Stalteri M A, Mather S J
Imperial Cancer Research Fund, Department of Nuclear Medicine, St. Bartholomew's Hospital, West Smithfield, London, U.K.
Bioconjug Chem. 1995 Mar-Apr;6(2):179-86. doi: 10.1021/bc00032a005.
Cross-linked F(ab')2 fragments derived from PR1A3, a murine monoclonal antibody used in radioimmunoscintigraphy of colorectal tumors, were produced using the bifunctional reagent bismaleimidohexane (BMH) as follows: Digestion of PR1A3 with pepsin gave F(ab')2 fragments which were purified by ion-exchange chromatography. Fab' was produced by reduction of F(ab')2 with cysteine. Following reaction with BMH, cross-linked F(ab')2 fragments, XL-F(ab')2, were isolated by preparative size-exclusion HPLC. Analysis by HPLC and SDS-PAGE demonstrated the presence of a molecule of approximately 100 kDa containing a nonreducible 50,000 MWt chain. Competitive and direct radioligand binding assays demonstrated that the XL-F(ab')2 had a capacity to bind to antigen similar to that of unmodified F(ab')2. The biodistribution of 125I-labeled XL-F(ab')2 and unmodified F(ab')2 was compared in a nude mouse human tumor xenograft model at 4, 24, and 48 h after injection. Differences between the two preparations were most significant after 24 or 48 h. Tumor uptake of the XL-F(ab')2 was greater and normal tissue retention less than with the unmodified fragment. Tumor to normal tissue ratios at 48 h ranged from 6.2 to 35.2 for XL-F(ab')2 while for the normal F(ab')2 they ranged from 1.5 to 14.2. These results suggest that cross-linked antibody fragments may produce better tumor targeting in clinical application.
源自PR1A3（一种用于结直肠癌肿瘤放射免疫闪烁成像的鼠单克隆抗体）的交联F(ab')2片段，是使用双功能试剂双马来酰亚胺己烷（BMH）按如下方法制备的：用胃蛋白酶消化PR1A3得到F(ab')2片段，该片段通过离子交换色谱法纯化。通过用半胱氨酸还原F(ab')2产生Fab'。与BMH反应后，通过制备型尺寸排阻高效液相色谱法分离交联的F(ab')2片段，即XL-F(ab')2。高效液相色谱法和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析表明存在一个约100 kDa的分子，其中包含一条不可还原的50,000 MWt链。竞争性和直接放射性配体结合试验表明，XL-F(ab')2与抗原结合的能力与未修饰的F(ab')2相似。在裸鼠人肿瘤异种移植模型中，比较了注射后4、24和48小时125I标记的XL-F(ab')2和未修饰的F(ab')2的生物分布。两种制剂之间的差异在24或48小时后最为显著。XL-F(ab')2的肿瘤摄取量大于未修饰片段，正常组织滞留量小于未修饰片段。XL-F(ab')2在48小时时的肿瘤与正常组织比率范围为6.2至35.2，而正常F(ab')2的该比率范围为1.5至14.2。这些结果表明，交联抗体片段在临床应用中可能产生更好的肿瘤靶向性。
