentation by APCs to generate an immune response. In one aspect, the amino acid or amino acid sequence is an amino acid sequence or a peptide sequence. In one embodiment, the amino acid sequence or peptide sequence is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the genome of the subject that encodes the epitope sequence. In another embodiment, the amino acid or amino acid sequence is continuous with the epitope sequence and is encoded by the genome of the subject that encodes the epitope sequence. For example, an amino acid or amino acid sequence that is continuous with the epitope sequence may contain one or more amino acid residues that enhance cleavage of the polypeptide (e.g., lysine). In such an embodiment, the polypeptide may contain an amino acid or amino acid sequence that is continuous with the epitope sequence, and may further contain an amino acid or amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of the nucleic acid sequence that encodes the epitope sequence in the genome of the subject.

In some embodiments, the epitope is presented by MHC class I APC. In some embodiments, the epitope is presented by MHC class II APC. In some embodiments, each amino acid of the epitope is an amino acid of a peptide sequence comprising any contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject. In some embodiments, the epitope comprises 8 to 12 contiguous amino acid residues and is presented by MHC class I APC. In some embodiments, the epitope comprises 8, 9, 10, 11, or 12 contiguous amino acid residues and is presented by MHC class I APC. In some embodiments, the epitope comprises 9 to 25 contiguous amino acid residues and is presented by MHC class II APC. In some embodiments, the epitope comprises 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acid residues and is presented by MHC class II APCs. In some embodiments, the epitope sequence comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acid residues of which one of several of 13-25 amino acids is optionally present and at least one amino acid is a mutant amino acid. In some embodiments, the epitope sequence comprises AA ₁ AA ₂ AA ₃ AA ₄ AA ₅ AA ₆ AA ₇ AA ₈ AA ₉ AA ₁₀ AA ₁₁ AA ₁₂ AA ₁₃ AA ₁₄ AA ₁₅ AA ₁₆ AA ₁₇ AA ₁₈ AA ₁₉ AA ₂₀ AA ₂₁ AA _{22 AA 23 AA 24} AA ₂₅ , wherein each AA is an amino acid, and one or more of AA ₉ , AA ₁₀ , AA ₁₁ , AA ₁₂ , AA ₁₃ , AA ₁₄ , AA ₁₅ , AA ₁₆ , AA ₁₇ , AA ₁₈ , AA ₁₉ , AA ₂₀ , AA ₂₁ , AA ₂₂ , AA ₂₃ , AA ₂₄ and AA ₂₅ are optionally present, and at least one AA is a mutant amino acid.

In some embodiments, a polypeptide comprising an epitope sequence and an amino acid or an amino acid sequence that is continuous with the epitope sequence and is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence that encodes the epitope in the genome of a subject may not comprise a linker. In some embodiments, a polypeptide comprising an epitope sequence and an amino acid or an amino acid sequence that is continuous with the epitope sequence and is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence that encodes the epitope in the genome of a subject may comprise a linker. In some embodiments, a polypeptide comprising an epitope sequence and an amino acid or an amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the genome of a subject that encodes the epitope sequence may further comprise a linker. In some embodiments, a polypeptide comprising an epitope sequence and an amino acid or amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope sequence may not comprise a linker.

In some embodiments, the amino acid or amino acid sequence comprises 0 to 1000 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence that is encoded by the nucleic acid sequence immediately upstream of the nucleic acid sequence in the genome of the subject that encodes the epitope comprises 0 to 1000 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence that is encoded by the nucleic acid sequence immediately downstream of the nucleic acid sequence in the genome of the subject that encodes the epitope comprises 0 to 1000 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence comprises more than 0, more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 55, more than 60, more than 65, more than 70, more than 75, more than 80, more than 85, more than 90, more than 95, more than 100, more than 150, more than 200, more than 250, more than 300, more than 350, more than 400, more than 450, more than 500, more than 550, more than 600, more than 650, more than 700, more than 750, more than 800, more than 850, more than 900 or more than 950 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence that is encoded by the nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes the epitope comprises more than 0, more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 55, more than 60, more than 65, more than 70, more than 75, more than 80, more than 85, more than 90, more than 95, more than 100, more than 150, more than 200, more than 250, more than 300, more than 350, more than 400, more than 450, more than 500, more than 550, more than 600, more than 650, more than 700, more than 750, more than 800, more than 850, more than 900, or more than 950 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence that is encoded by the nucleic acid sequence immediately downstream of the nucleic acid sequence in the genome of the subject that encodes the epitope comprises more than 0, more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 55, more than 60, more than 65, more than 70, more than 75, more than 80, more than 85, more than 90, more than 95, more than 100, more than 150, more than 200, more than 250, more than 300, more than 350, more than 400, more than 450, more than 500, more than 550, more than 600, more than 650, more than 700, more than 750, more than 800, more than 850, more than 900, or more than 950 amino acid residues in length.

In some embodiments, the amino acid or amino acid sequence comprises 1-5 or 7-1000 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence does not comprise 6 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence of the peptide sequence that is encoded by the nucleic acid sequence immediately upstream of the nucleic acid sequence in the genome of the subject that encodes the epitope comprises 1-5 or 7-1000 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence of the peptide sequence that is encoded by the nucleic acid sequence immediately upstream of the nucleic acid sequence in the genome of the subject that encodes the epitope does not comprise 6 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence of the peptide sequence that is encoded by the nucleic acid sequence located immediately downstream of the nucleic acid sequence in the genome of the subject that encodes the epitope comprises 1-4 or 6-1000 amino acid residues in length. In some embodiments, the amino acid or amino acid sequence of the peptide sequence that is encoded by the nucleic acid sequence located immediately downstream of the nucleic acid sequence in the genome of the subject that encodes the epitope does not comprise 5 amino acid residues in length.

In some embodiments, the polypeptide further comprises a linker. In some embodiments, the polypeptide does not consist of four different epitopes presented by MHC class I. In some embodiments, the polypeptide does not comprise four different epitopes presented by MHC class I. In some embodiments, the polypeptide comprises at least two different epitopes presented by MHC class I. In some embodiments, the polypeptide comprises at least three, at least five, or at least six different epitopes presented by MHC class I. In some embodiments, the epitope comprises at least one mutated amino acid. In some embodiments, the at least one mutated amino acid is encoded by an insertion, deletion, frameshift, neoORF, or point mutation in a nucleic acid sequence in the genome of the subject. In some embodiments, an amino acid or amino acid sequence of a peptide sequence that is not encoded by a nucleic acid sequence immediately downstream or upstream of a nucleic acid sequence in the subject's genome that encodes an epitope is cleaved from the epitope if the polypeptide is processed by an APC. In some embodiments, the polypeptide comprises at least two different polypeptide molecules. In some embodiments, the polypeptide comprises at least three, at least four, or at least five different polypeptide molecules.

In some embodiments, the present disclosure comprises a polypeptide comprising an amino acid or amino acid sequence of a peptide sequence that is not encoded by a nucleic acid sequence immediately downstream or upstream of a nucleic acid sequence in the subject's genome that encodes an epitope and/or a linker. The amino acid or amino acid sequence and/or a linker may provide the polypeptide with desirable properties, such as increased solubility, stability, immunogenicity, antigen processing, or antigen presentation. In some embodiments, the polypeptide may comprise an amino acid or amino acid sequence that enhances the processing and presentation of epitopes by APCs, such as to generate an immune response. In some embodiments, the polypeptide may comprise an amino acid or amino acid sequence at either the N- or C-terminus of the epitope sequence. In some embodiments, the amino acid or amino acid sequence may comprise polylysine (poly-Lys or polyK) or polyarginine (poly-Arg or polyR). In some embodiments, the amino acid or amino acid sequence may be from a polypeptide sequence of a protein not expressed in the subject that expresses the epitope (e.g., not encoded by the subject's genome encoding the epitope sequence). In another embodiment, the polypeptide may comprise a protease-cleavable linker. In some embodiments, the polypeptide may comprise both a protease-cleavable linker and an amino acid or amino acid sequence. In some embodiments, the invention relates to a polypeptide of formula (I), (II), (III), and/or (IV), or a pharmaceutically acceptable salt of a polypeptide of formula (I), (II), (III), and/or (IV), wherein the stereochemistry is undefined, such as a racemate or a mixture of diastereomers or individual diastereomers. It is known to a person skilled in the art that at any stage of the preparation of compounds of formula (I), (II), (III) and/or (IV), mixtures of isomers (e.g., racemates) of compounds corresponding to any of formulas (I), (II), (III), and/or can be used. At any stage of the preparation, a single stereoisomer can be obtained by isolating it from a mixture of isomers (e.g., a racemate) using, for example, chiral chromatographic separation.

In some embodiments, the linker comprises a non-polypeptide linker. In some embodiments, the linker comprises a chemical linker. In some embodiments, the linker comprises a non-naturally occurring amino acid. In some embodiments, the non-naturally occurring amino acid comprises β-γ-δ-amino acids. In some embodiments, the non-naturally occurring amino acid comprises L-α-amino acid derivatives. In some embodiments, the linker does not comprise an amino acid. In some embodiments, the linker does not comprise a natural amino acid. In some embodiments, the linker comprises a bond other than a peptide bond. In some embodiments, the linker comprises a disulfide bond. In some embodiments, a polypeptide described herein comprises more than one linker. In some embodiments, a polypeptide described herein comprises a first linker and a second linker, wherein the first linker is at the N-terminus of an epitope and the second linker is at the C-terminus of the epitope. In some embodiments, the first linker and the second linker are different. In some embodiments, the first linker and the second linker are the same.

In some embodiments, the polypeptide comprises a hydrophilic tail. In some embodiments, a polypeptide comprising an epitope sequence, an amino acid, or an amino acid sequence of a peptide sequence that is not encoded by a nucleic acid sequence immediately downstream or upstream of a nucleic acid sequence in the genome of a subject that encodes an epitope and/or a linker has increased solubility compared to a polypeptide comprising the same epitope sequence without the amino acid or amino acid sequence and/or a linker. In some embodiments, a polypeptide comprising an epitope sequence and an amino acid or amino acid sequence contiguous with an epitope sequence encoded by a nucleic acid sequence in the genome of a subject has increased solubility compared to a polypeptide comprising the same epitope sequence but without the amino acid or amino acid sequence. For example, an amino acid or amino acid sequence adjacent to an epitope sequence may comprise one or more amino acid residues that increase the solubility of the polypeptide (e.g., lysine). In such an embodiment, the polypeptide may comprise an amino acid or amino acid sequence that is continuous with the epitope sequence, and may further comprise an amino acid or amino acid sequence of a peptide sequence that is not encoded by a nucleic acid sequence immediately downstream or upstream of a nucleic acid sequence in the subject's genome that encodes the epitope.

In some embodiments, the epitope is released from a polypeptide comprising the epitope sequence if the polypeptide is processed by an APC. In some embodiments, the epitope is released at a higher rate if the polypeptide further comprises an amino acid or an amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or a linker, compared to a polypeptide that comprises the same epitope but does not comprise an amino acid or an amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or a linker. In some embodiments, the epitope is released at a higher rate if the polypeptide further comprises an amino acid or an amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or a linker, compared to a polypeptide that comprises the same epitope but does not comprise an amino acid or an amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or a linker. In some embodiments, the epitope is released at a higher rate if the amino acid or amino acid sequence does not relate to a peptide sequence of a protein expressed in the subject. In some embodiments, the epitope is released at a higher rate if the polypeptide contains a linker, compared to a polypeptide that contains the same epitope but does not contain a linker. In some embodiments, the epitope is released at a higher rate if the polypeptide contains a linker that is cleaved by a protease, compared to a polypeptide that contains the same epitope but does not contain a linker that is cleaved by a protease.

In some embodiments, the epitope is released at a higher rate if a polypeptide comprising the epitope and an amino acid or an amino acid sequence that comprises at least one additional amino acid that is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope further comprises an amino acid or an amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope, and/or a linker, compared to a corresponding polypeptide that comprises the same epitope and an amino acid or an amino acid sequence that comprises at least one additional amino acid that is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope, but does not comprise an amino acid or an amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope. and/or linker.

In some embodiments, a polypeptide is cleaved at a higher rate if the polypeptide comprises an amino acid or amino acid sequence that does not contain at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes an epitope and/or a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes an epitope. In some embodiments, the polypeptide is cleaved at a higher rate if the polypeptide comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes an epitope and/or a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the genome of the subject that encodes the epitope. In some embodiments, the polypeptide is cleaved at a higher rate if the amino acid or amino acid sequence does not belong to a peptide sequence of a protein expressed in the subject. In some embodiments, the polypeptide is cleaved at a higher rate if the polypeptide comprises a linker, compared to a polypeptide that comprises the same epitope but does not comprise a linker. In some embodiments, a polypeptide is cleaved at a higher rate if the polypeptide contains a linker that is cleaved by a protease, compared to a polypeptide that contains the same epitope but does not contain a linker that is cleaved by a protease.

In some embodiments, a polypeptide is cleaved at a higher rate if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes an epitope, compared to the cleavage of a corresponding polypeptide of the same length that comprises an epitope sequence and an amino acid or amino acid sequence continuous with the epitope sequence that is encoded by a nucleic acid sequence and does not comprise a linker. In some embodiments, a polypeptide is cleaved at a higher rate if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, compared to the cleavage of a corresponding polypeptide of the same length that comprises an epitope sequence and an amino acid or amino acid sequence contiguous with the epitope sequence that is encoded by a nucleic acid sequence and does not comprise a linker.

[0151] In some embodiments, a polypeptide is cleaved at a higher rate if the polypeptide comprises (i) an amino acid or amino acid sequence that is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, and (ii) an amino acid or amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, and/or (iii) a linker compared to a corresponding polypeptide of the same length and an epitope and an amino acid or amino acid sequence that is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope.

In some embodiments, the polypeptide is cleaved in the linker region if the polypeptide is processed by the APC. In some embodiments, the polypeptide is cleaved in the linker region at a higher rate if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes an epitope and a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes an epitope. In some embodiments, the polypeptide is cleaved in the linker region at a higher rate if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes an epitope and a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the subject's genome that encodes an epitope. In some embodiments, the polypeptide is cleaved in the linker region at a higher rate if the amino acid or amino acid sequence does not belong to a peptide sequence of a protein expressed in the subject.

In some embodiments, the presentation of an epitope by an APC is enhanced if the polypeptide is processed by an APC. In some embodiments, the presentation of an epitope by an APC is enhanced if the polypeptide comprising the epitope further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes the epitope and/or a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes the epitope. In some embodiments, the presentation of an APC epitope is enhanced if the polypeptide comprising the epitope further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes the epitope. In some embodiments, the presentation of an APC epitope is enhanced if the amino acid or amino acid sequence is not a peptide sequence of a protein expressed in the subject. In some embodiments, the presentation of an APC epitope is enhanced if the polypeptide comprises a linker, compared to a polypeptide that comprises the same epitope but does not comprise a linker. In some embodiments, presentation of an APC epitope is enhanced if the polypeptide comprises a protease-cleavable linker, compared to a polypeptide that comprises the same epitope but does not comprise a protease-cleavable linker.

In some embodiments, the presentation of an APC epitope is enhanced if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence located immediately upstream of the nucleic acid sequence in the genome of the subject that encodes the epitope, compared to a cleavage of a corresponding polypeptide of the same length that comprises an epitope sequence and an amino acid or amino acid sequence continuous with the epitope sequence that is encoded by the nucleic acid sequence and does not comprise a linker. In some embodiments, the presentation of an APC epitope is enhanced if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes the epitope, compared to a cleavage of a corresponding polypeptide of the same length that comprises an epitope sequence and an amino acid or amino acid sequence continuous with the epitope sequence that is encoded by a nucleic acid sequence and does not comprise a linker.

In some embodiments, the presentation of an APC epitope is enhanced if the polypeptide comprises (i) an amino acid or amino acid sequence that is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope, and (ii) an amino acid or amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope, and/or (iii) a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes the epitope.

In some embodiments, the immunogenicity is increased if the polypeptide is processed by an APC. In some embodiments, the immunogenicity is increased if the polypeptide comprising the epitope further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes the epitope. In some embodiments, the immunogenicity is increased if the polypeptide comprising the epitope further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of the nucleic acid sequence in the subject's genome that encodes the epitope and/or linker, compared to a corresponding polypeptide of the same length and epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately downstream of the nucleic acid sequence in the subject's genome that encodes the epitope. In some embodiments, the immunogenicity is increased if the amino acid or amino acid sequence is not a peptide sequence of a protein expressed in the subject. In some embodiments, the immunogenicity is increased if the polypeptide comprises a linker, compared to a polypeptide that comprises the same epitope but does not comprise a linker. In some embodiments, immunogenicity is increased if the polypeptide comprises a protease-cleavable linker, compared to a polypeptide that comprises the same epitope but does not comprise a protease-cleavable linker.

In some embodiments, immunogenicity is increased if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes an epitope, compared to a cleavage of a corresponding polypeptide of the same length that comprises an epitope sequence and an amino acid or amino acid sequence continuous with the epitope sequence that is encoded by a nucleic acid sequence and does not comprise a linker. In some embodiments, immunogenicity is enhanced if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, compared to a cleavage of a corresponding polypeptide of the same length that comprises an epitope sequence and an amino acid or amino acid sequence continuous with the epitope sequence that is encoded by a nucleic acid sequence and does not comprise a linker.

In some embodiments, immunogenicity is enhanced if the polypeptide comprises (i) an amino acid or amino acid sequence that is encoded by a nucleic acid sequence located immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, and (ii) an amino acid or amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, and/or (iii) a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope.

In some embodiments, the antitumor activity is enhanced if the polypeptide is processed by an APC. In some embodiments, the antitumor activity of an APC is enhanced if the polypeptide comprising the epitope further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes the epitope. In some embodiments, the antitumor activity is enhanced if the polypeptide comprising the epitope further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes the epitope and/or a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the subject's genome that encodes the epitope. In some embodiments, the antitumor activity is enhanced if the amino acid or amino acid sequence does not relate to a peptide sequence of a protein expressed in the subject. In some embodiments, the antitumor activity is enhanced if the polypeptide comprises a linker, compared to a polypeptide that comprises the same epitope but does not comprise a linker. In some embodiments, antitumor activity is enhanced if the polypeptide comprises a linker that is cleavable by a protease, compared to a polypeptide that comprises the same epitope but does not comprise a linker that is cleavable by a protease.

In some embodiments, the antitumor activity is enhanced if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence located immediately upstream of a nucleic acid sequence in the genome of the subject that encodes an epitope, compared to a cleavage of a corresponding polypeptide of the same length that comprises an epitope sequence and an amino acid or amino acid sequence continuous with the epitope sequence that is encoded by a nucleic acid sequence and does not comprise a linker. In some embodiments, the antitumor activity is enhanced if the polypeptide further comprises an amino acid or amino acid sequence that does not comprise at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of the nucleic acid sequence in the genome of the subject that encodes the epitope, compared to a cleavage of a corresponding polypeptide of the same length that comprises the epitope sequence and an amino acid or amino acid sequence continuous with the epitope sequence that is encoded by the nucleic acid sequence and does not comprise a linker.

In some embodiments, the antitumor activity is enhanced if the polypeptide comprises (i) an amino acid or amino acid sequence that is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, and (ii) an amino acid or amino acid sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope, and/or (iii) a linker, compared to a corresponding polypeptide of the same length and an epitope, and the amino acid or amino acid sequence is encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the subject's genome that encodes an epitope.

In some embodiments, an APC presents an epitope to an immune cell if the polypeptide is processed by an APC. In some embodiments, an APC presents an epitope preferentially or specifically to an immune cell if the polypeptide is processed by an APC. In some embodiments, an APC presents an epitope to a phagocytic cell if the polypeptide is processed by an APC. In some embodiments, an APC presents an epitope preferentially or specifically to a phagocytic cell if the polypeptide is processed by an APC. In some embodiments, an APC presents an epitope to a dendritic cell, a macrophage, a mast cell, a neutrophil, or a monocyte if the polypeptide is processed by an APC. In some embodiments, an APC presents an epitope preferentially or specifically to a dendritic cell, a macrophage, a mast cell, a neutrophil, or a monocyte.

In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of poly-Lys (polyK) and poly-Arg (polyR). In a preferred embodiment, the polypeptide comprises a polyK sequence. In some embodiments, the polypeptide comprises a sequence selected from the group consisting of polyK-AA-AA and polyR-AA-AA, wherein each AA is an amino acid or an analog or derivative thereof. In a preferred embodiment, the polypeptide comprises polyK-AA-AA. In some embodiments, polyK comprises poly-L-Lys. In some embodiments, polyK comprises at least two contiguous lysine residues. In some embodiments, polyK comprises at least three contiguous lysine residues, such as Lys-Lys-Lys. In a preferred embodiment, polyC comprises at least four contiguous lysine residues (SEQ ID NO: 1), such as Lys-Lys-Lys-Lys (SEQ ID NO: 1), also known as K4 (SEQ ID NO: 1). In some embodiments, polyC comprises at least five, at least six, at least seven, at least eight, at least nine, or at least 10 contiguous lysine residues (SEQ ID NO: 305). In some embodiments, polyR comprises poly-L-Arg. In some embodiments, polyR comprises at least two contiguous arginine residues. In some embodiments, polyR comprises at least three contiguous arginine residues, such as Arg-Arg-Arg. In some embodiments, polyR comprises at least four, at least five, at least six, or at least seven contiguous arginine residues (SEQ ID NO: 306). In some embodiments, polyR comprises at least eight contiguous arginine residues (SEQ ID NO: 307), such as Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 307), also known as R8 (SEQ ID NO: 307). In some embodiments, polyR comprises at least five, at least six, at least seven, at least eight, at least nine, or at least 10 contiguous arginine residues (SEQ ID NO: 308). In some embodiments, each of the lysine units in polyK and/or the arginine units in polyR can have an (L) stereochemical configuration, a (D) stereochemical configuration, or any mixture of (L) and (D) stereochemical configuration.

In some embodiments, the polypeptide comprises a linker selected from the group consisting of disulfide, p-aminobenzyloxycarbonyl (PABC), and AA-AA-PABC, wherein AA is an amino acid or an analog or derivative thereof. In some embodiments, AA-AA-PABC is selected from the group consisting of alanine-lysine-PABC (Ala-Lys-PABC), valine-citrulline-PABC (Val-Cit-PABC), and phenylalanine-lysine-PABC (Phe-Lys-PABC). In some embodiments, AA-AA-PABC is Ala-Lys-PABC. In some embodiments, AA-AA-PABC is Val-Cit-PABC. In some embodiments, AA-AA-PABC is Phe-Lys-PABC. In some embodiments, each of the valine and citrulline units of Val-Cit-PABC has an (L) stereochemical configuration. In some embodiments, each of the phenylalanine and lysine units of Phe-Lys-PABC has an (L) stereochemical configuration. In some embodiments, each of the valine and citrulline units of Val-Cit-PABC has a (D) stereochemical configuration. In some embodiments, each of the phenylalanine and lysine units of Phe-Lys-PABC has a (D) stereochemical configuration. In some embodiments, the valine and citrulline units of Val-Cit-PABC have a mixed (L) and (D) stereochemical configuration. In some embodiments, the phenylalanine and lysine units of Phe-Lys-PABC have a mixed (L) and (D) stereochemical configuration.

In some embodiments, the polypeptide comprises a linker having the following structure:

Formula (II).

In some embodiments, the polypeptide comprises a linker that is

Formula (III) or

Formula (IV),

where R ₁ and R ₂ are independently H or (C ₁ -C ₆ ) alkyl; j is 1 or 2; G ₁ is H or COOH; and i is 1, 2, 3, 4 or 5.

In some embodiments, A _r and/or A _s are formula (III) or formula (IV) wherein R ₁ and R ₂ are independently H or (C ₁ -C ₆ ) alkyl; j is 1 or 2; G ¹ is H or COOH; and i is 1, 2, 3, 4, or 5.

In some embodiments, the polypeptide comprises a linker of formula (III) or formula (IV).

Disulfide linkers of formula (IV) can be synthesized according to Zhang, Donglu, et al., ACS Med. Chem. Lett. 2016, 7, 988-993; and Pillow, Thomas H., et al., Chem. Sci., 2017, 8, 366-370. PABC-containing peptides can be synthesized according to Laurent Ducry (ed.), Antibody-Drug Conju gates, Methods in Molecular Biology, vol. 1045, DOI 10.1007/978-1-62703-541-5_5, Springer Science+Business Media, LLC 2013. In some embodiments, any resins manufactured for solid-phase peptide synthesis can be used.

Antigen processing pathways

The polypeptide described herein can be processed in a variety of ways to release an epitope for epitope presentation. To generate optimal peptide antigens, there are two key processing events in the antigen processing and presentation pathway. Cytosolic proteins are primarily processed by proteasomes. Short peptides are then transported to the endoplasmic reticulum (ER) via the transporter associated with antigen processing (TAP) for subsequent assembly with MHC class I molecules. Exogenous proteins are primarily presented by MHC class II molecules. Antigens are internalized via several pathways, including phagocytosis, macropinocytosis, and endocytosis, and are ultimately transported to the mature or late endosomal compartment where they are processed and loaded onto MHC class II molecules. Cytoplasmic/nuclear antigens can also be delivered to the endosomal network via autophagy for subsequent processing and presentation by MHC class II molecules.

Initial proteolysis of peptides occurs in the cytosol of the cell and cleaves larger protein fragments into smaller peptides by the proteasome or immunoproteasome. This processing event is often responsible for the formation of the last C-terminal residue of peptides that bind to MHC class I. The proteasome is a large proteolytic complex that contains several subunits, including two subunits, large multifunctional protease (LMP) 2 and LMP7. Proteins bound for degradation are targeted to the proteasome by covalent linkage to ubiquitin. LMP2 and LMP7 induce the proteolytic complex to create peptides that bind to MHC class I. Peptides formed in the cytosol are then transported via TAP to the ER. Because TAP preferentially transports peptides of 11-14 amino acids, peptides are often too long to stably bind MHC class I and require further processing upon entry into the ER. This processing involves trimming the N-terminal region of antigenic peptides by endoplasmic reticulum aminopeptidase (ERAP) 1 and ERAP2. This process creates a pool of peptides that have high affinity for MHC class I association.

In normal cellular environments, classical MHC class II molecules are expressed only on professional APCs such as dendritic cells (DCs) or macrophages. Exogenous or extracellular antigens that are internalized by phagocytosis, endocytosis, or pinocytosis are predominantly presented on MHC class II to CD4+ T cells. However, a small subset of cytosolic antigens are also expressed on MHC class II as a result of autophagy. Briefly, endocytosed antigens are processed in a vesicular pathway consisting of increasingly acidic and proteolytically active compartments classically described as early endosomes (pH 6.0 - pH 6.5), late endosomes or endolysosomes (pH 5.0 - pH 6.0), and lysosomes (pH 4.5 - pH 5.0). Antigens internalized by phagocytosis follow a similar pathway, ending up in phagolysosomes formed by the fusion of phagosomes and lysosomes. Lysosomes and phagolysosomes (pH 4.0 - pH 4.5) contain a number of pH-optimal acidic proteases, commonly called cathepsins. In highly degradative cells such as macrophages, sequential cleavage by these enzymes yields very short peptides and free amino acids that are translocated into the cytosol to replenish tRNA for new protein synthesis. In APCs, which are less proteolytically active, the larger intermediates form the dominant source of peptides for MHC class II binding, and these peptides are typically 13-18 amino acids long.

Both class I and class II MHCs can access peptides processed from endogenous and exogenous antigens. For example, class II MHCs bind peptides derived from endogenous membrane proteins that are degraded in the lysosome. Similarly, class I MHCs can bind peptides derived from exogenous proteins internalized by endocytosis or phagocytosis, a phenomenon called cross-presentation. Specific DC subsets are particularly adept at mediating this process, which is critical for initiating the primary response by naïve CD8+ T cells.

In one aspect, the invention relates to a method of cleaving a polypeptide comprising contacting a polypeptide described in the present invention with an APC. In some embodiments, the method can be performed in vivo. In some embodiments, the method can be performed in vitro.

In some embodiments, the polypeptide is ubiquitinated. In some embodiments, the polypeptide is ubiquitinated prior to cleavage. In some embodiments, the polypeptide is ubiquitinated prior to proteasomal and/or immunoproteasomal processing. In some embodiments, the polypeptide is ubiquitinated at a lysine residue. In some embodiments, the polypeptide is ubiquitinated at a lysine residue that is not in the epitope sequence. In some embodiments, the polypeptide is ubiquitinated at a lysine residue on polyC. In some embodiments, the polypeptide is ubiquitinated at the first lysine on polyC. In some embodiments, the polypeptide is ubiquitinated at the second lysine on polyC. In some embodiments, the polypeptide is ubiquitinated at the third lysine on polyC. In some embodiments, the polypeptide is ubiquitinated at the fourth lysine on polyC. In some embodiments, the polypeptide is ubiquitinated at the fifth, sixth, seventh, eighth, ninth, or tenth lysine on polyC. In some embodiments, the polypeptide is ubiquitinated at at least one lysine residue. In some embodiments, the polypeptide is ubiquitinated at more than one lysine residue. In some embodiments, the polypeptide is ubiquitinated at more than one lysine residue on polyC. In some embodiments, the polypeptide is ubiquitinated at every lysine residue. In some embodiments, the polypeptide is ubiquitinated at every lysine residue on polyC. In some embodiments, the polypeptide is ubiquitinated at two lysine residues on polyC. In some embodiments, the polypeptide is ubiquitinated at three lysine residues on polyC. In some embodiments, the polypeptide is ubiquitinated at four lysine residues on polyC. In some embodiments, the polypeptide is ubiquitinated at five, six, seven, eight, nine, or ten lysine residues on polyK. In some embodiments, the polypeptide is sequentially ubiquitinated at each lysine residue on polyK. In some embodiments, the polypeptide does not undergo sequential ubiquitination of each lysine residue on polyK.

In some embodiments, the polypeptide is ubiquitinated at a lysine residue on Ala-Lys-PABC. In some embodiments, the polypeptide is ubiquitinated at a lysine residue on Phe-Lys-PABC. In some embodiments, the polypeptide comprises polyK and AA-AA-PABC, wherein each AA is an amino acid, analog, or derivative thereof. In some embodiments, the polypeptide is ubiquitinated at at least one lysine residue on polyK and AA-AA-PABC. In some embodiments, the polypeptide is ubiquitinated at one or more lysine residues on polyK and AA-AA-PABC. In some embodiments, the polypeptide is ubiquitinated at one or more lysine residues on polyK and Ala-Lys-PABC. In some embodiments, the polypeptide is ubiquitinated at one or more lysine residues on polyK and Phe-Lys-PABC.

In some embodiments, the polypeptide is internalized by APC. In some embodiments, the polypeptide is internalized by APC via endocytosis. In some embodiments, the polypeptide is internalized by APC via phagocytosis. In some embodiments, the polypeptide is internalized by APC via pinocytosis. In some embodiments, the polypeptide is cleaved in the cytoplasm. In some embodiments, the polypeptide is cleaved in an endosome. In some embodiments, the polypeptide is cleaved in an endolysosome. In some embodiments, the polypeptide is cleaved in a lysosome. In some embodiments, the polypeptide is cleaved in the ER. In some embodiments, the polypeptide is cleaved by an aminopeptidase. In some embodiments, the aminopeptidase is insulin-regulated aminopeptidase (IRAP). In some embodiments, the aminopeptidase is endoplasmic reticulum aminopeptidase (ERAP). In some embodiments, the polypeptide is processed by a trypsin-like domain of a proteasome and/or an immunoproteasome. In some embodiments, the trypsin-like domain has trypsin-like activity. In some embodiments, the trypsin-like domain has chymotrypsin-like activity. In some embodiments, the trypsin-like activity comprises peptidyl glutamyl peptide hydrolase (PGPH) activity. In some embodiments, the polypeptide is cleaved by a protease. In some embodiments, the protease is a trypsin-like protease. In some embodiments, the protease is a chymotrypsin-like protease. In some embodiments, the protease is peptidyl glutamyl peptide hydrolase (PGPH). In some embodiments, the protease is selected from the group consisting of asparagine peptide lyase, aspartic protease, cysteine protease, glutamic protease, metalloprotease, serine protease, and threonine protease. In a preferred embodiment, the protease is a cysteine protease. In some embodiments, the cysteine protease is selected from the group consisting of calpain, caspase, cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, and cathepsin Z. In some embodiments, the protease is cathepsin B. In some embodiments, the protease is cathepsin C. In some embodiments, the protease is cathepsin F. In some embodiments, the protease is cathepsin Z.

In some embodiments, the polypeptide is cleaved at a lysine residue. In some embodiments, the polypeptide is cleaved at a lysine residue on polyK. In some embodiments, the polypeptide is cleaved at the first lysine residue on polyK. In some embodiments, the polypeptide is cleaved at the second lysine residue on polyK. In some embodiments, the polypeptide is cleaved at the third lysine residue on polyK. In some embodiments, the polypeptide is cleaved at the fourth lysine residue on polyK. In some embodiments, the polypeptide is cleaved at the fifth, sixth, seventh, eighth, ninth, or tenth lysine residue on polyK. In some embodiments, the polypeptide is cleaved at more than one lysine residue on polyK. In some embodiments, the polypeptide is cleaved at every lysine residue on polyK. In some embodiments, the polypeptide is cleaved sequentially at each lysine residue on polyK. In some embodiments, the polypeptide is not cleaved sequentially at each lysine residue on polyK.

In some embodiments, the polypeptide is cleaved at AA-AA-PABC, wherein each AA is an amino acid or an analog or derivative thereof. In some embodiments, the polypeptide is cleaved at Ala-Lys-PABC. In some embodiments, the polypeptide is cleaved at the lysine residue in Ala-Lys-PABC. In some embodiments, the polypeptide is cleaved at Phe-Lys-PABC. In some embodiments, the polypeptide is cleaved at the lysine residue in Phe-Lys-PABC. In some embodiments, the polypeptide is cleaved at Val-Cit-PABC. In some embodiments, the polypeptide is cleaved at the citrulline (Cit) residue in Val-Cit-PABC. In some embodiments, the epitope is released upon cleavage of the polypeptide.

One of the main disadvantages of peptide drugs, limiting their systemic therapeutic use, is the proteolytic degradation of peptides. Peptides administered by injection reach the bloodstream, which contains proteases involved in hemostasis, fibrinolysis and tissue transformation, i.e. important processes in the event of injury. Thus, it is important to stabilize the peptide against proteases present in the blood, serum or plasma. In one aspect, the polypeptide described in the present invention is stable in plasma, blood and/or serum. In some embodiments, the polypeptide is not cleaved prior to internalization by APC in the subject. In some embodiments, the polypeptide is not cleaved prior to processing by APC in the subject. In some embodiments, the polypeptide is not cleaved in the blood of the subject prior to internalization by APC. In some embodiments, the polypeptide is not cleaved in the blood of the subject prior to processing by APC. In some embodiments, the polypeptide is not cleaved by a blood protease. In some embodiments, the polypeptide is not cleaved by plasmin. In some embodiments, the polypeptide is not cleaved by plasma kallikrein. In some embodiments, the polypeptide is not cleaved by tissue kallikrein. In some embodiments, the polypeptide is not cleaved by thrombin. In some embodiments, the polypeptide is not cleaved by a coagulation factor. In some embodiments, the polypeptide is not cleaved by coagulation factor XII. In some embodiments, the polypeptide is stable in human plasma. In some embodiments, the polypeptide is stable in human blood. In some embodiments, the polypeptide is stable in human serum.

In some embodiments, the polypeptide has a half-life in human plasma of 1 hour to 5 days. In some embodiments, the polypeptide has a half-life of about 1 hour to about 120 hours. In some embodiments, the polypeptide has a half-life of about 1 hour to about 5 hours, about 1 hour to about 10 hours, about 1 hour to about 12 hours, about 1 hour to about 24 hours, about 1 hour to about 36 hours. from about 1 hour to about 48 hours, from about 1 hour to about 60 hours, from about 1 hour to about 72 hours, from about 1 hour to about 84 hours, from about 1 hour to about 96 hours, from about 1 hour to about 120 hours, from about 5 hours to about 10 hours, from about 5 hours to about 12 hours, from about 5 hours to about 24 hours, from about 5 hours to about 36 hours, from about 5 hours to about 48 hours, from about 5 hours to about 60 hours, about 5 hours to about 72 hours, from about 5 hours to about 84 hours, from about 5 hours to about 96 hours, from about 5 hours to about 120 hours, from about 10 hours to about 12 hours, from about 10 hours to about 24 hours, from about 10 hours to about 36 hours, from about 10 hours to about 48 hours, from about 10 hours to about 60 hours, from about 10 hours to about 72 hours, from about 10 hours to about 84 hours, from about 10 hours to about 96 hours, from about 10 hours to about 120 hours, from about 12 hours to about 24 hours, from about 12 hours to about 36 hours, from about 12 hours to about 48 hours, from about 12 hours to about 60 hours, from about 12 hours to about 72 hours, from about 12 hours to about 84 hours, from about 12 hours to about 96 hours, from about 12 hours to about 120 hours, from about 24 hours to about 36 hours, from about 24 hours to about 48 hours, from about 24 hours to about 60 hours, about 24 hours to about 72 hours, from about 24 hours to about 84 hours, from about 24 hours to about 96 hours, from about 24 hours to about 120 hours, from about 36 hours to about 48 hours, from about 36 hours to about 60 hours, from about 36 hours to about 72 hours, from about 36 hours to about 84 hours, from about 36 hours to about 96 hours, from about 36 hours to about 120 hours, from about 48 hours to about 60 hours, from about 48 hours to about 72 hours, from about 48 hours to about 84 hours, from about 48 hours to about 96 hours, from about 48 hours to about 120 hours, from about 60 hours to about 72 hours, from about 60 hours to about 84 hours, from 60 hours to 96 hours, from about 60 hours to about 120 hours, from about 72 hours to about 84 hours, from about 72 hours to about 96 hours, from about 72 hours to about 120 hours, from about 84 hours to about 96 hours, about 84 hours to about 120 hours, or from about 96 hours to about 120 hours. In some embodiments, the polypeptide has a half-life of about 1 hour, about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 120 hours. In some embodiments, the polypeptide has a half-life of at least about 1 hour, about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours. In some embodiments, the polypeptide has a half-life of at most about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 120 hours.

3. Neoantigens and their use

One of the critical hurdles in the development of therapeutic and tumor-specific immunotherapies is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise from genetic alterations (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) in 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 their identification, selection of optimized antigens, and production of neoantigens for use in vaccines or immunogenic compositions. These problems can be overcome by: identifying mutations in neoplasms/tumors that are present at the DNA level in the tumor but not in the corresponding germline samples from a large number of cancer subjects; analyzing the identified mutations using one or more peptide-MHC binding prediction algorithms to generate a plurality of neoantigenic T-cell epitopes that are expressed in the neoplasm/tumor and that bind to a high proportion of the patient's HLA alleles; and synthesizing a plurality of neoantigenic peptides selected from the sets of all neoantigenic peptides and the predicted binding peptides for use in a cancer vaccine or immunogenic composition suitable for treating a large number of subjects suffering from cancer.

For example, translation of peptide sequencing information into a therapeutic vaccine may involve prediction of mutant peptides that are likely to bind to HLA molecules in a large number of individuals. Efficient selection of specific mutations for use as an immunogen requires the ability to predict which mutated peptides will bind efficiently to a high proportion of a patient's HLA alleles. Recently, neural network-based learning approaches with validated binding and non-binding peptides have improved the accuracy of prediction algorithms for the major HLA-A and -B alleles. However, even with improved neural network-based algorithms for encoding HLA peptide binding rules, several factors limit the ability to predict peptides presented on HLA alleles.

Another example of translating peptide sequencing information into a therapeutic vaccine would involve formulating a drug as a multi-epitope long peptide vaccine. Targeting as many mutated epitopes as practicable exploits the vast capacity of the immune system, prevents immune evasion due to down-modulation of the immune-targeted gene product, and compensates for the known imprecision of epitope prediction approaches. Synthetic peptides provide useful tools for the efficient production of multiple immunogens and for the rapid translation of mutant epitope identification into an effective vaccine. Peptides can be readily synthesized chemically and are readily purified using reagents free of bacterial or animal contaminants. The small size allows for a precise focus on the mutated region of the protein and reduces irrelevant antigenic competition from other components (non-mutant protein or viral vector antigens).

Another example of translating peptide sequence information into a therapeutic vaccine may involve combination with a strong vaccine adjuvant. Effective vaccines may require a strong adjuvant to initiate an immune response. For example, poly-ICLC, an agonist of TLR3 and the RNA helicase domains of MDA5 and RIG3, has demonstrated several desirable properties for a vaccine adjuvant. These properties include induction of local and systemic immune cell activation in vivo, production of stimulatory chemokines and cytokines, and stimulation of antigen presentation by dendritic cells (DCs). In addition, poly-ICLC can induce robust CD4 ⁺ and CD8 ⁺ responses in humans. Importantly, striking similarities in the activation of transcription and signal transduction pathways were observed in subjects vaccinated with poly-ICLC and in volunteers receiving a highly potent replication-competent yellow fever vaccine. In addition, >90% of ovarian carcinoma patients immunized with poly-ICLC in combination with the NYESO-1 peptide vaccine (in addition to Montanide) showed induction of CD4 ⁺ and CD8 ⁺ T cells as well as antibody responses to the peptide in a recent phase 1 study. 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.

Peptides

[0186] In some aspects, the present invention provides isolated peptides that comprise a tumor-associated mutation. These peptides and polypeptides are referred to herein as "neoantigenic peptides" or "neoantigenic polypeptides." The term "peptide" is used interchangeably with "mutant peptide," "neoantigen peptide," and "neoantigenic peptide" herein to refer to a series of residues, typically L-amino acids, linked to each other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Similarly, the term "polypeptide" is used interchangeably with "mutant polypeptide," "neoantigen polypeptide," and "neoantigenic polypeptide" herein to refer to a series of residues, e.g., L-amino acids, linked to each other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The polypeptides or peptides may be of various lengths, either in their neutral (uncharged) forms or in forms that are salts, and either lack modifications such as glycosylation, side chain oxidation or phosphorylation, or contain these modifications, depending on the condition that the modification does not impair the biological activity of the polypeptides as described in the present invention.

In some embodiments, genomic or exome sequencing methods are used to identify tumor-specific mutations. Any suitable sequencing method can be used in accordance with the present disclosure, such as next-generation sequencing (NGS) technologies. In the future, third-generation sequencing methods may replace NGS technology to speed up the sequencing step of the method. For clarification purposes, the terms "next-generation sequencing" or "NGS" in the context of the present disclosure refer to all new high-throughput sequencing technologies that, in contrast to the "traditional" sequencing methodology known as Sanger chemistry, read nucleic acid profiles randomly, in parallel, across the entire genome, breaking the entire genome into small pieces. Such NGS technologies (also known as massively parallel sequencing technologies) are capable of delivering 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 very short periods of time, such as within 1-2 weeks, such as within 1-7 days or less than 24 hours, and in principle allow single-cell sequencing approaches. Several NGS platforms that are commercially available or that are mentioned in the literature can be used in the context of the present disclosure, such as those described in detail in WO 2012/159643.

In some embodiments, a polypeptide described herein may comprise, but is not limited to, 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 1000, about 1500, about 2000, about 2500, about 3000, about 4000, about 5000, about 7500, about 10,000 amino acids or more amino acid residues, and any range derived therefrom. In particular embodiments, the neoantigenic peptide molecule is equal to or less than 100 amino acids.

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

In some embodiments, the polypeptide can have a length of 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 some embodiments, the polypeptide can have a length of 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 some embodiments, the polypeptide may be no more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or fewer amino acid residues in length. In some embodiments, the polypeptide may be no more than 88, 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 fewer amino acid residues.

In some embodiments, the polypeptide has a total 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 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, at least 500, at least 1000 or at least 1500 amino acids.

In some embodiments, the polypeptide has a total length of no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 150, no more than 200, no more than 250, no more than 300, no more than 350, no more than 400, no more than 450, no more than 500, no more than 1000 or no more than 1500 amino acids.

In some embodiments, a polypeptide described herein may comprise an epitope. In some embodiments, the epitope can comprise, but is not limited to, 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 1000, about 1500, about 2000, about 2500, about 3000, about 4000, about 5000, about 7500, about 10,000 amino acids or more amino acid residues, and any range that can be obtained from them.

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

In some embodiments, the epitope may 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 epitope may 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 some embodiments, the epitope may be no more than 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 epitopes may be no more than 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 fewer amino acid residues.

