Targeting the DNA damage response enhances CD70 CAR-T cell therapy for renal carcinoma by activating the cGAS-STING pathway

Abstract

Chimeric antigen receptor T-cell (CAR-T) therapy has shown tremendous success in eradicating hematologic malignancies. However, this success has not yet been extrapolated to solid tumors due to the limited infiltration and persistence of CAR-T cells in the tumor microenvironment (TME). In this study, we screened a novel anti-CD70 scFv and generated CD70 CAR-T cells that showed effective antitumor functions against CD70⁺ renal carcinoma cells (RCCs) both in vitro and in vivo. We further evaluated the effect and explored the molecular mechanism of a PARP inhibitor (PARPi) in CAR-T cell immunotherapy by administering the PARPi to mouse xenografts model derived from human RCC cells. Treatment with the PARPi promoted CAR-T cell infiltration by stimulating a chemokine milieu that promoted CAR-T cell recruitment and the modulation of immunosuppression in the TME. Moreover, our data demonstrate that PARPi modulates the TME by activating the cGAS-STING pathway, thereby altering the balance of immunostimulatory signaling and enabling low-dose CAR-T cell treatment to induce effective tumor regression. These data demonstrate the application of CD70 CAR-T cell therapeutic strategies for RCC and the cross-talk between targeting DNA damage responses and antitumor CAR-T cell therapy. These findings provide insight into the mechanisms of PARPis in CAR-T cell therapy for RCC and suggest a promising adjuvant therapeutic strategy for CAR-T cell therapy in solid tumors.

Keywords: CAR; CD70; PARP; RCC; Renal carcinoma; Tumor microenvironment; cGAS-STING pathway.

Fig. 1

PARP inhibitors promote tumor regression for RCC models under low-dose CD70 CAR-T cell treatment. a Schemas of CD70-CARs incorporating different spacers [CD8α Signal peptide, CD8α Hinge, (G₄S)₃ Linker, and CD8α™] and costimulatory domains (4-1BB). b Lysis of spheres of 786-0 target cell cultures in the presence of CD70 CAR-T cells, or Mock CAR-T cells (control) at a 1:1, 2:1, 4:1, 8:1 effector/target ratio. (Scale bar: 250 μm) c A real-time cytotoxicity assay (xCElligence RTCA SP) was used to evaluate the lysis of the indicated tumor cells when treated with mock CAR-T (E⁻) cells or CAR-T (E⁺) cells at the indicated E/T ratios over a 50-h period. Representative of three independent experiments. d ELISA results showed the IL-2, TNF-α and IFN-γ secretion levels by CD70 CAR-T (E⁺), mock CAR-T (E⁻) cells encountering 786-0 cell. e Subcutaneous renal cell carcinoma tumor (786-0) development was monitored by in vivo bioluminescence imaging (5 × 10⁶ CAR-T cells/mice). Images taken at dpi. 0, 8, 10 are shown (exposure time of 40 s). f Data showing the tumor volume (mm³) change trend of B-NDG mice xenograft 786-0 tumor regression/growth in 4 different treat groups. g Kaplan–Meier survival curve was performed 140 days after 786-0 cells injection. Mice treated with CD70 CAR-T cells had a significantly longer survival probability in comparison with mice treated with PBS, T or mock CAR-T cells. h ELISA results showed the IL-2, TNF-α and IFN-γ secretion levels in mice blood treated by PBS, T, mock CAR-T, CD70 CAR-T cells. i Treatment scheme used in the 786-0 xenograft model treated with OLA and CAR-T cells. j B-NDG mice were treated with OLA and 2.5 × 10⁶ CAR-T cells/mice. Subcutaneous renal cell carcinoma tumor (786-0) development was monitored by in vivo bioluminescence imaging. Images taken at dpi. 0, 3, 6, 9, 20 are shown (exposure time of 40 s). k Data showing the tumor volume (mm³) change trend of B-NDG mice in 5 different treat groups. l Mice body weights monitored during treatment. m Kaplan–Meier survival curve was performed 150 days after 786-0 cells injection. Mice treated with CD70 CAR-T cells had a significantly longer survival probability in comparison with mice treated with T + Vehicle (DMSO), olaparib (OLA), T + OLA, CAR-T + Vehicle, CAR-T + OLA. n–p ELISA results showed the IL-2, TNF-α and IFN-γ secretion levels in mice blood treated by T + Vehicle, OLA, T + OLA, CAR-T + Vehiele, CAR-T + OLA. q CD70 CAR-T cells in peripheral blood were detected using flow cytometry on day15 after CAR-T inoculation. All error bars represent SD. In all plots, ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 2

Olaparib activation of cGAS-STING pathway is key to promote tumor killing of CAR-T cell. a Olaparib (OLA)- and vehicle (DMSO)-treated tumors were harvested 5 days post-treatment, detecting the phenotype of CAR-T/T cells infiltrating in tumor tissue by flow cytometry. b OLA- and DMSO-treated tumors were harvested 5 days post-treatment, subjected to immunofluorescence (IF) analysis for CD8. (Scale bar: 50 μm) c Statistical analysis of CD8⁺ T cells in the results of immunofluorescence analysis. d Representative images of the level of γH2AX and cytosolic double-stranded DNA (dsDNA) in 786-0 cells after OLA or DMSO treatment. Scale bar, 10 μm. e Immunoblots of markers in the cGAS-STING pathway including total and phospho (p) STING (S366), total and phospho TBK1 (S172), cGAS, total and phospho IRF3 (S396) in lysates collected from RCC cell lines treated with OLA or DMSO. TBB5 served as a loading control. f Representative images of the level of chemokines CCL5 in 786-0 cells after OLA or DMSO treatment. Scale bar, 10 μm. g The CCL5, CXCL10 and Granzyme B IF staining were performed in tumors from the resected tumors from Fig. 2b. Representative images of staining intensity are shown. (Scale bar, 20 μm). h–j Quantification of tumor sections immunostained for CCL5, CXCL10 and Granzyme B -positive areas quantified for each field (N = 5). k CAR-T- and OLA-treated 786-0^shSTING tumors were harvested 5 days post-treatment, quantification of CCL5, CXCL10 and Granzyme B in the results of IF analysis from Additional file 1: Fig. S6f (N = 3). l CAR-T- and OLA-treated 786-0^shSTING tumors were harvested 5 days post-treatment, statistical analysis of CD8⁺ T cells in the results of IF analysis from Additional file 1: Fig. S6g (N = 3). m Model for cGAS-STING pathway activation in response to DDR targeting in RCC. In the proposed model, targeting the DDR protein PARP with the small-molecule inhibitor OLA leads to cytosolic DNA in RCC models. The cytosolic DNA is then recognized by cGAS, which leads to activation of the STING/TBK1/IRF3 pathway. IRF activation leads to increased expression of IFNβ and enhanced expression of the chemokines CXCL10 and CCL5. STING pathway activation and increased chemokine expression lead to the recruitment and secretion of large amounts of Granzyme B by CD8⁺ CAR-T cells leads to enhanced antitumor immunity in RCC models. All error bars represent SD. In all plots, ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001