What are nanobodies (VHHs)?
VHH, the variable region of a heavy-chain only antibody, is the smallest antigen-binding domain with a molecule weight of only 1/10 of conventional antibodies (150-160 kDa), hence the name “nanobody”. VHHs are even smaller than Fab fragments (about 50 kDa, one light chain and one heavy chain) and scFv fragments (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain).
How were nanobodies discovered?
Naturally occurring heavy-chain only antibodies (HcAbs) were accidentally discovered in camels by immunologist Raymond Hamers in 1989. After a few years of research, Hamers confirmed this unexpected discovery and described them in Nature in 1993 (Hamers-Casterman et al., 1993).HcAbs are a feature of all camelids. In addition to the conventional IgG1 composed of two identical heavy chains and two identical light chains, camelids’ functional IgG2 and IgG3 lack the light chain and are composed of heavy-chain (lack CH1) dimers. The antigen-binding fragments of HcAbs in camelids are called VHHs or nanobodies. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, immunoglobulin new antigen receptor), from which antigen-binding fragments are called VNARs(Andrew S. Greenberg et al., 1995).
What are the advantages of nanobodies (VHHs)?
VHHs have many advantages over traditional antibodies or antibody fragments such as scFvs or Fabs.
Better targeting
Compared to monoclonal antibodies, VHHs are made of the antigen-binding fragment of HcAbs and also retain full antigen-binding activity. Due to their small sizes (~15kDa), VHHs can bind hidden epitopes not accessible to whole antibodies, so the targeting precision is higher compared to normal antibodies.
Easier production
Because VHHs are composed of one polypeptide chain, VHHs are relatively easy to produce in lower organisms (E. coli, yeast) up to very high amounts and purities. In addition, the hydrophilic side of nanobodies, which is not present in conventional antibodies, means they do not have issues with solubility and aggregation otherwise associated with conventional antibodies.
Better diffusion
VHHs have a high tissue penetration and are cleared from circulation rapidly. Some VHHs can even cross the blood-brain barrier.
Higher stability
VHHs are stable under a wide range of temperatures, extreme pH levels, and against proteases; hence, they keep their native folding and epitope binding capacity under very diverse experimental conditions.
How are nanobodies produced?
Alpaca or Llama is immunized with an antigen. After antigen immunization, the serum titer is sufficient, a small portion of blood would be obtained from the animal to construct a VHHs cDNA library/animal sacrifice in the process. Next, based on phage/yeast display technology, the whole immune repertoire of the immunized host is present in the phage/yeast display library, and following appropriate screening ensures the selection of the best VHH fragment against a dedicated target.
