A FACILE, WATER-SOLUBLE METHOD FOR MODIFICATION OF PROTEINS WITH DOTA - USE OF ELEVATED-TEMPERATURE AND OPTIMIZED PH TO ACHIEVE HIGH SPECIFIC ACTIVITY AND HIGH CHELATE STABILITY IN RADIOLABELED IMMUNOCONJUGATES
A FACILE, WATER-SOLUBLE METHOD FOR MODIFICATION OF PROTEINS WITH DOTA - USE OF ELEVATED-TEMPERATURE AND OPTIMIZED PH TO ACHIEVE HIGH SPECIFIC ACTIVITY AND HIGH CHELATE STABILITY IN RADIOLABELED IMMUNOCONJUGATES
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DOI：
10.1021/bc00030a012
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发表时间：
1994-11-01
影响因子：
4.7
通讯作者：
SHIVELY, JE
中科院分区：
文献类型：
--
作者：
LEWIS, MR;RAUBITSCHEK, A;SHIVELY, JE
我们开发了一种通过用N-羟基磺基琥珀酰亚胺（sulfo-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）在单一步骤中制备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缀合物的质谱显示，随着反应混合物中DOTA活性酯与蛋白质的摩尔比从10：1增加至100：1，连接至蛋白质分子的螯合剂的平均数从2.64增加至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3.8个螯合物。用In-111（III）和Y-90（III）放射性标记的抗体偶联物的免疫反应性保持定量。DOTA：磺基-NHS：EDC活化化学计量从2：2：1至10：10：1的变化揭示了放射性缀合物的动力学稳定性随着碳二亚胺相对于DOTA和磺基-NHS的摩尔比降低而增加。用In-111（III）和Y-90（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。标记反应的最适pH值对于每种蛋白质是不同的，并且可能与蛋白质的等电点有关。放射性金属掺入在高比活度完成在醋酸盐和Tris缓冲液，但柠檬酸盐的存在抑制了标记反应。将放射性标记反应的温度从25 ° C增加到43 ° C大大增加了放射性金属掺入的效率和放射性缀合物的动力学稳定性。在人血清中和存在5000- 250 000倍过量的二亚乙基三胺五乙酸（DTPA）的共轭物的稳定性研究表明，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，In-111（III）标记的抗体显示放射性金属损失率约为0.08%/天。在过量DTPA的存在下，两种缀合物以每天约0.3%的速率损失In-111（III）。在血清中没有观察到Y-90（III）从缀合物中的损失，但是在过量的DTPA中，两种Y-90（III）标记的蛋白质显示出每天约0.2%的放射性金属损失率。因此，与血清稳定性研究相比，在存在大量过量DTPA的情况下，放射性标记免疫偶联物的金属损失的动力学分析可提供该免疫偶联物的体内稳定性的更好指示。
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 In-111(III) and Y-90(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 In-111(III) and Y-90(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 In-111(III)-labeled antibody showed a rate of radiometal loss of approximately 0.08% per day. In the presence of excess DTPA, both conjugates lost In-111(III) at a rate of about 0.3% per day. No loss of Y-90(III) from the conjugates was observed in serum, but in excess DTPA, both Y-90(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.