Targeting PD-L1 and disrupting Siglec-9 cooperatively potentiate macrophage phagocytosis of breast cancer via a trispecific macrophage engager (TriME).

Breast cancer (BC) remains a leading cause of mortality, highlighting the need for novel immunotherapies capable of overcoming treatment resistance. Programmed death-ligand 1 (PD-L1) is recognized as a therapeutic target, particularly within aggressive subtypes like triple-negative breast cancer (TNBC). To effectively leverage the powerful phagocytic function of macrophages, we engineered a novel trispecific macrophage engager (TriME) designed for simultaneous engagement of three components: CD16 (macrophage activation), IL-15 (immune cell survival and activation), and PD-L1 (tumor recognition). The purified TriME protein demonstrated specific, dose-dependent binding to CD16+ primary monocyte-derived macrophages (MDMs) and PD-L1+ MDA-MB-231 breast cancer cells. Functional assays confirmed that TriME significantly enhanced MDM-mediated phagocytosis of PD-L1+ tumor cells and promoted NK92 cell activation via its IL-15 domain. To further eliminate an inhibitory checkpoint, we generated Siglec-9-null induced pluripotent stem cell (iPSC)-derived macrophages (S9-null iMacs) using CRISPR/Cas9. Interestingly, the combination of TriME with S9-null iMacs markedly enhanced tumor cell phagocytosis beyond the effects of Siglec-9 disruption alone. This functional enhancement was accompanied by increased phosphorylation of Syk (p-Syk), suggesting a strong association with the upregulation of pro-phagocytic signaling cascades. Together, these findings establish TriME as a highly promising therapeutic strategy and demonstrate that combining a potent TriME with checkpoint inhibition can cooperatively enhance macrophage-mediated tumor clearance.