WO2024173551A2 - Tryptophan neoantigen peptides for diagnostics, therapeutics, and vaccines - Google Patents

Abstract

Provided herein are RNA error derived neoantigen (REDN) peptides, and system and methods of use thereof, wherein the REDN peptides are the result of tryptophan stalling, resulting in tryptophan bump (W-bump REDN peptides). The systems relate to peptide arrays, and the composition and systems are used in methods for identifying neoantigens and for treating and preventing cancer. Also provided herein are methods of diagnosing cancer using W-bump REDN peptides, methods of predicting response to immunotherapy using W-bump REDN peptides, methods of predicting adverse immune responses to immunotherapy using W-bump REDN peptides, methods of treating and preventing cancer using vaccines composed of W-bump REDN peptides, or therapeutic compounds designed to bind W-bump REDN peptides. Also provided are compositions of vaccines that include W-bump REDN peptides and therapeutic compounds that bind W-bump REDN peptides.

Description

TRYPTOPHAN NEOANTIGEN PEPTIDES FOR DIAGNOSTICS, THERAPEUTICS, AND VACCINES

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. This application claims priority to U.S. Patent Application No. 63/485,735, filed February 17, 2023, which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a sequence listing in electronic format. The sequence listing is provided as a file entitled “2024-02- 12_SequenceListing-CALV037WO.xml”, created February 12, 2024, which is 229,957,632 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND

[0003] The present disclosure relates to the field of cancer diagnostics, therapeutics, and vaccines. More particularly, the present disclosure relates to cancer diagnostics, therapeutics, and vaccines related to RNA error derived neoantigen (REDN) peptides.

SUMMARY

[0004] Embodiments provided herein relate to RNA error derived neoantigen (REDN) peptides and methods of using such REDN peptides in diagnostics, therapeutics, and vaccines against cancer. In particular, the disclosure relates to vaccine compositions, therapeutic compositions, methods of diagnosis, and methods of treatment using REDN peptides disclosed herein. More particularly, the REDN peptides provided herein are tryptophan (W) bump REDN peptides (W-bump REDN peptides). Embodiments provided herein relate to REDNs produced at the RNA level used as a rich source for diagnostics, therapeutics, and vaccines.

[0005] Some embodiments provided herein relate to compositions. In some embodiments, the compositions included a plurality of RNA error derived neoantigen (REDN) peptides. In some embodiments, the plurality of REDN peptides include tryptophan bump REDN peptides. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons are less than 8 codons apart. In some embodiments, the plurality of REDN peptides include one or more peptides having a sequence or part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296. In some embodiments, the compositions further includes an adjuvant. In some embodiments, the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex Adjuvant Formulation (SAF), synthetic polynucleotides (poly IC/poly AU), TiterMax tomatine, Vaxfectin, Xtendlll, or zymosan.. In some embodiments, the plurality of REDN peptides include two or more pooled REDN peptides.

[0006] Some embodiments provided herein relate to peptide arrays. In some embodiments, the peptide arrays include a plurality of RNA error derived neoantigen (REDN) peptides. In some embodiments, the plurality of REDN peptides include tryptophan bump FSPs. In some embodiments, the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons are less than 8 codons apart. In some embodiments, the plurality of REDN peptides include one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296. In some embodiments, the plurality of REDN peptides is fixed on a substrate. In some embodiments, the substrate includes glass, silica, composite, resin, or combination thereof. In some embodiments, the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. In some embodiments, the peptide array includes between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides. In some embodiments, the plurality of REDN peptides include two or more pooled REDN peptides. In some embodiments, the REDN peptides are spaced between about 3 and about 9 pm apart. In some embodiments, the array is used to predict a response to an immunotherapy, to predict adverse responses to immunotherapy, to diagnose a cancer, to develop a vaccine, or to develop a therapeutic. In some embodiments, the array is used to detect binding of one or more antibodies against W-bump REDN peptides.

[0007] Some embodiments provided herein relate to therapeutic compounds. In some embodiments, the therapeutic compounds bind to one or more W-bump REDN peptides. In some embodiments, the therapeutic compound is an antibody or a synthetic antibody.

[0008] Some embodiments provided herein relate to methods of treating or preventing a disorder in a subject. In some embodiments, the methods include administering a composition as a vaccine. In some embodiments, the composition is any composition as described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig. In some embodiments, the disorder is a cancer. In some embodiments, the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor.

[0009] Some embodiments described herein relate to methods of detecting a disorder in a subject. In some embodiments, the methods include: (a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein the plurality of REDN peptides include tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array. In some embodiments, the disorder is a cancer. In some embodiments, the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons are less than 8 codons apart. In some embodiments, the plurality of REDN peptides include one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296. In some embodiments, the plurality of REDN peptides is fixed on a substrate. In some embodiments, the substrate include glass, silica, composite, resin, or combination thereof. In some embodiments, the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio- Layer Interferometry, and surface plasmon resonance. In some embodiments, the peptide array includes between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides. In some embodiments, the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof. In some embodiments, the biological sample includes an antibody. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig. In some embodiments, the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T- cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T- cell lymphoma, thyroid cancer, or Wilms' tumor. In some embodiments, the plurality of REDN peptides include two or more pooled REDN peptides. In some embodiments, the detecting the binding of the biological sample to the at least one peptide in the peptide array includes fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, or surface plasmon resonance assay.

[0010] Some embodiments provided herein relate to methods of measuring an immune response to a neoantigen peptide in a subject. In some embodiments, the methods include: (a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides include peptides including tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array. In some embodiments, the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons arc less than 8 codons apart. In some embodiments, the plurality of REDN peptides include one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296. In some embodiments, the plurality of REDN peptides is fixed on a substrate. In some embodiments, the substrate includes glass, silica, composite, resin, or combination thereof. In some embodiments, the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. In some embodiments, the peptide array includes between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides. In some embodiments, the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof. In some embodiments, the biological sample includes an antibody. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig. In some embodiments, the subject has or is suspected of having a cancer. In some embodiments, the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor. In some embodiments, the plurality of REDN peptides include two or more pooled REDN peptides.

[0011] Some embodiments provided herein relate to methods of predicting a response of a subject to an immunotherapy. In some embodiments, the methods include (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides include peptides include tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern prior to immunotherapy.

[0012] Some embodiments provided herein relate to methods of predicting adverse immune responses of a subject to an immunotherapy. In some embodiments, the methods include (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides include peptides including tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern indicative of adverse immune responses.

[0013] Some embodiments provided herein relate to methods of treating a disorder in a subject. In some embodiments, the methods include screening therapeutic compounds that bind to one or more W-bump REDN peptides; and administering a therapeutic compound that binds to one or more W-bump REDN peptides to the subject. In some embodiments, the therapeutic compound is an antibody or a synthetic antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [0015] Figure 1 depicts a comparison of normal translation of mRNA across tryptophan codons versus the situation in a tumor. In a tumor, interferon gamma leads to uncharged tryptophan codons leading to a RNA error derived neoantigen .

[0016] Figure 2 depicts screening of predicted REDN peptides that can result from mis-translation at W-bumps, which have potential in diagnostic, therapeutic, and vaccines.

[0017] Figure 3 illustrates a table depicting W-bump REDN coverage of an initial cohort of cohort of dogs diagnosed with pre-stage 1, stage 1 or stage 2 hemangiosarcoma (HSA) cancer.

DETAILED DESCRIPTION

[0018] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which are shown by way of illustration embodiments that may be practiced. It is to be understood that other embodiments may be utilized, and structural or logical changes may be made without departing from the scope. Therefore, the following detailed description is not to be taken in a limiting sense, and the scope of embodiments is defined by the appended claims and their equivalents.

[0019] Various operations may be described as multiple discrete operations in turn, in a manner that may be helpful in understanding embodiments; however, the order of description should not be construed to imply that these operations are order dependent.

[0020] For the purposes of the description, a phrase in the form “A/B” or in the form “A and/or B” means (A), (B), or (A and B). For the purposes of the description, a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C). For the purposes of the description, a phrase in the form “(A)B” means (B) or (AB) that is, A is an optional element.

DEFINITIONS

[0021] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Tnc., 1995 (ISBN 9780471185710); and other similar references.

[0022] The singular- terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0023] The description may use the terms “embodiment” or “embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments, are synonymous, and are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to”).

[0024] With respect to the use of any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0025] As used herein, the term “array” refers to an arrangement of molecules, such as biological macromolecules (such as peptides), in addressable locations on or in a substrate. A “microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. The array of molecules (“features”) makes it possible to carry out an exceptionally large number of analyses on a sample at one time. Within an array, each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array. The feature application location on an array can assume different shapes. For example, the array can be regular (such as arranged in uniform rows and columns) or irregular. Thus, in ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position. Often, ordered arrays are arranged in a symmetrical grid pattern, but samples can be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters). Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the subject features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer. In some cases, the methods provided herein involve multiplexed arrays in which a plurality of peptides or polypeptides attached to a solid support are contacted to a biological sample (for example, blood or other bodily tissue obtained from a subject).

[0026] In certain embodiments, the peptide array is a plurality of short linear peptides immobilized on a solid surface (for example, a polystyrene or other solid substrate). As used herein, the terms “peptide” and “polypeptide” refer to a polymer in which the monomers are alpha amino acids joined together through amide bonds. Peptides are two or often more amino acid monomers long. Standard abbreviations for amino acids are used herein (see Stryer, 1988, Biochemistry, Third Ed., incorporated herein by reference). In certain embodiments, random- sequence peptide arrays are used. As used herein, the term “substrate” refers to any type of solid support to which the peptides are immobilized. Examples of substrates include, but are not limited to, microarrays; beads; columns; optical fibers; wipes; nitrocellulose; nylon; glass; quartz; diazotized membranes (paper or nylon); silicones; polyformaldehyde; cellulose; cellulose acetate; paper; ceramics; metals; metalloids; semiconductive materials; coated beads; magnetic particles; plastics such as polyethylene, polypropylene, and polystyrene; gel-forming materials; silicates; agarose; polyacrylamides; methylmethacrylate polymers; sol gels; porous polymer hydrogels; nanostructured surfaces; nanotubes (such as carbon nanotubes); and nanoparticles (such as gold nanoparticles or quantum dots). When bound to a substrate, the peptides can be directly linked to the support, or attached to the surface via a linker. Thus, the solid substrate and/or the peptides can be derivatized using methods known in the art to facilitate binding of the peptides to the solid support, so long as the dcrivatization docs not eliminate detection of binding between the peptides and antibodies in the sera.

[0027] As used herein, “in-situ synthesis” refers to synthesis of peptides or polypeptides in situ on an array. This could be done with photoactivatable amino acids as done by Nimble Therapeutics (maskless photolithography), PEPperPRINT, a standard mask-based system (much like Intel uses to lay down circuits), BOC or FMOC peptide chemistry, or other synthesis methods known in the art.

[0028] As used herein, the term “detect,” “detection,” “detectable,” or “detecting” is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative, and qualitative measurements of measuring a cancer, using the methods and compositions as disclosed herein.

[0029] As used herein, the term “mammal” includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects, including dogs.

[0030] As used herein, the expression “a subject in need thereof’ means a human or non-human mammal that exhibits one or more symptoms or indications of cancer, , and/or who has been diagnosed with cancer. A cancer may include a solid tumor and treatment for the same. In many embodiments, the term “subject” may be interchangeably used with the term “patient”. For example, a human subject may be diagnosed with a primary or a metastatic tumor and/or with one or more symptoms or indications including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, swollen abdomen, chest pain/pressure, enlargement of spleen, and elevation in the level of a cancer-related biomarker.

