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

Key Points

• Daratumumab conjugated with ⁶⁴Cu efficiently binds to CD38 on myeloma cells and was mainly detected in the bones of mice.

• PET/CT based on ⁶⁴Cu-radiolabeled daratumumab displays a higher resolution and specificity for detecting myeloma than does ¹⁸F-FDG PET/CT.

Abstract

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (¹⁸F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (⁶⁴Cu; ⁶⁴Cu-DOTA-Dara). Here, we show that ⁶⁴Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on ⁶⁴Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does ¹⁸F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using ⁶⁴Cu-DOTA-Dara as a novel imaging agent for MM.

Introduction

Prognostic information to stratify multiple myeloma (MM) patients into distinct risk-based treatments is currently based on specific genetic and cytogenetic abnormalities and clinical characteristics.^1,2 However, the extensive heterogeneity in therapeutic response, even in patients sharing similar characteristics,^1,2 emphasizes the need for more accurate prognostic tools.

Positron emission tomography/computed tomography using fluorodeoxyglucose-18 (¹⁸F-FDG PET/CT) imaging in newly diagnosed MM patients correlates with progression-free survival.^3,4 18F-FDG PET/CT also predicts the progression rate from smoldering MM to active disease.⁵ Hence, most investigators now agree on the importance of using imaging as a prognostic criterion for MM.^6-8

However, despite widespread use of ¹⁸F-FDG PET/CT, its ability to detect malignancies is dependent on metabolic activity. Lesions with a low metabolic rate may therefore be undetectable. In fact, low expression of hexokinase-2 in MM cells is associated with false-negative ¹⁸F-FDG PET/CT in MM.⁹ By binding to a tumor antigen rather than relying on the metabolic state of a malignancy, antibody-based approaches may allow imaging of cancer cells with low metabolic activity. In support, the chimeric fibril-reactive monoclonal antibody can be used in patients with light chain–associated amyloidosis to identify candidates for passive immunotherapy.^10,11

Daratumumab (Dara), a human anti-CD38 immunoglobulin G₁ (ĸ subclass) antibody against the highly expressed plasma cell receptor CD38, has shown particularly efficacious clinical activity^12-16 and recently has been approved by the US Food and Drug Administration for the treatment of relapsed MM. Because almost all MM patients express CD38 on the surface of their cancer cells, we hypothesize that tracing MM cell dissemination by targeting CD38 could be a successful approach.

Study design

See the supplemental Methods section, available on the Blood Web site.

Results and discussion

The conjugation of Dara to DOTA was successful, as was demonstrated by gel electrophoresis (supplemental Figure 1A-B); DOTA-Dara was labeled with copper 64 (⁶⁴Cu) efficiently, with no evidence of aggregates (supplemental Figure 1C-D). The immunoreactivity of ⁶⁴Cu-DOTA-Dara preparations was >95% (supplemental Figure 1E). ⁶⁴Cu-DOTA-Dara was extremely stable up to 48 hours in saline solution and in mouse serum (supplemental Figure 2A-B). CD38-positive MM.1S green fluorescent protein/luciferase-positive (GFP⁺/Luc⁺) and CD38-negative U266 Luc⁺ MM cells,¹⁷ which express CD38 antigen at the same levels as do the respective parental cell lines (supplemental Figure 3A-B), were used to assess ⁶⁴Cu-DOTA-Dara specificity in vivo. Mice were analyzed 7 and 21 days after MM.1S GFP⁺/Luc⁺ cell injection. Each animal was then injected with the radiolabeled antibody 24 hours after bioluminescence (BLI) assessment and imaged immediately (T₀), at 4 hours and at days 1 and 2 after the injection (Figure 1A; supplemental Figure 4A). Both BLI and ⁶⁴Cu-DOTA-Dara PET/CT imaging of the same mice suggested absence of MM bone engraftment in the mice imaged at day 7, but both methods detected signals at day 21 (Figure 1A). No positive bone signs were indicated in nonengrafted mice or in mice engrafted with U266 Luc⁺ cells (Figure 1B; supplemental Figure 4B). Lack of bone signaling was also observed in mice engrafted with MM.1S GFP⁺/Luc⁺ cells but imaged by a MM nonspecific radiolabeled antibody (⁶⁴Cu-trastuzumab-DOTA [⁶⁴Cu-trast-DOTA]) (Figure 1C), and when unlabeled Dara (cold) (50:1 excess) was used in combination with ⁶⁴Cu-DOTA-Dara (Figure 1D-E; supplemental Figure 4C). Ex vivo biodistribution studies at 24 hours verified the trends observed through PET analysis (Figure 1F). ⁶⁴Cu-DOTA-Dara was significantly detectable in MM.1S-engrafted mice (n = 4) in comparison with the tumor-free mice (n = 4) and with the mice injected with ⁶⁴Cu-trast-DOTA (n = 3) in the femurs, tibias, humeri, and scapola, known sites of MM cell invasion (Figure 1F). The same biodistribution trend observed in the control mice was observed in the organs of the mice injected with U266 cells or with cold unlabeled Dara (1:50) (Figure 1F). Higher signals were also observed in all organs made of bone tissue such as sternum, skull, and spinal column and in the lung (supplemental Figure 5A-B). ⁶⁴Cu-DOTA-Dara was also significantly detected in the spleen of MM.1S GFP⁺/Luc⁺–engrafted mice (Figure 1F; supplemental Figure 5A), which was also confirmed by flow cytometry (supplemental Figure 5B). In contrast, the majority of ⁶⁴Cu-DOTA-Dara remained in the blood of the control mice (P < .0001) and was not significantly different in the other organs (supplemental Figure 5A). To assess the sensitivity of the ⁶⁴Cu-Dara signal in comparison with the standard ¹⁸F-FDG PET-CT scan, we analyzed an independent group of animals bearing CD38⁺ tumors. At 21 days after cell injection, mice were first assessed by BLI and then injected with ¹⁸F-FDG (Figure 2A-B). The same group of mice were then injected with ⁶⁴Cu-DOTA-Dara the next day, and PET imaging was performed (Figure 2C; supplemental video A). BLI and ⁶⁴Cu-DOTA-Dara PET/CT revealed the presence of MM cells, whereas tumors were almost undetectable with the ¹⁸F-FDG PET/CT (Figure 2C-D). The ¹⁸F-FDG PET signals were mainly associated with the nonspecific uptake of glucose in leg muscles and the brain. The ¹⁸F-FDG bone signal was almost equal to the background signal of ¹⁸F-FDG in the heart, a contrast to the highly specific bone signal detected by ⁶⁴Cu-DOTA-Dara (Figure 2D; see also supplemental videos A and B).

