Introduction
Multiple myeloma (MM) represents the second most common hematological malignancy, accounting for 2% of all cancers and 19% of hematological tumors. 1 While novel therapeutic approaches and new treatments have been successfully introduced in recent years, improving the prognosis and therapy of MM will depend on the accurate measurement of disease burden: MM remains incurable largely due to suboptimal means of measuring disease burden. 2 Only sensitive detection of disease burden allows for early intervention, which is directly associated with progression-free and overall survival. Furthermore, detection of minimal residual disease (MRD) following therapy is crucial to improving the prognosis of MM patients, as most eventually relapse. Thus, a sensitive method of detecting and measuring tumor burden is urgently needed. 3
The ability to assess the expression levels of selected targets in MM cells before and during treatment will also help to stratify patients according to their likelihood of responding to the corresponding therapy, in part by helping to identify dynamic expression that has been downregulated and thus contribute to disease resistance. 4 Likewise, baseline evaluation of and differentiation between active and inactive lesions after therapy is essential to evaluating therapeutic efficacy and better managing clinical decision-making. In this context, imaging plays a pivotal role in the management of the malignancy. More specifically, PET has proven to be a powerful molecular imaging tool for these unmet clinical needs, targeting different molecular pathways and facilitating the development of promising tracers. 5
Among the new generation of therapeutic targets, CD38, or cluster of differentiation 38, has attracted great attention in recent years due to its high expression in aberrant lymphoid and myeloid cells relative to normal cell populations. CD38 is a 45-kD, type II transmembrane glycoprotein that metabolizes NAD + , regulates cytoplasmic Ca 2+ flux, and mediates signal transduction. 6 Its expression is induced by inflammatory cytokines, endotoxins, interferons, and some nuclear receptors. Thus, an extensive range of (radio)pharmaceuticals have been designed to target and/or modulate its activity, some of which have already shown some success in early clinical trials in plasma cell malignancies. 7 This makes CD38 an excellent biomarker for targeted imaging and therapy in MM. 8
Certainly, anti-CD38 monoclonal antibodies have already shown high efficiency in the immunotherapy of MM, and preclinical studies highlight their potential use in other CD38-positive hematologic malignancies, including lymphomas and leukemias. 9 Daratumumab (Janssen Pharmaceuticals) was the first CD38-targeting antibody approved by the FDA as a single agent in MM immunotherapy treatment. 10 Labeling of the native daratumumab with positron-emitting radionuclides such as zirconium-89 has been exploited for immunoPET imaging in different models of disease, proving particularly successful in MM. 11 Our group previously reported the synthesis of 89 Zr-DFO-daratumumab for CD38-targeted immunologic PET imaging of MM in a murine model and a successful first-in-human imaging study. 12 89 Zr-DFO-daratumumab demonstrated successful uptake in osseous deposits in patients with MM and the ability to image myeloma that was not detected by 18F-FDG, the current standard-of-care imaging. Daratumumab has also been labeled with therapeutic nuclides, demonstrating the validity for CD38-targeted radioimmunotherapy in different preclinical models. 13 , 14 Finally, extension to other targeted molecular imaging modalities has been explored by conjugation to different dyes. 15
However, despite these promising results, daratumumab resistance, which occurs in approximately 60% of patients, is a recurrent problem hampering clinical response. 9 , 16 , 17 The underlying mechanisms of resistance include clone selection of CD38 dim myeloma cells; 9 CD38 reduction, for instance by CD38 endocytosis/internalization; 18 immunomodulation, for instance by downregulation of intracellular pathways in bone marrow stem/stromal cells and a decrease of effector memory T cells and M1 macrophages; 19 , 20 and prevention of complement-dependent cytotoxicity, for instance via CD55 and CD59 overexpression. 21
