CD125xCNE bispecific antibody development to treat bladder cancer

Abstract Background: Bladder cancer is one of the most prevalent cancers impacting adults worldwide.Although,successfully treated in its early stages, there are no effective therapies when the cancer has progressed into the surrounding muscle layer, clinically classified as muscle invasive bladder cancer (MIBC). CD125 was recently shown to be involved in MIBC progression and uniquely overexpressed in MIBC tumors relative to healthy urothelium or superficial tumors. Cyclin E (CNE) is a critical cell cycle protein and regulates progression of normal cells to replicate their DNA. There is a strong link between CNE dysregulation and tumorigenesis and was recently found to be overexpressed in MIBC. We characterized the monoclonal antibodies (mAbs) A14 specific for CD125, and HE-12 and HE-172 specific for CNE with a long-term purpose to develop a CD125xCNE therapeutic bispecific antibody. In order, for the antibody to efficiently target CNEour group developed a natural composite compound (termed Accum)that conjugates to surface lysines and enables mAbs to escape endosomal entrapment followed by active routing to and efficient accumulation in the nucleus. Methods: For Accum conjugation, maleimide groups were introduced into A14 by reaction with 10-to-100-fold molar excess of a PEGylated SMCC crosslinker at RT for 1 h. Purified and concentrated maleimide-derivatized A14 was reacted with 100-fold molar excess Accum for 18 h at 4 °C. Excess Accum-A14 was purified and concentrated. Conjugation was characterized by SDS-PAGE. CD125-positive MIBC cells were treated with Accum-A14 and nuclear localization efficiency determined by confocal microscopy.HE-12 and HE-172 mAbs were evaluated for binding nuclear CNE by flow cytometry using the whole cell and fractionated cell lysate along with saponin to enable the mAbs to diffuse into the cell. In addition, HE-12 and HE-172 mAb binding was evaluated by co-immunoprecipitation. PCR amplification of A14, HE-12, and HE-172 was performed. Results: A14 is readily loaded with Accum with minimal aggregation.Accum-A14 undergoesCD125-specific internalization and has 25-60-fold increased nuclear localization relative to A14 in MIBC cells.The binding of the intracellular CNE by mAb HE-12 and HE-172 was specific. Conclusion: This preliminary data demonstrates that Accum modification of A14 retains CD125 specificity and efficiently localizes to the nucleus of MIBC cells. HE-12 and HE172 recognized CNE. On going work will construct the CD125xCNE bispecific antibody. Future studies will determine the efficiency to target CNE and its impact to MIBC progression. Citation Format: Judit Hunyadkurti, Jeffrey Leyton, Laurent Fafard-Couture, Vincent Lacasse, Marc-Andre Bonin, Angel Lopez. CD125xCNE bispecific antibody development to treat bladder cancer [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 5610.