Part:BBa_K4387988

Single domain antibodies are also called VHH or Nanobody®, only consisting of a single variable domain, which is able to bind to a specific antigen. The small size of nanobodies (15 - 20kDa) compared to anibodies, give them special abilities, which are hard to reach with conventional antibodies. They can locally penetrate barriers (such as tissues) more easily and can withstand extreme environmental conditions, such as high temperatures and low pH. They show high affinity and stability and recombinant expression has revolutionized the biotechnology field. Nanobodies have been discovered in camelid animals, already back in the 90's. Usage of these nanobodies in the clinic often requires an additional step called "humanization" in order to reduce unwanted immunological reactions upon administration. This step describes the exchange of one or a few specific amino acids that are recognized as "foreign" by the human immune system. Still today, camelid animals are infected with the antigen of choice and effective nanobodies are obtained from their blood. However, new manufacturing technologies have been developed, allowing the screening of new candidates by using naive or synthetic libraries in combination with phage and ribsome display. The usage of synthetic libraries results in the generation of so called "sybodies". [2] Generating new nanobodies against a target is a time-consuming process with several selection steps. Therefore, we used the amino acid sequences for specific anti-tumour necrosis factor (TNF) nanobodies from a patent and converted them to DNA sequences. [3] The patent contains a variety of TNF-binding nanobodies. We selected 3 candidates (VHH#2B, VHH#3E and VHH#12B) based on their humanization characteristics. The patent described a complete humanization protocol for nanobody VHH3E which we applied. The candidates VHH2B and VHH12B were described as already suitable for clinical applications due to their >90% amino acid sequence homology to human VH framework regions and are therefore possibly "safe" for direct administration to patients. The nanobody described below is the bivalent construct of nanobody candidates VHH#3E and VHH#12B.