Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India.
Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India.
J Biol Chem. 2022 Apr;298(4):101772. doi: 10.1016/j.jbc.2022.101772. Epub 2022 Feb 24.
Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (K) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (K = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.
登革热是最主要的虫媒病毒病之一，每年在全球范围内感染至少 3.9 亿人。尽管如此，目前还没有有效的治疗方法，而唯一可用的疫苗由于严重的不良反应已被撤回。因此，目前正在寻求使用单克隆抗体进行被动免疫疗法作为一种治疗选择。迄今为止，已经鉴定出许多登革热单克隆抗体，其中大多数是血清型特异性的，只有少数是交叉反应的。此外，在血清型内发生交叉反应的抗体中和作用较弱，并且经常诱导抗体依赖性增强，从而促进病毒进入和复制。因此，需要具有无抗体依赖性增强风险的广泛中和抗体来治疗登革热。在这里，我们从抗融合环 E53 抗体（PDB：2IGF）中开发了一种单链可变片段（scFv）抗体。我们在编码 scFv 抗体的基因中引入了先前预测的有利互补决定区（CDR）突变，以进行亲和力成熟，并且所得变体在体外针对登革病毒包膜蛋白的高度保守融合和 bc 表位进行了测试。我们展示了其中一些 scFv 变体在三个不同的 CDR 中具有两个到三个取代突变，具有 20 到 200 nM 的亲和力常数（K）。包含 D31L、Y105W 和 S227W 取代的 scFv-mutant15 显示出最低的亲和力常数（K = 24 ± 7 nM），大约比其亲本构建体低 100 倍。我们提出，scFv 衍生抗体可能是开发有效和安全的免疫疗法的良好候选物。
