Kim Geun-Bae, Wang Zhirui, Liu Yuan Yi, Stavrou Scott, Mathias Askale, Goodwin K Jeanine, Thomas Judith M, Neville David M
Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Bldg 10, Room 3D46, 10 Center Drive, Bethesda, MD 20892-1216, USA.
Protein Eng Des Sel. 2007 Sep;20(9):425-32. doi: 10.1093/protein/gzm040. Epub 2007 Aug 10.
T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.
T细胞耗竭性抗CD3免疫毒素在移植耐受和自身免疫性疾病治疗的非人灵长类动物模型中具有应用价值。我们最近报道，一种亲和力成熟的单链（scFv）抗猴CD3抗体C207，与亲本抗体FN18相比，在基于白喉毒素（DT）的双价scFv免疫毒素中与T细胞的结合增加，生物活性增强。然而，FN18 scFv及其突变衍生物如C207在双价scFv形式中未表现出强大的二价特性。我们现在报道，双体形式的C207与scFv（C207）相比，与T细胞的结合增加了7倍，表明双体具有相当大的二价特性。该构建体通过将V（L）/V（H）连接子减少到五个残基形成，并作为非共价二聚体从毕赤酵母中分泌。基于这种双体形式的免疫毒素以非共价二聚体形式分泌，但缺乏生物活性且未能结合T细胞，这表明两个紧密相邻的大的截短DT部分存在空间位阻。我们通过将截短的DT C末端L1-VL-L1-VH-L2-VL-L1-VH融合构建了一种单链双体免疫毒素，其中L1是一个五残基连接子，L2是较长的（G4S）3连接子，允许远端和近端VL/VH结构域之间相互作用。这种“回折”免疫毒素主要以单体形式分泌，与DT390双价scFv（C207）相比，生物活性增加了5至7倍，并在体内耗尽了猴T细胞。
