Fig. 3: Masking of CD3 binder reversibly impairs binding to CD3ε antigen.

Single particle analysis of Prot-mαCD3 IgG. a Negative stain transmission electron microscopy (NS-TEM) of the Prot-mαCD3 IgG containing a matriptase cleavable linker. 2D class-averages, as they result from multivariate statistical analysis of raw micrographs, are displayed in boxes (box size 36 nm). The Fc parts have triangular shapes with a central hole and Fabs are more elongated. At the distal end of the Fab the protective scFv appears as a single small additional density (see white arrow). The complex formation between antibody and ligand is identified by the presence of two Fc-regions linked together by an elongated structure corresponding to Fab and bound ligand. Due to the flexibility of the complex, one of the Fc displays less details in most of the class-averages. b Time-lapsed tapping-mode AFM data of individual Prot-mαCD3 IgG molecules have been recorded before (left), during activation with matriptase (middle), as well as after complexation with CD3 antigen (right) in a qualitative manner. The process is accompanied by length and shape changes which are noticed in the morphology maps. The treatment of Prot-mαCD3 (left) with matriptase results in an activated and shorter molecule (middle). The complexation of the activated molecules with Fc-CD3 gives the expected elongated and larger molecule (right). The lengths changes are compared with cross-section profiles starting with the individual masked molecule (green) undergoing activation (red) and after been complexed (blue), the profiles match the particles dimensions measured with NS-TEM, as depicted in (a). Representative AFM images of n = 3 experiments shown.