A novel scaffold for nanobody selection and expression suitable for diagnostic and therapeutic applications

Abstract

A novelty in antibody field has recently been introduced after the discovery of the presence of heavy chain immunoglobulins G, lacking light chains molecules, in camelids blood. The potentiality of the variable domain of these heavy chain antibodies (called VHH or nanobody) was immediately exploited. These variable domains, singularly expressed, maintain a target affinity comparable to an entire antibody, are very soluble because of the absence of exposed hydrophobic amino acid residues and they are very small (~15 kDa), if compared to conventional antibodies (~150 kDa). All these features, combined with their easy expression as recombinant proteins in bacteria, determined their enrollment as new biotechnologial tools. During my Ph.D., I produced and validated a novel scaffold suitable for nanobody expression and selection: the scaffold consists of four framework regions (FRs, conserved in sequence) derived from the alignment of more than 750 llama nanobodies. Within this scaffold, complementary determining regions (CDRs) of natural VHH or artificial random sequences can be grafted. The scaffold leads to an improvement of solubility and stability of the nanobodies and is the potential basis for antibody libraries construction. I exploited the consensus framework scaffold (CFW) in the development of a platform for new nanobodies selection and for their improvement in order to render them diagnostic and therapeutic tools. Two artificial nanobody libraries are the basis of the platform. One, composed by the CFW and bearing semi-random artificial CDRs, presents a complexity of 10^12 different nanobody clones (comparable to the natural llama VHH repertoire) and is selectable through ribosome display technique. I validated this library by selecting new Maltose binding protein (MBP) binders. The second library is a humanized version of the previous one in which I introduced 12 point mutations in order to render CFW scaffold identical to human IgG VH III FRs. I confirmed the solubilizing and stabilizing effects of the humanized CFW scaffold and the library is now ready to be clone in a novel phage vector that I created to speed up phage display selection. From this humanized library potential directly human applicable nanobodies will be selected, avoiding any post-selection humanization which usually can lead to antibody target affinity alteration. Additionally, I developed methods to improve nanobodies for diagnostic applications. Since nanobodies are not detectable through secondary antibodies, I applied an in vitro peptide ligation system to conjugate them to immunoglobulins Fc regions. With this modular and stable conjugation approach, I demonstrated the possibility to combine any purified VHHs to any expressed Fc regions, obtaining antibodies that mimic monoclonal antibodies activity. In collaboration with Preclincs company, I further exploited my scaffold for the production of new in vivo imaging probes. Fusing nanobodies to a luciferase, a probe working for GFP imaging in mice has been validated, while other probes remain to be tested.