Influence of pelareorep on mutant KRAS-specific blood TIL clonal expansion.

2664 Background: Pelareorep (pela) is an intravenously delivered unmodified oncolytic reovirus that selectively infects cancer cells and is being developed as an immunotherapy for multiple cancers. We report here the analysis of tumor and blood samples from breast and pancreatic cancer patients that demonstrate a multi-step process of innate, viral, and tumor-specific immune activation culminating in the expansion of tumor-specific mutant KRAS (mKRAS) T cell clones that are associated with reductions in tumor volume. Methods: Translational samples were obtained from subjects enrolled in breast cancer (AWARE-1 ClinicalTrials.gov ID NCT04102618) and pancreatic (PDAC) cancer trials (GOBLET ClinicalTrials.gov ID NCT07280377). Analysis of tumor gene expression was performed on extracted RNA from Formalin-Fixed Paraffin-Embedded (FFPE) obtained from AWARE-1 clinical samples collected at baseline, day 3 and day 21 of therapy. Changes in tumor gene expression were determined using a customized code-set including the 50 PAM50 genes + other genes (as immune panels). Translational data from GOBLET PDAC subjects included anti-reovirus T cell responses, which were assessed by ELISPOT using whole inactivated reovirus antigen stimulation. Enumeration of the T cell fractions was performed by Adaptive Biotechnologies (Seattle, WA, USA). TCRβ CDR3 DNA was isolated from tissue and blood at baseline and from blood collected post-treatment from both studies. Antigenic specificity of selected T cell clones for mKRAS was determined by the MIRA assay (Adaptive Biotechnologies). Results: Sequential genetic analyses of breast cancer tumor biopsies pre- and post-pelareorep therapy demonstrated significant increases in anti-viral and immune gene expression consistent with the activation of toll-like receptor 3 (TLR3). Activation of TLR3 also induced the production of CXCL13, a chemokine critical for the formation of tertiary lymphoid structures (TLS). TLS in the tumor were confirmed by imaging mass cytometry of tumor biopsies following pelareorep treatment. Expansion of tumor-infiltrating lymphocytes (TILs) in both tumor and blood was also observed. Analysis of serial blood samples from a cohort of pelareorep-treated pancreatic cancer patients showed expansion of anti-viral T cells by ELISPOT. In addition, clonal expansion of tumor-specific T cells was observed after one cycle of treatment. The expansion of pre-existing TIL clones in the blood correlated with reductions in tumor volume in pancreatic cancer. Analysis of TCR sequences for antigen specificity confirmed the expansion of mKRAS clones in these samples. Conclusions: These findings suggest that pelareorep immunotherapy, through the combined activation of TLR3 and infection of the tumor, induces innate and adaptive antiviral and anti-tumor-specific immune responses capable of controlling tumor growth.