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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • A—HUMAN NECESSITIES
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  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
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  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
  • G01N33/56977—HLA or MHC typing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in epitope analysis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7023—(Hyper)proliferation
  • G01N2800/7028—Cancer

Definitions

• the present disclosure is directed to methods of identifying mutant peptides useful for developing immunotherapeutics.
• Cytotoxic T-lymphocytes involved in cell- mediated immunity monitor changes in cellular health by scanning peptide epitopes, or antigens, on cell surfaces.
• Peptide epitopes originate from cellular proteins and serve as a display mechanism that allows cells to present evidence of current cellular processes. Both native and non-native proteins (often referred to as self and non-self, respectively) are processed for peptide epitope presentation. Most self peptides are derived from natural protein turnover and defective ribosomal products. Non-self peptides may be derived from proteins produced in the course of events such as viral and bacterial infection, disease, and cancer.
• MHC class I molecules are responsible for peptide epitope presentation to cytotoxic T cells.
• HLA human leukocyte antigen
• HLA-A, - B, and -C genes code for MHC class I (MHCI) proteins.
• a peptide typically 8-11 amino acids in length, will bind an MHCI molecule through interaction with a groove formed by two alpha helices positioned above an antiparallel beta sheet.
• peptide-MHC class I (pMHCI) molecules involve a series of sequential stages comprising: a) pro tease-mediated digestion of proteins; b) peptide transport into the endoplasmic reticulum (ER) mediated by the transporter associated with antigen processing (TAP); c) formation of pMHCI using newly synthesized MHCI molecules; and, d) transport of pMHCI to the cell surface.
• ER endoplasmic reticulum
• TAP antigen processing
• T cell receptors TCRs
• identification of a non-self antigen may result in cytotoxic T cell activation through a series of biochemical events mediated by associated enzymes, co-receptors, adaptor molecules, and transcription factors.
• An activated cytotoxic T cell will proliferate to produce a population of effector T cells expressing TCRs specific to the identified immunogenic peptide epitope.
• the amplification of T cells with TCR specificity to the identified non-self epitope results in immune-mediated apoptosis of cells displaying the activating non-self epitope.
• tumor antigens can be classified into two categories: tumor-associated self-antigens (e.g., cancer-testis antigens, differentiation antigens) and antigens derived from shared or patient- specific mutant proteins. Since the presentation of self-antigens in the thymus may result in the elimination of high avidity T cells, mutant neoantigens are likely to be more immunogenic.
• tumor-associated self-antigens e.g., cancer-testis antigens, differentiation antigens
• antigens derived from shared or patient- specific mutant proteins Since the presentation of self-antigens in the thymus may result in the elimination of high avidity T cells, mutant neoantigens are likely to be more immunogenic.
• immunotherapeutic epitopes is a challenging pursuit and efficient methods useful for the identification of efficacious epitopes are yet to be developed.
• mutant neoepitopes require laborious screening of a patient's tumor infiltrating lymphocytes for their ability to recognize an antigen from libraries constructed based on information from that patient's tumor exome sequence.
• mutant neoepitopes may be detected by mass spectrometry.
• mutant sequences have evaded detection because use of public proteomic databases that do not contain patient- specific mutations do not allow for their identification.
• the present application in one aspect provides a method of identifying a disease-specific immunogenic mutant peptide, comprising a) providing a set of variant-coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample, and b) selecting immunogenic variant-coding sequences from the set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the variant-coding sequences, thereby identifying the disease-specific
• the method comprises a) obtaining a first set of variant-coding sequences based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample, b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequence of the disease tissue in the individual, c) selecting a third set of epitope variant-coding sequences from the second set based on predicted ability of the peptides encoded by the expression variant-coding sequences to bind to an MHC class I molecule (MHCI), and d) selecting immunogenic variant-coding sequences from the third set comprising predicting immunogenicity of the peptides comprising a variant amino acid encoded by the epitope variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• MHCI MHC class I molecule
• the method further comprises i) obtaining a plurality of peptides that are bound to an MHCI molecule from the disease tissue, ii) subjecting the MHCI-bound peptides to mass spectrometry-based sequencing, and iii) correlating the mass spectrometry-derived sequence information of the MHCI-bound peptides with the immunogenic variant-coding sequences.
• the present application in another aspect provides a method of identifying a disease-specific immunogenic mutant peptide, comprising a) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual, b) subjecting the MHC-bound peptides to mass spectrometry-based sequencing, and c) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with a set of variant- coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample, thereby identifying the disease- specific immunogenic mutant peptide.
• the method comprises a) obtaining a first set of variant- coding sequences based on the genomic sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample, b) selecting a second set of expression variant-coding sequences from the first set based on transcriptomic sequences of the disease tissue in the individual, c) selecting a third set of epitope variant-coding sequences from the second set based on predicted ability of the peptides encoded by the expression variant-coding sequences to bind to an MHC class I molecule (MHCI), d) obtaining a plurality of peptides that are bound to an MHCI molecule from the disease tissue, e) subjecting the MHCI-bound peptides to mass spectrometry-based sequencing, and f) correlating the mass spectrometry-derived sequence information of the MHCI-bound peptides with the third set of epitope variant-coding sequences, thereby identifying the MHC class I molecule (MH
• predicting immunogenicity is based on one or more of the following parameters: i) binding affinity of the peptide to the MHCI molecule; ii) protein level of a peptide precursor containing the peptide; iii) expression level of the transcript encoding the peptide precursor; iv) processing efficiency of the peptide precursor by an immunoproteasome; v) timing of the expression of the transcript encoding the peptide precursor; vi) binding affinity of the peptide to a TCR molecule; vii) position of a variant amino acid within the peptide; viii) solvent exposure of the peptide when bound to a MHCI molecule; ix) solvent exposure of the variant amino acid when bound to a MHCI molecule; x) content of aromatic residues in the peptide; xi) properties of the variant amino acid when compared to the wild type residue; and xii) nature of the following parameters: i) binding affinity of the peptide to the MHCI molecule; i
• the peptides bound to MHCI are obtained by isolating MHCI/peptide complexes from the disease tissue and eluting the peptides from the MHCI.
• the isolation of MHCI/peptide complexes is carried out by
• the immunoprecipitation is carried out using an antibody specific for MHCI.
• the isolated peptides are further separated by chromatography prior to being subjected to mass spectrometry.
• obtaining a first set of variant-coding sequences comprises i) obtaining a first set of variant sequences based on the genomic sequences of the disease tissue in the individual, each variant sequence having a variation in the sequence compared to a reference sample, and ii) identifying the variants coding- sequences from the first set of variant sequences.
• the method further comprises synthesizing a peptide based on the sequence of the identified disease-specific immunogenic mutant peptide. In some embodiments, according to any of the methods described above, the method further comprises synthesizing a nucleic acid encoding a peptide based on the sequence of the identified disease-specific immunogenic mutant peptide. In some embodiments, the method further comprises testing the synthesized peptide for immunogenicity in vivo.
• the disease is cancer. In some embodiments, according to any of the methods described above, the individual is human.
• the present application in another aspect also provides a disease- specific mutant peptide or compositions of a disease-specific mutant peptide identified by any of the methods described herein.
• the composition comprises two or more disease- specific immunogenic mutant peptides described herein.
• the composition further comprises an adjuvant.
• the present application in yet another aspect also provides a method of treating a disease in an individual, comprising administering to the individual an effective amount of a composition comprising a disease- specific mutant peptide identified with any of the methods for identifying a disease- specific immunogenic mutant peptide disclose herein.
• the individual is the same individual from whom the disease- specific immunogenic mutant peptide is identified.
• the present application also provides an immunogenic composition comprising at least one disease-specific peptide or a precursor of such disease-specific peptide, wherein said disease-specific peptide is identified by any of the methods described herein.
• the immunogenic composition comprises a plurality of disease-specific peptides.
• the present application also provides an immunogenic composition comprising at least one nucleic acid encoding a disease-specific peptide, wherein said disease- specific peptide is identified by any of the methods described herein.
• the immunogenic composition comprises a plurality of nucleic acids each encoding at least one disease-specific peptide.
• the immunogenic composition comprising a nucleic acid encoding two or more (such as any of 3, 4, 5, 6, 7, 8, 9, or more) disease-specific peptides.
• the present application in yet another aspect also provides a method of stimulating an immune response in an individual with a disease comprising administering any immunogenic compositions described herein.
• the method further comprises administering another agent.
• the other agent is an immunomodulator.
• the other agent is a checkpoint protein.
• the other agent is an antagonist of PD-1 (such as an anti-PDl antibody).
• the other agent is an antagonist of PD-L1 (such as an anti-PD-Ll antibody).
• the present application in yet another aspect also provides a method of stimulating an immune response in an individual with a disease comprising: a) identifying a disease- specific immunogenic mutant peptide from a disease tissue in the individual according to any one of the identification method described above; b) producing a composition comprising a peptide or a nucleic acid encoding the peptide based on the sequence of the identified disease-specific immunogenic mutant peptide; c) administering the composition to the individual.
• the method further comprises administering a PD-1 antagonist (such as anti-PDl antibody) to the individual.
• the method further comprises administering a PD-L1 antagonist (such as anti-PD-Ll antibody) to the individual.
• FIG. 1 illustrates exemplary methods of immunogenic peptide identification.
• FIG. 2A illustrates the distribution of identified genes that were identified as epitopes presented on MHC molecules of the MC-38 cell line in relation to the measured reads per kilobase of exon model per million mapped reads (RPKM).
• FIG. 2B illustrates the distribution of identified genes that were identified as epitopes presented on MHC molecules of the TRAMP-Cl cell line in relation to the measured RPKM.
• FIG. 3 illustrates structure modeling of peptides bound to MHC molecules.
• FIG. 4A illustrates percentage of peptide-specific CD8 T cells in wild type
• FIG. 4B illustrates percentage of dextramer positive CD8 T cells in the spleen and tumor.
• FIG. 4C shows measure of CD8 T cells and CD45 T cells in relation to tumor volume.
• FIG. 4D illustrates percentage of tumor- specific CD8 TILs co-expressing PD-1 and TIM-3 in the total CD8 TIL population and Adpgk positive CD8 TIL population.
