Immunogenic eradication of melanoma via ultrasmall lactoferrin-ICD inducer nanoconjugates integrated with BRAF-inhibitory liposomes

Approximately 50% of human melanoma is driven by B-Raf protooncogene mutation (BRAF-mutant). Tumors with such mutation are highly immunosuppressive, and often resistant to immune checkpoint inhibitor (ICI) therapies. Blocking the BRAF-MAPK pathway in the BRAF-mutant melanoma cells markedly increases the tumor antigen expression and hence enhances the ability of T cells to recognize autologous BRAFV600 mutant melanoma cells. In parallel, induction of immunogenic cell death (ICD) of melanoma cells can overcome immunosuppression and enhance the antigen presenting capacity of dendritic cells (DCs). Therefore, our goal is to engineer a novel nanomedicine for combined delivery of BRAF inhibitor and ICD inducer drugs for immunogenic eradication of melanoma cells and thus boost the efficacy of ICI therapy. First, we engineered liposomes encapsulating dabrafenib (DFN, BRAF inhibitor) and selumetinib (SLM, MEK inhibitor). To ensure their stability, we coupled DFN and SLM to cholesterol via pH-responsive carbamate and ester bonds, respectively prior to incorporation into liposomes. The liposomes showed uniform size, high drug loading and excellent physical stability as well as high internalization and cytotoxicity against A375 melanoma cells. Moreover, the expression of MHC I & II by A375 cells was enhanced after treatment with liposomes. Second, we synthesized ultrasmall lactoferrin (LF) nanoconjugates with ICD inducer drugs including doxorubicin (DOX), shikonin and bortezomib via a pH-responsive hydrazone bond to induce ICD of melanoma cells. The LF-DOX nanoconjugate showed a small size (38.4 nm) with efficient internalization into A375 cells resulting in high cytotoxicity. The engineered nanoconjugate successfully induced ICD of melanoma cells as evidenced by increased surface exposure of CRT and reduced intracellular expression of HMGB1 in A375 cells. Moreover, the ICD inducing nanoconjugate induced remarkable degree of apoptosis (33%) of A375 melanoma cells compared to control non-treated cells (5.7%). Finally, we encapsulated the ultrasmall ICD inducing LF-drug nanoconjugate into the aqueous core of the pH-responsive DFN/SLM liposomes decorated with alphaPD-L1 antibody. A mouse syngeneic melanoma model with BRAF mutation was established by subcutaneously implanting YUMM1.7 Melanoma cells (1×10 5 ) in the flanks of 4-6 weeks old C57BL/6 mice. The intravenously injected nanomedicine markedly increased the antitumor efficacy with significant reduction of tumor volume relative to vehicle treated group and mice treated with alphaPDL1 alone. In conclusion, the combined delivery of BRAF inhibitory and ICD inducing nanomedicine into one platform could boost the antitumor immune response to alphaPDL1 therapy and hence overcome the immune resistance of melanoma cells.