WO2018156645A1

WO2018156645A1 - Preventative cancer therapy

Info

Publication number: WO2018156645A1
Application number: PCT/US2018/019039
Authority: WO
Inventors: Timothy Webb
Original assignee: PrioBio, LLC
Priority date: 2017-02-21
Filing date: 2018-02-21
Publication date: 2018-08-30

Abstract

The present disclosure relates to methods of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies, methods of determining a signature of circulating cancer-associated autoantibodies in a subject, methods for identifying cancer-associated autoantibodies, methods of inducing protective immunity against a cancer in a subject, methods of identifying one or more peptide epitopes for inducing protective immunity against a cancer in a subject, and methods of monitoring a cancer in a subject.

Description

PREVENTATIVE CANCER THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the benefit of U.S. Provisional Patent Application No. 62/509,067 filed May 20, 2017 and U.S. Provisional Patent Application No. 62/461 ,569 filed February 21 , 201 7 the entire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present disclosure relates to methods and compositions for the induction of therapeutic autoimmunity in cancer.

BACKGROUND OF THE INVENTION

[0003] Cancer moves through "bottlenecks" in the genome, generally inactivation of tumor suppressors and upregulation/permanent expression of oncogenes. A large part of the evolution towards cancer in the body is through DNA mutation in these genes. Ideally, the body's immune system would rapidly identify and eliminate abnormalities in the bottleneck oncogene and tumor suppressor genes, but human evolution has not achieved optimal cancer immunosurveillance.

[0004] During the progression of cancer, there are often signs that the immune system has detected abnormal expression in the "bottlenecks." Such detection in part manifests in the form of circulating autoantibodies against the abnormally expressed tumor suppressor or oncogenic proteins. Furthermore, these autoantibodies are often correlated with prolonged survival, even though they can react against the normal version of these ubiquitous proteins. However, as cancer progresses, more mutations arise in the bottlenecks which may not be screened by the immune system, allowing cancer cells to continue multiplying.

[0005] Cells within tissues that are progressing towards cancer are often present for many years or even decades before undergoing all the changes necessary to grow dangerously out of control. These very minor changes in a single, or a few proteins are rarely screened by the immune system, and are not generally expected to elicit a reliable or strong immune response. Importantly, this indicates that there is a substantial window for targeting cells that are progressing towards cancer before they progress to full-fledged cancer.

[0006] There is a need for a cancer vaccine that targets the cancer early on, while number of mutations is low, and during the long window in which abnormal cells are present but go undetected by the immune system.

SUMMARY OF THE INVENTION

[0007] In one aspect, the present disclosure provides a method of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies. The method comprises the steps of (a) providing a plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences, wherein each autoantigen element further comprises at least one immunologic epitope; (b) providing a biological sample from one or more individuals having at least an early stage cancer; (c) contacting the peptide autoantigen elements with the biological sample under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies; and (d) selecting peptide autoantigen elements for the panel by selecting a plurality of peptide autoantigen elements each capable of producing a specific binding signal under the conditions of step (c).

[0008] The plurality of peptide autoantigen elements may be derived from a panel of cancer-associated protein sequences using a method comprising the steps of (a) isolating at least two isotypes of immunoglobulins from the biological sample to obtain a fraction for each isotype; (b) contacting a first concentration of each isotype fraction with a first set of cancer-associated autoantigen elements; (c) isolating from each isotype fraction each cancer-associated autoantibody that specifically binds to cancer-associated autoantigen elements in the first set of cancer-associated autoantigen elements to provide a reduced autoantibody pool; (d) subtracting a subset of cancer-associated autoantigen elements from the first set of cancer-associated autoantigen elements to obtain a remainder set, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the biological sample; and (e) obtaining a second set of cancer- associated autoantigen elements consisting of the remainder set of cancer- associated autoantigen elements, wherein the remainder set is the panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies.

[0009] The conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies may comprise identifying a maximum viable concentration of the biological sample, and contacting the peptide autoantigen elements with the maximum viable concentration of the biological sample. A maximum viable concentration of the biological sample may be identified by the steps of (a) contacting a first set of peptide autoantigen elements with a first concentration of the biological sample to identify a subset of peptide autoantigen elements, wherein the subset of peptide autoantigen elements produce a nonspecific signal; (b) diluting the first concentration of the biological sample to obtain a series of dilutions of the biological sample; (c) contacting the series of dilutions of the biological sample from step (b) with the subset of peptide autoantigen elements; and (d) identifying from the series of dilutions in step (b), a dilution comprising the highest concentration of the biological sample that does not produce a signal when the subset of peptide autoantigen elements is contacted with the series of dilutions in step (b), wherein the dilution of the biological sample identified in step (d) is the maximum viable concentration of the biological sample.

[00010] Conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies comprise sufficiently diluting the biological sample to produce the specific signal after contacting the peptide autoantigen elements at room temperature for about 78 hours. Additionally, the biological sample may be further processed to enhance the specific signal for detecting low abundance cancer-associated autoantibodies.

[0001 1 ] The panel of cancer-associated protein sequences may be selected from a group of protein sequences consisting of p53, PI3-kinase, K-ras, β-catenin, Smad4, B-raf, EGFR, FGRFR3, PDGFRA, SRSF2, EP300, NFE2L2, IDH1 , ATM, NOTCH1 , NOTCH2, FBXW7, MED1 2, H3F3A, SETBP1 , SF3B1 , ALK, CREBBP, CTNNB1 , FLT3, GNAS, JAK2, KIT, NRAS, PIK3CA, PTEN, TP53, VHL, and combinations thereof. Additionally, the cancer-associated protein sequences may be selected from the group consisting of wild type sequences of cancer-associated proteins, sequences of cancer-associated proteins having cancer-associated mutations, or combinations thereof. Peptide epitopes derived from one or more of the panel of peptide autoantigen elements may be capable of inducing therapeutic autoimmunity against cancer in a subject. The subject may be suspected of having cancer, may be at risk of developing cancer, or may be diagnosed with an early stage of cancer.

[00012] In another aspect, the present disclosure provides a composition for inducing protective immunity against a cancer in a subject. The composition comprises one or more peptide epitopes derived from one or more cancer- associated autoantigen elements identified in the method described above. The one or more peptide epitopes derived from one or more cancer-associated autoantigen elements may comprise native peptide sequences, mutant peptide sequences, engineered peptide sequences, or any combination thereof.

[00013] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a composition for inducing protective immunity against a cancer in a subject, and at least one pharmaceutically acceptable carrier, excipient or diluent. The composition may be as described above.

[00014] In another aspect, the present disclosure provides a method of inducing protective immunity against cancer in a subject. The method comprises administering to the subject a composition comprising one or more peptide epitopes derived from one or more peptide autoantigen element identified using the method of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies described above. The one or more peptide epitopes may each comprise a sequence modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject.

[00015] In an additional aspect, the present disclosure provides a method of determining a signature of circulating cancer-associated autoantibodies in a subject. The method comprises providing a biological sample from the subject; providing a panel of peptide autoantigen elements identified using the method of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer- associated autoantibodies described above; and contacting the peptide autoantigen elements with the biological sample under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies. The signature of circulating cancer-associated autoantibodies in a subject comprises a plurality of specific binding signals, each specific binding signal produced by each peptide probe.

[00016] In another aspect, the present disclosure provides a peptide array. The array comprises a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies. The panel of peptide autoantigen elements are identified using the method of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies described above.

[00017] In yet another aspect, the present disclosure provides a method for identifying cancer-associated autoantibodies. The method comprises the steps of (a) obtaining a serum sample from one or more subjects having at least an early stage cancer; (b) isolating at least two isotypes of immunoglobulins from the serum sample, to obtain a fraction for each isotype; (c) contacting a first concentration of each isotype fraction with a first autoantigen array comprising a first set of a plurality of different cancer-associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope; (d) isolating from each isotype fraction each cancer-associated autoantibody that specifically binds to a cancer-associated autoantigen element in the first autoantigen array to provide a reduced autoantibody pool; (e) subtracting a subset of cancer-associated autoantigen elements from the first set of cancer-associated autoantigen elements in the first array to obtain a remainder set, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample from (a); obtaining a second autoantigen array comprising the remainder set of cancer-associated autoantigen elements from (e); and contacting the second autoantigen array from (f) with an optimal dilution of the reduced autoantibody pool from (d), to produce a specific binding signal, wherein the autoantibodies that produce a specific binding signal are cancer-associated autoantibodies.