• FIG. 5A illustrates tumor volume of mice treated with a control and an
• the arrow indicates measurements from a single animal.
• FIG. 5B illustrates percentage of peptide-specific CD8 T cells in the spleen and tumor.
• FIG. 5C illustrates percentage of live cells in the tumor measured as CD45 expressing T cells and CD8 expressing T cells.
• FIG. 5D illustrates percentage of Adgpk- specific CD8 TILs co-expressing PD-1 and TEVI-3 in the total CD8 T cell population following vaccination.
• FIG. 5E illustrates level of PD-1 and TIM-3 surface expression following vaccination.
• FIG. 5F illustrates percentage of IFN-y-expressing CD8 and CD4 TILs in tumor and spleen following vaccination.
• FIG. 5G illustrates measurement of tumor volume following vaccination.
• the present application provides high-efficiency screening platforms for identifying disease-specific immunogenic mutant peptides.
• sequence -based variant identification methods with immunogenicity prediction and/or mass spectrometry, the methods described herein allow powerful and efficient identification of disease-specific immunogenic mutant peptides from the disease tissue (such as tumor cells) of an individual.
• These peptides, or nucleotide-based precursors e.g., DNA or RNA
• can be useful for a variety of different applications such as development of vaccines, development of mutant peptide- specific therapeutics (such as antibody therapeutics or T-cell receptor (“TCR”)-based therapeutics), as well as development of tools for monitoring the kinetics and distribution of T cell responses.
• TCR T-cell receptor
• individual peptide or peptide collections can be utilized to do comparative binding affinity measurements or multimerized to measure antigen- specific T cell responses by MHC multimer flow cytometry.
• the methods described herein are particularly useful in the context of personalized medicine, where mutant peptides identified from a diseased individual can be used for developing therapeutics (e.g., peptide-, DNA-, or RNA-based vaccines) for treating the same individual.
• the present application in one aspect provides a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue of an individual by combining sequence-based variant identification methods with immunogenicity prediction.
• the present application provides a method of identifying a disease-specific immunogenic mutant peptide from a diseased tissue of an individual by combining sequence-based variant identification methods with mass spectrometry.
• kits and systems useful for the methods described herein are also provided.
• immunogenic composition comprising peptides, cells presenting such peptides, and nucleic acids encoding such peptides identified.
• disease- specific mutant peptide refers to a peptide that comprises at least one mutated amino acid present in a disease tissue but not in a normal tissue.
• Disease- specific immunogenic mutant peptide refers to a disease- specific mutant peptide that is capable of provoking an immune response in an individual.
• Disease- specific mutant peptides can arise from, for example: non-synonymous mutations leading to different amino acids in the protein (e.g., point mutations); read-through mutations in which a stop codon is modified or deleted, leading to translation of a longer protein with a novel tumor- specific sequence at the C-terminus; splice site mutations that lead to the inclusion of an intron in the mature mRNA and thus a unique tumor- specific protein sequence; chromosomal rearrangements that give rise to a chimeric protein with tumor- specific sequences at the junction of 2 proteins (i.e., gene fusion); and frameshift mutations or deletions that lead to a new open reading frame with a novel tumor- specific protein sequence. See, e.g., Sensi and Anichini, Clin Cancer Res, 2006, v.12, 5023-5032.
• a "variant-coding sequence" as used herein refers to a sequence having a variation compared to a sequence in a reference sample, wherein the sequence variation results in a change in an amino acid sequence contained in or encoded by the variant-coding sequence.
• the variant-coding sequence can be a nucleic acid sequence having a mutation that results in an amino acid change in the encoded amino acid sequence.
• the variant-coding sequence can be an amino acid sequence containing an amino acid mutation.
• Expression variant-coding sequence refers to variant-coding sequences that are expressed in the disease tissue of the individual.
• a nucleic acid sequence "encoding" a peptide refers to a nucleic acid containing the coding sequence for the peptide.
• An amino acid sequence "encoding" a peptide refers to an amino acid sequence containing the sequence of the peptide.
• epitope variant-coding sequence refers to a variant-coding sequence that encodes a peptide that binds or is predicted to bind to an MHC molecule (such as MHC class I molecule, or MHCI).
• an "immunogenic variant-coding sequence” refers to a variant-coding sequence that encodes a peptide that is predicted to be immunogenic.
• disease tissue refers to the tissue associated with the disease in an individual, and includes a plurality of cells.
• Disease tissue sample refers to a sample of the disease tissue.
• polypeptide precursor used herein refers to a polypeptide present in the disease tissue of an individual that comprises the peptide of interest.
• the peptide precursor may be a polypeptide present in the disease tissue that can be process by an
• the methods of the present application in one aspect combine sequence- specific variant identification methods with methods of immunogenicity prediction. For example, in some embodiments, there is provided a method of identifying a disease-specific
• immunogenic mutant peptide from a disease tissue in an individual comprising: a) providing a first set of variant-coding sequences of the disease tissue in the individual, each variant- coding sequence having a variation in the sequence compared to a reference sample; and b) selecting immunogenic variant-coding sequences from the first set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual that serves as a neoepitope in a disease tissue is provided.
• the set of variant-coding sequences comprises more than 1, 10, 100, 1,000, or 10,000 different variant-coding sequences.
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; and b) selecting immunogenic variant-coding sequences from the first set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the variant- coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the selecting step comprises predicting immunogenicity of the peptides based on one or more (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) parameters: i) binding affinity of the peptide to the MHCI molecule; ii) protein level of a peptide precursor containing the peptide; iii) expression level of the transcript encoding the peptide precursor; iv) processing efficiency of the peptide precursor by an immunoproteasome; v) timing of the expression of the transcript encoding the peptide precursor; vi) binding affinity of the peptide to a TCR molecule; vii) position of a variant amino acid within the peptide; viii) solvent exposure of the peptide when bound to a MHCI molecule; ix) solvent exposure of the variant amino acid when bound to a MHCI molecule; x) content of aromatic residues in the peptide; xi) properties of the variant amino acid when compared to the wild type residue (e.g.,
• the first set of variant-coding sequences can first be filtered to obtain a smaller set of variant-coding sequences encoding peptides predicted to bind an MHC molecule (referred to as "epitope variant-coding sequences"), and the smaller set of variant-coding sequences are then subjected to selection based on prediction of
• the method may comprise: a) providing a first set of variant-coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), and c) selecting immunogenic variant-coding sequences from the second set of epitope variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the epitope variant-coding sequences, thereby identifying the disease-specific immunogenic mutant peptide.
• MHC molecule such as MHC class I molecule, or MHCI
• the method comprises: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual, each variant- coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), and c) selecting an MHC molecule (such as MHC class I molecule, or MHCI), and c) selecting MHC molecule (such as MHC class I molecule, or MHCI).
• MHC molecule such as MHC class I molecule, or MHCI
• the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the epitope variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the method further comprises validating the disease- specific immunogenic mutant peptides by functional analysis.
• the disease is cancer.
• the individual is a human individual (such as a human individual having cancer).
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; and b) selecting immunogenic variant-coding sequences from the first set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the genomic sequence is obtained by whole-genome sequencing.
• the genomic sequence is obtained by whole-exome sequencing. In some embodiments, the genomic sequence is obtained by targeted-genome or exome sequencing.
• the genomic sequences in the disease tissue and/or reference sample can first be enriched by a set of probes (for example probes specific for disease-associated genes) before being processed for variant identification.
• the first set of variant-coding sequences can be first filtered to obtain a smaller set of epitope variant-coding sequences, and the smaller set of variant-coding sequences is then subjected to selection based on prediction of
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), and c) selecting immunogenic variant-coding sequences from the second set of epitope variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the epitope variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the method further comprises
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the transcriptome sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; and b) selecting immunogenic variant-coding sequences from the first set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the transcriptome sequence is obtained by whole-transcriptome RNA-Seq sequencing. In some embodiments, the transcription sequence is obtained by targeted-transcriptome sequencing.
• the RNA or cDNA sequences in the disease tissue and/or reference sample can first be enriched by a set of probes (for example probes specific for disease-associated genes) before being processed for variant identification.
• the first set of variant-coding sequences can first be filtered to obtain a smaller set of epitope variant-coding sequences, and the smaller set of variant-coding sequences is then subjected to prediction of
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the transcriptome sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of epitope variant- coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), and c) selecting immunogenic variant-coding sequences from the second set of variant- coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the epitope variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• MHC molecule such as MHC class I molecule, or MHCI
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) providing a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; and c) selecting immunogenic variant- coding sequences from the second set of expression variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the expression variant-coding sequences, thereby identifying the disease-specific immunogenic mutant peptide.
• the method comprises: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the
• transcriptomic sequences of the disease tissue in the individual are selected from the group consisting of the following genes:
• immunogenic variant-coding sequences from the second set of expression variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the expression variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the second set of expression variant-coding sequences can be filtered to obtain a smaller set of epitope variant-coding sequences, and the smaller set of variant-coding sequences is then subjected to prediction of immunogenicity.
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) providing a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) selecting a third set of epitope variant- coding sequences from the second set based on predicted ability of the peptides encoded by the second set of expression variant-coding sequences to bind to an MHC molecule (such as MHC class
• the method comprises: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) selecting a third set of epitope variant-coding sequences from the second set based on predicted ability of the peptides encoded by the second set of expression variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), and d) selecting immunogenic variant-coding sequences from the third set of epitope variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the epitope variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the method comprises: a) obtaining
• the disease-specific immunogenic mutant peptides identified by the methods described herein are further validated by correlating the variant-coding sequence information with information of peptides physically bound to an MHC molecule.
• the methods for example can further comprise: obtaining a plurality of peptides that are bound to an MHC molecule from the disease tissue; subjecting the MHC-bound peptides to mass spectrometry-based sequencing; and correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with the peptides predicted to be immunogenic variant-coding sequences.
• the mass-spectrometry and correlation methods are further described in sections below.
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; b) subjecting the MHC-bound peptides to mass spectrometry-based sequencing; and c) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with a set of variant-coding sequences of the disease tissue in the individual, each variant- coding sequence having a variation in the sequence compared to a reference sample, thereby identifying the disease- specific immunogenic mutant peptide.
