Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells.
We have recently identified and validated the prostate cancer antigen Tomoregulin as a target for the radioimmunotherapy for prostate cancer. Here, we provide evidence that Tomoregulin is an internalizing antigen and a potential target for immunotoxins. First, the cell surface localization of Tomoregulin was confirmed by flow cytometry, and its expression levels were determined by whole-cell binding assays. Second, laser scanning confocal microscopy revealed Tomoregulin internalization into the cytoplasm on antibody binding at 37 degrees C. The internalized Tomoregulin was found to colocalize with acidic vesicles. Third, internalization kinetics assays using (125)I-labeled anti-Tomoregulin mouse monoclonal antibody 2H8 demonstrated that the amount of internalized antigen-antibody complexes increased with time and reached approximately 25% of the total surface antigen after 60 to 90 minutes. Because 2H8 is capable of binding to Tomoregulin on the cell surface and can be internalized, we finally evaluated 2H8 as a means of targeting toxic payloads to prostate cancer cells. 2H8 was coupled to the cytotoxin saporin through a secondary antibody (Mab-ZAP) in indirect immunotoxin assays. Cell killing occurred on Tomoregulin-positive cells (Clone69) at the immunotoxin concentrations not affecting the Tomoregulin-negative cells (PC-3). In contrast to 2H8, the control antibody (mouse anti-c-Myc antibody 9E10) had no effect on cells in the presence of Mab-ZAP. Thus, Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells, and Tomoregulin-mediated delivery of immunotoxin has potential as a prostate cancer therapy.
Zhao
Xiao-Yan
XY
Department of Cancer Research, Berlex Biosciences, Richmond, CA, USA.
Liu
Hsiao-Lai
HL
Liu
Bing
B
Willuda
Joerg
J
Siemeister
Gerhard
G
Mahmoudi
Mithra
M
Dinter
Harald
H