Targeted polymeric nanoconjugates for BBB penetration and time specific delivery of PDL1 blocking small peptides via LRP1 targeting in melanoma brain metastases

Background: Melanoma brain metastases (MBM) show high CNS tropism with limited benefit from single-agent ICB; a combination regimen with an AXL inhibitor may improve responses but carry substantial toxicity. The brain TiME features scarce T cells, abundant TAMs, and glial PD-L1, promoting immune evasion and poor ICB efficacy. BBB penetrant strategies via peptide-based Immuno-Nano-Conjugates (INCs) are under development, though intrathecal delivery has not improved survival. LRP-1-mediated INCs delivery may enable BBB transport and TAM-TiME reprogramming. Here, we report a targeted nanoplatform that crosses the BBB and delivers PD-L1-blocking peptides to the TiME. Methods: Immuno-Nano-Conjugates (INCs) were synthesized by conjugating a biocompatible malic acid polymer conjugated with (i) P-12, a PD-L1 blocking peptide, and (ii) Angiopep-2 (AP2), a ligand for LRP-1 mediated transcytosis. Therapeutic efficacy, safety, toxicity, and biodistribution studies were assessed using an in vitro static BBB-on-a-chip brain tumor model and in vivo in established murine cancer models. Clinical transcriptomic correlation data from metastatic melanoma (TCGA-SKCM, n = 471) were analyzed via the GDC legacy archive to construct Kaplan-Meier survival curves. Results: The INCs traversed the BBB and demonstrated penetration in a BBB-on-a-chip brain tumor model. Upon delivery, INCs were selectively internalized by the activated TiME environment, thereby promoting antitumor immune cell activation and proliferation. In vivo efficacy, safety, toxicity, and biodistribution revealed superior brain accumulation of INCs relative to ICB agents and significant efficacy and a better safety profile. Clinically, high AXL expression correlated with nonresponse to ICB in Stage IV melanoma and aligned with immunosuppressive myeloid signatures, demonstrating an association with reduced survival in patients with brain metastases. In subcutaneous melanoma models, the combination of P-12 (anti-PD-L1)- based peptides with the AXL inhibitor Bemcentinib yielded greater tumor burden reduction than anti-PD-1 monotherapy, suggesting the potential to circumvent ICB resistance in MBM settings. Conclusion: Our data indicate that BBB transcytosis of INCs via LRP-1 mediates enhanced delivery of PD-L1 blocking peptides to the TiME, supporting improved therapeutic exposure for MBM. AXL emerges as a critical therapeutic target and a rational axis for combination strategies with targeted nanoplatforms to treat resistant MBM. Ongoing studies across additional MBM-relevant models aim to validate these findings and further optimize second-generation bispecific INCs approaches.