Recurrent neoantigen-related peptides and related compositions and methods

Abstract

The current disclosure provides methods and compositions for treating and vaccinating individuals against cancer. Accordingly, aspects of the disclosure relate to an isolated peptide comprising at least 70% sequence identity to a peptide of one of SEQ ID NOS:1-192, and combinations thereof. In some embodiments, the peptide comprises at least 6 contiguous amino acids of a peptide of one of SEQ ID NOS:1-192. Further aspects relate to pharmaceutical compositions comprising the isolated peptide, nucleic acids encoding the peptide, and expression vectors and host cells comprising the nucleic acids of the disclosure. Also provided is an in vitro isolated dendritic cell comprising a peptide, nucleic acid, or expression vector of the disclosure.

Description

RECURRENT NEOANTIGEN-RELATED PEPTIDES AND RELATEDCOMPOSITIONS AND METHODSSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under grant numbers R01CA260761, R01CA257375, and U01CA233056 awarded by the US National Institutes of Health / National Cancer Institute. The government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Number 63 / 602,930, filed November 27, 2023, the entire contents of which are incorporated by reference.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on November 25, 2024, is named MDAC_P1376WO_Sequence_Listing.xml and is 167,670 bytes in size.FIELD

[0004] This invention relates to the field of treatment and diagnosis of cancer.BACKGROUND

[0005] Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC), and represents 2-4% of total CRC and at least 1 million carriers in the United States. LS patients carry heterozygous germline mutations in one of the four DNA mismatch repair (MMR) genes, conferring 50-80% lifetime risk of developing CRC, 40-60% risk of endometrial cancer, and heightened risk of multiple other tumor types. Improved treatments and diagnostics are needed for the significant population of individuals affected by LS and related cancers. Provided herein are solutions that address these challenges and other needs.BRIEF SUMMARY

[0006] The current disclosure fulfills a need in the art by providing methods and compositions for treating and vaccinating individuals against cancer through the use of newly identified immunogenic peptides (referred to interchangeably herein as neoantigens, neoAgs,frameshift neoAgs, FS-neoAgs, frameshift protein neoAgs, and FSP-neoAgs). In some aspects, the peptides are based on neoantigens identified subjects with LS.

[0007] In some aspects, provided herein is an isolated peptide comprising at least 70% sequence identity to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide comprises at least 6 contiguous amino acids of a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide is 25 amino acids or fewer in length. In some embodiments, the peptide is 8 amino acids or greater in length. In some embodiments, the peptide is 8 to 11 amino acids in length. In some embodiments, the peptide is 12 to 18 amino acids in length. In some embodiments, the peptide is immunogenic. In some embodiments, the peptide is modified. In some embodiments, the modification comprises conjugation to a molecule. In some embodiments, the molecule comprises an antibody, a lipid, an adjuvant, a cell penetrating peptide, or a detection moiety. In some embodiments, the peptide has at least 90% sequence identity to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide has 1, 2 or 3 substitutions relative to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide comprises 100% sequence identity to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the amino acid sequence of the peptide is identical to a sequence encoded by a mutated gene sequence in a precancer or cancer in at least 0.01%, at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 20%, or more, of subjects having Lynch Syndrome and a precancer or cancer. In some embodiments, the mutated gene sequence is the result of a frameshift mutation.

[0008] In some aspects, provided herein is a molecular complex comprising any peptide provided herein and an MHC polypeptide.

[0009] In some aspects, provided herein is a combination of peptides comprising two or more of any of the peptides provided herein. In some embodiments, the combination of peptides comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more than 200, of any of the peptides provided herein. In some embodiments, the combination of peptides comprises between about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 120, 120 to 140, 140 to 160, 160 to 180, 180 to 200, or 200 to 300, of any of the peptides provided herein. In some embodiments, the combination of peptides comprises at or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 of any of the peptides provided herein. In some embodiments, the combination of peptides comprises between 25 to 40 of any of the peptides provided herein. In some embodiments, the combination of peptides comprises at or about 30, 31, 32, 33, 34, 35, 36, 37,38, 39, or 40 of any of the peptides provided herein. In some embodiments, the combination of peptides comprises at or about 35 of any of the peptides provided herein. In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of precancers or cancers in subjects having Lynch Syndrome comprise a mutated gene sequence encoding an amino acid sequence that is identical to the sequence of one of the peptides in the combination of peptides. In some embodiments, in at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of subjects having Lynch Syndrome and a precancer or cancer, the precancer or cancer comprises a mutated gene sequence encoding an amino acid sequence that is identical to the sequence of one of the peptides in the combination of peptides. In some embodiments, for at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of subjects having Lynch Syndrome and a precancer or cancer, the combination of peptides comprises at least one peptide that is identical in sequence to a sequence encoded by a mutated gene sequence in the precancer or cancer. In some embodiments, the mutated gene sequence is the result of a frameshift mutation.

[0010] In some aspects, provided herein is one or more nucleic acids encoding any peptide or combination of peptides provided herein. In some embodiments, the nucleic acid(s) comprise DNA and / or RNA. In some embodiments, the nucleic acid(s) comprise mRNA.

[0011] In some aspects, provided herein is an expression vector comprising any nucleic acid(s) provided herein.

[0012] In some aspects, provided herein is a vector for intracellular delivery, comprising any peptide, molecular complex, combination of peptides, nucleic acid(s), or expression vector provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non- viral vector.

[0013] In some aspects, provided herein is a composition comprising any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, or vector for intracellular delivery provided herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutical carrier. In some embodiments, the composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. In some embodiments, the peptide, combination of peptides, nucleic acid(s), or expression vector, is comprised in a liposome, lipid-containing nanoparticle, or in a lipid-based carrier. In some embodiments, the composition is formulated for injection or inhalation as anasal spray. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition stimulates an immune response when administered to a subject. In some embodiments, the composition prevents colorectal cancer when administered to a subject having Lynch Syndrome.

[0014] In some aspects, provided herein is a method of stimulating an immune response in a subject, the method comprising administering to the subject any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein. In some aspects, provided herein is a method of treating or preventing cancer or precancer in a subject, the method comprising administering to the subject any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein. In some aspects, provided herein is a method of preventing cancer in a subject, the method comprising administering to the subject any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein. In some embodiments, the subject has been determined to have one or more precancers or cancers. In some embodiments, the subject is determined to be at risk of developing cancer. In some embodiments, the subject is determined to have a genetic predisposition for developing colorectal cancer. In some embodiments, the subject has Lynch Syndrome. In some embodiments, the cancer or precancer is colorectal. In some embodiments, the cancer or precancer is determined to comprise a mutated gene sequence encoding an amino acid sequence comprising the sequence set forth in any of SEQ ID NOS: 1-192. In some embodiments, the cancer or precancer is determined to comprise a mutated gene sequence encoding an amino acid sequence that is identical to the amino acid sequence of any peptide provided herein. In some embodiments, the mutated gene sequence is the result of a frameshift mutation.

[0015] In some aspects, provided herein is a vaccine for preventing colorectal cancer in subjects with Lynch Syndrome, comprising any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein. In some aspects, provided herein is a vaccine for preventing colorectal cancer in subjects with Lynch Syndrome, comprising one or more mRNA molecules encoding the amino acid sequences of the peptides of any of the combination of peptides provided herein.

[0016] Accordingly, aspects of the disclosure relate to an isolated peptide comprising or comprising at least 70% sequence identity to a peptide of one of SEQ ID NOS: 1-192. Aspects of the disclosure relate to an isolated peptide comprising 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% (or any derivable range therein) sequence identity to a peptide of one of SEQ ID NOS: 1-192. In some aspects, the peptide comprises or comprises at least 6 contiguous amino acids of a peptide of one of SEQ ID NOS: 1-192. Further aspects relate to pharmaceutical compositions comprising the isolated peptide, nucleic acids encoding the peptide, and expression vectors and / or host cells comprising the nucleic acids of the disclosure. Also provided is an in vitro-isolated dendritic cell comprising a peptide, nucleic acid, or expression vector of the disclosure.

[0017] Further aspects relate to a method of making a cell comprising transferring a nucleic acid or expression vector of the disclosure into a cell, such as a host cell. The method may further comprise isolating the expressed peptide or polypeptide. Other aspects of the disclosure relate to a method of producing cancer- specific immune effector cells comprising: (a) obtaining a starting population of immune effector cells; and (b) contacting the starting population of immune effector cells with a peptide of the disclosure, thereby generating peptide- specific immune effector cells.

[0018] The disclosure also describes peptide- specific engineered T cells produced according to the methods of the disclosure and pharmaceutical compositions comprising the engineered T cells. Further aspects relate to a method of treating or preventing cancer in a subject, the method comprising administering an effective amount of a peptide, pharmaceutical composition, nucleic acid, dendritic cell, or peptide- specific T cell of the disclosure. Yet further aspects relate to a method of cloning a peptide- specific T cell receptor (TCR), the method comprising (a) obtaining a starting population of immune effector cells; (b) contacting the starting population of immune effector cells with the peptide of the disclosure, thereby generating peptide- specific immune effector cells; (c) purifying immune effector cells specific to the peptide, and (d) isolating a TCR sequence from the purified immune effector cells. Also provide is a method for prognosing a patient or for detecting T cell responses in a patient, the method comprising: contacting a biological sample from the patient with a peptide of the disclosure.

[0019] Aspects of the disclosure also provide for a composition comprising at least one MHC polypeptide and a peptide of the disclosure and peptide- specific binding molecule that bind to a peptide of the disclosure or that bind to a peptide-MHC complex. Exemplary binding molecules include antibodies, TCR mimic antibodies, scFvs, nanobodies, camelids, aptamers, and DARPINs. Related methods provide for a method comprising contacting a compositioncomprising at least one MHC polypeptide and a peptide of the disclosure with a composition comprising T cells and detecting T cells with bound peptide and / or MHC polypeptide by detecting a detection tag. Further aspects relate to kits comprising a peptide, nucleic acid, expression vector, or composition of the disclosure.

[0020] In some aspects, the peptide is 13 amino acids in length or shorter. In some aspects, the peptide is 9 amino acids. The peptide may comprise, may be at least, may be at most, or may consist of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (or any range derivable therein). The peptide may consist of 9 amino acids or the peptide may consist of 15 amino acids.

[0021] In some embodiments, the peptide is 8 amino acids in length. In some embodiments, the peptide is 9 amino acids in length. In some embodiments, the peptide is 10 amino acids in length. In some embodiments, the peptide is 11 amino acids in length. In some embodiments, the peptide is 12 amino acids in length. In some embodiments, the peptide is 13 amino acids in length. In some embodiments, the peptide is 14 amino acids in length. In some embodiments, the peptide is 15 amino acids in length. In some embodiments, the peptide is 16 amino acids in length. In some embodiments, the peptide is 17 amino acids in length. In some embodiments, the peptide is 18 amino acids in length.

[0022] The peptide may be further described as being immunogenic. The term immunogenic refers to the production of an immune response, such as a protective immune response. The peptide may be modified. The modification may comprise conjugation to a molecule. The molecule may be an antibody, a lipid, an adjuvant, or a detection moiety (tag). In some aspects, the peptide comprises 100% sequence identity to a peptide of one of SEQ ID NOS: 1-192. Peptides of the disclosure also include those that have at least 90% sequence identity to a peptide of one of SEQ ID NOS: 1-192. The peptides of the disclosure may have 1, 2, or 3 substitutions relative to a peptide of one of SEQ ID NOS: 1-192. In some aspects, the peptide has at least or at most 1, 2, 3, 4, or 5 substitutions relative to a peptide of one of SEQ ID NOS: 1-192.

[0023] The pharmaceutical compositions of the disclosure may be formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. The peptides of the disclosure may be comprised in a liposome, lipid- containing nanoparticle, or in a lipid-based carrier. Pharmaceutical preparations may be formulated for injection or inhalation as a nasal spray. The compositions of the disclosure may be formulated as a vaccine. In some aspects, the composition may further comprise an adjuvant.

[0024] The dendritic cells of the disclosure may further be defined as being or as comprising mature dendritic cells. The cell may be a cell with an HLA-A type. The cell may also be a HLA A, HLA-B, or HLA-C. In some aspects, the cell is an HLA-A3 or HLA-A11 type. In some aspects, the cell is an HLA-A01, HLA-A02, HLA-A24, HLA-B07, HLA-B08, HLA-B 15, or HLA B40. The methods may further comprise isolating the expressed peptide or polypeptide. The T cell may comprise a CD8+ T cell. The cell may be a T cell is a CD4+ T cell, a Thl, Th2, Thl7, Th9, or Tfh T cell, a cytotoxic T cell, a memory T cell, a central memory T cell, or an effector memory T cell.

[0025] In methods of the disclosure, contacting may be further defined as co-culturing the starting population of immune effector cells with antigen presenting cells (APCs), artificial antigen presenting cells (aAPCs), or an artificial antigen presenting surface (aAPSs); wherein the APCs, aAPCs, or the aAPSs present the peptide on their surface. The APCs may be, for example, dendritic cells.

[0026] The immune effector cells may be T cells, peripheral blood lymphocytes, natural killer (NK) cells, invariant NK cells, or NKT cells. The immune effector cells may be ones that have been differentiated from mesenchymal stem cell (MSC) or induced pluripotent stem (iPS) cells. The T cell aspects include T cells that are further defined as CD8+ T cells, CD4+ T cells, or y5 T cells. The T cells may be defined as being cytotoxic T lymphocytes (CTLs).

[0027] The subject in the methods of the disclosure may be a human subject. In some embodiments, the subject has Lynch Syndrome and / or cancer. The subject may also be a laboratory animal, a mouse, rat, pig, horse, rabbit, or guinea pig. Methods may further comprise administration of at least a second therapeutic agent. The second therapeutic agent may be an anti-cancer agent. Treating, as defined in the methods of the disclosure, may comprise one or more of reducing tumor size; increasing the overall survival rate; reducing the risk of recurrence of the cancer; reducing the risk of progression; and / or increasing the chance of progression-free survival, relapse-free survival, and / or recurrence-free survival.

[0028] The composition of the disclosure may comprise or further comprise a MHC polypeptide and a peptide of the disclosure and wherein the MHC polypeptide and / or peptide is conjugated to a detection tag. As such, suitable detection tags include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The tag may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and / or other property. Inluminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent tags that produce signals include, but are not limited to bioluminescence and chemiluminescence. Examples of suitable fluorescent tags include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6. sup. th ed.). Detection tags also include streptavidin or its binding partner, biotin.

[0029] The MHC polypeptide and peptide may be operatively linked. The term “operatively linked” refers to a situation where two components are combined or capable of combining to form a complex. For example, the components may be covalently attached and / or on the same polypeptide, such as in a fusion protein or the components may have a certain degree of binding affinity for each other, such as a binding affinity that occurs through van der Waals forces. Accordingly, aspects of the disclosure relate to wherein the MHC polypeptide and peptide are operatively linked through a peptide bond. The MHC polypeptide and peptide may also be operatively linked through van der Waals forces. The peptide-MHC may be operatively linked to form a peptide-MHC (pMHC) complex. In some aspects, at least two pMHC complexes are operatively linked together. Other aspects include, include at least, or include at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes operatively linked to each other. In some aspects, at least two MHC polypeptides are linked to one peptide. In other aspects, the average ratio of MHC polypeptides to peptides is 1:1 to 4:1. In some aspects, the ratio or average ratio is at least, at most, or about 1, 2, 3, 4, 5, or 6 to about 1, 2, 3, 4, 5, or 6 (or any derivable range therein).

[0030] In some of the aspects of the disclosure, the peptide is complexed with MHC. In some embodiments, the MHC is a class I MHC (MHC-I). In some embodiments, the MHC is a class II MHC (MHC-II). In some aspects, the MHC comprises HLA-A type. The MHC may be further defined as HLA-A3 or HLA Al l type. The peptides may be loaded onto dendritic cells, lymphoblastoid cells, peripheral blood mononuclear cells (PBMCs), artificial antigen presentation cells (aAPC) or artificial antigen presenting surfaces. The artificial antigen presenting surface may comprise a MHC polypeptide conjugated or linked to a surface. Exemplary surfaces include a bead, microplate, glass slide, or cell culture plate.

[0031] Method of the disclosure may further comprise counting the number of T cells bound with peptide and / or MHC. The composition comprising T cells may be isolated from a patient having or suspected of having cancer. The cancer may comprise stage 0, I, II, III, or IV cancer. In some aspects, the cancer excludes stage 0, 1, II, III, or IV cancer. The cancer may be colorectal cancer. The colorectal cancer may comprise comprises mismatch repair deficient colorectal cancer (MMR-d) and / or micro satellite instability (MSI) positive colorectal cancer. The subject being diagnosed or treated may be treated for stage I or stage II cancer. The subject may be one that has been determined to have mismatch repair deficient colorectal cancer (MMR-d) and / or microsatellite instability (MSI) positive colorectal cancer. The cancer may comprise a cancer-specific peptide, such as the peptide of one of SEQ ID NOS: 1-192 or a peptide of the disclosure. The subject may be a subject that has been diagnosed and / or determined to have a cancer. The subject or patient may also be one that has been characterized as having a cancer with a cancer-specific peptide, such as a peptide of the disclosure or a peptide of one of SEQ ID NOS: 1-192. The methods of the disclosure may comprise or further comprise sorting the number of T cells bound with peptide and / or MHC. Methods of the disclosure may also comprise or further comprise sequencing one or more TCR genes from T cells bound with peptide and / or MHC. The methods may comprise or further comprise sequencing the TCR alpha and / or beta gene(s) from a TCR, such as a TCR that binds to a peptide of the disclosure. Methods may also comprise or further comprise grouping of lymphocyte interactions by paratope hotspots (GLIPH) analysis, for example as described in Glanville et al., Nature. 2017 Jul 6; 547(7661): 94 98, which is herein incorporated by reference.

[0032] The compositions of the disclosure may be serum-free, mycoplasma-free, endotoxin free, and / or sterile. The methods may further comprise culturing cells of the disclosure in media, incubating the cells at conditions that allow for the division of the cell, screening the cells, and / or freezing the cells. The methods may comprise or further comprise isolating the expressed peptide or polypeptide from a cell of the disclosure.

[0033] Methods of the disclosure may comprise or further comprise screening the dendritic cell for one or more cellular properties. The methods may comprise or further comprise contacting the cell with one or more cytokines or growth factors. The one or more cytokines or growth factors may comprise GM-CSF. The cellular property may comprise cell surface expression of one or more of CD86, HLA, and CD 14. The dendritic cell may be derived from a CD34+ hematopoietic stem or progenitor cell.

[0034] The contacting in the methods of the disclosure may be further defined as coculturing the starting population of immune effector cells with antigen presenting cells (APCs), wherein the APCs present the peptide on their surface. The APCs may be further defined as dendritic cells. The dendritic cell may be derived from a peripheral blood monocyte (PBMC). The dendritic cells may be isolated from PBMCs. The dendritic cells may also be cells in which the DCs are derived from are isolated by leukaphereses.

[0035] Peptide-MHC (pMHC) complexes of the disclosure may be made by contacting a peptide of the disclosure with a MHC complex. The peptide may be expressed in the cell and bind to endogenous MHC complex to form a pMHC. In some aspects, peptide exchange is used to make the pMHC complex. For example, cleavable peptides, such as pho tocleav able peptides may be designed that bind to and stabilize the MHC. Cleavage of the peptide (e.g. by irradiation for photocleavable peptides) dissociates the peptide from the HLA complex and results in an empty HLA complex that disintegrates rapidly, unless UV exposure is performed in the presence of a “rescue peptide.” Thus, the peptides of the disclosure may be used as “rescue peptides” in the peptide exchange procedure. Also described herein are pMHC complexes comprising a peptide of the disclosure. The pMHC complex may be operatively linked to a solid support or may be attached to a detectable moiety, such as a fluorescent molecule, a radioisotope, or an antibody. Peptide-MHC multimeric complexes may include, may include at least or may include at most 1, 2, 3, 4, 5, or 6 peptide-MHC molecules operatively linked together. The linkage may be covalent, such as through a peptide bond, or non-covalent. The pMHC molecules may be bound to a biotin molecule. Such pMHC molecules may be multimerized through binding to a streptavidin molecule. pMHC multermers may be used to detect antigen- specific T cells or TCR molecules that are in a composition or in a tissue. The multimers may be used to detect peptide- specific T cells in situ or in a biopsy sample. Multimers may be bound to a solid support or deposited on a solid support, such as an array or slide. Cells may then be added to the solid support (e.g. slide), and detection of the binding between the pMHC multimer and cell may be conducted. Accordingly, the pMHC molecules and multimers of the disclosure may be used to detect and diagnose cancer in subjects or to determine immune responses in individuals with cancer.

