PERSONALIZED CANCER VACCINES AND METHODS THEREFOR

Abstract

Methods of cane r treatment based, on personalized vaccines are disclosed. Individual amino acid substitutions from tumors are revealed using whole genome sequencing, and identified as neoantigens silico. Peptide sequences are then tested in vitro for ability to bind HLA molecules and to be presented to CD8+ T-cells. A vaccine is formed using neoantigen peptides and an adjuvant or dendritic cells (DC) autologous to a subject. In the latter, autologous DC are matured and contacted with the neoantigen peptides. The DC are then administered to the subject. PBMC are then obtained from the subject, and CD8+ T cells specific to the neoantigens are cultured and enriched. Enriched T-cells are then administered to the subject to treat cancer. Treatment resulted in tumor regression in mice bearing human melanomas, and complete or partial responses were observed in human patients.

Claims

1. A method of treating a cancer in a subject in need thereof, comprising: providing a neoantigen peptide encoded in DNA of a tumor of the subject, wherein the neoantigen peptide consists of from 8 to 13 amino acids, binds in silico to an HLA class I molecule with an affinity of <500 nM and a stability>2 h and binds in vitro to an HLA class I molecule with an affinity of <4.7 log (IC50, nM); transfecting at least one HLA class I positive cell with at least one tandem minigene construct comprising at least one sequence encoding the at least one neoantigen; identifying a complex comprising the at least one HLA molecule and the at least ogre neoantigen peptide produced by the at least one HLA class I positive cell; forming a vaccine comprising the at least one neoantigen; and administering the vaccine to the subject, wherein at least one tumor cell of the cancer comprises at least one polypeptide comprising at least one amino acid substitution.

2. A method in accordance with claim 1, wherein the at least one neoantigen peptide consists of 9 amino acids.

3. A method in accordance with claim 1, wherein the at least one neoantigen binds in silico to an HLA class I molecule with an affinity of <250 nM.

4. A method in accordance with claim 1, wherein the at least one neoantigen binds in vitro to an HLA class I molecule with an affinity of <3.8 log (IC50, nM).

5. A method in accordance with claim 1, wherein the vaccine comprises at least seven neoantigen peptides.

6. A method in accordance with claim 1, wherein the HLA class I molecule is selected from the group consisting of HLA-A*01:01, HLA-B*07:02, HLA-A*02:01, HLA-B*07:03, HLA-A*02:02, HLA-B*08:01, HLA-A*02:03, HLA-B*15:01, HLA-A*02:05, HLA-B*15:02, HLA-A*02:06, HLA-B*15:03, HLA-A*02:07, HLA-B*15:08, HLA-*03:01, HLA-B*15:12, HLA-A*11:01, HLA-B*15:16, HLA-A*11:02, HLA-B*15:18, HLA-A*24:02, HLA-B*27:03, HLA-A*29:01, HLA-B*27:05, HLA-A*29:02, HLA-B*27:08, HLA-A34:02, HLA-B*35:01, HLA-A*36:01, HLA-B*35:08, HLA-B*42:01, HLA-B*53:01, HLA-B*54:01, HLA-B*56:01, HLA-B*56:02, HLA-B*57:01, HLA-B*57:02, HLA-B*57:03, HLA-B*58:01, HLA-B*67:01 and HLA-B*81:01.

7. A method in accordance with claim 1, wherein the HLA class I molecule is selected from the group consisting of an HLA-A*02:01 molecule, an HLA-A*11:01 molecule and an HLA-B*08:01 molecule.

8. A method in accordance with claim 1, wherein the at least one HLA class I positive cell is at least one HLA class I positive melanoma cell.

9. A method in accordance with claim 1, wherein the cancer is selected from the group consisting of skin cancer, lung cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, esophageal cancer, gastric cancer, intestinal cancer, breast cancer, and a mismatch repair deficiency cancer.

10. A method in accordance with claim 1, wherein the cancer is a melanoma.

11. A method in accordance with claim 1, wherein the forming a vaccine comprises: providing a culture comprising dendritic cells obtained from the subject; and contacting the dendritic cells with the at least one neoantigen peptide, thereby forming dendritic cells comprising the at least one neoantigen peptide.

12. A method in accordance with claim 11, further comprising: administering to the subject the dendritic cells comprising the at least one neoantigen peptide; obtaining a population of CD8+ T cells from a peripheral blood sample from the subject, wherein the CD8+ cells recognize the at least one neoantigen; and expanding the population of CD8+ T cells that recognizes the neoantigen.

13. A method in accordance with claim 1, wherein the identifying a complex comprises performing an assay selected from the group consisting of an LC/MS assay, a reverse phase HPLC assay and a combination thereof.

14. A method of treating a cancer in a subject in need thereof, comprising: a) providing a sample of a tumor from a subject; b) performing exome sequencing on the sample to identify one or more amino acid substitutions comprised by the tumor exome; c) performing transcriptome sequencing on the sample to verify expression of the amino acid substitutions identified in b); and d) selecting at least one candidate neoantigen peptide sequence from amongst the amino acid substitutions identified in c) according to the following criteria: i) Exome VAF>10%; ii) Transcription VAF>10%; iii) Alternate reads>5; iv) FPKM>1; v) binds in silico to an HLA class I molecule with an affinity of <500 nM and a stability>2 h; e) performing an in vitro HLA class I binding assay; f) selecting at least one candidate neoantigen peptide sequence from amongst the amino acid substitutions identified in d) that bind HLA class one molecules with an affinity of <4.7 log (IC50, nM) in the assay performed in e) g) transfecting at least one HLA class I positive cell with at least one tandem minigene construct comprising at least one sequence encoding the at least one neoantigen; h) identifying a complex comprising the at least one HLA molecule and the at least one neoantigen peptide produced by the at least one HLA class I positive cell; i) forming a vaccine comprising the at least one neoantigen; and j) administering the vaccine to the subject, wherein at least one tumor cell of the cancer comprises at least one polypeptide comprising the one or more amino acid substitutions.

15. A method in accordance with claim 14, wherein the in vitro HLA class I binding assay is selected from the group consisting of a T2 assay and a fluorescence polarization assay.

16. A method in accordance with claim 14, wherein the forming a vaccine comprises: providing a culture comprising dendritic cells obtained from the subject; and contacting the dendritic cells with the at least one neoantigen peptide, thereby forming dendritic cells comprising the at least one neoantigen peptide.

17. A method in accordance with claim 16, further comprising: administering to the subject the dendritic cells comprising the at least one neoantigen peptide; obtaining a population of CD8+ T cells from a peripheral blood sample from the subject, wherein the CD8+ T cells recognize the at least one neoantigen; and expanding the population of CD8+ T cells that recognizes the neoantigen.

18. A method in accordance with claim 14, wherein the identifying a complex comprising the at least one HLA molecule and the at least one neoantigen peptide comprises performing an assay selected from the group consisting of a LC/MS assay, a reverse phase HPLC assay and a combination thereof.

19. A method of treating a cancer in a subject in need thereof, comprising: providing a neoantigen peptide encoded in DNA of a tumor of the subject, wherein the neoantigen peptide consists of from 8 to 13 amino acids, binds in silico to an HLA class I molecule with an affinity of <500 nM and a stability>2 h; performing an in vitro HLA class I molecule binding assay to identify at least one neoantigen peptide which binds in vitro to an HLA class I molecule with an affinity of <4.7 log (IC50, nM); transfecting at least one HLA class I positive cell with at least one tandem minigene construct comprising at least one sequence encoding the at least one neoantigen; identifying a complex comprising the at least one HLA molecule and the at least one neoantigen peptide produced by the at least one HLA class I positive cell; forming a vaccine comprising the at least one neoantigen; and administering the vaccine to the subject, wherein at least one tumor cell of the cancer comprises at least one polypeptide comprising at least one amino acid substitution.

20. A method in accordance with claim 19, wherein the in vitro HLA class I binding assay is selected from the group consisting of a T2 assay and a fluorescence polarization assay.

21. A method in accordance with claim 19, wherein the identifying a complex comprising the at least one HLA molecule and the at least one neoantigen peptide comprises performing an assay selected from the group consisting of an LC/MS assay, a reverse phase HPLC assay and a combination thereof.

22. A method in accordance with claim 19, wherein the forming a vaccine comprises: providing a culture comprising dendritic cells obtained from the subject; and contacting the dendritic cells with the at least one neoantigen peptide, thereby forming dendritic cells comprising the at least one neoantigen peptide.

23. A method in accordance with claim 22, further comprising: administering to the subject the dendritic cells comprising the at least one neoantigen peptide; obtaining a population of CD8+ T cells from a peripheral blood sample from the subject, wherein the CD8+ cells recognize the at least one neoantigen; and expanding the population of CD8+ T cells that recognizes the neoantigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 illustrates a work flow for identifying candidate neo-antigens and preparing a dendritic cell vaccine comprising the neo-antigens.

[0062] FIG. 2 illustrates the analytical steps and specific neo-antigen analysis for a melanoma patient.

[0063] FIG. 3 illustrates HLA binding on T-cell surfaces to various neo-antigens.

[0064] FIG. 4 illustrates a schematic representation of the steps for creating a dendritic cell based vaccine of the present teachings.

[0065] FIG. 5 illustrates T cell response in vaccinated patients for the listed neo-antigens using a dextramer assay.

[0066] FIG. 6 illustrates the in silico binding affinity (top) and stability (bottom) of peptides to T-cell HLA.

[0067] FIG. 7 illustrates the binding of immunogenic peptides to blood CD8 T cells following vaccination.

[0068] FIG. 8 illustrates antigen-specific T cell yields following vaccination.

[0069] FIG. 9 is a schematic diagram of a tandem mini-gene construct.

[0070] FIG. 10 illustrates ELISA-measured production of IFN- by T cells.

[0071] FIG. 11 illustrates that T cell specificity can detect a single amino acid change for AKAP13 and Sec24A.

[0072] FIG. 12 illustrates that T cells cannot discriminate between peptides with a single amino acid change for OR8B3.

[0073] FIG. 13 illustrates that vaccine-induced T cells produce large amounts of IFN- relative to IL-4, -5 and -13.

[0074] FIG. 14 illustrates tumor regression monitored by luciferase (photon flux).

[0075] FIG. 15 illustrates disease progression of mice inoculated with a luciferase expressing melanoma.

[0076] FIG. 16 illustrates the relationship between tumor regression and survival.

[0077] FIG. 17 illustrates immunological and clinical outcomes for patients treated with G209-2M and G2880-9V specific CD8+ T cells.

[0078] FIG. 18 illustrates ex-vivo IL-12 production and that Tc1 profile correlates with clinical outcome (TPP)

[0079] FIG. 19 illustrates that weak p35 transcription accounts for the IL-12p70 defect in non-responder patients.

[0080] FIG. 20 illustrates that impaired IL-12p70 production by a patient's dendritic cells is rescued by a combination of innate and adaptive signals.

[0081] FIG. 21 illustrates that a combination of innate and adaptive signals for dendritic cell maturation enhances the kinetics of the response.

[0082] FIG. 22 illustrates that a combination of innate and adaptive signals for dendritic cell maturation promotes Tc1 -polarized immunity.

[0083] FIG. 23 illustrates that cutaneous melanoma harbor a significant mutation burden.

[0084] FIG. 24 illustrates the translation of tumor missense mutations into patient-specific vaccines.

[0085] FIG. 25 illustrates discrimination between mutation and wild-type sequences and discrimination between antigens that are and are not presented to T-cells.

[0086] FIG. 26A-B illustrates clinical trial schema and ex-vivo IL-12p70 levels produced by mature DC.

[0087] FIG. 27 is a schematic representation of the selection of AAS peptides for use in experiments and vaccines.

[0088] FIG. 28 is a schematic representation of a strategy for neoantigen selection.

[0089] FIG. 29 illustrates AAS-comprising peptide binding to HLA-A*02:01.

[0090] FIG. 30A-C illustrate immune response to neoantigens.

[0091] FIG. 31 illustrates immune-monitoring of neoantigen-specific CD8+ T cell responses

[0092] FIG. 32 illustrates frequency of G209-2M- and G280-9V-specific T cells in CD8+ populations isolated directly from PBMC samples and after ex-vivo expansion using autologous DC and artificial antigen presenting cells.

[0093] FIG. 33 illustrates kinetics of immune responses to G209-2M and G280-9V peptides.

[0094] FIG. 34 illustrates antigenic determinants recognized by vaccine-induced T-cells

[0095] FIG. 35 illustrates cytokine production in neoantigen-specific T cells that were stimulated with artificial antigen presenting cells in the presence (open bar) or absence (close bar) of AAS-peptide.

[0096] FIG. 36 illustrates the Type 1/Type 2 phenotype of neoantigen-specific CD8+ T cells.

[0097] FIG. 37A-B illustrates the structure (A) and expression (B) of tandem mini-gene constructs (TMC) used for evaluating processing and presentation of neoantigens.

[0098] FIG. 38 illustrates neoantigen processing and presentation.

[0099] FIG. 39 illustrates interferon production in neoantigen-specific CD8 T cells cultured with neoantigen expressing DM6 cells.

[0100] FIG. 40A-H illustrates processing and presentation of tumor neoantigens.

[0101] FIG. 41A-D illustrates processing and presentation of to G280 and WNV SVG9 peptide controls.

[0102] FIG. 42 is a schematic diagram for analysis and identification of neoantigen-specific TCR clonotypes in CD8+ T cell populations isolated from PBMC samples obtained Pre- and Post-vaccination.

[0103] FIG. 43A-B illustrates profiles of purified neoantigen-specific CD8+ T cells used for the generation of TCR CDR3 reference libraries.

[0104] FIG. 44A-B illustrate that vaccination promotes a diverse neoantigen-specific T cell repertoire.

[0105] FIG. 45 depicts schematic diagrams of HLA-A*02:01 and HLA-B*08:01 neoantigen identification for patient MEL66.

[0106] FIG. 46 depicts schematic diagrams of HLA-A*02:01 and HLA-A*11:01 neoantigen identification for patient MEL69.

[0107] FIG. 47 depicts results of a dextramer assay to illustrate neoantigen response in T cells following administration of a vaccine in accordance with the present teachings.

DETAILED DESCRIPTION

[0108] The present teachings describe methods of creating vaccines for personalized cancer treatment. As used herein, a vaccine is a preparation that induces a T-cell mediated immune response. As used in the present description and the appended claims, the singular forms a, an and the are intended to include the plural forms as well, unless the context indicates otherwise.

[0109] In some embodiments, methods of the present teachings can comprise sequencing DNA from excised tumor tissue of a subject to identify amino acid substitutions, performing sequence capture to confirm the expression of the amino acid substitutions, selecting amino acid substitutions that bind or are likely to bind HLA molecules, transfecting nucleic acids encoding the selected amino acid substitutions into an HLA positive melanoma cell line, extracting HLA class I complexes from the transfected cells, identifying the sequence of neoantigens bound to the extracted HLA class one complexes, contacting dendritic cells obtained from the subject with the identified neoantigen peptides, thereby forming a dendritic cell vaccine, administering to the subject the dendritic cell vaccine, obtaining and enriching CD8+ T cells from the subject, and administering the enriched CD8+ T cells to the subject. In some embodiments, the neoantigen binding T cells can be used for adaptive T cell therapy. In some embodiments, a fluorescence polarization binding assay can be used to confirm the binding of neoantigen peptides to HLA molecules prior to selection for transfection.

[0110] In some configurations, the following criteria can be used to select the neoantigens for transfection into HLA class I positive cells; in the exome sequencing, the variant allele fraction of the neoantigen greater than 10%; in the transcript sequencing results the VAF greater than 10%, the alternate read counts greater than 5, and the FPKM greater than 1; the encoded peptides can be 9-11 amino acids in length; the predicted binding to any HLA class I allele can have following characteristics; the predicted MHC binding <250 nM (NetMHC3.4 algorithm), the predicted MHC stability>2 h (NetMHCStab, algorithm); the experimental MHC binding<3.2 log [IC.sub.50, nM] in the fluorescence polarization binding assay. In some embodiments, a personalized immunotherapy of the present teachings can be used in conjunction with check point inhibitors, such as but without limitation ipiplimumab therapy. In some configurations, a cancer vaccine can be generated by contacting dendritic cells obtained from the patient with at least one neoantigen peptide of the present teachings. In some configurations, the dendritic cell vaccine can then be administered to the subject. In some configurations, CD8+ T cells be obtained from PBMC samples from the subject, and CD8+ T cells that recognize the at least one neoantigen are isolated using cell sorting. In various configurations, the cell sorting can comprise using an affinity column or affinity beads. In some configurations, sorted CD8 + T cells that recognize neoantigens can be expanded using methods as described herein. In some configurations, the expanded T cells can then be administered to the subject.

[0111] In various configurations, the present teachings include a series of analytical steps for identification of neo-antigens from somatic tumor missense mutations, as illustrated in FIG. 2. In various embodiments, DNA isolated from tumor and matched PBMC can be subjected to exome sequencing in order to identify tumor somatic missense mutations. For example, in melanoma and lung cancer high number of missense mutations (>200) can be identified per tumor genome. Prediction algorithms such as, without limitation, PePSSI (Bui, H. H., et al., Proteins 63, 43-52, 2006) can be used for the identification of candidate tumor neoantigen epitopes presented in the context of the patient's HLA class I molecules. In various configurations, analysis of tumor transcriptome data can used for the identification and selection, among predicted candidates, of those epitopes that are expressed by the tumor.

Methods

[0112] The methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Methods In Molecular Biology, ed. Richard, Humana Press, NJ, 1995; Spector, D. L. et al, Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. Methods also are as described herein and in publications such as Linette, G. P. et al., Clin. Cancer Res. 11, 7692-7699, 2005; Carreno, B. M. et al., J. Immunol. 188, 5839-5849, 2012; and Carreno, B. M., et al., J. Clin. Invest. 123, 3383-3394, 2013.

[0113] In order to determine the safety, tolerability and immunological responses to Amino Acid Substitutions (AAS)-peptides formulated in an mDC vaccine, the following protocols were followed.

Human Subjects

EXAMPLES 1-10

[0114] Human subjects, Eligible adult patients with newly diagnosed treatment nave (ECOG performance status 0) stage IV cutaneous melanoma are enrolled in this clinical trial. All subjects are HLA-A*0201*, had gp100.sup.+ biopsy-proven (HMB45.sup.+, immunohistochemistry) melanoma metastases, have no evidence of autoimmune disorder, and are negative for HIV, HBV, and HCV. Leukapheresis was performed to obtain PBMCs from patients and healthy donors through the Barnes Jewish Hospital blood bank. For trial patients, leukapheresis is performed prior to treatment and after D3 and D6. Patients are not prescreened for IL-12p70 DC production prior to treatment. Prior to treatment, baseline imaging is performed by MRI scan of brain and CT scan of the chest/abdomen/pelvis with i.v. contrast.

EXAMPLES 11-15

[0115] All patients were enrolled in clinical trial (NCT00683670, BB-IND 13590) and signed informed consents that had been approved by the Institutional Review Board of Washington University. All subjects were HLA-A*02:01*, had no evidence of autoimmune disorder and were negative for HIV, HBV, and HCV. Leukapheresis was performed, prior to treatment and after the 3rd mature dendritic cell (DC) vaccination, at Barnes Jewish Hospital blood bank (Saint Louis, Mo.). Patients were not prescreened for interleukin (IL)-12p70 DC production prior to treatment. Prior to treatment, baseline imaging was performed by MRI scan of brain and CT scan of the chest, abdomen and pelvis with i.v. contrast. Toxicities and adverse effects were graded according to the National Cancer Institute Common Toxicity Scale (version 3.0). Informed consent for genome sequencing was obtained for all patients on protocols approved by the Institutional Review Board of Washington University.

Patient Information

[0116] Patient MEL21 was a 54-year-old man diagnosed with stage 3C cutaneous melanoma of the right lower extremity in 2010. The BRAF V600E mutation was detected. Surgery was performed to excise 2 cm inguinal lymph node and numerous in transit metastases. He developed recurrent in transit metastases and deep pelvic adenopathy in May 2012 and was given ipilimumab (3 mg/kg4 doses) with stable disease until late 2013. Disease progression was noted with increasing 2 cm external iliac, 1.2 cm inguinal, and 7 mm retrocrural adenopathy. Three surgically resected melanoma lesions (inguinal lymph node Jan. 30, 2011, leg skin May 10, 2012, leg skin Jun. 6, 2013) and PBMC were submitted for genomic analysis in order to identity somatic missense mutations. The patient provided written'informed consent for the study and underwent apheresis, and received cyclophosphamide 4 days prior to administration of the first vaccine dose. He received a <total of three vaccine doses without side effect or toxicity. Re-staging CT showed stable disease and be remains in follow up 9 months later.

[0117] Patient MEL38 was a 47-year-old woman diagnosed with stage 3C cutaneous flank melanoma and underwent surgical resection of an axillary lymph node in 2012. The BRAF V600E mutation was detected. She developed recurrent disease in the skin and axilla that was surgically resected. A few months later, CT imaging confirmed metastatic disease in the right lung and axilla and she was given ipilimumab (3 mg/kg4 doses) in May 2012 with complications of grade 2 autoimmune colitis requiring prednisone taper and later, grade 3 hypophysitis requiring replacement therapy with levothyroxine and hydrocortisone. Disease progression was noted 12 months later with new lung and skin metastases. Vemurafenib was administered for two months with no response in August 2013. Three surgically resected melanoma lesions (axilla lymph node Apr. 19, 2012, skin breast Feb. 14, 2013, skin abdominal wall Apr. 16, 2013) and PBMC were submitted for genomic analysis in order to identify somatic missense mutations. Further disease progression was evident with 3 lung nodules measuring 12 mm, 5 mm, and 5 mm in diameter. The patient provided written informed consent for the study and underwent apheresis, and received cyclophosphamide 4 days prior to the first vaccine dose. She received a total of three vaccine doses without side effect or toxicity. Re-staging CT showed 30% tumor reduction; however, the following CT examination 12 weeks later showed interval increase of tumor size back to baseline dimensions with no new sites of disease. The patient remains with stable disease for the past 8 months.

[0118] Patient MEL218 was a 52-year-old man diagnosed with stage 3C cutaneous melanoma on the left lower extremity in 2005. The BRAF mutation V600E mutation was detected when tested later on archived tumor. He underwent surgical resection and received adjuvant interferon for 6 months but had disease recurrence that was surgically resected on several occasions. In 2008, he developed disease progression with extensive in transit and subcutaneous metastases on the left leg with bulky inguinal nodal metastasis deemed unresectable. He received ipilimumab (10 mg/kg14 doses) on clinical trial from 2008-2012 with complete response. One surgical specimen (inguinal lymph node Apr. 4, 2005) and PBMC were submitted for genomic analysis to identify somatic missense mutations. The patient provided written informed consent for the study and underwent apheresis, and received cyclophosphamide 4 days prior to the first vaccine dose. He received a total of three vaccine doses administered in the adjuvant setting without side effect or toxicity. Re-staging PET-CT imaging confirms no evidence of recurrent or metastatic disease. The patient remains in complete remission and continues in follow up.

[0119] Patient MEL69 was a 61-year-old man diagnosed with stage 3C cutaneous melanoma in 2012. Surgery was performed to excise the primary site and the axillary adenopathy. A total of 3 lymph nodes contained metastatic melanoma. The BRAF V600E mutation was detected. The patient received adjuvant Interferon for 5 months but this was discontinued after progression and development of metastatic disease. The patient was given vemurafenib for 10 months but progressed with new sites of disease. Dabrafenib and trametinib combination systemic therapy was administered for 7 additional months until progression. Several new sites of metastatic disease including a solitary brain lesion were resected. His subsequent course was complicated by malignant pericardial effusion and deep venous thrombosis. After appropriate treatment, he improved. Two surgically resected melanoma lesions (MEL69A2, limb and MEL69B2, scalp) and PBMC were submitted for genomic analysis in order to identify somatic missense mutations. The patient provided written informed consent, underwent apheresis, and then received cyclophosphamide 4 d prior to the first vaccine dose. He received a total of 2 vaccines doses without side effect or toxicity. Re-staging CT examination confirmed disease progression and the patient was removed from the study and enrolled in hospice care.

