In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development.
In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development.
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DOI：
10.3389/fimmu.2017.01361
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发表时间：
2017
影响因子：
7.3
通讯作者：
Barelle CJ
中科院分区：
文献类型：
--
作者：
Steven J;Müller MR;Carvalho MF;Ubah OC;Kovaleva M;Donohoe G;Baddeley T;Cornock D;Saunders K;Porter AJ;Barelle CJ
通过分子工程提高非人治疗性抗体和支架与常见人免疫球蛋白序列的同一性百分比已成为标准实践。这种策略通常用于减少不期望的免疫原性应答，加速候选结构域的临床开发。Kovalenko及其同事报道了第一个人源化鲨鱼可变结构域（VNAR），并使用抗人血清白蛋白（HSA）结构域（克隆E06）作为模型构建了许多人源化版本，包括huE06v1.10。本研究通过使用huE06v1.10作为模板来分离具有改善的生物物理特性和降低的抗原性的结构域来扩展这项工作。对huE06v1.10进行随机诱变，然后通过基于解离速率排序的靶抗原选择来细化克隆。这些下一代结合剂中的许多保持了对靶标的高亲和力，以及良好的物种交叉反应性。在人树突状细胞试验中评估前导结构域的任何二聚化倾向、对N-和C-末端融合的耐受性、亲和力、稳定性和相对抗原性。通过延长典型药物形式的血清半衰期，在体内验证了候选克隆的功能。根据这些分析，BA 11结构域表现出可忽略的抗原性、高稳定性和对小鼠、大鼠和HSA的高亲和力。当这些属性与大鼠PK模型中可证实的功能相结合时，BA 11克隆被确定为我们的临床候选物。
Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.