Design and Construction of a Synthetic Nanobody Library: Testing Its Potential with a Single Selection Round Strategy.

Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 108 and an amino acid variability that matches the codon distribution set by design at each randomized position. We have explored the capabilities of the new library by selecting nanobodies specific for three antigens: vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and the glycoprotein complex (GnGc) of Andes virus. To test the potential of the library to yield a variety of antigen-specific Nbs, we introduced a biopanning strategy consisting of a single selection round using stringent conditions. Using this approach, we obtained several binders for each of the target antigens. The constructed library represents a promising nanobody source for different applications.

Selecting Nanobodies Specific for the Epidermal Growth Factor from a Synthetic Nanobody Library.

The epidermal growth factor (EGF) is one of the most critical ligands of the EGF receptor (EGFR), a well-known oncogene frequently overexpressed in cancerous cells and an important therapeutic target in cancer. The EGF is the target of a therapeutic vaccine aimed at inducing an anti-EGF antibody response to sequester this molecule from serum. However, strikingly, very few investigations have focused on EGF immunotargeting. Since the use of nanobodies (Nbs) for EGF neutralization may be an effective therapeutic strategy in several types of cancer, in this study, we decided to generate anti-EGF Nbs from a recently constructed, phage-displaying synthetic nanobody library. To our knowledge, this is the first attempt to obtain anti-EGF Nbs from a synthetic library. By applying a selection strategy that uses four different sequential elution steps along with three rounds of selection, we obtained four different EGF-specific Nb clones, and also tested their binding capabilities as recombinant proteins. The obtained results are very encouraging and demonstrate the feasibility of selecting nanobodies against small antigens, such as the EGF, from synthetic libraries.