Abstract 5765: Immunobiomarkers: Structural and functional characterization of single chain fragment variable (scFv) to ERG from a mouse monoclonal antibody
Authors: Tan Shyh-Han , Rastogi Anshu , Banerjee Sreedatta , Bagga Annie , Xavier Charles , Mohamed Ahmed , Young Denise , Petrovics Gyorgy , Dobi Albert , Sesterhenn Isabell A. , Kagan Jacob , Srivastava Sudhir , David David G. , Rosner Inger L. , Srivastava Shiv , Srinivasan Alagarsamy 
Published: August 2018

Abstract
Introduction: Biomarkers for early detection, disease stratification as well as treatment strategies are a major area of focus in prostate cancer (CaP) research. The gene fusion involving ERG and the TMPRSS2 gene promoter contributes to expression of ERG in CaP, serving as a biomarker for CaP. This led to the development of monoclonal or polyclonal antibodies against ERG protein for diagnostic or therapeutic purposes. As the use of intact monoclonal antibody is limited due to large size and low cell penetration, we hypothesized that single chain fragment variable (scFv) and single domain antibody (sdAb) known as minibodies will be valuable as therapeutic agents targeting ERG protein. Towards this goal, the present study addresses the following: i) Characterization of variable heavy (VH) and variable light (VL) chain sequences and generation of expression vectors; ii) Assessing the specificity of binding of scFv and sdAb to ERG protein; iii) Functional evaluation of scFv and sdAb in cellsMethods: We generated VH and VL encoding sequences using RNA derived from hybridoma cells secreting anti-ERG monoclonal antibody designated 9FY. scFv-9FY and sdAb-9FY fragments represent scFv and sdAb , respectively were cloned in an eukaryotic and prokaryotic expression vectors. The proteins (scFv and sdAb) were analyzed by western blot and ELISA. The binding of scFv and sdAb to ERG protein in live cells was evaluated by bimolecular fluorescence complementation (BiFC) assay. Biological effects of scFv and sdAb were assessed in HEK293, VCaP and LNCaP cells. Results: Analysis was carried out to determine the primary DNA sequence of scFv fragments. Using the predicted amino acid sequence, we identified CDR1, CDR2 and CDR3 regions. scFv expressed in prokaryotic vector was purified and used for the evaluation of immunoreactivity to ERG by ELISA. The affinity of scFv and sdAb to ERG was demonstrated through their binding to ERG protein in HEK293 cells by using BiFC assay. This involved the generation of chimeric proteins in which scFv or sdAb coding sequences were fused in-frame to the Venus N- or C-terminal segment separated by a flexible linker of 10 amino acids. Minibodies scFv and sdAb, upon introduction into VCaP cells through transfection, showed cytotoxic effects similar to the observations noted with siRNA against ERG.Conclusions: Here we showed that scFv and sdAb minibodies exhibit high affinity for ERG protein. This property enables them to disrupt the functions mediated by ERG in cells. Given the prevalence of ERG overexpression in prostate cancer, the novel agents based on scFv and sdAb have the potential to interfere with the effect or functions of ERG and are likely to be beneficial in the treatment of CaP.
Source of Funding: Center for Prostate Disease Research, USU Grant HU0001-10-2-0002, NCI/EDRN Grant ACN12011-001-0, and the NCI Grant R01CA162383.
Citation Format: Shyh-Han Tan, Anshu Rastogi, Sreedatta Banerjee, Annie Bagga, Charles Xavier, Ahmed Mohamed, Denise Young, Gyorgy Petrovics, Albert Dobi, Isabell A. Sesterhenn, Jacob Kagan, Sudhir Srivastava, David G. David, Inger L. Rosner, Shiv Srivastava, Alagarsamy Srinivasan. Immunobiomarkers: Structural and functional characterization of single chain fragment variable (scFv) to ERG from a mouse monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5765.