Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma

Abstract

Purpose: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells).

Experimental design: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids.

Results: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors.

Conclusions: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Figure 1.. Meso^FAP CAR T-cells specifically recognize their target antigens and eliminate mesothelin-expressing PDAC cells and FAP-expressing fibroblasts.

A. Schematic of meso^FAP CAR T-cells targeting PDAC and CAFs (created with BioRender.com). B. Graphic representation constructs used to make TEAM-secreting CAR T-cells. C. Representative histograms of flow cytometric analysis of mesothelin and FAP expression on PDAC cell lines, AsPC-1 and PDX-1294. D-E. CAR^TEAM cytotoxicity against AsPC-1 and PDX-1294 assessed by (D) a luciferase-based specific lysis assay and (E) a real-time impedance-based assay. F. Representative histograms of flow cytometric analysis of FAP expression on the fibroblast-cell line MRC-5 and HFF. G. Representative histograms including bar graph of flow cytometric analysis of FAP and mesothelin expression on CAF-1. H-I. Flow cytometric detection of His tag on TEAMs that recognize their specific target antigen on FAP+ HFF cells or CD19+ K562 cells. J. CAR^TEAM cytotoxicity against MRC-5 assessed by a luciferase-based specific lysis assay. All data represent mean +/−SEM and are representative of at least 3 independent experiments. Stars indicate significance between biological triplicates using a two-tailed t-test at the 10:1 ratio (panels D and I), and one-way ANOVA with Dunnett’s multiple comparison post hoc analysis (panel E), or HFF vs. K562 (panel H). *p<0.05, **p<0.01, ****p<0.0001, E:T = effector to target ratio. PDAC: pancreatic ductal adenocarcinoma, CAF: cancer-associated fibroblasts, UTD: untransduced T-cell.

Figure 2.. Meso^FAP CAR T-cells or UTD mixed with FAP TEAM specifically eliminate FAP-expressing fibroblasts and are superior to CARs targeting mesothelin only in PDAC + CAF co-cultures.

A. Schematic of meso^FAP CAR T-cells targeting HFF fibroblasts. B. Real-time impedance-based cytotoxicity assay of sorted CAR^TEAM or UTDs with supernatant from meso^FAP CAR T-cells against FAP-expressing HFF using an effector to target ratio (E:T) of 1:1. C. Schematic of transwell system containing UTDs and CAFs in the bottom well and TEAM-secreting CAR T-cells in the upper well. D. Real-time impedance-based cytotoxicity assay of sorted CAR^TEAM in the transwell system with CAF-1 and UTD cells in the lower well, E:T = 1:1. E. Schematic of co-culture system containing target-cells CAF-1 and AsPC-1 in a 1:1 ratio treated with CAR or UTD as effector cells (E:T = 1:5 = 0.2 CAR : (1 CAF : 1 PDAC)). F. Real-time impedance-based killing assay in the co-culture system (CAFs and AsPC-1 in 1:1 ratio) treated with meso^FAP, meso^CD19, or anti-mesothelin CAR T-cells or UTD controls. All data are plotted as the mean +/−SEM. Stars indicate significance between biological triplicates assessed by one-way ANOVA and Dunnett’s multiple comparison post hoc analysis. ***p<0.001, ****p<0.0001. PDAC: pancreatic ductal adenocarcinoma, CAF: cancer-associated fibroblasts, UTD: untransduced T-cell. Schematics were created with BioRender.com.

Figure 3.. Meso^FAP proliferate more in the presence of PDAC and CAF and demonstrate a different activation profile than control CAR T-cells.

