Characterization of a switchable chimeric antigen receptor platform in a pre-clinical solid tumor model

Abstract

The universal modular chimeric antigen receptor (UniCAR) platform redirects CAR-T cells using a separated, soluble targeting module with a short half-life. This segregation allows precise controllability and flexibility. Herein we show that the UniCAR platform can be used to efficiently target solid cancers in vitro and in vivo using a pre-clinical prostate cancer model which overexpresses prostate stem cell antigen (PSCA). Short-term administration of the targeting module to tumor bearing immunocompromised mice engrafted with human UniCAR-T cells significantly delayed tumor growth and prolonged survival of recipient mice both in a low and high tumor burden model. In addition, we analyzed phenotypic and functional changes of cancer cells and UniCAR-T cells in association with the administration of the targeting module to reveal potential immunoevasive mechanisms. Most notably, UniCAR-T cell activation induced upregulation of immune-inhibitory molecules such as programmed death ligands. In conclusion, this work illustrates that the UniCAR platform mediates potent anti-tumor activity in a relevant in vitro and in vivo solid tumor model.

Keywords: Chimeric antigen receptors; immune checkpoints; immunoevasion; prostate stem cell antigen; solid tumors; targeting module.

Figure 1.

Tumor biodistribution and plasma pharmacokinetics of _7KATM^PSCA in NSG mice (A) Tumor-bearing NSG mice ± UniCAR-T cells engraftment, were injected iv via tail vein with 50 µg fluorescence labeled _7KATM^PSCA. Tumor uptake was measured longitudinally by in vivo fluorescence imaging. Left: Images of 2 representative mice before, 28 h and 15 d after injection are shown. Right: the mean relative fluorescence intensity in the region of interest over time. Error bars represent SD (n = 3 per group). (B) Tumor-bearing NMRI-nu mice were injected iv via tail vein with 3.8 MBq ⁶⁴Cu-_7KATM^PSCA (NODAGA)_1.5. Radioactivity was determined longitudinally in the region of interest. Left: Representative maximum intensity projections over 2 h presented as summed images with midframe times of 5, 60, and 90 min after a single iv injection. Right: PET-kinetics in tumor bearing NMRI-nu mice after a single iv injection. Data are presented as logarithm of maximum activity concentration in the heart (representative for the blood), and the PC3^PSCA-tumor 2 h, 8 h, 24 h, and 56 h after injection. (C, D) NSG mice were injected either iv via tail vein (n = 3 for every time point) or ip (n = 3 for every time point) with 250 ng _7KATM^PSCA/g bw. PB was obtained at different time points for evaluation of _7KATM^PSCA plasma concentration. A terminal half-life time of 98 min and 16 min was calculated for _7KATM^PSCA after ip and iv injection, respectively.

Figure 2.

The UniCAR platform mediates tumor growth inhibition and prolongs survival of small tumor bearing NSG mice. Three to 4 weeks after iv injection of 1 × 10⁶ UniCAR-T cells, mice were sc transplanted with 1 × 10⁶ PC3^PSCA tumor cells. A separate group received only 1 × 10⁶ PC3^PSCA tumor cells. One week later 250 ng _7KATM^PSCA/g bw was ip injected bid for 7 consecutive days in the study cohort. Tumors were measured 2−3 times weekly with a digital caliper. Mice were killed when predefined end points were met. (A) Representative pictures of tumors from all 3 groups at the end of the experiment. (B) Tumor volume ± SD during the observation period of 13 weeks (last observation carried forward method). The tumor volume at the start of treatment did not differ significantly among the groups (PC3^PSCA: 39.2 ± 13.3 mm³, PC3^PSCA + UniCAR: 33.7 ± 17.8 mm³were PC3^PSCA + UniCAR + _7KATM^PSCA: 45.9 ± 36.2 mm³) (C) Kaplan-Meier plot for the treatment groups associated with survival. Data are pooled from 2 independent experiments with individual donors, n = 10 (*p < 0.05, ***p ≤ 0.001).

Figure 3.

