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Effects of masking and protease-dependent unmasking on anti-HER2 antibody activity. (A) Size exclusion chromatography (SEC) profiles of IGF-II-s-MMP2/9-Tras and native trastuzumab (Tras). UV absorbance at 280 nm was shown as normalized peak signal versus retention time (min). (B) Reducing SDS-PAGE analysis of heavy chain (HC) and light chain (LC) for IGF-II-s-MMP2/9-Tras before (“uncut”) and after (“cut”) MMP2 cleavage, with native Tras included for comparison. (C) ELISA binding assay measuring HER2 binding by masked IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) unmasked antibody and native Tras shown for comparison. The absorbance at 450 nm (OD 450 nm) was plotted versus antibody concentration (ng/mL) with the data presented as mean values ± sd ( n = 5). (D) SK-BR-3 cell viability assay measuring inhibition of cell viability by IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) antibody and native Tras shown for comparison. Percent cell viability was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd. (E, F) Antibody dependent cellular cytotoxicity (ADCC) (E) and antibody dependent cellular <t>phagocytosis</t> <t>(ADCP)</t> (F) reporter assays assessing IGF-II-s-MMP2/9-Tras and Tras in promoting engagement of SK-BR-3 target cells with effector cells and activation of CD16a (ADCC)- or CD32a (ADCP)-dependent reporter gene expression. Fold induction of reporter activity was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd.
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Effects of masking and protease-dependent unmasking on anti-HER2 antibody activity. (A) Size exclusion chromatography (SEC) profiles of IGF-II-s-MMP2/9-Tras and native trastuzumab (Tras). UV absorbance at 280 nm was shown as normalized peak signal versus retention time (min). (B) Reducing SDS-PAGE analysis of heavy chain (HC) and light chain (LC) for IGF-II-s-MMP2/9-Tras before (“uncut”) and after (“cut”) MMP2 cleavage, with native Tras included for comparison. (C) ELISA binding assay measuring HER2 binding by masked IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) unmasked antibody and native Tras shown for comparison. The absorbance at 450 nm (OD 450 nm) was plotted versus antibody concentration (ng/mL) with the data presented as mean values ± sd ( n = 5). (D) SK-BR-3 cell viability assay measuring inhibition of cell viability by IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) antibody and native Tras shown for comparison. Percent cell viability was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd. (E, F) Antibody dependent cellular cytotoxicity (ADCC) (E) and antibody dependent cellular phagocytosis (ADCP) (F) reporter assays assessing IGF-II-s-MMP2/9-Tras and Tras in promoting engagement of SK-BR-3 target cells with effector cells and activation of CD16a (ADCC)- or CD32a (ADCP)-dependent reporter gene expression. Fold induction of reporter activity was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd.

Journal: mAbs

Article Title: A novel insulin-like growth factor II-based masking domain for conditional activation of therapeutic antibodies

doi: 10.1080/19420862.2026.2668187

Figure Lengend Snippet: Effects of masking and protease-dependent unmasking on anti-HER2 antibody activity. (A) Size exclusion chromatography (SEC) profiles of IGF-II-s-MMP2/9-Tras and native trastuzumab (Tras). UV absorbance at 280 nm was shown as normalized peak signal versus retention time (min). (B) Reducing SDS-PAGE analysis of heavy chain (HC) and light chain (LC) for IGF-II-s-MMP2/9-Tras before (“uncut”) and after (“cut”) MMP2 cleavage, with native Tras included for comparison. (C) ELISA binding assay measuring HER2 binding by masked IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) unmasked antibody and native Tras shown for comparison. The absorbance at 450 nm (OD 450 nm) was plotted versus antibody concentration (ng/mL) with the data presented as mean values ± sd ( n = 5). (D) SK-BR-3 cell viability assay measuring inhibition of cell viability by IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) antibody and native Tras shown for comparison. Percent cell viability was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd. (E, F) Antibody dependent cellular cytotoxicity (ADCC) (E) and antibody dependent cellular phagocytosis (ADCP) (F) reporter assays assessing IGF-II-s-MMP2/9-Tras and Tras in promoting engagement of SK-BR-3 target cells with effector cells and activation of CD16a (ADCC)- or CD32a (ADCP)-dependent reporter gene expression. Fold induction of reporter activity was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd.

Article Snippet: Antibody-dependent cellular phagocytosis (ADCP) reporter assays were performed using Jurkat-LuciaTM NFAT-CD32 cells (InvivoGen, jktl-nfat-cd32), which express CD32A (FcγRIIa) and the same NFAT-inducible reporter.

Techniques: Activity Assay, Size-exclusion Chromatography, SDS Page, Comparison, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Viability Assay, Inhibition, Activation Assay, Gene Expression