Reist C J, Garg P K, Alston K L, Bigner D D, Zalutsky M R
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Cancer Res. 1996 Nov 1;56(21):4970-7.
Monoclonal antibodies (mAbs) that internalize following binding to cell-surface receptors require radiolabeling approaches that minimize loss of radioactivity from the cell after intracellular processing. One class of internalizing mAbs of great interest for imaging and radioimmunotherapy are those specific for EGFRvIII, a truncated form of the epidermal growth factor receptor found on gliomas, non-small cell lung carcinomas, breast carcinomas, and ovarian carcinomas. Because lysosomes are known to retain positively charged compounds, N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) might be ideal for radioiodination of these mAbs because of the positive charge on its pyridine ring. To investigate this hypothesis, the anti-EGFRvIII mAb L8A4 was labeled using SIPC, and internalization assays were performed using the EGFRvIII-positive cell lines HC2 20 d2 and NR6M. Compared with L8A4 labeled using Iodogen or N-succinimidyl 3-iodobenzoate, SIPC increased intracellular retention of activity by up to 65%. Reverse-phase high-performance liquid chromatography analyses indicated that a significantly higher fraction of the low molecular weight catabolites from mAbs labeled via SIPC were retained within cells (SIPC, 28.1%; Iodogen, 7.6% at 1 h). With SIPC, the primary labeled species in cell lysates was the 5-iodonicotinic acid (INA)-lysine conjugate, whereas in the supernatant, both INA-lysine and INA were seen. A 3-4-fold higher percentage of these catabolites were charged at lysosomal pH in comparison with those from mAb labeled using N-succinimidyl 3-iodobenzoate, in concert with the differences in cellular retention observed between these two labeling methods. In mice bearing HC2 20 d2 xenografts, a significant improvement in tumor retention of radioiodine and tumor:normal tissue ratios was seen when L8A4 was labeled using SIPC instead of the Iodogen method. These results suggest that SIPC is a promising reagent for the radioiodination of anti-EGFRvIII L8A4 and, possibly, other internalizing mAbs.
与细胞表面受体结合后会内化的单克隆抗体（mAb）需要采用放射性标记方法，以尽量减少细胞内处理后细胞中放射性的损失。一类对成像和放射免疫治疗极具吸引力的内化单克隆抗体是针对EGFRvIII的抗体，EGFRvIII是一种在神经胶质瘤、非小细胞肺癌、乳腺癌和卵巢癌中发现的表皮生长因子受体的截短形式。由于已知溶酶体能够保留带正电荷的化合物，N-琥珀酰亚胺基5-碘-3-吡啶羧酸盐（SIPC）可能是这些单克隆抗体放射性碘化的理想选择，因为其吡啶环上带有正电荷。为了验证这一假设，使用SIPC对抗EGFRvIII单克隆抗体L8A4进行标记，并使用EGFRvIII阳性细胞系HC2 20 d2和NR6M进行内化试验。与使用碘代甘氨酸或N-琥珀酰亚胺基3-碘苯甲酸酯标记的L8A4相比，SIPC使细胞内活性保留率提高了65%。反相高效液相色谱分析表明，通过SIPC标记的单克隆抗体产生的低分子量分解代谢产物中，有显著更高比例保留在细胞内（SIPC为28.1%；碘代甘氨酸在1小时时为7.6%）。使用SIPC时，细胞裂解物中的主要标记物种是5-碘烟酸（INA）-赖氨酸缀合物，而在上清液中，则同时可见INA-赖氨酸和INA。与使用N-琥珀酰亚胺基3-碘苯甲酸酯标记的单克隆抗体产生的分解代谢产物相比，这些分解代谢产物在溶酶体pH值下带电荷的百分比高3至4倍，这与这两种标记方法在细胞保留方面观察到的差异一致。在携带HC2 20 d2异种移植物的小鼠中，当使用SIPC而非碘代甘氨酸方法标记L8A4时，放射性碘在肿瘤中的保留以及肿瘤与正常组织的比率有显著改善。这些结果表明，SIPC是抗EGFRvIII L8A4以及可能其他内化单克隆抗体放射性碘化的一种有前景的试剂。
