Annotating CD38 Expression in Multiple Myeloma with [18F]F–Nb1053

The efficacy of daratumumab is partially dependent on CD38 expression on multiple myeloma (MM) cells. We have previously shown that ATRA upregulates CD38 expression and reverts daratumumab-resistance ex vivo. We therefore evaluated the optimal dose, efficacy and safety of daratumumab combined with ATRA in daratumumab-refractory MM patients in a phase 1/2 study (NCT02751255). In part A of the study, 63 patients were treated with daratumumab monotherapy. Fifty daratumumab-refractory patients were subsequently enrolled in part B, and treated with daratumumab (re-intensified schedule) combined with ATRA until disease progression. The recommended phase 2 dose of ATRA in combination with daratumumab was defined as 45 mg/m2. At this dose, the overall response rate (ORR) was 5%, indicating that the primary endpoint (ORR≥15%) was not met. However, the majority of patients (66%) achieved at least stable disease. After a median follow-up of 43 months, the median PFS for all patients was 2.8 months. Patients who previously achieved at least a partial response or minimal response/stable disease with prior daratumumab monotherapy had a significantly longer PFS, compared to those who immediately progressed during daratumumab as single agent (median PFS 3.4 and 2.8 versus 1.3 months). The median OS was 19.1 months. The addition of ATRA did not increase the incidence of adverse events. Flow cytometric analysis revealed that ATRA temporarily increased CD38 expression on immune cell subsets. In conclusion, the addition of ATRA and re-intensification of daratumumab had limited activity in daratumumab-refractory patients, which may be explained by the transient upregulation of CD38 expression.

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Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab

Leukemia ◽

10.1038/s41375-020-0840-y ◽

2020 ◽

Vol 35 (1) ◽

pp. 201-214 ◽

Cited By ~ 4

Author(s):

Estefanía García-Guerrero ◽

Ralph Götz ◽

Sören Doose ◽

Markus Sauer ◽

Alfonso Rodríguez-Gil ◽

...

Keyword(s):

Multiple Myeloma ◽

Class Effect ◽

Myeloma Cells ◽

Cd38 Expression

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Abstract 2966: Isatuximab-induced multiple myeloma cell killing through effector functions is dependent on CD38 expression and complement inhibitors

10.1158/1538-7445.am2019-2966 ◽

2019 ◽

Author(s):

Zhili Song ◽

Guang Yang ◽

Anlai Wang ◽

Rita Greco ◽

Joachim Theilhaber ◽

...

Keyword(s):

Multiple Myeloma ◽

Myeloma Cell ◽

Cell Killing ◽

Effector Functions ◽

Multiple Myeloma Cell ◽

Cd38 Expression ◽

Complement Inhibitors

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Panobinostat Induces Upregulation of CD38 and Augments the Anti-Myeloma Efficacy of Daratumumab in Pre-Clinical Models

Blood ◽

10.1182/blood.v128.22.4481.4481 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4481-4481 ◽

Cited By ~ 1

Author(s):