[0031] The term “malignancy” refers to a non-benign tumor or a cancer. As used herein, the term “cancer” refers to the broad class of disorders characterized by hyperproliferative cell growth, either in vitro (for example, transformed cells) or in vivo. Cancers appropriate for treatment with checkpoint inhibitor therapy include without limitation a variety of neoplasms, including benign or malignant tumors, a variety of hyperplasias, and the like. Exemplary cancers include carcinomas, sarcomas, leukemias, and lymphomas. Cancer includes primary malignant tumors (for example, those whose cells have not migrated to sites in the subject’s body other than the site of the original tumor) and secondary malignant tumors (for example, those arising from metastasis, the migration of tumor cells to secondary sites that arc different from the site of the original tumor). Non-limiting examples of cancer may include gastric, myeloid, colon, nasopharyngeal, esophageal, and prostate tumors, glioma, neuroblastoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (for example, adult T-cell leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, myelogenous leukemia, lymphocytic leukemia, acute myelogenous leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), hairy cell leukemia), lymphoma (Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma (NHL)), multiple myeloma, bladder, renal, gastric (for example, gastrointestinal stromal tumors (GIST)), liver, melanoma and pancreatic cancer, sarcoma, adenocarcinoma, astrocytoma, bone cancer, brain tumor, Burkitt’s lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin’s lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, pancreatic cancer, pituitary tumor, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, and Wilms’ tumor.

[0032] As used herein the term “frameshift mutation” is a mutation causing a change in the frame of the protein. Thus, a frameshift variant peptide is a peptide in which a frame has changed due to a frameshift mutation.

[0033] As used herein the term “RNA error derived neoantigen (REDN)” refers to a neoantigen that results from an RNA error, for example a frameshift, RNA mis-processing (including RNA mis- splicing), and RNA mis-translation.

[0034] A “W-bump” REDN peptide refers to a REDN peptide that is a result of tryptophan stalling. In some embodiments, the W-bump frameshift peptides include any one or more of the more than 66,000 REDN peptides mentioned by Bartok. In some embodiments, the W-bump REDN peptides include any one or more of the 177,361 REDN peptides as generated and disclosed herein (SEQ ID NOs: 1-177,361). In some embodiments, an array is prepared having one or more REDN peptides immobilized thereon having a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, a signature is detected on the array.

[0035] As used herein, the term “sample” means non-biological samples and biological samples. Non-biological samples include those prepared in vitro including varying concentrations of a target molecule of interest in solution. Biological samples include, without limitation, blood, lymph, urine, saliva, sputum, other bodily secretions, cells, and tissue specimens and dilutions of them. Any suitable biological sample can be used. For example, a biological sample can be a specimen obtained from a subject {for example, a mammal such as a human, canine, mouse, rat, pig, guinea pig, cow, monkey, or ape) or can be derived from such a subject. A subject can provide a plurality of biological sample, including a solid biological sample, from for example, a biopsy or a tissue. In some cases, a sample can be a tissue section or cells that are placed in or adapted to tissue culture. A biological sample also can be a biological fluid such as urine, blood, plasma, serum, saliva, tears, or mucus, or such a sample absorbed onto a paper or polymer substrate. A biological sample can be further fractionated, if desired, to a fraction containing particular cell types. In some embodiments, a sample can be a combination of samples from a subject (for example, a combination of a tissue and fluid sample). In some cases, sera are obtained from the individual using techniques known in the art. In some embodiments, a subject can, for example, use a “fingerstick”, or “fingerprick” to draw a small quantity of blood and add it to a surface, such as a filter paper or other absorbent source, or in a vial or container and optionally dried. A biological sample obtained, for example, from a drop of a subject’s blood and placed on a filter paper can be directly mailed to a provider of the methods of the invention without a processing of the sample. A biological sample provided by a subject can be concentrated or dilute.

[0036] As used herein, the term “contacting” includes placement in direct physical association, including solid or liquid forms. As used herein, “binding” refers to an association between two substances or molecules, such as the association of an antibody with a peptide. Binding can be detected by any procedure known to one skilled in the ail, such as by physical or functional properties of the formed complexes, such as a target/antibody complex.

[0037] As used herein, the term “control” means a sample or standard used for comparison with an experimental sample, such as a tumor sample obtained from a patient with a particular type of cancer. The control can be a sample obtained from a healthy patient or a non-tumor tissue sample obtained from a patient diagnosed with a particular type of cancer. A control can also be a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of cancer patients with poor prognosis, or group of samples that represent baseline or normal values). A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

[0038] The methods provided herein are sensitive and involve small quantities of biological samples from a subject. In some embodiments, biological samples from a subject are too concentrated and require a dilution prior to being contacted with an array of the invention. A plurality of dilutions can be applied to a biological sample prior to contacting the sample with an array of the invention. A dilution can be a serial dilution, which can result in a geometric progression of the concentration in a logarithmic fashion. For example, a ten-fold serial dilution can be 1 M, 0.01 M, 0.001 M, and a geometric progression thereof. A dilution can be, for example, a one-fold dilution, a two-fold dilution, a three-fold dilution, a four-fold dilution, a five-fold dilution, a six-fold dilution, a seven-fold dilution, an eight-fold dilution, a nine-fold dilution, a ten-fold dilution, a sixteen-fold dilution, a twenty-five-fold dilution, a thirty -two-fold dilution, a sixty-four-fold dilution, and/or a one-hundred-and-twenty-five-fold dilution.

[0039] The binding of a molecule to an array in accordance with certain embodiments of the methodology disclosed herein creates a pattern of binding that can be associated with a condition. The affinity of binding of a molecule to a peptide in the array can be mathematically associated with a condition. The off-target binding pattern of an antibody to a plurality of different peptides of the invention can be mathematically associated with a condition. The avidity of binding of a molecule to a plurality of different peptides can be mathematically associated with a condition. [0040] The peptide array can be contacted with the biological sample (for example, sera) under any suitable conditions to promote binding of antibodies in the sample to peptides immobilized on the array. Thus, the methods presented herein are not limited by any specific type of binding conditions employed. Such conditions will vary depending on the array being used, the type of substrate, the density of the peptides arrayed on the substrate, desired stringency of the binding interaction, and nature of the competing materials in the binding solution. In certain embodiments, the conditions include a step to remove unbound antibodies from the addressable array.

[0041] Similarly, any suitable detection technique can be used in the methods provided herein to detect binding of antibodies (“antibody reactivity”) in the biological sample to peptides on the array. Such reactivity may be measured or estimated in any operable way, such as, for example, by ELISA or by microarray assay. In one embodiment, any type of detectable label can be used to label peptides on the array, including but not limited to radioisotope labels, fluorescent labels, luminescent labels, and electrochemical labels (for example, ligand labels with different electrode mid-point potential, where detection includes detecting electric potential of the label). Alternatively, bound antibodies can be detected, for example, using a detectably labeled secondary antibody.

[0042] Immunogenicity, as used herein, refers to the ability of a substance, such as a peptide, to elicit an immune response, such as an antibody response or a T cell response, when administered to an individual, for example, in a vaccine formulation. As used herein, “immune response” means a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. For individuals with cancer, it is the immune response to the tumor. In some embodiments, a peptide that reacts with an antibody or elicits T cell activity in a biological sample from an individual is not immunogenic when administered in a vaccine formulation. In some embodiments, a peptide that reacts with an antibody or elicits T cell activity in a biological sample from an individual is immunogenic when administered in a vaccine formulation. Immunogenicity is determined by methods of those of skill in the art including in animal model testing and using in silico prediction of immunogenicity. In silico immunogenicity prediction tools are available for free to the public, for example at the Immune Epitope Database and Analysis Resource (www.iedb.org). As used herein, an immunogenic peptide is a peptide which includes an allele- specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response (for example, antibody production) against the antigen from which the immunogenic peptide is derived.

[0043] Immunogenic peptides include synthetic embodiments of peptides described herein. In addition, analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants (homologs) of these proteins can be utilized in the methods described herein. Each polypeptide of this disclosure is made of a sequence of amino acids, which may be either L- and/or D-amino acids, naturally occurring and otherwise.

[0044] Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, can be provided in the form of a salt of a pharmaceutically acceptable cation or esterified to form a Cl -Cl 6 ester, or converted to an amide of formula NR1R2 wherein R1 and R2 are each independently H or Cl -Cl 6 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups of the peptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or can be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.

[0045] Hydroxyl groups of the peptide side chains may be converted to Cl -Cl 6 alkoxy or to a C1-C16 ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability.

[0046] Peptidomimetic and organomimetic embodiments are envisioned, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of an immunogenic Brachyury polypeptide having measurable or enhanced ability to generate an immune response. For computer modeling applications, a pharmacophore is an idealized three- dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, “Computer- Assisted Modeling of Drugs,” in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interphann Press: Buffalo Grove, Ill., pp. 165-174 and Principles of Pharmacology, Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Also included are mimetics prepared using such techniques.

[0047] As used herein, the term “diagnose” or “diagnostic” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased subjects who test positive (percent of true positives). The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. “Prognostic” means predicting the probability of development (for example, severity) of a pathologic condition.

[0048] As used herein, the term “treat” or “treatment” refers to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. For example, a disclosed method for reducing the effects of a cancer is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease (for example, tumor size) in a subject with the disease when compared to native levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is also understood and contemplated herein that treatment can refer to any reduction in the progression of a disease or cancer. Thus, for example, methods of reducing the effects of a cancer are considered to be a treatment if there is a 10% reduction in the tumor growth rate relative to a control subject or tumor growth rates in the same subject prior to the treatment. It is understood that the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0049] As used herein the term “immunotherapeutic,” “immunotherapy,” or “IT” refers to a compound that is used to, in this case, treat cancer by inducing, enhancing, or suppressing the immune response. Immunotherapeutic s encompass immune checkpoint inhibitors, antibody-drug conjugates (ADCs), monoclonal antibodies, T-cell therapy, small molecules, and bispecific antibodies (bsAbs). Antibody-drug conjugates include monoclonal antibodies linked to biologically active drugs to combine the targeting ability of antibodies as well as the cytotoxic ability of the drug. T-cell therapy involves reprogramming a patient’s own immune T cells to attack tumors. One type of well-known T-cell therapy includes adoptive transfer of chimeric antigen receptor (CAR) T-cells. As used herein, the term “chimeric antigen receptor” refers to a fusion protein of the membrane or intracellular signaling region of T-cell activating proteins (for example, CD3-zeta chain, CD28, 41BBL, 0X40, ICOS, high-affinity receptor for IgE (FcsRI) and other T-cell activating proteins) and the antigen-binding site (for example, a single-chain Fv fragment) of a cancer antigen- specific antibody. Bispecific antibodies are recombinant proteins that can bind to two different types of antigens at the same time. For example, a bsAb can be engineered to bind a cytotoxic cell and a target tumor cell. That way, the bsAb brings the cytotoxic cell and the target tumor cell into close proximity and facilitates tumor treatment.

[0050] Immune checkpoint inhibitor therapy (CPI or ICI) is a form of cancer immunotherapy. The therapy targets immune checkpoints, key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function. Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD- 1 is the transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cell surface binds to PD1 on an immune cell surface, which inhibits immune cell activity. Among PD-L1 functions is a key regulatory role on T cell activities. It appears that (cancer mediated) upregulation of PD-L1 on the cell surface may inhibit T cells that might otherwise attack. Antibodies that bind to cither PD-1 or PD-L1 and therefore block the interaction may allow the T cells to attack the tumor.

[0051] Immune checkpoint inhibitors, such as anti-PD-1 antibodies, have been approved to treat different types of cancer (for example, bladder, lung, kidney, melanoma, head, neck, Hodgkin’s lymphoma, and solid tumors). PD-1 inhibitors include nivolumab, pembrolizumab, cemiplimab and spartalizumab. Additional CPIs include CTLA-4 blockage (for example, ipilimumab, such as for treatment of melanoma) and PD-LI inhibitors (for example, atezolizumab, avelumab, or durvalumab, such as for treatment of bladder cancer). Remarkably, the FDA for the first time gave tumor-type, agnostic approval to treat any latestage cancer that is MSI-H. This was based on the remarkably positive responses to treatment of not only cancers with frequent MSI-H phenotypes (colon, endometrial and stomach), but rare MSI-H patients in other cancers. For example, a woman with triple negative, metastatic breast cancer who was MSI-H had a complete remission, while most breast cancers have been unresponsive to CPI treatment.