A longer peptide can be created in several ways. In some embodiments, if HLA-binding peptides are predicted or known, the longer peptide comprises (1) individual binding peptides with extensions of 2-5 amino acids toward the N- and C-terminus of each corresponding gene product; or (2) a concatenation of some or all of the binding peptides with extended sequences for each. In other embodiments, if sequencing reveals a long (>10 residues) neoepitope sequence present in the tumor (e.g., due to a frameshift, read-through, or intron inclusion resulting in a new peptide sequence), the longer peptide can consist of the entire extension of the new tumor-specific amino acids as either a single longer peptide or multiple overlapping longer peptides. In some embodiments, it is believed that the use of a longer peptide allows for endogenous processing by the patient's cells and may result in more efficient antigen presentation and T cell response induction. In some embodiments, two or more peptides may be used, where the peptides overlap and are located on a long neoantigen peptide.

In some embodiments, the immunogenic antigen, neoantigenic peptide, or MHC class I epitope thereof is 12 or fewer amino acid residues in length and typically consists of about 8 to about 12 amino acid residues. In some embodiments, the immunogenic antigen, neoantigenic peptide, or MHC class I epitope thereof comprises about 8, about 9, about 10, about 11, or about 12 amino acid residues. In some embodiments, the immunogenic antigen, neoantigenic peptide, or MHC class II epitope thereof is 25 or fewer amino acid residues in length and typically consists of about 9 to about 25 amino acid residues. In some embodiments, the immunogenic antigen, neoantigenic peptide, or MHC class II epitope thereof comprises about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues.

In some embodiments, the antigen, neoantigenic peptide, or epitope binds an HLA protein (e.g., HLA MHC class I or HLA MHC class II). In particular embodiments, the antigen, neoantigenic peptide, or epitope binds the HLA protein with greater affinity than the corresponding wild-type peptide. In particular embodiments, the antigen, neoantigenic peptide, or epitope has an IC ₅₀ or K _D of at least less than 5000 nM, at least less than 500 nM, at least less than 100 nM, at least less than 50 nM, or less. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA class I MHC. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA class I MHC with an affinity of from 0.1 to 2000 nM. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA class I MHC with an affinity of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 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, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nM. In some embodiments, the antigen, neoantigenic peptide or epitope binds to HLA MHC class II. In some embodiments, the antigen, neoantigenic peptide or epitope binds to HLA MHC class II with an affinity of 0.1 nM to 2000 nM, 1 nM to 1000 nM, 10 nM to 500 nM, or less than 1000 nM. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA MHC class II with an affinity of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 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, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nM.

In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA class I MHC with a stability of 10 minutes to 24 hours. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA class I MHC with a stability of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA MHC class I with a stability of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA MHC class II with a stability of 10 minutes to 24 hours. In some embodiments, the antigen, neoantigen peptide, or epitope binds to HLA MHC class II with a stability of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the antigen, neoantigenic peptide, or epitope binds to HLA MHC class II with a stability of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

In some embodiments, the polypeptide may have a pI value of about 0.5 to about 12, about 2 to about 10, or about 4 to about 8. In some embodiments, the peptides may have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the polypeptide may have a pI value of no more than 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

In some embodiments, a polypeptide described in the present invention comprises an amino acid or an amino acid sequence of a peptide sequence that is not encoded by a nucleic acid sequence located immediately upstream of a nucleic acid sequence in the genome of a subject that encodes an epitope. In some embodiments, a polypeptide described in the present invention comprises an amino acid or an amino acid sequence of a peptide sequence that is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of a subject that encodes an epitope. In some embodiments, the amino acid or amino acid sequence comprises 0-1000, 1-900, 5-800, 10-700, 20-600, 30-500, 40-400, 50-300, 60-200, or 70-100 amino acid residues. In a preferred embodiment, the amino acid or amino acid sequence comprises 1 to 20 amino acid residues. In other preferred embodiments, the amino acid or amino acid sequence comprises 5 to 12 amino acid residues. In some embodiments, the amino acid or amino acid sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, at least 500, at least 1000, or at least 1500 amino acid residues. In some embodiments, the amino acid or amino acid sequence comprises about 1, about 2, about 3, about 4, 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 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 1000, or approximately 1500 amino acid residues.

In one aspect, the invention provides a method for producing a polypeptide comprising linking an amino acid or amino acid sequence and/or a linker to the N- and/or C-terminus of a sequence comprising an epitope sequence. In some embodiments, the polypeptide described herein may be in solution, lyophilized, or may be in crystalline form. In some embodiments, the polypeptide described herein may be produced synthetically, using recombinant DNA technology or chemical synthesis, or may be isolated from natural sources such as native tumors or pathogenic organisms. Epitopes or neoepitopes may be synthesized individually or directly or indirectly linked into a polypeptide. Although the polypeptide described herein may be substantially free of other native host cell proteins and fragments thereof, in some embodiments, the polypeptide may be synthetically conjugated to attach to native fragments or particles.

In some embodiments, the polypeptide described herein can be prepared by various methods. In some embodiments, the polypeptide can be synthesized in solution or on a solid support using conventional methods. Various 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. Additionally, an individual polypeptide can be joined using chemical ligation to produce larger polypeptides that are still within the scope of the present disclosure.

Alternatively, recombinant DNA technology can be used, in which a nucleotide sequence encoding a polypeptide or a portion of a polypeptide is 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 generally in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinant peptides that contain one or more neoantigenic peptides described in the present invention can be used to present an appropriate T-cell epitope.

In some embodiments, the polypeptide comprises at least one mutant amino acid. In some embodiments, the at least one mutant amino acid is encoded by an insertion of one or more nucleotides into a nucleic acid sequence in the genome of the subject. In some embodiments, the at least one mutant amino acid is encoded by a deletion of one or more nucleotides in a nucleic acid sequence in the genome of the subject. In some embodiments, the at least one mutant amino acid is encoded by a frameshift in a nucleic acid sequence in the genome of the subject. A frameshift occurs when a mutation disrupts the normal phase of codon periodicity of a gene (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame. In some embodiments, the at least one mutant amino acid is encoded by a neoORF in a nucleic acid sequence in the genome of the subject. In some embodiments, the at least one mutant amino acid is encoded by a point mutation in a nucleic acid sequence in the genome of the subject. In some embodiments, the at least one mutant amino acid is encoded by a gene with a mutation that results in a fusion polypeptide, an in-frame deletion, an insertion, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypeptides. In some embodiments, the at least one mutant amino acid is encoded by a fusion of a first gene to a second gene in the genome of the subject. In some embodiments, the at least one mutant amino acid is encoded by an in-frame fusion of the first gene to a second gene in the genome of the subject. In some embodiments, the at least one mutant amino acid is encoded by a fusion of the first gene to an exon of a splice variant of the first gene in the genome of the subject. In some embodiments, at least one mutant amino acid is encoded by a fusion of the first gene to a cryptic exon of the first gene in the genome of the subject.

In some aspects, the present disclosure relates to a polypeptide comprising at least two polypeptide molecules. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise an epitope. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise the same epitope. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise the same epitope of the same length. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise an amino acid or amino acid sequence that is a peptide sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the genome of the subject that encodes the epitope. In some embodiments, the amino acid or amino acid sequence, which is a peptide sequence that is not encoded by a nucleic acid sequence, immediately upstream of a nucleic acid sequence in the genome of the subject that encodes an epitope of two or more of the at least two polypeptides or polypeptide molecules, is the same. In some embodiments, the amino acid or amino acid sequence, which is a peptide sequence that is not encoded by a nucleic acid sequence, immediately downstream of a nucleic acid sequence in the genome of the subject that encodes an epitope of two or more of the at least two polypeptides or polypeptide molecules, is the same.

In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise a linker. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise a linker at the N- and/or C-terminus of the epitope. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise different linkers. In some embodiments, a first polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules does not comprise a linker, and a second polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules comprises a linker. In some embodiments, a first polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules does not comprise a linker at the N-terminus of the epitope, and a second polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules comprises a linker at the N-terminus of the epitope. In some embodiments, the first polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules does not comprise a linker at the C-terminus of the epitope, and the second polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules comprises a linker at the C-terminus of the epitope. In some embodiments, the first polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules comprises a linker, and the second polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules does not comprise a linker. In some embodiments, the first polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules comprises a linker at the N-terminus of the epitope, and the second polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules does not comprise a linker at the N-terminus of the epitope. In some embodiments, the first polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules comprises a linker at the C-terminus of the epitope, and the second polypeptide or polypeptide molecule of the at least two polypeptides or polypeptide molecules does not comprise a linker at the C-terminus of the epitope.

Disulfide linkers can be synthesized using methods well known in the art. For example, disulfide linkers can be synthesized according to Zhang, Donglu, et al., ACS Med. Chem. Lett. 2016, 7, 988-993; and Pillow, Thomas H., et al., Chem. Sci., 2017, 8, 366-370. An example of the synthesis of a disulfide linker and the synthesis of a disulfide-containing peptide is shown in Examples 3 and 4. PABC-containing peptides can be synthesized using methods well known in the art. For example, PABC-containing peptides can be synthesized according to Laurent Ducry (ed.), Antibody-Drug Conju gates, Methods in Molecular Biology, vol. 1045, DOI 10.1007/978-1-62703-541-5_5, Springer Science+Business Media, LLC 2013. An example of the synthesis of a peptide containing PABC is shown in Example 5. In some embodiments, any resins manufactured for solid-phase peptide synthesis can be used.

In some embodiments, the polypeptide comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more polypeptides or polypeptide molecules. For example, a polypeptide may comprise 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more polypeptides or polypeptide molecules.

In some embodiments, a polypeptide comprising an antigen, neoantigenic peptide, or epitope comprises a RAS epitope. In some embodiments, the peptide may be derived from a protein with a substitution mutation, such as a KRAS G12C, G12D, G12V, Q61H, or Q61L mutation, or a NRAS Q61K or Q61R mutation. The substitution may be located anywhere along the length of the peptide. For example, it may be located in the N-terminal third of the peptide, in the central third of the peptide, or in the C-terminal third of the peptide. In another embodiment, the replaced residue is located 2-5 residues from the N-terminus or 2-5 residues from the C-terminus. Peptides may similarly be derived from tumor-specific insertional mutations if the peptide contains one or more or all of the inserted residues. In some embodiments, the epitope comprises a mutant RAS sequence that comprises at least 8 contiguous amino acids of a mutant RAS protein comprising a mutation at G12, G13, or Q61 and a mutation at G12, G13, or Q61. In some embodiments, the at least 8 contiguous amino acids of the mutant RAS protein comprising a mutation at G12, G13, or Q61 comprise a G12A, G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K mutation. In some embodiments, the G12, G13, or Q61 mutation comprises a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation.

In some embodiments, the polypeptide comprising the RAS epitope further comprises an amino acid sequence. In some embodiments, the amino acid sequence is a cytomegalovirus (CMV) protein, such as pp65. In some embodiments, the amino acid sequence is a human immunodeficiency virus (HIV) protein. In some embodiments, the amino acid sequence is a MART-1 protein. In some embodiments, the amino acid sequence of a CMV protein, such as pp65, comprises 1, 2, 3, or more than 3 amino acid residues. In some embodiments, the amino acid sequence of a CMV protein, such as pp65, comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues. In some embodiments, the amino acid sequence of an HIV protein comprises 1, 2, 3, or more than 3 amino acid residues. In some embodiments, the amino acid sequence of the HIV protein comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues. In some embodiments, the amino acid sequence of the MART-1 protein comprises 1, 2, 3, or more than 3 amino acid residues. In some embodiments, the amino acid sequence of the MART-1 protein comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues.

In some embodiments, the RAS epitope binds to a protein encoded by an HLA allele. In some embodiments, the RAS epitope binds to a protein encoded by an HLA allele with an affinity of less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 950 nM, less than 900 nM, less than 850 nM, less than 800 nM, less than 750 nM, less than 600 nM, less than 550 nM, less than 500 nM, less than 450 nM, less than 400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, or less than 10 nM. In some embodiments, the RAS epitope binds to a protein encoded by an HLA allele with a stability of greater than 24 hours, greater than 23 hours, greater than 22 hours, greater than 21 hours, greater than 20 hours, greater than 19 hours, greater than 18 hours, greater than 17 hours, greater than 16 hours, greater than 15 hours, greater than 14 hours, greater than 13 hours, greater than 12 hours, greater than 11 hours, greater than 10 hours, greater than 9 hours, greater than 8 hours, greater than 7 hours, greater than 6 hours, greater than 5 hours, greater than 4 hours, greater than 3 hours, greater than 2 hours, greater than 1 hour, greater than 55 minutes, greater than 50 minutes, greater than 45 minutes, greater than 40 minutes, greater than 35 minutes, greater than 30 minutes, greater than 25 minutes, greater than 20 minutes, greater than 15 minutes, greater than 10 minutes, greater than 9 minutes, greater than 8 minutes, greater than 7 minutes, greater than 6 minutes, more than 5 minutes, more than 4 minutes, more than 3 minutes, more than 2 minutes, or more than 1 minute.

In some embodiments, the HLA allele is selected from the group consisting of HLA-A02:01 allele, HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, HLA-A74:01 allele and/or HLA-C08:02 allele, and any combination thereof. In some embodiments, the HLA allele is HLA-A02:01. In some embodiments, the HLA allele is HLA-A03:01 allele. In some embodiments, the HLA allele is HLA-A11:01 allele. In some embodiments, the HLA allele is the HLA-A03:02 allele. In some embodiments, the HLA allele is the HLA-A30:01 allele. In some embodiments, the HLA allele is the HLA-A31:01 allele. In some embodiments, the HLA allele is the HLA-A33:01 allele. In some embodiments, the HLA allele is the HLA-A33:03 allele. In some embodiments, the HLA allele is the HLA-A68:01 allele. In some embodiments, the HLA allele is the HLA-A74:01 allele. In some embodiments, the HLA allele is HLA-C08:02.

In some aspects, the present disclosure relates to a composition comprising one polypeptide comprising a first peptide and a second peptide, or one polynucleotide encoding the first peptide and the second peptide. In some embodiments, the composition provided in the present invention comprises one or more additional peptides, wherein the one or more additional peptides comprise a third neoepitope. In some embodiments, the first peptide and the second peptide are encoded by a sequence 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 is 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, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, or 10000 amino acids in length. 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 with 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 with 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 second peptide is longer than the first peptide. In some embodiments, the first peptide is longer than the second peptide. In some embodiments, the first peptide is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, or 10000 amino acids in length. In some embodiments, the second peptide is 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, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, or 10000 amino acids in length. In some embodiments, the first peptide comprises a sequence of at least 9 contiguous amino acids with 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 with 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 first peptide, the second peptide, or both comprise at least one flanking sequence, wherein the at least one flanking sequence is located upstream or downstream of the neoepitopes. 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 sequence different from the wild type. 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 flanking region of the first peptide differs from at least one flanking region of the second peptide. In some embodiments, at least one flanking residue comprises a mutation.

In some embodiments, the peptide comprises a neoepitope sequence comprising at least one mutated amino acid. In some embodiments, the peptide comprises a neoepitope sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutated amino acids. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising 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 neoepitope sequence derived from a protein comprising 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 and at least one mutant amino acid. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutated amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutated amino acid. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutated 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-mutated amino acids above 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-mutated amino acids below the at least one mutant amino acid.

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

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

In some embodiments, the epitope is a TMPRSS2:ERG epitope. In some embodiments, the TMPRSS2:ERG epitope comprises the amino acid sequence ALNSEALSV (SEQ ID NO: 304). In some embodiments, a polypeptide comprising a RAS epitope comprises the amino acid sequence GADGVGKSAL (SEQ ID NO: 4), GACGVGKSAL (SEQ ID NO: 5), GAVGVGKSAL (SEQ ID NO: 6), GADGVGKSA (SEQ ID NO: 7), GACGVGKSA (SEQ ID NO: 8), GAVGVGKSA (SEQ ID NO: 9), KLVVVGACGV (SEQ ID NO: 10), FLVVVGACGL (SEQ ID NO: 11), FMVVVGACGI (SEQ ID NO: 12), FLVVVGACGI (SEQ ID NO: 13), FMVVVGACGV (SEQ ID NO: 14), FLVVVGACGV (SEQ ID NO: 15), MLVVVGACGV (SEQ ID NO: 16), FMVVVGACGL (SEQ ID NO: ID NO: 17), YLVVVGACGV (SEQ ID NO: 18), KMVVVGACGV (SEQ ID NO: 19), YMVVVGACGV (SEQ ID NO: 20), MMVVVGACGV (SEQ ID NO: 21), DTAGHEEY (SEQ ID NO: 22), TAGHEEYSAM (SEQ ID NO: 23), DILDTAGHE (SEQ ID NO: 24), DILDTAGH (SEQ ID NO: 25), ILDTAGHEE (SEQ ID NO: 26), ILDTAGHE (SEQ ID NO: 27), DILDTAGHEEY (SEQ ID NO: 28), DTAGHEEYS (SEQ ID NO: 29), LLDILDTAGH (SEQ ID NO: 30), DILDTAGRE (SEQ ID NO: 31), DILDTAGR (SEQ ID NO: 32), ILDTAGREE (SEQ ID NO: 33), ILDTAGRE (SEQ ID NO: 34), CLLDILDTAGR (SEQ ID NO: 35), TAGREEYSAM (SEQ ID NO: 36), REEYSAMRD (SEQ ID NO: 37), DTAGKEEYSAM (SEQ ID NO: 38), CLLDILDTAGK (SEQ ID NO: 39), DTAGKEEY (SEQ ID NO: 40), LLDILDTAGK (SEQ ID NO: 41), ILDTAGKE (SEQ ID NO: 42), ILDTAGKEE (SEQ ID NO: 43), DTAGLEEY (SEQ ID NO: 44), ILDTAGLE (SEQ ID NO: 45), DILDTAGL (SEQ ID NO: 46), ILDTAGLEE (SEQ ID NO: 47), GLEEYSAMRDQY (SEQ ID NO: 48), LLDILDTAGLE (SEQ ID NO: 49), LDILDTAGL (SEQ ID NO: 50), DILDTAGLE (SEQ ID NO: 51), DILDTAGLEEY (SEQ ID NO: 52), AGVGKSAL (SEQ ID NO: 53), GAAGVGKSAL (SEQ ID NO: 54), AAGVGKSAL (SEQ ID NO: 55), CGVGKSAL (SEQ ID NO: 56), ACGVGKSAL (SEQ ID NO: 57), DGVGKSAL (SEQ ID NO: 58), ADGVGKSAL (SEQ ID NO: 59), DGVGKSALTI (SEQ ID NO: 60), GARGVGKSA (SEQ ID NO: 61), KLVVVGARGV (SEQ ID NO: 62), VVVGARGV (SEQ ID NO: 63), SGVGKSAL (SEQ ID NO: 64), VVVGASGVGK (SEQ ID NO: 65), GASGVGKSAL (SEQ ID NO: 66), VGVGKSAL (SEQ ID NO: 67), VVVGAGCVGK (SEQ ID NO: 68), KLVVVGAGC (SEQ ID NO: 69), GDVGKSAL (SEQ ID NO: 70), DVGKSALTI (SEQ ID NO: 71), VVVGAGDVGK (SEQ ID NO: 72), TAGKEEYSAM (SEQ ID NO: 73), DTAGHEEYSAM (SEQ ID NO: 74), TAGHEEYSA (SEQ ID NO: 75), DTAGREEYSAM (SEQ ID NO: 76), TAGKEEYSA (SEQ ID NO: 77), AAGVGKSA (SEQ ID NO: 78), AGCVGKSAL (SEQ ID NO: 79), AGDVGKSAL (SEQ ID NO: 80), AGKEEYSAMR (SEQ ID NO: 81), AGVGKSALTI (SEQ ID NO: 82), ARGVGKSAL (SEQ ID NO: 83), ASGVGKSA (SEQ ID NO: 84), ASGVGKSAL (SEQ ID NO: 85), AVGVGKSA (SEQ ID NO: 86), CVGKSALTI (SEQ ID NO: 87), DILDTAGK (SEQ ID NO: 88), DILDTAGREEY (SEQ ID NO: 89), DTAGHEEYSAMR (SEQ ID NO: 90), DTAGKEEYS (SEQ ID NO: 91), DTAGKEEYSAMR (SEQ ID NO: 92), DTAGLEEYS (SEQ ID NO: 93), DTAGLEEYSA (SEQ ID NO: 94), DTAGLEEYSAMR (SEQ ID NO: 95), DTAGREEYS (SEQ ID NO: 96), DTAGREEYSAMR (SEQ ID NO: 97), GAAGVGKSA (SEQ ID NO: 98), GACGVGKSA (SEQ ID NO: 8), GACGVGKSAL (SEQ ID NO: 5), GADGVGKS (SEQ ID NO: 99), GAGDVGKSA (SEQ ID NO: 100), GAGDVGKSAL (SEQ ID NO: 101), GASGVGKSA (SEQ ID NO: 102), GCVGKSAL (SEQ ID NO: 103), GCVGKSALTI (SEQ ID NO: 104), GHEEYSAM (SEQ ID NO: 105), GKEEYSAM (SEQ ID NO: 106), GLEEYSAMR (SEQ ID NO: 107), GREEYSAM (SEQ ID NO: 108), GREEYSAMR (SEQ ID NO: 109), HEEYSAMRD (SEQ ID NO: 110), KEEYSAMRD (SEQ ID NO: 111), KLVVVGASG (SEQ ID NO: 112), LDILDTAGR (SEQ ID NO: 113), LEEYSAMRD (SEQ ID NO: 114), LVVVGARGV (SEQ ID NO: 115), LVVVGASGV (SEQ ID NO: 116), REEYSAMRDQY (SEQ ID NO: 117), RGVGKSAL (SEQ ID NO: 118), TAGLEEYSA (SEQ ID NO: 119), TEYKLVVVGAA (SEQ ID NO: 120), VGAAGVGKSA (SEQ ID NO: 121), VGADGVGK (SEQ ID NO: 122), VGASGVGKSA (SEQ ID NO: 123), VGVGKSALTI (SEQ ID NO: 124), VVVGAAGV (SEQ ID NO: 125), VVVGAVGV (SEQ ID NO: 126), YKLVVVGAC (SEQ ID NO: 127), YKLVVVGAD (SEQ ID NO: 128), YKLVVVGAR (SEQ ID NO: 129) or DILDTAGKE (SEQ ID NO: 130).

In some embodiments, the polypeptide comprising a RAS epitope further comprises, e.g., at the N-terminus, the amino acid sequence K, K, KK, KKK, KKKK (SEQ ID NO: 1), KKKKK (SEQ ID NO: 3), KKKKKKK (SEQ ID NO: 223), KKKKKKKK (SEQ ID NO: 224), KTEY (SEQ ID NO: 225), KTEYK (SEQ ID NO: 226), KTEYKL (SEQ ID NO: 227), KTEYKLV (SEQ ID NO: 228), KTEYKLVV (SEQ ID NO: 229), KTEYKLVVV (SEQ ID NO: 230), KKTEY (SEQ ID NO: 231), KKTEYK (SEQ ID NO: 232), KKTEYKL (SEQ ID NO: 233), KKTEYKLV (SEQ ID NO: 234), KKTEYKLVV (SEQ ID NO: 235), KKTEYKLVVV (SEQ ID NO: 236), KKKTEY (SEQ ID NO: 237), KKKTEYK (SEQ ID NO: 238), KKKTEYKL (SEQ ID NO: 239), KKKTEYKLV (SEQ ID NO: 240), KKKTEYKLVV (SEQ ID NO: 241), KKKTEYKLVVV (SEQ ID NO: 242), KKKKTEY (SEQ ID NO: 243), KKKKTEYK (SEQ ID NO: 244), KKKKTEYKL (SEQ ID NO: 245), KKKKTEYKLV (SEQ ID NO: 246), KKKKTEYKLVV (SEQ ID NO: 247), KKKKTEYKLVVV (SEQ ID NO: 248), IDIIMKIRNA (SEQ ID NO: 131), FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC (SEQ ID NO: 132), FFFFFFFFFFFFFFFFFFFFFFFFAAFWFW (SEQ ID NO: 133), IFFIFFIIFFFFFFFFFFFIIIIIIWEC (SEQ ID NO: 134), FIFFFIIFFFFFIFFFFFIFIIIIIIFWEC (SEQ ID NO: 135), TEY, TEYK (SEQ ID NO: 249), TEYKL (SEQ ID NO: 250), TEYKLV (SEQ ID NO: 136), TEYKLVV (SEQ ID NO: 251), TEYKLVVV (SEQ ID NO: 252), WQAGILAR (SEQ ID NO: 137), HSYTTAE (SEQ ID NO: 138), PLTEEKIK (SEQ ID NO: 139), GALHFKPGSR (SEQ ID NO: 140), RRANKDATAE (SEQ ID NO: 141), KAFISHEEKR (SEQ ID NO: 142), TDLSSRFSKS (SEQ ID NO: 143), FDLGGGTFDV (SEQ ID NO: 144), CLLLHYSVSK (SEQ ID NO: 145), KKKKIIMKIRNA (SEQ ID NO: 146) or MTEYKLVVV (SEQ ID NO: 147).

In some embodiments, the polypeptide comprising a RAS epitope further comprises, e.g., at the C-terminus, the amino acid sequences K, KK, KKK, KKKK (SEQ ID NO: 1), KKKKK (SEQ ID NO: 3), KKKKKKK (SEQ ID NO: 223), KKKKKKKK (SEQ ID NO: 224), KKNKKDDI (SEQ ID NO: 148), KKNKKDDIKD (SEQ ID NO: 149), AGNDDDDDDDDDDDDDDDKKDKDDDDDD (SEQ ID NO: 150), AGNKKKKKKKKNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 151), AGRDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 152), SALTI (SEQ ID NO: 153), SALTIQL (SEQ ID NO: 154), GKSALTIQL (SEQ ID NO: 155), GKSALTI (SEQ ID NO: 156), SALTIK (SEQ ID NO: 253), SALTIQLK (SEQ ID NO: 254), GKSALTIQLK (SEQ ID NO: 255), GKSALTIK (SEQ ID NO: 256), SALTIKK (SEQ ID NO: 257), SALTIQLKK (SEQ ID NO: 258), GKSALTIQLKK (SEQ ID NO: 259), GKSALTIKK (SEQ ID NO: 260), SALTIKKK (SEQ ID NO: 261), SALTIQLKKK (SEQ ID NO: 262), GKSALTIQLKKK (SEQ ID NO: 263), GKSALTIKKK (SEQ ID NO: 264), SALTIKKKK (SEQ ID NO: 265), SALTIQLKKKK (SEQ ID NO: 266), GKSALTIQLKKKK (SEQ ID NO: 267), GKSALTI (SEQ ID NO: 156), KKKK (SEQ ID NO: 1), QGQNLKYQ (SEQ ID NO: 157), ILGVLLLI (SEQ ID NO: 158), EKEGKISK (SEQ ID NO: 159), AASDFIFLVT (SEQ ID NO: 160), KELKQVASPF (SEQ ID NO: 161), KKKLINEKKE (SEQ ID NO: 162), KKCDISLQFF (SEQ ID NO: 163), KSTAGDTHLG (SEQ ID NO: 164), ATFYVAVTVP (SEQ ID NO: 165), LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG (SEQ ID NO: 166) or TIQLIQNHFVDEYDPTIEDSYRKQVVIDGE (SEQ ID NO: 167).

In some embodiments, the polypeptide comprising a RAS epitope is selected from the group consisting of KTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 268), KTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 269), KTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 270), KTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 271), KKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 272), KKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 273), KKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 274), KKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 275), KKKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 276), KKKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 277), KKKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 278), KKKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 279), KKKKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 280), KKKKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 281), KKKKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 282), KKKKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 283) KKTEYKLVVVGAVGVGKSALTIQLKK (SEQ ID NO: 284), KKTEYKLVVVGADGVGKSALTIQLKK (SEQ ID NO: 285), KKTEYKLVVVGARGVGKSALTIQLKK (SEQ ID NO: 286), KKTEYKLVVVGACGVGKSALTIQLKK (SEQ ID NO: 287), TEYKLVVVGAVGVGKSALTIQLK (SEQ ID NO: 288), TEYKLVVVGADGVGKSALTIQLK (SEQ ID NO: 289), TEYKLVVVGARGVGKSALTIQLK (SEQ ID NO: 290), TEYKLVVVGACGVGKSALTIQLK (SEQ ID NO: 291), TEYKLVVVGAVGVGKSALTIQLKK (SEQ ID NO: 292), TEYKLVVVGADGVGKSALTIQLKK (SEQ ID NO: 293), TEYKLVVVGARGVGKSALTIQLKK (SEQ ID NO: 294), TEYKLVVVGACGVGKSALTIQLKK (SEQ ID NO: 295), TEYKLVVVGAVGVGKSALTIQLKKK (SEQ ID NO: 296), TEYKLVVVGADGVGKSALTIQLKKK (SEQ ID NO: 297), TEYKLVVVGARGVGKSALTIQLKKK (SEQ ID NO: 298), TEYKLVVVGACGVGKSALTIQLKKK (SEQ ID NO: 299), TEYKLVVVGAVGVGKSALTIQLKKKK (SEQ ID NO: 300), TEYKLVVVGADGVGKSALTIQLKKKK (SEQ ID NO: 301) and TEYKLVVVGARGVGKSALTIQLKKKK (SEQ ID NO: 302), TEYKLVVVGACGVGKSALTIQLKKKK (SEQ ID NO: 303) In some embodiments, the polypeptide comprising a RAS epitope is selected from the group consisting of KKKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 277), KKKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 278), KKKKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 280) and KKKKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 283). In some embodiments, the polypeptide comprising a RAS epitope is KKKTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 277). In some embodiments, the polypeptide comprising a RAS epitope is KKKTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 278). In some embodiments, the polypeptide comprising a RAS epitope is KKKKTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 280). In some embodiments, the polypeptide comprising a RAS epitope is KKKKTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 283).

In some embodiments, a peptide comprising a KRAS G12C mutation comprises the sequence MTEYKLVVVGACGVGKSALTIQLIQNHFVDEYDPTIEDS YRKQVVIDGETCLLDILDTAGQE (SEQ ID NO: 309). In some embodiments, a peptide comprising a KRAS G12 C mutation comprises the neoepitope sequence KLVVVGACGV (SEQ ID NO: 10). In some embodiments, a peptide comprising a KRAS G12 C mutation comprises the neoepitope sequence LVVVGACGV (SEQ ID NO: 310). In some embodiments, a peptide comprising a KRAS G12 C mutation comprises the neoepitope sequence VVGACGVGK (SEQ ID NO: 200). In some embodiments, a peptide comprising a KRAS G12 C mutation comprises the neoepitope sequence VVVGACGVGK (SEQ ID NO: 197).

In some embodiments, a peptide comprising a KRAS G12D mutation comprises the sequence MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDP TIEDSYRKQVVIDGETCLLDILDTAGQE (SEQ ID NO: 311). In some embodiments, a peptide comprising a KRAS G12D mutation comprises the neoepitope sequence VVGADGVGK (SEQ ID NO: 206). In some embodiments, a peptide comprising a KRAS G12D mutation comprises the neoepitope sequence VVVGADGVGK (SEQ ID NO: 203). In some embodiments, a peptide comprising a KRAS G12D mutation comprises the neoepitope sequence KLVVVGADGV (SEQ ID NO: 312). In some embodiments, a peptide comprising a KRAS G12D mutation comprises the neoepitope sequence LVVVGADGV (SEQ ID NO: 313).

In some embodiments, a peptide comprising a KRAS G12V mutation comprises the sequence MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDP TIEDSYRKQVVIDGETCLLDILDTAGQE (SEQ ID NO: 314). In some embodiments, a peptide comprising a KRAS G12V mutation comprises the neoepitope sequence KLVVVGAVGV (SEQ ID NO: 315). In some embodiments, a peptide comprising a KRAS G12V mutation comprises the neoepitope sequence LVVVGAVGV (SEQ ID NO: 316). In some embodiments, a peptide comprising a KRAS G12V mutation comprises the neoepitope sequence VVGAVGVGK (SEQ ID NO: 220). In some embodiments, a peptide comprising a KRAS G12V mutation comprises the neoepitope sequence VVVGAVGVGK (SEQ ID NO: 218).

In some embodiments, a peptide comprising a KRAS Q61H mutation comprises the sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV IDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM (SEQ ID NO: 317). In some embodiments, a peptide comprising a KRAS Q61H mutation comprises the neoepitope sequence ILDTAGHEEY (SEQ ID NO: 318).

In some embodiments, a peptide comprising a KRAS Q61L mutation comprises the sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV IDGETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM (SEQ ID NO: 319). In some embodiments, a peptide comprising a KRAS Q61L mutation comprises the neoepitope sequence ILDTAGLEEY (SEQ ID NO: 320). In some embodiments, a peptide comprising a KRAS Q61L mutation comprises the neoepitope sequence LLDILDTAGL (SEQ ID NO: 321).

In some embodiments, a peptide comprising a NRAS Q61K mutation comprises the sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV IDGETCLLDILDTAGKEEYSAMRDQYMRTGEGFLCVFAINNSSKSFADINLYREQIKRVKDSDDVPM (SEQ ID NO: 322). In some embodiments, a peptide comprising a NRAS Q61K mutation comprises the neoepitope sequence ILDTAGKEEY (SEQ ID NO: 323).

In some embodiments, a peptide comprising a NRAS Q61R mutation comprises the sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV IDGETCLLDILDTAGREEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM (SEQ ID NO: 324). In some embodiments, a peptide comprising a NRAS Q61R mutation comprises the neoepitope sequence ILDTAGREEY (SEQ ID NO: 325).

In some embodiments, a peptide comprising a RAS Q61H mutation comprises the sequence TCLLDILDTAGHEEYSAMRDQYM (SEQ ID NO: 326). In some embodiments, a peptide comprising a RAS Q61H mutation comprises a sequence as shown in Table 1. In some embodiments, a peptide sequence as shown in Table 1 binds or is predicted to bind to a protein encoded by an HLA allele, wherein said allele is shown in the corresponding column of Table 1 adjacent to the peptide sequence.

Table 1. Peptide sequences containing the RAS Q61H mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank ILDTAGHEEY 318 HLA-A36:01 1 ILDTAGHEEY 318 HLA-A01:01 2 DTAGHEEYSAM 74 HLA-A26:01 3 DTAGHEEYSAM 74 HLA-A25:01 4 GHEESYAM 105 HLA-B15:09 4 DTAGHEEY 22 HLA-A26:01 5 ILDTAGHEE 26 HLA-C08:02 5 AGHEESYAM 327 HLA-C01:02 6 AGHEESYAM 327 HLA-B46:01 6 DTAGHEEY 22 HLA-A25:01 6 DTAGHEEY 22 HLA-A01:01 6 DTAGHEEY 22 HLA-B18:01 7 DTAGHEEY 22 HLA-A36:01 7 ILDTAGHEE 26 HLA-C05:01 7 ILDTAGHEE 26 HLA-A02:07 7 ILDTAGHEEY 318 HLA-A29:02 7 ILDTAGHEEY 318 HLA-C08:02 7 HEEYSAMRD 110 HLA-B49:01 8 TAGHEEYSA 75 HLA-B35:03 8 DTAGHEEYS 29 HLA-A68:02 9 DTAGHEEYSAMR 90 HLA-A68:01 9 GHEESYAM 105 HLA-B39:01 9 ILDTAGHEE 26 HLA-A01:01 9 LDTAGHEEY 328 HLA-B53:01 9 HEEYSAMRD 110 HLA-B41:01 10 ILDTAGHEE 26 HLA-A36:01 10 DTAGHEEY 22 HLA-B58:01 11 LLDILDTAGH 30 HLA-A01:01 12 TAGHEESYAM 23 HLA-B35:03 12 LDTAGHEEY 328 HLA-B35:01 13 DILDTAGHE 24 HLA-A26:01 14 DTAGHEEY 22 HLA-C12:03 14 ILDTAGHEEY 318 HLA-C05:01 14 AGHEESYAM 327 HLA-A30:02 15 DILDTAGHEEY 28 HLA-A25:01 15 DTAGHEEY 22 HLA-C02:02 15 ILDTAGHEE 26 HLA-C04:01 15 DILDTAGH 25 HLA-A26:01 16 ILDTAGHEE 26 HLA-A02:01 16 LDTAGHEEY 328 HLA-A29:02 16 ILDTAGHE 27 HLA-A01:01 17 LDTAGHEEY 328 HLA-B18:01 17 AGHEESYAM 327 HLA-C14:03 18 DILDTAGHEEY 28 HLA-A29:02 18 DTAGHEEYS 29 HLA-A26:01 18 ILDTAGHEEY 318 HLA-B15:01 18 DTAGHEEYSA 329 HLA-A68:02 19 ILDTAGHE 27 HLA-C05:01 19 ILDTAGHEEY 318 HLA-A02:07 19 ILDTAGHEEY 318 HLA-A30:02 19 LDTAGHEEY 328 HLA-A36:01 19 AGHEESYAM 327 HLA-C14:02 20 AGHEESYAM 327 HLA-B15:03 20 LLDILDTAGH 30 HLA-A02:07 20

In some embodiments, a peptide comprising a RAS Q61R mutation comprises the sequence TCLLDILDTAGREEYSAMRDQYM (SEQ ID NO: 330). In some embodiments, a peptide comprising a RAS Q61R mutation comprises the sequence presented in Table 2. In some embodiments, the peptide sequence presented in Table 2 binds or is predicted to bind to a protein encoded by an HLA allele, where the allele is indicated in the corresponding column in Table 2 next to the peptide sequence.

Table 2. Peptide sequences containing the RAS Q61R mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank ILDTAGREEY 325 HLA-A36:01 1 ILDTAGREEY 325 HLA-A01:01 2 DTAGREEYSAM 76 HLA-A26:01 3 DILDTAGR 32 HLA-A33:03 4 DILDTAGR 32 HLA-A68:01 5 DTAGREY 331 HLA-A26:01 6 DTAGREEYSAM 76 HLA-A25:01 6 CLLDILDTAGR 35 HLA-A74:01 7 DTAGREY 331 HLA-A01:01 7 REEYSAMRD 37 HLA-B41:01 7 GREEYSAMR 109 HLA-B27:05 8 ILDTAGREE 33 HLA-C08:02 8 ILDTAGREEY 325 HLA-A29:02 8 REEYSAMRD 37 HLA-B49:01 8 AGREEYSAM 332 HLA-B46:01 9 DTAGREY 331 HLA-B18:01 9 DTAGREY 331 HLA-A25:01 9 DTAGREY 331 HLA-A36:01 9 DILDTAGR 32 HLA-A74:01 10 DILDTAGRE 31 HLA-A26:01 10 ILDTAGREE 33 HLA-C05:01 10 DILDTAGR 32 HLA-A26:01 11 GREEYSAM 108 HLA-B39:01 11 AGREEYSAM 332 HLA-B15:03 12 GREEYSAM 108 HLA-C07:02 12 ILDTAGREE 33 HLA-A01:01 12 TAGREEYSA 333 HLA-B35:03 12 ILDTAGREEY 325 HLA-A30:02 13 DTAGREEYS 96 HLA-A68:02 14 ILDTAGRE 34 HLA-A01:01 14 CLLDILDTAGR 35 HLA-A31:01 15 DTAGREEYSAMR 97 HLA-A68:01 15 LLDILDTAGR 334 HLA-A01:01 15 DTAGREY 331 HLA-B58:01 16 ILDTAGREEY 325 HLA-C08:02 16 DILDTAGR 32 HLA-A31:01 17 ILDTAGREE 33 HLA-C04:01 17 ILDTAGREEY 325 HLA-A32:01 17 LLDILDTAGR 334 HLA-A74:01 17 TAGREEYSAM 36 HLA-B35:03 17 DILDTAGREEY 89 HLA-A32:01 18 ILDTAGRE 34 HLA-C05:01 18 ILDTAGREE 33 HLA-A02:07 18 REEYSAMRD 37 HLA-B40:01 18 AGREEYSAM 332 HLA-B15:01 19 AGREEYSAMR 335 HLA-A31:01 19 ILDTAGRE 34 HLA-A36:01 19 LDILDTAGR 113 HLA-A68:01 19 LDTAGREEY 336 HLA-A29:02 19 LDTAGREEY 336 HLA-B35:01 19 REEYSAMRD 37 HLA-B45:01 19 REEYSAMRDQY 117 HLA-A36:01 19 DTAGREY 331 HLA-C02:02 20

In some embodiments, a peptide comprising a RAS Q61K mutation comprises the sequence TCLLDILDTAGKEEYSAMRDQYM (SEQ ID NO: 337). In some embodiments, a peptide comprising a RAS Q61K mutation comprises the sequence presented in Table 3. In some embodiments, the peptide sequence presented in Table 3 binds or is predicted to bind to a protein encoded by an HLA allele, said allele being presented in the corresponding column of Table 3 adjacent to the peptide sequence.

Table 3. Peptide sequences containing the RAS Q61K mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank ILDTAGKEEY 323 HLA-A36:01 1 ILDTAGKEEY 323 HLA-A01:01 2 DTAGKEEYSAM 38 HLA-A26:01 3 CLLDILDTAGK 39 HLA-A03:01 4 DTAGKEEY 40 HLA-A01:01 5 DTAGKEEY 40 HLA-A26:01 5 DTAGKEEYSAM 38 HLA-A25:01 5 AGKEEYSAM 338 HLA-B46:01 6 DILDTAGKE 130 HLA-A26:01 7 KEEYSAMRD 111 HLA-B41:01 7 DTAGKEEY 40 HLA-B18:01 8 GKEEYSAM 106 HLA-B15:03 8 ILDTAGKEE 43 HLA-C08:02 8 ILDTAGKEEY 323 HLA-A29:02 8 DTAGKEEYS 91 HLA-A68:02 9 LDTAGKEEY 339 HLA-B53:01 9 TAGKEEYSA 77 HLA-B35:03 9 DILDTAGK 88 HLA-A68:01 10 DTAGKEEY 40 HLA-A36:01 10 KEEYSAMRD 111 HLA-B49:01 10 LDTAGKEEY 339 HLA-C07:01 10 DTAGKEEYSAMR 92 HLA-A68:01 11 ILDTAGKEE 43 HLA-C05:01 11 ILDTAGKEEY 323 HLA-C08:02 11 LLDILDTAGK 41 HLA-A01:01 12 AGKEEYSAM 338 HLA-A30:02 13 DTAGKEEY 40 HLA-A25:01 13 DTAGKEEYS 91 HLA-A26:01 13 ILDTAGKE 42 HLA-C05:01 13 LDTAGKEEY 339 HLA-B35:01 13 AGKEEYSAMR 81 HLA-A31:01 14 DILDTAGK 88 HLA-A33:03 14 ILDTAGKE 42 HLA-A01:01 14 ILDTAGKEE 43 HLA-A01:01 14 ILDTAGKEE 43 HLA-A02:07 14 TAGKEEYSAM 73 HLA-B35:03 14 AGKEEYSAM 338 HLA-B15:01 15 ILDTAGKEEY 323 HLA-A30:02 15 LDTAGKEEY 339 HLA-B46:01 15 DTAGKEEY 40 HLA-B58:01 16 ILDTAGKEEY 323 HLA-C05:01 17 AGKEEYSAM 338 HLA-A30:01 18 AGKEEYSAM 338 HLA-B15:03 18 DTAGKEEY 40 HLA-C02:02 18 LDTAGKEEY 339 HLA-A29:02 18

In some embodiments, a peptide comprising a RAS Q61L mutation comprises the sequence TCLLDILDTAGLEEYSAMRDQYM (SEQ ID NO: 340). In some embodiments, a peptide comprising a RAS Q61L mutation comprises the sequence presented in Table 4. In some embodiments, the peptide sequence presented in Table 4 binds or is predicted to bind to a protein encoded by an HLA allele, wherein the allele is indicated in the corresponding column in Table 4 adjacent to the peptide sequence.