Isatuximab (Sanofi), the latest CD38-targeting antibody approved by the FDA (March 2021) for MM treatment, has shown encouraging results. In a phase 1b trial, combination of isatuximab with pomalidomide and dexamethasone showed an overall response rate of 53.2% in 47 MM patients, 14.9% of which were refractory to daratumumab. 22 In a multicentric phase 1/2 study in relapsed/refractory MM patients who had demonstrated disease progression under daratumumab mono- or combination therapy, isatuximab monotherapy enabled a ≥8-week disease control rate (stable disease or minor response) of 37.5%. 23
While belonging to the same IgG1-κ subclass and targeting the same receptor, very distinct mechanisms of action and binding modes have been reported between daratumumab and isatuximab, including the identification of their corresponding binding epitopes. 24 − 26 These structural and functional features have very important implications for the rational design of targeted pharmaceuticals, 27 sparking investigations into the possibility that isatuximab could overcome daratumumab resistance. 28 In this context, development of isatuximab as an imaging agent would expand the means to detect and quantify CD38-positive tumors and might potentially help elucidate these mechanisms of action and resistance, whether primary or acquired. A better understanding of the immune effector mechanism of each antibody is necessary to better predict patient response and treatment monitoring. 29 Additionally, some treatment features that greatly impact patient quality of life, such as administration route and regimes, have been reported as more favorable for isatuximab than daratumumab. 30 Accordingly, validation of isatuximab as an imaging and potentially therapeutic agent would have a high translational relevance for those patients who could benefit from this new mAb in the era of personalized medicine. 31
Here, we present the radiolabeling of the native isatuximab with the PET emitter radionuclide 89 Zr and the preclinical evaluation of its potency as an immunoPET tracer for CD38-targeted imaging. In this study, we detail the synthesis and the in vitro and in vivo comparison to 89 Zr-DFO-daratumumab in MM and lymphoma murine models.
Materials and Methods
Cell Culture
All cell lines were obtained from the American Type Culture Collection (ATCC). Cultures were grown in aseptic conditions at 37 °C and 5% CO 2 in a humidified atmosphere in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. MM.1S and Daudi cells were stably transfected with firefly luciferase (Luc) through a lentivirus, allowing for bioluminescence imaging (BLI). Successful transfection was confirmed by a luciferase reporter assay (Promega).
Flow Cytometry
The surface expression level of the CD38 receptor was evaluated and quantified before and after luciferase transfection by flow cytometry. Cells (5 × 10 5 cells) were incubated with FcR human blocking reagent (Milteny Biotech) in 50 μL of flow cytometry staining buffer (Invitrogen) for 15 min and subsequently stained with phycoerythrin (PE)-conjugated antihuman CD38 antibody (Biolegend clone HB-7) for 30 min. Dead cells were excluded using 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich). Cells were analyzed on a Fortessa flow cytometer, and data was processed with FlowJo V10 (treeStar). Gating of CD38 positive cells was performed using unstained controls.
Conjugation and Radiolabeling
Isatuximab (Sarclisa) and daratumumab (Darzalex) were conjugated to the chelator p -isothiocyanatobenzoyl desferrioxamine ( p -SCN-DFO, Macrocyclics, Inc.) The antibodies were prepared as a 2 mg/mL solution in PBS, and the solution pH was adjusted to 9 with 0.1 M Na 2 CO 3 . DFO-NCS was dissolved in dry DMSO at a concentration of 10 mg/mL, and a 4-fold molar excess was added to the corresponding solutions. The reaction was incubated at 37 °C for 1 h on a thermomixer. Purification of the immunoconjugates was performed with a prepacked disposable PD-10 desalting column (GE Healthcare) and concentrated with a 50,000 MWCO Amicon centrifugal filter (Millipore Sigma). Final concentrations were determined with a NanoDrop 2000 spectrometer (Thermo Fisher Scientific). Zirconium-89 ( 89 Zr) was supplied by 3D Imaging LLC (Little Rock, AR) in 0.1 M oxalic acid. After adjusting the pH of the solution to 7 with 0.1 M NaHCO 3 , each DFO-conjugated antibody was added to the neutralized 89 Zr and reacted at 37 °C for 1 h. Radiochemical purity (RCP) was assessed by radio-instant thin-layer chromatography (radio-iTLC) using 50 mM ethylenediaminetetraacetic acid (EDTA) as the eluent and was always greater than 99% before injection.
