Nanobodies specific for complement proteins

We continuously develop and produce llama-antibodies (nanobodies) specific for proteins from the complement system with the aim of using them for structural and functional studies. Nanobodies are single domain antibodies that we develop from immunization of a llama with a specific antigen, followed by a selection process involving phage display to obtain specific and tight binders. Upon identification of a nanobody candidate by phage display, we express a soluble version in bacteria and purify it in a simple manner by affinity chromatography, ion exchange and size exclusion chromatography.

We have developed nanobodies targeting a broad range of complement proteins of both human and murine origin. The nanobodies are easily modified, e.g. by fluorescent labeling and applicable in various functional and structural studies (Hansen et al. 2017, Acta Cryst D Struct Biol)

The following list contains the complement antigens we have used for immunization. For some of them we have already selected and characterized multiple antigens as exemplified by our selection of the C3 specific nanobody hC3Nb1 (Jensen et al. 2018, Journal of Biological Chemistry).

By determining the crystal structure of the hC3Nb1-C3b complex we found that it recognizes an epitope on the C3 MG7 domain. It binds with low-nanomolar to picomolar affinity to native C3, C3b, iC3b and the C3(H2O) mimic C3-methylamine. The nanobody inhibits alternative pathway dependent C3 deposition in both human and murine serum and to some degree also lectin pathway dependent C3 deposition in human serum. It functions by both preventing C3 binding to the convertase and by hindering convertase assembly as it inhibits factor B binding to C3b. Finally, it inhibits factor I mediated C3b degradation by competing with FH. These effects make hC3Nb1 are powerful tool to study the alternative pathway in vitro systems.

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Left panel of the figure shows the structure of our newly developed nanobody (magenta) bound to C3b (green) while the right panel reveals that the nanobody can inhibit the alternative pathway mediated cleavage of C3 in 15% human serum.

The IgE specific nanobody 026 sdab

In collaboration with the research team of Associate Professor Edzard Spillner at Department of Engineering, Aarhus University we have also used structural biology to investigate how a well-known anti-IgE nanobody 026 sdab patented in 2012 functions. The nanobody targets the Fc-region of human IgE. We crystallized a complex containing the IgE Fc fragment bound to two 026 sdab molecules. IgE in complex with antigen stimulates allergic reactions through interactions with the two receptors FcεRI and CD23 found on effector cells. The binding of FcεRI and CD23 to IgE are mutually exclusive. Our structural studies revealed that 026 sdab mimics and thereby inhibits IgE binding to CD23.  With respect to inhibition of FcεRI binding of 026 sdab, there is limited direct competition but the nanobody traps IgE in conformation incompatible with binding to FcεRI.

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The crystal structure of an IgE Fc fragment in complex with two 026 sdab molecules.