• the plurality of peptides bound to MHC are obtained by isolating MHC/peptide complexes (for example by immunoprecipitation) from the disease tissue and eluting the peptides from the MHC.
• the peptides are subjected to tandem mass spectrometry.
• the mass spectrometry-based sequencing comprises subjecting the peptides to mass spectrometry and comparing the mass spectrometry spectra with reference spectra (such as hypothetical mass spectrometry spectra of putative proteins encoded by sequences in a reference sample).
• the mass spectrometry sequence information is filtered by peptide length and/or the presence of anchor motifs prior to the correlation step.
• the method further comprises validating the disease- specific immunogenic mutant peptides by functional analysis.
• the disease is cancer.
• the individual is a human individual (such as a human individual having cancer).
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual, each variant- coding sequence having a variation in the sequence compared to a reference sample; b) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; c) subjecting the MHC -bound peptides to mass spectrometry-based
• the first set of variant- coding sequences can be filtered to obtain a smaller set of variant-coding sequences encoding peptides that is predicted to bind an MHC molecule (hereinafter referred to as "epitope variant-coding sequences"), and the smaller set of variant-coding sequences is then subjected to the correlation analysis.
• the method may comprise: a) providing a first set of variant-coding sequences of the disease tissue in the individual, each variant- coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC -bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry-derived sequence information of the MHC -bound peptides with the second set of epitope variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• MHC molecule such as MHC class I molecule, or MHCI
• the method comprises: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC -bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry-derived sequence information of the MHC -bound peptides with the second set of epitope variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• the method further comprises validating the disease-specific immunogenic mutant peptide
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; c) subjecting the MHC -bound peptides to mass spectrometry-based sequencing; and d) correlating the mass spectrometry-derived sequence information of the MHC -bound peptides with the first set of variant-coding sequences, thereby identifying the disease-specific immunogenic mutant peptide.
• the genomic sequence is obtained by whole-genome sequencing. In some embodiments, the genomic sequence is obtained by whole-exome sequencing. In some embodiments, the genomic sequence is obtained by targeted-genome or exome sequencing.
• the genomic sequences in the disease tissue and/or reference sample can be first be enriched by a set of probes (for example probes specific for disease-associated genes) before being processed for variant identification.
• the first set of variant-coding sequences can be filtered to obtain a smaller set of variant-coding sequences encoding peptides that is predicted to bind an MHC molecule (hereinafter referred to as "epitope variant-coding sequences"), and the smaller set of variant-coding sequences is then subjected to prediction of immunogenicity.
• epitopope variant-coding sequences a smaller set of variant-coding sequences encoding peptides that is predicted to bind an MHC molecule
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC-bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry- derived sequence information of the MHC-bound peptides with the second set of epitop
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the transcriptome sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; c) subjecting the MHC-bound peptides to mass spectrometry-based sequencing; and d) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with the first set of variant-coding sequences, thereby identifying the disease-specific immunogenic mutant peptide
• the transcriptome sequence is obtained by whole-transcriptome RNA-Seq sequencing.
• the transcriptome sequence is obtained by targeted-transcriptome sequencing.
• the RNA sequences or cDNA sequences in the disease tissue and/or reference sample can first be enriched by a set of probes (for example probes specific for disease-associated genes) before being processed for variant identification.
• the first set of variant- coding sequences can be filtered to obtain a smaller set of epitope variant-coding sequences, and the smaller set of variant-coding sequences are then subjected to prediction of immunogenicity.
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the transcriptome sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC -bound peptides to mass spectrometry-based
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) providing a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC- bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with the second set of expression variant-coding sequences, thereby identifying the disease-specific
• the method comprises: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC- bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with the second set of expression variant-coding sequences, thereby identifying the disease-specific
• the second set of expression variant-coding sequences can be filtered to obtain a smaller set of epitope variant-coding sequences, and the smaller set of variant-coding sequences is then subjected to prediction of immunogenicity.
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) providing a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) selecting a third set of epitope variant- coding sequences from the second set based on predicted ability of the peptides encoded by the second set of expression variant-coding sequences to bind to an MHC molecule (such as MHC class
• the method comprises: a) obtaining a first set of variant- coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant- coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) selecting a third set of epitope variant-coding sequences from the second set based on predicted ability of the peptides encoded by the second set of expression variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), d) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; e) subjecting the MHC-bound peptides to mass spectrometry-based sequencing; and f) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides
• the disease-specific immunogenic mutant peptides identified by the mass-spectrometry based methods described herein are further selected by predicting immunogenicity of the peptides.
• the selecting step comprises predicting immunogenicity of the peptides based on one or more (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) parameters: i) binding affinity of the peptide to the MHCI molecule; ii) protein level of a peptide precursor containing the peptide; iii) expression level of the transcript encoding the peptide precursor; iv) processing efficiency of the peptide precursor by an immunoproteasome; v) timing of the expression of the transcript encoding the peptide precursor; vi) binding affinity of the peptide to a TCR molecule; vii) position of a variant amino acid within the peptide; viii) solvent exposure of the peptide when bound to a MHCI molecule; ix) solvent exposure of the variant amino
• a method of identifying a disease-specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; b) subjecting the MHC-bound peptides to mass
• spectrometry-based sequencing and c) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with a set of variant-coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample to obtain a second set of variant-coding sequences, and d) selecting immunogenic variant-coding sequences from the second set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the second set of variant-coding sequences, thereby identifying the disease- specific immunogenic mutant peptide.
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) obtaining a set of variant-coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; c) subjecting the MHC -bound peptides to mass spectrometry-based sequencing; d) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with the first set of variant-coding sequences to obtain a second set of variant-coding sequences, and e) selecting immunogenic variant-coding sequences from the second set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino acid encoded by the second set of variant-coding sequences, thereby identifying the disease- specific immunogenic
• the method comprises: a) providing a first set of variant-coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC-bound peptides to mass spectrometry-based
• MHC molecule such as MHC class I molecule, or MHCI
• the method comprises: a) obtaining a first set of variant- coding sequences of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of epitope variant-coding sequences from the first set based on predicted ability of the peptides encoded by the first set of variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC-bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry- derived sequence information of the MHC-bound peptides with the set of epitope variant- coding sequences to obtain a third set of variant-coding sequences, and f) selecting immunogenic variant-coding sequences from the third set of variant-coding sequences, where
• the method further comprises validating the disease- specific immunogenic mutant peptides by functional analysis. In some embodiments, the method further comprises validating the disease-specific immunogenic mutant peptides by functional analysis. In some embodiments, the disease is cancer. In some embodiments, the individual is a human individual (such as a human individual having cancer).
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) providing a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC- bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with the second set of expression variant-coding sequences to obtain a third set of variant-coding sequences, and f) selecting immunogenic variant-coding sequences from the third set
• the method comprises: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; d) subjecting the MHC -bound peptides to mass spectrometry-based sequencing; and e) correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with the second set of expression variant-coding sequences to obtain a third set of variant-coding sequences, and f) selecting immunogenic variant-coding sequences from the third set of variant-coding sequences, wherein the selecting step comprises predicting immunogenicity of the peptides comprising a variant amino
• a method of identifying a disease- specific immunogenic mutant peptide from a disease tissue in an individual comprising: a) providing a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) selecting a third set of epitope variant- coding sequences from the second set based on predicted ability of the peptides encoded by the second set of expression variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI),d) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; e) subjecting the MHC-bound peptides to mass spectrometry-
• MHC molecule such as MHC class I
• the method comprises: a) obtaining a first set of variant-coding sequences of the disease tissue in the individual based on the genomic sequence of the disease tissue in the individual, each variant-coding sequence having a variation in the sequence compared to a reference sample; b) selecting a second set of expression variant-coding sequences from the first set based on the transcriptomic sequences of the disease tissue in the individual; c) selecting a third set of epitope variant-coding sequences from the second set based on predicted ability of the peptides encoded by the second set of expression variant-coding sequences to bind to an MHC molecule (such as MHC class I molecule, or MHCI), d) obtaining a plurality of peptides that are bound to an MHC molecule from a diseased tissue of an individual; e) subjecting the MHC -bound peptides to mass spectrometry-based sequencing; and f) correlating the mass spectrometry-derived sequence information of the MHC -bound peptides
• disease-specific immunogenic mutant peptides obtained by any one of the methods described herein.
• the disease- specific immunogenic mutant peptides can be used, for example, to make a composition (such as a vaccine composition) for treating the disease.
• the disease-specific immunogenic mutant peptide can be used for producing mutant-pep tide- specific therapeutics such as therapeutic antibodies.
• a method of treating a disease comprising: a) identifying a disease-specific, immunogenic mutant peptides in the individual; and b) synthesizing the peptide; and c) administering the peptide to the individual.
• a method of treating a disease (such as cancer) in an individual comprising: a) obtaining a disease tissue sample from the individual; b) identifying a disease- specific, immunogenic mutant peptides in the individual; and c) synthesizing the peptide; and d) administering the peptide to the individual.
• a method of treating a disease (such as cancer) in an individual comprising: a) identifying a disease- specific, immunogenic mutant peptide in the individual; b) producing an antibody (or a TCR analog, such as a soluble TCR) specifically recognizing the mutant peptide; and c) administering the peptide to the individual.
• a method of treating a disease comprising: a) obtaining a disease tissue sample from the individual; b) identifying a disease- specific, immunogenic mutant peptide in the individual; c) producing an antibody (or a TCR analog, such as a soluble TCR) specifically recognizing the mutant peptide; and d) administering the peptide to the individual.
• the identification step combines sequence- specific variant identification method with methods of immunogenicity prediction.
• the identification step combines sequence-specific variant identification method with mass spectrometry. Any methods of identifying a disease- specific, immunogenic mutant peptide described herein can be used for the treatment methods described herein.
• the methods described herein in various embodiments comprise providing and/or obtaining variant-coding sequences.
• the variant coding sequences can generally be obtained, for example, by sequencing the genomic or RNA sequences in the disease tissue sample of the individual and comparing the sequences to those obtained from a reference sample.
• the disease tissue is blood.
• the disease tissue is a solid tissue (such as solid tumor).
