On the Origin of VHHs: Student serendipity and coincidental camels

Sep 4, 2023

At Isogenica, we’re experts in discovering and developing VHHs – small format single chain antibodies. Unlike conventional IgG antibodies from humans or mice, VHHs (also known as nanobodies) are composed solely of the single variable domain of a heavy chain.  

Due to their unique properties and compact size, VHHs have been embraced by the biopharmaceutical industry as ideal building blocks for antibody engineering and are increasingly being used in therapeutic applications.  

But where did these unique antibodies come from? How were they discovered? And why did they lose their light chains in the first place?  


A serendipitous student discovery  

Open any immunology textbook and you’ll find one of the most famous biological structures in the science world – the Y-shaped antibody. The traditional view is that all antibodies are composed of two heavy chains and two light chains. However, thanks to one serendipitous discovery in 1989, we now know this isn’t true.  

In the late ‘80s, Professor Raymond Hamers and his team at the Vrije Universiteit Brussel (VUB) were looking into a diagnostic test for Trypanosome infection (sleeping sickness) in camels and water buffalos.  

As is the norm, Hamers also had a few students in his lab who were running some simpler experiments such as separating an antibody into its respective parts. To do this, the students needed to get their hands on some blood, but with HIV concerns rife at the time, human blood seemed risky. Thankfully, the Hamers lab had access to plentiful vials of an alternative – camel blood.  

The students set about analysing the total and fractionated immunoglobulin G (IgG) molecules in the serum of a dromedary camel. To their surprise, the camel’s blood seemed to be mostly composed of two subclasses of IgG molecules that appeared to lack a light chain.  

Until now, heavy-chain-only antibodies (HCAbs) had only been observed in the blood of patients with “heavy chain disease,” and the resulting antibodies had been mostly useless and couldn’t bind to antigens at all.  

The only logical explanation was that something had gone wrong. Perhaps the antibodies in the blood had degraded over time in the blood sample? Or the light chains were only weakly associated with the heavy chains in the first place and they had been lost during the purification step performed by the rookie students?  

To find out what was going on, fresh blood was gathered from some camels at the local zoo and the experiment was run a second time. Their analysis confirmed that HCAbs made up around 75% of the total serum IgG in camels. This was an extraordinary, if confusing, discovery.  

Still not convinced, Hamers and his team performed further experiments to try and discern whether these oddly shaped antibodies were at all functional. To their surprise, they found that the heavy chains alone could generate an extensive antigen binding repertoire in not only dromedary camels, but also related camelids such as llamas and alpacas.  


The camels of the sea? 

It was soon clear that this initially confusing discovery would turn out to be one of the biggest breakthroughs in the history of antibody therapeutics to date.  

Shortly after their discovery of HCAbs, the Hamers lab perfected the production of single-domain antibodies and patented their process. In 1994, they were the first to use the term VHH, which stands for variable heavy domain of heavy chain (Figure 1).  

Position of anti-PD-L1 VHH attached to pembrolizumab on both heavy chains (HC) and light chains (LC)
Isogenica VHH Antibody Libraries

Figure 1: Human IgG with VH and Heavy chain IgG with VHH 

A year later, the next big breakthrough in antibody research surfaced. In 1995, Greenberg and colleagues found a similar class of single domain heavy-chain immunoglobulins, named IgNAR, in the adaptive immune system of the nurse shark. These antibodies seemed to have similar properties to camelid HCAbs, including high affinities and specificities, small size, high thermal stability, and low production cost.  


The discovery of HCAbs in a distantly related species of shark pointed to one explanation – there must be some sort of evolutionary advantage to producing these small antibodies. But what could that be?  


A theory of evolution  

Since their initial discovery, hundreds of VHHs have been discovered that exhibit functional properties such as enzyme inhibition or the neutralisation of viral infections. The number of VHHs reported to be involved in such processes is much higher than the number of mAbs thought to perform similar functions – a surprising finding considering VHHs are a relatively recent discovery. All this has led researchers to suggest that the structural differences between conventional mAbs and VHHs may confer a selective advantage to the organism.  

Structural studies also suggest that the conventional role of the complementarity-determining regions (CDRs) in VHHs has been modified, with some binding interactions extended to non-CDR regions. CDR loops (especially CDR3) are also unusually long in VHHs, with occasional intradomain disulfide bridges adding further CDR3 loop stability.  

Overall, these structural differences seem to compensate for the loss of the VL domain, establishing the VHH as a novel antigen binding domain with a more diverse functional repertoire. The unique structure of VHHs, particularly the long CDR3 loops, makes them ideal for accessing certain epitopes that bulkier human antibodies are unable to reach, such as the catalytic clefts of enzymes or viral cavities.  

Higher stability could also make VHHs better at tolerating harsh conditions ranging from the sweltering climes of the Arabian desert to the deep-sea environments patrolled by bottom-dwelling nurse sharks.  


The future is nano 

Whatever the evolutionary advantage of VHHs, their benefits to modern medicine have become abundantly clear over the past few decades. After the primary patent on VHHs expired in 2013, there was a flurry of research into their use as biotherapeutics, imaging reagents and more 

The first single domain antibody therapeutic, Sanofi’s bi-valent caplacizumab, was approved in 2018 for rare blood clotting disorders, with the first VHH-targeted CAR-T therapy, the bi-paratopic BCMA binder ciltacabtagene autoleucel (CARVYKTI®), for multiple myeloma following in 2022. There are many more VHH-based therapies coming down the pipeline, showcasing the wide flexibility possible with these small format antibodies.   

VHHs are extremely useful in CAR engineering, can be chained together to create multi-specific antibodies, and have low immunogenicity compared to conventional monoclonals. It is now even possible to extend the half-life of these small molecules to improve the dosing frequency of drugs, cut healthcare costs and ultimately improve patient quality of life.  

With further key biologics patents expiring soon, the stage is set for VHHs to truly make an impact in the field of biotherapeutics and bring significant benefits to patients.   

Here at Isogenica, we are experts in discovering and developing single domain antibody therapeutics thanks to our large, highly diverse, fully synthetic libraries of camelid-based VHHs and decades of antibody engineering experience. If you want to learn more about how to harness their unique properties for your application, come and talk to our experts or head to our website 

Here at Isogenica, we have more than 20 years of experience in antibody discovery and engineering. If you want to discover how VHHs could supercharge your IgG-based drugs, get in touch with our team at info@isogenica.com.