[0052] Examples of immunotherapeutic include Tremelimumab (CTLA-4 blocking antibody), 0X40 agonists (for example, agonist antibodies), antibodies to B7 ligands (for example, anti-B7-Hl, anti-B7-H3, anti-B7-H3, anti-B7-H4), durvalumab (MEDI4736, anti-PD-Ll antibody), MK-3475 (PD-1 blocker), Nivolumab (anti-PD-1 antibody), Pembrolizumab (anti-PD-1 antibody), Pidilizumab/CT-Oi 1, BY55 monoclonal antibody, AMP224 (anti-PD-Ll antibody), BMS- 936559 (anti-PD-Ll antibody), MPLDL3280A (anti- PD-Ll antibody), MSB0010718C (anti-PD-Ll antibody), and Yervoy/ipilimumab (anti- CTLA-4 checkpoint inhibitor). Many new inhibitor targets are being investigated. In some cases, IT treatment includes a combination therapy in which two or more immunotherapeutic s are administered.

[0053] As used herein the terms “checkpoint inhibitor” and “checkpoint pathway inhibitor” are used interchangeably and refer to negative regulatory molecules, usually antibodies, that block or inhibit anti-T cell anti-tumor function to enhance tumor killing. Checkpoint inhibitors include, without limitation, CTLA-4, PD-LI, PD-L2, PD-1, B7-H3, B7- H4, BTLA, HVEM, TEVI3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and a B- 7 family ligand such as B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7- H3, B7-H4, B7-H5, B7-H6 and B7-H7 (or any combination thereof), or a combination thereof (for example, a combination of CTLA-4 and PD-L1 or PD-L2).

[0054] As used herein, the term “adverse event, “adverse reaction,” “adverse response,” “adverse immune response,” “side effect” or “side effects” refers to the unacceptable or undesirable adverse symptoms resulting from or associated with the administration of a particular treatment such as an immunotherapeutic therapy. Side effects specifically to immunotherapeutic s are termed “immune related adverse events” (irAE). While side effects vary by the type of therapy, common side effects of immunotherapeutic therapies include, without limitation fatigue, infusion related reactions, dermatological toxicity, diarrhea/colitis, hepatotoxicity, pneumonitis, hyper- and hypothyroidism. For review, see for example, uptodate.com/contents/patient-selection-criteria-and-toxicities-associated-with- checkpoint-inhibitor-immunotherapy on the World Wide Web. Immune-related adverse events are generally graded from 1-4. Grades 3 and 4 are considered serious and can require immunosuppression treatment. Patients with irAE are just as likely to have a positive response to treatment. Occurrence of Grade 3 or 4 event can prohibit the patient from further immunotherapeutic therapy. Therefore, knowing ahead of time which patients are more likely to have an event would allow closer monitoring to pre-empt a Grade 3 or 4 event.

[0055] Any appropriate criteria can be used to confirm a subject’s responsiveness to treatment with an immunotherapeutic. For example, in certain embodiments, responsiveness to treatment by an immunotherapeutic is measured by at least one criterion selected from the group consisting of clinical benefit rate, survival until mortality, pathological complete response, semi-quantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST criteria.

[0056] The methods described herein can be carried out using a computer programmed to receive data (for example, data from a W-bump REDN peptide array indicating whether a subject has a binding signature associated with cancer, associated with responsiveness to immunotherapeutic therapy, or associated with adverse reactions to immunotherapeutic therapy). The computer can output for display information related to a subject’s biomarkers, and the likelihood of the duration of time that the subject will be responsive to an immunotherapeutic therapy, suffer a side-effect, or the prognosis of survival.

[0057] After information regarding a subject’s biomarkers is reported, a professional can take one or more actions that can affect patient care (for example, administer a new treatment or modify an existing treatment). For example, a medical professional can record the information in a subject’s medical record and/or in an electronic database. In some cases, a medical professional can record that the subject is likely or not likely to respond to an immunotherapeutic therapy, or otherwise transform the patient’s medical record, to reflect the patient’s medical condition. In some cases, a medical professional can review and evaluate a patient’ s medical record, and can assess multiple treatment strategies for clinical intervention of a patient’s condition. The signature may indicate watchfulness or pre-treatment for a sideeffect or recommendation for a different treatment.

[0058] A professional (for example, medical professional) can communicate information regarding biomarker analysis to a subject or a subject’s family. In some cases, a professional can provide a subject and/or a subject’s family with information regarding an immunotherapeutic therapy, including treatment options and potential side effects. In some cases, a professional can provide a copy of a subject’s medical records to communicate information regarding biomarker analysis and/or disease states to a specialist.

[0059] A professional (for example, research professional) can apply information regarding a subject’s biomarkers to advance research into immunotherapeutic therapy. For example, a researcher can compile data on the presence of a particular signature with information regarding the efficacy of an immunotherapeutic therapy, or side effects associated with an immunotherapeutic therapy. In some cases, a research professional can obtain a subject’ s biomarker information to evaluate the subject’s enrollment, or continued participation in a research study or clinical trial. In some cases, a research professional can communicate a subject’ s biomarker information to a medical professional, or can refer a subject to a medical professional for clinical assessment and/or treatment.

[0060] Any appropriate method can be used to communicate information to another person (for example, a professional), and information can be communicated directly or indirectly. For example, a laboratory technician can input biomarker information or cancer diagnosis information into a computer-based record. In some cases, information can be communicated by making a physical alteration to medical or research records. For example, a medical professional can make a permanent notation or flag a medical record for communicating information to other medical professionals reviewing the record. Any type of communication can be used (for example, mail, e-mail, telephone, and face-to-face interactions). Information also can be communicated to a professional by making that information electronically available to the professional. For example, information can be placed on a computer database such that a medical professional can access the information. In addition, information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.

[0061] As used herein, the term “vaccine” means a composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen- specific immune response to an antigen of a pathogen, for example, a bacterial or viral pathogen, or to a cellular constituent correlated with a pathological condition, such as cancer. A vaccine may include a polynucleotide, a peptide or polypeptide, a virus, a bacterium, a cell or one or more cellular constituents. In some cases, the virus, bacteria or cell may be inactivated or attenuated to prevent or reduce the likelihood of infection, while maintaining the immunogenicity of the vaccine constituent. The immunogenic material may include live-attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them. In some cases, the vaccine is a subunit vaccine, which is an immunizing agent that has been treated to remove traces of nucleic acid (such as viral nucleic acid) so that only protein subunits remain. The subunits have less risk of causing adverse reactions. The vaccine can also be a live vaccine, which is a vaccine prepared from living attenuated organisms or from viruses that have been attenuated but can still replicate in the cells of the host organism. The immunogenic material for a cancer vaccine may include, for example, a protein or peptide expressed by a tumor or cancer cell. Vaccines may elicit both prophylactic (preventative) and therapeutic responses.

[0062] As used herein, the term “vector” means a virus, bacterium, or other microbe, or a nucleic acid, used to deliver an antigen or a gene for an antigen, as part of a vaccine. A nucleic acid vector is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

[0063] As used herein, the term “nucleic acid” refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral- methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (for example, A, T, G, C), this also includes an RNA sequence (for example, A, U, G, C) in which “U” replaces “T.” ’’Nucleotide” includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0064] Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single- stranded nucleotide sequence is the 5 ‘-end; the left-hand direction of a double- stranded nucleotide sequence is referred to as the 5’-direction. The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5’ to the 5’- end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3’ end of the coding RNA transcript are referred to as ’’downstream sequences.” “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0065] As used herein, “encode(s)” or “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0066] ‘Recombinant nucleic acid” refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors including an amplified or assembled nucleic acid, which can be used to transform a suitable host cell. A host cell that includes the recombinant nucleic acid is referred to as a ’’recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, such as a “recombinant polypeptide.” A recombinant nucleic acid may serve a non-coding function (such as a promoter, origin of replication, ribosome-binding site) as well.

[0067] As used herein, the term “adjuvant” means a vehicle used to enhance antigenicity; such as a suspension of minerals (alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in- oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Adjuvants also include immuno stimulatory molecules, such as cytokines, costimulatory molecules, and for example, immuno stimulatory DNA or RNA molecules. [0068] The composition also can be formulated to contain an adjuvant in order to enhance the immunological response. Suitable adjuvants include, but arc not limited to, lysolecithin, pluronic polyols, polyanions, other peptides, oil emulsions, and potentially useful human adjuvants such as Bacillus Calmette Guerin (BCG) and Corynebacterium parvum. Adjuvants for inclusion in the inventive composition desirably are safe, well tolerated, such as QS-21 , Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-1 , GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59 (as described in, for example, Kim et al., Vaccine, 18: 597 (2000)). Other adjuvants that can be administered to a mammal include lectins, growth factors, cytokines, and lymphokines (for example, alpha- interferon, gamma-interferon, platelet derived growth factor (PDGF), gCSF, gMCSF, TNF, epidermal growth factor (EGF), IL- 1, IL-2, IL- 4, IL-6, IL-8, IL- 10, and IL- 12), ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex Adjuvant Formulation (SAF), synthetic polynucleotides (poly IC/poly AU), TiterMax tomatine, Vaxfectin, Xtendlll, and zymosan.

[0069] Cosolvents may be added to a composition or formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. [0070] Supplementary compounds (for example, preservatives, antioxidants, antimicrobial agents including biocides and bio stats such as antibacterial, antiviral, and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions may therefore include preservatives, antioxidants, and antimicrobial agents.

[0071] Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.

[0072] In certain embodiments, the methods and compositions disclosed herein may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers of use are conventional. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19^th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0073] Any route of administration can be used to deliver the vaccine composition to the subject. Indeed, although more than one route can be used to administer the composition, a particular route can provide a more immediate and more effective reaction than another route. Exemplary routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, ophthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.

[0074] Formulations suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable, or synthetic oils.

[0075] In some examples, the composition is administered via intramuscular injection, for example, using a syringe or needleless delivery device. In this respect, this disclosure also provides a syringe or a needleless delivery device including the composition. The composition also can be applied or instilled into body cavities, absorbed through the skin (for example, via a transdermal patch), inhaled, ingested, topically applied to tissue, or administered parenterally via, for instance, intravenous, peritoneal, or intraarterial administration.

[0076] The composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant. Implants, such as an implantable device, for example, a mechanical reservoir or an implant or a device made of a polymeric composition, are particularly useful for administration of the composition. The composition also can be administered in the form of a sustained-release formulation including, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl- terephthalate (BHET), and/or a polylactic-glycolic acid. It can also be administered using a gene gun via microparticles.

[0077] The dose of the composition administered will depend on a number of factors, including the size of a target tissue, the extent of any side-effects, the particular route of administration, and the like. The dose ideally includes an “effective amount” of the composition, for example, a dose of composition, which provokes a desired immune response in the subject. As used herein, the term “effective amount” includes an amount of agent, such as an agent that is sufficient to generate a desired response, such an immune response. In some examples, an “effective amount” is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease, for example to treat and/or prevent cancer in a subject. In one example, an effective amount is a therapeutically effective amount. In one example, an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with cancer. The desired immune response can entail production of antibodies, protection upon subsequent challenge, immune tolerance, immune cell activation, and the like. One dose or multiple doses of the composition can be administered to a mammal to elicit an immune response with desired characteristics, including the production of specific antibodies, or the production of functional T cells.

[0078] Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms/disorder are/is affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counter indications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0079] Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 mg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. Following administration of a disclosed composition, such as a vaccine or an antibody, for treating, inhibiting, or preventing a cancer, the efficacy of the therapy or prophylaxis can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the ail will understand that a composition, such as a vaccine or an antibody, disclosed herein is efficacious in treating, inhibiting, or preventing a cancer in a subject by observing that the composition reduces tumor growth or prevents a further increase in tumor size.

[0080] As used herein, “inhibit,” “inhibiting,” and “inhibition” refer to decreasing an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0081] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term “antibody.” Antibodies may be chosen for their ability to interact with tumor- associated W-bump REDN peptides or other novopeptides or targets of interest, and in embodiments may be used such that tumor growth is inhibited. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities may be tested according to known clinical testing methods.

[0082] As used herein, the term “antibody” encompasses, but is not limited to, whole immunoglobulin (for example, an intact antibody) of any class. Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), for example, IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0083] The term “variable” is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each include four FR regions, largely adopting a beta sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0084] As used herein, the term “antibody” or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab, sFv, scFv, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain FS 1-78, FS 6-21, FS SMC1 A binding activity are included within the meaning of the term “antibody or fragment thereof’ Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0085] Also included within the meaning of “antibody or fragments thereof’ are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

[0086] The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site- specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M J. curr. Opin. Biotechnol. 3:348-354, 1992).

