Abstract 2796: Engineering multiple chimeric antigen receptors in T cells for the treatment of multiple myeloma
Authors: Chang Lung-Ji , Liu Yuchen , Moreb Jan S. 
Published: May 2015

Abstract
Multiple myeloma (MM) is an incurable plasma cell neoplasm accounting for 10% of all hematologic malignancies. With advanced treatments, the average survival has significantly improved. Recent progress in immunotherapy based on antibody and T cell receptor (TCR) engineering has shown great potential for treating cancer including MM. We hypothesize that immune cell engineering that overcomes immune suppression in the BM may completely eradicate residual cancer cells. To test our hypothesis, we have developed a streamlined approach to generate lentiviral constructs with chimeric antigen receptors (CAR) of T cells based on single chain antibodies that target multiple MM antigens including CD138, CD269 (mature B cell marker BCMA, or TNFRSF17), CD317 (hm1.24), and CD319 (CS1). To increase safety, we also incorporated a self-destructive, inducible suicide caspase 9 gene, which is fused with the CAR. Memory CD8 T cells were isolated from BM specimens of MM patients and immortalized for prolonged in vitro studies. These primary BM T cells were transduced using lentiviral vectors encoding the MM-targeting CARs, and co-cultured with MM cell lines to measure killing effect. The MM-specific killing activity was demonstrated based on annexin V and propidium iodide staining and flow cytometry analysis for quantitation of cell death. In transient (24 hours) killing assays, both CD138 and CD 319 CARs displayed moderate MM killing activities, at 48% and 36%, respectively, when using a CAR T effector to MM Target (E/T) ratio of 0.5/1. Interestingly, a synergistic MM killing effect (a five fold increase at an E/T ratio of 1/1) was observed when these two CARs were used in combination. On the other hand, CD269 and CD317 CAR T cells displayed marked MM killing activities, approximately 80% for both at an E/T ratio of 0.5/1 in 24 hours. In a long term CAR T/MM co-culture setting at E/T ratios of 0.5/1 and 1/1, we observed near complete MM killing (100%) by all four MM-targeting CAR T cells in two weeks. The self-destructive function of the inducible suicide gene in the CAR T cells was also demonstrated in the long-term co-cultures. Interestingly, upon killing of the CAR T cells by apoptotic induction, residual MM cells rebounded from the CD319 CAR T/MM co-culture, but not the other three CAR T/MM co-cultures, suggesting that rapid and efficient killing of MM cells by CAR T cells is necessary for complete eradication of MM cells. In summary, all four CARs demonstrated MM killing activities in short term and long term T cell co-cultures. Combination of multiple CARs may be necessary for effective suppression of MM residual disease in vivo and to prevent antigenic escape phenomenon. Together with the inducible apoptotic gene design, increased safety and efficiency for the CAR-based immunotherapy may be attained in future clinical trials.
Citation Format: Lung-Ji Chang, Yuchen Liu, Jan S. Moreb. Engineering multiple chimeric antigen receptors in T cells for the treatment of multiple myeloma. [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 2796. doi:10.1158/1538-7445.AM2014-2796