Generation of nanobodies for functional and structural studies

About half of the total antibody population in members of the Camelidae family (bactrian and dromedarian camel, llama, alpaca, vicugna, guanaco) consists of immunoglobulins that are missing CH1 regions of the heavy chains and as a result cannot not pair with light chains. They are called single domain heavy chain-only antibodies (hcAbs). These monospecific antibodies are ideally suited for construction of antibody libraries since they enable us to produce the antigen-binding domain of a heavy chain, using the genetic information obtained from immune cells. Large libraries of such antigen binding fragments, called VHH or nanobodies, can be engineered in phage or yeast display systems. These small fragments (~13 kDa) are fully functional entities that can recognize and bind their cognitive antigens with same affinities as much bigger conventional antibodies (~150 kDa). Due to their intrinsic properties like small size, increased solubility, excellent stability, ease and low cost of large-scale production, the camelid antibodies have become promising tool in a range of applications that deal with diverse aspects of cell biology, diagnostics and therapy. Their large therapeutic potential was recognized for the treatment of cancer, viral and parasitic diseases, and neutralizing toxins. In basic research, they are used for the targeting of antigens in live cells and tissues, as specific inhibitors, a tool in pull-down experiments, and protein crystallization chaperones, to mention just a few.

Since 2012 we established the production of nanobodies at our institute, which in 2016 became one of the University of Zurich Technology Platforms that serves the scientific community in the region (  Research groups from both Zurich’s universities, UZH and ETH, and as well from other big research centres, like Paul Scherrer Institute, use the nanobody technology to select specific binders for membrane proteins of medical relevance. Towards this end we are collaborating with the research groups of Prof. Raimund Dutzler and Prof. Markus Seeger (both UZH), Prof. Kaspar Locher (ETH), Prof. Eric R. Geertsma (Goethe-University Frankfurt), Dr. Volodymyr Korkhov and Dr. Jörg Standfuss (both PSI), who investigate structure and function of a variety of membrane proteins that catalyse important cellular transport reactions. The identification of high-affinity binders against purified membrane proteins provides important tools that aid the crystallization and structure determination of these important targets and facilitate the elucidation of transport mechanisms. Due to their high specificity and sutability for oral applications, nanobodies have great potential in diagnosis and therapy. At our institute we are developing nanobodies for different projects that deal with parasite biology (Toxoplasma gondii and Giardia lamblia projects), and identification of novel diagnostic and therapeutic targets for the control of parasitic zoonoses (Echinococcus multilocularis project). We hypothesise that the nanobodies can serve as ligands that can recognize and interfere with function of parasite molecules essential for the establishment and maintenance of parasitism. Because of their small size and long protruding variable loop, nanobodies may readily access receptor clefts and thus inhibit the target molecules, a task that is challenging to achieve with conventional antibodies.

During the past 6 years’ time we identified diverse high-affinity nanobodies that are currently being used as functional tools in basic research and many nanobodies were selected which greatly improved the quality of protein crystals. In several cases the identified nanobody helped determine the 3D structure of its membrane protein target. Part of this work is published in highly impacted journals and other manuscripts are in preparation.

Institute members: Saša Štefanić, (NSF manager and project leader), Hans-Peter Müller (animal caretaker), Peter Deplazes, (NSF Director)

Funding sources: The Nanobody Service Facility was supported by the Faculty of Sciences and the Vetsuisse Faculty of the University of Zurich, and by service fees.