[0087] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sd. USA, 81 :6851-6855 (1984)).

[0088] Administration of the antibodies can be carried out as disclosed herein. Nucleic acid approaches for antibody delivery also exist. Broadly neutralizing antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (for example, DNA or RNA) that encodes the antibody or antibody fragment, such that the patient’s or subject’s own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of the nucleic acid can be by any operable means, such as, for example, those disclosed herein.

[0089] Some embodiments provided herein relate to methods and compositions for preventing, treating, and diagnosing cancer, and predicting response to immunotherapies. The methods and compositions provided herein relate to tryptophan bump (W-bump) REDN peptides.

Compositions and Associated Methods of Manufacture

[0090] RNA error derived neoantigen (REDN) generated by RNA mis-processing, mis-transcription, or mis-translation in tumors are a rich source for creating cancer diagnostics, therapeutics, and vaccines. These REDN peptides are highly immunogenic, resulting in generation of antibodies and cellular responses against such REDN peptides in a subject. REDN errors are recurrent in different tumors and different patients, unlike most neoantigens produced from mutations in DNA that are personal. The raising of antibodies and the recurrence are the basis of creating diagnostics for cancer and predicting responses to treatments. Antibodies to the REDN peptide can be used as therapeutics to treat cancer. The REDN peptides themselves can be used as vaccines to treat, or to prevent cancer.

[0091] Accordingly, provided herein are tryptophan (W) bump REDN peptides (W-bump REDN peptides). The W-bump REDNs may be used to diagnose cancer , treat cancer with vaccine, or prevent cancer with a vaccine.

[0092] The success of checkpoint inhibitors in cancer therapy is largely attributed to activating the patient’s immune response to their tumor’s neoantigens arising from DNA mutations. Personal vaccines against cancer can be developed by sequencing the patient’s tumor DNA to identify neoantigens. Embodiments provided herein relate to an additional, unrecognized source of tumor ncoantigcns, W-Bump REDN peptides.

[0093] The least explored and potentially most useful subclass of tumor specific antigens is arguably that caused by RNA errors, resulting in RNA error derived neoantigens (REDNs). One of the consequences of transformation from a normal cell to a cancer cell is that DNA replication and RNA processing become more error prone while DNA repair becomes less robust. This causes an increase in the frequency of RNA-error derived mutations or valiants, such as frameshift mutations arising during transcription, where 1 or 2 (or other nonmultiple of three) new bases are inserted into or deleted from a gene. When such frameshift mutations occur in the coding regions of proteins, the resulting shift in reading frames gives rise to the synthesis of truncated genes that have lost their function. On average at least 20% of the FS variants would encode a new peptide of 9 or more amino acids. Since at least 8 amino acids are required to bind in the MHC I pocket for presentation to T cells (for example, 8, 9, 10, or 11 residues), many of the frameshift valiants could be presented. It will be seen that even short frameshift variants will present new 9-residue peptides by virtue of the fusion of wild-type and frameshift sequences.

[0094] Mutated proteins associated with frameshifts tend to be highly immunogenic and are expressed predominantly (if not exclusively) in tumor cells, making frameshift REDNs ideal vaccine candidates. In addition to frameshifts, an insertion or deletion of a nucleic acid sequence that is a multiple of three will produce an in-frame deletion or insertion. These will also lead to the production of novopeptides since the junction points will be new peptide sequence. Relative to oncogenesis, there are two classes of mutated proteins to consider, whether produced by frameshifts or other mechanisms: the first class, “oncogenic- related variants,” are those that result in or contribute to tumor formation or progression. The second class, “bystander variants,” are those that are not involved in oncogenesis but that happen to be altered simply because the cellular machinery is operating inefficiently. From the point of view of developing a vaccine, both are viable as vaccine candidates.

[0095] Previous studies by the inventors reveal that mis-processing of RNA produces another source of REDNs that are more frequent and recurrent across tumors and patients. These studies demonstrated REDN peptides produced by exon-mis-splicing, insertions and deletions (INDELs) introduced in transcription of coding microsatellites, and mis-initiation of translation at the exon Is.

[0096] A recent report set forth another way in which tumors create REDN peptides (Bartok, Nature, 590, 2021) in processing RNA. Previously, it was known that many tumors use the amino acid tryptophan to generate kynurenine, an inhibitor of T-cell function, which allows the tumor to escape T-cell killing. This process results in uncharged tRNAs, including uncharged tryptophan (W) tRNAs, which can cause ribosome stalling at W codons and especially at sites on mRNA having two closely spaced W codons (UGG). The ribosome can escape stalling by shifting frame, thereby generating an REDN peptide (Figure 1). Bartok termed these REDN peptide at W codons “W-bump” REDN peptides. W-bump REDN peptides are presented on a tumor cell and may be recognized by T-cells, which then kill the tumor cell. At least some of these W-bump REDN peptides are recurrently produced in different patients, though the numbers assayed were low. Bartok reported approximately 66,000 sequences identifies that could potentially be produced by this process.

[0097] Based on the research provided in Bartok, the present disclosure extends further to provide herein compositions and methods that utilize these W-bump REDN peptides in cancer vaccines, diagnostics, and treatment. In some embodiments, the W-bump REDN peptides are used in combination with other antigens, including, for example, REDN peptide antigens produced by other RNA processing errors. In some embodiments, REDN peptide vaccines in dogs reduce deaths from cancer. In some embodiments, REDN peptide vaccines are used to reduce the size of a tumor.

[0098] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular' carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor. In some embodiments, the plurality of REDN peptides include two or more pooled REDN peptides.

[0099] A process for making a composition for treating cancer may begin by identifying one or more W-bump REDN peptide sequences. By informatic analysis, all REDN peptides that may be produced by switching frames at W codons were determined herein. A peptide array of all possible predicted W-bump REDN peptides was designed. All possible REDN peptides downstream of W codons 10 aa or longer were bioinformatically determined. W-bump REDN peptides may be bioinformatically determined by identifying sequences that include two W codons within 8 amino acids of one another and are 10 aa or longer. All possible REDN peptides may include peptides with a (-2), (-1), (+1), or (+2) frameshift. Of note, the ribosome is much more likely to make an insertion (+1 REDN peptide) than a deletion (-1 REDN peptide) at these sites. In one example, 177,194 such peptides were identified, as set forth in the accompanying sequence listing (SEQ ID NOs: 1-177,194). The restriction of the peptide being longer than 10 amino acids for both sources of REDN peptides was applied. Each REDN peptide antigen that was longer than 15 aa was divided into 15 aa, non-overlapping peptides. Peptides that shared more than 10 aa identical sequences with any human reference proteins were excluded to avoid autoimmunity as a vaccine. Thus, W-bump REDN peptide sequences may be determined by using sequence information from publicly available human genome databases, and applying the informatic analysis described herein. All such sequences are incorporated herein by reference. Such sequences are described in the sequence listing.

[0100] Using the identified sequences of possible W-bump REDN peptides, a REDN peptide array may be prepared and processed to determine an IgG signal of each array protocols known to those of skill in the art. A peptide array that includes all of the identified W-bump REDN peptides may be created to identify W-bump REDN peptides as potential neoantigens for inclusion in an anti-cancer therapeutic composition. (Figure 2). A biological sample may be obtained from a subject and contacted with the REDN peptide array. The sample may be blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof. The REDN peptide array may be analyzed to identify W-bump REDN peptides that recurrently bind with antibodies in the biological sample. [0101] Each array may be normalized to its median florescence for analysis. Reactivity may be compared between the biological sample compared to a non-canccr control(s). Total reactivity on the arrays can be evaluated using at least two methods. The first method includes comparing the number of significant peptides in the biological sample and control samples using fold change and p- values. The second method uses a scoring method for each FS peptide. A peptide was scored as positive (red) if it was higher than six times the standard deviation (6SD) from the mean value of the control sample for the same peptide. W- bump peptides meeting these criteria may be forwarded for use in diagnostics, therapeutics, and vaccines.

[0102] In some embodiments, the methods include contacting a W-bump REDN peptide array including a plurality of W-bump REDN peptides with a first biological sample obtained from a first individual known to have a cancer, measuring binding of the first biological sample to the W-bump REDN peptide array, contacting the same type of W-bump REDN peptide array with a second biological sample obtained from a second individual, measuring binding of the second biological sample to the W-bump REDN peptide array, comparing the binding of the two biological samples to the W-bump REDN peptide array, and identifying one or more W-bump REDN peptides that are immunogenic, cancer-specific, and/or shared across cancers.

[0103] As used herein, the term “same type of W-bump REDN peptide array” refers to two or more W-bump FSP peptide arrays that include the same W-bump REDN peptides. The same type of W-bump REDN peptide array may be the same physical unit, or it may be a different physical unit with the same W-bump REDN peptides included on the array.

[0104] In some embodiments, measuring binding includes detecting antibody reactivity to the plurality of W-bump REDN peptides. In some embodiments, the second individual is a control individual without cancer. Comparison to this control screens out W- bump REDN peptides in which antibody reactivity is normal and unrelated to cancer. In some embodiments, the second individual is also known to have a cancer. In some embodiments, the second individual is known to have the cancer of the first individual. Use of a second individual with the same cancer as the first individual may provide confirmation that a “shared” W-bump REDN peptides (for example, a W-bump REDN peptide that is bound by samples of both the first and second individual) is common to a that particular type of cancer. In some embodiments, the second individual and the first individual have different types of cancer. In such an embodiment, comparison of binding between the first individual and the second individual may identify W-bump REDN peptides that are disease- specific because they are not shared by individuals with different cancers and/or may identify W-bump REDN peptides that are shared across multiple diseases when binding is observed with both the first and second individuals’ biological samples. These methods may be combined in that the binding of a first individual may be compared to a second individual who is a control with no cancer, to a third individual who is known to have cancerof the same type as the first individual, and/or a fourth individual who is known to have a different type of cancer. In some embodiments, comparison between groups is quantitative and/or qualitative. In some embodiments, each group (no disease, same disease, different disease) are represented by multiple members.

[0105] In some embodiments, the methods further include determining a nucleic acid sequence of said W-bump REDN peptides to identify one or more nucleic acids encoding W-bump REDN peptides that are immunogenic, cancer- specific, and/or shared across cancers. In some embodiments, determining a nucleic acid sequence of said W-bump REDN peptides includes identifying one or more W-bump REDN peptides that are immunogenic, cancerspecific, and/or shared across cancers, identifying the amino acid sequence of a W-bump REDN peptide, and then working backwards to determine a nucleic acid sequence that encodes said amino acid sequence, according to known mRNA codons which encode amino acids.

[0106] The identified W-bump REDN peptides may be used as a neoantigen in the anti-cancer therapeutic composition. In some embodiments, one or more W-Bump REDN peptides may be selected as a neoantigen based on the size of the W-bump REDN peptide or a predicted epitope binding. Two or more W-bump REDN peptides may be selected as neoantigens. Using the identified W-bump REDN peptides, the W-bump REDN peptide sequence may be manufactured into a vaccine using the W-bump REDN peptide and/or the W-bump REDN peptide sequence. The anti-cancer therapeutic may be the resulting vaccine and can be administered to the subject in a therapeutically effective dose to treat cancer.

[0107] The disclosed arrays have a variety of potential uses. In some embodiments, the array is used to identify one or more W-bump REDN peptides of interest, such as W-bump REDN peptides that are immunogenic, cancer- specific, and/or shared across cancers. In such an embodiment, the array may include a large number of W-bump REDN peptides, for example 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, or 200,000 or more W-bump REDN peptides, or an amount within a range defined by any two of the aforementioned values. This broad coverage of W-bump REDN peptides would allow for the potential of many W-bump REDN peptides to be explored and discovered.

[0108] In some embodiments, the array is used to diagnose cancer. In such an embodiment, the array may include W-bump REDN peptides which have been observed across many different cancers, in order to provide a broad panel coverage. In some embodiments, the arrays are used to predict response to immunotherapy. In some embodiments, the arrays are used to predict adverse responses to immunotherapy. In such an embodiment, the array may include W-bump REDN peptides which have been statistically correlated to a positive (desirable) or negative (undesirable) response to immunotherapy. In some embodiments, the array comprises one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 1-177,361, or a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361, and/or a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.

