Abstract 2797: Construction and validation of an activating and inhibitory chimeric antigen receptor (CAR) system
Authors: Chicaybam Leonardo , Bonamino Martin H. 
Published: May 2015

Abstract
Introduction: The use of adoptive transfer of T cells for the treatment of cancer has been hampered by the low persistence of the infused cells and the difficulty to isolate and expand tumor-specific lymphocytes. The use of chimeric antigen receptors (CARs) allows the circumvention of these problems. CARs consist of an antibody-derived scFv, a transmembrane region and an intracellular activating signaling domain, recognizing the target antigen with high affinity and in a MHC-independent fashion. Nonetheless, off-target responses owing to the recognition of the target antigen in healthy cells limit the application of this therapy. The antigen CD19 represents a good target for the elimination of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and anti-CD19 CAR+ lymphocytes are being used clinically. However, the expression of CD19 as a pan B-lineage marker leads to mature B cell depletion. Objective: Since mature B cells express both CD19 and CD20 antigens, we propose the creation of an inhibitory CAR directed to CD20, allowing the lymphocytes to discriminate between BCP-ALL and mature B cells. Methods: Jurkat cells expressing the plasmid pGL4.30 (luciferase controlled by a NFAT responsive element) were generated and used as a reporter cell line. As target cells we used the K562 cell line modified to express CD19 (K5-19), CD20 (K5-20) or CD19 and CD20 (K5-19/20). The expression of the activation marker CD69 was evaluated by flow cytometry. The Sleeping Beauty transposon system was used to modify primary human T lymphocytes and the effector activity was evaluated by cytotoxicity and IFNg production assays. Results: Three different inhibitory αCD20-CARs containing signaling domains CTLA-4, PD-1 or BTLA were constructed. Jurkat cells expressing the αCD19- activating CAR 19BBz showed high activity of luciferase when cultured with K5-19 or K5-19/20. However, Jurkat cells expressing both the activating and inhibitory CARs showed a potent inhibition of luciferase activity when incubated with K5-19/20, while maintaining a high activity when cultured with K5-19 cells. Moreover, the three inhibitory CARs were able to inhibit the de novo expression of CD69 induced by 19BBz in Jurkat cells. In primary T cells, the CAR 20PD1 was not able to inhibit the production of IFNg or the cytotoxic activity induced by 19BBz. Surprisingly, T cells expressing both 19BBz and 20PD1 showed preferential lysis of CD19+ CD20+ targets, suggesting that addition of PD1 signaling domain might augment T cell function in particular contexts. Conclusions: The results in Jurkat cells suggest that αCD20-CARs can inhibit the activation of lymphocytes mediated by αCD19-CAR. However, additional experiments are required to evaluate the inhibitory activity of 20PD1 in primary human T cells. Experiments with CARs 20CTLA4 and 20BTLA are in progress and might allow the validation of the dual CAR hypothesis in vitro and in vivo.
Citation Format: Leonardo Chicaybam, Martin H. Bonamino. Construction and validation of an activating and inhibitory chimeric antigen receptor (CAR) system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2797. doi:10.1158/1538-7445.AM2014-2797