Nanovesicles integrating PD-1-mediated targeting and CRISPR/Cas9-based CD47 editing for dual immune checkpoint blockade.

Immunotherapy with immune checkpoint inhibitors has revolutionized cancer treatment, yet many tumors evade immune surveillance through multiple suppressive mechanisms. In particular, the adaptive immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and the innate "don't eat me" signal CD47/signal-regulatory protein alpha (SIRPα) represent two distinct pathways that cancers exploit to avoid T-cell attack and macrophage phagocytosis, respectively. Herein, we present BITE (Biomimetic Immune Targeting and Editing), a genetically engineered biomimetic nanoplatform designed to concurrently blockade both pathways by combining PD-1-mediated tumor targeting with CRISPR/Cas9 gene editing of CD47. BITE nanovesicles display PD-1 on their surface, enabling selective binding to PD-L1-expressing tumor cells and local disruption of PD-1/PD-L1 signaling. Simultaneously, they deliver a CRISPR/Cas9 payload that knocks out the CD47 gene in tumor cells, abolishing the anti-phagocytic signal and thus activating innate immune clearance. We demonstrate that BITE efficiently homes to PD-L1-positive tumors in vitro and in vivo, achieves significant CD47 gene disruption in tumor cells, and triggers robust phagocytosis by macrophages. In a mouse tumor model, dual checkpoint blockade by BITE reshapes the tumor microenvironment, yielding increased infiltration of CD4 T cells, CD8 T cells, and M1 macrophages; treatment with BITE induces pronounced tumor regression and extended survival, outperforming single-target controls. Our results establish a proof-of-concept for this dual-function nanovesicle approach, highlighting its potential to engage both adaptive and innate immunity synergistically. The BITE platform offers a versatile and targeted strategy to overcome immune resistance in cancer, representing a promising therapeutic avenue in biomedical engineering and nanomedicine.