[0109] In some embodiments, the W-bump REDN peptides are used to design therapeutic or prophylactic vaccines. In some embodiments the arrays are used to determine targets for therapeutics to treat cancer. Certain embodiments of the arrays relate to arrays used in designing a personal vaccine, such as a cancer vaccine. Certain embodiments of the arrays relate to arrays used in designing a universal vaccine, such as a cancer vaccine.

[0110] In some embodiments, the arrays include at least about 100, about 200, about 300, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 7,500, about 10,000, about 12,500, about 15,000, about 17,500, about 20,000, about 22,500, about 25,000, about 27,500, about 30,000, about 32,500, about 35,000, about 37,500, about 40,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 1,500,000, about 2,000,000, about 2,500,000, about 3,000,000, about 3,500,000, about 4,000,000, about 4,500,000, or about 5,000,000 REDN peptides, or an amount within a range defined by any two of the aforementioned values. In some embodiments, the arrays include a range of peptides ranging from about 100 to about 2.5M peptides. In some embodiments, a plurality of W-bump REDN peptides are in-situ synthesized on the array. In some embodiments, the plurality of W- bump REDN peptides are fixed on a substrate. In some embodiments, the substrate includes glass, silica, composite, resin, or combination thereof.

[0111] In some embodiments, the array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. For example, after a biological sample is contacted with the array, binding of the biological sample to the array may be detected and/or measured by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. In some embodiments, the array is configured to facilitate detection of binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. In some embodiments, the plurality of W- bump REDN peptides include two or more pooled W-bump REDN peptides.

[0112] The arrays described herein may be combined with other embodiments. For example, the arrays described may be used in methods of identifying W-bump REDN peptides that are immunogenic, cancer-specific, and/or shared across cancers; methods of detecting cancer; methods of predicting immunotherapy response; methods of designing a cancer vaccine; methods of treating a subject with a vaccine; and methods of producing a therapeutic molecule designed to bind W-bump REDN peptides.

[0113] In some embodiments, vaccine composition comprises one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 1-177,361. In some embodiments, the vaccine compositions include one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361, and/or a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296. In some embodiments, the vaccine compositions further include an adjuvant. In some embodiments, the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51 , Montanide ISA-50, nor-MDP, oil-in-water emulsion, Pl 005 (non-ionic copolymer), Pam3Cys (lipoprotein), pertussis toxin, poloxamcr, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex Adjuvant Formulation (SAF), synthetic polynucleotides (poly IC/poly AU), TiterMax tomatine, Vaxfectin, Xtendlll, or zymosan. In some embodiments, the vaccine may include cosolvent(s), supplementary compound(s), preservative(s), and/or pharmaceutically acceptable carriers)

Methods of Preventing and/or Treating Cancer, Chronic Diseases, or to Counteract Aging

[0114] Checkpoint inhibitor immunotherapeutic s are revolutionizing cancer therapy. However, even in the most responsive cancers a substantial portion (50%- 80%) of the patients have poor to no positive response. A surprising finding in the analysis of these patients was that one of the best correlates of response has been the total number of neoantigens in the tumor. This is also the case for patients with high microsatellite instability (MSI) where the production of FS neoantigens drives the effective anti-tumor immune responses. The realization of the immunological importance of these DNA mutations has spawned the effort to develop personal vaccines. As promising as early studies are of these vaccines, a major problem is that the majority of tumors will not have enough neoantigen-generating mutations to sustain development of a personal vaccine. For example, melanoma tumors have a high mutational level with an average of 200 neoepitope mutations. This provides a substantial number to algorithmically screen for optimal antigenic presentation.

[0115] In recent reports of two Phase I clinical trials of personal melanoma vaccines, starting with 90-2,000 personal neoantigens, 10 or 20 were identified for the vaccine. However, in glioblastoma multiforme (GBM) only 3.5% patients had a high tumor mutation load, and further analysis showed that only a small subset of GBM patients would potentially benefit from checkpoint blockade treatment. This is also consistent with a lack of response in GBM patients to checkpoint inhibitors. Massive genomic sequencing results indicated that GBM, ovarian cancer, breast adenocarcinoma and many other cancer types had exceptionally low number non-synonymous mutations, which will make these cancers difficult targets for personalized cancer vaccines.

[0116] To solve this problem, methods and compositions are provided herein related to W-bump REDN peptides, which expand the scope of the application and efficacy of the neoantigen based cancer vaccines. Tn the process of becoming a tumor, not only does the DNA mutation rate increase with faster cell divisions, but also there is a disruption of basic cellular functions, including RNA transcription, splicing, translation, and the quality control system on peptides. The disrupted RNA processes increase the FS transcripts generated by RNA splicing errors, mis-translation, and the insertions and deletions (INDELs). These processes, combined with the disrupted quality control system in tumor cells, can lead to the production of FS peptides and exposure of the FS epitopes to the immune system. Embodiments provided herein relate to W-bump REDN peptides produced by errors in RNA translation as a source of cancer neoantigens and a simple system to detect and screen them.

[0117] The methods and compositions provided herein indicates that W-bump REDN peptides produced at the RNA level in tumor cells may be a reliable source of neoantigens for vaccines for several reasons. First, these W-bump REDN peptides produce neoantigens which are more likely to be immunogenic than neo-epitopes encoded by single nucleotide mutations. Second, W-bump REDN peptides are a particularly attractive source as are a limited number of possible variants (REDN peptides are downstream of W codons and can be further screened by length of peptide, +1 or -1 frame), thus reducing the search space for W-bump REDN peptides. Third, because of the predictable number of candidates it should be possible to use a peptide array to screen for immune reactive neoantigens. Not all of the predicted W-bump REDN peptides are expected to be immunogenic and only a subset of these have use as diagnostics, therapeutic targets, or vaccine components. As described above, the creation of the peptide arrays can be used to assess which of the W-bump REDN peptides are useful for these purposes. This approach is much simpler than sequencing tumor DNA obtained from a biopsy. Fourth, because any expressed gene has the potential to produce neoantigens, it may not be necessary to limit the vaccine to oncological driver genes. Finally, it should be difficult for the tumor cells to evolve away from the vaccine since these W-bump REDN peptides are variants, not heritable mutations. Particularly if the REDN was produced in RNA from an essential gene, the tumor cells would need to restrict MHC presentation or create an immune suppressive environment to escape an immune response.

[0118] As described above, a W-bump REDN peptide array may be created and used to identify W-bump REDN peptides that recurrently bind to antibodies from biological samples obtained from a subject. From, the identified W-bump REDN peptides, a vaccine may be created and administered to a subject in a therapeutically effective amount to treat cancer. In some embodiments, the vaccine may be administered in two or more doses across a time period.

[0119] In certain embodiments, the vaccine compositions include one or more W- bump REDN peptides. In some embodiments, biological samples, such as blood, from cancer patients are applied to the W-bump REDN peptide arrays described herein to determine reactivity of peptides for each patient. In some embodiments, W-bump REDN peptides unique to the patient are used in a personal vaccine. In some embodiments, W-bump REDN peptides shared between different patients are used for off-the-shelf therapeutic or preventative vaccines. In some embodiments, the vaccine compositions include W-bump REDN peptides resulting from translation of ncRNA.

[0120] An important aspect of this source of neoantigens is that it may allow extending the personal vaccines to more patients and tumor types. Many tumors have relatively low numbers of DNA mutations and probably could not support constructing a vaccine. Estimates from published mutational surveys of various tumors indicate that only 40% of patients could be treated with personal vaccines. However, the methods and compositions provided herein predicts that the RNA W-bump REDN peptides would be produced in any cancer type, even if the DNA mutation level is low.

[0121] In some embodiments, the vaccine compositions further include an adjuvant. In some embodiments, the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund’s adjuvant, Freund’s complete, Freund’s incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank’s Balanced Salt Solution), polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC, also known as Hiltonol), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex adjuvant formulation (SAF), synthetic polynucleotides (poly IC/poly AU), TiterMax tomatine, Vaxfcctin, Xtcndlll, or Zymosan.

Methods of Measuring or Predicting a Subject’s Response to Immunotherapy

[0122] The W-bump REDN peptide array can be used to detect the antibody response in a patient to the W-bump REDN peptides as a direct readout of the W-bump REDN peptide’s clinical potential as diagnostics, therapeutic targets, or vaccines. Methods, systems, and compositions provided herein are related to the analysis of a subject’s ability to generate antibodies to REDN peptide made from mis-processing/translating mRNA and are capable of making high density, high numbers of peptide arrays.

[0123] In some embodiments, the methods include obtaining a biological sample from a subject with cancer, contacting the biological sample to an array including a plurality of W-bump REDN peptides, and measuring binding of the biological sample to the plurality of W-bump REDN peptides. In some embodiments, measuring binding includes detecting antibody reactivity to at least one peptide of the array. In some embodiments, sera samples from patients may be collected before treatment with an immunotherapy, such as the compositions described herein. The sera may be applied to the arrays containing the W-bump peptides, and the signatures for each patient may be analyzed. Based on the patient’s antibody reactivity to 400 W bump REDN peptide, a therapy response may be predicted.

[0124] In some embodiments, the methods further include predicting a response of the subject to an immunotherapy. In some embodiments, the methods further include analyzing the binding to predict whether immunotherapy would be effective in treating the subject’s cancer. In some embodiments, the methods further include analyzing the binding to predict whether immunotherapy would elicit an adverse immune response to immunotherapy in the subject. In some embodiments, comparison is quantitative and/or qualitative. In some embodiments, analyzing includes comparing the binding of the biological sample to binding of another subject who responded positively to immunotherapy or experienced an adverse immune response in response to immunotherapy. In some embodiments, the binding is compared to another subject who had no response to immunotherapy (for example, immunotherapy was not effective in treating the subject’s cancer). In some embodiments the binding is compared to another subject who did not have an adverse event in response to an immunotherapy. In some embodiments, based on this analysis, the subject is classified as being likely to respond (positively) to treatment with the immunotherapeutic, or as being likely to have an adverse event in response to immunotherapy.

[0125] Certain embodiments relate to methods of predicting the response of a subject with cancer to an immunotherapy, and as described herein, can include, for example, obtaining the subject’s binding signature to a W-bump REDN peptides array using one or more biological samples obtained from the subject to determine whether the sample contains one or more indicators of favorable or unfavorable responses (for example, unfavorable side effects) to immunotherapeutic (“IT”) therapy. As used herein, “binding signature,” “binding profile,” or “W-bump REDN peptide signature” refers the observed binding of a subject’s biological sample to a W-bump REDN peptides array. A binding signature or binding profile may include qualitative information about whether a subject’s biological sample was observed to bind to a particular W-bump REDN peptide on a W-bump REDN peptide array and may include quantitative information such as the level or strength of observed binding to particular W-bump REDN peptides on a W-bump REDN peptide array.

[0126] The correlation between a binding signature or binding profile and responsiveness to IT therapy can be established by obtaining binding signatures for subjects having a known favorable response to IT treatment and for subjects that were unresponsive or had an unfavorable response to treatment using sera (or other bodily samples) collected before each subject received treatment. In some cases, a control includes non-disease sera contacted with an identical array under the same experimental conditions. The breadth of the binding profile can be quantified in multiple ways including, for example the number of motifs, the percentage of signature represented, and/or total immune reactivity. Once the quantitative correlate has been established, cancer patients can be classified according to a method provided herein by quantifying a subject’s signature for responsiveness to IT treatment, prognosis, or likelihood of experiencing serious side-effects of IT treatment. In such cases, the methods are useful for determining a subject’s responsiveness for IT treatment of a tumor (including early- stage tumor formation) associated with W-bump REDN peptide expression.