Table 4. Peptide sequences including the RAS Q61L mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank ILDTAGLEY 320 HLA-A36:01 1 ILDTAGLEY 320 HLA-A01:01 2 LLDILDTAGL 321 HLA-A02:07 3 GLEEYSAMRDQY 48 HLA-A36:01 4 DTAGLEY 44 HLA-A25:01 5 DTAGLEY 44 HLA-A26:01 5 DTAGLEEYSAM 341 HLA-A26:01 5 DTAGLEY 44 HLA-A01:01 6 ILDTAGLEE 47 HLA-C08:02 6 ILDTAGLEE 47 HLA-A01:01 6 CLLDILDTAGL 342 HLA-A02:04 7 ILDTAGLEE 47 HLA-A36:01 7 LLDILDTAGL 321 HLA-A01:01 7 DILDTAGL 46 HLA-B14:02 8 DILDTAGLEY 52 HLA-A25:01 8 DTAGLEEYS 93 HLA-A68:02 8 DTAGLEEYSAM 341 HLA-A25:01 8 GLEEYSAMR 107 HLA-A74:01 8 ILDTAGLE 45 HLA-A01:01 8 DILDTAGLEY 52 HLA-A26:01 9 DTAGLEY 44 HLA-A36:01 9 ILDTAGLEY 320 HLA-A29:02 9 DILDTAGL 46 HLA-B08:01 10 DTAGLEY 44 HLA-B18:01 10 ILDTAGLEE 47 HLA-A02:07 10 LDTAGLEEY 343 HLA-B35:01 10 CLLDILDTAGL 342 HLA-A02:01 11 DTAGLEY 44 HLA-C02:02 11 ILDTAGLEE 47 HLA-C05:01 11 ILDTAGLEY 320 HLA-C08:02 11 ILDTAGLEY 320 HLA-A02:07 11 LLDILDTAGL 321 HLA-C08:02 11 DILDTAGL 46 HLA-A26:01 12 LDTAGLEEY 343 HLA-B53:01 12 DTAGLEY 44 HLA-C03:02 13 DTAGLEY 44 HLA-B58:01 13 ILDTAGLEY 320 HLA-A30:02 13 LLDILDTAGL 321 HLA-C05:01 13 LLDILDTAGL 321 HLA-C04:01 13 DTAGLEEYSAMR 95 HLA-A68:01 14 ILDTAGLE 45 HLA-A36:01 15 LLDILDTAGL 321 HLA-A02:01 15 AGLEEYSAM 344 HLA-B15:03 16 DTAGLEEYSA 94 HLA-A68:02 16 GLEEYSAMRDQY 48 HLA-A01:01 16 ILDTAGLE 45 HLA-C04:01 16 ILDTAGLEY 320 HLA-B15:01 16 LDILDTAGL 50 HLA-B37:01 16 AGLEEYSAM 344 HLA-A30:02 17 AGLEEYSAM 344 HLA-B48:01 17 AGLEEYSAMR 345 HLA-A31:01 17 ILDTAGLEE 47 HLA-C04:01 17 LDTAGLEEY 343 HLA-C03:02 17 AGLEEYSAM 344 HLA-C14:02 18 GLEEYSAMR 107 HLA-A31:01 18 LEEYSAMRD 114 HLA-B41:01 18 LLDILDTAGLE 49 HLA-A01:01 18 AGLEEYSAM 344 HLA-C14:03 19 LDILDTAGL 50 HLA-B40:02 19 LDTAGLEEY 343 HLA-A29:02 19 DILDTAGLE 51 HLA-A26:01 20 DTAGLEY 44 HLA-B15:01 20 ILDTAGLEY 320 HLA-A02:01 20 LDTAGLEEY 343 HLA-A36:01 20 LDTAGLEEY 343 HLA-B46:01 20 DTAGLEY 44 HLA-A68:02 21 DTAGLEY 44 HLA-C12:03 21 ILDTAGLE 45 HLA-C05:01 21 LDTAGLEEY 343 HLA-B18:01 21 LEEYSAMRD 114 HLA-B49:01 21 TAGLEEYSA 119 HLA-B54:01 21 DILDTAGLEY 52 HLA-A29:02 22 GLEEYSAM 346 HLA-C05:01 22

In some embodiments, a peptide comprising a RAS G12A mutation comprises the sequence MTEYKLVVVGAAGVGKSALTIQL (SEQ ID NO: 347). In some embodiments, a peptide comprising a RAS G12A mutation comprises the sequence presented in Table 5. In some embodiments, the peptide sequence presented in Table 5 binds or is predicted to bind to a protein encoded by an HLA allele, the allele being indicated in the corresponding column in Table 5 next to the peptide sequence.

Table 5. Peptide sequences containing the RAS G12A mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank AAGVGKSAL 55 HLA-C03:04 1 VVVGAAGVGK 192 HLA-A11:01 1 VVGAAGVGK 194 HLA-A11:01 2 TEYKLVVVGAA 120 HLA-B50:01 3 VVGAAGVGK 194 HLA-A03:01 3 VVVGAAGVGK 192 HLA-A68:01 3 AAGVGKSAL 55 HLA-C08:02 4 AAGVGKSAL 55 HLA-C08:01 4 AAGVGKSAL 55 HLA-B46:01 4 AAGVGKSAL 55 HLA-B81:01 5 GAAGVGKSAL 54 HLA-B48:01 5 LVVVGAAGV 348 HLA-A68:02 5 AAGVGKSAL 55 HLA-C03:04 1 VVVGAAGVGK 192 HLA-A11:01 1 VVGAAGVGK 194 HLA-A11:01 2 TEYKLVVVGAA 120 HLA-B50:01 3 VVGAAGVGK 194 HLA-A03:01 3 VVVGAAGVGK 192 HLA-A68:01 3 AAGVGKSAL 55 HLA-C08:02 4 AAGVGKSAL 55 HLA-C08:01 4 AAGVGKSAL 55 HLA-B46:01 4 AAGVGKSAL 55 HLA-B81:01 5 AAGVGKSAL 55 HLA-C03:02 5 AAGVGKSAL 55 HLA-C01:02 5 GAAGVGKSAL 54 HLA-B48:01 5 LVVVGAAGV 348 HLA-A68:02 5 AAGVGKSAL 55 HLA-C03:03 6 VVGAAGVGK 194 HLA-A68:01 6 GAAGVGKSAL 54 HLA-B81:01 7 VVVGAAGVGK 192 HLA-A03:01 7 AAGVGKSAL 55 HLA-C05:01 8 AAGVGKSAL 55 HLA-C12:03 8 GAAGVGKSA 98 HLA-B46:01 8 VVGAAGVGK 194 HLA-A30:01 8 GAAGVGKSA 98 HLA-B55:01 9 KLVVVGAAGV 349 HLA-A02:01 9 AGVGKSAL 53 HLA-B08:01 10 GAAGVGKSAL 54 HLA-C03:04 10 AAGVGKSAL 55 HLA-C17:01 11 GAAGVGKSAL 54 HLA-C03:03 11 VVVGAAGV 125 HLA-A68:02 11 YKLVVVGAA 350 HLA-B54:01 11 AAGVGKSAL 55 HLA-B48:01 12 AGVGKSAL 53 HLA-C03:04 12 AGVGKSAL 53 HLA-C07:01 12 VVVGAAGVGK 192 HLA-A30:01 12 AAGVGKSA 78 HLA-B46:01 13 KLVVVGAAGV 349 HLA-A02:07 13 YKLVVVGAA 350 HLA-B50:01 13 AAGVGKSAL 55 HLA-B07:02 14 GAAGVGKSAL 54 HLA-A68:02 14 VVGAAGVGK 194 HLA-A74:01 14 AGVGKSAL 53 HLA-C08:01 15 GAAGVGKSAL 54 HLA-C17:01 15 GAAGVGKSAL 54 HLA-C08:01 16 GAAGVGKSAL 54 HLA-B35:03 16 AAGVGKSAL 55 HLA-C02:02 17 AAGVGKSAL 55 HLA-B35:03 17 AAGVGKSAL 55 HLA-C12:02 17 AAGVGKSAL 55 HLA-C14:03 17 GAAGVGKSA 98 HLA-B50:01 17 AGVGKSAL 53 HLA-C03:02 18 GAAGVGKSA 98 HLA-C03:04 18 LVVVGAAGV 348 HLA-B55:01 18 TEYKLVVVGAA 120 HLA-B41:01 18 AGVGKSAL 53 HLA-C01:02 19 GAAGVGKSA 98 HLA-B54:01 19 GAAGVGKSAL 54 HLA-B07:02 19 VGAAGVGKSA 121 HLA-B55:01 19 AGVGKSAL 53 HLA-B48:01 20 AGVGKSALTI 82 HLA-B49:01 20 VVVGAAGV 125 HLA-B55:01 20

In some embodiments, a peptide comprising a RAS G12C mutation comprises the sequence MTEYKLVVVGACGVGKSALTIQL (SEQ ID NO: 351). In some embodiments, a peptide comprising a RAS G12C mutation comprises the sequence presented in Table 6. In some embodiments, the peptide sequence presented in Table 6 binds or is predicted to bind to a protein encoded by an HLA allele, the allele being indicated in the corresponding column in Table 6 next to the peptide sequence.

Table 6. Peptide sequences containing the RAS G12C mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank VVVGACGVGK 197 HLA-A11:01 1 VVGACGVGK 200 HLA-A03:01 2 VVGACGVGK 200 HLA-A11:01 3 VVVGACGVGK 197 HLA-A68:01 4 VVGACGVGK 200 HLA-A68:01 5 VVVGACGVGK 197 HLA-A03:01 5 VVGACGVGK 200 HLA-A30:01 6 ACGVGKSAL 57 HLA-B81:01 7 ACGVGKSAL 57 HLA-C01:02 7 ACGVGKSAL 57 HLA-C14:03 8 ACGVGKSAL 57 HLA-C03:04 9 VVVGACGVGK 197 HLA-A30:01 9 ACGVGKSAL 57 HLA-C14:02 10 CGVGKSAL 56 HLA-B08:01 10 KLVVVGACGV 10 HLA-A02:01 10 ACGVGKSAL 57 HLA-B07:02 11 GACGVGKSAL 5 HLA-B48:01 12 GACGVGKSAL 5 HLA-C03:03 13 ACGVGKSAL 57 HLA-B48:01 14 ACGVGKSAL 57 HLA-B40:01 14 YKLVVVGAC 127 HLA-B48:01 14 YKLVVVGAC 127 HLA-B15:03 14 GACGVGKSA 8 HLA-B46:01 15 GACGVGKSAL 5 HLA-C03:04 15 GACGVGKSAL 5 HLA-C01:02 15 LvVVGACGV 310 HLA-A68:02 15 CGVGKSAL 56 HLA-C03:04 16 GACGVGKSAL 5 HLA-C08:02 16 VVGACGVGK 200 HLA-A74:01 16

In some embodiments, a peptide comprising a RAS G12D mutation comprises the sequence MTEYKLVVVGADGVGKSALTIQL (SEQ ID NO: 352). In some embodiments, a peptide comprising a RAS G12D mutation comprises the sequence presented in Table 7. In some embodiments, the peptide sequence presented in Table 7 binds or is suspected of binding to a protein encoded by an HLA allele, the allele being indicated in the corresponding column in Table 7 next to the peptide sequence.

Table 7. Peptide sequences containing the RAS G12D mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank GADGVGKSAL 4 HLA-C08:02 1 GADGVGKSAL 4 HLA-C05:01 2 VVVGADGVGK 203 HLA-A11:01 3 DGVGKSAL 58 HLA-B14:02 4 VVGADGVGK 206 HLA-A11:01 4 VVGADGVGK 206 HLA-A03:01 5 DGVGKSAL 58 HLA-B08:01 6 VVVGADGVGK 203 HLA-A68:01 6 GADGVGKSAL 4 HLA-C03:03 7 VVGADGVGK 206 HLA-A30:01 7 ADGVGKSAL 59 HLA-B37:01 8 GADGVGKSAL 4 HLA-C08:01 8 VVGADGVGK 206 HLA-A68:01 8 GADGVGKSA 7 HLA-C08:02 9 GADGVGKSAL 4 HLA-B35:03 9 GADGVGKS 99 HLA-C05:01 10 GADGVGKSA 7 HLA-C05:01 10 ADGVGKSAL 59 HLA-C07:01 11 VVVGADGVGK 203 HLA-A03:01 11 ADGVGKSAL 59 HLA-B40:02 12 ADGVGKSAL 59 HLA-B46:01 13 GADGVGKSAL 4 HLA-C03:04 13 ADGVGKSAL 59 HLA-B81:01 14 GADGVGKSAL 4 HLA-C17:01 14 VVVGADGVGK 203 HLA-A30:01 14 GADGVGKSA 7 HLA-B35:03 15 GADGVGKSA 7 HLA-B46:01 15 GADGVGKSAL 4 HLA-B48:01 15 KLVVVGADGV 312 HLA-A02:01 15 LvVVGADGV 313 HLA-A68:02 15 VGADGVGKSA 353 HLA-B55:01 15 VVGADGVGK 206 HLA-A74:01 16 GADGVGKSA 7 HLA-B53:01 17 KLVVVGADGV 312 HLA-A02:07 17 VGADGVGK 122 HLA-A68:01 17 YKLVVVGAD 128 HLA-B48:01 17 ADGVGKSAL 59 HLA-C14:03 18 DGVGKSALTI 60 HLA-B51:01 18 VGADGVGK 122 HLA-A11:01 18

In some embodiments, a peptide comprising a RAS G12R mutation comprises the sequence MTEYKLVVVGARGVGKSALTIQL (SEQ ID NO: 354). In some embodiments, a peptide comprising a RAS G12R mutation comprises the sequence presented in Table 8. In some embodiments, the peptide sequence presented in Table 8 binds or is predicted to bind to a protein encoded by an HLA allele, said allele being presented in the corresponding column of Table 8 adjacent to the peptide sequence.

Table 8. Peptide sequences containing the RAS G12R mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank VVGARGVGK 210 HLA-A11:01 1 VVVGARGVGK 208 HLA-A68:01 1 GARGVGKSA 61 HLA-B46:01 2 ARGVGKSAL 83 HLA-B27:05 3 GARGVGKSA 61 HLA-B55:01 3 RGVGKSAL 118 HLA-C07:01 4 VVGARGVGK 210 HLA-A30:01 5 ARGVGKSAL 83 HLA-B38:01 6 ARGVGKSAL 83 HLA-B14:02 6 VVGARGVGK 210 HLA-A68:01 6 VVVGARGVGK 208 HLA-A03:01 7 GARGVGKSAL 355 HLA-B48:01 8 RGVGKSAL 118 HLA-B48:01 8 RGVGKSALTI 356 HLA-A23:01 8 ARGVGKSAL 83 HLA-C06:02 9 GARGVGKSA 61 HLA-A30:01 9 GARGVGKSAL 355 HLA-B81:01 9 VVVGARGVGK 208 HLA-A30:01 9 GARGVGKSAL 355 HLA-B07:02 10 LVVVGARGV 115 HLA-C06:02 10 RGVGKSAL 118 HLA-B81:01 10 VVGARGVGK 210 HLA-A74:01 11 KLVVVGARGV 62 HLA-A02:01 12 LVVVGARGV 115 HLA-B55:01 12 YKLVVVGAR 129 HLA-A33:03 12 KLVVVGAR 357 HLA-A74:01 13 KLVVVGARGV 62 HLA-B13:02 13 RGVGKSAL 118 HLA-C01:02 13 LVVVGARGV 115 HLA-A68:02 14 VVVGARGV 63 HLA-B55:01 14 ARGVGKSAL 83 HLA-B15:09 15 ARGVGKSAL 83 HLA-C14:03 16 GARGVGKSA 61 HLA-B54:01 16 VVVGARGV 63 HLA-B52:01 16

In some embodiments, a peptide comprising a RAS G12S mutation comprises the sequence MTEYKLVVVGASGVGKSALTIQL (SEQ ID NO: 358). In some embodiments, a peptide comprising a RAS G12S mutation comprises the sequence presented in Table 9. In some embodiments, the peptide sequence presented in Table 9 binds or is predicted to bind to a protein encoded by an HLA allele, said allele being indicated in the corresponding column in Table 9 adjacent to the peptide sequence.

Table 9. Peptide sequences containing the RAS G12S mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank VVVGASGVGK 65 HLA-A11:01 1 VVGASGVGK 215 HLA-A11:01 2 VVGASGVGK 215 HLA-A03:01 3 VVVGASGVGK 65 HLA-A68:01 4 ASGVGKSAL 85 HLA-C03:04 5 ASGVGKSAL 85 HLA-B46:01 5 VVGASGVGK 215 HLA-A68:01 6 VVVGASGVGK 65 HLA-A03:01 6 ASGVGKSAL 85 HLA-C01:02 7 GASGVGKSAL 66 HLA-B48:01 7 ASGVGKSAL 85 HLA-C07:01 8 ASGVGKSAL 85 HLA-C08:02 9 GASGVGKSAL 66 HLA-B81:01 9 SGVGKSAL 64 HLA-B08:01 9 ASGVGKSAL 85 HLA-C03:03 10 ASGVGKSAL 85 HLA-C03:02 10 SGVGKSAL 64 HLA-B14:02 10 VVGASGVGK 215 HLA-A30:01 10 ASGVGKSAL 85 HLA-C08:01 11 VVVGASGVGK 65 HLA-A30:01 11 GASGVGKSAL 66 HLA-B35:03 12 SGVGKSAL 64 HLA-C07:01 12 ASGVGKSAL 85 HLA-B81:01 13 GASGVGKSA 102 HLA-B55:01 13 GASGVGKSAL 66 HLA-C03:03 13 KLVVVGASGV 359 HLA-A02:01 13 LvVVGASGV 116 HLA-A68:02 13 SGVGKSAL 64 HLA-C01:02 13 ASGVGKSA 84 HLA-B46:01 14 ASGVGKSAL 85 HLA-C15:02 14 GASGVGKSAL 66 HLA-C08:01 15 SGVGKSAL 64 HLA-C03:04 15 ASGVGKSAL 85 HLA-C05:01 16 GASGVGKSAL 66 HLA-C03:04 16 VVGASGVGK 215 HLA-A74:01 16 ASGVGKSAL 85 HLA-B48:01 17 GASGVGKSAL 66 HLA-C01:02 17 SGVGKSAL 64 HLA-C03:02 17 SGVGKSALTI 360 HLA-A23:01 17 VGASGVGKSA 123 HLA-B55:01 18 ASGVGKSAL 85 HLA-C12:03 19 ASGVGKSAL 85 HLA-B57:03 19 KLVVVGASGV 359 HLA-A02:07 19 SGVGKSAL 64 HLA-B81:01 19 ASGVGKSAL 85 HLA-C17:01 20 KLVVVGASG 112 HLA-A32:01 20

In some embodiments, a peptide comprising a RAS G12V mutation comprises the sequence MTEYKLVVVGAVGVGKSALTIQL (SEQ ID NO: 361). In some embodiments, a peptide comprising a RAS G12V mutation comprises a sequence presented in Table 10. In some embodiments, a peptide sequence presented in Table 10 binds or is predicted to bind to a protein encoded by an HLA allele, said allele being presented in the corresponding column of Table 10 adjacent to the peptide sequence.

Table 10. Peptide sequences containing the RAS G12V mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank VVGAVGVGK 220 HLA-A03:01 1 VVGAVGVGK 220 HLA-A11:01 2 VVVGAVGVGK 218 HLA-A11:01 2 VVVGAVGVGK 218 HLA-A68:01 3 VVGAVGVGK 220 HLA-A68:01 4 LvVVGAVGV 316 HLA-A68:02 5 VVGAVGVGK 220 HLA-A30:01 5 AVGVGKSAL 362 HLA-B81:01 6 KLVVVGAVGV 315 HLA-A02:01 6 AVGVGKSAL 362 HLA-B46:01 7 GAVGVGKSAL 6 HLA-C03:03 7 GAVGVGKSAL 6 HLA-B48:01 7 VVVGAVGVGK 218 HLA-A03:01 7 AVGVGKSAL 362 HLA-C03:04 8 GAVGVGKSAL 6 HLA-C03:04 8 KLVVVGAVGV 315 HLA-A02:07 9 VGVGKSAL 67 HLA-B08:01 9 VVVGAVGV 126 HLA-A68:02 9 AVGVGKSAL 362 HLA-C08:02 10 AVGVGKSAL 362 HLA-B07:02 10 GAVGVGKSAL 6 HLA-B35:03 10 AVGVGKSAL 362 HLA-C08:01 11 AVGVGKSAL 362 HLA-C01:02 11 GAVGVGKSA 9 HLA-B55:01 11 GAVGVGKSAL 6 HLA-B81:01 11 GAVGVGKSAL 6 HLA-C08:01 11 KLVVVGAVGV 315 HLA-B13:02 11 VGVGKSAL 67 HLA-C03:04 11 AVGVGKSAL 362 HLA-A32:01 12 GAVGVGKSA 9 HLA-B46:01 12 VGVGKSAL 67 HLA-C03:02 12 VGVGKSALTI 124 HLA-A23:01 12 GAVGVGKSA 9 HLA-B54:01 13 VGVGKSAL 67 HLA-C01:02 .3 AVGVGKSAL 362 HLA-B48:01 14 AVGVGKSAL 362 HLA-C03:03 14 AVGVGKSAL 362 HLA-B42:01 14 LvVVGAVGV 316 HLA-B55:01 14 VGVGKSAL 67 HLA-C08:01 14 VVGAVGVGK 220 HLA-A74:01 14 AVGVGKSAL 362 HLA-C05:01 15 AVGVGKSAL 362 HLA-C03:02 15 GAVGVGKSA 9 HLA-C03:04 15 KLVVVGAVGV 315 HLA-A02:04 15 LvVVGAVGV 316 HLA-A02:07 15 VGVGKSAL 67 HLA-B14:02 15 VVVGAVGVGK 218 HLA-A30:01 15 VVGAVGVGK 220 HLA-B81:01 16 VVVGAVGV 126 HLA-B55:01 16 AVGVGKSAL 362 HLA-C14:03 17 AVGVGKSAL 362 HLA-B15:01 17 LvVVGAVGV 316 HLA-B54:01 17 AVGVGKSA 86 HLA-B55:01 18 AVGVGKSAL 362 HLA-C17:01 18 GAVGVGKSA 9 HLA-B50:01 19 GAVGVGKSAL 6 HLA-C17:01 19 YKLVVVGAV 363 HLA-A02:04 19 GAVGVGKSAL 6 HLA-B35:01 20 VVGAVGVGK 220 HLA-A31:01 20 YKLVVVGAV 363 HLA-B51:01 20

In some embodiments, a peptide comprising a RAS G13C mutation comprises the sequence MTEYKLVVVGAGCVGKSALTIQL (SEQ ID NO: 364). In some embodiments, a peptide comprising a RAS G13C mutation comprises the sequence presented in Table 11. In some embodiments, the peptide sequence presented in Table 11 binds or is predicted to bind to a protein encoded by an HLA allele, the allele being indicated in the corresponding column in Table 11 next to the peptide sequence.

Table 11. Peptide sequences including the RAS G13C mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank VVVGAGCVGK 68 HLA-A11:01 1 VVGAGCVGK 365 HLA-A11:01 2 AGCVGKSAL 79 HLA-C01:02 3 VVGAGCVGK 365 HLA-A03:01 4 VVVGAGCVGK 68 HLA-A68:01 4 CVGKSALTI 87 HLA-B13:02 5 VVGAGCVGK 365 HLA-A68:01 5 VVGAGCVGK 365 HLA-A30:01 6 AGCVGKSAL 79 HLA-B48:01 7 AGCVGKSAL 79 HLA-C03:04 8 GCVGKSALTI 104 HLA-B49:01 8 AGCVGKSAL 79 HLA-C08:02 9 VVVGAGCVGK 68 HLA-A03:01 9 KLVVVGAGC 69 HLA-A30:02 10 GCVGKSAL 103 HLA-C07:01 11 VVGAGCVGK 365 HLA-A74:01 12 AGCVGKSAL 79 HLA-C14:03 13 KLVVVGAGC 69 HLA-B15:01 14

In some embodiments, a peptide comprising a RAS G13D mutation comprises the sequence MTEYKLVVVGAGDVGKSALTIQL (SEQ ID NO: 366). In some embodiments, a peptide comprising a RAS G13D mutation comprises the sequence presented in Table 12. In some embodiments, a peptide sequence presented in Table 12 binds or is predicted to bind to a protein encoded by an HLA allele, said allele being presented in the corresponding column of Table 12 adjacent to the peptide sequence.

Table 12. Peptide sequences containing the RAS G13D mutation, the corresponding HLA allele and the binding potential rank Peptide SEQ ID NO: Allele Binding potential rank AGDVGKSAL 80 HLA-C08:02 1 AGDVGKSAL 80 HLA-C05:01 2 VVGAGDVGK 367 HLA-A11:01 3 VVVGAGDVGK 72 HLA-A11:01 3 VVVGAGDVGK 72 HLA-A68:01 4 GAGDVGKSA 100 HLA-B46:01 5 GAGDVGKSAL 101 HLA-B48:01 5 VVGAGDVGK 367 HLA-A68:01 5 VVGAGDVGK 367 HLA-A03:01 5 AGDVGKSAL 80 HLA-C03:04 6 AGDVGKSAL 80 HLA-C04:01 6 AGDVGKSAL 80 HLA-C01:02 6 DVGKSALTI 71 HLA-B13:02 6 DVGKSALTI 71 HLA-A25:01 6 GDVGKSAL 70 HLA-C07:01 6 GDVGKSAL 70 HLA-B40:02 7 GDVGKSAL 70 HLA-B37:01 8 AGDVGKSAL 80 HLA-B48:01 9 DVGKSALTI 71 HLA-B51:01 10 VVGAGDVGK 367 HLA-A30:01 10 GAGDVGKSAL 101 HLA-C08:01 11 GAGDVGKSAL 101 HLA-B81:01 11 AGDVGKSAL 80 HLA-C08:01 12 GAGDVGKSAL 101 HLA-C03:04 12 DVGKSALTI 71 HLA-B53:01 13 AGDVGKSAL 80 HLA-B07:02 14 AGDVGKSAL 80 HLA-B46:01 14 DVGKSALTI 71 HLA-A26:01 14 VVGAGDVGK 367 HLA-A74:01 14 GAGDVGKSA 100 HLA-B54:01 15 DVGKSALTI 71 HLA-B38:01 16 GAGDVGKSAL 101 HLA-C03:03 16 VVVGAGDVGK 72 HLA-A03:01 16

In some embodiments, a polypeptide described herein does not comprise a RAS epitope. In some embodiments, the epitope is not a RAS epitope. In some embodiments, the polypeptide does not comprise KKKKKPKRDGYMFLKAESKIMFAT (SEQ ID NO: 168), KKKKYMFLKAESKIMFATLQRSS (SEQ ID NO: 169), KKKKKAESKIMFATLQRSSLWCL (SEQ ID NO: 170), KKKKKIMFATLQRSSLWCLCSNH (SEQ ID NO: 171), or KKKKMFATLQRSSLWCLCSNH (SEQ ID NO: 172).

In some embodiments, the polypeptide comprising the antigen, neoantigenic peptide, or epitope comprises a GATA3 epitope. In some embodiments, the GATA3 epitope comprises the amino acid sequence of MLTGPPARV (SEQ ID NO: 173), SMLTGPPARV (SEQ ID NO: 174), VLPEPHLAL (SEQ ID NO: 175), KPKRDGYMF (SEQ ID NO: 176), KPKRDGYMFL (SEQ ID NO: 177), ESKIMFATL (SEQ ID NO: 178), KRDGYMFL (SEQ ID NO: 179), PAVPFDLHF (SEQ ID NO: 180), AESKIMFATL (SEQ ID NO: 181), FATLQRSSL (SEQ ID NO: 182), ARVPAVPFD (SEQ ID NO: 183), IMKPKRDGY (SEQ ID NO: 184), DGYMFLKA (SEQ ID NO: 185), MFLKAESKIMF (SEQ ID NO: 186), AESKIMFATL (SEQ ID NO: 187), AESKIMFATL (SEQ ID NO: 188), AESKIMFATL (SEQ ID NO: 189), AESKIMFATL (SEQ ID NO: 200), AESKIMFATL (SEQ ID NO: 201), AESKIMFATL (SEQ ID NO: 202), AESKIMFATL (SEQ ID NO: 203), AESKIMFATL (SEQ ID NO: 204), AESKIMFATL (SEQ ID NO: 205), AESKIMFATL (SEQ ID NO: 206), AESKIMFATL (SEQ ID NO: 207), AESKIMFATL (SEQ ID NO: 208), AESKIMFATL (SEQ ID NO: 209), AESKIMFATL (SEQ ID NO: 210), AESKIMFATL (SEQ ID NO: 211), AESK NO: 186), LTGPPARV (SEQ ID NO: 187), ARVPAVPF (SEQ ID NO: 188), SMLTGPPAR (SEQ ID NO: 189), RVPAVPFDL (SEQ ID NO: 190) or LTGPPARVP (SEQ ID NO: 191).

Peptide modifications

In some embodiments, the present disclosure includes modified peptides. A modification may include a covalent chemical modification that does not alter the primary amino acid sequence of the antigenic peptide itself. Modifications may produce peptides with desirable properties, such as extending half-life in vivo, increasing stability, decreasing clearance, altering immunogenicity or allergenicity, allowing for an increase in certain antibodies, cell targeting, antigen uptake, antigen processing, HLA affinity, HLA stability, or antigen presentation. In some embodiments, a peptide may contain one or more sequences that enhance the processing and presentation of APC epitopes, such as to generate an immune response.

In some embodiments, the polypeptide can be modified to impart desired properties. For example, the ability of the peptides to induce cytotoxic T lymphocyte (CTL) activity can be enhanced by linking to a sequence that contains at least one epitope capable of inducing a T helper cell response. In some embodiments, the immunogenic peptide/T helper cell conjugates are linked by a spacer molecule. In some embodiments, the spacer comprises relatively small neutral molecules, such as amino acids or amino acid mimetics that are substantially uncharged under physiological conditions. Spacers can be selected from, for example, Ala, Gly, or other neutral spacers of non-polar amino acids or neutral polar amino acids. It should be understood that the optionally present spacer does not have to be composed of identical residues and thus can be a hetero- or homo-oligomer. The neoantigen peptide may be linked to the T-helper peptide either directly or through a spacer at either the amino or carboxyl terminus of the peptide. The amino terminus of either the neoantigen peptide or the T-helper peptide may be acylated. Examples of T-helper peptides include residues 830-843 of tetanus toxin, residues 307-319 of influenza, and residues 382-398 of malaria circumsporozoite protein and residues 378-389.

The peptide sequences of the present invention may optionally be altered through changes at the DNA level, in particular by mutating the DNA that encodes the peptide at pre-selected bases so that codons are generated that will translate into the desired amino acids.

In some embodiments, a peptide described herein may contain substitutions to modify a physical property (e.g., stability or solubility) of the resulting peptide. For example, peptides can be modified by replacing cysteine (C) with α-aminobutyric acid ("B"). Due to its chemical nature, cysteine has a tendency to form disulfide bridges and significantly alter the structure of the peptide to reduce binding affinity. Replacing C with α-aminobutyric acid not only alleviates this problem, but in some cases actually improves binding and cross-linking affinity. Substitution of cysteine with α-aminobutyric acid can occur at any residue of the neoantigenic peptide, such as at anchor or non-anchor positions of an epitope or analog within the peptide or at other positions of the peptide.

A peptide may also be modified by lengthening or shortening the amino acid sequence of the compound, such as by adding or removing amino acids. Peptides or analogs may also be modified by changing the order or composition of certain residues. Those skilled in the art will appreciate that certain amino acid residues essential for biological activity, such as those at critical contact sites or conserved residues, generally cannot be altered without adversely affecting biological activity. Non-critical amino acids are not limited to those naturally occurring in proteins, such as L-α-amino acids or their D-isomers, but may also include non-natural amino acids, such as β-γ-δ-amino acids, as well as many derivatives of L-α-amino acids.

In some embodiments, the peptide can be modified using a series of peptides with single amino acid substitutions to determine the effect of 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 can be made along the length of the peptide, revealing different sensitivity profiles to different HLA molecules and T cell receptors. In addition, multiple substitutions can be used using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues. The substitutions can be homo-oligomers or hetero-oligomers. The number and types of residues that are replaced or added depend on the desired distance between the main contact points and the particular functional attributes that are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for the HLA molecule or T-cell receptor may also be achieved by such substitutions compared to the affinity of the original peptide. In any case, such substitutions should use amino acid residues or other molecular fragments chosen to avoid, for example, spatial and charge interference that could impair binding. Amino acid substitutions are usually single residues. Substitutions, deletions, insertions, or any combination thereof may be combined to produce the final peptide.

In some embodiments, a peptide described herein may comprise amino acid mimetics or unnatural amino acid residues, such as D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1,2,3- or 4-pyrenylalanine; D- or L-3 thienylalanine; D- or L-(2-pyridinyl)alanine; D- or L-(3-pyridinyl)alanine; D- or L-(2-pyrazinyl)alanine; D- or L-(4-isopropyl)phenylglycine; D-(trifluoromethyl)phenylglycine; D-(trifluoromethyl)phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenylphenylalanine; D- or L-ρ-methoxybiphenylphenylalanine; D- or L-2-indole(allyl)alanines; and, D- or L-alkylalanines, wherein the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl or non-acidic amino acid residues. Aromatic rings of non-natural amino acid include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl and pyridyl aromatic rings. Modified peptides that have various amino acid mimetics or non-natural amino acid residues may have increased stability in vivo. Such peptides may also have improved storage or manufacturing properties.

In some embodiments, a peptide described herein may be modified by terminal-NH ₂ acylation, such as acetylation with an alkanoyl (C ₁ -C ₂₀ ) or thioglycolyl, amidation of the terminal carboxyl, such as with ammonia, methylamine, etc. In some embodiments, these modifications may provide sites for attachment to a support or another molecule. In some embodiments, a peptide described herein may contain modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of a surfactant such as a lipid, or may be chemically modified such as acetylation, etc. Moreover, the bonds in the peptide may be other than peptide bonds, such as covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

In some embodiments, the peptide described in the present invention may comprise carriers such as those well known in the art, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxin, polyamino acid residues such as poly-L-lysine and poly-L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.

Peptides can be further modified to contain additional chemical groups not normally part of the protein. These derivatized groups can improve solubility, biological half-life, protein absorption, or binding affinity. The groups can also reduce or eliminate any desirable side effects of the peptides, and the like. A review of these groups can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000). For example, neoantigen peptides having a desired activity can be modified as needed to provide certain desired properties, such as improved pharmacological characteristics, while increasing or at least maintaining substantially all of the biological activity of the unmodified peptide to bind a desired HLA molecule and activate the appropriate T cell. For example, a peptide may be subjected to various alterations such as substitutions, both conservative and non-conservative, where such alterations may provide certain advantages in their use, such as improved HLA binding. Such conservative substitutions may involve the replacement of an amino acid residue by another amino acid residue that is biologically and/or chemically similar, for example, one hydrophobic residue for another or one polar residue for another. The effect of single amino acid substitutions can also be studied using D-amino acids. Such modifications can be made 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).

In some embodiments, the peptide described in the present invention 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 viral particles; inactivated bacterial toxins such as diphtheria toxin, tetanus toxin, cholera toxin, leukotoxin molecules; inactivated bacteria; and dendritic cells.

Peptide alterations that may 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 fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant, addition of amino acid mimetics, or addition of non-natural amino acids.

Glycosylation can affect the physical properties of proteins and may also be important for protein stability, secretion, and subcellular localization. Proper glycosylation can be important for biological activity. In fact, some genes from eukaryotic organisms, when expressed in bacteria (e.g., E. coli) that lack the cellular processes for protein glycosylation, yield proteins that are reconstituted with little or no activity because they lack glycosylation. Addition of glycosylation sites can be accomplished by altering the amino acid sequence. Alteration of a peptide or protein can be accomplished, for example, by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). The structures of the N-linked and O-linked oligosaccharides and sugar residues found in each type may be different. One type of sugar that is commonly found in both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is typically the terminal residue of both N-linked and O-linked oligosaccharides, and due to its negative charge, can impart acidic properties to the glycoprotein. Embodiments of the present disclosure include the creation and use of N-glycosylation variants. Removal of carbohydrates can be accomplished chemically or enzymatically, or by replacing codons encoding amino acid residues that are glycosylated. Chemical deglycosylation methods are known, and enzymatic cleavage of carbohydrate groups on polypeptides can be achieved using a variety of endo- and exoglycosidases.

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

Another type of modification is the conjugation (e.g., linkage) of one or more additional components or molecules at the N- 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 in question) or a carrier molecule. Thus, an exemplary polypeptide sequence can be presented as a conjugate with another component or molecule. In some embodiments, the fusion of albumin to a peptide or protein according to the present disclosure can, for example, be achieved by genetic manipulation, such that DNA encoding HSA, or a fragment thereof, is joined to DNA that encodes one or more polypeptide sequences. A suitable host can then be transformed or transfected with the fused nucleotide sequences, for example, in the form of a suitable plasmid, to express the fused polypeptide. Expression may be in vitro, such as from prokaryotic or eukaryotic cells, or in vivo, such as from a transgenic organism. In some embodiments of the invention, the fusion protein is expressed in mammalian cell lines, such as CHO cell lines. Additionally, albumin itself may be modified to increase its circulating half-life. Fusion of the modified albumin to one or more polypeptides may be achieved by the genetic manipulation methods described above or by chemical conjugation; the resulting fusion molecule has a half-life that exceeds that of fusions to unmodified albumin (see, for example, WO2011/051489). Several albumin coupling strategies have been developed as alternatives to direct fusion, including coupling of albumin via a conjugated fatty acid chain (acylation). Since serum albumin is a transport protein for fatty acids, these natural ligands with albumin-binding activity are used to prolong the half-life of low molecular weight protein therapeutic agents.

Additional candidate components and molecules for conjugation include those that are suitable for isolation or purification. Non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), antibody, receptor, ligand, lectin, or molecules that comprise a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips and membranes. Purification methods such as cation exchange chromatography, which effectively separates conjugates by their different molecular weights, can be used to separate conjugates by charge differences. The content of fractions obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, such as mass spectroscopy, SDS-PAGE or other known methods for separating molecular units by molecular weight.

In some embodiments, the amino or carboxyl terminus of a peptide or protein sequence disclosed herein may be fused to an Fc region of an immunoglobulin (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 product may require less frequent administration. Fc binds to the neonatal Fc receptor (FcRn) on endothelial cells lining blood vessels, and upon Fc binding, the fusion molecule is protected from degradation and is re-released into the bloodstream, keeping the molecule in the bloodstream longer. Thus, Fc binding is believed to be the mechanism by which endogenous IgG maintains its long half-life in plasma. More recent Fc fusion technology links a single copy of a biopharmaceutical agent to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical agent compared to traditional Fc fusion conjugates.

The present disclosure contemplates the use of other modifications of the peptides, known or currently under development, to improve one or more properties. One such method for extending the circulating half-life, increasing stability, decreasing clearance, or altering the immunogenicity or allergenicity of a peptide of the present disclosure involves modifying the peptide sequences by hesylation, which uses hydroxyethyl starch derivatives linked to other molecules to alter a characteristic of the molecule. Various aspects of hesylation are described, for example, in U.S. Patent Application Nos. 2007/0134197 and 2006/0258607.

The stability of peptides can be assessed in several ways. For example, peptidases and various biological media such as human plasma and serum have been used for stability testing. 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% (v/v) human serum assay. The protocol is as follows: pooled human serum (type AB, not heat inactivated) is disrupted by centrifugation prior to use. The serum is then diluted to 25% with RPMI-1640 or other 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 (4°C) for 15 minutes and then centrifuged to pellet the precipitated serum proteins. The presence of peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.

Problems associated with short plasma half-life or susceptibility to protease degradation can be addressed by a variety of modifications, including conjugation or linkage of the peptide or protein sequence to any of a variety of non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes (see, e.g., typically via a linking group covalently linked to both the protein and the non-protein polymer, such as PEG). Such PEG-conjugated biomolecules have been shown to have clinically useful properties, including improved physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer circulating half-life in vivo and decreased clearance, decreased immunogenicity and antigenicity, and decreased toxicity.

PEGs suitable for conjugation to a polypeptide or protein sequence are typically soluble in water at room temperature and have the general formula R-(O- _CH2 - _CH2 ) _n -OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and where n is an integer from 1 to 1000. If R is a protecting group, it typically has from 1 to 8 carbon atoms. The PEG conjugated to the polypeptide sequence may be linear or branched. Branched derivatives of PEG, "star PEGs" and PEGs with multiple branches are contemplated by the present disclosure. Also contemplated by the present disclosure are conjugate compositions in which the PEGs have different n values and thus different different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates where n=1, 2, 3, and 4. In some compositions, the proportion of conjugates where n=1 is 18-25%, the proportion of conjugates where n=2 is 50-66%, the proportion of conjugates where n=3 is 12-16%, and the proportion of conjugates where n=4 is up to 5%. Such compositions can be prepared by reaction conditions and purification methods known in the art. For example, cation exchange chromatography can be used to separate the conjugates, and then identify a fraction that contains a conjugate having, for example, a desired amount of PEG attached, purified from unmodified protein sequences and conjugates having a different amount of PEG attached.

PEG can be linked to a peptide or protein of the present invention via a terminal reactive group ("spacer"). A spacer is, for example, a terminal reactive group that mediates the linkage between free amino or carboxyl groups of one or more polypeptide sequences and PEG. PEG having a spacer that can be linked to a free amino group comprises PEG N-hydroxysuccinylimide, which can be prepared by activating the succinic acid ester of PEG with N-hydroxysuccinylimide. Another activated PEG that can be linked to a free amino group is 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which can be prepared by reacting PEG monomethyl ether with cyanuric chloride. Activated PEG linked to a free carboxyl group includes polyoxyethylenediamine.

Conjugation of one or more peptide or protein sequences of the present invention to PEG having a spacer can be carried out by various conventional methods. For example, the conjugation reaction can be carried out in solution at a pH of 5 to 10, at a temperature of 4°C to room temperature for 30 minutes to 20 hours, using a molar ratio of reagent to peptide/protein of 4:1 to 30:1. Reaction conditions can be selected to direct the reaction toward obtaining predominantly the desired degree of substitution. In general, low temperature, low pH (e.g., pH=5) and short reaction time tend to decrease the amount of PEG attached, whereas high temperature, neutral to high pH (e.g., pH>7) and longer reaction time tend to increase the amount of PEG attached. Various means known in the art can be used to terminate the reaction. In some embodiments, the reaction is stopped by acidifying the reaction mixture and freezing, for example at -20°C.

Neoepitopes

A neoepitope comprises the neodeterminant portion of a neoantigenic peptide or neoantigenic polypeptide that is recognized by the immune system. A neoepitope refers to an epitope that is not present in a reference cell, such as a healthy cell, such as a non-malignant cell or a germline cell, but is found in a diseased cell, such as a cancer cell. Included herein are situations where the corresponding epitope is found in a normal healthy cell or a germline cell, but due to one or more mutations in a diseased cell, such as a cancer cell, the epitope sequence is altered such that a neoepitope results. The term "neoepitope" is used interchangeably with "tumor-specific epitope" or "tumor-specific neoepitope" herein to refer to a series of residues, typically L-amino acids, linked to each other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The neoepitope may be of various lengths, either in their neutral (uncharged) forms or in forms that are salts, and either lack modifications such as glycosylation, side chain oxidation, or phosphorylation, or contain these modifications, provided that the modification does not impair the biological activity of the polypeptides as described in the present invention. The present invention relates to isolated neoepitopes that contain a tumor-specific mutation of Tables 1-12.

In some embodiments, the neoepitopes described herein for HLA class I MHC are 12 or fewer amino acid residues in length, or less, and typically consist of about 8 to about 12 amino acid residues. In some embodiments, the neoepitopes described herein for HLA class I MHC comprise about 8, about 9, about 10, about 11, or about 12 amino acid residues. In some embodiments, the neoepitopes described herein for HLA class II MHC are 25 or fewer amino acid residues in length, and typically consist of about 9 to about 25 amino acid residues. In some embodiments, the neoepitopes described herein for HLA class II MHC comprise about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues.

In some embodiments, a composition described herein comprises a first peptide comprising a first neoepitope of a protein and a second peptide comprising a second neoepitope of the same protein, wherein the first peptide differs from the second peptide, and wherein the first neoepitope comprises a mutation and the second neoepitope comprises the same mutation. In some embodiments, a composition described herein 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, wherein the first region comprises at least one amino acid of the second region, wherein the first peptide differs from the second peptide, and wherein the first neoepitope comprises a first mutation and the second neoepitope comprises a second mutation. In some embodiments, the first and second mutations are the same. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a read-through mutation, a gene fusion mutation, and any combination thereof.