Figure 1.

⁶⁴Cu-DOTA-Dara detects MM cell dissemination in vivo. (A) The radiolabeled antibody ⁶⁴Cu-DOTA-Dara (3.7 MBq, 10 µg) was administered to 3 mice that had been transplanted by IV injection with a human MM cell line (MM.1S GFP⁺/Luc⁺) before (7 days post-IV) and after (21 days post-IV) signs of MM bone metastasis as detected by BLI. The mice were also imaged with ⁶⁴Cu-DOTA-Dara radiolabeled antibody on the following days, and PET imaging identified MM cell dissemination in the bone (yellow boxes), which overlap with the BLI signals in the same mouse. (B) ⁶⁴Cu-DOTA-Dara (3.7 MBq, 10 µg) was also administered to 2 mice that had been transplanted by IV injection with U266 Luc⁺ MM cells, when clear signs (5 weeks post-IV) of MM bone engraftment were detected by BLI. The mice were imaged with ⁶⁴Cu-DOTA-Dara radiolabeled antibody after 1 day from the radiolabeled Dara injection (see also supplemental Figure 4B). Of note, no sign of engraftment was observed by PET/CT scan. (C) ⁶⁴Cu-DOTA-trastuzumab (⁶⁴Cu-DOTA-trast) (3.7 MBq, 10 µg) was administrated to 3 MM.1S GFP⁺/Luc⁺–engrafted mice, as assessed by BLI imaging, and a PET/CT scan was performed 1 day after ⁶⁴Cu-DOTA-trast injection. No bone signals were observed, but only high background associated with the circulating radiolabeled antibody. (D-E) Mice bearing CD38⁺ MM cells (MM.1S GFP⁺/Luc⁺) with a high level of engraftment (see also supplemental Figure 4C), as assessed by BLI images, that were injected with ⁶⁴Cu-DOTA-Dara (3.7 MBq, 10 µg) without (D) and with (E) unlabeled Dara (500 µg) and imaged at day 1 by PET/CT. Almost no signs of engraftments were detected in the presence of cold Dara, independent of the BLI signals. (F) ⁶⁴Cu-DOTA-Dara PET/CT-positive mice (MM.1S GFP⁺/Luc⁺–engrafted mice: 4) and negative mice (tumor free mice: 4); ⁶⁴Cu-DOTA-trast–treated mice (3); unlabeled Dara (Block) treated mice (2); and U266-engrafted mice (2) were sacrificed, and organs were harvested at 24 hours and used for γ counting to determine their radioactive content, as is presented in the bar graphs as a percentage of the injected dose per gram (%ID/g). See also supplemental Figure 5A.

Figure 2.

