Discovery of Nanobodies

A short journey into the discovery of the Nanobody/ VHH technology

What’s so special about alpaca antibodies and how were they discovered? Alpacas aren’t typical lab animals, so these are legitimate questions...

As is often the case with important discoveries, chance helped scientists at the Free University of Brussels in the late 1980s. As Michael Gross remembers the story: During a practical course, a couple of biology students were to extract antibodies from human blood serum. They were not overly excited, on the one hand because they were concerned that the samples might be contaminated with HIV, on the other hand because this type of experiment had already been done numerous times before and the result was well documented in their text books. Their tutors then offered to sacrifice a few mice instead – not a very popular choice either. Eventually, a few liters of frozen dromedary serum were discovered in the lab freezer – this exotic example inspired the students to start working on the antibody separation.

In addition to the usual distribution of immunoglobins, they also discovered a group of smaller antibodies that did not correspond to anything known to science. This might have ended in obscurity, had not two researchers, Raymond Hamers and Cecile Casterman, investigated the matter more deeply. They did not believe that this species were just degraded variants of the “real” antibodies and therefore started to characterize them in more detail. Eventually, it became clear that they had discovered a new class of antibodies that were devoid of light chains and had a single antigen recognizing domain. These antibodies were later found in different camelid species, including llamas and alpacas. If you would like to learn more about the captivating story of this discovery, which includes travels to Morocco, a stolen camel and help from a Sheikh, you may read Michael Gross’ book “The birds, the Bees and the Platypuses”.

Based on their structure, these peculiar camelid antibodies have been named Heavy Chain Antibodies (hcAb), as they are composed of heavy chains only and are devoid   of light chains. HcAbs are not found in other mammals except in pathological cases. In 1995, Greenberg and colleagues found similar hcAbs in nurse sharks (Greenberg et al., 1995), but evolutionary analysis showed that camelid and shark hcAbs evolved independently (Nguyen et al., 2002). There are many speculations about the evolutionary driving force for the emergence of heavy chain antibodies in such distantly related species. A plausible explanation could be that, unlike conventional (comparably large) antibodies, these small single domain antigen binding fragments allow the targeting of otherwise inaccessible epitopes, e.g. catalytic centers of enzymes (Flajnik et al., 2011).

 

In the absence of light chains, the fragment-antigen-binding (Fab) part of these antibodies is reduced to a single domain. Therefore, hcAbs belong to the class of single domain antibodies (sdAbs). The single domain is called VHH (variable heavy domain of heavy chain antibodies) domain or Nanobody. The VHH domain contains a complete antigen binding site and is the smallest functional antigen binding fragment (around 15 kDa - only one tenth the size of a conventional antibody).

In fact, the many advantages of this novel class of antibodies range from research applications to drug development. The first approval of a Nanobody-based drug was in 2018, when Caplacizumab developed by Ablynx, now part of Sanofi, was launched for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP).

In general, Nanobodies can be readily selected and produced in bacteria, ensuring their virtually unlimited supply in consistent quality. In contrast to conventional antibodies, Nanobodies are also exceptionally stable, withstanding conditions of extreme temperatures, chaotropic reagents, detergents, glycerol, salt, reducing conditions, and pH. At ChromoTek, we thoroughly characterize our single domain antibody fragment derived products by function and structure: we prove applications and determine the chemical and thermal stability of every VHH individually. The stability of our products is outstanding: we have tested some Nanobody preparations that were more than five years old: they were still functional, with little or no loss of activity.