[0036] In the methods of the disclosure, obtaining may comprise isolating the starting population of immune effector cells from peripheral blood mononuclear cells (PBMCs). The starting population of immune effector cells may be obtained from a subject. The subject may be one that has a cancer, such as a cancer with a cancer- specific peptide. The subject may be one that has been determined to have a cancer that expresses a peptide of the disclosure. Themethods of the disclosure may comprise or further comprise introducing the peptides or a nucleic acid encoding the peptide into the dendritic cells prior to the co-culturing. The introduction of the peptide may be done by transfecting or infecting dendritic cells with a nucleic acid encoding the peptide or by incubating the peptide with the dendritic cells. The peptide or nucleic acids encoding the peptide may be introduced by electroporation. Other methods of transfer of nucleic acids are known in the art, such as lipofection, calcium phosphate transfection, transfection with DEAE-dextran, microinjection, and virus-mediated transduction. The peptide or nucleic acids encoding the peptide may be introduced by adding the peptide or nucleic acid encoding the peptide to the dendritic cell culture media. The immune effector cells may be co-cultured with a second population of dendritic cells into which the peptide or the nucleic acid encoding the peptide has been introduced. In the methods of the disclosure, a population of CD4-positive or CD8-positive and peptide MHC tetramer-positive T cells may be purified from the immune effector cells following the co-culturing. The population of CD4-positive or CD8-positive and peptide MHC tetramer-positive T cells may be purified by fluorescence activated cell sorting (FACS). A clonal population of peptidespecific immune effector cells may be generated by limiting or serial dilution followed by expansion of individual clones by a rapid expansion protocol.

[0037] In the methods of the disclosure, purifying may comprise or further comprise generation of a clonal population of peptide-specific immune effector cells by limiting or serial dilution of sorted cells followed by expansion of individual clones by a rapid expansion protocol. Methods of the disclosure may comprise or further comprise cloning of a T cell receptor (TCR) from the clonal population of peptide- specific immune effector cells. The term isolating in the methods of the disclosure may be defined as or may comprise cloning of a T cell receptor (TCR) from the clonal population of peptide- specific immune effector cells. Cloning of the TCR may comprise cloning of a TCR alpha and a beta chain. The TCR may be cloned using a 5 ’-Rapid amplification of cDNA ends (RACE) method. The TCR alpha and beta chains may be cloned using a 5 ’-Rapid amplification of cDNA ends (RACE) method. The cloned TCR may be subcloned into an expression vector. The expression vector may comprise a linker domain between the TCR alpha sequence and TCR beta sequence. The expression vector may be a retroviral or lentiviral vector. The vector may also be an expression vector described herein. The linker domain may comprise a sequence encoding one or more peptide cleavage sites. The one or more cleavage sites may be a Furin cleavage site and / or a P2A cleavage site. The TCR alpha sequence and TCR beta sequence may be linked by an IRES sequence.

[0038] A host cell of the disclosure may be transduced with an expression vector to generate an engineered cell that expresses the TCR alpha and / or beta chains. The host cell may be an immune cell. The immune cell may be a T cell and the engineered cell may be referred to as an engineered T cell. The T cell may be type of T cell described herein, such as a CD8+ T cell, CD4+ T cell, or y5 T cell. The starting population of immune effector cells may be obtained from a subject having a cancer or a peptide- specific cancer and the host cell is allogeneic or autologous to the subject. The peptide-specific T cells may be autologous or allogeneic. In the methods of the disclosure, a population of CD4-positive or CD8-positive and peptide MHC tetramer-positive engineered T cells may be purified from the transduced host cells. A clonal population of peptide specific engineered T cells may be generated by limiting or serial dilution followed by expansion of individual clones by a rapid expansion protocol. Purifying in the methods of the disclosure may be defined as purifying a population of CD4-positive or CD8-positive and peptide MHC tetramer-positive T cells from the immune effector cells following the co-culturing.

[0039] The peptide of the disclosure may be linked to a solid support. The peptide may be conjugated to the solid support or may be bound to an antibody that is conjugated to the solid support. The solid support may comprise a microplate, a bead, a glass surface, a slide, or a cell culture dish. The solid support may comprise a nanofluidic chip. In the methods of the disclosure, detecting T cell responses may comprise or further comprise detecting the binding of the peptide to the T cell or TCR. In the methods of the disclosure, detecting T cell responses may comprise or further comprise an ELISA, ELISpot, or a tetramer assay.

[0040] Kits of the disclosure may comprise a peptide of the disclosure in a container. The peptide may be comprised in a pharmaceutical preparation. The pharmaceutical preparation may be formulated for parenteral administration or inhalation. In some aspects, the peptide is comprised in a cell culture media.

[0041] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0042] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0043] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y orz.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0044] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0045] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0046] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0047] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be betterunderstood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0049] FIG. 1 shows a schematic illustrating the methods used herein to identify shared neoantigens. Colonic tumors and pre-cancers with their matching normal mucosa and blood were obtained from LS patients. Samples were sequenced at DNA and RNA level using WES and RNAseq, respectively. Using paired WES and RNAseq data neoAgs were predicted from frameshift indels and ranked using in-house formula. Predicted neoAgs were synthesized and their immunogenicity was tested using ELISPOT and tetramer assays. The described study provides a list of shared LS-neoAgs with confirmed immunogenicity for the development of a universal LS cancer vaccine, the somatic mutation landscape of the largest cohort of LS cancers and pre-cancers, and their landscape of immune activation at a transcriptomic level.

[0050] FIG. 2 shows a schematic illustrating the computational pipeline used to predict MHC-I and MHC-II restricted neoAgs from the analyzed samples. Blue boxes represent input data, green boxes with black outlines represent the analysis performed using the bioinformatics tools. The final product of this pipeline is a list of ranked neoantigens based on their ranking scores. These scores are obtained using the formula and filters shown in the green box with the red outline.

[0051] FIGS. 3A-B show variability in neoAg affinity to different HLA alleles. FIG. 3A shows an example of RNF43 neoantigen from which two neoepitopes were predicted: RNF43_2 and RNF43_3. The green and red line graphs with dots on the top show the mean binding affinity of each amino acid to the different HLA alleles shown below: the higher the association constant (1 / nM), the stronger the binding affinity. The green and red bar graphs show the percentage of samples within the cohort that are predicted to generate each of the neoantigens amino acid. The heatmap in the bottom shows the level of binding affinity (association constant) of each amino acid to the 14 Class I and 9 Class II HLA types used for the prediction. The higher the association constant (1 / nM), the stronger the binding affinity. FIG. 3B shows number of samples within the cohort covered by the HLA types used for neoantigen prediction.

[0052] FIG. 4 shows the number of neoantigens and their MHC binding affinity. The bar graph shows the number of predicted MHC-I and -II neoAgs with binding affinity <50 nM (blue), and 50 -100 nM (green). The bottom panel displays molecular and pathological characteristics of each sample: MSI status (top), disease category (middle), and tissue pathology (bottom) as covariate bars. MSLH, High micro satellite instability; MSLL, Low micro satellite instability; MSS, Micro satellite stable; PRECA, Precancer; ADVPRECA,Advanced Precancer; CANCER, Cancer; AP, Adenomatous polyp; ADCA, Adenocarcinoma (Stage III &IV); SSA, Sessile serrated adenoma; HP, Hyperplastic polyp; IP, Inflammatory polyp.

[0053] FIGS. 5A-E show the landscape of neoantigens produced from frameshift mutated proteins in the LS patient cohort. FIG. 5A shows there is a significant difference between the number of MHC-I and MHC-II neoAgs produced by the MSI-H samples compared to the MSI- L and MSS samples (Mann Whitney test ****P-value<0.0001). FIG. 5B shows there is a significant difference between the number of MHC-I and MHC-II neoAgs produced by the cancers compared to the advanced pre-cancers and pre-cancers (Mann Whitney test ****P- value<0.0001). FIG. 5C shows there is a significant difference between the number of MHC- I and MHC-II neoAgs produced by the cancers compared to the other pathology diagnoses (Mann Whitney test **P-value<0.01). FIG. 5D shows the number of both MHC-I and -II neoAgs detected per sample is significantly correlated with the mutational burden in each sample (Spearman P-value<0.0001). FIG. 5E shows a waterfall plot that shows the top 50 predicted MHC-I neoAgs with in silico analysis in MSI-H samples. The bar plot labeled Neoantigen per MB represents occurrence of neoAgs (neoAgs per Mb) in each sample. The grid panel shows the top 50 most shared MHC-I neoAgs based on their frequency among all samples (red bars adjacent to gene names) and MSI-H samples (blue bars adjacent to red bars). The ranking of the predicted immunogenic neoAgs is represented with the dark blue being the 1st percentile (highest ranked immunogenic neoAgs) and the yellow being the lowest-ranked immunogenic neoAgs. The bottom panel displays molecular and pathological characteristics of each sample: Disease category (Top), and tissue pathology (bottom) as covariate bars. MSI- H, High microsatellite instability; MSI-L, Low microsatellite instability; MSS, Microsatellite stable; PRECA, Precancer; ADVPRECA, Advanced Precancer; CANCER, Cancer; AP, Adenoma polyp; ADCA. Adenocarcinoma (Stage III &IV); SSA, Sessile serrated adenoma; HP, Hyperplastic polyp; IP, Inflammatory polyp.

[0054] FIG. 6 shows a waterfall plot for the top 50 MHC class II neoantigens predicted with the pipeline, showing only in MSI-H samples. The bar plot labeled Neoantigen per MB represents sample-wise neoAgs rate (neoAgs per Mb). The grid panel shows the top 50 most frequent MHC-II neoAgs organized by their frequency among all samples and MSI-H samples. The in silico neoAgs ranking is represented with the dark blue being the 1st percentile (highest ranked immunogenic neoAgs) and the yellow being the lowest ranked. The bottom panel displays molecular and pathological characteristics of each sample: Disease category (top) and tissue pathology (bottom) as covariate bars. MSI-H, High micro satellite instability; MSI-L,Low micro satellite instability; MSS, Microsatellite stable; PRECA, Precancer; ADVPRECA, Advanced Precancer; CANCER, Cancer; AP, = Adenoma polyp; ADC A, =Adenocarcinoma (Stage III &IV); SSA, Sessile serrated adenoma; HP, Hyperplastic polyp; IP, Inflammatory polyp.

[0055] FIGS. 7A-F show immunogenicity validation of predicted neoantigens using in vitro approaches. FIG. 7A shows a schematic of PBMC stimulation with peptide neoAgs and cell culture workflow for ELISpot assays. PBMCs from healthy human donors were stimulated with individual peptides in the presence of IL-7 for 3 d, followed by expansion of neoAgs- specific T cells in the presence of IL-2. On day 13, expanded cells (10A5 cells per well) were plated onto 96- well ELISpot plate coated with an IFNy antibody and re- stimulated with the respective peptide for 24 h. IFNy-secreting cells were analyzed as spot-forming units (SFUs). Peptides with significantly higher number of SFUs (Paired Wilcoxon Ranked Test, P- value<0.05) compared to DMSO control cells were defined as ELISpot-reactive or immunogenic. FIG. 7B shows quantification of IFNy-secreting cells (SFU) showing that ELISpot-reactive peptides had significantly higher number of mean SFUs compared to non- reactive peptides cells after averaging the SFUs of all donors and comparing the two groups with an unpaired T-test (****, P-value<0.00001). FIG. 7C shows representative well images showing the IFN-y secretion in PBMCs after stimulation with some of the ELISpot-reactive peptides. FIG. 7D shows donor A PBMCs (HLA-A2) were stained with anti-CD8 PerCP and a PE-conjugated RNF43_3- MHC-I tetramer (TQLARFFPI; left panel) to identify enrichment of specific HLA-A2-restricted T cell epitopes after stimulation. After gates were set on single lymphocytes and live CD3+ / CD8+ cells, the enrichment of RNF43_3-MHC-I tetramerpositive and T cell activation marker 4- IBB positive cells were shown to identify activated RNF43_3 reactive T cells (right panel). Unstimulated T cells were used as control. FIG. 7E shows levels of CD8+, CD4+, and 41BB+ cells in RNF43_3 neoAgs stimulated T cells. Each experiment was assessed through two independent assays. Results are represented as the mean percentage of CD8+, CD4+, and CD8+ / 41BB+ T cells in response to RNF43_3 antigenic peptide stimulation for all healthy human donors (n=6). Unstimulated T cells were used as control. FIG. 7F shows graphs showing the percentage of CD8+ (top panel), CD4+ (middle panel), and CD8+ / 41BB+ (bottom panel) T cells, quantified from flow cytometry analysis of FIG. 7E. Statistical significance for unstimulated versus stimulated T cells was evaluated by unpaired t-test for each healthy human donor. CD8+ T cells: Donor A ***P-value=0.0002; Donor B, C, D, and E: ****P-value<0.0001; Donor F *** P-value=0.0003. CD4+ T cells Donor A, E, F ****P-value<0.0001; Donor B **P-value=0.0177. CD8 / 4-1BB double positivecells: ****P-value<0.0001 for all donors. Each experiment was assessed from two independent assays.

[0056] FIGS. 8A-B show SFUs counts for each peptide after ELISPOT assay. FIG. 8A shows quantification of IFNy-secreting cells (SFU) showing that 65 out of 100 peptides were ELISpot-reactive showing significantly higher number of SFUs compared to DMSO control cells after Paired Wilcoxon Ranked Test or paired T-test (P-value<0.05). ConcA and DMSO served as positive and negative controls respectively. FIG. 8B shows quantification of IFNy- secreting cells (SFU) showing that 35 out of 100 peptides were EEISpot non-reactive with no significantly higher number of SFUs compared to DMSO control cells after Paired Wilcoxon Ranked Test or paired T-test (P-value<0.05). ConcA and DMSO served as positive and negative controls respectively.

[0057] FIGS. 9A-9E shows schematic representation of experiments as described herein, as well as data described herein. FIG. 9A shows experimental workflow as described herein. Select immunogenic FSP-neoAgs were selected for three phases of experiments using PBMCs of ES survivors and previvors. FIG. 9B shows timeline of stimulation of patient PBMCs for short, innate-like responses aimed at supporting the activation and development of antigen- presenting cells (APCs), and the second approach, characterized by an extended nine-day stimulation period to rapidly prime naive T cells, thereby evaluating the priming potential of each neoAg peptide. FIG. 9C shows quantification of SFUs per survivor and previvor in response to FSP-neoAg and respective controls. These data demonstrate, among other things, that all tested FSP-neoAgs induced T cell responses when averaging the responses from all ES carriers following short-term stimulation.. FIG. 9D shows quantification of PBMC SFUs in response to different FSP-neoAgs in both HEA matched and HEA unmatched groups. FIG. 9E shows quantification of PBMC SFUs in response to different FSP-neoAgs in HEA unmatched groups in after overnight stimulation and 9-day in vitro stimulation. These data showed that all four neoantigens have strong in vitro priming capabilities, leading to an enhanced IFN-gamma response. Most importantly, these data demonstrate that a 9-day in vitro priming period overcomes immunogenicity challenges often associated with HEA variability, thus enhancing the immune response to all tested antigens in patients, regardless of their HEA match, particularly compared to previous short-term stimulation results

[0058] FIG. 10 shows shows quantification of IFNy-secreting cells (SFU) from one previvor, Pl, and three survivors, S2, S4, and S8 in both baseline and follow-up PBMC samples.

[0059] FIGS. 11A-11G shows schematic representations and results from neoantigenstimulation experiments described herein. FIG. 11A shows a schematic representation of the physiologically relevant, gravity driven microfluidic co-culture system employed herein to assess tumor cytotoxicity. FIG. 11B shows genetically modified HCT116 colon cancer cells as described herein. MG1, minigene 1; MG2, minigene 2. FIG. 11C shows experimental results confirming expression of MG1 and MG2 constructs as described herein. FIGS. 11D- 11E shows experimental results of tumor cell death and apoptosis assays using baseline FSP- neoAg-stimulated PBMCs and follow-up FSP-neoAg-stimulated PBMCs. Assays demonstrated that all neo Ag- specific T cells exhibit significant tumor cell-killing potency. FIG. 11F-11G shows quantification of key effector molecules, IFN-gamma, IL-2, TNF-alpha, and Granzyme B, produced by PBMCs collected from co-cultures as described herein. These data demonstrate that, among other things, significant release of these cytokines and cytotoxic molecules was observed across all tested patients, (***p < 0.001; ****p < 0.0001). Notably, LS survivors exhibited elevated levels of activator cytokines (IFN-gamma and IL-2) as well as the pro-inflammatory cytokine TNF-alpha, indicating heightened immune activation.

[0060] FIGS. 12A-12C shows quantification of circulating FSP-neoAg-specific T cells in baseline and follow-up PBMC patient samples, as well as phenotypic and transcriptional states of these cells in LS carriers. FIG. 12A shows results of MHC multimer assays, as described herein, to detect circulating neo Ag- specific T cells responsive to each of the five FSP-neoAg peptides in PBMCs of HLA-matched patient samples collected at both baseline and follow-up. FIG. 12B shows select clusters resulting from unsupervised transcriptomic clustering of the combined 1,331,615 neoAg-specific and 464,619 pan-T cells, clusters revealed a range of transcriptomic states in circulating neoAg-specific CD8+ T cells, spanning from less differentiated, proliferative states (Cluster 0 and 3) to various differentiated states, including effector-like T cells (Cluster 4), effector memory T cells (Cluster 2), highly cytotoxic T cells (Cluster 5), and terminally differentiated effector T cells (Cluster 6) that expressed exhaustion markers and inhibitory molecules. FIG. 12C shows uniform manifold approximation and projection (UMAP) analysis of the combined 1,331,615 neoAg-specific and 464,619 pan-T cells. Mapping hyperexpanded neoAg-specific TCR clonotypes from all five neoAg peptides revealed that most of these clones localized within clusters associated with differentiated effector-like (Cluster 0), Proliferating T cells (Cluster 3), and terminally differentiated T cells (Cluster 6) (FIG. 12C, bottom).

[0061] FIGS. 13A-13D shows a characterization of neoAg specificity and tissue presence, as described herein. FIG. 13A shows cell number, diversity, and clonality of both pan-T cellsand neo Ag- specific T cells. Compared to pan-T cells, neo Ag- specific T cells show a markedly higher clonality, reflecting a repertoire dominated by hyperexpanded TCR clones. FIG. 13B shows quantification of clonotype characteristics, as described herein, focused on public clonse detected in LS carriers (either survivors, previvors, or both), with a further mphasis on clones present in LS carrier tissue. FIGS. 13C-13D shows the number of dMMR CRC associated TCR clones (FIG. 13C) and the number of tumor infiltrated TCR clones (FIG. 13D), as described herein.DETAILED DESCRIPTION

[0062] Lynch Syndrome (LS) is the leading cause of hereditary colorectal cancer (CRC) representing 2-4% of the total of colorectal cancers and affecting more than one million individuals in the United States (Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer 2015;15(3): 181-94 doi 10.1038 / nrc3878). LS patients carry heterozygous germline mutations in one of the four DNA mismatch repair (MMR) genes, conferring 50-80% lifetime risk of developing CRC, 40-60% risk of endometrial cancer, and heightened risk of multiple other tumor types (Bonadona V, Bonaiti B, Olschwang S, Grandjouan S, Huiart L, Longy M, et al. Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome. JAMA 2011;305(22):2304-10 doi 10.1001 / jama.2011.743). Normal colonic epithelial cells become MMR deficient (MMRd) upon acquisition of a second somatic hit in the alternate allele of the MMR gene that harbors the germline mutation. This second hit manifests into the accumulation of hundreds to thousands of base-to-base mismatches and insertion-deletion mutations (indels) in micro satellite sequences, which generate non-native frameshift proteins known as neoantigens (neoAgs). These mutated neoAgs are processed and presented as short peptides loaded onto major histocompatibility complexes (MHC-I / II) of antigen presenting cells / cancer cells that then interact with T cell receptors (TCRs) on cytotoxic CD8+ T cells. This stimulatory interaction promotes interferon y (IFNy) secretion and induces programmed cell death of neo Ag-expres sing cancer cells (Yarchoan M, Johnson BA, 3rd, Lutz ER, Laheru DA, Jaffee EM. Targeting neoantigens to augment antitumour immunity. Nat Rev Cancer 2017;17(9):569 doi 10.1038 / nrc.2017.74; Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 2017;547(7662):217-21 doi 10.1038 / nature22991). However, when cancer cells become capable of immune evasion, namely through upregulation of immune checkpoint molecules,neoplastic lesions (both pre-cancers and tumors) return to an unchecked and uncontrolled growth state. Thus, re-activation of CD8+ and CD4+ T cells (helper cells) that recognize neoAgs is important for adaptive immunity against tumors.

