Specific Imaging And Immunotherapy For Ovarian Cancer By Targeting TEM1

Tumor Endothelial Marker1(TEM1) has been identified as a microvascular marker of tumor angiogenesis in various human cancers. Our previous work showed that compared to normal ovary tissues, TEM1is overexpressed in malignant ovarian endothelial cells, suggesting that TEM1could be a potential vasculature target for tumor therapy.We have previously isolated human TEM1-specific single chain variable fragment (scFv) antibodies by screening yeast-display recombinant scFv libraries and found that scFv78was able to bind to the extracellular domain (aa.324-390) of both human and murine TEM1protein with Kd value of～2nM.To improve the TEM1-specific affinity and optimize its pharmacokinetic characters, we developed a series of new multivalent variants of scFv78including a dimeric Fc-fusion (from hu IgGl)(78Fc), a tetrameric CH2-fusion (78CH2),, a dimeric CH3-fusion (78mb) and a dimeric CHI-hinge-fusion (78F(ab’)2), to improve the thermal, serum stability and affinity. Those fusion proteins were generated by transicent transfection of293F cells and affinity purification from the cell culture supernatant. Live cell ELISA showed that the78Fc protein exhibited higher affinity (Kd=0.14±0.01nM,15fold of parental scFv78) and longer blood PK than scFv78. What’s more, its targeting only to TEM1+tumors, but not to normal organs in Naive mice has perfectly confirmed its specificity. We further characterized the pharmacokinetics of all the above five proteins in animal models and showed that Fc-based variants have relatively long serum half-lives compared to other variants. The application of78Fc derivatives in TEM1-targeted theranotics in ovarian and other cancers is also studied and well characterized by using Flurophores Near-Infrared fluorescence. At last, we demonstrated that78Fc conjugated immunotoxin (78Fc-MMAE) could specifically kill TEM1+cells, supported by a series of in vitro experiments. Taken together,78Fc is a favorable candidate for antibody-based imaging and therapy and has great potential for clinical application.