[0127] A W-bump REDN peptide signature is established by using a biological sample (for example, blood, sera, plasma) that may contain antibodies having affinity to peptides on the W-bump REDN peptide array. As further described herein, antibody reactivity to at least one peptide of the array may be detected. As described herein, antibodies are employed as biomarkers of disease, thus taking advantage of the immune system’s expansive antibody repertoire to identify a statistically significant pattern of peptides, each with specific binding values having predictive, prognostic, and diagnostic potential. In some cases, the biological sample is diluted. The sample is incubated long enough to allow cognate binding to approach equilibrium - usually overnight. The array is washed and then incubated with secondary antibody to quantify the amount of antibody bound to each peptide on the array. For each peptide, a quantitative amount of fluorescence is determined. These quantitative data can be analyzed in many different analytical and statistical approaches. In general, a patient’s W- bump REDN peptide signature for IT response, prognosis, or side-effects is determined by comparing two or more groups of interest. For example, a comparison may be made between patients who responded well to IT therapy and those that did not. Such comparisons are used to establish the classifier of interest. In some cases, because the W-bump REDN peptide arrays are directly measuring the immune response to tumor antigens, the difference in groups may be determined directly by quantifying total binding to the W-bump REDN peptides.

[0128] In some embodiments, the plurality of W-bump REDN peptides are fixed on a substrate. In some embodiments, the substrate includes glass, silica, composite, resin, or combination thereof. In some embodiments, the plurality of W-bump REDN peptides are in- situ synthesized on the array. In some embodiments, binding is detected by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, electro-interference, Bio-Layer Interferometry, and surface plasmon resonance. In some embodiments, the array includes at least about 100, about 200, about 300, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 7,500, about 10,000, about 12,500, about 15,000, about 17,500, about 20,000, about 22,500, about 25,000, about 27,500, about 30,000, about 32,500, about 35,000, about 37,500, about 40,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 1,500,000, about 2,000,000, about 2,500,000, about 3,000,000, about 3,500,000, about 4,000,000, about 4,500,000, or about 5,000,000 W-bump REDN peptides, or an amount within a range defined by any two of the aforementioned values. In some embodiments, the arrays include a range of peptides ranging from about 100 to about 2.5M peptides. In some embodiments, the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof. Tn some embodiments, the biological sample includes antibodies. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a Pig-

[0129] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt’s lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin’s lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, and Wilms’ tumor.

[0130] Methods of predicting response to an immunotherapy may be combined with other methods and embodiments disclosed herein. In some embodiments, the array may include a plurality of W-bump REDN peptides which have previously been identified as being immunogenic, cancer-specific, or shared across cancers according to the methods disclosed herein.

Methods of Diagnosing Cancer or Chronic Diseases

[0131] Provided herein are methods of detecting cancer in a subject. In some embodiments, the methods include obtaining a biological sample from a subject, contacting the biological sample from the subject with an array including a plurality of W-bump REDN peptides, measuring binding of the biological sample to the plurality of W-bump REDN peptides, and analyzing the binding to predict whether the subject has a cancer. In some embodiments, the W-bump REDN peptides include one or more peptides discovered by methods described in Zhao et al. (Translation of noncoding RNAs and cancer, Cancer Letters 497 (2021) 89-99, which is incorporated by reference in its entirety). In some embodiments, the W-bump REDN peptides result from non-convention al translation of a W-bump REDN peptide.

[0132] In some embodiments, measuring binding includes detecting antibody reactivity to at least one peptide of the array. In some embodiments, binding is detected by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, electrointerference, Bio-Layer Interferometry, and surface plasmon resonance.

[0133] In some embodiments, analyzing the binding to predict whether the subject has cancer, includes comparing the binding to previously measured binding patterns of other subjects who are known to have had cancer, or other subjects who did not have cancer (for example, positive and negative controls). For example, in some embodiments, binding is compared quantitatively and/or qualitatively to binding of a second subject, who is known to have cancer, to the same type of array. Sufficient similarity of binding is associated with having cancer, and it is predicted that the first subject has cancer.

[0134] In some embodiments, the plurality of W-bump REDN peptides are fixed on a substrate. In some embodiments, the substrate includes glass, silica, composite, resin, or combination thereof. In some embodiments, the plurality of W-bump REDN peptides are in- situ synthesized on the array. In some embodiments, the array includes at least about 100, about 200, about 300, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 7,500, about 10,000, about 12,500, about 15,000, about 17,500, about 20,000, about 22,500, about 25,000, about 27,500, about 30,000, about 32,500, about 35,000, about 37,500, about 40,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 1,500,000, about 2,000,000, about 2,500,000, about 3,000,000, about 3,500,000, about 4,000,000, about 4,500,000, or about 5,000,000 W-bump REDN peptides, or an amount within a range defined by any two of the aforementioned values. In some embodiments, the arrays include a range of peptides ranging from about 100 to about 2.5M peptides.

[0135] In some embodiments, the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof. In some embodiments, the biological sample includes antibodies. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a Pig- [0136] In some embodiments, the subject is suspected of having a cancer. In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt’ s lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin’s lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, and Wilms’ tumor.

[0137] Methods disclosed herein may be combined with other methods. For example, a subject’s biological sample may be contacted with a W-bump REDN peptide array, and binding of the sample to one or more peptides of the W-bump REDN peptide array may be used to simultaneously detect cancer, predict response to immunotherapy, identify immunogenic peptides for use in a preventative or therapeutic vaccine, and/or identify antibodies for use in treatment with a therapeutic molecule designed to bind a W-bump REDN peptide. In some embodiments, the W-bump REDN peptide array may include W-bump REDN peptides which have previously been identified as being immunogenic, cancer-specific, or shared across cancers according to the methods disclosed herein.

[0138] In some embodiments, if a W-bump REDN peptide is recurrent across different patients and/or tumor types, it affords the potential to make generally useful diagnostics, therapeutics, or vaccines. For example, if 20% of breast cancer patients generate antibodies to one W-bump REDN peptide, another 30% to another, and etc., then the collection of these W-bump REDN peptides could be used as a diagnostic for breast cancer. An antibody generated against a W-bump REDN peptide present in 30% of the breast cancer patients could be used as a therapeutic for these patients. In the same way, a vaccine composed of several recurrent W-bump REDN peptides could be manufactured as off-the-shelf, greatly reducing the cost and time of production relative to personal vaccines. [0139] Certain embodiments of the present disclosure are described in the following numbered alternatives. These embodiments arc illustrative only and not intended to be limiting in scope.

[0140] 1. A composition comprising a plurality of RNA error derived neoantigen

(REDN) peptides, wherein the plurality of REDN peptides comprise tryptophan bump REDN peptides.

[0141] 2. The composition of alternative 1, wherein the composition is formulated as a vaccine.

[0142] 3. The composition of any one of alternatives 1-2, wherein the plurality of

REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

[0143] 4. The composition of alternative 3, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

[0144] 5. The composition of any one of alternatives 1-4, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence or part of a sequence as set forth in SEQ ID NOs: 1-177,361.

[0145] 6. The composition of any one of alternatives 1-5, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.

[0146] 7. The composition of any one of alternatives 1-6, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence as set forth in any one of a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296

[0147] 8. The composition of any one of alternatives 1-7, further comprising an adjuvant.

[0148] 9. The composition of alternative 8, wherein the adjuvant is AB M2, AS01B,

AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS- 1313, Montanide ISA 206, Montanide ISA 720, Montanidc ISA-51, Montanide ISA-50, nor-MDP, oil-in-watcr emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex Adjuvant Formulation (SAF), synthetic polynucleotides (poly IC/poly AU), TiterMax tomatine, Vaxf ectin, Xtendlll, or zymosan.

[0149] 10. The composition of any one of alternatives 1-9, wherein the plurality of

REDN peptides comprise two or more pooled REDN peptides.

[0150] 11. A peptide array comprising a plurality of RNA error derived neoantigen

(REDN) peptides, wherein the plurality of REDN peptides comprise tryptophan bump REDN peptides.

[0151] 12. The peptide array of alternative 11, wherein the plurality of REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

[0152] 13. The peptide array of alternative 12, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

[0153] 14. The peptide array of any one of alternatives 11-13, wherein the plurality of REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361.

[0154] 15. The peptide array of any one of alternatives 10-13, wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.

[0155] 16. The peptide array of any one of alternatives 11-15, wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.

[0156] 17. The peptide array of any one of alternatives 11-16, wherein the plurality of REDN peptides is fixed on a substrate.

[0157] 18. The peptide array of alternative 17, wherein the substrate comprises glass, silica, composite, resin, or combination thereof.

[0158] 19. The peptide array of any one of alternatives 11-18, wherein the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. [0159] 20. The peptide array of any one of alternatives 11 -19, wherein the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides. [0159] 21. The peptide array of any one of alternatives 11-20, wherein the REDN peptides are spaced between about 3 and about 9 pm apart.

[0160] 22. The peptide array of any one of alternatives 11-23, wherein the array is used to predict a response to an immunotherapy, to predict adverse responses to immunotherapy, to diagnose a cancer, to develop a vaccine, or to develop a therapeutic.

[0161] 23. The peptide array of any one of alternatives 10-22, wherein the array is used to detect binding of one or more antibodies against W-bump REDN peptides.

[0162] 24. A therapeutic compound that binds to one or more W-bump REDN peptides.

[0163] 25. The therapeutic compound of alternative 24, wherein the therapeutic compound is an antibody or a synthetic antibody.

[0164] 26. A method of treating or preventing a disorder in a subject comprising administering a composition according to any one of alternatives 1-10 as a vaccine.

[0165] 27. The method of alternative 26, wherein the subject is a mammal.

[0166] 28. The method of any one of alternatives 26-27, wherein the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.

[0167] 29. The method of any one of alternatives 26-28, wherein the disorder is a cancer.

[0168] 30. The method of alternative 29, wherein the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor.

[0169] 31. A method of detecting a disorder in a subject, the method comprising:

(a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein the plurality of REDN peptides comprise tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array.

[0170] 32. The method of alternatives 1, wherein the disorder is a cancer.

[0171] 33. The method of any one of alternatives 31-32, wherein the plurality of

REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

[0172] 34. The method of alternative 33, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

[0173] 35. The method of any one of alternatives 31-34, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361.

[0174] 36. The method of any one of alternatives 31-35, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.

[0175] 37. The method of any one of alternatives 31-36, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.

[0176] 38. The method of any one of alternatives 31-37, wherein the plurality of

REDN peptides is fixed on a substrate.

[0177] 39. The method of alternative 38, wherein the substrate comprises glass, silica, composite, resin, or combination thereof.

[0178] 40. The method of any one of alternatives 31-39, wherein the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. [0179] 41 . The method of any one of alternatives 31-40, wherein the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.

[0180] 42. The method of any one of alternatives 31-41, wherein the biological sample comprises blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.

[0181] 43. The method of any one of alternatives 31-42, wherein the biological sample comprises an antibody.

[0182] 44. The method of any one of alternatives 31-43, wherein the subject is a mammal.

[0183] 45. The method of any one of alternatives 31-44, wherein the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.

[0184] 46. The method of alternative 32, wherein the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor.

[0185] 47. The method of any one of alternatives 31-46, wherein the plurality of

REDN peptides comprise two or more pooled REDN peptides.

[0186] 48. The method of any one of alternatives 31-47, wherein the detecting the binding of the biological sample to the at least one peptide in the peptide array comprises fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, or surface plasmon resonance assay.

[0187] 49. A method of measuring an immune response to a neoantigen peptide in a subject, the method comprising: (a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN), wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array.

[0188] 50. The method of alternative 49, wherein the plurality of REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

[0189] 51. The method of alternative 50, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

[0190] 52. The method of any one of alternatives 49-51, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361.

[0191] 53. The method of any one of alternatives 49-52, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.

[0192] 54. The method of any one of alternatives 49-53, wherein the plurality of

REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.

[0193] 55. The method of any one of alternatives 49-54, wherein the plurality of

REDN peptides is fixed on a substrate.

[0194] 56. The method of alternative 55, wherein the substrate comprises glass, silica, composite, resin, or combination thereof.

[0195] 57. The method of any one of alternatives 49-56, wherein the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance.

[0196] 58. The method of any one of alternatives 49-57, wherein the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.

[0197] 59. The method of any one of alternatives 49-58, wherein the biological sample comprises blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.

[0198] 60. The method of any one of alternatives 49-59, wherein the biological sample comprises an antibody.

[0199] 61. The method of any one of alternatives 49-60, wherein the subject is a mammal.

[0200] 62. The method of any one of alternatives 49-61, wherein the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.

[0201] 63. The method of any one of alternatives 49-62, wherein the subject has or is suspected of having a cancer.

