Vinyl sulfone bifunctional derivatives of DOTA allow sulfhydryl- or amino-directed coupling to antibodies. Conjugates retain immunoreactivity and have similar biodistributions
Vinyl sulfone bifunctional derivatives of DOTA allow sulfhydryl- or amino-directed coupling to antibodies. Conjugates retain immunoreactivity and have similar biodistributions
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DOI：
10.1021/bc015535b
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发表时间：
2002-01-01
影响因子：
4.7
通讯作者：
Shively, JE
中科院分区：
文献类型：
--
作者：
Li, L;Tsai, SW;Shively, JE
我们合成了 DOTA（1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）的双功能乙烯基砜-半胱氨酰胺衍生物，它可以在 pH 7 下与轻度还原重组抗体（嵌合抗 CEA 抗体 cT84.66）的巯基缀合，或在 pH 9 下与赖氨酸残基的氨基缀合。只要抗体没有游离的巯基，在 pH 7 下具有特异性（案例 1），在 pH 9 下具有氨基特异性（案例 2）。 BCA（双功能螯合剂）与 mAb 的摩尔比为 50 时，案例 1 中的螯合物数量为 0.8，案例 2 中的螯合物数量为 4.6。所得的偶联物可在 43°C 下 60 分钟内用 In-111 放射性标记至高比活度 (5 mCi/mg)，效率很高 (>95%)。放射性标记缀合物保留了 >95% 的免疫反应性，并且在含有 1mM DTPA 的血清中稳定超过 3 d。当将放射性标记的缀合物注射到携带LS174T人结肠肿瘤异种移植物的裸鼠中时，在24-72小时期间肿瘤中积累了超过40％ID/g。巯基偶联缀合物在 24、48 和 72 小时时的肿瘤与血液比率分别为 4.5、3.5 和 2.5，氨基偶联缀合物在相同时间点的肿瘤与血液比率分别为 2.7、2.5 和 2.3。对于其他器官，无论缀合物是通过巯基还是氨基连接，生物分布几乎相同。这些新型 BCA 易于合成，提供多种缀合选项，并具有等效的生物分布，从而导致高肿瘤摄取和良好的肿瘤与血液比率。
We have synthesized a bifunctional vinyl sulfone-cysteineamido derivative of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that can be conjugated to the sulfhydryls of mildly reduced recombinant antibody (chimeric anti-CEA antibody cT84.66) at pH 7 or to the amino groups of lysine residues at pH 9. The conjugation is sulfhydryl specific at pH 7 (case 1), and amino specific at pH 9 (case 2) as long as the antibody has no free sulhydryl groups. At a molar ratio of 50 BCA (bifunctional chelating agent) to mAb, the number of chelates conjugated is 0.8 for case 1, and 4.6 for case 2. The resulting conjugates can be radiolabeled with In-111 to high specific activity (5 mCi/mg) with high efficiency (>95%) at 43 degreesC in 60 min. The radiolabelod conjugates retained >95% immunoreactivity and are stable in serum containing 1mM DTPA over 3 d. When the radiolabeled conjugates were injected into nude mice bearing LS174T human colon tumor xenografts, over 40% ID/g accumulated in tumors during the period 24-72h. Tumor-to-blood ratios were 4.5, 3.5, and 2.5 for the sulfhydryl coupled conjugate at 24, 48, and 72 h, respectively, and 2.7, 2.5, and 2.3 for the amino-coupled conjugate at the same time points. For other organs the biodistributions were nearly identical whether the conjugates were attached via sulfhydryl or amino groups. These novel BCAs are easy to synthesize, offer versatile conjugation options, and give equivalent biodistributions that result in high tumor uptake and good tumor-to-blood ratios.