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 2

Murine expression of human antigen is stable and tunable using PiggyBac transposase gene transfer.

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Murine expression of human antigen is stable and tunable using PiggyBac ...
(A) Design of the luciferase and truncated human Her2 (hHer2) transposon vectors and the transposase vector used in the experiment. Group 1 mice were injected with 5 μg of the hHer2 transposon plasmid and 5 μg of the luciferase transposon plasmid, but no transposase plasmid. Group 2 mice were injected with 5 μg of the hHer2 transposon plasmid, 5 μg of the luciferase transposon plasmid, and 10 μg of the transposase plasmid, hyPBase. Group 3 mice were injected with the same plasmids as group 2 but at one-tenth the DNA concentration. (B) Comparison of hHer2 mRNA expression between mice that received higher versus lower concentrations of piggyBac transposon and transposase plasmids. hHer2 RNA was measured in murine livers using real-time PCR and normalized to mouse HPRT expression to calculate 2–ΔCt values. All data are shown as means ± SD (n = 4–7 mice per group). A 2-tailed Mann-Whitney U test of ΔCt values was used for statistical analysis. (C) Comparison of hHer2 DNA content and luciferase expression in the murine livers after hydrodynamic DNA injections (n = 15 mice). (D) In vivo imaging of luciferase expression in mice that either received piggyBac transposon but not transposase plasmid (group 1) or mice injected with either a higher or lower dose of piggyBac transposon and transposase plasmid (groups 2 and 3, respectively). (E) Mean ± SEM radiance over time with n = 9 mice per group. A 2-way repeated-measures ANOVA with Tukey’s multiple correction test was used for statistical analysis. Statistical significance for group 2 versus either group 1 (*) or group 3 (+) is denoted as */+P < 0.5 and **/++P < 0.01.