Augmenting the Protein C Pathway with Endothelial Targeted Biotherapeutics: Strategies to Promote Partnering of TM and EPCR

The design of targeted recombinant biotherapeutics is a rapidly growing area of translational biomedical research, with particular relevance to acute and life-threatening conditions, in which the available treatment options have narrow therapeutic indices. Although vascular immunotargeting typically has been thought of as a strategy for controlling and altering pharmacokinetics, in the context of biotherapeutic delivery, precise localization may be the primary goal, allowing optimal interaction of drug with endogenous partners. The protein C pathway has important protective roles in a variety of human illnesses, including sepsis and acute lung injury. We recently reported a strategy for augmenting this pathway by anchoring thrombomodulin (TM, CD141) to the endothelium via an affinity ligand to platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31). Endothelial PECAM-1, however, is believed to localize to a different portion of the cell membrane than the majority of endogenous TM and its key co-factor, the endothelial protein C receptor (EPCR, CD201). The current document includes new data indicating that recombinant TM anchored to endothelial PECAM-1 does not partner effectively with EPCR and describes the design, implementation, and validation of two strategies for more effectively replicating the enzymatic partnering of these two molecules. In both cases, proximity of these co-factors on the surface of the endothelial membrane appears to be the key variable and has significant implications, affecting not only functional activity in vitro but therapeutic efficacy in vivo. These findings underscore the complexity of targeting biotherapeutics to the plasmalemma, and suggest that precision on a nanometer scale is necessary for optimal biotherapeutic effect

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

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

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

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

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