In some embodiments, the first neoepitope binds to an HLA class I protein to form an HLA class I peptide complex. In some embodiments, the second neoepitope binds to an HLA class II protein to form an HLA class II peptide complex. In some embodiments, the second neoepitope binds to an HLA class I protein to form an HLA class I peptide complex. In some embodiments, the first neoepitope binds to an HLA class II protein to form an HLA class II peptide complex. In some embodiments, the first neoepitope activates CD8 ⁺ T cells. In some embodiments, the first neoepitope activates CD4 ⁺ T cells. In some embodiments, the second neoepitope activates CD4 ⁺ T cells. In some embodiments, the second neoepitope activates CD8 ⁺ T cells. In some embodiments, the TCR of the CD4 ⁺ T cell binds to the HLA class II peptide complex. In some embodiments, the TCR of the CD8 ⁺ T cell binds to an HLA class II-peptide complex. In some embodiments, the TCR of the CD8 ⁺ T cell binds to an HLA class I-peptide complex. In some embodiments, the TCR of the CD4 ⁺ T cell binds to an HLA class I-peptide complex.

In some embodiments, the second neoepitope is longer than the first neoepitope. In some embodiments, the first neoepitope is at least 8 amino acids long. In some embodiments, the first neoepitope is between 8 and 12 amino acids long. In some embodiments, the first neoepitope comprises a sequence of at least 8 contiguous amino acids, wherein at least 1 of the 8 contiguous amino acids differs at corresponding positions compared to the wild-type sequence. In some embodiments, the first neoepitope comprises a sequence of at least 8 contiguous amino acids, wherein at least 2 of the 8 contiguous amino acids differ at corresponding positions compared to the wild-type sequence. In some embodiments, the second neoepitope is at least 16 amino acids long. In some embodiments, the second neoepitope is between 16 and 25 amino acids long. In some embodiments, the second neoepitope comprises a sequence of at least 16 contiguous amino acids, wherein at least 1 of the 16 contiguous amino acids differs at corresponding positions from the wild-type sequence. In some embodiments, the second neoepitope comprises a sequence of at least 16 contiguous amino acids, wherein at least 2 of the 16 contiguous amino acids differ at corresponding positions from the wild-type sequence.

In some embodiments, the neoepitope comprises at least one anchor residue. In some embodiments, the first neoepitope, the second neoepitope, or both comprise at least one anchor residue. In one embodiment, the at least one anchor residue of the first neoepitope is in a canonical anchor position or a non-canonical anchor position. In another embodiment, the at least one anchor residue of the second neoepitope is in a canonical anchor position or a non-canonical anchor position. In another embodiment, the at least one anchor residue of the first neoepitope is different from the at least one anchor residue of the second neoepitope.

In some embodiments, at least one anchor residue is a wild-type residue. In some embodiments, at least one anchor residue is a substitution. In some embodiments, at least one anchor residue does not contain a mutation.

In some embodiments, the second neoepitope or both comprise at least one anchor residue flanking region. In some embodiments, the neoepitope comprises at least one anchor residue. In some embodiments, the at least one anchor residue comprises at least two anchor residues. In some embodiments, the at least two anchor residues are separated by a separation region comprising at least 1 amino acid. In some embodiments, the at least one anchor residue flanking region is outside the separation region. In some embodiments, the at least one anchor residue flanking region is (a) upstream of the N-terminal anchor residue of the at least two anchor residues; (b) downstream of the C-terminal anchor residue of the at least two anchor residues; or both (a) and (b).

In some embodiments, the neoepitopes bind an HLA protein (e.g., HLA MHC class I or HLA MHC class II). In some embodiments, the neoepitopes bind the HLA protein with greater affinity than the corresponding wild-type peptide. In some embodiments, the neoepitope has an IC ₅₀ of less than 5000 nM, less than 1000 nM, less than 500 nM, less than 100 nM, less than 50 nM, or less. In some embodiments, the neoepitope may have an HLA binding affinity of from about 1 pM to about 1 mM, from about 100 pM to about 500 μM, from about 500 pM to about 10 μM, from about 1 nM to about 1 μM, or from about 10 nM to about 1 μM. In some embodiments, the neoepitope may 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, 1000, 1500, or 2000 nM or more. In some embodiments, the neoepitope may have an HLA binding affinity of no more than 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, 1000, 1500, or 2000 nM.

In some embodiments, the first and/or second neoepitope binds to the HLA protein with greater affinity than the corresponding wild-type neoepitope. In some embodiments, the first and/or second neoepitope binds to the HLA protein with a _KD or _IC50 of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. In some embodiments, the first and/or second neoepitope binds to an HLA class I protein with a K _D or IC ₅₀ of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. In some embodiments, the first and/or second neoepitope binds to an HLA class II protein with a K _D or IC ₅₀ of less than 2000 nM, 1500 nM, 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 neoepitope binds to HLA MHC class I. In some embodiments, the neoepitope binds to HLA MHC class I with an affinity of 0.1 nM to 2000 nM. In some embodiments, the neoepitope binds to HLA MHC class I with an affinity of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 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, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 M. In some embodiments, the neoepitope binds to HLA MHC class II. In some embodiments, the neoepitope binds to HLA MHC class II with an affinity of 0.1 nM to 2000 nM, 1 nM to 1000 nM, 10 nM to 500 nM, or less than 1000 nM. In some embodiments, the neoepitope binds to HLA MHC class II with an affinity of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 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, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nM.

In some embodiments, the neoepitope binds to HLA MHC class I with a stability of 10 minutes to 24 hours. In some embodiments, the neoepitope binds to HLA MHC class I with a stability of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the neoepitope binds to HLA MHC class I with a stability of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the neoepitope binds to HLA MHC class II with a stability of 10 minutes to 24 hours. In some embodiments, the neoepitope binds to HLA MHC class II with a stability of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the neoepitope binds to HLA MHC class II with a stability of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

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 absent from 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 cancer cells of the subject. In some embodiments, the first neoepitope comprises a mutation as shown in column 1 of Tables 1-12. In some embodiments, the second neoepitope comprises a mutation as shown in column 1 of Tables 1-12. For example, the first neoepitope and the second neoepitope can comprise the sequence ALNSEALSVV (SEQ ID NO: 368). For example, the first neoepitope and the second neoepitope can comprise the sequence MALNSEALSV (SEQ ID NO: 369).

In some embodiments, the first neoepitope and the second neoepitope are from a KRAS protein. In some embodiments, the first neoepitope and the second neoepitope are from a NRAS protein. In some embodiments, the first neoepitope and the second neoepitope are from a KRAS protein comprising a G12C, G12D, G12V, Q61H, or Q61L substitution mutation. In some embodiments, the first neoepitope and the second neoepitope are from a NRAS protein comprising a Q61K or Q61R substitution mutation. In some embodiments, the neoepitope comprises a substitution mutation, such as a KRAS G12C, G12D, G12V, Q61H, or Q61L mutation or a NRAS Q61K or Q61R mutation. In some embodiments, the first neoepitope and the second neoepitope are derived from the KRAS or NRAS protein sequence MTEYKLVVVGACGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV IDGETCLLDILDTAGQE (SEQ ID NO: 309). For example, the first neoepitope and the second neoepitope can comprise the sequence KLVVVGACGV (SEQ ID NO: 10). For example, the first neoepitope and the second neoepitope can comprise the sequence LVVVGACGV (SEQ ID NO: 310). For example, the first neoepitope and the second neoepitope can comprise the sequence VVGACGVGK (SEQ ID NO: 200). For example, the first neoepitope and the second neoepitope can comprise the sequence VVVGACGVGK (SEQ ID NO: 197). In some embodiments, the first neoepitope and the second neoepitope are derived from the KRAS or NRAS protein sequence MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEVVGADGVGK (SEQ ID NO: 370). For example, the first neoepitope and the second neoepitope can comprise the sequence VVVGADGVGK (SEQ ID NO: 203). For example, the first neoepitope and the second neoepitope can comprise the sequence KLVVVGADGV (SEQ ID NO: 312). For example, the first neoepitope and the second neoepitope can comprise the sequence LVVVGADGV (SEQ ID NO: 313).

In some embodiments, the first neoepitope and the second neoepitope are derived from the KRAS or NRAS protein sequence MTEYKLVVVGAVGVG KSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE (SEQ ID NO: 314). For example, the first neoepitope and the second neoepitope can comprise the sequence KLVVVGAVGV (SEQ ID NO: 315). For example, the first neoepitope and the second neoepitope can comprise the sequence LVVVGAVGV (SEQ ID NO: 316). For example, the first neoepitope and the second neoepitope can comprise the sequence VVGAVGVGK (SEQ ID NO: 220). For example, the first neoepitope and the second neoepitope can comprise the sequence VVVGAVGVGK (SEQ ID NO: 218).

In some embodiments, the first neoepitope and the second neoepitope are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQ LIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM (SEQ ID NO: 317). For example, the first neoepitope and the second neoepitope can comprise the sequence ILDTAGHEEY (SEQ ID NO: 318).

In some embodiments, the first neoepitope and the second neoepitope are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQLIQ NHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGLEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM (SEQ ID NO: 319). For example, the first neoepitope and the second neoepitope can comprise the sequence ILDTAGLEEY (SEQ ID NO: 320). For example, the first neoepitope and the second neoepitope can comprise the sequence LLDILDTAGL (SEQ ID NO: 321).

In some embodiments, the first neoepitope and the second neoepitope are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQLIQ NHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGKEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINDSQUEQUEQUEQ (SEQ ID NO: 322). For example, the first neoepitope and the second neoepitope can comprise the sequence ILDTAGKEEY (SEQ ID NO: 323).

In some embodiments, the first neoepitope and the second neoepitope are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQLIQN HFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGREEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM (SEQ ID NO: 324). For example, the first neoepitope and the second neoepitope can comprise the sequence ILDTAGREEY (SEQ ID NO: 325).

In some embodiments, the neoepitope comprises a sequence selected from the group consisting of: DTAGHEEY (SEQ ID NO: 22), TAGHEEYSAM (SEQ ID NO: 23), DILDTAGHE (SEQ ID NO: 24), DILDTAGH (SEQ ID NO: 25), ILDTAGHEE (SEQ ID NO: 26), ILDTAGHE (SEQ ID NO: 27), DILDTAGHEEY (SEQ ID NO: 28), DTAGHEEYS (SEQ ID NO: 29), LLDILDTAGH (SEQ ID NO: 30), DILDTAGRE (SEQ ID NO: 31), DILDTAGR (SEQ ID NO: 32), ILDTAGREE (SEQ ID NO: 33), ILDTAGRE (SEQ ID NO: 34), CLLDILDTAGR (SEQ ID NO: 35), TAGREEYSAM (SEQ ID NO: 36), REEYSAMRD (SEQ ID NO: 37), DTAGKEEYSAM (SEQ ID NO: 38), CLLDILDTAGK (SEQ ID NO: 39), DTAGKEEY (SEQ ID NO: 40), LLDILDTAGK (SEQ ID NO: 41), ILDTAGKE (SEQ ID NO: 42), ILDTAGKEE (SEQ ID NO: 43), DTAGLEEY (SEQ ID NO: 44), ILDTAGLE (SEQ ID NO: 45), DILDTAGL (SEQ ID NO: 46), ILDTAGLEE (SEQ ID NO: 47), GLEEYSAMRDQY (SEQ ID NO: 48), LLDILDTAGLE (SEQ ID NO: 49), LDILDTAGL (SEQ ID NO: 50), DILDTAGLE (SEQ ID NO: 51), DILDTAGLEEY (SEQ ID NO: 52), AGVGKSAL (SEQ ID NO: 53), GAAGVGKSAL (SEQ ID NO: 54), AAGVGKSAL (SEQ ID NO: 55), CGVGKSAL (SEQ ID NO: 56), ACGVGKSAL (SEQ ID NO: 57), DGVGKSAL (SEQ ID NO: 58), ADGVGKSAL (SEQ ID NO: 59), DGVGKSALTI (SEQ ID NO: 60), GARGVGKSA (SEQ ID NO: 61), KLVVVGARGV (SEQ ID NO: 62), VVVGARGV (SEQ ID NO: 63), SGVGKSAL (SEQ ID NO: 64), VVVGASGVGK (SEQ ID NO: 65), GASGVGKSAL (SEQ ID NO: 66), VGVGKSAL (SEQ ID NO: 67), VVVGAGCVGK (SEQ ID NO: 68), KLVVVGAGC (SEQ ID NO: 69), GDVGKSAL (SEQ ID NO: 70), DVGKSALTI (SEQ ID NO: 71), VVVGAGDVGK (SEQ ID NO: 72), TAGKEEYSAM (SEQ ID NO: 73), DTAGHEEYSAM (SEQ ID NO: 74), TAGHEEYSA (SEQ ID NO: 75), DTAGREEYSAM (SEQ ID NO: 76), TAGKEEYSA (SEQ ID NO: 77), AAGVGKSA (SEQ ID NO: 78), AGCVGKSAL (SEQ ID NO: 79), AGDVGKSAL (SEQ ID NO: 80), AGKEEYSAMR (SEQ ID NO: 81), AGVGKSALTI (SEQ ID NO: 82), ARGVGKSAL (SEQ ID NO: 83), ASGVGKSA (SEQ ID NO: 84), ASGVGKSAL (SEQ ID NO: 85), AVGVGKSA (SEQ ID NO: 86), CVGKSALTI (SEQ ID NO: 87), DILDTAGK (SEQ ID NO: 88), DILDTAGREEY (SEQ ID NO: 89), DTAGHEEYSAMR (SEQ ID NO: 90), DTAGKEEYS (SEQ ID NO: 91), DTAGKEEYSAMR (SEQ ID NO: 92), DTAGLEEYS (SEQ ID NO: 93), DTAGLEEYSA (SEQ ID NO: 94), DTAGLEEYSAMR (SEQ ID NO: 95), DTAGREEYS (SEQ ID NO: 96), DTAGREEYSAMR (SEQ ID NO: 97), GAAGVGKSA (SEQ ID NO: 98), GACGVGKSA (SEQ ID NO: 8), GACGVGKSAL (SEQ ID NO: 5), GADGVGKS (SEQ ID NO: 99), GAGDVGKSA (SEQ ID NO: 100), GAGDVGKSAL (SEQ ID NO: 101), GASGVGKSA (SEQ ID NO: 102), GCVGKSAL (SEQ ID NO: 103), GCVGKSALTI (SEQ ID NO: 104), GHEEYSAM (SEQ ID NO: 105), GKEEYSAM (SEQ ID NO: 106), GLEEYSAMR (SEQ ID NO: 107), GREEYSAM (SEQ ID NO: 108), GREEYSAMR (SEQ ID NO: 109), HEEYSAMRD (SEQ ID NO: 110), KEEYSAMRD (SEQ ID NO: 111), KLVVVGASG (SEQ ID NO: 112), LDILDTAGR (SEQ ID NO: 113), LEEYSAMRD (SEQ ID NO: 114), LVVVGARGV (SEQ ID NO: 115), LVVVGASGV (SEQ ID NO: 116), REEYSAMRDQY (SEQ ID NO: 117), RGVGKSAL (SEQ ID NO: 118), TAGLEEYSA (SEQ ID NO: 119), TEYKLVVVGAA (SEQ ID NO: 120), VGAAGVGKSA (SEQ ID NO: 121), VGADGVGK (SEQ ID NO: 122), VGASGVGKSA (SEQ ID NO: 123), VGVGKSALTI (SEQ ID NO: 124), VVVGAAGV (SEQ ID NO: 125), VVVGAVGV (SEQ ID NO: 126), YKLVVVGAC (SEQ ID NO: 127), YKLVVVGAD (SEQ ID NO: 128), YKLVVVGAR (SEQ ID NO: 129) and DILDTAGKE (SEQ ID NO: 130).

In some embodiments, the neoepitope comprises a RAS epitope. In some embodiments, the neoepitope comprises a RAS mutant sequence that comprises at least 8 contiguous amino acids of a RAS mutant protein comprising a mutation at G12, G13, or Q61 and a mutation at G12, G13, or Q61. In some embodiments, the at least 8 contiguous amino acids of the RAS mutant protein comprising a mutation at G12, G13, or Q61 comprise a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation. In some embodiments, the mutation at G12, G13, or Q61 comprises a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation.

In some embodiments, the neoepitope comprising a mutant RAS sequence comprises the amino acid sequence GADGVGKSAL (SEQ ID NO: 4), GACGVGKSAL (SEQ ID NO: 5), GAVGVGKSAL (SEQ ID NO: 6), GADGVGKSA (SEQ ID NO: 7), GACGVGKSA (SEQ ID NO: 8), GAVGVGKSA (SEQ ID NO: 9), KLVVVGACGV (SEQ ID NO: 10), FLVVVGACGL (SEQ ID NO: 11), FMVVVGACGI (SEQ ID NO: 12), FLVVVGACGI (SEQ ID NO: 13), FMVVVGACGV (SEQ ID NO: 14), FLVVVGACGV (SEQ ID NO: 15), MLVVVGACGV (SEQ ID NO: 16), FMVVVGACGL (SEQ ID NO: 17), YLVVVGACGV (SEQ ID NO: 18), KMVVVGACGV (SEQ ID NO: 19), YMVVVGACGV (SEQ ID NO: 20), MMVVVGACGV (SEQ ID NO: 21), DTAGHEEY (SEQ ID NO: 22), TAGHEEYSAM (SEQ ID NO: 23), DILDTAGHE (SEQ ID NO: 24), DILDTAGH (SEQ ID NO: 25), ILDTAGHEE (SEQ ID NO: 26), ILDTAGHE (SEQ ID NO: 27), DILDTAGHEEY (SEQ ID NO: 28), DTAGHEEYS (SEQ ID NO: 29), LLDILDTAGH (SEQ ID NO: 30), DILDTAGRE (SEQ ID NO: 31), DILDTAGR (SEQ ID NO: 32), ILDTAGREE (SEQ ID NO: 33), ILDTAGRE (SEQ ID NO: 34), CLLDILDTAGR (SEQ ID NO: 35), TAGREEYSAM (SEQ ID NO: 36), REEYSAMRD (SEQ ID NO: 37), DTAGKEEYSAM (SEQ ID NO: 38), CLLDILDTAGK (SEQ ID NO: 39), DTAGKEEY (SEQ ID NO: 40), LLDILDTAGK (SEQ ID NO: 41), ILDTAGKE (SEQ ID NO: 42), ILDTAGKEE (SEQ ID NO: 43), DTAGLEEY (SEQ ID NO: 44), ILDTAGLE (SEQ ID NO: 45), DILDTAGL (SEQ ID NO: 46), ILDTAGLEE (SEQ ID NO: 47), GLEEYSAMRDQY (SEQ ID NO: 48), LLDILDTAGLE (SEQ ID NO: 49), LDILDTAGL (SEQ ID NO: 50), DILDTAGLE (SEQ ID NO: 51), DILDTAGLEEY (SEQ ID NO: 52), AGVGKSAL (SEQ ID NO: 53), GAAGVGKSAL (SEQ ID NO: 54), AAGVGKSAL (SEQ ID NO: 55), CGVGKSAL (SEQ ID NO: 56), ACGVGKSAL (SEQ ID NO: 57), DGVGKSAL (SEQ ID NO: 58), ADGVGKSAL (SEQ ID NO: 59), DGVGKSALTI (SEQ ID NO: 60), GARGVGKSA (SEQ ID NO: 61), KLVVVGARGV (SEQ ID NO: 62), VVVGARGV (SEQ ID NO: 63), SGVGKSAL (SEQ ID NO: 64), VVVGASGVGK (SEQ ID NO: 65), GASGVGKSAL (SEQ ID NO: 66), VGVGKSAL (SEQ ID NO: 67), VVVGAGCVGK (SEQ ID NO: 68), KLVVVGAGC (SEQ ID NO: 69), GDVGKSAL (SEQ ID NO: 70), DVGKSALTI (SEQ ID NO: 71), VVVGAGDVGK (SEQ ID NO: 72), TAGKEEYSAM (SEQ ID NO: 73), DTAGHEEYSAM (SEQ ID NO: 74), TAGHEEYSA (SEQ ID NO: 75), DTAGREEYSAM (SEQ ID NO: 76), TAGKEEYSA (SEQ ID NO: 77), AAGVGKSA (SEQ ID NO: 78), AGCVGKSAL (SEQ ID NO: 79), AGDVGKSAL (SEQ ID NO: 80), AGKEEYSAMR (SEQ ID NO: 81), AGVGKSALTI (SEQ ID NO: 82), ARGVGKSAL (SEQ ID NO: 83), ASGVGKSA (SEQ ID NO: 84), ASGVGKSAL (SEQ ID NO: 85), AVGVGKSA (SEQ ID NO: 86), CVGKSALTI (SEQ ID NO: 87), DILDTAGK (SEQ ID NO: 88), DILDTAGREEY (SEQ ID NO: 89), DTAGHEEYSAMR (SEQ ID NO: 90), DTAGKEEYS (SEQ ID NO: 91), DTAGKEEYSAMR (SEQ ID NO: 92), DTAGLEEYS (SEQ ID NO: 93), DTAGLEEYSA (SEQ ID NO: 94), DTAGLEEYSAMR (SEQ ID NO: 95), DTAGREEYS (SEQ ID NO: 96), DTAGREEYSAMR (SEQ ID NO: 97), GAAGVGKSA (SEQ ID NO: 98), GACGVGKSA (SEQ ID NO: 8), GACGVGKSAL (SEQ ID NO: 5), GADGVGKS (SEQ ID NO: 99), GAGDVGKSA (SEQ ID NO: 100), GAGDVGKSAL (SEQ ID NO: 101), GASGVGKSA (SEQ ID NO: 102), GCVGKSAL (SEQ ID NO: 103), GCVGKSALTI (SEQ ID NO: 104), GHEEYSAM (SEQ ID NO: 105), GKEEYSAM (SEQ ID NO: 106), GLEEYSAMR (SEQ ID NO: 107), GREEYSAM (SEQ ID NO: 108), GREEYSAMR (SEQ ID NO: 109), HEEYSAMRD (SEQ ID NO: 110), KEEYSAMRD (SEQ ID NO: 111), KLVVVGASG (SEQ ID NO: 112), LDILDTAGR (SEQ ID NO: 113), LEEYSAMRD (SEQ ID NO: 114), LVVVGARGV (SEQ ID NO: 115), LVVVGASGV (SEQ ID NO: 116), REEYSAMRDQY (SEQ ID NO: 117), RGVGKSAL (SEQ ID NO: 118), TAGLEEYSA (SEQ ID NO: 119), TEYKLVVVGAA (SEQ ID NO: 120), VGAAGVGKSA (SEQ ID NO: 121), VGADGVGK (SEQ ID NO: 122), VGASGVGKSA (SEQ ID NO: 123), VGVGKSALTI (SEQ ID NO: 124), VVVGAAGV (SEQ ID NO: 125), VVVGAVGV (SEQ ID NO: 126), YKLVVVGAC (SEQ ID NO: 127), YKLVVVGAD (SEQ ID NO: 128), YKLVVVGAR (SEQ ID NO: 129) or DILDTAGKE (SEQ ID NO: 130).

In some embodiments, a neoepitope comprising a mutant RAS sequence binds to a protein encoded by an HLA allele. In some embodiments, a neoepitope comprising a mutant RAS sequence binds to a protein encoded by an HLA allele with an affinity of less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 950 nM, less than 900 nM, less than 850 nM, less than 800 nM, less than 750 nM, less than 600 nM, less than 550 nM, less than 500 nM, less than 450 nM, less than 400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, or less than 10 nM. In some embodiments, a neoepitope comprising a mutant RAS sequence binds to a protein encoded by an HLA allele with a stability of greater than 24 hours, greater than 23 hours, greater than 22 hours, greater than 21 hours, greater than 20 hours, greater than 19 hours, greater than 18 hours, greater than 17 hours, greater than 16 hours, greater than 15 hours, greater than 14 hours, greater than 13 hours, greater than 12 hours, greater than 11 hours, greater than 10 hours, greater than 9 hours, greater than 8 hours, greater than 7 hours, greater than 6 hours, greater than 5 hours, greater than 4 hours, greater than 3 hours, greater than 2 hours, greater than 1 hour, greater than 55 minutes, greater than 50 minutes, greater than 45 minutes, greater than 40 minutes, greater than 35 minutes, greater than 30 minutes, greater than 25 minutes, greater than 20 minutes, greater than 15 minutes, greater than 10 minutes, greater than 9 minutes, greater than 8 minutes, greater than 7 minutes, more than 6 minutes, more than 5 minutes, more than 4 minutes, more than 3 minutes, more than 2 minutes, or more than 1 minute.

The substitution may be located anywhere along the neoepitope. For example, it may be located in the N-terminal third of the peptide, in the central third of the peptide, or in the C-terminal third of the peptide. In another embodiment, the substituted residue is located 2-5 residues from the N-terminus or 2-5 residues from the C-terminus. Peptides may similarly be derived from tumor-specific insertional mutations if the peptide contains one or more or all of the inserted residues.

In some embodiments, a peptide described herein can be readily synthesized chemically using reagents that are free of bacterial or animal contaminants (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963). In some embodiments, peptides are prepared by (1) parallel solid phase synthesis on multichannel instruments using the same synthesis and digestion conditions; (2) purification on an RP-HPLC column with column desorption; and repeated washing, but without displacement, between peptides; followed by (3) analysis with a limited set of the most informative assays. Good Manufacturing Practices (GMP) can be defined around a set of peptides for an individual patient, requiring changeover procedures only between peptide syntheses for different patients. In some embodiments, any resins designed for solid-phase peptide synthesis can be used.

Polynucleotides

Alternatively, a nucleic acid (e.g., a polynucleotide) encoding a peptide of the present invention can be used to produce a neoantigenic peptide in vitro. The polynucleotide can be, for example, DNA, cDNA, RNA, either single- and/or double-stranded, or native or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothioate backbone, or combinations thereof, and it may or may not contain introns as long as it encodes a peptide. In some embodiments, in vitro translation is used to produce the peptide.

The invention relates to neoantigenic polynucleotides encoding each of the neoantigenic polypeptides described in the present description. The term "polynucleotide", "nucleotides" or "nucleic acid" is used interchangeably with the terms "mutant polynucleotide", "mutant nucleotide", "mutant nucleic acid", "neoantigenic polynucleotide", "neoantigenic nucleotide" or "neoantigenic mutant nucleic acid" in the present description. Different nucleic acid sequences can encode the same peptide due to the repetitiveness of the genetic code. Each of these nucleic acids falls within the scope of the present invention. The nucleic acids encoding the peptides can be DNA or RNA, such as mRNA, or a combination of DNA and RNA. In some embodiments, the nucleic acid encoding the peptide is a self-amplifying mRNA (Brito et al., Adv. Genet. 2015; 89:179-233). Any suitable polynucleotide encoding a peptide described herein is within the scope of the present disclosure.

In some embodiments, the coding sequences for two consecutive antigenic peptides are separated by a spacer or linker. In some embodiments, the coding sequences for two consecutive antigenic peptides are adjacent to each other. In some embodiments, the coding sequences for two consecutive antigenic peptides are not separated by a spacer or linker.

In some embodiments, the spacer or linker comprises up to 5,000 nucleotide residues. An exemplary spacer sequence is GGCGGCAGCGGCGGCGGCGGCAGCGGCGGC (SEQ ID NO: 371). Another exemplary spacer sequence is GGCGGCAGCCTGGGCGGCGGCGGCAGCGGC (SEQ ID NO: 372). Another exemplary spacer sequence is GGCGTCGGCACC (SEQ ID NO: 373). Another exemplary spacer sequence is CAGCTGGGCCTG (SEQ ID NO: 374). Another exemplary spacer sequence is a lysine encoding sequence, such as AAA or AAG. Another exemplary spacer sequence is CAACTGGGATTG (SEQ ID NO: 375).

In some embodiments, the mRNA contains one or more additional structures to enhance the processing and presentation of the antigenic epitope by APC.

In some embodiments, the linker or spacer region may contain cleavage sites. The cleavage sites provide for cleavage of the protein product containing the epitope sequence strands into individual epitope sequences for presentation. Preferred cleavage sites are located near certain epitopes to avoid unintended cleavage of epitopes within the sequences. In some embodiments, the design of epitopes and cleavage regions on the epitope strands encoding mRNA is not random.

The term "RNA" includes and, in some embodiments, refers to "mRNA". The term "mRNA" means "messenger RNA" and refers to a "transcript" that is created using a DNA template and encodes a peptide or polypeptide. Typically, mRNA contains a 5'-UTR, a protein coding region, and a 3'-UTR. mRNA has a limited half-life in cells and in vitro. In some embodiments, mRNA is self-amplifying mRNA. As used herein, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription techniques are known to those skilled in the art. For example, many in vitro transcription kits are commercially available.

If desired, the stability and translation efficiency of the RNA can be altered. For example, the RNA can be stabilized and its translation can be increased by one or more modifications having stabilizing effects and/or increasing the translation efficiency of the RNA. Such modifications are described, for example, in PCT/EP2006/009448, incorporated herein by reference. To increase the expression of the 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, without changing the sequence of the expressed peptide or protein, to increase the GC content to increase the stability of the mRNA and to optimize the codons and thus to increase translation in cells.

The term "modification" in the context of RNA as used in the present invention includes any modification of RNA that is not naturally present in said RNA. In some embodiments, the RNA does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates 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 reduce cytotoxicity. In some embodiments, 5-methylcytidine can be partially or completely replaced in the RNA, for example, with cytidine. Alternatively, pseudouridine is partially or completely replaced, for example, with uridine.

In some embodiments, the term "modification" refers to providing an RNA with a 5' cap or a 5' cap analog. The term "5' cap" refers to the cap structure found at the 5' end of an mRNA molecule and typically consists of a guanosine nucleotide linked to the mRNA via an unusual 5'-5' triphosphate bond. In some embodiments, this guanosine is methylated at the 7 position. The term "conventional 5' cap" refers to the naturally occurring 5' cap of RNA, the 7-methylguanosine cap (mG). As used herein, the term "5' cap" includes a 5' cap analog that resembles the cap structure of RNA and is modified to have the ability to stabilize RNA and/or enhance translation of RNA when attached thereto, in vivo and/or in a cell.

In some embodiments, an mRNA encoding a neoantigen peptide as described herein is administered to a subject in need thereof. In some embodiments, the present disclosure relates to RNA, oligoribonucleotide, and polyribonucleotide molecules comprising a modified nucleoside, gene therapy vectors comprising them, gene therapy methods, and gene transcription suppression methods comprising them. In some embodiments, the administered mRNA comprises at least one modified nucleoside.

Polynucleotides encoding the peptides described in the present invention can be synthesized by chemical methods, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Polynucleotides encoding peptides containing or consisting of an analog can be obtained by simply replacing the appropriate and desired nucleic acid bases with those encoding the native epitope.

In some embodiments, polynucleotides may contain a coding sequence for a peptide or protein fused in frame with the polynucleotide that assists, 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 secretory sequence to control transport of the polypeptide from the cell). A polypeptide having a leader sequence is a pre-protein and may have a leader sequence that is cleaved by the host cell to form a mature form of the polypeptide.

In some embodiments, the polynucleotides may comprise a coding sequence for a peptide or protein fused in frame with a marker sequence, which allows, for example, purification of the encoded peptide, which can then be included in a personalized disease vaccine or immunogenic composition. For example, the marker sequence may be a hexahistidine tag (SEQ ID NO: 376) supplied by the pQE-9 vector to allow purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein if a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not limited to, Calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, SpyTag, biotin carboxyl carrier protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tags, TC tag, Ty tag, and the like.

In some embodiments, the polynucleotides may comprise a coding sequence for one or more peptides or proteins described herein fused in the same reading frame to create a single concatamerized neoantigen peptide construct capable of producing multiple neoantigen peptides.

In some embodiments, the DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence that encodes a wild-type protein of interest. Optionally, the sequence can be mutagenized using site-directed mutagenesis to produce functional analogs thereof. See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Patent No. 4,588,585. In another embodiment, the DNA sequence encoding the peptide or protein of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired peptide and the selection of those codons that are preferred in the host cell in which the recombinant polypeptide of interest is produced. Standard techniques can be used to synthesize an isolated polynucleotide sequence that encodes an isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a reverse-translatable gene. In addition, a DNA oligomer can be synthesized that contains a nucleotide sequence encoding a particular isolated polypeptide. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

After assembly (e.g., by synthesis, site-directed mutagenesis, or other method), polynucleotide sequences encoding a particular isolated polypeptide of interest are inserted into an expression vector and, optionally, operably linked to an expression control sequence suitable for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of the biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene can be operably linked to transcriptional and translational expression control sequences that are functional in the host for the selected expression. Accordingly, the present disclosure also relates to vectors and expression vectors useful for the production and administration of the neoantigenic polypeptides and neoepitopes described herein, as well as to host cells containing such vectors.

In some embodiments, an expression vector capable of expressing a peptide or protein as described herein can also be prepared. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation. Typically, the DNA is inserted into an expression vector, such as a plasmid, in the correct orientation and reading frame for expression. If desired, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences that are recognized by the desired host (e.g., bacteria), although such controls are typically available in the expression vector. The vector is then introduced into the host bacterium 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.).

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

Polynucleotides encoding neoantigenic peptides described in the present invention may also contain a ubiquitination signal sequence and/or a targeting sequence, such as an endoplasmic reticulum (ER) signal sequence, to facilitate translocation of the resulting peptide to the endoplasmic reticulum.

In some embodiments, the neoantigen peptide described herein can also be administered and/or expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia or fowlpox. Vaccinia vectors and methods used in 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 by Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization with the neoantigen polypeptides described herein, such as adeno- and adeno-associated viral vectors, retroviral vectors, Salmonella Typhimurium vectors, detoxified anthrax toxin vectors, Sendai virus vectors, poxvirus vectors, canarypox vectors, and the like, will be apparent to those skilled in the art from the disclosure provided herein. In some embodiments, the vector is a modified Ankara (VA) vaccine (e.g., Bavarian Noridic (MVA-BN)).

Various mammalian or insect cell culture systems have also been successfully used for recombinant protein expression. Expression of recombinant proteins in mammalian cells can be accomplished because such proteins are typically correctly folded, properly modified, and fully functional. Examples of suitable mammalian host cell lines include the monkey kidney COS-7 cell lines described by Gluzman (Cell 23:175, 1981) and other cell lines capable of expressing the appropriate vector, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, a suitable promoter and enhancer associated with the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and also 5' or 3' non-translated sequences such as essential ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcription termination sequences. A review of baculovirus systems for the production of heterologous proteins in insect cells is provided by Luckow and Summers, Bio/Technology 6:47 (1988).

The host cells are genetically engineered (transduced, transformed or transfected) with vectors, which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells may be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying polynucleotides. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to one of ordinary skill in the art.

Typical examples of suitable hosts include: bacterial cells such as E. coli, Bacillus subtilis, Salmonella typhimurium and various species of the genera Pseudomonas, Streptomyces and Staphylococcus; fungal cells such as yeast; insect cells such as Drosophila and Sf9; animal cells such as the COS-7 monkey kidney fibroblast lines described by Gluzman, Cell 23:175 (1981) and other cell lines capable of expressing a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines or Bowes melanoma; plant cells, etc. The selection of a suitable host is believed to be within the skill of the art, based on the teachings of the present invention.

The polynucleotides described in the present invention can be administered to and expressed in human cells (e.g., immune cells, including dendritic cells). The human codon usage table can be used to select a codon for each amino acid. Such polynucleotides contain spacer amino acid residues between epitopes and/or analogs, such as those described above, or can contain naturally occurring flanking sequences adjacent to the epitopes and/or analogs (and/or CTL (e.g., CD8 ⁺ ), Th (e.g., CD4 ⁺ ), and B-cell epitopes).

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

Vectors can be introduced into animal tissues by a variety of different routes. The two most popular approaches are injection of DNA in saline using a standard hypodermic needle and delivery by a gene gun. A schematic representation of the construction of a DNA vaccine plasmid and its subsequent delivery by these two routes to the host is shown in Scientific American (Weiner et al., (1999) Scientific American 281(1):34-41). Saline injection is usually administered intramuscularly (IM) into skeletal muscle or intradermally (ID), delivering DNA to extracellular spaces. This can be facilitated by electroporation, by transient injury of muscle fibers with myotoxins such as bupivacaine; or by application of hypertonic saline or sucrose solutions (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410). Immune responses to this delivery method can be influenced by many factors including needle type, needle position, injection speed, injection volume, muscle type, age, sex, and physiological state of the animal being injected (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410).

Gene gun delivery, another widely used delivery method, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten microparticles into target cells using compressed helium as an accelerator (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).

Alternative delivery methods may include aerosol instillation of naked DNA to mucosal surfaces such as the nasal and pulmonary mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88) and topical administration of pDNA to the eyes and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88). Delivery to mucosal surfaces has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA can also be delivered into cells following mild mechanical disruption of the cell membrane, transiently increasing cell permeability. Such gentle mechanical disruption of the membrane can be achieved by gently pushing cells through a small hole (Sharei et al., Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells, PLOS ONE (2015)).

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In the case of using a non-viral delivery system, the exemplary delivery vehicle is a liposome. "Liposome" is a general term covering a variety of mono- and multi-lamellar lipid carriers formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a bilayer phospholipid membrane and an internal aqueous environment. Multilamellar liposomes contain several lipid layers separated by an aqueous environment. They form spontaneously when phospholipids are suspended in excess aqueous solution. The fat components undergo self-rearrangement before forming closed structures and trap water and solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, compositions that have structures in solution other than the normal vesicular structure are also encompassed. For example, fats may have a micellar structure or simply exist as heterogeneous aggregates of fat molecules. Lipofectamine-nucleic acid complexes are also considered.

The use of lipid formulations for introducing nucleic acids into a host cell (in vitro, ex vivo, or in vivo) is contemplated. In another aspect, the nucleic acid can be linked to a fat. The lipid-linked nucleic acid can be encapsulated within an aqueous medium of a liposome, interspersed within a lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a fat, mixed with a fat, combined with a fat, contained as a suspension in a fat, contained or complexed with a micelle, or otherwise associated with a fat. Fat, fat/DNA, or fat/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or in a "folded" structure. They may also be simply mixed in solution, possibly forming aggregates that are heterogeneous in size or shape. Fats are fatty substances that may be natural or synthetic fats. For example, fats include the fat droplets that occur naturally in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Fats suitable for use can be obtained from commercial sources. Stock solutions of fats in chloroform or chloroform/methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol.

4. Antigen presenting cells (APC)

Antigen-presenting cells (APCs) present peptide fragments of protein antigens in association with MHC molecules on their cell surfaces. The presented peptide is associated with an MHC molecule as a peptide-MHC complex (pMHC) on the cell surface of the APC. Processing and presentation of peptide-MHC complexes may involve a series of sequential steps including: protease-mediated cleavage of proteins; transport of peptides into the endoplasmic reticulum (ER) mediated by the transporter associated with antigen processing (TAP); formation of peptide-MHC I molecules using newly synthesized MHC molecules; and transport of peptide-MHC molecules to the cell surface.

Some APCs can activate antigen-specific T cells. For example, a T cell containing a T cell receptor (TCR) that interacts with a pMHC can be activated, stimulated, induced, or expanded to form a TCR-pMHC. In some embodiments, an MHC (e.g., MHC class I or MHC class II) of an APC can be loaded with a peptide and presented to the APC by introducing into the APC a nucleic acid (e.g., RNA) that encodes an antigenic peptide or a polypeptide containing a presented peptide sequence.

From a biological perspective, for a somatic mutation to elicit an immune response, several criteria must be met: the allele containing the mutation must be expressed by the cell, the mutation must be in a protein coding region and non-synonymous, the translated protein must be degraded by the proteasome or other cellular protein degradation pathway, and the epitope containing the mutation must be presented by the MHC complex, the presented epitope must be recognized by the TCR, and finally, the TCR-pMHC complex must trigger a signaling cascade that activates the T cell.

Monocytes can circulate in the bloodstream and then move into tissues where they can differentiate into macrophages and dendritic cells. Classical monocytes are typically characterized by high levels of expression of the cell surface receptor CD14. Monocytes and B cells can be competent APCs, although their antigen-presenting abilities appear to be limited to reactivation of previously sensitized T cells. These cell types may be unable to directly activate functionally naive or unprimed T cell populations. Professional antigen-presenting cells are highly efficient at internalizing antigen, either by phagocytosis or receptor-mediated endocytosis, and then display the antigen fragment bound to an MHC molecule on their membrane. The T cell recognizes and interacts with the antigen-MHC molecule complex on the APC membrane. The APC then produces an additional costimulatory signal, resulting in T cell activation. Expression of costimulatory molecules is a typical feature of professional APCs.

Professional APCs can internalize antigen very efficiently, either by phagocytosis or receptor-mediated endocytosis, and then display the antigen fragment bound to an MHC molecule on their membrane. The T cell can recognize and interact with the antigen-MHC molecule complex on the APC membrane. The APC can then produce an additional costimulatory signal, leading to T cell activation. Expression of costimulatory molecules may be a defining feature of professional antigen-presenting cells. Examples of professional APCs may include, but are not limited to, dendritic cells (DCs), macrophages, and B cells. Professional APCs can express high levels of MHC class II, ICAM-1, and B7-2.

One of the major types of professional APCs are DCs, which have the broadest spectrum of antigen presentation. Other major types of professional APCs include macrophages, B cells, and some activated epithelial cells. DCs are leukocyte populations that present antigens (e.g., antigens captured in peripheral tissues) to T cells via the MHC class II and I antigen-presenting pathways. DCs are capable of activating both naive and pre-primed T cells (e.g., memory T cells). DCs may be leukocyte populations that present antigens captured in peripheral tissues to T cells via the MHC class I and II antigen-presenting pathways. DCs can be potent inducers of immune responses, and activation of these cells may be a critical step in the induction of antitumor immunity.

DC can be divided into “immature” and “mature” cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be construed as excluding all possible intermediate stages of differentiation. Immature DC can be characterized as APCs with high antigen uptake and processing capacity, which correlates with high expression of the Fcγ receptor and mannose receptor. The mature phenotype can generally be characterized by lower expression of these markers, but high expression of cell surface molecules responsible for T cell activation, such as MHC class I and class II, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Mature DCs can be CD11b ⁺ , CD11c ⁺ , HLA-DR ⁺ , CD80 ⁺ , CD86 ⁺ , CD54 ⁺ , ^CD3− , ^CD19− , ^CD14− , CD141 ⁺ (BDCA-3), and/or CD1a ⁺ . DC maturation can be indicated as the activation status of DCs, in which such antigen-presenting DCs lead to T cell priming, while presentation by immature DCs leads to tolerance. DC maturation can be induced by biomolecules with microbial signatures detected by innate receptors (e.g., bacterial DNA, viral RNA, endotoxins, etc.), proinflammatory cytokines (e.g., TNF, interleukins, and interferons), ligation of CD40 on the DC surface by CD40L, and substances released from cells that are dying. Additional non-limiting examples of cytokines that can induce DC maturation include IL-4, GM-CSF, TNF-α, IL-1β, PGE1, and IL-6. For example, DC can be generated by culturing bone marrow cells in vitro with cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-α). For example, DC can be generated from CD14+ monocytes isolated from PBMCs. Cytokines or growth factors that can be used to convert monocytes into DC include, but are not limited to, GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, and polyI:C.

Normally, non-professional antigen-presenting cells do not continually express MHC class II proteins. MHC class II proteins are normally expressed only upon stimulation of non-professional APCs by certain cytokines, such as IFN-γ.

The source of the APC may typically be a tissue source containing APCs or APC precursors that are capable of expressing and presenting antigenic peptides in vitro. In some embodiments, the APCs are capable of proliferating and becoming professional APCs when loaded with target RNA and/or treated with the necessary cytokines or factors.

In one aspect, the antigenic polypeptide or protein can be presented as a cell comprising such polypeptides, peptides, proteins or polynucleotides as described herein. In some embodiments, the cell is an antigen-presenting cell (APC). In some embodiments, the cell is a dendritic cell (DC). In some embodiments, the cell is a mature antigen-presenting cell. In some embodiments, the neoantigenic peptide or protein can be presented as APCs (e.g., dendritic cells) comprising such polypeptides, peptides, proteins or polynucleotides as described herein. In other embodiments, such APCs are used to stimulate T cells for use in patients. Thus, one embodiment of the present disclosure is a composition comprising at least one APC (e.g., dendritic cell) into which one or more neoantigenic peptides or polynucleotides described herein have been introduced or loaded. In some embodiments, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with neoantigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are reintroduced into the patient. In some embodiments, the APCs are dendritic cells. In related embodiments, the dendritic cells are autologous dendritic cells that have been introduced 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 that have been introduced with peptides from a tumor-associated antigen is described 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 ⁺ )).

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 APC-based immunogenic pharmaceutical composition can be formulated using any well-known techniques, carriers, and excipients, as appropriate and known in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. Sometimes, the APC-based immunogenic pharmaceutical composition is a dendritic cell-based immunogenic pharmaceutical composition.

