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

Pulmonary Embolism Mortality Prediction Using Multimodal Learning Based on Computed Tomography Angiography and Clinical Data

Purpose: Pulmonary embolism (PE) is a significant cause of mortality in the United States. The objective of this study is to implement deep learning (DL) models using Computed Tomography Pulmonary Angiography (CTPA), clinical data, and PE Severity Index (PESI) scores to predict PE mortality. Materials and Methods: 918 patients (median age 64 years, range 13-99 years, 52% female) with 3,978 CTPAs were identified via retrospective review across three institutions. To predict survival, an AI model was used to extract disease-related imaging features from CTPAs. Imaging features and/or clinical variables were then incorporated into DL models to predict survival outcomes. Four models were developed as follows: (1) using CTPA imaging features only; (2) using clinical variables only; (3) multimodal, integrating both CTPA and clinical variables; and (4) multimodal fused with calculated PESI score. Performance and contribution from each modality were evaluated using concordance index (c-index) and Net Reclassification Improvement, respectively. Performance was compared to PESI predictions using the Wilcoxon signed-rank test. Kaplan-Meier analysis was performed to stratify patients into high- and low-risk groups. Additional factor-risk analysis was conducted to account for right ventricular (RV) dysfunction. Results: For both data sets, the PESI-fused and multimodal models achieved higher c-indices than PESI alone. Following stratification of patients into high- and low-risk groups by multimodal and PESI-fused models, mortality outcomes differed significantly (both p<0.001). A strong correlation was found between high-risk grouping and RV dysfunction. Conclusions: Multiomic DL models incorporating CTPA features, clinical data, and PESI achieved higher c-indices than PESI alone for PE survival prediction

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

Fusion proteins and antibodies targeting human red blood cell antigens

Compositions and methods are provided for loading cargoes onto red blood cells. Provided herein are novel antibodies, fragments, fusion proteins and other conjugates which specifically bind red blood cells via RHCE or Band 3.http://deepblue.lib.umich.edu/bitstream/2027.42/176352/2/WO2019113224A1.pdfDescription of WO2019113224A1.pdf : Published versio

Abstract 6: Co-targeting of Thrombomodulin and the Endothelial Protein C Receptor Protects the Pulmonary Vasculature in a Mouse Model of Acute Endothelial Injury

Background Recombinant activated protein C is protective in animal models of sepsis, stroke, and focal thrombosis, but has a poor side effect profile. Thrombomodulin (TM) and the Endothelial Protein C Receptor (EPCR) are endothelial membrane proteins, which partner to bind thrombin and activate protein C at sites of inflammation, ischemia, and thrombosis. Both proteins can be lost from the endothelial surface in a variety of disease states. We have shown previously that targeted delivery of TM to the Platelet Endothelial Cell Adhesion Molecule (PECAM-1) on the endothelial surface is protective in mouse models of lung inflammation and ischemia. Unlike endogenous TM, however, recombinant TM targeted to PECAM-1 does not effectively partner with endogenous EPCR. Objective We hypothesized that co-delivery of TM and EPCR would enhance in vitro activity and therapeutic efficacy in vivo. Methods TM and EPCR were fused to single chain fragments of two anti-PECAM antibodies, which are known to demonstrate collaborative binding enhancement. TM and EPCR fusion proteins were tested on PECAM-expressing cells and mouse endothelial cells. Fusion proteins were injected intravenously in C57BL/6 mice prior to intratracheal injection of endotoxin. Expression of VCAM-1 was measured using qPCR and MIP-2 was quantified in bronchoalveolar lavage (BAL) fluid. Endothelial barrier dysfunction was quantified by measuring transendothelial leak of 125I-labeled albumin. Results TM and EPCR fusion proteins target PECAM-1 on endothelial cells and demonstrate collaborative binding. Independent of binding effects, the EPCR fusion protein results in a 50% increase in the activation of protein C by cell-bound TM, an effect blocked by anti-EPCR antibody. Consistent with in vitro results, the combination of TM and EPCR fusion proteins enhances protection in endotoxin-induced lung injury, reducing VCAM expression, BAL MIP-2, and transendothelial leak of albumin (p &lt; 0.001), as compared to TM fusion protein alone. Conclusion Co-targeting of TM and EPCR to PECAM-1 enhances activation of protein C and represents a novel and promising therapeutic strategy for the treatment of diseases involving acute endothelial injury. </jats:sec

Targeting Thrombomodulin to Endothelial ICAM-1 Rather Than PECAM-1 Allows Partnering with the Endothelial Protein C Receptor and Enhances Protection of Endothelial Barrier Function

Abstract Abstract 3351 The thrombomodulin-protein C pathway has important antithrombotic and anti-inflammatory roles in a variety of disease models and human illnesses, including severe sepsis, acute lung injury, and focal ischemia &amp; reperfusion. We recently reported that anchoring recombinant thrombomodulin (TM) to PECAM-1 on the luminal surface of the vascular endothelium is protective in a mouse model of inflammatory lung injury. To this point, it has been unclear whether targeted thrombomodulin is able to partner with the endothelial protein C receptor (EPCR) in the same way as its endogenous counterpart. Here we demonstrate that anchoring TM to endothelial ICAM-1, rather than PECAM-1, results in approximately 10-fold greater activation of protein C. Furthermore, blocking protein C binding to EPCR results in marked reduction of protein C activation when TM is targeted to ICAM-1, whereas protein C activation is largely independent of EPCR when PECAM-1 is used as the target ligand. Consistent with this in vitro observation, anti-ICAM/TM fusion protein provides greater in vivo protection in a mouse model of inflammatory lung injury than its PECAM-targeted analogue, more potently blocking expression of pro-inflammatory cytokines and more effectively stabilizing endothelial barrier function. Since ICAM, endogenous TM, and EPCR are all thought to localize to microdomains on the apical surface of endothelial cells, we hypothesize that ICAM-targeting more effectively mimics the natural configuration and brings TM within sufficient proximity to allow access to its membrane co-factor. These observations could have profound implications for the therapeutic efficacy of a whole series of endothelial-targeted proto-drugs and their potential for translational success. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Endothelial targeting of nanocarriers loaded with antioxidant enzymes for protection against vascular oxidative stress and inflammation

Endothelial-targeted delivery of antioxidant enzymes, catalase and superoxide dismutase (SOD), is promising strategy for protecting organs and tissues from inflammation and oxidative stress. Here we describe Protective Antioxidant Carriers for Endothelial Targeting (PACkET), the first carriers capable of targeted endothelial delivery of both catalase and SOD. PACkET formed through controlled precipitation loaded ~30% enzyme and protected it from proteolytic degradation, whereas attachment of PECAM monoclonal antibodies to surface of the enzyme-loaded carriers, achieved without adversely affecting their stability and functionality, provided targeting. Isotope tracing and microscopy showed that PACkET exhibited specific endothelial binding and internalization in vitro. Endothelial targeting of PACkET was validated in vivo by specific (vs IgG-control) accumulation in the pulmonary vasculature after intravenous injection achieving 33% of injected dose at 30 min. Catalase loaded PACkET protects endothelial cells from killing by H(2)O(2) and alleviated the pulmonary edema and leukocyte infiltration in mouse model of endotoxin-induced lung injury, whereas SOD-loaded PACkET mitigated cytokine-induced endothelial pro-inflammatory activation and endotoxin-induced lung inflammation. These studies indicate that PACkET offers a modular approach for vascular targeting of therapeutic enzymes