A single domain antibody (SdAb), sometimes referred to as a “nanobody,” represents a specialized fragment derived from larger antibodies. Unlike conventional antibodies, which are complex, multi-chain proteins, an SdAb consists of a single, functional binding unit. This compact structure, typically a solitary variable antibody domain, retains the full ability to selectively attach to a specific target molecule, known as an antigen. These miniature binding agents originate from unique antibody forms found in certain animals.
Unique Characteristics
Single domain antibodies differ from traditional antibodies, primarily due to their significantly smaller size. They weigh approximately 12 to 15 kilodaltons (kDa), about one-tenth the size of a conventional antibody (150-160 kDa). This reduced size stems from their composition as a single polypeptide chain, specifically a heavy-chain variable domain, lacking the light chains and first constant domain (CH1) found in larger antibodies.
This compact architecture provides single domain antibodies with remarkable stability across harsh conditions, including high temperatures, varying pH levels, detergents, and high urea concentrations. Their inherent solubility is also high, making them easier to handle and formulate. Their smaller size allows for superior tissue penetration, enabling them to reach targets within dense tissues or even inside cells inaccessible to larger antibody molecules. Furthermore, single domain antibodies often feature a longer and more flexible complementarity-determining region 3 (CDR3) loop, which can extend into concave or hidden pockets on antigens, known as cryptic epitopes, that conventional antibodies cannot reach.
Sources and Engineering
Single domain antibodies originate naturally in certain animal groups. Camelids, such as llamas, alpacas, and dromedary camels, produce unique heavy-chain-only antibodies that lack light chains. Cartilaginous fish, including sharks, also produce a distinct type of heavy-chain-only antibody, known as IgNAR, from which single domain antibodies can be derived. These naturally occurring single variable domains, referred to as VHH from camelids and VNAR from sharks, form the foundation for engineered single domain antibodies.
Laboratory production of single domain antibodies begins by immunizing a camelid or shark with the target antigen. After immunization, immune cells (lymphocytes) are collected from the animal, and their genetic material (mRNA) is extracted. This mRNA creates a library of antibody genes, which are displayed on the surface of bacteriophages, a technique known as phage display. Screening identifies specific single domain antibody clones that bind strongly to the target antigen. Identified genes are transferred into bacterial systems, such as E. coli, for cost-effective, large-scale production.
Diverse Applications
Single domain antibodies have diverse applications in medical and research fields. In diagnostics, their small size and stability make them suitable for rapid, point-of-care tests like lateral flow assays and ELISA, providing quick and accurate detection of various biomarkers or pathogens. Their ability to penetrate tissues and clear rapidly from the body also makes them excellent candidates for molecular imaging, where they can be labeled with imaging agents to visualize tumors or other disease sites with high resolution.
In therapeutics, single domain antibodies are being explored for their potential to deliver drugs directly to diseased cells, particularly in cancer treatment, by targeting specific markers on tumor cells. Their small size allows them to infiltrate solid tumors more effectively than larger antibodies, which can improve treatment efficacy. They are also being investigated for anti-viral therapies, where they can neutralize viruses by binding to accessible sites, and for neurodegenerative diseases like Alzheimer’s disease. Several single domain antibody-based drugs have already received approval, including Cablivi for a rare blood disorder, Nanozora for rheumatoid arthritis, Envafolimab for certain cancers, and Carvykti for multiple myeloma.
Beyond clinical uses, single domain antibodies serve as valuable tools in scientific research. Their capacity to bind to specific proteins with high affinity makes them useful for protein detection and purification in laboratory experiments. They are also increasingly employed in structural biology, acting as “chaperones” to stabilize challenging protein targets, thereby facilitating their structural analysis using techniques like X-ray crystallography and cryo-electron microscopy.