Background: The discovery of functional heavy chain-only antibodies devoid of
light chains in sera of camelids and sharks in the early nineties provided access to the generation
of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding
fragments, also known as Nanobodies.
Methods: Recombinant DNA technology and adaptation of phage display vectors form the
basis to construct large naïve, synthetic or medium sized immune libraries from where multiple
Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display,
bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify
Nanobodies. The antigen affinity, stability, expression yields and structural details of the
Nanobodies have been determined by standard technology. Nanobodies were subsequently
engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin
domains, or to add designed effector functions.
Results: Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from
various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm.
The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The
crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen
Conclusion: Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for
biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been
developed in the past decade.