Methods and Compositions Comprising MHC Class I Peptides

Abstract

The present disclosure provides methods and compositions for treating and vaccinating individuals against cancer. Accordingly, aspects of the present disclosure relate to peptides that comprise at least 70% sequence identity to one of the peptides of SEQ ID NOs: 1-776. In some embodiments, the peptide comprises at least 6 consecutive amino acids of one of the peptides of SEQ ID NOs: 1-776. Further aspects relate to pharmaceutical compositions that include the peptides, nucleic acids that encode the peptides, and expression vectors and host cells that include the nucleic acids of the present disclosure. In vitro dendritic cells that include the peptides, nucleic acids, or expression vectors of the present disclosure are also provided.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 171,137, filed April 6, 2021, which is incorporated by reference in its entirety herein.

[0002] I. FIELD OF THEINVENTION The present invention relates to the field of cancer treatment. [Background technology]

[0003] II. Background Lynch syndrome (LS), the most common cause of hereditary colorectal cancer (CRC), accounts for 2-4% of all CRCs and at least 1 million carriers in the United States (1). LS results from heterozygous germline mutations in DNA mismatch repair (MMR) genes, with MLH1 and MSH2 accounting for over 70% of LS cases. LS patients have an increased lifetime risk of developing CRC, which reaches 60% in MLH1 and MSH2 carriers (2). Normal colorectal cells become MMR-deficient (dMMR) when they acquire a second somatic hit in an alternative allele of the same MMR gene that harbors a germline mutation. This second hit manifests as a base-pair mismatch and an insertion-deletion mutation (indel) in homopolymeric microsatellite sequences that are susceptible to indels. These mutations alter the wild-type codon sequence and generate frameshifted peptides (FSPs) that differ from the wild-type protein and thus become neo-antigens (neo-Ags), which stimulate the adaptive immune system.

[0004] Tumor protein mutations (neo-antigens) are processed into short peptides and presented on the cell surface complexed with major histocompatibility complexes (MHC I / II). These peptides can bind to the T cell receptor (TCR) on cytotoxic CD8+ T cells, which promotes interferon-γ (IFNγ) secretion to kill cancer cells. Thus, activation of CD8+ and CD4+ T cells (helper cells) that recognize neo-antigens is critical for adaptive immunity against tumors. A wide range of systems biology platforms and computational algorithms have used next-generation sequencing to rapidly screen the mutational landscape of human cancers, including melanoma and colon (3-6). Such tests have identified a variety of nonsynonymous mutations that can be recognized as foreign antigens to the host immune system, providing promising avenues for further personalized and focused approaches to activate anti-tumor immunity (7). For example, recent vaccination approaches using mutant peptides to stimulate antitumor immunity have been successful in generating specific cytotoxic T lymphocyte (CTL) responses in human melanoma patients, and a similar approach in colorectal cancer has resulted in substantial tumor regression (4).

[0005] Targeted therapy against tumor-specific frameshift neoantigens using the host immune system offers several advantages over previous and current immunotherapy strategies. For example, autoimmunity and dose-limiting toxicities have been reported in CRC patients who received checkpoint inhibitors and adoptive T cell transfer against tumor-associated antigens (9-11). However, these immune-related adverse events are less of an issue when targeting foreign neoantigens and cancer antigens through strategies such as immune vaccination (7). Furthermore, it has been hypothesized that the significant deviation in sequence homology between frameshift neoantigens versus wild-type peptides will elicit stronger immunogenic responses than viral neoantigens and missense neoantigens, further supporting frameshift neoAg targeted therapy (12). Thus, there is a need in the art to develop compositions and methods for neoantigens identified from LS patients. Summary of the Invention

[0006] The present 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 neoantigens. Accordingly, aspects of the present disclosure relate to peptides that comprise at least 70% sequence identity to one of SEQ ID NOs: 1-776. In some aspects, the peptide comprises at least 70% sequence identity to one of SEQ ID NOs: 10, 323, 221, 44, 27, 156, 37, 168, 20, 163, 29, 136, 24, 62, 138, 157, 160, 151, 158, 23, 39, or 57. Aspects of the present disclosure relate to peptides that comprise at least 70% sequence identity to one of SEQ ID NOs: 10, 323, 221, 44, 27, 156, 37, 168, 20, 163, 29, 136, 24, 62, 138, 157, 160, 151, 158, 23, 39, or 57. 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) for one peptide among NO:1-776 ), or at most 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. In some aspects, the peptide comprises at least 6 contiguous amino acids with one of the peptides of SEQ ID NOs: 1-776. Aspects of the present disclosure relate to polypeptides that include a peptide of the present disclosure. Further aspects relate to pharmaceutical compositions comprising the peptides, polypeptides, viruses, nucleic acids encoding the peptides or polypeptides, and expression vectors and host cells comprising the nucleic acids of the disclosure. In some aspects, the host cells can be virus packaging cells. Aspects of the disclosure relate to viruses produced from the host cells of the disclosure.Also provided is an in vitro dendritic cell comprising a peptide, nucleic acid, or expression vector of the disclosure.

[0007] Further aspects relate to methods of making cells, comprising transferring a nucleic acid or expression vector of the disclosure into a cell, such as a host cell. The methods may comprise, or may further comprise, culturing a cell having a nucleic acid or expression vector encoding any of the proteins described herein, including, but not limited to, any of SEQ ID NOs: 1-776. The methods may comprise, or may further comprise, isolating the expressed peptide or polypeptide. Another aspect of the disclosure relates to methods of producing cancer-specific immune effector cells, comprising (a) obtaining a starting population of immune effector cells, and / or (b) contacting the starting population of immune effector cells with a peptide or polypeptide of the disclosure, thereby generating peptide-specific immune effector cells.

[0008] The present disclosure also describes peptide-specific engineered T cells produced according to the method of the present disclosure, and pharmaceutical compositions comprising engineered T cells. A further aspect relates to a method for treating or preventing cancer in a subject, comprising administering an effective amount of the peptide or polypeptide, pharmaceutical composition, nucleic acid, dendritic cell or peptide-specific T cell of the present disclosure. Yet a further aspect relates to a method for cloning peptide-specific T cell receptors (TCRs), comprising: (a) obtaining a starting population of immune effector cells; (b) contacting the starting population of immune effector cells with the peptide or polypeptide of the present disclosure, thereby generating peptide-specific immune effector cells; (c) purifying the immune effector cells specific for the peptide; and / or (d) isolating TCR sequences from the purified immune effector cells. Also provided is a method for predicting the prognosis of a patient or for detecting T cell responses in a patient, comprising contacting a biological sample from a patient with the peptide or polypeptide of the present disclosure.

[0009] Aspects of the present disclosure also provide compositions comprising at least one MHC polypeptide, a peptide of the present disclosure, and a peptide-specific binding molecule that binds to the peptide of the present disclosure or binds to a peptide-MHC complex. Exemplary binding molecules include antibodies, TCR mimic antibodies, scFvs, nanobodies, camelids, aptamers, and DARPINs. Related methods include contacting a composition comprising at least one MHC polypeptide and a peptide of the present disclosure with a composition comprising T cells, and detecting T cells with bound peptides and / or MHC polypeptides by detecting the detection tag. Further aspects relate to kits comprising the peptides, polypeptides, nucleic acids, expression vectors, or compositions of the present disclosure.

[0010] In some aspects, the peptide is 13 amino acids or less in length. In some aspects, the peptide is 9 amino acids. The peptide can be at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (or any range derivable therein), or can be at most 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (or any range derivable therein), or can 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 can consist of 9 amino acids, or the peptide can consist of 15 amino acids. The peptide can be further described as immunogenic. The term immunogenic refers to generating an immune response, such as a protective immune response. The peptide or polypeptide may be modified. Modification may include conjugation with a molecule. The molecule may be an antibody, lipid, adjuvant, or detection moiety (tag). In some aspects, the peptide comprises 100% sequence identity to one of the peptides of SEQ ID NOs: 1-776. The peptides of the present disclosure also include those having at least 90% sequence identity to one of the peptides of SEQ ID NOs: 1-776. The peptides of the present disclosure may have one, two, or three substitutions to one of the peptides of SEQ ID NOs: 1-776. In some aspects, the peptides have at least one, two, three, four, or five substitutions to one of the peptides of SEQ ID NOs: 1-776, or at most one, two, three, four, or five substitutions to one of the peptides of SEQ ID NOs: 1-776.

[0011] In some aspects, the nucleic acid of the present disclosure is DNA. In some aspects, the nucleic acid of the present disclosure is RNA. RNA can be further defined as mRNA. The expression vector can include an adenovirus backbone. The expression vector can be a simian adenovirus vector or its derivative. In some aspects, the expression vector includes a lentivirus expression vector.

[0012] The polypeptide may comprise at least two peptides of the present disclosure. In some aspects, the polypeptide comprises 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, or 40 peptides (or any derivable range therein) of the present disclosure, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 40 peptides of the present disclosure. , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 peptides (or any derivable range therein) of the present disclosure, or at most 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, or 40 peptides (or any derivable range therein) of the present disclosure. In some aspects, the polypeptide comprises four peptides of the present disclosure. The polypeptide may include, or may further include, a cell penetrating peptide (CPP). The CPP may include the Z13 variant of ZEBRA CPP Z12. In some aspects, the polypeptide includes or further includes one or more TLR agonists. The TLR agonist may include a TLR2 agonist, a TLR4 agonist, a TLR2 / 4 agonist, or a combination thereof. The TLR agonist may include one or both of Extra Domain A (EDA) and Anaxa. In some aspects, the polypeptide includes, from the amino-proximal position to the carboxy-proximal position, a cell-penetrating peptide, one or more peptides of claims 1-12, and a TLR agonist. In some aspects, the polypeptide further includes a TLR agonist amino-proximal to the cell-penetrating peptide. Further aspects are described in Belnoue et al., JCI Insight. 2019;4(11):e127305, which is incorporated herein by reference.

[0013] The pharmaceutical composition of the present disclosure can be formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection.The peptide or polypeptide of the present disclosure can be contained in liposome, lipid-containing nanoparticle, or lipid-based carrier.The pharmaceutical preparation can be formulated for injection or inhalation as a nasal spray.The composition of the present disclosure can be formulated as a vaccine.In some aspects, the composition can further comprise an adjuvant.In some aspects, the composition comprises at least two peptides of the present disclosure. In some aspects, a composition comprises 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, or 40 peptides (or any derivable range therein) of the present disclosure, or at least 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, or 40 peptides of the present disclosure. 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 peptides (or any derivable range therein), or at most 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, or 40 peptides (or any derivable range therein) of the present disclosure.

[0014] In some aspects, the polypeptide or composition comprises four different peptides, each peptide selected from the peptides of SEQ ID NOs: 10, 323, 221, 44, 27, 156, 37, 168, 20, 163, 29, 136, 24, 62, 138, 157, 160, 151, 158, 23, 39 and 57. In some aspects, a polypeptide or composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 peptides (or any derivable range therein), or comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 peptides (or any derivable range therein), or comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 peptides (or any derivable range therein), each peptide being selected from the group consisting of SEQ ID NO: NO:10, 323, 221, 44, 27, 156, 37, 168, 20, 163, 29, 136, 24, 62, 138, 157, 160, 151, 158, 23, 39, or 57.

[0015] The dendritic cells of the present disclosure may be further defined as being or including mature dendritic cells. The cells may be cells having HLA-A type. The cells may also be HLA-A, HLA-B or HLA-C. In some aspects, the cells are HLA-A3 or HLA-A11 type. In some aspects, the cells are HLA-A01, HLA-A02, HLA-A24, HLA-B07, HLA-B08, HLA-B15 or HLA-B40. The method may further comprise isolating the expressed peptide or polypeptide. The T cells may include CD8+ T cells. The T cells may be CD4+ T cells, Th1, Th2, Th17, Th9 or Tfh T cells, cytotoxic T cells, memory T cells, central memory T cells or effector memory T cells.

[0016] In the method of the present disclosure, the contacting step may be further defined as co-culturing the starting population of immune effector cells with an antigen presenting cell (APC), an artificial antigen presenting cell (aAPC), or an artificial antigen presenting surface (aAPS), where the APC, aAPC, or aAPS presents the peptide on its surface. The APC may be, for example, a dendritic cell.

[0017] The immune effector cells can be T cells, peripheral blood lymphocytes, natural killer (NK) cells, invariant NK cells, or NKT cells. The immune effector cells can be differentiated from mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells. Aspects of T cells include CD8 + T cells, CD4 + T cells, or T cells further defined as γδ T cells. T cells may be defined as being cytotoxic T lymphocytes (CTLs).

[0018] The subject in the method of the present disclosure can be a human subject. The subject can also be an experimental animal, mouse, rat, pig, horse, rabbit or guinea pig. The method can further comprise administering at least a second therapeutic agent. The second therapeutic agent can be an anti-cancer agent. Treating as defined in the method of the present disclosure can comprise one or more of: reducing tumor size, increasing overall survival rate, reducing the risk of cancer recurrence, reducing the risk of progression, and / or increasing the probability of progression-free survival, relapse-free survival, and / or recurrence-free survival.

[0019] The composition of the present disclosure may comprise or further comprise an MHC polypeptide and a peptide of the present disclosure, and the MHC polypeptide and / or peptide are conjugated to a detection tag. Thus, suitable detection tags include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The tag may be simply detected or quantified. A simply detected response generally includes a response that is merely confirmed to be present, whereas a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other properties. In a luminescent or fluorescent assay, the detectable response may be generated directly using a luminophore or fluorophore associated with the assay component that actually participates in the binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent tags that generate a signal include, but are not limited to, bioluminescence and chemiluminescence. Examples of suitable fluorescent tags include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in 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.

[0020] MHC polypeptides and peptides can be functionally linked. The term "functionally linked" refers to a situation where two components are combined or can be combined to form a complex. For example, the components can be covalently linked on the same polypeptide, e.g., in a fusion protein, or the components can have some degree of binding affinity for each other, e.g., binding affinity caused by van der Waals forces. Thus, aspects of the present disclosure relate to MHC polypeptides and peptides being functionally linked via peptide bonds. MHC polypeptides and peptides can also be functionally linked by van der Waals forces. Peptide-MHC can be functionally linked to form a pMHC complex. In some aspects, at least two pMHC complexes are functionally linked to each other. Other aspects include 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes operably linked to each other, or include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes operably linked to each other, or include at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes operably 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 polypeptide to peptide is 1:1 to 4:1. In some aspects, the ratio or average ratio is at least 1, 2, 3, 4, 5, or 6 to about 1, 2, 3, 4, 5, or 6 (or any derivable range therein), at most 1, 2, 3, 4, 5, or 6 to about 1, 2, 3, 4, 5, or 6 (or any derivable range therein), or about 1, 2, 3, 4, 5, or 6 to about 1, 2, 3, 4, 5, or 6 (or any derivable range therein).

[0021] In some aspects of the present disclosure, the peptide is complexed with MHC. In some aspects, the MHC comprises HLA-A type. The MHC can be further defined as HLA-A3 type or HLA-A11 type. The peptide can be loaded onto dendritic cells, lymphoblastoid cells, peripheral blood mononuclear cells (PBMCs), artificial antigen presenting cells (aAPCs), or artificial antigen presenting surfaces. The artificial antigen presenting surfaces can include MHC polypeptides conjugated or linked to the surface. Exemplary surfaces include beads, microplates, glass slides, or cell culture plates.

[0022] The method of the present disclosure may further comprise counting the number of T cells bound to the peptide and / or MHC. The composition comprising the 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, I, II, III, or IV cancer. The cancer may be colorectal cancer. The colorectal cancer may comprise mismatch repair deficient colorectal cancer (MMR-d) and / or microsatellite instability (MSI) positive colorectal cancer. The subject to be diagnosed or treated may be treated for stage I or stage II cancer. The subject may be a subject determined to have mismatch repair deficient colorectal cancer (MMR-d) and / or microsatellite instability (MSI) positive colorectal cancer. The cancer may comprise a peptide-specific cancer, which is a peptide of one of SEQ ID NOs: 1-776, or a peptide of the present disclosure. The subject may be a subject diagnosed with and / or determined to have cancer. The subject or patient may also be a subject or patient characterized as having a peptide-specific cancer, such as a peptide of the present disclosure or one of SEQ ID NOs: 1-776. The method of the present disclosure may include or further include a step of sorting the number of T cells bound to the peptide and / or MHC. The method of the present disclosure may also include or further include a step of sequencing one or more TCR genes from the T cells bound to the peptide and / or MHC. The method may also include or further include a step of sequencing TCR alpha and / or beta genes from a TCR, such as a TCR that binds to a peptide of the present disclosure. The method may also include or further include a step of grouping lymphocyte interactions by paratope hotspots (GLIPH) analysis. This is further described in Glanville et al., Nature. 2017 Jul 6;547(7661):94-98, which is incorporated herein by reference.

[0023] The composition of the present disclosure can be serum-free, mycoplasma-free, endotoxin-free and / or sterile.The method can further comprise culturing the cells of the present disclosure in culture medium, incubating the cells under conditions that allow the cells to divide, screening the cells, and / or freezing the cells.The method can comprise or further comprise isolating the expressed peptide or polypeptide from the cells of the present disclosure.

[0024] The method of the present disclosure may include or further include screening the dendritic cells for one or more cell characteristics. The method may include or further include contacting the cells with one or more cytokines or growth factors. The one or more cytokines or growth factors may include GM-CSF. The cell characteristics may include cell surface expression of one or more of CD86, HLA, and CD14. The dendritic cells may be derived from CD34+ hematopoietic stem cells or CD34+ hematopoietic progenitor cells.

[0025] The contacting step in the method of the present disclosure can be further defined as co-culturing the starting population of immune effector cells with antigen-presenting cells (APCs), and APCs present peptides on their surface. The APCs can be further defined as dendritic cells. The dendritic cells can be derived from peripheral blood monocytes (PBMCs). The dendritic cells can be isolated from PBMCs. The dendritic cells can also be the cells from which DCs are derived and isolated by leukapheresis.

[0026] The peptide-MHC (pMHC) complex of the present disclosure can be produced by contacting the peptide of the present disclosure with an MHC complex. The peptide can be expressed in a cell and bind to an endogenous MHC complex to form a pMHC. In some aspects, peptide exchange is used to produce a pMHC complex. For example, a cleavable peptide, such as a photocleavable peptide, can be designed to bind to and stabilize MHC. Cleavage of the peptide (e.g., by irradiation of the photocleavable peptide) dissociates the peptide from the HLA complex, resulting in an empty HLA complex that quickly disintegrates, unless UV exposure is performed in the presence of a "rescue peptide". Thus, the peptide of the present disclosure can be used as a "rescue peptide" in a peptide exchange procedure. Also described herein are pMHC complexes that include the peptides of the present disclosure. The pMHC complexes can be operably linked to a solid support or can be bound to a detectable moiety, such as a fluorescent molecule, a radioisotope, or an antibody. A peptide-MHC multimer complex may comprise 1, 2, 3, 4, 5, or 6 peptide-MHC molecules operably linked together, or may comprise at least 1, 2, 3, 4, 5, or 6 peptide-MHC molecules operably linked together, or may comprise at most 1, 2, 3, 4, 5, or 6 peptide-MHC molecules operably linked together. Linkage may be covalent, e.g., via peptide bonds, or may be non-covalent. pMHC molecules may be bound to biotin molecules. Such pMHC molecules may be multimerized by binding to streptavidin molecules. pMHC multimers may be used to detect antigen-specific T cells or TCR molecules in a composition or tissue. 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 slide, and detection of binding between pMHC multimers and cells may be performed. Thus, the pMHC molecules and pMHC multimers of the present disclosure may be used to detect and diagnose cancer in a subject or to determine the immune response in an individual with cancer.

[0027] In the method of the present disclosure, the obtaining step may include isolating a 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 a subject with cancer, such as a peptide-specific cancer. The subject may be a subject determined to have a cancer that expresses the peptide of the present disclosure. The method of the present disclosure may include, or may further include, a step of introducing the peptide, or a nucleic acid encoding the peptide, into the dendritic cells prior to co-culture. The introduction of the peptide may be performed by transfecting or infecting the dendritic cells with a nucleic acid encoding the peptide, or by incubating the peptide with the dendritic cells. The peptide or the nucleic acid encoding the peptide may be introduced by electroporation. Other methods of transferring 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 the nucleic acid encoding the peptide may be introduced by adding the peptide or the nucleic acid encoding the peptide to the dendritic cell culture medium. The immune effector cells can be co-cultured with a second population of dendritic cells into which a peptide or a nucleic acid encoding the peptide has been introduced. In the method of the present disclosure, after co-culture, a population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive T cells can be purified from the immune effector cells. The population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive T cells can be purified by fluorescence-activated cell sorting (FACS). Clonal populations of peptide-specific immune effector cells can be generated by limiting or serial dilution followed by expansion of individual clones by rapid expansion protocols.

[0028] In the method of the present disclosure, the purifying step may include or further include generating a clonal population of peptide-specific immune effector cells by limiting or serial dilution of the sorted cells followed by expansion of individual clones by a rapid expansion protocol. The method of the present disclosure may include or further include cloning of T cell receptors (TCRs) from a clonal population of peptide-specific immune effector cells. In the method of the present disclosure, the term isolating may be defined as or include cloning of T cell receptors (TCRs) from a clonal population of peptide-specific immune effector cells. Cloning of TCRs may include cloning of TCR alpha and beta chains. TCRs may be cloned using 5'-Rapid Amplification of cDNA Ends (RACE) method. TCR alpha and beta chains may be cloned using 5'-Rapid Amplification of cDNA Ends (RACE) method. The cloned TCRs may be subcloned into an expression vector. The expression vector may include a linker domain between the TCR alpha and TCR beta sequences. The expression vector may be a retroviral or lentiviral vector. The vector may be an expression vector as described herein. The linker domain may comprise a sequence encoding one or more peptide cleavage sites. The one or more cleavage sites may be furin cleavage sites and / or P2A cleavage sites. The TCR alpha sequence and the TCR beta sequence may be linked by an IRES sequence.

[0029] The host cells of the present disclosure may be transduced with an expression vector to generate engineered cells expressing TCR alpha and / or beta chains. The host cells may be immune cells. The immune cells may be T cells, and the engineered cells may be referred to as engineered T cells. The T cells may be any of the types of T cells described herein, e.g., CD8 +The starting population of immune effector cells may be T cells, CD4+ T cells, or γδ T cells. The starting population of immune effector cells may be obtained from a subject with cancer or peptide-specific cancer, and the host cells are allogeneic or autologous to the subject. In some, but not all, aspects, obtaining the starting population of immune effector cells refers to recovering them from the subject. The peptide-specific T cells may be autologous or allogeneic. In the methods of the present 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 rapid expansion protocols. In the methods of the present disclosure, the purifying step 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 after co-culture.

