A single-domain antibody (sdAb) is the smallest variable fragment from a common antibody that can selectively bind to a specific antigen. Single domain antibodies have an approximate molecular weight of 15 kDa and are considered the smallest naturally derived antigen-binding fragment. Due to their size in the nm range, the term ‘nanobody’ was coined by the Belgian company Ablynx (now part of Sanofi), which refers to the VHH domain from Camelidae species.


At Cortalix, we initially created immune libraries from llamas immunized with a mixture consisting of extracellular domains of membrane receptors overexpressed in fibrosis and various cell types present in fibrotic tissue and fibrotic cancers.
Starting from these immune sdAb libraries, we were able to select a large number of attractive sdAb candidates which can be developed into clinical candidates after various in vitro and in vivo studies.
Based on our previous experiences with immune libraries, we have invested in the design and construction of animal-free synthetic sdAb libraries.
For this purpose, we selected robust sdAb frameworks in which the antigen-binding regions in the three CDRs were (partially) randomized using trimeric phosphoramidites to avoid, for example, unwanted stop codons or cysteines.


It is actually not possible to make a 100% humanized VHH as that would disrupt the integrity and therefore the functionality of the single domain antibody. It is possible to approximate that situation as best as possible by replacing some amino acids of the framework, applying residue optimization, CDR grafting and finally evaluating the potential immunogenicity assessment with in silico prediction tools.
By following these activities, we ultimately end up with synthetic nanobody libraries with an optimized framework and variable CDRs that produce molecules with limited immunogenicity, while retaining their antigen-binding properties and good expression properties.


In the early days, when recombinant antibody technology – mainly focused on scFvsemerged, sdAbs did not attract much attention. However, over the years, a steadily growing number of publications illustrated the benefits of sdAbs in certain research niches. To identify practically applicable sdAbs, it is important to start from a high-quality gene bank of sdAbs, focused on the final application and with a great diversity. Our screening and selection procedure using phage display techonology allows us to extract the most promising nanobodies for a particular target from this diverse library. Then, various options for genetic engineering and post-translational modification, including specific spacers and linkers, can be used to tailor the sdAb for its final application. For instance, we are experienced with the conjugation of sdAbs to radionuclide chelators, including NOTA and DOTA derivatives. We use the latest techniques to control the PK and plasma clearance, which is different for a diagnostic and a therapeutic application/sdAb. 


Through 3D simulation we are able to make predictions, within certain limits, about the applicability of selected sdAbs. 
Now that the first therapeutic sdAbs have been admitted to the market, other pharmaceutical companies will quickly join in and this will certainly lead to even broader applications, including in the field of radiopharmaceuticals and immunotherapeutics. 
At Cortalix we want to participate in this development, and we will evolve into a party that will accelerate the application of sdAbs. Cortalix aims to present solutions that offer prospects for patients who currently do not have them.