• the disease tissue is a collection of cells (for example circulating cancer cells in the blood).
• the disease tissue is a collection of lymphocytes. In some embodiments, the disease tissue is a collection of leukocytes. In some embodiments, the disease tissue is a collection of epithelial cells. In some embodiments, the disease tissue is connective tissue. In some embodiments, the disease tissue is a collection of germ cells and/or pluripotent cells. In some embodiments, the disease tissue is a collection of blast cells.
• Suitable disease tissue samples include, but are not limited to, tumor tissue, normal tissue adjacent to the tumor, normal tissue distal to the tumor, or peripheral blood
• the disease tissue sample is a tumor tissue.
• the disease tissue sample is a biopsy containing cancer cells, such as fine needle aspiration of cancer cells (e.g., pancreatic cancer cells) or laparoscopy obtained cancer cells (e.g., pancreatic cancer cells).
• cancer cells such as fine needle aspiration of cancer cells (e.g., pancreatic cancer cells) or laparoscopy obtained cancer cells (e.g., pancreatic cancer cells).
• the biopsied cells are centrifuged into a pellet, fixed, and embedded in paraffin prior to the analysis.
• the biopsied cells are flash frozen prior to the analysis.
• the disease tissue sample comprises a circulating metastatic cancer cell.
• the disease tissue sample is obtained by sorting circulating tumor cells (CTCs) from blood.
• CTCs circulating tumor cells
• the CTCs have detached from a primary tumor and circulate in a bodily fluid.
• the CTCs have detached from a primary tumor and circulate in the bloodstream.
• the CTCs are an indication of metastasis.
• the CTCs are pancreatic cancer cells.
• the CTCs are colorectal cancer cells.
• the CTCs are non-small cell lung carcinoma cells.
• the variation can be identified based on the genomic sequence in the disease tissue in the individual.
• genomic DNA can be obtained from the disease tissue in the individual and subjected to sequencing analysis. The sequence so obtained can then be compared to those obtained from a reference sample.
• the disease sample is subjected to whole-genome sequencing.
• the disease sample is subjected to whole-exome sequencing, i.e., only exons in the genomic sequences are sequenced.
• the genomic sequences are "enriched" for specific sequences prior to the comparison to a reference sample.
• specific probes can be designed to enrich certain desired sequences (for example disease-specific sequences) before being subjected to sequencing analysis.
• the variations are identified based on the transcriptome sequences in the disease tissue in the individual.
• whole or partial transcriptome sequences can be obtained from the disease tissue in the individual and subjected to sequencing analysis. The sequence so obtained can then be compared to those obtained from a reference sample.
• the disease sample is subjected to whole-transcriptome RNA-Seq sequencing.
• the transcriptome sequences are "enriched" for specific sequences prior to the comparison to a reference sample.
• specific probes can be designed to enrich certain desired sequences (for example disease-specific sequences) before being subjected to sequencing analysis.
• transcriptomic sequencing techniques comprise, but are not limited to, RNA poly(A) libraries, microarray analysis, parallel sequencing, massively parallel sequencing, PCR, and RNA-Seq.
• RNA-Seq is a high-throughput technique for sequencing part of, or substantially all of, the transcriptome.
• an isolated population of transcriptomic sequences is converted to a library of cDNA fragments with adaptors attached to one or both ends. With or without amplification, each cDNA molecule is then analyzed to obtain short stretches of sequence information, typically 30-400 base pairs. These fragments of sequence information are then aligned to a reference genome, reference transcripts, or assembled de novo to reveal the structure of transcripts (i.e., transcription boundaries) and/or the level of expression.
• sequences in the disease tissue can be compared to the corresponding sequences in a reference sample.
• the sequence comparison can be conducted at the nucleic acid level, by aligning the nucleic acid sequences in the disease tissue with the corresponding sequences in a reference sample. Sequence variations that lead to one or more changes in the encoded amino acids are then identified.
• sequence comparison can be conducted at the amino acid level, that is, the nucleic acid sequences are first converted into amino acid sequences in silico before the comparison is carried out.
• comparison of a sequence from the disease tissue to those of a reference can be completed by techniques known in the art, such as manual alignment, FAST-A11 (FASTA), and Basic Local Alignment Search Tool (BLAST). Sequence comparison completed by BLAST requires input of a disease sequence and input of a reference sequence. BLAST compares a disease sequence to a reference database by first identifying short sequence matches between two sequences, a process referred to as seeding. Once a sequence match is found, expansion of the sequence alignment is performed using a scoring matrix.
• FAST-A11 FASTA11
• BLAST Basic Local Alignment Search Tool
• the reference sample is a matched, disease-free tissue sample.
• a "matched,” disease-free tissue sample is one that is selected from the same or similar tissue type as the disease tissue.
• a matched, disease-free tissue and a disease tissue may originate from the same individual.
• the reference sample described herein in some embodiments is a disease-free sample from the same individual.
• the reference sample is a disease-free sample from a different individual (for example an individual not having the disease).
• the reference sample is obtained from a population of different individuals.
• the reference sample is a database of known genes associated with an organism.
• a reference sample may be a combination of known genes associated with an organism and genomic information from a matched disease-free tissue sample.
• a variant-coding sequence may encode or comprise a point mutation in the amino acid sequence.
• the variant-coding sequence may encode or comprise an amino acid deletion or insertion.
• the set of variant-coding sequences are first identified based on genomic sequences. This initial set is then further filtered to obtain a narrower set of expression variant-coding sequences based on the presence of the variant-coding sequences in a transcriptome sequencing database (and is thus deemed “expressed”). In some embodiments, the set of variant-coding sequences are reduced by at least about 10, 20, 30, 40, 50, or more times by filtering through a transcriptome sequencing database.
• the variant-coding sequence is a sequence that results from a non-synonymous mutation leading to a different amino acid(s) in the protein (e.g., point mutations).
• the variant-coding sequence is a sequence that results from a read-through mutation in which a stop codon is modified or deleted, leading to translation of a longer protein with a novel tumor- specific sequence at the C-terminus.
• the variant-coding sequence is a sequence that results from a splice site mutation that leads to the inclusion of an intron in the mature mRNA and thus a unique tumor- specific protein sequence.
• the variant-coding sequence is a sequence that results from a chromosomal rearrangement that gives rise to a chimeric protein with tumor- specific sequences at the junction of 2 proteins (i.e., gene fusion). In some embodiments, the variant-coding sequence is a sequence that results from a frameshift mutation or deletion that leads to a new open reading frame with a novel tumor- specific protein sequence. In some embodiments, the variant-coding sequence is a sequence that results from more than one mutation. In some embodiments, the variant-coding sequence is a sequence that results from more than one mutation mechanism.
• the variant-coding sequences described herein in some embodiments are filtered to obtain a smaller set of variant-coding sequences encoding peptides that are predicted to bind an MHC molecule ("epitope variant-coding sequences").
• the set of variant-coding sequences are reduced by at least about 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, or more times by filtering through the MHC binding prediction process.
• NETMHC is an algorithm trained on quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from IEDB.
• IEDB Immune Epitope Database and Analysis Resource
• NETMHC uses predictors trained on 9-mer amino acid sequences from 55 MHC alleles (43 human and 12 non-human). To allow for the prediction of shorter input sequences, NETMHC will virtually extend an 8-mer amino acid sequence. For input sequences longer than a 9-mer, NETMHC will generate all possible 9-mer amino acid sequences contained within the input sequence. NETMHC then uses trained artificial neural networks and position specific scoring matrices to predict MHC binding.
• the methods provided herein in some embodiments further comprise selecting immunogenic variant-coding sequences comprising predicting immunogenicity of the peptides comprising a variant amino acid encoded by the variant-coding sequences.
• the prediction of immunogenicity can be carried out, for example, by a process (such as an in in silico process) which consider one or more parameters of the peptide and the corresponding peptide precursor to predict the likelihood that the peptide is immunogenic.
• these parameter include, but are not limited to, i) binding affinity of the peptide to the MHCI molecule; ii) protein level of a peptide precursor containing the peptide; iii) expression level of the transcript encoding the peptide precursor; iv) processing efficiency of the peptide precursor by an immunoproteasome; v) timing of the expression of the transcript encoding the peptide precursor; vi) binding affinity of the peptide to a TCR molecule; vii) position of a variant amino acid within the peptide; viii) solvent exposure of the peptide when bound to a MHCI molecule; ix) solvent exposure of the variant amino acid when bound to a MHCI molecule; x) content of aromatic residues in the peptide; and xi) nature of the peptide precursor.
• the immunogenicity is based on at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the parameters described herein.
• the binding affinity of the peptide to the MHC molecule is used for predicting immunogenicity.
• the binding affinity of peptide to an MHC molecule may be predictive of the stability of the pMHC, which in turn may allow for prolonged presentation of the pMHC, thus increasing cell surface exposure for potential interaction with immune cells. Binding affinity may be predicted using known techniques in the art, such as RankPep, MHCBench, nHLAPred, SVMHC, NETMHCpan, and POPI, which are based on methodology such as artificial neural networks, average relative binding matrices, quantitative matrices, and stabilized matrix methods.
• the binding affinity of a peptide to MHC is based on the presence of specific amino acid residues located in known anchor positions (amino acids involved in MHC binding). In some embodiments, each residue in the peptide is evaluated for its contribution to binding. In some embodiments, analysis systems are trained with peptides known to bind MHC. In some embodiments, the binding energy of a peptide-MHC molecule is calculated. In some embodiments, a binding threshold is used to evaluate predicted affinity of peptides to bind to MHC, such as an IC50 value ⁇ 500 nM.
• the expression level of the peptide precursor in the disease tissue is used for predicting immunogenicity.
• the protein level is measured biochemically (such as Western blot and ELISA).
• the protein level may be measured by known quantitative mass spectrometry techniques.
• La Gruta et al. high expression levels of peptide precursors in disease tissue can be correlated with predicted immunogenicity.
• the availability of larger quantities of peptide precursors that feed into epitope processing pathways has been positively correlated with increased epitope presentation and immunogenic response (La Gruta, N. L. et al., A virus- specific CD8+ T cell immunodominance hierarchy determined by antigen dose and precursor frequencies, Proceedings of the National Academy of Sciences, 2006, V.103, 994-999, which is hereby incorporated by reference).
