Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform

Abstract

Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purpura (TTP), as autoimmune disorders may produce self-reactive specificities. The yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage in the novel vector pAGA2 for yeast-display expression. The TTP yeast-display library (10⁹ members) was screened by magnetic and flow sorting with human TEM1 recombinant protein. A pool of yeast-display scFv able to detect 2 nM of TEM1 was obtained and transformed into yeast-secreted scFv by homologous recombination using the novel p416 BCCP vector for yeast secretion of biotinylated scFv. Anti-TEM1 yeast-secreted scFv were independently validated in vitro by flow cytometry analysis and ELISA assays, then in vivo biotinylated in N-termini to produce biobodies. Biobody-78 bound specifically to Endosialin/TEM1-expressing ovarian tumor in vivo, with functional stability over 48 h. Our results suggest that our novel paired display-secretory yeast libraries can serve as an ideal platform for the rapid isolation of high-affinity reagents, and that anti-TEM1 biobody-78 can be used for in vitro assays including flow cytometry analysis, as well as in vivo for targeted imaging and therapy of cancer.

Introduction

The growth of solid tumors beyond a diameter of 1–2 mm critically depends on the formation of a supporting stroma of newly formed blood vessels (Folkman, 1985). Tumor endothelial cells, stromal fibroblasts (activated fibroblast or myofibroblasts) and/or vascular pericytes acquire a phenotype different from that of normal stromal cells (Rettig et al., 1992, Christian et al., 2008) and express tumor vascular markers (TVM). TVM provide attractive targets for antibody-based tumor diagnosis and therapy (St Croix et al., 2000, Marty et al., 2006, Teicher, 2007, Rouleau et al., 2008) due to i) the relative stability of TVM-expressing cells comparing to tumor cells; ii) neovasculature essential function for tumor maintenance, as demonstrated by the widespread necrosis of solid tumor after destruction of their blood vessels (Hinnen and Eskens, 2007); iii) neovasculature leaky capillaries that permit circulating antibodies and antibody conjugates to easily access TVM.

Endosialin/Tumor Endothelial Marker 1 (TEM1 or CD248) is a TVM and a type I transmembrane protein which comprises an 80.9 kDa protein core modified by extensive sialylated O-linked glycosylation that gives rise to an approximately 175 kDa mature glycoprotein (Christian et al., 2001). Endosialin/TEM1 was originally discovered by an anti-fibroblast monoclonal antibody (FB5) as a glycoprotein expressed by the pericytes and myofibroblasts associated with tumor vasculature (MacFadyen et al., 2005, Christian et al., 2008, Rouleau et al., 2008) as well as by tumor-associated vascular endothelial cells in various human cancers (Rettig et al., 1992, Davies et al., 2004, Rmali et al., 2005, Becker et al., 2008), including ovarian cancer (Conejo-Garcia et al., 2005). Endosialin/TEM1 plays a unique role in tumor progression as a promoting factor of tumor angiogenesis (Bagley et al., 2008), proliferation, migration and metastasis through interaction with matrix proteins such as fibronectin, collagen type I and IV (Tomkowicz et al., 2007) and Mac-2 BP/90K (Becker et al., 2008). Importantly, mice without functional Tem1 gene present a striking reduction in tumor growth, invasiveness, and metastasis after tumor transplantation to abdominal sites (Nanda et al., 2006). Taken together, these results suggest that targeting Endosialin/TEM1 for diagnostic and/or therapy could provide a valuable strategy against cancer.

Isolation of antigen-specific antibodies has been achieved through a variety of methods, including screening of phage- and yeast-display recombinant antibody (scFv) libraries (Vaughan et al., 1996, Feldhaus et al., 2003, Paschke, 2006, Scholler et al., 2006, Scholler et al., 2008a, Scholler et al., 2008b, Bergan et al., 2007). Yeast display recently emerged as an efficient alternative strategy due to the advantages it offers over prokaryotic systems, including faster and more controlled flow cytometry-based selection compared to solid phase panning (Feldhaus et al., 2003, Bergan et al., 2007); a highly efficient sampling of the immune antibody repertoire (Bowley et al., 2007); post-translational modifications (glycosylation) due to the eukaryotic expression; and absence of scFv-induced growth bias because scFv are not displayed during the amplification step, when yeast multiply. Yet, transfer of scFv from displayed to secreted forms has often resulted in loss of antigen specificity and/or affinity, requiring additional time-consuming and costly steps, including in vitro maturation of scFv sequence and/or recloning of scFv fused to immunoglobulin (Ig) constant regions. Mechanisms underlying the loss of scFv function include changes in scFv conformation and post-translational modifications, due to the use of different expression systems for displayed and secreted forms.

We sought to generate a highly efficient system for high-throughput identification of antigen-specific affinity reagents. Because patients with autoimmune disorders produce large variety of antibodies, we hypothesized that a library derived from an autoimmune patient could contain high-affinity antibodies against various antigens, including tumor vascular markers. We also hypothesized that only one expression system (Saccharomyces cerevisiae) for both scFv display and secretion could eliminate changes in scFv post-translational modifications, while keeping the advantages of a eukaryotic system for the expression of high-affinity antibodies, and that conformational changes would be minimized during the shift from displayed to secreted scFv forms if both displayed and secreted scFv were modified only at the N-terminus, which binds to the yeast surface or to secondary reagents, respectively. To test our hypotheses, we generated a 1 × 10⁹ yeast-display scFv library by homologous recombination of our new vector pAGA2 with scFv amplified from a phage-display scFv library derived from a patient with thrombotic thrombocytopenic purpura (TTP), an autoimmune system disease related to the production of autoantibodies against coagulation factors (Siegel, 2008). We screened the TTP yeast-display scFv library in two steps using two complementary yeast systems of expression that permit to express yeast-display and yeast-secreted scFv with the same post-translational modifications while minimizing conformational changes. We identified several Endosialin/TEM1-specific scFv using human TEM1 recombinant protein, including one with affinity in the nanomolar range, that were further transformed in biobodies (Scholler et al., 2006). Antigen-specific binding of anti-TEM1 scFv and biobodies was characterized in vitro by flow cytometry analysis and ELISA assay, and in vivo by injection in an orthotopic mouse model of ovarian cancer. The anti-TEM1 biobody of highest affinity was able to bind specifically to both murine and human Endosialin/TEM1 and to target Endosialin/TEM1-expresser tumor cells in vivo, paving the way to the development of novel anti-angiogenesis targeted-theranostics.