Abstract 599: MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis

Abstract

Abstract Background: Gastric cancer is the primary cause of cancer-specific mortality worldwide. PODXL (podocalyxin-like protein, or podocalyxin) is a cell-surface glycoprotein that belongs to CD34 family of sialomucins. PODXL-v1 is widely expressed on vascular endothelium, mesothelial cells, and platelets; in contrast, PODXL-v2, a truncated short form of PODXL is specifically expressed or up-regulated in numerous cancer cell types as well as in small blood vessels in tumor tissues. There remains considerable interest in generating monoclonal antibodies (mAbs) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy. Methods: Mixed live cells from four human GC cell lines were used as the immunogen in A/J mice. MS17-38 mAb was generated by hybridoma technology and from high throughput screening (HTS), with GC cell lines versus normal human PBMC live cells. Purified MS17-38 mAb was used to confirm the target Ag through a combination of western blot (WB), immunoprecipitation (IP) and mass spectrometry (MS). Peptide microarrays of the identified Ag were layered on chips and double screening was performed to achieve epitope mapping of binding of the mAb its target. Dose-dependent treatment of GC cells with MS17-38 mAb and target siRNA-was followed by staining with CCK-8 and CyQuant GR dyes and read at 450nm and 480/520nm for cell proliferation and transwell migration assays. GC MKN-45 cells were injected subcutaneously (s.c.) into the flank of nu/nu mice treated with MS17-38 mAb or control mAb. In addition, tumor tissues from 42 human GC patients were stained by immunohistochemistry (IHC) and evaluated for PODXL expression using MS17-38, and the survival of the GC patients compared to target antigen expression levels. Results: MS17-38 mAb was generated by live cells HTS and was demonstrated to bind to a specific conformational epitope of PODXL-v2 on GC cells. PODXL-v2 expression inhibition by PODXL-v2 siRNA or the PODXL-v2-neutralizing antibody MS17-38 resulted in inhibition of GC cell growth and prevention of GC cell migration in vitro. Furthermore, in vivo studies demonstrated that MS17-38 mAb can potently inhibit tumor growth and prevent MKN-45 metastasis to the lungs in nu/nu mouse models. Finally, high expression of PODXL-v2 was associated with advanced GC stage and short survival time (P<0.01). Additional engineering of this mAb into chimeric or humanized forms could permit development of MS17-38 for diagnosis, staging or therapy of human malignancy. Conclusions: Our findings indicate that PODXL-v2 is specifically expressed in the extracellular matrix of GC, and MS17-38 mAb directed against a conformational epitope of PODXL-v2 identified through HTS can functionally inhibit GC cancer cell growth and migration/metastasis both in vitro and in vivo. MS17-38 is a promising functional antibody directed against GC that could potentially be developed into a therapeutic mAb for clinical application. Citation Format: Runhua Feng, Ming Li, Yuling Wang, Dongqing Zhang, Xiaolian Gao, Xuehua Chen, Joe X. Zhou, Victor G. Prieto, Jian H. Song, Shenying Fang, Binya Liu, Zhenggang Zhu, Xiangcang Ye, Lee M. Ellis, Mason Lu, Jeffery E. Lee. MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 599.