Background Chimeric antigen receptor (CAR) T cell therapy has shown limited success in treating solid tumors. One approach for improving antitumor efficacy in solid tumors is to equip CAR T cells with synthetic cytokine receptors to enhance cell-autonomous function. Our lab recently published that an orthologous IL-2/IL-9 receptor could augment antitumor immunity.1 Here we have engineered mouse and human CAR T cells targeting the tumor antigen mesothelin to express an authentic IL-9 receptor (IL-9R), providing them with an IL-9 signal (CAR-IL9R T cells).
Methods For in vitro studies, T cells were isolated from mouse splenocytes or human donors, activated, transduced, expanded and harvested for in vitro assays. For in vivo studies, we established a syngeneic model in which C57BL/6 mice were implanted with PDA7940b, a pancreatic ductal adenocarcinoma cell line derived from KPC mice. Animals were then treated with intravenous injection of CAR-IL9R T cells and intratumoral injection of an adenovirus encoding murine IL-9 (Ad-mIL9). Control groups included mice treated with CAR T cells, CAR-IL9R T cells, or Ad-mIL9. At the time of peak CAR T cell expansion, tumors were collected and analyzed by flow cytometry and immunohistochemistry. To determine the antitumor efficacy of human IL-9 signaling CAR T cells, we established a xenograft flank tumor model by implanting AsPC-1 tumor cells in NSG mice and treated them with CAR-IL9R T cells combined with an adenovirus encoding human IL-9 (Ad-hIL9).
Results CAR-IL9R T cells displayed a unique STAT signaling profile, a shift towards a stem cell-like memory T cell phenotype, and improved effector function in vitro compared with control CAR T cells. In our syngeneic model, tumor progression was significantly reduced, overall survival was improved, and higher numbers of CAR T cells were detected in tumors harvested from mice treated with CAR-IL9R + Ad-mIL9 compared with control groups. No significant changes in the tumor microenvironment were observed across the treatment groups, suggesting that the improved antitumor efficacy of IL-9 signaling CAR T cells is largely due to T cell-intrinsic mechanisms. Supporting this hypothesis, treatment with CAR-IL9R T cells + Ad-hIL9 significantly improved antitumor efficacy in immunodeficient NSG mice. Ongoing studies seek to understand the mechanisms underlying the gain-of-function of IL-9 signaling CAR T cells.
Conclusions CAR T cells provided with an IL-9 signal display a unique T cell phenotype combining the beneficial functional characteristics of stem cell-like memory and effector T cells, leading to enhanced in vivo antitumor activity supported by cell-intrinsic mechanisms.
Kalbasi A, Siurala M, Su LL, Tariveranmoshabad M, Picton LK, Ravikumar P, Li P, Lin JX, Escuin-Ordinas H, Da T, Kremer SV, Sun AL, Castelli S, Agarwal S, Scholler J, Song D, Rommel PC, Radaelli E, Young RM, Leonard WJ, Ribas A, June CH, and Garcia KC. Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature. 2022;607(7918):360–5.
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