Extended Data Fig. 3 ∣. Biochemical properties of 4T-Trap and control antibodies.

a, Schematic representation of ibalizumab Fab and TGF-βRII ECD fusion proteins in a murine IgG1 framework. The star indicates a D265A substitution in the CH2 domain, and the semicircle and moon shapes indicate knob-into-hole (KIH) modifications in the CH3 domain to enable heavy chain heterodimerization. The gray or colored parts indicate mouse or human sequences, respectively. b-c, Yield and aggregation percentage of ibalizumab Fab and TGF-βRII ECD fusion proteins produced in a FreeStyle HEK293-F cell transient expression system. FreeStyle HEK293-F cells transfected with plasmids encoding the indicated fusion antibodies were cultured for 4 days, and the supernatant was collected. Protein G affinity purification and size exclusion chromatography were used to purify these antibodies. d, Molecular weights of αCD4, mGO53, 4T-Trap and TGF-β-Trap antibodies detected by Coomassie Blue staining of samples run in a SDS-PAGE gel under non-reduced or reduced conditions. Molecular size markers (kDa) are shown on the left. HC, heavy chain; LC, light chain. e, Size exclusion chromatography analyses of mGO53, TGF-β-Trap, αCD4 and 4T-Trap antibodies. f, Schematic representation of human CD4 structure and purity examination of recombinant soluble CD4 (sCD4) by SDS-PAGE followed Coomassie Blue staining. g, The binding affinities of 4T-Trap and αCD4 to human CD4 as well as 4T-Trap and αTGF-β (1D11 clone) to human TGF-β1 were determined by surface plasmon resonance. h, Binding of 4T-Trap to human CD4 ectopically expressed on HEK293 cells. Cells were incubated with serial dilutions of 4T-Trap and αCD4 antibodies followed by a fluorophore-conjugated anti-mouse IgG secondary antibody. Samples were analyzed by flow cytometry. The measured mean fluorescence intensity (MFI) was quantified. i, TGF-β signaling inhibitory functions of 4T-Trap and αTGF-β. HEK293 cells transfected with a TGF-β/SMAD firefly luciferase reporter plasmid and a pRL-TK Renilla luciferase reporter plasmid were incubated with the indicated antibodies for 30 min and treated with 10 ng/mL recombinant human TGF-β1 for 12 hr before subject to the luciferase assay. RU, relative unit of normalized Firefly luciferase activity to Renilla luciferase activity. Data are representative of three independent experiments (b-f, h, i).