Selective Uptake of Macromolecules to the Brain in Microfluidics and Animal Models Using the HAVN1 Peptide as a Blood-Brain Barrier Modulator

Abstract

Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4-to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.

Exploring How Antibody Format Drives Clearance from the Brain

“…All IRdye-labeled mAbs were synthesized by conjugating IRDye800CW to lysine residues on mAbs . After conjugation and purification from the IRDye800CW, the antibody conjugates were evaluated with SDS-PAGE and the gels were scanned with an Odyssey CLx NIR imager.…”

“…Unfortunately, the BBB presents a formidable obstacle to the delivery of therapeutics to the brain. Previous studies have proposed alternative strategies to increase antibody permeability across the BBB, such as chemical modifications to facilitate receptor-mediated transcytosis mechanisms in BBB endothelial cells, , as well as enhancing paracellular transport through BBB osmotic disruption or BBB modulation by BBBM . Combining these delivery methods with Fab-VG1 could potentially enhance brain exposure when the drug is administered intravenously.…”

“…All IRdye-labeled anti-gD mAbs were generated and purified as in Schwinghamer et al The final antibody concentrations and degree of labeling (dye-to-protein molar ratio) were determined using formulas in which 0.03 was used as the correction factor for IRDye800CW, meaning that the absorbance at 280 nm is equal to 3% of the absorbance at A max (780 nm). ε and ε′ are the extinction coefficients of the antibody and fluorescent dye, respectively. a n t i b o d y c o n c e n t r a t i o n 0.25em ( M ) = A 280 − ( A max ) × C F ε × d i l u t i o n f a c t o r d e g r e e o f l a b e l i n g = A max 0.25em o f l a b e l e …”