• the expression levels of the transcript encoding the peptide precursor in the disease tissue may be used for predicting immunogenicity.
• the RNA expression level is measured by RT-PCR.
• the RNA expression level is measured by sequencing analysis. As discussed above, increasing the availability of peptide precursors for epitope processing pathways positively correlates with the immunogenic response associated with said epitopes resulting from said peptide precursors. Further, a positive correlation between mRNA levels and protein abundance has been observed (Ghaemmaghami, S. et al., Global analysis of protein expression in yeast, Nature, 2003, v.425, 737-741, which is hereby incorporated by reference). Therefore, expression levels of the transcript encoding the peptide precursor in the disease tissue may be used for predicting immunogenicity.
• the processing efficiency of the peptide precursor by an immunoproteasome is used for predicting immunogenicity.
• the "processing efficiency" of a peptide precursor refers to the efficiency in which source amino acid sequences (i.e., larger peptides or proteins) are expressed, translated, transcribed, digested, transported, and any further processing prior to binding an MHCI molecule.
• An "immunoproteasome” is a collection of proteases that enzymatically digest the peptide and/or protein precursors into small amino acid sequences for the ultimate purpose of epitope formation.
• the timing of expression of the peptide precursor may be used for predicting immunogenicity. Proteins expressed earlier in disease progression are more likely to be presented as MHC epitopes (Moutaftsi, M. et al., A consensus epitope prediction approach identifies the breadth of murine T CD8+-cell responses to vaccina virus, Nature Biotechnology, 2006, v.24, 817-819, which is hereby incorporated by reference).
• comparative analyses of disease tissue may be used to determine the temporal expression pattern of gene products.
• extrapolation of temporal expression patterns may allow for identification of expressed gene products that are presented in greater abundance at an early time point in comparison to other identified expressed gene products.
• the binding affinity of a peptide epitope with a T cell receptor may be used for predicting immunogenicity.
• Methods of predicting the binding affinity of a peptide epitope with a TCR are known in the art and reported, for example, in Tung, C.-W. et al., POPISK: T-cell reactivity prediction using support vector machines and string kernels, BMC Bioinformatics, 2011, v.12, 446, which is hereby incorporated by reference.
• the position of the variant amino acid in the peptide is used for predicting immunogenicity.
• a peptide epitope binds to a MHCI molecule at two distinct anchor positions. The span between anchor positions is separated by about 6-7 amino acid, as measured by the epitope peptide sequence, not inclusive of the amino acids occupying the anchor positions. It has been reported that mutations in the span of amino acids between the two MHCI anchor positions, namely amino acids in position 4-6, are more likely to positively correlate with an immunogenic response (Calis, J. J. A. et al., Properties of MHC class I presented peptides that enhance immunogenicity, PLOS Computational Biology, 2013, v.9, 1- 13, which is hereby incorporated by reference). The sequence position of an amino acid is determined by starting a sequence position count of 1 for a terminal amino acid.
• the structural characteristics of the peptide presented on the MHC presented epitope may be predictive of immunogenicity.
• Structural assessment of a MHC bound peptide may be conducted by in silico 3-dimensional analysis and/or protein docking programs. Methods of predicting the structure of a pMHC molecule are known in the art and reported, for example, in Marti-Renom, M. A. et al., Comparative protein structure modeling of genes and genomes, Annual Review of Biophysics and Biomolecular Structure, 2000, v.29, 291-325, Chivian, D.
• the solvent exposure of the entire peptide when presented on the MHC complex is used for predicting immunogenicity.
• the solvent exposure of the variant amino acid on the peptide is used for predicting immunogenicity.
• the epitope content of large and/or aromatic residues in the peptide may be used for predicting immunogenicity. Calis et al. observed a link between the presence of large and/or aromatic amino acid residue within the epitope amino acid sequence and immunogenicity. Specifically, it was reported that phenylalanine and isoleucine content was positively correlated with epitope immunogenicity. As discussed above, both location and structural assessment of variant-amino acids may be used to predict epitope
• the nature of the peptide precursor may be used to predict immunogenicity of a variant-coding amino acid sequence.
• peptide precursor sequences that are known to be associated with cancer can be useful when predicting immunogenicity.
• peptide precursor sequences from proteins not directly associated with cancer, for example, when mutated can also be useful within the methods of the present invention.
• At least two (such as at least any one 3, 4, 5, 6, 7, 8, 9, or 10) parameters described herein may be used to predict immunogenicity of a peptide.
• a first predictive assessment may be used to select a set of variant-coding amino acid sequences that are subsequently processed by a second predictive assessment resulting in a cumulative prediction of epitope immunogenicity. It is intended as part of the instant disclosure that multiple rounds of assessment may be used to predict epitope immunogenicity.
• multiple parameters are assessed in parallel, and a composite score based on the assessment of various parameters can be obtained. For example, a score can be calculated for each of the parameters, and each parameter can be assigned a percentage weight. A composite scope can then be calculated based on the scores and percentage weight of each of the parameters assessed.
• a combination of sequential assessment coupled with in-parallel parameter assessment can also be used.
• multiple overlapping putative mutant peptides of various lengths can be generated.
• these multiple overlapping putative mutant peptides can be ranked based on immunogenicity which can encompass one or more analyses as described herein.
• the ranking of putative mutant peptides can be accomplished by any one or multiple means well known in the art which can be included within the immunogenicity determination or be considered separate analyses depending on the preference of those conducting the ranking process. Some non- limiting examples of such means include abundance of the precursor protein, the abundance of the peptide within the processed proteome including the efficiency of this processing, and the abundance of the peptide within peptide-MHC complexes.
• peptides can be ranked based on correlation with data produced from mass spectrometry methods measuring these or other characteristics well known to one or ordinary skill or described herein. Some representative characteristics include the presence or absence of large or aromatic amino acids, which increases immunogenicity and the particular positions that these amino acids are found within the peptide, with immunogenicity impact preference given to those amino acids found in the middle positions of the peptide, for example, peptides four through six (see, e.g., Calis et al. PLOS, 9(10):el003266 (2013)).
• the prediction of immunogenicity further comprises HLA (human leukocyte antigen) -typing analysis.
• HLA human leukocyte antigen
• every person will express at least three different antigen-presenting MHC class I molecules and three (or sometimes four) MHC class II molecules on his or her cells.
• the number of different MHC molecules expressed on the cells of most people is greater because of the extreme polymorphism of the MHC and the codominant expression of MHC gene products.
• MHC haplotype The particular combination of MHC alleles found on a single chromosome is known as an MHC haplotype. Expression of MHC alleles is codominant, with the protein products of both the alleles at a locus being expressed in the cell, and both gene products being able to present antigens to T cells. The extensive polymorphism at each locus thus has the potential to double the number of different MHC molecules expressed in an individual and thereby increases the diversity already available through polygeny (see, e.g.
• HLA-typing can be accomplished using any one of several methods known in the prior art, such as DNA based histocompatability assays.
• DNA based histocompatability assays include polymerase chain reaction (PCR) product further analyzed such as PCR-RFLP (restriction fragment length polymorphism), PCR-SSO (sequence specific oligonucleotides), PCR-SSP (sequence specific primers), and PCR-SBT (sequence based typing) techniques.
• PCR-RFLP restriction fragment length polymorphism
• PCR-SSO sequence specific oligonucleotides
• PCR-SSP sequence specific primers
• PCR-SBT sequence based typing
• the methods provided herein comprising obtaining peptides bound to MHC molecules from the disease tissue of an individual.
• the MHC -bound peptides are isolated by immunoaffinity techniques.
• the MHC -bound peptides are isolated by affinity chromatography.
• the MHC -bound peptides are isolated by immunoaffinity affinity chromatography.
• the MHC -bound peptides are isolated by immunoprecipitation techniques.
• an anti-MHC antibody is used to capture the MHC/peptide molecule.
• multiple anti-MHC antibodies optionally with differing affinities and/or binding characteristics, may be used to capture the MHC/peptide complexes.
• Suitable antibodies include, but are not limited to, monoclonal antibody W6/32, specific for HLA class I, and monoclonal antibody BB7.2, specific for HLA-A2.
• a MHC/peptide complex may be first isolated, and the MHC -bound peptides are subsequently separated from the MHC molecule.
• a MHC -bound peptide is separated from a MHC molecule by acid elution.
• acid-mediated separation of a MHC-bound peptide from a MHC molecule may be performed on intact whole cells, optionally in the presence of lysed cells and/or cell remnants.
• a MHC-bound peptide may be separated from a MHC molecule following exposure of a pMHC to a buffer with an acidic pH.
• a MHC-bound peptide may be separated from a MHC molecule by mild acid elution (MAE). In some embodiments, a MHC-bound peptide may be separated from a MHC molecule by mild acid elution (MAE) of an extracellular surface. In some embodiments, a MHC-bound peptide may be separated from a MCH molecule by denaturation of the pMHC molecule.
• MAE mild acid elution
• the MHC-bound peptide may be further processed prior to mass spectrometry-based sequencing.
• the MHC-bound peptide may be concentrated prior to mass spectrometry-based sequencing.
• the MHC- bound peptide may be purified prior to mass spectrometry-based sequencing.
• the MHC-bound peptide may be fractionated prior to mass spectrometry-based sequencing.
• the MHC-bound peptide may be enriched prior to mass spectrometry-based sequencing.
• the MHC-bound peptide may be further enzymatically digested prior to mass spectrometry-based sequencing.
• a buffer in which the MHC-bound peptide may be contained, may be exchanged prior to mass spectrometry-based sequencing.
• the MHC- bound peptide may be labeled prior to mass spectrometry-based sequencing.
• the MHC-bound peptide may be covalently labeled prior to mass
• the MHC-bound peptide may be enzymatically labeled prior to mass spectrometry-based sequencing. In some embodiments, the MHC-bound peptide may be chemically labeled prior to mass spectrometry-based sequencing. In some embodiments, the MHC-bound peptide may be labeled to allow for enhanced ionization during mass spectrometry-based sequencing. In some embodiments, the MHC-bound peptide may be labeled to allow for quantification during mass spectrometry- based sequencing. In some embodiments, isolated MHC-bound peptides may originate from multiple sources and/or enrichment procedures and optionally may be collectively pooled prior to mass spectrometry-based sequencing.