[00018] Additionally, the method for identifying cancer-associated autoantibodies. The method comprises the steps of (a) obtaining a serum sample from one or more subjects having at least an early stage cancer; (b) isolating at least two isotypes of immunoglobulins from the serum sample, to obtain a fraction for each isotype; (c) contacting a high concentration of each isotype fraction with a first autoantigen array comprising a plurality of different cancer-associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope; (d) identifying any cancer-associated autoantigen elements in the array that specifically bind to autoantibodies in the serum sample from (a), wherein each cancer-associated autoantigen element that specifically binds to an autoantibody identifies a complete autoantigen protein; (e) using the complete autoantigen proteins identified from step (d) to identify and isolate target autoantibodies from each isotype fraction, to produce a reduced autoantibody pool for each isotype fraction; (f) identifying an optimal dilution of each reduced autoantibody pool from (e) for each isotype fraction; and (g) contacting the optimal dilution of each reduced autoantibody pool from (f), with a second autoantigen array comprising a plurality of different cancer-associated autoantigen elements, wherein the plurality of different cancer-associated autoantigen elements in the second autoantigen array is a subset of the plurality of different cancer associated- associated autoantigen elements in the first autoantigen array, the subset comprising the cancer-associated autoantigen elements that specifically bind to autoantibodies in step (d), to produce a specific binding signal, wherein the specific binding signal indicates target autoantibodies of interest.

[00019] Alternatively, the method for identifying cancer-associated autoantibodies. The method comprises the steps of (a) obtaining a serum sample from one or more subjects having at least an early stage cancer; (b) isolating at least two isotypes of immunoglobulins from the serum sample, to obtain a fraction for each isotype; (c) contacting a first concentration of each isotype fraction with a first autoantigen array comprising a first set of cancer-associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope; (d) isolating from each isotype fraction each cancer-associated autoantibody that specifically binds to a cancer-associated autoantigen element in the first autoantigen array to provide a reduced autoantibody pool; (e) subtracting a subset of cancer-associated autoantigen elements from the first set of cancer-associated autoantigen elements in the first autoantigen array to obtain a remainder set of cancer-associated autoantigen elements, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample from (a); (f) obtaining a second autoantigen array consisting of the remainder set of cancer-associated autoantigen elements from (e); (g) identifying a maximum viable concentration of each isotype fraction using the second autoantigen array; and (h) contacting the second autoantigen array from (f) with the maximum viable concentration of the biological sample from (g), to produce a specific binding signal, thereby identifying cancer-associated autoantibodies.

[00020] The autoantigen protein may be a complete autoantigen protein or linear peptides derived from the complete autoantigen protein. The method may further comprise comparing an immune response of the target autoantibodies of interest against corresponding native autoantigens, to the immune response of the target autoantibodies of interest against corresponding engineered variants of the autoantigens.

[00021 ] Contacting of an autoantigen array with any serum fraction or serum dilution may comprise contacting under conditions capable of producing a specific signal for detecting cancer-associated autoantibodies. The first concentration of each isotype fraction and the optimal dilution are identified using the method of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies; wherein conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies comprise identifying a maximum viable concentration of the biological sample, and contacting the peptide autoantigen elements with the maximum viable concentration of the biological sample; and wherein identifying a maximum viable concentration of the biological sample comprises contacting a first set of peptide autoantigen elements with a first concentration of the biological sample to identify a subset of peptide autoantigen elements, wherein the subset of peptide autoantigen elements produce a non-specific signal; diluting the first concentration of the biological sample to obtain a series of dilutions of the biological sample; contacting the series of dilutions of the biological sample from step (b) with the subset of peptide autoantigen elements; and identifying from the series of dilutions in step (b), a dilution comprising the highest concentration of the biological sample that does not produce a signal when the subset of peptide autoantigen elements is contacted with the series of dilutions in step (b), wherein the dilution of the biological sample identified in step (d) is the maximum viable concentration of the biological sample.

[00022] The optimal dilution may comprise a dilution sufficient to produce a specific binding signal after contact with the second microarray at room temperature for about 78 hours. Step (d) of the method may comprise using immunoprecipitation to bind free autoantibody in each isotype fraction to a plurality of complete autoantigen proteins fused to agarose beads, and eluting the bound autoantibodies. The plurality of different cancer-associated autoantigen elements in step (c) may comprise at least one sequence derived from p53, PI3-kinase, K-ras, β-catenin, Smad4, B-raf, EGFR, FGRFR3, PDGFRA, SRSF2, EP300, NFE2L2, IDH1 , ATM, NOTCH1 , NOTCH2, FBXW7, MED1 2, H3F3A, SETBP1 , SF3B1 , ALK, CREBBP, CTNNB1 , FLT3, GNAS, JAK2, KIT, NRAS, PIK3CA, PTEN, TP53, VHL, and combinations thereof. Additionally, the plurality of different cancer-associated autoantigen elements in step (c) may be selected from wild type sequences of cancer-associated proteins, sequences of cancer-associated proteins having naturally occurring or engineered cancer-associated mutations, or any combination thereof.

[00023] Epitopes derived from one or more of the cancer-associated autoantigen elements in step (c) may be capable of inducing therapeutic autoimmunity against cancer in a subject. The one or more subjects may primarily comprise subjects suspected of having a cancer, identified as at risk of developing a cancer, or has a diagnosis of a cancer. Alternatively, the one or more subjects may primarily consist of subjects suspected of having a cancer, identified as at risk of developing a cancer, or has a diagnosis of a cancer. The serum sample may be a pool of serum samples obtained from a population of subjects having at least an early stage cancer. The pool of identified cancer-associated autoantibodies are associated with a type of cancer, a class of cancer, or a stage in cancer development.

[00024] In another aspect, the present disclosure provides a method of inducing protective immunity against a cancer in a subject, the method comprising administering to the subject a composition comprising one or more peptide epitopes derived from one or more cancer-associated autoantigen elements identified from the specific binding signal from step (g) in the method for identifying cancer- associated autoantibodies described above. The one or more peptide epitopes may each comprise a sequence modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject. [00025] In another aspect, the present disclosure provides a method of determining a signature of circulating cancer-associated autoantibodies in a subject, the method comprising: a) providing a biological sample from the subject; b) providing a panel of cancer-associated autoantigen elements identified from step (g) in any of the methods described above; c) contacting the panel of cancer-associated autoantigen elements with the biological sample under conditions capable of producing a specific binding signal; wherein the signature of circulating cancer- associated autoantibodies in a subject comprises the specific binding signal produced by binding of circulating autoantibodies to one or more of the cancer- associated autoantigen elements in the panel.

[00026] In an additional aspect, the present disclosure provides a peptide array comprising a plurality of cancer-associated autoantigens capable of detecting early-stage cancer-associated autoantibodies and identified using any of the methods described above.

[00027] In another aspect, the present disclosure provides a composition for inducing protective immunity against a cancer in a subject or a pharmaceutical composition comprising the composition. The method comprises one or more peptide epitopes derived from one or more cancer-associated autoantigen elements identified from the specific binding signal from step (g) in any of the methods described above. The one or more peptide epitopes derived from one or more cancer-associated autoantigen elements may comprise native peptide sequences, mutant peptide sequences, engineered peptide sequences, or any combination thereof. The pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, excipient or diluent.

[00028] In yet another aspect, the present disclosure provides a method of identifying one or more peptide epitopes for inducing protective immunity against a cancer in a subject. The method comprises the steps of (a) providing a plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences; (b) modifying the peptide autoantigen elements of (a); and identifying from peptide autoantigen elements of (a) or (b), one or more peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies, thereby identifying one or more peptide epitopes for inducing protective immunity against a cancer in a subject. The plurality of peptide autoantigen elements may comprise overlapping peptides derived from a panel of cancer-associated protein sequences. Modifying the peptide autoantigen elements of (a) may comprise engineering the autoantigen elements to comprise a modification selected from the group consisting of amino acid substitutions with naturally-occurring amino acids, amino acid substitutions with unnatural amino acids, amino acid substitutions with amino acid analogs, amino acid substitutions with amino acid mimetics, amino acid substitutions with non-canonical amino acid residues, amino acid substitutions with amino acid derivatives, amino acid deletions, amino acid insertions, fusion with other amino acid sequences, chemical modification of amino acid residues, modification to side chains of amino acid residues, peptide circularization or cyclization, and combinations thereof. The one or more peptide epitopes may each be derived from a cancer-associated protein sequence having a cancer-associated mutation and a sequence modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject. The one or more peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies are identified by using any of the methods of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies, and step (g) in a method for identifying cancer-associated autoantibodies described above.

[00029] In yet another aspect, the present disclosure provides a method of monitoring a cancer. The method comprises identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies; and using the panel of peptide autoantigen elements of (a) to monitor the cancer-associated autoantibodies, thereby monitoring the cancer. The low abundance cancer-associated autoantibodies may be associated with a type of cancer, a class of cancer, or a stage in cancer development. Further, a cancer treatment may be monitored.

