Goldman Ellen R, Liu Jinny L, Zabetakis Dan, Anderson George P
Center for BioMolecular Science and Engineering, US Naval Research Laboratory, Washington, DC, United States.
Front Immunol. 2017 Jul 25;8:865. doi: 10.3389/fimmu.2017.00865. eCollection 2017.
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 (s) of sdAbs cover a wide range from below 50 to over 80°C. Many sdAbs have been engineered to increase their , 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 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.
单域抗体（sdAbs）正逐渐赢得作为卓越识别元件的声誉，因为它们兼具传统抗体的特异性和亲和力优势，同时还具有高稳定性和溶解性。单域抗体的解链温度范围很广，从低于50°C到超过80°C。许多单域抗体已经经过工程改造以提高其稳定性，使其在暴露于极端温度之前都保持稳定。源自骆驼科动物可变重链和仅具有重链的鲨鱼抗体的单域抗体分别称为VHH和VNAR，通常在热变性后表现出一定的重新折叠和结合抗原能力。这种重新折叠的能力从0到100%不等，并且是一种既取决于单域抗体内在因素又取决于外在条件（如样品缓冲液离子强度、pH值和单域抗体浓度）的特性。单域抗体也经过工程改造以提高其溶解性和重新折叠能力，这使它们即使在暴露于超过其熔点的温度后仍能发挥作用。此外，人们还努力提高它们在极端pH值以及存在化学变性剂或蛋白酶情况下的稳定性。已采用多种途径对单域抗体进行工程改造以获得这些增强的稳定性。用于实现这些目标的方法包括将互补决定区嫁接到稳定的框架上；引入非典型二硫键；随机诱变结合严格筛选；点突变（如引入负电荷）以及基因融合。已经实现了高达20°C的提升，使一些单域抗体的解链温度超过90°C。在此，我们概述了为稳定源自骆驼科动物和鲨鱼单域抗体所做的工作。利用这些各种策略，单域抗体在不显著损害其亲和力的情况下得到了稳定，从而为检测、诊断和治疗应用提供了卓越的试剂。
