Combining somatic mutations present in different in vivo affinity-matured antibodies isolated from immunized Lama glama yields ultra-potent antibody therapeutics
Combining somatic mutations present in different in vivo affinity-matured antibodies isolated from immunized Lama glama yields ultra-potent antibody therapeutics
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DOI：
10.1093/protein/gzw003
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发表时间：
2016-04-01
影响因子：
2.4
通讯作者：
Roovers, Rob C.
中科院分区：
文献类型：
--
作者：
Klarenbeek, Alex;Blanchetot, Christophe;Roovers, Rob C.
在治疗上，需要高效的人抗体来中和细胞因子，如白细胞介素-6 (IL-6)，白细胞介素-6与许多炎症性疾病和恶性肿瘤有关。虽然存在许多诱变方法来执行抗体亲和成熟，但这些方法可能导致抗体不稳定和生产问题。因此，一个强大的和容易的抗体亲和成熟策略，以增加抗体效力仍然是非常可取的。通过免疫羊驼，克隆“免疫”抗体库并使用噬菌体展示，我们选择了一组多样化的IL-6拮抗fab。在次品率最低的Fab上进行重链洗牌，导致次品率更低的变体面板。Fab: IL-6复合物的结构分析表明，亲和力的增加部分是由于HCDR2中丝氨酸到酪氨酸的开关。这在IL-6诱导的SAA产生的体内模型中转化为中和能力。最后，设计了一个新的Fab文库，编码在重链洗牌后鉴定的VH基因自然曲目中发现的所有变异。高严格性选择导致鉴定出在重新格式化为IgG1时效力增加250倍的Fab。与目前临床开发的一种高度工程化的抗il -6单克隆抗体相比，这种IgG至少具有同样的效力，表明工程化过程已经产生了一种高效的抗il -6抗体。
Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab: IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.