Benhar I, Brinkmann U, Webber K O, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Bioconjug Chem. 1994 Jul-Aug;5(4):321-6. doi: 10.1021/bc00028a007.
B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of monoclonal antibody B3 is connected to a truncated form of Pseudomonas exotoxin. It would be desirable to use the lysine residues of the molecule for chemical modification so that it can be derivatized with poly(ethylene glycol) to achieve reduced immunogenicity or with the Bolton-Hunter reagent for biodistribution studies. We found that derivatizing lysine residues of B3(Fv)-PE38 causes a marked loss of specific target cell cytotoxicity and/or immunoreactivity. Here we show that two lysine residues in the antibody-combining region of B3(Fv)-PE38 can be replaced with arginines, with only a small loss of cytotoxicity and no change in specificity. This mutant molecule is 3-fold more resistant to inactivation by derivatization with succinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) or Bolton-Hunter reagent.
B3(Fv)-PE38是一种重组单链免疫毒素，其中单克隆抗体B3的Fv区与铜绿假单胞菌外毒素的截短形式相连。使用该分子的赖氨酸残基进行化学修饰是可取的，这样它可以用聚乙二醇进行衍生化以降低免疫原性，或者用博尔顿-亨特试剂进行生物分布研究。我们发现对B3(Fv)-PE38的赖氨酸残基进行衍生化会导致特异性靶细胞细胞毒性和/或免疫反应性显著丧失。在此我们表明，B3(Fv)-PE38抗体结合区域中的两个赖氨酸残基可以被精氨酸取代，细胞毒性仅有少量损失且特异性无变化。该突变分子在用4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)或博尔顿-亨特试剂进行衍生化时，对失活的抵抗力提高了3倍。
