Fig. 2

Design and bifunctional target binding ability of a-CTLA4-TGFβRII. a Autocrine/paracrine TGFβ-induced expression of FOXP3, the signature transcription factor of the Treg lineage. FOXP3 induces the expression of CTLA-4 and Galectin-9, a ligand that engages TIM-3 and triggers exhaustion or apoptosis of effector T cells. Galectin-9 further interacts with TGFβ receptors to drive FOXP3 expression in a positive-feed forward autocrine loop involving SMAD3 activation to induce and maintain Tregs. Schematic representation of the structure and targets of a-CTLA4-TGFβRII are shown. a-CTLA4-TGFβRII was designed by fusing the C terminus of the heavy chain of a human a-CTLA-4 antibody with a ligand-binding sequence of the extracellular domain of TGFβ Receptor II (TGFβRII ECD) via a flexible linker peptide, (GGGGS)₃. b Amino acid sequences of the heavy chain and light chain of a-CTLA4-TGFβRII. c SDS-PAGE (unreduced-NR and reduced-R) comparing the molecular weight of a-CTLA4-TGFβRII and a-CTLA-4 antibody (FL, full-length; HC, heavy chain; LC, light chain). d,e Bifunctional ability of a-CTLA4-TGFβRII to simultaneously bind CTLA-4 and TGFβ1 using a ‘double-sandwich’ ELISA, wherein a-CTLA4-TGFβRII or a-CTLA-4 antibody was added to CTLA-4-Fc-coated plates, followed by rhTGFβ1 that was detected by a biotinylated anti-human TGFβ1 antibody. TGFβRII-Fc-coated plates were used as a TGFβ-binding positive control. f Standard ELISA showing the ability of a-CTLA4-TGFβRII to compete with a TGFβ capture antibody for binding to TGFβ1. For d–f, the data show the optical density (OD) values (mean of three replicate wells for each assay condition) from a representative of two independent experiments