How Did T Cell Immunotherapy Use Animal Testing

Human tumor antigens can be expressed in murine livers using AAV8 gene delivery. Human T cells respond differently than mouse T cells; thus, most preclinical studies use homo CARTs in immune-deficient mice (e.grand., NOD/scid/IL2rγ^−/− [NSG] mice) (15). The antigen recognition portion of CARs is commonly derived from monoclonal antibodies that are highly specific for their cognate antigen but are usually not species cross-reactive between mice and humans. The absence of man targets in mice poses a trouble for testing on-target, off-tumor toxicity of CARTs in preclinical mouse models. To accost this limitation, nosotros genetically introduced human tumor antigen targets into mouse livers using 2 different methods of factor transfer.

One method of gene transfer used adeno-associated virus serotype 8 (AAV8) that contained a truncated man Her2 gene (hHer2) and a fluorescent reporter (Katushka), which was then transduced into murine hepatocytes by i.v. tail vein injection (Figure 1A). The expression of the fluorescent reporter in murine hepatocytes was detected by ex vivo imaging of livers, which showed greater fluorescence intensity in the livers of mice that received a higher number of genomic copies (GCs) of AAV8 (Figure 1B). Similarly, the expression of the hHer2 antigen was detected by IHC and showed widespread expression with a more than pronounced perivascular staining blueprint (Figure 1C). Her2 staining of hepatocytes was quantified, and we observed a positive correlation with the number of GCs of AAV8 and the per centum of positive hepatocytes, thus demonstrating that the level of expression can be regulated through AAV8 dosing (Figure 1D). Our results ostend and extend previous in vivo studies that showed that transgene expression levels in hepatocytes straight correlate with AAV8 dosage ( sixteen).

Figure one

Hepatic factor transfer of hHer2 by AAV8 delivery and transposase cistron editing. (A) Design of the AAV8 vector, which includes a truncated homo Her2 gene (hHer2) and a fluorescent reporter gene (Katushka) that are expressed past the liver-specific thyroid hormone-binding globulin (TBG) promoter. Mice were i.v. injected with AAV8, and their livers were harvested 1 calendar 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 cess of hHer2 expression in mice that received either 0, 1.5 × 10¹⁰, or 1.5 × 10¹² GCs of AAV8. Darker Her2-stained cells take a perivascular pattern (blackness arrowhead) and are less frequent than the fainter Her2-stained cells (white arrowheads). Calibration bars: 400 μm (left) and 200 μm (right). (D) Mean hepatocytes ± SEM were quantified for night or faint Her2 staining by digitizing the IHC images using ImageScope and then analyzed using Aperio imaging software. Each grouping contained 4 mice except for one.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 cistron (IRFP720) that was expressed using the liver-specific TBG promoter. (F) Imaging of half-dozen mouse livers harvested 2 months later injection with the IRFP720 fluorescent reporter plasmid and either with or without the PiggyBac transposase Dna vector. (G) Detection of fluorescent reporter expression by menstruation cytometry in isolated mouse hepatocytes representative of the livers shown in F.

Transgene transposition by the piggyBac transposon organisation can create stable human antigen expression in murine livers. We next wanted to explore the suitability of transgene transposition using a transposon organisation delivered to hepatocytes in vivo by hydrodynamic injection. The advantages to this system are that it requires no virus production, generates no viral antigens, has a large cargo delivery size that could accommodate multiple antigen transgenes, and genetically integrates the transgene into the prison cell genome for stable expression. The piggyBac (PB) and Sleeping Beauty (SB) transposons are movable genetic elements that can efficiently transpose vector Deoxyribonucleic acid into mammalian genomes through a "cut-and-paste" mechanism that is effective in genetically modifying cells in vivo (Figure 1E, adapted from ref. 17). Although either the PB or SB system could be used in our model, nosotros chose the PB arrangement because information technology allows for a larger transgene cargo size ( 18, 19). We replaced the Katushka reporter from our AAV8 construct with the IRFP720 poly peptide since about-infrared fluorescent proteins are preferable for deep-tissue imaging ( 20). Injection of nontransposon plasmid Dna resulted in transient episomal expression that was ill-suited for prolonged mouse toxicity studies (Supplemental Effigy ane; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.136012DS1). To exam whether stable transgene expression could be achieved via the transposon system, mice were injected with 10 μg of the fluorescent reporter plasmid Dna pPB7-IRFP720 either with or without 10 μg of the Atomic number 82 transposase plasmid Deoxyribonucleic acid pCMV-HypPBase (Figure 1E). The murine livers were harvested 8 weeks after injection, and IRFP720 expression was measured by IVIS imaging, which showed fluorescence in the mice that was dependent on injection with the Pb transposase plasmids (Effigy 1F). One liver from each group was enzymatically digested, and dissociated hepatocytes were isolated and and then analyzed by period cytometry to find fluorescence in private hepatocytes (Figure 1G). We observed more IRFP720 expression in the mouse that received both the transposon and transposase plasmids versus the control mouse that but received the transposon plasmid, as evidenced by the proportion of positive cells (20% versus two%, respectively) and by the geometric hateful fluorescence intensity (MFI; 242 vs. 124, respectively).

