Chimeric Antigen Receptor Design in Human Mesenchymal Stromal Cells (CAR-MSCs) Mediates Homing to Target Sites and Downstream Immunosuppression for Immune Disease Treatment

• Published in: Transplantation and cellular therapy 2024-02, Vol.30 (2), p.S11-S12
• Main Authors: Sirpilla, Olivia, Sakemura, R. Leo, Hefazi, Mehrdad, Kimball, Brooke L, Huynh, Truc N, Siegler, Elizabeth L., Kenderian, Saad S.
• Format: Article
• Language: English
• ISSN: 2666-6367; 2666-6367
• DOI: 10.1016/j.jtct.2023.12.034

Allogeneic mesenchymal stromal cell (MSC) therapy for immune disease is generally safe but often ineffective in clinical trials, in part due to poor trafficking and limited immunosuppression. To overcome these obstacles, we have created novel chimeric antigen receptor (CAR)-MSCs. We hypothesized that CAR-MSCs would show superior 1) homing to target tissues due to a tissue-specific antigen binding domain and 2) immunosuppression due to inclusion of immunosuppressive intracellular signaling domains. We previously designed CAR-MSCs directed to human/mouse E-cadherin+(Ecad) tissues susceptible to immune attack in graft vs host disease (GvHD). CAR-MSCs showed a stable stem phenotype, superior suppression of T cells in vitro, and increased therapeutic efficacy in vivo compared to control MSCs. To characterize the mechanisms of CAR-MSC efficacy, we tested the homing of anti-Ecad CAR-MSCs to Ecad+ colonic tissue in GvHD xenograft models (a). To initiate inflammation, immunodeficient mice were irradiated and injected with 1.5 × 107 human PBMCs (B-cell depleted) intravenously. Mice received intraperitoneal injections of 1 × 106 luciferase-GFP+ Ecad-CAR-MSCs or nonspecific CD19-CAR-MSCs as a negative control for homing to the Ecad+ colon. One week later, mouse organs were harvested for bioluminescent and immunofluorescent quantification of MSCs to the colon, while additional mice were monitored long-term for GvHD symptoms, weight loss, and survival. Ecad-CAR-MSC groups showed enhanced MSC homing to the colon compared to CD19-CAR-MSC groups by bioluminescent MSC detection (b). CAR-MSCs also did not home to off-target tissues, such as the lungs. Immunofluorescent staining of mouse colons further supported that Ecad-CAR-MSCs preferentially homed to Ecad+ regions of the colon compared to control CD19-CAR-MSCs (c). Ecad-CAR-MSCs showed significant localization to Ecad+ colonic crypts (d). Ecad-CAR-MSCs also led to a superior prevention of weight loss (e), suppression of circulating human T cells (f), and survival (g) compared to CD19 CAR-MSC control groups. Next, we investigated the significance of CAR-MSC intracellular signaling domains downstream of antigen stimulation. We generated several anti-Ecad CAR-MSC subtypes containing CD28ζ, CD28, CD3ζ, or a null signaling domain as a negative control. These CAR-MSCs were tested in our GvHD mouse models. Our results indicated that CAR-MSCs with CD28ζ significantly outperformed all other subtypes in preventing weight loss (h),