Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

Abstract

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.

Figure 1. Stimulation of the CAR results in upregulation of the A_2AR, which limits CAR T cell cytokine production.

(A) 2 × 10⁶ murine anti-HER2 CAR T cells were stimulated overnight with either anti-CD3 (0.5 μg/ml)/anti-CD28 (0.5 μg/ml), anti-CMYC tag (1:1,000), or an isotype control. (B) 1 × 10⁶ CAR T cells were cocultured with 1 × 10⁶ 24JK or 24JK-HER2 tumor cells for 4 hours. (A and B) Stimulated cells were lysed and RNA isolated. Expression of A2aR and housekeeping gene L32 was determined by qPCR. (A) Data were normalized to unstimulated CAR T cells and are presented as the mean ± SEM of 4 individual experiments. (B) Data are presented as the mean ± SD from a representative experiment of n = 3. (C–F) 2 × 10⁵ CAR T cells were cocultured with 1 × 10⁵ 24JK/24JK-HER2 (C and E) or E0771/E0771-HER2 (D and F) tumor cells in the presence or absence of NECA (1 μM) and SCH58261 (1 μM). Supernatants were collected after 16 hours and the cytokine concentration determined. Data are represented as mean ± SD of triplicates from a representative experiment of n = 4. “θ” indicates that the recorded value was above the maximum indicated on the graph. “LXSN T cells” refers to T cells transduced with the empty retroviral vector (LXSN) control. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA; NS, not significant.

Figure 2. A_2AR is upregulated on CAR T cells in vivo and limits their antitumor efficacy.

C57BL/6-HER2 mice were injected with 1 × 10⁶ 24JK-HER2 (subcutaneous) (A) or 1 × 10⁵ E0771-HER2 tumor cells (B and D–F). (A, B, D, and E) Mice were injected with 1 × 10⁷ CAR T cells derived from WT or A_2AR^–/– splenocytes on days 7 and 8 after tumor injection following total-body irradiation (5 Gy) on day 7. Mice were injected with 50,000 IU IL-2 on days 0–4 after T cell transfer and, where indicated, with anti–PD-1 (200 μg/mouse) or 2A3 isotype control on days 0, 4, and 8 after T cell transfer. (C) 1 × 10⁶ 24JK-HER2 tumor cells were cocultured with 1 × 10⁶ anti-HER2 CAR T cells for 48 hours in the presence of anti–PD-1 (50 μg/ml) or 2A3 isotype control. A further 1 × 10⁶ 24JK-HER2 tumor cells were added after 24 hours to provide chronic stimulation. Cells were lysed after 48 hours. Expression of A_2AR is shown relative to T cells cocultured with 24JK parental tumor cells. (F) E0771-HER2 tumor–bearing mice were treated as above with Ly5.1⁺ CAR T cells. Nine days after T cell transfer, tumors and spleens of tumor-bearing mice were pooled, and CAR (Ly5.1⁺) and endogenous (CD45.2⁺) CD8⁺ T cells were FACS sorted. Expression of A2aR on these subsets was determined by qPCR in triplicate. (A, B, and D) Data are presented as the mean ± SEM of 6–8 mice from a representative experiment of n = 3. (C) Data are represented as the mean ± SD from a representative experiment of n = 2. (E) Survival endpoint was when tumor size reached >100 mm². Data are shown for pooled experiments with n = 14–24 per group. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way ANOVA (A, B, and D), 1-way ANOVA (C and F), or Mantel-Cox test (E).

Figure 3. A_2AR blockade enhances the efficacy of CAR T cells in combination with anti–PD-1 without inducing autoimmune pathology.

C57BL/6-HER2 mice were injected with 1 × 10⁶ 24JK-HER2 (subcutaneous) (A) or 1 × 10⁵ E0771-HER2 tumor cells (B–E) and treated with WT CAR T cells and anti–PD-1 or 2A3 per Figure 2. Mice were also treated daily with either 1 mg/kg SCH58261 (A–C), 1 mg/kg ZM241385 (D), or vehicle control (A, B, and D). Data are presented as the mean ± SEM of 6–14 mice. (C) Data are shown for n = 18–28 per group with tumors greater than 100 mm² taken as the cutoff for survival. (E) H&E sections were taken from the cerebellum and breast (non–tumor-inoculated) in E0771-HER2 tumor–bearing mice undergoing therapy at day 9 after T cell injection. Representative sections are shown at an original magnification of ×200. *P < 0.05, **P < 0.01 by 2-way ANOVA (A, B, and D) or Mantel-Cox test (C).

Figure 4. A_2AR^–/– CD8⁺ CAR T cells exhibit enhanced cytokine production in vivo when combined with PD-1 blockade.