Next, nosotros performed a longitudinal study of the PB gene editing organisation to evaluate the long-term kinetics of transgene expression (Effigy 2A). The versatility of this arrangement immune us to customize the pattern of hepatic protein expression by using unlike combinations and concentrations of plasmids. We created 3 groups of mice that differed in their hydrodynamic tail vein injections of hHer2 transposon DNA, luciferase transposon DNA, and PB transposase Deoxyribonucleic acid to inquire if we could maintain stable expression of the transgenes at various levels. Group i received injections of luciferase transposon DNA and hHer2 transposon Deoxyribonucleic acid but without transposase Deoxyribonucleic acid. Grouping two received injections that combined luciferase transposon Dna, hHer2 transposon DNA, and the Pb transposase DNA. Group 3 received the same plasmid combination as grouping 2 simply at one-tenth the DNA concentration.

Effigy 2

Murine expression of human antigen is stable and tunable using PiggyBac transposase cistron transfer. (A) Design of the luciferase and truncated homo Her2 (hHer2) transposon vectors and the transposase vector used in the experiment. Grouping ane 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, v μg of the luciferase transposon plasmid, and 10 μg of the transposase plasmid, hyPBase. Grouping 3 mice were injected with the aforementioned plasmids as group 2 simply at one-tenth the DNA concentration. (B) Comparison of hHer2 mRNA expression betwixt mice that received higher versus lower concentrations of piggyBac transposon and transposase plasmids. hHer2 RNA was measured in murine livers using existent-time PCR and normalized to mouse HPRT expression to calculate two^–ΔCt values. All information are shown as means ± SD (n = iv–seven mice per group). A 2-tailed Mann-Whitney U examination of ΔCt values was used for statistical analysis. (C) Comparison of hHer2 Deoxyribonucleic acid 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 two and iii, respectively). (Eastward) Hateful ± SEM radiance over fourth dimension with n = 9 mice per group. A 2-style repeated-measures ANOVA with Tukey's multiple correction exam was used for statistical assay. Statistical significance for group two versus either group ane (*) or group 3 (⁺) is denoted as *^/+ P < 0.5 and **^/++ P < 0.01.

hHer2 mRNA expression from the harvested murine livers was evaluated past real-time PCR using TaqMan assays for hHer2, which was normalized to liver RNA using the mouse housekeeping cistron HPRT (Figure 2B). Relative expression is shown every bit two^–ΔCt, where ΔCt is calculated as Ct HPRT minus Ct Her2 and was calculated for the mice that received all 3 of the plasmids (i.e., groups 2 and iii) and too for control mice that did not receive hHer2 transposon Dna, which were used to establish groundwork Ct levels. This showed that a higher level of Her2 expression was achieved in the mice from group 2 versus mice from Group 3 and, thus, confirmed that we could regulate antigen levels by the corporeality of transfected DNA, which is consistent with previous studies (18). We observed a positive correlation in the livers between luciferase radiance and Her2 mRNA expression, suggesting that the Her2 and the luciferase transposon plasmids were transfected with equal efficiency and, thus, luciferase measurements were indicative of Her2 antigen levels (Figure 2C).

