Nanobody or VHH – What’s the difference?
Discoveries beyond conventional antibodies
It all started with a surprising discovery. In 1989, Professor Raymond Hamers and his team at Vrije Universiteit Brussel found a unique type of antibody in camels infected with Trypanosoma evansi. Unlike conventional antibodies composed of heavy and light chains, these antibodies lacked light chains, consisting solely of heavy-chain fragments. These were later classified as Heavy-Chain Only Antibodies (HCAbs).
Following the discovery of HCAbs, Hamers’ team optimized the creation of single-domain antibodies and patented their method. In 1994, they pioneered the term VHH or VHH, which refers to the isolated variable domain of a heavy chain-only antibody.
Figure 1. An illustrative representation of different antibodies structure.
Coining the term ‘Nanobody’
Around the same time, VHH fragments began to attract significant attention for their potential use beyond the lab. Ablynx, the Belgian biotech company, trademarked the term Nanobody® in 2003 to represent the unique, nanometer-scale size of VHH fragments. These fragments measure approximately 2.5 nm x 4 nm and weigh around 15 kDa. That’s about 1/10th the size of a traditional monoclonal antibody (mAb).
Nanobody® remains a trademarked name today, but its use spread widely beyond Ablynx. The first FDA-approved nanobody-based drug was Caplacizumab, developed by Ablynx for the treatment of blood disorders, in 2019. Today, nanobodies (Nbs) is often used as a general term for these compact and versatile antibody fragments.
Although the term is used broadly in the antibody community, at Isogenica we use “VHH” instead of “nanobody” in order to respect its trademarked origins.
What sets VHHs apart?
When we think of conventional antibodies, we generally imagine Y-shaped structures made up of multiple parts. In these antibodies, both the heavy chain (VH) and the light chain (VL) are needed to effectively bind antigens. In total, six complementarity-determining regions (CDRs) are involved in the antigen-binding process.
In HCAbs, only 3 CDRs are responsible for equivalent antigen binding, all in the VHH region, which is the smallest and most compact functional unit of HCAbs. Their simpler and smaller structure gives them certain advantages, unique to the VHH modality.
Unique VHH structural features
Unlike conventional antibodies, where CDRs from both heavy and light chains share the burden of antigen binding, in VHHs, a unique CDR3 takes center stage as the primary player in antigen binding with immense variability in length and structure. However, it’s important to note that CDR1 and CDR2 are also crucial in VHHs. These regions often contribute significantly more to antigen binding than in human mAbs, possibly because VHHs lack a light chain and require all CDRs to work harder.
One of the characteristics that gives VHHs this ability is an unusually large CDR1. This increased size compensates for the absence of the light chain, allowing the VHH to have a broader and more versatile binding surface. Moreover, the architecture of the loops in these regions shows flexibility that isn’t found in traditional antibody structures. For these reasons, human CDRs cannot be simply transferred into VHH frameworks without compromising functionality In summary, what might seem like a limitation – having fewer CDRs and lacking a light chain – actually becomes an advantage for VHHs. Their compact and efficient design means they can be used readily as building blocks in complex therapies or expressed readily in vivo making them versatile and powerful tools in the world of biotechnology.
Comparing VHHs with Other Single-Domain Antibody Formats
The terms “nanobody”, “single domain antibody” or even “heavy chain antibody” can refer to several formats, sometimes causing confusion when differentiating between them. But what are the differences? Using synthetic or genetic engineering approaches has created a world of antibody fragments with almost identical shapes and basic structures, but very different strengths and weaknesses. As well as VHHs, other formats like VH domains and synthetic variants such as camelized VH domains or humanized VHHs also exist, each with distinct properties. Understanding these differences is essential when selecting the right format for your therapeutic development.
Figure 2. The structural differences among single domain antibody formats.
VHH Domains
Isogenica is focused on VHHs, the single variable domain derived from HCAbs found in llamas. Although we use synthetic scaffolds, some of which are humanized, these preserve the unique features of VHH antibodies that make the format so special:
- Soluble Framework 2
- Extended CDR lengths
- Hypervariable CDR3
- CDR sequence patterns based on natural llama distributions
VH Domains
VH domains represent the single heavy chain variable domain from a conventional monoclonal antibody (mAb). Unlike VHHs, VH domains evolved to work in tandem with a light chain, which affects their overall structure and stability. A notable challenge with VH domains is their hydrophobic framework 2 (FW2) region, which is naturally designed to pair with a light chain. This can result in aggregation when used as standalone domains, making them less favorable for certain therapeutic applications such as CAR-T ectodomains, where low-level electrostatic interaction can trigger tonic signaling and T cell exhaustion.
Camelized VH Domains
To address the hydrophobicity of these VH domains, some companies offer synthetic “camelized” VH domains. This process modifies the FW2 region to resemble the more hydrophilic nature of camelid antibodies, aiming to improve solubility and reduce aggregation. However, while camelization resolves some issues, the CDR regions are still fundamentally based on mAb sequence patterns, limiting the affinities that can be generated against most targets.
Humanized VHHs
The reverse approach is to generate humanized versions of camelid VHHs, which are altered to incorporate human sequences. However, antibody humanization can mean different things depending on the extent of modification, and overly humanizing the molecule can compromise the inherent strengths of VHHs, bringing them closer to a VH domain (*). With little evidence to suggest that even camelid VHHs pose a significant immunogenicity risk, it is important to consider this closely, and where risks might outweigh potential benefits. Isogenica retains the essential characteristics of VHHs during humanization, ensuring that the unique CDR loops and binding abilities are preserved. Some companies also offer fully human CDRs, but there’s not much evidence suggesting that this approach could be substantially beneficial. If you’re still curious, you can read more about Isogenica’s approach to humanization here. In conclusion, while the term “single domain antibody” encompasses many formats, each has its own strengths and limitations. For companies looking to develop cutting-edge therapies, choosing a VHH from Isogenica balances optimal binding performance, stability, and humanization, offering distinct advantages over other single-domain formats.
Current Nanobodies or VHHs in the market
As for VHH therapies on the market, some have already reached the end of the road and are approved, such as ciltacabtagene autoleucel for the treatment of multiple myeloma, and calplacizumab,approved in both the United States and Europe for a rare blood disease. In Japan, ozoralizumab is already offering relief to patients with rheumatoid arthritis. But the story doesn’t end here. With the regulatory success of pioneer therapies like these, the clinical pipeline keeps growing. Gefurulimab, is in crucial Phase 3 trials for myasthenia gravis, while vobarilizumab, sonelokimab and many other nanobody-based therapies continue their development, advancing in Phase 2 to demonstrate that they can transform the treatment of autoimmune diseases, infections and more.
Table 1. This table lists therapeutic products developed from nanobodies. It details the diseases or conditions they target, the specific antigens they aim at, their current clinical trial status, the companies developing them, and includes relevant references. Sources: Jin et al (2023), and The Antibody Society
As we can see, VHHs have now gone beyond being just another novel format and growing in clinical acceptance as regulatory precedents are set. With several in advanced stages and some already on the market, VHH-based medicines are not only transforming the treatment of complex diseases but are empowering the development of new and innovative therapeutic approaches such as in vivo delivery.
If you are looking to accelerate the development of cutting-edge therapies and take a decisive step towards innovative solutions, single-domain antibody technology is a great fit. But it’s worth doing your homework on what modalities you’re being offered.
With Isogenica’s vast synthetic libraries and tailor-made approach, we offer a fast, reliable, and low-risk approach to discover VHH antibodies that will revolutionize your projects and bring those disruptive treatments closer to those who need them most. Contact us today for your free expert consultation.