BRIEF DESCRIPTION OF THE DRAWINGS

[00030] The following drawings form part of the present disclosure and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein. [00031 ] FIG. 1 depicts a flow diagram describing a method of high- sensitivity signal detection for vaccine development. Determining signal to noise on peptide microarray and maximum viable concentrations of a biological sample may be as described in FIG. 2. Off-target antibodies may be depleted as described in FIGS. 3 and/or 4. Determining enhanced signal to noise on peptide microarray and enhanced maximum viable concentrations of a biological sample may be as described in FIG. 2.

[00032] FIG. 2 depicts flow diagrams describing a method of identifying a maximum viable concentration of the biological sample: (a) depicts a first array of cancer-associated autoantigen elements contacted with a high concentration of a sample to identify autoantigen elements that produce a non-specific background signal; and (b) depicts a second array comprising the peptide autoantigen elements that produce the non-specific signal as identified in FIG. 2a. The second array is contacted with a dilution series of the sample. The dilution with the highest concentration of the biological sample that does not produce a signal is the maximum viable concentration of the biological sample.

[00033] FIG. 3 depicts a flow diagram describing a method of capturing conformational epitope signatures of cancer-associated autoantigen elements to identifying cancer-associated autoantibodies.

[00034] FIG. 4 depicts a flow diagram describing a method of capturing linear epitope signatures of cancer-associated autoantigen elements to identifying cancer-associated autoantibodies.

[00035] FIG. 5 depicts a flow diagram method of generating a vaccine autoantibody signature. Peptide engineering may be as described in FIG. 6.

[00036] FIG. 6 depicts an example of genetic engineering of peptide cancer- associated autoantigen elements of EGFR. Shown are overlapping EGFR peptides (panel 1 ), overlapping peptides from EGFR having cancer-associated mutations (panel 2) with mutated residues highlighted in grey, and engineered peptides (panel 3) from EGFR showing mutated residues (grey) of cancer-associated mutations in EGFR and residue changes (clear rectangles) engineered to increase the binding of antibodies created from vaccine against peptides present in MHC cleft.

[00037] FIG. 7 depicts a flow diagram showing vaccine development wherein native, mutated, and/or engineered peptide cancer-associated autoantigen elements are used to develop a vaccine. The vaccine may be packaged into delivery systems such as a virion.

[00038] FIG. 8 depicts a flow diagram showing a method used to produce a specific signal for detecting low abundance cancer-associated autoantibodies.

[00039] FIG. 9 is a schematic diagram of an immune response sequence that can be harnessed for the preparation of cancer vaccines.

[00040] FIG. 10 is a schematic diagram of the immune response showing development of immune memory, which misses some subset of mutations.

[00041 ] FIG. 11 is a schematic diagram of the immune response showing accumulation of mutations in a cancerous progression wherein diverse cancer mutations not recognized by antibodies escape the immune system.

[00042] FIG. 12 is a schematic diagram illustrating how the vaccine compositions of the present disclosure provide immunity against proteins with key cancer mutations that are not otherwise recognized by the naive immune system, thereby allowing pre-cancerous and cancerous cells to be eliminated at a very early step in the development of cancer.

[00043] FIG. 13 is a schematic diagram illustrating how cancer-associated mutations are used to develop a cancer vaccine: (a) naturally occurring gatekeeper mutations, which are mutations identified in a high percentage of cancers, are identified as targets; (b) peptides are generated which incorporate at least one key mutation identified in a target identified in step (a); and (c) candidate peptides are diversified through further modifications, to increase safety and efficacy, and broaden scope of anti-cancer antibodies.

DETAILED DESCRIPTION

[00044] The present disclosure relates to methods and compositions directed to preventative cancer therapy wherein the immune response is manipulated to enhance the autoimmunity against a developing cancer, all while minimizing collateral damage that may be associated with autoimmunity. The present disclosure is based in part on the realization that the more diverse a cancer becomes within the body, the less likely it is that an autoantibody would be able to competently suppress growth of the tumor, even if the same autoantibody could have had an immunotherapeutic effect if it had been present earlier in sufficient amount. Given that there are fewer than 100 key tumor suppressors / oncogenes that are manipulated in the progression of cancer, the present disclosure describes use of this information to generate a preventative therapy which applies to most cancer types.

[00045] The present disclosure is based in part on the fact that autoantibodies are commonly found in healthy populations of all ages against a variety of proteins, some of which can be cancer associated. While some autoantibodies are correlated with certain diseases, other autoantibodies appear to have limited effect on health. Autoantibodies in the average adult may number in the thousands and are likely to increase in scope with age. Manipulating the pool of autoantibodies to include antibodies that may be protective against cancer (which are already present in some of the population with no associated disease state) should provide a net survival and quality of life benefit.

[00046] In essence, the inventors have discovered methods of identifying and producing peptide epitopes capable of enhancing therapeutic autoimmunity against cancer in a subject without, or while significantly minimizing, collateral damage that may be associated with autoimmunity. As such, peptide epitopes identified using methods of the instant disclosure can disengage therapeutic immunity against a cancer from deleterious effects normally associated with autoimmunity. Using the methods of the invention, therapeutic autoimmunity against cancer can be enhanced. For instance, methods of the instant disclosure may be used to develop a preventative cancer vaccine. An optimal preventative cancer vaccine using the induction of therapeutic autoimmunity may be relatively weak compared to the type of vaccine typically designed to prevent infection and the spread of virions, for example. However, a cancer preventative vaccine need only assist - and indeed may be better suited to - slow remodeling of tissues over the course of many years. As such, tissue damage and collateral damage from autoimmunity are minimized.

[00047] Methods of the disclosure may be better understood when referring to the figures. I. Method For Identifying Peptide Autoantigen Elements

[00048] In one aspect, the present disclosure provides a method of identifying peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies. As it will be recognized in the art, the term "autoantigen" refers to any antigen that stimulates autoantibodies in the organism that produced it, and the term "autoantibody" refers to an antibody formed in response to autoantigen.

[00049] The method comprises providing a plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences wherein each autoantigen element further comprises at least one immunologic epitope, providing a biological sample from one or more individuals having at least an early stage cancer, and contacting the peptide autoantigen elements with the biological sample under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies. The method further comprises selecting peptide autoantigen elements for the panel by selecting a plurality of peptide autoantigen elements, each capable of producing a specific binding signal under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies. The subjects have at least an early stage cancer, and may be subjects suspected of having cancer, are at risk of developing cancer, or are diagnosed with an early stage of cancer.

[00050] The plurality of peptide autoantigen elements may be derived from a panel of cancer-associated protein sequences using a method comprising: (a) isolating at least two isotypes of immunoglobulins from the biological sample to obtain a fraction for each isotype; (b) contacting a first concentration of each isotype fraction with a first set of cancer-associated autoantigen elements; (c) isolating from each isotype fraction each cancer-associated autoantibody that specifically binds to cancer-associated autoantigen elements in the first set of cancer-associated autoantigen elements to provide a reduced autoantibody pool; (d) subtracting a subset of cancer-associated autoantigen elements from the first set of cancer- associated autoantigen elements to obtain a remainder set, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the biological sample; and (e) obtaining a second set of cancer- associated autoantigen elements consisting of the remainder set of cancer- associated autoantigen elements, wherein the remainder set is the panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies.

[00051 ] Conditions capable of detecting low abundance antibodies are known in the art, any of which may be used to detect low abundance cancer- associated autoantibodies. In some embodiments, conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies may comprise identifying a maximum viable concentration of the biological sample, and contacting the peptide autoantigen elements with the maximum viable concentration of the biological sample. Identifying a maximum viable concentration of the biological sample comprises contacting a first set of peptide autoantigen elements with a first concentration of the biological sample to identify a subset of peptide autoantigen elements, wherein the subset of peptide autoantigen elements produce a non-specific signal. The first concentration of the biological sample is then diluted to obtain a series of dilutions of the biological sample before contacting the series of dilutions of the biological sample with the subset of peptide autoantigen elements to identify a dilution comprising the highest concentration of the biological sample that does not produce a signal when the subset of peptide autoantigen elements is contacted with the series of dilutions. The identified dilution of the biological sample is the maximum viable concentration of the biological sample. Identifying a maximum viable concentration of the biological sample may be as diagrammed in FIG. 2.