[0120] Patient MEL66 was a 43-year-old female diagnosed initially with stage 3B cutaneous melanoma in 2013. Surgery was performed to excise in transit metastases and the BRAF V600E mutation was detected. Subsequent imaging confirmed metastatic disease in the lung and retroperitoneal cavity deemed unresectable. She received several doses of ipilimumab and developed grade 3 autoimmune colitis treated with corticosteroids. After her recovery, disease progression was noted and combination therapy with dabrafenib/trametinib was begun. Disease progression was noted after 6 months of treatment. Surgical resection of several metastatic lesions was performed to render the patient disease-free. Two surgically resected melanoma lesions (ME1-66A, skin and. MEL66D, soft tissue) and PBMC were submitted for genomic analysis in order to identify somatic missense mutations. The patient provided written informed consent, underwent apheresis, and then received cyclophosphamide 4 d prior to the first vaccine dose. She received a total of 3 vaccine doses without side effect or toxicity. Re-staging Ct confirmed no evidence of disease recurrence and the patient remains in remission with no evidence of disease 4 months in follow up with no additional therapy.

Cyclophosphamide Treatment and DC Preparation (Examples 1-10)

[0121] Cyclophosphamide (300 mg/m.sup.2) was given 72 hours prior to D1 with the intention of eliminating Tregs (Hoons, D. S., et al., Cancer Res., 50, 5358-5364, 1990). All mature dendritic cell (mDC) vaccine doses were prepared at the time of immunization from either freshly isolated (D1) or cryopreserved (D2-D6) PBMCs (all derived from the same leukapheresis collection). A GMP-grade CD40Lexpressing K562 cell line (referred to as K463H), used for maturation of DCs, is generated, selected, and maintained under serum-free (Stemline, S1694 media) conditions. For each vaccine dose, monocyte-derived immature dendridic cells (iDCs) were generated as described previously (Linette, G. P., et al., Clin. Cancer Res., 11, 7692-7699, 2005) by culturing the PBMC adherent fraction in RPMI 1640 with 1% human AB-serum (DC media) supplemented with 100 ng/ml GM-CSF (Berlex) and 20 ng/ml IL-4 (CellGenix). 6 days after culture initiation, iDCs were harvested, washed in PBS, and cultured for an additional 24 hours in DC media (iDC control) or DC media with irradiated (100 Gy) K463H (5:1 DC/K463H ratio) and 100 U/ml IFN- (Actimmune; InterMune Inc.) to generate mDCs. 2 hours prior to infusion, mDCs were pulsed with (50 g/10.sup.6 cells/ml) peptide. For infusion, mDCs were resuspended in 50 ml normal saline supplemented with 5% human serum albumin and administered over 30 minutes by i.v. infusion after premedication with 650 mg acetaminophen.

DC Immunizations (Examples 1-10)

[0122] mDC infusions were given i.v. every 3 weeks for 6 doses in the outpatient clinic. A restaging CT scan of the chest/abdomen/pelvis with i.v. contrast was performed after D3 and D6 and then every 2 months thereafter until disease progression. If clinical or radiographic disease progression was evident, the patient was removed from the study. For D1, patients received 1.5+10.sup.7 DCs per peptide (610.sup.7 DCs total); for D2-D6, patients received 510.sup.6 DCs per peptide (210.sup.7 DCs total). Patients underwent clinical evaluation prior to each mDC infusion. Toxicities and adverse effects were graded according to the National Cancer Institute Common Toxicity Scale (version 3.0). Clinical response was assessed by measurement of assessable metastatic deposits by CT scan, MRI scan, or direct measure of cutaneous deposits. The RECIST (v1.0) group system was used (Therasse, P., et al., J. Nat'l. Cancer Inst., 92, 205-216, 2000).

[0123] Immunologic monitoring (Examples 1-10). Immunologic analysis to evaluate the kinetics and magnitude of T cell response to gp100 peptides was performed using PBMCs collected weekly (prior to vaccination and until week 21. Fresh PBMCs obtained by Ficoll-Hypaque gradient centrifugation were adjusted to 210.sup.6 cells/ml in Stemline media (Sigma-Aldrich) containing 5% human AB-serum, and dispersed at 1 ml/well in 24-well plates. Cultures were set up for the gp100 peptides and the CMV pp65 peptide (positive peptide control). Cultures were pulsed with 40 g/ml peptide and 50 U/ml IL-2 fed starting at 48 hours and every other day thereafter. On day 12 (peak of response; the inventors' unpublished observation), cultures were harvested, counted, and stained for flow cytometry analysis. To assess the antigen-specific T cell frequency, cells were stained with HLAA*0201/peptide tetramers (Beckman Coulter) for 30 minutes at room temperature, followed by addition of FITC-conjugated CD4, CD14, CD19, and CD56 and allophycocyanin-conjugated CD8 (Invitrogen) for 15 minutes at 4 C. Cells were washed and resuspended in FACS buffer, and 7AAD was added 5 minutes before analysis. Control CMV pp65-specific CD8+ T cells were detected in all CMV-seropositive patients before and after immunization. A negative HLA-A*0201/HIV gag peptide tetramer control was included. 25,000 events in the CD8+ gate were collected using a hierarchical gating strategy that included FSC/SSC and excluded 7AAD+ (dead) cells and CD4+CD14+CD19+CD56+ cells. Data were acquired and analyzed using Flow-Jo software.

DC Manufacturing and Vaccine (Examples 11-15)

[0124] Cyclophosphamide (300 mg/m.sup.2) was given 96 h prior to the first DC dose with the intention of eliminating Tregs. All mature DC (mDC) vaccine doses were prepared at time of immunization from either freshly isolated (D1) or cryopreserved (D2-3) PBMC (all derived from same leukapheresis collection). For each vaccine, dose, monocyte-derived immature DCs were generated in 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF, Berlex) and 20 ng/mL IL-4 (Miltenyi Biotec) as described (Carreno, B. M., et al., J. Clin. Invest. 123, 3383-3394, 2013; Linette G P, et al., Clin. Cancer Res 11, 7692-7699, 2005) by culturing the PBMC adherent fraction in RPMI 1640 with 1% human AB-serum (DC media) supplemented with 100 ng/ml GM-CSF (Berlex) and 20 ng/ml IL-4 (CellGenix). Six days after culture initiation, immature DCs were cultured with irradiated (10,000 rad) GMP-grade CD40L-expressing K562 cells (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013), 100 u/mL IFN- (Actimmune, InterMune Inc.), poly I:C (Invivogen, Inc) and R848 (Invivogen, Inc.) for 16 h to generate mDC. Two hours prior to infusion, mDC were pulsed (50 g/10.sup.6 cells/mL) separately with each peptide (7 AAS-peptides and 2 gp100 peptides, G209-2M and G280-9V) and, for dose 1 only, influenza virus vaccine (Fluvirin Novartis) was added to provide a source of recall antigen for CD4+ T cells. IL-12p70 production by vaccine DC was measured by ELISA (eBioscience) in accordance to the manufacturer's instructions. The initial priming dose was 1.510.sup.7 DC per peptide (1.3510.sup.8 DC total), in remaining doses, patients received 510.sup.6 DC per peptide (4.510.sup.7 DC total). mDC were resuspended in 50 mL normal saline supplemented with 5% human serum albumin and administered over 30 min by intravenous infusion after premedication with acetaminophen 650 mg. Patients underwent clinical evaluation prior to each mDC infusion.

[0125] Cytokine Production

[0126] DC IL-12p70 and IL-12p40 production is measured by ELISA (eBioscience) according to the manufacturer's instructions. Production of additional cytokines and chemokines by DCs is determined using MILLIPLEX map Human Cytokine Panels I and II (EMD Millipore). For production of cytokines by T cells, G280-9V-specific T cells are expanded using mDCs and AT-SCT as described previously (infra and Carreno, B. M., et al., J. Immunol. 188, 5839-5849, 2012). The frequency of antigen-specific T cells after secondary stimulation is 2%-52%, as determined by HLA-A*0201/peptide tetramers (NIH tetramers Facility or Beckman Coulter). T cells are restimulated as described infra (Carreno, B. M., et al., J. Immunol. 188, 5839-5849, 2012), supernatants are collected at 24 hours, and production of cytokines is determined using MILLIPLEX map Human Cytokine Panel I (EMD Millipore).

Generation and Expansion of Ag-Specific T Cells

[0127] CD8+ T cells were isolated from PBMCs using a CD82 negative-selection kit (Miltenyi Biotec, Auburn, Calif.). Purified CD8+ T cells were cultured at a 20:1 ratio with irradiated (2500 rad) autologous mature DC (mDC) pulsed with peptide in Stemline media (S1694; Sigma-Aldrich, St. Louis, Mo.) supplemented with pooled human sera (Stemline-5), Human IL-2 (10-50 U/ml; Chiron, Emeryville, Calif.) was added every 2 d starting 48 h after culture initiation. Fourteen days after DC stimulation, T cell cultures were harvested, characterized for neo-antigen specific frequencies using HLA/peptide tetramers (see below), and restimulated with irradiated (10,000 rad) Single Chain Trimers (SCT; U.S. Pat. No. 8,518,697; U.S. Pat. No. 8,895,020; Carreno, B. M., et al., J. Immunol., 188, 5839-5849, 2012) or amino-terminal extended peptide MHC class I single-chain trimer (AT-SCT)-expressing K562 cells at a 1:1 ratio. Cultures were initiated in either six-well plates (10.sup.6 each T and SCT or AT-SCT) or T25 flask (510.sup.6 each) using Stemline-5. Twenty-four hours after stimulation, cultures were supplemented with IL-2 (500 U/ml), and viable cell counts were performed daily.

[0128] Cell concentrations were maintained at 510.sup.5/ml throughout the culture period. For large-scale expansion, T cells were cultured in gas-permeable Lifecell bags (Nexell Therapeutics, Emeryville, Calif.). On days 10-14 of secondary stimulation, the percentage of tetramer+ cells and the number of viable cells were used to determine tetramer yields and tetramer folds.

[0129] For analysis of cytokines secreted by T cells upon SCT activation, cultures were activated 14 d after SCT or AT-SCT stimulation, T cells were restimulated with SCT at 1:1 ratio in RPMI 1640 supplemented with 5% pooled human sera (RPMI-5), supernatants were collected 24 h after activation and characterized using a MILLIPLEX cytokine kit (Millipore, Billerica, Mass.), per the manufacturer's instructions.

qRT-PCR

[0130] qRT-PCR was performed as described previously (Carreno, B. M., et al., Immunol. Cell Biol. 87: 167-177, 2009). cDNAs were prepared (2 g total RNA), and cDNA samples were amplified in triplicate using a GeneAmp 5700 sequencer detector (Applied Biosystems). Primers used are IL-12p35 (Hs00168405_m1) and ITGAX (integrin alpha X, referred to herein as CD11c; Hs01015070_m1). Transcript levels were calculated using the relative standard curve method, using CD11c transcript levels to normalize values.

.SUP.51.Cr Release and T2 Assays

[0131] .sup.51Cr release assays to measure specific lysis have been described previously (Carreno, B. M., et al., Immunol. Cell Biol., 87: 167-177, 2009; Linette, G. P. et al., Clin. Cancer Res. 11, 7692-7699, 2005). Melanoma cell lines DM6 (HLAA2+ gp100+) and A375 (HLA-A2+gp100) were labeled with 25 Ci .sup.51Cr for 1 hour, washed, and tested as targets in a standard 4-hour assay. Effectors were generated using PBMCs collected after D3 and cultured for 12 days in the presence of peptide (40 g/ml) and IL2 (50 U/ml every other day). Vaccine-induced antigen-specific T cells were characterized using HLAA*0201/peptide dextramers (Immudex). To determine the avidity (effective concentration at 50% maximal lysis) of vaccine-induced T cells for antigen, T2 cells were pulsed with titrated G209-2M or G280-9V peptide concentrations for 1 hour in serum-free media followed by 51Cr (25 Ci) labeling for 1 hour, washed twice, and tested using vaccine-induced gp100-specific T cells in a standard 4-hour assay.

Statistics

[0132] Student's t tests are 2-tailed (GraphPad Prism software, version 5.0). Data are presented as mean1 SD, unless otherwise indicated. Cox regression analysis followed by likelihood-ratio test is used to evaluate whether (loge) IL-12p70 (sum) production added statistically significant information to a model of time to progression (TTP). Kaplan-Meier TTP model is used to test whether cytokine ratios added statistically significant information to a model of TTP. Wilcoxon matched-pairs analysis is used to compare IL-12p70 production between patients and healthy donors (GraphPad Prism software, version 5.0). All P values less than 0.05 were considered significant, except the Cox proportional hazard model, which used a lower threshold of significance (P<0.048) to adjust for 1 interim analysis of this endpoint.

Peptides

[0133] Peptides were obtained lyophilized from American Peptide Company (>95% purity), dissolved in 10% DMSO in sterile water and tested for sterility, purity, endotoxin and residual organics. Peptide binding to HLA-A*02:01 was determined by T2 assay (Elvin et al. 1993 J. Immunol. Methods 158, 161) or using a fluorescence polarization assay (Pure Protein, L.L.C.) (Buchli, R., et al., Biochemistry 44, 12491-12507, 2005). The affinity scale of this latter assay is: high binders: log (IC.sub.50 nM)<3.7; intermediate binders: log (IC.sub.50 nM) 3.7-4.7; low binders: log (IC.sub.50 nM) 4.7-5.5; and very low binders: log (IC.sub.50 nM)6.0 (11).

Computer Algorithm

[0134] Burrows-Wheeler Aligner (BWA; Li, H. and Durbin R., Bioinformatics 25, 1754-1760, 2009) is a reference-directed aligner that is used for mapping low-divergent sequences against a large reference genome, and consists of separate algorithms designed for handling short query sequences up to 100 bp, as well as longer sequences ranged from 70 bp to 1 Mbp.

[0135] Picard (Broad Institute, Cambridge, Mass.) is a set of Java-based command-line tools for processing and analyzing high-throughput sequencing data in both Sequence Alignment/Map (SAM) text format and SAM binary (BAM) format. The MarkDuplicates utility within Picard examines aligned records in the supplied SAM or BAM file to locate duplicate molecule and can be used to flag and/or remove the duplicate records.

[0136] SAMtools (Li, H., et al., Bioinformatics, 25, 2078-2079, 2009) is a suite of programs for interacting with and post-processing alignments in the SAM/BAM format to perform a variety of functions like variant calling and alignment viewing as well as sorting, indexing, data extraction and format conversion.

[0137] Somatic Sniper (Larson, D. E., et al., Bioinformatics, 28, 311-317) is used to identify single nucleotide positions that are different between tumor and normal BAM files. It employs a Bayesian comparison of the genotype likelihoods in the tumor and normal, as determined by the germline genotyping algorithm implemented in the MAQ and then calculates the probability that the tumor and normal genotypes are different.

[0138] VarScan (Koboldt D. C., et al., Genome Research, 22, 568-576, 2012; Koboldt, D. C., et al., Bioinformatics 25, 2283-2285, 2009,) is a software program that detects somatic variants (SNPs and indels) using a heuristic method and a statistical test based on the number of aligned reads supporting each allele using an input SAMtools pileup/mpileup file. For tumor-normal pairs, it further classifies each variant as Germline, Somatic, or LOH, and also detects somatic copy number changes.

[0139] Strelka (Saunders, C. T., et al., Bioinformatics 28, 1811-1817, 2012) is an analysis package designed to detect SNVs and small indels from the sequencing data of matched tumor-normal samples. It is specifically designed to detect somatic variants at lower frequencies typically encountered in tumors due to high sample impurity or sub-clone variation, while maintaining sensitivity.

[0140] TopHat (Trapnell. C., et al., Bioinformatics, 25, 1105-1111, 2009; Kim, D., et al., Genome Biol., 14, R36, 2013) is a fast splice junction mapper for RNA-Seq reads that aligns reads to mammalian-sized genomes in order to identify exon-exon splice junctions. It uses the ultra high-throughput short read aligner Bowtie, and then analyzes the mapping results to identify splice junctions between exons.

[0141] Cufflinks (Trapnell, C., et al., Nat. Protoc., 7, 562-578, 2012) is a software program for transcriptome assembly and differential expression analysis for RNA-Seq data. It assembles transcripts from aligned RNA-Seq reads, estimates their abundances based on how many reads support each one, taking into account biases in library preparation protocols, and then tests for differential expression and regulation in RNA-Seq samples.

[0142] Flexbar (Dodt, M., et al., Biology (Basel), 1, 895-905, 2012) is a software package that preprocesses high-throughput sequencing data efficiently by demultiplexing barcoded runs and removing adapter sequences. Additionally, it supports trimming as well as filtering features; thereby aiming to increase read mapping rates and improve genome and transcriptome assemblies.

[0143] NetMHC 3.4 server (Nielsen, M., et al., Protein Sci., 12, 1007-1017, 2003; Lundegaard, C., et al., Nucleic Acids Res., 1, W509-512, 2008) makes high-accuracy predictions of major histocompatibility complex (MHC): peptide binding to a number of different HLA alleles. The predictions are based on artificial neural networks trained on different datasets (human and non-human) from several MHC alleles and position-specific scoring matrices (PSSMs).

[0144] In terms of additional filtering of variants from DNA/RNA data that would pass to analysis for identifying peptides, the following filters were used on coverage for tumor and normal, below which a variant is discarded from further consideration:

[0145] >=5 Normal coverage

[0146] >=10 Tumor coverage

[0147] <=2% Normal VAF

[0148] >=30% Tumor VAF

[0149] FPKM>1 (this is the only RNA-based filter).

[0150] In silico work flow.

[0151] The present inventors have developed an in silico automated pipeline for neoantigen prediction (pVAC-Seq) that can utilize several types of data input from next-generation sequencing assays. First a list of nonsynonymous mutations is identified by a somatic variant-calling pipeline using exomic sequencing and transcript sequencing of both normal and tumor tissue. This variant list can then be annotated with amino acid changes and transcript sequence. The HLA-haplotypes of the patient, can be derived through clinical genotyping assays or in silico approaches. These data can be input into the pVAC-Seq workflow which implements three steps: performing, epitope prediction, integrating sequencing-based information and lastly, filtering neoantigen candidates. The following paragraphs describe the analysis methodology from preparation of inputs to the selection of neoantigen vaccine candidates via pVAC-Seq.

[0152] Prepare Input Data: HLA-Typing, Alignment, Variant Detection and Annotation

[0153] As described above, pVAC-Seq utilizes input data generated from the analysis of next-generation sequence data that includes annotated nonsynonymous somatic variants that have been translated into mutant amino acid changes, as well as patient-specific HLA haplotypes. While these data could be obtained from any appropriate variant calling, annotation and HLA typing pipeline, the inventors' approach as disclosed herein utilized the following analysis methods for preparing these input data. In brief, BWA (version 0.5.9) (Li, H. and Durbin, R., Bioinformatics, 25, 1754-1760, 2009) was used as the aligner of choice with default parameters except the number of threads was set to 4 (t 4) for faster processing, and the quality threshold for read trimming to 5 (q 5). The resulting alignments were de-duplicated via Picard MarkDuplicates (version 1.46; Broad Institute, Cambridge, Mass.).

[0154] In cases where clinically genotyped HLA haplotyping calls were not available, the inventors used in silico HLA typing by HLAminer (Version1)(Warren, R. L., et al., Genome Med., 4, 95, 2012) to provide HLA haplotypes from either whole genome sequence data or RNA-seq data, or by Athlates (Liu, C., et al., Nucleic Acids Res, 41, e142, 2013) when exome data were available. Typing was performed on samples of the patient's normal cells, rather than cells from the tumor sample. The two software tools were >85% concordant in the inventors' test data; both algorithms were used in order to break ties reported by HLAminer (see below). [0155] 1. HLAminer for in silico HLA-typing using WGS data: When predicting HLA class I alleles tram WGS data, the inventors used HLAminer in de novo sequence alignment mode using TASR (Warren, R. L. and Holt, R. A., PLoS One., 6, e19816, 2011) (params: i 1 m 20) by running the script HPTASRwgs_classI.sh, provided in the download. (The download includes detailed instructions for customizing this script, and the scripts on which it depends, for the user's computing environment.) For each of the three HLA loci, HLAminer reports predictions ranked in decreasing order by score, where Prediction #1 and Prediction #2 are the most likely alleles for a given locus. When ties were present for Prediction 1 or Prediction 2, the inventors used all tied predictions downstream neo-epitope prediction. However, it should be noted that most epitope prediction algorithms, including NetMHC (Lundegaard, C., et al., Nucleic Acids Res., 36, 509-512, 2008; Nielsen, M., et al., Protein Sci., 12, 1007-1017, 2003), only work with an algorithm-specific subset of HLA alleles, so we are constrained to the set of NetMHC-compatible alleles. The current version NetMHC v3.4 supports 78 human alleles. [0156] II. Athlates for in silico HLA-typing using exome sequence data: The inventors diverged from the recommended procedure to run Athlates at two points in the procedure: 1) they performed the alignment step to align exome sequence data (corresponding to the normal tissue sample) against the HLA allele sequences present in the IMGT/HLA database (Robinson, J., et. al., Nucleic Acids Res., 41, D1222-D1227, 2013), using BWA with zero mismatches (params: bwa aln e 0 o 0 n 0) instead of NovoAlign (Hercus. C., Novocraft short read alignment package, 2009) with one mismatch, and 2) in the subsequent step, sequence reads that matched, for example, any HLA-A sequence from the database were extracted from the alignment using bedtools (Quinlan, A. R. and Hall, I. M., Bioinformatics 26, 841-842, 2010) instead of Picard. This procedure is resource-intensive, and may require careful resource management. Athlates reports alleles that have a Hamming distance of at most 2 and meet several coverage requirements. Additionally, it reports inferred allelic pairs, which are identified by comparing each possible allelic pair to a longer list of candidate alleles using a Hamming distance-based score. The inventors typically used the inferred allelic pair as input to subsequent steps in the neo-epitope prediction pipeline.

[0157] After alignments (and optional HLA typing) were completed, somatic mutation detection was performed using the following series of steps. (1) Samtools (Li, H., et al., Bioinformatics, 25, 2078-2079, 2009; Li, H. Bioinformatics, 27, 2987-2993, 2011) mpileup v0.1.16 was run with parameters A B with default setting for the other parameters. These calls were filtered based on GMS snp-filter v1 and were retained if they met all of the following rules: (a) Site is greater than 10 bp from a predicted indel of quality 50 or greater, (b) The maximum mapping quality at the site is 40, (c) Fewer than 3 SNV calls are present in a 10 bp window around the site, (d) The site is covered by at least 3 reads and less than 1109 reads, and (e) Consensus and SNP quality is 20. The filtered Samtools variant calls were intersected with those from Somatic Sniper version 1.0.2 (Larson, D. E., et al., Bioinformatics, 28, 311-317, 2012) (params: F vcf q 1 Q 15), and were further processed through the GMS false-positive filter v1 (params: -bam-readcount-version 0.4-bamreadcount-min-base-quality 15-min-mapping-quality 40-min-somatic-score 40). This filter used the following criteria for retaining variants: (a) 1% of variant allele support comes from reads sequenced on each strand, (b) variants have 5% Variant Allele Fraction (VAF) (c) more than 4 reads support the variant, (d) the average relative distance of the variant from the start/end of reads is greater than 0.1, (e) the difference in mismatch quality sum between variant and reference reads is less than 50, (f) the difference in mapping quality between variant and reference reads is less than 30, (g) the difference in average supporting read length between variant and reference reads is less than 25, (h) the average relative distance to the effective 3 end of variant supporting reads is at least 0.2, and (i) the variant is not adjacent to 5 or more bases of the same nucleotide identity (e.g. a homopolymer run of the same base), (2) VarScan Somatic version 2.2.6 (Koboldt, D. C., et al., Bioinformatics, 25, 2283-2285, 2009; Koboldt, D. C., et al., Genome Res., 22, 568-576, 2012) was run with default parameters and the variant calls were filtered by GMS filter varscan-high-confidence filter version v1. The varscan-high-confidence v1 filter employed the following rules to filter out variants (a) p-value (reported by Varscan) is greater than 0.07, (5) Normal VAF is greater than 5%, (c) Tumor VAF is less than 10% or (d) less than 2 reads support the variant. The remaining variant calls were then processed through false-positive filter v1 (params: -bam-readcount-version 0.4-bamreadcount-min-base-quality 15) as described above. (3) Strelka version 1.0.10 (Saunders, C. T., et al., Bioinformatics, 28, 1811-1817, 2012) (params: isSkipDepthFilters=1).

