Journal: Biological Chemistry

Article Title: Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies

doi: 10.1515/hsz-2021-0401

Figure Lengend Snippet: CH3-driven chain exchange enabled antibodies with and without interchain hinge-disulfides. (A) The Format Chain Exchange (ForCE) reaction. Two monospecific educts undergo chain exchange into bispecific antibodies under mild reducing conditions (in the presence of tris(2-carboxyethyl)phosphine (TCEP)). Educt A carries repulsive positive charges in the interface between the heavy chain and dummy chain, Educt B has two opposing negative charges. In the product, the attractive charges facilitate the stable formation of the bsAb, in which the hinge disulfide bridges re-oxidize (TCEP degrades over time). Fc portions (CH2/CH3) are depicted in gray. (B) The reduction-free ForCE reaction. The two monospecific educts undergo charge-mediated heavy chain recombination at physiological conditions and assemble into a bispecific antibody and a dummy-dimer molecule. (C) The CH3 domains of the crystal structure 4NQS representing the regular KiH (magenta: Knob [W366]; pink: Hole [S366, A368 and V407]) is superimposed with the single mutant of the Knob side (dark gray) and the Hole side (light gray) as present in educt B. In the latter, the gray ribbons are slightly more distant than seen in the reference KiH structure, reflecting the slightly destabilized nature of the prodrug (left panel). A zoom into the specific interaction of the KiH amino acids as well as on the single mutation K370E, that leads to a disruption of the salt bridge (yellow dotted line) with E357 in educt B (right panel). (D) Thermal stability of educt molecules with (traditional ForCE) and without disulfide-bridged hinge regions (ForCE under physiological conditions) was compared by differential scanning fluorimetry in three independent experiments, in triplicates. Depicted are mean melting temperatures and SD melting temperatures were greater than 60 °C, revealing an acceptable molecule stability. Educts contained Fabs that bind to conjugated fluorescein or conjugated biotin ( Dengl et al. 2015 ; Dengl et al. 2020 ). (E) A sandwich ELISA assay was performed to show the formation of the bsAb. BSA-Fluorescein was complexed on a plate. Educts A and B, with or without hinge disulfides (S–S), were mixed at 5 µM for 1 h at 37 °C and added to the plates. After several washing steps, biotinylated-Cy5 was added and unbound dye removed. The bsAb-mediated capture of Bio-Cy5 was detected in a microplate reader (ex 649 nm, em 670 nm). Results are expressed as mean and SD from triplicate wells. Representative plot of three independent experiments is shown.

Article Snippet: Total antibody-like protein (kappa(+)) was detected by capturing with a biotinylated anti-human kappa light chain Fab (Clone M-1.7.10), coated onto a Streptavidin microtiter plate (SA-MTP, MicroCoat Biotechnologie GmbH, Bernried) and detection was performed with a digoxigenin-labeled anti human Ch1 antibody (Clone M-1.19.31) Biotin-binding antibodies (formed if two prodrugs assemble into a functional -TriFab) were detected by capturing the antibody with biotinylated albumin, coated to a streptavidin microtiter plate.

Techniques: Mutagenesis, Disruption, Sandwich ELISA