Biohybrid M13 phage backpacks for active blood-brain barrier traversal and precision central nervous system leukemia treatment.

Central nervous system (CNS) leukemia remains a major clinical challenge in acute lymphoblastic leukemia, largely due to the restrictive nature of the blood-brain barrier (BBB), which severely limits therapeutic delivery and contributes to high relapse rates and poor prognosis. Here, we report ALB-phage, a bioengineered anti-leukemia backpack-phage system designed for active BBB navigation and intracranial drug delivery. M13 bacteriophages were genetically engineered to display BBB-homing ligands, forming a high-aspect-ratio biological transport platform (BTP). A leukemia-targeting therapeutic backpack (L-Pak) was constructed by encapsulating methotrexate within lipid nanoparticles fused with leukemia cell membranes, enabling homotypic targeting. The L-Pak was conjugated to the BTP via a matrix metalloproteinase-responsive PLGVR linker, allowing selective cleavage within the cerebrospinal fluid. Owing to its filamentous morphology, ALB-phage achieved efficient BBB translocation predominantly through energy-dependent macropinocytosis with an additional contribution from clathrin-mediated endocytosis. Both in vitro and in vivo studies demonstrate that ALB-phage effectively penetrates the BBB and selectively eradicates leukemia blasts, resulting in marked tumor suppression and a significant extension of median survival from 29 to 40 days in cell-line-derived xenograft models. Collectively, this M13 bacteriophage-based all-in-one platform integrates targeted BBB penetration with homology-mediated cytotoxicity, offering a promising strategy for the treatment of CNS leukemia and other central nervous system malignancies.