Precision targeting of tumor-associated macrophages via a 6-substituted pyrrolo[2,3-d]pyrimidine chimeric molecule for enhanced cancer immunotherapy

Small-molecule PI3Kγ inhibitors can reprogram immunosuppressive myeloid cells but are limited by rapid clearance, lack of cell-type selectivity, and systemic exposure to non-target tissues. Here, we extended a previously developed folate receptor (FR)-directed 6-substituted pyrrolo[2,3-d]pyrimidine ligand to the intracellular delivery of AS-252424. Comparative optimization of the spacer and payload-attachment architecture identified AS-6, which contains a glutathione-responsive disulfide and an N-methyl carbamate-containing release unit. AS-6 retained ligand-dependent FR-β recognition, exhibited greater stability in mouse plasma, and achieved 8.9-fold higher systemic exposure than the carbonate-linked AS-4 while releasing intact payload under reducing conditions. In the 4T1 orthotopic model, AS-6 drove a functional phenotypic shift in tumor-associated macrophages (TAMs) toward a pro-inflammatory state. This coordinated microenvironmental remodeling significantly suppressed primary tumor growth and distant lung metastasis. Furthermore, AS-6 significantly sensitized these resistant tumors to anti-PD-1 checkpoint blockade while effectively mitigating the severe systemic toxicities of the free inhibitor. Ultimately, this work establishes FR-β targeted chimeric molecules as a reliable chemical strategy to rescue highly potent yet systemically limited therapeutic agents, thereby enhancing cancer immunotherapy. The clinical application of small molecule immunomodulators is limited by poor selectivity for tumor-associated macrophages (TAMs) and systemic toxicities. Here, the authors develop a folate receptor-beta targeted chimeric molecule, AS-6, which selectively accumulates in TAMs, remodels the tumor microenvironment, and enhances the efficacy of anti-PD-1 therapy while reducing systemic side effects.