The immunogenic pharmaceutical composition based on dendritic cells can be prepared by any methods well known in the art. In some cases, the immunogenic pharmaceutical compositions based on dendritic cells can be prepared by an ex vivo or in vivo method. The ex vivo method can include the use of autologous DCs into which the polypeptides described in the present invention have been introduced ex vivo to activate or load the DCs prior to administration to a patient. The in vivo method can include targeting specific DC receptors using antibodies bound to the polypeptides described in the present invention. The immunogenic pharmaceutical composition based on DCs can further comprise DC activators, such as TLR3, TLR-7-8 and CD40 agonists. The immunogenic pharmaceutical composition based on DCs can further comprise adjuvants and a pharmaceutically acceptable carrier.

Antigen presenting cells (APCs) can be obtained from a variety of sources, including humans and non-human primates, other mammals, and vertebrates. In some embodiments, APCs can be obtained from human or non-human vertebrate blood. APCs can also be isolated from an enriched leukocyte population. Leukocyte populations can be obtained by methods known to those skilled in the art. Such methods typically include collecting heparinized blood, apheresis or leukophoresis, preparing a buffy coat, rosetting, centrifugation, density gradient centrifugation (e.g., using Ficoll, colloidal silica particles, and sucrose), differential lysis of non-leukocyte cells, and filtration. A leukocyte population can also be obtained by collecting blood from a subject, defibrillating to remove platelets, and lysing red blood cells. The leukocyte population may not necessarily be enriched in monocytic dendritic cell precursors.

The blood cell populations may be obtained from a plurality of subjects in accordance with the desired use of the enriched leukocyte population. The subject may be a healthy subject. Alternatively, the blood cells may be obtained from a subject in need of immunostimulation, such as, for example, a cancer patient or other patient for whom immunostimulation would be useful. Similarly, the blood cells may be obtained from a subject in need of immune suppression, such as, for example, a patient suffering from an autoimmune disease (e.g., rheumatoid arthritis, diabetes, lupus, multiple sclerosis, and the like). The leukocyte population may also be obtained from an HLA-matched healthy individual.

If blood is used as a source of APC, the 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 with a medium that may or may not contain heparin or another suitable anticoagulant. The volume of blood to medium can be from about 1 to 1. The cells can be concentrated by centrifuging the blood in the medium at about 1000 rpm (150 g) at 4 ° C. The platelets and red blood cells can be depleted by resuspending the cells in any number of solutions known in the art that lyse red blood cells, such as ammonium chloride. For example, the mixture can be a medium and contain ammonium chloride at a ratio of about 1:1 by volume. The cells can be concentrated by centrifugation and washed in the desired solution to obtain a population of leukocytes that is substantially free of platelets and red blood cells. Any isotonic solution commonly used in tissue culture can be used as a medium to separate blood leukocytes from platelets and red blood cells. Examples of such isotonic solutions include phosphate-buffered saline, Hanks' balanced salt solution, and complete growth medium. APCs and/or APC progenitor cells can also be purified by elutriation.

In one embodiment, the APCs may be non-nominal APCs under inflammatory or other activated conditions. For example, non-nominal APCs may include epithelial cells stimulated by interferon gamma, T cells, B cells, and/or monocytes activated by factors or conditions that induce APC activity. Such non-nominal APCs may be obtained according to methods known in the art.

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

In some embodiments, the APC can be cultured in a suitable culture or growth medium to maintain and/or expand the amount of APC in the preparation. Culture media can be selected based on the type of APC isolated. For example, mature APC, such as mature dendritic cells, can be cultured in a growth medium suitable for their maintenance and expansion. The growth medium can be supplemented with amino acids, vitamins, antibiotics, divalent cations, and the like. In addition, cytokines, growth factors, and/or hormones can be included in the growth medium. For example, cytokines such as granulocyte/macrophage colony stimulating factor (GM-CSF) and/or interleukin 4 (IL-4) can be added to maintain and/or expand mature dendritic cells. In other embodiments, immature APC can be cultured and/or expanded. Immature dendritic cells may retain the ability to capture target mRNA and process a new antigen. In some embodiments, immature dendritic cells can be cultured in media suitable for their maintenance and culture. The growth medium can be supplemented with amino acids, vitamins, antibiotics, divalent cations, and the like. In addition, cytokines, growth factors, and/or hormones can be included in the growth medium.

In a similar manner, other immature APCs can be cultured or expanded. Immature APC preparations can mature to form mature APCs. Maturation of APCs can occur during or after exposure to neoantigen peptides. In some embodiments, immature dendritic cell preparations can be matured. Suitable maturation factors include, for example, TNF-α cytokines, bacterial products (e.g., BCG), and the like. In another aspect, isolated APC precursors can be used to produce immature APC preparations. APC precursors can be cultured, differentiated, and/or matured. In some embodiments, monocytic dendritic cell precursors can be cultured in the presence of suitable culture media supplemented with amino acids, vitamins, cytokines, and/or divalent cations to stimulate differentiation of monocytic dendritic cell precursors into immature dendritic cells. In some embodiments, the APC precursors are isolated from PBMCs. The PBMCs can be obtained from a donor, such as a human donor, and can be used fresh or frozen for future use. In some embodiments, the APCs are obtained from one or more APC preparations. In some embodiments, the APCs comprise an APC loaded with first and second neoantigenic peptides comprising first and second neoepitopes, or polynucleotides encoding first and second neoantigenic peptides comprising first and second neoepitopes. In some embodiments, the APCs are autologous APCs, allogeneic APCs, or artificial APCs.

5. Adjuvants

An adjuvant can be used to enhance the immune response (humoral and/or cellular) elicited in a patient receiving a composition of the present invention. Sometimes adjuvants can elicit a Th1 type response. In other cases, adjuvants can elicit 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.

In some aspects, lipid-based adjuvants such as MPLA and MDP can be used with the immunogenic pharmaceutical compositions described herein. Monophosphoryl lipid A (MPLA), for example, is an adjuvant that causes increased presentation of liposomal antigen to specific T lymphocytes. In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant in combination with the immunogenic pharmaceutical compositions described herein.

Suitable adjuvants are known in the art (see WO 2015/095811) and include, but are not limited to, poly(I:C), poly-ICLC, hiltonol, STING agonist, 1018 ISS, aluminum salts, amplivax, AS15, BCG., CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanid IMS 1312, Montanid ISA 206, Montanid ISA 50V, Montanid 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, Pam2Cys, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil or Superfos. Adjuvants also include incomplete Freund's adjuvant or GM-CSF. Several immunological adjuvants (e.g. MF59) specific for dendritic cells and their production have been described previously (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 are directly associated with influencing the migration of dendritic cells into lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells into effective 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, incorporated herein by reference in its entirety), and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J. Immunother. Emphasis Tumor Immunol. 1996 (6):414-418).

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

Additional adjuvants include: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, 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 their functional fragments.

In some aspects, the adjuvant may be a toll-like receptor (TLR) modulator. Examples of TLR modulators include TLR-9 agonists and are not limited to small molecule TLR modulators such as Imiquimod. Other examples of adjuvants that are used in combination with the immunogenic pharmaceutical composition described herein may include, but are not limited to, saponin, CpG ODN, and the like. Sometimes the adjuvant is selected from bacterial toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. Sometimes the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion may include at least one oil and at least one surfactant, wherein the oils and surfactants are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion can have a diameter of less than 5 µm and even submicron diameter, and these small sizes are achieved using a microfluidizer to obtain stable emulsions. Droplets smaller than 220 nm can be sterilized on a filter.

6. Methods of treatment and pharmaceutical compositions

The neoantigen therapeutics (e.g., polypeptides or polynucleotides, APCs, or dendritic cells comprising the polypeptides or polynucleotides) described herein are useful in a variety of applications, including, but not limited to, therapeutic treatments, such as cancer treatment. In some embodiments, the therapeutic treatment methods include immunotherapy. In some embodiments, the neoantigen peptide is useful for activating, stimulating, enhancing, and/or improving an immune response, redirecting an existing immune response to a new target, increasing tumor immunogenicity, inhibiting tumor growth, reducing tumor volume, increasing tumor cell apoptosis, and/or reducing tumorigenicity of a tumor. The methods of use can be in vitro, ex vivo, or in vivo.

In some aspects, the present invention provides methods of activating an immune response in a subject using a polypeptide, a cell, or a pharmaceutical composition comprising a neoantigenic peptide or protein described herein. In some embodiments, the present invention provides methods of prophylaxis in a subject comprising contacting a cell of the subject with a polypeptide, a cell, or a pharmaceutical composition comprising a neoantigenic peptide or protein described herein. In some embodiments, the present invention provides methods of stimulating an immune response in a subject using a polypeptide, a cell, or a pharmaceutical composition comprising a neoantigenic peptide or protein described herein. In some embodiments, the present invention provides methods of enhancing an immune response in a subject using a polypeptide, a cell, or a pharmaceutical composition comprising a neoantigenic peptide or protein described herein. In some embodiments, the present invention provides methods of enhancing an immune response using a polypeptide, a cell, or a pharmaceutical composition comprising a neoantigenic peptide or protein described herein.

In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises enhancing cell-mediated immunity. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises enhancing T cell activity or humoral immunity. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises enhancing cytotoxic T lymphocyte (CTL) or helper T lymphocyte (Th) activity. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises enhancing natural killer (NK) activity. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises enhancing T cell activity and enhancing NK cell activity. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises enhancing CTL activity and enhancing NK cell activity. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises inhibiting or reducing the suppressive activity of T-regulatory (Treg) cells. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises increasing antitumor activity. In some embodiments, activating, stimulating, enhancing and/or improving the immune response comprises increasing immunogenicity. In some embodiments, the immune response is the result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer.

In some embodiments, the present invention provides methods for activating, stimulating, enhancing and/or improving an immune response using a polypeptide, cell, or pharmaceutical composition comprising a neoantigenic peptide or protein described herein. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a polypeptide that delivers a neoantigenic peptide or polynucleotide to a tumor cell. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigenic polypeptide internalized by a tumor cell. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigenic polypeptide that is internalized by a tumor cell, and the neoantigenic peptide is processed by the cell. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigenic polypeptide that is internalized by a tumor cell, and the neoepitope is presented on the surface of the tumor cell. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigenic polypeptide that is internalized by a tumor cell, processed by the cell, and the antigenic peptide is presented on the surface of the tumor cell.

In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigenic polypeptide or polynucleotide described herein that delivers an exogenous polypeptide comprising at least one neoantigenic peptide to a tumor cell, wherein at least one neoepitope derived from the neoantigenic peptide is presented on the surface of the tumor cell. In some embodiments, the antigenic peptide is presented on the surface of the tumor cell in complex with an MHC class I molecule. In some embodiments, the antigenic peptide is presented on the surface of the tumor cell in complex with an MHC class II molecule.

In some embodiments, the method comprises contacting a tumor cell with a neoantigenic polypeptide or polynucleotide described herein, which delivers an exogenous polypeptide comprising at least one neoantigenic polypeptide to the tumor cell, wherein at least one neoepitope derived from the at least one neoantigenic polypeptide is presented on the surface of the tumor cell. In some embodiments, the neoepitope is presented on the surface of the tumor cell in a complex with an MHC class I molecule. In some embodiments, the neoepitope is presented on the surface of the tumor cell in a complex with an MHC class II molecule.

In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigenic polypeptide or polynucleotide described herein that delivers an exogenous polypeptide comprising at least one antigenic peptide to a tumor cell, wherein the epitope or neoepitope is presented on the surface of the tumor cell, and an immune response against the tumor cell is induced. In some embodiments, the immune response to the epitope or neoepitope is enhanced. In some embodiments, the immune response against the tumor cell is enhanced. In some embodiments, the neoantigenic polypeptide or polynucleotide delivers an exogenous polypeptide comprising at least one neoantigenic peptide to a tumor cell, wherein the epitope or neoepitope is presented on the surface of the tumor cell, and tumor growth is inhibited.

In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a neoantigenic polypeptide or polynucleotide described in the present invention that delivers an exogenous polypeptide comprising at least one neoantigenic peptide to a tumor cell, wherein a neoepitope derived from the at least one neoantigenic peptide is presented on the surface of the tumor cell and T cell killing directed against the tumor cell is induced. In some embodiments, T cell killing directed against the tumor cell is enhanced. In some embodiments, T cell killing directed against the tumor cell is increased.

In some embodiments, a method for enhancing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a neoantigen therapeutic agent described in the present invention, wherein the agent is an antibody that specifically binds a neoantigen described in the present invention. In some embodiments, a method for enhancing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an antibody.

The present disclosure relates to methods of redirecting an existing immune response to a tumor. In some embodiments, a method of redirecting an existing immune response to a tumor comprises administering to a subject a therapeutically effective amount of a neoantigen therapeutic agent described herein. In some embodiments, the existing immune response is directed against a virus. In some embodiments, the virus is selected from the group consisting of: measles virus, varicella zoster virus (VZV; chickenpox virus), influenza virus, mumps virus, poliovirus, rubella virus, rotavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). In some embodiments, the virus is varicella virus. In some embodiments, the virus is cytomegalovirus. In some embodiments, the virus is measles virus. In some embodiments, the existing immune response was acquired following a natural viral infection. In some embodiments, the existing immune response was acquired after vaccination against the virus. In some embodiments, the existing immune response is a cell-mediated response. In some embodiments, the existing immune response includes CTL or Th cells.

In some embodiments, a method of redirecting an existing immune response to a tumor in a subject comprises administering a fusion protein comprising (i) an antibody that specifically binds a neoantigen and (ii) at least one neoantigen peptide described herein, wherein (a) the fusion protein is internalized by a tumor cell upon binding to a tumor-associated antigen or neoepitope; (b) the neoantigen peptide is processed and presented on the surface of the tumor cell associated with an MHC class I molecule; and (c) the neoantigen peptide/MHC Class I complex is recognized by CTLs. In some embodiments, the CTLs are memory T cells. In some embodiments, the memory T cells are the result of vaccination with the neoantigen peptide.

The present disclosure relates to methods for enhancing the immunogenicity of a tumor. In some embodiments, a method for enhancing the immunogenicity of a tumor comprises contacting a tumor or tumor cells with an effective amount of a neoantigen therapeutic agent described in the present invention. In some embodiments, a method for enhancing the immunogenicity of a tumor comprises administering to a subject a therapeutically effective amount of a neoantigen therapeutic agent described in the present invention.

The present disclosure also relates to methods of inhibiting tumor growth using a neoantigen therapeutic agent described herein. In some embodiments, the method of inhibiting tumor growth comprises contacting a mixture of cells with a therapeutic neoantigen in vitro. For example, an immortalized cell line or a cancer cell line mixed with immune cells (e.g. _, T cells) is cultured in a medium supplemented with a neoantigen peptide. In some embodiments, tumor cells are isolated from a patient sample, such as a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., T cells), and cultured in a medium supplemented with a neoantigen therapeutic agent. In some embodiments, the neoantigen therapeutic agent increases, stimulates, and/or enhances the activity of immune cells. In some embodiments, the neoantigen therapeutic agent suppresses the growth of tumor cells. In some embodiments, the neoantigen therapeutic agent activates tumor cell killing.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a tumor or the subject had a tumor that was at least partially removed.

In some embodiments, a method for inhibiting tumor growth comprises redirecting an existing immune response to a new target, including administering to a subject a therapeutically effective amount of a neoantigenic therapeutic agent, wherein the existing immune response is directed against an antigenic peptide delivered to a tumor cell by the neoantigenic peptide.

In some embodiments, the tumor contains cancer stem cells. In some embodiments, the frequency of cancer stem cells in the tumor is reduced by administering a neoantigen therapeutic agent. In some embodiments, a method is provided for reducing the frequency of cancer stem cells in a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a neoantigen therapeutic agent.

Additionally, in some aspects, the present disclosure relates to a method for reducing tumorigenicity of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a neoantigen therapeutic agent described herein. In some embodiments, the tumor comprises cancer stem cells. In some embodiments, tumorigenicity of the tumor is reduced by reducing the frequency of cancer stem cells in the tumor. In some embodiments, the methods comprise using a neoantigen therapeutic agent described herein. In some embodiments, the frequency of cancer stem cells in the tumor is reduced by administering a neoantigen therapeutic agent described herein.

In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a tumor selected from the group consisting of: a colorectal tumor, a pancreatic tumor, a lung tumor, an ovarian tumor, a liver tumor, a breast tumor, a kidney tumor, a prostate tumor, a neuroendocrine tumor, a gastrointestinal tumor, a melanoma, a cervical tumor, a bladder tumor, a glioblastoma, and a head and neck tumor. In some embodiments, the tumor is a colorectal tumor. In some embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a breast tumor. In some embodiments, the tumor is a lung tumor. In some embodiments, the tumor is a pancreatic tumor. In some embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a solid tumor.

The present disclosure further provides methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a neoantigen therapeutic agent described in the present disclosure. In some embodiments, a method of treating cancer comprises redirecting an existing immune response to a new target, the method comprising administering to the subject a therapeutically effective amount of a neoantigen therapeutic agent, wherein the existing immune response is directed against an antigenic peptide delivered to a cancer cell by the neoantigen peptide.

The present disclosure relates to methods of treating cancer comprising administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of a neoantigen therapeutic agent described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a cancerous tumor. In some embodiments, the subject has had the tumor at least partially removed.

Subjects may be, for example, mammals, humans, pregnant women, elderly humans, adults, young adults, adolescents, children, toddlers, infants, babies, or neonates. A subject may be a patient. In some cases, a subject may be a human. In some cases, a subject may be a child (i.e., a young adult who has not yet reached puberty). In some cases, a subject may be an infant. In some cases, a subject may be a formula-fed infant. In some cases, a subject may be a human being enrolled in a clinical trial. In some cases, a subject may be a laboratory animal, such as a mammal or rodent. In some cases, a subject may be a mouse. In some cases, a subject may be obese or overweight.

In some embodiments, the subject has previously been treated with one or more different cancer treatments. In some embodiments, the subject has previously been treated with one or more of radiotherapy, chemotherapy, or immunotherapy. In some embodiments, the subject has previously been treated with one, two, three, four, or five lines of therapy. In some embodiments, the prior therapy is cytotoxic therapy.

In some embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, uterine cancer, glioblastoma, and head and neck cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a solid tumor.

In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).

In some embodiments, the neoantigen therapeutic agent is administered as a combination therapy. Combination therapy of two or more therapeutic agents uses agents that act by different mechanisms of action, although this is not necessarily the case. Combination therapy using agents with different mechanisms of action can provide additive or synergistic effects. Combination therapy can allow for the use of a lower dose of each agent than monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agents. Combination therapy can reduce the likelihood of developing resistant cancer cells. In some embodiments, combination therapy includes a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) tumor/cancer cells.

In some cases, the immunogenic pharmaceutical composition may be administered with an additional agent. The choice of additional agent may depend, at least in part, on the condition being treated. The additional agent may include, for example, a checkpoint inhibitor agent such as an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 agent (e.g., an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 antibody); or any agents that have a therapeutic effect on a pathogenic infection (e.g., a viral infection), including, for example, drugs used to treat inflammatory conditions, such as NSAIDs, e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, the checkpoint inhibitor may be a PD-1/PD-L1 antagonist selected from the group consisting of: nivolumab (ONO-4538/BMS-936558, MDX1 106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA (ROCHE). As another example, the formulations may further comprise one or more additives such as vitamin C, E, or other antioxidants.

The methods of the present disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancer treatable by the methods of the present disclosure can include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-resistant prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, squamous cell cancer of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other malignancies.

In addition, the disease or condition provided by the present invention includes refractory or recurrent malignancies whose growth can be inhibited by the treatment methods described herein. In some embodiments, the cancer to be treated by the treatment methods of the present invention is selected from the group consisting of carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, anogenital squamous cell carcinoma, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, squamous cell lung cancer, gastric cancer, bladder cancer, gallbladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma and combinations thereof. In some embodiments, a cancer treatable by the methods disclosed herein includes, for example, carcinoma, squamous cell carcinoma (e.g., cervical canal, eyelid, conjunctiva, vagina, lung, oral cavity, skin, bladder, tongue, larynx, and esophagus) and adenocarcinoma (e.g., prostate, small intestine, endometrium, cervical canal, colon, lung, pancreas, esophagus, rectum, uterus, stomach, breast, and ovary). In some embodiments, a cancer treatable by the methods disclosed herein further includes sarcoma (e.g., myogenic sarcoma), leukemia, neuroma, melanoma, and lymphoma. In some embodiments, a cancer treatable by the methods disclosed herein is breast cancer. In some embodiments, the cancer to be treated by the treatment methods described herein is triple-negative breast cancer (TNBC). In some embodiments, the cancer to be treated by the treatment methods described herein is ovarian cancer. In some embodiments, the cancer to be treated by the treatment methods described herein is colorectal cancer.

In some embodiments, the patient or patient population to be treated with the pharmaceutical composition of the present disclosure has a solid tumor. In some embodiments, the solid tumor is melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gallbladder cancer, laryngeal cancer, liver cancer, thyroid cancer, gastric cancer, salivary gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma. In some embodiments, the patient or patient population to be treated with the pharmaceutical composition of the present disclosure has a hematological cancer. In some embodiments, the patient has a hematological cancer, such as diffuse large B-cell lymphoma ("DLBCL"), Hodgkin lymphoma ("HL"), non-Hodgkin lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML"), or multiple myeloma ("MM"). In some embodiments, the patient or population of patients to be treated has a cancer selected from the group consisting of ovarian cancer, lung cancer, and melanoma.

Specific examples of cancers that can be prevented and/or treated according to the present disclosure include, but are not limited to, the following: renal cell carcinoma, renal cell carcinoma, glioblastoma multiforme, metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; childhood tumors; neuroblastoma; malignant melanoma; epidermal carcinomas; leukemias such as but not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemic leukemia and myelodysplastic syndrome; chronic leukemias such as but not limited to chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as, but not limited to, sarcoma of bone, myeloma disease of bone, multiple myeloma, osteosarcoma of bone due to cholesteatoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as, but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary lymphoma of the brain; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease), and inflammatory breast cancer; adrenal cancer such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer such as but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancer such as but not limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye cancer such as but not limited to melanoma of the eye such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer such as but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer such as but not limited to epithelial ovarian carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; Esophageal cancer such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, warty carcinoma, and oat cell (small cell) carcinoma; Gastric cancer such as but not limited to adenocarcinoma, fungal (polypoid), ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; Colon cancer; Colorectal cancer, KRAS-mutated colorectal cancer; Colon carcinoma; Rectal cancer; liver cancer such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancer such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; lung carcinoma; testicular cancer such as but not limited to germ cell tumor, seminoma, anaplastic, classic (typical), spermatocytic, non-seminoma, embryonal cancer, teratoma, choriocarcinoma (yolk sac tumor); prostate cancer such as but not limited to androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penitentiary cancers; oral cancer such as but not limited to squamous cell carcinoma; basal cell carcinoma; salivary gland cancer such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; Pharyngeal cancer such as but not limited to squamous cell carcinoma and warty carcinoma; Skin cancer such as but not limited to basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acralentiginous melanoma; Kidney cancer such as but not limited to renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell carcinoma (of the renal pelvis and/or uterus); Renal carcinoma; Wilms' tumor; Bladder cancer such as but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In addition, cancer includes myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma.

Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone-resistant prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid cancer, adrenal glands, osteosarcoma, chondrosarcoma, hematological tissue cancer and the like. Other non-limiting examples of cancer types applicable to the methods covered by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias such as acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the method of the present disclosure is an epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancers, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer or colon cancer. In other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian carcinoma (e.g., serous ovarian cancer), or breast carcinoma. Epithelial cancer can be characterized in various other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated. In some embodiments, the present invention is used to treat, diagnose, and/or prognosticate lymphoma or subtypes thereof, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered proliferative diseases.

In some embodiments, the combination of an agent described in the present invention and at least one additional therapeutic agent provides additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of additional therapeutic agents. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of additional therapeutic agents.

In some embodiments, in addition to administering a neoantigen therapeutic agent described in the present invention, the method or treatment further comprises administering at least one additional therapeutic agent. The additional therapeutic agent may be administered before, simultaneously with, and/or after administration of the agent. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

Therapeutic agents that can be administered in combination with a neoantigen therapeutic agent described in the present invention include chemotherapeutic agents. Thus, in some embodiments, the method or treatment comprises administering an agent described in the present invention in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with the agent can occur before, simultaneously, or after chemotherapy. Combination administration can include co-administration either in a single pharmaceutical formulation or using separate formulations, or sequential administration in any order, but typically for a period of time such that all active agents can exert their biological activity simultaneously. The preparation and dosing regimens of such chemotherapeutic agents can be used in accordance with the manufacturers' instructions or as determined empirically by an experienced practitioner. Preparation and dosing regimens for such chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

Suitable classes of chemotherapeutic agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapeutic sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like. In some embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.

Chemotherapeutic agents that may be used in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quulamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folic acid replenishers such as folinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elphormitin; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK; Razoxane; Sizofuran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosin; arabinoside (Ara-C); taxoids such as paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Chemotherapeutic agents also include antihormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. In some embodiments, the additional therapeutic agent is cisplatin. In some embodiments, the additional therapeutic agent is carboplatin.

In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In some embodiments, the additional therapeutic agent is irinotecan.

In some embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, but different enough to interfere with one or more normal cellular functions, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, sodium methotrexate, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In some embodiments, the additional therapeutic agent is gemcitabine.

In some embodiments, the chemotherapeutic agent is an antimitotic agent, including but not limited to tubulin binding agents. In some embodiments, the agent is a taxane. In some embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In some embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In some alternative embodiments, the antimitotic agent comprises a vinca alkaloid such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some embodiments, the antimitotic agent is an Eg5 kinesin inhibitor or a mitotic kinase inhibitor such as Aurora A or Plk1. In some embodiments, the additional therapeutic agent is paclitaxel. In some embodiments, the additional therapeutic agent is albumin-bound paclitaxel.

In some embodiments, the additional therapeutic agent comprises an agent such as a small molecule. For example, treatment can include combined administration of an agent of the present invention with a small molecule that acts as an inhibitor against tumor-associated antigens, including but not limited to EGFR, HER2 (ErbB2), and/or VEGF. In some embodiments, the agent of the present invention is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor. In another embodiment, the additional therapeutic agent is chemotherapy or other inhibitors that reduce the number of Treg cells. In some embodiments, the therapeutic agent is cyclophosphamide or an anti-CTLA4 antibody. In another embodiment, the additional therapeutic agent reduces the presence of myeloid-derived suppressor cells. In another embodiment, the additional therapeutic agent is carbotaxol. In another embodiment, the additional therapeutic agent shifts cells to a T helper 1 response. In another embodiment, the additional therapeutic agent is ibrutinib.

In some embodiments, the additional therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment can include combined administration of an agent of the present invention with antibodies against tumor-associated antigens, including, but not limited to, antibodies that bind EGFR, HER2/ErbB2 and/or VEGF. In some embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an antibody against VEGF or the VEGF receptor). In some embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

The agents and compositions of the present invention can be used alone or in combination with conventional therapeutic regimens such as surgery, radiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated). A tumor antigen array can be useful, for example, for a large proportion of cancer patients.

In some embodiments, at least one or more chemotherapeutic agents may be administered in addition to a composition comprising an immunogenic vaccine. In some embodiments, the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents.

Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan); nitrosoureas (e.g., N-nitroso-N-methylurea, streptozocin, carmustine (BCNU), lomustine, and semustine); alkyl sulfonates (e.g., busulfan); tetrazines (e.g., dacarbazine (DTIC), mitozolomide, and temozolomide (Temodar®)); aziridines (e.g., thiotepa, mitomycin, and diaziquone); and platinum agents (e.g., cisplatin, carboplatin, and oxaliplatin); non-classical alkylating agents such as procarbazine and altretamine (hexamethylmelamine); antimetabolite agents such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), decitabine, floxuridine, fludarabine, nelarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, thioguanine, Vidaza; tubulin polymerization inhibitors such as vinca alkaloids (eg, vincristine, vinblastine, vinorelbine, vindesine, and vinflunine); taxanes (eg paclitaxel (Taxol®), docetaxel (Taxotere®)); podophyllotoxin (eg etoposide and teniposide); epothilones (eg, ixabepilone (Ixempra®)); estramustine (Emcyt®); antineoplastic antibiotics such as anthracyclines (eg, daunorubicin, doxorubicin (Adriamycin®, epirubicin, idarubicin); actinomycin-D, and bleomycin; topoisomerase I inhibitors such as topotecan and irinotecan (CPT-11); topoisomerase II inhibitors such as etoposide (VP-16), teniposide, mitoxantrone, novobiocin, merbarone, and aclarubicin; corticosteroids such as prednisone, methylprednisolone (Solumedrol®), and dexamethasone (Decadron®); L-asparaginase; bortezomib (Velcade®); immunotherapy agents such as rituximab (Rituxan®), alemtuzumab (Campath®), thalidomide, lenalidomide (Revlimid®), BCG, interleukin-2, interferon-alpha, and cancer vaccines such as Provenge®; hormonal therapeutic agents such as fulvestrant (Faslodex®), tamoxifen, toremifene (Fareston®), anastrozole (Arimidex®), exemestane (Aromasin®), letrozole (Femara®), megestrol acetate (Megace®), estrogens, bicalutamide (Casodex®), flutamide (Eulexin®), nilutamide (Nilandron®), leuprolide (Lupron®), and goserelin (Zoladex®); differentiating agents such as retinoids, tretinoin (ATRA or Atralin®), bexarotene (Targretin®), and arsenic trioxide (Arsenox®); and targeted therapeutic agents such as imatinib (Gleevec®), gefitinib (Iressa®), and sunitinib (Sutent®). In some embodiments, the chemotherapy is a cocktail therapy. Examples of cocktail therapy include, but are not limited to, CHOP/R-CHOP (rituxan, cyclophosphamide, hydroxydoxorubicin, vincristine, and prednisone), EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, hydroxydoxorubicin), Hyper-CVAD (cyclophosphamide, vincristine, hydroxydoxorubicin, dexamethasone), FOLFOX (fluorouracil (5-FU), leucovorin, oxaliplatin), ICE (ifosfamide, carboplatin, etoposide), DHAP (high-dose cytarabine [ara-C], dexamethasone, cisplatin), ESHAP (etoposide, methylprednisolone, cytarabine [ara-C], cisplatin), and CMF (cyclophosphamide, methotrexate, fluuracil).

In some embodiments, the additional therapeutic agent comprises a second immunotherapeutic agent. In some embodiments, the additional immunotherapeutic agent includes, but is not limited to, a colony stimulating factor, an interleukin, an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-CD3 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody), an antibody that enhances immune cell functions (e.g., an anti-GITR antibody, an anti-OX-40 antibody, an anti-CD40 antibody, or an anti-4-1BB antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), a soluble ligand (e.g., GITRL, GITRL-Fc, OX-40L, OX-40L-Fc, CD40L, CD40L-Fc, 4-1BB ligand, or 4-1BB-Fc ligand), or a member of the B7 family (e.g., CD80, CD86). In some embodiments, the additional immunotherapeutic agent targets CTLA-4, CD28, CD3, PD-1, PD-L1, TIGIT, GITR, OX-40, CD-40, or 4-1BB.

In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-4-1BB antibody, or an anti-OX-40 antibody. In some embodiments, the additional therapeutic agent is an anti-TIGIT antibody. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody selected from the group consisting of: nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilzumab, MEDI0680, REGN2810, BGB-A317, and PDR001. In some embodiments, the additional therapeutic agent is an anti-PD-L1 antibody selected from the group consisting of: BMS935559 (MDX-1105), atexolizumab (MPDL3280A), durvalumab (MEDI4736), and avelumab (MSB0010718C). In some embodiments, the additional therapeutic agent is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab (YERVOY) and tremelimumab. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from the group consisting of: BMS-986016 and LAG525. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody selected from the group consisting of: MEDI6469, MEDI0562, and MOXR0916. In some embodiments, the additional therapeutic agent is an anti-4-1BB antibody selected from the group consisting of: PF-05082566.

In some embodiments, the therapeutic neoantigen can be administered in combination with a biologic molecule selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMP, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, PlGF, gamma-IFN, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15 and IL-18.

In some embodiments, treatment with a neoantigen therapeutic agent described herein may be accompanied by surgical removal of tumors, removal of cancer cells, or any other surgical therapy deemed necessary by the treating physician.

In some embodiments, the treatment comprises administering a neoantigen therapeutic agent described in the present invention in combination with radiation therapy. Treatment with the agent may occur before, simultaneously, or after radiation therapy. The dosing schedule for such radiation therapy may be determined by a qualified practitioner.

Combination administration may involve co-administration either in a single pharmaceutical formulation or using separate formulations, or sequential administration in any order, but usually over a period of time such that all active agents can exert their biological activity simultaneously.

It should be understood that the combination of a neoantigen therapeutic agent described in the present invention and at least one additional therapeutic agent can be administered in any order or simultaneously. In some embodiments, the agent will be administered to patients previously treated with a second therapeutic agent. In some other embodiments, the neoantigen therapeutic agent and the second therapeutic agent will be administered substantially simultaneously or concurrently. For example, the subject can be given the agent while undergoing treatment with a second therapeutic agent (e.g., chemotherapy). In some embodiments, the neoantigen therapeutic agent will be administered within 1 year of treatment with the second therapeutic agent. Furthermore, it should be understood that two (or more) agents or treatments can be administered to the subject within a few hours or minutes (i.e., substantially simultaneously).

For the treatment of a disease, the appropriate dose of the neoantigen therapeutic agent described in the present invention depends on the type of disease to be treated, the severity and course of the disease, the response to the disease, whether the agent is administered for therapeutic or prophylactic purposes, previous therapy, the patient's medical history, etc., all at the discretion of the attending physician. The neoantigen therapeutic agent can be administered once or over a series of treatment courses lasting from several days to several months, or until a cure or a decrease in the disease state (e.g., a decrease in tumor size) occurs. Optimal dosing regimens can be calculated based on measurements of drug accumulation in the patient's body and will vary depending on the relative potency of the individual agent. The attending physician can determine optimal dosages, dosing techniques, and frequency of repetition.

In some embodiments, the neoantigen therapeutic agent may be administered at an initial higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also vary. In some embodiments, the dosing regimen may include administering an initial dose, followed by additional doses (or "maintenance" doses) once per week, once every two weeks, once every three weeks, or once per month. For example, the dosing regimen may include administering an initial loading dose, followed by a weekly maintenance dose, such as half the initial dose; the dosing regimen may include administering an initial loading dose, followed by maintenance doses, such as half the initial dose every two weeks; or the dosing regimen may include administering three initial doses over a period of 3 weeks, followed by maintenance doses, such as every two weeks.

As is known to those skilled in the art, administration of any therapeutic agent may result in side effects and/or toxicity. In some cases, the side effects and/or toxicity are so severe that they preclude administration of a particular agent at a therapeutically effective dose. In some cases, therapy must be discontinued and other agents may be tried. However, many agents in the same therapeutic class exhibit similar side effects and/or toxicity, meaning that the patient must either discontinue therapy or, if possible, suffer from the unpleasant side effects associated with the therapeutic agent.

In some embodiments, the dosing regimen may be limited to a certain number of administrations or "cycles." In some embodiments, the agent is administered for 3, 4, 5, 6, 7, 8 or more cycles. For example, the agent is administered every 2 weeks for 6 cycles, the agent is administered every 3 weeks for 6 cycles, the agent is administered every 2 weeks for 4 cycles, the agent is administered every 3 weeks for 4 cycles, etc. Dosing regimens may be determined and subsequently modified by those skilled in the art.

The present disclosure relates to methods of administering to a subject a neoantigen therapeutic agent described herein comprising using an intermittent dosing strategy for administering one or more agents that can reduce side effects and/or toxicity associated with administration of the agent, a chemotherapeutic agent, etc. In some embodiments, a method of treating cancer in a human comprises administering to the subject a therapeutically effective dose of a neoantigen therapeutic agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both agents are administered according to an intermittent dosing strategy. In some embodiments, a method of treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of a neoantigen therapeutic agent in combination with a therapeutically effective dose of a second immunotherapeutic agent, wherein one or both agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the neoantigen therapeutic agent and administering subsequent doses of the agent about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the neoantigen therapeutic agent and administering subsequent doses of the agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the neoantigen therapeutic agent and administering subsequent doses of the agent about once every 4 weeks. In some embodiments, the agent is administered using an intermittent dosing strategy and the additional therapeutic agent is administered weekly.

The present disclosure relates to compositions comprising a neoantigen therapeutic agent described herein. The present disclosure also relates to pharmaceutical compositions comprising a neoantigen therapeutic agent described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions find use in immunotherapy. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).

The formulations are prepared for storage and use by combining the neoantigen therapeutic agent described herein with a pharmaceutically acceptable carrier (e.g., a carrier or excipient). Those skilled in the art generally consider pharmaceutically acceptable carriers, excipients, and/or stabilizers to be non-active ingredients of the formulation or pharmaceutical composition. Exemplary formulations are listed in WO 2015/095811.

Suitable pharmaceutically acceptable carriers include, but are not limited to, non-toxic buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London). In some embodiments, the carrier is a 5% dextrose solution in water.

In one aspect, the invention relates to pharmaceutically acceptable or physiologically acceptable compositions comprising solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic agents and agents promoting or delaying absorption, compatible with pharmaceutical administration. Thus, pharmaceutical compositions or pharmaceutical formulations refer to a composition suitable for pharmaceutical use in a subject. The compositions can be formulated to be compatible with a particular route of administration (i.e., systemic or local). Thus, the compositions include carriers, diluents or excipients suitable for administration by various routes.

In some embodiments, the composition may further comprise an acceptable additive to enhance the stability of the immune cells in the composition. Acceptable additives cannot alter the specific activity of the immune cells. Examples of acceptable additives include, but are not limited to, sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Acceptable additives can be combined with acceptable carriers and/or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to enhance the stability of the peptide and reduce gelation of the solution. The surfactant can be added to the composition in an amount of from 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

The pharmaceutical compositions described herein may be administered by any number of routes for both local and systemic treatment. Administration may be local, via epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary, via inhalation or insufflation of powders or aerosols, including via a nebulizer, intratracheally and intranasally; oral; or parenteral, including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion) or intracranial (e.g., intrathecal or intraventricular).

The pharmaceutical composition can be administered, for example, by injection. The administration can be by intradermal injection, intranasal spray, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration or subcutaneous injection. Compositions for injection include aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, for example, sugars, polyhydric alcohols such as mannitol, sorbitol and sodium chloride, can be included in the composition. The resulting solutions can be packaged for use as is or lyophilized; the lyophilized preparation can later be combined with a sterile solution before administration. For intravenous administration, injection or injection into the lesion site, the active ingredient will be in the form of a parenterally acceptable pyrogen-free aqueous solution that has a suitable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included if necessary. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, if necessary, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient in a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation may be vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions can generally be administered intravenously, for example, by injection of a unit dose. For injection, the active ingredient can be in the form of a parenterally acceptable aqueous solution that is essentially pyrogen-free and has a suitable pH, isotonicity and stability. Suitable solutions can be prepared using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives can be included. In addition, the compositions can be administered by aerosolization.

If the compositions are contemplated for use in a medicament or in any of the methods provided herein, it is contemplated that the composition may be substantially non-pyrogenic, such that the composition will not cause an inflammatory response or an unsafe allergic reaction when administered to a human patient. Testing of compositions for pyrogens and preparing compositions that are substantially non-pyrogenic are well understood by those skilled in the art and can be accomplished using commercially available kits.

Suitable carriers may contain a compound that stabilizes, increases, or delays absorption, or increases or delays clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buffers. Absorption-retarding agents include, for example, aluminum monostearate and gelatin. Detergents may also be used to stabilize or increase or decrease absorption of a pharmaceutical composition, including liposomal carriers. To protect against digestion, a compound may form a complex with the composition to make it resistant to acid and enzymatic hydrolysis, or the compound may form a complex with a suitable stable carrier such as a liposome. Means for protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135; and U.S. Patent No. 5,391,377).

The compositions may be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount administered depends on the subject being treated, the ability of the subject's immune system to utilize the active ingredient, and the degree of binding capacity desired. The precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are individualized for each individual. Suitable schedules for initial administration and repeat injections also vary, but typical are initial administration followed by repeat doses at intervals of one or more hours, followed by an injection or other administration. Alternatively, continuous intravenous infusions sufficient to maintain blood concentrations are contemplated.

In some cases, pharmaceutical compositions containing one or more agents exert local and regional effects when locally administered or injected into or near specific sites of infection. Direct topical application, for example, of a viscous liquid, solution, suspension, dimethyl sulfoxide (DMSO) based solutions, liposomal formulations, gel, jelly, cream, lotion, ointment, suppository, foam or aerosol spray, can be used for topical administration to produce, for example, local and/or regional effects. Pharmaceutically suitable carriers for such a formulation include, for example, lower aliphatic alcohols, polyglycols (e.g., glycerol or polyethylene glycol), fatty acid esters, oils, fats, silicones and the like. Such preparations may also include preservatives (e.g., esters of p-hydroxybenzoic acid) and/or antioxidants (e.g., ascorbic acid and tocopherol). See also Dermatological Formulations: Percutaneous absorption, Barry (Ed.), Marcel Dekker Incl, 1983. In another embodiment, topical/topical formulations containing a transporter, carrier, or ion channel inhibitor are used to treat epidermal or mucosal viral infections.

In some cases, the immunogenic pharmaceutical composition may include carriers and excipients (including, but not limited to, buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils, including oils of petroleum, animal, vegetable or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline solutions, aqueous dextrose and glycerol solutions, flavors, colors, anti-tack agents and other acceptable additives, adjuvants or binders, other pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene glycol, water, ethanol, and the like. In other cases, the pharmaceutical preparation is substantially free of preservatives. In other cases, the pharmaceutical preparation may contain at least one preservative. It should be understood that while any suitable carrier known to those skilled in the art may be used to administer the pharmaceutical compositions described herein, the type of carrier will vary depending on the route of administration.

The immunogenic pharmaceutical composition may include preservatives such as thiomersal or 2-phenoxyethanol. In some cases, the immunogenic pharmaceutical composition is substantially free (e.g., <10 μg/mL) of mercury material, such as thiomersal. Alpha-tocopherol succinate may be used as an alternative to mercury compounds.

To control tonicity, a physiological salt such as sodium salt may be included in the immunogenic pharmaceutical composition. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate and/or magnesium chloride and the like.

The immunogenic pharmaceutical composition may have an osmolality of 200 mOsm/kg to 400 mOsm/kg, 240-360 mOsm/kg, or within the range of 290-310 mOsm/kg.

The immunogenic pharmaceutical composition may contain one or more buffers such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (especially with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers are in some cases included in the range of 5-20 or 10-50 mM.

The immunogenic pharmaceutical composition may comprise a pH modifier. In some embodiments, the pH modifier is present at a concentration of less than 1 mM or greater than 1 mM. In some embodiments, the pH modifier is present at a concentration of less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 nM or 1 mM. In some embodiments, the pH modifier is present at a concentration of greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mM. In some embodiments, the pH modifier is a dicarboxylate. In some embodiments, the pH modifier is a tricarboxylate. In some embodiments, the pH modifier is succinic acid dicarboxylate. In some embodiments, the pH modifier is a disuccinate. In some embodiments, the pH modifier is citric acid tricarboxylate. In some embodiments, the pH modifier is tricitrate. In some embodiments, the pH modifier is disodium succinate. In some embodiments, succinic acid dicarboxylate is present in the pharmaceutical composition at a concentration of 0.1 mM to 1 mM. In some embodiments, the disuccinate is present in the pharmaceutical composition at a concentration of 0.1 mM to 1 mM. In some embodiments, succinic acid dicarboxylate is present in the pharmaceutical composition at a concentration of 1 mM to 5 mM. In some embodiments, the disuccinate is present in the pharmaceutical composition at a concentration of 1 mM to 5 mM. The pH of the immunogenic pharmaceutical composition can be from about 5.0 to about 8.5, from about 6.0 to about 8.0, from about 6.5 to about 7.5, or from about 7.0 to about 7.8.

The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be non-pyrogenic, for example, containing <1 EU (endotoxin unit, a standard measure) per dose, and may contain <0.1 EU per dose. The composition may be gluten-free.