Binding Studies
Cell binding assays were performed by incubating the different cell lines with 1 μCi of 89 Zr-DFO-mAb in PBS with 1% BSA for 2 h at 4 °C. Excess of unlabeled DFO-mAb was added in the blocking group to demonstrate the specificity of the binding. Unbound radiotracer was removed by washing the cells twice with ice-cold PBS and centrifuging (600 g for 5 min). The radioactivity of the different fractions was measured using a gamma counter.
Animals and Tumor Models
All animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee and Research Animal Resource Center at Memorial Sloan Kettering Cancer Center. In vivo imaging and tissue biodistribution studies were performed in female NSG mice (NOD.Cg -Prkdcscid Il2rgtm1Wjl/ SzJ; 6–8 weeks old; Jackson Laboratories, Bar Harbor, Maine). MM.1S and K562 cells (5 × 10 6 cells) were subcutaneously (SQ) injected in a 1:1 mixture of culture medium and Matrigel into the right flank to establish the SQ model. MM.1S-Luc and Daudi-Luc cells (1 × 10 6 cells) were intravenously injected in the tail vein for the disseminated model of disease. Solid SQ tumors were regularly measured with a caliper. Disseminated tumor burden was monitored by BLI. Mice were injected with 100 μL of 15 mg/mL luciferin 10 min prior to imaging with an IVIS SpectrumCT (PerkinElmer, Melville, NY).
ImmunoPET/CT Imaging Studies and Analysis
For PET imaging studies, tumor-bearing mice were intravenously injected with 89 Zr-DFO-isatuximab or 89 Zr-DFO-daratumumab ( n = 4/group) (5–7.5 MBq; 18.7–25 μg). For blocking studies ( n = 4) blocked mice were co-injected with a 12-fold excess of unlabeled DFO-isatuximab to block relevant receptors and demonstrate specific binding of the tracer to CD38. An additional control group of healthy mice ( n = 3) was also included in the experiment. To avoid anomalous biodistribution caused by the lack of endogenous immunoglobulin G in NSG mice, 500 μg of nonspecific human immunoglobulin G was either coinjected with the radiolabeled antibody or, in the case of the blocking study, injected 3 days before the radiotracer. In vivo small animal PET/CT was performed on an Inveon micro PET/CT instrument (Siemens, Erlangen, Germany). Mice were anesthetized with 1–2% isoflurane, and static images were collected for a maximum of 45 min. Images were analyzed using the Inveon Research Workstation (IRW) software.
Ex Vivo Biodistribution Studies
Biodistribution studies were performed by euthanizing five mice at discrete time points after injection of the tracer (0.37–1.1 MBq; 2.0–2.5 μg). Acute biodistribution of the imaging groups (5–7.5 MBq; 18.7–25 μg) was performed at the terminal time point of the corresponding study (144 h). Relevant organs including bone marrow were harvested and weighed, and radioactivity was counted on an automated gamma counter (PerkinElmer). Bone marrow was extracted by dissecting out both mouse hind limbs and centrifuging the femurs and tibias. Biodistribution values are presented as the percent of the injected dose per gram (%ID/g), determined by counting relevant standards along with organ samples and normalizing the counts per sample to the total amount of activity injected.
Autoradiography Imaging
Autoradiography was performed on frozen bone sections. Femurs, tibias, and hips were sliced in sections of 300–500 μm, using a Vantik Qs11 Cryostat, and exposed to the same phosphor screen for 24 h. The film was read on Typhoon FLA 7000 (GE Healthcare) and produced images analyzed with ImageJ software.
