Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate.
Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate.
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发表时间：
1996-11
期刊：
影响因子：
11.2
通讯作者：
C. Reist;P. Garg;K. Alston;D. Bigner;M. Zalutsky
中科院分区：
文献类型：
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作者：
C. Reist;P. Garg;K. Alston;D. Bigner;M. Zalutsky
与细胞表面受体结合后内化的单克隆抗体（mAb）需要放射性标记方法，以最大限度地减少细胞内加工后细胞的放射性损失。一类对成像和放射免疫治疗非常感兴趣的内化mAb是对EGFRvIII特异性的那些，EGFRvIII是在神经胶质瘤、非小细胞肺癌、乳腺癌和卵巢癌上发现的表皮生长因子受体的截短形式。由于已知溶酶体保留带正电荷的化合物，因此5-碘-3-吡啶羧酸N-琥珀酰亚胺酯（SIPC）可能是这些mAb放射性碘标记的理想选择，因为其吡啶环上带正电荷。为了研究这一假设，使用SIPC标记抗EGFRvIII mAb L 8A 4，并使用EGFRvIII阳性细胞系HC 2 20 d2和NR 6 M进行内化测定。与使用Iodogen或N-琥珀酰亚胺基3-碘苯甲酸酯标记的L 8A 4相比，SIPC使细胞内活性保留增加高达65%。反相高效液相色谱分析表明，通过SIPC标记的mAb的低分子量催化剂的显著更高部分保留在细胞内（SIPC，28.1%; Iodogen，1 h时7.6%）。使用SIPC，细胞裂解物中的主要标记物质是5-碘烟酸（INA）-赖氨酸缀合物，而在上清液中，可以看到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mAb相比，在溶酶体pH下，这些催化剂的电荷百分比高3-4倍，这与在这两种标记方法之间观察到的细胞保留差异一致。在携带HC 2 20 d2异种移植物的小鼠中，当使用SIPC而不是Iodogen方法标记L 8A 4时，观察到放射性碘的肿瘤保留和肿瘤：正常组织比率的显著改善。这些结果表明，SIPC是一种有前途的试剂，用于抗EGFRvIII L 8A 4的放射性碘化，并可能，其他内化单克隆抗体。
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.