[0030] The peptide of the present disclosure can be linked to a solid support. The peptide can be conjugated to a solid support or can be bound to an antibody conjugated to a solid support. The solid support can include a microplate, a bead, a glass surface, a slide, or a cell culture dish. The solid support can include a nanofluidic chip. In the method of the present disclosure, the step of detecting T cell response can include or further include detecting the binding of the peptide to T cell or TCR. In the method of the present disclosure, the step of detecting T cell response can include or further include ELISA, ELISPOT, or tetramer assay.

[0031] The kit of the present disclosure may include one or more peptides of the present disclosure in a container. The peptide may be included in a pharmaceutical preparation. The pharmaceutical preparation may be formulated for parenteral administration or inhalation. In some aspects, the peptide is included in a cell culture medium.

[0032] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the field 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.

[0033] The use of the words "a" or "an" when used in conjunction with the term "comprising" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0034] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination with or exclusive of each other. 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 or z". It is specifically contemplated that x, y or z can be specifically excluded from an embodiment or aspect.

[0035] The terms "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), "characterized by" (and any form of including, e.g., "characterized as"), or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0036] The compositions and methods of their use can "comprise", "consist essentially of", or "consist" of any of the components or steps disclosed throughout this specification. The phrase "consisting of" excludes any element, step, or ingredient not specified. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps, and those that do not materially affect the basic and novel characteristics. It is contemplated that an embodiment or aspect described in conjunction with the term "comprising" may also be implemented in conjunction with the term "consisting of" or "consisting essentially of".

[0037] It is specifically contemplated that any limitation described with respect to one embodiment or aspect of the present invention may be applied to any other embodiment or aspect of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to produce or utilize any composition of the present invention. Aspects of the embodiments described in the examples are also embodiments that may be practiced in conjunction with the embodiments described elsewhere in different examples, or elsewhere in this application, for example, in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legend Descriptions.

[0038] Other objects, features and advantages of the present invention will become apparent from the following detailed description, but it should be understood that the detailed description and 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]

[0039] 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 better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0040] [Figure 1]FIG. 1 is a schematic showing the in silico neoantigen prediction process. [Diagram 2] 1 shows the in vitro validation pipeline. [Diagram 3] Analyses from whole exosome and RNA sequencing are shown. [Figure 4] FIG. 1 shows a waterfall plot of the most repetitive neoantigens derived from MHC class I. [Figure 5A] Figures 5A-D show validation of neo-antigen immunogenicity. Figure 5A shows the results of MHC tetramer staining. Figure 5B shows quantification of INFγ secreting cells. Figure 5C shows cytotoxic gene expression. Figure 5D shows quantification of secreted cytokines from a multiplex ELISA-based cytokine profile obtained from CD8+ T cells after stimulation with neo-Ag-MHC. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6] Schematic diagram of the test. [Figure 7A]Figure 7A-B. Mutational landscape in LS samples. A) In the top panel, the absolute number of each type of mutation per sample is shown on the left y-axis, and the mutational burden (mutations / MB) of each sample is shown on the right x-axis. The central grid panel shows a summary of mutations in selected genes. Each horizontal row is a gene and each vertical row is a sample. Mutations are colored by type as shown in the legend on the right. The bar graph to the left of this summary of mutations represents the proportion of individuals in which each particular gene is mutated. The bottom panel shows the molecular and pathological features of each sample as covariate bars: MSI status (top), disease category (middle) and histopathology (bottom). MSI-H=microsatellite instability high, MSI-L=microsatellite instability low, MSS=microsatellite stable, PRECA=precancerous, ADVPRECA=high-grade precancerous, CANCER=cancer, AP=adenomatous polyp, ADVCA=adenocarcinoma (stages III and IV), SSA=sessile serrated adenoma, HP=hyperplastic polyp, IP=inflammatory polyp, MB=megabase. B) Significant differences in mutation rates are observed when comparing samples by MSI status (Mann-Whitney test****P-value>0.0001), disease category (Mann-Whitney test**P-value>0.01) and histopathology (Mann-Whitney test***P-value>0.001). [Figure 7B] See legend to Figure 7A. [Figure 8A]Figure 8A-E. Neo-antigen landscape produced from mutant proteins in LS patient cohorts. A) There is a significant difference between the number of MHC I neo-Ags and MHC II neo-Ags produced by MSI samples compared to MSI-L and MSS samples (Mann-Whitney test****P-value<0.0001). B) There is a significant difference between the number of MHC I neo-Ags and MHC II neo-Ags produced by cancer compared to high-grade precancerous and precancerous conditions (Mann-Whitney test***P-value<0.001). C) There is a significant difference between the number of MHC I neo-Ags and MHC II neo-Ags produced by cancer compared to other histopathologies (Mann-Whitney test**P-value<0.01). D) The number of both MHC-I and MHC-II neo-Ags detected per sample is significantly correlated with the mutation burden in each sample (Spearman P-value>0.0001). E) Waterfall plot of the most recurrent MHC class I neo-antigens in the discovery set. The top bar plot represents the neo-Ag rate (neo-Ag / Mb) per sample. The grid panel shows the top 50 most frequent MHC-I neo-Ags from the discovery set organized by the percentage of MSI-H sites in each gene (represented as a color scheme on the left). In silico neo-Ag immunogenicity ranking is represented, with dark grey being the 1st percentile (highest predicted immunogenicity) and light grey being the lowest rank. The bottom panel shows the molecular and pathological features of each sample as covariate bars: MSI status (top), disease category (middle) and histopathology (bottom). MSI-H=microsatellite instability high, MSI-L=microsatellite instability low, MSS=microsatellite stable, PRECA=premalignant, ADVPRECA=high-grade premalignant, carcinoma=carcinoma, AP=adenomatous polyp, ADVCA=adenocarcinoma (stages III and IV), SSA=sessile serrated adenoma, HP=hyperplastic polyp, IP=inflammatory polyp. Magenta asterisks indicate whether a particular neoAg meets one TESLA presentation criterion (*), two TESLA presentation criteria (**), and three TESLA presentation criteria (***). TESLA presentation criteria are binding affinity <34 nM, tumor abundance >33 TPM, and binding stability >1.4 h. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 9] Figure 9A-C. NeoAgs shared between discovery and validation sets. A) Venn diagram showing the number of predicted neoAgs from each set. B) Waterfall plot of the top 50 most repetitive MHC class I neo-antigens from the validation set. The top bar plot represents the neoAg rate (neoAg / Mb) per sample. The grid panel shows the top 50 most repetitive MHC I neo-Ags from the validation set organized by the percentage of MSI-H sites within that gene (represented as a color scheme on the left). In silico neoAg immunogenicity ranking is represented as a greyscale, with dark grey being the 1st percentile (highest predicted immunogenicity) and light grey being the lowest ranked. NeoAgs from genes in light font were also present in the discovery set. The bottom panel shows the molecular and pathological features of each sample as covariate bars: MSI status (top), disease category (middle) and histopathology (bottom). MSI-H=microsatellite instability high, MSI-L=microsatellite instability low, MSS=microsatellite stable, PRECA=precancerous, ADVPRECA=high-grade precancerous, CANCER=cancer, AP=adenomatous polyp, ADVCA=adenocarcinoma (stages III and IV), SSA=sessile serrated adenoma, HP=hyperplastic polyp, IP=inflammatory polyp. C) Each category of predicted neoags in the discovery set indicates the percentage also present in the validation set. [Figure 10A]FIG. 10A-D. In vitro validation of predicted neo-Ag immunogenicity using ELISpot IFNy assay. A) Schematic of peptide pool, stimulation and cell culture workflow for ELISpot assay. PBMCs were exposed to individual peptides from the immunogenic pool in the presence of IL-7 for 3 days, followed by expansion of neo-Ag-specific T cells in the presence of IL-2. On day 13, expanded cells (105 cells / well) were seeded in 96-well ELISpot plates coated with IFNy antibody and restimulated with the respective peptide for 24 h. IFNy secreting cells were analyzed as spot forming units (SFU) and we selected ≧15 SFU produced by peptide stimulated cells over DMSO control cells as an indicator of peptide immunogenicity. B) Quantification of IFNy secreting cells (SFU) obtained from ELISpot assay of 12 neo-Ag stimulated PBMCs. ConcA and DMSO served as positive and negative controls. The bottom of the bars shows representative images of triplicate wells containing IFNγ-secreting cells from three donors. C) Selection of 110 predicted MHC-I neoAgs from three different categories and percentage of immunogenicity in vitro validation. The numbers shown in brackets () refer to the percentage of tested neoAgs that showed immunogenicity in the ELISpot assay. "Most immunogenic" refers to neoAgs selected from the predicted list of the top 100 most immunogenic MHC-I neoAgs. "Most repetitive" refers to neoAgs selected from the predicted list of the top 100 most repetitive MHC-I neoAgs. "Other" refers to neoAgs predicted to have low immunogenicity and no repetitiveness. A total of 44 MHC-II neoAgs from two different categories were tested. The numbers shown in brackets () refer to the percentage of tested neoAgs that showed reactivity in the ELISpot assay. "Most immunogenic" refers to neoAgs selected from a predicted list of the top 100 most immunogenic MHC-II neoAgs. "Most repetitive" refers to neoAgs selected from a predicted list of the top 100 most repetitive MHC-II neoAgs. D) pMHC-pentamer staining.Expanded Pan-T cells from healthy human donors were isolated and stained with WDTC1 neo-Ag peptide / A*02:01 pentamer complex and PerCP-conjugated CD8 antibody, followed by flow cytometry analysis. Negative magnetic selection was used to isolate Pan-T cells from healthy human donor PBMCs (HLA-A*02:01). Opto-antigen presenting beads conjugated with WDTC1 neo-Ag-peptide were used to stimulate and expand isolated Pan-T cells. Viable Pan-T cells were gated based on FSC and SSC scatter and SYTOX Blue dead cell staining after double exclusion. CD8 positive cells (x-axis) and PE-biotin-pMHC-pentamer positive cells (y-axis); flow cytometry plots representing unstained, unstimulated, and WDTC1 Opto-antigen presenting beads stimulated cells (left to right). Percentages of live CD8+ cells and CD8 / WDTC1 neo-Ag loaded MHC pentamer positive cells are shown. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 11A] Figure 11A-C. Differential gene expression analysis between cancer and precancerous conditions. A) Transcriptional expression profile of 78 genes differentially expressed between cancer and precancerous conditions. Genes in light grey font are part of the immune response. B) Pathway enrichment analysis showing pathways activated and suppressed in cancer compared to precancerous conditions. Pathways in light grey font are part of the immune response. C) Immune cells showing significant increase or decrease between cancer, high-grade precancerous conditions and precancerous conditions after immune cell deconvolution. (Mann-Whitney test, *=p<0.05, **=p<0.01). [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 12]Figure 12A-B. MSI status derived from MSI sensor results. A) Bar graphs show the number of microsatellite sites (left y-axis) with unstable sites as purple stacked bars and stable sites as grey stacked bars. MSI scores are shown as dark grey circles (right y-axis). Samples with an MSI score ≥ 10% are considered MSI-H. The bottom panel displays MSI status as covariate bars. B) MSI score distribution by tissue category. MSI score >= 10% means MSI-H, MSI score < 10% and >= 3.5% means MSI-L, and MSI score < 3.5% means MSS. [Figure 13] Status of second somatic hits in MMR genes. The grid panel shows the type of germline mutation present in one of the MMR genes for each sample (pastel colors) and the type of second somatic mutation in the same sample as white and black symbols. The bottom panel shows the molecular and pathological features of each sample as covariate bars: MSI status (top), disease category (middle) and pathology (bottom). MSI-H=high microsatellite instability, MSI-L=low microsatellite instability, MSS=microsatellite stable, PRECA=precancerous, ADVPRECA=high-grade precancerous, cancer=cancer, AP=adenomatous polyp, ADVCA=adenocarcinoma (stages III and IV), SSA=sessile serrated adenoma, HP=hyperplastic polyp, IP=inflammatory polyp. [Figure 14] Neo-antigen prediction pipeline. Schematic of the computational pipeline used to predict MHC-I and MHC-II neo-Ags from each sample. The final product of this pipeline is a list of neo-epitopes ranked based on their immunogenicity score. [Figure 15] The most frequent HLA alleles in the LS patient cohort. Proportion of samples covered by the top 80 most frequent HLA alleles. [Figure 16]Number of neo-antigens, and their MHC binding affinity. Bar graphs show the number of predicted MHC-I neo-Ags and predicted MHC-II neo-Ags with binding affinities of <500nM, 50-100nM, 100-500nM. The bottom panel shows the molecular and pathological features of each sample as covariate bars: MSI status (top), disease category (middle) and histopathology (bottom). MSI-H=high microsatellite instability, MSI-L=low microsatellite instability, MSS=microsatellite stable, PRECA=precancerous, ADVPRECA=high-grade precancerous, CANCER=carcinoma, AP=adenomatous polyp, ADVCA=adenocarcinoma (stages III and IV), SSA=sessile serrated adenoma, HP=hyperplastic polyp, IP=inflammatory polyp. [Figure 17] Waterfall plot of the top 50 most repetitive MHC class II neo-antigens from the discovery set. The top bar plot represents the neo-Ag rate (neo-Ag / Mb) per sample. The grid panel shows the top 50 most repetitive MHC II neo-Ags from the discovery set organized by the proportion of MSI-H sites within their genes. In silico predicted neo-Ag immunogenicity ranking is represented as a scale, with dark grey being the 1st percentile (highest predicted immunogenicity) and light grey being the lowest rank. The bottom panel shows the molecular and pathological features of each sample as covariate bars: MSI status (top), disease category (middle) and histopathology (bottom). MSI-H=microsatellite instability high, MSI-L=microsatellite instability low, MSS=microsatellite stable, PRECA=precancerous, ADVPRECA=high-grade precancerous, CANCER=cancer, AP=adenomatous polyp, ADVCA=adenocarcinoma (stages III and IV), SSA=sessile serrated adenoma, HP=hyperplastic polyp, IP=inflammatory polyp. [Figure 18]The top 100 most immunogenic MHC-I predicted neo-antigens from the discovery set meet the TESLA presentation and recognition criteria. Five different peptide features or criteria were determined by TESLA that improve the performance of neo-antigen prediction. These are binding affinity (Best.MTScore) < 34 nM, tumor abundance (mt_allele_exp) > 33 TPM, binding stability (Thalf(h)) > 1.4 h, agretopicity < 0.1 and heterogeneity > 10-16. The upset plots show the number of neo-antigens passing various combinations of the five criteria. The aquamarine bar graphs on the left show the total number of neo-ags passing each criterion alone. [Figure 19] Figure 19A-B. In vitro validation of pooled predicted neoAg immunogenicity using ELISpot IFNγ assay. A) Schematic of peptide pool, stimulation and cell culture workflow for ELISpot assay. All 154 peptides were grouped into 15 peptide pools as indicated to stimulate PBMCs from three healthy human donors. IFNγ-secreting cells, indicating immunogenicity, were analyzed as spot-forming units (SFU). B) Quantification of IFNγ-secreting cells (SFU) obtained from ELISpot assay. Pools 2, 3, 5, 6, 9 and 12 produced at least 15 SFU / 105 cells from two different PBMCs. The bottom of the bar shows a representative image of triplicate wells containing IFNγ-secreting cells. ConcA and DMSO served as positive and negative controls. [Figure 20] Other elispot reactive peptides. IFN-g ELISPOT SFU counts and well images for neoAgs that showed lower ELISPOT reactivity against human PBMC from normal donors. [Figure 21]MSI-H sample coverage of the most immunogenic MHC-I predicted neoags. The graph on the left shows the percentage of MSI-H samples covered by the top 100 most immunogenic MHC-I neoags when ranked by repetition, with the most repetitive ones considered first in the list. The graph on the right shows the percentage of MSI-H samples covered by the top 100 most immunogenic MHC-I neoags when ranked by immunogenicity score only, with the most immunogenic ones considered first in the list even if they are not repetitive. [Figure 22A] Figure 22A-B. Validation of predicted neo-Ags in LS rhesus macaques. A) Immunogenicity of neo-Ag peptide pools and deconvoluted neo-Ags by ELISpot assay. PBMCs from LS rhesus macaques (n=4) were stimulated for 48 hours with 10 peptide pools and 12 individual peptides from the pools, concavalin A (+ve control), and DMSO (-ve control). B) ELISpot images. All deconvoluted peptides were determined to be immunogenic in stimulated rhesus PBMCs, except for PLOD1 and CELSR2. Spot forming units of IFNγ secretion were analyzed and quantified by ELISpot plate reader. [Figure 22B] See legend to Figure 22A. [Figure 23A] Figures 23A-B. Unsupervised principal component analysis of all samples with gene expression results. A) Unsupervised PCA analysis of samples labeled based on tissue category. B) Unsupervised PCA analysis of samples labeled based on MSI status. [Figure 23B] See legend to Figure 23A. [Figure 24A]Figure 24A-C. Differential gene expression analysis between MSI-H and MSS samples. A) Transcriptional expression profile of 44 genes differentially expressed between MSI-H and MSS samples. Genes in light grey font are part of the immune response. B) Pathway enrichment analysis showing pathways activated and suppressed in MSI-H compared to MSS samples. Pathways in light grey font are part of the immune response. C) Immune cells showing significant increase or decrease between MSI-H, MSI-L and MSS samples after immune cell deconvolution. (Mann-Whitney test, *=p<0.05, **=p<0.01). [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description of the Invention Lynch syndrome (LS) patients constitute a well-defined population likely to benefit from cancer immune blockade strategies, given that they develop DNA mismatch repair-deficient tumors that generate a high burden of neoantigens (neo-Ags). In the present application examples, whole-exome sequencing and mRNA-seq of colorectal cancer (CRC) and precancerous conditions of a LS patient cohort (N=46) to identify somatic and genomic mutation variant landscapes, as well as predictions of highly immunogenic and recurrent neo-antigens (neo-Ags), based on immunogenicity scores using in silico computational methods. The analysis revealed a positive correlation between microsatellite instability (MSI) and high neo-antigen burden in precancerous and cancerous colorectal lesions. Furthermore, we tested 154 highly immunogenic and recurrent predicted neo-Ags in vitro from peripheral blood mononuclear cells (PBMCs) of six healthy donors using an ELISpot assay. These results showed that up to 50% of predicted MHC-I frameshifted neoAgs retained their immunogenicity, thus validating the neoAg prediction pipeline. Overall, the results obtained from the mutational and gene expression analysis of classified neoAgs in this application will help improve our understanding of LS-derived cancers, which will guide the future development of immunoprophylactic vaccine strategies.

[0042] I. Immunotherapy Using the Peptides of the Disclosure The peptides described herein (e.g., one of SEQ ID NOs: 1-776) can be used in cancer immunotherapy. For example, one of SEQ ID NOs: 1-776 can be contacted with or used to stimulate a population of T cells to induce proliferation of T cells that recognize or bind to the peptide. In other aspects, the peptides of the present disclosure can be administered to a subject, such as a human patient, to enhance the subject's immune response against cancer.

[0043] The peptides of the present disclosure can be included in active immunotherapy (e.g., cancer vaccines) or passive immunotherapy (e.g., adoptive immunotherapy). Active immunotherapy includes immunizing a subject with purified peptide antigens or immunodominant peptides (natural or modified). Alternatively, antigen-presenting cells pulsed with the peptides of the present disclosure (or transfected with a gene encoding an antigen comprising the peptide) can be administered to the subject. The peptides can be modified or contain one or more mutations, such as, for example, substitution mutations. Passive immunotherapy includes adoptive immunotherapy. Adoptive immunotherapy generally involves administering cells to a subject, where the cells (e.g., cytotoxic T cells) have been sensitized in vitro to the peptides of the present disclosure (see, for example, U.S. Pat. No. 7,910,109).

[0044] In some aspects, flow cytometry can be used in adoptive immunotherapy for rapid isolation of human tumor antigen-specific T cell clones, for example, by using T cell receptor (TCR) Vβ antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assays. See, for example, Lee et al., J.Immunol.Methods, 331:13-26, 2008, which is incorporated by reference for all purposes. In some aspects, tetramer-guided cell sorting can be used, for example, the method described in Pollack, et al., J Immunother Cancer.2014;2:36, which is incorporated by reference for all purposes. Various culture protocols are also known for adoptive immunotherapy and can be used in the aspects of the present disclosure. In some aspects, cells can be cultured under conditions that 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 for all purposes). In other aspects, T cells may be expanded under culture conditions utilizing antigen-presenting cells such as dendritic cells (Nestle et al., 1998, incorporated by reference), and in some aspects, 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), incorporated by reference, which may be used with aspects of the present disclosure. Various methods are known and may be used for cloning and expanding human antigen-specific T cells (see, for example, Riddell et al., 1990, incorporated by reference herein).

[0045] In certain aspects, the following protocol may be used to generate T cells that selectively recognize the peptides of the present disclosure: Peptide-specific T cell lines can be generated from normal donors or HLA-restricted normal donors and patients using previously reported methods (Hida et al., 2002). ENREF 32Briefly, PBMCs (1 × 10 5 The T cells (cells / well) can be stimulated with 50% AIM-V medium (Invitrogen), 50% RPMI1640 medium (Invitrogen), 10% human AB serum (Valley Biomedical, Winchester, VA) and 100 IU / ml interleukin-2 (IL-2). Cells can be restimulated with the corresponding peptide about every 3 days. After 5 stimulations, T cells from each well can be washed and incubated with T2 cells in the presence or absence of the corresponding peptide. After about 18 hours, production of interferon (IFN)-γ in the supernatant can be determined by ELISA. T cells secreting large amounts of IFN-γ can be further expanded by a rapid expansion protocol (Riddell et al., 1990; Yee et al., 2002b).

[0046] In some aspects, immunotherapy may utilize the peptides of the present disclosure in association with cell-penetrating substances, such as liposomes or cell-penetrating peptides (CPPs). Antigen-presenting cells (such as dendritic cells) pulsed with peptides may be used to enhance antitumor immunity (Celluzzi et al., 1996; Young et al., 1996). Liposomes and CPPs are described in more detail below. In some aspects, immunotherapy may utilize nucleic acids encoding the peptides of the present disclosure, the nucleic acids being delivered, for example, by viral or non-viral vectors.

[0047] In some aspects, the peptides of the present disclosure may be used in immunotherapy to treat cancer in a mammalian subject, such as a human patient.

