Monoclonal antibody (mAb) ligands recognizing distinct extracellular epitopes of PECAM-1.

<p>(<b>A</b>) MAbs investigated in this study to probe the affinity and accessibility to distinct epitopes of human PECAM-1 (huPECAM-1; mAbs 62 and 37) and mouse PECAM-1 (muPECAM-1; mAbs 390 and MEC13.3). Listed is the effect of various anti-PECAM-1 mAbs on PECAM-1-dependent homophilic adhesion, as defined by the aggregation of L-cells fibroblast transfectants expressing PECAM-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Nakada1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Yan3" target="_blank">[50]</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Yan1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Nakada1" target="_blank">[22]</a>. (<b>B</b>–<b>C</b>) Diagram of immunoreactive regions within PECAM-1 domains 1 and 2. (<b>B</b>) Amino acid (AA) location of distinct non-overlapping epitopes for binding of mAbs 62 and 37 on Ig-domain 1 (IgD1) of huPECAM-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Nakada1" target="_blank">[22]</a>. (<b>C</b>) AA location of epitopes for mAbs 390 and MEC13.3 on Ig-domain 2 (IgD2) of muPECAM-1 (H. DeLisser, unpublished results). Peptide sequence recognized by mAbs are colored in red.</p

Anti-PECAM-1 [¹²⁵I]-mAb binding in live cells is enhanced by paired mAb directed to adjacent PECAM-1 epitope.

<p>The modulation of PECAM-1 binding was determined by co-incubation of [¹²⁵I]-mAb with indicated concentrations of unlabeled self-paired mAb or paired mAb with cells for 2 h at 4°C. Binding data were plotted as [¹²⁵I]-mAb molecules bound per cell (mAb/cell) and data points were fit as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s4" target="_blank">Methods</a>.” (<b>A</b> and <b>B</b>) Unlabeled mAb 62 competitively inhibits binding of [¹²⁵I]-mAb 62 to huPECAM-1 in HUVEC. However, mAb 37 enhances [¹²⁵I]-mAb 62 binding to huPECAM-1 in HUVEC by 1.5−fold over binding of [¹²⁵I]-mAb 62 alone. Interestingly, mAb 62 does not enhance the binding of [¹²⁵I]-mAb 37 (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958.s003" target="_blank">Figure S3</a></b>). (<b>C</b>–<b>D</b>) Collaborative binding studies of mAbs 390 and MEC13.3 with REN-muP cells as described in panel A. Unlabeled self-paired mAb 390 and mAb MEC13.3 competitively inhibit binding of [¹²⁵I]-mAb390 and [¹²⁵I]-mAb MEC13.3 to REN-muP cells, respectively. In contrast, mAb pairs [¹²⁵I]-mAb 390/MEC13.3 and [¹²⁵I]-mAb MEC13.3/390 enhance binding by ∼1.5−fold and ∼2.7−fold, respectively, over [¹²⁵I]-mAb alone (***, P<0.001, <i>n</i> = 3–4).</p

In vitro enhancement of binding, accessibility and therapeutic output of anti-PECAM-1 390 scFv-TM fusion protein <i>via</i> dual epitope-engagement of muPECAM-1.

<p>(<b>A</b>) Cell surface binding of the therapeutic fusion protein 390 scFv-TM to REN-muP cells was assessed in the presence of 200 nM self-paired parental mAb 390 or paired mAb MEC13.3 by ELISA. The curves shown are representative ELISA. Only binding to REN-muP cells shown; there was no significant binding detected using control REN cells lacking muPECAM-1. Binding affinity of 390 scFv-TM, reflected by IC₅₀, increases 3.8−fold when paired with MEC13.3. The IC₅₀ is reported as the mean IC₅₀ value ± SD of three independent experiments performed in triplicate. (<b>B</b>) Generation of activated protein c (APC), a cell-protective species, on the surface of REN-muP cells is initiated by targeted binding of 390 scFv-TM (+thrombin). APC generation is augmented up to 5−fold when 390 scFv-TM binding is enhanced with paired mAb MEC13.3 compared to 390 scFv-TM alone. (<b>C</b>) Co-IP of the MEC13.3/muPECAM-1/390 scFv-TM-FLAG complex in REN-muP cells. REN-muP cells were treated with muPECAM-1 targeted rat anti-mouse IgG MEC13.3 and anti-mouse 390 scFv-TM-FLAG combinations. Cell lysates were immunoprecipitated with Protein G agarose beads to MEC13.3 and analyzed by SDS-PAGE and immunoblotting (IB) using anti-muPECAM-1, anti-FLAG, and rat polyclonal anti-mouse antibodies, as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s4" target="_blank">Methods</a>.” For controls, REN-muP cells ±390 scFv-TM FLAG were incubated with Protein G beads alone (lanes 1 and 5, 3 and 7). 390 scFv-TM-FLAG was only detected in the IP for REN-muP cells co-treated with MEC13.3 and 390 scFv-TM-FLAG (lane 6), indicating an interaction between MEC13.3 and 390 scFv-TM through muPECAM-1. Data are representative of two independent experiments.</p

