Weisbart Richard H, Wakelin Rika, Chan Grace, Miller Carl W, Koeffler Phillip H
Department of Medicine, Division of Rheumatology, Veterans Affairs Greater Los Angeles Health Care System, Sepulveda, CA 91343, USA.
Int J Oncol. 2004 Oct;25(4):1113-8.
A bispecific, single-chain antibody Fv fragment (Bs-scFv) was constructed from a single-chain Fv fragment of mAb 3E10 that penetrates living cells and localizes in the nucleus, and a single-chain Fv fragment of a non-penetrating antibody, mAb PAb421 that binds the C-terminal of p53. PAb421 binding restores wild-type functions of some p53 mutants, including those of SW480 human colon cancer cells. The Bs-scFv penetrated SW480 cells and was cytotoxic, suggesting an ability to restore activity to mutant p53. COS-7 cells (monkey kidney cells with wild-type p53) served as a control since they are unresponsive to PAb421 due to the presence of SV40 large T antigen that inhibits binding of PAb421 to p53. Bs-scFv penetrated COS-7 cells but was not cytotoxic, thereby eliminating non-specific toxicity of Bs-scFv unrelated to binding p53. A single mutation in CDR1 of PAb421 VH eliminated binding of the Bs-scFv to p53 and abrogated cytotoxicity for SW480 cells without altering cellular penetration, further supporting the requirement of PAb421 binding to p53 for cytotoxicity. Our study demonstrates the use of an antibody that penetrates living cells in the design of a bispecific single chain antibody to target and restore the function of an intracellular protein.
一种双特异性单链抗体Fv片段（Bs-scFv）由可穿透活细胞并定位于细胞核的单克隆抗体3E10的单链Fv片段，以及与p53 C末端结合的非穿透性抗体单克隆抗体PAb421的单链Fv片段构建而成。PAb421的结合可恢复某些p53突变体的野生型功能，包括SW480人结肠癌细胞的功能。Bs-scFv可穿透SW480细胞并具有细胞毒性，表明其具有恢复突变型p53活性的能力。COS-7细胞（具有野生型p53的猴肾细胞）用作对照，因为由于存在抑制PAb421与p53结合的SV40大T抗原，它们对PAb421无反应。Bs-scFv可穿透COS-7细胞但无细胞毒性，从而消除了与结合p53无关的Bs-scFv的非特异性毒性。PAb421 VH的CDR1中的单个突变消除了Bs-scFv与p53的结合，并消除了对SW480细胞的细胞毒性，而不改变细胞穿透性，进一步支持了PAb421与p53结合对细胞毒性的必要性。我们的研究证明了在设计双特异性单链抗体以靶向和恢复细胞内蛋白质功能时使用可穿透活细胞的抗体。
