A logic-gated trispecific engager enhances macrophage killing of cancer cells in solid tumors.

The antitumor efficacy of immune cell engagers that bind two targets on the same immune cell is limited by structural constraints, leading to incomplete coengagement and uncoordinated signaling. Here, we develop a trispecific macrophage engager (TrME) that both activates the prophagocytic receptor lipoprotein receptor-related protein 1 (LRP1) and blocks the antiphagocytic receptor signal regulatory protein alpha (SIRPα). This 'activate and block' AND logic gate, when coupled to a tumor-targeting moiety, enables coordinated signaling that enhances macrophage cytotoxicity against solid tumors. The TrME tandemly links monovalent LRP1 activator calreticulin, anti-SIRPα scFv and a tumor-associated antigen (TAA)-targeting arm through flexible linkers. Computational modeling and screening of tandem constructs revealed an optimal conformation for robust cis-targeting, allowing logic-gated control of ratiometric prophagocytic and antiphagocytic signaling. In situ generation of TrME by delivering mRNA encoding TAA-targeting TrME through an optimized lipid nanoparticle system activates macrophages and induces antitumor responses, significantly inhibiting tumor growth and prolonging survival in multiple solid tumor mouse models.