The immunogenic pharmaceutical composition may include a detergent, such as a polyoxyethylene sorbitan ester surfactant (known as "Tweens") or an octoxynol (such as octoxynol-9 (Triton X-100) or tert-octylphenoxypolyethoxyethanol). The detergent may be present only in trace amounts. The immunogenic pharmaceutical composition may include less than 1 mg/mL of each of octoxynol-10 and polysorbate 80. Other residual components in trace amounts may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

The immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension, in suitable carriers well known in the art. The pharmaceutical compositions can be sterilized by conventional, well-known sterilization methods, or can be sterilized by filtration. The resulting aqueous solutions can be packaged for use as is or lyophilized, where the lyophilized preparation is combined with a sterile solution before administration.

Pharmaceutical compositions comprising, for example, an active agent, such as the immune cells described herein, in combination with one or more adjuvants, can be formulated to have certain molar ratios. For example, molar ratios of from about 99:1 to about 1:99 of an active agent, such as an immune cell described herein, in combination with one or more adjuvants can be used. In some cases, a range of molar ratios of the active agent, such as an immune cell described herein, in combination with one or more adjuvants can be selected from about 80:20 to about 20:80; from about 75:25 to about 25:75, from about 70:30 to about 30:70, from about 66:33 to about 33:66, from about 60:40 to about 40:60; about 50:50; and from about 90:10 to about 10:90. The molar ratio of the active agent, such as an immune cell described herein, in combination with one or more adjuvants can be about 1:9, and in some cases can be about 1:1. The active agent, such as an immune cell described herein, in combination with one or more adjuvants can be formulated together in the same dosage unit, such as in a single vial, suppository, tablet, capsule, aerosol spray; or each agent, form and/or compound can be formulated as separate units, such as two vials, suppositories, tablets, two capsules, a tablet and a vial, aerosol spray and the like.

The therapeutic composition may be in unit dosage form. Such compositions include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories.

The neoantigen peptides described in the present invention can also be enclosed in microcapsules. Such microcapsules are prepared, for example, by coacervation or interfacial polymerization methods, for example hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, as described in Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.

In some embodiments, the pharmaceutical compositions include a neoantigen therapeutic agent described in the present invention in complex with liposomes. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be prepared by reverse phase evaporation with a fat composition containing phosphatidylcholine, cholesterol, and a PEG derivative of phosphatidylethanolamine (PEG-PE). The liposomes can be extruded through filters with a certain pore size to obtain liposomes of a desired diameter.

In some embodiments, sustained release preparations comprising the neoantigen peptides described herein may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent, wherein the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable copolymers of lactic and glycolic acids such as LUPRON DEPOT™ (injectable microspheres consisting of a copolymer of lactic and glycolic acids and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

The present disclosure relates to methods of treatment comprising an immunogenic vaccine. Methods of treating a disease (e.g., cancer or a viral infection) are provided. The method may comprise administering to a subject an effective amount of a composition comprising an immunogenic antigen. In some embodiments, the antigen comprises a viral antigen. In some embodiments, the antigen comprises a tumor antigen.

Non-limiting examples of vaccines that can be prepared include a peptide-based vaccine, a nucleic acid-based vaccine, an antibody-based vaccine, and an antigen-presenting cell-based vaccine.

Vaccine compositions may be formulated using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate processing of the active agents into preparations that can be used pharmaceutically. The proper formulation may depend on the route of administration selected. Any of the well-known techniques, carriers and excipients may be used as appropriate and as understood in the art.

In some cases, the vaccine composition is formulated as a peptide-based vaccine, a nucleic acid-based vaccine, an antibody-based vaccine, or a cell-based vaccine. For example, the vaccine composition can include naked cDNA in cationic fat formulations; lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), naked cDNAs or peptides encapsulated in, for example, poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al, Vaccine 12:299-306, 1994; Jones et al, Vaccine 13:675-681, 1995); peptide composition contained in immunostimulatory complexes (ISCOMS) (e.g., Takahashi et al, Nature 344:873-875, 1990; Hu et al, Clin. Exp. Immunol. 113:235-243, 1998); or multiple antigen peptide (MAP) systems (see, e.g., Tam, J. P., Proc. Natl Acad. Sci. U.S.A. 85:5409-5413, 1988; Tarn, J. P., J. Immunol. Methods 196:17-32, 1996). Sometimes the vaccine is formulated as a peptide-based vaccine or a nucleic acid-based vaccine in which the nucleic acid codes for polypeptides. Sometimes the vaccine is formulated as an antibody-based vaccine. Sometimes the vaccine is formulated as a cell-based vaccine.

The amino acid sequence of the identified disease-specific immunogenic neoantigen peptide can be used to develop a pharmaceutically acceptable composition. The source of the antigen can be, but is not limited to, natural or synthetic proteins, including glycoproteins, peptides and superantigens; antibody/antigen complexes; lipoproteins; RNA or a translation product thereof; and DNA or a polypeptide encoded by DNA. The source of the antigen can also include untransformed, transformed, transfected or transduced cells or cell lines. The cells can be transformed, transfected or transduced using any of a variety of expression vectors or retroviral vectors known to those skilled in the art that can be used to express recombinant antigens. Expression can also be achieved in any suitable host cell that has been transformed, transfected, or transduced with an expression vector or retroviral vector containing a DNA molecule encoding the recombinant antigens. Any number of transfection, transformation, and transduction protocols known to those skilled in the art can be used. Recombinant vaccinia vectors and vaccinia vector-infected cells can be used as a source of antigen.

The pharmaceutical composition may comprise a synthetic disease-specific immunogenic neoantigenic peptide. The pharmaceutical composition may comprise two or more disease-specific immunogenic neoantigenic peptides. The pharmaceutical composition may comprise a disease-specific immunogenic peptide precursor (such as a protein, peptide, DNA, and RNA). The disease-specific immunogenic peptide precursor may be created or created for an identified disease-specific immunogenic neoantigenic peptide. In some embodiments, the therapeutic composition comprises an immunogenic peptide precursor. The disease-specific immunogenic peptide precursor may be a prodrug. In some embodiments, the pharmaceutical composition comprising the disease-specific immunogenic neoantigenic peptide may further comprise an adjuvant. For example, the neoantigenic peptide may be used as a vaccine. In some embodiments, the immunogenic vaccine may comprise a pharmaceutically acceptable immunogenic neoantigenic peptide. In some embodiments, the immunogenic vaccine may comprise a pharmaceutically acceptable precursor of an immunogenic neoantigenic peptide (such as a protein, peptide, DNA, and RNA). In some embodiments, the method of treatment comprises administering to a subject an effective amount of an antibody that specifically recognizes the immunogenic neoantigenic peptide.

The methods described in the present invention are applicable in the context of personalized medicine, where immunogenic neoantigenic peptides are used to develop therapeutic agents (such as vaccines or therapeutic antibodies) for the same individual. Thus, a method of treating a disease in a subject may include identifying an immunogenic neoantigenic peptide in the subject according to the methods described in the present invention; and synthesizing the peptide (or a precursor thereof); and administering the peptide or an antibody specifically recognizing the peptide to the subject.

In some embodiments, identifying an epitope expressed by tumor cells of a subject or an immunogenic neoantigen peptide comprises selecting a plurality of nucleic acid sequences from a pool of nucleic acid sequences sequenced from tumor cells of the subject that encode a plurality of candidate peptide sequences comprising one or more different mutations not present in a pool of nucleic acid sequences sequenced from non-tumor cells of the subject, wherein the pool of nucleic acid sequences sequenced from tumor cells of the subject and the pool of nucleic acid sequences sequenced from non-tumor cells of the subject are sequenced by whole genome sequencing or whole exome sequencing. In some embodiments, identifying an epitope expressed by the subject's tumor cells or by an immunogenic neoantigenic peptide further comprises predicting or measuring which candidate peptide sequences from a plurality of possible peptide sequences form a complex with a protein encoded by an HLA allele of the same subject by means of an HLA peptide binding assay. In some embodiments, identifying an epitope expressed by the subject's tumor cells or by an immunogenic neoantigenic peptide further comprises selecting a plurality of selected tumor-specific peptides or one or more polynucleotides encoding a plurality of selected tumor-specific peptides from the candidate peptide sequences based on the HLA peptide binding assay. In some embodiments, the epitope expressed by the tumor cells of the subject is a neoantigen, a tumor-associated antigen, an antigen associated with a mutated tumor, and/or wherein the expression of the epitope in the tumor cells of the subject is higher than the expression of the epitope in normal cells of the subject.

In some embodiments, the expression profile of the immunogenic neoantigen can serve as an important basis for creating patient-specific vaccines. In some embodiments, the expression profile of the immunogenic neoantigen can serve as an important basis for creating a vaccine for a group of patients with a particular disease. Thus, specific diseases, such as certain types of tumors, can be selectively treated in a group of patients.

In some embodiments, the peptides described in the present invention are structurally normal antigens that can be recognized by autologous anti-disease T cells in a large group of patients. In some embodiments, an antigen expression profile is determined in a group of diseased subjects whose disease expresses structurally normal neoantigens.

In some embodiments, a pharmaceutical composition described herein comprises at least two polypeptides comprising at least two polypeptide molecules. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise the same epitope of the same length. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise the same amino acid or amino acid sequence, which refers to a peptide sequence that is not encoded by a nucleic acid sequence immediately upstream or downstream of a nucleic acid sequence in the genome of the subject that encodes the epitope. In some embodiments, two or more of the at least two polypeptides or polypeptide molecules comprise a different linker. In some embodiments, a first polypeptide of the at least two polypeptides or polypeptide molecules does not comprise a linker, and a second polypeptide of the at least two polypeptides or polypeptide molecules comprises a linker. In some embodiments, the first polypeptide of the at least two polypeptides or polypeptide molecules does not comprise a linker at the N-terminus of the epitope, and the second polypeptide of the at least two polypeptides or polypeptide molecules comprises a linker at the N-terminus of the epitope. In some embodiments, the first polypeptide of the at least two polypeptides or polypeptide molecules does not comprise a linker at the C-terminus of the epitope, and the second polypeptide of the at least two polypeptides or polypeptide molecules comprises a linker at the C-terminus of the epitope. In some embodiments, the first polypeptide of the at least two polypeptides or polypeptide molecules comprises a linker, and the second polypeptide of the at least two polypeptides or polypeptide molecules does not comprise a linker. In some embodiments, the first polypeptide of the at least two polypeptides or polypeptide molecules comprises a linker at the N-terminus of the epitope, and the second polypeptide of the at least two polypeptides or polypeptide molecules does not comprise a linker at the N-terminus of the epitope. In some embodiments, the first polypeptide of the at least two polypeptides or polypeptide molecules comprises a linker at the C-terminus of the epitope, and the second polypeptide of the at least two polypeptides or polypeptide molecules does not comprise a linker at the C-terminus of the epitope.

In some embodiments, the epitope is present in the pharmaceutical composition in an amount of 1 ng to 10 mg or 5 μg to 1.5 mg. In some embodiments, the epitope is present in an amount of 1 ng to 10 mg. In some embodiments, the epitope is present in an amount of 1 ng to 100 ng, 10 ng to 200 ng, 20 ng to 300 ng, 30 ng to 400 ng, 40 ng to 500 ng, 50 ng to 600 ng, 60 ng to 700 ng, 70 ng to 800 ng, 80 ng to 900 ng, 90 ng to 1 μg, 100 ng to 2 μg, 200 ng to 3 μg, 300 ng to 4 μg, 400 ng to 5 μg, 500 ng to 6 μg, 600 ng to 7 μg, 700 ng to 8 mcg, 800 ng to 9 mcg, 900 ng to 10 mcg, 1 mcg to 100 mcg, 20 mcg to 200 mcg, 30 mcg to 300 mcg, 40 mcg to 400 mcg, 50 mcg to 500 mcg, 60 mcg to 600 mcg, 70 mcg to 700 mcg, 80 mcg to 800 mcg, 90 mcg to 900 mcg, 100 mcg to 1 mg, 200 mcg to 1.1 mg, 300 mcg to 1.2 mg, 400 mcg to 1.3 mg, 500 mcg to 1.4 mg, 600 mcg to 1.5 mg, 700 mcg to 2 mg, 800 mcg to 3 mg, 900 mcg to 4 mg, 1 mg to 5 mg, 1.3 mg to 6 mg, 1.5 mg to 7 mg, 2 mg to 8 mg, 3 mg to 9 mg, or 4 mg to 10 mg. In some embodiments, the epitope is present in an amount of about 1, 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, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 ng. In some embodiments, the epitope is present in an amount of about 1, 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, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 μg. In some embodiments, the epitope is present in an amount of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mg.

There are many methods for producing immunogenic neoantigens. Proteins or peptides can be produced by any method known to those skilled in the art, including expression of proteins, polypeptides or peptides using standard molecular biology techniques, isolation of proteins or peptides from natural sources, in vitro translation, or chemical synthesis of proteins or peptides. In general, such disease-specific neoantigens can be produced either in vitro or in vivo. Immunogenic neoantigens can be produced in vitro as peptides or polypeptides, which can then be included in a personalized vaccine or immunogenic composition and administered to a subject. Production of immunogenic neoantigens in vitro can include peptide synthesis or expression of a peptide/polypeptide from a DNA or RNA molecule in any of a variety of bacterial, eukaryotic or viral recombinant expression systems, followed by purification of the expressed peptide/polypeptide. Alternatively, immunogenic neoantigens can be produced in vivo by administering to a subject molecules (e.g., DNA, RNA, and viral expression systems) that encode an immunogenic neoantigen, after which the encoded immunogenic neoantigens are expressed. In some embodiments, a polynucleotide encoding an immunogenic neoantigenic peptide can be used to produce a neoantigenic peptide in vitro.

In some embodiments, the polynucleotide comprises a 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%, 99%, or 100% sequence identity to a polynucleotide encoding an immunogenic neoantigen. The polynucleotide may be, for example, DNA, cDNA, single- and/or double-stranded, native or stabilized forms of polynucleotides, or combinations thereof. The nucleic acid encoding the immunogenic neoantigen peptide may or may not contain introns as long as it encodes the peptide. In some embodiments, in vitro translation is used to produce the peptide.

Also contemplated are expression vectors comprising sequences encoding a neoantigen, as well as host cells comprising expression vectors. Expression vectors suitable for use herein may comprise at least one expression control element operably linked to a nucleic acid sequence. Expression control elements are incorporated into the vector to control and regulate expression of the nucleic acid sequence. Examples of expression control elements are well known in the art and include, for example, the lac system, the lambda phage operator and promoter regions, yeast promoters, and promoters derived from polyoma, adenovirus, retrovirus, or SV40. Additional functional elements include, but are not limited to, leader sequences, termination codons, polyadenylation signals, and any other sequences necessary or preferred for proper transcription and subsequent translation of the nucleic acid sequence in the host system. One skilled in the art will appreciate that the proper combination of expression control elements will depend on the host system selected. In addition, it will be understood that the expression vector must contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers.

The neoantigenic peptides may be provided as RNA or cDNA molecules encoding the desired neoantigenic peptides. One or more neoantigenic peptides as described herein may be encoded by a single expression vector. Typically, the DNA is inserted into an expression vector, such as a plasmid, in the correct orientation and reading frame for expression, and the DNA may optionally be linked to appropriate transcriptional and translational control nucleotide sequences recognized by the desired host (e.g., a bacterium), although such controls are typically available in the expression vector. The vector is then introduced into the host bacterium for cloning using standard techniques. Useful expression vectors for eukaryotic hosts, especially mammals or humans, include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as those from E. coli including pCR 1, pBR322, pMB9 and derivatives thereof, broader host range plasmids such as M13 and filamentous single stranded DNA phages. Suitable host cells for expression of the polypeptide are discussed under Polynucleotides [0250]. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian host cells are well known in the art.

The proteins produced by the transformed host may be purified by any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and calibration column chromatography, and the like), centrifugation, differential solubility, or any other standard protein purification technique. Affinity tags such as hexahistidine (SEQ ID NO: 376), maltose binding domain, influenza virus envelope sequence, glutathione S-transferase, and the like may be attached to the protein to allow easy purification by passage through a column of appropriate affinity. The isolated proteins may also be physically characterized by methods such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

The vaccine may comprise a substance that binds a polypeptide sequence described in the present invention. The substance may be an antibody. The antibody-based vaccine may be formulated using any well-known techniques, carriers and excipients, if they are suitable and known in the art. In some embodiments, the peptides described in the present invention can be used to create neoantigen-specific therapeutic agents, such as antibody-based therapeutic agents. For example, neoantigens can be used to generate and/or identify antibodies that specifically recognize neoantigens. These antibodies can be used as therapeutic agents. The antibody can be a natural antibody, a chimeric antibody, a humanized antibody, or can be an antibody fragment. The antibody can recognize one or more polypeptides described in the present invention. In some embodiments, the antibody can recognize a polypeptide that has a sequence with no more than 40%, 50%, 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 polypeptide described in the present invention. In some embodiments, the antibody can recognize a polypeptide that has a sequence with at least 40%, 50%, 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 polypeptide described in the present invention. In some embodiments, the antibody can recognize a polypeptide sequence that is at least 30%, 40%, 50%, 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% of the length polypeptide described in the present invention. In some embodiments, the antibody may recognize a polypeptide sequence that is no more than 30%, 40%, 50%, 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% of the length polypeptide described in the present invention.

The present disclosure also relates to the use of nucleic acid molecules as carriers for delivering neoantigenic peptides/polypeptides to a subject in need thereof in vivo, such as in the form of DNA vaccines.

In some embodiments, the vaccine is a nucleic acid-based vaccine. In some embodiments, the nucleic acid encodes an immunogenic peptide or a precursor peptide. In some embodiments, the nucleic acid-based vaccine comprises sequences flanking a sequence encoding an immunogenic peptide or a precursor peptide. In some embodiments, the nucleic acid-based vaccine comprises more than one immunogenic epitope. In some embodiments, the nucleic acid-based vaccine is a DNA-based vaccine. Delivery methods are discussed in the Polynucleotides section [0250].

The polynucleotide may be substantially pure or contained in a suitable vector or delivery system. Suitable vectors and delivery systems include viral ones, such as those based on adenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associated virus, or hybrids containing elements of more than one virus. Non-viral delivery systems include cationic lipids and cationic polymers (e.g., cationic liposomes).

One or more neoantigen peptides can be encoded and expressed in vivo using a viral system. Viral vectors can be used as recombinant vectors herein, wherein a portion of the viral genome is removed to introduce new genes without destroying the infectivity of the virus. The viral vector of the present disclosure is a non-pathogenic virus. In some embodiments, the viral vector has a tropism for a particular type of mammalian cells. In another embodiment, the viral vector of the present disclosure is capable of infecting professional antigen-presenting cells, such as dendritic cells and macrophages. In another embodiment of the present disclosure, the viral vector is capable of infecting any mammalian cell. The viral vector can also infect tumor cells. Viral vectors used herein include, but are not limited to, poxvirus such as vaccinia virus, avipox virus, fowlpox virus, and highly attenuated vaccinia virus (Ankara or MVA), retrovirus, adenovirus, baculovirus, and the like.

The vaccine may be delivered by a variety of routes. Routes of delivery may include oral (including buccal and sublingual), rectal, nasal, topical, transdermal patch, pulmonary, vaginal, suppository, or parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous, and intravenous) administration, or in a form suitable for administration by aerosolization, inhalation, or insufflation. General information on drug delivery systems can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999). The vaccine described herein can be administered into muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can be used. The vaccine described herein can be administered by intradermal injection, intranasal spray, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, or subcutaneous injection.

In some cases, the vaccine may also be formulated for administration through the nasal passages. Formulations suitable for nasal administration, where the carrier is a solid, may include a coarse powder having a particle size of, for example, in the range of about 10 to about 500 microns, which is administered in the manner in which tobacco is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose. The formulation may be a nasal spray, nasal drops, or for administration of an aerosol using a nebulizer. The formulation may include aqueous or oily solutions of the vaccine.

A vaccine may be a liquid preparation, such as a suspension, syrup, or elixir. A vaccine may also be a preparation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injection), such as a sterile suspension or emulsion.

The vaccine may comprise a single immunization material, or may comprise a multiple immunization material (i.e., a "multi-dose" kit). In multi-dose embodiments, it is preferred to include a preservative. Alternatively (or in addition) to including a preservative in multi-dose compositions, the compositions may be contained in a container having an aseptic adapter for release of the product.

The vaccine can be administered in a dose volume of approximately 0.5 ml, although children may be given half the dose (i.e. approximately 0.25 ml). Sometimes the vaccine may be administered in a higher dose, such as approximately 1 ml.

The vaccine may be given as a course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses. Sometimes the vaccine is given as a course of 1, 2, 3, or 4 doses. Sometimes the vaccine is given as a course of 1 dose. Sometimes the vaccine is given as a course of 2 doses.

The administration of the first dose and the second dose can be separated by approximately 0 days, 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, 30 days, 2 months, 4 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3 years, 4 years or more.

The vaccine described in the present invention can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. Sometimes, the vaccine described in the present invention is administered every 2, 3, 4, 5, 6, 7 or more years. Sometimes, the vaccine described in the present invention is administered every 4, 5, 6, 7 or more years. Sometimes, the vaccine described in the present invention is administered once.

The dosage examples are not limiting and are used only to illustrate specific dosage regimens for administering the vaccine described in the present invention. An effective amount for use in humans can be determined in animal models. For example, a dose for humans can be formulated to achieve blood, liver, site, and/or gastrointestinal tract concentrations that have proven effective in animals. Based on animal data and other types of similar data, those skilled in the art can determine effective amounts of a vaccine composition suitable for humans.

An effective amount, when referring to an agent or combination of agents, generally means the dosage ranges, routes of administration, formulations, etc., that have been recommended or approved by any of the various medical or pharmaceutical regulatory or advisory organizations (e.g., FDA, AMA) or by the manufacturer or supplier.

In some aspects, the vaccine and kit described in the present invention can be stored at a temperature of 2°C to 8°C. In some cases, the vaccine is not stored frozen. In some cases, the vaccine is stored at a temperature of -20°C to -80°C. In some cases, the vaccine is stored away from sunlight.

7. Sets

The therapeutic neoantigen described in the present invention may be presented in the form of a kit together with instructions for administration. Typically, the kit contains the desired therapeutic neoantigen in a container, in a unit dosage form and instructions for administration. Additional therapeutic agents, such as cytokines, lymphokines, checkpoint inhibitors, antibodies, may also be included in the kit. Other components of the kit that may also be desirable include, for example, a sterile syringe, booster doses and other desired excipients.

The invention also relates to kits and articles of manufacture for use with one or more of the methods described in the present invention. The kits may comprise one or more neoantigenic polypeptides comprising one or more neoepitopes. The kits may also comprise nucleic acids that encode one or more peptides or proteins described in the present invention, antibodies that recognize one or more peptides described in the present invention, or APC-based cells activated by one or more peptides described in the present invention. The kits may further comprise adjuvants, reagents, and buffers necessary for the preparation and delivery of vaccines.

The kits may also include a carrier, packaging or container divided into compartments to accommodate one or more containers such as vials, tubes and the like, each of the containers containing one of the individual elements such as peptides and adjuvants for use in the method described in the present invention. Suitable containers include, for example, bottles, vials, syringes and tubes. The containers may be made of various materials such as glass or plastic.

The finished articles of the present invention comprise packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, vials, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and the intended route of administration and treatment. A kit typically includes labels indicating the contents and/or instructions for use, and package inserts with instructions for use. A set of instructions is also typically included.

The present description will be described in more detail by means of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present description in any way. Those skilled in the art will readily recognize many non-critical parameters that can be changed or modified to obtain alternative embodiments according to the present description. All patents, patent applications and printed publications listed in the present invention are incorporated herein by reference in their entirety.

EXAMPLES

These examples are provided for illustrative purposes only and do not limit the scope of the claims presented in the present invention.

Example 1 - Evaluation of enhanced cleavage and processing of polypeptides

T cell receptor (TCR)-transduced cells are used to screen polypeptides for epitope processing and presentation in vitro. As effector cells, engineered Jurkat cells expressing CD8 together with validated TCRs are prepared. For target cells, peripheral blood mononuclear cells (PBMCs) with specific HLA alleles are stimulated with FLT3 ligand overnight, loaded with polypeptides containing the epitope of interest in various contexts for an hour, and matured with cytokines. Engineered Jurkat cells and PBMCs are cocultured for 48 hours, and the level of IL-2 secreted by the engineered Jurkat cells is measured as an indicator of TCR peptide recognition. The experimental setup is shown in Fig. 3, and the results are shown in Figs. 4 and 5.

Example 2 - Immunogenicity of a neoantigen polypeptide

The immunogenicity of a variety of polypeptides designed around defined epitopes, as well as the quality of T cell responses to these polypeptides, are studied. Eighty-four 8-12-week-old female C57BL/6 mice (Taconic Biosciences) are randomly and prospectively assigned to treatment groups upon arrival. Animals are acclimated for three days before study entry. Animals are maintained on sterile LabDiet™ 5053 rodent chow and sterile water ad libitum. Twelve animals in group 1 serve as unvaccinated controls. Twelve animals in each group 2-7 receive 50 μg polyIC:LC and 10 μg of each polypeptide (defined in Table 13, bolded sequence represents minimal epitope) or the molar equivalent of alternative peptide designs (defined in Table 14). Kif18b is used as a CD4 helper peptide and is used unmodified in all mice in groups 2-7. Blood is collected by retro-orbital bleeding on days 7, 14, and 21. Animals are weighed and general health is monitored daily. Animals are sacrificed by CO ₂ overdose on day 21 of the study completion if the animal has lost >30% of its body weight compared to its weight on day 0; or if the animal is considered moribund.

Table 13. Peptides used in the study Antigen Subsequence SEQ ID NO: Limitation Allele Source model Alg8 AVGITYTWTRLYASVLTGSLVSKTKK 377 MHCI H-2Kb T3 Lama4 IQKISFFDGFEVGFNFRTLQPNGLLFYYT 378 MHCI H-2Kb T3 Adpgk GIPVHLELASMTNMELMSSIVHQQVFPT 379 MHCI H-2Db MC38 Reps1 GRVLELFRAAQLANDVVLQIMELCGATR 380 MHCI H-2Db MC38 Irgq KARDETAALLNSAVLGAAPLFVPPAD 381 MHCI H-2Db MC38 Obsl1 REGVELCPGNKYEMRRHGTTHSLVIHD 382 MHCI H-2Db B16F10 Kif18b PSKPSFQEFVDWENVSPELNSTDQPFL 383 MHCII I-Ab B16F10

Table 14. Experimental design (see also Fig. 6) (the table describes “K4” as SEQ ID NO: 1 and “K4-Val-Cit” as SEQ ID NO: 384) Group Number of mice Care Antigens (base of tail on the left) Antigens (scruff of the neck) Vaccine dose Treatment with vaccines Collection of fabrics 1 12 untreated N/A N/A N/A N/A N/A 2 12 Strong SLP+CD4+hiltonol Kif18b* Alg8, Lama4, Obsl1 10 mcg of each SLP or equimolar SSP
50 mcg Hiltonol Path:
s.c. base of tail on the left in 100 µl/injection
Scheme:
Days -21,
-14, -7 OO blood flow:
Days -14,
-7, 0 3 12 SLP+CD4+hiltonol Reps1, Adpgk, Irgq, Kif18b* Alg8, Lama4, Obsl1 4 12 SSP+CD4+hiltonol 5 12 K4-SSP+CD4+hiltonol 6 12 K4-Val-Cit-PABC-SSPs+CD4+hiltonol 7 12 K4-disulfide-SSP+CD4+hiltonol

MHC tetramers are produced in house and used to measure the expansion of peptide-specific T cells in immunogenicity assays. For assessment, the tetramer is added to 1 x 10 ⁵ cells in PBS containing 1% FCS and 0.1% sodium azide (FACS buffer). The cells are incubated in the dark for 15 min at 37°C. Antibodies specific for T cell markers such as CD8 and for irrelevant cell types such as CD4/CD11b/CD11c/CD19 are then added to a final concentration recommended by the manufacturer and the cells are incubated in the dark at 4°C for 20 min. The cells are washed with cold FACS buffer, immediately analyzed on an LSR2 instrument (Becton Dickinson) and analyzed using FacsDiva software (Becton Dickinson). For analysis of tetramer-positive cells, lymphocyte gates were taken from the forward and side scatter plots. Data are presented as the proportion of cells that were CD4 ^- CD11b ^- CD11c ^- CD19 ^- CD8 ⁺ /tetramer ⁺ .

Immunization with the K4 epitope (“K4” is described as SEQ ID NO: 1) significantly enhances the immune response to 5 of the 6 epitopes assessed. Immune responses to Alg8, Lama4, Reps1, Adpgk, and Obsl1 are significantly enhanced. Immunization with the K4 epitope (“K4” is described as SEQ ID NO: 1) enhances the immunogenicity of poorly immunogenic epitopes (e.g., Obsl1). Immunization with the K4-Val-Cit-PABC epitope (“K4-Val-Cit” is described as SEQ ID NO: 384) enhances Alg8-specific immune responses. The results are shown in Figs. 7–9.

Example 3 - Synthesis of a disulfide linker (compound 5)

Stage 1

2,2'-Bis(5-nitropyridyl)disulfide 2 (2 mmol) was suspended in 10 ml of dichloromethane and the corresponding mercapto alcohol 1 (1 mmol, where R ¹ and R ² have the meanings specified in the present invention) in dichloromethane (4 ml) was added to the suspension. The resulting suspension was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure. The resulting residue was redissolved in 5 ml of dimethylformamide and purified using a C18 reverse phase column with a gradient of acetonitrile and water containing 0.05% TFA. The desired fractions were combined and lyophilized to give (5-nitropyridin-2-yl)disulfanylalkyl alcohol 3 (yield ~65-82%, purity >90%, UPLC-MS/UV analysis at 220 nm).

Stage 2

To a solution of (5-nitropyridin-2-yl)disulfanylalkyl alcohol 3 (0.5 mmol, where R ¹ and R ² have the meanings given herein) in dimethylformamide (2 mL) was added N,N'-diisopropylethylamine (1.5 mmol), and then 4-nitrophenyl chloroformate 4 (0.55 mmol) was added. This solution was stirred at room temperature for 16 h, then purified using a C18 reverse phase column with a gradient of acetonitrile and water containing 0.05% TFA. The desired fractions were combined and lyophilized to give 4-nitrophenyl-(5-nitropyridin-2-yl)disulfanylalkyl carbonate 5 (yield ~90-98%, purity >90%, UPLC-MS/UV analysis at 220 nm).

Example 4 - Synthesis of disulfide-containing peptides

Step 1: Preparation of 4-nitro-2-pyridylthioactivated disulfide peptide 8.

According to the above scheme, the N-terminus of the peptide resin 6 (any resin manufactured for solid-phase peptide synthesis can be used) is acylated using a linker 5 (where R ¹ and R ² have the meanings specified in the present invention) manually, or programmed accordingly on an automatic peptide synthesizer. More specifically, the resin 6 (0.05 mmol) is swollen in dimethylformamide for 5 minutes and dried. The corresponding 4-nitrophenyl-(5-nitropyridin-2-yl)disulfanyl alkyl carbonate 5 (0.2 mmol) and Oxyma Pure Novabiochem® (also known as Oxyma Pure, 0.3 mmol) are dissolved in 1 ml of dimethylformamide added to the swollen resin 6, and then N,N'-diisopropylethylamine (0.3 mmol) is added. The resulting resin suspension was stirred for 3 hours, dried, and then the resulting peptide-linked resin 7 was washed with dimethylformamide (5x, 5 ml), dichloromethane (5x, 5 ml), and methanol (2x, 5 ml). The peptide-bound resin 7 was dried under reduced pressure for 1 hour and cleaved using 3 mL of 95% trifluoroacetic acid (TFA), 2.5% water, 2.5% triisopropylsilane (TIPS) at room temperature for 3 hours to form a cleavage solution (“A”) containing both unbound peptide 8 and the cleaved resin from 7. This cleavage solution A was then filtered and dried into a 50 mL conical tube, the cleaved resin from 7 was washed with 95:5 TFA:water (1 mL), filtered, dried, and combined to form a filtered peptide solution (“B”). Unbound peptide 8 was isolated from the filtered peptide B solution by precipitation with ice-cold diethyl ether, centrifuged at 3600 rpm for 5 min, and the diethyl ether distilled off. The resulting peptide pellet was then washed with 20 ml of ice-cold diethyl ether to obtain a suspension, which was then shaken and centrifuged again at 3600 rpm for 3 min. This was repeated for a total of 3 washes to thoroughly wash the pellet to yield 4-nitrophenyl-(5-nitropyridin-2-yl)disulfanyl alkylcarbamate peptide 8, which was used in the next step of the synthesis without further purification.

Step 2: Disulfide exchange reaction to form disulfide-containing peptide 10.

As described in the scheme above, crude 4-nitrophenyl-(5-nitropyridin-2-yl)disulfanylalkylcarbamate peptide 8 (where R ¹ and R ² are as defined herein) is subjected to disulfide exchange with a desired thiol-containing molecule 9 (where G ¹ and j are as defined herein). More specifically, 4-nitrophenyl-(5-nitropyridin-2-yl)disulfanylalkylcarbamate peptide 8 (0.05 mmol) is dissolved in dimethylformamide (1 ml), then a solution of the desired thiol-containing compound 9 (0.05 mmol) in 1:1 dimethylformamide-1 M Tris buffer is added. The resulting yellow solution was stirred for 2 h and purified on a C18 reversed-phase column with a gradient of acetonitrile and water containing 0.05% TFA. The desired fractions were pooled and lyophilized to give the disulfide-containing peptide 10 (yield ~10-30%, purity >95%, UPLC-MS/UV analysis at 220 nm, starting from solid-phase peptide synthesis).

Example 5 - Synthesis of PABC Containing Peptide (13)

As described in the scheme above, the N-terminus of peptide resin 6 is acylated using Fmoc-AA-AA-PAB-PNP 11 manually or appropriately programmed on an automated peptide synthesizer. Specifically, resin 6 (0.05 mmol) is swollen in dimethylformamide for 5 min and dried. The appropriate Fmoc-AA-AA-PAB-PNP 11 (0.2 mmol) and Oxyma Pure Novabiochem® (also known as Oxyma Pure, 0.3 mmol) are dissolved in 1 ml of dimethylformamide, added to resin 6, and then N,N'-diisopropylethylamine (0.3 mmol) is added. The resulting resin suspension is stirred for 3 h, dried, and the resulting Fmoc-protected resin 12 is then washed with dimethylformamide (5x, 5 ml). The final N-terminal α-Fmoc is removed with 20% piperidine in dimethylformamide (2x, 5 min). At this point, deprotected intermediate 12 is optionally reacted with additional amino acid residues at the N-terminus of 12 using standard solid phase Fmoc peptide syntheses, followed by deprotection of the N-terminal α-Fmoc using a similar procedure as discussed immediately above. After the desired Fmoc deprotection is complete, resin 12 (or analogs with extended amino acids) is washed with dimethylformamide (5x, 5 mL), dichloromethane (5x, 5 mL), and then methanol (2x, 5 mL). The peptide-bound resin 12 is dried under reduced pressure for 1 hour and cleaved using 3 mL of 70% trifluoroacetic acid (TFA), 10% phenol, 10% triisopropylsilane (TIPS), and 10% thioanisole at room temperature for 30 minutes to form a cleavage solution (“A”) containing both unbound peptide 13 and cleaved resin from 12. This cleavage solution A is then filtered and dried to yield a filtered peptide solution (“B”) in a 50 mL conical tube. The cleaved resin from 12 was washed with 95:5 TFA:aqueous solution (1 mL), filtered, dried and combined with the filtered solution of peptide B. Unbound peptide 13 was isolated from the filtered solution of peptide B by precipitation with ice-cold diethyl ether, centrifuged at 3600 rpm for 5 min and the diethyl ether distilled off. The resulting peptide pellet was then washed with 20 mL of ice-cold diethyl ether to obtain a suspension which was then vortexed and centrifuged again at 3600 rpm for 3 min. This was repeated for a total of 3 washes to thoroughly wash the pellet to afford compound 13 (~10-30% yield, >95% purity, UPLC-MS/UV analysis at 220 nm, starting from solid phase peptide synthesis).

Example 6 - Evaluation of TMPRSS2::ERG epitope processing

T cell receptor (TCR)-transduced cells are used to assess the processing and presentation of the TMPRSS2::ERG epitope on HLA-A02:01 in vitro. Engineered Jurkat cells expressing CD8 together with validated TCRs are generated as effector cells. For target cells, 293T cells that naturally express HLA-A02:01 were i) loaded with peptides containing the TMPRSS2::ERG epitope for 24 h only or ii) stably transduced with a plasmid that encodes a peptide containing the TMPRSS2::ERG epitope in different contexts (epitope in a natural context, i.e., the peptide further comprises an amino acid or amino acid sequence that naturally flank the epitope sequence at the N- and/or C-terminus of the epitope, in a non-natural context, i.e., the peptide further comprises an amino acid or amino acid sequence that does not naturally flank the epitope sequence (e.g., the CMVpp65 sequence), or a plasmid encoding a peptide containing an irrelevant epitope in a non-natural context (as a control). The engineered Jurkat cells and 293T cells were co-cultured for 24 hours, and the level of IL-2 secreted by the engineered Jurkat cells was measured as an indicator of TCR peptide recognition. The results are shown in Fig. 10.

Example 7 - Comparison of enhanced cleavage and processing and immunogenicity of polypeptides

T cell receptor (TCR)-transduced cells are used for in vitro comparison of RAS-G12V-HLA-A11:01 epitope processing from peptides containing the RAS-G12V epitope only, the RAS-G12V epitope and additional amino acid sequence flanking the epitope at the N-terminus only, or the RAS-G12V epitope and additional amino acid sequences flanking the epitope at both the N- and C-terminus for epitope processing and presentation. Engineered Jurkat cells expressing CD8 together with validated TCRs are generated as effector cells. For target cells, peripheral blood mononuclear cells (PBMCs) with specific HLA alleles are stimulated with FLT3 ligand overnight, loaded with polypeptides containing the RAS-G12V epitope in various contexts for an hour, and allowed to mature with cytokines. Engineered Jurkat cells and PBMCs were co-cultured for 48 h, and the level of IL-2 secreted by engineered Jurkat cells was measured as an indicator of TCR peptide recognition. The results are shown in Fig. 11.

Example 8 - Evaluation of the immunogenicity of mutant RAS peptides with different contexts around epitopes

Materials:

AIM V medium (Invitrogen)

Human FLT3L, preclinical CellGenix #1415-050 Stock 50 ng/µL

TNF-α, preclinical CellGenix # 1406-050 Stock 10 ng/µL

IL-1β, preclinical CellGenix #1411-050 Stock 10 ng/µL

PGE1 or Alprostadil - Cayman from Czech republic Stock 0.5 mcg/mcl

R10 medium - RPMI 1640 glutamax + 10% human serum + 1% PenStrep

20/80 Medium - 18% AIM V+72% RPMI 1640 glutamax+10% human serum+1% PenStrep

IL7 Stock 5 ng/µl

IL15 Stock 5 ng/µl

Procedure:

Step 1: Seed 5 million PBMCs (or cells of interest) into each well of a 24-well FLT3L plate in 2 ml AIMV medium.

Stage 2: Peptide loading and maturation - in AIMV

1. Mix the peptide pool of interest (except for no peptide conditions) with PBMCs (or cells of interest) in the appropriate wells.

2. Incubate for 1 hour.

3. Mix the maturation cocktail (including TNF-α, IL-1β, PGE1 and IL-7) into each well after incubation.

Step 3: Add human serum to each well to a final concentration of 10% by volume and mix.

Stage 4. Replace the medium with fresh RPMI+10% HS medium supplemented with IL7+IL15.

Step 5: Replace the medium with fresh 20/80 medium supplemented with IL7+IL15 every 1-6 days during the incubation period.

Step 6: Seed 5 million PBMCs (or cells of interest) into each well of a new 6-well FLT3L plate in 2 ml AIM V medium.

Stage 7: Peptides are loaded and allowed to mature for re-stimulation (new plates)

1. Mix the pool of peptides of interest (except no peptides) with PBMCs (or cells of interest) in the appropriate wells.

2. Incubate for 1 hour.

3. Add maturation cocktail to each well after incubation.

Stage 8: Re-stimulation:

1. Count FLT3L cultures after the first stimulation and add 5 million cultured cells to new plates for re-stimulation.

2. Bring the culture volume to 5 ml (AIM V) and add 500 µl of human serum (10% by volume).

Step 9: Remove 3 ml of medium and add 6 ml of RPMI+10% HS medium supplemented with IL7+IL15.

Stage 10: Change 75% of the medium to fresh 20/80 medium with the addition of IL7+IL15.

Stage 11: Repeat re-stimulation if necessary.

Antigen-specific induction assay

MHC tetramers are commercially available or produced in-house and used to measure peptide-specific T cell expansion in immunogenicity assays. For assessment, the tetramer is added to 1 x 10 ⁵ cells in PBS containing 1% FCS and 0.1% sodium azide (FACS buffer) according to the manufacturer's instructions. Cells are incubated in the dark for 20 min at room temperature. Antibodies specific for T cell markers such as CD8 are then added to a final concentration recommended by the manufacturer and cells are incubated in the dark at 4°C for 20 min. Cells are washed with cold FACS buffer and resuspended in buffer containing 1% formaldehyde. Cell data are collected on an LSR Fortessa instrument (Becton Dickinson) and analyzed using FlowJo software (Becton Dickinson). For analysis of tetramer-positive cells, lymphocyte gates are taken from the forward and side scatter plots. Data are presented as the proportion of cells that are CD8+/Tteramer+.

The peptide immunogenicity workflow (i.e., T cell induction and tetramer assay) was used to evaluate the relative immunogenicity of the three peptide constructs described in Fig. 11. Exemplary data showing the increased immunogenicity of the peptide with the C-terminal epitope compared to the peptide with the middle epitope, based on hit rates across 3 donors, are shown in Fig. 12, top. The same three peptide constructs were also evaluated using the in vivo mouse vaccination strategy described in Example 2. Exemplary data showing the increased immunogenicity of the peptide with the C-terminal epitope compared to the peptide with the middle epitope are shown in Fig. 12, bottom.

Example 9 - High CD8 hit rate when APCs are stimulated with mRNA encoding peptides

Shortmers (9-10 amino acids) or longmers (25 amino acids) are designed to form a middle neoantigen strand as shown graphically in Fig. 13A. Antigen sequences are represented as colored rectangles. Linker sequences (K, QLGL (SEQ ID NO: 385) or GVGT (SEQ ID NO: 386) - represented as blue circles) are added between antigen sequences as predicted by NetChop (an algorithm for predicting human proteome cleavage). If a sequence is predicted to cleave within an antigen sequence, cleavage sites are added to promote cleavage between antigen sequences. PBMCs are then nucleofected with the aforementioned multi-antigen encoding mRNA constructs and used to stimulate T cells. Side-by-side comparisons are performed with pools of congruent peptides, and their length and sequence are found to be the same as those encoded in the RNA strands. Short and long RNA sequences enhance similar CD8+ T cell responses to multimers (Table 15). Notably, robust CD8 responses are observed using mRNA encoding longmers and shortmers.

Table 15. Comparison of longmer- and shortmer-mediated activation of peptide and RNA CD8 Hit Rate (%) Mean neoantigen+ frequency (% CD8 cells) Diversity of answers (out of 6) CD4 answers Donor 1 Peptide short 7 0.03% 1 n.d. Long peptide 19 0.09% 2 0 RNA short 11 1.50% 2 n.d. RNA long 8 0.36% 2 0 Donor 2 Peptide short 11 0.03% 2 n.d. Long peptide 17 0.21% 3 1 RNA short 19 0.39% 2 n.d. RNA long 20 0.05% 2 0

As shown in Fig. 13B, the Gli3 epitope is well expressed and presented by peptides as well as mRNA, but PBMCs encoded by mRNA loaded with the shorter Gli3 epitope yield higher Gli3-specific CD8+ T cells (as determined by multimeric analysis). Representative flow cytometry results for the multimeric analysis are shown in Fig. 13C. In this strand, the sequence preceding the Gli3 sequence is from a non-native context. It may have improved processing and presentation of Gli3 from the polypeptide strand and an increased response compared to the peptide. In addition, the shorter mRNA strand elicits an ME-1 T cell response that is not present in the pool of congruent short peptides. In our strands, ME-1 has cleavage sites before and after the epitope sequence, and such enhanced processing and presentation on the epitope may lead to superior T cell responses.

