X-ray Crystallography of a Nanobody/JAM-1 Complex Informs Barrier Weakening and Enhanced Drug Permeability in a Cellular Blood–Brain Barrier Model

Abstract For pharmacological treatments of brain disorders, the blood–brain barrier (BBB) poses a formidable obstacle, tightly restricting the cerebral uptake of therapeutic agents. Tight junction (TJ) binders, including peptides, proteins, and monoclonal antibodies, that specifically bind to the extracellular domains (ECDs) of TJ proteins are increasingly being explored as BBB-weakening agents for enhanced drug delivery to the brain. Given that nanobodies (Nbs) can access epitopes that are often inaccessible to conventional antibodies, we obtained 16 Nbs with unique CDRs via immunization of alpaca with the ECD of either human (h) or murine JAM-1 (mJAM-1), a major TJ protein. Twelve Nb candidates exhibited high-affinity binding to mJAM-1, as measured by surface plasmon resonance (SPR), and these interactions were further validated for 9 Nbs by immunostaining of JAM-1-overexpressing cells. Five Nbs were found to weaken the barrier of the eGFP-hCldn5-MDCKII cellular monolayer, as shown by reduced transendothelial electrical resistance (TEER). X-ray crystallography revealed that the lead barrier-weakening Nb, E6, binds to the membrane-proximal nondimeric D2 domain of mJAM-1. Mutating key residues at this interface eliminated the BBB-weakening effect of E6, as evidenced by unaltered sodium fluorescence (NaFI) leakage in both eGFP-hCldn5-MDCKII and mJAM1-mGL-MDCKII cellular monolayers compared with the nontreated control. Our study highlights the potential of JAM-1-targeting Nbs as BBB modulators, offering a promising approach for generating TJ binders to enhance brain drug delivery.