Abstract

Several monoclonal antibodies targeting the CD20 have been produced and Ofatumumab is a case in point. Although whole antibodies target cancer cells effectively, their applications are restricted in some ways. Single-chain fragment variable antibodies, rather than employing the entire structure of antibodies, have proven a practical approach for creating completely functional antigen-binding fragments. In current research, the DNA coding sequence of ${\mathrm{V}}_{\mathrm{L}}$ and ${\mathrm{V}}_{\mathrm{H}}$ of the wild and mutant forms of ofatumumab were joined with a flexible linker ${(\mathrm{GGGGS})}_3$ separately. Using the E. coli BL21 (DE3) expression system, the ${\mathrm{V}}_{\mathrm{L}}$-linker-${\mathrm{V}}_{\mathrm{H}}$ genes were cloned into the pET-28 a (+), and the associated recombinant proteins were produced. Purified and refolded scFvs (scFv-C and scFv-${\mathrm{V}}_3$) represented a concentration of around 0.7 mg/ml from 1 L of initial E. coli culture with a molecular weight of about 27 kDa. Affinity measurement disclosed anti-CD20 scFv-${\mathrm{V}}_3$ possesses a higher affinity constant compared to anti-CD20 scFv-$\mathrm{C}.$ The recombinant scFvs exclusively attach to Raji cells but not to Jurkat cells, according to a cell-ELISA analysis. The MTT test signified anti-CD20 scFvs could affect cell viability in Raji cells but had no impact on Jurkat cells and also, Raji cells viability was affected more significantly by anti-CD20 scFv-${\mathrm{V}}_3.$

摘要

已经产生了几种靶向 CD20 的单克隆抗体，Ofatumumab 就是一个很好的例子。尽管全抗体有效靶向癌细胞，但它们的应用在某些方面受到限制。单链片段可变抗体，而不是使用抗体的整个结构，已被证明是一种产生完全功能性抗原结合片段的实用方法。在目前的研究中，DNA编码序列${\mathrm{V}}_{\mathrm{大号}}$和${\mathrm{V}}_{\mathrm{H}}$的野生和突变形式的 ofatumumab 与灵活的接头连接${(\mathrm{格格格斯})}_{\mathrm{3个}}$分别地。使用大肠杆菌BL21 (DE3) 表达系统，${\mathrm{V}}_{\mathrm{大号}}$-链接器-${\mathrm{V}}_{\mathrm{H}}$基因被克隆到 pET-28 a (+) 中，并产生了相关的重组蛋白。纯化并重新折叠的 scFv（scFv-C 和 scFv-${\mathrm{V}}_{\mathrm{3个}}$) 表示来自 1 L 初始大肠杆菌培养物的浓度约为 0.7 mg/ml，分子量约为 27 kDa。亲和力测量显示抗 CD20 scFv-${\mathrm{V}}_{\mathrm{3个}}$与抗 CD20 scFv 相比具有更高的亲和力常数$\mathrm{C}.$根据细胞 ELISA 分析，重组 scFvs 仅附着于 Raji 细胞，但不附着于 Jurkat 细胞。MTT 测试表明抗 CD20 scFv 可以影响 Raji 细胞的细胞活力，但对 Jurkat 细胞没有影响，而且抗 CD20 scFv 对 Raji 细胞活力的影响更显着。${\mathrm{V}}_{\mathrm{3个}}.$