[00052] Conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies may also comprise sufficiently diluting the biological sample to produce a specific signal after contacting the peptide autoantigen elements at room temperature for an extended period of time. For instance, the biological sample may be sufficiently diluted to produce a specific signal for detecting low abundance cancer-associated autoantibodies after the peptide autoantigen elements are contacted with diluted sample for 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or longer. Preferably, the peptide autoantigen elements are contacted with diluted sample for about 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, or about 78 hours or longer. Additionally, conditions may further comprise contacting the peptide autoantigen elements with a fresh aliquot of the biological sample throughout the period wherein the peptide autoantigen elements are contacted with the biological sample. For instance, the peptide autoantigen elements may be contacted with a first biological sample for a first period of time, followed by removal of the first biological sample and contacting the peptide autoantigen elements with a second fresh aliquot of the biological sample for a second period of time, and repeating the contacting the peptide autoantigen elements with an n^th fresh aliquot of the biological sample n number of times for an m^th period of time, wherein n and m are independently integers from 0 to 10. A method of repeated contacting the peptide autoantigen elements with a fresh aliquot of the biological sample may be as diagrammed in FIG. 8. Preferably, the peptide autoantigen elements are contacted with a first biological sample for about 6, 12, or about 18 hours, followed by removal of the first biological sample and contacting the peptide autoantigen elements with a second fresh aliquot of the biological sample for a second period of time, and repeating the contacting the peptide autoantigen elements 2, 3, or 4 times.

[00053] The biological sample may further be processed to enhance the specific signal for detecting low abundance cancer-associated autoantibodies. For instance, the biological sample may further be processed to discard antibodies that do not bind peptide autoantigen elements of the disclosure, thereby reducing the background signal that may develop when low abundance antibodies such as cancer-associated autoantibodies are to be detected. Preferably, the biological sample is further processed as described in FIGS. 3 and 4, and Section II below.

[00054] The method further comprises providing a plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences. Cancer associated protein sequences are known in the art, and may be any known or yet to be discovered tumor suppressor or oncogene. The cancer-associated protein sequences may be selected from the group consisting of wild type sequences of cancer-associated proteins, sequences of abnormally expressed proteins, sequences of cancer-associated proteins having cancer-associated mutations, or combinations thereof. Preferably, the cancer-associated protein sequences are protein sequences capable of inducing a cancer-therapeutic autoimmune response during early stages of cancer. Preferably, the panel of cancer-associated protein sequences may be selected from the group of protein sequences consisting of p53, PI3-kinase, K-ras, β-catenin, Smad4, B-raf, EGFR, FGRFR3, PDGFRA, SRSF2, EP300, NFE2L2, IDH1 , ATM, NOTCH 1 , NOTCH2, FBXW7, MED1 2, H3F3A, SETBP1 , SF3B1 , ALK, CREBBP, CTNNB1 , FLT3, GNAS, JAK2, KIT, NRAS, PIK3CA, PTEN, TP53, VHL, and combinations thereof.

[00055] Peptide epitopes may be derived from a panel of peptide autoantigen elements described above. For instance, peptide epitopes are derived from a panel of peptide autoantigen elements as described in Section V and FIG. 6. Such peptide epitopes may be capable of inducing therapeutic autoimmunity against cancer in a subject. Preferably, the subject is suspected of having cancer, is at risk of developing cancer, or is diagnosed with an early stage of cancer.

II. Identify Cancer-associated Autoantibodies.

[00056] In another aspect, the present disclosure provides a method for identifying cancer-associated autoantibodies. The method comprises obtaining a serum sample from one or more subjects having at least an early stage cancer. At least two isotypes of immunoglobulins are isolated from the serum sample, to obtain a fraction for each isotype. A first concentration of each isotype fraction is contacted with a first autoantigen array comprising a first set of a plurality of different cancer- associated autoantigen elements. Each autoantigen element further comprises at least one immunologic epitope. Cancer-associated autoantibodies that specifically bind to cancer-associated autoantigen elements in the first autoantigen array are isolated from isotype fraction to provide a reduced autoantibody pool. A subset of cancer-associated autoantigen elements is then subtracted from the first set of cancer-associated autoantigen elements in the first array to obtain a remainder set, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample. A second autoantigen array is also obtained. The second set consists of the remainder set of cancer-associated autoantigen elements. Contacting the second autoantibody array with an optimal dilution of the reduced autoantibody pool to produce a specific binding signal identifies the cancer-associated antibodies.

[00057] Alternatively, a method for identifying cancer-associated autoantibodies comprises obtaining a serum sample from one or more subjects having at least an early stage cancer, and isolating at least two isotypes of immunoglobulins from the serum sample, to obtain a fraction for each isotype. A high concentration of each isotype fraction is contacted with a first autoantigen array comprising a plurality of different cancer-associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope(s). The method further comprises identifying any cancer-associated autoantigen elements in the array that specifically bind to autoantibodies in the serum sample, wherein each cancer-associated autoantigen element that specifically binds to an autoantibody identifies a complete autoantigen protein. The complete autoantigen proteins identified are used to identify and isolate target autoantibodies from each isotype fraction, to produce a reduced autoantibody pool for each isotype fraction. Additionally, an optimal dilution of each reduced autoantibody pool is identified for each isotype fraction. The optimal dilution of each reduced autoantibody pool is then contacted with a second autoantigen array comprising a plurality of different cancer- associated autoantigen elements. The plurality of different cancer-associated autoantigen elements in the second autoantigen array is a subset of the plurality of different cancer associated-associated autoantigen elements in the first autoantigen array, the subset consisting of the cancer-associated autoantigen elements that specifically bind to identified autoantibodies, to produce a specific binding signal. The specific binding signal indicates target autoantibodies of interest.

[00058] A method for identifying cancer-associated autoantibodies may also comprise obtaining a serum sample from one or more subjects having at least an early stage cancer, and isolating at least two isotypes of immunoglobulins from the serum sample, to obtain a fraction for each isotype. A first concentration of each isotype fraction is contacted with a first autoantigen array comprising a first set of a plurality of different cancer-associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope(s). Each cancer-associated autoantibody that specifically binds to cancer-associated autoantigen elements in the first autoantigen array is then isolated from each isotype fraction to provide a reduced autoantibody pool, and a subset of cancer-associated autoantigen elements from the first set of cancer-associated autoantigen elements in the first array is subtracted to obtain a remainder set. The subset consists of cancer- associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample. Next, a second autoantigen array consisting of the remainder set of cancer-associated autoantigen elements is obtained, and a maximum viable concentration of each isotype fraction using the second array is identified. Finally, the second autoantigen array is contacted with the maximum viable concentration of the biological sample to produce a specific binding signal, thereby identifying cancer- associated autoantibodies.

[00059] A method may further comprise comparing the immune response of the target autoantibodies of interest against corresponding native autoantigens, to the immune response of the target autoantibodies of interest against corresponding engineered variants of the autoantigens. Additionally, contacting of an autoantigen array with any serum fraction or serum dilution comprises contacting under conditions capable of producing a specific signal for detecting cancer-associated autoantibodies.

[00060] Conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies comprise sufficiently diluting the biological sample to produce a specific signal after contacting the peptide autoantigen at room temperature for an extended period of time, and may be as described in Section I above.

[00061 ] Identifying any cancer-associated autoantigen elements in the array that specifically bind to autoantibodies in the serum sample may comprise using immunoprecipitation to bind free autoantibody in each isotype fraction to a plurality of complete autoantigen proteins or linear peptides derived from the complete autoantigen proteins, wherein the proteins or peptides are fused to agarose beads. The bound autoantibodies may then be eluted to produce a reduced autoantibody pool for each isotype fraction. Preferably, the immunoprecipitation may be as diagrammed in FIGS. 3 and 4.

[00062] The cancer-associated autoantigen elements may be selected from the group consisting of wild type sequences of cancer-associated proteins, sequences of cancer-associated proteins having cancer-associated mutations, or combinations thereof. Preferably, the panel of cancer-associated autoantigen elements may be sequences derived from the group of protein sequences consisting of p53, PI3-kinase, K-ras, β-catenin, Smad4, B-raf, EGFR, FGRFR3, PDGFRA, SRSF2, EP300, NFE2L2, IDH1 , ATM, NOTCH1 , NOTCH2, FBXW7, MED1 2, H3F3A, SETBP1 , SF3B1 , ALK, CREBBP, CTNNB1 , FLT3, GNAS, JAK2, KIT, NRAS, PIK3CA, PTEN, TP53, VHL, and combinations thereof. [00063] Epitopes derived from one or more of the cancer-associated autoantigen elements may be capable of inducing therapeutic autoimmunity against cancer in a subject. As it will be recognized by a skilled artisan, the term "therapeutic autoimmunity against cancer" refers to a sufficient autoimmune reaction to produce a measurable effect on a cancer. The population of subjects primarily comprises or consists of subjects suspected of having a cancer, identified as at risk of developing a cancer, or has a diagnosis of a cancer.