The UniCAR platform mediates potent anti-tumor activity in a high tumor burden model: Mice were transplanted sc with 1 × 10⁶ PC3^PSCA and 4 weeks later 1 × 10⁶ UniCAR-T cells were iv injected. Eight weeks tumor post-inoculation, mice were treated with ip injections of 250 ng _7KATM^PSCA/g bw bid for 7 consecutive days. Tumors were measured twice weekly. Mice were killed when predefined end points were met. (A) The tumor volume at the start of treatment was 362 ± 220 mm³, 265 ± 103.1 mm³ and 561.6 ± 386.5 mm³ in PC3^PSCA + UniCAR, PC3^PSCA + UniCAR + _7KATM^PSCA and PC3^PSCA, respectively. Tumor volume progress was significantly delayed by administration of _7KATM^PSCA in tumor-bearing mice engrafted with UniCAR-T cells. (B) Tumor volume doubling time (VDT) was significantly increased in the study cohort compared with the controls. Bars represent mean ± SD. (C) Administration of _7KATM^PSCA led to a significant improved overall survival of tumor-bearing, UniCAR-T cell engrafted NSG mice. Data are pooled from 2 independent experiments with individual donors, n = 8−12 mice per cohort (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 4.

Upregulation of Th1 and Th2 cytokine production in the plasma of UniCAR-T cell engrafted mice treated with _7KATM^PSCA. The plasma of UniCAR-T cell engrafted, tumor-bearing mice was analyzed for human Th1/Th2 cytokines in dependence of _7KATM^PSCA administration. INF-γ (A), TNF-α (B), IL-18 (C) and IL-4 (D) were the only detectable human cytokines. All of them were increased in mice receiving _7KATM^PSCA, while only the increment of TNF-α and IL-18 was statistically significant. Bars represent mean ± SD. Data pooled from 3 independent experiments with individual donors (n = 13–14). (LLOQ = lower limit of quantification). Values below the LLOQ were set as zero for the statistical analysis (*p < 0.05).

Figure 5.

Phenotypic and functional characterization of effector and target cells in vivo. Tumors were excised right before start of the treatment and immediately after one week therapy with _7KATM^PSCA and were analyzed for various factors associated with activity of the UniCAR platform. (A) The proportion of viable tumor cells (hCD45⁻hCD3⁻hEpCAM⁺), which did not express PSCA, was numerically increased in the “PC3^PSCA + UniCAR + _7KATM^PSCA group,” however the difference did not reach statistical significance. (B) Administration of _7KATM^PSCA to UniCAR-T cell engrafted, PC3^PSCA tumor-bearing NSG mice led to a significant increase in tumor-infiltrating viable UniCAR-T cells (hCD45⁺hCD3⁺GFP⁺) (PC3^PSCA + UniCAR: 1.9 ± 2.3, PC3^PSCA + UniCAR (early): 2,1 ± 2.4, PC3^PSCA + UniCAR + _7KATM^PSCA: 9.5 ± 8,3) (C) Representative dot blots of post-thymic differentiation of CD8⁺ T cells and CD8⁺ UniCAR-T cells. The addition of _7KATM^PSCA in vivo did not further alter the differentiation phenotype. Most CD8⁺ effector cells were effector memory cells (CCR7⁻CD45RO⁺): graft 45.3 ± 19.7%, early group 83.5 ± 8.8%, PC3^PSCA + UniCARs 87.5 ± 4.2%, PC3^PSCA + UniCARs + _7KATM^PSCA 90.3 ± 4.4%. (D) Tumor-infiltrating UniCAR-T cells were sorted from NSG mice immediately after the treatment period. Purified UniCAR-T cells were co-incubated for 24 h with ⁵¹Cr labeled PC3^PSCA in vitro in the presence or in the absence of _7KATM^PSCA. Most analyses were performed in triplicates. Data were pooled for e:t < 5:1 (range 1:1–4:1) and e:t ≥ 5:1 (range 5:1–24:1). At low e:t ratios, in both cohorts ( ± preceding exposition toward _7KATM^PSCA in vivo) no specific lysis could be induced by the addition of _7KATM^PSCA. At higher e:t ratios, addition of _7KATM^PSCA still induced specific lysis, irrespective of the preceding exposition to _7KATM^PSCA in vivo, which proves remaining functionality of the UniCAR platform after in vivo passage. Bars represent mean ± SD. Data pooled from 2 independent experiments with individual donors (*p < 0.05, **p ≤ 0.01).

