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).

Keywords: Cancer immunotherapy; Immunology; Therapeutics.

Figure 1. Hepatic gene transfer of hHer2 by AAV8 delivery and transposase gene editing.

(A) Design of the AAV8 vector, which includes a truncated human Her2 gene (hHer2) and a fluorescent reporter gene (Katushka) that are expressed by the liver-specific thyroid hormone-binding globulin (TBG) promoter. Mice were i.v. injected with AAV8, and their livers were harvested 1 month later. (B) IVIS imaging of fluorescence in ex vivo livers harvested from mice that received either no genomic copies (GCs), 1.5 × 10¹⁰ GCs, or 1.5 × 10¹² GCs of AAV8. (C) Immunohistological assessment of hHer2 expression in mice that received either 0, 1.5 × 10¹⁰, or 1.5 × 10¹² GCs of AAV8. Darker Her2-stained cells have a perivascular pattern (black arrowhead) and are less frequent than the fainter Her2-stained cells (white arrowheads). Scale bars: 400 μm (left) and 200 μm (right). (D) Mean hepatocytes ± SEM were quantified for dark or faint Her2 staining by digitizing the IHC images using ImageScope and then analyzed using Aperio imaging software. Each group contained 4 mice except for 1.5 × 10¹² GC, which had an n of 1. (E) Overview of gene editing using the piggyBac transposase system. The transposon vector contained a fluorescent reporter gene (IRFP720) that was expressed using the liver-specific TBG promoter. (F) Imaging of 6 mouse livers harvested 2 months after injection with the IRFP720 fluorescent reporter plasmid and either with or without the PiggyBac transposase DNA vector. (G) Detection of fluorescent reporter expression by flow cytometry in isolated mouse hepatocytes representative of the livers shown in F.

Figure 2. Murine expression of human antigen is stable and tunable using PiggyBac transposase gene transfer.

(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.

Figure 3. CARTs recognize cognate human antigen in mice.

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.

Figure 4. CARTs cause lethal on-target, off-tumor toxicity in mice.

(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.

Figure 5. The low-affinity CAR has better tumor control than the high-affinity CAR when antigen is also expressed in normal tissue.

(A) Overview of the experimental design for comparing Her2⁺ tumor control between affinity-tuned Her2 CARTs. All mice received 1.5 × 10¹⁰ GCs of Her2-AAV8 and were implanted with 5 × 10⁶ Her2⁺ SKOV3 tumor cells. Then, 3 groups were injected with either 5 × 10⁶ high-affinity (HA) or low-affinity (LA) Her2-CARTs or no CAR control T cells. (B) The Her2⁺ tumor cells, SKOV3, were genetically modified to express the fluorescent reporter, IRFP720, for in vivo imaging. Tumor xenograft fluorescence is shown in a yellow-to-red spectrum. Lateral views of fluorescent tumor imaging. (C) Mean tumor volume ± SEM measured by calipers in n = 6 mice per group. A 2-way repeated measures ANOVA with Bonferroni’s multiple comparisons test was used for statistical analysis. Statistical significance is denoted as *P < 0.5 and ****P < 0.0001.

Figure 6. 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.