[00064] The serum sample may be a pool of serum samples obtained from a population of subjects having at least an early stage cancer. Alternatively, the serum sample may be a serum sample obtained from a single subject having at least an early stage cancer. Further, the pool of identified cancer-associated autoantibodies may be associated with a type of cancer, a class of cancer, or a stage in the development of cancer. Alternatively, the pool of identified cancer- associated autoantibodies may not be specific to any one type of cancer, a class of cancer, or a stage in the development of cancer, thereby identifying autoantibodies generally associated with cancer.

A. Cancer

[00065] As it will be recognized by individuals skilled in the art, cancer as used throughout the instant disclosure may be one or more neoplasm or cancer. The neoplasm may be malignant or benign, the cancer may be primary or metastatic; the neoplasm or cancer may be early stage or late stage. Non-limiting examples of neoplasms or cancers that may be treated include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas (childhood cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial adenomas/carcinoids, Burkitt lymphoma, carcinoid tumors (childhood, gastrointestinal), carcinoma of unknown primary, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancers (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumors (childhood extracranial, extragonadal, ovarian), gestational trophoblastic tumor, gliomas (adult, childhood brain stem, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemias (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip and oral cavity cancer, liver cancer (primary), lung cancers (non-small cell, small cell), lymphomas (AIDS-related, Burkitt, cutaneous T- cell, Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia (Waldenstrom), malignant fibrous histiocytoma of bone/osteosarcoma, medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cell carcinoma, mesotheliomas (adult malignant, childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia (chronic), myeloid leukemias (adult acute, childhood acute), multiple myeloma, myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer, sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sezary syndrome, skin cancers (nonmelanoma, melanoma), skin carcinoma (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary (metastatic), stomach cancer, supratentorial primitive neuroectodermal tumor (childhood), T-Cell lymphoma (cutaneous), testicular cancer, throat cancer, thymoma (childhood), thymoma and thymic carcinoma, thyroid cancer, thyroid cancer (childhood), transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor (gestational), enknown primary site (adult, childhood), ureter and renal pelvis transitional cell cancer, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (childhood), vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor (childhood).

III. Signature of Cancer-associated Autoantibodies

[00066] In yet another aspect, the present disclosure provides a method of determining a signature of circulating cancer-associated autoantibodies in a subject. The method comprises providing a biological sample from the subject and a panel of cancer-associated autoantigen elements. The panel of cancer-associated autoantigen elements may be identified as described in any of Section I or Section II above. The panel of cancer-associated autoantigen elements is contacted with the biological sample under conditions capable of producing a specific binding signal to produce a signature of circulating cancer-associated autoantibodies in a subject. The signature of circulating cancer-associated autoantibodies comprises the specific binding signal produced by binding of circulating autoantibodies to one or more of the cancer-associated autoantigen elements in the panel. A signature of cancer- associated autoantibodies may be used for diagnostic or prognostic applications. IV. Array

[00067] The disclosure also provides a peptide array. The array comprises a panel of two or more peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies. The peptide autoantigens are identified using a method for identifying peptide autoantigens as described in Section I or Section II above. Additionally, peptide autoantigen elements may comprise native peptide sequences, peptide sequences comprising cancer- associated mutations, engineered peptide sequences, or any combination thereof. Engineered peptide sequences may be as described in Section V below.

V. Inducing Protective Immunity Against Cancer

[00068] In another aspect, the present disclosure provides a method of inducing protective immunity against cancer in a subject. The method comprises administering to the subject a composition comprising one or more peptide epitopes derived from one or more peptide autoantigen elements identified using a method for identifying peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies. A method for identifying peptide autoantigen elements may be as described in any of Section I or Section II above.

[00069] The present disclosure also provides a pharmaceutical composition comprising the composition of peptide epitopes, and at least one pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutically acceptable carrier, excipient or diluent can be, for example, an adjuvant as described below.

[00070] The present disclosure also provides peptide vaccines. Peptide vaccines comprise at least one peptide or a combination of two, three, four, five, six, seven, eight, nine, ten or more than ten peptides, each acting as an autoantigen, in combination with a pharmaceutically acceptable carrier, excipient or diluent. For instance, peptide vaccines may be loaded into delivery vehicle for administration. Vaccine delivery vehicles are known in the art. Non-limiting examples of delivery vehicles include a liposome or poloxamer, or a viral delivery vehicle such as an HPV virion lacking genetic material.

[00071 ] Additionally, in one aspect, a pharmaceutically acceptable carrier, excipient, or diluent is or includes a vaccine adjuvant. Vaccine adjuvants include any compound, or composition that potentiates the protective immune response to the autoantigen. The adjuvant may also stabilize the vaccine formulation. Compounds and compositions suitable for use as vaccine adjuvants are well known in the art, and generally include aqueous adjuvants such as aluminum or calcium salts and oils. Suitable vaccine adjuvants for example include but are not limited to aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, mineral oil, emulsifiers, cytokines (such as but not limited to 11-1 , IL-2 and 11-12), chitosan-based adjuvants, saponins, Freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FIA), and combinations thereof.

[00072] The one or more peptide epitopes derived from one or more cancer-associated autoantigen elements may comprise native peptide sequences, peptide sequences comprising cancer-associated mutations, engineered peptide sequences, or any combination thereof. For instance, the one or more peptide epitopes of the instant disclosure may each be engineered to comprise a modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject.

[00073] Non-limiting examples of modifications that may be suitable in a peptide epitope of the present disclosure include amino acid substitutions with naturally-occurring amino acids, unnatural amino acids, amino acid analogs, amino acid mimetics, non-canonical amino acid residues, or amino acid derivatives, amino acid deletions, amino acid insertions, fusion with other amino acid sequences, chemical modification of amino acid residues including crosslinking of amino acid residues, modifications to side chains, peptide circularization or cyclization, or any other method of modifying peptides that may result in enhanced immunogenicity and reduced autoreactivity in a subject.

[00074] The term "Naturally occurring amino acids" as used herein refers to those twenty L-amino acids encoded by the universal genetic codes and appearing in proteins or peptides, as well as selenocysteine and pyrrolysine that are incorporated into proteins by distinctive biosynthetic mechanisms. They include Histidine, Alanine, Isoleucine, Arginine, Leucine, Asparagine, Lysine, Aspartic acid, Methionine, Cysteine, Phenylalanine, Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine, selenocysteine and pyrrolysine. The term "Naturally occurring amino acids" also refers to those produced by the body, but are not encoded by the universal genetic codes, such as β-Alanine, Ornithine, and citrulline. The term "Naturally occurring amino acids" further includes those naturally occurring L-amino acids that are later modified (e.g., via post- translational modification by enzymes) in the body, such as hydroxyproline, v- carboxyglutamate, and O-phosphoserine.

[00075] The term "unnatural amino acids" as used herein is intended to include the "D" stereochemical form of the naturally occurring amino acids described above. It is further understood that the term "unnatural amino acids" includes homologues of the natural L-amino acids or their D isomers, and synthetically modified forms of the natural L-amino acids or their D isomers. The synthetically modified forms include, but are not limited to, amino acids having side chains shortened or lengthened by up to two carbon atoms, amino acids comprising optionally substituted aryl groups, and amino acids comprised halogenated groups, preferably halogenated alkyl and aryl groups and also N substituted amino acids e.g. N-methyl-Histidine, N-methyl-Alanine, N-methyl-lsoleucine, N-methyl-Arginine, N- methyl-Leucine, N-methyl-Asparagine, N-methyl-Lysine, N-methyl-Aspartic acid, N- methyl-Methionine, N-methyl-Cysteine, N-methyl-Phenylalanine, N-methyl-Glutamic acid, N-methyl-Threonine, N-methyl-Glutamine, N-methyl-Tryptophan, N-methyl- Glycine, N-methyl-Valine, N-methyl-Proline, N-methyl-Serine, N-methyl-Tyrosine, N- methyl-selenocysteine, and N-methyl-pyrrolysine, each including an L or D isomer.

[00076] The term "amino acid analogs" as used herein refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. They include 3-aminoalanine, 3-dimethylaminoalanine, 2-amino-4- (dimethylamino)butanoic acid, 2,4-diaminobutanoic acid, 2-amino-6- (dimethylamino)hexanoic acid, 2-amino-5-(dimethylamino)pentanoic acid, homoserine, norleucine, cysteine sulfonic acid, cysteine sulfinic acid, methionine sulfoxide, and methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid analogs also include D isomers of the above-referenced L-isomers.

[00077] The term "amino acid mimetics" as used herein refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

[00078] For example, one or more amino acid residues may be replaced with amino acid derivatives wherein replacing the one or more amino acid residues results in enhanced immunogenicity and reduced autoreactivity in a subject. Another example of a modification that may be used to in enhanced immunogenicity and reduced autoreactivity in a subject is cyclization or circularization of a peptide. Cyclization or circularization of a peptide can increase the peptide's antigenic and immunogenic potency by mimicking a naturally-occurring conformation of the peptide in the context of the native protein in order to optimize the effector immune responses that are elicited. See, e.g., U.S. Pat. No. 5,001 ,049 which is incorporated by reference herein.