Weekly IVIS imaging of the murine liver was measured to determine longitudinal luciferase expression. This imaging revealed that Group one mice initially had the highest BLI levels (i.e., high concentration of the transposon Deoxyribonucleic acid but no transposase DNA), merely and then bioluminescence was found to decrease dramatically during the first two weeks and then continued to reject during the following 6 months (Figure 2, D and E). The mice from groups 2 and 3, which received the boosted transposase plasmid, showed a luciferase expression blueprint that decreased markedly in the start calendar week just and so increased steadily before stabilizing approximately one month later (Figure two, D and E). A comparing of groups ii and three showed the group that received the higher concentration of DNA had a higher final average bioluminescent bespeak (i.e., transgene expression), which was consequent with the previous hHer2 mRNA expression results. Our findings demonstrate that either the PB transposon system or AAV8 tin can exist used to finer obtain stable man antigen expression in the mouse liver and at predetermined levels.

Human antigens in the murine liver tin can be targeted by CARTs. We next tested whether the hHer2 antigen that was expressed in the mouse livers could provoke on-target toxicity post-obit infusion of anti–Her2-CARTs. Her2 CARTs with two dissimilar scFvs (4D5, high analogousness [HA]; 4D5-v, depression analogousness [LA]) were i.v. infused into mice that had hepatic expression of hHer2 antigen, which was established using our Lead transfection method. To find a Her2-independent T cell response in the livers, nosotros included i negative control group that expressed Her2 merely received untransduced T cells and a second negative control grouping that received Her2-CARTs just in Her2-negative mice. The presence of CARTs in the murine livers were measured i week after injection using a CAR Dna TaqMan assay, which was normalized confronting a mouse PTGER2 TaqMan assay. Her2-CART (HA or LA) DNA was elevated only in mice that had hHer2 expression (Figure 3A). The mice that had CARTs present in their livers too had elevated hepatic expression of human IFN-γ mRNA, every bit measured using a TaqMan assay normalized against mouse HPRT mRNA (Figure 3B). The serum cytokine levels in the 4 groups revealed that simply mice that were injected with Her2-CARTs and expressed hepatic hHer2 antigen had elevated human cytokines associated with T cell activation (i.e., IFN-γ, GM-CSF, IL-2, IL-5, and MIP-1b), which suggests antigen-dependent activation and no response to hydrodynamic injection (Effigy 3C). Serum levels of IFN-γ and GM-CSF were significantly higher in the mice that received the HA versus LA CARTs, consistent with increased activation. To demonstrate that hepatic antigen expression delivered past AAV8 could also arm-twist an antigen-specific allowed response, mice were injected with Her2-AAV8 and then infused with luciferase-expressing Her2 CARTs (Supplemental Figure two). Mouse livers were analyzed by bioluminescent imaging and IHC, which showed an infiltration of Her2 CARTs. These results confirmed that the human antigen nosotros delivered to the liver can human activity as a target for CARTs and that presence of this human antigen was sufficient for CART infiltration and activation. We ended that either AAV8 or hydrodynamic tail vein injection of transposon DNA were suitable methods for promoting an antigen-dependent allowed response in hepatocytes.

Figure 3

CARTs recognize cognate human being antigen in mice. hHer2 antigen was expressed in mouse hepatocytes following piggyBac factor transfer. Mice were then injected with ii.5 × 10^{half dozen} anti-hHer2 CART (either HA-Car or LA-Auto), and livers were harvested 1 calendar week later for assay. Command groups included mice that had hepatic Her2 expression but untransduced T cells and mice that received Her2 CART just lacked hepatic Her2 expression due to empty transposon vector transfections. (A) Her2 CARTs were detected in mouse livers (due north = 4–8) by performing existent-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-fourth dimension PCR and normalized to mouse HPRT expression. Kruskal-Wallis test with Dunn's multiple comparisons tests was used for statistical analysis of real-fourth dimension PCR information. (C) Systemic cytokine release by T cells was detected in mouse serum (n = 4–8) past Luminex assay. A 2-way ANOVA with Tukey's multiple comparison exam was performed, and comparisons are shown between all groups and the untransduced (UTD) group (*) or between the HA-Auto and LA-Machine groups (⁺). Statistical significance is denoted as *P < 0.five, **P < 0.01,⁺⁺⁺ P < 0.001, and ****^/++++ P < 0.0001.

