Single Domain Antibody VS Conventional Antibody
Advantages of Single Domain Antibody Compared to Conventional Antibody
Since the discovery of heavy-chain only antibodies in 1993, the field of single domain antibodies (sdAbs) has been rapidly developing. Compared to conventional antibody, sdAbs have many clear advantages for biotechnological applications, mainly including their small size (2.5 nm in diameter and about 4 nm in length; ~15 kDa), strictly monomeric behavior, high affinity and specificity, high thermostability, good solubility, relatively low production costs, ease of genetic engineering, format flexibility or modularity, and a high penetration rate into tissues.
- Small size leading to rapid tissue penetration and fast clearance
Because of their small size of about 15 kDa in comparison to full-size conventional antibody, sdAbs have short serum half-life of about 2 hours and allow rapidly pass the renal filter resulting in rapid blood clearance and fast tissue penetration such as the blood-brain barrier. It is advantageous for targeting of sdAbs coupled with toxic substances to tumors, in vivo diagnosis using imaging, and therapy of neurotropic virus infections. However, for other therapeutic applications, such as treatment of infectious or inflammatory diseases, sdAbs may not cross the endothelial barriers to clear out the virus, limiting the effect of sdAb treatment in infection.
- The unique structure and extended flexible CDR3 for recognition of hidden antigenic sites
Due to the small size and the ability of the extended CDR3 loop to form the convex conformations, sdAbs can recognize unique and cryptic antigenic sites that are typically not recognized by conventional antibodies, such as enzyme active sites and conserved cryptic epitopes. It facilitates their use as enzyme inhibitors, targeting intracellular targets or epitopes concealed in protein structures. However, their single-domain nature could be a disadvantage for binding to small antigens such as haptens and peptides because they typically bind in a groove or cavity at the VH-VL interface.
Fig.1 Structure and features of sdAb. (Wu, 2017)
- High solubility and high physicochemical stability
Compared to the conventional antibody, a few critical conserved amino acids are replaced by some amino acids of sdAbs, including Leu12Ser, Val42Phe/Tyr, Gly49Glu, Leu50Arg/Cys, and Trp52Gly. These residue positions are critical to the interaction of VH-VL of conventional antibody; thus, these substitutions and the additional disulfide linkages render the overall structure of sdAbs more hydrophilic and contribute to high stability, solubility and resistance against aggregation. Moreover, sdAbs possess resistance to high temperatures or extreme pH; the high stability of sdAbs is attributed to their efficient refolding after thermal or chemical denaturation. These features show that sdAbs are ideal candidates for developing viable treatment strategies in harsh environments.
- Well expressed, facile genetic manipulation and production
Owing to the stability, increased hydrophilicity, and single-domain nature, sdAbs are efficiently suited for production in bacteria, yeast, mammalian cells, and plant cells, enabling large-scale production at reasonable costs. The majority of sdAbs do not contain N-glycosylation sites due to the devoid of the Fc domain, which makes sdAbs not only suitable for production by the prokaryotic expression system but also by the eukaryotic expression system. N-glycosylation is rare in sdAbs, and O-glycosylation is never observed so that these typical posttranslational modifications do not affect the folding, functionality, or activity of the sdAb fragments.
Nevertheless, about 10% of the sdAbs still contain potential N-glycosylation sites, and they may occasionally be N-glycosylated through the secretory pathway. As a result, N-glycosylation of sdAbs that have an average size of 15 kDa, can result in a large increase in molecular mass and even contributes to increasing their functional toxin- and virus- neutralization capacity. Thus, the neutralization capacity of sdAbs could be possibly increased by the introduction of potential N-glycosylation sites on single or even multiple sites that are located distant from the sdAb antigen-binding site.
- Facile engineering and production of multivalent formats
The single-domain nature of sdAbs facilitates subsequent molecular manipulation and engineering, such as the generation of bispecific constructs, multispecific antibodies, or enzyme fusions. It has advantages to engineer monovalent antibody fragments into multivalent formats to increase functional affinity or to produce bispecific antibody fragments that can simultaneously bind to different antigens. As opposed to conventional antibodies, sdAbs are more suitable for the production of such formats because they allow more flexible linker design without the problems posed by domain mispairing, or the side effects by the target cross-linking, or the presence of the Fc region.
Fig.2 Unique characteristics of sdAb. (Pain, 2015)
Application of sdAbs
Several sdAbs are now being studied against more than 120 therapeutically relevant targets for use in various disease areas, including oncology, hematology, infectious diseases, inflammatory disorders, and neurodegenerative diseases. The remarkable preference of sdAbs for binding into clefts and cavities on antigen surfaces offers the possibility for efficient inhibition of enzymes, neutralization of toxins, activity modulation of cell surface proteins (such as receptors and ion channels) and targeting some intracellular antigens. The ability of sdAbs to transmigrate across the blood-brain barrier could be utilized in the diagnostics and therapy of neurodegenerative diseases. Moreover, the high stability and resistance to extreme conditions make them especially suited for the immunotherapy of gastrointestinal disorders using oral administration. Furthermore, the modular single-domain nature of sdAbs allows easy engineering of bispecific or multispecific constructs that show an increased therapeutic potency compared with monovalent sdAbs.
The small size of sdAbs is an advantage in different analytical and diagnostic applications. Conventional monoclonal antibodies conjugated to different probes have been used for tumor imaging, but their large size (~150 kDa) and Fc regions impair tumor penetration. By contrast, sdAbs show effective penetration into tissues resulting in enhanced imaging signal and reduced accumulation of labeled fragments in vivo and lower radiation burden. On the other hand, sdAbs enable a rapid biodistribution and homogeneous tumor labeling, whereas the unbound fraction is rapidly cleared by renal filtration. The unique features of sdAbs make them good candidates for the construction of imaging probes used for in vivo monitoring of tumors, delivery of toxin, and radioisotopes to diseased tissues where rapid clearance is required.
Fig.3 Strategies for intracellular tumor targeting. (Hu, 2017)
References
- Wu, Y.; et al. Single-domain antibodies as therapeutics against human viral diseases. Frontiers in immunology. 2017, 8: 1802.
- Pain, C.; et al. Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena. Biochimie. 2015, 111: 82-106.
- Hu, Y.; et al. sdAb-based delivery systems for diagnosis and targeted tumor therapy. Frontiers in immunology. 2017, 8: 1442.
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