[00079] When the one or more peptide epitopes is engineered to comprise a modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject, a peptide epitope may comprise 1 , 2, 3, 4, 5, or more modifications. Preferably, a peptide epitope comprises one modification in every 15 amino acid residues of a peptide epitope. One example of a peptide epitope and modifications that may be suitable for such a peptide epitope is shown in FIG. 6. Additionally, it will be recognized that suitable modifications of peptide epitopes may be identified using the methods disclosed herein. For instance, modifications of a peptide epitope may be assessed using methods as described in Sections I and II. As such, the methods disclosed herein may also be used in successive iterations for developing and evolving peptide autoantigen elements having enhanced therapeutic autoimmunity against cancer without, or while significantly minimizing collateral damage that may be associated with autoimmunity. Similarly, the methods disclosed herein may also be used to assess the efficacy of a vaccine composition. For instance, the efficacy of a vaccine composition may be assessed by identifying the cancer associated antibodies in a subject before administering a vaccine composition and after administration of the vaccine composition, and comparing the obtained results.

[00080] In some embodiments, the one or more peptide epitopes comprise cancer-associated mutations. Preferably, the cancer-associated mutations are known, or have been determined to generate autoantibodies against cancer. In other embodiments, the one or more peptide epitopes comprise cancer-associated mutations, and are further engineered to comprise a modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject. The modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject may be any modification to a peptide or amino acid residue as described above. Preferably, the modification comprises one or more minor amino acid substitutions resulting in enhanced immunogenicity and reduced autoreactivity in the subject. As used herein, the term "minor amino acid substitution" refers to substitutions of an amino acid residue with an amino acid residue having similar properties such as hydrophobicity, the molecular bulk of the side chain, the location of the core structural functional group (alpha- (a-), beta- (β-), gamma- (γ-) or delta- (δ-)) of the amino acid residue, polarity, pH level, and side-chain group type (aliphatic, acyclic, aromatic, containing hydroxyl or sulfur, etc.).

[00081 ] The one or more peptide epitopes that may be used in a composition or method for inducing protective immunity may be selected based on the frequency in which a cancer associated mutation that may be present in the peptide epitopes may be present in a population, and/or the frequency of autoantibodies generated against the peptide epitopes. Preferably, peptide epitopes are selected based on the frequency a cancer-associated mutation and the frequency of autoantibodies generated against the peptide epitopes.

[00082] In another aspect of the disclosure, a method of identifying one or more peptide epitopes for inducing protective immunity against a cancer in a subject is provided. The method comprises providing a plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences. The plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences may be derived as described above. The method further comprises modifying the peptide autoantigen elements. Modifying the peptide autoantigen elements may be as described above, and may comprise engineering the autoantigen elements to comprise a modification selected from the group consisting of amino acid substitutions with naturally-occurring amino acids, amino acid substitutions with unnatural amino acids, amino acid substitutions with amino acid analogs, amino acid substitutions with amino acid mimetics, amino acid substitutions with non-canonical amino acid residues, amino acid substitutions with amino acid derivatives, amino acid deletions, amino acid insertions, fusion with other amino acid sequences, chemical modification of amino acid residues, modification to side chains of amino acid residues, peptide circularization or cyclization, and combinations thereof. Finally, the method comprises identifying from the peptide autoantigen elements, one or more peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies. The identified peptide autoantigen elements are the one or more peptide epitopes for inducing protective immunity against a cancer in a subject.

[00083] The plurality of peptide autoantigen elements may comprise overlapping peptides derived from a panel of cancer-associated protein sequences. Additionally, the one or more peptide epitopes may each be derived from a cancer- associated protein sequence having a cancer-associated mutation and a sequence modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject. The one or more peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies may be identified by using any of the methods described in the instant disclosure.

[00084] A method of monitoring a cancer is also provided. The method comprises identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies, and using the panel of peptide autoantigen elements to monitor the cancer-associated autoantibodies, thereby monitoring the cancer. As used herein, the term "monitoring a cancer" refers to monitoring a type of cancer, a class of cancer, a stage in the development of cancer, or any other aspect of cancer diagnosis, prognosis, treatment, or development. For instance, the treatment of a cancer may be monitored throughout the duration of the treatment.

VI. PEPTIDES

[00085] In any of the embodiments described in the foregoing, consistent with the definition of "peptide" herein below, it should be understood that peptides of varying lengths can be used. At the same time, the inventors have found that using peptides of no more than eleven (1 1 ) amino acid residues in length helps prevent the immune system from mounting a response to the peptides without consideration for the mutant residue of interest. In view of the observation that antibodies usually form against peptides having five (5) or more amino acid residues, for example, a peptide having eleven (1 1 ) amino acid residues and including a key cancer mutation centrally located at position 6 in the peptide chain increases the likelihood that when antibodies form they have increased specificity for the mutant versions over native versions. Accordingly, preferred peptides for arrays and vaccine candidates are 7, 8, 9, 10 or 1 1 amino acids in length. A preferred peptide includes a key cancer mutation located centrally within any sequence of 7, 8, 9, 10 or 1 1 amino acids. For example, non-limiting examples of preferred peptides include a peptide with a key cancer mutation located at position 4 within a sequence of 7 amino acids; at position 4 or 5 within a sequence of 8 amino acids; at position 5 within a sequence of 9 amino acids, or at position 5 or 6 within a sequence of 10 amino acids.

DEFINITIONS

[00086] As used herein, the term "peptide" includes any moiety comprising amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (5-20 amino acids) and to longer chain oligopeptides (20-500 amino acids). The peptide may comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 or at least 45 amino acids joined to each other by peptide bonds or modified peptide bonds.

[00087] As used herein, the term "epitope" refers to a short peptide sequence derived from a protein antigen, which specifically binds to an MHC molecule and is specifically recognized by a particular T cell.

[00088] As used herein, the term "subject" may be utilized for any mammalian subject. Such mammalian subjects include, but are not limited to, humans and companion animals. Exemplary companion animals may include domesticated mammals (e.g., dogs, cats, horses), mammals with significant commercial value (e.g., dairy cows, beef cattle, sporting animals), mammals with significant scientific value (e.g., captive or free specimens of endangered species), or mammals which otherwise have value.

[00089] As used herein, the term "biological sample" refers to liquid or non- liquid samples from a wide variety of sources. Representative types of biological samples include tissue scrapings, whole blood, urine, cervical secretions, bronchial aspirates, sputum, saliva, feces, serum, synovial and cerebrospinal fluid, as well as laboratory preparations such as purified or partially purified macromolecules and cell culture materials. By way of example, the biological sample may be body fluid, body excretion, a population of cells, saliva, urine, mucus, tissue, or other biological sample type known in the art. Further examples of biological samples include, physiological/pathological body liquids (e.g., secretions, excretions, exudates and transudates) or cell suspensions (e.g., blood, lymph, synovial fluid, semen, saliva containing buccal cells, skin scrapings, hair root cells, etc.) of humans and animals; liquid extracts or homogenates of human or animal body tissues (e.g. , bone, liver, kidney, etc.).

EXAMPLES

Example 1. Identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies.

[00090] A plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences may be provided. Each autoantigen element may comprise at least one immunologic epitope. Additionally, a biological sample from one or more individuals having at least an early stage cancer may be provided. The peptide autoantigen elements may be contacted with the biological sample under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies. Peptide autoantigen elements may be selected for the panel by selecting a plurality of peptide autoantigen elements each capable of producing a specific binding signal.

[00091 ] One or more peptide epitopes may be derived from one or more cancer-associated autoantigen elements identified above. A composition for inducing protective immunity against a cancer in a subject may be prepared using one or more of the peptide epitopes.

Example 2. Determining a signature of circulating cancer-associated autoantibodies in a subject.

[00092] A biological sample from a subject may be provided, and a panel of peptide autoantigen elements may be identified as described in Example 1 . The peptide autoantigen elements may be contacted with the biological sample under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies. The signature of circulating cancer-associated autoantibodies in a subject comprises a plurality of specific binding signals, each specific binding signal produced by each peptide probe.

Example 3. Identifying cancer-associated autoantibodies.

[00093] A biological sample may be obtained from one or more subjects having at least an early stage cancer, and at least two isotypes of immunoglobulins may be isolated from the serum sample, to obtain a fraction for each isotype.