A. Fold change in T-cell expansion when CAR^TEAM or UTD were repeatedly stimulated with AsPC-1 and CAF-1. (AsPC-1:CAF-1:T-cell ratio of 1:1:2). B. Exhaustion markers expressed by T-cells in the co-culture at day 13 as measured by flow cytometry. Data is from one representative donor. C. Schematic of co-culture set-up (created with BioRender.com). T-cells (live, CD45+, CD3+, and mCherry+) were sorted 24h after the 3rd stimulation (day 14) and RNA analysis was performed by NanoString. D. Volcano plot of differential RNA expression of flowcytometric sorted (expressing the same level of mCherry) meso^FAP CAR T-cells versus meso^CD19 CAR T-cells following co-culture with AsPC-1 and CAFs. The top 20 genes are annotated. The grey horizontal line indicates an adjusted p-value of 0.05 and 0.001. E. Pathway analysis of NanoString data comparing meso^FAP and meso^CD19 CAR T-cells from 3 independent healthy donors (ND218, ND210, and ND100). All data represent mean +/−SEM of at least biological triplicates. MFI-sorted CAR T cells or UTDs were added in biological triplicates per condition. Stars indicate significance as determined by one-way ANOVA and Dunnett’s multiple comparison post hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001, E:T = effector to target ratio. PDAC: pancreatic ductal adenocarcinoma, CAF: cancer-associated fibroblasts, UTD: untransduced T-cell, TEMRA: T-effector memory and CD45+/CCR7− subset, ND: normal donor.

Figure 4.. Live cell, time-lapse microscopy shows reduced edge dynamics and acoustic force microscopy reveals increased binding avidity of meso^FAP in comparison to controls.

A. Representative image of sorted meso^FAP CAR T-cells (pink/salmon) interacting with AsPC-1 (cyan), CAFs (magenta), or AsPC-1 and CAFs. B. Representative images visualizing edge dynamics (percentage change of CAR T-cell area over time) of meso^FAP CAR T-cells over 20 frames (frame 1 dark blue to frame 20 white). C. Percentage change of meso^FAP CAR T-cell area over time (number of cell interactions counted: PDAC, n=28; P+C, n=25; CAF, n=42; not on target, n=25). D,F. Representative images visualizing edge dynamics of D. meso^CD19 CAR T-cells or F. UTD T-cells. E,G. Percentage change of E. meso^CD19 CAR T-cells over time (number of cell interactions counted: PDAC, n=28; P+C, n=16; CAF, n=18; not on target, n=23) or G. UTDs over time (number of cell interactions counted: PDAC n=12; P+C, n=8; CAF, n=10; not on target, n=14). H-J. Schematic showing 1:1 ratio of AsPC-1 and CAFs in the flow chamber to measure cell binding of meso^FAP (H), meso^CD19 (I), and UTD (J) avidity by acoustic force microscopy (created with BioRender.com). K. Percentage of meso^FAP, meso^CD19, or UTD bound to 1:1 AsPC-1 and CAF mixture (left) with an increasing amount of acoustic force (pN) and (right) at 1000pN as measured by z-Movi. L. Fold increase of bound meso^FAP CAR T-cells or UTD cells with the addition of FAP-TEAM on HFF fibroblasts using z-Movi. All data represent mean +/−SEM. Representative images have been chosen within 1 standard deviation from the median. Results were obtained from biological duplicates, from at least 3 different locations on the slide. Stars represent significance as determined by unpaired two-tailed t-tests. *p < 0.05, **p < 0.01 ***p < 0.001****p < 0.0001. UTD: untransduced T-cell, pN: picoNewton, P+C: PDAC+CAF.

Figure 5.. meso^FAP CAR T-cells are efficacious against PDAC and CAFs in vivo.

A. Schematic of experimental setup in which CAF-1 and AsPC-1 (9:1 ratio) were subcutaneously implanted in the flank of NSG mice. Mice were treated intravenously with UTD, meso^FAP, or meso^CD19 CAR T-cells 2 days after tumor-CAF implantation. B. Tumor volume over time as measured by caliper. C. Representative hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining for GFP (AsPC-1), and CD3 (T-cells) on tumors harvested on day 18 from mice treated with UTD, meso^CD19 or meso^FAP CAR T-cells. D. Quantification of C as % area positive for each marker. E. Schematic of experimental setup with AsPC-1 and AsPC-1 mesothelin −/− FAP+ in a 1:1 ratio being subcutaneously implanted into the flank of NSG mice. Mice were treated intravenously with UTD, meso^FAP, or meso^CD19 CAR T-cells 7 days after tumor implantation. F. Tumor volume over time as measured by caliper. G. Schematic of experimental hemi-spleen model for liver metastasis. H. Representative immunofluorescence images of PDAC (AsPC-1 GFP+) and CAFs (mCherry+) metastasis in murine liver. I. Kaplan-Meier curve of survival cohort of mice (n=7 mice carried out through death). J. IHC representation of sequential cut slides from liver metastasis from mice sacrificed on day 21. K. Quantification of J as percent positive cell for each marker (n=3). Data represent mean +/−SEM. Stars indicate significance as determined by unpaired two-tailed t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (panels D and K). N=4-5 animals/group, repeated with T-cells from two healthy donors. Data represent two-way ANOVA with correction for multiple comparisons, individual p-values are indicated *p < 0.05, ****p < 0.0001 (panel B and F). Data represent survival analysis (Kaplan-Meier) individual p-values are indicated *p < 0.05, ns = not significant (panel I). Schematics were created with BioRender.com.

