B-CLL Mediated Resistance to CAR T Cell Therapy Via Insufficient Activation Is CAR-Independent

Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, a