Targeting B- and T-cell lymphomas with anti-CXCR5 CAR T cell therapy

Abstract Chimeric antigen receptor (CAR) T cell therapy has shown unprecedented efficacy in B-cell leukemias and lymphomas. However, >50% of these patients treated with anti-CD19 CAR T cell therapy relapse with CD19 loss or downregulation as a frequent mechanism of escape. These results indicate that novel CAR T cell targets are needed to further improve outcomes in these patients. Development of CAR T cell therapy approaches is also needed for patients with T-cell lymphomas, most of whom have poor long-term survival. However, this has been challenging due to difficulty in identifying appropriate target antigens for CAR T cell therapy. Targeting pan-T-cell antigens such as CD5 and CD7 may lead to compromised T cell immunity and increase the risk of life threatening infections. Here, we describe the development of a novel anti-CXCR5 CAR T cell product for targeting B- and T-cell lymphomas as a potential strategy to address some of the current challenges. CXCR5 is a G protein-coupled chemokine receptor whose expression is restricted to mature B cells and follicular helper T cells (TFH) in normal human tissues. Among lymphoid malignancies, it has been reported to be highly expressed in multiple B-cell neoplasms including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and chronic lymphocytic leukemia (CLL). Consistent with their cell-of-origin, CXCR5 is also reported to be highly expressed in TFH-derived T-cell lymphomas including angioimmunoblastic T-cell lymphoma, TFH lymphoma follicular type, and TFH lymphoma not otherwise specified. Together, the TFH-derived T-cell lymphomas are the largest subgroup of non-cutaneous T-cell lymphomas accounting for ~35% of these malignancies. The restricted expression in normal tissues and high expression in certain subtypes of B- and T-cell malignancies suggest that CXCR5 could be a safe and effective target for CAR T cell therapy in these lymphoproliferative disorders. A prior study demonstrated efficacy of a CXCR5-targeting CAR-T in preclinical models of B-cell lymphoma but its efficacy in T-cell lymphomas is unknown. We generated a novel anti-CXCR5 monoclonal antibody and demonstrated by flow cytometry that it specifically binds to CXCR5-expressing B- and T-cell lymphoma cell lines but not their isogenic counterparts that lack CXCR5 expression. Using the variable heavy (VH) and light (VL) chains of this antibody, we developed a single-chain variable fragment (scFv)-based CAR construct with CD28 and CD3z signaling domains. Next, we generated anti-CXCR5 CAR T cells by lentiviral transduction of the CAR molecule into primary human T cells and evaluated their function against CXCR5-expressing B- and T-cell lymphoma cell lines/clones with varying antigen density. As compared to untransduced T cells, anti-CXCR5 CAR T cells were specifically cytotoxic to CXCR5+ B- and T-cell lymphoma tumor cells at high efficiency but not their CXCR5-negative isogenic counterparts (P<0.001). At a low effector to target ratio of 1:2, the anti-CXCR5 CAR T cells eliminated over 95% of CXCR5+ B- and T-cell lymphoma tumor cells, including tumor cells with very low antigen density (2000-3000 CXCR5 molecules per cell). In a B-cell lymphoma xenograft mouse model, adoptive transfer of anti-CXCR5 CAR T cells but not untransduced T cells rapidly and significantly induced regression of tumors (total flux P<0.001) and prolonged survival of mice (P<0.01). Evaluation of the anti-CXCR5 CAR T cells in T-cell lymphoma xenograft models is ongoing. In conclusion, our novel anti-CXCR5 CAR T cell therapy product is specific and highly effective against preclinical models of both B- and T-cell lymphomas. Successful development of such a CAR-T product may be applicable for the treatment of various B-cell malignancies and TFH-derived T-cell lymphomas, the largest subgroup of systemic T-cell lymphomas with a significant unmet clinical need. CXCR5 CAR-T can also simultaneously target the tumor-promoting normal TFH cells in B-cell lymphomas and the tumor-promoting normal B cells in TFH-derived T-cell lymphomas. This dual targeting of the tumor and the supporting CXCR5+ normal cells in the tumor microenvironment can make the CXCR5 CAR-T particularly potent.