Relationship between Targeting Efficacy of Liposomes and the Dosage of Targeting Antibody Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) was used in this research to investigate the targeting efficacy (i.e., the binding affinity) of antibody-modified liposomes. The results indicated that liposomes modified by targeting antibodies exhibited an increase in apparent binding affinity, a result attributed to the avidity effect. More specifically, the targeting effect improved as the surface density of the targeting antibody increased, an increase primarily attributed to the decrease of the dissociation rate. However, this trend stopped when the surface density reached a threshold of approximately 1.5 × 10⁸ antibody/mm². This surface density was found to be quite consistent regardless of the liposome size and the type of targeting antibody. In addition, a traditional cell binding experiment was conducted to confirm the saturation point obtained from SPR

Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1.

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo <i>in vitro</i> and <i>in vivo</i>

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in purified muPECAM.

<p>Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in purified muPECAM.</p

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb 390 in live cells.

<p>Binding curves of anti- [125I]-mAb 390 alone (“solo”) or in the presence of unlabeled mAb Mec13.3 to paired epitope (”paired”) in live cells stably expressing recombinant muPECAM-1 (RmP) (A) and mutant form of muPECAM-1 (RmPK89A) (B) determined by RIA-based method. Increasing concentrations of [125I]-mAb treatments solo or with a paired mAb were added to cells and incubated at 4°C for 2h. The results are presented as total binding corrected for nonspecific binding on REN-WT cells. The effect of co-incubation with paired mAb vs solo was not significant. Yet, Kd changed 0.86 ±0.07-fold in RmP and 0.83±0.05 in RmPK89A. The insets show Scatchard analysis of experiments.</p

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in cellular homogenates of live cells stably expressing muPECAM-1.

<p>Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in cellular homogenates of live cells stably expressing muPECAM-1.</p

Anti-muPECAM-1 [125I]-mAb binding to purified muPECAM is enhanced by paired mAb.

<p>Binding parameters of anti-muPECAM-1 mAbs (A) Mec13.3 and (B) 390 with recombinant purified muPECAM-1. Millipore Multiscreen filter plates were coated with 5 μg/ml of recombinant muPECAM-1 and incubated for 2 h at 37°C. Radioligand binding assay was performed as described for cellular homogenates. The results are presented as total binding corrected for nonspecific binding on Filter Plates coated with 1% BSA. Kd of Mec13.3 sees 0.53 ± 0.03-fold change in solo vs paired. Again, Bmax values increase for both mAbs Mec13.3 and 390 co-incubated with a paired mAb. The insets show Scatchard analysis of experiments.</p

Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in live cells stably expressing full-length (RmP) and mutant forms (RmP_K89A) of muPECAM-1.

<p>Collaborative enhancement binding of anti-muPECAM-1 [125I]-mAb in live cells stably expressing full-length (RmP) and mutant forms (RmP_K89A) of muPECAM-1.</p

Anti-muPECAM-1 [125I]-mAb binding in cellular homogenates of live cells is enhanced by paired mAb.

<p>Binding parameters of anti-muPECAM-1 mAbs (A) Mec13.3 and (B) 390 in cellular homogenates of live cells stably expressing muPECAM. Membrane preparations were added to antibody cocktail [125I]-mAb solo or with paired mAb and incubated for 2h, followed by filtering through Millipore Multiscreen Filter Plates using vacuum manifold. The results are presented as total binding corrected for nonspecific binding on membrane preparations of REN-WT cells. Kd of Mec13.3 sees 0.77 ± 0.02-fold change in solo vs paired. Notably, Bmax values increase for both mAbs Mec13.3 and 390 co-incubated with a paired mAb. The insets show Scatchard analysis of experiments.</p