Ex vivo Flow Cytometry
Ex vivo flow cytometry of the bone marrow was performed to quantify CD38 expression. Bone marrow was obtained from the femurs using a centrifugation protocol.
Tissue Immunohistochemistry Staining
The immunohistochemistry detection of CD38 was performed at Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center, using a Discovery XT processor (Ventana Medical Systems, Roche, AZ). Femurs were decalcified prior to sectioning.
Statistical Analysis
All data presented are expressed as mean ± SD. To evaluate the blocking and healthy cohort, a 2-way ANOVA test using GraphPad Prism 9 software was performed with a threshold for statistical significance set at p < 0.05. A correction for multiple comparisons was performed using the Holm–Sidak method to determine statistical significance (α = 0.05). Automated image processing and automated analysis were used where possible to eliminate human bias.
Results
89 Zr-DFO-Isatuximab Synthesis and In Vitro Assessment of CD38 Targeting
Development of 89 Zr-DFO-isatuximab as an immunoPET agent was achieved by incorporation of the PET radioisotope ( Figure 1 A). Chemical modification of the native commercial antibody by covalent conjugation of the chelator DFO on nonsite specific lysine residues allowed the radiolabeling with 89 Zr. Synthesis of 89 Zr-DFO-isatuximab was successfully accomplished with yields higher than 92% ( n = 10). Purified radioimmunoconjugate had a radiochemical purity greater than 99%, with no detectable free 89 Zr, and specific activities of 33.3 ± 3.7 MBq/mg. Additionally, 89 Zr-DFO-isatuximab exhibited an excellent in vitro stability, displaying >90% in PBS and human serum over 144 h ( Figure S1 ). The same methodology was followed for the synthesis of 89 Zr-DFO-daratumumab.
Binding of the new radioimmunoconjugate to the target was first validated in a magnetic bead assay. 32 89 Zr-DFO-isatuximab exhibited successful binding to CD38, comparable to that of 89 Zr-DFO-daratumumab, and was blockable in the presence of a 1000-fold excess of unlabeled DFO-isatuximab ( Figure 1 C).
For cellular studies, human MM, MM.1S cell line, Burkitt’s lymphoma, Daudi cell line, and myelogenous leukemia, K562 cell line, were used. Cellular expression of CD38 receptor for the three lines was validated by flow cytometry ( Figure 1 B). In vitro cell assays confirmed that 89 Zr-DFO-isatuximab binds to the cell surface receptor on the CD38+ cell lines and displays a better performance on Daudi cells, which is in accordance with the higher density of CD38 in this cell line. Binding remained baseline and non-blockable on the CD38– cell line ( Figure 1 D).
In Vivo Evaluation of 89 Zr-DFO-isatuximab in Subcutaneous Xenograft Models
After in vitro validation of its CD38-targeting potential, we evaluated the imaging performance of 89 Zr-DFO-isatuximab in a subcutaneous xenograft model of human MM. PET/CT and maximum-intensity-projection images (MIP) with 89 Zr-DFO-isatuximab showed a high delineation of the tumor at 144 h after injection of the tracer ( Figure 2 A). For the blocking group, tumor uptake was significantly reduced in comparison with the control (26 ± 4.1% vs 14.1 ± 1.7%ID/g), which proved the specificity of the tracer for the CD38 receptor. A subcutaneous xenograft of K562, which does not express CD38, was used as a negative model. Immunohistochemistry analysis of resected MM.1S and K562 tumors confirmed expression or lack of CD38, respectively ( Figures 2 B and S2 ).
