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. 2023 Jun 9;22(9):999–1012. doi: 10.1158/1535-7163.MCT-22-0786

Figure 2.

Figure 2. In vitro characterization of B7-H4 ADCs. A, The anti–B7-H4 antibody does not block the functional activity of B7-H4 ligand. HEK293 or HEK293-B7-H4 cells were incubated with 10 nmol/L antibody (B7-H4 or nonbinding control) prior to the addition of CellTrace Violet-labeled CD3+ T cells and CD3/CD28 T-cell Activator. T cell proliferation was determined after 4-day incubation. Data represent the mean (± standard deviation) of triplicate samples. B, Schematics of the B7-H4–targeted ADCs evaluated in this study: site-specific DS DAR 2 and DAR 6, and DF DAR 12. C, Binding of ADCs to HEK293-B7-H4 cells and MX-1 breast cancer cells. Flow cytometry analysis was performed with B7-H4 ADCs, unconjugated antibody, and nonbinding control ADCs. D, Binding of ADCs to recombinant human B7-H4 protein. ELISA was performed with B7-H4 ADCs, unconjugated antibody, and non-binding control ADCs. E, B7-H4 ADCs elicit target-dependent cytotoxicity. HEK293-B7-H4 cells were incubated with test article for 3 days, and viability was measured with CellTiter-Glo.

In vitro characterization of B7-H4 ADCs. A, The anti–B7-H4 antibody does not block the functional activity of B7-H4 ligand. HEK293 or HEK293-B7-H4 cells were incubated with 10 nmol/L antibody (B7-H4 or nonbinding control) prior to the addition of CellTrace Violet-labeled CD3+ T cells and CD3/CD28 T-cell Activator. T cell proliferation was determined after 4-day incubation. Data represent the mean (± standard deviation) of triplicate samples. B, Schematics of the B7-H4–targeted ADCs evaluated in this study: site-specific DS DAR 2 and DAR 6, and DF DAR 12. C, Binding of ADCs to HEK293-B7-H4 cells and MX-1 breast cancer cells. Flow cytometry analysis was performed with B7-H4 ADCs, unconjugated antibody, and nonbinding control ADCs. D, Binding of ADCs to recombinant human B7-H4 protein. ELISA was performed with B7-H4 ADCs, unconjugated antibody, and non-binding control ADCs. E, B7-H4 ADCs elicit target-dependent cytotoxicity. HEK293-B7-H4 cells were incubated with test article for 3 days, and viability was measured with CellTiter-Glo.