VpreB1 is expressed across a spectrum of B-ALLs and can be targeted with an antibody. (A) Schematic illustrating the expression of the SLC, consisting of VpreB1 (CD179a) and IGLL1 (CD179b or λ5), during B-cell and B-ALL development. (B) Gene expression of pre-BCR component-encoding genes was analyzed across 3532 B-ALL samples, classified into 21 molecular subtypes, with counts normalized using counts per million.²⁴ (C) The binding and dissociation of varying recombinant VpreB1/λ5 concentrations to anti-VpreB1 antibody were measured with biolayer interferometry. The experiment was performed in triplicate, and representative data are revealed. The association and dissociation transients were used to estimate a binding affinity K_d = 26.7 nM (±2.5 nM). (D) MFI of NALM-6 and REH leukemia cells incubated with increasing concentrations of VpreB1-AF647 antibody. (E) Internalization of cell surface VpreB1 protein on NALM-6 cells was measured by labeling the cells with both a fluorescent (AF647) and biotin conjugated anti-VpreB1 antibody. Internalization was then either blocked by incubation on ice or allowed to proceed for 1 hour at 37°C. Cells were then labeled with fluorescein isothiocyanate (FITC)-Neutravidin and assessed by flow cytometry. FITC fluorescence corresponds to relative cell surface anti-VpreB1 and APC (AF647) fluorescence to total cell surface and intracellular anti-VpreB1. Error bars represent the mean ± standard deviation (SD). ∗∗∗∗P < .0001 by t test. MFI, mean fluorescent intensity; ns, not significant; RFU, relative fluorescence units.