Evaluation of the cyclic single-chain Fv antibody derived from nivolumab by biophysical analyses and in vitro cell-based bioassay

Sena Kamesawa, Mizuki Ogawa, Yoshiki Funakoshi, Masaya Kato, Shosei Kai, Mana Namikawa, Kyo Okazaki, Takashi Sato, Yoshihiro Kobashigawa, Hiroshi Morioka
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Abstract

Single-chain Fv (scFv) is a recombinant small antibody in which a polypeptide linker connects the variable regions of the light chain (VL) and the heavy chain (VH). The practical use of scFv, however, has been prevented by its tendency to aggregate due to interchain VL–VH interactions. We recently developed a cyclic scFv whose N-terminus and C-terminus were connected by protein ligation techniques. Biophysical comparisons between cyclic and linear scFv have been conducted, but cell biological evaluations remain unexplored. Here we studied the properties of cyclic and linear scFv derived from nivolumab. Biophysical studies revealed that the thermal stability was not changed but that the antigen-binding activity was approximately 3-fold higher as a result of circularization. A cell-based PD-1/PD-L1 interaction inhibitory assay revealed that the biological activity of scFv was markedly higher in the circularized form. In addition, biophysical analysis of scFv proteins incubated in the presence of serum revealed that circularization suppressed the decrease in antigen-binding activity. It could be assumed that circularization of scFv improved stability in the presence of serum, which in turn would suggest the applicability of cyclic scFv as a biopharmaceutical format.
通过生物物理分析和体外细胞生物测定评估由 nivolumab 衍生的环状单链 Fv 抗体
单链 Fv(scFv)是一种重组小抗体,其轻链(VL)和重链(VH)的可变区之间由多肽连接。然而,由于 VL-VH 链间的相互作用,scFv 容易聚集,这阻碍了它的实际应用。我们最近开发了一种环状 scFv,其 N 端和 C 端通过蛋白质连接技术连接起来。我们已对环状 scFv 和线性 scFv 进行了生物物理比较,但细胞生物学评估仍未进行。在此,我们研究了从 nivolumab 提取的环状和线性 scFv 的特性。生物物理研究表明,热稳定性没有改变,但抗原结合活性因环化而提高了约 3 倍。基于细胞的 PD-1/PD-L1 相互作用抑制试验显示,环化后的 scFv 生物活性明显更高。此外,在有血清存在的情况下培养 scFv 蛋白的生物物理分析表明,环化抑制了抗原结合活性的降低。可以认为,scFv 的环化提高了其在血清存在下的稳定性,这反过来又表明了环状 scFv 作为生物制药形式的适用性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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