[0158] The consolidated list of somatic mutations identified from these different variant-callers was then annotated using our internal annotator as part of the GMS pipeline. This annotator leverages the functionality of the Ensembl database (Flicek, P, et al., Nucleic Acids Res., 41, D48-55, 2013) and Variant Effect Predictor (VEP)(McLaren, W., et al., Bioinformatics, 26, 2069-2070, 2010).

[0159] From the annotated variants, there are two components that are needed for pVAC-Seq: amino acid change and transcript sequence. Even a single amino acid change in the transcript arising from missense mutations can alter the binding affinity of the resulting peptide with the MHC Class I molecule. Larger insertions and deletions, such as, for example, those arising from frameshift and truncating mutations, splicing aberrations or gene fusions can also result in potential neoantigens. However, for the present iterations of pVAC-Seq, the inventors chose to focus their analysis on only missense mutations.

[0160] One feature of the inventor's pipeline is the ability to compare the differences between tumor neo-antigens and normal peptides in terms of the peptide binding affinity. Additionally, it leverages RNA-Seq data to incorporate isoform-level expression information and to quickly cull variants that are not expressed in the tumor. To integrate RNA-Seq data, both transcript ID as well as the entire wild-type transcript amino acid sequence can be used as part of the annotated variant file.

Perform Epitope Prediction

[0161] One component of pVAC-Seq is predicting epitopes that result from mutations by calculating their binding affinity against the Class I MHC molecule. This process involves the following steps for effectively preparing the input data as well as parsing the output.

Generate FASTA File of Peptide Sequences:

[0162] Peptide sequences are an input to the MHC binding prediction tool, and the existing process to compare the germline normal with the tumor can be very onerous. To streamline the comparison, the inventors first build a FASTA file that consists of two amino acid sequences per variant sitewild-type (normal) and mutant (tumor). The FASTA sequence can be built using approximately 8-10 flanking amino acids on each side of the mutated amino acid. However, if the mutation is towards the end or beginning of the transcript, then the preceding or succeeding 16-20 amino acids can be taken respectively, as needed, to build the FASTA sequence. Subsequently, a key file can be created with the header (name and type of variant) and order of each FASTA sequence in the file. This can be done to correlate the output with the name of the variant protein, as subsequent epitope prediction software strips off each name.

Run Epitope Prediction Software:

[0163] To predict high affinity peptides that bind to the HLA class I molecule, the standalone version of NetMHC 3.4 is used. The input to this software is the HLA type of the patient, determined via genotyping or using in silico methods, as well as the FASTA file generated in the previous step comprised of mutated and wild-type 17-21-mer sequences. Typically, antigenic epitopes presented by MHC class I molecules can vary in length from 8 to 13 or 8 to 11 amino acids. Therefore, specifying the same range when running epitope prediction software is recommended.

Parse and Filter the Output:

[0164] Starting with the output list of all possible epitopes from the epitope prediction software, the inventors apply specific filters to choose the best mutant peptide incorporating candidates. First, further consideration is restricted to strong to intermediate binding peptides by focusing on candidates with a mutant (MT) binding score of less than 500 nM or less than 250 nM. Second, epitope binding calls are evaluated only for those peptides that contain the mutant amino acid (localized peptides). This filter eliminates any wild-type (WT) peptides that may overlap between the two FASTA sequences. The pVAC-seq workflow enables screening across multiple lengths and multiple alleles very efficiently. If predictions are run to assess multiple epitope lengths (e.g., 9-mer, 10-mer, etc.), and/or to evaluate all different patient HLA allele types, the inventors review all localized peptides and choose the single best binding value representative across lengths (9aa, 10aa, etc.) based on lowest binding score for MT sequence. Furthermore, they choose the best candidate (lowest MT binding score) per mutation between all independent HLA allele types that were used as input.

Integrate Expression and Coverage Information

[0165] Subsequently several filters are applied to ensure that the predicted neoantigens are expressed as RNA variants, and are predicted correctly based on coverage depth in the normal and tumor tissue data sets. Specifically, gene expression levels from RNA-Seq data measured as Fragments per kilobase of exon per million reads mapped (FPKM) provide a method to filter only the expressed transcripts. We used the tuxedo suiteTophat (Trapnell, C. et al., Bioinformatics, 25, 1105-1111, 2009; Kim, D., et al., Genome Biol., 14, R36, 2013) and Cufflinks (Trapnell, C., et al., Nat. Protoc., 7, 562-578, 2012) as part of the GMS to align RNA-Seq data and subsequently infer gene expression for our in-house sequencing data. Depending on the type of RNA prep kit, OVATION RNA-Seq System V2 (NuGEN Technologies, Inc. San Carlos, Calif.) or TRUSEQ Stranded Total RNA Sample Prep kit (ILLUMINA, Inc. San Diego, Calif.), used, Tophat was run with the following parameters: Tophat v2.0.8 -bowtie-version-2.1.0 for OVATION, and -library-type fr-firststrand-bowtie-version=2.1.0 for TRUSEQ. For OVATION data, prior to alignment, paired 2100 bp sequence reads were trimmed with Flexbar version 2.21 (Dodt, M., et al. Biology (Basel), 1, 895-905, 2012.) (params: -adapter CTTTGTGTTTGA (SEQ. ID NO: 474)-adapter-trim-end LEFT-nono-length-dist-threads 4-adapter-min-overlap 7-maxuncalled 150-min-readlength 25) to remove single primer isothermal amplification adapter sequences. Expression levels (FPKM) were calculated with Cufflinks v2.0.2 (params-max-bundle-length=10000000-num-threads 4).

[0166] For selecting unique vaccine candidates, targeting the best quality of mutations is an important factor for prioritizing peptides. Sequencing depth as well as the fraction of reads containing the variant allele (VAF) are used as criteria to filter or prioritize mutations. This information was added in our pipeline via bam-readcount (Larson, D., The Gnome Institute at Washington University). Both tumor (from DNA as well as RNA) and normal coverage are calculated along with the VAF from corresponding DNA and RNA-Seq alignments.

Filter Neoepitope Candidates

[0167] Since manufacturing antigenic peptides can be one of the most expensive steps in vaccine development and efficacy depends on selection of the best neoantigens, the inventors filter the list of predicted high binding peptides to the most highly confident set, primarily with expression and coverage based filters.

The Filters can be Employed as Follows:

[0168] Depth based filters: any variants with normal coverage <=5 and normal VAF of >=2% can be filtered out. The normal coverage cutoff can be increased up to 20 to eliminate occasional misclassification of germline variants as somatic. Similarly, the normal VAF cutoff can be increased based on suspected level of contamination by tumor cells in the normal sample. For tumor coverage from DNA and/or RNA, a cutoff can be placed at >=10 with a VAF of >=10% or 30%. This can ensure that neoantigens from the major clones in the tumor are included, but the tumor VAF can be lowered to capture more variants, which may or may not be present in all tumor cells. Alternatively, if the patients are selected based on a pre-existing disease-associated mutation such as BRAF V600E in the case of melanoma, the VAF of the specific presumed driver mutation can be used as a guide for assessing clonality of other mutations.

[0169] Expression based filters: as a standard, genes with FPKM values of greater than zero are considered to be expressed. The inventors slightly increase this threshold to 1, to eliminate noise. Alternatively, the FPKM distribution (and the corresponding standard deviation) can be analyzed over the entire sample, to determine the sample-specific cutoffs for gene expression. Spike-in controls can also be added to the RNA-Seq experiment to assess quality of the sequencing library and to normalize gene expression data. This filtered list of mutations can be manually reviewed via visual inspection of aligned reads in a genome viewer like IGV (Robinson, J. T., et al., Nat Biotechnol., 29, 24-26, 2011; Thorvaldsdottir, H., et al. Brief Bioinform., 14, 178-192, 2013) to reduce the retention of obvious false positive mutations.

Analysis of T Cell Responses

[0170] For functional characterization, neoantigen-specific T cell lines were generated using autologous mDC and antigen loaded artificial antigen presenting cells at a ratio of 1:1 as previously described (Carreno, B. M., et al., J. Immunol., 188, 5839-5849, 2012). To determine the peptide avidity (effective concentration at 50% maximal lysis, EC50) of neoantigen-specific T cells, T2 cells were pulsed with titrated peptide concentrations for 1 h, followed by .sup.51Cr (25Ci) labeling for 1 h, washed twice and tested in a standard 4 h .sup.51Cr release assay using neoantigen-specific cells as effectors. For production of cytokines, neoantigen-specific T cells were restimulated using artificial antigen presenting cells in the presence or absence of peptide, supernatants collected at 24 h and cytokine produced determined using MILLIPLEX MAP Human Cytokine Panel I (EMD Millipore).

Overview of the Present Teachings

[0171] FIG. 4 illustrates a scheme showing neo-antigen identification and its incorporation into a personalized dendritic cells vaccine. The upper diagram depicts a pipeline for neoantigen identification. Tumor cells and matched peripheral blood mononuclear cells (PBMC) are subjected to whole exome sequencing to identify somatic missense mutations. Missense mutations are evaluated as peptides (8-13 aa long) through MHC class I binding and algorithms to identify potential candidate neoantigens and the expression of transcripts encoding mutated protein is confirmed by transcriptome sequencing. Synthetic peptides encoding candidate neoantigens can be tested experimentally for MHC class I binding and vaccine candidates can be selected using characteristics described infra. The lower diagram represents a vaccination process whereby dendritic cells (DC) can be generated from monocytes using GM-CSF and IL-4, and matured using CD40L/IFN-g/poly IC and R848. Mature DC can be pulsed with candidate neoantigen peptides and infused in order to generate mutation (missense)-specific T cells.

EXAMPLES

[0172] The present teachings it descriptions that are not intended to limit the scope of any aspect or claim. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The examples and methods are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

Example 1

[0173] This example illustrates the clinical use of common cancer antigen peptides and the difficulties of using matured dendritic cells in cancer vaccines.

[0174] Vaccination was performed with HLA-A*0201-restricted gp100 melanoma antigen-derived peptides (G209-2M, and G280-9V) (Carreno, B. M., et al., J. Clin. Investigation, 123, 3383-3394, 2013; Kawakami, Y., et al., J. Immunol., 154, 3961-3968, 1995; Skipper, J. C., et al., Int. J. Cancer, 82, 669-677, 1999) using autologous peptide-pulsed, CD40L/IFN--activated mature DCs (mDCs). The top of FIG. 17 illustrates the comparison of gp100 (G209-2M and G280-9V)-specific T cell frequencies observed pre- and post-vaccine. Statistical assessment was performed using paired two-tail t-test; p values are indicated in figure. The table on FIG. 17 bottom left summarizes the characteristics of patients enrolled in the trial and details their clinical outcomes: CR, complete response; PR, partial response: PD, progressive disease.

[0175] The bottom left of FIG. 17 illustrates radiologic studies (FDG-PET/CT imaging) that were obtained on Patient 1 before vaccination, 11 months and 21 months after treatment. Coronal whole body PET images show complete regression of left supra-clavicular and hilar lymph nodes as well as multiple subcutaneous lesions on the right leg. P1 remains in remission as of December 2012.

[0176] FIG. 18 illustrates that ex-vivo dendritic cell (DC) IL-12 production and Tc1 profile correlates with clinical outcome (TTP, time to progression) (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013). A Cox regression analysis followed by likelihood-ratio test revealed a positive correlation between IL-12 production and TTP (FIG. 18, top; p=0.0198, log rank). Filled (dark) circles indicate patients that had a confirmed clinical response (P1, CR; P5 and P6, PR; FIG. 17, bottom left) with disease progression observed at or after 11.5 months of treatment initiation. The open (white) circles represent patients with rapid disease progression. The analysis was performed on Aug. 5, 2012, P1 remains in complete remission 4 years after initiation of treatment. No correlation was observed between IL-12 production and immune response or immune response and clinical outcome. Cytokine ratios differed among clinical responders (Clin Resp) and non-responder (Clin non-Resp) patients and demonstrate a Tc1 profile (FIG. 18, bottom; high IFN-g, low IL-5 or IL-13) among responders. p values are indicated in figure, unpaired two-tailed t-test.

[0177] FIG. 19 illustrates that weak p35 transcription accounts for the IL-12p70 defect in clinical non-responder patients (Carreno, B. M., et al., J. Clin. Invest. 123, 3383-3394, 2013). FIG. 19 top, left DC from age and gender matched healthy (H) donors and melanoma (M) patients were activated with CD40L/IFN- for 24 h, supernatants harvested and assayed for IL-12 production by ELISA. Horizontal lines and whiskers indicated median and interquartile range. p=0.0420, Wilcoxon matched-pairs test. Healthy individuals;produced on average 10 more IL12p70 than melanoma patients. Patient DC were activated with CD40L/IFN- for 24 h, supernatants were collected and IL-12p40 (circles) and IL-12p70 (squares) production measured by ELISA (FIG. 19, top right). Results are shown for 10 melanoma patients. Horizontal lines and whiskers indicated median and interquartile range. Results demonstrate a defect on IL-12p70 (p40/p35) but not in IL-12p40 suggesting defect lies in induction of IL-12p35. To examine IL-12p35 gene activation, DC were activated with CD40L/IFN- for 6 h, cells harvested, washed and total RNA prepared. Total RNA was also prepared from immature DC. Using p35 and CD11c (DC lineage marker) specific primers, qRT-PCR was performed and analyzed using the relative standard method. Values shown in FIG. 19 (bottom) were normalized to expression CD11c and p35 fold induction in mature DC calculated relative to immature DC. Results decreased IL-12p35 induction in clinical non-responding patients (P2, P3, P7).

Example 2

[0178] This example illustrates techniques of maturing DC that overcome the limitations discussed in Example 1.

[0179] Based on the results obtained in Example 1, different DC maturation techniques were required to increase clinical response to cancer antigens. The inventors therefore tested maturation signals for dendritic cells. Immature DC were stimulated with a combination of CD40L/IFN- plus poly I:C (30 ug/mL, TLR3 agonist) and R848 (5 g/mL, TLR8 agonist) (P8-P10) for 24 h and supernatants assayed for IL-12. As a control, data from immature dendritic cells stimulated with CD40L/IFN-(patients P1-P7; Carreno, B. M., et al., J. Clin. Invest. 123, 3383-3394, 2013) were plotted on the same graph. The results depicted in FIG. 20 demonstrate that a combination of all 4 signals enhances IL-12p70 production to levels similar to those observed m healthy individuals (see FIG. 19 top left for the baseline).

[0180] A combination of innate and adaptive signals for DC maturation enhances the kinetics of the immune responses to gp100 (g209-2M and G280-9V) antigens. FIG. 21, left demonstrates that gp100-specific T cell responses can be detected in patients vaccinated with CD40L/IFN-/TLR3/8 agonist-matured DC as early as one week after vaccination (bottom left). In contrast, two vaccinations with CD41/IFN-g matured DCs are required for detection of gp100-specific cell responses (FIG. 21, top left). Time is recorded in weeks. Antigen-specific numbers were calculated based on dextramer percentage and total live cell yields. The dot plots (FIG. 21, right) depict frequencies of gp100-specific T cells in ex-vivo expanded peripheral blood mononuclear cells obtained pre- and post-vaccination. FIG. 22 illustrates that a combination of innate and adaptive signals for DC maturation promotes Tc1-polarized immunity. Purified CD8+ T cells were stimulated twice in vitro and antigen-specific frequencies determined by peptide/HLA-A*0201 tetramers. T cells were adjusted to 10.sup.6 cell/mL, stimulated with antigen and supernatants harvested at 20 h. Cytokine production was determined using MILLIPLEX MAP Human Cytokine Panel I (FIG. 22, top). To compare production of Tc1 (IFN-) and Tc2 (IL-5, IL-13) cytokines among patients, a cytokine ratio was derived by dividing pg/mL IFN- by pg/mL IL-5 or IL-13. Ratios>1 indicate a Tc1 phenotype (FIG. 21, bottom).

Example 3

[0181] This example illustrates in silico analysis of missense mutations found in melanoma tumors.

[0182] FIG. 23 illustrates that cutaneous melanoma harbors a significant mutation burden and hence continues a cancer model to study tumor somatic mutations as neoantigens. Mutation pattern, spectrum and clinical features in 15 metastases from 13 WGS melanoma cases are illustrated. Numbers and frequencies of Tier 1 transitions and transversions events identified in all 15 tumors are shown. Hence, melanoma patients were chosen for further study of personalized vaccines.

[0183] The diagram in FIG. 2 illustrates an example derived from analysis of a tumor/PBMC matched pair derived from a melanoma patient. As depicted multiple candidate patient-specific tumor-derived epitopes can be identified per HLA-class 1 molecule; in this particular case, those presented by HLA-A*0201 are shown. The analysis depicted here can be performed for each of the HLA class I alleles (n=3-6) expressed by the patient.

[0184] In various embodiments, the present teachings include analysis of missense mutations by prediction algorithms for binding to HLA-A*0201. Table 1 shows the chromosomal (CHR) location, genomic alignment position and nucleotide change encoding missense mutation in metastases (breast, abdominal wall) derived from a patient. Exomic variant allele fraction (under exome column) for each mutation as well as gene encoding mutation and amino acid change are shown. One mutation in OR5K2 is unique to breast metastasis, while mutations in CCDC57 and IL17Ra are unique to abdominal wall metastasis. Proteins encoding missense mutations were analyzed using the NetMHC and NetMHCstab algorithms in order to predict mutation-containing peptides (9-11 amino acid in length) that may bind to any of patient's HLA-class I molecules. Candidate peptides to consider for a vaccine are selected based on variant frequencies (exome, transcriptome>10), expression (FPKM>1) and HLA class I affinity (<250 nM0 and stability (>2 h). In Table 1, mutated peptides fulfilling these criteria are highlighted in bold. NR=not recorded.

Example 4

[0185] This example illustrates the in vitro binding of neoantigen peptides to HLA class I molecules.

[0186] In some embodiments, the present teachings disclose HLA class I binding capacity of peptides containing tumor-specific missense mutations. The binding capacity of missense mutation-containing peptides is experimentally evaluated using a flow cytometric assay. Peptide binding to cell surface HLA class I can lead to stable peptide/HLA class I complexes that can be detected using a HLA-class I allele specific antibody. Four control peptides can be included in the assay, two known HLA-A*0201 binding peptides (FluM1,G280-9V) and 2 negative controls (G17, NP265). In the graph shown in FIG. 3, binding of mutation-containing peptides to HLA-A*0201 expressed on the surface of T2 cells is examined. Nine of the 15 mutation-containing peptides tested bound to HLA-A*0201 and all these peptides show affinities<250 nM.

Example 5

[0187] This example illustrates the translation of tumor missense imitations into patient-specific vaccines. FIG. 24 (top) illustrates the distribution of somatic missense mutations identified in a melanoma patient (MEL38) tumor. HLA-A*02:01-binding candidate peptides were in silico identified among amino acid substituted peptides and expression of gene encoding mutated protein determined from cDNA capture data. FIG. 24 (bottom) illustrates the immune-monitoring of neoantigen-specific CD8+ cell responses. Results are derived from PBMC isolated before DC vaccination (Pre-vaccine) and at peak (Post-Vaccine). PBMCs were cultured in vitro in the presence of peptide and IL-2 for 10 days followed by HLA-A*02:01/neoantigen-peptide dextramer assay. This immune monitoring strategy allows the reliable detection, as well as, the assessment of replicative potential of vaccine-induced T cell responses. Numbers within dot plots represent percent neoantigen-specific T cells in lymph+/CD8+ gated cells. A pre-existing response to one neoantigen (SEC24A) was observed; vaccination enhance this response and reveal two additional ones (AKAP13 and OR8B3). Demonstrating that tumor somatic mutations can be immunogenic and that vaccination can expand the antigenic diversity of such response.

Example 6

[0188] This example illustrates CD8+ T cell response to mutation containing peptides.

[0189] In some embodiments, the present teachings include vaccination with tumor-specific missense mutations to elicit CD8+ T cell immunity. As shown in FIG. 5, a dextramer assay (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013) was used to monitor development of CD8+ T cell immunity to mutation-containing peptides. Dot plots show frequencies of CD8+ T cells specific for the mutation-containing peptides prior to vaccination (pre-vacc) and after 2-3 vaccinations (post-vacc). In all 3 patients, responses to 3 of the 7 peptides are observed as demonstrated by an increase in the frequency of dextramer+ T cells.

[0190] In some embodiments, predicted affinities (FIG. 6 top) and stabilities (FIG. 6 bottom) of mutated peptides and their wild-type counterparts can be compared. In FIG. 6, mutated peptides (two-antigens) that elicited CD+ T cell immunity are indicated by rectangles. All immunogenic peptides display HLA-A*0201 affinities of <50 nM and stabilities>3 h. These characteristics can be important as determinants of immunogenicity. These characteristics can be taken into consideration when choosing mutation-containing peptides to incorporate in a vaccine.

[0191] In some embodiments, the present teachings include vaccine-induced CD8+ T cells directed at tumor missense mutations display high replicative potential. As shown in FIG. 7 and FIG. 8, after 3 DC vaccinations, leukapheresis was performed in patients in order to obtain PBMC. CD8+ T cells purified from PBMC were stimulated with neo-antigen-peptide pulsed autologous DC and cultured in the presence of IL-2 for 10 days. These primary cultures were re-stimulated with peptide-pulsed K562-expressing HLA-class I single-chain dimer (SCD) as described (Carreno, B. M., et al., J. Immunol., 188, 5839-5849, 2012). Cultures were maintained for an additional 10 day period in the presence of IL-2. FIG. 7 depicts results from the dextramer assay, the frequencies (%) neo-antigen specific T cells found in the CD8+ T cell population at initiation of cultures (Blood, day 0) and after DC/SCD stimulation (Expanded, day 20) were determined. FIG. 8 illustrates that based on viable cell counts and antigen-specific T cell frequencies, at initiation and termination of cultures, antigen-specific T cell yields and expansion folds were calculated. Antigen-specific yields were calculated as the % of HLA/Ag dextramer+ CD8+ T cellstotal CD8+ T cell numbers at day 20. Antigen-specific cell folds represented (% of HLA/Ag dextramer+ CD8+ T cellstotal CD8+ T cell numbers at day 20)/(% of HLA/Ag dextramer+ CD8+ T cellstotal CD8+ T cell numbers at day 0). Results demonstrated that this method allows the expansion of vaccine-induced T cells over 10.sup.4 fold (FIG. 8, right panel). A 10.sup.4 fold expansion yields 10.sup.8 antigen-specific T cells from a starting population with <10.sup.4 antigen-specific T cells.

Example 7

[0192] This example illustrates the specificity of neoantigen peptide recognition by CD8+ T cells.

[0193] In various embodiments, the present teachings include disclosure of discrimination between mutated and wild-type sequences by vaccine-induced CD8+ T cells.

[0194] As illustrated in FIG. 9 and FIG. 10, to determine whether vaccine-induced T cells could recognize naturally processed antigen, the melanoma tumor cell line DM6 was transduced with a multi-mini-gene construct encoding mutated (MUT) or wild-type (WT) sequences of peptides incorporated into a vaccine. FIG. 9 illustrates that each minigene consists of 21 aa encoding either the MUT or WT sequences. A scheme depicting minigene construct characteristics and a representative MUT 21-mer aa sequence encoded in construct is shown. Vaccine-induced T cells, specific for AKAP13 containing the Q285K mutation, were incubated with MUT or WT expressing DM6 cells, supernatants collected after 24 h of incubation, and IFN- produced by T cells was measured in supernatants by ELISA (FIG. 10). Results indicate that the AKAP13 (Q285K) neo-antigen is processed, presented and recognized by vaccine-induced T cells. The results indicate that a vaccine comprising mutation-containing peptides plus autologous DC can induce cells that will recognize processed and presented antigens on the tumor cell surface.

[0195] For therapeutic use of vaccine-induced T cells, it can be important to determine whether responses elicited by MUT peptides can cross-react with WT sequences. T cell responses that cannot discriminate between MUT and WT sequences may have adverse effects if given to patients as part of adoptive cell therapy.