⁶⁴Cu-DOTA-Dara PET/CT shows higher resolution than does ¹⁸F-FDG-PET/CT. (A-C) Representative images of 2 NSG mice (mouse 1 and mouse 2), which were injected with 5 × 10⁶ MM.1S GFP⁺/Luc⁺ cells. After 2 weeks from the injection, the mice were imaged using BLI (A), and after 24 hours, the same mice were imaged by ¹⁸F-FDG-PET (B). The day after, ⁶⁴Cu-DOTA-Dara (3.7 MBq, 10 µg) was administered to the same mice imaged with ¹⁸F-FDG-PET and BLI (C). (D) Graphical representation of the intensity of the signal of ⁶⁴Cu-DOTA-Dara PET/CT in comparison with ¹⁸F-FDG-PET/CT, expressed as a percentage of the increase in percentage of the injected dose per gram (%ID/gr) of bone organs in comparison with the nonspecific signal of each radio nucleotide (%ID/gr) detected in the heart. A radiolabeled antibody identified MM cancer cells, which were undetectable by ¹⁸F-FDG-PET scan. (E) Mice with minimum BLI signals were IV-injected with ⁶⁴Cu-DOTA-Dara (3.7 MBq, 10 µg) and then imaged by PET/CT, which identified MM cell dissemination in the bone (yellow boxes). (F-G) Representative images at different magnifications of tissue sections stained by hematoxylin and eosin stain (F) and CD138 immunohistochemical stain (G) showing MM cell accumulation (black dashed ovals) in the left and right femurs and in the vertebral body (lower lumbar), but not in the left humerus, which did not show PET/CT signals. H&E, hematoxylin and eosin; IHC, immunohistochemical.

To assess the ability of ⁶⁴Cu-DOTA-Dara to recognize MM cells in the bones when the BLI signal was minimal or completely absent, we used an independent group of mice for histology analysis and for immunohistochemical staining for the MM cell marker CD138. Step section of the bones show foci of MM cells, as is also assessed by CD138 staining in the right and left femurs, confirming the presence of MM cells in the location where both BLI and ⁶⁴Cu-DOTA-Dara signals were present (Figure 2E-G). We also detected MM cells in the lower vertebral body, which had no BLI signal (Figure 2E-G) and no cells in the humerus, which was also found negative by PET/CT scan (Figure 2E-G).

The relatively short half-life of ⁶⁴Cu (12.7 hours) may be ideal for patient safety and is sufficiently long for PET/CT imaging up to 48 hours after administration. We have used ⁶⁴Cu-labeled trastuzumab to safely detect human epidermal growth factor receptor 2 (HER2)–positive tissue in metastatic breast cancer (MBC) patients.^{18 64}Cu was safe, and by day 1 after injection, ⁶⁴Cu-trastuzumab-DOTA was able to detect MBC, its binding correlating with patient HER2 status. Although Dara has been conjugated with the radionuclide ⁸⁹zirconium (⁸⁹Zr),¹⁹ the tendency of ⁸⁹Zr to accumulate in the bones,^19-21 associated with a longer time requested for imaging in contrast to ⁶⁴Cu, may be a limitation in specifically detecting MM cell dissemination in the bones. Our data clearly show higher sensitivity of ⁶⁴Cu-Dara-DOTA not only in comparison with ¹⁸F-FDG signal but also in comparison with BLI imaging, an effect that was not seen when Dara was conjugated with ⁸⁹Zr.¹⁹ Because our studies show a high sensitivity of ⁶⁴Cu-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,^22-24 because effective therapies necessitate 5 to 10 years of follow-up to detect differences in survival.^25,26 However, irrespective of its sensitivity, this technique is marrow based and cannot account for a nonhomogenous bone marrow infiltration and extramedullary involvement. In support of Dara as a unique tool for imaging in MM, our group developed a ⁶⁴Cu antibody (elotuzumab [Elo]) against the highly specific MM receptor CS1 (⁶⁴Cu-DOTA-Elo). Although ⁶⁴Cu-DOTA-Elo bound the CS1 antigen in vitro, it did not identify bone engraftment of CS1-positive MM cells in in vivo studies (data not shown). We have begun a ⁶⁴Cu-DOTA-Dara PET/CT imaging trial that has the potential to affect MM clinical cell detection.

Supplementary Material

The online version of this article contains a data supplement.

Authorship

Contribution: E.C., S.H., J.K., S.R., J.S., N.C., D.C., A.K., and F.P. designed the research; E.C., J.C., M.M., D.V., S.V., J.K., P.Y., Y.K., and R.B. performed the research; E.C., J.F.S., J.M.P., G.M., J.S., D.C., and F.P. analyzed the data; J.F.S. and F.P. wrote the manuscript; and all authors reviewed the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Flavia Pichiorri, Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, 1500 E Duarte Rd, Duarte, CA 91010; e-mail: fpichiorri@coh.org; and David Colcher, Department of Molecular Immunology, City of Hope, 1500 E Duarte Rd, Duarte, CA 91010; e-mail: dcolcher@coh.org.