[0063] Extensive computational algorithms have used next-generation sequencing (NGS) data to rapidly screen the mutational landscape of human cancers, primarily in melanoma for which neoAg-targeting therapies have already shown clinical activity in patients (Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 2017;547(7662):217-21 doi 10.1038 / nature22991; Nielsen M, Andreatta M. NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets. Genome Med 2016;8(l):33 doi 10.1186 / sl3073-016-0288-x; Hundal J, Carreno BM, Petti AA, Linette GP, Griffith OL, Mardis ER, et al. pVAC-Seq: A genome-guided in silico approach to identifying tumor neoantigens. Genome Med 2016;8(l): 11 doi 10.1186 / sl3073-016-0264-5). Such studies have identified a variety of neoAgs that may be recognized by the host’s immune system, thus providing promising avenues to activate anti-tumor immunity (Cohen CJ, Gartner JJ, Horovitz-Fried M, Shamalov K, Trebska-McGowan K, Bliskovsky VV, et al. Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes. J Clin Invest 2015; 125(10):3981-91 doi 10.1172 / JCI82416). Given the propensity for an increased mutational burden in MMRd cancers, putative neoAgs characterized from genomic and transcriptomic data in LS patients may provide a similar opportunity to develop novel immunoprevention approaches such as neoAgs-based vaccines. However, most prediction algorithms focus primarily on parameters associated with the immunogenicity of the epitopes and identification of neoAgs that are personalized as opposed to those that are shared among different individuals (Wells DK, van Buuren MM, Dang KK, Hubbard- Lucey VM, Sheehan KCF, Campbell KM, et al. Key Parameters of Tumor Epitope Immunogenicity Revealed Through a Consortium Approach Improve Neoantigen Prediction. Cell 2020; 183(3): 818-34 el3 doi 10.1016 / j. cell.2020.09.015). Considering the high rate of shared neoAgs among different LS patients due to micro satellite- specific mutations recurring in specific genes within ‘prone’ loci (Schwitalle Y, Linnebacher M, Ripberger E, Gebert J, von Knebel Doeberitz M. Immunogenic peptides generated by frameshift mutations in DNA mismatch repair-deficient cancer cells. Cancer Immun 2004;4:14), there is a need to better identify highly immunogenic and commonly shared neoAgs for developing a universal vaccine for the LS population.

[0064] The inventors acquired paired whole-exome sequencing (WES) and mRNAseq data from early-stage LS CRC and precancers to predict and identify the most immunogenic andrecurrent frameshift-neoAgs (FS-neoAgs) present in LS colorectal carcinogenesis using systems biology approaches. The inventors established a computational pipeline that accurately identified somatic micro satellite (MS) indels by overcoming errors produced during short read sequencing, PCR amplification, and other sources of noise. The pipeline also accounted for the allele frequency of a given mutated frameshift protein at both DNA and RNA levels, the binding affinity and stability of the FS-neoAgs, and the most frequent human leukocyte antigen (HLA) genotypes. Immunological assays validated the predicted immunogenicity of FS- neoAgs from the computational methods, thus moving the field closer towards development of a FS-neoAgs vaccine for immune interception of LS cancers.

[0065] In some aspects, provided herein are immunogenic peptides based on the neoantigens identified herein that may be used in therapy for treating or preventing cancer, such as colorectal cancer in subjects with LS.I. PEPTIDES AND NEOANTIGENS

[0066] In some aspects, provided herein are peptides, such as any of the peptides set forth in SEQ ID NOs: 1-192, or a peptide having at least 70% sequence identity thereto. In some embodiments, the peptide is set forth in any one of SEQ ID NOS: 1-192. In some embodiments, the peptide is an isolated peptide. In some embodiments, the peptide and / or a contiguous amino acid sequence thereof is present in a composition, such as any of the compositions provided herein. In some aspects, the peptide is immunogenic, either alone or in combination with one or more additional agents. For example, in some embodiments, the peptide is capable of eliciting an immune response in the presence of immune cells, such as an in vivo and / or in vitro response.

[0067] In some embodiments, the peptide may be used as, in, or in the development of a therapy for preventing or treating cancer, such as any described herein. Thus, in some embodiments, provided herein is a method of stimulating an immune response in an individual. In some embodiments, the method comprises providing to the individual a therapeutically effective amount of a composition comprising a peptide comprising a sequence set forth in any one of SEQ ID NOS: 1-192. In some embodiments, provided herein is a composition comprising a peptide comprising a sequence set forth in any of one of SEQ ID NOS: 1-192. In some embodiments, provided herein are methods of obtaining and / or engineering T cells that have a receptor specific for any of the peptides provided herein, such as the peptide of any of SEQ ID NOS: 1-192.

[0068] In some embodiments, the peptide is an MHC-I (MHC class I) restricted peptide. In some embodiments, the peptide is presented by both MHC-I (MHC class I) and MHC-II (MHC class II). In some embodiments, the peptide has at least 70% sequence identity to any one of SEQ ID NOS: 1-100. In some embodiments, the peptide is set forth in any one of SEQ ID NOS: 1-100. In some embodiments, the peptide has at least 70% sequence identity to any one of SEQ ID NOS: 1-64. In some embodiments, the peptide is set forth in any one of SEQ ID NOS: 1-64. In some embodiments, the peptide has at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the peptide is set forth in SEQ ID NO: 1. In some embodiments, the peptide is 8 to 11 amino acids in length. In some embodiments, the peptide is 8 amino acids in length. In some embodiments, the peptide is 9 amino acids in length. In some embodiments, the peptide is 10 amino acids in length. In some embodiments, the peptide is 11 amino acids in length.

[0069] In some embodiments, the peptide is an MHC-II (MHC class II) restricted peptide. In some embodiments, the peptide is presented by both MHC-II and MHC-I. In some embodiments, the peptide has at least 70% sequence identity to any one of SEQ ID NOS: 101 - 192. In some embodiments, the peptide is set forth in any one of SEQ ID NOS: 101-192. In some embodiments, the peptide is 12 to 18 amino acids in length. In some embodiments, the peptide is 12 amino acids in length. In some embodiments, the peptide is 13 amino acids in length. In some embodiments, the peptide is 14 amino acids in length. In some embodiments, the peptide is 15 amino acids in length. In some embodiments, the peptide is 16 amino acids in length. In some embodiments, the peptide is 17 amino acids in length. In some embodiments, the peptide is 18 amino acids in length.

[0070] In some aspects, the peptide corresponds to and / or is based on (e.g. has the same or similar sequence as) a neoantigen. In some aspects, a neoantigen is an amino acid sequence encoded by a mutated gene sequence, for example in a cancer or precancer. In some embodiments, the amino acid sequence of the peptide is identical to a sequence encoded by a mutated gene sequence, such as in a precancer or cancer. The mutated gene sequence can be the result of any mutation that gives rise to a neoantigen. One of skill in the art would readily know and recognize various classes of mutations that may give rise to neoantigens. In some embodiments, the mutated gene sequence is the result of a frameshift mutation. In some embodiments, the neoantigen can be a recurrent neoantigen, such that the peptide corresponds to and / or is based on a neoantigen that occurs in a percentage of individuals with a particular cancer. For example, in some embodiments, the amino acid sequence of the peptide is identical to a sequence encoded by a mutated gene sequence in a precancer or cancer in at least 0.01%,at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 20%, or more, of subjects having Lynch Syndrome and a precancer or cancer.

[0071] In some embodiments, the peptide corresponds to a neoantigen resulting from a mutation (e.g. frameshift mutation) in a gene, such as in any one of the genes set forth in Table 1. For example, in some embodiments, the peptide corresponds to a neoantigen resulting from a frameshift mutation in the gene RNF43. In some aspects, the neoantigen is present in a subject with Lynch Syndrome (LS), colorectal cancer, and / or a cell therefrom. In some embodiments, the neoantigen is present in a mutated allele of any of the genes set forth in Table 1. In some embodiments, the neoantigen is a result of a frameshift mutation.

[0072] In some embodiments, the neoantigen (e.g. that the peptide corresponds to) is a recurrent, or shared, neoantigen. For example, the recurrent neoantigen can be present in at least two different subjects and / or two different samples, such as a subject having colorectal cancer and / or Lynch Syndrome or a sample therefrom. In some embodiments, the neoantigen is present in at least two different subjects having Lynch Syndrome. In some embodiments, the neoantigen is present in at least two different samples derived from subject(s) having Lynch Syndrome. In some embodiments, the neoantigen is present in a percentage of subjects having Lynch Syndrome and a cancer or precancer, such as a colorectal cancer or precancer. In some embodiments, the neoantigen is present in at least 0.1%, at least 0.5%, 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 15%, at least 20%, or more, of subjects having colorectal precancer or cancer and / or Lynch Syndrome. In some embodiments, the neoantigen is present in at least 0.1%, at least 0.5%, 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 15%, at least 20%, or more, of subjects having colorectal precancer or cancer and Lynch Syndrome. In some embodiments, the amino acid sequence of the peptide is identical to a sequence encoded by a mutated gene sequence (e.g. a neoantigen) in a precancer or cancer in at least 0.01%, at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 20%, or more, of subjects having Lynch Syndrome and a precancer or cancer. In some embodiments, the mutated gene sequence is the result of a frameshift mutation.

[0073] Peptides based on recurrent neoantigens provide several advantages. For example, it can be seen that the therapeutic potential of peptides based on recurrent neoantigens is not restricted to individual patients. In some aspects, the peptides based on neoantigens (e.g. recurrent neoantigens) provided herein may be used in connection with therapeutic strategies that have the potential to benefit a significant population of individuals. For example, thepeptides may be used in cancer treatments, or cancer preventative vaccines, for LS patients based on recurrent and immunogenic neoantigens.

[0074] Although peptides based on recurrent neoantigens provide certain advantages, the peptide does not need to be based on a recurrent neoantigen. In some embodiments, the peptide is based on a neoantigen that is not a recurrent neoantigen, or that is not known to be a recurrent neoantigen.

[0075] Also provided herein are combinations of peptides, such as any of the peptides provided herein. In some aspects, the combination of peptides comprises two or more peptides of SEQ ID Nos: 1-192. In some embodiments, the combination of peptides comprises two or more peptides with 70% identity to peptides of SEQ ID Nos: 1-192.

[0076] In some aspects, provided herein is an isolated peptide comprising at least 70% sequence identity to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide comprises at least 6 contiguous amino acids of a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide is 25 amino acids or fewer in length. In some embodiments, the peptide is 8 amino acids or greater in length. In some embodiments, the peptide is 8 to 11 amino acids in length. In some embodiments, the peptide is 12 to 18 amino acids in length. In some embodiments, the peptide is immunogenic. In some embodiments, the peptide is modified. In some embodiments, the modification comprises conjugation to a molecule. In some embodiments, the molecule comprises an antibody, a lipid, an adjuvant, a cell penetrating peptide, or a detection moiety. In some embodiments, the peptide has at least 90% sequence identity to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide has 1, 2 or 3 substitutions relative to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the peptide comprises 100% sequence identity to a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the amino acid sequence of the peptide is identical to a sequence encoded by a mutated gene sequence in a precancer or cancer in at least 0.01%, at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 20%, or more, of subjects having Lynch Syndrome and a precancer or cancer. In some embodiments, the mutated gene sequence is the result of a frameshift mutation. In some aspects, provided herein is a molecular complex comprising any peptide provided herein and an MHC polypeptide.

[0077] In some aspects, provided herein is a combination of peptides comprising two or more of any of the peptides provided herein. The combination of peptides can comprise any number of peptides. In some embodiments, any combination of peptides provided herein can include or exclude any of the peptides provided herein, such as any of the peptides set forth in SEQ ID NOS: 1-192. In some embodiments, the combination of peptides comprises at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more than 200, of any of the peptides provided herein. In some embodiments, the combination of peptides comprises between about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 120, 120 to 140, 140 to 160, 160 to 180, 180 to 200, or 200 to 300, of any of the peptides provided herein. In some embodiments, the combination of peptides comprises at or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 of any of the peptides provided herein. In some embodiments, the combination of peptides comprises between 25 to 40 of any of the peptides provided herein. In some embodiments, the combination of peptides comprises at or about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 of any of the peptides provided herein. In some embodiments, the combination of peptides comprises at or about 35 of any of the peptides provided herein. In some embodiments, the combination of peptides comprises a plurality of peptides, each corresponding to recurrent neoantigens that are present in Lynch Syndrome precancers or cancers. In some embodiments, the combination of peptides will have at least two different peptides that each correspond to a different recurrent neoantigen present in Lynch Syndrome cancers or precancers. In this way, the combination of peptides can correspond to neoantigens in a larger proportion of a population of individuals with Lynch Syndrome than any of the peptides alone. In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of precancers or cancers in subjects having Lynch Syndrome comprise a mutated gene sequence encoding an amino acid sequence that is identical to the sequence of one of the peptides in a combination of peptides provided herein. In some embodiments, in at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of subjects having Lynch Syndrome and a precancer or cancer, the precancer or cancer comprises a mutated gene sequence encoding an amino acid sequence that is identical to the sequence of one of the peptides in a combination of peptides provided herein. In some embodiments, for at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of subjects having Lynch Syndrome and a precancer or cancer, the combination of peptides comprises at least one peptide that is identical in sequence to a sequence encoded by a mutated gene sequence in the precancer or cancer. The mutated gene sequence can be any mutated gene sequence. In some embodiments, the mutated gene sequence is the result of a frameshift mutation.

[0078] In some aspects, provided herein is a combination of a first, second, third, fourth, fifth, sixth, or more peptides (e.g., a frameshift protein (FSP)-neoantigen peptide (FSP- neoAg)), or a nucleic acid encoding said peptides. In some embodiments, the combination of peptides comprises a combination of individual peptides. In some embodiments, the combination comprises a single peptide comprising a concatenation of the neoAgs by covalent bond, optionally separated by short non-neoAg linker sequences. In some embodiments provided herein are peptides modified to include one or more additional sequences, including one or more subcellular trafficking sequence (e.g., secretory signal), and / or MHC trafficking signal (MITD), and / or fluorescent protein sequence (e.g., GFP). In some embodiments, the combination provided herein comprises RNF43_2 (SEQ ID NO: 9), or a nucleic acid encoding therefor. In some embodiments, the combination provided herein comprises RNF43_3 (SEQ ID NO: 1), or a nucleic acid encoding therefor. In some embodiments, the combination provided herein comprises TGFBR2_4 (SEQ ID NO: 8), or a nucleic acid encoding therefor. Unless stated otherwise or clear from context, the term “TGFBR2” is used herein synonymously with “TGFBR2_4” and “TGFBR4”, each of which correspond to SEQ ID NO: 8 (i.e., VPVALMSAM). In some embodiments, the combination provided herein comprises BMPR2 (SEQ ID NO: 6), or a nucleic acid encoding therefor. In some embodiments, the combination provided herein comprises MSH3 (SEQ ID NO: 5), or a nucleic acid encoding therefor. In some embodiments, the combination provided herein comprises RNF43_2 (SEQ ID NO: 9), or a nucleic acid encoding therefor, RNF43_3 (SEQ ID NO: 1), or a nucleic acid encoding therefor, TGFBR2_4 (SEQ ID NO: 8), or a nucleic acid encoding therefor, BMPR2 (SEQ ID NO: 6), or a nucleic acid encoding therefor, and / or MSH3 (SEQ ID NO: 5), or a nucleic acid encoding therefor. In some embodiments, the combination excludes one or more of SEQ ID NOs: 1, 5-6, and / or 8-9. In some embodiments, the combination provided herein comprises MIS18BP1 (SEQ ID NO: 12), or a nucleic acid encoding therefor. In some embodiments, the combination provided herein comprises AXIN2 (SEQ ID NO: 13), or a nucleic acid encoding therefor. In some embodiments, the combination provided herein comprises SLC25A6 (SEQ IDNO: 17), or a nucleic acid encoding therefor. In some embodiments, the combination comprises PUM3_1 (SEQ ID NO: 21), or a nucleic acid encoding therefor. In some embodiments, the combination comprises PUM3_2 (SEQ ID NO: 22), or a nucleic acid encoding therefor. In some embodiments, the combinations provided herein comprise one or more of (i) MIS18BP1 (SEQ ID NO: 12), AXIN2 (SEQ ID NO: 13), SLC25A6 (SEQ IDNO: 17), PUM3_1 (SEQ ID NO: 21), PUM3_2 (SEQ ID NO: 22), or nucleic acid(s) encoding therefor, with one or more of (ii) RNF43_2 (SEQ ID NO: 9),RNF43_3 (SEQ ID NO: 1), TGFBR2_4 (SEQ ID NO: 8), BMPR2 (SEQ ID NO: 6), and / or MSH3 (SEQ ID NO: 5), or nucleic acids encoding therefor.

[0079] In some aspects, provided herein is one or more nucleic acids encoding any peptide or combination of peptides provided herein. In some embodiments, the nucleic acid(s) comprise DNA and / or RNA. In some embodiments, the nucleic acid(s) comprise mRNA. In some aspects, provided herein is an expression vector comprising any nucleic acid(s) provided herein.

[0080] In some aspects, provided herein is a vector for intracellular delivery, comprising any peptide, molecular complex, combination of peptides, nucleic acid(s), or expression vector provided herein. In some embodiments, the vector is a viral vector. Any suitable viral vector may be used to deliver any of the compositions provided herein to a cell. One skilled in the art would be familiar with various such viral vectors, such as adeno-associated virus (AAV) vectors. In some embodiments, the vector is a non- viral vector. Any suitable non-viral vector may be used, such as a liposome, lipid-containing nanoparticle, or other lipid-based carrier.

[0081] In some aspects, provided herein is a composition comprising any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, or vector for intracellular delivery provided herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutical carrier. The composition can be formulated for any suitable method of administration, for example to a subject. In some embodiments, the composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. In some embodiments, the peptide, combination of peptides, nucleic acid(s), or expression vector, is comprised in a liposome, lipid-containing nanoparticle, or in a lipid-based carrier. In some embodiments, the composition is formulated for injection or inhalation as a nasal spray. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition stimulates an immune response when administered to a subject. In some embodiments, the composition prevents colorectal cancer when administered to a subject having Lynch Syndrome.

[0082] In some aspects, provided herein is a method of stimulating an immune response in a subject, the method comprising administering to the subject any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein. In some aspects, provided herein is a method of treating or preventing cancer or precancer in a subject, the method comprising administering to the subject- l-any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein. In some aspects, provided herein is a method of preventing cancer in a subject, the method comprising administering to the subject any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein.

[0083] Disclosed herein are methods of stimulating an immune response, methods of provoking an immune response, methods of treating cancer, methods of preventing cancer, methods of enhancing a subject’s responsiveness to a cancer therapy, methods of enhancing the effectiveness of a cancer therapy, methods of sensitizing a subject to a cancer therapy, methods of increasing an immune response against a cancer, methods of decreasing cancer progression, and methods of improving a subject’s prognosis. The method can comprise any number (e.g. 1, 2, 3, 4, 5, 6, 7, or more) of any of the following steps: administering an effective amount of any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, composition, pharmaceutical composition, vaccine, dendritic cell, or peptide- specific T cell provided herein to a subject; administering a cancer therapy to a subject; administering a pharmaceutical composition to the subject; administering a vaccine to a subject; monitoring a subject; evaluating a sample from a subject; diagnosing a subject; and determining the presence of one or more neoantigens or mutations in a subject. It is contemplated that the preceding steps can occur in any order and each step may be performed one or more times, in any order relative to the other steps. It is also specifically contemplated, in certain aspects, that any of the preceding steps may be excluded from the methods. In certain aspects, the subject is a human. In some embodiments, the subject has been determined to have Lynch Syndrome, and / or one or more cancers or precancers, such as colorectal cancers or precancers.

