Department of Cancer Research, Berlex Biosciences, Richmond, CA, USA.
Transl Oncol. 2008 Jul;1(2):102-9. doi: 10.1593/tlo.08124.
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.
我们最近确定并验证了前列腺癌抗原 Tomoregulin 是前列腺癌放射免疫治疗的靶标。在这里，我们提供的证据表明 Tomoregulin 是一种内化抗原，也是免疫毒素的潜在靶标。首先，通过流式细胞术证实了 Tomoregulin 的细胞表面定位，并通过全细胞结合测定法确定了其表达水平。其次，激光扫描共聚焦显微镜显示 Tomoregulin 在抗体结合时在 37°C 内化到细胞质中。发现内化的 Tomoregulin 与酸性囊泡共定位。第三，使用 (125)I 标记的抗 Tomoregulin 小鼠单克隆抗体 2H8 进行内化动力学测定表明，随着时间的推移，内化的抗原-抗体复合物的数量增加，60 至 90 分钟后达到总表面抗原的约 25%。由于 2H8 能够结合细胞表面上的 Tomoregulin 并被内化，我们最终评估了 2H8 作为将毒性有效负载靶向前列腺癌细胞的手段。在间接免疫毒素测定中，2H8 通过第二抗体（Mab-ZAP）与细胞毒素丝裂霉素偶联。在免疫毒素浓度下，细胞杀伤发生在 Tomoregulin 阳性细胞（Clone69）上，而不影响 Tomoregulin 阴性细胞（PC-3）。与 2H8 相反，在 Mab-ZAP 存在的情况下，对照抗体（鼠抗 c-Myc 抗体 9E10）对细胞没有影响。因此，Tomoregulin 内化使免疫毒素对前列腺癌细胞具有选择性细胞毒性，并且 Tomoregulin 介导的免疫毒素传递有可能成为前列腺癌治疗的一种方法。
