Additional file 1 of A lesion-selective albumin-CTLA4Ig as a safe and effective treatment for collagen-induced arthritis

Additional file 1: Figure S1. The N-terminal Ab lock and VpreB were unable to mask the binding activity of CTLA4Ig. (A) Binding activity of Ab lock-CTLA4Ig (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to HEK-293 cells overexpressing CD80 (CD80 cells), detected by FITC-conjugated goat anti-mouse Fcγ in flow cytometry. Gray line: unstained cells. (B) Binding activity of VpreB-CTLA4Ig (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to CD80 cells, detected by FITC-conjugated goat anti-mouse Fcγ antibodies by flow cytometry. Gray line: unstained cells. VpreB: immunoglobulin iota chain. (C) Simulation of Ab lock-mCTLA4Ig by the computer software BIOVIA Discovery Studio 2019 (Discovery Studio v19.1.0.18287). The structures of CTLA-4, the CDR3-like domain and the Ab lock are shown in magenta, yellow and light blue, respectively. Figure S2. Full recovery of the binding activity of mAlb-CTLA4Ig after MMP2/9 digestion. Nondigested, MMP-digested mAlb-CTLA44Ig, and conventional mCTLA4Ig (all at 1 nM) were added to the ELISA. Binding of the fusion proteins on the plate was detected by an HRP-conjugated anti-mouse IgG Fcγ secondary antibody. Figure S3. Characterization of an alternative Alb-CTLA4Ig with MMP substrate linker between albumin and CTLA4Ig (mAlb-MMP-CTLA4Ig). (A) Schematic representations of mAlb-MMP-CTLA4Ig constructs. MMP: MMP substrate sequence (GPLGMWSR) linker, eCTLA4: extracellular domain of CTLA4. P: promoter in the expression vector. (B) Reducing SDS-PAGE (left) and western blot analysis (right) of purified mAlb-MMP-CTLA4Ig. (C) The stability of mAlb-MMP-CTLA4Ig in DMEM containing 10% fetal bovine sera for seven days. (D) mAlb-MMP-CTLA4Ig were digested with the indicated amount of MMP2/9 and analyzed by western blot. (E) mAlb-MMP-CTLA4Ig were subjected to varying degrees of digestion by MMP2/9. Part of the digestion was analyzed by western blot to determine the degree of cleavage. The percent (%) cleaved Alb-MMP-CTLA4Ig was quantitated and is indicated below each lane. The digestion was added to the cell-based ELISA. The binding of CD80 by 1 nM conventional mCTLA4Ig was used as a positive control. (F) Binding kinetics of mAlb-MMP-CTLA4Ig before (blue curve) and after MMP digestion (orange curve). The binding kinetics of conventional mCTLA4Ig are shown in the black curve. Figure S4. Lacking an N-terminal albumin and a C-terminal Fc decreases masking efficiency and stability. (A) Competitive binding of Ig-CTLA4 ECD (0.5 μg/ml, blue line) or conventional CTLA4Ig (0.5 μg/ml, black line) against PE-conjugated anti-mouse CD80 antibodies (1 μg/ml) to HEK-293 cells overexpressing CD80 (CD80 cells). Red line: CD80-expressing cells stained with PE-conjugated anti-CD 80 antibodies alone (1 μg/ml). Gray line: unstained cells. Digestion products of the IgG1 Fc-CTLA4 ECD by the indicated amounts of MMP2/9 are shown in the western blot using anti-mouse Fcγ antibodies (right panel). (B) Competitive binding activity of Alb-CTLA4 ECD (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to CD80 cells in the presence of PE-conjugated anti-mouse CD80 antibodies (1 μg/ml) by flow cytometry. Red line: CD80 cells stained with PE-conjugated anti-CD80 antibody alone (1 μg/ml). Gray line: unstained cells. Digestion products of the Alb-CTLA4 ECD by the indicated amounts of MMP2/9 are shown in the western blot using anti-mouse CTLA4 antibodies (right panel). (C) Digestion products of conventional human CTLA4Ig (3.6 picomole) by 2 units of MMP2/9 is shown in the western blot using anti-human Fcγ antibodies. (D) Overdigestion of hAlb-CTLA4Ig (1.35 picomole) in higher amounts (9 units) of MMP2/9. Figure S5. The stability of hAlb-CTLA4Ig in sera. hAlb-CTLA4Ig incubated in RPMI 1640 containing 10% fetal bovine sera for seven days was analyzed by western blot using an anti-human Fcγ secondary antibody. Med: medium alone. Figure S6. Differential levels of MMP9 and MMP3 in normal control mice and CIA mice. Protein levels of MMP9 (A) and MMP3 (B) in the synovial fluid lavages, sera, and paws of normal control mice and CIA mice were measured by ELISA. Protein levels are expressed as ng/ml in synovial fluid lavages and sera, ng/mg protein in protein extracts of the paws. Figure S7. Histopathological microphotographs of the digits of a normal mouse or CIA mice. The area enclosed by red rectangles at low magnification (5X objective, upper row) is shown at higher magnification (20X objective, lower row) for inflammatory cells. Figure S8. Splenocyte proliferation in response to different concentrations of M. tuberculosis restimulation. Splenocytes from a normal (preimmune) mouse and a CIA mouse were stimulated with 0 μg/ml 5μg/ml or 25 μg/ml M. tuberculosis extracts for 72 h. BrdU was added at the final 2 hours of stimulation. Proliferation (BrdU incorporation) of the CD45+ splenocytes was analyzed by flow cytometry.