Binding parameters of anti-PECAM-1 [¹²⁵I]-mAbs to live cells expressing PECAM-1.

<p>Cell surface binding parameters (K_d and B_max) of [¹²⁵I]-mAbs to PECAM-1 was determined by RIA-based method with (<b>A</b>) native huPECAM-1 on HUVECs, and (<b>B</b>) recombinant muPECAM-1 on REN-muP cells. Serial dilutions of [¹²⁵I]-mAbs were added to confluent cellular monolayers and incubated for 2 h at 4°C. The results shown are from a representative experiment, with the inset showing Scatchard plot of binding data. Note that total binding was corrected for NSB using 100−fold excess of unlabeled mAb for HUVECs or using parent REN cells for REN-muP binding. (<b>C</b>–<b>D</b>) K_d and B_max Binding parameters are for [¹²⁵I]-mAbs to huPECAM-1 and muPECAM-1 are listed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s2" target="_blank">Results</a> were determined by three independent RIA experiments performed in quadruplicate, with data expressed as mean ± SD.</p

<i>In vitro</i> binding properties of mAb to live cells expressing PECAM-1.

<p>Cell surface binding of mAbs to PECAM-1 was determined by ELISA-based method with (<b>A</b>) HUVECs, (<b>B</b>) REN-muP cells. Proteins were added to confluent cellular monolayers at the indicated dilutions and incubated for 2 h at 4°C. The results shown are from a representative experiment. Non-targeted IgG or non-PECAM-1 expressing cells were used as negative control. Representative plots for mAb binding to MS1 cells are available in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958.s002" target="_blank">Figure S2</a></b>. (<b>C</b>) Analysis of the relative binding affinity of anti-PECAM-1 mAbs, when binding to cells is half-maximal (IC₅₀). Data points were fit as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s4" target="_blank">Methods</a>.” The IC₅₀ is reported as the mean IC₅₀ value ± SD of three independent experiments performed in triplicate.</p

Targeting to Endothelial Cells Augments the Protective Effect of Novel Dual Bioactive Antioxidant/Anti-Inflammatory Nanoparticles

Oxidative stress and inflammation are intertwined contributors to numerous acute vascular pathologies. A novel dual bioactive nanoparticle with antioxidant/anti-inflammatory properties was developed based on the interactions of tocopherol phosphate and the manganese porphyrin SOD mimetic, MnTMPyP. The size and drug incorporation efficiency were shown to be dependent on the amount of MnTMPyP added as well as the choice of surfactant. MnTMPyP was shown to retain its SOD-like activity while in intact particles and to release in a slow and controlled manner. Conjugation of anti-PECAM antibody to the nanoparticles provided endothelial targeting and potentiated nanoparticle-mediated suppression of inflammatory activation of these cells manifested by expression of VCAM, E-selectin, and IL-8. This nanoparticle technology may find applicability with drug combinations relevant for other pathologies

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>

Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging

Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate ∼14 small-molecule fluorophores (AlexaFluor 488) and 3–4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging

APC generation by TM fusion proteins on non-endothelial REN cells with and without EPCR expression.

<p>(a) anti-PECAM scFv/TM and anti-ICAM scFv/TM activate protein C while bound to PECAM and ICAM-expressing cells, respectively. Minimal APC is generated on wild type REN cells, presumably due to lack of binding. (b) A ∼4-fold increase in APC generation is seen when PECAM and ICAM-targeted TM fusion proteins are anchored to cells which stably express mouse EPCR (i.e. REN-PECAM-EPCR and REN-ICAM-EPCR cells), as compared to EPCR-negative counterparts. All experiments were done in triplicate. Data shown are mean ± SD.</p

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