[0202] 64. The method of alternative 63, wherein the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor.

[0203] 65. The method of any one of alternatives 49-64, wherein the plurality of

REDN peptides comprise two or more pooled REDN peptides.

[0204] 66. A method of predicting a response of a subject to an immunotherapy, the method comprising: (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN), wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern prior to immunotherapy.

[0205] 67. A method of predicting adverse immune responses of a subject to an immunotherapy, the method comprising: (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN), wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern indicative of adverse immune responses.

[0206] 68. A method of treating a disorder in a subject, comprising: screening therapeutic compounds that bind to one or more W-bump REDN peptides; and administering a therapeutic compound that binds to one or more W-bump REDN peptides to the subject.

[0207] 69. The method of alternative 68, wherein the therapeutic compound is an antibody or a synthetic antibody.

EXAMPLES

[0208] Some aspects of the embodiments provided herein are disclosed in further detail in the following examples, which are not intended in any way to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the disclosure as it is described herein above and/or in the claims.

Example 1: Detection of Antibodies to W-Bump Frameshift Peptides

[0209] An array of all possible predicted W-bump REDN peptides was designed, starting with a bioinformatic determination of all possible REDN peptides downstream of W codons 10 aa or longer. 177,194 such peptides were identified, as set forth in the accompanying sequence listing (SEQ ID NOs: 1-177,194). The restriction of the peptide being longer than 10 amino acids for both sources of REDN peptides was applied. Each REDN peptide antigen that was longer than 15 amino acids was divided into 15 amino acid, non-overlapping peptides. Peptides that shared more than 10 amino acid identical sequences with any human reference proteins were excluded to avoid autoimmunity as a vaccine. [0210] The REDN peptide array was prepared and processed to determine an IgG signal of each array using standard protocols. The specific IgG reactivities were analyzed in 64 non-cancer control samples and a total of 85 cancers from 5 different late-stage cancer types with 17 samples each (LC: lung cancer, BC: breast cancer, GBM: glioblastoma, GC: gastric cancer, PC: pancreatic cancer) and 12 stage I pancreatic cancer samples. The purpose was to broadly screen against different cancers to determine which peptides were of value. Typically, only about 10-20% of REDN peptides resulting from W-bumps were immunoreactive.

[0211] Total reactivity on the arrays was evaluated in the 5 cancer types and noncancer samples with at least two methods. The first method compared the number of significant peptides in the cancer and control samples using fold change and p-values. The second method used a scoring method for each REDN peptide. A peptide was scored as positive (red) if it was higher than six times the standard deviation (6SD) from the mean value of non-cancers for the peptide. All 5 cancer types had more positive REDN peptides than the non-cancer controls. W-bump peptides meeting these criteria were then forwarded to testing in diagnostics, therapeutics, and vaccines.

Example 2

[0212] 3 peptide array slides containing 70,000 unique human cancer W-bump

REDN peptides in duplicate were produced. Human serum samples (cancer and non-cancer) were incubated with arrays at 1:1000 dilution for overnight at 4°C. Human IgG binding was detected by the Dy light 550 labeled goat anti human IgC Fc antibody with 1:5000 dilution for 45 minutes at room temperature. After thorough washing and drying, the slides were scanned by InnoScan 910 instrument, and the relative fluorescence unit (RFU) for each peptide was extracted using Mappix software for subsequent analysis. 89 W-Bump REDNs (10‘³) had positive reactivity with the sera antibodies in cancer samples but not in non-cancer samples. The 89 W-bump REDNs are listed below in Table 1:

Table 1

Example 3

[0213] Arrays were synthesized displaying 1.2 million dog cancer REDN peptides. 277,000 of these were W-bump REDN peptides. Dog serum samples (21 non-cancer and 22 hemangiosarcoma (HSA) cancer) were incubated with arrays at 1:400 dilution for 24 hours at

4 °C. Dog IgG binding was detected by the biotin labeled anti dog IgG antibody and Alex 555 labeled Streptavidin. The slides were scanned by InnoScan 910 instrument, and the relative fluorescence unit (RFU) for each peptide was extracted using Mappix software for subsequent analysis. 78 (2xl0‘⁴) W-Bump REDN peptides had significant reactivity in the cancer versus non-cancer samples. The amino acid sequences of the 78 W-Bump REDNs are shown in Table 2 below, along with the number of non-canccr and HSA samples which showed immunoreactivity with each peptide.

[0214] As shown in Figure 3, using a threshold of 20,000 RFUs for positivity, a set of 13 W-bump REDN peptides were identified that were positive in at least 7 of the HSA serum samples and none of the control samples. Figure 3 depicts detection of immunoreactivity of the 13 peptides across the 22 HSA samples. In Figure 3, Pep_l through Pep_13 refer to SEQ ID NOs: 177284-177296 in Table 2 below.

Table 2

Example 4: W-Bump Frameshift Peptides as Vaccines

[0215] W-bump REDN peptides that recurrently bind to antibodies from sera of cancer patients are encoded on a plasmid in a standard genetic immunization vector and introduced with a gene gun. IxlO⁵ B16F10 tumor cells are injected and animals vaccinated 4 weeks later.

[0216] Plasmids for Genetic Immunization

[0217] The DNA fragments encoding W-bump REDNs are cloned as C-terminal fusions into the genetic immunization vectors pCMVi-UB and pCMVi-LSrCOMPTT with Bgl II and Hind III and mixed with a 1:1 ratio as the vaccine antigen. Three adjuvants are encoded by genetic immunization vectors. The pCMVi-mGM-CSF vector expresses the adjuvant mouse granulocyte/macrophage colony-stimulating factor (mGM-CSF) under the control of the human cytomegalovirus (CMV) promoter. LT AB indicates immunization with a 1:5 ratio by weight of two plasmids, pCMVi-LTA and pCMVi-LTB, expressing the heat-labile enterotoxins LTA and LTB from Escherichia coli. These plasmids express LTA and LTB as C-terminal fusions to the secretion leader sequence from the human al antitrypsin gene. Additional adjuvants are the class A CpG 2216 single- stranded oligodeoxynucleotide obtained from Sigma and alum from Pierce.

[0218] Bullet Preparation for Genetic Immunization with Gene Gun [0219] Bullets for biolistic genetic immunization use the gold micronanoplex approach and arc prepared as described with the following changes. Two grams of 1 -micron gold are used. Prior to the addition of N-hydroxysuccinimide and N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide hydrochloride, the gold is resuspended in 20 mL of a 0.1 M solution of 2-(N-morpholino) ethanesulfonic acid (MES), pH 6.0. DNA-gold micronanoplexes are prepared by combining, per bullet, 57 pL of cysteamine-gold solution with precipitated DNA (<10 pg) that has been resuspended in <15 pL of water, and then vortexing for 10 min. To the DNA-cysteamine-gold, 6 pL/bullet of a freshly made solution of PEI-micron gold (167 mg/mL in 0.1 M MES, pH 6, without NaCl) is added. The pelleted micronanoplexes are washed with ethanol prior to resuspension in n-butanol (55 pL/bullet), followed by bullet formation under nitrogen gas.

[0220] Immunization Dosage and Regime and Tumor Challenge

[0221] C57BL/B 16-F10 Mouse Melanoma Model

[0222] Six-week-old mice (n = 10 per group) receive one genetic immunization with the Gene Gun in the pinna of the ear (4 shots/mouse) with 20 ng of antigen (W-bump REDN and non-protective Cowpox viral antigen CPV 172 (31)) in pCMVi vectors plus the adjuvants pCMVi-mGM-CSF (0.5 pg) and CpG 2216 (5 pg) for each shot. All of the mice are challenged with IxlO⁵ B16-F10 cells 4 weeks after the immunization.

[0223] B ALB/C-4T 1 Mouse Breast Tumor Model

[0224] For a first W-bump REDN experiment, all mice (n = 10 per group) are genetically immunized in the ear by Gene Gun at 8 weeks of age (2 shots/mouse, 60 ng pooled antigens plus 0.25 pg LTAB and 2.5 pg CpG2216 as the adjuvant for each shot) and boosted twice (two days apart) in three weeks with 1 pg pooled antigens plus the same adjuvants dosage. All mice are boosted again in two weeks with 50 pg KLH conjugated W-bump REDN peptides with 50 pg CpG 2216 and 50 pl alum in total 100 pl PBS. The negative groups are immunized with the empty vectors and KLH protein with the same dosage. All mice are challenged with 5xl0³ 4T1 cells two weeks after the last immunization.

[0225] For a second W-bump REDN experiment, all mice (n = 10 per group) are genetically immunized in the ear by Gene Gun at 8 weeks of age (2 shots/mouse, 1 pg antigen plus 0.25 pg LTAB and 2.5 pg CpG2216 as the adjuvant for each shot), and boosted in two weeks with KLH conjugated W-Bump REDN peptide plus 50 pg Poly:IC (Sigma) in 100 pl PBS. The same regime is repeated in two weeks. The negative groups are immunized with the empty vectors and KLH protein with the same dosage. All mice arc challenged with 5xl0³4T1 cells 4 weeks after the last immunization. The CD8 and CD4 T cell depletion stalls 2 weeks after the last immunization by i.p injection of 100 pg antibody (anti CD8, clone 2.43; anti CD4, clone GK 1.5; BioXCell, West Lebanon, NH) every 3 days until the end of the experiment.

[0226] BALB-neuT Mice

[0227] Mice are genetically immunized by Gene Gun at 4-6 weeks with 100 ng of antigen(s) in pCMVi vectors, boosted twice (3-4 days apart) at 9-10 weeks with 1 pg of the same antigen(s), and boosted once at 13-14 weeks with protein. Genetic immunizations include adjuvants LTAB (0.5 pg) and CpG 2216 (5 pg). Protein boosts are 50 pg of KLH conjugated W-bump REDN peptides (first W-Bump REDN peptide, n=32; second W-Bump REDN peptide, n=22; third W-bump REDN peptide, n=14 and pool of the three W-bump REDN peptides, n=37). The protein boost includes 50 pg CpG 2216 and 50 pl alum in 100 pl PBS as the adjuvant. The negative groups (n=30) are immunized with the empty vectors and GST or KLH protein with the same adjuvants and dosage.

[0228] ELISPOT

[0229] Peptides used in the ELISPOT assays are synthesized in-house. The Mouse IFN y ELISPOT Set (BD Biosciences) is used according to the manufacturer’s directions except that blocking is at 37°C.

[0230] Tumor volume is monitored and compared to control mice receiving a mock vaccination. The vaccine confers significant retardation of tumor growth. The pooled vaccine confers an even greater retardation of tumor grown than vaccines which include only one W- Bump REDN peptide.

Example 5: W-Bump Frameshift Peptide Arrays

[0231] The array of all possible predicted tryptophan bump RNA error derived neoantigen (REDN) peptides created in Example 1 is used in this example.

[0232] Sets of sera samples from people with and without various cancers are tested for the diagnostic potential of the arrays. Serum is diluted 1: 100 in binding buffer (0.01M Tris- HC1, pH 7.4, 1% alkali- soluble casein, 0.05% Tween-20), and 150 pl diluted samples are loaded into each compartment of the 12-plex array and incubated overnight at room temperature or 4 °C. After sample binding, the arrays are washed 3X in wash buffer (lx TBS, 0.05% Twccn-20), 10 minutes per wash. Primary sample binding is detected via Alexa Fluor® 647 -conjugated goat anti-human IgG secondary antibody (Jackson ImmunoResearch # 109- 605- 098). The secondary antibody is diluted 1:10,000 (final concentration 0.15 ng/pl) in secondary binding buffer (lx TBS, 1% alkali-soluble casein, 0.05% Tween-20). Arrays are incubated with the secondary antibody for 3 hours at room temperature, washed 3X in wash buffer (10 minutes per wash), 30 seconds in reagent-grade water, and then dried by centrifuging at 690 RPM for 5 minutes. All washes and centrifugations are done on a Little Dipper 650C Microarray Processor (SciGene) with preset programs. The fluorescent signal of the secondary antibody is detected by scanning at 635 nm at 2 pm resolution and 15% gain, using an MS200 microarray scanner (Roche NimbleGen).

