Background Chimeric antigen receptor (CAR) T cell therapies show remarkable progress in treating liquid tumors, with a complete remission rate of over 57%.¹ Translating the success of CAR T cells to solid tumors will need an understanding of the key mechanisms responsible for the cytotoxicity of CAR T cells. The primary factors contributing to tumor resistance against CAR T therapies are widely contested², therefore, we seek to explore the impact of different CAR T cell killing mechanisms of tumors.

Methods We examine CAR T cell killing of a leukemic cell line, NALM6, and an ovarian cancer cell line, SkOV3-CD19, in the presence of Granzyme B inhibitors and a Fas ligand inhibitor. We develop a fluorescent membrane reporter that translocates to the nucleus upon specific proteolytic cleaving by Granzyme A and B.

Results

• Overexpressing native Granzyme B inhibitor, Protease Inhibitor-9 (PI-9), in NALM6 and SkOV3-CD19 does not affect killing frequencies in CAR(19-41BBζ and 19-28ζ) T cell cytotoxicity assays.

• Treating 19-41BBζ with a small molecule inhibitor of Granzyme B does not impact killing frequencies in cytotoxicity assays against NALM6 and SkOV3-CD19.

• Overexpressing PI-9 in NALM6 and SkOV3-CD19 does not affect 19-41BBζ CAR T killing frequencies or killing kinetics in single cell time-lapse assays.

• Inhibition of Fas ligand on 19-41BBζ CAR T cells does not impact killing frequencies against NALM6 and SkOV3-CD19.

Conclusions Our findings suggest that suppressing Granzyme B activity with small molecules or native proteins does not impair killing frequencies of 19-41BBζ CAR T cells en masse or at the single cell level. We hypothesize that Granzyme A facilitates CAR T killing in the absence of Granzyme B, implying redundancy in granzyme expression. This study provides a comprehensive understanding of the main mechanisms associated with CAR T cell-mediated killing.

References

1. Xu X, Huang S, Xiao X, Sun Q, Liang X, Chen S, Zhao Z, Huo Z, Tu S and Li Y. Challenges and clinical strategies of CAR T-cell therapy for acute lymphoblastic leukemia: overview and developments. Front Immunol. 2021; 11:569117.

2. Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol. 2019;16(6):372–385.