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 3

CARTs recognize cognate human antigen in mice.

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CARTs recognize cognate human antigen in mice. hHer2 antigen was express...
hHer2 antigen was expressed in mouse hepatocytes following piggyBac gene transfer. Mice were then injected with 2.5 × 106 anti-hHer2 CART (either HA-CAR or LA-CAR), and livers were harvested 1 week later for analysis. Control groups included mice that had hepatic Her2 expression but untransduced T cells and mice that received Her2 CART but lacked hepatic Her2 expression due to empty transposon vector transfections. (A) Her2 CARTs were detected in mouse livers (n = 4–8) by performing real-time PCR assays for CAR DNA. 2–ΔCt values for CARTs were calculated using PCR assays that amplify the CAR intracellular signaling domain, 4-1BBz-CD3z, and were normalized to mouse PTGER2 genomic DNA content. (B) Expression of human IFN-γ mRNA from T cells was measured in murine livers (n = 4–8) using real-time PCR and normalized to mouse HPRT expression. Kruskal-Wallis test with Dunn’s multiple comparisons tests was used for statistical analysis of real-time PCR data. (C) Systemic cytokine release by T cells was detected in mouse serum (n = 4–8) by Luminex assay. A 2-way ANOVA with Tukey’s multiple comparison test was performed, and comparisons are shown between all groups and the untransduced (UTD) group (*) or between the HA-CAR and LA-CAR groups (+). Statistical significance is denoted as *P < 0.5, **P < 0.01,+++P < 0.001, and ****/++++P < 0.0001.