[0084] In some embodiments, the subject has been determined to have one or more precancers or cancers. In some aspects, a precancer can be any precancerous cell or group of cells, such as a dysplasia, hyperplasia, or neoplasia. In some embodiments, the subject is determined to be at risk of developing cancer. In some embodiments, the subject is determined to have a genetic predisposition for developing colorectal cancer. In some embodiments, the subject has Lynch Syndrome. In some embodiments, the cancer or precancer is colorectal. In some embodiments, the cancer or precancer is determined to comprise a mutated gene sequence encoding an amino acid sequence comprising the sequence set forth in any of SEQ ID NOS:1- 192. In some embodiments, the cancer or precancer is determined to comprise a mutated gene sequence encoding an amino acid sequence that is identical to the amino acid sequence of anypeptide provided herein. In some embodiments, the mutated gene sequence is the result of a frameshift mutation.

[0085] In some aspects, provided herein is a vaccine for preventing colorectal cancer in subjects with Lynch Syndrome, comprising any peptide, molecular complex, combination of peptides, nucleic acid(s), expression vector, vector for intracellular delivery, or composition provided herein. In some aspects, provided herein is a vaccine for preventing colorectal cancer in subjects with Lynch Syndrome, comprising one or more mRNA molecules encoding the amino acid sequences of the peptides of any of the combination of peptides provided herein. In some aspects, the vaccine can be a preventative vaccine. Thus, in some embodiments, the vaccine can be administered to a subject having Lynch Syndrome but that has not been diagnosed with cancer. In some embodiments, the vaccine comprises a combination of peptides or one or more nucleic acids encoding a combination of peptides, such as any of the combination of peptides provided herein. In some aspects, the combination of peptides comprises a plurality of peptides corresponding to recurrent neoantigens that are present in Lynch Syndrome cancers and precancers. In this way, the vaccine does not need to be tailored to individual cancers or subjects, but can be effective in preventing the development of cancer in a significant proportion of individuals with Lynch Syndrome. This is because the combination of peptides collectively comprises peptides corresponding to at least one neoantigen in a significant proportion of cancers or precancers in individuals with Lynch Syndrome (e.g. the combination of peptides and / or the vaccine provides coverage for a large proportion of Lynch Syndrome cancers or precancers). In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of precancers or cancers in subjects having Lynch Syndrome comprise a mutated gene sequence encoding an amino acid sequence that is identical to the sequence of one of the peptides in a combination of peptides provided herein, such as the combination of peptides provided in or encoded by a vaccine provided herein.

[0086] Aspects and embodiments of the present disclosure may include or exclude any of the peptides provided herein. Aspects and embodiments of the present disclosure may include or exclude any peptide comprising a sequence set forth in any of SEQ ID NOS: 1-192, or any peptide having at least 70% sequence identity thereto.IL IMMUNOTHERAPIES USING PEPTIDES OF THE DISCLOSURE

[0087] A peptide as described herein (e.g., a peptide of one of SEQ ID NOS: 1-192) may be used for immunotherapy of a cancer. For example, a peptide of one of SEQ ID NOS: 1- 192may be contacted with or used to stimulate a population of T cells to induce proliferation of the T cells that recognize or bind said peptide. In other embodiments, a peptide of the disclosure may be administered to a subject, such as a human patient, to enhance the immune response of the subject against a cancer.

[0088] A peptide of the disclosure may be included in an active immunotherapy (e.g., a cancer vaccine) or a passive immunotherapy (e.g., an adoptive immunotherapy). Active immunotherapies include immunizing a subject with a purified peptide antigen or an immunodominant peptide (native or modified); alternatively, antigen presenting cells pulsed with a peptide of the disclosure (or transfected with genes encoding an antigen comprising the peptide) may be administered to a subject. The peptide may be modified or contain one or more mutations such as, e.g., a substitution mutation. Passive immunotherapies include adoptive immunotherapies. Adoptive immunotherapies generally involve administering cells to a subject, wherein the cells (e.g., cytotoxic T cells) have been sensitized in vitro to a peptide of the disclosure (see, e.g., US 7910109).

[0089] In some embodiments, flow cytometry may be used in the adoptive immunotherapy for rapid isolation of human tumor antigen- specific T cell clones by using, e.g., T cell receptor (TCR) VP antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay. See, e.g., Lee et al., J. Immunol. Methods, 331:13-26, 2008, which is incorporated by reference for all purposes. In some embodiments, tetramer-guided cell sorting may be used such as, e.g., the methods described in Pollack, et al., J Immunother Cancer. 2014; 2: 36, which is herein incorporated by reference for all purposes. Various culture protocols are also known for adoptive immunotherapy and may be used in embodiments of the disclosure. In some embodiments, cells may be cultured in conditions which do not require the use of antigen presenting cells (e.g., Hida et al., Cancer Immunol. Immunotherapy, 51 :219-228, 2002, which is incorporated by reference). In other embodiments, T cells may be expanded under culture conditions that utilize antigen presenting cells, such as dendritic cells (Nestle et al., 1998, incorporated by reference), and in some embodiments artificial antigen presenting cells may be used for this purpose (Maus et al., 2002 incorporated by reference). Additional methods for adoptive immunotherapy are disclosed in Dudley et al. (2003), which is incorporated by reference, that may be used with embodiments of the current disclosure. Various methods are known and may be used for cloning and expanding human antigen- specific T cells (see, e.g., Riddell et al., 1990, which is herein incorporated by reference).

[0090] In certain embodiments, the following protocol may be used to generate T cells that selectively recognize peptides of the disclosure. Peptide- specific T cell lines may be generatedfrom normal donors or HLA-restricted normal donors and patients using methods previously reported (Hida et al., 2002). Briefly, PBMCs (l x 10A5 cells / well) can be stimulated with about 10 pg / ml of each peptide in quadruplicate in a 96-well, U-bottom-microculture plate (Corning Incorporated, Lowell, MA) in about 200 pl of culture medium. The culture medium may consist of 50% AIM-V medium (Invitrogen), 50% RPMI1640 medium (Invitrogen), 10% human AB serum (Valley Biomedical, Winchester, VA), and 100 lU / ml of interleukin-2 (IL- 2). Cells may be restimulated with the corresponding peptide about every 3 days. After 5 stimulations, T cells from each well may be washed and incubated with T2 cells in the presence or absence of the corresponding peptide. After about 18 hours, the production of interferon (IFN)-y may be determined in the supernatants by ELISA. T cells that secret large amounts of IFN-y may be further expanded by a rapid expansion protocol (Riddell et al., 1990; Yee et al., 2002b).

[0091] In some embodiments, an immunotherapy may utilize a peptide of the disclosure that is associated with a cell penetrator, such as a liposome or a cell penetrating peptide (CPP). Antigen presenting cells (such as dendritic cells) pulsed with peptides may be used to enhance antitumour immunity (Celluzzi et al., 1996; Young et al., 1996). Liposomes and CPPs are described in further detail below. In some embodiments, an immunotherapy may utilize a nucleic acid encoding a peptide of the disclosure, wherein the nucleic acid is delivered, e.g., in a viral vector or non-viral vector.

[0092] In some embodiments, a peptide of the disclosure may be used in an immunotherapy to treat cancer in a mammalian subject, such as a human patient.III. CELL PENETRATING PEPTIDES

[0093] A peptide of the disclosure may also be associated with or covalently bound to a cell penetrating peptide (CPP). Cell penetrating peptides that may be covalently bound to a peptide of the disclosure include, e.g., HIV Tat, herpes virus VP22, the Drosophila Antennapedia homeobox gene product, signal sequences, fusion sequences, or protegrin I. Covalently binding a peptide to a CPP can prolong the presentation of a peptide by dendritic cells, thus enhancing antitumour immunity (Wang and Wang, 2002). In some embodiments, a peptide of the disclosure (e.g., comprised within a peptide or polyepitope string) may be covalently bound (e.g., via a peptide bond) to a CPP to generate a fusion protein. In other embodiments, a peptide or nucleic acid encoding a peptide, according to the current disclosure,may be encapsulated within or associated with a liposome, such as a mulitlamellar, vesicular, or multivesicular liposome.

[0094] As used herein, “association” means a physical association, a chemical association or both. For example, an association can involve a covalent bond, a hydrophobic interaction, encapsulation, surface adsorption, or the like.

[0095] As used herein, “cell penetrator” refers to a composition or compound which enhances the intracellular delivery of the peptide / polyepitope string to the antigen presenting cell. For example, the cell penetrator may be a lipid which, when associated with the peptide, enhances its capacity to cross the plasma membrane. Alternatively, the cell penetrator may be a peptide. Cell penetrating peptides (CPPs) are known in the art, and include, e.g., the Tat protein of HIV (Frankel and Pabo, 1988), the VP22 protein of HSV (Elliott and O'Hare, 1997) and fibroblast growth factor (Lin et al., 1995).

[0096] Cell-penetrating peptides (or “protein transduction domains”) have been identified from the third helix of the Drosophila Antennapedia homeobox gene (Antp), the HIV Tat, and the herpes virus VP22, all of which contain positively charged domains enriched for arginine and lysine residues (Schwarze et al., 2000; Schwarze et al., 1999). Also, hydrophobic peptides derived from signal sequences have been identified as cell-penetrating peptides. (Rojas et al., 1996; Rojas et al., 1998; Du et al., 1998). Coupling these peptides to marker proteins such as P-galactosidase has been shown to confer efficient internalization of the marker protein into cells, and chimeric, in frame fusion proteins containing these peptides have been used to deliver proteins to a wide spectrum of cell types both in vitro and in vivo (Drin et al., 2002). Fusion of these cell penetrating peptides to a peptide of the disclosure may enhance cellular uptake of the polypeptides.

[0097] In some embodiments, cellular uptake is facilitated by the attachment of a lipid, such as stearate or myristilate, to the polypeptide. Lipidation has been shown to enhance the passage of peptides into cells. The attachment of a lipid moiety is another way that the present invention increases polypeptide uptake by the cell.

[0098] A peptide of the disclosure may be included in a liposomal vaccine composition. For example, the liposomal composition may be or comprise a proteoliposomal composition. Methods for producing proteoliposomal compositions that may be used with the present invention are described, e.g., in Neelapu et al. (2007) and Popescu et al. (2007). In some embodiments, proteoliposomal compositions may be used to treat a cancer.

[0099] By enhancing the uptake of a polypeptide of the disclosure, it may be possible to reduce the amount of protein or peptide required for treatment. This in turn can significantlyreduce the cost of treatment and increase the supply of therapeutic agent. Lower dosages can also minimize the potential immunogencity of peptides and limit toxic side effects.

[0100] In some embodiments, a peptide of the disclosure may be associated with a nanoparticle to form nanoparticle-polypeptide complex. In some embodiments, the nanoparticle is a liposomes or other lipid-based nanoparticle such as a lipid-based vesicle (e.g., a DOTAP:cholesterol vesicle). In other embodiments, the nanoparticle is an iron-oxide based superparamagnetic nanoparticles. Superparamagnetic nanoparticles ranging in diameter from about 10 to 100 nm are small enough to avoid sequestering by the spleen, but large enough to avoid clearance by the liver. Particles this size can penetrate very small capillaries and can be effectively distributed in body tissues. Superparamagnetic nanoparticles-polypeptide complexes can be used as MRI contrast agents to identify and follow those cells that take up the peptide. In some embodiments, the nanoparticle is a semiconductor nanocrystal or a semiconductor quantum dot, both of which can be used in optical imaging. In further embodiments, the nanoparticle can be a nanoshell, which comprises a gold layer over a core of silica. One advantage of nanoshells is that polypeptides can be conjugated to the gold layer using standard chemistry. In other embodiments, the nanoparticle can be a fullerene or a nanotube (Gupta et al., 2005).

[0101] Peptides are rapidly removed from the circulation by the kidney and are sensitive to degradation by proteases in serum. By associating a peptide with a nanoparticle, the nanoparticle-polypeptide complexes of the present invention may protect against degradation and / or reduce clearance by the kidney. This may increase the serum half-life of polypeptides, thereby reducing the polypeptide dose need for effective therapy. Further, this may decrease the costs of treatment, and minimizes immunological problems and toxic reactions of therapy.IV. POLYEPITOPE STRINGS

[0102] In some embodiments, a peptide described herein (e.g., SEQ ID NOs: 1-192) is included or comprised in a polyepitope string. A polyepitope string is a peptide or polypeptide containing a plurality of antigenic epitopes from one or more antigens linked together. A polyepitope string may be used to induce an immune response in a subject, such as a human subject. Polyepitope strings have been previously used to target malaria and other pathogens (Baraldo et al., 2005; Moorthy et al., 2004; Baird et al., 2004). A poly epitope string may refer to a nucleic acid (e.g., a nucleic acid encoding a plurality of antigens including a peptide of the disclosure) or a peptide or polypeptide (e.g., containing a plurality of antigens including apeptide of the disclosure). A polyepitope string may be included in a cancer vaccine composition.V. APPLICATIONS OF ANTIGENIC PEPTIDES

[0103] Various embodiments are directed to development of and use of antigenic peptides that that are useful for treating and preventing certain cancers. In many embodiments, antigenic peptides are produced by chemical synthesis or by molecular expression in a host cell. Peptides can be purified and utilized in a variety of applications including (but not limited to) assays to determine peptide immunogenicity, assays to determine recognition by T cells, peptide vaccines for treatment of cancer, development of modified TCRs of T cells, and development of antibodies.

[0104] Peptides can be synthesized chemically by a number of methods. One common method is to use solid-phase peptide synthesis (SPPS). Generally, SPPS is performed by repeating cycles of alternate N-terminal deprotection and coupling reactions, building peptides from the c-terminus to the n-terminus. The c-terminus of the first amino acid is coupled the resin, wherein then the amine is deprecated and then coupled with the free acid of the second amino acid. This cycle repeats until the peptide is synthesized.

[0105] Peptides can also be synthesized utilizing molecular tools and a host cell. Nucleic acid sequences corresponding with antigenic peptides can be synthesized. In some embodiments, synthetic nucleic acids synthesized in in vitro synthesizers (e.g., phosphoramidite synthesizer), bacterial recombination system, or other suitable methods. Furthermore, synthesized nucleic acids can be purified and lyophilized, or kept stored in a biological system (e.g., bacteria, yeast). For use in a biological system, synthetic nucleic acid molecules can be inserted into a plasmid vector, or similar. A plasmid vector can also be an expression vector, wherein a suitable promoter and a suitable 3’-polyA tail is combined with the transcript sequence.

[0106] Embodiments are also directed to expression vectors and expression systems that produce antigenic peptides or proteins. These expression systems can incorporate an expression vector to express transcripts and proteins in a suitable expression system. Typical expression systems include bacterial (e.g., E. coli), insect (e.g., SF9), yeast (e.g., S. cerevisiae), animal (e.g., CHO), or human (e.g., HEK 293) cell lines. RNA and / or protein molecules can be purified from these systems using standard biotechnology production procedures.

[0107] Assays to determine immunogenicity and / or TCR binding can be performed. One such as is the dextramer flow cytometry assay. Generally, custom-made HLA-matched MHC Class I dextramer :peptide (pMHC) complexes are developed or purchased (Immudex, Copenhagen, Denmark). T cells from peripheral blood mononuclear cells (PBMCs) or tumorinfiltrating lymphocytes (TILs) are incubated the pMHC complexes and stained, which are then run through a flow cytometer to determine if the peptide is capable of binding a TCR of a T cell.

[0108] The peptides of the disclosure can also be used to isolate and / or identify T cell receptors that bind to the peptide. T cell receptors comprise two different polypeptide chains, termed the T cell receptor a (TCRa) and P (TCRP) chains, linked by a disulfide bond. These a:P heterodimers are very similar in structure to the Fab fragment of an immunoglobulin molecule, and they account for antigen recognition by most T cells. A minority of T cells bear an alternative, but structurally similar, receptor made up of a different pair of polypeptide chains designated y and 5. Both types of T cell receptor differ from the membrane-bound immunoglobulin that serves as the B-cell receptor: a T cell receptor has only one antigenbinding site, whereas a B-cell receptor has two, and T cell receptors are never secreted, whereas immunoglobulin can be secreted as antibody.

[0109] Both chains of the T cell receptor have an amino-terminal variable (V) region with homology to an immunoglobulin V domain, a constant (C) region with homology to an immunoglobulin C domain, and a short hinge region containing a cysteine residue that forms the interchain disulfide bond. Each chain spans the lipid bilayer by a hydrophobic transmembrane domain, and ends in a short cytoplasmic tail.

[0110] The three-dimensional structure of the T cell receptor has been determined. The structure is indeed similar to that of an antibody Fab fragment, as was suspected from earlier studies on the genes that encoded it. The T cell receptor chains fold in much the same way as those of a Fab fragment, although the final structure appears a little shorter and wider. There are, however, some distinct differences between T cell receptors and Fab fragments. The most striking difference is in the Ca domain, where the fold is unlike that of any other immunoglobulin-like domain. The half of the domain that is juxtaposed with the CP domain forms a P sheet similar to that found in other immunoglobulin-like domains, but the other half of the domain is formed of loosely packed strands and a short segment of a helix. The intramolecular disulfide bond, which in immunoglobulin-like domains normally joins two P strands, in a Ca domain joins a P strand to this segment of a helix.

[0111] There are also differences in the way in which the domains interact. The interface between the V and C domains of both T cell receptor chains is more extensive than in antibodies, which may make the hinge joint between the domains less flexible. And the interaction between the Ca and CP domains is distinctive in being assisted by carbohydrate, with a sugar group from the Ca domain making a number of hydrogen bonds to the CP domain. Finally, a comparison of the variable binding sites shows that, although the complementaritydetermining region (CDR) loops align fairly closely with those of antibody molecules, there is some displacement relative to those of the antibody molecule. This displacement is particularly marked in the Va CDR2 loop, which is oriented at roughly right angles to the equivalent loop in antibody V domains, as a result of a shift in the P strand that anchors one end of the loop from one face of the domain to the other. A strand displacement also causes a change in the orientation of the VP CDR2 loop in two of the seven VP domains whose structures are known. As yet, the crystallographic structures of seven T cell receptors have been solved to this level of resolution.

[0112] Embodiments of the disclosure relate to engineered T cell receptors that bind a peptide of the disclosure, such as a peptide of one of SEQ ID NOS: 1-192. The term “engineered” can refer to T cell receptors that have TCR variable regions grafted onto TCR constant regions to make a chimeric polypeptide that binds to peptides and antigens of the disclosure. In certain embodiments, the TCR comprises intervening sequences that are used for cloning, enhanced expression, detection, or for therapeutic control of the construct, but are not present in endogenous TCRs, such as multiple cloning sites, linker, hinge sequences, modified hinge sequences, modified transmembrane sequences, a detection polypeptide or molecule, or therapeutic controls that may allow for selection or screening of cells comprising the TCR.

[0113] In some embodiments, the TCR comprises non-TCR sequences. Accordingly, certain embodiments relate to TCRs with sequences that are not from a TCR gene. In some embodiments, the TCR is chimeric, in that it contains sequences normally found in a TCR gene, but contains sequences from at least two TCR genes that are not necessarily found together in nature.VI. ANTIBODIES

[0114] Aspects of the disclosure relate to antibodies that target the peptides of the disclosure, or fragments thereof. The term “antibody” refers to an intact immunoglobulin ofany isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity.

[0115] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess one or more epitopes that are capable of interacting with different antibodies.

[0116] The term “epitope” includes any region or portion of molecule capable eliciting an immune response by binding to an immunoglobulin or to a T cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.

[0117] The epitope regions of a given polypeptide can be identified using many different epitope mapping techniques are well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0118] The term “immunogenic sequence” means a molecule that includes an amino acid sequence of at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host. The term “immunogenic composition” means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate).

[0119] An intact antibody is generally composed of two full-length heavy chains and two full length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, differentportions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human. The antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013; 4: 302; 2013).

[0120] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (K) and lambda ( ). The term “VL fragment” means a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region, including CDRs. A VL fragment can further include light chain constant region sequences. The variable region domain of the light chain is at the amino-terminus of the polypeptide.