[00094] A first concentration of each isotype fraction may be contacted with a first autoantigen array comprising a first set of a plurality of different cancer- associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope. Each cancer-associated autoantibody that specifically binds to a cancer-associated autoantigen elements in the first autoantigen array may be isolated from each isotype fraction to provide a reduced autoantibody pool. A subset of cancer-associated autoantigen elements may be subtracted from the first set of cancer-associated autoantigen elements in the first array to obtain a remainder set, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample.

[00095] A second autoantigen array may be obtained comprising the remainder set of cancer-associated autoantigen elements from the previous step, and the second autoantigen array may be contacted with an optimal dilution of the reduced autoantibody pool, to produce a specific binding signal, wherein the autoantibodies that produce a specific binding signal are cancer-associated autoantibodies.

[00096] Alternatively, after contacting a high concentration of each isotype fraction with a first autoantigen array, any cancer-associated autoantigen elements may be identified in the array that specifically bind to autoantibodies in the serum sample, wherein each cancer-associated autoantigen element that specifically binds to an autoantibody identifies a complete autoantigen protein. The complete autoantigen proteins identified may be used to identify and isolate target autoantibodies from each isotype fraction, to produce a reduced autoantibody pool for each isotype fraction. An optimal dilution of each reduced autoantibody pool may be identified for each isotype fraction, and the optimal dilution of each reduced autoantibody pool may be contacted with a second autoantigen array comprising a plurality of different cancer-associated autoantigen elements, wherein the plurality of different cancer-associated autoantigen elements in the second autoantigen array is a subset of the plurality of different cancer associated-associated autoantigen elements in the first autoantigen array, the subset comprising the cancer-associated autoantigen elements that specifically bind to autoantibodies, to produce a specific binding signal, wherein the specific binding signal indicates target autoantibodies of interest.

[00097] In another alternative, after contacting a high concentration of each isotype fraction with a first autoantigen array, each cancer-associated autoantibody that specifically binds to a cancer-associated autoantigen element in the first autoantigen array may be isolated from each isotype fraction to provide a reduced autoantibody pool. A subset of cancer-associated autoantigen elements may be subtracted from the first set of cancer-associated autoantigen elements in the first autoantigen array to obtain a remainder set of cancer-associated autoantigen elements, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample. A second autoantigen array may be obtained consisting of the remainder set of cancer-associated autoantigen elements, and a maximum viable concentration of each isotype fraction may be identified using the second autoantigen array. The second autoantigen array may be contacted with the maximum viable concentration of the biological sample to produce a specific binding signal, thereby identifying cancer-associated autoantibodies.

Claims

CLAIMS What is claimed is:

1 . A method of identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies, the method comprising:

a. providing a plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences, wherein each autoantigen element comprises at least one immunologic epitope;

b. providing a biological sample from one or more individuals having at least an early stage cancer;

c. contacting the peptide autoantigen elements with the biological sample under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies; and d. selecting peptide autoantigen elements for the panel by selecting a plurality of peptide autoantigen elements each capable of producing a specific binding signal under the conditions of step (c).

2. The method of claim 1 , wherein the plurality of peptide autoantigen elements are derived from a panel of cancer-associated protein sequences using a method comprising:

a. isolating at least two isotypes of immunoglobulins from the biological sample to obtain a fraction for each isotype;

b. contacting a first concentration of each isotype fraction with a first set of cancer-associated autoantigen elements;

c. isolating from each isotype fraction each cancer-associated autoantibody that specifically binds to cancer-associated autoantigen elements in the first set of cancer-associated autoantigen elements to provide a reduced autoantibody pool;

d. subtracting a subset of cancer-associated autoantigen elements from the first set of cancer-associated autoantigen elements to obtain a remainder set, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the biological sample; and e. obtaining a second set of cancer-associated autoantigen elements consisting of the remainder set of cancer-associated autoantigen elements, wherein the remainder set is the panel of peptide autoantigen elements capable of detecting low abundance cancer- associated autoantibodies.

3. The method of claim 1 , wherein conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies comprise:

a. identifying a maximum viable concentration of the biological sample; and

b. contacting the peptide autoantigen elements with the maximum viable concentration of the biological sample.

4. The method of claim 3, wherein identifying a maximum viable concentration of the biological sample comprises:

a. contacting a first set of peptide autoantigen elements with a first concentration of the biological sample to identify a subset of peptide autoantigen elements, wherein the subset of peptide autoantigen elements produce a non-specific signal;

b. diluting the first concentration of the biological sample to obtain a series of dilutions of the biological sample;

c. contacting the series of dilutions of the biological sample from step (b) with the subset of peptide autoantigen elements; and

d. identifying from the series of dilutions in step (b), a dilution comprising the highest concentration of the biological sample that does not produce a signal when the subset of peptide autoantigen elements is contacted with the series of dilutions in step (b);

wherein the dilution of the biological sample identified in step (d) is the maximum viable concentration of the biological sample.

5. The method of claim 1 , wherein conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies comprise sufficiently diluting the biological sample to produce the specific signal after contacting the peptide autoantigen elements at room temperature for about 78 hours.

6. The method of claim 1 , wherein the biological sample is further processed to enhance the specific signal for detecting low abundance cancer-associated autoantibodies.

7. The method of claim 1 , wherein the panel of cancer-associated protein sequences is selected from a group of protein sequences consisting of p53, PI3-kinase, K-ras, β-catenin, Smad4, B-raf, EGFR, FGRFR3, PDGFRA, SRSF2, EP300, NFE2L2, IDH1 , ATM, NOTCH 1 , NOTCH2, FBXW7, MED1 2, H3F3A, SETBP1 , SF3B1 , ALK, CREBBP, CTNNB1 , FLT3, GNAS, JAK2, KIT, NRAS, PIK3CA, PTEN, TP53, VHL, and combinations thereof.

8. The method of claim 1 , wherein the cancer-associated protein sequences are selected from the group consisting of wild type sequences of cancer- associated proteins, sequences of cancer-associated proteins having cancer- associated mutations, or combinations thereof.

9. The method of claim 1 , wherein peptide epitopes derived from one or more of the panel of peptide autoantigen elements are capable of inducing therapeutic autoimmunity against cancer in a subject.

10. The method of claim 9, wherein the subject is suspected of having cancer, is at risk of developing cancer, or is diagnosed with an early stage of cancer.

1 1 . A composition for inducing protective immunity against a cancer in a subject, comprising one or more peptide epitopes derived from one or more cancer- associated autoantigen elements identified in the method of claim 1 .

12. The composition of claim 1 1 , wherein the one or more peptide epitopes derived from one or more cancer-associated autoantigen elements comprise native peptide sequences, mutant peptide sequences, engineered peptide sequences, or any combination thereof.

13. A pharmaceutical composition comprising the composition of claim 1 1 and at least one pharmaceutically acceptable carrier, excipient or diluent.

14. A method of inducing protective immunity against cancer in a subject, the method comprising administering to the subject a composition comprising one or more peptide epitopes derived from one or more peptide autoantigen element identified using the method of claim 1 .

15. The method of claim 14, wherein the one or more peptide epitopes each comprise a sequence modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject.

16. A method of determining a signature of circulating cancer-associated autoantibodies in a subject, the method comprising:

(a) providing a biological sample from the subject;

(b) providing a panel of peptide autoantigen elements identified using the method of claim 1 ; and

(c) contacting the peptide autoantigen elements with the biological sample under conditions capable of producing a specific signal for detecting low abundance cancer-associated autoantibodies; wherein the signature of circulating cancer-associated autoantibodies in a subject comprises a plurality of specific binding signals, each specific binding signal produced by each peptide probe.

17. A peptide array, the array comprising a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies, wherein the panel of peptide autoantigen elements are identified using the method of claim 1 .

18. A method for identifying cancer-associated autoantibodies, comprising:

a. obtaining a biological sample from one or more subjects having at least an early stage cancer;

b. isolating at least two isotypes of immunoglobulins from the serum sample, to obtain a fraction for each isotype;

c. contacting a first concentration of each isotype fraction with a first autoantigen array comprising a first set of a plurality of different cancer- associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope;

d. isolating from each isotype fraction each cancer-associated autoantibody that specifically binds to a cancer-associated autoantigen elements in the first autoantigen array to provide a reduced autoantibody pool;

e. subtracting a subset of cancer-associated autoantigen elements from the first set of cancer-associated autoantigen elements in the first array to obtain a remainder set, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample from (a);

f. obtaining a second autoantigen array comprising the remainder set of cancer-associated autoantigen elements from (e); and

g. contacting the second autoantigen array from (f) with an optimal dilution of the reduced autoantibody pool from (d), to produce a specific binding signal, wherein the autoantibodies that produce a specific binding signal are cancer-associated autoantibodies.

