Abstract B68: Targeted theranostics of tumor vasculature by multivalent scFv constructs against TEM1

Abstract

Abstract Background: It is direly needed to develop new therapy and diagnosis for ovarian cancer. There are molecular markers on tumor vessels but not normal; thus provide strong rationale to develop early detection and targeting agents for such “signature”. One such protein, Tumor endothelial marker-1 (TEM1), is overexpressed in tumor endothelial cells as a suitable marker for targeting tumor vasculature. Objective: To better assist TEM1-specific theranostics, we sought to 1)develop TEM1-specific affinity agents from scFv-display library by protein engineering, 2)develop immunoPET/optical imaging; and 3)develop TEM1-targeting immunotherapies such as radioimmunotherapy, antibody-drug conjugate and bispecific antibodies. Methods: To determine the expression of TEM1 in tumor vasculature, 53 cases of metastatic serous ovarian cancer (stages IIIC and IV) resected at HUP were studied by immunohistochemistry. We previously isolated TEM1-specific scFv,”scFv78”, binding to extracellular domain (aa.324-390) of TEM1 with Kd ~2 nM. To improve thermal/serum stability and affinity, a series of multivalent variants were engineered, including scFv78 fusion with huIgG1 Fc region, or partial Fc (CH2-, CH3-, or hinge). Protein production was carried out with 293F cells and purified with affinity chromatography. These variants are characterized in vitro for Kd, serum and thermo stability. In vivo PK/PD and biodistribution studies were carried out in naïve and TEM1+ tumor bearing animals. Protein with the most favorable affinity, PK/PD and stability was developed into agent for in vivo imaging, such as NIR optical imaging and immunoPET by 124-Iodine labeling. Antibody-drug conjugate (ADC) was designed and tested by conjugation to MMAE. Results: Over 99% of patients have positive staining on either fibroblast or vessel. To develop TEM1 antibody for theranostics, we characterized a panel of multivalent scFv78 variants. All variants exhibit comparable thermo and serum stability. ScFv78-Fc exhibited higher affinity (KD=0.15nM, 15 fold of parental scFv78) to TEM1. In vivo pharmacokinetics study of these variants suggested that scFv78-Fc is suitable for further development as targeted therapeutics and imaging. In pilot optical and PET imaging study, florouphore-labeled and radiolabeled scFv78-Fc showed enrichment in TEM1+ tumor, but not control tumor or normal organs. Pilot experiment showed specific killing of TEM1+ cells by 78Fc-MMAE conjugate. Conclusions: We developed an innovative approach of tumor therapy by targeting tumor vasculature marker such as TEM1, which is common on ovarian cancer and some other solid tumors. Citation Format: Chunsheng Li, Jia Hu, Ann Marie Chacko, Junying Wang, Aizhi Zhao, Xingmei Duan, Vladimir Muzykantov, George Coukos. Targeted theranostics of tumor vasculature by multivalent scFv constructs against TEM1. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B68.