Off-tumor toxicity tin can be reduced using a lower-affinity CART. The amount of toxicity in Her2-expressing livers was compared between mice that received a HA versus a LA CART (4D5 and 4D5-5 scFv, respectively). We postulated that the HA Automobile would cause more liver damage than its LA analogue when hepatic Her2 expression was low, since the Her2 levels would be below the limit of detection for the LA CARTs. Nosotros as well hypothesized that, when Her2 expression in the murine livers was high, hepatocytes would be recognized by both the HA and LA CARTs and, thus, the caste of liver damage would exist equivalent. To test our assumption, nosotros injected 1 group of mice with a high dose of Her2-AAV8 to create livers in mice with high antigen levels, while another group of mice received a comparatively low dose of Her2-AAV8 to generate low-antigen livers (Figure 4A). To determine if the AAV8 viral antigens alone would arm-twist T cell–mediated toxicity, a Her2-negative control grouping received a loftier dose of AAV8 that expressed GFP instead of Her2; so, these mice were injected with HA Her2-CARTs. As predicted, there was severe toxicity in mice with loftier antigen levels due to both the HA and LA CARTs, as observed by markedly increased mortality (Figure 4B) and postmortem analysis of liver pathology (Supplemental Table i). In addition to liver damage, cytokine release as shown in Figure 3C may have also contributed to morbidity in these mice. In contrast, the HA CARTs were nonlethal in mice that lacked hepatic Her2 expression, excluding xenoreactivity equally a crusade of morbidity (Effigy 4B).

Figure 4

CARTs cause lethal on-target, off-tumor toxicity in mice. (A) Overview of the experimental design for comparing on-target liver toxicity betwixt analogousness-tuned Her2 CARTs. Two groups of mice received either one.5 × x¹⁰ or vii.v × ten¹¹ GCs of Her2-AAV8 and then were infused with either 5 × ten⁶ loftier-analogousness (HA) or low-affinity (LA) Her2-CARTs. A command group of mice received four × 10¹¹ GCs of GFP-AAV8 (i.eastward., no Her2) and 5 × 10^{half dozen} HA CARTs. n = vi mice per grouping are shown in each panel, unless stated otherwise. (B) Survival curves of mice that received the vii.5 × ten¹¹ GCs of Her2-AAV8 then CART injection. Statistical analysis was performed using a log-rank Mantel-Cox test. (C) Survival curves of mice that received the 1.5 × 10^ten GCs of Her2-AAV8 and and then CART injections. (D) Liver part profile every bit determined by serum ALT levels nerveless 25 days later T cell injection. Mean ALT ± SEM in mice (n = 4–half-dozen) that received 1.5 × 10^ten GCs of Her2-AAV8 and either HA-Motorcar or LA-CAR. A one-tailed unpaired 2-sample t examination of ALT was used for statistical assay. (East) Weight modify shown by pct modify from initial weight ± SD in mice that received either 7.five × 10¹¹ (dashed lines) or 1.five × 10^ten (solid lines) GCs of Her2-AAV8 so either HA-Automobile or LA-Machine. (F) Hateful total flux ± SD for whole torso bioluminescence imaging (BLI) of T jail 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.

Toxicity was decreased in mice with low antigen levels, every bit seen by reduced mortality (Figure 4C) and liver pathology (Supplemental Table 1). The low-antigen mice had no significant divergence in bloodshed between affinity-tuned CARTs (Figure 4C), but more liver damage was acquired by the HA versus LA CARTs, according to elevated serum ALT levels (Effigy 4D). The mean ALT from the mice that received HA CARTs is 84 U/50 with a range of 64–118 U/50 versus the negative control mice with a mean ALT of 46 U/L and a range of 34–66 U/Fifty. The resulting fold change for the HA CART grouping is about double the normal values, which would be considered serious in humans. According to Hy'due south law, drug-induced hepatocellular injury that is three times or greater above the upper limit of normal presents a high risk of fatal drug-induced liver injury. Toxicity effects were also credible by mouse weight loss, which was profound in the high-antigen groups that received either the LA or HA CARTs and also occurred in the depression-antigen mice that were infused with the HA but not LA CARTs (Figure 4E). Differences in affluence between the HA and LA CARTs were assessed by bioluminescence imaging (BLI) of their luciferase reporter gene (Figure 4F). In the Her2-negative control mice (i.e., AAV8-GFP group), the number of HA Her2 CARTs remained constant during the first two weeks after T cell injection, indicating an absence of antigen-dependent activation (Figure 4F). In the low-antigen mice, we initially observed an increase in abundance via luminescence for both the HA and LA CARTs, followed by a subtract afterwards 4 days by the LA CARTs. In contrast, the HA CARTs continued to increase until day 8 and remained higher than the LA CARTs, until dropping to equivalent values by day 22. This suggests that the HA CARTs remained activated longer due to prolonged antigen recognition. All three groups had similar upwards trends in T cell abundance starting at solar day 22, which was presumably due to xenogeneic graft-versus-host affliction (GVHD). Overall, the LA CARs were better able to distinguish between low- and loftier-antigen density tissues in our safety model.