[0196] To examine cross-reactivity, T2 cells were pulsed with MUT or WT peptide at the indicated concentrations, labeled with .sup.51CR-chromium and used as target in a cytotoxic assay. Vaccine-induced T cells were incubated with peptide-pulsed T2 cells and .sup.51Cr-Chromium release measured at 4 h. Results obtained with T cell lines specific for 3 mutated peptides are shown in FIG. 11-12. The results indicate that T cells can display exquisite antigen specificity and can discriminate between peptide sequence containing single an changes, as shown for AKAP13 and Sec24A (FIG. 11). Only peptides containing the mutated aa can induce lysis of targets. On the other hand, other T cell lines cannot discriminate between MUT and WT sequences as shown for responses directed at OR8B3 (FIG. 12). Thus, screening for cross reactivity can be important in the selection of mutation-specific vaccine-induced T cells to be incorporated in adoptive T cell therapies, only those free of reactivity to WT sequences should be considered.

Example 8

[0197] This example illustrates that vaccine-induced mutation-specific T cells discriminate between mutated (MUT) and wild type (WT) sequences and recognized processed and presented antigens. Neoantigen-specific T cells recognition of mutated (closed circles) and wild type (open circles) peptides was determined in a standard 4 h .sup.51Cr-release assay using peptide titrations on T2 (HLA-A*02:01) cells. Percent specific lysis of triplicates (meanstandard deviation) is shown in FIG. 25 (left) for each peptide concentration; spontaneous lysis was <5%. Results are shown at 10:1 E:T ratio. T cells generated against mutated sequences do not recognize wild-type sequences. Thus, T cells induced by vaccine demonstrate an exquisite specificity for mutated antigen. Neoantigen-specific T cells were co-cultured with DM6 expressing mutated(closed rectangles) or wild type(closed circles) tandem mini-gene constructs in a 4 h .sup.51Cr-release assay. Media represent lysis obtained with parental DM6 cells. Percent specific lysis of triplicates (meanstandard deviation) is shown in FIG. 25 (right) for each E:T ratio; spontaneous lysis was <5%. Therefore, immunization with autologous mature IL-12p70 producing DC elicits shared self-antigen specific T cell responses in humans with cancer. Collectively, these data show that clinical benefit correlates with IL-12p70 which dictates lineage commitment to type-I T cell immunity.

Example 9

[0198] This example illustrates cytokine production in response to neoantigen peptides.

[0199] In various embodiments, a vaccine of the present teachings can induce CD8+ T cells to display a Tc1 profile.

[0200] Substantial evidence supports the hypothesis that Th2/Tc2 immune polarization correlates with worse disease outcome in patients with cancer (Fridman, W. H., et al., Nat. Rev. Cancer, 12, 298-306, 2012). In our previous study (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013) the inventors demonstrated that patients presenting vaccine-induced T cells displaying a Tc1 (high IFN-, low IL-4, -5, -13 production) benefited from vaccine as determined by an increased time to progression. Thus, we determined production of cytokines upon antigen stimulation as described above. In these studies, neo-antigen-specific AKAP13 (Q285K) T cells were incubated with peptide-pulsed SCD-expressing cells and supernatants collected 24 h after stimulation. Cytokine production was determined using a multi-plex bead assay. Results illustrated in FIG. 13 indicate that vaccine-induced T cells produced large amounts of IFN- relative to IL-4, -5 and -13 and hence display a Tc1 phenotype.

Example 10

[0201] This example illustrates successful treatment of melanoma in mice using a vaccine of the present teachings.

[0202] In some embodiments, the present teachings disclose that adoptive transfer of human antigen-specific T cells can lead to melanoma rejection. In investigations by the inventors, humanized mice were inoculated i.v. with luciferase-expressing melanoma. Ten days later (indicated by vertical arrows FIG. 14-15) mice received a single dose of melanoma-specific human T cells (n=5 mice/treatment). FIG. 14 depicts tumor regression monitored by luciferase (photon flux). As shown in FIG. 14 and FIG. 15, in untreated mice luciferase signal increases with time as a result of tumor growth. Conversely in mice treated with T cells, a decrease in luciferase signal was observed. This signal decrease is proportional to the number on cells transferred. These data demonstrate the T cell transfer can result in tumor regression. Importantly, tumor regression can lead to increased survival (FIG. 16). In some configurations, concentration of >10.sup.7 T cells/mouse can lead to significant changes in survival rates in this model. Adoptive transfer of mutation-specific T cells can lead to tumor regression in this animal model. Furthermore, these pre-clinical results can translate into therapeutic benefit for cancer patients.

Example 11

[0203] This example illustrates selection of neoantigens for further study.

[0204] Tumor missense mutations (MM), translated into amino acid substitutions (AAS), may provide a form of antigens that the immune system perceives as foreign, which elicits tumor-specific T cell immunity (Wlfel, T., et al., Science, 269, 1281-1284, 1995; Coulie, P. G., et al., Proc. Nat'l. Acad. Sci. USA 92, 7976-7980, 1995; van Rooij, N. et al., J. Clin. Oncol., 31, e439-e442, 2013; Robbins, P. F., et al., Nat. Med., 19, 747-752, 2013). In these experiments, three patients (MEL21, MEL38 and MEL218) with stage III resected cutaneous melanoma were consented for genomic analysis of their surgically excised tumors and subsequently enrolled in a phase 1 clinical trial with autologous, functionally mature, interleukin (IL)-12p70-producing dendritic cell (DC) vaccine (FIG. 26A-B) (Carreno, B. M., et al., J. Clin. Invest., 123, 3381-3394, 2013). FIG. 26A illustrates that dendritic cells (DC) were matured with CD40L, IFN- plus TLR3 (poly I:C) and TLR8 (R848) agonists in order to optimize the production of IL-12p70. Results shown are the ex-vivo IL-12p70 levels produced by patient-derived mature DC used for manufacturing vaccines doses D1-D3 (each symbol represents a vaccine dose). DC supernatants were harvested 24 h after activation and IL-12p70 production levels determined by ELISA. Results represent meanSEM. FIG. 26B illustrates that study timelines depicting cyclophosphamide treatment (300 mg/m.sup.2 i.v), DC vaccinations (D1-D3), PBMC sampling for immune monitoring and leukapheresis collections. The vaccine dosing schedule was altered from every 3 weeks to every 6 weeks based on the kinetics of the T cell response previously reported (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013).

[0205] All tumor samples were flash frozen except one from MEL 21 (skin, Jun. 6, 2013), which was formalin-fixed paraffin embedded. Peripheral blood mononuclear cells (PBMC) were cryopreserved as cell pellets. DNA samples were prepared using QIAAMP DNA Mini Kit (Qiagen) and RNA using High Pure RNA Paraffin kit (Roche), DNA and RNA quality was determined by NANODROP 2000 and quantitated by the QUBIT Fluorometer (Life Technologies). For each patient, tumor/PBMC (normal) matched genomic DNA samples were processed for exome sequencing with one normal and two tumor libraries, each using 500 ng DNA input (Service, S. K. et al., P.L.o.S. Genet., 10, e1004147, 2014). Exome sequencing was performed to identify somatic mutations in tumor samples.

[0206] Tumor M M, translated as AAS-encoding nonamer peptides, were filtered through in silico analysis to assess HLA-A*02:01 peptide binding affinity (Nielsen, M. et al., Protein Sci., 12, 1007-1017, 2003). Alignment of exome reads was performed using the inventors' Genome Modeling System (GMS) processing-profile. This pipeline uses BWA (version 0.5.9) for alignment with default parameters except for the following: t 4 q 5. All alignments were against GRCh37-lite-build37 of the human reference genome and were merged and subsequently de-duplicated with Picard (version 1.46). Detection of somatic mutations was performed using the union of three variant callers: 1) SAMtools version r963 (params: A B) filtered by snp-filter v1 and further intersected with Somatic Sniper version 1.0.2 (params: F vcf q 1 Q 15) and processed through false-positive filter v1 (params: -bam-readcount-version 0.4-bamreadcount-min-base-quality 15 min-mapping-quality 40-min-somatic-score 40) 2) VarScan Somatic version 2.2.6 filtered by varscan-high-confidence filter version v1 and processed through false-positive filter v1 (params, -bam-readcount-version 0.4bamreadcount-min-base-quality 15), and 3) Strelka version 1.0.10 (params: isSkipDepthFilters=1). Amino acid substitutions (AAS) corresponding to each of the coding missense mutations (MM) were translated into a 21-mer amino acid FASTA sequence, with ideally 10 amino acids flanking the substituted amino acid on each side.

[0207] Each 21-mer amino acid sequence was then evaluated through the HLA class I peptide binding algorithm NetMHC 3.4 to predict high affinity HLA-A*02:01 nonamer peptides for the AASas well as the WT sequence to calculate differences in binding affinities (8, 32). Any peptides with binding affinity IC.sub.50 value<500 nM were considered for further analysis.

[0208] Experimental expression of genes encoding predicted HLA-A*02:01 peptide candidates was determined by cDNA capture. All RNA samples were DNase-treated with TURBO DNA-FREE kit (Invitrogen) according to the manufacturer's instructions; RNA integrity and concentration were assessed using Agilent Eukaryotic Total RNA 6000 assay (Agilent Technologies) and QUANT-IT RNA assay kit on a QUBIT Fluorometer (Life Technologies Corporation).

[0209] Given the dynamic nature of genomic technologies, multiple overlapping methods were tested. However, results for tumors within a patient (Tables 2-4) are consistent with one methodology: NuGen OVATION V2 for MEL38 and MED218, Illumina TRUSEQ Stranded for MEL21. The MicroPoly(A)PURIST Kit (Ambion) was used to enrich for poly(A) RNA from MEL218 and MEL38 DNAse-treated total RNA; MEL21 RNA was ribo-depleted using the RIBO-ZERO Magnetic Gold Kit (EpiCeture, Madison Wis.) following the manufacturer protocol. The inventors used either the OVATION RNA-Seq System V2 (NuGen, 20 ng of either total or polyA RNA), or the OVATION RNA-Seq FFPE System (NuGen, 150 ng of DNase-treated total RNA) or the TRUSEQ Stranded Total RNA Sample Prep kit (Illumina, 20 ng ribosomal RNA-depleted total RNA) for cDNA synthesis. All NuGen cDNA sequencing libraries were generated using NEBNEXT ULTRA DNA Library Prep Kit for ILLUMINA with minor modifications.

[0210] All NuGEN generated cDNA was processed as described previously (Cabanski, C. R., et al., J. Mol. Diagn., 16, 440-451, 2014). Briefly, 500 ng of cDNA was fragmented, end-repaired, and adapter-ligated using IDT synthesized dual same index adapters. The TRUSEQ stranded cDNA was also end-repaired and adapter-ligated using IDT synthesized dual same index adapters. These indexed adapters, similar to Illumina TRUESEQ HT adapters, contain the same 8 bp index on both strands of the adapter. Binning reads requires 100% identity from the forward and reverse indexes to minimize sample crosstalk in pooling strategies. Each library ligation reaction was PCR-optimized using the Eppendorf Epigradient SqPCR instrument, and PCR-amplified for limited cycle numbers based on the Ct value in the optimization step.

[0211] Libraries were assessed for concentration using the QUANT-IT dsDNA HS Assay (Life Technologies) and for size using the BioAnalyzer 2100 and the Agilent DNA 1000 Assay (Agilent Technologies). The ILLUMINA-ready libraries were enriched using the Nimblegen SeqCap EZ Human. Exome Library v3.0 reagent. The targeted genomic regions in this kit cover 63.5 Mb or 2.1% of the human reference genome, including 98.8% of coding regions, 23.1% of untranslated regions (UTRs), and 55.5% of miRNA bases (as annotated by Ensembl version 73 (Flicek. P., et al., Nucleic Acids Res., 41, D48-55, 2013)). Each hybridization reaction was incubated at 47 C. for 72 hours, and single-stranded capture libraries were recovered and PCR-amplified per the manufacturer's protocol. Post-capture library pools were sized and mixed at a 1:0.6 sample: Ampure XP magnetic head ratio to remove residual primer-dimers and to enrich for a library fragment distribution between 300 and 500 bp. The pooled capture libraries were diluted to 2 nM for Illumina sequencing.

[0212] For cDNA-capture data were aligned with Tophat v2.0.8 (params: version=2.1.0 for OVATION; -library-type fr-firststrand-bowtie-version=2.1.0 for TRUSEQ). For OVATION data, prior to alignment, paired 2100 bp sequence reads were trimmed with flexbar v 2.21 (params: -adapter CTTTGTGTTTGA (SEQ ID NO: 474-adapter-trim-end LEFT-nono-length-dist-threads 4-adapter-min-overlap 7-maxuncalled 150-min-readlength 25) to remove single primer isothermal amplification adapter sequences. In seqcap, the relative expression of a transcript is proportional to the number of cDNA fragments that originate from it. Therefore, expression levels expressed as fragments per kilobase of exon per million fragments mapped (FPKM) were calculated with Cufflinks v2.0.2 (Trapnell et al. 2010, Nature Biotechnology 28, 511; params-max-bundle-length=10000000-num-threads 4). A visual review step of cDNA capture data was performed to evaluate for expression of MM identified by exome data. Both cDNA-capture and FPKM values were considered for candidate prioritization.

[0213] FIG. 27 illustrates distribution of somatic (exomic and missense) mutations identified in patients MEL21 and MEL38 metachronous tumors (anatomical location and date of collection indicated) and patient MEL218 tumor are shown. HLA-A*02:01-binding candidate peptides were identified among AAS and expression of gene encoding mutated protein determined from cDNA capture data (Tables 2-4) as discussed supra. Venn diagrams show expression, among metachronous tumors, of mutated genes encoding vaccine neoantigens. The identities of the three immunogenic neoantigens identified in each patient are depicted in diagrams; type style identifies naturally occurring (italics) and vaccine-induced (bold) neoantigens.

[0214] Peptide candidates for experimental validation were selected according to the strategy described in FIG. 28: Tumor-specific missense mutations (MM) in melanoma samples were detected using exome sequencing and identified using the union of three variant calling algorithms. BRAF allelic frequency (Tables 2-4) was considered the upper limit variant allelic fraction fix each tumor and used as a comparator to assess the clonality of other MM-encoding genes. Amino acid substitutions (AAS) corresponding to each of the coding MM were translated into a 21-mer amino acid FASTA sequence and evaluated through the HLA class I peptide binding algorithm NetMHC 3.4 to predict HLA-A*02:01 nonamer AAS-encoding peptides with EC.sub.50<500 nM. Transcriptional status of genes encoding AAS candidates was determined by cDNA-capture and their expression levels determined using Cufflinks. Filters were applied to deprioritize those with low cDNA-capture (Alt_reads<5) and prioritized those with high numbers of Atl_reads and/or FPKM>1. For MEL21 and MEL38 patients, candidates were prioritized if expressed by more than one metachronous tumor. For experimental validation, candidates were further prioritized on the basis of predicted HLA-A*02:01 binding affinity and/or HLA-A*02:01 affinity differential between AAS- and WT-peptide (Tables 2-4). Only those peptides with confirmed HLA-A*02:01 binding as determined by T2 assay (FIG. 29) and fluorescence polarization assay [log(IC.sub.50 nM)<4.7, Table 5]were prioritized for vaccine formulation.

[0215] HLA-A*02:01 binding was evaluated using the T2 assay (See Analysis of T cell responses) (FIG. 29) (Elvin, J., et al., J. Immunol. Methods, 158, 161-171, 1993) and confirmed in the fluorescence polarization-based competitive peptide binding assay (Buchli, R., et al., Biochemistry, 44, 12491-12507, 2005). FIG. 29 illustrates AAS-encoding peptide binding to HLA-A*02:01. T2 cells were incubated with 100 uM of the indicated peptide for 16 h, washed and stained with PE-conjugated anti-HLA-A*02:01 (clone BB7.2) monoclonal antibody. Melanoma G280-9V and Influenza NP265 peptides represent positive and negative controls, respectively. Binding fold are calculated as MFI experimental peptide/MFI NP265 peptide. Data are representative of 3 independent experiments. Peptides selected for incorporation in the vaccine formulation are indicated with an asterisk. Per patient, 7 AAS peptide candidates were selected among validated HLA-A*02:01 binders (Table 5) for incorporation into a personalized vaccine formulation along with the melanoma gp100-derived peptides G209-2M and G280-9V (as positive controls for vaccination) (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013). The expression pattern of mutated genes encoding vaccine candidates is shown in Venn diagrams in FIG. 27.

Example 12

[0216] This example illustrates the effectiveness of personalized dendritic vaccines.

[0217] To examine the kinetics and magnitude of T cell immunity to AAS peptides upon vaccination, peripheral blood mononuclear cells (PBMC) were collected prior to vaccination and weekly thereafter. The CD8+ T cell response to each peptide was analyzed using a HLA-A*02:01/AAS-peptide dextramer assay after a single round of in vitro stimulation. FIG. 30A illustrates kinetics of immune responses to neoantigens. Time is recorded in weeks (0 indicates pre-vaccination). Culture conditions and staining details are described infra. Antigen-specific numbers were calculated based on dextramer percentage and total live cell yields. Immunologic analysis to evaluate the kinetic and magnitude of T cell response to AAS-encoding and gp100-derived peptides was performed using PBMC collected weekly, starting before DC vaccination (Pre-vaccine in the figures) as described (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013). Briefly, fresh PBMC obtained by Ficoll-Paque PLUS gradient centrifugation were cultured with 40 ug/mL peptide and IL-2 (50 U/mL). On day 10 (peak of response, unpublished data, labeled Post-Vaccine in the figures), neoantigen specific T cell frequencies were determined by staining with HLA-A*02:01/peptide dextramers (Immudex), followed by addition of FITC-CD4, -CD14, -CD19 (Invitrogen) and ALEXA 488-CD56 (BD Pharmigen), APC-CD8 (Invitrogen). Cells were washed, resuspended in FACS buffer containing 7AAD. Twenty five thousand events in the CD8+ gate were collected using a hierarchical gating strategy that included FSC/SSC and excluded 7AAD-positive (dead cells) and CD4/14/19/56-positive cells. PBMC/CD8+ T cells derived from an unrelated HLA-A*02:01 patient were used as negative controls for assessing specificity of HLA-A*02:01/AAS-peptide dextramers (data not shown). Data were acquired and analyzed using Flow-Jo software. Immune monitoring demonstrated that in each patient, T cell immunity to one AAS peptide could be detected in pre-vaccine PBMC samples after in vitro stimulation (FIG. 31, MEL21:TMEM48 F169L; MEL38; SEC24A P469L and MEL218: EXOC8 Q656P, type style identifies naturally occurring (italics) and vaccine-induced (bold) neoantigens) although not directly from the blood. FIG. 30B illustrates the frequency of neoantigen specific T cells in CD8+ populations isolated directly from PBMC samples and after ex-vivo expansion using autologous DC and artificial antigen presenting cells. For dominant neoantigens TMEM48 F169L, SEC24A P469L and EXOC8 Q656, results are shown for samples obtained before vaccination (Pre-vaccine) and after 3 vaccine doses (Post-vaccine). For remaining neoantigens, results obtained with post-vaccine PBMC samples are shown. Percentage of neoantigen-specific CD8+ T cells is indicated in the right upper quadrant of the plot. A representative experiment of two performed is shown. Pre-existing immunity to these three neoantigens was confirmed in ex-vivo expanded pre-vaccine purified CD8+ T cells using dextramer assay (FIG. 30B) and interferon (IFN)- production. FIG. 30C illustrates ex-vivo expanded pre-vaccine neoantigen-specific T cells (dextramer % shown in FIG. 30B) were stimulated with artificial antigen presenting cells in the presence (closed bar) or absence (open bar) of AAS-peptide and supernatants were harvested at 24 h. IFN- production was determined using ELISA assay. Mean values+/standard deviation (SD) of duplicates are shown. Cytokine production by T cells in the absence of any stimuli was <100 pg/mL.

[0218] Vaccination augmented the cell response to these neoantigens with observed frequencies of 23% TMEM48 F169L+ CD8+ T cells, 64% SEC24A P469L+ CD8+T cells and 89% EXOC8 Q656P+ CD8 T cells detected, upon culture, at the peak of response (FIG. 31). Immune monitoring also revealed vaccine-induced T cell immunity to two additional neoantigens per patient: TKT R438W and CDKN2A E153K (55% and 12%, respectively) in patient MEL21; AKAP13 Q285K and OR8B3 T190I (47% and 42%, respectively) in patient MEL38, and MRPS5 P59L and PABC1 R520Q (58% and 84%, respectively) in patient MEL218 (FIG. 31). Two (MEL21 and MEL218) of the three patients had pre-existing immunity to G209-2M and G280-9V peptides, as determined by the presence of gp100-specific T cells in pre-vaccine PBMC samples and their ex-vivo expansion upon antigen stimulation. FIG. 32 illustrates the frequency of G209-2M- and G280-9V-specific T cells in CD8+ populations isolated directly from PBMC samples and after ex-vivo expansion using autologous DC and artificial antigen presenting cells. Results are shown for samples obtained before vaccination (Pre-vaccine) and at peak post vaccination (Post-vaccine). Percentage of antigen-specific CD8+ T cells is indicated in the right upper quadrant of the plot. A representative experiment of three performed is shown. Upon vaccination, these T cell responses were enhanced in patients MEL21 and MEL218 and revealed in patient MEL38. FIG. 33 illustrates the kinetics of immune responses to G209-2M and G280-9V peptides. Time is recorded in weeks (0 indicates prevaccination). Culture conditions and staining details are described supra. Antigen specific numbers were calculated based on dextramer percentage and total live cell yields. No T cell immunity was detected to the remaining 12 AAS peptides. Overall, robust neoantigen T cell immunity was detectable as early as week 2 and peaked at week 8-9 after the initial vaccine dose (FIG. 30A). Neoantigen-specific CD8+ T cells are readily identified by dextramer assay directly in post-vaccine PBMC samples (FIG. 30B) and memory T cells are detected up to 4 months after the final vaccine dose.

[0219] Analysis of T cell reactivity among the three patients indicated no preferential skewing towards AAS at specific positions in the peptide sequencethat is towards TCR, contact residues or primary anchor residues (Kim, Y., et al., J. Immunol. Methods, 374, 62-69, 2011). Rather, in each patient, T cell immunity appeared to focus on the 3 AAS candidates exhibiting the highest HLAA*02:01 binding affinity while the remaining medium-high affinity peptides were nonimmunogenic (Table 5) (Nielsen M., et al., Protein Sci., 12, 1007-1017, 2003; Buchli, R., et al., Biochemistry, 44, 12491-12507, 2005). Immunogenic AAS peptides (FIG. 27) were not preferentially derived from genes with high allelic frequency or expression levels (Tables 2-4).

[0220] To characterize the function of vaccine-induced neoantigen-specific T cells, short-term expanded CD8+ T cell lines were established and antigen specificity confirmed by dextramer assay (FIG. 30B) (Carreno B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013; Carreno, B. M. et al., J. Immunol, 188, 5839-5849, 2012). Neoantigen-specific T cell lines were generated using autologous mDC and antigen loaded artificial antigen presenting cells at a ratio of 1:1 as previously described (Carreno, B. M. et al., J. Immunol. 188, 5839-5849, 2012); antigen-specific frequencies in cell lines are shown in FIG. 30B. To determine the peptide avidity (effective concentration at 50% maximal lysis, EC.sub.50) of neoantigen-specific T cells, T2 cells were pulsed with titrated peptide concentrations for 1 h, followed by .sup.51Cr (25Ci) labeling for 1 h, washed twice and tested in a standard 4 h .sup.51Cr release assay using neoantigen-specific T cells as effectors. For production of cytokines, neoantigen-specific T cells were restimulated using artificial antigen presenting cells in the presence or absence of peptide, supernatants collected at 24 h and cytokine produced determined using MILLIPLEX MAP Human Cytokine Panel I (EMD Millipore).

