Bera T K, Williams-Gould J, Beers R, Chowdhury P, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Mol Cancer Ther. 2001 Dec;1(2):79-84.
We have used protein engineering to generate a stable bivalent fragment variable (Fv) molecule from the antimesothelin antibody SS, in which the VH and VL domains of the Fv are linked to each other by a disulfide bond, and the two Fvs are connected by a flexible 15-amino acid (Gly4-Ser)3 linker. The SS (dsFv)2 molecule is fused to a M(r) 38,000 truncated form of Pseudomonas exotoxin to generate a bivalent, disulfide stabilized, (dsFv)2 immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro, and purified to approximately 95% purity with a high yield of > 10%. Binding studies demonstrated that the (dsFv)2 molecule has 40 times higher apparent affinity for recombinant mesothelin than a monovalent dsFv molecule. The (dsFv)2 immunotoxin was 4-10-fold more cytotoxic to three mesothelin antigen-positive cell lines than the monovalent dsFv immunotoxin. However, when tested in mice bearing tumor cells expressing mesothelin, the antitumor activity of the bivalent immunotoxin is very similar to the activity of the lower affinity monovalent immunotoxin. Our data indicate that increasing affinity of an antibody fragment does not necessarily lead to higher antitumor activity of an immunotoxin in vivo.
我们利用蛋白质工程技术，从抗间皮素抗体SS构建了一个稳定的二价可变区片段（Fv）分子，其中Fv的重链可变区（VH）和轻链可变区（VL）通过二硫键相互连接，两个Fv通过一个由15个氨基酸组成的柔性（Gly4-Ser）3接头相连。将SS（dsFv）2分子与截短至分子量38,000的铜绿假单胞菌外毒素融合，构建成一种二价、二硫键稳定的（dsFv）2免疫毒素。该免疫毒素在大肠杆菌中表达，经体外重折叠，并以大于10%的高产率纯化至纯度约95%。结合研究表明，（dsFv）2分子对重组间皮素的表观亲和力比单价dsFv分子高40倍。（dsFv）2免疫毒素对三种间皮素抗原阳性细胞系的细胞毒性比单价dsFv免疫毒素高4至10倍。然而，在携带表达间皮素的肿瘤细胞的小鼠中进行测试时，二价免疫毒素的抗肿瘤活性与亲和力较低的单价免疫毒素的活性非常相似。我们的数据表明，增加抗体片段的亲和力并不一定能在体内提高免疫毒素的抗肿瘤活性。
