Abstract 5575: Pre-clinical validation of a LGR5-targeting CAR-T against colorectal cancer

Abstract Chimeric antigen receptor T cells (CAR-T) have demonstrated robust clinical activity against B-cell malignancies, and rapid translation to approval for use in man has inspired the search for CAR-T cells against solid tumors. Cancer stem cells are a small population of cells within a tumor that have the capacity to self-renew, differentiate, and initiate new tumors. Cancer stem cells are known to be resistant to chemotherapy and radiotherapy and are enriched in residual disease which allows relapse. Targeting cancer stem cells will reduce the tumors’ ability to generate new cells resulting in tumor remission. We have tested a novel CAR-T cell that has the potential to target cancer stem cells. Leucine-rich G protein-coupled receptor 5 (LGR5) expression is restricted to stem cell populations in multiple tissues in adults. In addition, LGR5 is a marker of cancer stem cells and LGR5+ cells are implicated in tumor progression and metastasis. We designed six lentiviral CAR-T constructs targeting the extra cellar domain of LGR5 with differing linker lengths and binding domain orientation, and tested gene delivery to purified human CD3 T cells and in vitro cytotoxicity. All constructs were expressed robustly (>90%), and four of six LGR5-targeting CAR-T cells showed significant antigen-specific cytotoxicity against colorectal cancer cell lines in vitro. We confirmed the on-target specificity of the LGR5 targeting CAR-T using a cell line over-expressing the LGR5 protein and antibody competition assays, which blocked cytotoxicity. Using optimized gene delivery and CAR-T manufacturing protocols, we generated pre-clinical scale batches of the four lead constructs to evaluate their potency in a pre-clinical colorectal cancer xenograft mouse model. Post expansion our cell products expressed markers of self-renewing naïve-like and central memory phenotype and three out of four LGR5-targeting CAR-T cells were able to significantly inhibit the growth of human colorectal tumors. These results suggest a CAR-T targeting LGR5 may provide long-term protection against tumor growth. Our data positions LGR5-targeting CAR-T therapy as a viable therapeutic for human metastatic colorectal cancer types with minimal off-target effects, which we now aim to test in human clinical trials. Citation Format: Veronika Bandara, Stuart Mills, Emma Thompson, Lih Tan, Batjargal Gundsambuu, Silvana Napoli, Jade Foeng, Dylan McPeake, Justin Coombs, Allison Cowin, Claudine Bonder, Timothy Sadlon, Shaun McColl, Simon Barry. Pre-clinical validation of a LGR5-targeting CAR-T against colorectal 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 5575.