[0121] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CHI, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs. A VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxy-terminus, with the CH3 being closest to the — COOH end. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (p), delta (5), gamma (y), alpha (a), or epsilon (a) chains, respectively. IgG has several subtypes, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM subtypes include IgMl and IgM2. IgA subtypes include IgAl and IgA2.VII. ANTIBODY CONJUGATES

[0122] Embodiments of the disclosure relate to antibodies against a peptide of the disclosure, generally of the monoclonal type, that are linked to at least o

[0123] ne agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.

[0124] Any antibody of sufficient selectivity, specificity or affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. Sites for binding to biological active molecules in the antibody molecule, in addition to the canonical antigen binding sites, include sites that reside in the variable domain that can bind pathogens, B-cell superantigens, the T cell co-receptor CD4 and the HIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993; Kreier et al., 1991). In addition, the variable domain is involved in antibody self-binding (Kang et al., 1988), and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

[0125] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds and / or elements that can be detected due to their specific functional properties, and / or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and / or further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti cellular agent, and may be termed "immunotoxins".

[0126] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in avariety of immunoassays, and / or those for use in vivo diagnostic protocols, generally known as "antibody directed imaging".

[0127] Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patent Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.

[0128] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

[0129] In the case of radioactive isotopes for therapeutic and / or diagnostic application, one might mention astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodinel23, iodinel25, iodinel31, indiuml l l, 59iron, 32phosphorus, rheniuml86, rheniuml88, 75selenium, 35sulphur, technicium99m and / or yttrium90. 1251 is often being preferred for use in certain embodiments, and technicium99m and / or indium 111 are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present invention may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and / or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC12, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).

[0130] Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0131] Another type of antibody conjugates contemplated in the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

[0132] Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, haptenbased affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.

[0133] Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et al., 1989) and may be used as antibody binding agents.

[0134] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and / or tetrachloro-3(X-6(X-diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and thedetectable imaging moieties are bound to the antibody using linkers such as methyl-p- hy droxybenzimidate or N- succinimidy 1- 3 -(4-hy droxypheny l)propionate .

[0135] In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.

[0136] In another embodiment of the disclosure, the antibody may be linked to semiconductor nanocrystals such as those described in U.S. Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which are incorporated herein in their entireties); as well as PCT Publication No. 99 / 26299 (published May 27, 1999). In particular, exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to those described above, including group II- VI, III-V and group IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AIS, A1P, AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures thereof. Methods for linking semiconductor nanocrystals to antibodies are described in U.S. Patent Nos. 6,630,307 and 6,274,323.

[0137] In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and / or otherwise generally detecting biological components such as T cells or that selectively bind or recognize a peptide of the disclosure. In some embodiments, a tetramer assay may be used with the present invention. Tetramer assays generally involve generating soluble peptide-MHC tetramers that may bind antigen specific T lymphocytes, and methods for tetramer assays are described, e.g., in Altman et al. (1996). Some immunodetection methods that may be used include, e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, tetramer assay, and Western blot. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev, 1999; Gulbis and Galand, 1993; De Jager et al., 1993; and Nakamura et al., 1987, each incorporated herein by reference.VIII. MHC POLYPEPTIDES

[0138] Embodiments of the disclosure relate to compositions comprising MHC polypeptides. In some embodiments, the MHC polypeptide comprises at least 2, 3, or 4 MHC polypeptides that may be expressed as separate polypeptides or as a fusion protein. Presentation of antigens to T cells is mediated by two distinct classes of molecules MHC class I (MHC-I) and MHC class II (MHC-II) (also identified as “pMHC” herein), which utilize distinct antigen processing pathways. Peptides derived from intracellular antigens are presented to CD8+ T cells by MHC class I molecules, which are expressed on virtually all cells, while extracellular antigen-derived peptides are presented to CD4+ T cells by MHC-II molecules. In certain embodiments, a particular antigen is identified and presented in the antigen-MHC complex in the context of an appropriate MHC class I or II polypeptide. In certain aspects, the genetic makeup of a subject may be assessed to determine which MHC polypeptide is to be used for a particular patient and a particular set of peptides. In certain embodiments, the MHC class I polypeptide comprises all or part of a HLA-A, HLA-B, HLA- C, HLA-E, HLA-F, HLA-G or CD-I molecule. In embodiments wherein the MHC polypeptide is a MHC class II polypeptide, the MHC class II polypeptide can comprise all or a part of a HLA-DR, HLA-DQ, or HLA-DP.

[0139] Non-classical MHC polypeptides are also contemplated for use in MHC complexes of the invention. Non-classical MHC polypeptides are non-polymorphic, conserved among species, and possess narrow, deep, hydrophobic ligand binding pockets. These binding pockets are capable of presenting glycolipids and phospholipids to Natural Killer T (NKT) cells or certain subsets of CD8+ T cells such as Qal, HLA-E-restricted CD8+ T cells, or MAIT cells. NKT cells represent a unique lymphocyte population that co-express NK cell markers and a semi-invariant T cell receptor (TCR). They are implicated in the regulation of immune responses associated with a broad range of diseases.IX. HOST CELLS

[0140] As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include both freshly isolated cells and ex vivo cultured, activated or expanded cells. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence,“host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

[0141] In certain embodiments transfection can be carried out on any prokaryotic or eukaryotic cell. In some aspects electroporation involves transfection of a human cell. In other aspects electroporation involves transfection of an animal cell. In certain aspects transfection involves transfection of a cell line or a hybrid cell type. In some aspects the cell or cells being transfected are cancer cells, tumor cells or immortalized cells. In some instances tumor, cancer, immortalized cells or cell lines are induced and in other instances tumor, cancer, immortalized cells or cell lines enter their respective state or condition naturally. In certain aspects the cells or cell lines can be A549, B-cells, B16, BHK-21, C2C12, C6, CaCo-2, CAP / , CAP-T, CHO, CHO2, CHO-DG44, CHO-K1, COS-1, Cos-7, CV-1, Dendritic cells, DLD-1, Embryonic Stem (ES) Cell or derivative, H1299, HEK, 293, 293T, 293FT, Hep G2, Hematopoietic Stem Cells, HOS, Huh-7, Induced Pluripotent Stem (iPS) Cell or derivative, Jurkat, K562, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, Mesenchymal Cells, Min-6, Monocytic cell, Neuro2a, NIH 3T3, NIH3T3L1, K562, NK cells, NSO, Panc-1, PC12, PC-3, Peripheral blood cells, Plasma cells, Primary Fibroblasts, RBL, Renca, RLE, SF21, SF9, SH-SY5Y, SK-MES- 1, SK-N-SH, SL3, SW403, Stimulus-triggered Acquisition of Pluripotency (STAP) cell or derivate SW403, T cells, THP-1, Tumor cells, U2OS, U937, peripheral blood lymphocytes, expanded T cells, hematopoietic stem cells, or Vero cells.X. ADDITIONAL AGENTSA. Immunostimulators

[0142] In some embodiments, the method further comprises administration of an additional agent. In some embodiments, the additional agent is an immuno stimulator. The term “immuno stimulator” as used herein refers to a compound that can stimulate an immune response in a subject, and may include an adjuvant. In some embodiments, an immuno stimulator is an agent that does not constitute a specific antigen, but can boost the strength and longevity of an immune response to an antigen. Such immunostimulators may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combinedwith monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL.RTM. (ASO4), MPL A of above-mentioned bacteria separately, saponins, such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.), liposomes and liposomal formulations such as AS01, synthesized or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic block co-polymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide 4 phosphates, such as RC529, or proteins, such as bacterial toxoids or toxin fragments.

[0143] In some embodiments, the additional agent comprises an agonist for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and / or combinations thereof. In some embodiments, additional agents comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited immunostimulators comprise imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381, U.S. Published Patent Application 2010 / 0075995, or WO 2010 / 018132; immuno stimulatory DNA; or immunostimulatory RNA. In some embodiments, the additional agents also may comprise immunostimulatory RNA molecules, such as but not limited to dsRNA, poly I:C or poly Lpoly C12U (available as Ampligen.RTM., both poly I:C and poly I:polyC12U being known as TLR3 stimulants), and / or those disclosed in F. Heil et al., "Species-Specific Recognition of Single Stranded RNA via Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway" WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity" U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections" WO 2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods" WO 2003086280 A2. In some embodiments, an additional agent may be a TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB-1. In some embodiments, additional agents may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725.

[0144] In some embodiments, additional agents may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals). In some embodiments, additional agents may be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some embodiments, additional agents may be activated components of immune complexes. Additional agents also include complement receptor agonists, such as a molecule that binds to CD21 or CD35. In some embodiments, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some embodiments, immuno stimulators are cytokines, which are small proteins or biological factors (in the range of 5 kD-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. In some embodiments, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.B. Immunotherapies

[0145] In some embodiments, the additional therapy comprises a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour- associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immumotherapies are known in the art, and some are described below.1. Inhibition of co-stimulatory molecules

[0146] In some embodiments, the immunotherapy comprises an inhibitor of a costimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.2. Dendritic cell therapy

[0147] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalianimmune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0148] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).

[0149] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0150] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0151] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.3. CAR-T cell therapy

[0152] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0153] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T cell activating functions. The general premise of CAR-T cells is to artificially generate T cells targeted to markers found on cancer cells. Scientists can remove T cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognitiondomain to an intracellular signalling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.

[0154] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19.4. Cytokine therapy

[0155] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0156] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFN ).

[0157] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.5. Adoptive T cell therapy

[0158] Adoptive T cell therapy is a form of passive immunization by the transfusion of T cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumour death.

[0159] Multiple ways of producing and obtaining tumour targeted T cells have been developed. T cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.6. Checkpoint Inhibitors and Combination Treatment

[0160] In some embodiments, the additional therapy comprises immune checkpoint inhibitors. Certain embodiments are further described below. a. PD- 1 , PDL 1 , and PDL2 inhibitors

[0161] PD- 1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL 1 activity.

[0162] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0163] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD 1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL 1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0164] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments,the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7 DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0165] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0166] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. b. CTLA-4, B7-l, and B7-2

[0167] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T cell co- stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activationthrough the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0168] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0169] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0170] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0171] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.C. Oncolytic virus

[0172] In some embodiments, the additional therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumour. Oncolytic viruses are thought not only to cause direct destruction of the tumour cells, but also to stimulate host anti-tumour immune responses for long-term immunotherapy.D. Polysaccharides

[0173] In some embodiments, the additional therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.E. Chemotherapies

[0174] In some embodiments, the additional therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon- a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0175] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral orintraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg / m2 to about 20 mg / m2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments. In some embodiments, the amount of cisplatin delivered to the cell and / or subject in conjunction with the construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.

[0176] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter / TNFa construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and / or TNF-a, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a.

[0177] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain embodiments, appropriate intravenous doses for an adult include about 60 mg / m2 to about 75 mg / m2 at about 21 -day intervals or about 25 mg / m2 to about 30 mg / m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone- marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.

[0178] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (E-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria), is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0179] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorideoxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0180] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.

[0181] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.F. Radiotherapy

[0182] In some embodiments, the additional therapy or prior therapy comprises radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0183] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18,19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any derivable range therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the does may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0184] In some embodiments, the amount of IR may be presented as a total dose of IR, which is then administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 (or any derivable range therein). In some embodiments, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein. In some embodiments, at least, at most, or exactly 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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 fractionated doses are administered (or any derivable range therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some embodiments, at least, at most, or exactly 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, or 30 (or any derivable range therein) fractionated doses are administered per week.G. Surgery

[0185] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as thetreatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electro surgery, and microscopically-controlled surgery (Mohs’ surgery).

[0186] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.H. Other Agents

[0187] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.XL PROTEINACEOUS COMPOSITIONS

[0188] As used herein, a “protein” “peptide” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemicalstructure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.

[0189] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In particular embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0190] In certain embodiments the size of a peptide, protein, or polypeptide (wild-type or modified), such as a peptide or protein of the disclosure comprising a peptide of one of SEQ ID NOS:1-192 may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). It is specifically contemplated that any one or more peptides of one of SEQ ID NOS: 1-192 may be excluded in one or more embodiments.

[0191] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may include 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids or nucleic acid substitutions or be 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% (or any derivable range therein) similar, identical, or homologous in sequence to at least, or at most 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of a peptide of one of SEQ ID NOS: 1-192 or nucleic acids encoding a peptide of one of SEQ ID NOS: 1-192. In certain embodiments, the peptide or polypeptide is not naturally occurring and / or is in a combination of peptides or polypeptides.

[0192] In some embodiments, the protein or polypeptide may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or any derivable range therein) of a peptide of one of SEQ ID NOS: 1- 192. In some embodiments, the peptides of the disclosure comprise at least, at most, or exactly 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) flanking the caboxy and / or flanking the amino end of a peptide comprising or consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of a peptide of one of SEQ ID NOS:1-192.

[0193] In some embodiments, the protein, polypeptide, or nucleic acid may comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or any derivable range therein) contiguous amino acids of a peptide of one of SEQ ID NOS: 1-192.

[0194] In some embodiments, the polypeptide, protein, or nucleic acid may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or any derivable range therein) contiguous amino acids of a peptide of one of SEQ ID NOS: 1- 192 that are at least, at most, or exactly 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% (or any derivable range therein) similar, identical, or homologous to a peptide of one of SEQ ID NOS:1-192.

[0195] In some aspects there is a polypeptide (or a nucleic acid molecule encoding such a polypeptide) starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of a peptide of one of SEQ ID NOS: 1-192 and comprising at least, at most, or exactly 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 (or any derivable range therein) contiguous amino acids of a peptide of one of SEQ ID NOS: 1-192.

[0196] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0197] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’ s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0198] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0199] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type (or any range derivable therein). A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0200] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminalsequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0201] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0202] Insertional mutants typically involve the addition of amino acid residues at a non terminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0203] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0204] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.

[0205] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. In further embodiments, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0206] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathy indices are within +2 is included. In some aspects of the invention, those that are within +1 are included, and in other aspects of the invention, those within +0.5 are included.

[0207] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity valueshave been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (_0.5); histidine (_0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within +2 are included, in other embodiments, those which are within +1 are included, and in still other embodiments, those within +0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0208] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0209] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of a polypeptide with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy . org / proteomic s / protein_s tructure .

[0210] In some embodiments of the invention, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5)confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).XII. NUCLEIC ACIDS

[0211] In certain embodiments, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding peptides and polypeptides of the disclosure, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein. Nucleic acids encoding fusion proteins that include these peptides are also provided. The nucleic acids can be single-stranded or double- stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0212] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.

[0213] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may beadapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0214] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0215] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.A. Hybridization

[0216] Nucleic acids can hybridize, for example to other nucleic acids, under particular hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, a moderately stringent hybridization condition uses a prewashing solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6xSSC, and a hybridization temperature of 55° C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42° C), and washing conditions of 60° C. in 0.5xSSC, 0.1% SDS. A stringent hybridization condition hybridizes in 6xSSC at 45° C., followed by one or more washes in O.lxSSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequence that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other typically remain hybridized to each other.

[0217] The parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by, for example, Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are herein incorporated by reference in their entirety for all purposes) and can be readily determined by those having ordinary skill in the art based on, for example, the length and / or base composition of the DNA.B. Mutation

[0218] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigenic peptide or polypeptide) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site- directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0219] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, eg., Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.C. Probes

[0220] In another aspect, nucleic acid molecules are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences. A nucleic acid molecule can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion of a given polypeptide.

[0221] In another embodiment, the nucleic acid molecules may be used as probes or PCR primers for specific nucleic acid sequences. For instance, a nucleic acid molecule probe may be used in diagnostic methods or a nucleic acid molecule PCR primer may be used to amplify regions of DNA that could be used, inter alia, to isolate nucleic acid sequences for use in producing the engineered cells of the disclosure. In a preferred embodiment, the nucleic acid molecules are oligonucleotides.

[0222] Probes based on the desired sequence of a nucleic acid can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of interest. The probe can comprise a label group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide. XIII. POLYPEPTIDE EXPRESSION

[0223] In some aspects, there are one or more nucleic acid molecule encoding polypeptides or peptides of the disclosure (e.g antibodies, TCR genes, MHC molecules, and immunogenic peptides). These may be generated by methods known in the art, e.g., isolated from B cells of mice that have been immunized and isolated, phage display, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules or by recombinant methods.

[0224] The nucleic acid molecules may be used to express large quantities of polypeptides. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for humanization of the antibody or TCR genes.A. Vectors

[0225] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigen binding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, antigenic peptides and polypeptides, TCR genes, MHC molecules, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0226] To express the polypeptides or peptides of the disclosure, DNAs encoding the polypeptides or peptides can be inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In some aspects, a vector that encodes a functionally complete human TCR alpha or TCR beta sequence with appropriate restriction sites engineered so that any variable sequence or CDR1, CDR2, and / or CDR3 can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art.B. Expression Systems

[0227] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and / or eukaryote-based systems can beemployed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.C. Methods of Gene Transfer

[0228] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.D. Host Cells

[0229] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. Polypeptides can be expressed in a variety of cell types. An expression construct encoding a polypeptide or peptide of the disclosure can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0230] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.XIV. FORMULATIONS AND CULTURE OF THE CELLS

[0231] In particular embodiments, the cells of the disclosure may be specifically formulated and / or they may be cultured in a particular medium. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.

[0232] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.

[0233] The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The serum-free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0234] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98 / 30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0235] In certain embodiments, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and / or T3 (triodo-I-thyronine). . In specific embodiments, one or more of these may be explicitly excluded.

[0236] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises proteins. In some embodiments, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin,transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, or combinations thereof. In some embodiments, the medium comprises or futher comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific embodiments, one or more of these may be explicitly excluded.

[0237] The medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts. . In specific embodiments, one or more of these may be explicitly excluded.

[0238] One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, pg / ml, mg / ml, or any range derivable therein.

[0239] In specific embodiments, the cells of the disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases the cells are formulated for storage prior to use, and the cell formulation may compriseone or more cryopreservation agents, such as DMSO (for example, in 5% DMSO). The cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. The cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In particular embodiments the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing.

[0240] In some embodiments, the method further comprises priming the T cells. In some embodiments, the T cells are primed with peptides disclosed herein, and / or antigen presenting cells. In some embodiments, the antigen presenting cells present tumor antigens or peptides, such as those disclosed herein.

[0241] In particular embodiments, the cells of the disclosure comprise an exogenous TCR, which may be of a defined antigen specificity, such as defined antigen specificity to any one of SEQ ID NOS: 1-192. In some embodiments, the TCR can be selected based on absent or reduced alloreactivity to the intended recipient (examples include certain virus- specific TCRs, xeno-specific TCRs, or cancer testis antigen- specific TCRs). In the example where the exogenous TCR is non alloreactive, during T cell differentiation the exogenous TCR suppresses rearrangement and / or expression of endogenous TCR loci through a developmental process called allelic exclusion, resulting in T cells that express only the non- alloreactive exogenous TCR and are thus non-alloreactive. In some embodiments, the choice of exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some embodiments, the endogenous TCR genes have been modified by genome editing so that they do not express a protein. Methods of gene editing such as methods using the CRISPR / Cas9 system are known in the art and described herein.XV. ADMINISTRATION OF THERAPEUTIC COMPOSITIONS

[0242] Methods of the disclosure relate to the treatment of subjects with cancer. In some embodiments, the treatment may be directed to those that have or have been determined to have a cancer expressing a neoantigen having the sequence of a particular peptide of the disclosure, such as a peptide of one of SEQ ID NOS: 1-192. In some embodiments, the methods may be employed with respect to individuals who have tested positive for such cancer, who have one or more symptoms of a cancer, or who are deemed to be at risk for developing such a cancer.

[0243] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first anti-cancer therapy and a second anti-cancer therapy. The therapies may be administered in any suitable manner known in the art. For example, the firstand second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the first and second cancer treatments are administered in a separate composition. In some embodiments, the first and second cancer treatments are in the same composition.

[0244] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0245] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0246] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0247] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg / kg to 200 mg / kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg / kg, mg / kg, pg / day, or mg / day or any range derivable therein. Furthermore, suchdoses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0248] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 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 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0249] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0250] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of pg / ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0251] In select embodiments, it is contemplated that a peptide of the disclosure may be comprised in a vaccine composition and administered to a subject to induce a therapeutic immune response in the subject towards a cancer. A vaccine composition for pharmaceutical use in a subject may comprise a peptide composition disclosed herein and a pharmaceutically acceptable carrier.

