Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74

Abstract

Purpose: Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T-targeted antigens in tumors and accurate monitoring of response.

Experimental design: We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer.

Results: [18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses.

Conclusions: This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy. See related commentary by Weber et al., p. 5241.

Figure 1.. Structure of [¹⁸F]AlF-FAPI-74 and its uptake in FAP-expressing cells in vitro.

(A) Structure of FAPI-74 and radiolabeled [¹⁸F]AlF-FAPI-74. (B) 1×10⁶ I45 WT and human FAP-transduced I45 cells (I45 huFAP) were incubated with [¹⁸F]AlF-FAPI-74 for 1 hour at 37°C in the presence or absence of unlabeled FAPI (10μM). The in vitro uptake study demonstrated a greater than 100-fold increased uptake of the tracer in I45 huFAP cells relative to WT and blocked controls. n=3, data points are mean ± SD. Uptake was measured as percent injected dose per gram (%ID/g) with a gamma counter. Groups were compared using a two-way ANOVA with Tukey’s multiple comparisons test. ****p<0.0001, ns = not significant.

Figure 2.. Imaging of FAP Expression in a I45 Mesothelioma Xenograft Model In Vivo

(A) Representative [¹⁸F]AlF-FAPI-74 PET images of 3 different NSG mice (M742, M743, M744 refer to mouse number) xenografted with I45 WT (left side, blue ROI) and I45 huFAP mesothelioma tumor (right side, red ROI) showed a selective uptake of the tracer in the FAP-expressing, I45 huFAP tumor. [¹⁸F]AlF-FAPI-74 PET/CT images were acquired 1 hour post-radiotracer administration. (B) For [¹⁸F]AlF-FAPI-74 PET quantification, ROIs were drawn around I45 tumors and background muscle. The left panel shows the raw tumor uptake in SUV_mean and SUV_max, and the right panel shows the tumor-to-muscle ratio calculated by dividing the signal from the tumors by the signal from the muscle. I45 huFAP tumor demonstrated 7 to 8-fold higher tracer uptake relative to the WT tumor. n=10, data points are mean ± SD. Groups were compared using an unpaired t-test (two-tailed). ****p<0.0001. (C) Ex vivo biodistribution analysis performed following terminal [¹⁸F]AlF-FAPI-74 imaging (approximately 1.5 hours post-radiotracer administration) showed 12 and 15-fold increased tracer retention in FAP-expressing huFAP tumor relative to the WT tumor and background organs, respectively. n=6, data points are mean ± SD. Groups were compared using a one-way ANOVA with Tukey’s multiple comparison test. ****p<0.0001, ns = not significant. (D) Representative IF images of A549 tumor sections stained with antibodies against FAP, α-SMA, PDGFRα, and DAPI, demonstrated robust FAP expression in the I45 huFAP tumor. Scale = 100μm.

Figure 3.. Imaging of Mouse Stromal FAP Expression in an A549 Lung Adenocarcinoma Xenograft Model In Vivo

(A) Representative [¹⁸F]AlF-FAPI-74 PET images of 2 different NSG mice (M4922 and M4923 refer to mouse number) xenografted with A549 lung adenocarcinoma tumor (right side, pink ROI) 1 hour post-radiotracer administration demonstrated retention of [¹⁸F]AlF-FAPI-74 tracer in the tumor area. (B) [¹⁸F]AlF-FAPI-74 PET quantification, with the left panel showing the raw tumor uptake in SUV_mean and SUV_max, and the right panel shows the tumor-to-muscle ratio. Target-to-background ratio demonstrated 4 (SUV_mean) to 6.5-fold (SUV_max) higher uptake in the A549 tumor relative to muscle. n=12, data points are mean ± SD. (C) Representative IF images of A549 tumor sections stained with antibodies against FAP, α-SMA, PDGFRα, and DAPI, demonstrate robust FAP expression in the tumor, validating PET imaging findings. Scale = 100μm.

