Novel antibody probes for the characterization of endosialin/TEM-1.

Daniel J O’Shannessy,Young Park,Stephen M Jackson,Xin Cheng,Brad Kline,Michael F Smith,Brian Tomkowicz,Andrew Milinichik,Earl Albone,Elizabeth B Somers,Regan Fulton,Danielle Fernando,Pankaj Oberoi,Nicholas C Nicolaides

doi:10.18632/oncotarget.11018

Abstract

Endosialin (Tumor Endothelial Marker-1 (TEM-1), CD248) is primarily expressed on pericytes of tumor-associated microvasculature, tumor-associated stromal cells and directly on tumors of mesenchymal origin, including sarcoma and melanoma. While the function of endosialin/TEM-1 is incompletely understood, studies have suggested a role in supporting tumor growth and invasion thus making it an attractive therapeutic target. In an effort to further understand its role in cancer, we previously developed a humanized anti-endosialin/TEM-1 monoclonal antibody (mAb), called ontuxizumab (MORAb-004) for testing in preclinical and clinical studies. We herein report on the generation of an extensive panel of recombinant endosialin/TEM-1 protein extracellular domain (ECD) fragments and novel mAbs against ECD motifs. The domain-specific epitopes were mapped against ECD sub-domains to identify those that can detect distinct structural motifs and can be potentially formatted as probes suitable for diagnostic and functional studies. A number of mAbS were shown to cross-react with the murine and human protein, potentially allowing their use in human animal models and corresponding clinical trials. In addition, pairing of several mAbs supported their use in immunoassays that can detect soluble endosialin/TEM-1 (sEND) in the serum of healthy subjects and cancer patients.

Highlights

Maintenance of epithelial tissues including those involved in malignant diseases requires interactions with neighboring cells that form tissue stroma
These results indicate that endosialin/Tumor Endothelial Marker-1 (TEM-1) contains at least one trypsin-sensitive cleavage site within its extracellular domain
We have described the development and characterization of a panel of reagents, both monoclonal antibodies and recombinant proteins, that should prove useful for further elucidating endosialin/TEM-1 expression and biological function in cells/tissues from healthy and disease states, especially cancer

Summary

Introduction

Maintenance of epithelial tissues including those involved in malignant diseases requires interactions with neighboring cells that form tissue stroma. Experimental animal models have demonstrated that tumor invasion is stimulated by stromal microenvironments similar to those present in wound healing [5] This observation suggests that growth factors implicated in wound healing such as transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF) may play a role in changing the stromal host www.impactjournals.com/oncotarget compartment in cancer [6]. In both wound healing and tumorigenesis, the fibroblast to myofibroblast transition marks the stromal alteration that leads to the biological functions needed to support the lesion and perturbation of this process using angiogenic inhibitors, such as the antiVEGF antibody bevacizumab, suppress the natural wound healing process

Methods

Results

Conclusion