• mass spectrometry-based sequencing refers to the technique of identifying an amino acid sequence of a peptide and/or protein by use of mass spectrometry.
• a mass spectrometer is an instrument capable of measuring the mass-to-charge (m/z) ratio of individual ionized molecules, allowing researchers to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules.
• the methods provided herein may be used to obtain sequence information of a peptide epitope bound to a MHCI molecule. In some embodiments, the entire sequence of a peptide epitope may be determined.
• a partial sequence of the peptide epitope may be determined.
• the peptides are subjected to tandem mass spectrometry such as tandem chromatography mass spectrometry (for example LC-MS or LC-MS-MS).
• the MHC-bound peptide may be chromatographically processed prior to mass spectrometry analysis.
• chromatography is liquid chromatography.
• chromatography is reverse phase chromatography.
• the MHC-bound peptide may be
• chromatographic separation may be online, wherein peptides eluting from the chromatography source enter directly into mass spectrometer.
• chromatographic separation may be offline.
• offline chromatographic separation may be used to fractionate isolate MHC- bound peptides.
• Offline chromatographic separation typically involves separation and/or fractionation of a mass spectrometry sample wherein the resulting separated and/or fractionated sample is not immediate introduced into the mass spectrometer as the sample exits the chromatographic system.
• the MHC-bound peptide may be sequenced using known mass spectrometry ionization techniques (such as matrix-assisted laser desorption/ionization, electrospray ionization, and/or nano-electrospray ionization, atmospheric pressure chemical ionization).
• mass spectrometry ionization techniques such as matrix-assisted laser desorption/ionization, electrospray ionization, and/or nano-electrospray ionization, atmospheric pressure chemical ionization.
• the MHC-bound peptide may be ionized outside, inside, and/or as they enter the mass spectrometer.
• a positive ion of the MHC-bound peptide may be analyzed in the mass spectrometer. Subsequently, the ions are separated according to their mass-to-charge ratio via exposure to a magnetic field.
• a sector instrument is used, and the ions are quantified according to the magnitude of the deflection of the ion's trajectory as it passes through the instrument's electromagnetic field, which is directly correlated to the ions mass-to-charge ratio.
• ion mass-to-charge ratios are measured as the ions pass through quadrupoles, or based on their motion in three dimensional or linear ion traps or Orbitrap, or in the magnetic field of a Fourier transform ion cyclotron resonance mass spectrometer.
• the instrument records the relative abundance of each ion, which is used to determine the chemical, molecular and/or isotopic composition of the original sample.
• a time-of-flight instrument is used, and an electric field is utilized to accelerate ions through the same potential, and measures the time it takes each ion to reach the detector.
• This approach depends on the charge of each ion being uniform so that the kinetic energy of each ion will be identical.
• the only variable influencing velocity in this scenario is mass, with lighter ions traveling at larger velocities and reaching the detector faster consequently.
• the resultant data is represented in a mass spectrum or a histogram, intensity vs. mass-to- charge ratio, with peaks representing ionized compounds or fragments.
• Mass spectra data can be obtained by tandem mass spectrometry.
• a mass spectrometry acquisition technique for acquiring information for peptide sequencing of the MHC-bound peptide may be data-dependent.
• a mass spectrometry acquisition technique for acquiring information for peptide sequencing of the MHC-bound peptide may be data-independent. In some embodiments, a mass spectrometry acquisition technique for acquiring information for peptide sequencing of the MHC-bound peptide may be based on measured accurate mass mass spectrometry. In some embodiments, a mass spectrometry acquisition technique for acquiring information for peptide sequencing of the MHC-bound peptide may be peptide mass fingerprinting. Mass spectra data useful in this invention can be obtained by peptide mass fingerprinting.
• Peptide mass fingerprinting involves inputting the observed mass from a spectrum of the mixture of peptides generated by proteolytic digestion into a database and correlating the observed masses with the predicted masses of fragments arising from digestions of known proteins in silico. Known masses corresponding to sample masses provide evidence that the known protein is present in the sample tested.
• tandem mass spectrometry includes a process that causes peptide ions to collide with gas and to fragment (e.g., due to vibrational energy imparted by the collision).
• the fragmentation process produces cleavage products that break at the peptide bonds at various sites along the protein.
• the observed fragments' masses may be matched with a database of predicted masses for one of many given peptide sequences, and the presence of a protein may be predicted.
• a mass spectrometry acquisition technique may utilize fragmentation techniques (such as collision-induced dissociation, pulsed-Q dissociation, higher-energy collisional dissociation, electron-transfer dissociation, and electron-transfer dissociation, infrared multiphoton dissociation).
• data acquired from the mass spectrometer may be used to identify a peptide sequence.
• a search algorithm such as SEQUEST and Mascot
• assigned peptide sequences may have a false discovery rate of less than about 5%.
• the assigned peptide sequences may have a false discovery rate of less than about 1%.
• the assigned peptide sequences may have a false discovery rate of less than about 0.5%.
• a database may be used by a search algorithm to make peptide sequence assignments of an acquired spectrum.
• a database used by a search algorithm to make a peptide sequence assignment of an acquired spectrum may be a database of known sequences of an organism. In some embodiments, a database used by a search algorithm to make a peptide sequence assignment of an acquired spectrum may be a database of known proteins of an organism. In some embodiments, a database used by a search algorithm to make a peptide sequence assignment of an acquired spectrum may be a database of known genomic sequences of an organism. In some embodiments, a database used by a search algorithm to make a peptide sequence assignment of an acquired spectrum may comprise sequence information obtained from a disease tissue. In some embodiments, a database used by a search algorithm to make a peptide sequence assignment of an acquired spectrum may comprise sequence information obtained from a disease-free tissue.
• a sequence assigned spectrum may be manually validated to confirm correct fragment ion assignments by the algorithm.
• a synthetic peptide standard may be used to confirm an algorithm-assigned sequence.
• a spectrum generated from the MHC -bound peptide may be compared to a spectrum generated from a peptide standard. For example, comparison may involve matching the pattern of fragment ions, and optionally fragment abundance or intensity, based on mJz values of the spectrum acquired from a disease tissue source to that of a reference.
• manual validation may confirm a sequence assignment of the complete peptide sequence.
• manual validation may confirm the sequence assignment of a partial segment of a peptide sequence.
• the methods provided herein in some embodiments comprise correlating the mass spectrometry-derived sequence information of the MHC-bound peptides with a set of variant- coding sequences to identify disease- specific immunogenic mutant peptides.
• the mass spectrometry sequence of a MHC-bound peptide may be used to further select a population of predicted disease-specific immunogenic mutant peptides.
• mass spectrometry-based epitope identification may supplement genomic- based immunogenic epitope identification and/or prediction. In some embodiments, mass spectrometry-based epitope identification may confirm genomic-based immunogenic epitope identification and/or prediction.
• the data obtained from a mass spectrometry analysis may be correlated with immunogenic peptides predictions based on genomic and/or transcriptomic sequence analysis of a disease tissue.
• the amino acid sequence identified from mass spectrometry- based sequencing is compared with amino acid sequences of predicted immunogenic peptides to find regions comprising partial sequence alignment.
• the peptide identified via mass spectrometry will be an exact sequence match to the sequence of the predicted immunogenic peptide.
• the length of the amino acid sequence may vary between the peptide identified via mass spectrometry and that of the predicted immunogenic peptide.
• a peptide identified via mass spectrometry may contain additional amino acids amended to the C- and/or N-terminus of the peptide as compared to the predicted immunogenic peptide.
• a peptide identified via mass spectrometry comprising a variant amino acid may have fewer amino acids on the C- and/or N-terminus as compared to the predicted immunogenic peptide.
• the variant amino acid and the sequence surrounding the variant amino acid must be the same in both the peptide identified via mass spectrometry and the peptide predicted to be
• the result obtained from correlating mass spectrometry- based sequences with immunogenic peptide predictions is to match predicted immunogenic sequences with sequences that are confirmed, via mass spectrometry-based sequencing, to be physically presented by MHC molecules.
• the acquired mass spectrometry sequence identifications may be further filtered by peptide length.
• the population of MHC -bound peptides identified by mass spectrometry may be further filter to include only those identified peptide sequences that are 8 or 9 amino acids in length.
• the disease- specific, immunogenic mutant peptides identified by the methods described herein can further be validated by functional studies.
• the peptide may be synthesized and tested based on the ability to activate a targeted immune response (such as that mediated by cytotoxic T cells).
• the peptide is synthesized chemically.
• a peptide is synthesized by recombinant methods.
• a peptide is synthesized by first expressing a peptide precursor molecule which is then processed (for example by an immunoproteasome) to produce the peptide of interest.
• the synthesized peptides can be subjected to further purification before being subjected to functional analysis.
• a synthetic predicted disease- specific immunogenic peptide is used in vitro to test for cytotoxic T cell response. In some embodiments, a synthetic predicted disease- specific immunogenic peptide is used in vivo to test for cytotoxic T cell response.
• immunogenicity of a disease-specific peptide may be tested by immunization of a mouse. In some embodiments, immunogenicity of a disease-specific peptide may be tested following immunization by measuring CD8 T cell response. In some embodiments, a CD8 T cell response may be measured using MHCI/peptide-specific dextramers. In some embodiments, immunogenicity of a disease-specific peptide may be tested by analyzing tumor-infiltrating cells (TILs).
• TILs tumor-infiltrating cells
• the presence of specific epitopes and/or cell surface proteins may be measured.
• the presence of epitopes originating from the vaccination may be measured.
• interferon gamma IFN- ⁇
• IFN- ⁇ interferon gamma
• PD-1 programmed cell death 1
• TIM-3 T cell immunoglobulin mucin-3
• cytotoxic T cells expressing specific proteins and/or epitopes may be measured.
• cytotoxic T cells displaying specific epitopes may be measured.
• the immunogenicity of a disease-specific peptide may be tested by first expressing the peptide in a dendritic cell and then testing for the ability of the presented antigen to be recognized by a T cell.