Figure 4:. In Vitro Characterization and Validation of FAP CAR T Cell Effector Function

(A) Schematic of pTRPE L2HG FAP CAR-T2A-mCherry backbone. CD3 and mCherry (marker gene for assessment of transduction efficiency and flow-based sorting) are separated by a T2A cleavage site. (B) Primary human T cells were transduced with pTRPE L2HG FAP CAR-T2A-mCherry lentivirus at MOI of 5 and transduction efficiency was assessed with flow cytometry using mCherry and AF647-conjugated F(ab’)₂ fragment. Transduced cells were sorted on mCherry expression and AF647 stain for downstream assays. (C) Target-specific cytolytic activity of FAP CAR T cells were tested by co-incubating them with I45 WT and I45 huFAP target cells overnight. The assay demonstrated an Effector-to-Target ratio (E:T)-dependent killing of the target cells. Percent specific cytotoxicity was determined using MTS assay. n=3, data points are mean ± SD. Groups were compared using a two-way ANOVA with Šídák’s multiple comparisons test. ****p<0.0001, ns = not significant. (D) IFNγ secretion from effector FAP CAR T cells following an overnight exposure to target I45 cells. The level of cytokine secretion was determined by ELISA. Data points are mean ± SD and groups were compared using a two-way ANOVA with Tukey’s multiple comparisons test. ****p<0.0001.

Figure 5:. Monitoring of Therapeutic Response to FAP CAR T Cell Therapy

(A) Schematic of experimental timeline. NSG immunodeficient mice were subcutaneously xenografted with A549 tumor. The tumors were grown for 3 weeks, and all animals were imaged on small animal PET/CT 1 hour following [¹⁸F]AlF-FAPI-74 administration to establish a baseline uptake. Animals were then randomized to receive either 5×10⁶ CAR⁺ FAP CAR T cells or cell number-matched non-transduced control (NTD) T cells via tail vein. The mice were imaged again with [¹⁸F]AlF-FAPI-74 2 weeks following T cell injection for terminal PET imaging and downstream tissue processing. (B) Tumor volume data between CAR T cell injection (Day 0) and follow-up [¹⁸F]AlF-FAPI-74 PET/CT scan (Day 14) demonstrated a statistically significant decrease in tumor volume for the FAP CAR T cell-treated group relative to the NTD control T cell-treated group. n=4 for NTD control T cell group, n=8 for FAP CAR T cell group. Data points are mean ± SD. Data for Day 14 between the two groups were compared using an unpaired t-test (two-tailed). **p=0.0011. (C) Representative [¹⁸F]AlF-FAPI-74 PET/CT images showed statistical differences in tracer uptake between FAP CAR T cell-treated (right, M4917) and non-transduced (NTD) T cell-treated (left, M4916) animals. (D) [¹⁸F]AlF-FAPI-74 PET quantification demonstrated a 2 (SUV_mean) to 3-fold (SUV_max) reduction in target tumor-to-muscle uptake ratio for FAP CAR T cell-treated group relative to the baseline scan. n=4 for NTD control T cell group, n=8 for FAP CAR T cell group. Data points are mean ± SD. Groups were compared using a one-way ANOVA with Tukey’s multiple comparison test. *p=0.0151, ****p<0.0001.

Figure 6:. Ex Vivo Biodistribution and Tumor Analysis for Correlation with Radiologic Findings

(A) Ex vivo biodistribution analysis following the terminal [¹⁸F]AlF-FAPI-74 PET/CT imaging time point on Day 14 showed around 3-fold higher tracer retention in the NTD control T cell-treated tumor compared to the FAP CAR T cell-treated tumor. Data points are mean ± SD and groups were compared using a two-way ANOVA with Šídák’s multiple comparisons test. **p=0.0065, ****p<0.0001, ns = not significant. (B) Flow analysis of cell population in the A549 tumor treated with FAP CAR T cell vs. NTD control T cells showed a higher percentage of total CD45⁺ in FAP CAR T cell-treated tumor. Groups were compared using a Mann-Whitney test. **p=0.0040. (C) Representative IF images of A549 tumor sections stained with antibodies against FAP, α-SMA, PDGFRα, and DAPI. A549 tumor harvested from animals treated with FAP CAR T cells showed lack of FAP expression and patchy areas of apoptotic cells, whereas tumor harvested from NTD-treated animals showed intact and robust expression of FAP, validating PET imaging findings. Scale = 100μm.