Estefania Garcia-Guerrero ◽

Tea Gogishvili ◽

Sophia Danhof ◽

Martin Schreder ◽

Celine Pallaud ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Line ◽

Myeloma Cell ◽

Target Cells ◽

Myeloma Cell Line ◽

Myeloma Cells ◽

Cd38 Expression ◽

Clinical Models ◽

Pre Treatment

Abstract Background: Immunotherapy with monoclonal antibodies (mAbs) has recently entered the clinical arena in multiple myeloma, including Daratumumab that targets CD38 on malignant plasma cells. The efficacy of mAbs depends on antigen density and expression of accessory ligands on target cells to initiate cell- and complement-dependent effector mechanisms. Here, we investigate the use of the histone deacetylase inhibitor (HDACi) Panobinostat to modulate target antigen expression and ligand profile on myeloma in favor of potent mAb-mediated recognition and destruction. We show that Panobinostat augments CD38 expression specifically on myeloma cells and demonstrate powerful synergy with anti-CD38 mAb Daratumumab in pre-clinical models. Methods: The myeloma cell line MM1.S and primary myeloma cells were treated with titrated doses of Panobinostat (0, 10, 25 nM) and expression of CD38 and a panel of additional target molecules including B-cell maturation antigen (BCMA) and SLAMF7, as well as accessory ligands analyzed by flow cytometry at 24, 48 and 72 hours. Antibody-dependent cellular cytotoxicity (ADCC) against Panobinostat treated and untreated myeloma cells was analyzed at 4 and 20 hours after addition of PBMC at an effector to target ratio of 25:1 in the presence of Daratumumab (1, 10, 50 ug/mL) or an isotype control antibody. Results: We first treated the myeloma cell line MM1.S with Panobinostat and analyzed its direct cytotoxic anti-myeloma effect. Consistent with previous work, the percentage of live MM1.S myeloma cells had decreased to 85% and 50% after 48 hours of exposure to 10 and 25 nM respectively. We analyzed expression of CD38 on residual live, i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 1.5 (10 nM) and 2-fold (25 nM) increase of CD38 expression by mean fluorescence intensity (MFI) compared to baseline levels and untreated control cells. The increase in CD38 expression was already detectable after 24 hours and plateaued between 48 and 72 hours. We confirmed our observation in primary myeloma cells from multiple donors (n=4) and detected an even stronger increase to 2 (10 nM) and 4-fold (25 nM) higher CD38 expression compared to untreated cells at 48 hours. Interestingly, expression of BCMA and SLAMF7 was not increased after Panobinostat treatment at all tested concentrations and time points in both MM1.S and primary myeloma. We confirmed that Panobinostat-induced upregulation of CD38 specifically occurred in myeloma, and neither observed this phenomenon in a panel of leukemia and lymphoma cell lines including Raji (Burkitt) and JeKo-1 (mantle cell), nor on resting/activated primary CD8+ and CD4+ T cells that we isolated from peripheral blood of several donors (n=3). Next, we were interested in determining whether the increase in CD38 expression enabled superior anti-myeloma activity of the anti-CD38 mAb Daratumumab. Panobinostat pre-treatment was done for 48 hours at 10 nM as this is a clinically achievable serum level with currently approved regimens. Indeed, significantly higher ADCC was mediated by Daratumumab at all tested concentrations (1, 10 and 50 ug/mL) against MM1.S that we had exposed to Panobinostat. At 4 hours, ADCC was 45% and 25% in Panobinostat-treated and untreated MM1.S respectively, and at 20 hours, near-complete, >90% ADCC of Panobinostat-pre-treated MM1.S had occurred, whereas only 65% of MM1.S were eliminated by Daratumumab without Panobinostat pre-treatment. These data were confirmed in multiple experiments with MM1.S and PBMC from different donors, and with primary myeloma cells. Experiments to evaluate synergy of Panobinostat and Daratumumab therapy in a xenograft model (NSG/MM1.S) are ongoing. Conclusions: Our data demonstrate that the HDACi Panobinostat induces upregulation of CD38 on myeloma and a subsequent dramatic increase of Daratumumab-mediated ADCC in pre-clinical models. These data suggest that Panobinostat could be used synergistically with Daratumumab in a clinical setting to increase response rates and extend duration of responses to Daratumumab. Panobinostat has a known ability to modulate the transcriptional profile of myeloma cells and our data demonstrate for the first time that this ability can be utilized to augment the therapeutic index of antibody-based immunotherapy in multiple myeloma. Disclosures Pallaud: Novartis: Employment. Lehmann:Novartis: Employment. Hudecek:Novartis: Research Funding.

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All-Trans Retinoic Acid (ATRA) up-Regulates Cell Surface CD38 Expression Which Promotes Pro-Tumoral Mitochondrial Trafficking from Stromal Cells to Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-110323 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3153-3153

Author(s):

Christopher Richard Marlein ◽

Rebecca H Horton ◽

Rachel E Piddock ◽

Jayna J Mistry ◽

Charlotte Hellmich ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Stromal Cells ◽

Plasma Cells ◽

Atra Treatment ◽

Cd38 Expression ◽

Mitochondrial Transfer ◽

Mitotracker Green

Abstract Background Multiple myeloma (MM) is malignancy highly reliant on its microenvironment. In this study, we investigated whether mitochondrial transfer occurred between bone marrow stromal cells (BMSC) and malignant plasma cells. We then used our observations as a platform to investigate the mechanisms controlling pro-tumoral mitochondrial transfer with a view to identifying druggable targets. Methods Primary MM cells were obtained from patients' bone marrow after informed consent and under approval from the United Kingdom Health Research Authority. Animal experiments were conducted under approvals from the UK Home Office and the University of East Anglia Animal Welfare and Ethics Review Board. Primary BMSC were also obtained from patient bone marrow, using adherence and characterised using flow cytometry. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining of the BMSC (in-vitro), rLV.EF1.AcGFP-Mem9 labelling of the MM plasma membrane with MitoTracker CMXRos staining of the BMSC (in-vitro) and an in vivo MM NSG xenograft model. CD38 expression on MM cells was tested after ATRA treatment, using RT-qPCR and flow cytometry. Mitochondrial transfer levels were assessed when CD38 was over expressed using ATRA or inhibited using lentivirus targeted shRNA. Results We report that mitochondria are transferred from BMSC to MM cells. First, we cultured MM cells on MitoTracker Green labelled BMSC and found increased MitoTracker Green fluorescence in the MM cells. We then transduced MM with rLV.EF1.AcGFP-Mem9 lentivirus and stained BMSC with MitoTracker CMXRos and used wide field microscopy to show MM derived tunnelling nanotubles (TNT) formed between MM cells and BMSC, with red mitochondria located within the GFP-tagged TNT. Next, we engrafted the MM cell lines MM1S and U266 into NSG mouse, after isolation we detected the presence of mouse mitochondrial DNA in the purified MM population. Together, these data show that mitochondria are transferred from BMSC to MM cells. We next analysed OXPHOS levels in MM cells grown on BMSC, using the seahorse extracellular flux assay. We found that the MM cells had increased levels of OXPHOS after culture with BMSC, which was also the case for MM cell lines analysed after isolation from NSG mice, showing the micro-environment of MM can alter the metabolism of the malignant cell. To examine whether the mitochondrial transfer process was controlled by CD38, we knocked down CD38 in MM cells using lentiviral targeted shRNA. We found reduced levels of mitochondrial transfer in CD38KD MM cells, with a consequent reduction of OXPHOS in the malignant cells. Finally, as ATRA has previously been shown to increase CD38 expression in AML, we next quantified CD38 mRNA and surface glycoprotein level on malignant plasma cells with and without ATRA treatment. We found ATRA increased CD38 expression at the mRNA and protein levels and this resulted in an increase in mitochondrial transfer from BMSC to MM cells. Conclusion Here we show that CD38 mediated mitochondrial transfer in the MM micro-environment forms part of the malignant phenotype of multiple myeloma. This finding develops our understanding of the mechanisms which underpin the efficacy of CD38 directed therapy in MM. Disclosures No relevant conflicts of interest to declare.

