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 6

Low-affinity CARTs spend less time off -tumor than high-affinity CARTs.

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Low-affinity CARTs spend less time off -tumor than high-affinity CARTs. ...
In vivo CART kinetics were captured using IVIS imaging for n = 6 mice per group. (A) T cells were engineered to express a luciferase gene for in vivo luminescent imaging. The dorsal views of the mice that were kept in the same order as in Figure 5B, and luminescence intensity is shown in a blue-to-red spectrum. In addition to luciferase expression, the T cells contained either no CAR expression (negative control) or they were engineered to express a high-affinity (HA) or low-affinity (LA) Her2 CAR. (B) Whole body bioluminescent imaging (BLI) of T cell luciferase. Statistical significance for HA-CAR versus LA-CAR (*) or HA-CAR vs. No CAR (+) was compared by 2-way repeated measures ANOVA with a Tukey’s multiple comparison test. (C) Spatial luciferase expression was measured along a line that starts in the upper left thorax (point A) and ends in the lower right abdomen (point B). Luminescence from the spleen, liver, and tumor appear at the beginning (~0–1.5 cm), middle (~1–3 cm), and end (~2.5–4 cm) of the line, respectively. Mean luminescence along the line was compared between groups by 2-way repeated measures ANOVA with Bonferroni’s multiple comparisons test. Statistical significance is denoted as **P < 0.01 and ++++P < 0.0001.