[0221] FIG. 34 illustrates that neoantigen-specific T cells recognition of AAS (closed circles) and WT (open circles) peptides was determined in a standard 4 h .sup.51Cr-release assay using peptide titrations on T2 (HLAA*02:01) cells. Percent specific lysis of triplicates (mean+standard deviation) is shown for each peptide concentration; spontaneous lysis was <5%. Results are shown at 10:1 E:T ratios for all cell lines except TMEM48 F169L and CDKN2A E153K cells which are shown at 60:1 E:T ratio. A representative experiment of two independent evaluations is shown. Neoantigen-specific T cells displayed significant levels of cytotoxic activity at AAS peptide concentrations of 1 to 10 nM, a finding that is consistent with high avidity cell recognition of antigen (FIG. 34). OR8B3 T190I-specific T cells could not discriminate between AAS and wild-type (WT) peptide when presented on T2 cells, while all of the remaining cell lines showed clear specificity for AAS peptide sequences (FIG. 34).

[0222] The cytokine production profile of these cells was characterized as previously described (Carreno, B. M., et al., J. Clin. Invest., 123, 3383-3394, 2013; Fridman, W. H., et al., Nat. Rev. Cancer, 12, 298-306, 2012). This characterization is illustrated in FIG. 35: Neoantigen-specific T cells were stimulated with artificial antigen presenting cells in the presence (open bar) or absence (close bar) of AAS-peptide and supernatants were harvested at 24 h. Cytokine production was determined using MILLIPLEX MAP Human Cytokine Panel I. Mean values+/SD of duplicates are shown. Cytokine production by T cells in the absence of any stimuli was <100 pg/mL. A representative experiment of 2 performed is shown. FIG. 36 illustrates a comparison of production of Type 1 (IFN-) and Type 2 (IL-4, IL-5, IL-13) cytokines among neoantigen-specific T cells, a cytokine index was derived by dividing pg/mL IFN- by pg/mL IL-13, IL-5 or IL-4. IFN-/IL-13, IFN-/IL-5 and IFN-/IL-4 ratios above 1 are indicative of Type 1 phenotype. Results are representative of two experiments. Upon antigen stimulation, most vaccine-induced neoantigen-specific T cells produced high amounts of IFN- relative to IL-4, IL-5 and IL-13, a pattern that is indicative of a type 1 phenotype (FIG. 35-36). However, SEC24A P469L specific cells exhibited a type 2-skewed phenotype (high IL-4, IL-5 and IL-13 levels relative, to IFN-), and TMEM48 F169L specific T cells showed a mixed phenotype with only higher IL-13 (hut not IL-4 or IL-5) levels relative to IFN- (FIG. 35-36).

Example 13

[0223] This example illustrates the in vitro detection of neoantigens that are presented to immune cells in vivo.

[0224] Tandem mini-gene constructs (TMC) were used for evaluating processing and presentation of neoantigens. The structure of a representative TMC (MEL21 AAS sequences) is shown in FIG. 37A. All constructs were 19-21-mers encoding AAS- or WT-sequences for peptides included in vaccine. No spacers are present between sequences. A ubiquitination signal and two mini-gene controls (encoding G280 and WNV SVG9 peptides) were included to monitor processing and presentation. The amino acid sequence of a 21-mer encoding TMEM48 F169L is shown with mutated amino acid residue underlined. TMC also encoded the West Nile Virus (WNV) SVG9 (McMurtrey, C. P., et al., P.N.A.S., 105, 2981-2986, 2008) and melanoma G280 (Cox, A. L., et al., Science, 264, 716-719, 1994) antigenic determinants as controls (Table 6).

[0225] TMC were cloned into pMX (GFP+), expressed as retrovirus and used to transfect the HLA-A*02:01+ melanoma lines DM6 (Darrow, T. L., et al., J. Immunol., 142, 3329-3335, 1989) or A375 (obtained from ATCC and mycoplasma free). TMC expressing cells were selected by sorting for GFP+ cells expressing cell surface HLA-A*02:01/SVG9 peptide complexes as detected by a T cell receptor mimic (TCRm) monoclonal antibody (Kim S., et al., J. Immunol., 184, 4423-4430, 2010). AAS- and WT-TMC reactivity with the HLA-A*02:01/SVG9 peptide complex specific TCRm monoclonal antibody validated expression of the mini-gene constructs. FIG. 37B demonstrates that expression of AAS- and WT-TMC constructs was determined using a TCR-mimic monoclonal antibody that detects HLAA*02:01/SVG9 (SVGGVFTSV SEQ ID NO: 31) complexes Kim S., et al., J. Immunol., 184, 4423-4430, 2010). Results are shown for parental DM6 (shaded histogram) and DM6 cells expressing AAS(dashed line) and WT (solid line) TMC constructs. A representative experiment of four performed is shown.

[0226] DM6 cells expressing TMC were labeled with 25Ci .sup.51Cr for 1 h, washed and tested as targets in a standard 4 h assay using neoantigen-specific T cells as effectors (Carreno B. M. et al. 2012 J Immunol 188, 5839). DM6 cells expressing AAS(closed rectangles) or WT(closed circles) TMC were co-cultured with neoantigen-specific T cells at a 1:1 ratio, supernatants harvest at 16 h and IFN- production evaluated by ELISA as described (Carreno, B. M., et al., J Immunol., 188, 58395849, 2014; plots in FIG. 38). Open triangles represent lysis obtained with parental DM6 cells. Percent specific lysis of triplicates (mean+standard deviation) is shown for each E:T ratio; spontaneous lysis was <5%. A representative experiment of two independent evaluations is shown.

[0227] FIG. 39 illustrates that neoantigen-specific CD8 T cells were co-cultured with DM6 expressing AAS- or WT-encoding TMC for 20 h and IFN- production determined by ELISA. T cells cultured with parental DM6 cells are indicated as media. Mean values+/SD of duplicates are shown. Results are representative of 2 experiments performed. Seven (TMEM48 F169L, TKT R438W, CDKN2A E153K, SEC24A P469L, AKAP13 Q285K, EXOC8 Q656P and PABPC1 R520Q) of the nine immunogenic neoantigens are processed and presented as evidenced by cytotoxic activity (FIG. 38) and IFN- production (FIG. 39) by corresponding neoantigen-specific T cells upon co-culture with DM6 expressing AAS-encoding TMC. In contrast, neither cytotoxic activity (FIG. 38) nor IFN- production (FIG. 39) was observed upon co-culture of OR8B3 T190I- and MRPS5 P59L-specific T cells with DM6 expressing AAS-encoding TMC showing that these neoantigens are not processed and presented from endogenously expressed protein. None of the neoantigen-specific T cells recognized WT-encoding TMC (FIGS. 38 and 39). Based on these findings and the immune monitoring results (FIG. 31), the nine neoantigens identified in this study fall into three distinct antigenic determinant categories (Sercarz, E. E., et al., Annu. Rev. Imunol., 11, 729-766, 1993; Assarsson, E., et al., J. Immunol., 178, 7890-7901, 2007). TMEM48 F169L, SEC24A P469L, and EXOC8 Q656P represent dominant antigens as T cell immunity was detected prior to vaccination (naturally occurring) (FIG. 31 and these neoantigens are processed and presented from endogenously expressed protein (FIG. 38). TKT R438W, CDKN2A E153K, AKAP13 Q285K and PABPC1 R520Q are characterized as subdominant antigens as T cell immunity required peptide vaccination (FIG. 31) and these neoantigens are processed and presented from endogenously expressed protein (FIG. 38). And finally, OR8B3 T190I and MRPS5 P59L constitute cryptic antigens since peptide vaccination elicited T cell immunity but these neoantigens are not processed from endogenously expressed protein.

Example 14

[0228] This example illustrates the use of proteomic techniques to determine which neoantigens are presented to cells in vivo.

[0229] To validate neoantigen processing and presentation, proteomic analysis was performed on peptides eluted from soluble HLA-A*02:01 molecules isolated from melanoma cells expressing a TMC encoding AAS candidates from patient MEL218 tumor (Sercarz, E. E., et al., Annu. Rev. Imunol., 11, 729-766, 1993; Assarsson, E., et al., J. Immunol., 178, 7890-7901, 2007). TMC expressing A375 melanoma cells were transfected with soluble HLA-A*02:01(sHLA-A*02:01) and single cell sorted for a high (>1000 ng/ml in static culture) sHLAA*02:01 producing clone. The sHLA-A*02:01 construct includes a C-terminal VLDLr epitope purification tag (SVVSTDDDLA SEQ ID NO. 32) that is recognized by the anti-VLDLr mAb (ATCC CRL-2197). This antibody was also used for quantification of sHLA production as the capture antibody in a sandwich ELISA, with an antibody directed against 2-microglobulin (Dako Cytomation) as the detector antibody. Cells were grown in roller bottles and sHLA/peptide complexes were purified from supernatants by affinity chromatography with the anti-VLDLr antibody (Kaabinejadian, S., et al., P.L.o.S. One, 8, e66298, 2013). Eluate fractions containing sHLA/peptide complexes were brought to a final acetic acid concentration of 10%, pooled, and heated to 78 C. in a water bath. Peptides were purified through a 3 kDa molecular weight cutoff cellulose membrane (EMD Millipore) and lyophilized.

[0230] Synthetic peptides corresponding to the mutant sequences were resuspended in 10% acetic acid in water at 1 M, and fractionated by RP-HPLC with an acetonitrile gradient in 10 mM ammonium formate at pH 10. Peptide-containing fractions were dried and resuspended in 25 ul of 10% acetic acid and subjected to nanoscale RP-HPLC at pH 2.5 utilizing an Eksigent nanoLC coupled to a TripleTOF 5600 (AB Sciex) quadrupole time-of-flight mass spectrometer (LC/MS). Information dependent acquisition (IDA) was used to obtain MS and MS/MS fragment spectra for peptide ions. The sequence of each peptide was determined by observed mass and fragment ions, and the 1st dimension fraction number and LC/MS retention times were recorded.

[0231] Next, peptides purified from TMC expressing A375 melanoma cells were resuspended in 10% acetic acid and HPLC fractionated under the same conditions and gradient method. Reverse phase HPLC was used to reduce the complexity and determine the elution profile of the pool of soluble HLA-A*02:01 restricted peptides presented by melanoma cells, as well as, the synthetic AAS peptide mixture. FIGS. 40A and 40E illustrate RP-HPLC fractionation of HLA-A*02:01 peptides eluted from the AAS-TMC expressing melanoma cell line (solid trace) and the synthetic peptide mixture containing MEL218 neoantigen candidates (dashed trace), with fraction 50 (FIG. 40A) and fraction 44 (FIG. 40E) indicated. The HPLC fractions corresponding to those containing the synthetic peptides were then subjected to the same LC/MS conditions. Resulting spectra were found positive for the presence of the mutant peptides if the following criteria were met: 1. The observed fragment ions were in the same RP-HPLC fraction as the synthetic, 2. LC/MS elution time was within 2 minutes of the synthetic, and 3. Fragment ion masses matched those of the synthetic with an accuracy of 25 ppm. PEAKVIEW Software version: 1.2,0,3 was used for exploring and interpreting of the LC/MS data.

[0232] Separation and sequencing of peptides were carried out by two-dimensional liquid chromatography, followed by information dependent acquisition (IDA) generated tandem MS (MS/MS). For the first dimension, the peptide sample was loaded on a reverse-phase C.sup.18 column (pore size, 110 ; particle size, 5 m; 2 mm i.d. by 150 mm long Gemini column; Phenomenex) with a Michrom BioResources Paradigm MG4 high performance liquid chromatograph (HPLC) with UV detection at 215 nm wavelength. Elution was at pH 10 using 10 mM ammonium formate in 2% acetonitrile/98% water as solvent A and 10 mM ammonium formate in 95% acetonitrile/5% water for solvent B. The 1st dimension HPLC column was preequilibrated at 2% solvent B, then the peptide sample, dissolved in 10% acetic acid/water, was loaded at a flow rate of 120 l/min over an 18 minute period. Then a two segment gradient was performed at 160 l/min; the 1st segment was a 40 minute linear gradient from 4% B to 40% B, followed by an eight minute linear gradient from 40% B to 80% B. Forty peptide-rich fractions were collected and dried by vacuum centrifugation.

[0233] For the second dimension chromatography, each dried fraction was resuspended in 10% acetic acid and subjected to nano-scale RP-HPLC (Eksigent nanoLC415, AB Sciex). The second dimension nano-HPLC setup included a C.sup.18 trap column (350 m i.d. by 0.5 mm long; ChromXP (Eksigent) with 3 m particles and 120 pores and a ChromXP, C.sup.18 separation column with dimensions of 75 m i.d. by 15 cm long packed with the same medium. A two-solvent system was utilized, where solvent A is 0.1% formic acid in water and solvent B contains 0.1% formic acid in 95% acetonitrile/5% water. Samples were loaded at 5 L/min flow rate on the trap column and at 300 nL/min flow rate on the separation column that was equilibrated in 2% solvent B. The separation was performed by a program with two linear gradients: 10% to 40% solvent B for 70 min and then 40% to 80% solvent B for 7 min. The column effluent was connected to the nanospray III ion source of an AB Sciex TripleTOF 5600 quadrupole-time of flight mass spectrometer with the source voltage set to 2400 v.

[0234] Extracted ion chromatograms revealed the presence of an eluted peptide with a retention time within 2 minutes of synthetic EXOC8 Q656P peptide in fraction 50. FIG. 40B illustrates an extracted ion chromatogram of the parent ion with the theoretical m/z of 480.8156 (+2) in HPLC fraction 50 from the HLA-A*02:01 eluted peptides (solid line) overlaid with the EXOC8 Q656P synthetic peptide (dashed line). MS/MS fragmentation pattern comparison of the eluted and the synthetic peptides ensured EXOC8 Q656P sequence identity and confirmed HLA-A*02:01 presentation of this dominant neoantigen. The eluted EXOC8 Q656P peptide MS/MS fragmentation pattern is illustrated in FIG. 40C and that of the corresponding synthetic peptide is illustrated in FIG. 40D. A similar analysis of fraction 44 demonstrated the HLA-A*02:01 presentation of subdominant neoantigen PABPC1 R520Q. FIG. 40F illustrates the extracted ion chromatogram of the parent ion (depicted in FIG. 40E, supra) with the theoretical m/z 524.2808 (+2) in HPLC fraction 44 from the HLA-A*02:01 elated peptides (solid line) overlaid with the PABPC1 R520Q synthetic peptide (dashed line). The MS/MS fragmentation pattern of the eluted peptide is shown in FIG. 40G and that of the corresponding synthetic peptide is shown in 3H. Altogether, these results show that two of the 7 neoantigens included inpatient MEL218 vaccine, along with antigen controls WNV SVG9 and G280, are processed and presented in the context of HLA-A*02:01 molecules. MS/MS fragmentation pattern of the peptide elated from HLA-A*02:01 identified as YLEPGPVTA (SEQ ID No. 165) (FIG. 41A), and the corresponding G280 synthetic peptide. MS/MS fragmentation pattern (FIG. 41C) of the peptide elated from HLA-A*02:01 identified as SVGGVFTSV (SEQ ID No. 33) (FIG. 41B), and the corresponding WNV SVG9 synthetic peptide (FIG. 41D).

Example 15

[0235] This example illustrates characterization of the composition and diversity of neoantigen-specific T cells and the effect vaccination can have on these repertoires.

[0236] Short-term ex-vivo expanded neoantigen-specific T cells were purified to 97-99% purity by cell sorting in a Sony SY3200 BSC (Sony Biotechnology) fitted with a 100 um nozzle, at 30 psi, using 561 nm (585/40) and 642 nm (665/30) lasers and cell pellets were prepared. DNA isolation and TCR sequencing was performed by Adaptive Biotechnologies and The Genome Institute at Washington University. Sequencing was performed at either survey (for neoantigen-specific TCR reference libraries) or deep (for pre- and post-vaccine CD8+ T cell populations) level (Robins, H., et al., J. Immunol. Methods, 375, 14-19, 2012; Carlson, C. S., et al., Nat. Commun., 4, 2680, 2013). TCR V-, D-, J-genes of each CDR3 regions were defined using IMGT (ImMunoGeneTics)/Junctional algorithms and data uploaded into the ImmunoSeq Analyzer (Adaptive Biotechnologies) for analysis. Complete amino acid identity between the reference library and pre- and post-vaccine CD8 samples was required for assigning a TCR match. In the reference library, TCR clonotypes with frequencies above 0.1% (>100-fold sequencing depth) were set as a threshold for identification of neoantigen-specific TCR CDR3 sequences within pre- and post-vaccine CD8+ T cell populations.

[0237] Reference T cell receptor- (TCR) complementarity-determining region 3 (CDR3) sequence libraries (shown schematically in FIG. 42, Tables 7-11) were generated from short-term expanded sorted neoantigen-specific T cells (97-99% dextramer-positive), in Tables 7-11, TCRBV, TCRBD and TCRBJ are shown according to consensus nomenclature and CDR3 sequence for each clonotype indicated. Read counts indicates the number of times a given CDR3 sequence was found in the short term ex-vivo expanded neoantigen population. TCR clonotypes with frequencies above 0.1% (>100-fold sequencing depth), in reference library, were set as a threshold for identification of neoantigenspecific TCR CDR3 sequences within CD8+ T cell populations isolated from PBMC obtained pre- and post-vaccination. FIG. 43A illustrates profiles purified neoantigen-specific CD8+ T cells used for the generation of TCR CDR3 reference libraries. In FIG. 43A, purified CD8+ T cells isolated from PBMC obtained after vaccination were stimulated in an antigen-specific manner as described supra. Cells were stained using HLA-A*02:01/AAS-peptide dextramers and anti-CD8 monoclonal antibody; neoantigen-specific CD8+ cells were sorted in a Sony SY3200 BSC Cell Sorter. Purity of post-sort populations is shown in dot plots (upper right quadrants, 97-99% purity). FIG. 43B illustrates the comparison of clonotype distribution in sorted/expanded dominant and subdominant neoantigen-specific CD8 T cells obtained from each of the indicated patients. These clonotypes represent the TCR CDR3 reference libraries used for probing pre- and post-vaccine CD8+ T cell populations. Frequencies are shown as percent of total reads. Reference library comprised clonotypes with frequencies of 0.1 or above (Lossius, A., et al., Eur. J. Immunol., 44, 3439-3452, 2014). The total number of clonotypes in each antigen population is indicated in the x- and y-axis and CDR3 sequences arc listed in Tables 7-11. The one clonotype that overlapped between EXOC8 Q656P and PABPC1 R520Q (indicated by circle) was excluded from analysis. These sequence libraries were used to characterize neoantigen TCR clonotypes in purified CD8+ T cells isolated from pre- and post-vaccine PBMC samples (Robins, et al., J. Immunol Methods, 375, 14-19, 2012; Lossius, A., et al., Eur. J. Immunol., 44, 3439-3452, 2014; Robins, H. S., et al., Sci. Transl. Med., 5, 214ra169, 2013). In pre-vaccination CD8+ T cell populations, as few as one and as many as 10 unique TCR clonotypes per neoantigen were identified. FIG. 44A summarizes the TCR clonotypes identified, using neoantigen-specific TCR CDR3 reference libraries (see Tables 7-11), in CD8+ T cell populations isolated from PBMC obtained before and after vaccination. Each symbol represents a unique TCR sequence and its frequency (%) in pre- and post-vaccine samples. Wilcoxon-signed rank test was performed and p values indicated in figure. Thus, vaccination increased the frequency of most existing pre-vaccine TCR clonotypes and revealed new clonotypes for all 6 neoantigens (FIG. 44A). For both dominant and subdominant neoantigens, the TCR repertoire was increased significantly after vaccination. FIG. 44B illustrates TCR CDR3 sequence of clonotypes (Tables 7-11) identified in pre(black bars) and post(white bars) vaccine CD8+ T cell populations for neoantigens TKT R438W (pre=5, post=84 clonotypes); SEC24A P469L (pre=9, post=61) and EXOC8 Q656P (pre=2, post=12). Frequency of each unique clonotype is reported as percentage of total read counts. 84 clonotypes representing TCR families are detected for TKT R438W, 61 clonotypes representing 12 TCR families are detected for SEC:24A P469L and 12 clonotypes representing 8 TCR families are detected for EXOC8 Q656P (FIG. 44B). Thus, peptide vaccination with functionally mature DC can promote the expansion of a highly diverse neoantigen TCR repertoire.

Example 16

[0238] This example illustrates vaccination of patients using multiple HLA cell types.

[0239] Distribution of somatic (exomic and missense) mutations was identified in metachronous tumors of patients MEL66 is illustrated in FIG. 45 (anatomical location and date of collection indicated). HLA-A*02:01- and HLA-B*08:01-binding candidate peptides were identified in silico according to the methods of the present teachings among amino acid substitutions present in the patient's tumor; expression of genes encoding mutated proteins was determined from cDNA capture data. Venn diagrams show expression, among metachronous tumors, of mutated genes encoding vaccine neoantigens. The identities of the 6 immunogenic neoantigens identified among the 10 included in vaccine are indicated; type style identifies naturally occurring (italics) and vaccine-induced (bold) neoantigens.

[0240] Distribution of somatic (exomic and missense) mutations identified in metachronous tumors of patients MEL69 is illustrated in FIG. 46 (anatomical location and date of collection indicated). HLA-A*02:01- and HLA-A*11:01-binding candidate peptides were identified in among amino acid substitutions in the patient's tumor according to a method of the present teachings; expression of genes encoding mutated proteins was determined from cDNA capture data (Table 12). Venn diagrams show expression, among metachronous tumors, of mutated genes encoding vaccine neoantigens. The identities of the 5 immunogenic neoantigens identified among the 10 included in vaccine are indicated; type style identifies naturally occurring (italics) and vaccine-induced (bold) neoantigens.

[0241] The vaccine for patient MEL66 included neoantigens that bound to HLA-A*02:01 and HLA-B*08:01 molecules. The vaccine for MEL69 included neoantigens that bound to HLA-A*03:01 and HLA-A*11:01 molecules. Both vaccines were prepared by contacting the neoantigens with the patient's own dendritic cells and maturing them prior to administration in accordance with the present teachings. Representative results (dextramer assay) to neoantigens restricted to these alleles are shown (FIG. 47) before DC vaccination (pre-vaccine) and at peak of immune response (post-vaccine). Numbers within dot plots represent percentage neoantigen-specific T cells within the lymph+/CD8+ gated cells. A naturally occurring response to HLA-A*11:01-restricted neoantigen ERCC6L V476I was observed in patient MEL69.

[0242] All cited publications are hereby incorporated by reference, each in its entirety.

