Biochemistry, structure, and cellular internalization of a four nanobody‐bearing Fc dimer

Abstract

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2‐Fc‐VHH2. The structure of VHH2‐Fc‐VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2‐Fc‐VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti‐HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2‐Fc). This new type of molecules, VHH2‐Fc‐VHH2, could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

Genetic Incorporation of Non-canonical Amino Acids in Anti-HER2 VHH: Expression and Characterization

“…Note that in some preparations (Figure 3b, lanes D and G), a contaminant appears at twice the molecular weight value, suggesting dimerization. In lane I, the very high molecular weight might be a polymer of the VHH, a pattern already detected in previous experiments with the WT anti-HER2 VHH [17].…”

“…As shown and discussed in our preceding publications [17,23,24,39], VHH might be a step towards "easier-touse-and-produce" short proteins with therapeutical and imaging (diagnostic) potential. We developed an approach to producing VHH proteins containing ncAA in E. coli with specific incorporation of the two ncAAs at the two targeted positions.…”

“…The present report was part of a more global program aiming at the use of VHH in different contexts, including the research of specific ways to ultimately target cancerous cells. Considering the timeline at which this program started, it was necessary to address some basic questions about VHHs: their production [24], their total chemical synthesis [23], and even their complexification as potential multitask therapeutical molecules [17].…”

“…We targeted the standard anti-HER2 VHH that we explored before [17,23,24] in order to compare and ease our understanding of the production systems. We chose two ncAAs as models for this approach, namely N ε -2-[(azidoethyloxy)carbonyl]-L-lysine (AzK) and O-propargyl-L-tyrosine (PrY), mostly because the incorporation of these two ncAAs has been widely described [19,30,31].…”

“…One can also choose to label the VHH with radioactive atoms either in a positron emission tomography (PET) context (see Bao et al [10] for review), or with iodine125 [15,16] for traditional binding experiments that can lead to the quantification of the VHH/target complex. A third way that we also explored was to build a tetrameric VHH molecule that could be labeled on one of the VHH units [17].…”