CD38-Targeted PET Imaging of 89 Zr-DFO-Isatuximab in a Disseminated Murine Model of Multiple Myeloma
We aimed to evaluate 89 Zr-DFO-isatuximab in a disseminated model of disease which is more consistent with the real pathology of hematological malignancies. A disseminated MM model was generated by intravenous injection of MM1.S-Luc cells into NSG mice. Mice were monitored by BLI. After photon flux reached an average of 1 × 10 7 p/sec/cm 2 /sr, approximately 3–4 weeks after cell injection, they were randomized into the different groups. Diseased mice received 89 Zr-DFO-isatuximab, 89 Zr-DFO-isatuximab coinjected with a blocking dose of DFO-isatuximab, and 89 Zr-DFO-daratumumab, respectively. Additionally, 89 Zr-DFO-isatuximab was administered to a group of healthy NSG mice. Mice were imaged at 24, 48, 72, 96, 120 and 144 h upon tracer injection ( Figure 3 ). A complete biodistribution study was performed at the last time point (144 h) to quantify the radiotracer uptake in major organs for each cohort ( Figure 4 A and Table S1 ). Bone marrow was extracted from the femurs by centrifugation and counted separately from the bone (femur or tibia), which was measured unprocessed. %ID/g values confirmed high uptake of 89 Zr-DFO-isatuximab in bone and bone marrow (23.0 ± 6.5% and 21.7 ± 6.8%ID/g, respectively), which was statistically significant compared to the uptake in the blocking group (9.8 ± 2.6%ID/g; P < 0.0005 and 9.2 ± 1.3%ID/g; P < 0.05, respectively), and as good as uptake with 89 Zr-DFO-daratumumab (20.4 ± 2.7% and 23.4 ± 9.3%ID/g). Additionally, a control experiment was conducted to compare the uptake of 89 Zr-DFO-isatuximab in bone and bone marrow between diseased mice, a blocking group, and a healthy cohort ( Figure 4 B). This study revealed a minimal amount of tracer accumulation in bone and marrow (6.7 ± 0.6%ID/g; P < 0.0005, 11.1 ± 2.7%ID/g; P < 0.05), one of the main concerns when working with 89 Zr-mAbs, which increases background-to-tumor signal.
Ex Vivo Biodistribution Study
The time-course biodistributon study ( Figure 4 C) confirmed the progressive increase of 89 Zr-DFO-isatuximab uptake in bone and in bone marrow, which peaks at 120 h (23.6 ± 0.3 and 33.9 ± 21.2%ID/g). As expected, the highest uptake in blood pool and lungs was seen at the earliest time point, decreasing gradually (4.8 ± 0.3 and 6.1 ± 0.6%ID/g at 48 h, respectively), while uptake in liver remained relatively constant (18–10%ID/g).
Autoradiography Imaging and Ex Vivo Tissue Staining
Autoradiography imaging of select bones further demonstrated the specific uptake shown by the imaging data ( Figure 5 A). Finally, ex vivo flow cytometry analysis of extracted bone marrow cells confirmed the expression of CD38 ( Figure 5 B). Analogous data was obtained by immunohistochemistry staining of dissected femurs ( Figures 5 C and S3 ). CD38 expression was not detectable in healthy control femurs ( Figure S4 ).
CD38-Targeted PET Imaging of 89 Zr-DFO-Isatuximab in a Disseminated Murine Model of Non-Hodgkin Lymphoma
To demonstrate that the use of 89 Zr-DFO-isatuximab as an immunoPET agent can be extended to other hematological malignancies, we conducted the same studies in a disseminated model of Burkitt’s lymphoma, a very well-established model of CD38+ lymphoma. As shown by the PET/CT images ( Figure 6 A), uptake of 89 Zr-DFO-isatuximab by the tumor increases over time, displaying an exceptional contrast and delineation of the lymphoma lesions that matches the bioluminescence imaging. In fact, the contrast is almost superior to that obtained in the MM.1S-model, an outcome in accordance with the superior expression of the CD38 receptor, as consistenly determined by different in vitro evaluations. Regions of interest (ROI) on the corresponding PET images were used to determine the time-course tracer uptake by the organs of interest ( Figure 6 B). Slow clearance is observed from heart, lungs, and liver while a relatively constant uptake is observed for leg and lumbar spine, peaking at 120 h (15.4 ± 5.1 and 16.8 ± 3.1%ID/g, respectively).
