Cancer vaccines have been envisioned as a key tool for generating effective cancer therapy. Tumor neoantigens are ideal targets because of their exquisite tumor-specific expression (arising from somatic mutations of the tumor) and high level of immunogenicity (lacking of central tolerance against them). Recently, we and others have demonstrated that personalized neoantigen-targeting vaccines are safe, feasible and highly immunogenic in phase I trials of stage III/IV resected high-risk melanoma (Ott & Hu, Nature 2017; Sahin, Nature 2017). Our neoantigen vaccine (NeoVax), consisting of up to 20 long peptides and poly-ICLC, induced strong polyfunctional neoantigen-specific T-cells that recognized patient tumor in vitro. In addition, 2 patients who were vaccinated and received anti-PD1 checkpoint blockade (CPB) therapy upon relapse had durable complete responses (CRs). Thus far, these vaccine studies have been performed in the adjuvant setting, preventing direct assessment of on-target tumor killing in vivo due to the lack of evaluable tumor. On the other hand, the detection of epitope spreading (the broadening of the immune response from the initially targeted epitope to others) would indirectly suggest therapy-induced tumor lysis, whereby the release of additional tumor antigens leads to further tumor-specific T-cell activation. To explore the hypothesis that NeoVax+/- CPB generates epitope spreading, we evaluated the T-cell responses against neoantigens and tumor associated antigens (TAAs) that were not included in the original vaccine in 3 patients. We performed experiments for a patient with stage III melanoma who has remained disease-free (Pt.3) after vaccination and 2 patients with resected stage IV disease who recurred but achieved CR after CPB (Pts. 2&6). For the assessment of CD8+ T-cell responses, we designed 9-10 aa epitope length peptides (predicted by NetMHCpan and/or a mass spectrometry [MS]-based prediction algorithm (Abelin, Immunity 2017) or detected physically on the tumor’s surface class I complexes by MS) arising from 3 categories of antigens: (i) neoantigen peptides; (ii) TAA peptides based on high tumor gene expression; (iii) TAA peptides, detected on the tumor by MS (available for 2 of the 3 patients). For testing of CD4+ T-cell responses, we designed 15-16 aa peptides that spanned predicted neoepitopes from category i. Per patient, we designed peptides against up to 70 genes (~20 for each category). PBMCs from pre- , week 16 post-vaccination and post-CPB were stimulated with peptide pools (~10 peptides/pool) for 2 weeks, followed by restimulation with individual peptides in IFN-γ ELISPOT assays to deconvolute the peptides. Thus far, we have tested CD8+ T-cells against 71 neoantigens (category i) and 22 TAAs (ii) from Pts. 2 and 6, and CD4+ T-cells against 30 neoantigens from all 3 patients. We identified CD4+ T-cells specific for 3 peptides (mut-AGAP3 [Pt.2], -EYA3 and -P2RY4 [Pt.3]) in the week 16 samples that were not included in the original respective vaccines; these populations were expanded only post, but not pre-vaccination. For Pt.2, an additional CD4+ T-cell response against a different neoantigen peptide derived from mut-AGAP3 was detected only after CPB therapy. Lastly, all four lines of CD4+ T-cells reactive against these identified neoantigens were able to discriminate between the mutated and wild-type forms of the peptides, suggesting tumor specificity and lack of cross reactivity with normal tissues. Therefore, our results demonstrate that epitope spreading occurred in 2 patients after vaccination, and further spreading was detected in one of the two following CPB therapy. Ongoing studies are focused on screening additional peptides and investigating the association of epitope spreading and any residual tumor burden. The newly activated antigen-specific T-cells can target additional tumor antigens provided by epitope spreading, thus potentially enhancing therapeutic efficacy.

Citation Format: Zhuting Hu, Donna Leet, Siranush Sarkizova, Rebecca Holden, Jing Sun, Susan Klaeger, Karl R. Clauser, Sachet A. Shukla, Wandi Zhang, Steven A. Carr, Edward F. Fritsch, Bradley L. Pentelute, Nir Hacohen, Derin B. Keskin, Patrick A. Ott, Catherine J. Wu. Personalized neoantigen-targeting vaccines for high-risk melanoma generate epitope spreading [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A010.