[0048] II. Cell-penetrating peptides The peptides of the present disclosure may also be associated or covalently linked to cell penetrating peptides (CPPs). Cell penetrating peptides that may be covalently linked to the peptides of the present disclosure include, for example, HIV Tat, herpes virus VP22, Drosophila antennapedia homeobox gene product, signal sequence, fusion sequence, or protegrin I. Covalently linking a peptide to a CPP can prolong the presentation of the peptide by dendritic cells, thus enhancing anti-tumor immunity (Wang and Wang, 2002). In some aspects, the peptides of the present disclosure (e.g., peptides or polyepitope strings contained within) can be covalently linked (e.g., via peptide bonds) to a CPP to generate a fusion protein. In other aspects, the peptides of the present disclosure or nucleic acids encoding the peptides can be encapsulated or associated with liposomes, such as multilamellar, vesicular, or multivesicular liposomes.

[0049] As used herein, "association" refers to a physical association, a chemical association, or both. For example, the association can involve covalent bonding, hydrophobic interactions, encapsulation, surface adsorption, and the like.

[0050] As used herein, "cell-permeable agent" refers to a composition or compound that enhances the intracellular delivery of peptide / polyepitope string to antigen-presenting cells. For example, cell-permeable agent can be lipid that enhances the ability to cross plasma membrane when associated with peptide. Alternatively, cell-permeable agent can be peptide. Cell-permeable peptides (CPPs) are known in the art and include, for example, 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).

[0051] Cell-penetrating peptides (or "protein transduction domains") have been identified from the third helix of the Drosophila Antennapedia homeobox gene (Antp), HIV Tat, and herpesvirus VP22, all of which contain positively charged domains rich in arginine and lysine residues (Schwarze et al., 2000; Schwarze et al., 1999). Hydrophobic peptides derived from signal sequences have also been identified as cell-penetrating peptides (Rojas et al., 1996; Rojas et al., 1998; Du et al., 1998). Coupling of these peptides to marker proteins such as β-galactosidase has been shown to result in efficient internalization of the marker proteins into cells, and chimeric in-frame fusion proteins containing these peptides have been used to deliver proteins to a wide range of cell types both in vitro and in vivo (Drin et al., 2002). Fusion of these cell-penetrating peptides to the peptides of the present disclosure can enhance cellular uptake of the polypeptide.

[0052] In some aspects, cellular uptake is promoted by conjugation of lipids, such as stearate or myristilate, to the polypeptide. Lipidation has been shown to enhance the passage of peptides into cells. Conjugation of lipid moieties is another way in which the present invention increases cellular uptake of polypeptides.

[0053] The peptide of the present disclosure can be included in a liposomal vaccine composition. For example, the liposomal composition can be or include a proteoliposome composition. Methods for producing proteoliposome compositions that can be used with the present invention are described, for example, in Neelapu et al. (2007) and Popescu et al. (2007). In some aspects, the proteoliposome composition can be used to treat cancer.

[0054] By enhancing uptake of the polypeptides of the present disclosure, it may be possible to reduce the amount of protein or peptide required for treatment, which can significantly reduce treatment costs and increase supply of therapeutic agents. Lower dosages can also minimize potential immunogenicity of the peptides and limit toxic side effects.

[0055] In some aspects, the peptides of the present disclosure may be associated with nanoparticles to form nanoparticle-polypeptide complexes. In some aspects, the nanoparticles are liposomes or other lipid-based nanoparticles, such as lipid-based vesicles (e.g., DOTAP:cholesterol vesicles). In other aspects, the nanoparticles are iron oxide-based superparamagnetic nanoparticles. Superparamagnetic nanoparticles, with diameters ranging from about 10-100 nm, are small enough to avoid capture by the spleen, but large enough to avoid clearance by the liver. Particles of this size can penetrate very small capillaries and can be effectively distributed in body tissues. The superparamagnetic nanoparticle-polypeptide complexes can be used as MRI contrast agents to identify and track cells that take up the peptide. In some aspects, the nanoparticles are semiconductor nanocrystals or semiconductor quantum dots, both of which can be used for optical imaging. In a further aspect, the nanoparticles can be nanoshells that include a gold layer over a silica core. One advantage of nanoshells is that the polypeptide can be conjugated to the gold layer using standard chemistry. In other aspects, the nanoparticles can be fullerenes or nanotubes (Gupta et al., 2005).

[0056] Peptides are rapidly removed from circulation by the kidney and are susceptible to degradation by proteases in serum.By associating peptides with nanoparticles, the nanoparticle-polypeptide complex of the present invention can protect against degradation and / or reduce clearance by the kidney.This can increase the serum half-life of polypeptide, thereby reducing the polypeptide dose required for effective treatment.In addition, this can reduce the cost of treatment and minimize the immunological problems and toxic reactions of treatment.

[0057] III. Polyepitope Strings In some aspects, the peptide is included or comprised in a polyepitope string. A polyepitope string is a peptide or polypeptide that contains multiple 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 used previously to target malaria and other pathogens (Baraldo et al., 2005; Moorthy et al., 2004; Baird et al., 2004). A polyepitope string may refer to a nucleic acid (e.g., a nucleic acid that encodes multiple antigens, including the peptides of the present disclosure) or a peptide or polypeptide (e.g., that contains multiple antigens, including the peptides of the present disclosure). A polyepitope string may be included in a cancer vaccine composition.

[0058] IV. Uses of Antigenic Peptides Various aspects relate to the development and use of antigenic peptides useful for treating and preventing certain cancers.In many aspects, antigenic peptides are produced by chemical synthesis or by molecular expression in host cells.Peptides can be purified and utilized in various applications, including (but not limited to) the assay for determining peptide immunogenicity, the assay for determining T cell recognition, peptide vaccines for treating cancer, the development of modified TCR of T cells, and the development of antibodies.

[0059] Peptides can be chemically synthesized by several methods. One common method is to use solid phase peptide synthesis (SPPS). In general, SPPS is performed by building up a peptide from c-terminus to n-terminus through repeated cycles of alternating N-terminal deprotection and coupling reactions. The c-terminus of the first amino acid is coupled to the resin, then the amine is deprotected, and then coupled to the free acid of the second amino acid. This cycle is repeated until the peptide is synthesized.

[0060] Peptides can also be synthesized using molecular tools and host cells. Nucleic acid sequences corresponding to antigenic peptides can be synthesized. In some aspects, synthetic nucleic acids synthesized using an in vitro synthesizer (e.g., phosphoramidite synthesizer), bacterial recombinant system or other suitable methods. Furthermore, the synthesized nucleic acid can be purified and lyophilized or can remain stored in a living system (e.g., bacteria, yeast). For use in a living system, the synthetic nucleic acid molecule can be inserted into a plasmid vector, etc. The plasmid vector can also be an expression vector in which a suitable promoter and a suitable 3'-polyA tail are combined with the transcript sequence.

[0061] Aspects also relate to expression vectors and systems that produce antigenic peptides or proteins. These expression systems can incorporate expression vectors to express transcripts and proteins in a suitable expression system. Typical expression systems include bacterial cell lines (e.g., E. coli), insect cell lines (e.g., SF9), yeast cell lines (e.g., S. cerevisiae), animal cell lines (e.g., CHO) or human cell lines (e.g., HEK 293). Standard biotechnology production procedures can be used to purify RNA and / or protein molecules from these systems.

[0062] Assays can be performed to determine immunogenicity and / or TCR binding. One such is the dextramer flow cytometry assay. Typically, custom 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 tumor infiltrating lymphocytes (TILs) are incubated with the pMHC complexes, stained, and then run through a flow cytometer to determine whether the peptides can bind to the TCR of the T cells.

[0063] The peptides of the present disclosure may also be used to isolate and / or identify T cell receptors that bind to the peptides. T cell receptors contain two distinct polypeptide chains, called the T cell receptor alpha (TCRα) and beta (TCRβ) chains, linked by disulfide bonds. These alpha:beta heterodimers are very similar in structure to the Fab fragments of immunoglobulin molecules and are responsible for antigen recognition by most T cells. A minority of T cells have alternative but structurally similar receptors composed of a different pair of polypeptide chains, called gamma and delta. Both types of T cell receptors differ from membrane-bound immunoglobulins that function as B cell receptors: T cell receptors have only one antigen-binding site, whereas B cell receptors have two, and T cell receptors are never secreted, whereas immunoglobulins can be secreted as antibodies.

[0064] Both chains of the T cell receptor have an amino-terminal variable (V) region with homology to immunoglobulin V domains, a constant (C) region with homology to immunoglobulin C domains, and a short hinge region containing cysteine ​​residues that form interchain disulfide bonds. Each chain spans the lipid bilayer by a hydrophobic transmembrane domain and terminates in a short cytoplasmic tail.

[0065] The three-dimensional structure of the T-cell receptor has been determined. Its structure is indeed similar to that of an antibody Fab fragment, as suspected from previous studies of the gene that codes for it. The T-cell receptor chains fold in much the same manner as those of the Fab fragment, but the final structure appears to be a bit shorter and wider. However, there are some distinct differences between the T-cell receptor and the Fab fragment. The most notable difference is in the Cα domain, where the fold is different from that of other immunoglobulin-like domains. The half of the domain juxtaposed with the Cβ domain forms a β-sheet similar to that found in other immunoglobulin-like domains, while the other half of the domain is formed from loosely packed strands and a short segment of an α-helix. The intramolecular disulfide bond that normally links two β-strands in immunoglobulin-like domains links the β-strand to this segment of an α-helix in the Cα domain.

[0066] There are also differences in the manner in which the domains interact. The interface between the V and C domains of both T cell receptor chains is more extensive than that of antibodies, which may reduce the flexibility of the hinge bond between the domains. Also, the interaction between the Cα and Cβ domains is unique in that it is carbohydrate-assisted, with sugar groups from the Cα domain forming several hydrogen bonds to the Cβ domain. Finally, a comparison of the variable binding sites shows that the complementarity determining region (CDR) loops are aligned fairly closely with those of the antibody molecule, but there is some displacement compared to the loops of the antibody molecule. This displacement is particularly pronounced in the Vα CDR2 loop, which is oriented at nearly right angles to the equivalent loop of the antibody V domain, as a result of a shift in the β-strand that anchors one end of the loop from one face of the domain to the other. Strand displacement also causes a change in the orientation of the Vβ CDR2 loop in two of the seven Vβ domains for which structures are known. So far, the crystal structures of seven T cell receptors have been solved to this level of resolution.

[0067] Aspects of the present disclosure relate to engineered T cell receptors that bind to a peptide of the present disclosure, for example, one of SEQ ID NOs: 1-776. The term "engineered" refers to a T cell receptor having a TCR variable region grafted onto a TCR constant region to create a chimeric polypeptide that binds to a peptide and antigen of the present disclosure. In certain aspects, the TCR includes an intervening sequence that is used for cloning, expression enhancement, detection, or therapeutic control of the construct, but is not present in the endogenous TCR, such as a multiple cloning site, a linker, a hinge sequence, a modified hinge sequence, a modified transmembrane sequence, a detection polypeptide or molecule, or a therapeutic control that may allow for selection or screening of cells containing the TCR.

[0068] In some aspects, the TCR comprises non-TCR sequences. Thus, certain aspects relate to TCRs with sequences that are not derived from TCR genes. In some aspects, the TCR is chimeric in that it contains sequences that are normally found in TCR genes, but that are derived from at least two TCR genes that are not necessarily found together in nature.

[0069] V. Antibodies An aspect of the present disclosure relates to an antibody that targets the peptide of the present disclosure or a fragment thereof.The term "antibody" refers to any isotype of intact immunoglobulin or fragment thereof that can compete with intact antibody for specific binding to target antigen, including chimeric antibody, humanized antibody, fully human antibody and bispecific antibody.As used herein, the term "antibody" or "immunoglobulin" is used interchangeably and refers to any of several classes of structurally related proteins that function as part of the immune response of animals, including IgG, IgD, IgE, IgA, IgM and related proteins, and polypeptides that contain antibody CDR domains that retain antigen-binding activity.

[0070] 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 can have one or more epitopes that can interact with different antibodies.

[0071] The term "epitope" includes any region or portion of a molecule that can induce an immune response by binding to an immunoglobulin or 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. In general, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture.

[0072] Epitope regions of a given polypeptide can be identified using many different epitope mapping techniques well known in the art, including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, and protein display arrays, see, for example, Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are known in the art and are described, for example, in U.S. Patent 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 a protein can also be predicted and identified using standard antigenicity and hydropathy plots.

[0073] The term "immunogenic sequence" refers to a molecule that contains the 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" refers to a composition that contains at least one immunogenic molecule (e.g., an antigen or carbohydrate).

[0074] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but may include naturally occurring antibodies in camelids, which may in some instances include fewer chains, e.g., only heavy chains. The antibodies disclosed herein may be derived from only a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. For example, the variable region or CDR region may be derived from a rat source or a mouse source, while the constant region is derived from a different animal source, such as a human. The antibody or binding fragment may be produced in a hybridoma, by recombinant DNA technology, or by enzymatic or chemical cleavage of an intact antibody. Unless otherwise specified, the term "antibody" includes its derivatives, variants, fragments and muteins, examples of which are described below (Sela-Culang et al., Front Immunol. 2013;4:302;2013).

[0075] The term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of about 25,000 daltons and include a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classes of light chains identified as kappa (κ) and lambda (λ). The term "VL fragment" refers to a fragment of the light chain of a monoclonal antibody that includes all or a portion of the light chain variable region, including the CDRs. The VL fragment may further include a light chain constant region sequence. The variable region domain of the light chain is at the amino terminus of the polypeptide.

[0076] The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of about 50,000 daltons and includes a variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2 and CH3). The term "VH fragment" refers to a fragment of a heavy chain of a monoclonal antibody that includes all or a portion of the heavy chain variable region including the CDRs. A VH fragment can further include a heavy chain constant region sequence. The number of heavy chain constant region domains depends on the isotype. The VH domain is at the amino-terminus of the polypeptide, the CH domain is at the carboxy-terminus, and CH3 is closest to the -COOH-terminus. The isotype of an antibody is defined by the heavy chain present, which can be IgM, IgD, IgG, IgA or IgE, and there are five classifications: mu (μ), delta (δ), gamma (γ), alpha (α) or epsilon (ε) chains. IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0077] VI. Antibody Conjugates An aspect of the present disclosure relates to an antibody, generally of monoclonal type, against the peptide of the present disclosure, which is linked to at least one agent to form an antibody conjugate. To enhance the effect of an antibody molecule as a diagnostic or therapeutic agent, it is conventional to link or covalently bind or conjugate at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, antitumor agents, therapeutic enzymes, radiolabeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or colored ligands, e.g., biotin.

[0078] Any antibody of sufficient selectivity, specificity or affinity can be used as the basis for antibody conjugates. Such properties can be evaluated using conventional immunological screening methods known to those skilled in the art. Binding sites for biologically active molecules in antibody molecules include sites present in the variable domain that can bind pathogens, B cell superantigens, T cell co-receptor CD4 and HIV-1 envelope in addition to the canonical antigen binding site (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 the self-binding of antibodies (Kang et al., 1988) and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

[0079] A specific example of antibody conjugate is a conjugate 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 and / or chemical properties, and their use allows them to detect and / or optionally even quantify the antibody to which they are bound. Another such example is the formation of a conjugate that includes an antibody linked to a cytotoxic or anti-cellular agent, which can be called an "immunotoxin".

[0080] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics are generally divided into two classes: those for use in in vitro diagnostics, such as various immunoassays, and / or those for use in in vivo diagnostic protocols commonly known as "antibody-directed imaging."

[0081] Many suitable imaging agents are known in the art, as are methods for their attachment to antibodies (see, for example, U.S. Patent No. 5,021,236, U.S. Patent No. 4,938,948 and U.S. Patent No. 4,472,509, each of which is incorporated herein by reference). The imaging moiety used may be a paramagnetic ion, a radioisotope, a fluorescent dye, an NMR detectable substance, or X-ray imaging.

[0082] In the case of paramagnetic ions, examples include 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).

[0083] For radioisotopes for therapeutic and / or diagnostic use, astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, Copper 67 , 152 Eu, gallium 67 , 3 Hydrogen, Iodine 123 , iodine 125 , iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, Technetium 99m and / or yttrium 90 may be mentioned. 125 I is often preferred for use in certain situations, whereas technetium 99m and / or indium 111 are also often preferred due to their low energy and suitability for long distance detection. Radiolabeled monoclonal antibodies of the invention can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodized by contact with sodium iodide 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 can be iodized with technitium by a ligand exchange process, for example, by reducing pertechnate with a stannous solution, chelating the reduced technitium on a Sephadex column and applying the antibody to the column. 99m Alternatively, the antibody may be labeled with, for example, pertechnate, SNC1 2Direct labeling techniques may be used by incubating the antibody with a reducing agent such as , a buffer such as sodium-potassium phthalate solution, etc. Intermediary functional groups often used to attach radioisotopes that exist as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0084] 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.

[0085] Another type of antibody conjugate contemplated in the present invention is an antibody intended primarily for in vitro use that is linked to a secondary binding ligand and / or enzyme (enzyme tag) that produces a colored product when contacted with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin compounds and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent No. 3,817,837, U.S. Patent No. 3,850,752, U.S. Patent No. 3,939,350, U.S. Patent No. 3,996,345, U.S. Patent No. 4,277,437, U.S. Patent No. 4,275,149 and U.S. Patent No. 4,366,241, each of which is incorporated herein by reference.

[0086] Yet another known method of site-specific binding of molecules to antibodies involves the reaction of the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids in the antigen-binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous as it will result in loss of antigen binding by the antibody conjugate.

[0087] Molecules containing azide groups can also be used to form covalent bonds to proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-specific optical probes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 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 can be used as antibody binding agents.

[0088] Several methods are known in the art for binding or conjugating an antibody to its conjugate moiety. Some binding methods include, for example, the use of organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA) bound to the antibody; ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or metal chelate complexes using tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Pat. Nos. 4,472,509 and 4,938,948, each of which is incorporated herein by reference). Monoclonal antibodies may be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, and a detectable imaging moiety is attached to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0089] In another aspect, derivatization of immunoglobulins is contemplated by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method have been disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific conjugation of effector or reporter molecules has also been disclosed in the literature, where the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region (O'Shannessy et al., 1987). This approach has been reported to generate diagnostically and therapeutically promising antibodies that are currently undergoing clinical evaluation.

[0090] In another aspect of the disclosure, the antibody may be linked to a semiconductor nanocrystal, such as those described in U.S. Pat. No. 6,048,616, U.S. Pat. No. 5,990,479, U.S. Pat. No. 5,690,807, U.S. Pat. No. 5,505,928, U.S. Pat. No. 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, II-VI, III-V and 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, AlS, AlP, AlSb, PbS, PbSe, Ge and Si, as well as the above-mentioned materials, including ternary and quaternary mixtures thereof. Methods for linking semiconductor nanocrystals to antibodies are described in U.S. Patent No. 6,630,307 and U.S. Patent No. 6,274,323.

[0091] In yet another aspect, the present invention relates to immunodetection methods for binding, purifying, removing, quantifying and / or otherwise generally detecting biological components such as T cells, or selectively binding to or recognizing the peptides of the present disclosure. In some aspects, tetramer assays may be used with the present invention. Tetramer assays generally involve generating soluble peptide-MHC tetramers that can bind to antigen-specific T lymphocytes, and methods of tetramer assays are described, for example, in Altman et al. (1996). Some immunodetection methods that can be used include, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescence assay, bioluminescence assay, tetramer assay and Western blot. Steps of various useful immunodetection methods are described in the scientific literature, e.g., Doolittle and Ben-Zeev, 1999; Gulbis and Galand, 1993; De Jager et al., 1993; and Nakamura et al., 1987, each of which is incorporated herein by reference.

[0092] VII. MHC Polypeptides Aspects of the present disclosure relate to compositions comprising MHC polypeptides. In some aspects, the MHC polypeptide comprises at least two, three, or four MHC polypeptides that can be expressed as separate polypeptides or as fusion proteins. 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 herein as "pMHC"), which utilize distinct antigen processing pathways. Peptides derived from intracellular antigens are presented to CD8+ T cells by MHC class I molecules expressed on virtually every cell, and peptides derived from extracellular antigens are presented to CD4+ T cells by MHC-II molecules. In certain aspects, a particular antigen is identified and presented in an antigen-MHC complex in association with an appropriate MHC class I or MHC class II polypeptide. In certain aspects, the genetic makeup of a subject may be evaluated to determine which MHC polypeptide should be used for a particular patient and a particular set of peptides. In certain aspects, the MHC class 1 polypeptide comprises all or a portion of an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G or CD-1 molecule. In aspects where the MHC polypeptide is an MHC class II polypeptide, the MHC class II polypeptide can comprise all or a portion of an HLA-DR, HLA-DQ or HLA-DP.

[0093] Non-classical MHC polypeptides are also contemplated for use in the MHC complexes of the present invention. Non-classical MHC polypeptides are non-polymorphic, conserved among species, and have narrow, deep, hydrophobic ligand-binding pockets. These binding pockets can present glycolipids and phospholipids to natural killer T (NKT) cells, or specific subsets of CD8+ T cells, such as Qa1, HLA-E-restricted CD8+ T cells, or MAIT cells. NKT cells represent a unique lymphocyte population that co-expresses NK cell markers and semi-invariant T cell receptors (TCRs). They are involved in regulating immune responses associated with a wide range of diseases.

[0094] VIII. Host cells As used herein, the terms "cell", "cell line" and "cell culture" may be used interchangeably. All of these terms include freshly isolated cells and cells cultured, activated or expanded ex vivo. All of these terms include their progeny, which are 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 heterologous nucleic acid sequences, "host cells" refers to prokaryotic or eukaryotic cells, including any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector. Host cells can be and have been used as recipients of vectors or viruses. Host cells can be "transfected" or "transformed", which refers to the process by which exogenous nucleic acid, such as a recombinant protein coding sequence, is transferred or introduced into a host cell. Transformed cells include the primary subject cell and its progeny.

[0095] In certain aspects, transfection can be performed on any prokaryotic or eukaryotic cell. In some aspects, electroporation involves the transfection of human cells. In other aspects, electroporation involves the transfection of animal cells. In certain aspects, transfection involves the transfection of cell lines or hybrid cell types. In some aspects, the cell or cells to be transfected are cancer cells, tumor cells, or immortalized cells. In some examples, the tumor cells or tumor cell lines, cancer cells or cancer cell lines, immortalized cells or immortalized cell lines are induced, and in other examples, the tumor cells or tumor cell lines, cancer cells or cancer cell lines, immortalized cells or immortalized cell lines naturally enter into their respective states or conditions. In certain aspects, the cells or cell lines are selected from the group consisting of 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) cells or derivatives, H1299, HEK, 293, 293T, 293FT, Hep G2, hematopoietic stem cells, HOS, Huh-7, induced pluripotent stem (iPS) cells or derivatives, Jurkat, K562, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, mesenchymal cells, Min-6, monocytic cells, Neuro2a, NIH The cells may be 3T3, NIH3T3L1, K562, NK cells, NS0, 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-induced acquisition of pluripotency (STAP) cells or derivatives SW403, T cells, THP-1, tumor cells, U2OS, U937, peripheral blood lymphocytes, expanded T cells, hematopoietic stem cells or Vero cells.