TABLE-US-00001 TABLE1 AnalysisofmissensemutationsbypredictionalgorithmsforbindingtoHLA-A*0201 Protein MUTATED WILD-TYPE AA Sequence Binding Affinity Sequence Binding Affinity CHR Gene Change AAseq Listing (nM) (h) AAseq Listing (nM) (h) 15 AKAP13 Q285K KLMNIQQKL SEQID 19 5.02 KLMNIQQQL SEQID 17 4.72 NO:1 NO:16 8 ARFGEF1 R782C FVSALCMFL SEQID 19 3.09 FVSALRMFL SEQID 88 0.88 NO:2 NO:17 17 CCDC57 R353C QLCEDASTV SEQID 352 2.77 QLREDASTV SEQID 2265 1.02 NO:3 NO:18 8 CPNE3 P448L LMSIIIVGV SEQID 16 6.98 PMSIIIVGV SEQID 817 1.77 NO:4 NO:19 14 DICER1 Y153C LIMTCCVAL SEQID 46 4.99 LIMTCYVAL SEQID 43 1.88 NO:5 NO:20 16 GLYR1 P386L ALVSGNQQL SEQID 273 1.05 APVSGNQQL SEQID 25384 0.3 NO:6 NO:21 1 HSD17B7 H108Y YISKCWDYA SEQID 233 0.94 YISKCWDHA SEQID 971 0.78 NO:7 NO:22 22 IL17RA T362M FIMGISILL SEQID 4 7.46 FITGISILL SEQID 24 3.58 NO:8 NO:23 1 KIF14 G842W IQLSWVLIA SEQID 144 0.7 IQLSGVLIA SEQID 658 0.59 NO:9 NO:24 12 MED13L G2045W ILMTWNLHS SEQID 259 0.97 ILMTGNLHS SEQID 1243 0.78 NO:10 NO:25 1 OR5K2 G64E YIFLENLAL SEQID 55 1.15 YIFLGNLAL SEQID 38 1.02 NO:11 NO:26 11 OR8B3 T190I QLSCISTYV SEQID 18 6.54 QLSCTSTYV SEQID 35 5.06 NO:12 NO:27 5 SEC24A P469L FLYNLLTRV SEQID 4 19.62 FLYNPLTRV SEQID 6 13.57 NO:13 NO:28 17 TAOK1 A196V WMAPEVILV SEQID 7 4.32 WMAPEVILA SEQID 40 1.32 NO:14 NO:29 6 UTRN Q1058K QLDKCSAFV SEQID 12 6.63 QLDQCSAFV SEQID 22 7.65 NO:15 NO:30 abdominalwall breast(Feb.14,2013) (Apr.16,2013) Protein Transcrip- Protein Transcrip- AA Exome tomeVar AA Exome tomeVar CHR Gene Change VarFreq Freq FPKM CHR Gene Change VarFreq Freq FPKM 15 AKAP13 Q285K 13.97 23.49 NR 15 AKAP13 Q285K 25.13 26 NR 8 ARFGEF1 R782c 19.17 15.07 23.73 8 ARFGEF1 R782C 11.65 10.79 17.51 17 CCDC57 R353C 23.97 30.23 0.79 17 CCDC57 R353C 8 CPNE3 P448L 15.49 17.46 0.29 8 CPNE3 P448L 16.11 16.87 2.27 14 DICER1 Y153C 39.34 49.55 7.21 14 DICER1 Y153C 31.03 31.48 8.05 16 GLYR1 P386L 48.64 42.81 35.963 16 GLYR1 P386L 43.18 38.21 32.52 1 HSD17B7 H108Y 17.89 19.97 0.11 1 HSD17B7 H108Y 18.41 17.86 0.2 22 IL17RA T362M 30.97 26.83 0.22 22 IL17RA T362M 1 KIF14 G842W 20.97 22.92 3.63 1 KIF14 G842W 16.27 22.22 2.1 12 MED13L G2045W 44.44 43.58 13.64 12 MED13L G2045W 30.43 28.1 14.97 1 OR5K2 G64E 29.67 63.64 0.47 1 OR5K2 G64E 11 OR8B3 T190I 60.52 NR NR 11 048B3 T190I 20.23 NR NR 5 SEC24A P469L 37.5 42.48 1.34 5 SEC24A P469L 24.05 20.12 0.39 17 TAOK1 A196V 30.8 35.31 11.32 17 TAOK1 A196V 31.57 29 8.28 6 UTRN Q1058K 58.33 81.5 15.94 6 UTRN Q1058K 38.98 57.43 12.56

TABLE-US-00002 TABLE2 MEL21 PredictedAffinity(nM).sup.a Hugo wild-type wild- AminoAcid CHR Symbol AAS-peptide AAS-SEQID peptide WTSEQID mutated tpe Substitution(AAS) 1 AGMAT NLSGNTALL SEQID.35 DLSGNTALL SEQID.36 247 8129 D326N 8 ARFGEF1 QTIDNIVFL SEQID.37 QTIDNIVFF SEQID.38 387 10867 F1637L 9 CDKN2A KMIGNHLWV SEQID.39 EMIGNHLWV SEQID.40 14 1044 E153K 19 CYP2S1 FTMLALQDL SEQID.41 FTMLALRDL SEQID.42 287 1164 R136Q 7 FBXL13 SLWNAIDFF SEQID.43 SLWNAIDFS SEQID.44 414 348 S201F 4 FHDC1 ELQDEVYTL SEQID.45 ELQDEAYTL SEQID.46 111 518 A426V 5 GPX8 LLSIVPCTV SEQID.47 LLSIVLCTV SEQID.48 52 33 L27P 6 KDM1B IIGAGPAEL SEQID.49 IIGAGPAGL SEQID.50 469 928 G394E 13 LCP1 NLFNRYLAL SEQID.51 NLFNRYPAL SEQID.52 57 30 P375L 2 LRP1B WLTRNFYFV SEQID.53 WLTRNLYFV SEQID.54 9 7 L297F 18 NPC1 MLSSVACSL SEQID.55 VLSSVACSL SEQID.56 21 55 V664M 12 OASL ILNPADPTL SEQID.57 ILDPADPTL SEQID.58 71 40 D305N 5 PCDHB3 FLFLVLLFV SEQID.59 FLFSVLLFV SEQID.60 6 3 S704L 5 PCDHB11 MLLEISENS SEQID.61 MLLEIPENS SEQID.62 252 210 P143S X PHKA2 LLSIIFFPA SEQID.63 LLSIISFPA SEQID.64 23 25 S264F 6 PTPRK PLANSIWNV SEQID.65 PLANPIWNV SEQID.66 34 106 P137S 5 SH3RF2 HIVEISTPV SEQID.67 HMVEISTPV SEQID.68 27 6 M320I 3 TKT AMFWSVPTV SEQID.69 AMFRSVPTS SEQID.70 4 1525 R438W 1 TMEM48 CLNEYHLFL SEQID.71 CLNEYHLFF SEQID.72 23 3442 F169L 7 BRAF.sup.d V600E LymphNode(Jan.30,2011) Hugo EXOME cDNA-capture CHR Symbol Altre Refre VAF.sup.b Altre Refre VAF FPKM.sup.c 1 AGMAT 16 49 24.62 1 22 4.35 0.38 8 ARFGEF1 21 129 14.00 64 240 20.98 31.37 9 CDKN2A 13 49 20.97 162 38 81.00 0.18 19 CYP2S1 3 68 4.23 0 12 0.00 0.12 7 FBXL13 12 44 21.43 2 6 25.00 0.00 4 FHDC1 22 93 18.97 0 3 0.00 0.39 5 GPX8 7 63 10.00 20 62 24.39 15.02 6 KDM1B 15 55 21.43 23 24 48.94 7.33 13 LCP1 12 82 12.77 36 766 4.47 49.11 2 LRP1B 11 38 22.45 0 5 0.00 0.00 18 NPC1 4 24 14.29 54 36 60.00 36.55 12 OASL 3 35 7.89 0 23 0.00 1.62 5 PCDHB3 46 225 16.97 24 2 92.31 7.05 5 PCDHB11 0 40 15.69 1 7 12.50 5.25 X PHKA2 13 25 34.21 13 21 38.24 4.60 6 PTPRK 14 89 13.59 118 297 28.43 0.00 5 SH3RF2 14 61 18.67 49 207 18.99 10.19 3 TKT 10 45 18.18 124 190 39.49 0.64 1 TMEM48 7 40 14.89 292 382 43.13 0.17 7 BRAF.sup.d 10 55 15.38 Skin(May10,2012) Hugo EXOME cDNA-capture CHR Symbol Altre Refre VAF Altre Refre VAF FPKM 1 AGMAT 51 50 50.50 5 2 71.43 0.14 8 ARFGEF1 109 154 41.44 140 177 44.03 34.67 9 CDKN2A 30 17 63.83 168 26 86.60 0.05 19 CYP2S1 41 50 45.05 0 1 0.00 0.05 7 FBXL13 15 50 22.39 0 1 0.00 1.61 4 FHDC1 53 52 50.48 0 0 0.00 0.40 5 GPX8 35 27 56.45 30 12 71.43 6.92 6 KDM1B 35 51 40.70 34 28 54.84 12.67 13 LCP1 30 88 25.42 2 189 1.05 16.73 2 LRP1B 39 50 43.82 34 122 21.79 9.23 18 NPC1 0 51 0.00 0 255 0.00 0.103 12 OASL 26 19 57.78 6 16 27.27 2.96 5 PCDHB3 155 124 55.36 50 1 98.04 10.89 5 PCDHB11 17 40 29.82 4 16 20.00 5.64 X PHKA2 31 5 86.11 47 11 81.03 6.98 6 PTPRK 61 75 44.85 172 144 54.43 0.02 5 SH3RF2 43 35 55.13 101 71 58.72 6.82 3 TKT 36 25 59.02 129 122 51.19 128.54 1 TMEM48 20 24 45.45 430 263 61.52 0.24 7 BRAF.sup.d 49 48 50.52 Skin(Jun.6,2013) Hugo EXOME cDNA-capture CHR Symbol Altre Refre VAF Altre Refre VAF FPKM 1 AGMAT 42 62 40.38 1 7 12.50 0.3 8 ARFGEF1 31 103 23.13 69 195 25.84 34.23 9 CDKN2A 19 18 51.35 30 27 52.63 0.83 19 CYP2S1 31 54 36.47 0 14 0.00 0.11 7 FBXL13 6 33 15.38 0 6 0.00 0.00 4 FHDC1 33 52 38.82 3 14 17.65 7.24 5 GPX8 18 45 28.57 17 47 26.56 0.16 6 KDM1B 17 37 31.48 10 37 21.28 12.01 13 LCP1 8 75 9.64 8 284 2.73 23.56 2 LRP1B 16 49 24.62 22 47 31.88 4.57 18 NPC1 0 53 0.00 0 203 0.00 44.81 12 OASL 12 27 30.77 0 16 0.00 0.89 5 PCDHB3 59 94 38.06 39 7 84.78 5.65 5 PCDHB11 4 27 12.90 2 10 16.67 4.10 X PHKA2 11 12 45.83 41 26 61.19 7.46 6 PTPRK 26 69 27.37 58 149 38.02 0.23 5 SH3RF2 28 49 36.36 47 76 38.21 7.63 3 TKT 21 21 50.00 173 338 33.86 0.93 1 TMEM48 12 15 44.44 40 72 34.19 0.43 7 BRAFd 23 49 31.94 .sup.aPredicted affinity as determined using NetMHC3.4 algorithm. .sup.bVAF = Variant Allelic Fraction as determined from exome sequencing. BRAF VAF are reported as these were used as comparator to assess clonality of other mutations. Candidates formulated in vaccine are shown bolded. .sup.cFPKM = Fragment Per Kilobase of transcript per Million per transcriptome as determined from cDNA-capture data .sup.dBRAF VAF values are reported and were used as comparator to interpret frequencies of remaining MM-genes.

TABLE-US-00003 TABLE3 PatientMel38 PredictedAffinity(nM).sup.a Amino Hugo AAS- wild-type wild- Acid CHR Symbol peptide SEQID peptide SEQID mutated type Substitution 15 AKAP13 KLMNIQQKL SEQIDNO:1 KLMNIQQQL SEQIDNO:16 19 17 Q285K 8 ARPGEF1 FVSALCMFL SEQIDNO:2 FVSALRMFL SEQIDNO:17 19 88 R792C 17 CCDC57 QLCHDASTV SEQIDNO:3 QLRSDASTV SEQIDNO:18 352 2265 R353C 8 CPNE3 LMSIIIVGV SEQIDNO:4 PMSIIIVGV SEQIDNO:19 18 817 F448L 14 DICER1 LIMTCCVAL SEQIDNO:5 LIMTCYVAL SEQIDNO:20 45 43 Y153C 16 GLYR1 ALVSGNQQL SEQIDNO:6 APVSGNQQL SEQIDNO:21 273 25384 P386L 1 HSD17B7 YISKCWDYA SEQIDNO:7 YISKCWDHA SEQIDNO:22 233 971 N108Y 22 IL17RA FIMGISILL SEQIDNO:8 FITGISILL SEQIDNO:23 4 24 T326M 1 KIP14 IQLSWVLIA SEQIDNO:9 IQLSGVLIA SEQIDNO:24 144 658 G842W 12 MED13L ILMTWNLRS SEQIDNO:10 ILMTGNLHS SEQIDNO:25 259 1243 G2045W 3 OR5K2 YIFLENLAL SEQIDNO:11 YIFLGNLAL SEQIDNO:26 55 38 G64E 11 OR8B3 QLSCISTYV SEQIDNO:12 QLSCTSTYV SEQIDNO:27 18 35 T190I 11 PSKCDBP CLPPQTLAA SEQIDNO:73 CLSPQTLAA SEQIDNO:74 81 694 S153F 5 SEC24A FLYNLLTRV SEQIDNO:13 FLYNPLTRV SEQIDNO:28 4 6 P469L 17 TAOK1 MMAPEVILV SEQIDNO:14 MMAPEVILA SEQIDNO:29 7 40 A196V 6 UTRN QLDKCSAFV SEQIDNO:15 QLDQCSAFV SEQIDNO:30 21 22 Q1058K 2 WDR35 FLNCNSSRL SEQIDNO:75 SLNCNSSRL SEQIDNO:76 38 616 S550F 7 ZYX SLKGTSFIV SEQIDNO:77 PLEGTSFIV SEQIDNO:78 64 5774 P329S 7 BRAF.sup.d V600E Acilla(Apr.19,2012) EXOME Hugo Alt_ Ref_ cDNA-capture CHR Symbol reads reads VAF.sup.b Alt_reads Refre VAF FPKM.sup.c 15 AKAP13 20 50 28.57 4 13 23.53 54.3 8 ARPGEF1 23 81 22.12 60 161 27.15 7.1 17 CCDC57 35 41 26.78 53 351 13.12 9.5 8 CPNE3 31 127 19.62 113 536 17.41 14.3 14 DICER1 10 21 32.26 2 4 33.33 4.1 16 GLYR1 21 25 45.65 124 150 45.26 155.5 1 HSD17B7 52 183 22.13 68 228 22.97 29.7 22 IL17RA 12 28 30 4 26 13.33 1.9 1 KIP14 23 68 25.27 5 25 16.67 2.2 12 MED13L 12 8 60 71 81 46.71 8.8 3 OR5K2 57 64 47.11 3 0 100 0.1 11 OR8B3 15 0 100 13 1 92.88 0.6 11 PSKCDBP 13 0 100 24 0 100.00 0.0 5 SEC24A 22 25 46.81 50 56 46.73 2.6 17 TAOK1 23 33 41.07 23 29 44.23 3.0 6 UTRN 22 0 100 44 1 97.78 6.9 2 WDR35 34 15 69.39 90 41 58.7 15.2 7 ZYX 18 48 27.27 26 67 27.96 6.7 7 BRAF.sup.d 58 14 80 Breast(Feb.14,2013) EXOME cDNA-capture Hugo Alt_ Ref_ Alt_ Ref_ CHR Symbol reads reads VAF reads reads VAF FPKM 15 AKAP13 19 117 14.0 31 101 23.5 1.47 8 ARPGEF1 46 194 19.2 206 1161 15.1 23.73 17 CCDC57 29 92 24.0 91 210 30.2 0.79 8 CPNE3 42 229 15.5 608 2833 17.5 0.29 14 DICER1 24 37 39.3 65 56 49.6 7.21 16 GLYR1 54 57 48.7 384 513 42.8 35.63 1 HSD17B7 102 467 17.9 411 1644 20.0 0.11 22 IL17RA 35 77 31.3 33 90 26.8 0.22 1 KIP14 35 132 22.0 22 74 20.9 3.63 12 MED13L 20 25 44.4 156 202 43.6 13.64 3 OR5K2 125 227 35.5 0 20 0.0 0.00 11 OR8B3 40 21 65.8 3 0 100.0 0.35 11 PSKCDBP 21 6 77.8 161 11 93.6 0.64 5 SEC24A 33 55 37.5 127 172 42.5 1.34 17 TAOK1 37 83 30.8 185 339 35.3 11.32 6 UTRN 35 25 58.3 207 46 81.5 15.94 2 WDR35 56 50 52.8 389 247 61.8 0.04 7 ZYX 27 104 20.6 115 405 22.1 14.64 7 BRAF.sup.d 103 45 69.38 Abd.wall(Apr.16,2013) EXOME cDNA-capture Hugo Alt_ Ref_ Alt_ Ref_ CHR Symbol reads reads VAF reads reads VAF FPKM 15 AKAP13 39 116 25.16 13 37 26.00 0.14 8 ARPGEF1 29 219 11.65 56 460 10.79 17.51 17 CCDC57 32 85 27.35 45 170 20.93 2.23 8 CPNE3 38 203 16.12 342 1684 16.86 2.27 14 DICER1 18 40 31.03 17 27 31.48 8.05 16 GLYR1 38 50 43.18 214 246 38.21 32.52 1 HSD17B7 100 443 18.42 195 896 17.86 0.20 22 IL17RA 22 69 24.18 7 83 7.78 0.27 1 KIP14 28 143 16.28 6 21 22.22 2.10 12 MED13L 14 32 30.43 77 197 26.10 14.97 3 OR5K2 105 246 29.83 14 8 63.64 0.47 11 OR8B3 17 52 24.64 1 2 33.33 0.25 11 PSKCDBP 17 9 65.38 112 13 88.89 1.94 5 SEC24A 19 60 24.05 34 134 20.12 0.39 17 TAOK1 30 65 31.58 78 191 29.00 8.28 6 UTRN 23 36 38.98 58 42 57.43 12.56 2 WDR35 59 62 48.76 239 365 59.16 0.02 7 ZYX 22 81 19.47 44 477 8.43 20.16 7 BRAF.sup.d 69 56 55.20 .sup.aPredicted affinity as determined using NetMHC3.4 algorithm. .sup.bVAF = variant Allelic Fraction as determined from exome sequencing. BRAF VAF are reported as tehse were used as comparator to assess clonality of other mutations. .sup.cFPKM = Fragment Per Kilobase of transcript per Million per transcriptome as determined from cDNA-capture data. .sup.dBRAF VAF values are reported and were used as comparator to interpret frequencies of remaining MM-genes

TABLE-US-00004 TABLE4 MEL218 PredictedAffinity(nM) Amino wild- Acid Hugo AAS- type wild- Substitution CHR Symbol peptide SEQID peptide SEQID mutated type (AAS) X ABCD1 GMHLLITGL SEQIDNO:79 GMHLLITGP SEQIDNO:80 202 18419 P508L 2 ALMS1 VLAVSVLAA SEQIDNO:81 VSAVSVLAA SEQIDNO:82 170 13703 S934L 15 BTBD1 FMLLTQARI SEQIDNO:83 FMLLTQARL SEQIDNO:84 52 36 L189T 9 CDC14B IQYFRNHNV SEQIDNO:85 IQYFKNHNV SEQIDNO:86 93 93 K253R 15 DMXL2 SVMIMAFSV SEQIDNO:87 SDMIMAFSV SEQIDNO:88 19 6986 D2662V 1 EIF2B3 SISKPLLPV SEQIDNO:89 STPKPLLPV SEQIDNO:90 105 166 P24S 1 EXOC8 IILVAVPHV SEQIDNO:91 IILVAVQHV SEQIDNO:92 25 143 Q656P 22 FBXO7 LMLESGYIL SEQIDNO:93 LMLESGYIP SEQIDNO:94 10 5952 P100L 7 GET4 AVDDGKLTV SEQIDNO:95 AVDGGKLTV SEQIDNO:96 357 1067 G196D 15 HERC1 SLLLLSVSV SEQIDNO:97 SLLLLPVSV SEQIDNO:98 20 24 P1074S 6 HLA-DRB5 YMAELTVTL SEQIDNO:99 YMAKLTVTL SEQIDNO:100 4 7 K14E 8 KAT6A KLSREIKPV SEQIDNO:101 KLSREIMPV SEQIDNO:102 62 6 M1180K 4 LARP7 AVIDAYTEI SEQIDNO:103 AVINAYTEI SEQIDNO:104 213 775 N515D 7 MRPS17 VLLRALPVL SEQIDNO:105 VLLRALPVP SEQIDNO:106 24 11696 P167L 2 MRPS5 HLYASLSRA SEQIDNO:107 HPYASLSRA SEQIDNO:108 116 23536 P59L 12 OSBPL8 FCFKLSHPL SEQIDNO:109 FCFKLFHPL SEQIDNO:110 174 126 P213S 8 PABPC1 MLGEQLFPL SEQIDNO:111 MLGERLFPL SEQIDNO:112 3 3 R520Q 3 PLA1A FIWGDAPPT SEQIDNO:113 SIWGDAPPT SEQIDNO:114 41 744 S6F 17 RNASEK RLLCPPARA SEQIDNO:115 RPLCPPARA SEQIDNO:116 432 22016 P10L 20 SMOX KLANPLPYT SEQIDNO:117 KLAKPLPYT SEQIDNO:118 38 63 K499N 1 SRP9 IMAHCILDL SEQIDNO:119 IIAHCILDL SEQIDNO:120 22 250 I64M 13 TPP2 SLAETFLET SEQIDNO:121 SLAETFWET SEQIDNO:122 82 17 W1168L 1 VANGL1 FVFCALLLV SEQIDNO:123 FVFRALLLV SEQIDNO:124 6 10 R186C 16 ZFP90 FTQEKWYHV SEQIDNO:125 FTQEEWYHV SEQIDNO:126 22 20 E27K 7 BRAF.sup.d V600E LymphNode(Apr.4,2005) Hugo EXOME cDNA-capture CHR Symbol Altre Refre VAF.sup.b Alt_reads Refreads VAF FPKM.sup.c X ABCD1 23 38 37.7 156 12 92.86 10.65 2 ALMS1 5 11 31.25 20 20 50 5.74 15 BTBD1 6 17 26.09 170 358 32.14 18.84 9 CDC14B 6 67 8.11 27 136 16.56 10.73 15 DMXL2 10 46 17.86 102 704 12.64 50.71 1 EIF2B3 5 24 17.24 55 111 32.93 13.83 1 EXOC8 7 26 21.21 145 300 32.37 4.83 22 FBXO7 12 45 21.05 900 1597 36.04 87.45 7 GET4 20 27 42.55 57 51 52.78 5.2 15 HERC1 12 55 17.91 68 162 29.57 71.99 6 HLA-DRB5 81 85 48.8 573 1645 25.8 247.95 8 KAT6A 25 116 17.73 261 463 36 27.21 4 LARP7 6 36 14.29 30 50 37.5 10.15 7 MRPS17 5 71 6.58 29 75 27.88 1.48 2 MRPS5 10 58 14.49 60 125 32.43 14.55 12 OSBPL8 6 35 14.63 341 614 35.63 105.47 8 PABPC1 16 44 26.67 4073 11235 26.6 1180.59 3 PLA1A 18 79 18.56 4 10 28.57 4.07 17 RNASEK 9 58 13.43 9 18 33.33 109.67 20 SMOX 131 0 100 11 50 18.03 3.01 1 SRP9 0 58 0* 43 41 51.19 2.31 13 TPP2 10 98 9.26 98 265 26.92 25.93 1 VANGL1 22 159 12.15 289 714 28.76 26.52 16 ZFP90 11 70 13.58 22 53 29.33 4.29 7 BRAF.sup.d 13 47 21.67 .sup.aPredicted affinity as determined using NetMHC3.4 algorithm. .sup.dBRAF VAF values are reported and were used as comparator to interpret frequencies of remaining MM-genes. (*) Expression of mutated gene was validated by cDNA-capture and Sanger sequencing. Candidates formulated in vaccine are shown in bold.

TABLE-US-00005 TABLE5 AnalysisofHLA-A*02:01restrictedAAS-directedCD8+ Tcellresponses Recog- Amino Experi- nition Acid mental of Substi- Predicted Affinity Sponta- pro- Hugo tution Mutated affinity log(IC50, neous Immuno- cessed Antigenic Patient symbol (AAS) Peptide.sup.a SEQID (nM) nM).sup.b Immunity.sup.c genicity antigen Determinant.sup.d MEL21 ARFGEF1 F1637L QTIDNIVFL SEQID37 67 3.19 No No CDKN2A K153K KMIGNHLWV SEQID39 14 3.18 No Yes Yes SUBDOMINANT GPY8 L27P LLSIVPCTV SEQID47 52 3.09 No No KDM1B G394E IIGAGPAEV* SEQID166 111 3.82 No No PHKA2 S264F LLSIIFFPA SEQID63 23 3.90 No No TKT R438W AMFWSVPTV* SEQID69 4 2.35 No Yes Yes SUBDOMINANT TMEM48 P169L CLNEYHLFL SEQID71 23 3.09 Yes Yes Yes DOMINANT MKL38 AKAP13 Q285K KLMNIQQKL SEQID1 19 3.07 No Yes Yes SUBDOMINANT ARFGEF1 R782C FVSALQMFL SEQID2 19 3.18 No No HSD1787 H108Y YISKCWDYA SEQID7 233 4.28 No No OR8B3 T190I QLSCISTYV SEQID12 18 3.10 No Yes No CRYPTIC PRKCDEP S153F CLFPQTLAA SEQID73 81 3.53 No No SKC24A P469L FLYNLLTRV SEQID13 4 2.68 Yes Yes Yes DOMINANT UTRN Q1058K QLDKCSAFV SEQID15 21 3.36 No No MEL218 EXOC8 Q656P IILVAVPKV SEQID91 25 3.06 Yes Yes Yes DOMINANT LARP7 N518D AVIDAYTEI SEQID103 213 4.41 No No MRPS5 P59L HLYASLSRV* SEQID167 19 3.28 No Yes No CRYPTIC MRPS17 P167L VLLRALPVL SEQID105 24 3.05 No No PABPC1 R520Q MLGEQLFPL SEQID111 3 2.35 No Yes Yes SUBDOMINANT SMOX K499N KLANPLPYT SEQID117 134 3.73 No No SRP8 I64M IMAHCILDL SEQID119 37 4.02 No No .sup.aMutated residues are underlined and peptides that elicited immune responses are itacized (naturally-occurring) and bold (vaccine-induced). *Indicates anchor-modified peptides at P9 (Tables 2-4). .sup.bAffinity experimentally determined using fluorescence polarization-based competitive peptide-binding assay, high affinity binding peptides in this assay are log(IC50; nM) <3.7 (11). .sup.cAs determined by immune monitoring assay (FIG. 31, FIG. 30B), .sup.dAntigenic determinant classification according to Sercarz et al. Annu. Rev. Immunol. 11, 729-766 (1993).

