Multiplexed mAbs: a new strategy in preclinical time-domain imaging of acute myeloid leukemia

Antibodies play a fundamental role in diagnostic immunophenotyping of leukemia's and in cell targeting therapy. However, this versatility is not reflected in imaging diagnostics. We monochromatically labeled anti-human monoclonal antibodies (mAbs) against selected human myeloid markers expressed on acute myeloid leukemia cells, all with the same near-infrared fluorochrome. In a novel "multiplexing" strategy we then combined these mAbs to overcome limiting target-to-background ratio to image multiple xenografts of acute myeloid leukemia (AML). Time domain imaging was employed to discriminate autofluorescence from the distinct fluorophore-conjugated antibodies. Imaging with multiplexed mAbs demonstrated superior imaging of AML to GFP or bioluminescence and permitted evaluation of therapeutic efficacy with the standard combination anthracycline and cytarabine in primary patient xenografts. Multiplexing mAbs against CD11b and CD11c, provided for surrogate imaging biomarkers of differentiation therapy in an acute promyelocytic leukemia model treated with all-trans retinoic acid combined with the histone-deacetylase inhibitor valproic acid. Together, we present an optimized application of multiplexed immunolabeling in vivo for optical imaging of AML cell xenografts that provides reproducible, highly accurate disease staging and monitoring of therapeutic effect.