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

Abstract 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 a nti- l eukemia b ackpack–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.