An LNP-mRNA–based in vivo cell engager as a producer of T macrophage tumor triads in situ for targeted anti-solid tumor therapies.

2547 Background: The solid tumor microenvironment promotes infiltration of myeloid cells, with tumor-associated macrophages as the most abundant innate immune population. A recent study showed tumor-reactive T cells enriched in functional clusters with tumor or antigen-presenting cells, and T cells expanded from these clusters ex vivo exhibit enhanced tumor-killing activity. Bispecific and trispecific antibodies have emerged to link T cells with tumor and myeloid cells, improving antitumor immunity. However, their clinical use is limited by structural complexity, manufacturing challenges, and immunogenicity risks. Here, we developed a “live-cell engager” by harnessing and engineering macrophages in vivo to form T-macrophage-tumor triads, activating both T cells and macrophages near tumor cells to boost antitumor responses. Methods: We engineered macrophages to express a single-chain variable fragment (scFv) targeting CD3 (αCD3), a tumor-specific chimeric antigen receptor (CAR) and CD80 on their surface. Each was encoded by separate mRNAs encapsulated within one lipid nanoparticle formulation (LNP-C-3-80). Mouse bone marrow-derived macrophages (BMDMs) were used ex vivo to evaluate T cell activation and antitumor function. The efficacy of LNP-C-3-80 was further tested in immunocompetent syngeneic models including ID8 ovarian cancer and Hepa1-6 hepatocellular carcinoma. Results: In vitro, BMDMs engineered with LNP-3-80 effectively engaged T cells with macrophages, significantly boosting T cell activation compared to LNP-3 alone, as shown by increased CD69 expression and elevated secretion of IFN-γ, IL-2, and Granzyme B. Furthermore, BMDMs engineered with LNP-C-3-80 were able to engage both T cells and tumor cells, and when co-cultured with T cells, showed tsuperior antitumor activity over control groups (LNP-3-80, LNP-3, LNP-GFP). The LNP-delivered αCD3 and CD80 mRNAs were mainly expressed on macrophages but not on T cells, facilitating macrophage-T cell-tumor cell cluster formation. This proximity enabled mutual activation and synergistic tumor cell killing. In vivo, LNP-C-3-80 achieved nearly complete tumor regression and strong prevention of recurrence in ovarian and liver cancer models after intraperitoneal or intravenous administration. Toxicity studies in healthy C57BL/6J mice showed no organ damage or adverse serum biochemical changes, indicating a favorable safety profile. Conclusions: In summary, in vivo macrophage engineering promotes the formation of T cell–macrophage–tumor cell clusters, reducing immune cell–cancer cell distance and concurrently activating T cells. LNP-C-3-80 shows significantly enhanced antitumor efficacy in syngeneic solid tumor models with minimal toxicity. These results highlight LNP-C-3-80’s strong potential as a promising therapeutic strategy against diverse solid tumors.