Preclinical Development of CD38-Targeted [⁸⁹Zr]Zr-DFO-Daratumumab for Imaging Multiple Myeloma.

Abstract

Multiple myeloma (MM) is a plasma B-cell hematologic cancer that causes significant skeletal morbidity. Despite improvements in survival, heterogeneity in response remains a major challenge in MM. Cluster of differentiation 38 (CD38) is a type II transmembrane glycoprotein overexpressed in myeloma cells and is implicated in MM cell signaling. Daratumumab is a U.S. Food and Drug Administration-approved high-affinity monoclonal antibody targeting CD38 that is clinically benefiting refractory MM patients. Here, we evaluated [⁸⁹Zr]Zr-desferrioxamine (DFO)-daratumumab PET/CT imaging in MM tumor models. <b>Methods:</b> Daratumumab was conjugated to DFO-<i>p</i>-benzyl-isothiocyanate (DFO-Bz-NCS) for radiolabeling with ⁸⁹Zr. Chelator conjugation was confirmed by electrospray ionization-mass spectrometry, and radiolabeling was monitored by instant thin-layer chromatography. Daratumumab was conjugated to Cyanine5 (Cy5) dye for cell microscopy. In vitro and in vivo evaluation of [⁸⁹Zr]Zr-DFO-daratumumab was performed using CD38⁺ human myeloma MM1.S-<i>luciferase</i> (MM1.S) cells. Cellular studies determined the affinity, immunoreactivity, and specificity of [⁸⁹Zr]Zr-DFO-daratumumab. A 5TGM1-<i>luciferase</i> (5TGM1)/KaLwRij MM mouse model served as control for imaging background noise. [⁸⁹Zr]Zr-DFO-daratumumab PET/CT small-animal imaging was performed in severe combined immunodeficient mice bearing solid and disseminated MM tumors. Tissue biodistribution (7 d after tracer administration, 1.11 MBq/animal, <i>n</i> = 4-6/group) was performed in wild-type and MM1.S tumor-bearing mice. <b>Results:</b> A specific activity of 55.5 MBq/nmol (0.37 MBq/μg) was reproducibly obtained with [⁸⁹Zr]Zr-daratumumab-DFO. Flow cytometry confirmed CD38 expression (>99%) on the surface of MM1.S cells. Confocal microscopy with daratumumab-Cy5 demonstrated specific cell binding. Dissociation constant, 3.3 nM (±0.58), and receptor density, 10.1 fmol/mg (±0.64), was obtained with a saturation binding assay. [⁸⁹Zr]Zr-DFO-daratumumab/PET demonstrated specificity and sensitivity for detecting CD38⁺ myeloma tumors of variable sizes (8.5-128 mm³) with standardized uptake values ranging from 2.1 to 9.3. Discrete medullar lesions, confirmed by bioluminescence images, were efficiently imaged with [⁸⁹Zr]Zr-DFO-daratumumab/PET. Biodistribution at 7 d after administration of [⁸⁹Zr]Zr-DFO-daratumumab showed prominent tumor uptake (27.7 ± 7.6 percentage injected dose per gram). In vivo blocking was achieved with a 200-fold excess of unlabeled daratumumab. <b>Conclusion:</b> [⁸⁹Zr]Zr-DFO- and Cy5-daratumumab demonstrated superb binding to CD38⁺ human MM cells and significantly low binding to CD38^low cells. Daratumumab bioconjugates are being evaluated for image-guided delivery of therapeutic radionuclides.