[0096] IX. Additional Medications A. Immunostimulants In some aspects, the method further comprises administering an additional agent. In some aspects, the additional agent is an immunostimulant. The term "immunostimulant" as used herein refers to a compound that can stimulate immune response in a subject, and can include adjuvants. In some aspects, an immunostimulant is a drug that does not constitute a specific antigen, but can enhance the strength and longevity of the immune response to the antigen. Such immunostimulants include, but are not limited to, stimulators of pattern recognition receptors such as Toll-like receptors, RIG-1-like receptors and NOD-like receptors (NLRs), mineral salts, such as alum, alum in combination with monophosphoryl lipid (MPL) A of enterobacteria such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium or Shigella flexneri, or in particular MPL.® (ASO4), alum in combination separately with MPL A of the above bacteria, saponins, such as QS-21, Quil-A, ISCOM, ISCOMATRIX, emulsions, such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21 + squalene + MPL.), liposomes and liposomal formulations such as AS01, synthetic or specifically prepared microparticles and microcarriers such as bacterial derived outer membrane vesicles (OMVs) from N. gonorrhoeae, Chlamydia trachomatis, or chitosan particles, depot forming agents such as Pluronic block copolymers, specifically modified or prepared peptides such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphate such as RC529, or proteins such as bacterial toxoids or toxin fragments.

[0097] In some aspects, the additional agent includes an agonist for a pattern recognition receptor (PRR), including, but not limited to, a Toll-like receptor (TLR), specifically TLR 2, 3, 4, 5, 7, 8, 9, and / or a combination thereof. In some aspects, the additional agent includes an agonist for Toll-like receptor 3, an agonist for Toll-like receptors 7 and 8, or an agonist for Toll-like receptor 9. Preferably, the recited immunostimulants include imidazoquinolines, such as R848, adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381, U.S. Patent Application Publication No. 2010 / 0075995, or WO 2010 / 018132, immunostimulatory DNA, or immunostimulatory RNA.In some aspects, the additional agent also includes an immunostimulatory RNA molecule, such as, but not limited to, dsRNA, poly I:C or poly I:poly C12U (available as Ampligen.®, both poly I:C and poly I:poly C12U are known TLR3 stimulators), and / or a nucleotide sequence similar to those described 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; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s)and targeting the Toll-like receptor 8 pathway" These may include those disclosed in International Publication No. 2007062107; E. Uhlmann et al., "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity" U.S. Patent Application Publication No. 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections" International Publication No. 2005097993; G. Lipford et al., "Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods" International Publication No. 2003086280.In some aspects, the additional agent may be a TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB-1. In some aspects, the additional agent may include a TLR-5 agonist, such as flagellin or a portion or derivative thereof, including, but not limited to, those disclosed in U.S. Patent No. 6,130,082, U.S. Patent No. 6,585,980, and U.S. Patent No. 7,192,725.

[0098] In some aspects, the additional agent can be a pro-inflammatory stimulus released from necrotic cells (e.g., uric acid crystals). In some aspects, the additional agent can be an activated component of the complement cascade (e.g., CD21, CD35, etc.). In some aspects, the additional agent can be an activated component of an immune complex. The additional agent also includes a complement receptor agonist, such as a molecule that binds to CD21 or CD35. In some aspects, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some aspects, the immunostimulant is a cytokine, which is a small protein or biological factor (range 5 kD-20 kD) that is released by cells and exerts specific effects on cell-cell interactions, cell-cell communication, and the behavior of other cells. In some aspects, the cytokine receptor agonist is a small molecule, an antibody, a fusion protein, or an aptamer.

[0099] B. Immunotherapy In some aspects, the additional therapy includes cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapies can be classified as active, passive or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAA). They are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy makes the immune system attack tumor cells by targeting TAA. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapies are known in the art, and some are described below.

[0100] 1. Inhibition of costimulatory molecules In some aspects, the immunotherapy comprises inhibitors of costimulatory molecules. In some aspects, the inhibitors comprise inhibitors of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, inhibitory polypeptides, inhibitory compounds, and inhibitory nucleic acids.

[0101] 2. Dendritic cell therapy Dendritic cell therapy uses dendritic cells to present tumor antigens to lymphocytes, thereby activating lymphocytes and priming them to kill other cells that present antigens, thereby inducing antitumor responses. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, they assist in cancer antigen targeting. One example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0102] One way to induce dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small portions of proteins that correspond to protein antigens on cancer cells). These peptides are often administered in combination with adjuvants (highly immunogenic substances) to increase immune and antitumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0103] Dendritic cells can also be activated in vivo by expressing GM-CSF in tumor cells, which can be accomplished by genetically engineering the tumor cells to produce GM-CSF or by infecting the tumor cells with an oncolytic virus that expresses GM-CSF.

[0104] Another strategy is to remove dendritic cells from the patient's blood and activate them ex vivo. The dendritic cells are activated in the presence of tumor antigens, which can be single tumor-specific peptides / proteins or tumor cell lysates (a solution of destroyed tumor cells). These cells (along with any adjuvants) are injected and induce an immune response.

[0105] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, and dendritic cells can be induced to mature and provide immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

[0106] 3. CAR-T cell therapy Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine immune cells with new specificities to target cancer cells. Typically, these receptors transfer the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because parts from different sources are fused together. CAR-T cell therapy refers to the treatment of using such transformed cells for cancer treatment.

[0107] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding and T-cell activation functions. The general premise of CAR-T cells is to artificially generate T cells that target markers found on cancer cells. Scientists can remove T cells from humans, genetically alter them, and return them to the patient to attack cancer cells. Once a T cell is engineered to become a CAR-T cell, it acts as a "living drug". CAR-T cells create a link between an extracellular ligand recognition domain and an intracellular signaling molecule that activates the T cell. 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 and not normal cells are targeted. The specificity of CAR-T cells is determined by the choice of the molecule that is targeted.

[0108] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtageneciloreucel (Yescarta). In some aspects, the CAR-T therapy targets CD19.

[0109] 4. Cytokine therapy Cytokines are proteins produced by many types of cells present in tumors. Cytokines can regulate the immune response. Tumors often use cytokines to allow the tumor to grow and dampen the immune response. These immunomodulatory effects allow cytokines to be used as drugs to induce immune responses. Two commonly used cytokines are interferons and interleukins.

[0110] Interferons are produced by the immune system. They are usually involved in antiviral responses, but are also used in cancer. Interferons are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ).

[0111] Interleukins have a range of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0112] 5. Adoptive T cell therapy Adoptive T cell therapy is a form of passive immunization by injecting T cells (adoptive cell transfer). They are found in the blood and tissues and are usually activated when they find a foreign pathogen. Specifically, they are activated when the surface receptors of the T cells encounter cells that present a portion of a foreign protein on their surface antigen. These can be either infected cells or antigen-presenting cells (APCs). They are found in normal and tumor tissues and 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 are capable of attacking tumors, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0113] Several methods have been developed to produce and obtain tumor-targeting T cells. T cells specific for tumor antigens can be removed from tumor samples (TILs) or filtered from blood. Subsequent activation and culture are performed ex vivo, and the resulting product is reinfused. Activation can occur through gene therapy or by exposing T cells to tumor antigens.

[0114] 6. Checkpoint Inhibitors and Combination Treatments In some aspects, the additional therapy comprises an immune checkpoint inhibitor. Certain aspects are further described below.

[0115] a. PD-1 inhibitors, PDL1 inhibitors, and PDL2 inhibitors PD-1 can act within the tumor microenvironment where T cells encounter infection or tumors. Activated T cells upregulate PD-1 and continue to express PD-1 in peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial 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 tissues during immune responses. The inhibitors of the present disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0116] 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 aspects, PD-1, PDL1 and PDL2 are human PD-1, human PDL1 and human PDL2.

[0117] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another aspect, the PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Pat. No. 8,735,553, U.S. Pat. No. 8,354,509, and U.S. Pat. No. 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art, such as those described in U.S. Patent Application Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0118] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular binding portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some aspects, 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 WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA® and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. Pidilizumab, also known as CT-011, hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0119] In some aspects, 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 a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor, such as rHIgM12B7.

[0120] In some aspects, the inhibitor comprises the heavy chain CDR or heavy chain VR and the light chain CDR or light chain VR of nivolumab, pembrolizumab or pidilizumab. Thus, in one aspect, the inhibitor comprises the CDR1 domain, the CDR2 domain and the CDR3 domain of the VH region of nivolumab, pembrolizumab or pidilizumab, and the CDR1 domain, the CDR2 domain and the CDR3 domain of the VL region of nivolumab, pembrolizumab or pidilizumab. In another aspect, the antibody competes for binding to and / or binds to the same epitope on PD-1, PDL1 or PDL2 as the above antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) of variable region amino acid sequence identity to the above antibody.

[0121] b. CTLA-4, B7-1, and B7-2 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and functions as an "off" switch when it binds 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 inhibitory signals to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found on regulatory T cells and may be important for their function. T cell activation via T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitor of the present disclosure can block one or more functions of CTLA-4 activity, B7-1 activity, and / or B7-2 activity. In some aspects, the inhibitor blocks the interaction between CTLA-4 and B7-1. In some aspects, the inhibitor blocks the interaction between CTLA-4 and B7-2.

[0122] In some aspects, 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 an oligopeptide.

[0123] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the methods disclosed herein can be used with anti-CTLA-4 antibodies disclosed in U.S. Patent No. 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. The teachings of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application No. WO2001 / 014424, International Patent Application No. WO2000 / 037504, and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0124] An additional 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., WO 01 / 14424).

[0125] In some aspects, the inhibitor comprises the heavy chain CDR or heavy chain VR and the light chain CDR or light chain VR of tremelimumab or ipilimumab. Thus, in one aspect, 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 aspect, the antibody competes for binding to and / or binds to the same epitope on PD-1, B7-1 or B7-2 as the above antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) of variable region amino acid sequence identity to the above antibody.

[0126] C. Oncolytic Viruses In some aspects, the additional therapy includes an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. When infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are believed to not only cause direct destruction of tumor cells, but also stimulate a host anti-tumor immune response for long-term immunotherapy.

[0127] D. Polysaccharide In some aspects, the additional therapy includes polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can upregulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory tests to stimulate macrophages, NK cells, T cells and immune system cytokines, and are being investigated in clinical trials as immune adjuvants.

[0128] E. Chemotherapy In some aspects, the additional therapy comprises chemotherapy. Suitable classes of chemotherapeutic agents include (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (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 substances (e.g., 6 -mercaptopurine, 6-thioguanine, pentostatin), (c) natural products such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α), and (d) various pharmaceutical agents such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenal cortex suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly preferred chemotherapeutic agent.

[0129] Cisplatin is widely used to treat cancers such as, for example, metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must be delivered via other routes, such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and in certain aspects, effective doses including about 15 mg / m2 to about 20 mg / m2 for 5 days every 3 weeks for a total of 3 courses used in clinical applications are contemplated. In some aspects, the amount of cisplatin delivered to cells and / or subjects with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount delivered when using cisplatin alone.

[0130] Other suitable chemotherapeutic agents include anti-microtubule agents, such as paclitaxel ("taxol") and doxorubicin hydrochloride ("doxorubicin"). The combination of an Egr-1 promoter / TNFα construct delivered via an adenoviral vector with doxorubicin has been determined to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combined treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0131] Doxorubicin is poorly absorbed and is preferably administered intravenously. In certain aspects, suitable intravenous doses for adults 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 two or three consecutive days repeated at about 3-4 week intervals, or about 20 mg / m2 once a week. The lowest dose should be used in elderly patients if myelosuppression has previously been caused by previous chemotherapy, or if there is neoplastic bone marrow infiltration, or if the drug is combined with other myelopoiesis-suppressing drugs.

[0132] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards can include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) available from Mead Johnson and NEOSTAR® available from Adria) are another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous doses include, for example, about 40 mg / kg to about 50 mg / kg initially in divided doses for a period of about 2 to about 5 days, or about 10 mg / kg to about 15 mg / kg every about 7 to about 10 days, or about 3 mg / kg to about 5 mg / kg twice weekly, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse gastrointestinal effects, the intravenous route is preferred. Drugs may also be administered intramuscularly, by infiltration, or into body cavities.

[0133] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to a subject at a dosage of about 7.5 to about 1000 mg / m2. Furthermore, 5-FU administration schedules may be for various periods of time, such as up to 6 weeks, or as determined by one of skill in the art to which this disclosure pertains.

[0134] Another suitable chemotherapeutic agent, gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), is recommended for the treatment of advanced and metastatic pancreatic cancer and thus may also be useful in the present disclosure for these cancers.

[0135] The amount of chemotherapeutic agent delivered to the patient may vary. In a preferred aspect, the chemotherapeutic agent may be administered in an amount effective to cause the arrest or regression of cancer in the host when the chemotherapeutic agent is administered with the construct. In other aspects, the chemotherapeutic agent may be administered in an amount that is between 1 / 2 and 1 / 10,000 of the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 1 / 20, about 1 / 500, or even about 1 / 5000 of the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure may be tested in vivo to determine the desired therapeutic activity in combination with the construct, and the effective dosage. For example, such compounds may be tested in suitable animal model systems, including, but not limited to, rats, mice, chickens, cows, monkeys, rabbits, and the like, prior to testing in humans. As described in the examples, in vitro testing may be used to determine suitable combinations and dosages.

[0136] F. Radiation Therapy In some aspects, the additional or previous therapy comprises radiation, such as ionizing radiation. As used herein, "ionizing radiation" refers to radiation that includes particles or photons that have sufficient energy or can generate sufficient energy through nuclear interaction to cause ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is X-rays. Means for delivering X-rays to target tissue or target cells are well known in the art.

[0137] In some aspects, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose, hi some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some aspects, the amount of ionizing radiation is at least 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) and at most 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). 2, 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), 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 aspects, ionizing radiation is administered in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein). When multiple doses are administered, the doses may be about every 1, 4, 8, 12, or 24 hours, or about every 1, 2, 3, 4, 5, 6, 7, or 8 days, or about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks, or any derivable range therein.

[0138] In some aspects, the amount of IR may be referred to as a total dose of IR, where the total dose of IR is administered in fractionated doses. For example, in some aspects, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some aspects, the total dose is 50-90 Gy administered in 20-60 fractionated doses of 2-3 Gy each. In some aspects, the total dose of IR is at least 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), at most 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, 10 0, 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), 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 aspects, the total dose is administered in fractions of at least 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), at most 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), 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 aspects, at least 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 fractions, at most 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 or just 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 (or any derivable range therein). In some aspects, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses, 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 aspects, the present invention provides a method for administering at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses, but not more than 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. 1, 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, 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.

[0139] G. Surgery Approximately 60% of cancer patients undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised and / or destroyed, and may be used in combination with other therapies, such as treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microscopically controlled surgery (Mohs surgery).

[0140] Removal of part or all of a cancerous cell, tissue, or tumor may result in the formation of a cavity in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with 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. Dosages may also vary in these treatments.

[0141] H. Other Medications It is contemplated that other agents may be used in combination with certain aspects of this aspect to improve the therapeutic effect of the 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 hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other aspects, cytostatic or differentiation agents can be used in combination with certain aspects of this aspect to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are contemplated to improve the effect of this aspect. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of this aspect to improve the effect of the treatment.

[0142] X. Protein Composition As used herein, "protein," "peptide," or "polypeptide" refers to a molecule that contains 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 aspects, a wild type version of a protein or polypeptide is used, but in many aspects of the disclosure, a modified protein or polypeptide is used to generate an immune response. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered relative to the wild type protein or polypeptide. In some aspects, a modified / variant protein or modified / variant polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or modified / variant polypeptide may be altered with respect to one activity or function, but may retain wild type activity or function in other respects, such as immunogenicity.

[0143] When a protein is specifically mentioned herein, it generally refers to a natural (wild type) or recombinant (modified) protein, or a protein with any signal sequence removed, if desired. Proteins can be directly isolated from the organism in which they are naturally occurring, can be produced by recombinant DNA / exogenous expression methods, or can be produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term "recombinant" can be used with a polypeptide, or with the name of a particular polypeptide, generally referring to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is the product of replication of such a molecule.

[0144] In certain aspects, the size of a peptide, protein or polypeptide (wild type or modified), such as a peptide or protein of the disclosure, including any one of SEQ ID NOs: 1-1245, can be, 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 110 9, 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, 8 2, 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, 52 The polypeptides may comprise 5, 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 more, and any range derivable therein. It is contemplated that the polypeptides may be mutated by truncation to make them shorter than their corresponding wild-type forms, and may also be altered by fusing or conjugating heterologous proteins or heterologous polypeptide sequences with specific functions (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: 1-1245 may be excluded in one or more aspects.

[0145] A polypeptide, protein, or polynucleotide encoding such a polypeptide or protein of the disclosure may contain 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 acid or nucleic acid substitutions, or a substitution of at least 3, 4, 5, 6, 7, 8, or 9, or at most 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids or nucleic acid substitutions of one of the peptides of SEQ ID NOs: 1-1245. The sequence is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous to a nucleic acid encoding a peptide of one of NO:1-1245. In certain aspects, the peptide or polypeptide is not naturally occurring and / or is a combination of peptides or polypeptides.

[0146] In some aspects, a 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: 1-1245. In some aspects, a peptide of the disclosure comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of a peptide of one of SEQ ID NOs: 1-1245 adjacent to the carboxy terminus and / or adjacent to the amino terminus 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 consecutive amino acids of a peptide of one of SEQ ID NOs: 1-1245. , 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), at most 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 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).

[0147] In some aspects, a protein, polypeptide or nucleic acid may comprise 1, 2, 3, 44, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids (or any derivable range therein) of a peptide of one of SEQ ID NOs: 1-1245.

[0148] In some aspects, the polypeptide, protein or nucleic acid is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 1 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein), or at most 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or 83% , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein), 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). 4%, 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 SEQ ID NO:1 or SEQ ID NO:2. It may comprise at least 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) of a peptide of ID NOs: 1-1245, at most 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), 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) of consecutive amino acids.

[0149] In some aspects, the peptide of SEQ ID NO: 1-1245 begins at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the peptide of SEQ ID NO: 1-1245. There is a polypeptide (or a nucleic acid molecule encoding such a polypeptide) that includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 (or any derivable range therein), at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 (or any derivable range therein), 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) consecutive amino acids of one of the peptides of NO:1-1245.

[0150] It is contemplated that compositions of the present disclosure have from about 0.001 mg to about 10 mg of total polypeptide, peptide and / or protein per ml. The concentration of protein in the composition may be at least 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), , 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), 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).

[0151] The following is a description of changing the amino acid subunits of a protein to produce equivalent or even improved second generation variant polypeptides or peptides. For example, certain amino acids can be substituted for other amino acids in a protein or polypeptide sequence, with or without discernible loss of interactive binding ability with structures such as, for example, the antigen-binding region of an antibody or a binding site on a substrate molecule. Since it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in a protein sequence and its corresponding DNA coding sequence, and still produce a protein with similar or desirable properties. Thus, the inventors believe that various changes can be made to the DNA sequence of a gene that codes for a protein without obviously losing their biological usefulness or activity.

[0152] The term "functionally equivalent codon" is used herein to refer to a codon that codes for the same amino acid, for example, the six different codons for arginine. "Neutral substitutions" or "neutral mutations" are also contemplated, which refer to changes in one or more codons that code for biologically equivalent amino acids.

[0153] The amino acid sequence variants of the present disclosure can be substitution, insertion or deletion variants. Mutations in the polypeptides of the present disclosure can 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 a protein or polypeptide (or any range derivable therein) compared to the wild type. A variant can contain an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90% identical (including all values ​​and ranges therebetween) to any sequence provided or referenced herein. A variant can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0154] In some aspects, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, twenty-nine, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six ... 0, 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, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 1 35, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 19 The amino acid at position 2, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, or 214 is substituted with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0155] It will also be understood that amino acid sequences and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, but may still be essentially identical as described in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including the maintenance of the biological protein activity to which protein expression pertains. The addition of terminal sequences applies particularly to nucleic acid sequences, which may contain, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0156] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues can be deleted, or several consecutive amino acids can be deleted. Stop codons can be introduced (by substitution or insertion) into the coding nucleic acid sequence to generate truncated proteins.

[0157] Insertional variants typically involve the addition of amino acid residues at non-terminal points in the polypeptide. This may involve the insertion of one or more amino acid residues. Terminal additions may also be produced, including fusion proteins that are multimers or concatamers of one or more peptides or polypeptides described or referenced herein.

[0158] Substitution variants typically involve the exchange of one amino acid for another at one or more sites in a protein or polypeptide, and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, i.e., one amino acid is replaced by one with similar chemical properties. "Conservative amino acid substitutions" can involve the exchange of a member of an amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, 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 can include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in living systems. These include peptidomimetics, or other reversed or inverted forms of amino acid moieties.

[0159] Alternatively, the substitution can be "non-conservative" so that the function or activity of the polypeptide is affected. Non-conservative changes typically involve replacing amino acid residues with chemically different ones, for example, replacing polar or charged amino acids with non-polar or uncharged amino acids, or vice versa. Non-conservative substitutions can involve exchanging a member of one amino acid class for a member of another class.

[0160] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein.Those skilled in the art can identify suitable regions of molecules that can be changed without destroying activity by targeting regions that are not considered important for activity.Those skilled in the art can also identify amino acid residues and parts of molecules that are conserved between similar proteins or polypeptides.In a further aspect, regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without significantly changing biological activity or adversely affecting the structure of protein or polypeptide.

[0161] In making such changes, the hydropathic index of amino acids may be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then averaging these values ​​repeatedly along the peptide chain. Each amino acid is 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 hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is recognized that the relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein or polypeptide, thereby defining the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is also known that certain amino acids may be substituted with other amino acids having similar hydropathic indices or hydropathic scores and still retain similar biological activity. In making changes based on hydropathic index, certain aspects include substitution of amino acids with hydropathic indices within ±2. Some aspects of the invention include those within ±1, and other aspects of the invention include those within ±0.5.

