Shinozaki Naoya, Hashimoto Ryuji, Noda Masanori, Uchiyama Susumu
Modality Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., Shinagawa R&D Center, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Modality Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., Shinagawa R&D Center, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
J Biosci Bioeng. 2018 Jun;125(6):654-661. doi: 10.1016/j.jbiosc.2018.01.006. Epub 2018 Feb 15.
Recently, we showed that immunized rabbit heavy chain variable regions (rVHs) can have strong antigen binding activity comparable to that of the camelid variable domain of the heavy chain of heavy chain antibody (VHH). These rVHs lack the light chain variable regions (rVLs), which exist in the authentic Fab format; thus, molecular surfaces at the interface region of rVHs are exposed to solvent. This physical feature may change physicochemical properties, such as causing reduced stability. By overcoming potential physicochemical issues through engineering the interface region, rVHs could become more useful as single-domain antibodies. In this study, we substituted amino acid residues conserved at the interface region of rVHs with those of VHHs. These substitutions included V37F, involving substitution of a residue in the hydrophobic core with a bulkier hydrophobic amino acid, and G44E/L45R, involving double substitutions of highly exposed residues with more hydrophilic ones. As expected, biophysical and structural characterizations showed that the V37F substitution markedly enhanced the thermal stability through increased hydrophobic packing, while G44E/L45R substitutions greatly reduced hydrophobicity of the interface. The quadruple substitutions of V37F/G44E/L45R/F91Y resulted in not only enhancements of thermal stability and reduction in hydrophobicity, both in an additive manner, but also synergistic improvement of purification yield. This quadruple mutant exhibited greatly reduced non-specific binding with improved colloidal stability owing to the reduced hydrophobicity. The approach used in this study should further enhance the utility of rVHs and promote research and development of single-domain antibodies.
最近，我们发现免疫兔重链可变区（rVHs）可具有与重链抗体（VHH）重链的骆驼科动物可变结构域相当的强抗原结合活性。这些rVHs缺乏存在于正宗Fab形式中的轻链可变区（rVLs）；因此，rVHs界面区域的分子表面暴露于溶剂中。这种物理特征可能会改变物理化学性质，例如导致稳定性降低。通过对界面区域进行工程改造来克服潜在的物理化学问题，rVHs作为单域抗体可能会变得更有用。在本研究中，我们用VHHs的氨基酸残基取代了rVHs界面区域保守的氨基酸残基。这些取代包括V37F，即将疏水核心中的一个残基替换为一个更大的疏水氨基酸，以及G44E/L45R，即将高度暴露的残基双重替换为更亲水的残基。正如预期的那样，生物物理和结构表征表明，V37F取代通过增加疏水堆积显著提高了热稳定性，而G44E/L45R取代大大降低了界面的疏水性。V37F/G44E/L45R/F91Y的四重取代不仅以累加方式提高了热稳定性并降低了疏水性，还协同提高了纯化产率。由于疏水性降低，这种四重突变体表现出非特异性结合大大减少，胶体稳定性得到改善。本研究中使用的方法应进一步提高rVHs的实用性，并促进单域抗体的研发。
