Targeting frameshift mutations with a Listeria monocytogenesimmunotherapy drives neoantigen-specific antitumor immunity in the MC38 and CT26 mouse tumor models

Introduction: Neoantigens derived from tumor-specific mutations have been shown to drive tumor specific CD8+ T cell responses leading to tumor regression and extending overall survival. Frameshift mutations are estimated to generate up to nine times more neoantigens per mutation compared to in-frame mutations. However, it is not clear if vaccination against frameshift mutations induces neoantigen-specific CD8+ T cell responses that result in control of tumor growth. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to target mutation-derived tumor-specific neoantigens. Advaxis9 Lm-based immunotherapies consist of live attenuated bacterial vectors that are bioengineered to secrete an antigen-adjuvant fusion protein consisting of a truncated non-hemolytic fragment of listeriolysin O, which has adjuvant properties, and tumor-specific antigens. Here, we demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific frameshift mutations in order to generate neoantigen-specific T cells that control tumor growth. Results: Whole-exome sequencing of the CT26 and MC38 mouse tumor cell lines identified 30 and 31 unique frameshift mutations respectively. Individual frameshift mutations ranged in size from 12 to as many as 150 amino acids (aa). Lm vectors targeting the two longest frameshift mutations were constructed for each tumor model. The therapeutic efficacy of Lm vectors expressing either a single 57 aa (Lm-57) or a single 150 aa (Lm-150) MC38 frameshift mutation were evaluated in C57BL/6J mice. Both Lm vectors generated multiple unique frameshift-specific TILs and slowed tumor growth. Furthermore, we evaluated the tumor microenvironment following Lm-57 or Lm-150 treatment and observed a decrease in the frequency and absolute number of Tregs, TAMs, and MDSCs and an increase in the frequency and absolute number of total cytotoxic granzyme A+ effector CD8+ T cells. Similarly, Lm vectors expressing either a 64 aa (Lm-64) or a 93 aa (Lm-93) CT26 frameshift mutation were evaluated in the CT26 tumor model. Both Lm-64 and Lm-93 significantly controlled tumor growth. Additionally, an influx of neoantigen-specific TILs and a significant decrease in the frequency of intratumoral Tregs was observed. Conclusion: ADXS-NEO induced potent immune responses against tumor-specific frameshift mutations and controlled tumor growth. Advaxis9 Lm platform is able to target frameshift mutations ≥150 aa and generate multiple neoantigen-specific T cells per frameshift. ADXS-NEO controls tumor growth via multiple mechanisms, including the generation of tumor-specific cytotoxic TILs, by secreting tumor-derived neoantigens directly into dendritic cells and by attenuating the suppressive tumor microenvironment. Citation Format: Brandon Coder, Daniel O. Villarreal, Susan Armington, Elena Filippova, Andrew L9Huillier, Dipti Kelkar, Xiaoming Ju, Cristina Mottershead, David Balli, Kim Ramos, Hyewon Phee, Jim Johnston, Robert Petit, Michael Princiotta. Targeting frameshift mutations with a Listeria monocytogenes immunotherapy drives neoantigen-specific antitumor immunity in the MC38 and CT26 mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-148.