MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER

Abstract

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2018MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER Bernhard Kiss, Kelly McKenna, Robert Ertsey, Nynke Sjoerdtje Van den Berg, Kathleen Mach, Eben Rosenthal, and Joseph Liao Bernhard KissBernhard Kiss More articles by this author , Kelly McKennaKelly McKenna More articles by this author , Robert ErtseyRobert Ertsey More articles by this author , Nynke Sjoerdtje Van den BergNynke Sjoerdtje Van den Berg More articles by this author , Kathleen MachKathleen Mach More articles by this author , Eben RosenthalEben Rosenthal More articles by this author , and Joseph LiaoJoseph Liao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2075AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Near infrared photoimmunotherapy (PIT) is a new molecular-targeted cancer therapy that uses a specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IRDye700DX, conjugated to a monoclonal antibody. CD47 is a cell surface protein that elicits a ″don′t eat me signal″ to prevent macrophage engulfment and is expressed on over 80% of bladder cancer tumors but absent on the luminal cell layer of normal bladder urothelium. Thus, targeting CD47 for PIT has the potential to selectively induce cell death in CD47 expressing tumor cells as well as increase phagocytosis of tumor cells. METHODS The anti-CD47 antibody (B6H12) was conjugated to IRDye700DX NHS ester and purified using a dialysis spin column. Anti-CD47-IRDye700 concentration was determined with a Coomassie Plus protein assay. The molecular weight of the conjugated antibody was verified by SDS-page and IRDye700 conjugation verified using the 700nm fluorescence channel of a PEARL imager. Binding of anti-CD47-IRDye700DX to CD47 was verified by ELISA. Three different human urothelial cell lines (UMUC3, HT-1376 and 639V) were used for in vitro NIR-PIT experiments. 639V cells were stably transfected with GFP using a lentiviral vector. After plating in a 96well plate and incubation with 10 µg/ml of anti-CD47-IRDye700 or unconjugated anti-CD47 antibody (1h on ice), cells were irradiated with a red light-emitting diode (LED) at 670-710nm wavelength at different energy levels. The cytotoxic effects of NIR-PIT with anti-CD47-IRDye700DX were determined by propidium iodide staining and flow cytometry. To evaluate macrophage activity, a phagocytosis assay using human macrophages and analyzed by flow cytometry was performed. RESULTS Cancer-cell death significantly increased with anti-CD47-IRDye700DX in a light-dose dependent manner in all 3 human bladder cancer cell lines. Significant cancer-cell death was first observed with 2J exposure and when exposed to 40J of NIR light over 90% of 639V and UMUC3 cells and over 50% of HT1376 died. Phagocytosis of cancer-cells incubated with anti-CD47-IRDye700DX and irradiated with 8J was significantly increased compared to anti-CD47-antibody alone (p=0.0002). There was no cytotoxicity associated with anti-CD47 antibody alone with NIR light, with NIR light alone or with anti-CD47-IRDye700DX without NIR light. CONCLUSIONS NIR-PIT with anti-CD47-IRDye700 shows significantly increased cancer-cell cytotoxicity and significantly increased cancer-cell phagocytosis making this combination a highly promising novel approach for molecular targeted bladder cancer therapy. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e862 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Bernhard Kiss More articles by this author Kelly McKenna More articles by this author Robert Ertsey More articles by this author Nynke Sjoerdtje Van den Berg More articles by this author Kathleen Mach More articles by this author Eben Rosenthal More articles by this author Joseph Liao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...