Bivalent engagement of endothelial surface antigens is critical to prolonged surface targeting and protein delivery in vivo

Abstract

Targeted drug delivery to the endothelium has the potential to generate localized therapeutic effects at the blood‐tissue interface. For some therapeutic cargoes, it is essential to maintain contact with the bloodstream to exert protective effects. The pharmacokinetics (PK) of endothelial surface‐targeted affinity ligands and biotherapeutic cargo remain a largely unexplored area, despite obvious translational implications for this strategy. To bridge this gap, we site‐specifically radiolabeled mono‐ (scFv) and bivalent (mAb) affinity ligands specific for the endothelial cell adhesion molecules, PECAM‐1 (CD31) and ICAM‐1 (CD54). Radiotracing revealed similar lung biodistribution at 30 minutes post‐injection (79.3% ± 4.2% vs 80.4% ± 10.6% ID/g for αICAM and 58.9% ± 3.6% ID/g vs. 47.7% ± 5.8% ID/g for αPECAM mAb vs. scFv), but marked differences in organ residence time, with antibodies demonstrating an order of magnitude greater area under the lung concentration vs. time curve (AUCinf 1698 ± 352 vs. 53.3 ± 7.9 ID/g*hrs for αICAM and 1023 ± 507 vs. 114 ± 37 ID/g*hrs for αPECAM mAb vs scFv). A physiologically based pharmacokinetic model, fit to and validated using these data, indicated contributions from both superior binding characteristics and prolonged circulation time supporting multiple binding‐detachment cycles. We tested the ability of each affinity ligand to deliver a prototypical surface cargo, thrombomodulin (TM), using one‐to‐one protein conjugates. Bivalent mAb‐TM was superior to monovalent scFv‐TM in both pulmonary targeting and lung residence time (AUCinf 141 ± 3.2 vs 12.4 ± 4.2 ID/g*hrs for ICAM and 188 ± 90 vs 34.7 ± 19.9 ID/g*hrs for PECAM), despite having similar blood PK, indicating that binding strength is more important parameter than the kinetics of binding. To maximize bivalent target engagement, we synthesized an oriented, end‐to‐end anti‐ICAM mAb‐TM conjugate and found that this therapeutic had the best lung residence time (AUCinf 253 ± 18 ID/g*hrs) of all TM modalities. These observations have implications not only for the delivery of TM, but also potentially all therapeutics targeted to the endothelial surface.

Generation of high affinity ICAM-1-specific nanobodies and evaluation of their suitability for allergy treatment

“…SPR-based affinity measurements showed high affinity binding of Nb44 to ICAM-1. The dissociation constants, calculated as the ratios of off/on-rate, were in the (sub)nanomolar range (10 -9 to 10 -10 M), which was in or above the range determined for other highly affine antibodies directed at ICAM-1 ( 49 – 51 ). Importantly, we measured slow off-rates of 10 -4 /s.…”

Physiologically based modeling of LNP-mediated delivery of mRNA in the vascular system

“…To account for differences between ICAM and PECAM expression, relative estimates of accessible target were obtained from our recent publication. 15 These values were then scaled to relevant values for LNPs based on the estimate of LNP-accessible PECAM obtained in this study. All other parameters were fixed to those values used for PECAM-targeted LNPs.…”

Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs

“…Proteins were directly radioiodinated with [ 125 I]NaI (Perkin Elmer) using Pierce iodination reagent (Thermo Fisher, 28601) and purified using Zeba desalting columns. Radiochemical purity was assessed via thin layer chromatography (TLC) performed using aluminum TLC silica gel 60 F254 plates (Millipore Sigma, 105554) and a 75%:25% mixture of methanol and 1 M sodium acetate (pH 6.8) as a mobile phase, as previously reported 87 . Radiolabeling efficiency was typically >75% and proteins were only used for radiotracing if free 125 I was <5% following purification.…”