Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA); Present address: California Institute for Biomedical Research (Calibr), 11119 N. Torrey Pines Road, La Jolla, CA 92037 (USA).
Angew Chem Int Ed Engl. 2014 Jan 3;53(1):132-5. doi: 10.1002/anie.201307939. Epub 2013 Nov 19.
The X-ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent-exposed β-strand "stalk" fused to a disulfide crosslinked "knob" domain. We have substituted an antiparallel heterodimeric coiled-coil motif for the β-strand stalk in this antibody. The resulting antibody (Ab-coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H-D exchange supports the coiled-coil structure of the substituted peptides. Substitution of the knob-domain of Ab-coil with bovine granulocyte colony-stimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS-60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled-coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.
牛抗体 (BLV1H12) 的 X 射线晶体结构揭示了其超长重链互补决定区 3 (CDR3H) 中独特的结构，该结构折叠成一个暴露在溶剂中的 β-链“茎”，融合到一个二硫键交联的“旋钮”结构域。我们已经在该抗体中用一个反平行的异源二聚体螺旋线圈模体替代了β-链茎。由此产生的抗体 (Ab-coil) 在哺乳动物细胞中表达，并具有与亲本牛抗体相似的稳定性。H-D 交换的 MS 分析支持取代肽的螺旋线圈结构。用牛粒细胞集落刺激因子 (bGCSF) 替换 Ab-coil 的旋钮结构域，导致稳定表达的嵌合抗体，其对小鼠 NFS-60 细胞的增殖活性与 bGCSF 相当。这项工作证明了这种新颖的螺旋线圈 CDR3 基序作为产生具有有用药理学性质的稳定、有效抗体融合蛋白的一种手段的实用性。
