Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
J Biosci Bioeng. 2024 Nov;138(5):439-444. doi: 10.1016/j.jbiosc.2024.07.009. Epub 2024 Aug 28.
In this study, we have demonstrated a complementary-determining region (CDR) grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the CDR/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries. The new version successfully maintained the antigen binding affinity. CDR-grafted scFvs possessing a common CDR sequence and different FR sequences were successfully generated based on this new CDR/FR definition, and their physicochemical properties were further investigated. The antigen-binding activities of rabbit scFvs on Maxisorp varied between the tested clones in sandwich ELISA, supporting the idea that the combination of CDR with different FRs might change the physicochemical properties of scFvs on a solid material. The CDR-grafted scFvs possessing a frame sequence of anti-CRP scFv C2R maintained the ability to bind to protein L and were successfully purified. Expression titers showed improved solubility by diminishing the amount of insoluble scFvs. Thus, the method developed in this study is promising for generating alternatives with strict antigen binding recognition and different physicochemical properties.
在这项研究中，我们展示了一种互补决定区（CDR）移植技术，用于生成具有不同抗原识别和物理化学性质的兔 scFv。与原始克隆 C1R 相比，基于传统编号规则的 CDR/FR 定义生成的抗 CRP scFv，C1R/B1R（V1）的抗原结合亲和力不足，这表明原始 CDR 以外的氨基酸残基可能对兔 scFv 的抗原识别有重要贡献。我们根据从噬菌体文库中分离出的兔 scFv 的氨基酸序列比对，重新定义了新的 CDR 和 FR，通过延长 CDRH1 和 CDRH2 的多个氨基酸残基来保持抗原结合亲和力。新版本成功地保持了抗原结合亲和力。基于这种新的 CDR/FR 定义，成功地生成了具有共同 CDR 序列和不同 FR 序列的 CDR 移植 scFv，并进一步研究了它们的物理化学性质。在夹心 ELISA 中，测试克隆之间兔 scFv 的抗原结合活性不同，这支持了这样一种观点，即 CDR 与不同 FR 的结合可能会改变 scFv 在固体材料上的物理化学性质。具有抗 CRP scFv C2R 框架序列的 CDR 移植 scFv 保持了与蛋白 L 结合的能力，并成功地进行了纯化。表达滴度表明通过减少不溶性 scFv 的量提高了溶解度。因此，本研究中开发的方法有望生成具有严格抗原结合识别和不同物理化学性质的替代物。