[0162] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity.In U.S. Patent No. 4,554,101, which is incorporated herein by reference, it is described that the maximum local average hydrophilicity of a protein controlled by the hydrophilicity of adjacent amino acids correlates with the biological properties of the protein.In certain aspects, the maximum local average hydrophilicity of a protein controlled by the hydrophilicity of adjacent amino acids correlates with its immunogenicity and antigen binding, i.e., the biological properties of the protein. The following hydrophilicity values ​​have 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 on similar hydrophilicity values, certain aspects include substitutions of amino acids whose hydrophilicity values ​​are within ±2, other aspects include those within ±1, and still other aspects include those within ±0.5. In some instances, epitopes can be identified from a primary amino acid sequence based on hydrophilicity. These regions are also called "epitope 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.

[0163] Furthermore, one skilled in the art can outline structure-function studies to identify residues in similar polypeptides or proteins that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to the amino acid residues that are important for the activity or structure of similar proteins. One skilled in the art can select chemically similar amino acid replacements for such predicted important amino acid residues.

[0164] Also, the skilled person can analyze the three-dimensional structure and amino acid sequence related to that structure in similar proteins or polypeptides. In view of such information, the skilled person can predict the alignment of amino acid residues of a polypeptide to its three-dimensional structure. The skilled person can select not to cause 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. Furthermore, the skilled person can generate test variants containing single amino acid substitutions at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus obtaining information gathered from such routine experiments, which may enable the skilled person to determine amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0165] In some aspects of the present invention, amino acid substitutions are made to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity to form protein complexes, (4) change ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) can be made to naturally occurring sequences. Substitutions can be made to portions of antibodies that are outside the domains that form intermolecular contacts. In such aspects, conservative amino acid substitutions that do not substantially change the structural properties of proteins or polypeptides can be used (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes natural antibodies).

[0166] XI. Nucleic acids In certain aspects, the nucleic acid sequence may be present in various instances, such as an isolated segment of an integrated sequence and a recombinant vector, or a recombinant polynucleotide encoding the peptides and polypeptides of the present disclosure or fragments, derivatives, muteins or variants thereof, a polynucleotide sufficient for use as a hybridization probe, a PCR or sequencing primer for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, an antisense nucleic acid for inhibiting expression of a polynucleotide, and the aforementioned complementary sequences described herein. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids may be single-stranded or double-stranded, and may include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0167] The term "polynucleotide" refers to a nucleic acid molecule that is recombinant or isolated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors including, for example, plasmids, cosmids, phages, viruses, and the like. Polynucleotides, in certain aspects, include control sequences that are isolated substantially away from their naturally occurring genes or protein coding sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may be present in the polynucleotide, but need not be present in the polynucleotide.

[0168] In this regard, 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 necessary for proper transcription, post-translational modification or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins and variants. A nucleic acid that encodes all or a portion of a polypeptide may contain a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid that contains a mutation having a slightly different nucleic acid sequence, but still encodes the same or a substantially similar protein.

[0169] In certain aspects, there are polynucleotide variants that have substantial identity to the sequences disclosed herein, and comprise at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity (including all values ​​and ranges therebetween) when compared to the polynucleotide sequences provided herein using methods described herein (e.g., BLAST analysis using standard parameters).In certain aspects, the isolated polynucleotide comprises a nucleotide sequence that encodes a polypeptide that has at least 90%, preferably 95% or more identity to the amino acid sequence described herein over the entire length of the sequence, or a nucleotide sequence that is complementary to the isolated polynucleotide.

[0170] Regardless of the length of the coding sequence itself, the nucleic acid segment may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and its total length may vary considerably. The nucleic acids may be of any length. They may 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 may include one or more additional sequences, such as regulatory sequences, and / or may be part of an even larger nucleic acid, such as a vector. Thus, it is contemplated that nucleic acid fragments of almost any length may be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow purification, transport, secretion, post-translational modification of the polypeptide, or for therapeutic benefits such as targeting or efficacy. As noted above, tags or other heterologous polypeptides may be added to sequences encoding the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0171] A. Hybridization A nucleic acid that hybridizes to another nucleic acid under specific hybridization conditions. The method of hybridizing nucleic acids is well known in the art. For example, see Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions containing about 50% formamide, and a hybridization temperature of 42°C), and a washing condition of 60°C in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions are hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate the hybridization and / or wash conditions to increase or decrease the stringency of hybridization, such that nucleic acids containing nucleotide sequences 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 will typically remain hybridized to each other.

[0172] Parameters influencing the selection of hybridization conditions, and guidelines for devising suitable conditions, are described, for example, by Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 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 incorporated by reference in their entireties for all purposes), and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA.

[0173] B. Mutation Changes can be introduced by mutation into the nucleic acid, thereby resulting in changes in the amino acid sequence of the polypeptide (e.g., antigenic peptide or polypeptide) that it encodes. Any technique known in the art can be used to introduce the mutation. In one aspect, for example, a site-directed mutagenesis protocol is used to change one or more specific amino acid residues. In another aspect, for example, a random mutagenesis protocol is used to change one or more randomly selected residues. Regardless of how it is done, the mutant polypeptide can be expressed and screened for desired properties.

[0174] Mutations can be introduced into a nucleic acid without significantly changing the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that result in 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 the polypeptide it encodes. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, mutations can quantitatively or qualitatively change biological activity. Examples of quantitative changes include increasing, decreasing or eliminating activity. Examples of qualitative changes include changing the antigen specificity of an antibody.

[0175] C. Probe In another aspect, the nucleic acid molecule is suitable for use as a primer or hybridization probe for the detection of a nucleic acid sequence. The nucleic acid molecule can contain 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 that encodes an active portion of a given polypeptide.

[0176] In another aspect, nucleic acid molecule can be used as a probe or PCR primer for specific nucleic acid sequence.For example, nucleic acid molecule probe can be used in diagnostic method, or nucleic acid molecule PCR primer can be used to amplify the region of DNA that can be used to isolate the nucleic acid sequence for use in producing the engineered cell of the present disclosure.In a preferred aspect, nucleic acid molecule is an oligonucleotide.

[0177] A probe based on the desired sequence of the nucleic acid can be used to detect the nucleic acid or a similar nucleic acid, for example, a transcript that encodes a polypeptide of interest.The probe can contain a label group, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.Such a probe can be used to identify cells that express the polypeptide.

[0178] XII. Polypeptide Expression In some aspects, there are nucleic acid molecules encoding the polypeptides or peptides of the present disclosure (e.g., antibodies, TCR genes, MHC molecules, and immunogenic peptides). These can be generated by methods known in the art, such as isolated from B cells of immunized and isolated mice, phage display, expressed in any suitable recombinant expression system and assembled to form antibody molecules, or by recombinant methods.

[0179] The nucleic acid molecule can be used to express large amounts of the polypeptide. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule can be used for humanizing the antibody or TCR gene.

[0180] A. Vector In some aspects, expression vectors are contemplated that contain nucleic acid molecules that encode a polypeptide of desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs, or one or more variable region domains). The expression vectors that contain nucleic acid molecules can encode the heavy chain, light chain, or antigen-binding portion thereof. In some aspects, the expression vectors that contain nucleic acid molecules can encode fusion proteins, antigenic peptides and polypeptides, TCR genes, MHC molecules, modified antibodies, antibody fragments, and probes thereof. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors can contain nucleic acid sequences that serve other functions.

[0181] To express the polypeptide or peptide of the present disclosure, the DNA encoding the polypeptide or peptide is inserted into an expression vector such that the gene region is operably linked to transcriptional and translational control sequences. In some aspects, the vector encodes a functionally complete human CH or CL immunoglobulin sequence with suitable restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In some aspects, the vector encodes a functionally complete human TCR alpha or beta sequence with suitable restriction sites engineered so that any variable sequence or CDR1, CDR2 and / or CDR3 can be easily inserted and expressed. Typically, the expression vector used in any of the host cells contains sequences for plasmid or viral maintenance, as well as sequences for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences", typically include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding an expressed polypeptide, and a selectable marker element. Such sequences and methods for their use are well known in the art.

[0182] B. Expression Systems There are many expression systems that contain at least some or all of the above expression vectors. Prokaryotic and / or eukaryotic based systems can be adopted for use with the aspect of producing nucleic acid sequences or their cognate polypeptides, proteins and peptides. Commercially widely available systems include, but are not limited to, bacterial, mammalian, yeast and insect cell systems. Various host cells have characteristic and specific mechanisms for post-translational processing and post-translational modification of proteins. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of expressed foreign proteins. Those skilled in the art can use appropriate expression systems to express the vectors for producing nucleic acid sequences or their cognate polypeptides, proteins or peptides.

[0183] C. Methods of gene transfer Suitable methods for nucleic acid delivery to result in expression of the compositions are expected to include virtually any method by which a nucleic acid (e.g., DNA, including viral and non-viral vectors) can be introduced into a cell, tissue or organism, as described herein or known to one of skill in the art. Such methods include, but are not limited to, injection, including, for example, microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference), (U.S. Pat. Nos. 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 of which is incorporated herein by reference); electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 2001; al., 1990); using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); direct acoustic loading (Fechheimer et al., 1987); 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. al., 1991); particle bombardment (PCT Application Nos. WO 94 / 09699 and WO 95 / 06128, each of which is incorporated herein by reference; U.S. Patent No. 5,610,042; U.S. Patent No. 5,322,783, U.S. Patent No. 5,563,055, U.S. Patent No. 5,550,318, U.S. Patent No. 5,538,877, and U.S. Patent No. 5,538,880); agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent No. 5,302,523 and U.S. Patent No. 5,464,765, each of which is incorporated herein by reference; Agrobacterium-mediated transformation (U.S. Patent No. 5,591,616 and U.S. Patent No. 5,563,055, each of which is incorporated herein by reference); or PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent No. 4,684,611 and U.S. Patent No. 4,952,500, each of which is incorporated herein by reference); direct delivery of DNA by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0184] D. host cell In another aspect, the use of a host cell into which a recombinant expression vector is introduced is contemplated. Polypeptides can be expressed in various cell types. Expression constructs encoding the polypeptides or peptides of the present disclosure can be transfected into cells according to various methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors can use control sequences that can replicate and / or express in prokaryotic and eukaryotic cells. Those skilled in the art will understand the conditions for incubating host cells to maintain and allow vector replication. The techniques and conditions that allow large-scale production of vectors and the production of nucleic acids encoded by the vectors and their cognate polypeptides, cognate proteins, or cognate peptides are also understood and known.

[0185] It is known that for stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small portion of cells can integrate foreign DNA into their genome.In order to identify and select these integrants, a selectable marker (for example, for antibiotic resistance) is generally introduced into host cells together with the gene of interest.The cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (for example, the cells that have integrated the selectable marker gene survive, while other cells die), among other methods known in the art.

[0186] XIII. Cell preparation and culture In certain aspects, the cells of the present disclosure may be specifically formulated and / or cultured in a specific medium. The cells may be formulated so as to be suitable for delivery to a recipient without adverse effects.

[0187] In certain aspects, the medium may be prepared using any of the media used to culture animal cells, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640 and Fischer medium, and any combination thereof, as their basal medium, but the medium is not particularly limited thereto, as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0188] The medium can be serum-containing or serum-free, or xenogeneic component-free.From the aspect of preventing contamination with xenogeneic animal-derived components, serum can be derived from the same animal as stem cells.Serum-free medium refers to the medium that does not contain raw or unpurified serum, and therefore can include the medium that contains purified blood-derived components or animal tissue-derived components (such as growth factors).

[0189] The medium may or may not contain a serum replacement. Serum replacement may include materials that suitably contain albumin (lipid-rich albumin, bovine albumin, albumin substitutes, such as recombinant or humanized albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or their equivalents. Serum replacement may be prepared, for example, by the method disclosed in WO 98 / 30679, which is incorporated herein in its entirety. Alternatively, for further convenience, any commercially available material may be used. Commercially available materials include knockout serum replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0190] In certain aspects, the medium may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty 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 HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triiodo-I-thyronine). In specific aspects, one or more of these may be explicitly excluded.

[0191] In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, vitamin B12 (and any range derivable therein), or the medium comprises a combination thereof or a salt thereof. In some aspects, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamin comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises a protein. In some aspects, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, simple sugars, inorganic ions, In some aspects, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof.In some aspects, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some aspects, the medium further includes one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins described herein, and / or one or more proteins described herein, and / or one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodo-I-thyronine, B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (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 aspects, one or more of these may be explicitly excluded.

[0192] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, of which in specific aspects one or more may be explicitly excluded.

[0193] One or more of the medium components may have a concentration of at least 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, μg / ml, mg / ml, or any derivable range therein, and at most 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, μg / ml, mg / ml, or any derivable range therein, It may be added at a concentration of 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein, 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, μg / ml, mg / ml, or any range derivable therein.

[0194] In a specific aspect, the cells of the present disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In a specific case, 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 include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, with a specific formulation including 2.5% human albumin. The cells may be specifically formulated for intravenous administration. For example, they are formulated for intravenous administration for less than 1 hour. In certain aspects, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from the time of thawing.

[0195] In some aspects, the method further comprises priming T cells. In some aspects, the T cells are primed by antigen-presenting cells. In some aspects, the antigen-presenting cells present tumor antigens or peptides such as those disclosed herein.

[0196] In certain aspects, the cells of the present disclosure include an exogenous TCR that may be of defined antigen specificity, such as a defined antigen specificity for SEQ ID NO:1. In some aspects, the TCR may be selected based on lack or reduced alloreactivity to the intended recipient (examples include a specific virus-specific TCR, a xenospecific TCR, or a cancer testis antigen-specific TCR). In examples where the exogenous TCR is non-allo-reactive, during T cell differentiation, the exogenous TCR suppresses rearrangement and / or expression of the endogenous TCR locus through a developmental process called allelic elimination, resulting in T cells that express only non-allo-reactive exogenous TCRs and are therefore non-allo-reactive. In some aspects, the selection of the exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some aspects, the endogenous TCR gene is modified by genome editing so that it does not express the protein. Methods of gene editing, such as those using the CRISPR / Cas9 system, are known in the art and described herein.

[0197] XIV. Administration of Therapeutic Compositions The method of the present disclosure relates to the treatment of subjects with cancer. In some aspects, the treatment can be directed to those who have or have been determined to have cancer related to a particular peptide of the present disclosure, for example, one of the peptides of SEQ ID NO: 1-776. In some aspects, the method can be used with individuals who have tested positive for such cancer, who have one or more symptoms of cancer, or who are considered to be at risk of developing such cancer.

[0198] The treatment provided herein may comprise a combination of therapeutic agents, for example, administering a first anti-cancer therapy and a second anti-cancer therapy.The treatment may be administered in any suitable manner known in the art.For example, the first and second cancer treatments may be administered sequentially (at different times) or concurrently (at the same time).In some aspects, the first and second cancer treatments are administered in separate compositions.In some aspects, the first and second cancer treatments are in the same composition.

[0199] Aspects of the present disclosure relate to compositions and methods, including therapeutic compositions.Different treatments can be administered in one composition or in multiple compositions, for example, two compositions, three compositions or four compositions.Various combinations of drugs can be used.

[0200] The therapeutic agents of the present disclosure may be administered by the same or different routes of administration. In some aspects, the cancer treatment is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intraventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implant, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history, and response to treatment, as well as the judgment of the attending physician.

[0201] Treatments may include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount administered, as well as the specific route and formulation, are within the skill of those skilled in the clinical arts to determine. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some aspects, a unit dose includes a single administrable dose.

[0202] The amount to be administered depends on the desired treatment effect, depending on both the number of treatments and the unit dose. Effective dose is understood to refer to the amount required to achieve a specific effect. In certain aspects of implementation, it is contemplated that a dose ranging from 10mg / kg to 200mg / kg can affect the protective capacity of these agents. Thus, doses are contemplated to 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 μg / kg, mg / kg, μg / day or mg / day, or any range derivable therein. Furthermore, such doses can be administered multiple times during the day and / or on multiple days, weeks or months.

[0203] In certain aspects, an effective dose of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In other aspects, an effective dose provides a blood level of about 4 μM to 100 μM, or about 1 μM to 100 μM, or about 1 μM to 50 μM, or about 1 μM to 40 μM, or about 1 μM to 30 μM, or about 1 μM to 20 μM, or about 1 μM to 10 μM, or about 10 μM to 150 μM, or about 10 μM to 100 μM, or about 10 μM to 50 μM, or about 25 μM to 150 μM, or about 25 μM to 100 μM, or about 25 μM to 50 μM, or about 50 μM to 150 μM, or about 50 μM to 100 μM (or any range derivable therein). In other aspects, the dose is 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 5, 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 100μ M, or any derivable range therein, at least 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 101, 102, 103, 104, 105, 106, 107, 2, 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 μM, or any derivable range therein;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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 ... 7, 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 μM, or any range derivable therein. In certain aspects, the therapeutic agent administered to the subject is metabolized in the body to a metabolized therapeutic agent, in which case blood levels may refer to the amount of the agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, blood levels as described herein may refer to the unmetabolized therapeutic agent.

[0204] The exact amount of the therapeutic composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended goal of the treatment (alleviation of symptoms versus cure), and the efficacy, stability and toxicity of the particular therapeutic agent or other treatment that the subject may be subjected to.

[0205] It is understood and appreciated by those skilled in the art that dosage units of μg / kg body weight or mg / kg body weight can be converted and expressed in equivalent concentration units of μg / ml or mM (blood levels), for example, 4 μM to 100 μM. It is also understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions made regarding uptake and concentration measurements are well known, allowing those skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0206] In selected aspects, it is contemplated that the peptides of the present disclosure can be included in a vaccine composition and administered to a subject to induce a therapeutic immune response against cancer in the subject. Vaccine compositions for pharmaceutical use in a subject can include the peptide compositions disclosed herein and a pharmaceutically acceptable carrier.

[0207] The phrases "pharmaceutical", "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not cause adverse, allergic or other untoward reactions when administered to animals, such as humans, as appropriate. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials and combinations thereof, as known to those skilled in the art (see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Pharmaceutical Press, 2011, which is incorporated herein by reference). Except where any conventional carrier is incompatible with the active ingredient, its use in the vaccine composition of the present invention is contemplated.

[0208] 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 can provide a therapeutic effect for the treatment of cancer, such as reducing tumor size, increasing survival rates, and the like.

[0209] In some aspects, the vaccine composition can be administered by microstructured transdermal delivery or ballistic particle delivery. Microstructures as carriers for vaccine formulations are desirable configurations for vaccine applications and are widely known in the art (Gerstel and Place 1976 (US Pat. No. 3,964,482); Ganderton and McAinsh 1974 (US Pat. No. 3,814,097); US Pat. Nos. 5,797,898, 5,770,219 and 5,783,208 and US Patent Application No. 2005 / 0065463). Such vaccine compositions formulated for ballistic particle delivery can include the isolated peptides disclosed herein immobilized on the surface of a support substrate. In these aspects, the support substrate can include, without limitation, microcapsules, microparticles, microspheres, nanocapsules, nanoparticles, nanospheres, or combinations thereof.

[0210] In other aspects, the vaccine composition comprises the immobilized or encapsulated peptide or antibody disclosed herein and a support substrate.In these aspects, the support substrate can include, but is not limited to, lipid microspheres, lipid nanoparticles, ethosomes, liposomes, niosomes, phospholipids, sphingosomes, surfactants, transferosomes, emulsions, or combinations thereof.The formation and use of liposomes and other lipid nanocarrier and microcarrier formulations are generally known to those skilled in the art, and the use of liposomes, microparticles, nanocapsules, etc. is widely used to deliver therapeutic agents (e.g., U.S. Patent No. 5,741,516, the entirety of which is specifically incorporated herein by reference). Numerous methods for the preparation of liposomes and liposome-like preparations as potential drug carriers, including the encapsulation of peptides, have been reviewed (U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; ​​U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868; and U.S. Pat. No. 5,795,587, each of which is specifically incorporated by reference in its entirety).

[0211] In addition to the delivery methods described herein, some alternative techniques are also contemplated for administering the disclosed vaccine composition.As non-limiting examples, the vaccine composition can be administered by sonophoresis (i.e., ultrasound), which is used and described in U.S. Patent No. 5,656,016; intraosseous injection (U.S. Patent No. 5,779,708) or feedback control delivery (U.S. Patent No. 5,697,899) to enhance the speed and effectiveness of drug penetration into and through circulatory system, and each of the patents in this paragraph is specifically incorporated herein in its entirety by reference.

[0212] XV. Detection and Vaccination Kits The peptide or antibody of the present disclosure can be included in a kit. The peptide or antibody in the kit can be detectably labeled or immobilized on the surface of a support substrate that is also included in the kit. The peptide or antibody can be provided in the kit in a suitable form, such as, for example, sterile, lyophilized, or both.

[0213] The support substrate included in the kit of the present invention can be selected based on the method to be performed. As non-limiting examples, the support substrate can be a multi-well plate or microplate, a membrane, a filter, 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 device, a microchip, a comb, a silica particle, a magnetic particle, or a self-assembled monolayer.

[0214] As appropriate for the method to be carried out, the kit may further comprise one or more devices for delivering the composition to a subject or for otherwise handling the composition of the present invention.As non-limiting examples, the kit may comprise the device of syringe, eye dropper, ballistic particle applicator (e.g., the applicator disclosed in U.S. Patent No. 5,797,898, U.S. Patent No. 5,770,219 and U.S. Patent No. 5,783,208 and U.S. Patent Application No. 2005 / 0065463), spoon, microslide cover, test strip holder or cover, etc.

[0215] The kit for carrying out the method of the present invention may include a detection reagent for labeling the components of the kit.In a particular aspect, the labeling or detection reagent is selected from a group including those commonly used in the art, including but not limited to radioactive elements, enzymes, molecules that absorb light in the UV range, and fluorophores, such as fluorescein, rhodamine, auramine, Texas Red, AMCA Blue, and Lucifer Yellow.In another aspect, a kit is provided that includes one or more container means and a BST protein agent that is already labeled with a detection reagent selected from a group including radioactive elements, enzymes, molecules that absorb light in the UV range, and fluorophores.

[0216] When the reagents and / or components constituting the kit are provided in lyophilized form (lyophilizate) or as a dry powder, the lyophilizate or powder can be reconstituted by adding a suitable solvent. In certain aspects, the solvent can be a sterile pharma- ceutically acceptable buffer and / or other diluent. It is contemplated that such solvents can also be provided as part of the kit.

[0217] When the components of the kit are provided in one and / or multiple liquid solutions, the liquid solution can be, by way of non-limiting example, a sterile aqueous solution.The composition can also be formulated into a composition for administration.In this case, the container means can itself be a syringe, a pipette, a topical applicator, etc., from which the formulation can be applied to an affected area of ​​the body, injected into a subject, and / or applied to or mixed with other components of the kit.

