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. 2020 Apr 14;11(15):1344–1357. doi: 10.18632/oncotarget.27544

Figure 2. In vitro assays to evaluate the effect of labeling of VivoTag®680XL to P-cadherin LP-DART.

Figure 2

ELISA based assay was used to evaluate the effect of VT680 labeling on binding of P-cadherin LP-DART to human P-cadherin and human CD3. The proteins were coated on to the plates and incubated with serial dilutions of P-cadherin LP-DART-VT680 or Control LP-DART-VT680 for 1 hr at 37°C. The bound P-cadherin LP-DART was quantified using IgG-HRP conjugate followed by calorimetric quantitation. (A) The labeling of VT680 to P-cadherin LP-DART had a DOL dependent effect on binding to human P-cadherin and Control LP-DART had no binding to human P-cadherin. (B) The labeling of VT680 to P-cadherin LP-DART and Control LP-DART affected binding to human CD3. P-cadherin LP-DART retained moderate binding even at a DOL of 2.0, whereas Control LP-DART lost its binding to human CD3 protein. (C) CTL Assay: Firefly luciferase expressing HCT116 cells and expanded human CD3+ T lymphocytes were co-incubated with increasing concentrations of P-cadherin LP-DART with different DOL of VT680. After 24 hr the remaining viable cells were quantified by measuring the luciferase activity. The relative cytotoxicity observed at various concentrations of P-cadherin LP-DART-VT680 was plotted against the PBS treated samples. VT680 conjugation decreased the cytotoxic ability in a DOL dependent manner.