[0252] The phrases "pharmaceutical,” “pharmaceutically acceptable,” or “pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Pharmaceutical Press, 2011, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the vaccine compositions of the present invention is contemplated.

[0253] As used herein, a "protective immune response" refers to a response by the immune system of a mammalian host to a cancer. A protective immune response may provide a therapeutic effect for the treatment of a cancer, e.g., decreasing tumor size, increasing survival, etc.

[0254] In some embodiments, the vaccine composition may be administered by microstructured transdermal or ballistic particulate delivery. Microstructures as carriers for vaccine formulation are a desirable configuration for vaccine applications and are widely known in the art (Gerstel and Place 1976 (U.S. Patent 3,964,482); Ganderton and McAinsh 1974 (U.S. Patent 3,814,097); U.S. Patents 5,797,898, 5,770,219 and 5,783,208, and U.S. Patent Application 2005 / 0065463). Such a vaccine composition formulated for ballistic particulate delivery may comprise an isolated peptide disclosed herein immobilized on a surface of a support substrate. In these embodiments, a support substrate can include, but is not limited to, a microcapsule, a microparticle, a microsphere, a nanocapsule, a nanoparticle, a nanosphere, or a combination thereof.

[0255] In other embodiments, a vaccine composition comprises an immobilized or encapsulated peptide or antibody as disclosed herein and a support substrate. In theseembodiments, a support substrate can include, but is not limited to, a lipid microsphere, a lipid nanoparticle, an ethosome, a liposome, a niosome, a phospholipid, a sphingosome, a surfactant, a transferosome, an emulsion, or a combination thereof. The formation and use of liposomes and other lipid nano- and microcarrier formulations is generally known to those of ordinary skill in the art, and the use of liposomes, microparticles, nanocapsules and the like have gained widespread use in delivery of therapeutics (e.g., U.S. Patent 5,741,516, specifically incorporated herein in its entirety by reference). Numerous methods of liposome and liposomelike preparations as potential drug carriers, including encapsulation of peptides, have been reviewed (U.S. Patents 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587, each of which is specifically incorporated in its entirety by reference).

[0256] In addition to the methods of delivery described herein, a number of alternative techniques are also contemplated for administering the disclosed vaccine compositions. By way of nonlimiting example, a vaccine composition may be administered by sonophoresis (i.e., ultrasound) which has been used and described in U.S. Patent 5,656,016 for enhancing the rate and efficacy of drug permeation into and through the circulatory system; intraosseous injection (U.S. Patent 5,779,708), or feedback-controlled delivery (U.S. Patent 5,697,899), and each of the patents in this paragraph is specifically incorporated herein in its entirety by reference.XVI. DETECTION AND VACCINATION KITS

[0257] A peptide or antibody of the disclosure may be included in a kit. The peptide or antibody in the kit may be detectably labeled or immobilized on a surface of a support substrate also comprised in the kit. The peptide(s) or antibody may, for example, be provided in the kit in a suitable form, such as sterile, lyophilized, or both.

[0258] The support substrate comprised in a kit of the invention may be selected based on the method to be performed. By way of nonlimiting example, a support substrate may be a multi- well plate or microplate, a membrane, a filter, a paper, an emulsion, a bead, a microbead, a microsphere, a nanobead, a nanosphere, a nanoparticle, an ethosome, a liposome, a niosome, a transferosome, a dipstick, a card, a celluloid strip, a glass slide, a microslide, a biosensor, a lateral flow apparatus, a microchip, a comb, a silica particle, a magnetic particle, or a selfassembling monolayer.

[0259] As appropriate to the method being performed, a kit may further comprise one or more apparatuses for delivery of a composition to a subject or for otherwise handling a composition of the invention. By way of nonlimiting example, a kit may include an apparatus that is a syringe, an eye dropper, a ballistic particle applicator (e.g., applicators disclosed inU.S. Patents 5,797,898, 5,770,219 and 5,783,208, and U.S. Patent Application 2005 / 0065463), a scoopula, a microslide cover, a test strip holder or cover, and such like.

[0260] A detection reagent for labeling a component of the kit may optionally be comprised in a kit for performing a method of the present invention. In particular embodiments, the labeling or detection reagent is selected from a group comprising reagents used commonly in the art and including, without limitation, radioactive elements, enzymes, molecules which absorb light in the UV range, and fluorophores such as fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. In other embodiments, a kit is provided comprising one or more container means and a BST protein agent already labeled with a detection reagent selected from a group comprising a radioactive element, an enzyme, a molecule which absorbs light in the UV range, and a fluorophore.

[0261] When reagents and / or components comprising a kit are provided in a lyophilized form (lyophilisate) or as a dry powder, the lyophilisate or powder can be reconstituted by the addition of a suitable solvent. In particular embodiments, the solvent may be a sterile, pharmaceutically acceptable buffer and / or other diluent. It is envisioned that such a solvent may also be provided as part of a kit.

[0262] When the components of a kit are provided in one and / or more liquid solutions, the liquid solution may be, by way of non-limiting example, a sterile, aqueous solution. The compositions may also be formulated into an administrative composition. In this case, the container means may itself be a syringe, pipette, topical applicator or the like, from which the formulation may be applied to an affected area of the body, injected into a subject, and / or applied to or mixed with the other components of the kit.XVII. SEQUENCESTable 1. PeptidesXVIII. EXAMPLES

[0263] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0264] Lynch syndrome (LS) carriers develop mismatch repair deficient (MMRd) tumors, which generate high loads of neoantigens (neoAgs) and subsequent immune activation. As described in Examples 1-3, and elsewhere herein, paired whole exome and transcriptomic sequencing data from colorectal lesions (13 cancers and 61 pre-cancers) of an institutional LS cohort (N=46) were used to identify shared neoAgs derived from insertion-deletion mutations in microsatellites (referred to interchangeably herein as neoantigens, neoAgs, frameshift neoAgs, FS-neoAgs, frameshift protein neoAgs, and FSP-neoAgs). A custom computational pipeline was used to rank candidate neoAgs based on their recurrence and predicted immunogenicity. The inventors demonstrated that 65% of the top predicted neoAgs were immunogenic in vitro via ELISpot assays. The results demonstrated that the in silico computational pipelines accurately predict the immunogenicity of LS neoAgs, and that the predicted neoAgs have the potential to drive an effective anti-cancer immune response. The results support that the predicted neoAg peptides provide the foundation for developing treatments for LS carriers, including an immunoprevention vaccine.

[0265] As described in Examples 1-3, and elsewhere herein, the inventors acquired paired whole-exome sequencing (WES) and mRNAseq data from early-stage LS CRC and precancers to predict and identify the most immunogenic and recurrent frameshift-neoAgs (FS-neoAgs) present in LS colorectal carcinogenesis using systems biology approaches, as illustrated in FIG. 1. The analysis pipeline accurately identified somatic micro satellite (MS) indels by overcoming errors produced during short read sequencing, PCR amplification, and othersources of noise. It also accounted for the allele frequency of a given mutated frameshift protein at both DNA and RNA levels, the binding affinity and stability of the FS-neoAgs, and the most frequent human leukocyte antigen (HLA) genotypes. Immunological assays validated the predicted immunogenicity of FS-neoAgs from the computational methods. The results provide immunogenic peptides based on recurrent neoantigens present in subjects with LS, and support the utility of the peptides in therapies for preventing and treating LS cancers.

[0266] Identifying and characterizing neoantigen-reactive T cells and their T cell receptors (TCRs) is crucial for mapping neoantigen-driven T cell immunity, understanding mechanisms of immune evasion, and optimizing therapeutic interventions targeting LS tumors. As described in Examples 4-12, and elsewhere herein, the inventors further evaluated immunologically relevant characteristics of select FSP-neoAgs, including RNF43_2, RNF43_3, TGFBR2_4, BMPR2, and MSH3, using PBMCs of 20 LS carriers, including 10 previvors (LS carriers with no history of cancer) and 10 survivors (LS carriers with a history of cancer). Among other things, the inventors demonstrated that: i. all tested FSP-neoAgs were immunogenic and induced T cell responses when averaging responses from all LS carriers following short-term stimulation, including in both survivors and previvors (see, e.g., Example 4); ii. some FSP-neoAgs identified herein are presented across a range of HLA types, and are thus of broader immunogenic potential (see, e.g., Example 5); iii. as measured in a 9-day in vitro priming period, all four FSP-neoAgs tested overcame immunogenicity challenges associated with HLA variability, thus enhancing the immune response to all tested antigens in patients, regardless of their HLA match (see, e.g., Example 6); iv. LS carriers exhibit enhanced FSP-neoAg-specific immunogenic responses over time, regardless of status as survivor or previvor (see, e.g., Example 7); v. FSP-neoAg-stimulated T cells from LS carriers, including both survivors and previvors, exhibit significant tumor cytotoxicity in vitro using microfluidic co-culture systems (see, e.g., Example 8); vi. both LS survivors and previvors harbor circulating T cells responsive to each of the five FSP-neoAgs tested, suggesting an active state of immunosurveillance in both previvors and survivors (see, e.g., Example 9); vii. in both LS previvors and survivors, FSP-neoAg -responsive T cells exhibit distinct gene signatures; andviii. FSP-neoAgs described herein, and T cells reactive thereto, are valuable biomarkers for early detection of cancer in LS carriers.

[0267] Examples 4-11, and as described elsewhere herein, demonstrate identification of circulating neo Ag- specific T cell transcriptomic states and TCRs in the peripheral blood of LS patients, providing critical insights into the immunobiological mechanisms underlying cancer susceptibility in LS. Examples 4-11, and as described elsewhere herein, offers a comprehensive snapshot of the neo antigen- specific circulating T cell landscape and TCR repertoire in LS carriers across various stages of disease progression, advancing the understanding of T cell immune evolution and mechanisms of immune escape. The identification of neoantigenspecific hyperexpanded and tumor-targeted TCR clones establishes a foundation for immune monitoring in LS patients post-vaccination and highlights the importance of neoantigen quality and evolution in designing effective vaccines and selecting biomarkers for immunoprevention.EXAMPLE 1: DEMOGRAPHIC AND GENOMIC CHARACTERISTICS OF LS SAMPLES

[0268] Analysis was performed on a total of 74 colorectal lesions (61 pre-cancers and 13 tumors) from 46 LS patients with matched adjacent normal mucosa and peripheral blood. Mean age was 52 years (range, 20 - 80) and the majority of patients harbored germline mutations in MSH2 and MSH6 (Table 2). Two patients met Amsterdam Criteria but had no detectable germline mutation. Thirty-six patients (78%) had a history of cancer at the time of diagnosis, with CRC being the most commonly diagnosed cancer followed by endometrial cancer. Among the 61 precancers, 44 were confirmed to be early tubular adenomas, two tubulovillous adenomas, four sessile serrated adenomas (SSA), nine hyperplastic polyps (HP), and two inflammatory polyps (IP). All cancers were confirmed to be stage I to III adenocarcinomas (n=13). Microsatellite instability (MSI) status was determined using MSIsensor2 (Niu B, Ye K, Zhang Q, Lu C, Xie M, McLellan MD, et al. MSIsensor: micro satellite instability detection using paired tumor-normal sequence data. Bioinformatics 2014;30(7): 1015-6 doi 10.1093 / bioinformatics / btt755) with 27 samples classified as MSLH, 15 as MSLL, and the remaining as MSS. Notably, the median MSIscore for precancers was 13.6% with 23% (12 / 53) of them displaying MSLH and 28% (15 / 53) MSLL. Lor advanced pre-cancers, the median MSIscore was 30%, with 50% of them displaying MSLH. Most cancers (11 / 13) were MSLH with a median MSIscore of 65.2%. The top 10 most unstable MS sites detected from MSLH lesions generated novel shorter alleles compared with matched normal samples and included loci within CREBBP, ERBB2, GTF2IRD, ACVR2A.

[0269] To identify somatic hits in MMR genes, germline mutations detected in clinical genetic testing were confirmed in LS patients using HaplotypeCaller. The types of detected heterozygous germline MMR mutations consisted of nonsense mutations (26%), frameshift indels (24%), splicing events (13%), exon deletions (11%), and missense mutations (9%). Somatic hits were identified in the alternate allele of the gene harboring the germline mutation in more than 66% (18 / 27) of the MSI-H samples, thus explaining the MMRd phenotype of these lesions.Table 2. Summary of patient demographics and characteristics of lesionsEXAMPLE 2: IN SILICO PREDICTION GENERATED RANKED LISTS OF NEOANTIGENS BASED ON IMMUNOGENICITY

[0270] A systems biology neoantigens (neoAgs) prediction pipeline was developed to identify and catalogue those neoAgs produced from frameshift mutations in pre-cancers and tumors from the LS cohort by applying a series of computational methods that are publicly available (FIG. 2). First, MHC class I and II typing of all samples from WES data was performed using PHLAT (Bai Y, Ni M, Cooper B, Wei Y, Fury W. Inference of high resolution HLA types using genome-wide RNA or DNA sequencing reads. BMC Genomics 2014;15:325 doi 10.1186 / 1471-2164-15-325). Among HLAs, the two most frequently found in the sample cohort were HLA-A*01:01 and HLA-A*02:01, present in 72 / 74 and 61 / 74 samples,respectively (Table 3). Second, prediction of potential neoAgs in each gene with frameshift mutation in MS loci was calculated using the NetMHCpan algorithm (Reynisson B, Alvarez B, Paul S, Peters B, Nielsen M. NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res 2020;48(Wl):W449-W54 doi 10.1093 / nar / gkaa379). The analysis identified several potential neoepitopes predicted to bind different HLAs (FIG. 3A). Those epitopes with highest affinity to 14 HLA class I and 8 class II types were selected. These 22 HLA alleles covered 97% of the LS sample cohort, had an allele frequency (AF)>0.1 in any population (race) and had a sample size > 100,000 in the HLA database from the National Marrow Donor Program (Fig. 3B) (Ballen KK, King RJ, Chitphakdithai P, Bolan CD, Jr., Agura E, Hartzman RJ, et al. The national marrow donor program 20 years of unrelated donor hematopoietic cell transplantation. Biol Blood Marrow Transplant 2008; 14(9 Suppl):2-7 doi 10.1016 / j.bbmt.2008.05.017). Third, neoepitopes were ranked based on the AF of the mutation at the DNA level and the number of transcripts per million (TPM) at the RNA level, respectively, and considering the frequency of these neoAgs in the patient population. The final ranking of the predicted immunogenic epitopes was based on a combination of four criteria: binding affinity (<100 nM), binding stability (>1.5 h), a foldchange of wildtype over mutant of > 10, and a dissimilarity score when compared to the human reference proteome of >0 (FIG. 2).Table 3. Frequency of HLA allelels that are present within the cohort of LS patients.

[0271] The prediction identified a total number of MHC-I and MHC-II neoAgs per sample ranging from 0 to -1320 and 0 to -9300, respectively, with a majority of neoAgs bearing a high predicted binding affinity (<50 nM) (FIG. 4). In concordance with the mutational rate, MSLH samples showed significantly higher numbers of MHC-I and MHC-II neoAgs (Mann- Whitney test, P<0.0001; FIG. 5A). Similarly, cancers showed significantly more neoAgs than pre-cancers, when compared based on tissue category or pathology (Mann-Whitney test, P<0.01; FIG. 5B and FIG. 5C). Nonetheless, a remarkable proportion of pre-cancers (19%) and advanced lesions (50%) displayed high numbers of predicted neoantigens. This was further evidenced by comparing the number of neoAgs identified per sample with their mutational rate, which showed a significant positive correlation for both MHC-I and MHC-II neoAgs (Pearson, P<0.001; FIG. 5D).

[0272] The top 100 MHC-I predicted frameshift neoantigens (FS-neoAgs) are shown in Table 4 and Table 5. Of these, the genes from which the top 50 most recurrent and immunogenic neoAgs were predicted are shown in FIG. 5E. The pipeline identified a set of novel recurrent MHC-I restricted neoAgs with high immunogenic potential, predicted to be generated in genes that include USP35, TCF20, OR4M2, PDS5B, and DAZAP1, among others. It also identified highly recurrent and immunogenic neoAgs from previously reported genes, including SETD1B, RNF43, TGFBR2, BMPR2, MSH3, and TMEM97 (Roudko V, Bozkus CC, Orfanelli T, McClain CB, Carr C, O'Donnell T, et al. Shared Immunogenic Poly-Epitope Frameshift Mutations in Microsatellite Unstable Tumors. Cell 2020; 183(6): 1634-49 el7 doi 10.1016 / j.cell.2020.11.004; Ballhausen A, Przybilla MJ, Jendrusch M, Haupt S, Pfaffendorf E, Seidler F, et al. The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution. Nat Commun 2020;l l(l):4740 doi 10.1038 / s41467-020-18514-5; Leoni G, D'Alise AM, Cotugno G, Langone F, Garzia I, De Lucia M, et al. A Genetic Vaccine Encoding Shared Cancer Neoantigens to Treat Tumors withMicrosatellite Instability. Cancer Res 2020;80(18):3972-82 doi 10.1158 / 0008-5472. CAN-20- 1072). These predicted neoAgs originated primarily in MS-sites of mononucleotide repeats that acquired frameshift mutations, thus leading to the reduction of the specific MS length. Similarly, the MHC-II restricted neoAgs were also predicted and catalogued (Table 6). The landscape of the genes that generated the top 50 most recurrent MHC-II restricted neoAgs included 17 originating from the same genes that generated top MHC-I neoAgs (FIG. 6), thus suggesting that the same MS loci contribute to both MHC-I and MHC-II epitopes. Of importance, the same MSI-H pre-cancers with high mutational burden consistently showed increased levels of predicted MHC-I and MHC-II neoAgs. In this context, 12 / 27 (44%) of all MSI-H samples that showed high production of predicted and shared neoAgs were pre-cancers, specifically tubular adenomas (FIG. 5E and FIG. 6). Overall, 12 / 39 (30%) of tubular adenomas without signs of high-grade dysplasia or other high-risk features displayed MSI-H and high neoAgs loads, thus stimulating the immune system.Table 4. MHC-I restricted frameshift neoantigen peptide characteristicsTable 5. MHC-I restricted frameshift neoantigen peptide characteristics and rankingTable 6. MHC-II restricted neoantigen peptide characteristicsEXAMPLE 3: IN SILICO PREDICTED NEOANTIGENS DEMONSTRATE A HIGH VALIDATION RATE OF IN VITRO IMMUNOGENICITY WITH CLONAL EXPANSION OF ACTIVATED CD8+ AND CD4+ T CELLS

[0273] To validate in vitro the immunogenicity of the list of predicted neoAgs, the top 100 MHC-I restricted neoAgs were selected, and each individual peptide was synthesized with purity >95%. Healthy donor PBMCs with different HLA types were stimulated with individual peptides to expand neo Ag- specific T cells, followed by quantitative ELISpot assays to assess the level of immunogenicity (FIG.7A). A neoantigen peptide was considered ELIspot-reactivewhen it generated: i) at least five times more spot-forming units (SFUs) / 10A5 cells compared to the DMSO control, when averaging the responses from all human donors, and ii) a significantly higher number of SFUs / 10A5 cells compared to the DMSO control, using a paired Wilcoxon test. Overall, reactive peptides generated significantly higher mean SFUs compared to non-reactive peptides, when averaging the responses from all healthy donors (FIG. 7B). The data showed a very high validation rate, with 65 of the 100 peptides eliciting significant secretion of IFNy compared to unexposed control cells (FIG. 7C, FIG. 8A, and Table 7). The remaining 35 neoAgs were non- significant in eliciting IFNy-secretion in donor PBMCs (FIG. 8B and Table 7), which might be partly due to their affinity to rare HLA alleles, low expression of those HLA genes in donors, or poor stimulation in expansion of CD8+ T cells, although the mechanism is not clear. Several ELISpot-reactive neoAgs were derived from genes involved in different types of cancer development pathways including TGFBR2, MSH3, AX1N2, TP53, HOXA11, TTK, and SMC3. A summary of the ELISpot assay results for each of the tested MHC-I restricted neoantigen peptides is shown in Table 7.Table 7. MHC-I restricted neoantigen ELISPOT assay results

[0274] The RNF43_3 epitope (TQLARFFPI; SEQ ID NO:1) was selected, given that this was the neoAg with the highest ELISpot-reactivity, to assess the level of activation of CD8+ T cells. After T cell staining using PE-conjugated RNF43_3 HLA-A*02:01 tetramers, anti-human CD8-PerCP, and 41BB-APC-Cy7 (a marker for activated CD8+ T cells) antibodies, up to 5% of the T cell population as RNF43_3 tetramer- specific CD8+ T cells were enriched, and 1% activated RNF43_3 tetramer positive T cells based on 4- IBB labeling were enriched (FIG. 7D). The RNF43_3 epitope was also used to determine the percentage of CD8+ and CD4+ T cells in stimulated PBMCs from the six donors used for the ELISpot experiments. The results showed that for all donors the percentage of CD8+ and CD4+ T cells was significantly higher in RNF43_3-stimulated PBMCs, compared to unstimulated controls (FIGS. 7E-F). Furthermore, staining with 41BB-APC-Cy7 antibody indicated that the percentage of CD8 / 41BB double positive T cells was significantly higher in PBMCs after stimulation with RNF43_3 compared to unstimulated controls (FIGS. 7E-F). Overall, these results confirm that the predicted neoAgs can elicit an immunogenic response with clonal expansion of activated CD8+ and CD4+ T cells.

