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 (TAAs).
Here, we hypothesize that targeting three distinct leukemia antigens 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) fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta chain (2nd generation). 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). 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). 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 ratios (E:T) in a 51Cr release cytotoxicity assay. TriCAR and SideCAR T cells secreted similar levels of IFN-gamma; when compared to CD19 CAR T cells demonstrating a safety profile very 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-gamma; and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells of varying antigen profiles and mitigating CD19 negative relapse. This strategy has potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse.
Citation Format: Kristen Fousek, Junji Watanabe, Xingyue An, Ann George, Heba Samaha, Shoba Navai, Tiara T. Byrd, Jonathan Kirzner, Hye Na Kim, Albert Jang, Sujith Joseph, Matthew Baker, Meenakshi Hegde, Navin Varadarajan, Nora Heisterkamp, Hisham Abdel-Azim, Nabil Ahmed. Trivalent CAR T cells mitigate CD19-negative relapse and improve tumor control in primary pre-B cell acute lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A50.