Figure 1

Biophysical properties of Expamers. (A) Schematic visualization of Expamers mode of action. Expamers spontaneously assemble form single components (anti-CD3 and anti-CD28 Fab fragments as well as Strep-Tactin multimer backbone). Assembled Expamers interact with target T cells by binding to and cross-linking TCR and CD28 surface receptors. Subsequently, Expamers dissociate upon addition of D-biotin. Dissociation results in removing of Expamers from the T cell surface and termination of the activation signal. (B) Histogram overlay of radius measurements of three different Strep-Tactin multimer backbone lots (green, yellow, blue). Graphs depict average hydrodynamic radii from cumulative fits of 19 individual Strep-Tactin multimer backbones as well as polydispersity measurements for different manufacturing lots. (C) The confirmation plot plotting the variation of the molar mass (an average molar mass of 1.29 × 10⁸ Da corresponds to an average amount of approx. 2400 Strep-Tactin tetramers) against the variation of the radius of gyration (92 nm in average). The slope of 0.54 indicates the conformation of a random coil. (D) Electron microscopy images of negatively stained Strep-Tactin multimer show a pleomorphic backbone with mesh sizes ranging from 30 to 300 nm depending on the crosslinking conditions. Individual Strep-Tactins can be observed on the surface of negatively stained multimer (zoom-in). A streptavidin (PDynabeads: 6J6J, red) filtered to 20 Å is inserted for reference. The scale-bars are 100 nm. (E) Graph displays the change of signal intensity over time of CD3⁺ cells (pre-gated on live, single cells) interacting with fluorescently labelled Strep-Tactin (Strep-Tactin-PE) pre-assembled with anti-CD3 and anti-CD28 Fab fragments. Within seconds all cells became Strep-Tactin-PE-positive underlying the speed of Expamers activation potential. Graph is a representative of three independent measurements. (F) Representative histograms of calcium flux measurements of five independent experiments as shown. Time point of Expamer addition is indicated by green arrows. Time point of D-biotin addition is indicated by red arrows. Ionomycin addition is indicated by a grey arrow and addition of Fab fragments only is indicated by a blue arrow. (G) Graphs display residual content of Expamer components; either Fab fragments detected using Strep-Tactin-PE (left panel) or Strep-Tactin multimer backbone detected using an anti-streptavidin antibody (right panel) 8 days after activation. ‘Expamers’ column represents T cells cultured in the presence of Expamers for 8 days. Dynabeads and Strep-Tactin multimer backbone only were used as a negative controls and Expamer-stained T cells (without dissociation) as a positive control (pos ctrl). Graphs show cumulative data from four independent experiments. Lines represent mean ± SD.