Abstract 6097: FcRγ and TLR4 based CARs in DCs modulate antitumor T cell priming
Authors: Shelby Namen, Alex Kim, Gaurav Pandey, Carl DeSelm
Journal: Cancer Research (2025)

Abstract Background: Immunotherapies have advanced the field of cancer therapy for hematologic malignancies but have not had similar success in solid tumors. Due to their abundance in solid tumors and ability to phagocytose, we developed a chimeric antigen receptor (CAR) macrophage (M) that reduced tumor burden in syngeneic mice. However, in syngeneic mice lacking conventional type 1 dendritic cells (cDC1) the effect was lost suggesting that a mechanism of CAR-M function is through antigen transfer to cDC1s, which then prime an antitumor T cell response. Purpose: To directly induce antitumor T cell responses, we tested different antitumor CAR constructs in cDC1s (DCs). Toll-like receptors (TLRs) and Fc receptors (FcRs) are major types of receptors in phagocytes, with different downstream signaling; we focused on understanding the impact of TLR4 and FcRγ signaling CARs on DC function. We compared the impact of each CAR on the DC’s ability to phagocytose tumor cells, cross-present or degrade the captured antigen, induce costimulatory molecule expression, and cross-prime tumor-specific T cells. Methods: Mouse bone marrow derived DCs were electroporated with mRNA encoding for anti-B7H3 (B7) TLR4, FcRγ, or truncated (t) CAR. The CAR DCs were cultured with AF488-ovalbumen (ova) loaded MCA-induced sarcoma tumor that expressed human B7 (B7+) or was B7 negative (B7-) to assess uptake of tumor antigen. To determine the impacts on cross-priming, the CAR DCs were cultured with B7+ and B7- ova loaded MHCI negative tumor cells and naïve CD8 OTI T cells. Results: The FcRγ CAR improved target-specific tumor antigen uptake by &amp;gt;10x compared to CAR(t), while the TLR4 CAR improved tumor uptake by 1.6x. Although the TLR4 CAR DCs did not take up as much antigen, it cross presented the antigen more effectively, as it had 4x more surface expression of MHCI:SIINFEKL compared to the FcRγ CAR. The TLR4 CAR induced slower degradation of antigen compared to the FcRγ CAR. The TLR4 CAR also induced higher levels of CD70, CCR7, and CD86 expression compared to the FcRγ CAR. Both the TLR4 and the FcRγ CAR DCs induced greater proliferation of OTI T cells relative to CAR(t) DCs. The proliferated OTI T cells from the TLR4 condition had a 2.8x increase in tumor killing of ova-loaded, B7- tumor cells, indicating that the quality of the T cell response may differ if the T cells are cross-primed by a DC experiencing TLR4 versus FcRγ signaling at the time of tumor antigen capture. Conclusion: This data suggests that engineered DCs with either FcRγ or TLR4 CAR led to increased tumor uptake and cross presentation compared to a truncated CAR; however, the two CARs lead to different downstream signaling pathways that impact the amount of antigen cross-presented, activation marker expression, and the quality of the antitumor T cell response. These findings suggest de novo antitumor T cell responses can be enhanced and shaped by DCs engineered with CARs having different signaling domains. Citation Format: Shelby Namen, Alex Kim, Gaurav Pandey, Carl DeSelm. FcRγ and TLR4 based CARs in DCs modulate antitumor T cell priming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6097.