Abstract 3140: Antigen-induced downmodulation is associated with diminished efficacy of a novel chimeric antigen receptor targeting anaplastic lymphoma kinase
Authors: Walker Alec J. , Zhang Ling , Long Adrienne H. , Orentas Rimas J. , Mackall Crystal L. 
Published: August 2015

Abstract
CD19 directed Chimeric Antigen Receptor (CAR) T cell therapies have shown clinical responses against B cell malignancies. However, CARs targeting other tumor-associated antigens have been less successful. Factors limiting CAR efficacy are not well understood. Anaplastic lymphoma kinase (ALK) is a cell-surface protein overexpressed in a large fraction of neuroblastomas, the most common extracranial solid tumor of childhood. We constructed MSGV1.ALK.BBZ retroviral vectors using single-chain variable fragment (scFv) sequences from murine monoclonal antibodies targeting ALK and transduced these into human PBMCs. ALK-CAR T cells lysed ALK+ tumor lines, but produced limited amounts of IFN-g and IL-2 upon co-culture with ALK+ tumor cells compared to CD19-CARs co-cultured with CD19+ targets. In order to identify potential differences between ALK-CAR and CD19-CAR induced signaling we employed a reporter system in which GFP expression was directed by an NFAT-responsive promoter and could be visualized by flow cytometry. T cells were co-transduced with lentiviral NFAT-GFP and MSGV1.ALK.BBZ or MSGV1.CD19.BBZ. Transduced T cells were co-cultured with tumor lines expressing ALK or CD19 antigen. GFP expression was induced within 4h of antigen exposure in double transduced T cells and persisted for at least 24h. The magnitude of GFP expression correlated positively with antigen density on tumor targets. Additionally, &gt;50% of GFP+ cells retained CD19-CAR surface expression after antigen encounter. However, we observed drastically reduced surface ALK-CAR expression on GFP+ T cells after encounter with tumor targets. Upon further examination, we noticed that ALK-CARs were down-modulated from the T cell surface within 1h of exposure to antigen, and remained internalized for at least 24h. The magnitude of NFAT translocation in ALK-CAR T cells, as measured by GFP intensity, was also markedly lower than the magnitude of NFAT translocation in CD19-CAR T cells. These results suggest that limited numbers of CARs remaining on the T cell surface after initial antigen encounter may contribute to diminished ALK-CAR T cell efficacy, possibly by preventing temporal summation of repeated CAR signaling. Work is underway to prevent CAR down-modulation after antigen encounter, and to evaluate the impact of increased surface CAR retention on CAR functionality. In summary, optimal design of new CAR therapeutics requires a better understanding of essential factors limiting CAR efficacy. Through evaluation of a novel CAR targeting ALK, we have identified CAR down-modulation as one factor that may influence CAR-T efficacy, and may be amenable to modulation in order to improve CAR functionality.
Citation Format: Alec J. Walker, Ling Zhang, Adrienne H. Long, Rimas J. Orentas, Crystal L. Mackall. Antigen-induced downmodulation is associated with diminished efficacy of a novel chimeric antigen receptor targeting anaplastic lymphoma kinase. [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 3140. doi:10.1158/1538-7445.AM2015-3140