Figure 6.

Upregulation of immune checkpoint molecules as a function of the activity of the UniCAR platform. PC3^PSCA cells were (co)-incubated with/without UniCAR-T cells, non-signaling UniCAR-T cells (UniCAR^stop) and T cell^vectorCTR in the presence or in the absence of _7KATM^PSCA (EC₉₀) at an e:t ratio of 1:1 for 24 h, 48 h and 72 h. The proportion of target (A, B) and effector cells (C, D), which expressed certain immune checkpoint molecules during co-incubation is shown. Upregulation of the mentioned immune checkpoint molecules was always restricted to the cohort encompassing all components necessary for activity of the UniCAR platform (target cell + TM + functional effector cell). (A) PD-L1 expression on tumor cells significantly increased in the presence of the active UniCAR platform. PD-L1 expression peaked at 48 h of co-incubation (13.9 ± 10.1% of PC3^PSCA at baseline compared with 72.9 ± 24.1% at 24 hours, p ≤ 0.001). (B) PD-L2 expression on tumor cells was also significantly upregulated in the presence of the active UniCAR platform reaching its maximum at 48 h of co-incubation (4.1 ± 3.6% of PC3^PSCA at baseline compared with 42.8 ± 16.1% at 24 hours, p ≤ 0.001). (C) PD-1 upregulation peaked after 24 h of co-incubation and did not further change during the observation period (12.8 ± 10.4% of UniCAR-T cells at baseline compared with 41.4 ± 14.8% at 24 hours, p < 0.05). (D) CD80 upregulation continually increased during the observation period (14.4 ± 3.5% of UniCAR-T cells at baseline compared with 21.2 ± 2.0% at 24 hours, p ≤ 0.01). Bars represent mean ± SD, data pooled from 4–5 independent experiments with individual donors (*p < 0.05, **p ≤ 0.01, ***p ≤ 0.0001).

Figure 7.

Activity of the UniCAR platform in solid tumors is associated with upregulation of PD-L1 and PD-L2 on tumor cells. PC3^PSCA tumor-bearing NSG mice engrafted with UniCAR-T cells were treated for one week with _7KATM^PSCA. Mice were either killed right before and after the treatment period (A,B) or at later time points due to the tumor size (C,D). Tumor cells were analyzed by flow cytometry. The mere presence of UniCAR-T-cells led to a significant upregulation of PD-L1 in vivo. PD-L1 expression was significantly further amplified after treatment with _7KATM^PSCA (UniCAR-T cell engrafted mice without _7KATM^PSCA administration killed after the treatment period compared with UniCAR-T cell engrafted mice receiving _7KATM^PSCA, p ≤ 0.001) (A). An upregulation of PD-L2 on tumor cells was only found in association with the active UniCAR platform comprising target cells, effector cells and the TM (UniCAR-T cell engrafted mice without _7KATM^PSCA administration compared with UniCAR-T cell engrafted mice receiving _7KATM^PSCA, p < 0.05). (B). With time delay to the administration of _7KATM^PSCA, PD-L1 expression declined comparing to the mice killed right after therapy but still remained significantly elevated compared with mice without UniCAR-T cell engraftment. The significant difference observed between both UniCAR-T cell engrafted cohorts right at the end of the treatment period had vanished. PD-L1 expression of both cohorts was still numerically elevated compared with UniCAR-T cell engrafted mice before the treatment period ( = early group) (C). PD-L2 expression did not decline and also remained significantly elevated in those mice which had been treated with _7KATM^PSCA compared with mice without UniCAR-T cell engraftment. In contrast to earlier time points, there was no significant difference between both UniCAR-T cell engrafted cohorts anymore (D). Bars represent mean ± SD. Data are pooled from 2 independent experiments with individual donors (n = 10) (*p < 0.05, **p ≤ 0.001,***p ≤ 0.001, ****p ≤ 0.0001).