Discussion
Current measurements of MM disease burden—whether by blood analysis, imaging, or blind bone marrow biopsy—are suboptimal, greatly hampering clinical care. 8 For example, the limited sensitivity of standard 18F-FDG imaging for MM detection is well-recognized. Several studies have shown that at least 11% of patients are false negative on 18F-FDG due to the low hexokinase-2 expression, 33 , 34 and this false-negative rate can increase up to 60% in pretreated myeloma patients. 35 Thus, there exists an urgent need for the development of a superior, noninvasive PET imaging agent that can not only determine tumor burden but also predict the effectiveness of a selected therapy, monitor treatment response, and rule out MRD, improving the prognosis of patients. Ultimately, the use of such an agent could be extended to the targeted imaging and treatment of other hematologic malignancies such as lymphomas and leukemias.
We used isatuximab, the latest FDA-approved CD38-targeting antibody for MM treatment, for the synthesis and preclinical validation of ( 89 Zr)-labeled isatuximab as an immunoPET agent in one negative and two positive models of disease. We successfully demonstrated that 89 Zr-DFO-isatuximab is a highly selective and specific agent for MM imaging, capable of delivering results of equal quality to those previously obtained with 89 Zr-DFO-daratumumab—the reference in the literature for this malignancy in recent years. 12 Finally, we demonstrated 89 Zr-DFO-isatuximab’s excellent imaging performance in a preclinal model of lymphoma, opening the door to further extending its application to this hematological malignancy.
The development and validation of this new radioagent could contribute greatly to MM research. Despite daratumumab’s success both as an immunotherapy and immunoPET agent, acquired resistance is a recurrent problem among many MM patients. Introducing a substitute agent with a different mechanism of action may offer numerous patients a suitable therapeutic alternative. In fact, clinical trials are currently evaluating the value of isatuximab in daratumumab-refractory patients ( NCT02514668 ). Thus, a matching imaging pair could contribute to the development of new and personalized diagnostic and treatment strategies.
More importantly, the faster tumor accumulation observed for 89 Zr-DFO-isatuximab and the fact that native isatuximab does not induce CD38 release from the cell surface, as daratumumab does, could allow for shorter imaging times via pretargeting strategies, reducing radiation exposure to patients and improving quality of life.
Overall, the high specificity and sensitivity of 89 Zr-DFO-isatuximab supports its clinical translation as a new imaging agent. As isatuximab is already an FDA-approved antibody, we expect this will ease and speed up the first-in-human clinical trial of the derived PET and future therapeutic agents.
Conclusion
The findings of this study suggest 89 Zr-DFO-isatuximab can be used as a noninvasive diagnostic PET agent to detect the presence of CD38-positive lesions, opening the door to an optimized and/or alternative agent for patients suffering from these malignancies that could improve their prognosis and survival.
Acknowledgments
The authors would like to acknowledge the Radiochemistry and Molecular Imaging Probes Core Facility, the Small Animal Imaging Facility, and the Molecular Cytology Core Facility. The study was supported in part by R35 CA232130 (J.S.L.). The Radiochemistry and Molecular Imaging Probes Core Facility, the Small Animal Imaging Facility, and the Molecular Cytology Core Facility were supported in part by NIH P30 CA08748.
Supporting Information Available
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c00624 .
Detailed information on antibody characterization, in vitro binding assays, immunohistochemistry, flow cytometry, and biodistribution studies ( PDF )
Author Contributions
Conception and design: N.H.A. and J.S.L. Development and methodology: N.H.A. and J.S.L. Acquisition of data: N.H.A., A.L.M., and T.D.V. Analysis and interpretation of data: N.H.A. Writing, review, and/or revision of the manuscript: N.H.A., M.E.M., and J.S.L. Study supervision: J.S.L.
The authors declare no competing financial interest.
Supplementary Material
ao3c00624_si_001.pdf
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Associated Data
Supplementary Materials
ao3c00624_si_001.pdf