Unbiased identification of tumor rejection mediating neoepitopes

1. An unbiased method of identifying tumor rejection mediating neoepitopes (TRMNs), comprising:

comparing a cancer cell exome sequence from a cancer patient to a reference exome sequence and identifying single nucleotide variants (SNVs) in the cancer cell exome sequence compared to the reference exome sequence;

validating the SNVs using nucleic acid sequencing;

identifying 8-14 amino acid putative neoepitopes including the validated SNVs, wherein the putative neoepitopes are unbiased by MHC binding and/or CD8T* reactivity;

calculating an IC 50 for an MHC allele for each 8-14 amino acid putative neoepitope including the SNVs and calculating an IC 50 for the MHC allele for a corresponding non-mutated amino acid sequence for each validated SNV;

plotting the putative neoepitope IC 50 s on one axis, and the non-mutated amino acid sequence IC 50 s on a perpendicular axis to provide a bivariate scatter plot;

clustering the putative neoepitopes in the bivariate scatter plot using model-based clustering based on parameterized finite Gaussian mixture models using the IC 50 s;

selecting as TRMNs the neoepitopes in the bivariate scatter plot which are clustered putative neoepitopes in the space greater than 501 nM on the x-axis and greater than 501 nM on the y-axis

wherein the TRMNs are in an elliptical cluster encompassed by a circle having a center at 27,176.9 nM for the x-axis and 33,556.51 nM for the y-axis, and a radius of 33,195 nM from the center, or

wherein the TRMNs are in an elliptical cluster encompassed by a circle having a center at 27,176.9 nM for the x-axis and 33,556.51 nM for the y-axis and a radius of 22,430 nM from the center;

producing a peptide population or a nucleic acid population for expressing the peptide population, the peptide population comprising 15-100 different amino acid peptides, the peptides including one or more of the TRMNs;

producing a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptide population or nucleic acid population; and

optionally administering the pharmaceutical composition to the cancer patient.

2. The method of claim 1 , further comprising normalizing the putative neoepitope IC 50 s and the non-mutated amino acid sequence IC 50 s prior to plotting.

3. The method of claim 1 , wherein the TRMNs have IC 50 values for the MHC allele of 2,000 nM to 33,000 nM.

4. The method of claim 1 , wherein the TRMNs do not produce a statistically significant CD8 + T cell response, measured by ELISpot, cytotoxicity or FACS assays.

5. The method of claim 1 , wherein NetMHC 4.0 is used to determine the IC 50 s for the MHC allele.

6. The method of claim 1 , wherein the cancer cell exome sequence is from cancer cells that are solid tumor cancer cells.

7. The method of claim 6 , wherein the solid tumor cancer cells are from breast, prostate, ovaries, lungs or brain, and the liquid cancer cells are from a leukemia or a lymphoma.

8. The method of claim 1 , wherein the cancer cell exome sequence includes all potential neoepitopes in the cancer cells.

9. The method of claim 1 , wherein the reference exome is from a subject of the same species as the cancer cells.

10. The method of claim 1 , wherein the pharmaceutical composition further comprises an adjuvant.

11. The method of claim 1 , wherein the pharmaceutical composition further comprises one or more immune-modulating agents.

12. The method of claim 11 , wherein the immune-modulating agent is a TLR ligand or an antibody.

13. A method of treating a cancer patient comprising identifying an unbiased population of tumor rejection mediating neoepitopes (TRMNs) by;

comparing a cancer cell exome sequence from the cancer patient to a reference exome sequence and identifying single nucleotide variants (SNVs) in the cancer cell exome sequence compared to the reference exome sequence;

validating the SNVs using nucleic acid sequencing;

identifying 8-14 amino acid putative neoepitopes including the validated SNVs, wherein the putative neoepitopes are unbiased by MHC binding and/or CD8T* reactivity;

calculating an IC 50 for an MHC allele for each 8-14 amino acid putative neoepitope including the SNVs and calculating an IC 50 for the MHC allele for a corresponding non-mutated amino acid sequence for each validated SNV;

plotting the putative neoepitope IC 50 s on the x-axis, and the non-mutated amino acid sequence IC 50 s on the y-axis to provide a bivariate scatter plot;

clustering the putative neoepitopes in the bivariate scatter plot using model-based clustering based on parameterized finite Gaussian mixture models using the IC 50 s

selecting as TRMNs the neoepitopes in the bivariate scatter plot which are clustered putative neoepitopes in the space greater than 501 nM on the x-axis and greater than 501 nM on the y-axis

wherein the TRMNs are in an elliptical cluster encompassed by a circle having a center at 27,176.9 nM for the x-axis and 33,556.51 nM for the y-axis, and a radius of 33,195 nM from the center, and/or

wherein the TRMNs are in an elliptical cluster encompassed by a circle having a center at 27,176.9 nM for the x-axis and 33,556.51 nM for the y-axis and a radius of 22,430 nM from the center;

producing a peptide population or a nucleic acid population for expressing the peptide population, the peptide population comprising 15-100 different amino acid peptides, the peptides including one or more of the TRMNs;

producing a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptide population or nucleic acid population; and

administering the pharmaceutical composition to the cancer patient.