Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview.
Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview.
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DOI：
10.3389/fimmu.2017.00865
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发表时间：
2017
影响因子：
7.3
通讯作者：
Anderson GP
中科院分区：
文献类型：
--
作者：
Goldman ER;Liu JL;Zabetakis D;Anderson GP
单结构域抗体（sdAb）作为上级识别元件获得了声誉，因为它们联合收割机结合了在常规抗体中发现的特异性和亲和力的优点以及高稳定性和溶解性。sdAb的熔化温度（Tms）覆盖从低于50 ° C到超过80°C的宽范围。许多sdAb被设计成增加它们的Tm，使它们在暴露于极端温度之前保持稳定。来源于骆驼科动物的可变重链的SdAb和仅鲨鱼重链抗体分别被称为VHH和VNAR，并且通常在热变性后表现出一定的重折叠和结合抗原的能力。这种再折叠能力在0至100%之间变化，并且是依赖于sdAb的内在因素和外在条件（例如样品缓冲液离子强度、pH和sdAb浓度）的性质。SdAb还被设计成增加其溶解度和再折叠能力，这使它们即使在暴露于超过其熔点的温度后也能发挥作用。此外，已经进行了改善其在极端pH下和在化学变性剂或蛋白酶存在下的稳定性的努力。已经采用多种途径来工程化具有这些增强的稳定性的sdAb。用于实现这些目标的方法包括将互补决定区移植到稳定框架上，引入非典型二硫键，随机诱变与严格选择相结合，点突变如包含负电荷和遗传融合。已经实现了高达20°C的增加，将一些sdAb的Tm推高到90°C以上。在本文中，我们概述了为稳定源自骆驼科动物和鲨鱼的sdAb所做的工作。利用这些不同的策略，sdAb已经被稳定化而不显著损害它们的亲和力，从而提供用于检测、诊断和治疗应用的上级试剂。
Single domain antibodies (sdAbs) are gaining a reputation as superior recognition elements as they combine the advantages of the specificity and affinity found in conventional antibodies with high stability and solubility. Melting temperatures (Tms) of sdAbs cover a wide range from below 50 to over 80°C. Many sdAbs have been engineered to increase their Tm, making them stable until exposed to extreme temperatures. SdAbs derived from the variable heavy chains of camelid and shark heavy chain-only antibodies are termed VHH and VNAR, respectively, and generally exhibit some ability to refold and bind antigen after heat denaturation. This ability to refold varies from 0 to 100% and is a property dependent on both intrinsic factors of the sdAb and extrinsic conditions such as the sample buffer ionic strength, pH, and sdAb concentration. SdAbs have also been engineered to increase their solubility and refolding ability, which enable them to function even after exposure to temperatures that exceed their melting point. In addition, efforts to improve their stability at extreme pH and in the presence of chemical denaturants or proteases have been undertaken. Multiple routes have been employed to engineer sdAbs with these enhanced stabilities. The methods utilized to achieve these goals include grafting complementarity-determining regions onto stable frameworks, introduction of non-canonical disulfide bonds, random mutagenesis combined with stringent selection, point mutations such as inclusion of negative charges, and genetic fusions. Increases of up to 20°C have been realized, pushing the Tm of some sdAbs to over 90°C. Herein, we present an overview of the work done to stabilize sdAbs derived from camelids and sharks. Utilizing these various strategies sdAbs have been stabilized without significantly compromising their affinity, thereby providing superior reagents for detection, diagnostic, and therapeutic applications.
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