C57BL/6-HER2 mice were injected with 1 × 10⁵ E0771-HER2 tumor cells and treated per Figure 2. On day 6 after T cell injection, TILs and splenocytes were isolated. (A) CD45.2⁺CD3⁺CD8⁺ T cells were FACS sorted 6 days after injection and lysed. IFN-g mRNA was determined by qPCR. Data are expressed relative to IFN-g mRNA content of splenic CD8⁺ T cells. Samples from 3–5 mice were pooled, and qPCR was run in triplicate. (B) TILs were restimulated with PMA (20 ng/ml) and ionomycin (1 μg/ml) for 3 hours in the presence of GolgiPlug/GolgiStop. (B–D) Proportion of CD3⁺CD8⁺ (B and C) or CD3⁺CD4⁺ (D) cells that were IFN-γ⁺, TNF-α⁺, or IFN-γ⁺TNF-α⁺. ICS, intracellular cytokine staining. (C) Representative FACS plots gated on CD3⁺CD8⁺ TILs. (E and F) Proportion (E) and absolute number (F) of splenic CD8⁺ or CD4⁺ cells expressing CD44. (B, D, E, and F) Data are shown as the mean ± SEM for at least 5 mice. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA.

Figure 5. Combined A_2AR and PD-1 blockade enhances the antitumor efficacy of anti-HER2 CAR T cells by increasing CD8⁺ IFN-γ production.

C57BL/6-HER2 mice were injected with E0771-HER2 tumor cells and treated with WT (B and C) or Ly5.1⁺ (A and D) CAR T cells, anti–PD-1 or 2A3, and SCH58261 or vehicle control per Figures 2 and 3. (B and C) Where indicated, mice were also treated with anti–IFN-γ (250 μg/mouse) on days 0, 1, 4, and 8 after T cell transfer. (C) Mean tumor size at day 14 is shown for each group. (B and C) Data are shown as the mean ± SEM for 5 mice per group from a representative experiment of n = 2. (A and D) Day 9 after T cell transfer, TILs were analyzed by FACS. (A) Proportion of CD3⁺CD8⁺ cells that were IFN-γ⁺. (D) Proportion of CD3⁺CD8⁺ or CD3⁺CD4⁺ cells expressing Ki-67. (A and D) Data are shown as the mean ± SEM for at least 3 mice from a representative experiment of n = 3. “LXSN T cells” refers to T cells transduced with the empty retroviral vector (LXSN) control. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA (A, C, and D) or 2-way ANOVA (B).

Figure 6. Targeting the A_2AR with shRNA retroviral technology enhances CAR T cell function.

CAR T cells were transduced with retroviruses encoding A_2AR-directed shRNA or scrambled shRNA control and then selected with 2 μg/ml puromycin from day 4 to day 6. Eight days after activation, CAR T cells were then assessed for function. (A) 1 × 10⁶ CAR T cells were reactivated with plate-bound anti-CD3/anti-CD28 (0.5 μg/ml) for 4 hours and then lysed for RNA analysis. Data are presented as the mean ± SD of triplicates and expressed relative to the production of IFN-γ of control cultures for each shRNA-transduced CAR T cell. (B) 1 × 10⁵ CAR T cells were cocultured with 24JK/24JK-HER2 tumor cells at a 1:1 ratio in the presence or absence of NECA at indicated concentrations. Supernatants were collected after 16 hours and analyzed for concentration of IFN-γ. Data are presented relative to the IFN-γ production of control cultures and as the mean ± SD of quadruplicates from a representative experiment of n = 2. *P < 0.05, **P < 0.01 by 1-way ANOVA.

Figure 7. A_2AR blockade enhances the activity of patient-derived CAR T cells against HER2-expressing autologous melanocytes.

(A) Expression of anti-HER2 CAR on patient-derived CAR T cells. (B) Expression of HER2 and CD73 on primary melanoma samples used as targets in coculture assays. Dashed histograms indicate isotype control staining. (C and D) 2 × 10⁵ human anti-HER2 CAR T cells were cocultured with 1 × 10⁵ autologous primary-derived melanocytes in the presence of NECA (1 μM), SCH58261 (1 μM), CGS21680 (10 μM), or TGF-β (10 ng/ml) where indicated. After 16 hours, supernatants were analyzed for IFN-γ content by cytometric bead array. (A–C) Data are shown from a representative patient of n = 4. (C) Data are the mean ± SD of triplicates. (D) Data are pooled from 4 individual patients and are shown as percentage IFN-γ production normalized to control cultures in each experiment. *P < 0.05 by 1-way ANOVA.