[0218] XVI. Sequence Table 1 Peptides TIFF2024516542000001.tif154166TIFF2024516542000002.tif161128TIFF2024516542000003.tif241121TIFF2024516542000004.tif24 2121TIFF2024516542000005.tif242121TIFF2024516542000006.tif242121TIFF2024516542000007.tif242121TIFF2024516542000008.t if242121TIFF2024516542000009.tif242121TIFF2024516542000010.tif242121TIFF2024516542000011.tif242121TIFF20245165420000 12.tif242121TIFF2024516542000013.tif242121TIFF2024516542000014.tif242121TIFF2024516542000015.tif242121TIFF20245165420 00016.tif242121TIFF2024516542000017.tif242121TIFF2024516542000018.tif246121TIFF2024516542000019.tif242121TIFF2024516 542000020.tif242121TIFF2024516542000021.tif242121TIFF2024516542000022.tif242121TIFF2024516542000023.tif242121TIFF202 4516542000024.tif242121TIFF2024516542000025.tif244121TIFF2024516542000026.tif245121TIFF2024516542000027.tif242121TIF F2024516542000028.tif241121TIFF2024516542000029.tif242121TIFF2024516542000030.tif242121TIFF2024516542000031.tif192128 EXAMPLES

[0219] XVII. Working Examples The following examples are given for the purpose of illustrating various aspects of the present invention and are not meant to limit the present invention in any way. Those skilled in the art will easily understand that the present invention is well adapted to carry out the objects and obtain the results and advantages mentioned, as well as the inherent objects, results and advantages herein. The examples, together with the methods described herein, are currently representative of preferred aspects and are illustrative and are not intended to limit the scope of the present invention. Modifications therein and other uses encompassed within the spirit of the invention as defined by the claims will occur to those skilled in the art.

[0220] Example 1: Identification and validation of frameshift neoantigens for mismatch repair deficient Lynch syndrome Lynch syndrome (LS) is responsible for approximately 2.5% of all diagnosed colorectal cancer (CRC) cases. Patients with LS are at high risk for developing CRC, with an estimated lifetime risk of 70-80%. Patients with LS have a germline mutation in one of the mismatch repair (MMR) system genes (MLH1, MSH2, MSH6, PMS2, or TACSTD1 / EPCAM). MMR deficiency (dMMR) is manifested in microsatellite instability (MSI) and the generation of frameshifted peptides (FSPs) that become neo-antigens (neo-Ags). Neo-Ags are presented by MHC classes I and II and recognized by the adaptive immune system. Immunogenic neo-Ags are likely to have an effective avenue for CRC immune blockade strategies, such as immunoprophylactic vaccines for LS carriers.

[0221] We utilized paired whole-exome sequencing and mRNAseq in LS CRC (stages I-III) and precancerous conditions to classify and identify the most frequently recurrent neoAgs present in LS patients and predicted their immunogenicity using in silicometrics such as HLA genotype, mutation frequency, HLA binding affinity and expression levels. To validate the computational predictions, we harvested cytotoxic lymphocytes from a total of three LS patients and generated neoAg-loaded tetramers to mimic the MHC-I presentation of 10 different neoAgs from the predicted list. After tetramer staining, counting and isolating neoAg-specific CTLs using ELISpot, a 15-plex cytokine profiling ELISA assay was used to confirm the immunogenic potential of each neoAg.

[0222] MHC-tetramer staining revealed that neoAg-specific CTLs constituted approximately 0.5-1% of the total peripheral CTL population, which is consistent with previous studies. ELISpot performed with CTLs showed a significant secretion of IFNγ (spot-forming units) upon overnight stimulation with neoAg-loaded tetramers compared to controls. A 15-plex cytokine profile using CTLs from one patient identified a significant activation of pro-inflammatory cytokines (IL-1a, IL-1b, IL-12, IL-17, IL-23) and pro-proliferative cytokines (IL-2, IL-15) upon neoAg stimulation compared to unstimulated controls.

[0223] These results provide strong evidence that the in silico computational pipeline accurately predicts the immunogenicity of LS neoAgs and suggests that these neoAgs have the potential to initiate immune responses consistent with previously published studies performed in other cancers. This study provides the basis for developing an immunoprophylactic vaccine for LS carriers.

[0224] Patients and specimen collection: All patients in this study had a confirmed diagnosis of LS (n=28). Patient characteristics are shown in the table below.

[0225] TIFF2024516542000032.tif23298TIFF2024516542000033.tif32128*A patient may have multiple cancers or neoplasms.

[0226] The strategy for in silico neoantigen prediction is depicted in Figure 1, and the in vitro validation pipeline is shown in Figure 2. Figures 3-4 show the mutation frequency and neoantigen sequencing. Figures 5A-D show the validation of neoantigen immunogenicity.

[0227] In conclusion, we performed paired whole exome sequencing (WES) and mRNAseq of LS CRC (stages I-III) and precancerous conditions from a LS patient cohort. A state-of-the-art bioinformatics pipeline predicted a classification of recurrent and highly immunogenic neoAgs. We validated the immunogenicity of several peptides using MHC class I tetramers and ELISPOT. In vitro validation confirms the accuracy of the in silico prediction of immunogenic neoAgs. The data support the use of these neoAgs as a vaccine-based immunoprophylactic strategy for LS patients to prevent the development of CRC.

[0228] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024516542000034.tif92160

[0229] Example 2: Identification and validation of frameshift neoantigens for mismatch repair deficient Lynch syndrome Lynch syndrome (LS) patients constitute a well-defined population likely to benefit from cancer immune blockade strategies, given that they develop DNA mismatch repair-deficient tumors that generate a high burden of neoantigens. We used paired whole-exome sequencing and mRNAseq to perform in silico prediction, immunogenicity ranking and in vitro validation of highly immunogenic and repetitive frameshift neoantigens (FS-neoAgs) from colorectal cancer (CRC) (n=13) and precancerous conditions (n=61) in a LS patient cohort (N=46). We showed that mutational burden derived from microsatellite instability positively correlated with high FS-neoAg burden even in precancerous conditions. After testing 154 predicted FS-neoAgs, we demonstrated an in vitro validation rate of up to 50% in MHC-I-restricted FS-neoAgs, when the high predicted immunogenicity and repetitiveness of FS-neoAgs in the cohort were considered factors for their selection. Overall, the mutational data, gene expression data and FS-NeoAg classification improve our understanding of LS-derived cancers, which will guide the future development of immunoprophylactic vaccine strategies.

[0230] This study provides the largest LS somatic mutation, gene expression and FS-neoAg landscape report currently available, with supporting evidence of a computational pipeline that accurately predicts the immunogenicity of tumor-derived FS-neoAg. This computational platform will inform the future development and discovery of a universal LS cancer vaccine.

[0231] A. Introduction Lynch syndrome (LS), the leading cause of hereditary colorectal cancer (CRC), accounts for 2-4% of all CRC cases in the United States, affecting more than one million carriers (1). LS results from heterozygous germline mutations in DNA mismatch repair (MMR) genes, with MLH1 and MSH2 accounting for more than 70% of LS cases. LS patients have an increased lifetime risk of developing CRC, which reaches 60% in MLH1 and MSH2 carriers (2). Normal colorectal cells become MMR-deficient (dMMR) when they acquire a second somatic hit in an alternative allele of an MMR gene that harbors a germline mutation. This second hit manifests itself in the accumulation of base-pair mismatches and insertion-deletion mutations (indels) in microsatellite sequences that generate neoantigens (neoAgs). These tumor-specific antigens are processed and presented as short peptides loaded onto major histocompatibility complexes (MHC I / II) to the T cell receptor (TCR) on cytotoxic CD8+ T cells, which promotes interferon-γ (IFNγ) secretion and kills neoAg-producing cancer cells (2). However, if cancer cells are able to evade the immune system through upregulation of immune checkpoint molecules, tumors revert to a state of uncontrolled proliferation. Therefore, activating CD8+ and CD4+ T cells (helper cells) that recognize neoantigens is critical for adaptive immunity against tumors.

[0232] Extensive computational algorithms have used next-generation sequencing (NGS) data to rapidly screen the mutational landscape of human cancers, including melanoma and colon (4-7). Such tests have identified a variety of neo-antigens that can be recognized by the host immune system, providing promising avenues for further personalized and focused approaches to activate antitumor immunity (8). Given the trend for increased mutational burden in dMMR cancers, putative neo-Ags characterized from genomic and transcriptomic data of LS patients may provide similar opportunities to develop novel immunoprophylactic therapies, such as neo-antigen peptide vaccines.

[0233] In this study, we used innovative bioinformatics to acquire genomic data using paired whole-exome sequencing (WES) and mRNAseq in LS CRC (stages I-III) and precancerous conditions (advanced adenomas and adenomas) to classify and identify the most immunogenic and recurrent frameshift-neoAgs (FS-neoAgs) present in LS colorectal precancerous conditions and tumors. This established pipeline accurately identifies somatic microsatellite (MS) indels by estimating and reducing read-length-related sequencing errors, PCR amplification errors, and other noise sources. Also, the frequency of a given peptide within the test cohort, its binding affinity, expression levels, and the HLA genotypes of the individuals are accounted for. Finally, we used immunological assays to validate the predicted immunogenicity of FS-neoAgs from the computational method, thus bringing the field closer to improved immunoprophylactic therapy for LS cancer. An overview of this study is shown in Figure 6.

[0234] B. Results 1. Demographics and characteristics of the LS patient cohort We analyzed a total of 74 colorectal adenoma (polyp) or tumor samples obtained from the lower gastrointestinal tract of 46 LS patients with matched normal mucosa and peripheral blood. Patient demographics and clinical characteristics are summarized in Table 1, and pathological characteristics of each polyp or tumor are found in Supplementary Table 1. The mean age of the patient cohort was 52 years (range, 20-80). The majority of patients had germline pathogenic mutations in MSH2 (N=14) and MSH6 (N=14), followed by MLH1 (N=11), and PMS2 (N=5). Two patients fulfilled the Amsterdam Criteria but no germline mutations were detected. Of the precancerous lesions, 44 were identified as early tubular adenomas, 9 as hyperplastic polyps (HPs), 2 as inflammatory polyps (IPs), 2 as tubulovillous adenomas, and 4 as sessile serrated adenomas (SSAs). All cancerous lesions were identified as adenocarcinomas of different stages (n=13). Based on the MSI sensor score, the MSI status of the samples was determined. Of the 74 samples, the scores ranged from 0% to 33.67%, with 21 samples classified as MSI-H, 22 as MSI-L, and the rest as MSS (Figure 12A). The median MSI score of the precancerous lesions was 4.1%, with almost half exhibiting MSI-H (n=9) and MSI-L (n=19). The majority of cancers (n=10) were MSI-H, and as expected, the median MSI score was 15.3% (Figure 12B).

[0235] 2. Germline mutations and second somatic hit analysis of MMR genes in a cohort of LS patients Identification of germline mutations was performed using HaplotypeCaller as described in Methods. The types of germline mutations in the MMR genes in 46 patients consisted of splicing events (13%), frameshift indels (24%), nonsense mutations (26%), exon deletions (11%), missense mutations (9%), and unknown type in the remaining patients (17%). These germline mutations are shown in Figure 13 along with the second somatic hits that we were able to detect. As expected, more than 60% (13 / 21) of the MSI-H samples had a detectable second somatic hit. The remaining MSI-H samples with undetected second somatic hits and patients with unknown type of germline mutations could potentially be explained by the lack of sensitivity that WES has in detecting structural mutations, intronic variants, and variants present in distal control elements of the genome.

[0236] 3. LS patients with MSI-H have somatic mutations in CRC-related genes We determined the somatic mutational landscape in the LS cohort using WES data from 74 lesion-normal pairs by combining Mutect2 and MSmutect outputs. We observed a range of (2-2862) mutations per sample, with most of these mutations being missense and frameshift indels (Figure 7A). Furthermore, we detected recurrent deleterious mutations (frameshift indels, nonsense and stop-loss) in several genes of canonical CRC-associated pathways, including WNT, chromatin remodelers, DNA repair and TGFβ / BMP. For example, within Wnt pathway genes, APC mutations were identified in 33 / 74 samples, of which 27 were adenomas (with 15% MSI-H), 5 were adenocarcinomas (with 100% MSI-H) and 1 was HP (without MSI-H). Additionally, BCL9 was mutated in 17 / 74 samples, of which 8 were adenomas (with 75% MSI-H), 7 were adenocarcinomas (with 86% MSI-H), and 2 were SSAs (without MSI-H). Furthermore, this analysis determined that mutations in CRC-associated genes were widely present within the 74 samples, including 14 samples with ARID1A mutations, 11 with TGFBR2, 10 with ATM, CTNNB1, KRAS, SOX9, and TCF7L2, 9 with PIK3CA, 7 with TP53, 6 with PTEN, and 4 with BRAF mutations. BRAF mutations were only detected in 4 SSAs in this sample cohort, consistent with previous studies (10). Overall, when considering MSI status, histology category, or pathology, mutation burden was significantly increased at the most advanced levels of each of these features (Figure 7B). MSI-H samples showed the highest mutation rates when MSI status was considered, and cancers had significantly higher mutation rates when looking at histology categories, with adenocarcinomas, followed by adenomatous polyps, showing the highest levels of mutation burden by pathology.

[0237] 4. In silico neoantigen prediction by immunogenicity ranking We applied a series of computational and bioinformatics methods to identify and classify neoAgs generated from frameshift mutations in the LS sample cohort, as summarized in (Figure 14), and developed a neoAg prediction pipeline. To do this, we first performed MHC class I and II typing from the WES data using PHLAT (11) and determined the ranking of the frequency of HLA alleles within the LS cohort in this study (Supplementary Table 2). The typing results showed that the most frequent HLA class I alleles were HLA-A*02:01, HLA-B*07:02, and HLA-C*07:02 for each locus, covering 32%, 34%, and 34% of the cohort, respectively. For HLA class II alleles, the most frequent were HLA-DQA1*01:02, HLA-DQB1*06:02, and HLA-DRB1*15:01 for each locus, covering 49%, 36%, and 34% of the patient cohort, respectively. Furthermore, more than 90% of the patient population within the cohort contained the top 10 most frequent HLA class I alleles (Figure 15).

[0238] As described in the Methods section, the NetMHCpan algorithm was used to calculate immunogenicity predictions for potential neo-Ags bound to HLA class I and II epitopes. The total number of predicted MHC-I and MHC-II neo-antigens per sample ranged from 0 to approximately 3500, with the majority of neo-Ags having high predicted binding affinity (<50 nM) (Figure 16). When considering MSI status, tissue category and pathology, consistent with mutation rates, the number of neo-Ags increased significantly at the most advanced levels of each of these features (Figure 8A, B and C). MSI-H samples showed significantly higher numbers of MHC-I and MHC-II neo-Ags (Mann-Whitney test, P<0.0001; Figure 8A), as well as cancer, when compared between tissue categories or pathologies (Mann-Whitney test, p<0.01; Figure 8B and C). This was further demonstrated by comparing the number of neoAgs identified per sample with the mutation rate of each sample, which showed a significant positive correlation for both MHC-I and MHC-II neoAgs (Pearson, P value = <0.001; Figure 8D ).

[0239] Since the aim of this study was to discover a set of neoAgs that could potentially be used as LS cancer vaccines, and any discovery requires its independent corroboration, we divided the sample cohort into a discovery set and a validation set (Supplementary Table 3). For each set, predictions of MHC-I and MHC-II neoAgs derived from frameshift indels were performed separately. MHC-I neoAgs from the discovery set were ranked based on the formula described in Figure 14 and the immunogenicity score obtained from "Methods" (Supplementary Table 4). Separately, these MHC-I predicted neoAgs were ranked based on their repetitiveness within the sample cohort (Supplementary Table 5). Figure 8E shows the status of the top 50 genes generating the most repetitive MHC-I restricted neoAgs in the discovery sample cohort, along with the calculated immunogenicity score (blue scale). The pipeline identified a set of novel repetitive MHC-I-restricted neo-Ags with high immunogenic potential predicted to be generated by genes including RNF43, ACVR2A, BCORL1, BMPR2 and TCF20. Notably, predicted neo-Ags produced from mutant MARCKS, TGFBR2, TCF7L2 and ASTE1 proteins have been previously reported in LS patients (11), and we also predicted and classified MHC-II-restricted neo-Ags based on their immunogenicity scores (Supplementary Table 6) and repetitions (Supplementary Table 7). The landscape of the top 50 genes generating the most repetitive MHC-II-restricted neo-Ags in the discovery sample cohort is shown in Figure 17. Several genes generated repetitive and potentially immunogenic neo-Ags restricted to both MHC-I and MHC-II molecules.

[0240] To validate the performance of our neoAg prediction pipeline in silico, we first benchmarked MHC-I-restricted neoantigen predictions from our discovery set to that of the Tumor Neoantigen Selection Alliance (TESLA). The TESLA platform used a common dataset of three melanomas and three non-small cell lung cancers, followed by an in vitro validation platform using MHC-I multimer-based assays to evaluate the concordance level of 25 different pipelines and ranking systems (12). In those studies, five robust immunogenicity criteria were proposed to rank the potential of predicted neoAgs for immune system presentation and recognition: 1. binding affinity < 34 nM; 2. tumor abundance > 33 TPM (transcripts per million); 3. binding stability > 1.4 h; agretopic < 0.1 and heterogeneity > 10. -16 From the top 100 most immunogenic MHC-I indel-derived predicted neoAgs, 25% met all three presentation criteria, including binding affinity, tumor abundance, and binding stability, and 13% met all five criteria, including the three presentation criteria plus the recognition criteria (agretopic and heterogeneous) (Figure 18).

[0241] To further validate the in silico predictions made on the discovery set (n=43), we utilized a validation set of samples (n=31) (Supplementary Table 4) to perform neoAg predictions and evaluate the level of concordance between the two datasets in terms of shared neoAgs. For MHC-I neo-antigens, we found 130 shared neo-Ags between the discovery and validation sets. Conversely, for MHC-II neo-antigens, we found 142 shared neo-antigens between the discovery and validation sets (Figure 9A). Notably, among the top 50 predicted neo-Ags from the validation set, 10 were also present in the discovery set, including CNOT1, ACVR2A, MARCKS, MXRA8, RNF43, BCORL1, and CAMTA2 (Figure 9B, light grey font). Of the 100 most immunogenic MHC-I and MHC-II neo-Ags in the discovery set, 6% were also found in the validation set. For the most repetitive MHC-I and MHC-II neo-Ags, 14% and 18%, respectively, were also present in the validation set (Figure 9C). We classified the predicted MHC-I restricted neo-Ags from the validation set based on immunogenicity score (Supplementary Table 8) and repetition (Supplementary Table 9). 10% of the top 100 most immunogenic MHC-I restricted neo-ags predicted in the validation set were also present in the discovery set, and 18% of the top 100 most repetitive MHC-I neo-ags from the validation set were also present in the discovery set. The list of MHC-II restricted neo-Ags based on immunogenicity and repetition in the validation set is included in Supplementary Tables 10 and 11, respectively. From the neo-ag predictions in the validation set, 17% of the top 100 most immunogenic and 18% of the top 100 most repetitive were also present in the discovery set.

[0242] 5. Selection of predicted neoAgs for in vitro validation of immunogenicity in human donors To validate the immunogenicity of predicted neo-Ags in silico, we selected a total of 154 neo-Ags from the discovery set to test the immunogenic response of pooled and individual peptides in ELISpot assays using PBMCs from healthy donors. These MHC-I peptides were selected as follows: 10 were randomly selected from the top 100 most immunogenic predicted neo-Ags, 55 were selected from the top 100 most repetitive, 14 were part of both the top 100 most immunogenic and top 100 most repetitive MHC-I neo-ags (Supplementary Tables 4 and 5; column: tested by ELISPOT), and 31 were not part of either group, had low immunogenicity, and were not repetitive (Supplementary Table 12). For MHC-II peptides, 20 were randomly selected from the top 100 most immunogenic MHC-II predicted neo-Ags and 17 were randomly selected from the top 100 most repetitive MHC-II neo-Ags, with 7 being part of both groups and being the most immunogenic and most repetitive MHC-II neo-Ags (Supplementary Tables 6 and 7; columns: tested by ELISPOT).

[0243] PBMCs from healthy donors were stimulated with 15 peptide pools (Supplementary Table 13) to generate neoAg-specific CD8 +T cells were expanded and subsequently subjected to quantitative ELISpot assays (Figure 19A) to measure immunogenicity. The results showed that peptide pools 1, 2, 3, 4, 5, 8, 9 and 12 induced high secretion of IFNγ in PBMCs of one or more healthy donors when compared to DMSO control cells (Figure 19B). To confirm the immunogenicity of individual peptides within each immunogenic pool, a deconvolution protocol (see Methods) was performed (Figure 10A). The data showed that a total of 20 MHC-I predicted neo-Ags and 2 MHC-II predicted neo-Ags (from 8 immunogenic pools) induced significant secretion of IFNγ in PBMCs of healthy donors compared to unexposed control cells (Supplementary Table 14). The top 12 most reactive (immunogenic) frameshift antigenic peptides were generated from the following genes: BCORL1, TTLL10, R3HDM3, CRIM1, WDTC1, USP9Y, AASDH, HOXA11, TCF20, CCDC186, RNF43 and UBR5 (Figure 10B) and the other 10 reactive peptides are shown in Figure 20. ELISpot data showed that at least 10% of the most immunogenic, 16% of the most repetitive, and 50% of peptides that were part of both the most immunogenic and most repetitive MHC-I neo-Ags showed in vitro reactivity, thus validating the in silico neo-Ag prediction pipeline (Figure 10C). As assessed by ELISpot assay, 3% of MHC-I neo-Ags that were not predicted to be highly immunogenic or repetitive (others), 5% of the top 100 most immunogenic MHC-II predicted neo-Ags, and 6% of the top 100 most repetitive MHC-II predicted neo-Ags induced immunogenic responses. Moreover, 18% of these total elispot reactive neo-Ags were also predicted from the validation set (Figure 9C). Based on these results, the highest percentage of in vitro validation was achieved with MHC-I predicted neo-Ags that were some of the most immunogenic and most repetitive neo-Ags.These two factors, when combined, also showed the best population coverage in silico, with the top 10 most immunogenic + repetitive MHC-I neoags present in >85% of the cohort and the top 10 most immunogenic (only) covering <50% (Figure 21).

