LAUSR

Chimeric antigen receptor (CAR) T cells have demonstrated robust clinical efficacy against B-cell malignancies, and rapid translation to approval for use in man has inspired the search for CAR-T cells against solid tumours. Cancer stem cells are a small population of cells within a tumour that have the capacity to self-renew, differentiate, and initiate new tumours. 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 is desirable as it will reduce a tumours’ ability to generate new cells, resulting in tumour remission. Leucine-rich G protein-coupled receptor 5 (LGR5) expression is restricted to stem cell populations in several tissues in adults. In addition, LGR5 is a marker of cancer stem cells and LGR5+ cancer cells are implicated in tumour progression and metastasis. We have designed and tested a CAR targeting the extracellular domain of LGR5, and optimised gene delivery to purified human CD3+ T cells showed robust CAR expression. Having confirmed on target specificity and cytotoxicity we show antigen-specific IFNγ release against colorectal cancer (CRC) and neuroblastoma cell lines in vitro. We confirmed the specificity and safety of the LGR5 targeting CAR-T cells by testing against normal human tissues from colon, liver, kidney, lung and heart. Using optimised gene delivery and CAR-T manufacturing protocols, we generated clinical scale batches to evaluate their potency in a preclinical CRC xenograft mouse model. A protocol for clinical scale manufacture of LGR5-targeting CAR-T cells has been developed using CD3+ T cells from healthy donors and now we have successfully tested this protocol using CRC patient-derived CD3+ T cells. This protocol is GMP ready and has been tested in a GMP manufacturing facility. Using T cells from healthy donors and CRC patients we were able to generate optimal cell numbers (80-fold expansion) with high CAR expression. These CAR-T cells showed high in vitro cytotoxicity and IFNγ release when co-cultured with LGR5 expressing CRC cell line. Post expansion, CAR-T cell products expressed markers of self-renewing naïve-like and central memory phenotype and showed very low percentage of cells that were triple-positive for PD1, TIM3 and LAG3. These results suggest a CAR-T targeting LGR5 may provide long-term protection against tumour 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, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Emma Thompson, Lih Tan, Jade Foeng, Dylan McPeake, Timona Tylis, Caitlin Abbott, Allison Cowin, Claudine Bonder, Timothy Sadlon, Shaun McColl, Simon C. Barry. Development and in vitro validation of an LGR-5 targeting CAR-T against colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4085.