TABLE-US-00006 TABLE6 CompositionofTMCconstructs MutAA Tumor Gene Position Nucleotidesequence* SeqIDNo. MEL21 ARFGEF1 1637 CAGCTGGAGCTGATCCAGACGATAGACAACATCGTGTTCGTGCCTGCAACTAGTAAG SEQIDNO:168 GPX8 27 AAAGTTTTCGCTGTCTTGCTCTCCATTGTGCCGTGCACAGTGACACTTTTTCTGCTT SEQIDNO:169 KDM1B 384 AACAAGAGCGTGATAATTATAGGAGCTGGCCCAGCAGAAGTGGCAGCAGCTAGACAA SEQIDNO:170 PHKA2 264 GAGATCGATGCTGGACTGCTTAGCATAATCTTTTTTCCTGCTTTTGCGGTAGAGGAT SEQIDNO:171 TKT 438 GCGCTGGAAGACCTGGCTATGTTTTGGTCAGTGCCCACAGTGACAGTCTTCTACCCTTCTGAT SEQIDNO:172 TMEM48 168 CCTGCAGCTCAGACCTGTCTCAACGAGTATCACCTGTTCCTGCTTCTCACAGGTGCC SEQIDNO:173 CDKN2A 153 GCTGAGGGACCCTCCAAAATGATAGGTAACCATCTGTGGGTATGTCGGAGTCGCCAT SEQIDNO:174 MEL38 AKAP13 285 ACTGGCCCTATTTTTAAGCTCATGAATATCCAGCAAAAGCTTATGAAAACAAATCTGAAG SEQIDNO:175 ARFGEF1 782 TTTAGCGGAAAAGATTTTGTGAGCGCACTCTGCATGTTTCTCGAGGGATTCAGGCTGCCA SEQIDNO:176 PRKCDEP 153 GTGCCCAGTCATGCGTGTCTTTTCCCCCAAACTCTGGCCGCTGAGGAGGAGGGCGAGGTG SEQIDNO:177 SKC24A 469 GATGTGCCAGAGGAGTTTCTCTATAATCTGCTTACACGCGTCTACGGAGAGGCACACCGG SEQIDNO:178 UTRN 1058 GCTGGATTGCAGCGGCAGCTGGACAAATGCAGCGCATTCGTAAATGAGATCGAAACCATA SEQIDNO:179 OR8B3 190 ATCCTGCCACTGCTGCAACTGTCTTGCATTCTACCTACGTGAATGAAGTCGTGGTGCTC SEQIDNO:180 HSD1787 108 TCCGAGATCAGACCATACATTAGCAAGTGCTGGGACTATGCC SEQIDNO:181 MEL218 MRPS17 167 ACAGTGGGGGACATTGTGCTGCTCCGAGCACTGCCCGTACTTCGAGCAAAACACGTGAAG SEQIDNO:182 MRPS5 59 GGCACACGGGACACACATCTGTATGCCAGCTTGAGCCGCGCACTCCAAACACAGTGCTGC SEQIDNO:183 SRP8 64 CAATGCTCCGGTATGATCATGGCCCACTGTATCCTCGACTTGTTGGCAGCAGCGGGCCC SEQIDNO:184 LARP7 528 CCAGAGGACGCACAGGCAGTGATCGACGCCTACACCGAGATAAACAAGAAACATTGCTGG SEQIDNO:185 EXOC8 654 GAATCCCTGGTCGAGATCATCCTGGTAGCTGTTCCACATGTCGATTACAGCCTTAGGTGT SEQIDNO:186 SMOX 499 GGTGCCGATGTCGAAAAGCTCGCCAACCCTCTCCCTTATACGGAATCAAGCAAAACCGCG SEQIDNO:187 PABPC1 538 CCACAGGAGCAAAAAATGTTGGGCGAACAATTGTTCCCGCTGATTCAGGCGATGCACCCG SEQIDNO:188 Contol MutAA Ag Gene Position Nucleotidesequence SeqIDNo. G380 GP100 N/A GTGGTGACACACACCTATCTCGAGCCGGGCCCCGTGACAGCCCAGGTAGTTCTGCAGGCC SEQIDNO:189 SVG8 KNV N/A GCTTGGGATTTCGGGAGCGTGGGTGGCGTCTTCACATCTGTTGGCAAGGCAGTGCATCAG SEQIDNO:190 *nucleotide sequences encoding 19-21-mer amino acid sequence containing missense mutation targeted by peptides included in vaccine.

TABLE-US-00007 TABLE7 ReferenceTCRCDR3libraryfromdominantTMEM4F169LexpandedCD8+ Tcells(MEI CDR3aminoacidsequence SEQID TCRBV TCRBD TCRBJ Frequency ReadCounts CASSQDLSGGVYYGYTF EQIDNO:19 TCRBV04-01 TCRBJ01-02 18.24 494191 CSTLLAGGGDEQYV EQIDNO:19 TCRBV29-01 TCRBD02 TCRBJ02-07 3.96 107162 CASSPTGLGETQYF EQIDNO:19 TCRBV10-02 TCRBD01 TCRBJ02-05 2.97 80581 CSAPPGPLAHTQYF EQIDNO:19 TCRBV20 TCRBD02 TCRBJ02-03 2.14 58087 CASSFKGTGPNQPQHF EQIDNO:19 TCRBV27-01 TCRBD01 TCRBJ01-05 0.98 26493 CASSFGGPPNTGELFF EQIDNO:19 TCRBV06 TCRBD02 TCRBJ02-02 0.88 23788 CASSIGPVNTEAFF EQIDNO:19 TCRBV19-01 TCRBD01 TCRBJ01-01 0.21 5787 CASSVAASPSGNTIYF EQIDNO:19 TCRBV09-01 TCRBJ01-03 0.19 5051 CASSPYRAGYEQYF EQIDNO:19 TCRBV03 TCRBD01 TCRBJ02-07 0.11 3056 CASSRTGITDTQYF EQIDNO:20 TCRBV03 TCRBD01 TCRBJ02-03 0.06 1619

TABLE-US-00008 TABLE8 ReferenceTCRBCDR3libraryfromsubdominantTXTR438WexpandedCD8+ Tcells(MEL21) CDR3aminoacidsequence SeqIDNo. TCRBV TCRBD TCRBJ Frequency ReadCounts CASSIASGIYEQYF SEQIDNO:201 TCRBV19-01 TCRBD02 TCRBJ02-07 4.97 112412 CASSISSSEKLFF SEQIDNO:202 TCRBV19-01 TCRBD02 TCRBJ01-04 4.79 108219 CASSLVVGLALEQYF SEQIDNO:203 TCRBV12 TCRBD02 TCRBJ02-07 2.96 66048 CASSFWGLSTEAFF SEQIDNO:204 TCRBV12 TCRBD02 TCRBJ01-01 2.75 62085 CASSSDLYEQYF SEQIDNO:205 TCRBV05-04 TCRBJ02-03 2.38 53716 CASSQEVGSGNTIYF SEQIDNO:206 TCRBV04-03 TCRBJ01-03 2.00 45241 CASSSAGGGGNTIYF SEQIDNO:207 TCRBV07-08 TCRBD01 TCRBJ01-07 1.97 44623 CASSIAGGYEQYV SEQIDNO:208 TCRBV19-01 TCRBD01 TCRBJ02-01 1.86 42081 CSVVGGLLEAFF SEQIDNO:209 TCRBV19-01 TCRBD02 TCRBJ01-01 1.84 41524 CASSSDWGLMNTEAFF SEQIDNO:210 TCRBV05-06 TCRBD01 TCRBJ01-01 1.78 40297 CASSAVDRVTSYNEQFF SEQIDNO:211 TCRBV27-01 TCRBD01 TCRBJ02-03 1.67 37852 CASSLIAGNSDTQYF SEQIDNO:212 TCRBV27-01 TCRBD02 TCRBJ02-05 1.64 37136 CASRLTAGEYQETQYF SEQIDNO:213 TCRBV12-02 TCRBD02 TCRBJ02-02 1.64 36999 CASSLWDYGYTF SEQIDNO:214 TCRBV05-06 TCRBJ01-01 1.59 35919 CASSLWGVGTEAFF SEQIDNO:215 TCRBV12 TCRBD02 TCRBJ01-06 1.54 34759 CASSYFGVNSPLHF SEQIDNO:216 TCRBV06 TCRBD02 TCRBJ01-01 1.48 33424 CATSALAGQGRDEQFF SEQIDNO:217 TCRBV24 TCRBD01 TCRBJ02-03 1.42 32032 CASSRLAGTDTQYF SEQIDNO:218 TCRBV12 TCRBD02 TCRBJ02-01 1.36 30644 CASSFPGYGLNTEAFF SEQIDNO:219 TCRBV06 TCRBD02 TCRBJ01-01 1.59 36045 CASSVLAGGLDTQYF SEQIDNO:220 TCRBV10-02 TCRBD02 TCRBJ02-03 1.15 26035 CASSYMLQTFNTEAFF SEQIDNO:221 TCRBV06 TCRBJ01-01 1.00 22716 CASSPGLLAGGSSWETQYF SEQIDNO:222 TCRBV07-02 TCRBD02 TCRBJ02-05 0.99 22276 CASTSTPGQVGQPQHF SEQIDNO:223 TCRBV27-01 TCRBD01 TCRBJ01-05 0.95 21583 CASKGLAGAYTDTQYF SEQIDNO:224 TCRBV12 TCRBD02 TCRBJ02-03 0.87 19584 CASSLGGNEQYF SEQIDNO:225 TCRBV07-08 TCRBJ02-07 0.86 19499 CASSFTAGLNTEAFF SEQIDNO:226 TCRBV12 TCRBD01 TCRBJ01-01 0.83 18659 CASSLVWGLGTEAFF SEQIDNO:227 TCRBV28-01 TCRBJ01-01 0.80 18100 CASSLGLSGESF SEQIDNO:228 TCRBV07-08 TCRBD02 unresolved 0.78 17740 CASSKLAGGLDTQYF SEQIDNO:229 TCRBV10-02 TCRBD02 TCRBJ02-03 0.78 17662 CASTHRTGLNTEAFF SEQIDNO:230 TCRBV12 TCRBD01 TCRBJ01-01 0.77 17470 CASSIGGQEETQYF SEQIDNO:231 TCRBV03 TCRBD01 TCRBJ02-05 0.76 17163 CASSLEIVGETEAFF SEQIDNO:232 TCRBV05-06 TCRBJ01-01 0.68 15460 CASSISGGYEQYV SEQIDNO:233 TCRBV19-01 TCRBD01 TCRBJ02-07 0.68 15403 CSARTLAGFTDTQYF SEQIDNO:234 TCRBV20 TCRBD02 TCRBJ02-03 0.65 14669 CASSDLLTGELFF SEQIDNO:235 TCRBV06-01 TCRBD03 TCRBJ02-02 0.58 13155 CASSSGLAGYLM SEQIDNO:236 TCRBV07-08 TCRBD02 TCRBJ02-03 0.55 12339 CASSHRTTDEETQYF SEQIDNO:237 TCRBV23-01 TCRBD01 TCRBJ02-05 0.54 12253 CASSYPGYGLNTEAFF SEQIDNO:238 TCRBV06 TCRBJ01-01 0.49 11037 CASSLDLYEQYF SEQIDNO:239 TCRBV05-04 TCRBJ02-07 0.44 9958 CASSWTGFGLNTEAFF SEQIDNO:240 TCRBV06 TCRBD01 TCRBJ01-01 0.44 9860 CASSLITGLSYEQYF SEQIDNO:241 TCRBV12 TCRBD01 TCRBJ02-07 0.42 9469 CASSTWTGMNTEAFF SEQIDNO:242 TCRBV28-01 TCRBD01 TCRBJ01-01 0.40 9127 CASSELWGAGDNEQFF SEQIDNO:243 TCRBV10-02 TCRBD02 TCRBJ02-01 0.39 8722 CASSFITTSLNVEQYF SEQIDNO:244 TCRBV28-01 TCRBD02 TCRBJ02-07 0.38 8666 CSAQQGIQPQHF SEQIDNO:245 TCRBV20 TCRBD01 TCRBJ01-05 0.38 8478 CASSLVGGLAETQYF SEQIDNO:246 TCRBV27-01 TCRBJ02-05 0.35 7817 CASSFSGGLTHEQYV SEQIDNO:247 TCRBV06 TCRBD02 TCRBJ02-07 0.35 7808 CASSLGAGEQYF SEQIDNO:248 TCRBV07-08 TCRBJ02-07 0.33 7414 CASSPIFGLTNEQYF SEQIDNO:249 TCRBV02-01 TCRBD02 TCRBJ02-07 0.31 6910 CASSYFGGEQFF SEQIDNO:250 TCRBV06 TCRBD02 TCRBJ02-01 0.30 6856 CASSQDWGLNYEQYF SEQIDNO:251 TCRBV04-01 TCRBJ02-07 0.30 6776 CASSTSGGYEQYF SEQIDNO:252 TCRBV19-01 TCRBD02 TCRBJ02-07 0.28 6396 CASSRLAGGLDTQYF SEQIDNO:253 TCRBV10-02 TCRBD02 TCRBJ02-03 0.28 6392 CASSGLITDTQYF SEQIDNO:254 TCRBV19-01 TCRBD02 TCRBJ02-03 0.26 5848 CSARELAGFQETQYF SEQIDNO:255 TCRBV20 TCRBD02 TCRBJ02-05 0.25 5732 CSPIRGIEQYV SEQIDNO:256 TCRBV20-01 TCRBD02 TCRBJ02-07 0.24 5486 CAIGPQGGFYEQYF SEQIDNO:257 TCRBV10-02 TCRBD01 TCRBJ02-07 0.24 5364 CATSSAILAGVKETQYF SEQIDNO:258 TCRBV15-01 TCRBD02 TCRBJ02-05 0.24 5313 CASSEGVGLAFEQFF SEQIDNO:259 TCRBV02-01 TCRBD02 TCRBJ02-01 0.23 5254 CAIGLAGAYEQYF SEQIDNO:260 TCRBV10-03 TCRBD02 TCRBJ02-07 0.23 5123 CASSSWTGLSLSFYGYTF SEQIDNO:261 TCRBV28-01 TCRBD01 TCRBJ01-02 0.22 5077 CASSEPGTVEAFF SEQIDNO:262 TCRBV02-01 TCRBD02 TCRBJ01-01 0.21 4771 CSVEEGIDEQYF SEQIDNO:263 TCRBV29-01 TCRBJ02-07 0.20 4627 CASSLGAGEQFF SEQIDNO:264 TCRBV07-08 TCRBD02 TCRBJ02-01 0.20 4549 CASSFQGGTGNTIYF SEQIDNO:265 TCRBV07-08 TCRBD02 TCRBJ01-03 0.20 4505 CASSLALPYEQYF SEQIDNO:266 TCRBV12 TCRBD02 TCRBJ02-07 0.18 4029 CASSPTQGLAITGELFF SEQIDNO:267 TCRBV19-01 TCRBD02 TCRBJ02-02 0.18 3969 CASSQTHPPGELFF SEQIDNO:268 TCRBV04-03 TCRBJ02-02 0.17 3928 CASSISAGYEQYV SEQIDNO:269 TCRBV19-01 TCRBD02 TCRBJ02-07 0.16 3684 CASSVDGAYNEQFF SEQIDNO:270 TCRBV09-01 TCRBD02 TCRBJ02-01 0.16 3650 CAFGVNWDLPHSGNTIYF SEQIDNO:271 TCRBV30-01 TCRBJ01-03 0.15 3435 CASSFTWGLNTEAFF SEQIDNO:272 TCRBV12 TCRBJ01-01 0.14 3276 CASSYFSYEQYF SEQIDNO:273 TCRBV06 TCRBJ02-04 0.14 3150 CASSSDRGLPSGNTIYF SEQIDNO:274 TCRBV28-01 TCRBD01 TCRBJ01-03 0.13 2973 CSAHEGLEQYF SEQIDNO:275 TCRBV20-01 TCRBJ02-07 0.13 2906 CASSASWTDYYGYTF SEQIDNO:276 TCRBV27-01 TCRBD01 TCRBJ01-02 0.13 2902 CASSTGTGSYEQYF SEQIDNO:277 TCRBV06 TCRBJ02-07 0.12 2718 CASSLWYNQPQHF SEQIDNO:278 TCRBV27-01 TCRBJ01-05 0.12 2715 CASSPLAAPGSFETQYF SEQIDNO:279 TCRBV06 TCRBD02 TCRBJ02-05 0.11 2420 CASSVDGDYNEQFF SEQIDNO:280 TCRBV09-01 TCRBD02 TCRBJ02-01 0.11 2406 CASSPTPSGLWWELFF SEQIDNO:281 TCRBV12 TCRBD02 TCRBJ02-02 0.11 2400 CASSTGTGLNTEAFF SEQIDNO:282 TCRBV02-01 TCRBD01 TCRBJ01-01 0.10 2348 CATSALPGQETTDTQYF SEQIDNO:283 TCRBV24 TCRBD01 TCRBJ02-03 0.10 2267 CASSLVGGLSNQPQHF SEQIDNO:284 TCRBV27-01 TCRBD02 TCRBJ01-05 0.10 2265

TABLE-US-00009 TABLE9 ReferenceTCRBCDR3libraryfromdominantSEC24AP469LexpandedCD8+ Tcells (MEL38) CDR3aminoacidsequence SEQIDNo. TCRBV TCRBD TCRBJ Frequency ReadCounts CASSQQAGGITYNEQFF SEQIDNO:285 TCRBV03 TCRBD01 TCRBJ02-01 13.04 142392 CASSYSTAGQPQHF SEQIDNO:286 TCRBV06-05 TCRBD01 TCRBJ01-05 6.25 68241 CASSPTGAGYEQYF SEQIDNO:287 TCRBV06-05 TCRBD01 TCRBJ02-07 3.96 43243 CASSLLSGSTEAFF SEQIDNO:288 TCRBV28-01 TCRBD02 TCRBJ01-01 3.83 41830 CASSYGTSTNEQFF SEQIDNO:289 TCRBV06-05 TCRBD02 TCRBJ02-01 3.26 35641 CASSQGDSGTDTQYF SEQIDNO:290 TCRBV03 TCRBD01 TCRBJ02-03 1.57 17192 CASSFSNQPQHF SEQIDNO:291 TCRBV28-01 TCRBJ01-05 1.57 17171 CASSGGQGTQPQHF SEQIDNO:292 TCRBV28-01 TCRBJ01-05 1.49 16310 CASSYSGAGQPQHF SEQIDNO:293 TCRBV06-05 TCRBD01 TCRBJ01-05 1.42 15495 CASSLLQGAESPLHF SEQIDNO:294 TCRBV13-01 TCRBD01 TCRBJ01-06 1.39 15226 CASSPQDRGPNYGYTF SEQIDNO:295 TCRBV28-01 TCRBD01 TCRBJ01-02 1.21 13219 CASSFDYSYEQYF SEQIDNO:296 TCRBV05-04 TCRBD02 TCRBJ02-07 0.88 9558 CAAGGVNQPQHF SEQIDNO:297 TCRBV28-01 TCRBJ01-05 0.84 9144 CASSLLAGELFF SEQIDNO:298 TCRBV06-05 TCRBD02 TCRBJ02-02 0.76 8282 CASSPSSPYEQYF SEQIDNO:299 TCRBV12 TCRBD02 TCRBJ02-07 0.72 7894 CASSEGTDTQYF SEQIDNO:300 TCRBV10-02 TCRBJ02-03 0.67 7299 CASGISNQPQHF SEQIDNO:301 TCRBV28-01 TCRBJ01-05 0.66 7225 CASSLDPPFDRQNYGYTF SEQIDNO:302 TCRBV28-01 TCRBD01 TCRBJ01-02 0.59 6456 CASSYGDMAYNEQFF SEQIDNO:303 TCRBV06-05 TCRBJ02-01 0.59 6440 CATMGTGGSLYYGYTF SEQIDNO:304 TCRBV28-01 TCRBD01 TCRBJ01-02 0.59 6433 CASSVSNQPQHF SEQIDNO:305 TCRBV28-01 TCRBJ01-05 0.58 6305 CASSFTSGGYNEQFF SEQIDNO:306 TCRBV28-01 TCRBD02 TCRBJ02-01 0.55 6055 CASSLYRANTGELFF SEQIDNO:307 TCRBV28-01 TCRBD01 TCRBJ02-02 0.53 5747 CASSLTSLTDTQYF SEQIDNO:308 TCRBV06-05 TCRBD02 TCRBJ02-03 0.51 5617 CASSKSKGSPLHF SEQIDNO:309 TCRBV21-01 TCRBJ01-06 0.42 4580 CASSLAGQGPNSPLHF SEQIDNO:310 TCRBV05-06 TCRBD01 TCRBJ01-06 0.41 4470 CASSPTGAGQPQHF SEQIDNO:311 TCRBV06-05 TCRBD01 TCRBJ01-05 0.40 4417 CASSSGTSGSDTQYF SEQIDNO:312 TCRBV28-01 TCRBD02 TCRBJ02-03 0.35 3791 CASSFSGPRSPQHF SEQIDNO:313 TCRBV12 TCRBJ01-05 0.33 3592 CASNLQGLDYEQYF SEQIDNO:314 TCRBV12 TCRBD01 TCRBJ02-07 0.32 3519 CASSLGQGNQPQHF SEQIDNO:315 TCRBV28-01 TCRBD01 TCRBJ01-05 0.32 3486 CASSFWGANEKLFF SEQIDNO:316 TCRBV28-01 TCRBD02 TCRBJ01-04 0.32 3474 CASSYSVGVNTEAFF SEQIDNO:317 TCRBV06-05 TCRBD02 TCRBJ01-01 0.31 3419 CASRYRAAPNQPQHF SEQIDNO:318 TCRBV28-01 TCRBD01 TCRBJ01-05 0.30 3235 CASSQDAGGVFGNTIYF SEQIDNO:319 TCRBV03 TCRBD02 TCRBJ01-03 0.27 2894 CASSLYSNQPQHF SEQIDNO:320 TCRBV28-01 TCRBJ01-05 0.25 2744 CATAPINSPLHF SEQIDNO:321 TCRBV28-01 TCRBD02 TCRBJ01-06 0.24 2636 CASSPPNQPQHF SEQIDNO:322 TCRBV28-01 TCRBJ01-05 0.21 2262 CASSFNNQPQHF SEQIDNO:323 TCRBV28-01 TCRBD02 TCRBJ01-05 0.21 2255 CASGVSNQPQHF SEQIDNO:324 TCRBV28-01 TCRBD01 TCRBJ01-05 0.20 2180 CASSYESNYGYTF SEQIDNO:325 TCRBV06 TCRBD02 TCRBJ01-02 0.19 2093 CASSLDVATNEKLFF SEQIDNO:326 TCRBV06-05 TCRBJ01-04 0.18 2018 CSDSSTGGAGFTF SEQIDNO:327 TCRBV29-01 TCRBD01 TCRBJ01-02 0.17 1868 CASSESGGGYRWTEAFF SEQIDNO:328 TCRBV10-01 TCRBD02 TCRBJ01-01 0.17 1839 CASSEGPSGYTF SEQIDNO:329 TCRBV09-01 TCRBJ01-02 0.17 1838 CASSPGLGEQYF SEQIDNO:330 TCRBV28-01 TCRBD02 TCRBJ02-07 0.16 1777 CASSLEGVYGYTF SEQIDNO:331 TCRBV06 TCRBJ01-02 0.16 1758 CASTIGPGITDTQYF SEQIDNO:332 TCRBV05-06 TCRBJ02-03 0.16 1715 CASSPRDRGPRSPQHF SEQIDNO:333 TCRBV28-01 TCRBD01 TCRBJ01-05 0.16 1714 CASSRTGAGEKLFF SEQIDNO:334 TCRBV06-05 TCRBD01 TCRBJ01-04 0.16 1705 CASSLGIAGPYNEQFF SEQIDNO:335 TCRBV07-06 TCRBD02 TCRBJ02-01 0.15 1634 CAGGLLNQPQHF SEQIDNO:336 TCRBV28-01 TCRBD02 TCRBJ01-05 0.14 1520 CASSLGQGAQPQHF SEQIDNO:337 TCRBV28-01 TCRBD01 TCRBJ01-05 0.14 1497 CASSPMNTEAFF SEQIDNO:338 TCRBV28-01 TCRBD02 TCRBJ01-01 0.14 1493 CASSLSSHGYTF SEQIDNO:339 TCRBV28-01 TCRBD02 TCRBJ01-02 0.13 1397 CASSFATVGEKLFF SEQIDNO:340 TCRBV06-05 TCRBD01 TCRBJ01-04 0.12 1364 CASTLYTGDNEQFF SEQIDNO:341 TCRBV06-05 TCRBD02 TCRBJ02-01 0.12 1358 CASSYSAGGYYGYTF SEQIDNO:342 TCRBV06-05 TCRBD01 TCRBJ01-02 0.12 1310 CASSYQQGSQPQHF SEQIDNO:343 TCRBV28-01 TCRBD01 TCRBJ01-05 0.11 1212 CASSPLNTEAFF SEQIDNO:344 TCRBV19-01 TCRBJ01-01 0.11 1198 CASSWSNQPQHF SEQIDNO:345 TCRBV28-01 TCRBJ01-05 0.10 1072