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DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-114206 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5618-5618 ◽

Cited By ~ 1

Author(s):

Priya Choudhry ◽

Margarette C. Mariano ◽

Arun P Wiita

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Cpg Island ◽

Ex Vivo ◽

Single Agent ◽

Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

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Preclinical Development of an Anti-CD38 Antibody-Drug Conjugate for Treatment of Hematological Malignancies

Blood ◽

10.1182/blood-2019-132062 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5621-5621 ◽

Cited By ~ 1

Author(s):

Lingna Li ◽

Wenyong Tong ◽

Megan Lau ◽

Katherine Fells ◽

Tong Zhu ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Cell Binding ◽

Antibody Drug Conjugate ◽

Employment Equity ◽

Cd38 Expression ◽

Equity Ownership ◽

Anti Tumor Activity ◽

Antibody Drug

CD38 is a validated target for the treatment of multiple myeloma (MM). Daratumumab (Darzalex®), an anti-CD38 monoclonal antibody (mAb), has shown great clinical efficacy and has been approved for multiple myeloma treatment. However, both primary refractoriness and development of resistance to daratumumab therapy have been reported. Based on the therapeutic benefits of this CD38 antibody, we developed a CD38-targeting antibody-drug conjugate (ADC), employing a fully human anti-CD38 antibody STI-6129, identified from Sorrento's G-MAB® antibody library, and proprietary linker-toxin technology. The toxin payload is duostatin 5.2 (Duo.5.2), a microtubule inhibitor, conjugated to STI-6129 via a non-polyethylene glycol linker resulting in our lead ADC CD38-077. Cell binding studies showed that it specifically binds to CD38-positive tumor cells but not CD38-negative cell lines. The cell binding was proportional to the CD38 expression level on the cell surface. The ADC was internalized into CD38-positive cells at a rate comparable to that of the unconjugated antibody, indicating that conjugation did not change the binding characteristics of STI-6129 to its antigen. In cytotoxicity studies, CD38-077 exhibited a CD38-dependent cytotoxic activity against a panel of CD38-expressing tumor cell lines and was more potent in cells with high CD38 expression. The cytotoxic effect of CD38-077 was also examined against human PBMC cells, as it has been reported that certain types of the immune cells express CD38. The result indicated that normal PBMC cells were generally insensitive to the ADC up to 1 µM following 120 hr exposure. We investigated the anti-tumor activity of CD38-077 in xenograft animal models of Burkitt's lymphoma and two different multiple myeloma (MM) cell lines. The studies evaluated different dose levels and dosing regimens, including single dose and multiple doses at various intervals. The data showed that the ADC has a broad, potent and CD38-dependent in vivo efficacy in all three xenograft tumor models examined. In a pharmacokinetic study in naïve mice, CD38-077 was found to be stable, with T1/2 of about 7-11 days, comparable to that of the unconjugated STI-6129 antibody. In summary, CD38-077 exhibits strong anti-tumor activity in vitro and in vivo. The ADC showed specific activity towards CD38-expressing tumors but was less active against CD38-expressing normal PBMC cells, which express relatively low levels of CD38 level and where internalization was not detectable. These results warrant further development exploration of CD38-077. Disclosures Li: Concortis Biotherapeutics: Employment, Equity Ownership. Lau:Levena Biopharma: Employment, Equity Ownership. Fells:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhu:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties. Sun:Levena Biopharma: Employment, Equity Ownership. Kovacs:Levena Biopharma: Employment, Equity Ownership. Khasanov:Levena Biopharma: Employment, Equity Ownership. Yan:Levena Biopharma: Employment, Equity Ownership. Deng:Levena Biopharma: Employment, Equity Ownership. Takeshita:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Li:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties; Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Concortis Biotherapeutics: Employment, Equity Ownership, Patents & Royalties.

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