• the dendritic cell is obtained from a patient, wherein the disease-specific peptide was identified in said patient.
• the immunogenicity of a disease- specific peptide may be tested by first expressing the peptide in a B-lymphocyte and then testing for the ability of the presented antigen to be recognized by a T cell.
• the B-lymphocyte is obtained from a patient, wherein the disease- specific peptide was identified in said patient. See, e.g., United States Patent No. 8,349,558.
• the present disclosure provides methods of identifying a disease- specific immunogenic peptide.
• An immunogenic peptide may be identified based on the ability to activate a targeted immune response (such as that mediated by cytotoxic T cells).
• the amino acid sequence of an identified disease-specific immunogenic peptide may be used develop a pharmaceutically acceptable composition.
• a composition may comprise a synthetic disease-specific immunogenic peptide.
• a composition may comprise a synthetic disease- specific immunogenic mutant peptide.
• a composition may comprise two or more disease- specific immunogenic peptides.
• a composition may comprise two or more disease-specific immunogenic mutant peptides.
• the two or more disease-specific immunogenic peptides may activate a cytotoxic T cell response to two or more unique epitopes.
• a composition may comprise a precursor to a disease-specific immunogenic peptide (such as a protein, peptide, DNA and RNA).
• a precursor to a disease-specific immunogenic peptide may generate or be generated to the identified disease-specific immunogenic peptide.
• the precursor to a disease- specific immunogenic peptide may be a pro-drug.
• the composition comprising a disease-specific immunogenic peptide may be pharmaceutically acceptable.
• the composition comprising a disease specific immunogenic peptide may further comprise an adjuvant.
• the mutated peptide can be utilized as a vaccine (see, Sahin et al., Int. J. Cancer, 78:387-9 (1998); Stumiolo et al., Nature Biotechnol, 17:555-61 (1999); Rammensee et al., Immunol Rev 188: 164-76 (2002); and Hannani et al. Cancer J 17:351-358 (2011)).
• the vaccine may contain individualized components, according to the personal needs of the particular patient.
• the vaccine may be specific to an immunogenic peptide predicted in a particular patient.
• the vaccine will contain more than one immunogenic peptide or peptide precursor.
• the length of the peptide used in the vaccine may vary in length.
• the peptide is about 7 to 50 amino acids in length (such as about any of 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 22, 25, 30, 35, 40, 45, or 50 amino acids in length).
• the peptide is about 8 to 12 amino acids in length.
• the peptide is about 8 to 10 amino acids in length.
• the peptide can be utilized in its isolated form, or alternatively, peptides can be added to the ends of the MHC isolated peptide to produce a "long peptide" that may prove more immunogenic (see, e.g. Castle et al., Cancer Res 72: 1081-1091 (2012)).
• the peptide may also be tagged, or be a fusion protein, or be hybrid molecule.
• the peptide is in the form of a pharmaceutically acceptable salt.
• the vaccine is a nucleic acid vaccine.
• the nucleic acid encodes an immunogenic peptide or peptide precursor.
• the nucleic acid vaccine comprises sequences flanking the sequence coding the immunogenic peptide or peptide precursor. In some embodiments, the nucleic acid vaccine comprises more than one immunogenic epitope. In some embodiments, the nucleic acid vaccine is a DNA-based vaccine. In some embodiments, the nucleic acid vaccine is a RNA- based vaccine. In some embodiments, the RNA-based vaccine comprises mRNA. In some embodiments, the RNA-based vaccine comprises naked mRNA. In some embodiments, the RNA-based vaccine comprises modified mRNA (e.g., mRNA protected from degradation using protamine. mRNA containing modified 5 'CAP structure, or mRNA containing modified nucleotides).
• modified mRNA e.g., mRNA protected from degradation using protamine. mRNA containing modified 5 'CAP structure, or mRNA containing modified nucleotides).
• the RNA-based vaccine comprises single- stranded mRNA.
• the polynucleotide may be substantially pure, or contained in a suitable vector or delivery system.
• Suitable vectors and delivery systems include viral, such as systems based on adenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associated virus or hybrids containing elements of more than one virus.
• Non- viral delivery systems include cationic lipids and cationic polymers (e.g., cationic liposomes).
• physical delivery such as with a 'gene-gun' may be used.
• the peptides described herein can be used for making mutant peptide specific therapeutics such as antibody therapeutics.
• the mutant peptides can be used to raise and/or identify antibodies specifically recognizing the mutant peptides. These antibodies can be used as therapeutics.
• Synthetic short peptides have been used to generate protein-reactive antibodies. The advantage of immunizing with synthetic peptides is that unlimited quantity of pure stable antigen can be used. This approach involves
• the properties of antibodies are dependent on the primary sequence information. A good response to the desired peptide usually can be generated with careful selection of the sequence and coupling method. Most peptides can elicit a good response.
• the advantage of anti-peptide antibodies is that they can be prepared immediately after determining the amino acid sequence of a mutant peptide and the particular regions of a protein can be targeted specifically for antibody production. Since the mutant peptides have been screened for high immunogenicity there is a high chance that the resulting antibody will recognize the native protein in the tumor setting.
• peptide length is another important factor to consider. Approximately, a peptide of 10-15 residues is optimal for anti-peptide antibody production; longer peptides are better since the number of possible epitopes increases with peptide length. However, long peptides increase the difficulties in synthesis, purification, and coupling to carrier proteins. The quality of an antibody is dependent upon the quality of the peptide. Side products contained in peptide products can lead to low-quality antibodies.
• Peptide-carrier protein coupling is another factor involved in the production of high titer antibodies. Most coupling methods rely on the reactive functional groups in amino acids, such as -NH2, -COOH, -SH, and phenolic -OH. Site-directed coupling is the method of choice. Any suitable method used in anti-peptide antibody production can be utilized with the peptides identified by the methods of the present invention. Two such known methods are the Multiple Antigenic Peptide system (MAPs) and the Lipid Core Peptides (LCP method). The advantage of MAPs is that the conjugation method is not necessary. No carrier protein or linkage bond is introduced into the immunized host. One disadvantage is that the purity of the peptide is more difficult to control. In addition, MAPs can bypass the immune response system in some hosts. The LCP method is known to provide higher titers than other anti-peptide vaccine systems and thus can be advantageous.
• MAPs Multiple Antigenic Peptide system
• LCP method Lipid Core Peptides
• MHC/peptide complexes comprising the disease specific immunogenic mutant peptides disclosed herein.
• Such MHC/peptide complexes can be used, for example, for identifying antibodies, soluble TCRs, or TCR analogs.
• TCR mimics are antibodies that bind peptides from tumor associated antigens in the context of specific HLA environments. This type of antibody has been shown to mediate the lysis of cells expressing the complex on their surface as well as protect mice from implanted cancer cells lines that express the complex (see, e.g., Wittman et al., J. of Immunol. 177:4187-4195 (2006)).
• TCR mimics as IgG mAbs is that affinity maturation can be performed and the molecules are coupled with immune effector functions through the present Fc domain.
• These antibodies can also be used to target therapeutic molecules to tumors, such as toxins, cytokines, or drug products.
• Other types of molecules that have been developed using peptides such as those selected using the methods of the present invention using non-hybridoma based antibody production or production of binding competent antibody fragments such as anti-peptide Fab molecules on bacteriophage.
• These fragments can also be conjugated to other therapeutic molecules for tumor delivery such as anti-peptide MHC Fab-immunotoxin conjugates, anti-peptide MHC Fab-cytokine conjugates and anti-peptide MHC Fab-drug conjugates.
• the present disclosure provides methods of treatment comprising an immunogenic vaccine.
• a method of treatment for disease such as cancer
• a method of treatment for a disease such as cancer
• a method of treatment for a disease such as cancer
• a method of treatment for a disease such as cancer
• an immunogenic vaccine may comprise a pharmaceutically acceptable disease- specific immunogenic peptide.
• an immunogenic vaccine may comprise a pharmaceutically acceptable precursor to a disease- specific immunogenic peptide (such as a protein, peptide, DNA and RNA).
• a method of treatment for a disease (such as cancer) is provided, which may include administering to an individual an effective amount of an antibody specifically recognizing the disease-specific, immunogenic mutant peptide.
• a method of treatment for a disease (such as cancer) is provided, which may include administering to an individual an effective amount of a soluble TCR or TCR analog specifically recognizing the disease- specific, immunogenic mutant peptide.
• the cancer is any one of: carcinoma, lymphoma, blastema, sarcoma, leukemia, squamous cell cancer, lung cancer (including small cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, head and neck cancer, colorectal cancer, rectal cancer, soft-tissue sarcoma, Kaposi's sarcoma, B-cell lymphoma (including low grade/follicular non-Hodgkin's lymph
• intermediate grade/follicular NHL intermediate grade diffuse NHL
• high grade follicular NHL intermediate grade diffuse NHL
• immunoblastic NHL high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and
• ALL lymphoblastic leukemia
• PTLD post-transplant lymphoproliferative disorder
• a method of treating a disease comprising: a) identifying a disease-specific, immunogenic mutant peptides in the individual; and b) synthesizing the peptide or peptide precursor; and c) administering the peptide to the individual.
• a method of treating a disease comprising: 1) identifying a disease- specific, immunogenic mutant peptide in the individual; b) producing an antibody specifically recognizing the mutant peptide; and c) administering the peptide to the individual.
• the identification step combines sequence- specific variant identification method with methods of immunogenicity prediction.
• the identification step combines sequence-specific variant identification method with mass spectrometry. Any methods of identifying a disease- specific, immunogenic mutant peptide described herein can be used for the treatment methods described herein.
• the method further comprises obtaining a sample of the disease tissue from the individual.
• an individual e.g., human who has been diagnosed with or is suspected of having cancer.
• an individual may be a human.
• an individual may be at least about any of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years old.
• an individual may be a male.
• an individual may be a female.
• an individual may have refused surgery.
• an individual may be medically inoperable.
• an individual may be at a clinical stage of Ta, Tis, Tl, T2, T3a, T3b, or T4.
• a cancer may be recurrent.
• an individual may be a human who exhibits one or more symptoms associated with cancer.
• an individual may be genetically or otherwise predisposed (e.g., having a risk factor) to developing cancer.
