Akiyama Yasuto, Miyata Haruo, Komiyama Masaru, Nogami Masahiro, Ozawa Kazumichi, Oshita Chie, Kume Akiko, Ashizawa Tadashi, Sakura Naoki, Mochizuki Tohru, Yamaguchi Ken
Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007 Shimonagakubo, Nagaizumicho,Sunto-gun, Shizuoka 411-8777, Japan.
Biomed Res. 2014;35(2):105-16. doi: 10.2220/biomedres.35.105.
Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becoming the dominant focus of clinical research. In particular, smaller recombinant antibody fragments such as single-chain variable fragments (scFv) have become the subject of intense focus. However, an efficient affinity ligand for antibody fragment purification has not been developed. In the present study, we designed a consensus sequence for the human antibody heavy or light chain-variable regions (Fv) based on the antibody sequences available in the ImMunoGeneTics information system (IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screened peptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody using a 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VH template and 8 peptides for the VK template were selected as the candidate ligands after 4 rounds of panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 and code-13 peptides showed recovery rates of the VH and VK templates that were 20-30% and 40-50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately 40%). If it were possible to identify the best combination of VH and VK-binding peptides among the ligand peptides suitable for the human mAb Fv sequence, the result could be a promising purification tool that might greatly improve the cost efficiencies of the purification process.
在所有潜在的生物治疗药物中，基于单克隆抗体（mAb）的疗法正成为临床研究的主要焦点。特别是，较小的重组抗体片段，如单链可变片段（scFv），已成为研究的热点。然而，尚未开发出一种用于抗体片段纯化的高效亲和配体。在本研究中，我们基于免疫遗传学信息系统（IMGT）中可用的抗体序列，设计了人抗体重链或轻链可变区（Fv）的共有序列，并将这些共有序列合成为模板Fv抗体。然后，我们使用表达噬菌体展示法的12肽文库筛选与源自库的人Fv共有抗体特异性结合的肽配体。随后，在对噬菌体展示进行4轮淘选后，选择了1个针对VH模板的肽和8个针对VK模板的肽作为候选配体。使用基于肽珠的免疫沉淀法，代码4和代码13的肽对VH和VK模板的回收率分别为20%-30%和40%-50%。两种肽对曲妥珠单抗scFv的回收率均较好（约40%）。如果能够在适合人源单克隆抗体Fv序列的配体肽中确定VH和VK结合肽的最佳组合，结果可能是一种有前景的纯化工具，有望大大提高纯化过程的成本效益。
