Targeting Primary Pre-B Cell Acute Lymphoblastic Leukemia and CD19-Negative Relapses Using Trivalent CAR T Cells

Abstract B-Acute Lymphoblastic Leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19, have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens. Here, we hypothesize that targeting three distinct leukemia antigens including CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse. We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR), and the second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). All CARs are fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta (ζ) chain (2nd generation). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target (E:T) ratios. TriCAR and SideCAR T cells secreted similar levels of IFN-γ/TNF-α when compared to CD19 CAR T cells suggesting a safety profile similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-γ and TNF-α and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. Finally, we interrogated immune synapse (IS) microcluster formation and actin dynamics during interactions between CAR T cells and primary B-ALL cells by quantitative imaging flow cytometry. All CAR T cells exhibited higher actin polymerization compared to non-transduced T cell controls. TriCAR T cells formed significantly higher number of IS microclusters with different morphologies of actin polymerization compared to CD19 CAR T cells, suggesting distinct remodeling and dynamics of T cell polarization and immunoactivity, when interacting with TXL-2 primary B-ALL cells. TriCAR T cells formed IS microclusters, while CD19 CAR T cells failed to form IS with CD19 knockout TXL-2 cells. In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells with varying antigen profiles and at mitigating CD19-negative relapse. This strategy has the potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. Disclosures No relevant conflicts of interest to declare.