Abstract 1775: Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer
Authors: Kono Evelyn A. , Kobayashi Naoko , Zettlitz Kirstin , Li Keyu , Yamashiro Joyce , Wang Chun , Wu Anna , Reiter Robert E. 
Published: August 2018

Enzalutamide (ENZ) is an androgen receptor (AR) antagonist that targets various stages of the AR signaling pathway, and approved for the treatment of castration resistant prostate cancer (CRPC). Studies show increased survival in men with metastatic CRPC after chemotherapy when treated with ENZ. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance and resistance to AR inhibitors may lead to increase in N-cadherin. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR positive and negative prostate cancers. Co-targeting AR and N-cadherin with ENZ and mAb respectively show promise in addressing not only castration resistant tumor progression but also ENZ resistance. Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with ENZ. In vivo studies on promising candidates were performed using castration resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to ENZ, mAb alone, or in combination. At end-point, tumors were harvested and further analyzed. Final candidate mAbs were then selected and re-formatted into fully humanized N-cadherin monoclonal antibodies and further evaluated. A molecular model of the mouse chimeric antibodies were built based on structure of similar antibodies and the CDR loops were grafted to the human framework region and back-translated into nucleotide sequences. Codon-optimized DNA encoding the humanized variable domains connected by a 15 glycine-rich amino acid linker was synthesized by GeneArt and sub-cloned into expression vectors containing light and heavy chain constant regions. Clones were then screened and selected. Candidates were transiently expressed in 293 cells and supernatant were collected and purified for further analysis. These newly generated humanized mAbs engineered from the promising human anti-mouse N-cadherin antibody candidates exhibited similar outcomes to their chimeric counterparts, revealing potential in providing methods for diagnosis and treatment of advanced diseases.

Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Keyu Li, Joyce Yamashiro, Chun Wang, Anna Wu, Robert E. Reiter. Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate 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 1775.