Application Notes

1 Antibody-Antigen Interaction Analysis Application Note NT-MO-011 Using MST to analyse the binding of Nanobodies and Nanobody-Fc fusion proteins to human CD38 Mandy Unger 1 , Friedrich Koch-Nolte 1 , Hon Cheung Lee 2 , Stephen Blanke 3 , Moran Jerabek-Willemsen 3 1 Department of Immunology, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany 2 Department of Physiology, University of Hong Kong, Hong Kong, China 3 NanoTemper Technologies GmbH, Munich, Germany Abstract Antibody affinities play an important role in diagnostic, basic research and medical applications. Conventional antibodies are composed of two heavy and two light chains. Both chains contribute to binding of the antigen by their variable domains. In addition to conventional antibodies, llamas produce functional heavy chain antibodies that lack light chains and interact with antigens solely via the variable domain of the heavy chain, designated VHH. Recombinant VHHs are also called single domain antibodies or Nanobodies because of their size in the nm range. In this study we compared the binding of Nanobodies and Nanobody-Fc fusion proteins to human CD38 using MicroScale Thermophoresis (MST). This study highlights the potential of MST Technology in affinity determination of antibody- antigen interactions. Introduction CD38 is a multifunctional cell surface ecto-enzyme involved in diverse functions (1). Clinically, CD38 is used as a prognostic marker of B-cell chronic lymphatic leukemia (2). Conventional antibodies consist of two heavy and two light chains (Fig. 1). Both chains contribute to the antigen-binding site. In addition to these conventional antibodies, llamas and other camelids produce antibodies composed only of heavy chains (3). The antigen-binding domain of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH. VHHs are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or Nanobodies (Nb) (4-6). The CDR3 of these sdAbs possesses the extraordinary capacity to form long protruding extensions that can extend into cavities on antigens, e.g., the active site of enzymes (7). Other advantageous features of Nb include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo (5). The aim of this study was to analyse the binding of a monovalent Nb and a bivalent Nb-Fc fusion protein to human CD38 using MicroScale Thermophoresis or MST (11- 13). Fig. 1 Schematic diagram of the 3D-structures of a Nb-Fc fusion protein, Nanobody, and a conventional antibody. The three CDR regions of the Nanobody are depicted in red (CDR1), green (CDR2) and blue (CDR3), and the canonical disulfide bond in yellow. The figure was assembled with Pymol using the coordinates of pdb codes 3kik (lama VHH "GFP- enhancer") and 1igt (mouse IgG2a mAb "231"). Results and Discussion In order to investigate the binding of a Nb and a bivalent Nb-Fc fusion protein to purified CD38 using MicroScale Thermophoresis, the two proteins were mixed in different molar concentrations. The concentration of Alexa 647 - labeled CD38 was kept constant at 5 nM while the concentration of the unlabeled binding partner was varied. To this end, the Nb, Nb-Fc fusion protein, or a conventional control monoclonal antibody (rat-α-GFP mAb) were diluted in MST optimized buffer and incubated for 10 min at room temperature. Samples were loaded into Monolith

• Cover