Figure 6.. Meso^FAP CAR T-cells outperform control CAR T-cells in PDX in vivo and ex vivo models.

A. Schematic of experimental design in which CAFs and PDX1291 cell line (9:1 ratio) were subcutaneously implanted in the flank of NSG mice. Mice were treated intravenously on day 2 post-tumor-CAF implantation with UTD, meso^FAP, and meso^CD19 CAR T-cells. B. Transduction efficiencies of normalized CAR^TEAM including UTD of in vivo experiment from 3 independent experiments. C. Tumor volume over time as measured by caliper (n=5 mice per group). D. Representative hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining for GFP (PDX1294), FAP (CAF), and CD3 (T-cells) on tumors harvested on day 14. Scale bar indicates 100mm. E. Quantification of D. as percent area positive for each marker; 4-6 representative examples quantified. F. Schematic of PDOTS experimental setup including the 3D microfluidic co-culture system with a side channel (where CAR ^TEAM are added with an E:T of 1:3) and center/gel region (where tumor spheroids are grown in collagen hydrogels). G. Evaluation of live/dead cells on day 5 of meso^FAP-, meso^CD19-, and CD19^FAP-treated or tumor-only PDOTS (n=3). H. Secreted cytokine and chemokines analysis as Log₂ fold change (relative to control samples) on day 5 of meso^FAP, meso^CD19, CD19^FAP, and tumor-only PDOTS from F. Data represents mean +/−SEM. Stars indicate significance as determined by unpaired two-tailed t-tests (panels B and D) or one-way ANOVA with Tukey’s multiple comparisons test (panel F). *p < 0.05, **p < 0.01 ***p < 0.001****p < 0.0001, ns: not significant. Schematics were created with BioRender.com.

Figure 7.. Efficacy of meso^FAP CAR T-cell in patient-derived organoids (PDO) with matching CAFs.

A. PDAC patient information corresponding to PDO and CAF co-cultures. B. Schematic of meso^FAP CAR T-cells targeting organoid co-cultures consisting of PDAC PDO and CAF. C. Co-culture transillumination at day 2 and day 5 of co-culture visualized by Nikon ECLIPSE Ti2 microscope at 20X. Transillumination and RPF images merged by NIS-Elements AR Software. D. CD3+ recovery acquired at both day 2 and day 5. E. Representative flow cytometry plots of specific cytotoxicity of meso^FAP targeting JHH317 FAP+ CAF and PDO co-culture at day 5. F. Flow cytometric analysis showing PDO count recovered on day 2 and day 5. G. Flow cytometric analysis showing CAF count recovered on day 2 and day 5. H. Flow cytometric analysis of activation and exhaustion markers on CD3+ CART-cells at day 2 and day 5. I. VITAL CTL killing assay of target PDO with CAR T effector cells, and UTD T-cells in increasing ratios of effector : target/T cells (technical duplicate; unpaired, two-tailed t-test, p=0.09 at 10:1:1 ratio). Data represent means of biological and technical triplicates. Stars indicate significance as determined by Wilcoxon test (panels D, F, and G) or paired, two-tailed t-test (panel H and I). *p <0.05, ****p <0.0001. PDO: pancreatic ductal adenocarcinoma organoids, Dx: diagnosis, NGS: next-generation sequencing. Schematics were created with BioRender.com.