A rational mouse model to detect on-target, off-tumor CAR T cell toxicity
Mauro Castellarin, Caroline Sands, Tong Da, John Scholler, Kathleen Graham, Elizabeth Buza, Joseph A. Fraietta, Yangbing Zhao, Carl H. June
Mauro Castellarin, Caroline Sands, Tong Da, John Scholler, Kathleen Graham, Elizabeth Buza, Joseph A. Fraietta, Yangbing Zhao, Carl H. June
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Research Article Immunology Therapeutics

A rational mouse model to detect on-target, off-tumor CAR T cell toxicity

Abstract

Off-tumor targeting of human antigens is difficult to predict in preclinical animal studies and can lead to serious adverse effects in patients. To address this, we developed a mouse model with stable and tunable human Her2 (hHer2) expression on normal hepatic tissue and compared toxicity between affinity-tuned Her2 chimeric antigen receptor T cells (CARTs). In mice with hHer2-high livers, both the high-affinity (HA) and low-affinity (LA) CARTs caused lethal liver damage due to immunotoxicity. In mice with hHer2-low livers, LA-CARTs exhibited less liver damage and lower systemic levels of IFN-γ than HA-CARTs. We then compared affinity-tuned CARTs for their ability to control a hHer2-positive tumor xenograft in our model. Surprisingly, the LA-CARTs outperformed the HA-CARTs with superior antitumor efficacy in vivo. We hypothesized that this was due, in part, to T cell trafficking differences between LA and HA-CARTs and found that the LA-CARTs migrated out of the liver and infiltrated the tumor sooner than the HA-CARTs. These findings highlight the importance of T cell targeting in reducing toxicity of normal tissue and also in preventing off-tumor sequestration of CARTs, which reduces their therapeutic potency. Our model may be useful to evaluate various CARTs that have conditional expression of more than 1 single-chain variable fragment (scFv).

Authors

Mauro Castellarin, Caroline Sands, Tong Da, John Scholler, Kathleen Graham, Elizabeth Buza, Joseph A. Fraietta, Yangbing Zhao, Carl H. June

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Figure 4

CARTs cause lethal on-target, off-tumor toxicity in mice.

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CARTs cause lethal on-target, off-tumor toxicity in mice. (A) Overview o...
(A) Overview of the experimental design for comparing on-target liver toxicity between affinity-tuned Her2 CARTs. Two groups of mice received either 1.5 × 1010 or 7.5 × 1011 GCs of Her2-AAV8 and then were infused with either 5 × 106 high-affinity (HA) or low-affinity (LA) Her2-CARTs. A control group of mice received 4 × 1011 GCs of GFP-AAV8 (i.e., no Her2) and 5 × 106 HA CARTs. n = 6 mice per group are shown in each panel, unless stated otherwise. (B) Survival curves of mice that received the 7.5 × 1011 GCs of Her2-AAV8 and then CART injection. Statistical analysis was performed using a log-rank Mantel-Cox test. (C) Survival curves of mice that received the 1.5 × 1010 GCs of Her2-AAV8 and then CART injections. (D) Liver function profile as determined by serum ALT levels collected 25 days after T cell injection. Mean ALT ± SEM in mice (n = 4–6) that received 1.5 × 1010 GCs of Her2-AAV8 and either HA-CAR or LA-CAR. A 1-tailed unpaired 2-sample t test of ALT was used for statistical analysis. (E) Weight change shown by percent change from initial weight ± SD in mice that received either 7.5 × 1011 (dashed lines) or 1.5 × 1010 (solid lines) GCs of Her2-AAV8 and then either HA-CAR or LA-CAR. (F) Mean total flux ± SD for whole body bioluminescence imaging (BLI) of T cell luciferase. A 2-way repeated measures ANOVA with Bonferroni’s multiple comparison test was used for statistical analysis of weight change and BLI. Statistical significance for D–F is denoted as *P < 0.5, **P < 0.01, ***P < 0.001, and ****/++++P < 0.0001.