Abstract 34: Improving CAR T cell efficacy in pancreatic cancer using an in vivo CRISPR Cas9 screen

Abstract

Abstract Introduction: Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable success in the treatment of certain hematologic malignancies. However, efficacy against solid tumors has remained limited and is associated with relatively low T cell persistence. Here, we perform an in vivo CAR T cell CRISPR screen in pancreatic adenocarcinoma using a curated guide library to identify genes that enhance CAR T cell persistence in vivo. Material and Methods: The Mario library consists of 1180 total guide RNAs targeting 135 genes that have been shown to modulate T cell functions in other models. To generate Mario-CART cells, primary human T cells were transduced with two lentiviral vectors, the first encoding an anti-mesothelin CAR and the second containing a fixed guide targeting the TCR alpha gene constant region (TRAC) as well as the Mario library. Mario-CART cells were transduced at low MOI, expanded in vitro and selected for loss of CD3 expression using magnetic selection, to enrich for cells that had a successful gene-edited event. Purified Mario-CART cells were intravenously injected into NSG mice bearing subcutaneous pancreatic adenocarcinoma (ASPC1). Tumor and spleen were harvested 2 or 4 weeks after Mario-CART injection, and engrafted T cells were isolated by CD45 positive selection. Genomic DNA was prepared from retrieved cells and illumina sequencing was used to evaluate guide abundance to identify gene knockouts that had been enriched after tumor exposure in vivo. Results: The transcription factor, GATA3, was consistently identified as the most enriched hit across two donor T cells when Mario-CART was recovered at the 2-week timepoint. When compared to control or a depleted hit, GATA3 KO CAR showed early improvements in tumor clearance in vivo. However, relapse occurred at similar timepoints across groups. At the 4-week timepoint, the most enriched guides comprised a different set, including genes involved in transforming growth factor beta and JAK-STAT signaling pathways as well as T cell trafficking. In vivo anti-tumor control data are being collected. Conclusions: Our findings highlight the dynamic nature of CART-tumor interactions. Gene knockouts that confer enhancements may change over time with varying context (such as in vitro vs in vivo conditions). It may be difficult to identify single gene knockouts that enhance CAR-T cell functions across different conditions and over time, but rather it may be necessary to exert more dynamic functional control of CAR T cells to enhance their function in different settings. Citation Format: Tamina Kienka, Felix Korell, Nelson Knudsen, Kathleen Yates, Andi Cheng, Debattama Sen, Marcela Maus, Robert Manguso. Improving CAR T cell efficacy in pancreatic cancer using an in vivo CRISPR Cas9 screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 34.