[0244] 6. Validation of immunogenicity of predicted neoantigens in the rhesus macaque LS model Since the ultimate goal is to develop a universal LS cancer vaccine, we tested the immunogenicity of ELISpot-reactive peptides on PBMCs from LS rhesus macaques carrying pathogenic mutations in the MLH1 gene, which have pathology similar to human LS. These animals, housed at MD Anderson Cancer Center, spontaneously develop CRC and serve as an ideal preclinical LS model for immune blockade strategies with neo-Ag vaccines. As shown in Figure 22A, PBMCs from four different LS rhesus macaques were stimulated with four peptide pools and 12 individual peptides for ELISpot assays. These results demonstrate that all four donor PBMCs yielded 80-600 SFU for peptide pools 1 and 2 compared to DMSO control cells, suggesting that pools 1 and 2 are immunogenic based on the ELISpot assay (Figure 22B). Further ELISpot assays with individual peptides demonstrated that predicted neoAgs derived from TTL10, WDTC1, SPECC1, BCORL1, AASDH, R3HDM2, CCDC186 and HOXA11 (8 of 12) were more abundant than predicted in at least two donors. 5 We demonstrated that these antibodies appear to be highly immunogenic, as they yielded SFUs ranging from 25 to 400 per cell. These results, together with the human in vitro validation, demonstrate that the computational pipeline performs well in predicting MHC-I neoAgs.

[0245] 7. Transcriptome landscape and immune cell profile Given the increased number of neoags produced in cancer and certain precancerous conditions, we decided to evaluate the level of immune activation at the transcriptome level using mRNAseq data in both discovery and validation cohorts. Unsupervised clustering analysis showed even more effective grouping of samples when considering tissue category compared to MSI status (Figure 23A and B). We found 78 genes significantly dysregulated between cancer and precancerous conditions (Figure 11A), of which 7 were genes involved in immune response (APLN, IGKV1D-17, TRBV5-4, ABI3BP, IGLV10-54, TRBV9, CD300LG, CEACAM7). Interestingly, most of these immune genes were downregulated in cancer compared to precancerous conditions. However, after performing pathway enrichment analysis, we found activation of antigen processing and presentation pathway, IL-17 signaling pathway, TNF signaling pathway as well as several other pathways (Figure 11B). Regarding immune cell infiltration, we found that naive B cells, macrophages M0 and CD8+ T cells were significantly decreased in cancer compared to precancerous conditions, whereas monocytes were significantly increased (Figure 11C). Taken together, these results could potentially be explained by the fact that LS patients have a genetic predisposition to dMMR in all cells of their organism, which could ensure a higher immune infiltration in normal tissues leading to a more pronounced immune response against tumor development, even at early stages (precancerous and MSS stages). This surveillance level is potentially reduced at MSI-H and cancerous stages due to the capacity of these cells for immune evasion and destruction (13).

[0246] We further confirmed this hypothesis by performing differential gene expression analysis between MSI-H and MSS samples. We observed fewer significantly dysregulated genes in MSI-H versus MSS (44 genes) compared to the cancer versus precancerous analysis. Only two of the 44 genes were involved in immune response (IGHA2 and ABI3BP), which showed downregulation in MSI-H samples (Figure 24A). Furthermore, IL-17, p53, and cell cycle signaling pathways were enriched in MSI-H samples. However, many more pathways, including chemokine signaling pathway, B cell receptor signaling pathway, gut IgA production, and other pathways involved in immune response, were suppressed in MSI-H samples compared to the cancer versus precancerous analysis (Figure 24B). After immune cell deconvolution, we found significantly higher resting NK cells and CD8+ T cells in MSS samples than in MSI-H samples. Additionally, resting CD4+ T cells were significantly more abundant in MSI-L samples compared to MSI-H (Figure 24C).

[0247] C. Discussion LS-associated tumorigenesis is characterized for dMMR, MSI, and the generation of a high burden of neoags that can be recognized by the host immune system. LS patients are a defined population at high risk for cancer development, especially CRC, at an early age. This makes them a distinctive population for evaluating preventative cancer vaccines. To this end, several attempts have been made towards the identification of cancer-derived epitopes, and their vaccine potential (9). Despite this, there is still a large room for improvement in the in silico prediction of candidate neoags combined with in vitro validation of immunogenicity, especially in MSI cancers and MSI precancerous conditions.

[0248] In this study, we used a combined approach of WES and mRNAseq data to identify a classification of immunogenic and recurrent indel-derived MHC-I and II-restricted neo-ags from a cohort of precancerous conditions from CRC and LS patients routinely followed at MDACC. Various systems biology platforms have been developed using next-generation sequencing (NGS) paired with bioinformatics pipelines to predict and classify tumor-associated antigens from synonymous and nonsynonymous mutations as foreign antigens (neo-antigens) to the host immune system (15, 16). However, in MSI cancers, accurately predicting immunogenic frameshift peptides in homopolymer stretches of coding microsatellite (cMS) mutations has always been challenging for computational analysis (17). These concerns are mainly due to the limited sensitivity of short-read next-generation approaches, as well as ambiguities in the alignment and assembly of short repetitive DNA that create biases and errors for accurately predicting FSP neo-antigens in cMS mutations (18, 19). To address some of these unmet challenges, the pipeline incorporates mutation calling by combining the output from two different tools, Mutect2 (33) and MsMutect (34). MsMutect allows careful realignment of reads that contain MS and nominate MS indels, specifically by applying an empirical noise profile based on the motif and length of the repetitive DNA sequence. Thus, the rate of false-positive MS indel calls is significantly reduced and neoags are predicted more accurately (34).

[0249] In silico prediction and in vitro validation identified a series of recurrent and immunogenic neoantigens generated in previously reported MS hotspots in genes including RNF43, SEC31A, and ASTE1 ( 9 ), as well as novel MS hotspots including BCORL1, TTLL10, R3HDM2, CRIM1, WDTC1, USP9Y, HOXA11, UBR5, and SPINK5, among others.

[0250] To strengthen the robustness of the predictions, we tested the strength of the pipeline against the TESLA benchmark (12), and we observed that 25% of the top 100 predicted most immunogenic MHC-I neo-Ags met all presentation criteria, and 13% met all five presentation and recognition criteria. The prediction pipeline exceeded the performance cutoffs established in the TESLA analysis of 10% for predicted peptides that passed presentation criteria and 5% for predicted peptides that passed recognition criteria (12). These results suggest that the pipeline, which generates predictions with 25% of the most immunogenic neo-Ags meeting presentation criteria and 13% meeting recognition criteria, has strong performance compared to most pipelines analyzed by TESLA.

[0251] Importantly, in vitro immunogenicity evaluation of 154 predicted neo-Ags (MHC-I and MHC-II) by performing large-scale ELISpot assays using peptide pools and individual peptides allowed us to identify several predicted neo-Ags that were highly immunogenic in PBMCs from healthy humans and LS rhesus macaques. Although several previous reports have demonstrated the in vitro immunogenicity of neo-Ags in MSI tumors, the number of neo-Ags selected for this validation was relatively small compared to this study (25, 26). One of the limitations of the in vitro validation included the exclusion of LS patient PBMCs due to the unavailability of specimens to perform ELISpot assays, which may be considered for future experiments. Utilizing PBMCs with LS rhesus macaques demonstrated higher reactivity of neo-Ag peptides in the ELISpot assay than human PBMCs, thus validating the in vitro immunogenicity of these antigenic peptides. Thus, similar to human LS, the LS model of rhesus macaques harboring pathogenic mutations in MLH1 provides an ideal preclinical animal model for immune blockade strategies by neo-Ag peptide vaccination.

[0252] The utility of immune blockade strategies as cancer prevention has gained significant traction in recent years. We recently reported a phase 1b clinical trial of long-term exposure of nonsteroidal anti-inflammatory drug (NSAID) naproxen to LS patients and observed that naproxen exposure led to an increase in resident immune cells in the mucosal tissue of the colon (27). Therefore, it is reasonable to hypothesize that an immune stimulant, e.g., naproxen, when combined with a neoantigen vaccine, could lead to a favorable immune response in the mucosal tissue, which would result in durable immune prevention for LS cancers, including CRC, EC, and other GI cancers. Further testing is required to support this hypothesis. This data demonstrates the powerful approach of bioinformatics to identify, predict, and rank candidate neoantigens for the future development of LS-specific immunotherapy.

[0253] In summary, we report a novel, validated computational algorithm that predicts the immunogenicity and recurrent nature of frameshift neo-antigen mutations in a cohort of LS patients routinely cared for at MDACC. This pipeline offers the ability to accurately identify candidate neo-Ags suitable for developing cancer preventive vaccines and other durable blockade modalities, which remains a huge unmet clinical need in the field of oncology.

[0254] D. Method 1. Patients and specimen collection All patients included in this study had a confirmed diagnosis of Lynch syndrome (n=46) and provided written informed consent at the University of Texas MD Anderson Cancer Center (MDACC). All samples were obtained from study participants through a protocol approved by the MDACC Institutional Review Board (IRB) (Protocol PA12-0327). Seventy-four paired snap-frozen or formalin-fixed paraffin-embedded (FFPE) tissue biopsies from polyps or tumors of the lower gastrointestinal tract with matching normal mucosa and peripheral blood were collected from 46 LS patients who presented to MDACC for standard-of-care surveillance colonoscopy (Supplementary Table 2). Pathological diagnoses of all tissue samples were confirmed by a gastrointestinal pathologist (MWT) at MDACC. DNA and RNA were extracted from snap-frozen and FFPE tissue samples using the Quick-DNA / RNA Miniprep Kit (Zymo Research, CA) and AllPrep DNA / RNA FFPE Kit (Qiagen, MD), respectively. Genomic DNA was obtained from peripheral blood using the Gentra Puregene Blood Kit (Qiagen).

[0255] 2. Whole-exon sequencing, mRNA sequencing, and bioinformatics analysis Library construction and sequencing were performed at the MDACC Advanced Technology Genomics Core and the MDACC Cancer Genomics Laboratory. Samples were grouped into discovery and validation sets (Supplementary Table 4). RNA and DNA samples obtained from polyps, tumors, and matched normal mucosa were sequenced using HiSeq4000 sequencers (Illumina) and NovaSeq 6000 sequencers (Illumina) for the discovery and validation sets, respectively. Alignment of WES data was performed using BWA mem 0.7.17 with default parameters against the human genome reference hg19 [arXiv:1303.3997, Li, 2013]. Picard 2.9.0 [Picard Toolkit." 2018. Broad Institute, GitHub Repository [ http: / / broadinstitute.github.io / picard / Duplicate reads were marked using [ 28 ]. Base quality recalibration was performed using GATK Apply BQSR 4.1.2.0. Alignment of mRNA-seq data to the human genome reference hg19 was performed using STAR ( 29 ) and bowtie 1.2.2.

[0256] 3. Determining MSI Status MSI sensor was used to predict MSI status from WES data in both discovery and validation sets as previously described (30). Duplicate reads were physically removed from normal and tumor BAM files using samtools (31, 32). Microsatellite loci were first identified in the hg19 reference genome by MSI sensor scanning (30). Pearson's chi-square test was used to compare the distribution of expected (normal) and observed (tumor) lengths of repeat sequences per microsatellite after coverage normalization. In this Pearson's chi-square test, the default FDR=0.05 was used as the cutoff for identifying somatic microsatellite sites by MSI sensor msi (30). MSI score was defined as the percentage of somatic sites relative to the total number of microsatellite sites with a minimum coverage of 20 in normal and paired tumor samples. Samples were then classified as MSI-H if MSI score >=10%, as MSI-L if MSI score <10% and >=3.5%, and as MSS if MSI score <3.5%, based on the recommended cutoffs from the MSI sensor (30). The number of somatic and non-somatic sites above threshold for all samples was plotted as stacked bar graphs. Sample classification based on MSI score was shown as a covariate bar in a waterfall plot.

[0257] 4. Detection of somatic mutations Somatic mutations from both the discovery and validation sets were detected using Mutect2 4.0.8.1 following GATK best practices [(33)]. MSMuTect (34) was used to identify somatic indels located at microsatellite loci identified in the hg19 reference genome by Phobos using default parameters [Mayer, Christoph, Phobos 3.3.11, 2006-2010, <found on the World Wide Web at: rub.de / ecoevo / cm / cm_phobos.htm>]. After MS-specific alignment, alleles were inferred by an empirical noise model, followed by mutation calling using Akaike's Information Criterion (AIC) and the Kolmogorov-Smirnov (KS) test. Somatic mutations above the threshold were annotated by Oncotator (35) using the default cutoffs in MSMuTect and used as part of the input for the neoantigen discovery pipeline, together with mutations detected using Mutect2.

[0258] 5. Transcriptomics analysis All samples from the discovery and validation cohorts were included in the analysis, except those without paired normal tissue. Quality control of RNAseq results was assessed by FASTQC software version 0.11.5 (36). Adapters and low-quality bases were trimmed using Trimmomatic version 0.39 (37) with default parameters. Reads were mapped using Spliced ​​Transcripts Alignment to a Reference (STAR ​​version 2.7.9a) (29) and counted using RNA-Seq by Expectation Maximization (RSEM version 1.3.1) (38). Raw counts were normalized by the trimmed mean of M values ​​method (39). Differentially expressed genes (DEGs) were determined by genewise negative binomial generalized linear model with quasi-likelihood test in EdgeR package Ver. 3.36.0 (40) with 0.05 as the Log2FC and Benjamini-Hochberg (BH) adjusted P-value cutoff. Normalized counts per million (CPM) of each sample were used for cell deconvolution in the CIBERSORT-abs algorithm (41) implemented by Immunedeconv package Ver. 2.0.4 (42). Gene set enrichment analysis (43) of KEGG pathways from the Kyoto encyclopedia of genes and genomes pathways (KEGG) (44) was preshaped by ClusterProfiler Ver. 4.0.5 (45). Batch effect-corrected DEGs were visualized by ComplexHeatmap Ver. 2.8.0 (46).

[0259] 6. HLA typing MHC class I and II HLA alleles were detected for each patient from the WES data using PHLAT with default settings ( 47 ).

[0260] 7. Bioinformatics methods for neoantigen prediction Germline mutations were detected by GATK HaplotypeCaller 4.1.2.0 following GATK best practices using SNV sensitivity threshold = 99.9 and indel sensitivity threshold = 98.0 (28) [biorxiv:201178v2, Poplin, 2017]. Somatic mutations passed by Mutect2 and MsMutect were annotated by VEP version 98.3 (48). Somatic and germline mutations were phased using GATK ReadBackedPhasing 3.8. Corresponding RNA depth and variant allele frequency (VAF) of somatic mutations were collected using bam read count helper [https: / / github.com / genome / bam-readcount]. We ran pVACseq 1.5.3 (7) to generate neo-antigen predictions for staged somatic mutations and sample-specific MHC class I and II HLA alleles using NetMHCpan 4.0 with epitope lengths of 8, 9, 10, and 11 amino acids for MHC I peptides, and NetMHCIIpan 3.2 with epitope lengths of 15 for MHC II peptides (49, 50). Predicted neo-antigens with binding affinity >500 nM and DNA VAF <0.05 were removed. Each predicted neo-antigen was assigned an immunogenicity score obtained by combining a composite of the HLA binding affinity score, a binding score fold change rank derived by dividing the binding affinity score of the wild-type protein by the neo-Ag, an allele expression rank (tumor RNA variant allele frequency * gene expression in transcripts / million), the tumor DNA variant allele frequency of each neo-Ag, and a Non-NA feature, which is a neo-Ag that did not have measurements for all previous variables. (Figure 143). The binding stability of each predicted epitope was calculated using NetMHCStabPan with default parameters (51).

[0261] 8. Selection and preparation of neo-Ag peptides for in vitro validation Using the predicted immunogenicity scores, we filtered the top ranked neoAgs generated from each mutation across all samples from the discovery set and selected 154 of them based on their predicted immunogenicity scores (most immunogenic) and their repetitiveness (most repetitive) within the sample cohort (Figure 20). We randomly selected 10 neoAgs from the top 100 MHC class I binder neoAgs with the highest immunogenic scores, even if they were not repetitive (Supplementary Table 5). We randomly selected 55 neoAgs from the top 100 MHC class I binder neoAgs with the highest repetitiveness within the sample cohort, even if they were not predicted to be highly immunogenic (Supplementary Table 6). 14 were MHC-I neoAgs from both the top 100 most immunogenic and top 100 most repetitive lists. We selected 20 neoAgs from the top 100 MHC class II neoAgs with the highest immunogenicity scores (Supplementary Table 7), 17 from the top 100 MHC class II neoAgs with the highest repetitiveness within the sample cohort (Supplementary Table 8), and 7 were from MHC-II neoAgs from both the top 100 most immunogenic and top 100 most repetitive lists. Finally, 31 MHC-I neoAgs with low predicted immunogenicity scores and no repetitiveness were also selected for validation (Supplementary Table 9). All selected peptides were synthesized by GenScript Biotech with a purity of >95%. Peptides were randomly grouped into 15 pools (Supplementary Table 13).

[0262] 9. T Cell Culture and Expansion Proliferation, and ELISpot Assay In a 12-well plate (1.5 × 10 cells / well), 10% heat-inactivated FBS (catalog no. SH30070.03, HyClone), 10 mM Hepes buffer (catalog no. 25060-CI, Corning), and 1X pen / strep (catalog no. 30002CI, Corning) were cultured in R10 medium [RPMI 1640 with L-glutamine (catalog no. 10040CV, Corning), 10% heat-inactivated FBS (catalog no. SH30070.03, HyClone), 10 mM Hepes buffer (catalog no. 25060-CI, Corning), and 1X pen / strep (catalog no. 30002CI, Corning)] supplemented with recombinant human IL-7 (R&D Systems Biotechne, 330 U / ml). 6 PBMCs from healthy donors were cultured on 1x10 wells of 10x10 PBS / well. PBMCs were stimulated with peptide pools or individual peptides (5 μg / ml of each peptide individually or in the pool). Concavalin A and DMSO were used as positive and negative controls, respectively. On days 3, 7 and 10 of culture, cells were fed with R10 medium in the presence of IL-2 (R&D Systems Biotechne, 20 U / mL). On day 12, cells were harvested and placed overnight at 37°C in R10 medium. On day 13, cells were plated in triplicate (1x10 PBS / well) onto 96-well ELISpot plates (Mabtech catalogue no. 3420-2apt-10) pre-coated with human IFNγ antibody. 5 Cells were seeded at 1000 x 1000 / well. Cells were restimulated with the respective peptide pools or individual peptides (3 μg / mL of each peptide) and cultured for 16–20 h. Where indicated, cells were also stimulated with concavalin A (Invitrogen, 0.25 μg / mL) as a positive control. After incubation, secreted IFN-γ was detected according to the manufacturer's instructions (Mabtech) and SFU cells were measured using an ImmunoSpot S6 UNIVERSAL analyzer (Cellular Technolgy Limited, OH). We determined total counts of 1 × 10 5Spot count normalization was performed to account for cell concentration differences by factoring into spots per cell. To determine the immunogenicity of peptide pools or individual peptides, we performed ELISpot assays on six healthy donors (n=6) obtained from Stemcell Technologies Inc (cat. no. 70025.3). Immunogenic pools were defined as those that yielded ≥15 spot-forming units (SFU) compared to the negative control (DMSO) after averaging the results from all donors.

[0263] 10. Isolation and Expansion of Pan T Cells Uninfected T cells (purity >96%) were isolated from PBMCs of HLA-A*02:01 positive healthy human donors using the Pan T Cell Isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany). Briefly, non-T cells were depleted from PBMCs using biotin-conjugated Abs to CD14, CD16, CD19, CD36, CD56, CD123 and glycophorin A anti-biotin-labeled magnetic beads and LS columns. After isolation, 10x106 cells were cultured for 3 days with Opto™ Antigen-Presenting Beads conjugated with WDTC1 neo-Ag peptide (Berkeley Lights, Emeryville, CA, USA) in advanced RPMI supplemented with 10% FBS, 1% GlutaMAX, 1% penicillin / streptomycin (Thermo Fisher Scientific), 55nM 2-mercaptoethanol (Sigma-Aldrich), and 30ng / ml IL-21 (catalog number: 8879-IL-010) (R&D Systems). On day 3, a final concentration of 150ng / ml IL-21 was added and cultured for 5 days. On day 8, the frequency of WDTC1-specific CD8+ T cells was analyzed by a CYTOFLEX SRT flow cytometer (Beckman Coulter, USA).

[0264] 11. Flow cytometry analysis and cell sorting For each healthy human donor, expanded Pan T cells were suspended in Ca2+Mg2+-free phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) (washing buffer) and stained with R-phycoerythrin (PE)-labeled multimeric Pro5 pentamer HLA-A*02:01 / FLADSGIDPV (Proimmune) and Peridinin-Chlorophyll-Protein (PerCP) mouse anti-human CD8 antibody (catalog no. 347314) (BD Biosciences, San Jose, CA, USA) to determine the number of WDTC1 neoAg peptide-specific CD8+ T cells. For pentamer staining, cells were incubated for 10 min in the dark at RT (22 °C) at the manufacturer's recommended concentrations. After pentamer staining, cells were washed twice with 2 ml of wash buffer, centrifuged at 1,200 rpm for 5 min at 4°C, resuspended in the residual volume, and incubated with anti-CD8 antibody on ice for 20 min. Dead cells were excluded by Sytox Blue staining (1 mM, Molecular Probes, Carlsbad, CA, USA). Unstained Pan-T cells were used to detect autofluorescence or background staining. Stained cells were analyzed and sorted using a CytoFLEX SRT flow cytometer (Beckman Coulter, USA) under sterile conditions, and the results were analyzed by FlowJo Software version 10.8.1 (Tree Star, Inc., Ashland, OR, USA).

[0265] 12. Statistical analysis Statistical analysis was performed using PRISM8. Nonparametric Mann-Whitney two-tailed test was used to infer the statistical significance of the differences between tissue categories and MSI status for MSI score (Figure 12B), mutation rate (Figure 7B and Figure 132), number of neo-Ags (Figure 8A), and immune cell expression (Figure 11C and Figure 24C). Nonparametric Spearman's rank correlation coefficient was used to infer the statistical significance of the correlation between mutation rate and number of neo-Ags (Figure 7B). For all tests, significance was defined by a P value <0.05.

[0266] E.Table Table 1. Summary of patient demographics and lesion characteristics TIFF2024516542000035.tif224138TIFF2024516542000036.tif77138*A patient may have multiple cancers or neoplasms.

