54 research outputs found
Bivalent engagement of endothelial surface antigens is critical to prolonged surface targeting and protein delivery in vivo
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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156501/3/fsb220760_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156501/2/fsb220760-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156501/1/fsb220760.pd
Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting
Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by “vascular immunotargeting.” To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The “collaborative enhancement” of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The “collaborative enhancement” phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents
The requirement for macrophage-lymphocyte interaction in t lymphocyte proliferation induced by generation of aldehydes on cell membranes.
Targeted Delivery of Human Exosomes via Bispecific Antibody Platform
Background: Exosomes are cell-derived nanovesicles which have been proposed as potential drug carriers. Fusion of affinity ligands to exosome surface proteins has been shown to enhance uptake by target tissues, but requires genetic modification of parent cells. Similarly, direct chemical conjugation of affinity ligands can damage exosome structure and lacks reproducible surface modification. We present a novel bispecific antibody platform, which enables reproducible surface decoration of exosomes, increasing uptake by target cells and altering tissue distribution in animals. Methods: Exosome Targeting bispecific Antibodies (ExTAbs) were designed by fusing single-chain variable fragments (scFv) to the C-terminus of the heavy chain of a recombinant, anti-CD63 monoclonal antibody. ExTAbs were expressed in HEK cells, purified using affinity chromatography, and confirmed via SDS-PAGE and size exclusion HPLC. Binding affinities to CD63 and target antigens were determined using flow cytometry and/or ELISA. Decoration of exosomes was quantified via competitive-binding assay using radiolabeled 125 I-anti-CD63. For in vitro targeting experiments, fluorescent exosomes were decorated with ExTAbs or anti-CD63 as a non-targeting control. For in vivo biodistribution studies, radiolabeled exosomes were decorated with ExTAbs (or anti-CD63 as untargeted control) and injected intravenously in mice. Results: ExTAbs targeting mouse ICAM-1, PECAM-1, and CD98hc were synthesized with > 95% purity and demonstrated nanomolar binding affinity to both CD63 (K d = 4.47 ± 0.47nM) and their respective target antigens (e.g., PECAM-1 Kd = 4.63 ± 0.61 nM). Target cells showed an ~20-fold increase in uptake of targeted vs. untargeted exosomes following 30-minute incubation at 37C. Uptake of PECAM-targeted exosomes was significantly higher than untargeted exosomes in heavily-endothelialized organs (e.g., lung, kidney, and heart), including a 10-fold increase in lung uptake (14.8 ± 2.7 v.s. 1.4 ± 0.2 % ID/g, p = 0.001). Conclusion: CD63 bispecific antibodies enable straightforward and reproducible surface decoration of human exosomes. Targeted exosomes demonstrate increased uptake in target cells and organs.http://deepblue.lib.umich.edu/bitstream/2027.42/176966/1/Honors_Capstone_Report_-_Kaitlyn_Pierpont.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/176966/2/Design_Expo_Poster_-_Kaitlyn_Pierpont.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/176966/3/ACS_Spring_2023_ExTAbs_Presentation_-_Kaitlyn_Pierpont.ppt
GENETIC CONTROL OF ALLOGENEIC INTERACTIONS IN THE GUINEA-PIG: II. EVIDENCE FOR ALLOANTIGEN-SPECIFIC SUPPRESSOR CELLS IN THE GUINEA-PIG MIXED LEUKOCYTE CULTURE
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