An increase in off-tumor targeting is associated with a filibuster in tumor CART infiltration and a decrease in antitumor efficacy. We next wanted to test our analogousness-tuned CART treatments for their tumor control in a mouse model that false a common clinical scenario, in which antigen is overexpressed in a patient's tumor but is besides found at lower levels in some of their healthy tissue. To achieve this, mice were engrafted with a loftier Her2–expressing tumor xenograft and additionally injected with a low dose of Her2-AAV8 to produce a low Her2–expressing liver (Effigy 5A). The mice were then infused with either HA or LA CARTs. Surprisingly, the mice that received the LA CARTs showed significantly better antitumor efficacy than the ones that were treated with the HA CARTs (Figure five, B and C). Tumor size was visualized by the expression of the fluorescent reporter IRFP720, which was congruent with our caliper measurements (Figure 5C and Supplemental Figure 3).

Figure 5

The low-analogousness CAR has ameliorate tumor command than the loftier-affinity CAR when antigen is also expressed in normal tissue. (A) Overview of the experimental design for comparison Her2⁺ tumor control between affinity-tuned Her2 CARTs. All mice received 1.5 × 10^ten GCs of Her2-AAV8 and were implanted with 5 × ten^{half dozen} Her2⁺ SKOV3 tumor cells. Then, three groups were injected with either 5 × 10⁶ loftier-analogousness (HA) or low-affinity (LA) Her2-CARTs or no Machine command 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-ruby spectrum. Lateral views of fluorescent tumor imaging. (C) Mean tumor volume ± SEM measured past calipers in due north = 6 mice per group. A 2-mode repeated measures ANOVA with Bonferroni's multiple comparisons test was used for statistical analysis. Statistical significance is denoted as *P < 0.v and ****P < 0.0001.

To investigate whether differences in tumor control betwixt the 2 groups were due to differences in T jail cell abundance and/or trafficking, nosotros observed luciferase-expressing T cells using in vivo imaging (Figure 6A). Slight differences in overall abundance between the HA and LA CARTs were seen by whole body BLI measurements in the start calendar week so became comparable throughout the rest of the experiment (Effigy 6B). Migratory behavior betwixt the T prison cell treatment groups was similar at day ane, in which the HA and LA CARTs were both seen in the liver while the T cells in the control group were observed in the spleen (Effigy 6C). However, by day eight, there were striking differences in the trafficking of the CARTs as the HA CARTs remained in the mouse livers, whereas the LA CARTs had emigrated from the liver and homed to the tumor. Past day 12, the HA CARTs had left the liver and infiltrated the tumor, while the LA CARTs had already caused measurable tumor regression. By mean solar day 43, the LA CART group had no measurable tumors by either fluorescent reporter expression or caliper readings (Figure 5, B and C). Conversely, the HA CART group at 24-hour interval 43 had detectable tumor in 4 of the half dozen mice and less tumor infiltration of CARTs in those tumors compared with earlier time points. In tumor-bearing mice without hepatic antigen expression, no deviation between affinity-tuned CARTs was observed in trafficking (Supplemental Effigy iv) or tumor control ( 11). Thus, in our mouse model, the LA CARTs were amend able to discriminate between low-antigen healthy tissue and high-antigen tumor tissue, which resulted in a better therapeutic outcome.

Effigy 6

Low-affinity CARTs spend less fourth dimension off -tumor than high-affinity CARTs. In vivo CART kinetics were captured using IVIS imaging for n = 6 mice per grouping. (A) T cells were engineered to limited a luciferase cistron 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 improver to luciferase expression, the T cells independent either no Car expression (negative control) or they were engineered to limited a high-affinity (HA) or low-analogousness (LA) Her2 Motorcar. (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 past two-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 (indicate A) and ends in the lower right belly (point B). Luminescence from the spleen, liver, and tumor appear at the showtime (~0–1.5 cm), middle (~ane–3 cm), and end (~ii.v–4 cm) of the line, respectively. Hateful brilliance forth 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.

Source: https://insight.jci.org/articles/view/136012

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