Melanoma remains a challenging malignancy despite the significant outcomes achieved with immune checkpoint inhibitor (ICI) monotherapy. Here, we investigated a polymer-based chemo-immunotherapy strategy combining KT-1, a backbone-degradable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–epirubicin conjugate that induces immunogenic cell death (ICD), with MPPA, a multivalent HPMA copolymer–peptide antagonist of PD-L1 (PPA: (NYSKPTDRQYHF). In B16F10 melanoma, a 3-day dosing schedule significantly outperformed 7-day dosing. KT-1 monotherapy induced CD8+ T cell–mediated immunity through increased infiltration and upregulation of effector genes (Prf1, Gzmk, Eomes, Xcl1, Cxcl10), with depletion studies confirming CD8+ T cell dependence.

Concurrent KT-1 + MPPA administration proved superior to sequential dosing. Single-cell RNA sequencing revealed that KT-1 promoted dendritic cell maturation and CD8+ T cell activation, while MPPA selectively reversed KT-1–induced PD-L1 upregulation on tumor cells. The combination enhanced dendritic cell activation, CD8+ T cell cytotoxicity, and reduced regulatory T cell immunosuppression. Importantly, MPPA did not induce autoimmune diabetes in NOD mice, in contrast to conventional anti–PD-L1 antibodies, and showed no observed immune-related adverse effects highlighting the safety. These findings support HPMA copolymer-based chemo-immunotherapy as a safer, effective alternative to traditional ICI regimens for treating immune-excluded tumors.