Benhar I, Padlan E A, Jung S H, Lee B, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12051-5. doi: 10.1073/pnas.91.25.12051.
B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of carcinoma-specific antibody B3 is fused to a truncated form of Pseudomonas exotoxin (PE). The efficacy of monoclonal antibody B3 and B3 immunotoxins in cancer therapy and diagnosis may be limited by the human anti-mouse response. Here we describe the humanization of the Fv of B3(Fv)-PE38 by "framework exchange." The variable domains of the heavy (VH) and light (VL) chains were aligned with their best human homologs to identify framework residues that differ. Initially, 11 framework residues in VH and six in VL were changed by site-specific mutagenesis to human residues and introduced simultaneously into a preassembled single-chain Fv expression cassette. Six VH and five VL residues that differ were not changed because they were buried, in the interdomain interface, or previously found to result in decreased affinity when mutated. This basic design resulted in some 20-fold loss of activity. Changing VL residues at the interdomain interfacial position 100 and at the buried position 104 to the human sequence increased the activity 8-fold. Changing VH residue at position 82b from the human sequence back to that of the mouse restored the activity 2- to 3-fold to the full binding and cytotoxic activity of the mouse sequence. Humanized B3(Fv)-PE38 lost immunogenic epitopes recognized by sera from monkeys that had been immunized with B3(Fv)-PE38.
B3(Fv)-PE38是一种重组单链免疫毒素，其中癌特异性抗体B3的Fv区与铜绿假单胞菌外毒素（PE）的截短形式融合。单克隆抗体B3和B3免疫毒素在癌症治疗和诊断中的疗效可能会受到人抗鼠反应的限制。在此，我们描述了通过“框架交换”对B3(Fv)-PE38的Fv进行人源化改造。重链（VH）和轻链（VL）的可变结构域与其最佳人源同源物进行比对，以确定不同的框架残基。最初，通过定点诱变将VH中的11个框架残基和VL中的6个框架残基替换为人源残基，并同时引入预先组装好的单链Fv表达盒中。6个不同的VH残基和5个不同的VL残基未作改变，因为它们位于结构域间界面处被掩埋，或者先前已发现突变后会导致亲和力下降。这种基本设计导致活性约损失20倍。将结构域间界面位置100和掩埋位置104处的VL残基替换为人源序列，可使活性提高8倍。将位置82b处的VH残基从人源序列改回小鼠序列，可使活性恢复至小鼠序列的完全结合和细胞毒性活性的2至3倍。人源化的B3(Fv)-PE38失去了用B3(Fv)-PE38免疫的猴子血清所识别的免疫原性表位。