19. A method for identifying cancer-associated autoantibodies, comprising:

a. obtaining a serum sample from one or more subjects having at least an early stage cancer;

b. isolating at least two isotypes of immunoglobulins from the serum sample, to obtain a fraction for each isotype; c. contacting a high concentration of each isotype fraction with a first autoantigen array comprising a plurality of different cancer-associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope;

d. identifying any cancer-associated autoantigen elements in the array that specifically bind to autoantibodies in the serum sample from (a), wherein each cancer-associated autoantigen element that specifically binds to an autoantibody identifies a complete autoantigen protein; e. using the complete autoantigen proteins identified from step (d) to identify and isolate target autoantibodies from each isotype fraction, to produce a reduced autoantibody pool for each isotype fraction;

f. identifying an optimal dilution of each reduced autoantibody pool from

(e) for each isotype fraction; and

g. contacting the optimal dilution of each reduced autoantibody pool from

(f) , with a second autoantigen array comprising a plurality of different cancer-associated autoantigen elements, wherein the plurality of different cancer-associated autoantigen elements in the second autoantigen array is a subset of the plurality of different cancer associated-associated autoantigen elements in the first autoantigen array, the subset comprising the cancer-associated autoantigen elements that specifically bind to autoantibodies in step (d), to produce a specific binding signal, wherein the specific binding signal indicates target autoantibodies of interest.

A method for identifying cancer-associated autoantibodies, comprising:

c. contacting a first concentration of each isotype fraction with a first autoantigen array comprising a first set of cancer-associated autoantigen elements, wherein each autoantigen element further comprises at least one immunologic epitope; d. isolating from each isotype fraction each cancer-associated autoantibody that specifically binds to a cancer-associated autoantigen element in the first autoantigen array to provide a reduced autoantibody pool;

e. subtracting a subset of cancer-associated autoantigen elements from the first set of cancer-associated autoantigen elements in the first autoantigen array to obtain a remainder set of cancer-associated autoantigen elements, the subset consisting of cancer-associated autoantigen elements that do not specifically bind to autoantibodies in the serum sample from (a);

f. obtaining a second autoantigen array consisting of the remainder set of cancer-associated autoantigen elements from (e);

g. identifying a maximum viable concentration of each isotype fraction using the second autoantigen array; and

h. contacting the second autoantigen array from (f) with the maximum viable concentration of the biological sample from (g), to produce a specific binding signal, thereby identifying cancer-associated autoantibodies.

21 . The method of claim 18, 19 or 20, wherein the autoantigen protein is a complete autoantigen protein or linear peptides derived from the complete autoantigen protein.

22. The method of claim 18, 19 or 20, further comprising comparing an immune response of the target autoantibodies of interest against corresponding native autoantigens, to the immune response of the target autoantibodies of interest against corresponding engineered variants of the autoantigens.

23. The method of claim 18, 1 9 or 20, wherein the contacting of an autoantigen array with any serum fraction or serum dilution comprises contacting under conditions capable of producing a specific signal for detecting cancer- associated autoantibodies.

24. The method of claim 18, 1 9 or 20, wherein the first concentration of each isotype fraction and the optimal dilution are identified using the method described in claim 4.

25. The method of claim 18, 1 9 or 20, wherein the optimal dilution comprises a dilution sufficient to produce a specific binding signal after contact with the second microarray at room temperature for about 78 hours.

26. The method of claim 1 8, 1 9 or 20, wherein step (d) comprises using immunoprecipitation to bind free autoantibody in each isotype fraction to a plurality of complete autoantigen proteins fused to agarose beads, and eluting the bound autoantibodies.

27. The method of claim 1 8, 19 or 20, wherein the plurality of different cancer- associated autoantigen elements in step (c) comprises at least one sequence derived from p53, PI3-kinase, K-ras, β-catenin, Smad4, B-raf, EGFR, FGRFR3, PDGFRA, SRSF2, EP300, NFE2L2, IDH1 , ATM, NOTCH 1 , NOTCH2, FBXW7, MED1 2, H3F3A, SETBP1 , SF3B1 , ALK, CREBBP, CTNNB1 , FLT3, GNAS, JAK2, KIT, NRAS, PIK3CA, PTEN, TP53, VHL, and combinations thereof.

28. The method of claim 1 8, 19 or 20, wherein the plurality of different cancer- associated autoantigen elements in step (c) are selected from wild type sequences of cancer-associated proteins, sequences of cancer-associated proteins having naturally occurring or engineered cancer-associated mutations, or any combination thereof.

29. The method of claim 18, 19 or 20, wherein epitopes derived from one or more of the cancer-associated autoantigen elements in step (c) are capable of inducing therapeutic autoimmunity against cancer in a subject.

30. The method of claim 18, 19 or 20, wherein the one or more subjects primarily comprises or consists of subjects suspected of having a cancer, identified as at risk of developing a cancer, or has a diagnosis of a cancer.

31 . The method of claim 30, wherein the serum sample is a pool of serum samples obtained from a population of subjects having at least an early stage cancer.

32. The method of claim 18, 19 or 20, wherein the pool of identified cancer- associated autoantibodies are associated with a type of cancer, a class of cancer, or a stage in cancer development.

33. A method of inducing protective immunity against a cancer in a subject, the method comprising administering to the subject a composition comprising one or more peptide epitopes derived from one or more cancer-associated autoantigen elements identified from the specific binding signal from step (g) in the method of claim 18, 19 or 20.

34. The method of claim 33, wherein the one or more peptide epitopes each comprise a sequence modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject.

35. A method of determining a signature of circulating cancer-associated autoantibodies in a subject, the method comprising: a) providing a biological sample from the subject; b) providing a panel of cancer-associated autoantigen elements identified from step (g) in the method of claim 1 8, 19 or 20 or of claim 1 ; c) contacting the panel of cancer-associated autoantigen elements with the biological sample under conditions capable of producing a specific binding signal; wherein the signature of circulating cancer-associated autoantibodies in a subject comprises the specific binding signal produced by binding of circulating autoantibodies to one or more of the cancer-associated autoantigen elements in the panel.

36. A peptide array comprising a plurality of cancer-associated autoantigens capable of detecting early-stage cancer-associated autoantibodies and identified using the method of claim 1 8, 19 or 20 or claim 1 .

37. A composition for inducing protective immunity against a cancer in a subject, comprising one or more peptide epitopes derived from one or more cancer-associated autoantigen elements identified from the specific binding signal from step (g) in the method of claim 1 8, 1 9 or 20 or from claim 1 .

38. The composition of claim 37, wherein the one or more peptide epitopes derived from one or more cancer-associated autoantigen elements comprise native peptide sequences, mutant peptide sequences, engineered peptide sequences, or any combination thereof.

39. A pharmaceutical composition comprising the composition of claim 37 and at least one pharmaceutically acceptable carrier, excipient or diluent.

40. A method of identifying one or more peptide epitopes for inducing protective immunity against a cancer in a subject, the method comprising:

a. providing a plurality of peptide autoantigen elements derived from a panel of cancer-associated protein sequences;

b. modifying the peptide autoantigen elements of (a); and

c. identifying from peptide autoantigen elements of (a) or (b), one or more peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies, thereby identifying one or more peptide epitopes for inducing protective immunity against a cancer in a subject.

41 . The method of claim 40, wherein the plurality of peptide autoantigen elements comprises overlapping peptides derived from a panel of cancer- associated protein sequences.

42. The method of claim 40, wherein modifying the peptide autoantigen elements of (a) comprises engineering the autoantigen elements to comprise a modification selected from the group consisting of amino acid substitutions with naturally-occurring amino acids, amino acid substitutions with unnatural amino acids, amino acid substitutions with amino acid analogs, amino acid substitutions with amino acid mimetics, amino acid substitutions with non- canonical amino acid residues, amino acid substitutions with amino acid derivatives, amino acid deletions, amino acid insertions, fusion with other amino acid sequences, chemical modification of amino acid residues, modification to side chains of amino acid residues, peptide circularization or cyclization, and combinations thereof.

43. The method of claim 40, wherein the one or more peptide epitopes are each derived from a cancer-associated protein sequence having a cancer- associated mutation and a sequence modification, the modification resulting in enhanced immunogenicity and reduced autoreactivity in the subject.

44. The method of claim 40, wherein the one or more peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies are identified by using any of the methods of claims 1 -1 0, and step (g) in the method of claim 18, 19 or 20.

45. A method of monitoring a cancer in a subject, the method comprising:

a. identifying a panel of peptide autoantigen elements capable of detecting low abundance cancer-associated autoantibodies; and b. using the panel of peptide autoantigen elements of (a) to monitor the cancer-associated autoantibodies, thereby monitoring the cancer.

46. The method of claim 45, wherein the low abundance cancer-associated autoantibodies are associated with a type of cancer, a class of cancer, or a stage in cancer development.

47. The method of claim 45, wherein a cancer treatment is monitored.