Therapeutic Tumor Macrophage Reprogramming in Breast Cancer Through a Peptide-Drug Conjugate

In triple negative breast cancer (TNBC), pro-tumoral macrophages promote metastasis and suppress the immune response. To target these cells, we engineered a previously identified CD206 (mannose receptor)-binding peptide, mUNO, to enhance its affinity and proteolytic stability. The new rationally designed peptide, MACTIDE, includes a trypsin inhibitor loop, from the Sunflower Trypsin Inhibitor-I. Binding studies to recombinant CD206 revealed a 15-fold lower K _D for MACTIDE compared to parental mUNO. Additionally, mass spectrometry showed a 5-fold increase in half-life in tumor lysate for MACTIDE compared to mUNO. Homing studies in TNBC-bearing mice showed that fluorescein (FAM)-MACTIDE precisely targeted CD206 ⁺ tumor-associated macrophages (TAMs) upon intravenous, intraperitoneal and even oral administration, with no significant accumulation in liver. We coupled MACTIDE to the FDA-approved drug Verteporfin, an established photosensitizer for photodynamic therapy and inhibitor of the YAP/TAZ pathway, to generate a conjugate here referred to as MACTIDE-V. In the orthotopic 4T1 TNBC mouse model, non-irradiated MACTIDE-V-treated mice unexpectedly showed a similar anti-tumoral effect and fewer signs of toxicity as irradiated MACTIDE-V-treated mice, leading to subsequent studies on the laser-independent activity of this conjugate. In vitro studies using bone-marrow derived mouse macrophages showed that MACTIDE-V excluded YAP from the nucleus, increased the phagocytic activity and upregulated several genes associated with cytotoxic anti-tumoral macrophages. In mouse models of TNBC, MACTIDE-V slowed primary tumor growth, suppressed lung metastases, increased markers of phagocytosis and antigen presentation in TAMs and monocytes, increasing the tumor infiltration of several lymphocyte subsets. We therefore propose MACTIDE-V as a useful peptide-drug conjugate to modulate macrophage function in the context of breast tumor immunotherapy.