Lewis M R, Raubitschek A, Shively J E
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Bioconjug Chem. 1994 Nov-Dec;5(6):565-76. doi: 10.1021/bc00030a012.
We have developed a method for attachment of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane N,N',N",N"'-tetraacetic acid (DOTA) to proteins by activation of a single carboxyl group with N-hydroxysulfosuccinimide (sulfo-NHS). The sulfo-NHS active ester of DOTA was prepared in a single step using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and DOTA conjugates of cytochrome c and the anti-carcinoembryonic antigen chimeric monoclonal antibody cT84.66 were prepared by adding the DOTA active ester reaction mixture to the proteins at pH 8.5-9.0. Mass spectrometry of the cytochrome c conjugates showed that as the molar ratio of DOTA active ester to protein in the reaction mixture was increased from 10:1 to 100:1, the average number of chelators attached to the protein molecule increased from 2.64 to 8.79. When DOTA active ester reacted with the antibody at a molar ratio of 100:1, the conjugate averaged 3.8 chelates per antibody. Immunoreactivity of the antibody conjugate radiolabeled with 111In(III) and 90Y(III) remained quantitative. Variation of the DOTA:sulfo-NHS:EDC activation stoichiometry from 2:2:1 to 10:10:1 revealed that the kinetic stability of the radioconjugates increased as the molar ratio of carbodiimide, relative to DOTA and sulfo-NHS, was decreased. Radiolabeling of the protein conjugates with 111In(III) and 90Y(III) proved to be sensitive to pH, buffer, and temperature effects. The optimum pH for the labeling reaction was different for each protein and may be related to the isoelectric point of the protein. Radiometal incorporation at high specific activity was accomplished in acetate and Tris buffers, but the presence of citrate inhibited the labeling reaction. Increasing the temperature of the radiolabeling reaction from 25 to 43 degrees C greatly increased both the efficiency of radiometal incorporation and the kinetic stability of the radioconjugates. Stability studies of the conjugates in human serum and in the presence of a 5000- to 250,000-fold excess of diethylenetriaminepentaacetic acid (DTPA) demonstrated that the radiolabeled proteins are kinetically inert under physiological conditions. In serum, the 111In(III)-labeled antibody showed a rate of radiometal loss of approximately 0.08% per day. In the presence of excess DTPA, both conjugates lost 111In(III) at a rate of about 0.3% per day. No loss of 90Y(III) from the conjugates was observed in serum, but in excess DTPA, both 90Y(III) labeled proteins showed a rate of radiometal loss of approximately 0.2% per day. Therefore, kinetic analysis of metal loss from a radiolabeled immunoconjugate in the presence of a vast excess of DTPA may provide a better indication of the in vivo stability of that immunoconjugate than serum stability studies.
我们开发了一种通过用N-羟基磺基琥珀酰亚胺（磺基-NHS）活化单个羧基将大环螯合剂1,4,7,10-四氮杂环十二烷N,N',N",N"'-四乙酸（DOTA）连接到蛋白质上的方法。使用1-乙基-3-[3-(二甲氨基)丙基]碳二亚胺（EDC）一步制备DOTA的磺基-NHS活性酯，并通过在pH 8.5-9.0下将DOTA活性酯反应混合物加入蛋白质中来制备细胞色素c和抗癌胚抗原嵌合单克隆抗体cT84.66的DOTA缀合物。细胞色素c缀合物的质谱分析表明，随着反应混合物中DOTA活性酯与蛋白质的摩尔比从10:1增加到100:1，连接到蛋白质分子上的螯合剂平均数量从2.64增加到8.79。当DOTA活性酯与抗体以100:1的摩尔比反应时，缀合物平均每个抗体有3.8个螯合物。用111In(III)和90Y(III)放射性标记的抗体缀合物的免疫反应性保持定量。将DOTA:磺基-NHS:EDC活化化学计量比从2:2:1改变为10:10:1表明，随着碳二亚胺相对于DOTA和磺基-NHS的摩尔比降低，放射性缀合物的动力学稳定性增加。用111In(III)和90Y(III)对蛋白质缀合物进行放射性标记被证明对pH、缓冲液和温度效应敏感。标记反应的最佳pH因每种蛋白质而异，可能与蛋白质的等电点有关。在乙酸盐和Tris缓冲液中实现了高比活度的放射性金属掺入，但柠檬酸盐的存在会抑制标记反应。将放射性标记反应的温度从25℃提高到43℃大大提高了放射性金属掺入效率和放射性缀合物的动力学稳定性。在人血清中以及在存在5000至250,000倍过量二乙烯三胺五乙酸（DTPA）的情况下对缀合物进行的稳定性研究表明，放射性标记的蛋白质在生理条件下是动力学惰性的。在血清中，111In(III)标记的抗体显示放射性金属损失率约为每天0.08%。在过量DTPA存在下，两种缀合物以约每天0.3%的速率损失111In(III)。在血清中未观察到缀合物中90Y(III)的损失，但在过量DTPA中，两种90Y(III)标记的蛋白质显示放射性金属损失率约为每天0.2%。因此，在大量过量DTPA存在下对放射性标记免疫缀合物中金属损失的动力学分析可能比血清稳定性研究更好地表明该免疫缀合物的体内稳定性。
