Figures & data
Figure 1. The amino acid sequences of the heavy chain variable region of murine 37E1B5. The CDR regions are shown in italic and underlined. The N-linked glycosylation signals, NLS and NYT at the positions 19-21 and 59-61, are high-lighted as yellow, respectively.
![Figure 1. The amino acid sequences of the heavy chain variable region of murine 37E1B5. The CDR regions are shown in italic and underlined. The N-linked glycosylation signals, NLS and NYT at the positions 19-21 and 59-61, are high-lighted as yellow, respectively.](/cms/asset/7e42a2a9-511a-4074-951d-cd90b60993d0/kmab_a_1255390_f0001_c.gif)
Figure 2. Characterization of Beta8 glycosylation. Relative quantitation of released glycans (Fc and Fab) by 2-aminobenzamide (2-AB)-labeled oligosaccharide profiling (A). Site-specific glycan analysis by reduced antibody LC-MS of heavy chain combined, de-convoluted mass spectra for Beta8 (B) and the aglycosylated Beta8-AG (N59Q) variant (C).
![Figure 2. Characterization of Beta8 glycosylation. Relative quantitation of released glycans (Fc and Fab) by 2-aminobenzamide (2-AB)-labeled oligosaccharide profiling (A). Site-specific glycan analysis by reduced antibody LC-MS of heavy chain combined, de-convoluted mass spectra for Beta8 (B) and the aglycosylated Beta8-AG (N59Q) variant (C).](/cms/asset/b908dcbb-6007-4557-8b9c-201eb5d32ac1/kmab_a_1255390_f0002_c.gif)
Figure 3. Binding of Beta8 and Beta8-AG to αvβ8 (A). Recombinant αvβ8 was coated to the ELISA wells. Antibody binding was detected using a HRP-conjugated goat anti-human Fc antibody. Inhibition of αvβ8 binding to LAP ((B) and C). Recombinant LAP was coated to the ELISA wells. The amount of the recombinant αvβ8 protein that generated 75% maximum binding signals was mixed with serially diluted Beta8 and Beta8-AG (B) or Fabs of Beta8 and Beta8-AG (C), and the reaction was incubated at room temperature. The ability of αvβ8 binding was detected by an HRP-conjugated anti-αvβ8 antibody.
![Figure 3. Binding of Beta8 and Beta8-AG to αvβ8 (A). Recombinant αvβ8 was coated to the ELISA wells. Antibody binding was detected using a HRP-conjugated goat anti-human Fc antibody. Inhibition of αvβ8 binding to LAP ((B) and C). Recombinant LAP was coated to the ELISA wells. The amount of the recombinant αvβ8 protein that generated 75% maximum binding signals was mixed with serially diluted Beta8 and Beta8-AG (B) or Fabs of Beta8 and Beta8-AG (C), and the reaction was incubated at room temperature. The ability of αvβ8 binding was detected by an HRP-conjugated anti-αvβ8 antibody.](/cms/asset/cba5892a-77ea-408f-b86e-731fb40d132f/kmab_a_1255390_f0003_b.gif)
Figure 4. Inhibition of TGF-β activation by 37E1B5 requires a complex glycan. Schematic overview of the different antibody glycoforms assessed for bioactivity and PK properties, illustrated for the binantennary glycan FA2G2S2 (A). TMLC-HeLa cell co-culture TGF-β signaling reporter assay. Relative luciferase units (RLU) from TMLC reporter cells show baseline luciferase reporter activity (“TMLC”). Relative luciferase units (RLU) from TMLC and HeLa αvβ8+ cell co-culture show maximal luciferase reporter activity (“TMLC + HeLa); and the anti-TGF-β mAb 1D11 (“anti-TGFb-1D11,” 7.5 µg/mL) effect on the TGF-β dependent luciferase reporter activity in the TMLC and HeLa αvβ8+ co-culture. Mouse IgG1 (“mIgG1,” 7.5 µg/mL) was used as the isotype control. (B). Maximum response of TGF-β dependent luciferase reporter activity by 37E1B5 variants in the TMLC co-culture assay with HeLa αvβ8+ cells. (C). Data shown is the mean ±SEM from 3 independent experiments.
![Figure 4. Inhibition of TGF-β activation by 37E1B5 requires a complex glycan. Schematic overview of the different antibody glycoforms assessed for bioactivity and PK properties, illustrated for the binantennary glycan FA2G2S2 (A). TMLC-HeLa cell co-culture TGF-β signaling reporter assay. Relative luciferase units (RLU) from TMLC reporter cells show baseline luciferase reporter activity (“TMLC”). Relative luciferase units (RLU) from TMLC and HeLa αvβ8+ cell co-culture show maximal luciferase reporter activity (“TMLC + HeLa); and the anti-TGF-β mAb 1D11 (“anti-TGFb-1D11,” 7.5 µg/mL) effect on the TGF-β dependent luciferase reporter activity in the TMLC and HeLa αvβ8+ co-culture. Mouse IgG1 (“mIgG1,” 7.5 µg/mL) was used as the isotype control. (B). Maximum response of TGF-β dependent luciferase reporter activity by 37E1B5 variants in the TMLC co-culture assay with HeLa αvβ8+ cells. (C). Data shown is the mean ±SEM from 3 independent experiments.](/cms/asset/a1b9dd3e-2bde-4635-9adc-c4cd0bfa0fdc/kmab_a_1255390_f0004_c.gif)
Table 1. Model-based pharmacokinetic parameter estimates for comparison of the wild type (WT), fully glycosylated (AG) and the desialylated (DE) Beta8 in mice. PK parameters derived from the non-linear mixed effects software NONMEM (Icon Development Solutions, Version 7.3).