A comparison of affinity-tuned Her2 CARs using a new mouse model for on-target off-tumor CAR T cell cytotoxicity
Mauro Castellarin1, Joseph Fraietta1, Tong Da1, John Scholler1, Yangbing Zhao1,2, Carl H. June1,2,3
1Center for Cellular Immunotherapies, University of Pennsylvania (Penn); 2Pathology and Laboratory Medicine, Penn; 3Abramson Cancer Center, Penn
Chimeric antigen receptor T cells (CARTs) can target malignant cells by recognizing tumor-associated antigens (TAA) but autoimmune toxicity can occur if TAAs are expressed on normal cells even at low levels. We developed a mouse model, in which human TAA transgenes are expressed at varying levels in the liver and can be used as targets for CAR T cells. We chose human Her2 as a TAA, and injected mice IV with AAV8-Her2 at varying titers, and observed the expected dose-dependent Her2 expression in murine hepatocytes. This model was used to compare toxicity between T cells that expressed either high- or low-affinity Her2 CARs. In mice expressing high levels of hepatic Her2 (Her2high), both the high- and low-affinity CARTs caused severe liver toxicity as indicated by a four-fold increase in serum ALT levels and uniform mortality relative to Her2-negative controls (100% vs 0% 22 days post-CART infusion). In mice expressing low levels of hepatic Her2 (Her2low), the high-affinity CARTs caused significantly more liver toxicity than the low-affinity CARTs, suggesting that low-affinity CARTs were better tolerated.
Next, we compared the anti-tumor effects of high- and low-affinity CARTs in mice with both Her2high tumor xenografts and Her2low livers, as a model for relative TAA expression in patient tumors and normal tissues. In vivo imaging revealed that high-affinity CARTs exhibited longer retention time in the liver compared to low affinity CARTs and to negative controls (12, 8 and 4 days, respectively). Consequently, the low-affinity CARTs were observed infiltrating the tumor earlier than the high-affinity CARTs (8 vs 12 days) and the low affinity CARTs cleared the tumor more effectively. Overall, our model clearly shows a higher therapeutic index for the low-affinity CAR T cells versus high-affinity CAR T cells.
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