[0267] (Supplementary Table 1) Clinical, pathological, and demographic characteristics of each specimen taken from LS patients included in this study TIFF2024516542000037.tif247142TIFF2024516542000038.tif247155TIFF2024516542000039.tif247158TIFF2024516542000040.tif247139

[0268] (Supplementary Table 1) (continued) TIFF2024516542000041.tif22711TIFF2024516542000042.tif227157TIFF2024516542000043.tif227157TIFF2024516542000044.tif22768

[0269] (Supplementary Table 2) Frequencies of various HLA alleles present within the cohort of LS patients. TIFF2024516542000045.tif21280TIFF2024516542000046.tif212157TIFF20245165420 00047.tif212157TIFF2024516542000048.tif212158TIFF2024516542000049.tif212141

[0270] (Supplementary Table 3) Distribution of samples for discovery and validation sets TIFF2024516542000050.tif239164TIFF2024516542000051.tif239157TIFF2024516542000052.tif23999

[0271] (Supplementary Table 4) List of the top 100 most immunogenic predicted MHC-I neoAgs obtained from computational methods in the discovery set. TIFF2024516542000053.tif24738TIFF2024516542000054.tif247158TIFF2024516542000055.tif247158TIFF20245165420 00056.tif247158TIFF2024516542000057.tif247158TIFF2024516542000058.tif247158TIFF2024516542000059.tif24717

[0272] (Supplementary Table 4) (continued) TIFF2024516542000060.tif229132TIFF2024516542000061.tif229152TIFF2024516542000062.tif229152TIFF2024516542000063.tif229152TIFF202 4516542000064.tif229156TIFF2024516542000065.tif229152TIFF2024516 542000066.tif229152TIFF2024516542000067.tif229152TIFF20245165420 00068.tif229148TIFF2024516542000069.tif229156TIFF2024516542000070.tif229148TIFF2024516542000071.tif229148TIFF2024516542000072.t if229148TIFF2024516542000073.tif229148TIFF2024516542000074.tif229148TIFF2024516542000075.tif229152TIFF2024516542000076.tif229127

[0273] (Supplementary Table 5) List of the top 100 most recurrent predicted MHC-I neoAgs with higher immunogenicity obtained from computational methods in the discovery set. TIFF2024516542000077.tif24713TIFF2024516542000078.tif247158TIFF2024516542000079.tif247158TIFF20245165420 00080.tif247158TIFF2024516542000081.tif247158TIFF2024516542000082.tif247158TIFF2024516542000083.tif24742

[0274] (Supplementary Table 5) (continued) TIFF2024516542000084.tif24799TIFF2024516542000085.tif247148TIFF2024516542000086.tif247148 TIFF2024516542000087.tif247148TIFF2024516542000088.tif247148TIFF2024516542000089.tif247148 TIFF2024516542000090.tif247148TIFF2024516542000091.tif247148TIFF2024516542000092.tif24714 8TIFF2024516542000093.tif247148TIFF2024516542000094.tif247148TIFF2024516542000095.tif24783

[0275] (Supplementary Table 6) List of the top 100 most immunogenic predicted MHC-II neoAgs obtained from computational methods in the discovery set. TIFF2024516542000096.tif24763TIFF2024516542000097.tif247158TIFF2024516542000098.tif247158 TIFF2024516542000099.tif247158TIFF2024516542000100.tif247158TIFF2024516542000101.tif247150

[0276] (Supplementary Table 6) (continued) TIFF2024516542000102.tif247141TIFF2024516542000103.tif247149TIFF202 4516542000104.tif247149TIFF2024516542000105.tif247153TIFF2024516542 000106.tif247149TIFF2024516542000107.tif247157TIFF2024516542000108. tif247149TIFF2024516542000109.tif247153TIFF2024516542000110.tif24775

[0277] (Supplementary Table 7) List of the top 100 most recurrent predicted MHC-II neoAgs with higher immunogenicity obtained from computational methods in the discovery set. TIFF2024516542000111.tif24771TIFF2024516542000112.tif247158TIFF2024516542000113.tif247158 TIFF2024516542000114.tif247158TIFF2024516542000115.tif247158TIFF2024516542000116.tif247142

[0278] (Supplementary Table 7) (continued) TIFF2024516542000117.tif247141TIFF2024516542000118.tif247153TIFF202 4516542000119.tif247153TIFF2024516542000120.tif247149TIFF2024516542 000121.tif247149TIFF2024516542000122.tif247153TIFF2024516542000123. tif247149TIFF2024516542000124.tif247153TIFF2024516542000125.tif24779

[0279] (Supplementary Table 8) List of the top 100 most immunogenic predicted MHC-I neoAgs with higher immunogenicity obtained from the computational method in the validation set. TIFF2024516542000126.tif24763TIFF2024516542000127.tif247158TIFF2024516542000128.tif247158 TIFF2024516542000129.tif247158TIFF2024516542000130.tif247158TIFF2024516542000131.tif247150

[0280] (Supplementary Table 8) (continued) TIFF2024516542000132.tif247148TIFF2024516542000133.tif247148TIFF2024516542000134.tif24714 8TIFF2024516542000135.tif247148TIFF2024516542000136.tif247148TIFF2024516542000137.tif24714 8TIFF2024516542000138.tif247148TIFF2024516542000139.tif247148TIFF2024516542000140.tif24714 8TIFF2024516542000141.tif247148TIFF2024516542000142.tif247148TIFF2024516542000143.tif24734

[0281] (Supplementary Table 9) List of the top 100 most recurrent predicted MHC-I neoAgs with immunogenicity scores obtained from the computational method in the validation set. TIFF2024516542000144.tif247113TIFF2024516542000145.tif247158TIFF2024516542000146.tif247158 TIFF2024516542000147.tif247158TIFF2024516542000148.tif247158TIFF2024516542000149.tif247100

[0282] (Supplementary Table 9) (continued) TIFF2024516542000150.tif24750TIFF2024516542000151.tif247148TIFF2024516542000152.tif247148 TIFF2024516542000153.tif247148TIFF2024516542000154.tif247148TIFF2024516542000155.tif247148 TIFF2024516542000156.tif247148TIFF2024516542000157.tif247148TIFF2024516542000158.tif247148 TIFF2024516542000159.tif247148TIFF2024516542000160.tif247148TIFF2024516542000161.tif247132

[0283] (Supplementary Table 10) List of the top 100 most immunogenic predicted MHC-II neoAgs with higher immunogenicity obtained from the computational method in the validation set. TIFF2024516542000162.tif24713TIFF2024516542000163.tif247158TIFF2024516542000164.tif247158TIFF20245165420 00165.tif247158TIFF2024516542000166.tif247158TIFF2024516542000167.tif247158TIFF2024516542000168.tif24742

[0284] (Supplementary Table 10) (continued) TIFF2024516542000169.tif247104TIFF2024516542000170.tif247149TIFF202 4516542000171.tif247149TIFF2024516542000172.tif247149TIFF20245165420 00173.tif247149TIFF2024516542000174.tif247157TIFF2024516542000175.t if247149TIFF2024516542000176.tif247157TIFF2024516542000177.tif247120

[0285] (Supplementary Table 11) List of the top 100 most recurrent predicted MHC-II neoAgs with immunogenicity scores obtained from the computational method in the validation set. TIFF2024516542000178.tif24726TIFF2024516542000179.tif247158TIFF2024516542000180.tif247158TIFF20245165420 00181.tif247158TIFF2024516542000182.tif247158TIFF2024516542000183.tif247158TIFF2024516542000184.tif24726

[0286] (Supplementary Table 11) (continued) TIFF2024516542000185.tif247116TIFF2024516542000186.tif247157TIFF202 4516542000187.tif247157TIFF2024516542000188.tif247157TIFF20245165420 00189.tif247153TIFF2024516542000190.tif247157TIFF2024516542000191.t if247149TIFF2024516542000192.tif247153TIFF2024516542000193.tif247145

[0287] (Supplementary Table 12) List of other MHC-I neoAgs with low predicted immunogenicity and low or no repetitiveness obtained from computational methods in the discovery set. TIFF2024516542000194.tif247146TIFF2024516542000195.tif247133

[0288] (Supplementary Table 12) (continued) TIFF2024516542000196.tif24713TIFF2024516542000197.tif247149TIFF2024516542000198.tif247149TIFF2024516542000199.tif24799

[0289] (Supplementary Table 13) Pool of selected neoAgs for in vitro validation of immunogenicity using ELISpot assay TIFF2024516542000200.tif22950TIFF2024516542000201.tif229158TIFF2024 516542000202.tif229158TIFF2024516542000203.tif229158TIFF20245165420 00204.tif229158TIFF2024516542000205.tif229158TIFF2024516542000206.t if229158TIFF2024516542000207.tif229158TIFF2024516542000208.tif229125

[0290] (Supplementary Table 14) List of validated ELISpot reactive peptides TIFF2024516542000209.tif24721TIFF2024516542000210.tif247158TIFF2024516542000211.tif24717

[0291] (Supplementary Table 14) (continued) TIFF2024516542000212.tif247128TIFF2024516542000213.tif247149TIFF2024516542000214.tif24713

[0292] F. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024516542000215.tif209160TIFF2024516542000216.tif231160TIFF2024516542 000217.tif245160TIFF2024516542000218.tif238160TIFF2024516542000219.tif34160

[0293] All methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments or aspects, it will be apparent to those skilled in the art that modifications may be applied to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutes and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims. References and patent applications cited herein are specifically incorporated herein by reference.

Claims

1. A peptide comprising at least 70% sequence identity to (a) one of the peptides of SEQ ID NOs: 24, 77, 117, 415, 10, 323, 221, 44, 27, 156, 37, 168, 20, 163, 29, 136, 62, 138, 157, 160, 151, 158, 23, 39, or 57, or (b) one of the peptides of SEQ ID NOs: 1-776, optionally comprising: (i) the peptide comprises at least 6 consecutive amino acids of one of the peptides of SEQ ID NOs: 24, 77, 117, 415, 1-23, 25-76, 78-116, 118-414, or 416-776; and / or (ii) the peptide is 15 amino acids or less in length, optionally (A) consisting of 9 amino acids, or (B) consisting of 15 amino acids; and / or (iii) the peptide is immunogenic; and / or (iv) the peptide is optionally modified: (A) the modification comprises conjugation with a molecule; and / or (B) the molecule comprises an antibody, a lipid, an adjuvant, or a detection moiety; and / or (v) the peptide has at least 90% sequence identity to one of the peptides of SEQ ID NOs: 24, 77, 117, 415, 1-23, 25-76, 78-116, 118-414, or 416-776; and / or (vi) the peptide is: (A) having one, two, or three substitutions in one peptide of SEQ ID NOs: 24, 77, 117, 415, 1-23, 25-76, 78-116, 118-414, or 416-776; or (B) Contains 100% sequence identity to one peptide of SEQ ID NOs: 24, 77, 117, 415, 1-23, 25-76, 78-116, 118-414, or 416-776; The peptide.

2. 10. A polypeptide comprising the peptide of claim 1, optionally comprising: (a) the polypeptide comprises at least two peptides according to claim 1; and / or (b) the polypeptide comprises a cell-penetrating peptide (CPP), the CPP comprising the Z13 variant of ZEBRA CPP Z12, and optionally the polypeptide comprises, from amino-proximal to carboxy-proximal positions, a cell-penetrating peptide, one or more peptides of claim 1, and a TLR agonist, and optionally the polypeptide further comprises a TLR agonist amino-proximal to the cell-penetrating peptide; and / or (c) the polypeptide further comprises one or more TLR agonists, wherein the TLR agonist comprises a TLR2 agonist, a TLR4 agonist, a TLR2 / 4 agonist, or a combination thereof, and optionally the TLR agonist comprises one or both of extra domain A (EDA) and Anaxa; optionally, the polypeptide comprises, from an amino-proximal position to a carboxy-proximal position, a cell-penetrating peptide, one or more peptides of claim 1, and a TLR agonist; and further optionally, the polypeptide further comprises a TLR agonist at the amino-proximal position of the cell-penetrating peptide. The polypeptide.

3. A molecular complex comprising the peptide of claim 1 and an MHC polypeptide.

4. A pharmaceutical composition comprising: (i) one or more peptides of claim 1, one or more polypeptides of claim 2, or a molecular complex of claim 3; and (ii) a pharmaceutical carrier, optionally comprising: (a) the pharmaceutical composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection; and / or (b) the pharmaceutical composition comprises at least two peptides; and / or (c) the peptide is contained within a liposome, lipid-containing nanoparticle, or lipid-based carrier, and optionally, the pharmaceutical composition is formulated for injection or for inhalation as a nasal spray; and / or (d) the pharmaceutical composition is formulated as a vaccine; and / or (e) the pharmaceutical composition further comprises an adjuvant; The pharmaceutical composition.

5. A nucleic acid encoding the peptide of claim 1 or the polypeptide of claim 2, said nucleic acid optionally being (a) DNA or (b) RNA.

6. 6. An expression vector comprising the nucleic acid of claim 5, optionally comprising an adenovirus backbone, optionally wherein the viral backbone comprises a simian adenovirus backbone.

7. A host cell comprising the nucleic acid of claim 5 or an expression vector comprising said nucleic acid, said host cell optionally comprising a viral packaging cell.

8. A virus produced from the host cell of claim 7.

9. 1. An in vitro dendritic cell comprising a peptide according to claim 1, a nucleic acid encoding the peptide according to claim 1 or the polypeptide according to claim 2, or an expression vector comprising said nucleic acid, optionally comprising: (a) is a mature dendritic cell; and / or (b) cells having HLA-A, HLA-B, or HLA-C types; The dendritic cells.

10. 10. A peptide-specific binding molecule that specifically binds to the peptide of claim 1, the polypeptide of claim 2, or the molecular complex of claim 3, wherein the binding molecule is optionally an antibody, a TCR mimetic antibody, an scFV, a camelid-derived antibody, an aptamer, or a DARPIN.

11. A method for producing a cell, comprising the step of introducing into a cell (i) a nucleic acid encoding the peptide described in claim 1 or the polypeptide described in claim 2, or (ii) an expression vector containing the nucleic acid, said method optionally further comprising the step of isolating the expressed peptide or polypeptide.

12. A method for producing cancer-specific immune effector cells, comprising contacting a starting population of immune effector cells with a peptide of claim 1, a polypeptide of claim 2, or a molecular complex of claim 3, thereby generating peptide-specific immune effector cells, and optionally: (a) the contacting step is further defined as co-culturing the starting population of immune effector cells with antigen-presenting cells (APCs), artificial antigen-presenting cells (aAPCs), or artificial antigen-presenting surfaces (aAPSs), wherein the APCs, aAPCs, or aAPSs present the peptides on their surfaces, and the APCs are dendritic cells; or (b) the immune effector cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, NKT cells, optionally wherein the T cells are: (i) CD8 + T cells, CD4 + T cells, or γδ T cells; or (ii) is a cytotoxic T lymphocyte (CTL); or (c) the immune effector cells are differentiated from mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells; or (d) the starting population of immune effector cells is a starting population of immune effector cells isolated from peripheral blood mononuclear cells (PBMCs); or (e) the starting population of immune effector cells is a starting population of immune effector cells obtained from a subject, wherein the subject is a human, and the subject has cancer, wherein the cancer comprises tumor cells that are positive for expression of the peptide, and optionally, the cancer comprises leukemia, lung cancer, or skin cancer; or (f) the method further comprises the step of introducing the peptide or a nucleic acid encoding the peptide into the dendritic cells prior to co-culture, optionally comprising: (i) the peptide or a nucleic acid encoding the peptide is introduced by electroporation; or (ii) the peptide or a nucleic acid encoding the peptide is introduced by adding the peptide or a nucleic acid encoding the peptide to the culture medium of the dendritic cells; or (iii) the immune effector cells are co-cultured with a second population of dendritic cells transfected with the peptide or the nucleic acid encoding the peptide; or (iv) after co-culture, a population of CD8-positive or CD4-positive peptide-MHC tetramer-positive T cells is purified from the immune effector cells, and optionally: (A) A clonal population of peptide-specific immune effector cells is generated by limiting or serial dilution followed by expansion of individual clones by a rapid expansion protocol, optionally the method further comprising cloning T cell receptors (TCRs) from the clonal population of peptide-specific immune effector cells, and further optionally: (I) the cloning of the TCR is cloning of a TCR alpha chain and a beta chain; and / or (II) the TCR is cloned using rapid amplification of 5'-cDNA ends (RACE), optionally the cloned TCR is subcloned into an expression vector, the expression vector being a retroviral or lentiviral vector, optionally a host cell is transduced with the expression vector to generate an engineered cell that expresses the TCR, optionally the host cell is an immune cell; or (B) Clonal populations of peptide-specific engineered T cells are generated by limiting or serial dilution followed by expansion of individual clones by rapid expansion protocols; or (g) the immune cells are T cells and the engineered cells are engineered T cells, optionally, the T cells are CD8 + T cells, CD4+ T cells, or γδ T cells, and the engineered cells are engineered T cells, optionally: (i) the starting population of immune effector cells is a starting population of immune effector cells obtained from a subject with cancer, and the host cells are allogeneic or autologous to the subject, optionally, the cancer being positive for expression of the peptide; or (ii) purifying a population of CD8-positive or CD4-positive peptide-MHC tetramer-positive engineered T cells from the transduced host cells; The method.

13. 13. A peptide-specific engineered T cell produced according to the method of claim 12.

14. 13. A pharmaceutical composition comprising peptide-specific T cells produced according to the method of claim 12.

15. A pharmaceutical composition comprising the host cell of claim 7.

16. A pharmaceutical composition comprising the virus described in claim 8.

17. A pharmaceutical composition (a) for use in a method of treating or preventing cancer in a subject, or (b) for use in a method of stimulating an immune response in a subject, comprising: (1) the peptide of claim 1; (2) the polypeptide of claim 2; (3) the molecular complex of claim 3; (4) a nucleic acid encoding the peptide of claim 1 or the polypeptide of claim 2; (5) an expression vector comprising said nucleic acid; (6) a virus produced from a host cell comprising said nucleic acid or said expression vector; or (7) a dendritic cell comprising a nucleic acid encoding the peptide of claim 1, the peptide of claim 1, or the polypeptide of claim 2, or an expression vector comprising said nucleic acid. Including, The pharmaceutical composition.

18. The pharmaceutical composition of claim 14 for use in (a) a method for treating or preventing cancer in a subject, or (b) a method for stimulating an immune response in a subject.

19. A method for cloning a peptide-specific T cell receptor (TCR), comprising the steps of: (a) contacting a starting population of immune effector cells with a peptide of claim 1 or a polypeptide of claim 2, thereby generating peptide-specific immune effector cells; (b) purifying immune effector cells specific for the peptide; and (c) isolating TCR sequences from the purified immune effector cells; and optionally, （i）： (A) the contacting step is further defined as co-culturing the starting population of immune effector cells with antigen-presenting cells (APCs), artificial antigen-presenting cells (aAPCs), or artificial antigen-presenting surfaces (aAPSs), wherein the APCs, aAPCs, or aAPSs present the peptides on their surfaces, and optionally, the APCs are dendritic cells; or (B) the immune effector cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, or NKT cells; optionally, the T cells are: (I) CD8 + T cells, CD4 + T cells, or γδ T cells; or (II) is a cytotoxic T lymphocyte (CTL); or (C) the immune effector cells are differentiated from mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells; and / or (ii) the starting population of immune effector cells is a starting population of immune effector cells isolated from peripheral blood mononuclear cells (PBMCs); and / or (iii) the starting population of immune effector cells is a starting population of immune effector cells obtained from a subject, optionally: (A) the subject is a human, optionally the subject has cancer, further optionally the cancer comprises a cancer that is positive for expression of the peptide; and / or (B) the subject: (I) has been diagnosed with cancer; or (II) has not been diagnosed with cancer; and / or (C) the subject has been determined to have Lynch syndrome; and / or (D) the cancer comprises colorectal cancer, optionally wherein the colorectal cancer comprises mismatch repair deficient colorectal cancer (MMR-d) and / or microsatellite instability (MSI) positive colorectal cancer; and / or (E) the subject is being treated for stage I or stage II cancer; and / or (F) the subject has been determined to have mismatch repair-deficient colorectal cancer (MMR-d) and / or microsatellite instability (MSI)-positive colorectal cancer; and / or (G) the cancer (I) includes stage 0, I, II, III, or IV cancer, or (II) excludes stage 0, I, II, III, or IV cancer; The method.

20. 10. A method for detecting a T cell response in a patient, comprising contacting a biological sample from the patient with the peptide of claim 1, the polypeptide of claim 2, or the molecular complex of claim 3, optionally comprising: (a) the biological sample comprises a blood sample or a fraction thereof, the biological sample comprising lymphocytes, and optionally the biological sample comprises a lymphocyte-containing fraction; and / or (b) the peptide is linked to a solid support, optionally: (i) the peptide is conjugated to the solid support or bound to an antibody conjugated to the solid support; or (ii) the solid support comprises a microplate, a bead, a glass surface, a slide, or a cell culture dish; and / or (c) detecting a T cell response comprises detecting binding of the peptide to the T cell or TCR; and / or (d) detecting the T cell response comprises an ELISA, ELISPOT, or tetramer assay; The method.

21. A composition for use in a method for predicting the prognosis of a patient or a method for detecting a T cell response in a patient, comprising the peptide of claim 1, the polypeptide of claim 2, or the molecular complex of claim 3.

22. 10. A composition comprising at least one MHC polypeptide and a peptide according to claim 1, optionally comprising: (a) the MHC polypeptide and / or the peptide is conjugated to a detection tag; and / or (b) the MHC polypeptide and the peptide are operably linked to form a peptide-MHC complex, optionally: (i) the MHC polypeptide and the peptide are operably linked via a peptide bond or by van der Waals forces; and / or (ii) at least two peptide-MHC complexes are operably linked to each other, optionally at least three or four peptide-MHC complexes are operably linked to each other; and / or (c) an average ratio of MHC polypeptide to peptide of 1:1 to 4:1; The composition.

23. 23. A method comprising contacting the composition of claim 22 with a composition comprising T cells, and detecting T cells having bound peptides and / or MHC polypeptides by detecting the detection tag, optionally comprising: (a) the method further comprises the step of counting the number of T cells that have bound the peptide and / or MHC; and / or (b) the composition comprising T cells is a composition comprising T cells isolated from a patient having or suspected of having cancer, optionally comprising: (i) the cancer comprises a peptide-specific cancer; or (ii) the peptide is selected from one of the peptides of SEQ ID NOs: 24, 77, 117, 415, 1-23, 25-76, 78-116, 118-414, or 416-776; and / or (c) the method further comprises sorting T cells that bind the peptide and / or MHC; optionally, the method further comprises sequencing one or more TCR genes from T cells that bind the peptide and / or MHC; and optionally, the method further comprises grouping lymphocyte interactions by paratope hotspot (GLIPH) analysis; The method.

24. A kit comprising the peptide of claim 1 or the polypeptide of claim 2 in a container, optionally comprising: (a) the peptide is contained in a pharmaceutical preparation, optionally, the pharmaceutical preparation is formulated for parenteral administration or inhalation; or (b) the peptide is contained in a cell culture medium; The kit.

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