The body’s immune system defends against foreign invaders like viruses and bacteria. Antibodies are specialized proteins that identify and neutralize threats. While conventional antibodies are well-known, VHH antibodies have emerged as an important area of research. These smaller, more versatile antibody fragments are opening new avenues in medicine and biotech.
Understanding VHH Antibodies
VHH antibodies originate from a distinct class of antibodies found naturally in camelids (camels, llamas, and alpacas). Unlike typical antibodies in humans and other mammals, which are composed of four protein chains (two heavy and two light chains), camelids produce a significant proportion of “heavy-chain-only antibodies” (HCAbs). These HCAbs lack the light chains and the first constant domain (CH1) of the heavy chain, simplifying their structure.
The antigen-binding portion of these unique HCAbs is a single, small variable domain, which is the Variable domain of a Heavy chain antibody (VHH). This VHH domain, also commonly called a nanobody, weighs approximately 12-15 kilodaltons (kDa), making it about one-tenth the size of a conventional antibody. Despite their small size, VHH domains bind specifically and tightly to their target molecules. Their single-domain nature and unique structural adaptations, like longer complementarity-determining region (CDR3) loops, enable access to hidden or recessed binding sites inaccessible to larger antibodies.
Advantages Over Conventional Antibodies
The small size of VHH antibodies provides distinct advantages over conventional antibodies. At about 15 kDa, they are significantly smaller than typical antibodies (around 150 kDa). This allows VHH antibodies to penetrate tissues more effectively, potentially crossing biological barriers like the blood-brain barrier, a significant hurdle for many therapeutics. Their compact nature also enables them to reach “hidden” or recessed binding sites on target molecules, such as enzyme active sites or viral cavities, that larger antibodies cannot access.
VHH antibodies also exhibit high stability. They are resilient to a wide range of challenging conditions, including extreme temperatures, pH levels, and denaturing agents. This robustness makes them easier to store and handle, allowing for their use in diverse environments that would degrade conventional antibodies. Their high solubility further contributes to their stability and ease of formulation.
Beyond their physical resilience, VHH antibodies demonstrate high affinity and specificity, meaning they bind tightly and precisely to their intended targets. This strong binding is comparable to that of conventional antibodies, despite their simpler structure. The single-domain nature of VHH antibodies also simplifies their engineering and manipulation. This characteristic, coupled with efficient production in cost-effective microbial systems like bacteria and yeast, makes their manufacturing more scalable and economical compared to the more complex production of traditional antibodies in mammalian cell systems.
Applications Across Fields
VHH antibodies have diverse applications across scientific and medical fields.
Therapeutics
VHH antibodies show promise in treating a range of conditions. They are explored for cancer therapies, engineered into formats like VHH-based chimeric antigen receptors (VHH-CARs) or bispecific killer cell engagers (BiKEs) to eliminate cancer cells. They also hold potential for infectious diseases, with examples targeting SARS-CoV-2 and respiratory syncytial virus (RSV). VHH antibodies are also investigated for autoimmune disorders, such as Caplacizumab, and for neurological conditions due to their ability to potentially cross the blood-brain barrier for targeted drug delivery.
Diagnostics
In diagnostics, VHH antibodies enhance the precision of various tests. Their stability and ability to bind specific markers make them suitable for rapid diagnostic tests, improving sensitivity and specificity. They are also employed as molecular imaging agents, visualizing disease-related biomolecules. For tumor imaging, their small size allows for deeper penetration and faster clearance, leading to better image quality and reduced patient radiation exposure. VHH antibodies are also integrated into biosensors for real-time detection.
Fundamental Research
VHH antibodies serve as tools in fundamental research. Researchers use them as specific probes for studying protein interactions, isolating target molecules, or creating novel biosensors. Their small size and robust nature make them suitable for intracellular imaging, enabling the tracking of antigens within living cells, often by fusing them with fluorescent proteins to create “chromobodies.” They are also useful in structural biology, assisting in protein crystallization and providing insights into molecular structures. VHH antibodies also have potential in biotechnology and industrial processes, such as enzyme immobilization and protein purification.
Developing VHH Antibodies
The journey of discovering and engineering VHH antibodies begins with immunizing camelids (llamas or alpacas) with a specific target molecule. This encourages the animal’s immune system to produce heavy-chain-only antibodies, from which VHHs are derived. Following immunization, B cells are isolated from the camelid.
After isolating B cells, common laboratory techniques select and isolate specific VHH antibodies with desired binding properties. Phage display is a widely used method, where VHH genes are inserted into bacteriophages, causing the VHH antibodies to be displayed on the phage surface. These phages are then screened against the target antigen, allowing for the selection of high-affinity binders through biopanning. Yeast display is another technique, where VHH fragments are presented on the surface of yeast cells, enabling the screening and isolation of specific binders using flow cytometry.
Once specific VHH antibodies are identified, they can be further engineered for various applications. For therapeutic use, humanization strategies might modify the camelid VHH sequence to be more human-like, reducing the risk of an immune reaction when administered to humans. This can involve grafting antigen-specific VHH sequences onto human antibody scaffolds or replacing camelid-derived complementarity-determining regions (CDRs) with human CDRs while preserving binding affinity. VHH antibodies can also be fused to other molecules, such as an Fc region, to extend their half-life or to toxins for targeted drug delivery.