Breast Cancer Neoantigens Can Induce CD8+ T-Cell Responses and Antitumor Immunity

Abstract

Next-generation sequencing technologies have provided insights into the biology and mutational landscape of cancer. Here, we evaluate the relevance of cancer neoantigens in human breast cancers. Using patient-derived xenografts from three patients with advanced breast cancer (xenografts were designated as WHIM30, WHIM35, and WHIM37), we sequenced exomes of tumor and patient-matched normal cells. We identified 2,091 (WHIM30), 354 (WHIM35), and 235 (WHIM37) nonsynonymous somatic mutations. A computational analysis identified and prioritized HLA class I-restricted candidate neoantigens expressed in the dominant tumor clone. Each candidate neoantigen was evaluated using peptide-binding assays, T-cell cultures that measure the ability of CD8⁺ T cells to recognize candidate neoantigens, and preclinical models in which we measured antitumor immunity. Our results demonstrate that breast cancer neoantigens can be recognized by the immune system, and that human CD8⁺ T cells enriched for prioritized breast cancer neoantigens were able to protect mice from tumor challenge with autologous patient-derived xenografts. We conclude that next-generation sequencing and epitope-prediction strategies can identify and prioritize candidate neoantigens for immune targeting in breast cancer. Cancer Immunol Res; 5(7); 516-23. ©2017 AACR.

Fig. 1. Identification and validation of candidate neoantigens

Autologous PBMCs were stimulated with candidate breast cancer neoantigens for 12 days. CD8⁺ T-cell IFNγ ELISPOT assays were performed on day 12 by co-culturing stimulated PBMC overnight with autologous, irradiated PBMCs pulsed with the candidate neoantigens (black) or irradiated PDX tumor cells (white). The immune response induced by candidate neoantigens and PDX tumor cells is shown in (A) WHIM30, (B) WHIM35 and (C) WHIM37. Negative controls in the ELISPOT assays included responder T cells cultured with no peptide (number of spot forming cells per 10⁶ cells was 50–120) or irrelevant peptide (number of spot forming cells per 10⁶ cells was 250–400). The background with irrelevant peptide was subtracted from the experimental condition in each case. To confirm the specificity of the immune response induced by candidate neoantigens, CD8⁺ T-cell IFNγ ELISPOT assays were performed against mutant (black) and wildtype peptides (white) after 12 day stimulation with mutant peptides. The results are shown in (D) WHIM30, (E) WHIM35 and (F) WHIM37. Data are presented as means ± s.e.m (n = 3 wells per peptide in ELISpot assay) and are representative of three independent experiments. Samples were compared using unpaired, two tailed Student’s test (^*P < 0.05, ^** P < 0.01), SFC is spot forming cells.

Fig. 2. Adoptive transfer of neoantigen-stimulated T cells inhibits growth of PDXs in vivo

PDXs were established by injection of 1 × 10⁶ tumor cells subcutaneously into NSG mice. 5–10 × 10⁶ neoantigen-stimulated T cells and control viral-antigen stimulated T cells were adoptively transferred into tumor-bearing mice after the tumor became palpable. T cells were transferred at days 0, 7, and 14. Tumor size was measured every two days. (A) WHIM30 tumor growth following treatment with PBMC stimulated with mutant PALB2/ROBO3 (black solid circle) or CMV peptides (black solid square). (B) WHIM35 tumor growth following treatment with PBMC stimulated with mutant PTPRS (black solid circle) or FluM1 peptides (black solid square). (A) and (B) Data are presented as tumor size (mm²) ± s.e.m. of 5 mice per group (^*P < 0.05). (C) WHIM30 tumor size (52 days after tumor challenge) following treatment with PBMC stimulated with mutant PALB2/ROBO3 or CMV peptides. (D) WHIM35 tumor size (62 days after tumor challenge) following treatment with PBMC stimulated with mutant PTPRS or CMV peptides. (E, G) TILs were isolated from WHIM30 tumors following treatment with PBMC stimulated with mutant PALB2/ROBO3 (black) or negative control mutant E1F5B and CMV (white) peptides. CD8⁺ T-cell IFNγ ELISPOT assay and flow cytometry were performed with experimental and control peptides as indicated. (F, H) TILs were isolated from WHIM30 tumors following treatment with PBMC stimulated with mutant PTPRS (black) or negative control mutant EML2 and FluM1 peptides (white). CD8⁺ T-cell mIFNγ ELISPOT assay and flow cytometry were performed with experimental and control peptides as indicated. (E) and (F) Data shown are mean ± s.e.m (n = 3 wells per peptide in ELISpot assay). Data shown are representative of three independent experiments. Samples were compared using an unpaired, two-tailed Student t test (P < 0.05).