TABLE-US-00010 TABLE10 ReferenceTCRBCDR3libraryfromsubdominantAKAP13Q285KexpandedCD8+ Tcells (MEL38) CDR3aminoacidsequence SEQIDNo. TCRBV TCRBD TCRBJ Frequency ReadCounts CASSPVTGGDNSPLHF SEQIDNO:346 TCRBV13-01 TCRBD01 TCRBJ01-06 8.80 69934 CASSSGNYEQYF SEQIDNO:347 TCRBV13-01 TCRBJ02-07 8.52 67687 CASSLGLSGAYNEQFF SEQIDNO:348 TCRBV13-01 TCRBD01 TCRBJ02-01 7.87 62566 CAWSVASGNEQFF SEQIDNO:349 TCRBV30-01 TCRBD02 TCRBJ02-01 6.44 51166 CASSWGQGGYEQYF SEQIDNO:350 TCRBV13-01 TCRBD01 TCRBJ02-07 4.66 37068 CAWSVGVSNQPQHF SEQIDNO:351 TCRBV30-01 TCRBJ01-05 4.36 34646 CASSLGQGGELFF SEQIDNO:352 TCRBV13-01 TCRBD01 TCRBJ02-02 4.30 34205 CASSLGNYEQYF SEQIDNO:353 TCRBV13-01 TCRBD01 TCRBJ02-07 2.10 16658 CAWSAGTGGNEKLFF SEQIDNO:354 TCRBV30-01 TCRBD01 TCRBJ01-04 1.82 14434 CAWSVAGGHEQYF SEQIDNO:355 TCRBV30-01 TCRBD01 TCRBJ02-07 1.49 11869 CASSLGQGYEQYF SEQIDNO:356 TCRBV13-01 TCRBD01 TCRBJ02-07 0.98 7807 CASSFGQRETEAFF SEQIDNO:357 TCRBV05-06 TCRBJ01-01 0.86 6805 CASSQGTGVTEAFF SEQIDNO:358 TCRBV13-01 TCRBD01 TCRBJ01-01 0.85 6761 CASSFGTGYEQYF SEQIDNO:359 TCRBV06-05 TCRBD01 TCRBJ02-07 0.81 6446 CASSLNPDTQYF SEQIDNO:360 TCRBV05-06 TCRBJ02-03 0.33 2657 CAWSPGQGGTNEKLFF SEQIDNO:361 TCRBV30-01 TCRBD01 TCRBJ01-04 0.29 2319 CAWSAYGGELFF SEQIDNO:362 TCRBV30-01 TCRBD01 TCRBJ02-02 0.23 1846 CAWSVGAGVGEQYF SEQIDNO:363 TCRBV30-01 TCRBD02 TCRBJ02-07 0.20 1625 CAWSGDRPLAFF SEQIDNO:364 TCRBV30-01 TCRBJ01-01 0.18 1470

TABLE-US-00011 TABLE11 ReferenceTCRBCDR3libraryfromdomiantEXOC8Q656PandsubdominantPABPC1R520Q expandedCD8+ Tcells(MEL218) CDR3aminoacidsequence SEQIDNo. TCRBV TCRBD TCRBJ Frequency ReadCounts EXOC8Q656P CASSVGLSETTALYNEQFF SEQIDNO:365 TCRBV25 TCRBD02 TCRBJ02-01 4.85 15597 CASSLEVVQETQYF SEQIDNO:366 TCRBV11-02 TCRBJ02-05 3.64 11717 CSARDPASWGEKLFF SEQIDNO:367 TCRBV20 TCRBJ01-04 2.75 8846 CASSVAGLQGAEQYF SEQIDNO:368 TCRBV09-01 TCRBJ02-07 2.5 8039 CASSYEQGSYEQYF SEQIDNO:369 TCRBV06-05 TCRBD01 TCRBJ02-07 1.87 6014 CASSFGPLGMNWAEAFF SEQIDNO:370 TCRBV06 TCRBJ01-01 1.53 4914 CASSYLSVQETQYF SEQIDNO:371 TCRBV11-02 TCRBD02 TCRBJ02-05 0.33 1061 CASSLETGYGEQYF SEQIDNO:372 TCRBV05-05 TCRBD01 TCRBJ02-07 0.33 1062 CASSVFGLAGAEQYF SEQIDNO:373 TCRBV09-01 TCRBD02 TCRBJ02-07 0.32 1033 CASSEFGGGSPDTQYF SEQIDNO:374 TCRBV09-01 TCRBD02 TCRBJ02-03 0.21 661 CASSVYGGAEAFF SEQIDNO:375 TCRBV09-01 TCRBD02 TCRBJ01-01 0.12 370 CASSTYGLAGETQYF SEQIDNO:376 TCRBV09-01 TCRBD02 TCRBJ02-05 0.1 322 PABPC1R520Q CSVENRVIYGYTF SEQIDNO:377 TCRBV29-01 TCRBD01 TCRBJ01-02 16.65 28165 CSVEDPTFYGYTF SEQIDNO:378 TCRBV29-01 TCRBJ01-02 15.13 25599 CASSLGSSGNTIYF SEQIDNO:379 TCRBV09-01 TCRBJ01-03 9.83 16628 CSVEGQIAGKYGYTF SEQIDNO:380 TCRBV29-01 TCRBJ01-02 8.42 14240 CASSYGTSGTEQFF SEQIDNO:381 TCRBV07-06 TCRBD02 TCRBJ02-01 3.20 5412 CSVEDGAAKQIYGYTF SEQIDNO:382 TCRBV29-01 TCRBJ01-02 0.47 797 CASSVEYSNQPQHF SEQIDNO:383 TCRBV02-01 TCRBD02 TCRBJ01-05 .27 457 CSVEDRVNYGYTF SEQIDNO:384 TCRBV29-01 TCRBD01 TCRBJ01-02 0.16 275 CASSQWSSTNEKLFF SEQIDNO:385 TCRBV14-01 TCRBJ01-04 0.12 199 CARNHDRDRLYEQYF SEQIDNO:386 TCRBV02-01 TCRBD01 TCRBJ02-07 0.11 185 CASSSWGTSDEQYF SEQIDNO:387 TCRBV07-09 TCRBD02 TCRBJ02-07 0.10 172

TABLE-US-00012 TABLE12 MEL69HLAA2 Predicted Affinity(nM) Amino Acid Substi- Hugo AAS- wild-type wild- tution CHR Symbol peptide AAS-SEQID peptide WTSEQID mutated type (AAS) 2 MPV17 VLDGFIPGT SEQIDNO:127 VLDRFIPGT SEQIDNO:128 51 233 R75G 5 RUFY1 KLADYLNVL SEQIDNO:129 KLADYLKVL SEQIDNO:130 5 15 K225N 7 LANCL2 YSFLFLYRL SEQIDNO:131 YSFLSLYRL SEQIDNO:132 71 213 S370F 12 UBE3B HLGFLSPRV SEQIDNO:133 HLGSLSPRV SEQIDNO:134 60 42 S321F 16 AARS RVVFIGVPV SEQIDNO:136 RVVSAGVPV SEQIDNO:136 488 237 S698F 17 CASC3 SMSPGQPPL SEQIDNO:137 SMSPGQPPP SEQIDNO:138 17 8696 P513L X ZMYM3 VVDFTESIPV SEQIDNO:139 VVDSTESIPV SEQIDNO:140 444 360 S258F 2 GPC1 RLFGEAPREIL SEQIDNO:141 RPFGEAPREL SEQIDNO:142 83 21700 P201L 1 SRSF11 ALAALGLSGA SEQIDNO:143 ALAALGLPGA SEQIDNO:144 176 73 P137S 12 OASL TIPSEIQIFV SEQIDNO:145 TIPSEIQVFV SEQIDNO:146 274 470 V438I 19 SIPA1L3 ILGIFNEFV SEQIDNO:147 ILGISNEFV SEQIDNO:148 45 118 S893F 18 NPC1 FVGALSFSI SEQIDNO:149 FVGVLSFSI SEQIDNO:150 23 88 V845A 10 MARCH5 YYLDLANRL SEQIDNO:151 YVLDLADRL SEQIDNO:152 37 54 D90N 11 SCYL1 FLFELIPEP SEQIDNO:153 FPFELIPEP SEQIDNO:154 21 12401 P13L 5 PRRC1 QMIYSAARV SEQIDNO:155 QMIYSAARA SEQIDNO:156 79 1783 A431V 13 LMO7 SLVEEQSPA SEQIDNO:157 SPVEEQSPA SEQIDNO:158 79 21881 P583L 19 HSD11B1L MAFPEAPESV SEQIDNO:159 MASPEAPESV SEQIDNO:160 156 1145 S90F 19 PPAN SLVRDVFSSL SEQIDNO:161 SLVRDVVSSL SEQIDNO:162 106 135 V69F 7 BRAF LATEKSRWS SEQIDNO:163 LATVKSRWS SEQIDNO:164 24853 27478 V600E MEL69A.2 MEL69A.2 MEL69A.2 MEL69B.2 MEL69B.2 Hugo (Limb) (Limb) (Limb) MEL69B.2(Scalp) Scalp)RNA (Scalp) CHR Symbol ExomeVAF RNAVAF FPKM ExomeTumorVAF TumorVAF FPKM 2 MPV17 34.78 31.51 44.1711 36.59 37.87 44.5254 5 RUFY1 32.5 17.95 10.8626 23.81 42.05 12.321 7 LANCL2 16.07 31.86 15.3511 31.58 42.57 15.187 12 UBE3B 28.57 41.94 13.1866 37.68 42.11 18.9171 16 AARS 13.51 43.51 21.7187 39.02 48.85 44.5936 17 CASC3 21.05 26.79 6.77417 33.33 28.81 8.93879 X ZMYM3 35.29 51.81 9.72465 80.95 75.44 14.715 2 GPC1 28.12 30 7.40362 33.33 38.89 9.89646 1 SRSF11 11.76 26.4 63.5826 46.15 44.17 62.4002 12 OASL 16.36 14.79 10.8827 40.43 27.56 9.78642 19 SIPA1L3 8.33 29.41 1.41955 30 64.71 3.27408 18 NPC1 30.77 32 32.9957 46.67 48.27 48.3298 10 MARCH5 0 0 9.44984 30.43 37.8 11.4002 11 SCYL1 15.38 27.54 29.3756 46.15 37.41 48.8269 5 P44C1 11.11 26.14 26.921 30.56 36.17 31.9828 13 LMO7 23.68 0 12.5597 30.25 13.04 8.01764 19 HSD11B1L 18.52 0 0.551889 33.33 100 0.367626 19 PPAN 0 0 7.52204 34.29 43.53 11.0531 7 BRAF 30 67.67 13.3533 56.25 56.1 14.5002 Predicted Affinity(nM) Amino Acid Substi- Hugo AAS- AAS- wild-type wild- tution CHR Symbol peptide SEQID peptide WTSEQID mutated type (AAS) 5 ZSWIM6 LSALTRCEK SEQIDNO:388 LSALTLCEK SEQIDNO:389 295 215 L1002R 12 KIAA0528 LSACNSPSK SEQIDNO:390 LPACNSPSK SEQIDNO:391 91 14975 P256S 12 SMARCC2 KVFEHVGSR SEQIDNO:392 KVSEHVGSR SEQIDNO:393 69 390 S624F 19 PIP5K1C FISNTVFRK SEQIDNO:394 FMSNTVFRK SEQIDNO:395 21 25 M439I 20 PPP1R16B HQCCIDNFK SEQIDNO:396 HQCCIDNFE SEQIDNO:397 162 21019 E114K 22 RHBDD3 SSAAGSFGY SEQIDNO:398 SSAAGSCGY SEQIDNO:399 51 668 C119F X ERCC6L KIYRRQIFK SEQIDNO:400 KIYRRQVFK SEQIDNO:401 12 13 V476I 7 BRAF LATEKSRWS SEQIDNO:163 LATVKSRWS SEQIDNO:164 24853 27478 V600E MEL69A.2 MEL69A.2 MEL69A.2 MEL69B.2 MEL69B.2 Hugo (Limb) (Limb) (Limb) MEL69B.2(Scalp) (Scalp)RNA (Scalp) CHR Symbol ExomeVAF RNAVAF FPKM ExomeTumorVAF TumorVAF FPKM 5 ZSWIM6 25.49 43.75 9.3725 33.33 51.16 11.045 12 KIAA0528 28.57 11.96 24.255 50 25 20.069 12 SMARCC2 27.66 17.78 14.734 26.83 41.77 20.227 19 PIP5K1C 22.5 23.81 6.1374 24 38.57 11.467 20 PPP1R16B 18.92 15.79 2.8959 25.81 45.16 2.8599 22 RHBDD3 30 57.14 11.48 66.67 83.33 8.2471 X ERCC6L 55.56 69.23 2.4877 43.24 63.64 2.4041 7 BRAF 30 67.67 13.353 56.25 56.1 14.6 Predicted affinity (MT and WT score) as determined using NetMCH3.4 algorithm. VAF = Variant Allelic Fraction as determined from exome sequencing. BRAF VAF are reported as these were used as comparator to assess clonality of other mutations FPKM = Fragment Per Kilobase of transcript per Million per transcriptome as determined from cDNA-capture data. BRAF VAF values are reported and were used as comparator to interpret frequencies of remaining missense mutation encoding-genes. Candidates formulated in vaccine are shown bolded.

TABLE-US-00013 TABLE13 MEL66HLAA2 Predicted Affinity(nM) Amino Acid Substi- Hugo AAS- wild-type wild- tution CHR Symbol peptide AAS-SEQID peptide WTSEQID mutated type (AAS) 7 LMBR1 LLLLLCTSV SEQIDNO:402 LLLLLCTPV SEQIDNO:403 19 10 P210S 2 SH3BP4 RLIQGFVLL SEQIDNO:404 RLIQDFVLL SEQIDNO:405 41 51 D843G 1 ATP2B4 QLIVIFIFV SEQIDNO:406 QLIVIFILV SEQIDNO:407 34 60 L934F 2 MGAT4A ALAFITFFL SEQIDNO:408 ALAFITSFL SEQIDNO:409 7 26 S17F X PORCN LLHGFSFYL SEQIDNO:410 LLHGFSFHL SEQIDNO:411 5 11 H346Y 7 PHKG1 TLFENTPKA SEQIDNO:412 ALFENTPKA SEQIDNO:413 18 14 A401T 14 ATG2B KLNLVCCEL SEQIDNO:414 KLMPVCCEL SEQIDNO:415 95 23 P679L 12 CAMKK2 YLGMESFIV SEQIDNO:416 HLGMESFIV SEQIDNO:417 9 111 H46Y 2 ZDBF2 YILKYSVFL SEQIDNO:418 YISKYSVFL SEQIDNO:419 5 11 S2228L 11 EXT2 VLQEATICV SEQIDNO:420 VLQEATFCV SEQIDNO:421 13 7 F350I 9 ZNF658 GLYDKAICI SEQIDNO:422 GLYDKTICI SEQIDNO:423 25 13 T228A 14 PLEKHH1 YLLKIGSQV SEQIDNO:424 YLLKMGSQV SEQIDNO:425 15 18 M588I 17 GAS7 FLGEAWAQV SEQIDNO:426 SLGEAWAQV SEQIDNO:427 11 32 S270F 20 SLC2A10 FLSSMACCI SEQIDNO:428 SLSSMACCI SEQIDNO:429 27 232 S113F 3 LMLN SLVVTLWPL SEQIDNO:430 SLVVTLWLL SEQIDNO:431 12 36 L637P 2 CERS6 SMWRFTFYL SEQIDNO:432 SMWRFSFYL SEQIDNO:433 3 3 S140T 6 CUL9 CLLQLCPRL SEQIDNO:434 RLLQLCPRL SEQIDNO:435 64 25 R1335C 12 GCN1L1 SLLRSLENV SEQIDNO:436 SLLRSPENV SEQIDNO:437 21 59 P274L 20 SLC13A3 FLISILYSA SEQIDNO:438 FLISIPYSA SEQIDNO:439 3 4 P239L 8 ARHGEF10 YLLRWSVPL SEQIDNO:440 YLLKWSVPL SEQIDNO:441 3 3 K697R 22 SF3A1 MLTTAIPKV SEQIDNO:442 MPTTAIPKV SEQIDNO:443 5 12945 P6L 1 WDR63 HILEILWTL SEQIDNO:444 HILEIPWTL SEQIDNO:445 7 11 P793L 14 SLC24A4 NMFDILVGL SEQIDNO:446 NVFDILVGL SEQIDNO:447 6 53 V527M 6 PDE7B RMWDFDIFL SEQIDNO:448 GMWDFDIFL SEQIDNO:449 3 3 G113R 1 RASAL2 IMSSSLFNL SEQIDNO:450 IMSPSLFNL SEQIDNO:451 6 8 P637S 7 AKAP9 RLSDFSEQL SEQIDNO:452 RLSDLSEQL SEQIDNO:453 30 52 L974F Hugo MEL66A MEL66A MEL66A MEL66DExome MEL66DRNA MEL66D CHR Symbol ExomeVAF RNAVAF FPKM VAF VAF FPKM 7 LMBR1 66.07 95.59 133.906 31.17 64.38 32.8169 2 SH3BP4 51.72 38.56 24.5197 29.41 41.53 27.9068 1 ATP2B4 48.48 36.47 37.9108 25.81 35.89 36.7154 2 MGAT4A 48 17.12 34.4185 23.08 7.38 61.5058 X PORCN 47.37 89.68 22.2618 8.86 78.2 17.7896 7 PHKG1 47.06 52.94 1.77883 17.86 34.78 1.61569 14 ATG2B 46.15 36.41 40.641 17.14 37.26 38.7526 12 CAMKK2 43.59 47.62 15.4478 14.89 19.78 14.1399 2 ZDBF2 42.22 89.47 7.94103 14.74 53.97 11.8555 11 EXT2 42 38.9 53.8156 10 40.85 37.7597 9 ZNF658 40.37 48.09 17.1165 20.83 33.77 13.9748 14 PLEKHH1 40 50.88 14.6035 46.67 41.96 25.1339 17 GAS7 38.48 19.74 10.3323 31.82 26.24 31.4939 20 SLC2A10 36.59 46.15 1.86998 21.43 63.33 2.29521 3 LMLN 36.17 45.45 7.56894 25.93 52.17 6.56604 2 CERS6 36.11 42.02 10.198 14.81 33.53 7.74818 6 CUL9 36 38.6 7.63523 22.58 26.88 13.4072 12 GDN1L1 34.78 33.67 38.6382 19.15 28.24 45.0198 20 SLC13A3 34 59.26 4.30641 15.94 62.79 5.58358 8 ARHGEF10 33.33 43.24 13.6682 19.57 35.42 14.6281 22 SF3A1 32.56 37.95 32.8032 15.58 32.72 56.3619 1 WDR63 31.82 46.82 41.4768 11.94 36.11 3.23577 14 SLC24A4 29.82 53.04 72.1497 11.54 56.82 3.81134 6 PDE7B 26.91 32.69 6.92805 14.29 34.88 6.604 1 RASAL2 33.33 31.23 21.9958 16.07 38.83 26.2991 7 AKAP9 71.05 86.04 60.8703 26.56 26.56 26.56 Predicted Affinity(nM) Amino Acid Substi- Hugo AAS- wild-type wild- tution CHR Symbol peptide AAS-SEQID peptide WTSEQID mutated type (AAS) 14 AHNAK2 MPKFKMSSF SEQIDNO:454 MPKFKMPSF SEQIDNO:455 9 14 P3151S 4 DDX60 LPSMHRHQI SEQIDNO:456 LPSMYRHQI SEQIDNO:457 35 90 Y194H 19 TLE2 LPRAKKLIL SEQIDNO:458 LPRAKELIL SEQIDNO:459 14 40 E288K 9 DMRTA1 FSNYRRSRL SEQIDNO:460 FPNYRRSRL SEQIDNO:461 80 14 P338S 3 WDR52 QLILRTKAF SEQIDNO:462 QPILRTKAF SEQIDNO:463 38 41 P264L 7 FKBP3 YLKYHCNAS SEQIDNO:464 YLKYHYNAS SEQIDNO:465 62 32 Y449C 18 SOCS6 SLRSHHYSL SEQIDNO:466 SLRSHHYSP SEQIDNO:467 6 75 P134L 2 CHPF FFSMHFQAF SEQIDNO:468 FFPMHFQAF SEQIDNO:469 20 49 P641S 2 DUSP2 LFRYKSISV SEQIDNO:470 LFRYKSIPV SEQIDNO:471 95 120 P223S 1 LRRC42 NLRYFAKSL SEQIDNO:472 NLRYSAKSL SEQIDNO:473 26 40 S85F 7 BRAF LATEKSRWS SEQIDNO:163 LATVKSRWS SEQIDNO:164 24853 27478 V600E MEL66A MEL66A MEL66D MEL66D Hugo Exome RNA MEL66A Exome RNA MEL66D CHR Symbol VAF VAF FPKM VAF VAF FPKM 14 AHNAK2 74.74 95.54 14.8985 35.24 93.66 40.6564 4 DDX60 41.51 30.09 35.1655 28.26 24.84 72.2322 19 TLE2 42 38.6 4.18558 27.59 42.86 2.88573 9 DMRTA1 31.25 29.61 16.3335 24.19 35.14 2.76312 3 WDR52 40 48.95 28.3206 22.22 26.32 12.81 7 FKBP3 40.19 45.63 210.808 19.42 44.71 167.962 18 SOCS6 39.13 27.48 30.9938 16.67 27.97 23.4984 2 CHPF 40 47.62 32.2709 15.73 48.12 27.2727 2 DUSP2 41.98 19.78 5.98827 14.63 15.14 19.9318 1 LRRC42 32.53 39.61 27.2896 12.05 36.05 25.2227 7 BRAF 66.67 33.33