Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview

Abstract

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.

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

“…This suggests that antibody libraries designed with sequence variation only in heavy chain CDR3 have a reduced risk for displaying affinity/stability trade-offs. More generally, these and other findings [18–20] suggest that affinity-enhancing mutations have an increased risk of destabilization and that compensatory mutations are frequently required to maintain antibody thermodynamic stability.…”

Protein binders and their applications in developmental biology

“…1). In the past decade, tremendous progress has been made in refining these scaffolds for better stability, higher affinity and easier handling (Goldman et al, 2017;Schilling et al, 2014;Schmidt et al, 2016), and libraries of increasing complexity have been generated and characterized (Moutel et al, 2016;Tiede et al, 2017;Yan et al, 2014).…”

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

“…Camelids and sharks have surprisingly something in common: the way their antibody system is designed. 8 Indeed, besides the classical double-chain antibodies, shared by all living animals, they also have single-chain ones. Even more interesting is the fact that the variable portion of their heavy chain bears the full antigen-recognition site.…”