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 [Zr]Zr-desferrioxamine (DFO)-daratumumab PET/CT imaging in MM tumor models. Daratumumab was conjugated to DFO--benzyl-isothiocyanate (DFO-Bz-NCS) for radiolabeling with Zr. 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 [Zr]Zr-DFO-daratumumab was performed using CD38 human myeloma MM1.S- (MM1.S) cells. Cellular studies determined the affinity, immunoreactivity, and specificity of [Zr]Zr-DFO-daratumumab. A 5TGM1- (5TGM1)/KaLwRij MM mouse model served as control for imaging background noise. [Zr]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, = 4-6/group) was performed in wild-type and MM1.S tumor-bearing mice. A specific activity of 55.5 MBq/nmol (0.37 MBq/μg) was reproducibly obtained with [Zr]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. [Zr]Zr-DFO-daratumumab/PET demonstrated specificity and sensitivity for detecting CD38 myeloma tumors of variable sizes (8.5-128 mm) with standardized uptake values ranging from 2.1 to 9.3. Discrete medullar lesions, confirmed by bioluminescence images, were efficiently imaged with [Zr]Zr-DFO-daratumumab/PET. Biodistribution at 7 d after administration of [Zr]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. [Zr]Zr-DFO- and Cy5-daratumumab demonstrated superb binding to CD38 human MM cells and significantly low binding to CD38 cells. Daratumumab bioconjugates are being evaluated for image-guided delivery of therapeutic radionuclides.

Copper 64–labeled daratumumab as a PET/CT imaging tracer for multiple myeloma

“…Our data clearly show higher sensitivity of 64 Cu-Dara-DOTA not only in comparison with 18 F-FDG signal but also in comparison with BLI imaging, an effect that was not seen when Dara was conjugated with 89 Zr. 19 Because our studies show a high sensitivity of 64 Cu-Dara-DOTA in detecting MM cells, we believe that this imaging approach for MM may also be clinically relevant for detecting minimal residual disease (MRD). MRD, as detected by high throughput bone marrow DNA sequencing, is being considered as a surrogate endpoint in clinical trials, [22][23][24] because effective therapies necessitate 5 to 10 years of follow-up to detect differences in survival.…”

“…18 64 Cu was safe, and by day 1 after injection, 64 Cu-trastuzumab-DOTA was able to detect MBC, its binding correlating with patient HER2 status. Although Dara has been conjugated with the radionuclide 89 zirconium ( 89 Zr), 19 the tendency of 89 Zr to accumulate in the bones, [19][20][21] associated with a longer time requested for imaging in contrast to 64 Cu, may be a limitation in specifically detecting MM cell dissemination in the bones. Our data clearly show higher sensitivity of 64 Cu-Dara-DOTA not only in comparison with 18 F-FDG signal but also in comparison with BLI imaging, an effect that was not seen when Dara was conjugated with 89 Zr.…”

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion

“…Here, we show that the CD38/Dara complex can be rapidly internalized in MM cells, and, although it is not certain how internalization process affects anti-neoplastic efficacy, we believe it may have contributed to positive results obtained when Dara was used for imaging studies. [41][42][43] There have been several reports that end-stage MM patients are now more likely to have extramedullary disease after becoming resistant to combinations of IMiDs, proteasome inhibitors, and CD38 antibody combinations. 44 Clearly, clones that are more resistant to Dara combination treatments are often independent of the BM-ME, 45 and one could extrapolate from our study that prolonged Dara exposure may support resistance.…”

Poly(ADP-Ribose) Polymerases in Host-Pathogen Interactions, Inflammation, and Immunity

“…The antibody exhibited high efficacy in an animal model of multiple myeloma (434). CD38 has also been developed as a target for positron emission tomography (PET) imaging using daratumumab antibody (435,436). Other antibodies, such as MOR202, a human antibody, and isatuximab, a humanized antibody, are in various stages of clinical trials (437,438).…”