Compositions and methods of identifying tumor specific neoantigens

A method of identifying subject-specific peptides and preparing a subject-specific immunogenic composition comprising said subject-specific peptides that upon administration presents said subject-specific peptides to the subject's immune system, wherein the subject has a tumor and said subject-specific peptides are specific to the subject and the subject's tumor, said method comprising:

• sequencing nucleic acid sample of the subject's tumor and of a non-tumor sample of the subject,
• identifying 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 sequences comprising tumor-specific non-silent mutations not present in the non-tumor sample;
• producing 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subject-specific peptides encoded by said 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 sequences comprising tumor-specific non-silent mutations non present in the non-tumor sample
• measuring binding of said produced subject-specific peptides to a HLA protein of said subject,
• wherein each of said subject-specific peptides has a different tumor neo-epitope that is an epitope specific to the tumor of the subject, from the neo-epitopes identified in tumor specific mutations,
• wherein each neo-epitope is an expression product of a tumor-specific non-silent mutation not present in the non-tumor sample and each neo-epitope binds to a HLA protein of the subject,
• wherein said subject-specific peptides have a point mutation and bind to HLA proteins of the subject with an IC50 less than 500 nM;
• formulating said subject-specific immunogenic composition for administration to the subject so that upon administration said 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subject-specific peptides are presented to the subject's immune system,
• wherein said immunogenic composition comprises:
• said 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subject-specific peptides, comprising:
• (1) a peptide that has a non-synonymous mutation leading to different amino acids in comparison with a protein of the non-tumor sample;
• or
• (2) a peptide having a read-through mutation in which a stop codon is modified or deleted, leading to translation of a longer protein in comparison with a protein of the non-tumor sample with a novel tumor-specific sequence at the C-terminus;
• or
• (3) a peptide that has a splice site mutation that leads to the inclusion of an intron in the mature mRNA and thus has a unique tumor-specific protein sequence;
• or
• (4) a peptide representing a chromosomal rearrangement that has Given rise to a chimeric protein with tumor-specific sequences at the junction of two proteins of the non-tumor sample and thus represents a gene fusion;
• or (5) a peptide representing in comparison with a protein of the non-tumor sample a frameshift mutation or deletion that leads to a new open reading frame and a novel tumor-specific protein sequence.