Abstract B247: Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications.

Abstract

Abstract While monoclonal antibodies remain highly effective anticancer agents, their complex post-translational processing preclude their use in multi-effector gene therapy platforms. Breast and ovarian cancers often overexpress HER family receptors and consequently the receptors are targets for effective therapeutics such as the monoclonal antibodies trastuzumab and cetuximab. To address the need for new immune modulating formats that can be encoded in a single therapy, we have designed a platform for rapid conversion and assay of monoclonal antibodies into functional single chain variable fragment-Fc fusion proteins. Here we demonstrate results with trastuzumab and cetuximab as examples of the platform scFv-Fc conversion. The scFv-Fc trastuzumab displays efficient expression from transfected cell lines and high-affinity, high specificity binding to HER2 in plate-based screens with measured KD of 0.15nM compared to commercial trastuzumab's KD of 0.08nM in the same assay. scFv-Fc trastuzumab was shown to bind the HER2+ breast cancer cell lines BT-474 and SKBR3 in FACS-based assays while showing no affinity towards HER2- control cells SHP-77. Antibody-dependent cell-mediated cytotoxicity assays (ADCC) were performed to evaluate ADCC potency of the scFv-Fc trastuzumab. Using NK92/CD16A(158V/V) cells as effectors and BT474 cells as targets, the EC50 of the scFv-Fc trastuzumab was 3.6ng/ml, compared to the EC50 of monoclonal format trastuzumab at 0.94ng/ml. The data provide a direct demonstration of the scFv-Fc format as a viable alternative for achieving molecular targeting in a single chain entity at a level commensurate with KD values of full-length monoclonal antibodies while maintaining ADCC activity. In addition, functional scFv-Fc trastuzumab was expressed in combination with scFv-Fc cetuximab from a single vector. scFv-Fc molecules generated using this modular protein engineering platform were cloned into plasmid DNA vectors controlled by the RheoSwitch Therapeutic System®. Transient transfections into differentiated C2C12 myocytes demonstrated the production of scFv-Fc proteins from muscle cells under control of the RheoSwitch® Activator Ligand. The integrated scFv-Fc optimization platform and inducible multigenic systems described here provides support for future work for the treatment of cancer using multigenic therapeutic antibodies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B247. Citation Format: Charles Reed, Vernon Dailey, Anissa Elayadi, Lindsay Williams, Dina Schneider, Thomas Reed, Jonathan Lewis, Richard Einstein, Samuel Broder. Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B247.