[0275] In conclusion, a state-of-the-art bioinformatics pipeline predicted a catalog of recurrent and immunogenic neoAgs. The inventors validated the immunogenicity of the MHC- I peptides. The in vitro validation confirms the accuracy of in silico prediction of the immunogenic neoAg. The data support using these neoAgs in vaccine-based immunoprevention strategies for LS patients to prevent the development of CRC.Example 4. Immunogenic Profiles of Neoantigen-Specific T Cells demonstrates that Lynch Syndrome (LS) Carrier T cells respond to FSP-neoAgs independent of disease status

[0276] One of the primary challenges in frame shift protein (FSP)-neoAg discovery is accurately identifying peptides that elicit a strong immunogenic response. To enhance the precision of NeoAg- specific T cell discovery and immunogenicity assessments described elsewhere herein (e.g., Examples 1-3), five immunogenic and recurrent FSP-neoAg peptides from CRC-associated genes were selected. These FSP-neoAgs were highly immunogenic, derived from genes exhibiting a high frequency of non-silent mutations in microsatellite loci within the MSI-H phenotype, and served as the primary focus of Examples 4-1 IX. Specific T cell responses to these five recurrent FSP-neoAgs were evaluated in 20 Lynch syndrome (LS) carriers - 10 previvors (LS carriers with no history of cancer) and 10 survivors (LS carriers with a history of cancer) - using peripheral blood mononuclear cells (PBMCs)(FIG. 9A). Specifically, as illustrated in FIG. 9B, first, short-term stimulation responses, similar to innate- like reactions, were assessed by exposing the cells to FSP-neoAg peptides overnight within athree-day culture period, aimed at supporting the activation and development of antigen- presenting cells (APCs). In the second approach, an extended nine-day stimulation period to rapidly prime naive T cells was applied, thereby evaluating the priming potential of each neoAg peptide (FIG. 9B).

[0277] Following the two types of stimulation, neoAg-peptide-specific T cell responses from PBMCs were assessed through IFN-y production using the ELISPOT assay. A neoAg- peptide was defined as immunogenic by ELISPOT if it met two criteria: (1) a response of at least five times the number of spot-forming units (SFUs) per 10A5 cells compared to the DMSO control, when averaged across all human donors, and (2) a significantly higher SFU count per 10A5 cells than the DMSO control, determined using a paired Wilcoxon test. For individual analysis, a neoAg-peptide was considered immunogenic if (1) it produced five or more times the SFUs per 10A5 cells relative to the DMSO control, and (2) demonstrated a significantly elevated SFU count per 10A5 cells compared to the control in a paired T-test or the paired Wilcoxon sign-ranked test, when results were not normally distributed.

[0278] As shown in FIG. 9C, all tested FSP-neoAgs induced T cell responses when averaging the responses from all LS carriers following short-term stimulation. Furthermore, stratifying the analysis by disease status demonstrated that BMPR2 (B07:02), with a median of 81.83 SFU, and MSH3 (A*02:01), with a median of 95.70 SFU, was significantly more immunogenic in LS previvors compared to survivors (***p < 0.001). Conversely, RNF43-3 (A*02:01) demonstrated significantly higher immunogenicity in LS survivors ( **p < 0.01), with a median of 80.83 SFU (FIG. 9C). In contrast, the tumor suppressor gene RNF43, which regulates Wnt signaling, accumulates non-silent mutations in micro satellite loci, leading to aberrant pathway activity. While such mutations may influence early LS prognosis, they appear to play a more substantial role later in tumor development by promoting progression and supporting tumor survival through sustained Wnt signaling activation.

[0279] These data demonstrate that FSP-neoAgs are immunogenic, even in premalignant lesions, and that all LS survivors demonstrated immunogenicity to at least two neoAg peptides, with two survivors showing immunogenic responses to all tested neoAgs. In the previvor group, all patients except one exhibited immunogenic response to the tested neoAg peptides. Specifically, two patients displayed immunogenicity exclusively to RNF43-3, while the remaining previvors responded to multiple peptides, with one patient demonstrating immunogenicity across all tested neoAg peptides (Table 8).Table 8. Clinical and Demographic Characteristics of Each Patient, Including Immunogenic NeoantigenSExample 5. FSP-neoAgs identified herein are presented across a range of HLA types, and thus exhibit broader immunogenic potential.

[0280] To investigate the extent to which immunogenic responses to FSP-neoAgs used herein depend on HLA type, ELISPOT results were analyzed based on HLA matching. For peptides restricted to HLA B*07:02 (RNF43_02, TGFBR2, and BMPR2), no significant difference in immunogenicity between HLA-matched and unmatched groups was observed, as both demonstrated significant immunogenic responses (*p < 0.05) (FIG. 9D, left). These data demonstrate that neoAgs identified by the methods described herein are presented across a range of HLA types, broadening their immunogenic potential.

[0281] In contrast, HLA A*02:01 -restricted neoantigens (RNF43_03 and MSH3) exhibited a clear HLA-dependent effect. Specifically, RNF43_3 elicited a higher immunogenic response in the HLA-matched group (**p < 0.001), whereas MSH3 demonstrated significant immunogenicity exclusively within the HLA-matched group (**p < 0.01). The difference in immunogenicity between matched and unmatched groups for MSH3 was statistically significant (**p < 0.01) (FIG. 9D, right). Notably, the observed disparity in responses for the HLA A*02:01 -restricted peptides may be partially attributed to the smaller sample size of unmatched individuals (n=5), which could limit the observed range of immune responses among unmatched patientsExample 6. Extended Stimulation of NeoAg Peptides Boosts IFN-y Responses and Reduces HLA-Related Variability

[0282] Following the observation of innate-like responses to the tested neoAg peptides in short-term stimulation, all of which were identified as immunogenic, their in vitro priming capacity was assessed using an extended 9-day stimulation protocol. This extended stimulation showed that all four neoantigens have strong in vitro priming capabilities, leading to an enhanced IFN-gamma response. Most importantly, these data demonstrate that a 9-day in vitro priming period overcomes immunogenicity challenges often associated with HLA variability, thus enhancing the immune response to all tested antigens in patients, regardless of their HLA match, particularly compared to previous short-term stimulation results (FIG. 9E).Example 7. LS Carriers Exhibit Enhanced Neoantigen-Specific Immunogenic Responses Over Time, Regardless of Disease Status

[0283] To evaluate changes in the FSP-neoAg-mediated immune response over time in LS carriers, IFN-gamma responses were assessed in four patients (one previvor, Pl, and threesurvivors, S2, S4, and S8) with baseline and follow-up PBMC samples. Both LS previvor and survivors showed increased immunogenicity to neoantigen peptides over time, with a particularly pronounced response in survivors diagnosed with cancer during the study (FIG. 10). This elevated response may be due to repeated neoantigen exposure from FSP-neoAg mutations, continuously priming the immune system even before malignancy develops. In survivors, tumor presence likely further boosted immune recognition, serving as an additional antigen source.Example 8. Neoantigen-Stimulated T Cells Eliminate LS Tumors Using Microfluidic CoCulture Systems

[0284] A critical characteristic of candidate neoAgs is their ability to elicit immune responses by engaging cognate TCRs in CD8+ T cells, facilitating the elimination of tumor or dysplastic cells. Baseline and follow-up PBMC samples from available patient samples were used to assess the tumor-targeting efficacy of neoAg-stimulated T cells (FIG. 10).

[0285] To assess tumor cytotoxic activity, a physiologically relevant, gravity-driven microfluidic co-culture system was employed (FIG. 11A). In this setup, neo Ag-expres sing tumor organoids (Targets, T) derived from LS colon cancer (confirmed adenocarcinoma) were co-cultured with baseline and follow-up PBMCs stimulated with the RNF43-3 peptide (Effectors, E) from two HLA-matched LS survivors (HLA-A*02:01). Co-culture was conducted at a 5:1 E:T ratio over a overnight incubation period, with PTDOs alone as negative controls. Post- incubation, organoids were assessed for cytotoxicity using the CellTiter-Glo kit (Promega), and apoptotic cell death was quantified via caspase 3 / 7 activity assays.

[0286] For one LS survivor and one LS previvor with baseline PBMC samples but without matching tumor organoids, we developed genetically modified HCT116 colon cancer cells (MLH1 -mutated) to express multiple HLA alleles along with a neo Ag- specific minigene (FIG. 11B). These HCT116 cells were stably transfected to express HLA-A24:01 and B07:02 alongside their naturally expressed HLA-A02:01, A01:01, B45:01, and B45:01 alleles (FIG. 11B). The expression of these HLA alleles was confirmed via western blotting with a c-Myc tag antibody (FIG. 11C, left). Following HLA transfection, these cells were further modified with a plasmid encoding 14 neoAg peptides (9-l lmer) restricted to HLA-B07:02 and HLA- A*03:01 (Minigene 1, MG1), creating HCT-MG1 cells (FIG. 11B). MG1 expression in HCT- MG1 cells was validated by qRT-PCR with primers targeting neoAg sequences (FIG. 11C, middle). For cell tracking, MG1 also included GFP as a reporter (FIG. 11B). Another construct, Minigene 2 (MG2), contained neoAg peptides restricted to HLA-A24:01 and HLA-A02:01 and was stably transfected into HCT116 cells to create HCT-MG2 (FIG. 11B). This modified HCT116 MG1 line was used to evaluate TGFBR2-stimulated T cells from LS survivors and BMPR2- stimulated T cells from LS previvors for cytotoxicity and apoptosis induction.

[0287] These assays demonstrated that all neoantigen- specific T cells exhibit significant tumor cell-killing potency. Both baseline and follow-up RNF43-3- stimulated T cell-enriched PBMCs from LS survivors, TGFBR2-stimulated T cell-enriched PBMCs from LS survivors, and BMPR2- stimulated T cell-enriched PBMCs from LS previvors demonstrated significant tumor cell killing and apoptosis capacities (****p < 0.0001) (FIG. 11D-11E). LS survivor 2 (S2) exhibited a marked reduction in tumor cell killing and apoptosis potential between baseline and follow-up PBMC samples, suggesting a potential decline in immune potency over time. Given S2’s cancer history between the two blood draws, this reduction may reflect ongoing immune surveillance that could be influencing T cell efficacy.

[0288] In contrast, BMPR2- stimulated T cells from LS previvors showed a significant increase in T cell-mediated apoptosis (**p < 0.01) and an upward trend in T cell-mediated cell death, suggesting an enhanced immune response in the absence of active cancer. This contrasting immune response between LS survivors and previvors underscores the dynamic nature of immune surveillance and activation in relation to neoantigen exposure, highlighting variations in immune functionality at different stages of disease progression.

[0289] To further elucidate the dynamics of neo Ag- stimulated T cell tumor targeting, PBMCs collected from these co-cultures were re-plated onto multiplex FluoroSpot plates (Immunospot, OH) to measure the secretion of key effector molecules — IFN-gamma, IL-2, TNF-alpha, and Granzyme B — using a 4-plex ELISpot plate reader. Across all tested patients, significant release of these cytokines and cytotoxic molecules was observed (***p < 0.001; ****p < 0.0001) (FIG. 11F-11G). Notably, LS survivors exhibited elevated levels of activator cytokines (IFN-gamma and IL-2) as well as the pro-inflammatory cytokine TNF-alpha, indicating heightened immune activation. However, the cytotoxic protease Granzyme B, essential for effective tumor cell elimination, decreased over time in LS survivors, suggesting a decline in cytotoxic potential.

[0290] In contrast, the LS previvor patient showed only a slight increase in Granzyme B levels over the same period, maintaining a relatively stable cytotoxic profile. These findings demonstrate distinct immunological dynamics between LS survivors and previvors, with survivors displaying an increased pro-inflammatory response but a reduction in cytotoxic capacity over time.Example 9. LS Survivors and Previvors Harbor Detectable Circulating Neoantigen- Specific T Cells

[0291] Following assessment of tumor-targeting potency in neo Ag- specific T cells, a screen was performed to identify circulating neo Ag- specific T cells in the PBMCs of LS carriers. Using MHC-I dextramer staining, specific to all five neoAg peptides, short-term stimulation was conducted to simulate innate-like responses (as in the ELISpot assay, FIG. 9B), aiming to detect any prior exposure to these neoAg peptides in LS carriers. NeoAg- specific T cells exhibiting >1% MHC dextramer positivity in LS PBMCs were selected for downstream single-cell RNA and TCR sequencing to profile their single-cell transcriptomics. Samples were classified as follows: those with MHC dextramer positivity below 0.5% were considered negative, those between 0.5% and 1% were considered positive but were not used in single-cell sequencing due to insufficient cell yield (lower than 30.000 cells), and those with MHC dextramer positivity >1% (yielding approximately 30,000-50,000 cells) were directed to downstream single-cell sequencing.

[0292] MHC multimer assays detected circulating neoAg- specific T cells responsive to each of the five neoAg peptides in HLA-matched patient samples. Among these samples, six patients showed >1% circulating neoAg- specific T cells, with two patients exhibiting >1% T cells specific to two distinct neoAgs. One patient displayed >1% positivity at both baseline and follow-up (FIG. 12A). Additionally, nine patients exhibited circulating neoAg-specific T cells with positivity between 0.5% and 1%, of whom three showed reactivity to multiple neoAgs. Five patients presented MHC dextramer positivity below 0.5%, suggesting an absence of specific reactivity. Among the detected neoAg peptides, RNF43-2 and BMPR2 were the most frequently observed, with >1% MHC multimer positivity (FIG. 12A). Overall, LS survivors exhibited higher frequencies of dextramer-positive cells compared to previvors, although this difference did not reach statistical significance.

[0293] These findings have significant implications for understanding immune dynamics in LS carriers. The presence of circulating neoAg-specific T cells may indicate prior immune recognition of tumor-associated neoAgs, suggesting an active state of immuno surveillance in both previvors and survivors. Early detection of these circulating neoAg-specific T cells provides insight into the immune system’s capacity to recognize and respond to emerging malignancies in LS carriers, which could enable earlier intervention before tumor progression. Moreover, tracking these neoAg-specific T cells over time allows for a deeper understanding of immune adaptability in response to persistent neoAg exposure in LS carriers.

[0294] In survivors, particularly those with a history of cancer, circulating neo Ag- specific T cells may also serve as indicators of residual immune memory or ongoing immune surveillance, potentially aiding in early detection of recurrence. The identification of these circulating neo Ag- specific T cells highlights their potential as biomarkers for personalized immunomonitoring in LS carriers. Such profiles could be instrumental in guiding targeted immunotherapies and surveillance strategies tailored to individual neoAg profiles, advancing precision management for LS carriers.Example 10. Single-Cell Analysis of Circulating NeoAg-Specific T Cells Reveals Immune Surveillance Signatures in LS Carriers

[0295] Following the identification of circulating neoAg-specific T cells using MHC dextramer assays, the phenotypic and transcriptional states of these cells in LS carriers was evaluated through single-cell omics analysis. This investigation focused on six LS carriers (three LS previvors: Pl, P5, P7, and three LS survivors: S2, S8, S10) who exhibited more than 1% circulating neoAg-specific T cells in their PBMCs. Both neoAg-specific and pan-T cells from these individuals were included in order to construct a comprehensive phenotypic map of circulating T cells in LS carriers and to discern their role in immune surveillance and potential reactivity against neoantigen-expressing cells. High-throughput single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing was applied to characterize phenotypes, clonality, and neoAg-specific TCR sequences in circulating CD8+ T cells.

[0296] Included in the dataset were transcriptomes from 1,331,615 neoAg-specific T cells, targeting five neoAg peptides (RNF43_2, RNF43_3, TGFBR4, BMPR2, MSH3), and 464,619 pan-T cells derived from PBMCs of the six LS carriers. Unsupervised transcriptomic clustering was performed using the combined 1,331,615 neoAg-specific and 464,619 pan-T cells (FIG. 12B), followed by uniform manifold approximation and projection (UMAP) analysis (FIG. 12C), which delineated nine distinct cell clusters. These clusters revealed a range of transcriptomic states in circulating neoAg-specific CD8+ T cells, spanning from less differentiated, proliferative states (Cluster 0 and 3) to various differentiated states, including effector-like T cells (Cluster 4), effector memory T cells (Cluster 2), highly cytotoxic T cells (Cluster 5), and terminally differentiated effector T cells (Cluster 6) that expressed exhaustion markers and inhibitory molecules (FIG. 12B). Mapping hyperexpanded neoAg-specific TCR clonotypes from all five neoAg peptides revealed that most of these clones localized within clusters associated with differentiated effector-like (Cluster 0), Proliferating T cells (Cluster 3), and terminally differentiated T cells (Cluster 6) (FIG. 12C, bottom). This distribution ofhyperexpanded clonotypes suggests a dynamic progression from antigen encounter through proliferation and differentiation into memory and effector states, potentially reflecting prior exposure to neoAgs. Furthermore, to better characterize the transcriptomic states of neoAg- specific clones, a correlation analysis was conducted using established gene signatures from tumor-infiltrating neoAg-specific T cells and circulating neo Ag- specific T cells. Notably, Clusters 3 and 6, which were enriched in hyperexpanded neoAg-specific TCR clones, showed significant correlation with known neoAg-specific gene sets from Lowrey et al (Lowery, F.J. et al. Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers. Science 375, 877-884 (2022)), aligning closely with gene signatures characteristic of both circulating and tumor- infiltrating T cells(Yossef, R. et al. Phenotypic signatures of circulating neoantigen-reactive CD8(+) T cells in patients with metastatic cancers. Cancer Cell 41, 2154-2165 e2155 (2023)).

[0297] These findings hold profound implications for understanding immune surveillance in LS carriers. The detection of distinct gene signatures in circulating neoAg-specific T cells, especially those aligning with tumor-infiltrating gene sets, underscores the potential of these cells as a unique immune compartment actively primed against neo Ag-expres sing cells (e.g., tumor cells). For LS previvors, the presence of neoAg-specific T cells, likely associated with premalignant lesions, supports the notion of ongoing immune surveillance, positioning these clones as possible early biomarkers for cancer risk. In LS survivors, hyperexpanded neoAg- specific T cells enriched with tumor-like gene signatures suggest a role in recognizing and potentially eliminating cancer cells, as well as persistent immune memory that may aid in detecting minimal residual disease or early recurrence. Together, these observations highlight the importance of circulating neoAg-specific T cells as indicators of immune readiness in LS carriers, with potential applications for personalized immunomonitoring and early intervention in high-risk individuals.Example 11. Public NeoAg-specific TCR Clonotypes in Lynch Syndrome: NeoAg Specificity and Tissue Presence

[0298] The TCR repertoires of neoAg-specific T cells exhibit significantly reduced diversity and increased clonality, as indicated by the Simpson index, which accounts for both the number of clonotypes and their degree of expansion. Compared to pan-T cells, neoAg- specific T cells show a markedly higher clonality, reflecting a repertoire dominated by hyperexpanded TCR clones. This elevated clonality likely stems from a greater prevalence of hyperexpanded and extensively proliferated TCRs within the neoAg-specific population,highlighting their focused and robust response to antigenic stimulation (FIG. 13A). To define the specificity of the identified TCRs more precisely, we first filtered out clones associated with known pathogens using public databases. Next, we prioritized clones linked to dMMR CRC by leveraging TCRP- sequencing data from nine MMRd colorectal adenocarcinomas previously reported in the literature. Neo Ag- specific TCR clonotypes among LS carriers (shared by at least two individuals) were identified by integrating archival public bulk TCR sequencing data from 277 PBMC samples and 14 colorectal mucosal biopsies. This prioritization process focused on public clones detected in LS carriers (either survivors, previvors, or both), with a further emphasis on clones present in LS carrier tissue (FIG. 13B).

