Abstract 5370: CARG-2020 a novel immunemodulatory approach to prevent recurrent ovarian cancer
Authors: Alvero Ayesha B. , Fox Alexandra , Madina Bhaskara , Krady Marie , Yarovinski Timur O. , Nakaar Valerian , Almassian Bijan , Mor Gil 
Published: June 2022

Abstract
Background: Immune therapy has been proven successful in multiple types of cancer, but not ovarian. Indeed, ovarian cancer is classified as a “cold tumor” with most tumors exhibiting low levels of T cell infiltrates and poor responses to immune checkpoint inhibitors. It is therefore important to develop better immune modulatory modalities that can improve clinical responses. In this study we evaluated the efficacy of CARG-2020, a virus-like vesicle (VLV) delivering three immune modulators (single chain IL-12, IL-17 antagonist and shRNA for PD-L1) in eliciting an effective and sustained anti-tumoral response to prevent recurrent disease in a syngeneic mouse model of ovarian cancer.
Materials and Methods: Triple knockout (TKO; p53LSL-R172H/Dicerflox/flox/Ptenflox/flox) mouse ovarian cancer cells stably expressing the mCherry fluorescent protein were injected i.p. in C57BL/6 mice. Tumor growth was monitored by live imaging using mCherry fluorescence as surrogate for i.p. tumor burden. Treatment commenced when mCherry region of interest (ROI) area reached 20,000 photons/sec. Treatment groups were as follows (n=6/group): 1) PBS Control; 2) 1x10^6 PFU CARG-2020; 3) 1x10^6 PFU VLV-GFP (vector only control). All treatments were given i.p. for a total of 3 doses given 72h apart. Percentage of effector memory, central memory, and naïve CD8 T cells were measured by flow cytometry by staining for CD8, CD44, and CD62L.
Results: Treatment with CARG-2020 induced a significant decrease in i.p. tumor burden compared to PBS Control (p=0.0044) and VLV-GFP (p=0.0011). Complete disease regression was observed in all CARG-2020-treated mice with a significant impact on overall survival conferring 100% protection (p=0.0008). Rechallenge of these animals with the same cancer cells failed to form tumors and analysis of splenocytes showed high levels of CD8+/CD44+/CD62- cytolytic effector memory T cells compared to tumor-bearing control and tumor-bearing VLV-GFP-treated mice (p=0.0011). Transfer of splenocytes from long term survivor mice into a new set of tumor-bearing, treatment-naïve mice, induced complete disease regression (p&lt;0.0001, compared to PBS Control) and improved overall survival (p=0.0019, compared to PBS Control).
Conclusion: We report the successful anti-tumoral effect of CARG-2020 by modulating the immune response in a syngeneic mouse model of recurrent ovarian cancer. CARG-2020, by enhancing the expression of IL-12 and blocking IL-17 and PD-L1, provided a long-term protection associated with the expansion of cytolytic effector memory CD8+T cells, which in addition to its antitumoral effect, provides protection against recurrent disease. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients.
Citation Format: Ayesha B. Alvero, Alexandra Fox, Bhaskara Madina, Marie Krady, Timur O. Yarovinski, Valerian Nakaar, Bijan Almassian, Gil Mor. CARG-2020 a novel immunemodulatory approach to prevent recurrent ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5370.