Abstract 3156: Construction and validation of an activating and inhibitory chimeric antigen receptor (CAR) system

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Abstract Introduction: Chimeric antigen receptors (CARs) consists a promising type of adoptive immunotherapy. CARs consist of an antigen recognition unit (scFv), a transmembrane (TM) region and an intracellular activation domain. However, on target/off tumor responses due to target recognition in normal cells limits the widespread application of this therapy. In B-cell precursor acute lymphoblastic leukemia, lymphocytes expressing anti-CD19 CARs (19BBz) are being used in the clinical setting. However, the expression of CD19 as a pan-B marker can lead to undesired side effects such as depletion of mature B cells. As mature B cells express both CD19 and CD20 antigens, we propose the creation of an inhibitory anti-CD20 CAR, that when used in combination with 19BBz would be able to discriminate between leukemic blasts and normal B cells. Methods and results: Three inhibitory CARs were constructed containing signaling domains of CTLA-4, PD-1 or BTLA. Jurkat T cells expressing the plasmid pGL4.30 - expressing luciferase controlled by a NFAT responsive promoter - were generated. K562 cell line was used as target cells and modified to express CD19 (K5-19), CD20 (K5-20) or CD19 and CD20 (K5-19/20). In coculture experiments, Jurkat cells expressing 19BBz showed induction of luciferase activity when cultured with K5-19 or K5-19/20. However, Jurkat expressing both activation and inhibitory CAR showed inhibition of luciferase activity when incubated with K5-19/20 while maintained high activity when cultured with K5-19 cells. Furthermore, all three inhibitory CARs were able to inhibit the expression of the activation marker CD69 induced by 19BBz. In primary human T lymphocytes, the 20PD1 CAR did not inhibit proliferation or cytotoxicity induced by 19BBz. Surprisingly, T cells expressing 19BBz and 20PD1 showed preferential lysis of NALM6 CD19+ CD20+ cells, suggesting that the addition of PD1 domain may increase T-cell function in specific contexts. This effect was partially reversed by using a mutant PD1 domain where the tyrosines were replaced by phenylalanine and was completely reversed by using an anti-CD20 CAR truncated at the TM region, suggesting that this phenomenon is dependent of signaling. Conclusions: Additional experiments are needed to evaluate the activity of 20PD1 in primary human T cells. Experiments with inhibitory CARs 20LAG3 and 20PD1LAG3 are ongoing and can serve as an alternative to CAR 20PD1 in the construction of a conditional response system. 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 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3156. doi:10.1158/1538-7445.AM2015-3156