• the methods provided herein may be practiced in an adjuvant setting.
• the method is practiced in a neoadjuvant setting, i.e., the method may be carried out before the primary/definitive therapy.
• the method is used to treat an individual who has previously been treated. Any of the methods of treatment provided herein may be used to treat an individual who has not previously been treated.
• the method is used as a first line therapy. In some embodiments, the method is used as a second line therapy.
• a method of reducing incidence or burden of preexisting cancer tumor metastasis comprising administering to the individual an effective amount of a composition comprising an immunogenic vaccine.
• a method of prolonging time to disease progression of cancer in an individual comprising administering to the individual an effective amount of a composition comprising an immunogenic vaccine.
• a method of prolonging survival of an individual having cancer comprising administering to the individual an effective amount of a composition comprising an immunogenic vaccine.
• At least one or more chemotherapeutic agents may be administered in addition to the composition comprising an immunogenic vaccine.
• the one or more chemotherapeutic agents may (but not necessarily) belong to different classes of chemotherapeutic agents.
• a method of treating a disease (such as cancer) in an individual comprising administering: a) an immunogenic vaccine, and b) an immunomodulator.
• a method of treating a disease (such as cancer) in an individual comprising administering: a) an immunogenic vaccine, and b) an antagonist of a checkpoint protein.
• a method of treating a disease (such as cancer) in an individual comprising administering: a) an immunogenic vaccine, and b) an antagonist of programmed cell death 1 (PD-1), such as anti- PD-1.
• PD-1 programmed cell death 1
• a method of treating a disease (such as cancer) in an individual comprising administering: a) an immunogenic vaccine, and b) an antagonist of programmed death-ligand 1 (PD-L1), such as anti-PD-Ll.
• PD-L1 programmed death-ligand 1
• a method of treating a disease (such as cancer) in an individual comprising administering: a) an immunogenic vaccine, and b) an antagonist of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), such as anti-CTLA-4.
• CTLA-4 cytotoxic T-lymphocyte- associated protein 4
• TILs tumor- infiltrating cells
• tumor-specific CD8 TILs co-expressed PD-1 and TEVI-3, the markers of T cell exhaustion compared to bulk TILs (52.6+3.6%) (FIG. 4D). Tumor- specific CD8 TILs also expressed higher level of surface PD-1.
• the neoepitope- specific CD8 T cell responses were evaluated to see if they could be further amplified in tumor-bearing mice upon immunization.
• the frequency of Adpgk-reactive CD8 T cells increased remarkably in the spleen of tumor-bearing mice compared to naive healthy animals (FIG 4B). It was also observed nearly three-fold increase in accumulation of Adgpk- specific CD8 T cells among total CD8 TILs in the tumors (FIG. 5B).
• Peptide vaccination also increased overall infiltration of CD45 + cells and CD8 + T cells in tumors, which resulted into nearly 20-fold increase in the frequency of neoepitope- specific CD8 T cells among the total live cells in the tumor (FIG. 5C).
• MHCI peptide profiling was conducted for the H-2Kb and H-2Db ligandome of two murine cell lines of H-2b-background: TRAMP-C1 (ATCC) and MC-38 (Academisch Ziekenhuis Leiden). Cells derived from C57BL/6 mice were prepared as previously described. Reference is made to U.S. patent application Ser. No. 13/087,948 and U.S. patent application Ser. No. 11/00,474 for complete description of methods of preparing cell lines. MHCI molecules of each sample were immunoprecipitated using two different antibodies to extract H-2Kb specific and H-2Db specific peptides, respectively.
• Peptides were separated by reversed-phase chromatography (nanoAcquity UPLC system, Waters, Milford, MA) using a 180-minute gradient.
• the eluted peptides were analyzed by data-dependent acquisition (DDA) in an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ionization (ESI) source.
• DDA data-dependent acquisition
• LTQ-Orbitrap Velos hybrid mass spectrometer Thermo Fisher Scientific, Bremen, Germany
• ESI electrospray ionization
• Synthetic peptides corresponding to identify mutant MC-38 and TRAMP-C1 antigen peptides were analyzed on an LTQ-Orbitrap Elite mass spectrometer (ThermoFisher, Bremen, Germany) and ionized using an ADVANCE source (Michrom-Bruker, Fremont, CA) at a spray voltage of 1.2 kV. Mass spectral data were acquired using a method consisting of one full MS scan (375-1600 m/z) in the Orbitrap at resolution of 60,000 ⁇ / ⁇ at m/z 400, followed by MS/MS (centroid) scans in the LTQ of the peptide fragment ions.
• RNA-Seq libraries 1 ⁇ g of total RNA from MC-38 and TRAMP-Cl cancer cell lines was used to generate RNA-Seq libraries using TruSeq RNA sample preparation kit (Illumina, CA). Total RNA was purified from cell lines and fragmented to 200-300 base pairs (bp), with an average length of 260 bp. RNA-Seq libraries were multiplexed (two per lane) and sequenced on HiSeq 2000 as per manufacturer's recommendation (Illumina, CA).
• RNA fragments were sequenced from MC-38 and 65.3 M from TRAMP-Cl.
• 60M reads were sequenced from each cell line. Reads were mapped to the mouse genome (NCBI build 37 or mm9) using GSNAP (Wu and Nacu, Bioinformatics, 2010, v.26, 873-881). Only uniquely mapped reads were retained for further analysis. 80.6 M RNA fragments were uniquely mapped in the MC-38 sample and 57.6 M in the TRAMP-Cl sample. 50.9 M exome fragments in MC-38 and 52 M fragments in TRAMP- Cl were uniquely mapped. To obtain mouse gene models, Refseq mouse genes were mapped to the mm9 genome using GMAP, and the genomic sequence was then used for making the gene models.
• Exome-seq based variants were called using GATK1. Variants with 10% or greater allelic frequency were retained. Variants were annotated for effects on transcripts using the variant effect predictor tool 2. Only the variants for which an amino acid change can be interpreted were retained. In order to obtain variants with evidence of expression, the exome- based variant positions were checked for evidence of variation with RNA-Seq read alignments. Variants that were corroborated by more than 2 RNA-Seq reads and expressed at 10% or more allelic frequency based on RNA-Seq were retained.
• Fragment ion mass tolerance was specified at 0.8 Da or 0.05 Da for MS/MS data acquired in the LTQ or Orbitrap, respectively. Search results were filtered using a linear discriminant algorithm (LDA) to an estimated peptide false discovery rate (FDR) of 5%. For higher confidence in mutant peptide identifications, the data was further filtered either by peptide length, 8 for H-2Kb data and 9 for H-2Db or employing regular expressions to isolate peptides with the following well characterized anchor motifs H-2Kb: XXXX[FY]XX[MILV] (SEQ ID NO. 22) and H-2Db: XXXX[N]XXX[MIL] (SEQ ID NO. 23). Synthetic peptides were generated to validate the sequences.
• LDA linear discriminant algorithm
• FDR estimated peptide false discovery rate
• peptide-MHC complex structures were chosen from the PDB based on sequence similarity between the mutant peptide and peptide in the model structure.
• the following PDB code was used: Repsl, 2ZOL 4; Adpgk, 1HOC 5; Dpagtl, 3P9L 6; Cpnel, 1JUF7; Irgq, 1FFN 8; Aatf, 1BZ9.
• the Medl2 peptide was not modeled due to a lack of a published H-2Kb crystal structure in complex with 10-mer peptide that could be used as a reasonable starting model.
• the peptide was then modified to the mutant form using COOT 10.
• These first models were then optimized using the Rosetta FlexPepDock web server 11, and the top scoring model chosen for display.
• mice Age-matched 6-8 weeks old C57BL/6 mice (The Jackson Laboratory) were injected intraperitonealy with 50mg long peptide each in combination with adjuvant (50 ⁇ g anti-CD40 Ab clone FJK45 plus lOOmg poly(LC) (Invivogen)) in PBS. Mice were immunized on day 0 and day 14 and one week following the last injection, either blood or splenocytes were used for detection of Ag-specific CD8 T cells. To identify peptide-specific T cells, cells were stained with PE-conjugated dextramers (MHCI/peptide complex; Immudex, Denmark) for 20 min followed by staining with cell surface markers CD3, CD4, CD8 and B220 (BD
• GIPVHLELASMTNMELMSSIVHQQVFPT SEQ ID NO. 2; Dpagtl:
• C57BL/6 mice were implanted subcutaneously on the right flank with 1X105 MC- 38 tumor cells. The whole tumor was isolated and digested with collagenase and DNAase to isolate TILs. TILs were stained with dextramers (as described above) followed by antibodies against CD45, Thyl.2, CD4, CD 8 (BD Biosciences), PD-1 (eBiosciences) and TIM-3 (R&D Systems). Live/dead stain was used to gate on live cells.
• mice were inoculated subcutaneously (right hind flank) with lxlO 5 MC-38 cells in a suspension of Hanks' balanced salt solution (HBSS) and phenol red-free matrigel (Becton Dickinson Bioscience, San Jose, CA).
• HBSS Hanks' balanced salt solution
• phenol red-free matrigel Becton Dickinson Bioscience, San Jose, CA.
• mice were immunized with adjuvant (50mg anti-CD40 plus lOOmg poly(LC) or adjuvant with 50 ⁇ g Repsl, Adpgk and Dpagtl peptide each 3 weeks before the tumor inoculation. Induction of peptide- specific CD8 T cells was measured in blood a day prior to inoculation with tumor cells.
• mice For vaccination in tumor bearing mice, 10 days after inoculation with 1x105 MC-38 tumor cells (only tumor with volume of approximately 100-150 mm at day 10 were included in the study) mice were injected with adjuvant or adjuvant with 50 ⁇ g Repsl, Adpgk, and Dpagtl peptide each. Measurements and weights were collected twice a week. Animals exhibiting weight loss of more than 15% of their first body weight were weighed daily and euthanized if they lose more than 20% of their first body weight.
• mice that exhibited adverse clinical issues were observed more frequently, up to daily depending on the severity, and euthanized if moribund. Mice were euthanized if tumor volumes exceeded 3,000 mm , or after 3 months if tumors did not form. Throughout the entire study, clinical observations of all mice were performed twice a week. Tables

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