[0233] The binding of the samples to the array (including quantitative levels of binding) is compared for cancer and non-cancer samples, and among samples from different types of cancer. Sets of W-bump REDN peptides are identified that have different levels of binding between cancer/non-cancer groups, and/or between different types of cancer. The identified W-bump REDN peptides may be used to classify various cancers from non-cancer and distinguish cancer types. The identified W-bump REDN peptides are included in diagnostics arrays for the detection of cancer and/or diagnosis of cancer type.

Example 6: Using W bump REDN peptides to predict response to immunotherapy

[0234] Sera samples from patients taken before treatment with an immunotherapy are collected. Some of the patients responded well to treatment and others did not. The sera were applied to the arrays containing the W-bump REDN peptides and the signatures for each patient analyzed.

[0235] Seventy-eight blood samples are obtained from human patients having various cancers that are being treated with an immunotherapeutic agent that inhibits PD- 1. The samples are obtained before treatment starts. 30 patients are then monitored long enough to be designated as “Responder” or “Non-Responder” to PD-1 inhibitor treatment. All 78 samples are analyzed on peptide arrays including W-bump REDN peptides. Results of the arrays can be used to distinguish human cancer patients that respond (n=10, green bar) or do not respond (n=20, red bar) to IT treatment. Leave-one-out validation reveals about 90% accuracy in predicting responders to the IT treatment. Different types of analysis applied to the W-bump signature data yield different accuracies.

[0236] It is found that antibody reactivity to certain W bump REDN peptide is associated with whether a patient will respond to therapy or not, for example whether the immunotherapy will be effective in treating a disease such as cancer, or whether the patient is likely to experience an adverse event in response to immunotherapy. These REDN peptides are subsequently used in a classifier to predict responses in patients before therapy.

[0237] Based on this data, a physician could take a small sample of blood from a patient before treatment and determine with high accuracy whether the patient is likely to respond to that particular therapy. If the likelihood is low, a different therapy is recommended. If the patient is predicted to be a responder but likely to have an immune-related adverse event (irAE), W-bump signature analysis is integrated into the treatment plan, and a different therapy is recommended or the treatment plan is modified. irAEs are generally graded from 1-4. Grades 3 and 4 are considered serious and can require immunosuppression treatment. Patients with irAEs are just as likely to have a positive response to treatment. The occurrence of a Grade 3 or 4 event can prohibit the patient from further checkpoint therapy. Therefore, knowing ahead of time which patients are more likely to have an event allows closer monitoring to preempt a Grade 3 or 4 event, such as hypothyroidism, diarrhea, elevated ALT/AST (hepatotoxicity), colitis, diabetes, rash, or fatigue. It is noteworthy that, even though patients suffer from a variety of events, patients who have an irAE have a common predictive W-bump signature.

Example 7: Classifying Responders to Combination Immunotherapy Treatments

[0238] Following intravenous (i.v.) injection of cells of the K7M2 osteosarcoma cell line, mice are treated with 3 dosages of anti-PDLl plus anti-CTLA4, and 2 additional dosages of anti-PLDl treatment. Treatments are spaced three days apart. Binding of samples from the mice to peptide arrays including W-bump REDN peptides (W-bump signatures) are analyzed at the following time-points: (1) prior to the tumor injection and the treatment, (2) right after the treatment, and (3) at the end of the experiment after non-responder mice die from lung metastasis. At each time point, W-bump signatures significantly distinguish the responder mice from the non-responder mice. Remarkably, W-bump signatures can predict the response even before the tumor is injected. [0239] Further study of immunotherapeutic (“IT”) response prediction is performed using a mammary tumor mouse model and an array to measure specific binding to W-bump REDN peptides (W-bump signatures). The 4T-1 mammary tumor cell line is used. Four groups of mice are assayed. Group 1: No Treatment (28 mice). Group 2: Early treatment group (1^st treatment at 16 weeks) (16 mice). Group 3: Treat at first palpable tumor (1^st treatment at -33 weeks). Group 4: Late treatment group (1st treatment at 24-26 weeks) (15 mice). IT treatment is 100 pg anti-CTLA4 (UC10-4F10-11) plus 200 pg anti-PD-Ll (10F.9G2). Five doses are administered, with each dose administered every 3 days, and then two additional doses, with one every week. Palpable tumors are monitored following the treatment period.

[0240] In the Responder group, palpable tumor initiation is significantly delayed by early IT treatment. Late IT treatment has no effect. In the Non-Responder group, IT treatment does not slow tumor growth.

[0241] Samples from Responders and Non-Responders from the Early Treatment group are tested on a W-bump REDN peptide array and W-bump signatures are compared. Hierarchical clustering reveals a number of significant W-bump REDN peptides from the array capable of distinguishing Responders from Non-responders in the early treatment group. These data demonstrate that W-bump signatures are able to distinguish Responders from Non-Responders.

Example 8: Therapeutic Antibody

[0242] Patients with a particular type of cancer, such as breast cancer, are screened for having antibodies to W-bump REDN peptides. Biological samples from patients are applied a peptide array that includes W-bump REDN peptides. W-bump REDN peptides are identified that are associated with an immune response in most of the patients that are tested, for example W-bump REDN peptides on the array which most samples from breast cancer patients react to, and most samples from patients without cancer don’t have binding to.

[0243] Monoclonal antibodies to a chosen set of these immunoreactive peptides are selected. For example, the criteria for choice includes frequency of reactivity in the patients or the level of antibody reactivity, such as compared to healthy control samples. The monoclonal antibodies are tested in vitro for their ability to bind and or kill cancer cells (cell lines or tumor cells) that express the W-bump REDN peptides. [0244] Antibody candidates which show in vitro activity are further screened in mouse tumor models for therapeutic effects when injected. Based on this data, one or more of the antibodies are produced on large scale by Good Manufacturing Practice to enter into clinical trials in patients with the relevant cancer. If the antibody is shown to be safe and efficacious it is used to treat cancer in patients having a cancer.

[0245] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0246] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0247] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0248] As will be understood by one of skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0249] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those of skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT TS CLAIMED TS:

1. A composition comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein the plurality of REDN peptides comprise tryptophan bump REDN peptides.

2. The composition of claim 1, wherein the composition is formulated as a vaccine.

3. The composition of claim 1, wherein the plurality of REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

4. The composition of claim 3, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

5. The composition of claim 1, wherein the plurality of REDN peptides comprise one or more peptides having a sequence or part of a sequence as set forth in SEQ ID NOs: 1-

177,361.

6. The composition of claim 1, wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-

177,361.

7. The composition of claim 1, wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284- 177,296.

8. The composition of claim 1, further comprising an adjuvant.

9. The composition of claim 8, wherein the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide ISA 206, Montanide ISA 720, Montanide ISA-51, Montanide ISA-50, nor-MDP, oil-in-water emulsion, P1005 (non-ionic copolymer), Pam3Cys (lipoprotein), pertussis toxin, poloxamer, QS21, RaLPS, Ribi, saponin, Seppic ISA 720, soybean oil, squalene, Syntex Adjuvant Formulation (SAF), synthetic polynucleotides (poly IC/poly AU), TitcrMax tomatine, Vaxfcctin, Xtcndlll, or zymosan.

10. The composition of claim 1, wherein the plurality of REDN peptides comprise two or more pooled REDN peptides.

11. A peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein the plurality of REDN peptides comprise tryptophan bump REDN peptides.

12. The peptide array of claim 11 , wherein the plurality of REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

13. The peptide array of claim 12, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

14. The peptide array of claim 11 , wherein the plurality of REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-

177,361.

15. The peptide array of claim 11 , wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-

177,361.

16. The peptide array of claim 11 , wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284- 177,296.

17. The peptide array of claim 11, wherein the plurality of REDN peptides is fixed on a substrate.

18. The peptide array of claim 17, wherein the substrate comprises glass, silica, composite, resin, or combination thereof.

19. The peptide array of claim 11, wherein the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance.

20. The peptide array of claim 11, wherein the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.

21. The peptide array of claim 11, wherein the REDN peptides are spaced between about 3 and about 9 pm apart.

22. The peptide array of claim 11, wherein the array is used to predict a response to an immunotherapy, to predict adverse responses to immunotherapy, to diagnose a cancer, to develop a vaccine, or to develop a therapeutic.

23. The peptide array of claim 11, wherein the array is used to detect binding of one or more antibodies against W-bump REDN peptides.

24. A therapeutic compound that binds to one or more W-bump REDN peptides.

25. The therapeutic compound of claim 24, wherein the therapeutic compound is an antibody or a synthetic antibody.

26. A method of treating or preventing a disorder in a subject comprising administering a composition according to claim 1 as a vaccine.

27. The method of claim 26, wherein the subject is a mammal.

28. The method of claim 26, wherein the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.

29. The method of claim 26, wherein the disorder is a cancer.

30. The method of claim 29, wherein the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor.

31. A method of detecting a disorder in a subject, the method comprising: (a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein the plurality of RNA error derived neoantigen (REDN) peptides comprise tryptophan bump REDN peptides; and

(b) detecting binding of the biological sample to at least one peptide in the peptide array.

32. The method of claim 31, wherein the disorder is a cancer.

33. The method of claim 31, wherein the plurality of REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

34. The method of claim 33, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

35. The method of claim 31 , wherein the plurality of REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1- 177,361.

36. The method of claim 31, wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.

37. The method of claim 31 , wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.

38. The method of claim 31, wherein the plurality of REDN peptides is fixed on a substrate.

39. The method of claim 38, wherein the substrate comprises glass, silica, composite, resin, or combination thereof.

40. The method of claim 31, wherein the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance.

41. The method of claim 31, wherein the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.

42. The method of claim 31 , wherein the biological sample comprises blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.

43. The method of claim 31, wherein the biological sample comprises an antibody.

44. The method of claim 31, wherein the subject is a mammal.

45. The method of claim 31, wherein the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.

46. The method of claim 32, wherein the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor.

47. The method of claim 31 , wherein the plurality of REDN peptides comprise two or more pooled REDN peptides.

48. The method of claim 31, wherein the detecting the binding of the biological sample to the at least one peptide in the peptide array comprises fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, or surface plasmon resonance assay.

49. A method of measuring an immune response to a neoantigen peptide in a subject, the method comprising:

(a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array.

50. The method of claim 49, wherein the plurality of REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.

51. The method of claim 50, wherein the two closely spaced tryptophan codons are less than 8 codons apart.

52. The method of claim 49, wherein the plurality of REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1- 177,361.

53. The method of claim 49, wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.

54. The method of claim 49, wherein the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.

55. The method of claim 49, wherein the plurality of REDN peptides is fixed on a substrate.

56. The method of claim 55, wherein the substrate comprises glass, silica, composite, resin, or combination thereof.

57. The method of claim 49, wherein the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance.

58. The method of claim 49, wherein the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.

59. The method of claim 49, wherein the biological sample comprises blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.

60. The method of claim 49, wherein the biological sample comprises an antibody.

61. The method of claim 49, wherein the subject is a mammal.

62. The method of claim 49, wherein the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.

63. The method of claim 49, wherein the subject has or is suspected of having a cancer.

64. The method of claim 63, wherein the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, retinoblastoma, skin cancer, small cell lung cancer, squamous cell carcinoma, stomach cancer, T-cell leukemia, T-cell lymphoma, thyroid cancer, or Wilms' tumor.

65. The method of claim 49, wherein the plurality of REDN peptides comprise two or more pooled REDN peptides.

66. A method of predicting a response of a subject to an immunotherapy, the method comprising:

(a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN)peptides, wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides;

(b) detecting binding of the biological sample to at least one peptide in the peptide array; and

(c) comparing a binding pattern following immunotherapy to a binding pattern prior to immunotherapy.

67. A method of predicting adverse immune responses of a subject to an immunotherapy, the method comprising: (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides;

(b) detecting binding of the biological sample to at least one peptide in the peptide array; and

(c) comparing a binding pattern following immunotherapy to a binding pattern indicative of adverse immune responses.

68. A method of treating a disorder in a subject, comprising: screening therapeutic compounds that bind to one or more W-bump RNA error derived neoantigen (REDN) peptides; and administering a therapeutic compound that binds to one or more W-bump REDN peptides to the subject.

69. The method of claim 67, wherein the therapeutic compound is an antibody or a synthetic antibody.