190. Adaptive CAR T Cell Design

Abstract

Chimeric antigen receptor (CAR) T cell therapy has recently emerged as an attractive approach for the treatment of CD19-expressing hematological malignancies. However extending the success of this strategy to other targets has proven to be more complicated that simply replacing the scFv. To address this issue we have implemented a form of adaptive CAR design whereby a series of sequential modifications are made to a single domain and subsequently tested in vitro and in vivo to assess activity. We illustrate the utility of such a strategy using our published CAR-PSCA (Anurathapan U. et al, Mol Ther. 2014) as a template (CAR-PSCA v1.0) with subsequent iterations coded as versions 2.0, 3.0, 4.0 and 5.0. We now demonstrate how modifications made to a single CAR structural domain can result in enhanced (i) T cell migration, (ii) antigen recognition, and (iii) cell phenotype, ultimately producing superior anti-tumor effects. First, with CAR v2.0 and v3.0 we were able to improve T cell migration, which was evident in NSG mice engrafted s.c. with Capan-1 and treated i.v. with FFluc+ T cells. Ten days post CAR administration we saw a 2 log increase in the T cell signal at the tumor site (4.5±2.3×105 p/s vs 4.8±0.5×107 p/s vs 4.0±1.1×107 p/s, CAR v1.0, v2.0 and v3.0 respectively). Subsequently to enhance in vivo T cell persistence, we next generated CAR v4.0, which resulted in a less differentiated T cell phenotype (Tnaive: 1.8±0.6% to 19.2±4.0%, TCM: 10.4±1.4% to 14.1±3.0%, TEM: 83.5±1.2% to 53.7±6.9% and TEMRA: 4.3±0.9% to 12.9±1.7% - CAR v3.0 and CAR v4.0, respectively). When administered to Capan-1-engrafted NSG mice CAR v4.0 T cells exhibited enhanced in vivo longevity as measured using bioluminescence imaging (7.3±4.6×107 p/s CAR v3.0 vs 2.8±1.7×108 p/s CAR v4.0 T cells - day 35 post-administration). Finally, antigen recognition of CAR-PSCA was further improved in v5.0 where a final modification to the same domain produced superior anti-tumor effects against a PSCA-dim target tumor cell line (DU145) in a 6hr 51Cr-release assay (20.7±5.8% vs 48.4±5.2%, CAR v4.0 vs CAR v5.0, 40:1 E:T). Overall, therefore, implementation of this adaptive design produced a CART cell product with enhanced in vivo anti-tumor activity. This was clearly illustrated when we compared the tumor volume of NSG mice treated with CAR v1.0 or CAR v5.0 T cells (1309±143 mm3 vs 510±53 mm3 on Day 66). Specific details of the modifications conducted in this adaptive CAR design will be presented.