Paragraphs of embodiments

[0492] A polypeptide comprising an epitope presented by MHC class I or MHC class II of an antigen-presenting cell (APC), the polypeptide having a structure of formula (I):

Y _n -B _t -A _r -X _m -A _s -C _u -Z _p

Formula (I),

or a pharmaceutically acceptable salt thereof,

(i) where X _m is an epitope, where each X is independently an amino acid of a contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject,

and where (a) MHC is MHC class I and m is an integer from 8 to 12, or

(b) MHC is MHC class II and m is an integer from 9 to 25;

(ii) where each Y is independently an amino acid, its analogue or derivative, and where:

(A) if the variable r of A _r in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t -A _r -X _m ,

(B) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m, or

(C) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 1 or greater, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t ; and

additionally, where n is an integer from 0 to 1000;

(iii) where each Z is independently an amino acid, its analogue or derivative, and where:

(A) if variable s of A _s in Formula (I) is 0, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m -A _s -C _u ,

(B) if variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 0, Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m, or

(C) if variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 1 or more, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the genome of the subject that encodes _Cu ; and

additionally, where p is an integer from 0 to 1000;

and also where

if n is 0, p is an integer between 1 and 1000; and

if p is 0, n is an integer between 1 and 1000;

(iv) where A _r is a linker and r is 0 or 1;

(v) where A _s is a linker and s is 0 or 1;

(vi) where each B is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m,

and where t is an integer between 0 and 1000; and

(vii) where each C is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m,

and where u is an integer between 0 and 1000;

and also where

(a) the polypeptide does not consist of four different epitopes presented by MHC class I;

(b) the polypeptide comprises at least two different polypeptide molecules;

(c) the epitope comprises at least one mutated amino acid; and/or

(d) Y _n and/or Z _p are cleaved from the epitope if the polypeptide is processed by the APC.

[0493] The polypeptide of paragraph [0487], wherein the epitope is presented by MHC class II.

[0494] The polypeptide of paragraph [0487] or [0488], wherein m is an integer from 9 to 25.

[0495] The polypeptide of any one of paragraphs [0487]-[0489], wherein t is 1, 2, 3, 4, or 5 or more, and r is 0.

[0496] The polypeptide of any one of paragraphs [0487]-[0490], wherein u is 1, 2, 3, 4, or 5 or more, and s is 0.

[0497] The polypeptide of any one of paragraphs [0487]-[0491], wherein t is 1 or more, r is 0, and n is from 1 to 1000.

[0498] The polypeptide of any one of paragraphs [0487]-[0492], wherein u is 1 or greater, s is 0, and p is 1-1000.

[0499] The polypeptide of any one of paragraphs [0487]-[0493], wherein t is 0.

[0500] The polypeptide of any one of paragraphs [0487]-[0494], wherein u is 0.

[0501] The polypeptide of any one of paragraphs [0487]-[0495], wherein t is at least 1 and B _t comprises lysine.

[0502] The polypeptide of any one of paragraphs [0487]-[0496], wherein u is at least 1 and C _u comprises lysine.

[0503] The polypeptide of any one of paragraphs [0487]-[0497], wherein B _t is cleaved from the epitope if the polypeptide is processed by the APC.

[0504] The polypeptide of any one of paragraphs [0487]-[0498], wherein C _u is cleaved from the epitope if the polypeptide is processed by APC.

[0505] The polypeptide of any one of paragraphs [0487]-[0499], wherein n is an integer from 1 to 5 or from 7 to 1000.

[0506] The polypeptide of any one of paragraphs [0487]-[0500], wherein p is an integer from 1 to 4 or from 6 to 1000.

[0507] The polypeptide of any one of paragraphs [0487]-[0501], wherein the polypeptide does not consist of four different epitopes presented by MHC class I.

[0508] The polypeptide of any one of paragraphs [0487]-[0502], wherein the polypeptide does not comprise four different epitopes presented by MHC class I.

[0509] The polypeptide of any one of paragraphs [0487]-[0503], wherein the polypeptide comprises at least two different polypeptide molecules.

[0510] The polypeptide of any one of paragraphs [0487]-[0504], wherein the epitope comprises at least one mutant amino acid.

[0511] The polypeptide of paragraph [0505], wherein at least one mutant amino acid is encoded by an insertion, deletion, frameshift, neoORF, or point mutation in a nucleic acid sequence in the genome of the subject.

[0512] The polypeptide of any one of paragraphs [0487]-[0506], wherein Y _n and/or Z _p are cleaved from the epitope if the polypeptide is processed by the APC.

[0513] The polypeptide of any one of paragraphs [0487]-[0507], where m is X_mis not less than 8 and where X_mis AA₁AA₂AA₃AA₄AA₅AA₆AA₇AA₈AA₉AA₁₀AA₁₁AA₁₂AA₁₃AA₁₄AA₁₅AA₁₆AA₁₇AA₁₈AA₁₉AA₂₀AA₂₁AA₂₂AA₂₃AA₂₄AA₂₅, where each AA is an amino acid, and where one or more of the AA₉, AA₁₀, AA₁₁, AA₁₂, AA₁₃, AA₁₄, AA₁₅, AA₁₆, AA₁₇, AA₁₈, AA₁₉, AA₂₀, AA₂₁, AA₂₂, AA₂₃, AA₂₄and AA₂₅are optionally present, and in addition at least one AA is a mutant amino acid.

[0514] The polypeptide of any one of paragraphs [0487]-[0508], wherein r is 1.

[0515] The polypeptide of any one of paragraphs [0487]-[0509], wherein s is 1.

[0516] The polypeptide of any one of paragraphs [0487]-[0510], wherein r is 1 and s is 1.

[0517] The polypeptide of any one of paragraphs [0487]-[0511], wherein r is 0.

[0518] The polypeptide of any one of paragraphs [0487]-[0512], wherein s is 0.

[0519] The polypeptide of any one of paragraphs [0487]-[0513], wherein r is 0 and s is 0.

[0520] The polypeptide of any one of paragraphs [0487]-[0514], wherein A _r and/or A _s is a non-polypeptide linker.

[0521] The polypeptide of any one of paragraphs [0487]-[0515], wherein A _r and/or A _s is a chemical linker.

[0522] The polypeptide of any one of paragraphs [0487]-[0516], wherein A _r and/or A _s comprise a non-naturally occurring amino acid.

[0523] The polypeptide of any one of paragraphs [0487]-[0517], wherein A _r and/or A _s do not comprise an amino acid.

[0524] The polypeptide of any one of paragraphs [0487]-[0518], wherein A _r and/or A _s do not comprise a natural amino acid.

[0525] The polypeptide of any one of paragraphs [0487]-[0519], wherein A _r and/or A _s comprise a bond other than a peptide bond.

[0526] The polypeptide of any one of paragraphs [0487]-[0520], wherein A _r and/or A _s comprise a disulfide bond.

[0527] The polypeptide of any one of paragraphs [0487]-[0521], wherein A _r and/or A _s are different.

[0528] The polypeptide of any one of paragraphs [0487]-[0522], wherein A _r and/or A _s are the same.

[0529] The polypeptide of any one of paragraphs [0487]-[0523], wherein the polypeptide comprises a hydrophilic tail.

[0530] The polypeptide of any one of paragraphs [0487]-[0524], wherein Y _n -B _t -A _r and/or A _s -C _u -Z _p increases the solubility of the polypeptide compared to a corresponding peptide that does not contain Y _n -B _t -A _r and/or A _s -C _u -Z _p.

[0531] The polypeptide of any one of paragraphs [0487]-[0525], wherein each X of X _m is a naturally occurring amino acid.

[0532] The polypeptide of any one of paragraphs [0487]-[0526], wherein the epitope is released from Y _n -B _t -A _r and/or A _s -C _u -Z _p if the polypeptide is processed by APC.

[0533] The polypeptide of any one of paragraphs [0487]-[0527], wherein the polypeptide is cleaved into A _r and/or A _s .

[0534] The polypeptide of any one of paragraphs [0487]-[0528], wherein the polypeptide is cleaved at a higher rate when n is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m ; and/or

wherein the polypeptide is cleaved at a higher rate when p is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of the nucleic acid sequence in the subject's genome that encodes X _m .

[0535] The polypeptide of any one of paragraphs [0487]-[0528], wherein the polypeptide is cleaved at a higher rate when n is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that comprises B _t -X _m , where t is at least one and r of the variable A _r in Formula (I) is 0; and/or

where the polypeptide is cleaved at a higher rate when p is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains X _m -C _u , where u is at least one and s of the variable A _s in formula (I) is 0.

[0536] The polypeptide of any one of paragraphs [0487]-[0530], wherein the polypeptide cleaves into A _r at a higher rate when n is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m ; and/or

wherein the polypeptide is cleaved into A _s at a higher rate when p is an integer from 1 to 1000, compared to the cleavage of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded immediately by a nucleic acid sequence downstream of a nucleic acid sequence in the subject's genome that encodes X _m .

[0537] The polypeptide of any one of paragraphs [0487]-[0531], wherein the presentation of the epitope by the APC is enhanced when n is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m ; and/or

wherein the presentation of the APC epitope is enhanced if p is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of the nucleic acid sequence in the subject's genome that encodes X _m .

[0538] The polypeptide of any one of paragraphs [0487]-[0531], wherein the presentation of the APC epitope is enhanced when n is an integer from 1 to 1000, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises B _t -X _m , where t is at least one and r of the variable A _r in Formula (I) is 0; and/or

wherein the presentation of the epitope by the APC is enhanced if p is an integer from 1 to 1000, compared to the presentation of the epitope by a corresponding polypeptide of the same length that contains X _m -C _u , where u is at least one and s of the variable A _s in formula (I) is 0.

[0539] The polypeptide of any one of paragraphs [0487]-[0533], wherein the APC presents an epitope to an immune cell.

[0540] The polypeptide of any one of paragraphs [0487]-[0534], wherein the APC presents an epitope to a phagocytic cell.

[0541] The polypeptide of any one of paragraphs [0487]-[0535], wherein the APC presents an epitope to a dendritic cell, a macrophage, a mast cell, a neutrophil, or a monocyte.

[0542] The polypeptide of any one of paragraphs [0487]-[0536], wherein the APC presents an epitope predominantly or specifically to an immune cell, a phagocytic cell, a dendritic cell, a macrophage, a mast cell, a neutrophil, or a monocyte.

[0543] The polypeptide of any one of paragraphs [0487]-[0537], wherein the immunogenicity is increased when n is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m ; and/or

wherein the immunogenicity is increased if p is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence located immediately downstream of the nucleic acid sequence in the subject's genome that encodes X _m .

[0544] The polypeptide of any one of paragraphs [0487]-[0537], wherein the immunogenicity is increased when n is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that comprises B _t -X _m , where t is at least one and r of the variable A _r in formula (I) is 0; and/or

wherein the immunogenicity is increased if p is an integer from 1 to 1000, compared to the immunogenicity of a corresponding polypeptide of the same length that contains X _m -C _u , where u is at least one and s of the variable A _s in formula (I) is 0.

[0545] The polypeptide of any one of paragraphs [0487]-[0539], wherein the anti-tumor activity is enhanced when n is an integer from 1 to 1000, compared to the anti-tumor activity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m ; and/or

wherein the antitumor activity is enhanced when p is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately downstream of the nucleic acid sequence in the genome of the subject that encodes X _m .

[0546] The polypeptide of any one of paragraphs [0487]-[0539], wherein the anti-tumor activity is increased when n is an integer from 1 to 1000, compared to the anti-tumor activity of a corresponding polypeptide of the same length that comprises B _t -X _m , where t is at least one and r of the variable A _r in Formula (I) is 0; and/or

wherein the antitumor activity is increased if p is an integer from 1 to 1000, compared to the antitumor activity of a corresponding polypeptide of the same length that contains X _m -C _u , where u is at least one and s of the variable A _s in Formula (I) is 0.

[0547] The polypeptide of any one of paragraphs [0487]-[0541], wherein Y _n and/or Z _p comprises a sequence selected from the group consisting of poly-Lys (polyK) and poly-Arg (polyR).

[0548] The polypeptide of paragraph [0542], wherein Y _n and/or Z _p comprises a sequence selected from the group consisting of polyK-AA-AA and polyR-AA-AA, wherein each AA is an amino acid, an analog or a derivative thereof.

[0549] The polypeptide of paragraph [0542] or [0543], wherein the polyK comprises poly-L-Lys.

[0550] The polypeptide of paragraph [0542] or [0543], wherein polyR comprises poly-L-Arg.

[0551] The polypeptide of any one of paragraphs [0542]-[0545], wherein the polyK or polyR comprises at least three or four consecutive lysine (SEQ ID NO: 1) or arginine (SEQ ID NO: 2) residues, respectively.

[0552] The polypeptide of any one of paragraphs [0487]-[0546] wherein A _r and/or A _s is selected from the group consisting of disulfide; p-aminobenzyloxycarbonyl (PABC); and AA-AA-PABC, wherein each AA is an amino acid, analog, or derivative thereof.

[0553] The polypeptide of paragraph [0547], wherein AA-AA-PABC is selected from the group consisting of Ala-Lys-PABC, Val-Cit-PABC, and Phe-Lys-PABC.

[0554] The polypeptide of any one of paragraphs [0487]-[0546], wherein A _r and/or A _s is

Formula (II).

[0555] The polypeptide of any one of paragraphs [0487]-[0546], wherein A _r and/or A _s is

Formula (III) or

Formula (IV),

Where,

R ₁ and R ₂ are independently H or (C ₁ -C ₆ ) alkyl;

j is 1 or 2;

G ₁ is H or COOH; and

i is 1, 2, 3, 4, or 5.

[0556] The polypeptide of any one of paragraphs [0487]-[0550], wherein the polypeptide is ubiquitinated.

[0557] The polypeptide of paragraph [0551], wherein the polypeptide is ubiquitinated prior to cleavage.

[0558] The polypeptide of paragraph [0551] or [0552], where the polypeptide is ubiquitinated at a lysine residue.

[0559] The polypeptide of any one of paragraphs [0487]-[0553], wherein the polypeptide is not cleaved prior to processing by APC or prior to internalization of APC in the subject.

[0560] The polypeptide of any one of paragraphs [0487]-[0554], wherein the polypeptide is not cleaved in the blood of the subject prior to processing by APC or prior to internalization by APC.

[0561] The polypeptide of any one of paragraphs [0487]-[0555], wherein the polypeptide is not cleaved by a blood protease.

[0562] The polypeptide of any one of paragraphs [0487]-[0556], wherein the polypeptide is not cleaved by plasmin, plasma kallikrein, tissue kallikrein, thrombin, or a clotting factor.

[0563] The polypeptide of any one of paragraphs [0487]-[0557], wherein the polypeptide is stable in human plasma.

[0564] The polypeptide of any one of paragraphs [0487]-[0558], wherein the half-life of the polypeptide in human plasma is from 1 hour to 5 days.

[0565] The polypeptide of any one of paragraphs [0487]-[0559], wherein the polypeptide is cleaved in a lysosome, endolysosome, endosome, or endoplasmic reticulum (ER).

[0566] The polypeptide of any one of paragraphs [0487]-[0560], wherein the polypeptide is cleaved by an aminopeptidase.

[0567] The polypeptide of paragraph [0561], wherein the aminopeptidase is insulin-regulated aminopeptidase (IRAP) or endoplasmic reticulum aminopeptidase (ERAP).

[0568] The polypeptide of any one of paragraphs [0487]-[0560], wherein the polypeptide is processed by a trypsin-like domain of the proteasome and/or immunoproteasome.

[0569] The polypeptide of paragraph [0563], wherein the trypsin-like domain has trypsin-like activity, chymotrypsin-like activity, or peptidyl glutamyl peptide hydrolase (PGPH) activity.

[0570] The polypeptide of any one of paragraphs [0487]-[0560], wherein the polypeptide is cleaved by a protease.

[0571] The polypeptide of paragraph [0565], wherein the protease is a trypsin-like protease, a chymotrypsin-like protease, or a peptidyl glutamyl peptide hydrolase (PGPH).

[0572] The polypeptide of paragraph [0565], wherein the protease is selected from the group consisting of asparagine peptide lyase, aspartic protease, cysteine protease, glutamic protease, metalloprotease, serine protease, and threonine protease.

[0573] The polypeptide of paragraph [0567], wherein the protease is a cysteine protease selected from the group consisting of calpain, caspase, cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, and cathepsin Z.

[0574] The polypeptide of any one of paragraphs [0487]-[0568], wherein the subject is a mammal.

[0575] The polypeptide of any one of paragraphs [0487]-[0569], wherein the subject is a human.

[0576] The polypeptide of any one of paragraphs [0487]-[0570], wherein the epitope binds to HLA MHC class I.

[0577] The polypeptide of paragraph [0571], wherein the epitope binds to HLA MHC class I with a stability of from 10 minutes to 24 hours.

[0578] The polypeptide of paragraph [0571], wherein the epitope binds to HLA MHC class I with an affinity of from 0.1 nM to 2000 nM.

[0579] The polypeptide of any one of paragraphs [0487]-[0570], wherein the epitope binds to HLA MHC class II.

[0580] The polypeptide of paragraph [0574], wherein the epitope binds to HLA MHC class II with a stability of from 10 minutes to 24 hours.

[0581] The polypeptide of paragraph [0574], wherein the epitope binds to HLA MHC class II with an affinity of from 0.1 nM to 2000 nM, from 1 nM to 1000 nM, from 10 nM to 500 nM, or less than 1000 nM.

[0582] The polypeptide of any one of paragraphs [0487]-[0576], wherein n is an integer from 1 to 20 or from 5 to 12.

[0583] The polypeptide of any one of paragraphs [0487]-[0577], wherein p is an integer from 1 to 20 or from 5 to 12.

[0584] The polypeptide of any one of paragraphs [0487]-[0578], wherein the epitope comprises a tumor-specific epitope.

[0585] The polypeptide of any one of paragraphs [0487]-[0579], wherein the polypeptide comprises at least two polypeptides, wherein two or more of the at least two polypeptides have the same formula Y _n -B _t -A _r -X _m -A _s -C _u -Z _p.

[0586] The polypeptide of paragraph [0580], wherein the polypeptide comprises at least two polypeptide molecules.

[0587] The polypeptide of paragraph [0580] or [0581], wherein X _m two or more of the at least two polypeptides or polypeptide molecules are the same.

[0588] The polypeptide of any one of paragraphs [0580]-[0582], wherein Y _n of two or more of the at least two polypeptides or polypeptide molecules are the same.

[0589] The polypeptide of any one of paragraphs [0580]-[0583], wherein Z _p of two or more of the at least two polypeptides or polypeptide molecules are the same.

[0590] The polypeptide of any one of paragraphs [0580]-[0584], wherein A _r and/or A _s of two or more of the at least two polypeptides or polypeptide molecules are different.

[0591] The polypeptide of any one of paragraphs [0580]-[0585], wherein r=0 for the first of the at least two polypeptides or polypeptide molecules, and r=1 for the second of the at least two polypeptides or polypeptide molecules.

[0592] The polypeptide of any one of paragraphs [0580]-[0586], wherein s=0 for the first of the at least two polypeptides or polypeptide molecules and s=1 for the second of the at least two polypeptides or polypeptide molecules.

[0593] The polypeptide of any one of paragraphs [0487]-[0587], wherein the polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides or polypeptide molecules.

[0594] The polypeptide of any one of paragraphs [0487]-[0588], wherein the epitope is a RAS epitope.

[0595] The polypeptide of paragraph [0589], wherein the epitope comprises a mutant RAS peptide sequence that comprises at least 8 contiguous amino acids of a mutant RAS protein comprising a mutation at G12, G13, or Q61 and a mutation at G12, G13, or Q61.

[0596] The polypeptide of paragraph [0590], wherein at least 8 contiguous amino acids of the mutant RAS protein comprising a mutation at G12, G13, or Q61 comprise a G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K, or Q61R mutation.

[0597] The polypeptide of paragraph [0590] or [0591], where the mutation G12, G13 or Q61 contains G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61H, Q61L, Q61K or Q61R mutation.

[0598] The polypeptide of any one of paragraphs [0487]-[0592], wherein Y _n and/or Z _p comprise an amino acid sequence of a CMV protein, such as pp65, HIV, or MART-1.

[0599] The polypeptide of any one of paragraphs [0487]-[0593], wherein n and/or p is 1, 2, 3, or an integer greater than 3.

[0600] The polypeptide of any one of paragraphs [0487]-[0594], wherein the epitope binds to a protein encoded by an HLA allele with an affinity of less than 10 μM, less than 1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, or less than 50 nM.

[0601] The polypeptide of any one of paragraphs [0487]-[0595], wherein the epitope binds to a protein encoded by an HLA allele with a stability of greater than 24 hours, greater than 12 hours, greater than 9 hours, greater than 6 hours, greater than 5 hours, greater than 4 hours, greater than 3 hours, greater than 2 hours, greater than 1 hour, greater than 45 minutes, greater than 30 minutes, greater than 15 minutes, or greater than 10 minutes.

[0602] The polypeptide of paragraph [0595] or [0596], wherein the HLA allele is selected from the group consisting of the HLA-A02:01 allele, the HLA-A03:01 allele, the HLA-A11:01 allele, the HLA-A03:02 allele, the HLA-A30:01 allele, the HLA-A31:01 allele, the HLA-A33:01 allele, the HLA-A33:03 allele, the HLA-A68:01 allele, the HLA-A74:01 allele, and/or the HLA-C08:02 allele, and any combinations thereof.

[0603] The polypeptide of any one of paragraphs [0487]-[0597], wherein the epitope comprises the amino acid sequence GADGVGKSAL (SEQ ID NO: 4), GACGVGKSAL (SEQ ID NO: 5), GAVGVGKSAL (SEQ ID NO: 6), GADGVGKSA (SEQ ID NO: 7), GACGVGKSA (SEQ ID NO: 8), GAVGVGKSA (SEQ ID NO: 9), KLVVVGACGV (SEQ ID NO: 10), FLVVVGACGL (SEQ ID NO: 11), FMVVVGACGI (SEQ ID NO: 12), FLVVVGACGI (SEQ ID NO: 13), FMVVVGACGV (SEQ ID NO: 14), FLVVVGACGV (SEQ ID NO: 15), MLVVVGACGV (SEQ ID NO: ID NO: 16), FMVVVGACGL (SEQ ID NO: 17), YLVVVGACGV (SEQ ID NO: 18), KMVVVGACGV (SEQ ID NO: 19), YMVVVGACGV (SEQ ID NO: 20), MMVVVGACGV (SEQ ID NO: 21), DTAGHEEY (SEQ ID NO: 22), TAGHEEYSAM (SEQ ID NO: 23), DILDTAGHE (SEQ ID NO: 24), DILDTAGH (SEQ ID NO: 25), ILDTAGHEE (SEQ ID NO: 26), ILDTAGHE (SEQ ID NO: 27), DILDTAGHEEY (SEQ ID NO: 28), DTAGHEEYS (SEQ ID NO: 29), LLDILDTAGH (SEQ ID NO: 30), DILDTAGRE (SEQ ID NO: 31), DILDTAGR (SEQ ID NO: 32), ILDTAGREE (SEQ ID NO: 33), ILDTAGRE (SEQ ID NO: 34), CLLDILDTAGR (SEQ ID NO: 35), TAGREEYSAM (SEQ ID NO: 36), REEYSAMRD (SEQ ID NO: 37), DTAGKEEYSAM (SEQ ID NO: 38), CLLDILDTAGK (SEQ ID NO: 39), DTAGKEEY (SEQ ID NO: 40), LLDILDTAGK (SEQ ID NO: 41), ILDTAGKE (SEQ ID NO: 42), ILDTAGKEE (SEQ ID NO: 43), DTAGLEEY (SEQ ID NO: 44), ILDTAGLE (SEQ ID NO: 45), DILDTAGL (SEQ ID NO: 46), ILDTAGLEE (SEQ ID NO: 47), GLEEYSAMRDQY (SEQ ID NO: 48), LLDILDTAGLE (SEQ ID NO: 49), LDILDTAGL (SEQ ID NO: 50), DILDTAGLE (SEQ ID NO: 51), DILDTAGLEEY (SEQ ID NO: 52), AGVGKSAL (SEQ ID NO: 53), GAAGVGKSAL (SEQ ID NO: 54), AAGVGKSAL (SEQ ID NO: 55), CGVGKSAL (SEQ ID NO: 56), ACGVGKSAL (SEQ ID NO: 57), DGVGKSAL (SEQ ID NO: 58), ADGVGKSAL (SEQ ID NO: 59), DGVGKSALTI (SEQ ID NO: 60), GARGVGKSA (SEQ ID NO: 61), KLVVVGARGV (SEQ ID NO: 62), VVVGARGV (SEQ ID NO: 63), SGVGKSAL (SEQ ID NO: 64), VVVGASGVGK (SEQ ID NO: 65), GASGVGKSAL (SEQ ID NO: 66), VGVGKSAL (SEQ ID NO: 67), VVVGAGCVGK (SEQ ID NO: 68), KLVVVGAGC (SEQ ID NO: 69), GDVGKSAL (SEQ ID NO: 70), DVGKSALTI (SEQ ID NO: 71), VVVGAGDVGK (SEQ ID NO: 72), TAGKEEYSAM (SEQ ID NO: 73), DTAGHEEYSAM (SEQ ID NO: 74), TAGHEEYSA (SEQ ID NO: 75), DTAGREEYSAM (SEQ ID NO: 76), TAGKEEYSA (SEQ ID NO: 77), AAGVGKSA (SEQ ID NO: 78), AGCVGKSAL (SEQ ID NO: 79), AGDVGKSAL (SEQ ID NO: 80), AGKEEYSAMR (SEQ ID NO: 81), AGVGKSALTI (SEQ ID NO: 82), ARGVGKSAL (SEQ ID NO: 83), ASGVGKSA (SEQ ID NO: 84), ASGVGKSAL (SEQ ID NO: 85), AVGVGKSA (SEQ ID NO: 86), CVGKSALTI (SEQ ID NO: 87), DILDTAGK (SEQ ID NO: 88), DILDTAGREEY (SEQ ID NO: 89), DTAGHEEYSAMR (SEQ ID NO: 90), DTAGKEEYS (SEQ ID NO: 91), DTAGKEEYSAMR (SEQ ID NO: 92), DTAGLEEYS (SEQ ID NO: 93), DTAGLEEYSA (SEQ ID NO: 94), DTAGLEEYSAMR (SEQ ID NO: 95), DTAGREEYS (SEQ ID NO: 96), DTAGREEYSAMR (SEQ ID NO: 97), GAAGVGKSA (SEQ ID NO: 98), GACGVGKSA (SEQ ID NO: 8), GACGVGKSAL (SEQ ID NO: 5), GADGVGKS (SEQ ID NO: 99), GAGDVGKSA (SEQ ID NO: 100), GAGDVGKSAL (SEQ ID NO: 101), GASGVGKSA (SEQ ID NO: 102), GCVGKSAL (SEQ ID NO: 103), GCVGKSALTI (SEQ ID NO: 104), GHEEYSAM (SEQ ID NO: 105), GKEEYSAM (SEQ ID NO: 106), GLEEYSAMR (SEQ ID NO: 107), GREEYSAM (SEQ ID NO: 108), GREEYSAMR (SEQ ID NO: 109), HEEYSAMRD (SEQ ID NO: 110), KEEYSAMRD (SEQ ID NO: 111), KLVVVGASG (SEQ ID NO: 112), LDILDTAGR (SEQ ID NO: 113), LEEYSAMRD (SEQ ID NO: 114), LVVVGARGV (SEQ ID NO: 115), LVVVGASGV (SEQ ID NO: 116), REEYSAMRDQY (SEQ ID NO: 117), RGVGKSAL (SEQ ID NO: 118), TAGLEEYSA (SEQ ID NO: 119), TEYKLVVVGAA (SEQ ID NO: 120), VGAAGVGKSA (SEQ ID NO: 121), VGADGVGK (SEQ ID NO: 122), VGASGVGKSA (SEQ ID NO: 123), VGVGKSALTI (SEQ ID NO: 124), VVVGAAGV (SEQ ID NO: 125), VVVGAVGV (SEQ ID NO: 126), YKLVVVGAC (SEQ ID NO: 127), YKLVVVGAD (SEQ ID NO: 128), YKLVVVGAR (SEQ ID NO: 129) or DILDTAGKE (SEQ ID NO: 130).

[0604] The polypeptide of any one of paragraphs [0487]-[0598], wherein Y _n comprises the amino acid sequence of IDIIMKIRNA (SEQ ID NO: 131), FFFFFFFFFFFFFFFFFFFFFFIIFFIFFWMC (SEQ ID NO: 132), FFFFFFFFFFFFFFFFFFFFFFFFFFAAFWFW (SEQ ID NO: 133), IFFIFFIIFFFFFFFFFFFFIIIIIIIWEC (SEQ ID NO: 134), FIFFFIIFFFFFIFFFFFIFIIIIIIFWEC (SEQ ID NO: 135), TEY, TEYKLV (SEQ ID NO: 136), WQAGILAR (SEQ ID NO: 137), HSYTTAE (SEQ ID NO: 138), PLTEEKIK (SEQ ID NO: 139), GALHFKPGSR (SEQ ID NO: 140), RRANKDATAE (SEQ ID NO: 141), KAFISHEEKR (SEQ ID NO: 142), TDLSSRFSKS (SEQ ID NO: 143), FDLGGGTFDV (SEQ ID NO: 144), CLLLHYSVSK (SEQ ID NO: 145), KKKKIIMKIRNA (SEQ ID NO: 146) or MTEYKLVVV (SEQ ID NO: 147).

[0605] The polypeptide of any one of paragraphs [0487]-[0599], wherein Z _p comprises the amino acid sequence of KKNKKDDI (SEQ ID NO: 148), KKNKKDDIKD (SEQ ID NO: 149), AGNDDDDDDDDDDDDDDDKKDKDDDDDD (SEQ ID NO: 150), AGNKKKKKKKKNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 151), AGRDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 152), SALTI (SEQ ID NO: 153), SALTIQL (SEQ ID NO: 154), GKSALTIQL (SEQ ID NO: 155), GKSALTI (SEQ ID NO: 156), QGQNLKYQ (SEQ ID NO: 157), ILGVLLLI (SEQ ID NO: 158), EKEGKISK (SEQ ID NO: 159), AASDFIFLVT (SEQ ID NO: 160), KELKQVASPF (SEQ ID NO: 161), KKKLINEKKE (SEQ ID NO: 162), KKCDISLQFF (SEQ ID NO: 163), KSTAGDTHLG (SEQ ID NO: 164), ATFYVAVTVP (SEQ ID NO: 165), LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG (SEQ ID NO: 166) or TIQLIQNHFVDEYDPTIEDSYRKQVVIDGE (SEQ ID NO: 167).

[0606] The polypeptide of any one of paragraphs [0487]-[0588], wherein the epitope is not a RAS epitope.

[0607] The polypeptide of any one of paragraphs [0487]-[0601], wherein the polypeptide is not KKKKKPKRDGYMFLKAESKIMFAT (SEQ ID NO: 168), KKKKYMFLKAESKIMFATLQRSS (SEQ ID NO: 169), KKKKKAESKIMFATLQRSSLWCL (SEQ ID NO: 170), KKKKKIMFATLQRSSLWCLCSNH (SEQ ID NO: 171), or KKKKMFATLQRSSLWCLCSNH (SEQ ID NO: 172).

[0608] The polypeptide of any one of paragraphs [0487]-[0588], wherein the epitope is a GATA3 epitope.

[0609] The polypeptide of paragraph [0603], where the GATA3 epitope contains the amino acid sequence MLTGPPARV (SEQ ID NO: 173), SMLTGPPARV (SEQ ID NO: 174), VLPEPHLAL (SEQ ID NO: 175), KPKRDGYMF (SEQ ID NO: 176 ), KPKRDGYMFL (SEQ ID NO: 177), ESKIMFATL (SEQ ID NO: 178), KRDGYMFL (SEQ ID NO: 179), PAVPFDLHF (SEQ ID NO: 180), AESKIMFATL (SEQ ID NO: 181), FATLQRSSL (SEQ ID NO: 182), ARVPAVPFD (SEQ ID NO : 183), IMKPKRDGY (SEQ ID NO: 184), DGYMFLKA (SEQ ID NO: 185), MFLKAESKIMF (SEQ ID NO: 186), LTGPPARV (SEQ ID NO: 187), ARVPAVPF (SEQ ID NO: 188), SMLTGPPAR (SEQ ID NO: 189), RVPAVPFDL (SEQ ID NO: 190) or LTGPPARVP ( SEQ ID NO: 191).

[0610] A cell comprising a polypeptide according to any one of paragraphs [0487]-[0604].

[0611] A cell according to paragraph [0605], wherein the cell is an antigen-presenting cell.

[0612] A cell according to paragraph [0606], wherein the cell is a dendritic cell.

[0613] The cell of paragraph [0605], wherein the cell is a mature antigen-presenting cell.

[0614] A method of cleaving a polypeptide comprising contacting the polypeptide of any one of paragraphs [0487]-[0604] with an APC.

[0615] The method of paragraph [0609], wherein the method is carried out in vivo.

[0616] The method of paragraph [0609], wherein the method is carried out ex vivo.

[0617] A method for producing a polypeptide comprising linking Y _n -A _r and/or A _s -Z _p to a sequence comprising an epitope sequence, wherein the epitope sequence is represented by MHC class I or MHC class II antigen-presenting protein (APP); and wherein

(i) each Y is independently an amino acid, an analogue or a derivative thereof and where Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes an epitope,

and where n is an integer from 0 to 1000;

(ii) each Z is independently an amino acid, an analogue or a derivative thereof and where Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes an epitope,

and where p is an integer between 0 and 1000; and

(iii) A _r is a linker and A _s is a linker, where at least one of r and s is 1;

and additionally where

(a) the polypeptide does not consist of four different epitopes presented by MHC class I;

(b) the polypeptide comprises at least two different polypeptide molecules;

(c) the epitope comprises at least one mutated amino acid; and/or

(d) Y _n and/or Z _p are cleaved from the epitope if the polypeptide is processed by the APC.

[0618] A method for producing a polypeptide comprising linking Y _n to B _t -X _m and/or Z _p to X _m -C _u , where X _m is an epitope sequence presented by MHC class I or MHC class II of an antigen-presenting cell (APC); and where

(i) each B is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m ,

and where t is an integer between 0 and 1000;

(ii) each C is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m ,

and where u is an integer between 0 and 1000;

(iii) each Y is independently an amino acid, an analogue or a derivative thereof and where Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the genome of the subject that encodes B _t -X _m ,

and where n is an integer between 0 and 1000; and

(4) each Z is independently an amino acid, an analogue or a derivative thereof and where Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m -C _u ,

and where p is an integer between 0 and 1000;

and additionally where

(a) the polypeptide does not consist of four different epitopes presented by MHC class I;

(b) the polypeptide comprises at least two different polypeptide molecules;

(c) the epitope comprises at least one mutated amino acid; and/or

(d) Y _n -B _t and/or C _u -Z _p are cleaved from the epitope if the polypeptide is processed by APC.

[0619] The method of paragraph [0612] or [0613], wherein if n is 0, p is an integer from 1 to 1000; and if p is 0, n is an integer from 1 to 1000.

[0620] The method of any one of paragraphs [0612]-[0614], wherein each X is independently an amino acid peptide sequence comprising any contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject, and wherein (a) the MHC is MHC class I and m is an integer from 8 to 12 or (b) the MHC is MHC class II and m is an integer from 9 to 25.

[0621] A pharmaceutical composition comprising a polypeptide according to any one of paragraphs [0487]-[0604] and a pharmaceutically acceptable excipient.

[0622] The pharmaceutical composition according to [0616], further comprising an immunomodulatory agent or adjuvant.

[0623] The pharmaceutical composition of [0617], wherein the immunomodulatory agent or adjuvant is selected from the group consisting of poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonists, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, 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, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam2Cys, Pam3Cys, Pam3CSK4 and Aquila QS21 stimulon.

[0624] The pharmaceutical composition of [0617] or [0618], wherein the immunomodulatory agent or adjuvant comprises poly-ICLC.

[0625] The pharmaceutical composition of any one of paragraphs [0616]-[0619], wherein the pharmaceutical composition is a vaccine composition.

[0626] The pharmaceutical composition of any one of paragraphs [0616]-[0620], wherein the pharmaceutical composition is aqueous or liquid.

[0627] The pharmaceutical composition of any one of paragraphs [0616]-[0621], wherein the epitope is present in the pharmaceutical composition in an amount of from 1 ng to 10 mg or from 5 μg to 1.5 mg.

[0628] The pharmaceutical composition of any one of paragraphs [0616]-[0622], further comprising DMSO.

[0629] The pharmaceutical composition of any one of paragraphs [0616]-[0623], wherein the pharmaceutically acceptable excipient comprises water.

[0630] The pharmaceutical composition of any one of paragraphs [0616]-[0624], wherein the pharmaceutical composition comprises a pH modifier present at a concentration of less than 1 mM or greater than 1 mM.

[0631] The pharmaceutical composition of paragraph [0625], wherein the pH modifier is a dicarboxylate or tricarboxylate.

[0632] The pharmaceutical composition according to paragraph [0625], wherein the pH modifier is succinic acid dicarboxylate or disuccinate.

[0633] The pharmaceutical composition of paragraph [0625], wherein the pH modifier is citric acid tricarboxylate or tricitrate.

[0634] The pharmaceutical composition of paragraph [0625], wherein the pH modifier is disodium succinate.

[0635] The pharmaceutical composition of paragraph [0627], wherein the succinic acid dicarboxylate or disuccinate is present in the pharmaceutical composition at a concentration of 0.1 mM - 1 mM.

[0636] The pharmaceutical composition of paragraph [0627], wherein the succinic acid dicarboxylate or disuccinate is present in the pharmaceutical composition at a concentration of 1 mM - 5 mM.

[0637] The pharmaceutical composition of any one of paragraphs [0616]-[0631], wherein the immune response to the epitope is enhanced upon administration to a subject.

[0638] A method of treating a disease or condition comprising administering a therapeutically effective amount of a pharmaceutical composition according to any of paragraphs [0616]-[0632] to a subject in need thereof.

[0639] The method of paragraph [0633], wherein the disease or condition is cancer.

[0640] The method of paragraph [0634], wherein the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, colorectal cancer, uterine cancer, and liver cancer.

[0641] The method of any one of paragraphs [0633]-[0635], wherein the administration comprises intradermal injection, intranasal spray, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, or subcutaneous injection.

[0642] A method of prophylaxis of a subject comprising contacting a cell of the subject with a polypeptide, cell, or pharmaceutical composition of any one of paragraphs [0487]-[0608] or [0616]-[0632].

[0643] A method comprising identifying an epitope expressed by tumor cells of a subject and obtaining a polypeptide comprising the epitope, wherein the polypeptide has a structure of formula (I):

Y _n -B _t -A _r -X _m -A _s -C _u -Z _p

Formula (I),

or a pharmaceutically acceptable salt thereof,

(i) where X _m is an epitope, where each X is independently an amino acid of a contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject,

and where (a) MHC is MHC class I and m is an integer from 8 to 12, or

(b) MHC is MHC class II and m is an integer from 9 to 25;

(ii) where each Y is independently an amino acid, its analogue or derivative, and where:

(A) if the variable r of A _r in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t -A _r -X _m ,

(B) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 0, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m, or

(C) if variable r of A _r in Formula (I) is 1 and variable t of B _t in Formula (I) is 1 or greater, Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t ; and

additionally, where n is an integer from 0 to 1000;

(iii) where each Z is independently an amino acid, its analogue or derivative, and where:

(A) if variable s of A _s in Formula (I) is 0, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m -A _s -C _u ,

(B) if variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 0, Z _p is not encoded by a nucleic acid sequence located immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m, or

(C) if variable s of A _s in formula (I) is 1 and variable u of _Cu in formula (I) is 1 or more, Z _p is not encoded by a nucleic acid sequence immediately downstream of a nucleic acid sequence in the genome of the subject that encodes _Cu ; and

additionally, where p is an integer from 0 to 1000;

and also where

if n is 0, p is an integer between 1 and 1000; and

if p is 0, n is an integer between 1 and 1000;

(iv) where A _r is a linker and r is 0 or 1;

(v) where A _s is a linker and s is 0 or 1;

(vi) where each B is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately upstream of a nucleic acid sequence in the genome of the subject that encodes X _m,

and where t is an integer between 0 and 1000; and

(vii) where each C is independently an amino acid encoded by a nucleic acid sequence in the genome of the subject that is immediately downstream of a nucleic acid sequence in the genome of the subject that encodes X _m,

and where u is an integer between 0 and 1000;

and also where

(a) the polypeptide does not consist of four different epitopes presented by MHC class I;

(b) the polypeptide comprises at least two different polypeptide molecules;

(c) the epitope comprises at least one mutated amino acid; and/or

(d) Y _n and/or Z _p are cleaved from the epitope if the polypeptide is processed by the APC.

[0644] The method of paragraph [0638], wherein identifying comprises selecting a plurality of nucleic acid sequences from a pool of nucleic acid sequences sequenced from tumor cells of the subject that encode a plurality of candidate peptide sequences comprising one or more different mutations not present in a pool of nucleic acid sequences sequenced from non-tumor cells of the subject, wherein the pool of nucleic acid sequences sequenced from tumor cells of the subject and the pool of nucleic acid sequences sequenced from non-tumor cells of the subject are sequenced by whole genome sequencing or whole exome sequencing.

[0645] The method of paragraph [0638] or [0639], wherein identifying further comprises predicting or measuring which candidate peptide sequences from the plurality of candidate peptide sequences form a complex with a protein encoded by an HLA allele of the same subject using an HLA peptide binding assay.

[0646] The method of any one of paragraphs [0638]-[0640], wherein identifying further comprises selecting a plurality of selected tumor-specific peptides or one or more polynucleotides encoding a plurality of selected tumor-specific peptides from candidate peptide sequences based on an HLA peptide binding assay.

[0647] The method of any one of paragraphs [0638]-[0641], further comprising administering the polypeptide to the subject.

[0648] The method of paragraph [0642], wherein the administration comprises intradermal injection, intranasal spray, intramuscular injection, intraperitoneal injection, intravenous injection, oral administration, or subcutaneous injection.

[0649] The method of any one of paragraphs [0638]-[0643], wherein an immune response is elicited in the subject.

[0650] The method of any one of paragraphs [0638]-[0644], wherein the epitope expressed by the tumor cells of the subject is a neoantigen, a tumor-associated antigen, a mutant tumor-associated antigen, and/or wherein the expression of the epitope is higher in the tumor cells of the subject compared to the expression of the epitope in a normal cell of the subject.

--->

LIST OF SEQUENCES

<110> BIONTECH US INC.

