Serpin B9 expression in malignant cells confers resistance to Granzyme B-mediated killing by engineered T or NK cells.
Chimeric antigen receptor (CAR) T cell therapies, are an effective form of immune therapy that found its way to the clinic. These gene modified T cells use granzyme B to kill tumor cells. Importantly, it was recently reported that expression of granzyme B-inhibitor serpin B9 promotes T cell dysfunction in case of cancer. The goal of our study is to test the effect of serpin B9 expression on the killing capacity of cytotoxic cells, including CAR T cells and NK cells.
Therefore, Serpin B9 of tumor cells was manipulated either by lentiviral-transduction to overexpress Serpin B9 or by siRNA transfection to transient knockdown serpin B9. We generated CD20 CAR T cells by retroviral transduction of primary T cells. We analyzed serpin B9 expression by Western blot or intracellular flow cytometry. And finally assessed cell death of tumor cells by flow cytometry after a co-culture killing assay with CD20 CAR T cells or NK cells.
First, we confirmed that serpin B9-overexpressing melanoma cells are less sensitive to recombinant granzyme B or to killing by NK cells. Next, we reveal that serpin B9 is expressed abundantly in different B cell malignancies and especially in chronic lymphocytic leukemia (CLL). Knock-down of serpin B9 in diffuse large B cell lymphoma (DLBCL) cells rendered them more sensitive towards killing by CD20 CAR T cells or NK cells. Finally, CD20 CAR T cells select for survival of high serpin B9-expressing primary CLL cells.
Combined, our data exposes serpin B9 as a potential mediator underlying resistance towards engineered T or NK cells and provides a rationale to improve their killing capacity by circumventing serpin B9-mediated inhibition of granzyme B.