BAT1 binds tightly to CXCR4, leading to dose-dependent inhibition of antibody binding and delivery of fluorescent cargo. (A) Phycoerythrin-CXCR4 antibody competition assay against BAT1 (red line) and plerixafor (blue line), assessed using flow cytometry. Primary CLL cells from a representative case were incubated with BAT1 or plerixafor at concentrations between 0 and 20 µM for 3 hours and then stained with phycoerythrin-conjugated CXCR4 antibodies. Experiment was performed in triplicate, and the mean was taken of the medians of each fluorescent distribution; errors were calculated as standard deviation of the mean. Dose-response curve fitted using a modified Hill equation, as detailed in supplemental Methods and data. A dose-dependent reduction in fluorescence was observed for BAT1 and plerixafor, with IC₅₀(BAT1) = 138 nM and IC₅₀(plerixafor) = 11.7 nM. (B) Analogous PerCP-CXCR7 antibody competition assay against BAT1 (red line) and plerixafor (black line). A dose-dependent reduction in antibody-staining was not observed for BAT1 or plerixafor. Fluorescence due to bound anti-CXCR7 antibody decreased at the highest concentrations of plerixafor or BAT1, but this decrease is within the measurement error, determined using the standard deviation of the mean. (C) Flow cytometric analysis of dose-dependent and selective BAT1-Cy5 targeting to CLL cells. Primary CLL cells were incubated with Cy5-conjugated BAT1 at 5 µM (solid red line) and 10 µM (solid blue line) for 3 hours. To demonstrate selectivity, the cells were also preincubated with 20 µM plerixafor as a comparison (dashed lines). BAT1-Cy5 was found to bind to CLL cells in a dose-dependent manner, and cells preincubated with 20 µM plerixafor present significantly reduced fluorescence, as indicated by the arrow. These data indicate that BAT1 can be used to specifically target functional molecules, such as dyes, to CXCR4-expressing cells.