<120> NEOANTIGENIC COMPOSITIONS AND THEIR USE

<130> 50401-737.601

<140> PCT/US2020/037019

<141> 2020-06-10

<150> 62/860,493

<151> 2019-06-12

<160> 395

<170> PatentIn version 3.5

<210> 1

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 1

Lys Lys Lys Lys

1

<210> 2

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 2

Arg Arg Arg Arg

1

<210> 3

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 3

Lys Lys Lys Lys Lys

1 5

<210> 4

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 4

Gly Ala Asp Gly Val Gly Lys Ser Ala Leu

1 5 10

<210> 5

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 5

Gly Ala Cys Gly Val Gly Lys Ser Ala Leu

1 5 10

<210> 6

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 6

Gly Ala Val Gly Val Gly Lys Ser Ala Leu

1 5 10

<210> 7

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 7

Gly Ala Asp Gly Val Gly Lys Ser Ala

1 5

<210> 8

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 8

Gly Ala Cys Gly Val Gly Lys Ser Ala

1 5

<210> 9

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 9

Gly Ala Val Gly Val Gly Lys Ser Ala

1 5

<210> 10

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 10

Lys Leu Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 11

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 11

Phe Leu Val Val Val Gly Ala Cys Gly Leu

1 5 10

<210> 12

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 12

Phe Met Val Val Val Gly Ala Cys Gly Ile

1 5 10

<210> 13

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 13

Phe Leu Val Val Val Gly Ala Cys Gly Ile

1 5 10

<210> 14

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 14

Phe Met Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 15

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 15

Phe Leu Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 16

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 16

Met Leu Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 17

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 17

Phe Met Val Val Val Gly Ala Cys Gly Leu

1 5 10

<210> 18

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 18

Tyr Leu Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 19

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 19

Lys Met Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 20

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 20

Tyr Met Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 21

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 21

Met Met Val Val Val Gly Ala Cys Gly Val

1 5 10

<210> 22

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 22

Asp Thr Ala Gly His Glu Glu Tyr

1 5

<210> 23

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 23

Thr Ala Gly His Glu Glu Tyr Ser Ala Met

1 5 10

<210> 24

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 24

Asp Ile Leu Asp Thr Ala Gly His Glu

1 5

<210> 25

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 25

Asp Ile Leu Asp Thr Ala Gly His

1 5

<210> 26

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 26

Ile Leu Asp Thr Ala Gly His Glu Glu

1 5

<210> 27

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 27

Ile Leu Asp Thr Ala Gly His Glu

1 5

<210> 28

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 28

Asp Ile Leu Asp Thr Ala Gly His Glu Glu Tyr

1 5 10

<210> 29

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 29

Asp Thr Ala Gly His Glu Glu Tyr Ser

1 5

<210> 30

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 30

Leu Leu Asp Ile Leu Asp Thr Ala Gly His

1 5 10

<210> 31

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 31

Asp Ile Leu Asp Thr Ala Gly Arg Glu

1 5

<210> 32

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 32

Asp Ile Leu Asp Thr Ala Gly Arg

1 5

<210> 33

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 33

Ile Leu Asp Thr Ala Gly Arg Glu Glu

1 5

<210> 34

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 34

Ile Leu Asp Thr Ala Gly Arg Glu

1 5

<210> 35

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 35

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg

1 5 10

<210> 36

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 36

Thr Ala Gly Arg Glu Glu Tyr Ser Ala Met

1 5 10

<210> 37

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 37

Arg Glu Glu Tyr Ser Ala Met Arg Asp

1 5

<210> 38

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 38

Asp Thr Ala Gly Lys Glu Glu Tyr Ser Ala Met

1 5 10

<210> 39

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 39

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Lys

1 5 10

<210> 40

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 40

Asp Thr Ala Gly Lys Glu Glu Tyr

1 5

<210> 41

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 41

Leu Leu Asp Ile Leu Asp Thr Ala Gly Lys

1 5 10

<210> 42

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 42

Ile Leu Asp Thr Ala Gly Lys Glu

1 5

<210> 43

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 43

Ile Leu Asp Thr Ala Gly Lys Glu Glu

1 5

<210> 44

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 44

Asp Thr Ala Gly Leu Glu Glu Tyr

1 5

<210> 45

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 45

Ile Leu Asp Thr Ala Gly Leu Glu

1 5

<210> 46

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 46

Asp Ile Leu Asp Thr Ala Gly Leu

1 5

<210> 47

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 47

Ile Leu Asp Thr Ala Gly Leu Glu Glu

1 5

<210> 48

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 48

Gly Leu Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr

1 5 10

<210> 49

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 49

Leu Leu Asp Ile Leu Asp Thr Ala Gly Leu Glu

1 5 10

<210> 50

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 50

Leu Asp Ile Leu Asp Thr Ala Gly Leu

1 5

<210> 51

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 51

Asp Ile Leu Asp Thr Ala Gly Leu Glu

1 5

<210> 52

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 52

Asp Ile Leu Asp Thr Ala Gly Leu Glu Glu Tyr

1 5 10

<210> 53

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 53

Ala Gly Val Gly Lys Ser Ala Leu

1 5

<210> 54

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 54

Gly Ala Ala Gly Val Gly Lys Ser Ala Leu

1 5 10

<210> 55

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 55

Ala Ala Gly Val Gly Lys Ser Ala Leu

1 5

<210> 56

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 56

Cys Gly Val Gly Lys Ser Ala Leu

1 5

<210> 57

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 57

Ala Cys Gly Val Gly Lys Ser Ala Leu

1 5

<210> 58

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 58

Asp Gly Val Gly Lys Ser Ala Leu

1 5

<210> 59

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 59

Ala Asp Gly Val Gly Lys Ser Ala Leu

1 5

<210> 60

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 60

Asp Gly Val Gly Lys Ser Ala Leu Thr Ile

1 5 10

<210> 61

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 61

Gly Ala Arg Gly Val Gly Lys Ser Ala

1 5

<210> 62

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 62

Lys Leu Val Val Val Gly Ala Arg Gly Val

1 5 10

<210> 63

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 63

Val Val Val Gly Ala Arg Gly Val

1 5

<210> 64

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 64

Ser Gly Val Gly Lys Ser Ala Leu

1 5

<210> 65

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 65

Val Val Val Gly Ala Ser Gly Val Gly Lys

1 5 10

<210> 66

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 66

Gly Ala Ser Gly Val Gly Lys Ser Ala Leu

1 5 10

<210> 67

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 67

Val Gly Val Gly Lys Ser Ala Leu

1 5

<210> 68

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 68

Val Val Val Gly Ala Gly Cys Val Gly Lys

1 5 10

<210> 69

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 69

Lys Leu Val Val Val Gly Ala Gly Cys

1 5

<210> 70

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 70

Gly Asp Val Gly Lys Ser Ala Leu

1 5

<210> 71

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 71

Asp Val Gly Lys Ser Ala Leu Thr Ile

1 5

<210> 72

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 72

Val Val Val Gly Ala Gly Asp Val Gly Lys

1 5 10

<210> 73

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 73

Thr Ala Gly Lys Glu Glu Tyr Ser Ala Met

1 5 10

<210> 74

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 74

Asp Thr Ala Gly His Glu Glu Tyr Ser Ala Met

1 5 10

<210> 75

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 75

Thr Ala Gly His Glu Glu Tyr Ser Ala

1 5

<210> 76

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 76

Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala Met

1 5 10

<210> 77

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 77

Thr Ala Gly Lys Glu Glu Tyr Ser Ala

1 5

<210> 78

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 78

Ala Ala Gly Val Gly Lys Ser Ala

1 5

<210> 79

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 79

Ala Gly Cys Val Gly Lys Ser Ala Leu

1 5

<210> 80

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 80

Ala Gly Asp Val Gly Lys Ser Ala Leu

1 5

<210> 81

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 81

Ala Gly Lys Glu Glu Tyr Ser Ala Met Arg

1 5 10

<210> 82

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 82

Ala Gly Val Gly Lys Ser Ala Leu Thr Ile

1 5 10

<210> 83

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 83

Ala Arg Gly Val Gly Lys Ser Ala Leu

1 5

<210> 84

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 84

Ala Ser Gly Val Gly Lys Ser Ala

1 5

<210> 85

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 85

Ala Ser Gly Val Gly Lys Ser Ala Leu

1 5

<210> 86

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 86

Ala Val Gly Val Gly Lys Ser Ala

1 5

<210> 87

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 87

Cys Val Gly Lys Ser Ala Leu Thr Ile

1 5

<210> 88

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 88

Asp Ile Leu Asp Thr Ala Gly Lys

1 5

<210> 89

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 89

Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr

1 5 10

<210> 90

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 90

Asp Thr Ala Gly His Glu Glu Tyr Ser Ala Met Arg

1 5 10

<210> 91

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 91

Asp Thr Ala Gly Lys Glu Glu Tyr Ser

1 5

<210> 92

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 92

Asp Thr Ala Gly Lys Glu Glu Tyr Ser Ala Met Arg

1 5 10

<210> 93

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 93

Asp Thr Ala Gly Leu Glu Glu Tyr Ser

1 5

<210> 94

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 94

Asp Thr Ala Gly Leu Glu Glu Tyr Ser Ala

1 5 10

<210> 95

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 95

Asp Thr Ala Gly Leu Glu Glu Tyr Ser Ala Met Arg

1 5 10

<210> 96

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 96

Asp Thr Ala Gly Arg Glu Glu Tyr Ser

1 5

<210> 97

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 97

Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala Met Arg

1 5 10

<210> 98

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 98

Gly Ala Ala Gly Val Gly Lys Ser Ala

1 5

<210> 99

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 99

Gly Ala Asp Gly Val Gly Lys Ser

1 5

<210> 100

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 100

Gly Ala Gly Asp Val Gly Lys Ser Ala

1 5

<210> 101

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 101

Gly Ala Gly Asp Val Gly Lys Ser Ala Leu

1 5 10

<210> 102

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 102

Gly Ala Ser Gly Val Gly Lys Ser Ala

1 5

<210> 103

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 103

Gly Cys Val Gly Lys Ser Ala Leu

1 5

<210> 104

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 104

Gly Cys Val Gly Lys Ser Ala Leu Thr Ile

1 5 10

<210> 105

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 105

Gly His Glu Glu Tyr Ser Ala Met

1 5

<210> 106

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 106

Gly Lys Glu Glu Tyr Ser Ala Met

1 5

<210> 107

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 107

Gly Leu Glu Glu Tyr Ser Ala Met Arg

1 5

<210> 108

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 108

Gly Arg Glu Glu Tyr Ser Ala Met

1 5

<210> 109

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 109

Gly Arg Glu Glu Tyr Ser Ala Met Arg

1 5

<210> 110

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 110

His Glu Glu Tyr Ser Ala Met Arg Asp

1 5

<210> 111

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 111

Lys Glu Glu Tyr Ser Ala Met Arg Asp

1 5

<210> 112

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 112

Lys Leu Val Val Val Gly Ala Ser Gly

1 5

<210> 113

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 113

Leu Asp Ile Leu Asp Thr Ala Gly Arg

1 5

<210> 114

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 114

Leu Glu Glu Tyr Ser Ala Met Arg Asp

1 5

<210> 115

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 115

Leu Val Val Val Gly Ala Arg Gly Val

1 5

<210> 116

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 116

Leu Val Val Val Gly Ala Ser Gly Val

1 5

<210> 117

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 117

Arg Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr

1 5 10

<210> 118

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 118

Arg Gly Val Gly Lys Ser Ala Leu

1 5

<210> 119

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 119

Thr Ala Gly Leu Glu Glu Tyr Ser Ala

1 5

<210> 120

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 120

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Ala

1 5 10

<210> 121

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 121

Val Gly Ala Ala Gly Val Gly Lys Ser Ala

1 5 10

<210> 122

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 122

Val Gly Ala Asp Gly Val Gly Lys

1 5

<210> 123

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 123

Val Gly Ala Ser Gly Val Gly Lys Ser Ala

1 5 10

<210> 124

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 124

Val Gly Val Gly Lys Ser Ala Leu Thr Ile

1 5 10

<210> 125

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 125

Val Val Val Gly Ala Ala Gly Val

1 5

<210> 126

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 126

Val Val Val Gly Ala Val Gly Val

1 5

<210> 127

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 127

Tyr Lys Leu Val Val Val Gly Ala Cys

1 5

<210> 128

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 128

Tyr Lys Leu Val Val Val Gly Ala Asp

1 5

<210> 129

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 129

Tyr Lys Leu Val Val Val Gly Ala Arg

1 5

<210> 130

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 130

Asp Ile Leu Asp Thr Ala Gly Lys Glu

1 5

<210> 131

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 131

Ile Asp Ile Ile Met Lys Ile Arg Asn Ala

1 5 10

<210> 132

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 132

Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe

1 5 10 15

Phe Phe Phe Phe Ile Ile Phe Phe Ile Phe Phe Trp Met Cys

20 25 30

<210> 133

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 133

Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe

1 5 10 15

Phe Phe Phe Phe Phe Phe Phe Phe Ala Ala Phe Trp Phe Trp

20 25 30

<210> 134

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 134

Ile Phe Phe Ile Phe Phe Ile Ile Phe Phe Phe Phe Phe Phe Phe Phe

1 5 10 15

Phe Phe Phe Phe Ile Ile Ile Ile Ile Ile Ile Trp Glu Cys

20 25 30

<210> 135

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 135

Phe Ile Phe Phe Phe Ile Ile Phe Phe Phe Phe Phe Ile Phe Phe Phe

1 5 10 15

Phe Phe Ile Phe Ile Ile Ile Ile Ile Ile Phe Trp Glu Cys

20 25 30

<210> 136

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 136

Thr Glu Tyr Lys Leu Val

1 5

<210> 137

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 137

Trp Gln Ala Gly Ile Leu Ala Arg

1 5

<210> 138

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 138

His Ser Tyr Thr Thr Ala Glu

1 5

<210> 139

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 139

Pro Leu Thr Glu Glu Lys Ile Lys

1 5

<210> 140

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 140

Gly Ala Leu His Phe Lys Pro Gly Ser Arg

1 5 10

<210> 141

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 141

Arg Arg Ala Asn Lys Asp Ala Thr Ala Glu

1 5 10

<210> 142

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 142

Lys Ala Phe Ile Ser His Glu Glu Lys Arg

1 5 10

<210> 143

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 143

Thr Asp Leu Ser Ser Arg Phe Ser Lys Ser

1 5 10

<210> 144

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 144

Phe Asp Leu Gly Gly Gly Thr Phe Asp Val

1 5 10

<210> 145

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 145

Cys Leu Leu Leu His Tyr Ser Val Ser Lys

1 5 10

<210> 146

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 146

Lys Lys Lys Lys Ile Ile Met Lys Ile Arg Asn Ala

1 5 10

<210> 147

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 147

Met Thr Glu Tyr Lys Leu Val Val Val

1 5

<210> 148

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 148

Lys Lys Asn Lys Lys Asp Asp Ile

1 5

<210> 149

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 149

Lys Lys Asn Lys Lys Asp Asp Ile Lys Asp

1 5 10

<210> 150

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 150

Ala Gly Asn Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp

1 5 10 15

Asp Asp Asp Asp Lys Lys Asp Lys Asp Asp Asp Asp Asp Asp

20 25 30

<210> 151

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 151

Ala Gly Asn Lys Lys Lys Lys Lys Lys Lys Asn Asn Asn Asn Asn Asn

1 5 10 15

Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn

20 25 30

<210> 152

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 152

Ala Gly Arg Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp

1 5 10 15

Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp

20 25 30

<210> 153

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 153

Ser Ala Leu Thru Ile

1 5

<210> 154

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 154

Ser Ala Leu Thr Ile Gln Leu

1 5

<210> 155

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 155

Gly Lys Ser Ala Leu Thr Ile Gln Leu

1 5

<210> 156

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 156

Gly Lys Ser Ala Leu Thr Ile

1 5

<210> 157

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 157

Gln Gly Gln Asn Leu Lys Tyr Gln

1 5

<210> 158

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 158

Ile Leu Gly Val Leu Leu Leu Ile

1 5

<210> 159

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 159

Glu Lys Glu Gly Lys Ile Ser Lys

1 5

<210> 160

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 160

Ala Ala Ser Asp Phe Ile Phe Leu Val Thr

1 5 10

<210> 161

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 161

Lys Glu Leu Lys Gln Val Ala Ser Pro Phe

1 5 10

<210> 162

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 162

Lys Lys Lys Leu Ile Asn Glu Lys Lys Glu

1 5 10

<210> 163

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 163

Lys Lys Cys Asp Ile Ser Leu Gln Phe Phe

1 5 10

<210> 164

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 164

Lys Ser Thr Ala Gly Asp Thr His Leu Gly

1 5 10

<210> 165

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 165

Ala Thr Phe Tyr Val Ala Val Thr Val Pro

1 5 10

<210> 166

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 166

Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr Asp Pro

1 5 10 15

Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly

20 25 30

<210> 167

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 167

Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr Asp Pro Thr

1 5 10 15

Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly Glu

20 25 30

<210> 168

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 168

Lys Lys Lys Lys Lys Pro Lys Arg Asp Gly Tyr Met Phe Leu Lys Ala

1 5 10 15

Glu Ser Lys Ile Met Phe Ala Thr

20

<210> 169

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 169

Lys Lys Lys Lys Tyr Met Phe Leu Lys Ala Glu Ser Lys Ile Met Phe

1 5 10 15

Ala Thr Leu Gln Arg Ser Ser

20

<210> 170

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 170

Lys Lys Lys Lys Lys Ala Glu Ser Lys Ile Met Phe Ala Thr Leu Gln

1 5 10 15

Arg Ser Ser Leu Trp Cys Leu

20

<210> 171

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 171

Lys Lys Lys Lys Lys Ile Met Phe Ala Thr Leu Gln Arg Ser Ser Leu

1 5 10 15

Trp Cys Leu Cys Ser Asn His

20

<210> 172

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 172

Lys Lys Lys Lys Met Phe Ala Thr Leu Gln Arg Ser Ser Leu Trp Cys

1 5 10 15

Leu Cys Ser Asn His

20

<210> 173

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 173

Met Leu Thr Gly Pro Pro Ala Arg Val

1 5

<210> 174

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 174

Ser Met Leu Thr Gly Pro Pro Ala Arg Val

1 5 10

<210> 175

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 175

Val Leu Pro Glu Pro His Leu Ala Leu

1 5

<210> 176

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 176

Lys Pro Lys Arg Asp Gly Tyr Met Phe

1 5

<210> 177

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 177

Lys Pro Lys Arg Asp Gly Tyr Met Phe Leu

1 5 10

<210> 178

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 178

Glu Ser Lys Ile Met Phe Ala Thr Leu

1 5

<210> 179

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 179

Lys Arg Asp Gly Tyr Met Phe Leu

1 5

<210> 180

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 180

Pro Ala Val Pro Phe Asp Leu His Phe

1 5

<210> 181

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 181

Ala Glu Ser Lys Ile Met Phe Ala Thr Leu

1 5 10

<210> 182

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 182

Phe Ala Thr Leu Gln Arg Ser Ser Leu

1 5

<210> 183

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 183

Ala Arg Val Pro Ala Val Pro Phe Asp

1 5

<210> 184

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 184

Ile Met Lys Pro Lys Arg Asp Gly Tyr

1 5

<210> 185

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 185

Asp Gly Tyr Met Phe Leu Lys Ala

1 5

<210> 186

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 186

Met Phe Leu Lys Ala Glu Ser Lys Ile Met Phe

1 5 10

<210> 187

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 187

Leu Thr Gly Pro Pro Ala Arg Val

1 5

<210> 188

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 188

Ala Arg Val Pro Ala Val Pro Phe

1 5

<210> 189

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 189

Ser Met Leu Thr Gly Pro Pro Ala Arg

1 5

<210> 190

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 190

Arg Val Pro Ala Val Pro Phe Asp Leu

1 5

<210> 191

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 191

Leu Thr Gly Pro Pro Ala Arg Val Pro

1 5

<210> 192

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 192

Val Val Val Gly Ala Ala Gly Val Gly Lys

1 5 10

<210> 193

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 193

Val Val Val Gly Ala Ala Gly Val Gly

1 5

<210> 194

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 194

Val Val Gly Ala Ala Gly Val Gly Lys

1 5

<210> 195

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 195

Val Val Gly Ala Ala Gly Val Gly

1 5

<210> 196

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 196

Val Gly Ala Ala Gly Val Gly Lys

1 5

<210> 197

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 197

Val Val Val Gly Ala Cys Gly Val Gly Lys

1 5 10

<210> 198

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 198

Val Val Val Gly Ala Cys Gly Val Gly

1 5

<210> 199

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 199

Val Val Val Gly Ala Cys Gly Val

1 5

<210> 200

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 200

Val Val Gly Ala Cys Gly Val Gly Lys

1 5

<210> 201

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 201

Val Val Gly Ala Cys Gly Val Gly

1 5

<210> 202

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 202

Val Gly Ala Cys Gly Val Gly Lys

1 5

<210> 203

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 203

Val Val Val Gly Ala Asp Gly Val Gly Lys

1 5 10

<210> 204

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 204

Val Val Val Gly Ala Asp Gly Val Gly

1 5

<210> 205

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 205

Val Val Val Gly Ala Asp Gly Val

1 5

<210> 206

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 206

Val Val Gly Ala Asp Gly Val Gly Lys

1 5

<210> 207

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 207

Val Val Gly Ala Asp Gly Val Gly

1 5

<210> 208

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 208

Val Val Val Gly Ala Arg Gly Val Gly Lys

1 5 10

<210> 209

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 209

Val Val Val Gly Ala Arg Gly Val Gly

1 5

<210> 210

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 210

Val Val Gly Ala Arg Gly Val Gly Lys

1 5

<210> 211

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 211

Val Val Gly Ala Arg Gly Val Gly

1 5

<210> 212

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 212

Val Gly Ala Arg Gly Val Gly Lys

1 5

<210> 213

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 213

Val Val Val Gly Ala Ser Gly Val Gly

1 5

<210> 214

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 214

Val Val Val Gly Ala Ser Gly Val

1 5

<210> 215

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 215

Val Val Gly Ala Ser Gly Val Gly Lys

1 5

<210> 216

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 216

Val Val Gly Ala Ser Gly Val Gly

1 5

<210> 217

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 217

Val Gly Ala Ser Gly Val Gly Lys

1 5

<210> 218

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 218

Val Val Val Gly Ala Val Gly Val Gly Lys

1 5 10

<210> 219

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 219

Val Val Val Gly Ala Val Gly Val Gly

1 5

<210> 220

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 220

Val Val Gly Ala Val Gly Val Gly Lys

1 5

<210> 221

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 221

Val Val Gly Ala Val Gly Val Gly

1 5

<210> 222

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 222

Val Gly Ala Val Gly Val Gly Lys

1 5

<210> 223

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 223

Lys Lys Lys Lys Lys Lys Lys

1 5

<210> 224

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 224

Lys Lys Lys Lys Lys Lys Lys Lys

1 5

<210> 225

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 225

Lys Thr Glu Tyr

1

<210> 226

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 226

Lys Thr Glu Tyr Lys

1 5

<210> 227

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 227

Lys Thr Glu Tyr Lys Leu

1 5

<210> 228

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 228

Lys Thr Glu Tyr Lys Leu Val

1 5

<210> 229

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 229

Lys Thr Glu Tyr Lys Leu Val Val

1 5

<210> 230

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 230

Lys Thr Glu Tyr Lys Leu Val Val Val

1 5

<210> 231

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 231

LysLysThrGluTyr

1 5

<210> 232

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 232

LysLysThrGluTyrLys

1 5

<210> 233

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 233

LysLysThrGluTyrLysLeu

1 5

<210> 234

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 234

Lys Lys Thr Glu Tyr Lys Leu Val

1 5

<210> 235

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 235

Lys Lys Thr Glu Tyr Lys Leu Val Val

1 5

<210> 236

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 236

Lys Lys Thr Glu Tyr Lys Leu Val Val Val

1 5 10

<210> 237

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 237

LysLysLysThrGluTyr

1 5

<210> 238

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 238

Lys Lys Lys Thr Glu Tyr Lys

1 5

<210> 239

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 239

Lys Lys Lys Thr Glu Tyr Lys Leu

1 5

<210> 240

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 240

Lys Lys Lys Thr Glu Tyr Lys Leu Val

1 5

<210> 241

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 241

Lys Lys Lys Thr Glu Tyr Lys Leu Val Val

1 5 10

<210> 242

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 242

Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val

1 5 10

<210> 243

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 243

Lys Lys Lys Lys Thr Glu Tyr

1 5

<210> 244

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 244

Lys Lys Lys Lys Thr Glu Tyr Lys

1 5

<210> 245

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 245

Lys Lys Lys Lys Thr Glu Tyr Lys Leu

1 5

<210> 246

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 246

Lys Lys Lys Lys Thr Glu Tyr Lys Leu Val

1 5 10

<210> 247

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 247

Lys Lys Lys Lys Thr Glu Tyr Lys Leu Val Val

1 5 10

<210> 248

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 248

Lys Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val

1 5 10

<210> 249

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 249

Thr Glu Tyr Lys

1

<210> 250

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 250

Thr Glu Tyr Lys Leu

1 5

<210> 251

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 251

Thr Glu Tyr Lys Leu Val Val

1 5

<210> 252

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 252

Thr Glu Tyr Lys Leu Val Val Val

1 5

<210> 253

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 253

Ser Ala Leu Thre Ile Lys

1 5

<210> 254

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 254

Ser Ala Leu Thr Ile Gln Leu Lys

1 5

<210> 255

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 255

Gly Lys Ser Ala Leu Thr Ile Gln Leu Lys

1 5 10

<210> 256

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 256

Gly Lys Ser Ala Leu Thr Ile Lys

1 5

<210> 257

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 257

Ser Ala Leu Thre Ile Lys Lys

1 5

<210> 258

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 258

Ser Ala Leu Thr Ile Gln Leu Lys Lys

1 5

<210> 259

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 259

Gly Lys Ser Ala Leu Thr Ile Gln Leu Lys Lys

1 5 10

<210> 260

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 260

Gly Lys Ser Ala Leu Thr Ile Lys Lys

1 5

<210> 261

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 261

Ser Ala Leu Thr Ile Lys Lys Lys

1 5

<210> 262

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 262

Ser Ala Leu Thr Ile Gln Leu Lys Lys Lys

1 5 10

<210> 263

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 263

Gly Lys Ser Ala Leu Thr Ile Gln Leu Lys Lys Lys

1 5 10

<210> 264

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 264

Gly Lys Ser Ala Leu Thr Ile Lys Lys Lys

1 5 10

<210> 265

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 265

Ser Ala Leu Thr Ile Lys Lys Lys Lys

1 5

<210> 266

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 266

Ser Ala Leu Thr Ile Gln Leu Lys Lys Lys Lys

1 5 10

<210> 267

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 267

Gly Lys Ser Ala Leu Thr Ile Gln Leu Lys Lys Lys Lys

1 5 10

<210> 268

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 268

Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 269

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 269

Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 270

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 270

Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 271

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 271

Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 272

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 272

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu

20

<210> 273

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 273

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu

20

<210> 274

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 274

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu

20

<210> 275

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 275

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu

20

<210> 276

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 276

Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val

1 5 10 15

Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 277

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 277

Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val

1 5 10 15

Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 278

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 278

Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val

1 5 10 15

Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 279

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 279

Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val

1 5 10 15

Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 280

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 280

Lys Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly

1 5 10 15

Val Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 281

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 281

Lys Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly

1 5 10 15

Val Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 282

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 282

Lys Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly

1 5 10 15

Val Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 283

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 283

Lys Lys Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly

1 5 10 15

Val Gly Lys Ser Ala Leu Thr Ile Gln Leu

20 25

<210> 284

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 284

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu Lys Lys

20 25

<210> 285

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 285

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu Lys Lys

20 25

<210> 286

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 286

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu Lys Lys

20 25

<210> 287

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 287

Lys Lys Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly

1 5 10 15

Lys Ser Ala Leu Thr Ile Gln Leu Lys Lys

20 25

<210> 288

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 288

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys

20

<210> 289

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 289

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys

20

<210> 290

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 290

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys

20

<210> 291

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 291

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys

20

<210> 292

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 292

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys

20

<210> 293

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 293

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys

20

<210> 294

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 294

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys

20

<210> 295

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 295

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys

20

<210> 296

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 296

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys

20 25

<210> 297

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 297

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys

20 25

<210> 298

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 298

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys

20 25

<210> 299

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 299

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys

20 25

<210> 300

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 300

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys Lys

20 25

<210> 301

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 301

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys Lys

20 25

<210> 302

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 302

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys Lys

20 25

<210> 303

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 303

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thr Ile Gln Leu Lys Lys Lys Lys

20 25

<210> 304

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 304

Ala Leu Asn Ser Glu Ala Leu Ser Val

1 5

<210> 305

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<220>

<221> OTHER_SIGN

<222> (1)..(10)

<223> This region may span 5-10 residues

<220>

<223> See the submitted specification for a detailed description of the replacements and

preferred embodiments

<400> 305

Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

1 5 10

<210> 306

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<220>

<221> OTHER_SIGN

<222> (1)..(7)

<223> This region may span 4-7 residues

<220>

<223> See the submitted specification for a detailed description of the replacements and

preferred embodiments

<400> 306

Arg Arg Arg Arg Arg Arg Arg

1 5

<210> 307

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 307

Arg Arg Arg Arg Arg Arg Arg Arg

1 5

<210> 308

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<220>

<221> OTHER_SIGN

<222> (1)..(7)

<223> This region may span 5-10 residues

<220>

<223> See the submitted specification for a detailed description of the replacements and

preferred embodiments

<400> 308

Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg

1 5 10

<210> 309

<211> 62

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 309

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr

20 25 30

Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly

35 40 45

Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu

50 55 60

<210> 310

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 310

Leu Val Val Val Gly Ala Cys Gly Val

1 5

<210> 311

<211> 62

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 311

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr

20 25 30

Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly

35 40 45

Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu

50 55 60

<210> 312

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 312

Lys Leu Val Val Val Gly Ala Asp Gly Val

1 5 10

<210> 313

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 313

Leu Val Val Val Gly Ala Asp Gly Val

1 5

<210> 314

<211> 62

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 314

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr

20 25 30

Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly

35 40 45

Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu

50 55 60

<210> 315

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 315

Lys Leu Val Val Val Gly Ala Val Gly Val

1 5 10

<210> 316

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 316

Leu Val Val Val Gly Ala Val Gly Val

1 5

<210> 317

<211> 101

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 317

Ala Gly Gly Val Gly Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn

1 5 10 15

His Phe Val Asp Glu Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys

20 25 30

Gln Val Val Ile Asp Gly Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr

35 40 45

Ala Gly His Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Met Arg Thr

50 55 60

Gly Glu Gly Phe Leu Cys Val Phe Ala Ile Asn Asn Thr Lys Ser Phe

65 70 75 80

Glu Asp Ile His His Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp Ser

85 90 95

Glu Asp Val Pro Met

100

<210> 318

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 318

Ile Leu Asp Thr Ala Gly His Glu Glu Tyr

1 5 10

<210> 319

<211> 101

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 319

Ala Gly Gly Val Gly Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn

1 5 10 15

His Phe Val Asp Glu Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys

20 25 30

Gln Val Val Ile Asp Gly Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr

35 40 45

Ala Gly Leu Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Met Arg Thr

50 55 60

Gly Glu Gly Phe Leu Cys Val Phe Ala Ile Asn Asn Thr Lys Ser Phe

65 70 75 80

Glu Asp Ile His His Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp Ser

85 90 95

Glu Asp Val Pro Met

100

<210> 320

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 320

Ile Leu Asp Thr Ala Gly Leu Glu Glu Tyr

1 5 10

<210> 321

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 321

Leu Leu Asp Ile Leu Asp Thr Ala Gly Leu

1 5 10

<210> 322

<211> 101

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 322

Ala Gly Gly Val Gly Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn

1 5 10 15

His Phe Val Asp Glu Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys

20 25 30

Gln Val Val Ile Asp Gly Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr

35 40 45

Ala Gly Lys Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Met Arg Thr

50 55 60

Gly Glu Gly Phe Leu Cys Val Phe Ala Ile Asn Asn Ser Lys Ser Phe

65 70 75 80

Ala Asp Ile Asn Leu Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp Ser

85 90 95

Asp Asp Val Pro Met

100

<210> 323

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 323

Ile Leu Asp Thr Ala Gly Lys Glu Glu Tyr

1 5 10

<210> 324

<211> 101

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 324

Ala Gly Gly Val Gly Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn

1 5 10 15

His Phe Val Asp Glu Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys

20 25 30

Gln Val Val Ile Asp Gly Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr

35 40 45

Ala Gly Arg Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Met Arg Thr

50 55 60

Gly Glu Gly Phe Leu Cys Val Phe Ala Ile Asn Asn Ser Lys Ser Phe

65 70 75 80

Ala Asp Ile Asn Leu Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp Ser

85 90 95

Asp Asp Val Pro Met

100

<210> 325

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 325

Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr

1 5 10

<210> 326

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 326

Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly His Glu Glu Tyr Ser

1 5 10 15

Ala Met Arg Asp Gln Tyr Met

20

<210> 327

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 327

Ala Gly His Glu Glu Tyr Ser Ala Met

1 5

<210> 328

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 328

Leu Asp Thr Ala Gly His Glu Glu Tyr

1 5

<210> 329

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 329

Asp Thr Ala Gly His Glu Glu Tyr Ser Ala

1 5 10

<210> 330

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 330

Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser

1 5 10 15

Ala Met Arg Asp Gln Tyr Met

20

<210> 331

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 331

Asp Thr Ala Gly Arg Glu Glu Tyr

1 5

<210> 332

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 332

Ala Gly Arg Glu Glu Tyr Ser Ala Met

1 5

<210> 333

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 333

Thr Ala Gly Arg Glu Glu Tyr Ser Ala

1 5

<210> 334

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 334

Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg

1 5 10

<210> 335

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 335

Ala Gly Arg Glu Glu Tyr Ser Ala Met Arg

1 5 10

<210> 336

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 336

Leu Asp Thr Ala Gly Arg Glu Glu Tyr

1 5

<210> 337

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 337

Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Lys Glu Glu Tyr Ser

1 5 10 15

Ala Met Arg Asp Gln Tyr Met

20

<210> 338

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 338

Ala Gly Lys Glu Glu Tyr Ser Ala Met

1 5

<210> 339

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 339

Leu Asp Thr Ala Gly Lys Glu Glu Tyr

1 5

<210> 340

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 340

Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Leu Glu Glu Tyr Ser

1 5 10 15

Ala Met Arg Asp Gln Tyr Met

20

<210> 341

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 341

Asp Thr Ala Gly Leu Glu Glu Tyr Ser Ala Met

1 5 10

<210> 342

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 342

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Leu

1 5 10

<210> 343

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 343

Leu Asp Thr Ala Gly Leu Glu Glu Tyr

1 5

<210> 344

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 344

Ala Gly Leu Glu Glu Tyr Ser Ala Met

1 5

<210> 345

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 345

Ala Gly Leu Glu Glu Tyr Ser Ala Met Arg

1 5 10

<210> 346

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 346

Gly Leu Glu Glu Tyr Ser Ala Met

1 5

<210> 347

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 347

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Ala Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 348

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 348

Leu Val Val Val Gly Ala Ala Gly Val

1 5

<210> 349

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 349

Lys Leu Val Val Val Gly Ala Ala Gly Val

1 5 10

<210> 350

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 350

Tyr Lys Leu Val Val Val Gly Ala Ala

1 5

<210> 351

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 351

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 352

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 352

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 353

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 353

Val Gly Ala Asp Gly Val Gly Lys Ser Ala

1 5 10

<210> 354

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 354

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Arg Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 355

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 355

Gly Ala Arg Gly Val Gly Lys Ser Ala Leu

1 5 10

<210> 356

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 356

Arg Gly Val Gly Lys Ser Ala Leu Thr Ile

1 5 10

<210> 357

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 357

Lys Leu Val Val Val Gly Ala Arg

1 5

<210> 358

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 358

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Ser Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 359

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 359

Lys Leu Val Val Val Gly Ala Ser Gly Val

1 5 10

<210> 360

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 360

Ser Gly Val Gly Lys Ser Ala Leu Thr Ile

1 5 10

<210> 361

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 361

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 362

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 362

Ala Val Gly Val Gly Lys Ser Ala Leu

1 5

<210> 363

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 363

Tyr Lys Leu Val Val Val Gly Ala Val

1 5

<210> 364

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 364

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Cys Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 365

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 365

Val Val Gly Ala Gly Cys Val Gly Lys

1 5

<210> 366

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 366

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Asp Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu

20

<210> 367

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 367

Val Val Gly Ala Gly Asp Val Gly Lys

1 5

<210> 368

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 368

Ala Leu Asn Ser Glu Ala Leu Ser Val Val

1 5 10

<210> 369

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 369

Met Ala Leu Asn Ser Glu Ala Leu Ser Val

1 5 10

<210> 370

<211> 71

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 370

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr

20 25 30

Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly

35 40 45

Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Val Val

50 55 60

Gly Ala Asp Gly Val Gly Lys

65 70

<210> 371

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 371

ggcggcagcg gcggcggcgg cagcggcggc 30

<210> 372

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 372

ggcggcagcc tgggcggcgg cggcagcggc 30

<210> 373

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 373

ggcgtcggca cc 12

<210> 374

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 374

cagctgggcc tg 12

<210> 375

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

oligonucleotide

<400> 375

caactgggat tg 12

<210> 376

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

6xHis mark

<400> 376

His His His His His

1 5

<210> 377

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 377

Ala Val Gly Ile Thr Tyr Thr Trp Thr Arg Leu Tyr Ala Ser Val Leu

1 5 10 15

Thr Gly Ser Leu Val Ser Lys Thr Lys Lys

20 25

<210> 378

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 378

Ile Gln Lys Ile Ser Phe Phe Asp Gly Phe Glu Val Gly Phe Asn Phe

1 5 10 15

Arg Thr Leu Gln Pro Asn Gly Leu Leu Phe Tyr Tyr Thr

20 25

<210> 379

<211> 28

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 379

Gly Ile Pro Val His Leu Glu Leu Ala Ser Met Thr Asn Met Glu Leu

1 5 10 15

Met Ser Ser Ile Val His Gln Gln Val Phe Pro Thr

20 25

<210> 380

<211> 28

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 380

Gly Arg Val Leu Glu Leu Phe Arg Ala Ala Gln Leu Ala Asn Asp Val

1 5 10 15

Val Leu Gln Ile Met Glu Leu Cys Gly Ala Thr Arg

20 25

<210> 381

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 381

Lys Ala Arg Asp Glu Thr Ala Ala Leu Leu Asn Ser Ala Val Leu Gly

1 5 10 15

Ala Ala Pro Leu Phe Val Pro Pro Ala Asp

20 25

<210> 382

<211> 27

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 382

Arg Glu Gly Val Glu Leu Cys Pro Gly Asn Lys Tyr Glu Met Arg Arg

1 5 10 15

His Gly Thr Thr His Ser Leu Val Ile His Asp

20 25

<210> 383

<211> 27

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 383

Pro Ser Lys Pro Ser Phe Gln Glu Phe Val Asp Trp Glu Asn Val Ser

1 5 10 15

Pro Glu Leu Asn Ser Thr Asp Gln Pro Phe Leu

20 25

<210> 384

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<220>

<221>MOD_RES

<222> (6)..(6)

<223> Cit

<400> 384

Lys Lys Lys Lys Val Xaa

1 5

<210> 385

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 385

Gln Leu Gly Leu

1

<210> 386

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 386

Gly Val Gly Th

1

<210> 387

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 387

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser

1 5 10 15

Ala Leu Thru Ile

20

<210> 388

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 388

Lys Lys Lys Lys Ile Ile Met Lys Ile Arg Asn Ala Val Val Gly Ala

1 5 10 15

Val Gly Val Gly Lys Lys Lys Asn Lys Lys Asp Asp Ile

20 25

<210> 389

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 389

Val Val Gly Ala Val Gly Val Gly Lys Ser Ala Leu Thr Ile Gln Leu

1 5 10 15

<210> 390

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 390

Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys

1 5 10 15

<210> 391

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 391

Trp Gln Ala Gly Ile Leu Ala Arg Val Val Gly Ala Val Gly Val Gly

1 5 10 15

Lys Gln Gly Gln Asn Leu Lys Tyr Gln

20 25

<210> 392

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 392

His Ser Tyr Thr Thr Ala Glu Val Val Gly Ala Val Gly Val Gly Lys

1 5 10 15

Ile Leu Gly Val Leu Leu Leu Ile

20

<210> 393

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 393

Pro Leu Thr Glu Glu Lys Ile Lys Val Val Gly Ala Val Gly Val Gly

1 5 10 15

Lys Glu Lys Glu Gly Lys Ile Ser Lys

20 25

<210> 394

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 394

Gln Leu Gly Leu Lys

1 5

<210> 395

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 395

Lys Gln Leu Gly Leu

1 5

<---

Claims (36)

1. A polypeptide for obtaining antigen-specific T cells, comprising an epitope, wherein said epitope is presented by MHC class I or MHC class II of an antigen-presenting cell (APC), having a structure of Formula (I): Y _n -B _t -A _r -X _m -A _s -C _u -Z _p Formula (I), or a pharmaceutically acceptable salt thereof, where X _m is the specified epitope, where each X independently is an amino acid of a contiguous amino acid sequence encoded by a nucleic acid sequence in the genome of the subject, and where X _m is a mutant RAS protein comprising a mutation in G12, wherein X _m is selected from the group consisting of VVGA V GVGK (SEQ ID NO: 220), VVGA D GVGK (SEQ ID NO: 206), VVGA R GVGK (SEQ ID NO: 210) and VVGA C GVGK (SEQ ID NO: 200), wherein the underlined amino acid residue is a mutant amino acid at G12; where each Y independently represents an amino acid, and where: Y _n is not encoded by a nucleic acid sequence immediately upstream of a nucleic acid sequence in the subject's genome that encodes B _t -A _r -X _m , wherein n is additionally equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; where each Z independently represents an amino acid, and where: Z _p is not encoded by a nucleic acid sequence located immediately downstream of the nucleic acid sequence in the subject's genome that encodes X _m -A _s -C _u , and p is 0; where A _r is a linker and r is 0; where A _s is a linker and s is 0; where each B independently represents an amino acid encoded by a nucleic acid sequence in the subject's genome that is immediately upstream of a nucleic acid sequence in the subject's genome that encodes X _m , and where t is 1, 2, 3, 4, or 5, or more; and where each C independently represents an amino acid encoded by a nucleic acid sequence in the subject's genome that is immediately downstream of a nucleic acid sequence in the subject's genome that encodes X _m , and where u is 1, 2, 3, 4, or 5, or more; and additionally, (a) the polypeptide does not consist of four distinct epitopes presented by MHC class I; and/or (b) Y _n and/or Z _p is cleaved from the specified epitope when the polypeptide is processed by the APC; wherein the subject is a human, and wherein Y _n -B _t -A _r and/or A _s -C _u -Z _p increases the solubility of the polypeptide compared to a corresponding peptide that does not contain Y _n -B _t -A _r and/or A _s -C _u -Z _p. 2. The polypeptide of claim 1, wherein (a) the polypeptide is cleaved at a higher rate when n is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, compared to the cleavage of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m ; (b) the presentation of the APC epitope is enhanced when n is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, compared to the presentation of the epitope of a corresponding polypeptide of the same length that comprises X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m ; and/or (c) the immunogenicity is increased when n is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, compared to the immunogenicity of a corresponding polypeptide of the same length that contains X _m and at least one additional amino acid encoded by a nucleic acid sequence immediately upstream of the nucleic acid sequence in the subject's genome that encodes X _m . 3. The polypeptide of any one of claims 1 or 2, wherein Y _n comprises poly-Lys (polyK), wherein polyK comprises at least two, three or four consecutive lysine residues, respectively; optionally wherein poly-K comprises poly-L-Lys. 4. The polypeptide of any one of claims 1 to 3, wherein said epitope binds to a protein encoded by an HLA allele, wherein the HLA allele is selected from the group consisting of the HLA-A02:01 allele, the HLA-A03:01 allele, the HLA-A11:01 allele, the HLA-A03:02 allele, the HLA-A30:01 allele, the HLA-A31:01 allele, the HLA-A33:01 allele, the HLA-A33:03 allele, the HLA-A68:01 allele, the HLA-A74:01 allele and/or the HLA-C08:02 allele and any combination thereof. 5. The polypeptide of any one of claims 1 to 4, wherein Y _n is poly-Lys (polyK), which has three or four consecutive lysine residues. 6. The polypeptide of any one of claims 1-5, wherein X _m is VVGAVGVGK (SEQ ID NO: 220). 7. The polypeptide of any one of claims 1-5, wherein X _m is VVGADGVGK (SEQ ID NO: 206). 8. The polypeptide according to any one of paragraphs 1-5, where X_mis VVGARGVGK (SEQ ID NO: 210). 9. The polypeptide of any one of claims 1-5, wherein X _m is VVGACGVGK (SEQ ID NO: 200). 10. The polypeptide according to any one of claims 1-5, where the polypeptide contains the amino acid sequence KTEYKLVVVGA V GVGKSALTIQL, KTEYKLVVVGA D GVGKSALTIQL, KTEYKLVVVGA R GVGKSALTIQL, KTEYKLVVVGA C GVGKSALTIQL, KKTEYKLVVVGA V GVGKSALTIQL, KKTEYKLVVVGA D GVGKSALTIQL, KKTEYKLVVVGA R GVGKSALTIQL, KKTEYKLVVVGA C GVGKSALTIQL, KKKTEYKLVVVGA V GVGKSALTIQL, KKKTEYKLVVVGA D GVGKSALTIQL, KKKTEYKLVVVGA R GVGKSALTIQL, KKKTEYKLVVVGA C GVGKSALTIQL, KKKKTEYKLVVVGA V GVGKSALTIQL, KKKKTEYKLVVVGA D GVGKSALTIQL, KKKKTEYKLVVVGA R GVGKSALTIQL or KKKKTEYKLVVVGA C GVGKSALTIQL. 11. Polypeptide according to any one of paragraphs. 1-5, where the polypeptide contains the amino acid sequence KKKTEYKLVVVGA D GVGKSALTIQL, KKKTEYKLVVVGA R GVGKSALTIQL, KKKKTEYKLVVVGA V GVGKSALTIQL or KKKKTEYKLVVVGA C GVGKSALTIQL. 12. A polynucleotide for producing antigen-specific T cells, comprising a sequence encoding a polypeptide according to any one of claims 1-11; optionally, wherein the polynucleotide is mRNA. 13. A method for producing antigen-specific T cells, comprising stimulating T cells with antigen-presenting cells containing a polypeptide according to any one of claims 1-11 or a polynucleotide according to claim 12, wherein the method is carried out ex vivo.

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