CLINICAL CANCER RESEARCH | CLINICAL TRIALS: IMMUNOTHERAPY Phase I Trial of Viral Vector-Based Personalized Vaccination Elicits Robust Neoantigen-Speci ﬁc Antitumor T-Cell Responses Anna Morena D ’Alise1, Guido Leoni1, Gabriella Cotugno1, Loredana Siani1, Rosa Vitale1, Valentino Ruzza1, Irene Garzia1, Laura Antonucci1, Elisa Micarelli1, Veronica Venafra2, Sven Gogov3, Alessia Capone1, Sarah Runswick3, Juan Martin-Liberal4, Emiliano Calvo5, Victor Moreno6, Stefan N. Symeonides7, Elisa Scarselli1, and Oliver Bechter8 ABSTRACT ◥ Purpose: Personalized vaccines targeting multiple neoantigens (nAgs) are a promising strategy for eliciting a diversi ﬁed antitumor T-cell response to overcome tumor heterogeneity. NOUS-PEV is a vector-based personalized vaccine, expressing 60 nAgs and consists of priming with a nonhuman Great Ape Adenoviral vector (GAd20) followed by boosts with Modi ﬁed Vaccinia Ankara. Here, we report data of a phase Ib trial of NOUS-PEV in combination with pembrolizumab in treatment-na €/C16ve patients with metastatic mela- noma (NCT04990479). Patients and Methods: The feasibility of this approach was demonstrated by producing, releasing, and administering to 6 patients 11 of 12 vaccines within 8 weeks from biopsy collection to GAd20 administration. Results: The regimen was safe, with no treatment-related serious adverse events observed and mild vaccine-related reactions.Vaccine immunogenicity was demonstrated in all evaluable patients receiving the prime/boost regimen, with detection of robust neoan- tigen-speci ﬁc immune responses to multiple neoantigens compris- ing both CD4 and CD8 T cells. Expansion and diversi ﬁcation of vaccine-induced T-cell receptor (TCR) clonotypes was observed in the posttreatment biopsies of patients with clinical response, pro- viding evidence of tumor in ﬁltration by vaccine-induced neoanti- gen-speci ﬁc T cells. Conclusions: These ﬁndings indicate the ability of NOUS-PEV to amplify and broaden the repertoire of tumor-reactive T cells to empower a diverse, potent, and durable antitumor immune response. Finally, a gene signature indicative of the reduced presence of activated T cells together with very poor expression of the antigen- processing machinery genes has been identi ﬁed in pretreatment biopsies as a potential biomarker of resistance to the treatment. Introduction Theﬁeld of immunotherapy has revolutionized the treatment of many tumors with the development and approval of immune check- point inhibitors (CPI). The activity of CPI relies mostly on the reinvigoration of T cells against tumor antigens, which are sponta- neously induced by the tumor. Tumor neoantigens (nAgs) are con- sidered a central target of such spontaneous antitumor T-cell responses and several studies have shown that cytotoxic CD8 T cellstargeting tumor neoantigens play a critical role in mediating the response to checkpoint blockade (1, 2). Neoantigens are mutated self-antigens arising from somatic mutations unique to the tumor cells and not expressed on normal cells. As such, they are recognized by the immune system as nonself and capable of triggering a strong and tumor-speci ﬁc immune response (3). These features make them an attractive target for cancer vaccines. The advance of next-generation sequencing (NGS) techniques has provided opportunities to rapidly identify these tumor-speci ﬁc mutations in each individual patient, making the approach of personalized nAgs-based vaccines feasible. Personalized neoantigen vaccines aim to expand and broaden the repertoire of tumor antigen-speci ﬁc T-cell responses by boosting tumor-induced T cells and by priming de novo T-cell responses against additional cancer nAgs, thus increasing the potency and breadth of antitumor immunity. In this scenario, the combination of neoantigen cancer vaccines with CPI may improve antitumor ef ﬁcacy through complementary mechanisms of action (4). The choice of the vaccine platform, and associated number of nAgs that can be vaccinated, is key to elicit a proper magnitude, quality, and breadth of T-cell responses to achieve effective and durable antitumor responses. Indeed, various vaccine platform approaches are being pursued in the ﬁeld of per- sonalized vaccines (PEV; ref. 5). Viral vector-based vaccines represent an attractive choice as antigen delivery system. In particular, heter- ologous prime/boost with adenoviruses derived from nonhuman Great Apes (GAd), followed by Modi ﬁed Vaccinia Ankara (MVA), is a powerful vaccine platform that has demonstrated robust and durable T-cell response in humans, both in the context of the infec- tious disease ﬁeld and more recently also in cancer (6, 7). Moreover, these viral vectors have the capability to encode large gene inserts and, therefore, in the context of neoantigen vaccines, to target many1Nouscom Srl, Rome, Italy.2Department of Biology, University of Rome “Tor Vergata, ”Rome, Italy.3Nouscom AG, Basel, Switzerland.4Catalan Institute of Oncology (ICO), Barcelona, Spain.5START Madrid-CIOCC, Centro Integral Oncol /C19ogico Clara Campal, Madrid, Spain.6START Madrid-FJD, Hospital Funda- cion Jimenez Díaz, Madrid, Spain.7Edinburgh Experimental Cancer Medicine Centre, University of Edinburgh, Edinburgh, United Kingdom.8Leuven Cancer Institute, Department of General Medical Oncology, University Hospitals Leuven, Leuven, Belgium. A.M.