[0299] Through this pipeline, ten clonotypes were identified, with 4 clones present in active cancer tissue (FIG. 13D) and two clones detected in tissue from three LS previvors with hyperplastic polyps. Additionally, an RNF43-3-specific TCR clonotype associated with cancer was identified (FIG. 13C).

[0300] These findings are particularly significant in the context of immune surveillance and early detection in LS carriers. The presence of neoAg-specific clones in premalignant tissue from previvors suggests an active immune response against early-stage or potentially tumorigenic lesions, underscoring the potential of these clones as early biomarkers for cancer risk. The tissue-specific detection of neoAg-specific TCR clonotypes, even in non-malignant lesions, provides a unique insight into ongoing immune activation in LS carriers, highlighting the immune system's role in monitoring precancerous changes. Most importantly, the identification of cancer-tis sue- specific TCR clonotypes reinforces the relevance of these T cells in targeting malignant cells, positioning these clones as potential biomarkers for active disease and as candidates for immunotherapeutic development in Lynch syndrome carriers.References

[0301] Abu-Ghazaleh, N., Kaushik, V., Gorelik, A., Jenkins, M. & Macrae, F. Worldwide prevalence of Lynch syndrome in patients with colorectal cancer: Systematic review and metaanalysis. Genet Med 24, 971-985 (2022).

[0302] Dominguez- Valentin, M. et al. Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database. Genet Med 22, 15-25 (2020).

[0303] Lynch, H.T., Snyder, C.L., Shaw, T.G., Heinen, C.D. & Hitchins, M.P. Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer 15, 181-194 (2015).

[0304] Ballhausen, A. et al. The shared frameshift mutation landscape of microsatellite- unstable cancers suggests immunoediting during tumor evolution. Nat Commun 11, 4740 (2020).

[0305] Bolivar, A.M. et al. Genomic Landscape of Lynch Syndrome Colorectal Neoplasia Identifies Shared Mutated Neoantigens for Immunoprevention. Gastroenterology 166, 787-801 e711 (2024).

[0306] Pinheiro, M. et al. Target gene mutational pattern in Lynch syndrome colorectal carcinomas according to tumour location and germline mutation. Br J Cancer 113, 686-692 (2015).* * *

[0307] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

CLAIMS1. An isolated peptide comprising at least 70% sequence identity to a peptide of one of SEQ ID NOS: 1-192, a polyepitope string of peptides comprising at least 70% sequence identity to two or more peptides of SEQ ID NOs: 1-192, or nucleic acid encoding therefor, further comprising one or more (i) amino acid modification, (ii) protein fusion, and / or (iii) structural modification, wherein the amino acid modification comprises one or more amino acid substitution, deletion or addition; the protein fusion comprises a covalent bond to a cell penetrating peptide, a secretory signal peptide, an MHC class I binding domain, an MHC class II binding domain, an antibody or antigen-binding fragment thereof, a cytokine, a lipid, a polysaccharide, an N- or C-terminal polyethylene glycol (PEG), an adjuvant, an amino acid linker sequence separating the isolated peptides in a poly epitope string of isolated peptides; and the structural modification comprises a post-translational modification comprising phosphorylation, glycosylation, ubiquitination.

2. The peptide of claim 1, wherein the isolated peptide, polyepitope string of isolated peptides, or nucleic acid encoding therefor, comprises one or more of SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 6, and / or SEQ ID NO: 5.

3. The peptide of claim 1 or 2, wherein the isolated peptide, poly epitope string of isolated peptides, or nucleic acid encoding therefor, comprises two or more of SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 6, and / or SEQ ID NO: 5.

4. The peptide of any of claims 1-3, wherein the peptide comprises at least 6 contiguous amino acids of a peptide of one of SEQ ID NOS: 1-192, optionally wherein the peptide is 25 amino acids or fewer in length, optionally wherein the peptide is 8 amino acids or greater in length.

5. The peptide of any of claims 1-4, wherein the peptide is 8 to 11 amino acids in length.

6. The peptide of any of claims 1-4, wherein the peptide is 12 to 18 amino acids in length.

7. The peptide of any one of claims 1-6, wherein the peptide is immunogenic, optionally wherein the peptide is presented on MHC class I and MHC class II molecules.

8. The peptide of any one of claims 1-7, wherein the peptide is modified.

9. The peptide of claim 8, wherein the modification comprises conjugation to a molecule.

10. The peptide of claim 8 or 9, wherein the molecule comprises an antibody, a lipid, an adjuvant, a cell penetrating peptide, or a detection moiety.

11. The peptide of any of claims 1-10, wherein the peptide has at least 90% sequence identity to a peptide of one of SEQ ID NOS: 1-192.

12. The peptide of any of claims 1-11, wherein the peptide has 1, 2 or 3 substitutions relative to a peptide of one of SEQ ID NOS: 1-192.

13. The peptide of any one of claims 1-11, wherein the peptide comprises 100% sequence identity to a peptide of one of SEQ ID NOS: 1-192.

14. The peptide of any of claims 1-13, wherein the amino acid sequence of the peptide is identical to a sequence encoded by a mutated gene sequence in a precancer or cancer in at least 0.01%, at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 20%, or more, of subjects having Lynch Syndrome and a precancer or cancer.

15. The peptide of claim 14, wherein the mutated gene sequence is the result of a frameshift mutation.

16. A molecular complex comprising the peptide of any one of claims 1-15 and an MHC polypeptide.

17. A combination of peptides comprising two or more of the peptides of any of claims 1- 15.

18. The combination of peptides of claim 17, wherein the combination of peptides comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more than 200, of any of the peptides of claims 1-15.

19. The combination of peptides of claim 17 or 18, wherein the combination of peptides comprises between about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 120, 120 to 140, 140 to 160, 160 to 180, 180 to 200, or 200 to 300, of any of the peptides of claims 1-15.

20. The combination of peptides of any of claims 17-19, wherein the combination of peptides comprises at or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 of any of the peptides of claims 1-15.

21. The combination of peptides of any of claims 17-20, wherein the combination of peptides comprises between 25 to 40 of any of the peptides of claims 1-15.

22. The combination of peptides of any of claims 17-21, wherein the combination of peptides comprises at or about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 of any of the peptides of claims 1-15.

23. The combination of peptides of any of claims 17-22, wherein the combination of peptides comprises at or about 35 of any of the peptides of claims 1-15.

24. The combination of peptides of any of claims 17-23, wherein at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of precancers or cancers in subjects having Lynch Syndrome comprise a mutated gene sequence encoding an amino acid sequence that is identical to the sequence of one of the peptides in the combination of peptides.

25. The combination of peptides of any of claims 17-24, wherein in at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of subjects having Lynch Syndrome and a precancer or cancer, the precancer or cancer comprises a mutated gene sequence encoding an amino acid sequence that is identical to the sequence of one of the peptides in the combination of peptides.

26. The combination of peptides of any of claims 17-25, wherein for at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of subjects having Lynch Syndrome and a precancer or cancer, the combination of peptides comprises at least one peptide that is identical in sequence to a sequence encoded by a mutated gene sequence in the precancer or cancer.

27. The combination of peptides of any of claims 24-26, wherein the mutated gene sequence is the result of a frameshift mutation.

28. One or more nucleic acids encoding the peptide of any of claims 1-15, or the combination of peptides of any of claims 17-27.

29. The nucleic acid(s) of claim 28, wherein the nucleic acid(s) comprise DNA and / or RNA.

30. The nucleic acid(s) of claim 28 or 29, wherein the nucleic acid(s) comprise mRNA.

31. An expression vector comprising the nucleic acid(s) of any of claims 28-30.

32. A vector for intracellular delivery, comprising the peptide of any of claims 1-15, the molecular complex of claim 16, the combination of peptides of any of claims 17-27, the nucleic acid(s) of any of claims 28-30, or the expression vector of claim 31.

33. The vector for intracellular delivery of claim 32, wherein the vector is a viral vector.

34. The vector for intracellular delivery of claim 32, wherein the vector is a non-viral vector.

35. A composition comprising the peptide of any of claims 1-15, the molecular complex of claim 16, the combination of peptides of any of claims 17-27, the nucleic acid(s) of any ofclaims 28-30, the expression vector of claim 31, or the vector for intracellular delivery of any of claims 32-34.

36. The composition of claim 35, wherein the composition is a pharmaceutical composition.

37. The composition of claim 35 or 36, wherein the composition comprises a pharmaceutical carrier.

38. The composition of any of claims 35-37, wherein the composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection.

39. The composition of any of claims 35-38, wherein the peptide, combination of peptides, nucleic acid(s), or expression vector, is comprised in a liposome, lipid-containing nanoparticle, or in a lipid-based carrier.

40. The composition of any of claims 35-39, wherein the composition is formulated for injection or inhalation as a nasal spray.

41. The composition of any of claims 35-40, wherein the composition is formulated as a vaccine.

42. The composition of any of claims 35-41, wherein the composition further comprises an adjuvant.

43. The composition of any of claims 35-42, wherein the composition stimulates an immune response when administered to a subject.

44. The composition of any of claims 35-43, wherein the composition prevents colorectal cancer when administered to a subject having Lynch Syndrome.

45. A method of stimulating an immune response in a subject, the method comprising administering to the subject the peptide of any of claims 1-15, the molecular complex of claim 16, the combination of peptides of any of claims 17-27, the nucleic acid(s) of any of claims 28-30, the expression vector of claim 31, the vector for intracellular delivery of any of claims 32-34, or the composition of any of claims 35-44.

46. A method of treating or preventing cancer or precancer in a subject, the method comprising administering to the subject the peptide of any of claims 1-15, the molecular complex of claim 16, the combination of peptides of any of claims 17-27, the nucleic acid(s) of any of claims 28-30, the expression vector of claim 31, the vector for intracellular delivery of any of claims 32-34, or the composition of any of claims 35-44.

47. A method of preventing cancer in a subject, the method comprising administering to the subject the peptide of any of claims 1-15, the molecular complex of claim 16, thecombination of peptides of any of claims 17-27, the nucleic acid(s) of any of claims 28-30, the expression vector of claim 31, the vector for intracellular delivery of any of claims 32-34, or the composition of any of claims 35-44.

48. The method of any of claims 45-47, wherein the subject has been determined to have one or more precancers or cancers.

49. The method of any of claims 45-48, wherein the subject is determined to be at risk of developing cancer.

50. The method of any of claims 45-49, wherein the subject is determined to have a genetic predisposition for developing colorectal cancer.

51. The method of any of claims 45-50, wherein the subject has Lynch Syndrome.

52. The method of any of claims 46-51, wherein the cancer or precancer is colorectal.

53. The method of any of claims 46-52, wherein the cancer or precancer is determined to comprise a mutated gene sequence encoding an amino acid sequence comprising the sequence set forth in any of SEQ ID NOS: 1-192.

54. The method of any of claims 45-53, wherein the cancer or precancer is determined to comprise a mutated gene sequence encoding an amino acid sequence that is identical to the amino acid sequence of the peptide of any of claims 1-15.

55. The method of claim 53 or 54, wherein the mutated gene sequence is the result of a frameshift mutation.

56. A vaccine for preventing colorectal cancer in subjects with Lynch Syndrome, comprising the peptide of any of claims 1-15, the molecular complex of claim 16, the combination of peptides of any of claims 17-27, the nucleic acid(s) of any of claims 28-30, the expression vector of claim 31, the vector for intracellular delivery of any of claims 32-34, or the composition of any of claims 35-44.

57. A vaccine for preventing colorectal cancer in subjects with Lynch Syndrome, comprising one or more mRNA molecules encoding the amino acid sequences of the peptides of the combination of peptides of any of claims 17-27.

58. A host cell comprising the nucleic acid(s) of any of claims 28-30 or the expression vector of claim 31.

59. An in vitro isolated dendritic cell comprising the peptide of any one of claims 1-15, the nucleic acid(s) of any of claims 28-30, or the expression vector of claim 31.

60. The dendritic cell of claim 59, wherein the dendritic cell is a mature dendritic cell.

61. The dendritic cell of claim 59 or 60, wherein the cell is a cell with an HLA-A, HLA-B, or HLA-C type.

62. A peptide- specific binding molecule, wherein the molecule specifically binds to a peptide of any one of claim 1-15 or the molecular complex of claim 16.

63. The binding molecule of claim 62, wherein the binding molecule is an antibody or antigen binding fragment thereof, TCR mimic antibody, scFV, camelid, aptamer, or DARPIN.

64. A method of making a cell comprising transferring the nucleic acid(s) of any of claims 28-30 or the expression vector of claim 31 into the cell.

65. The method of claim 64, wherein the method further comprises isolating the expressed peptide or polypeptide.

66. A method of producing cancer- specific immune effector cells comprising:(a) obtaining a starting population of immune effector cells; and(b) contacting the starting population of immune effector cells with a peptide of any one of claims 1-15 or the molecular complex of claim 16, thereby generating peptidespecific immune effector cells.

67. The method of claim 66, wherein contacting is further defined as co-culturing the starting population of immune effector cells with antigen presenting cells (APCs), artificial antigen presenting cells (aAPCs), or an artificial antigen presenting surface (aAPSs); wherein the APCs, aAPCs, or the aAPSs present the peptide on their surface.

68. The method of claim 67, wherein the APCs are dendritic cells.

69. The method of any one of claims 66-68, wherein the immune effector cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, NKT cells.

70. The method of any one of claims 66-69, wherein the immune effector cells have been differentiated from mesenchymal stem cell (MSC) or induced pluripotent stem (iPS) cells.

71. The method of claim 69, wherein the T cells are CD8+T cells, CD4+T cells, or y5 T cells.

72. The method of claim 69, wherein the T cells are cytotoxic T lymphocytes (CTLs).

73. The method of any one of claims 66-72, wherein obtaining comprises isolating the starting population of immune effector cells from peripheral blood mononuclear cells (PBMCs).

74. The method of any one of claims 66-73, wherein the starting population of immune effector cells is obtained from a subject.

75. The method of claim 74, wherein the subject is a human.

76. The method of claim 74 or 75, wherein the subject has a cancer and / or has been determined to have Lynch Syndrome.

77. The method of claim 76, wherein the cancer comprises tumor cells that are positive for expression of the peptide.

78. The method of claim 77, wherein the cancer comprises colorectal cancer, leukemia, lung cancer, or skin cancer.

79. The method of any one of claims 68-78, wherein the method further comprises introducing the peptide or a nucleic acid encoding the peptide into the dendritic cells prior to the co-culturing.

80. The method of claim 79, where the peptide or nucleic acids encoding the peptide are introduced by electroporation.

81. The method of claim 79, wherein the peptide or nucleic acids encoding the peptide are introduced by adding the peptide or nucleic acid encoding the peptide to the dendritic cell culture media.

82. The method of claim 79, wherein the immune effector cells are co-cultured with a second population of dendritic cells into which the peptide or the nucleic acid encoding the peptide has been introduced.

83. The method of claim 79, wherein a population of CD8 or CD4-positive and peptide MHC tetramer-positive T cells are purified from the immune effector cells following the coculturing.

84. The method of claim 83, wherein a clonal population of peptide- specific immune effector cells are generated by limiting or serial dilution followed by expansion of individual clones by a rapid expansion protocol.

85. The method of claim 84, wherein the method further comprises cloning of a T cell receptor (TCR) from the clonal population of peptide- specific immune effector cells.

86. The method of claim 85, wherein cloning of the TCR is cloning of a TCR alpha and a TCR beta chain.

87. The method of claim 85 or 86, wherein the TCR is cloned using a 5 ’-Rapid amplification of cDNA ends (RACE) method.

88. The method of claim 87, wherein the cloned TCR is subcloned into an expression vector.

89. The method of claim 88, wherein the expression vector is a retroviral or lentiviral vector.

90. The method of claim 89, where a host cell is transduced with the expression vector to generate an engineered cell that expresses the TCR.

91. The method of claim 90, wherein the host cell is an immune cell.

92. The method of any one of claims 68-91, wherein the immune cell is a T cell and the engineered cell is an engineered T cell.

93. The method of claim 92, wherein the T cell is a CD8+T cell, CD4+ T cell, or y5 T cell and the engineered cell is an engineered T cell.

94. The method of claim 93, wherein the starting population of immune effector cells is obtained from a subject with cancer and the host cell is allogeneic or autologous to the subject.

95. The method of claim 94, wherein the cancer is positive for expression of the peptide.

96. The method of claim 92 or 93, wherein a population of CD8 or CD4-positive and peptide MHC tetramer-positive engineered T cells are purified from the transduced host cells.

97. The method of claim 83, wherein a clonal population of peptide- specific engineered T cells are generated by limiting or serial dilution followed by expansion of individual clones by a rapid expansion protocol.

98. A peptide- specific engineered T cell produced according to any one of the methods of claims 66-77 or 90-97.

99. A pharmaceutical composition comprising the peptide- specific T cells produced according to any one of the methods of claims 66-77 or 90-97.

100. A method of treating or preventing cancer in a subject, the method comprising administering an effective amount of the peptide of any of claims 1-15, the molecular complex of claim 15, the combination of peptides of any of claims 17-27, the nucleic acid(s) of any of claims 28-30, the expression vector of claims 31, the vector for intracellular delivery of any of claims 32-34, the composition of any of claims 35-44, the vaccine of claims 56 or 57, the dendritic cell of any of claims 59-61, or the peptide- specific T cell of claim 98.

101. A method of stimulating an immune response in a subject, the method comprising administering an effective amount of the peptide of any of claims 1-15, the molecular complex of claim 15, the combination of peptides of any of claims 17-27, the nucleic acid(s) of any of claims 28-30, the expression vector of claims 31, the vector for intracellular delivery of any of claims 32-34, the composition of any of claims 35-44, the vaccine of claims 56 or 57, the dendritic cell of any of claims 59-61, or the peptide- specific T cell of claim 98.

102. The method of claim 100 or 101, wherein the subject is a human.

103. The method of any one of claims 100-102, wherein the peptide- specific T cells are autologous or allogeneic.

104. The method of any one of claims 100-103, further comprising administering at least a second therapeutic agent.

105. The method of claim 104, wherein the second therapeutic agent is an anti-cancer agnt.

106. The method of any one of claims 100-105, wherein the subject has been diagnosed with cancer.

107. The method of any one of claims 100-105, wherein the subject has not been diagnosed with cancer.

108. The method of any one of claims 100-107, wherein the subject has been determined to have Lynch Syndrome.

109. The method of claim 106, wherein the cancer comprises a cancer that is positive for expression of the peptide.

110. The method of any one of claims 100-109, wherein the cancer comprises colorectal cancer.

111. The method of claim 110, wherein the colorectal cancer comprises mismatch repair deficient colorectal cancer (MMR-d) and / or micro satellite instability (MSI) positive colorectal cancer.

112. The method of any one of claims 100-111, wherein the subject is treated for stage I or stage II cancer.

113. A method of diagnosing colorectal cancer in a subject with LS, comprising:(a) obtaining a sample from a subject, wherein the sample comprises peripheral blood mononuclear cells (PBMCs);(b) contacting the sample with a peptide or combination of peptides of any one of claims 1-27; and(c) detecting an immune response in the sample; wherein the subject is said to have colorectal cancer if an immune response is detected.

114. The method of claim 113 or 114, wherein the immune response comprises production of IFN-gamma, IL-2, TNF-alpha, and / or granzyme B.

115. The method of any one of claims 113-114, wherein the immune response comprises detection of T cells in the PBMCs specific with TCRs specific to one or more of the peptide or combination of peptides.

116. The method of any one of claims 113-115, wherein the immune response comprises determining that T cells exhibit gene signatures characteristic of (i) differentiated effectorlike cells, (ii) proliferating T cells, and / or (iii) terminally differentiated T cells.

117. The method of any one of claims 113-116, further comprising administering cancer therapy to subjects said to have colorectal cancer.

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