Background
CD38 is a type II membrane protein active in receptor-mediated adhesion, calcium mobilization, formation of cyclic ADP-ribose (ADPR) from nicotinamide adenine dinucleotide (NAD
+), and hydrolysis of cADPR into ADP-ribose [
1‐
3]. CD38 also mediates activation and proliferation of lymphocytes and regulates extracellular NAD
+ levels [
4]. Over several decades, monoclonal antibodies to CD38 had been developed for use against hematological malignancies without success until the identification of daratumumab, a monoclonal anti-CD38 approved for myeloma in late 2015 [
5‐
8].
Daratumumab’s mechanisms of action include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytic cytotoxicity (ADPC) and enzymatic interference triggering apoptosis. CD38 is also found on normal human marrow and mobilized hematopoietic progenitor cells, particularly lineage committed CD34+ cells, where its expression is responsive to various cytokines [
9‐
11]. In view of the role of autologous SCT in patients with multiple myeloma [
12], we investigated CD38 expression on mobilized CD34+ cells from myeloma patients and the binding and effect of daratumumab on mobilized CD34+ cells in vitro.
Methods
Patients and cells
On an IRB approved study requiring informed consent, myeloma patients undergoing SCT (none of whom had ever been treated with daratumumab) donated mobilized blood cells for research, used fresh after collection or thawed from cryopreserved products. Patients were mobilized with G-CSF and plerixafor and cells collected by leukapheresis. Cells were used after Ficoll-Pague separation or CD34+ cell selection with MiniMACS (Miltenyi Biotec, Auburn, CA). Controls were Daudi, IM-9 and KG-1 cells from American Type Culture Collection (Manassas, VA) cultured as directed.
Antibodies and flow cytometry
Daratumumab was from Janssen Pharmaceuticals (Titusville, NJ), isotype control (human IgG1 kappa) from Sigma-Aldrich (St Louis MO), and anti-CD38-APC, anti-CD34-PerCP, anti-CD59-FITC (H19 clone) and isotype controls from BioLegend (San Diego, CA). Second antibody for daratumumab binding was mouse anti-human IgG Fc APC-conjugated (HP6017, BioLegend). The anti-C1q was a rabbit polyclonal FITC-conjugated (Abcam, Cambridge, MA) used with an appropriate isotype control. BRIC 229, a CD59 neutralizing antibody, was obtained from the International Blood Group Reference Laboratory of the Bristol Institute for Transfusion Sciences (NHS Blood and Transplant, Bristol, UK), and the anti-CD46 monoclonal GB24 was kindly provided by Dr. J. Aktinson, Washington University, St. Louis, MO, USA. Antibodies were titrated for optimal use and analyses performed on a BD Accuri flow cytometer (BD Biosciences, San Jose, CA).
CD38 quantitation and daratumumab binding assay
The phycoerythrin (PE) fluorescence quantitation kit Quantibrite™ with anti-CD38-PE (clone HB7), both from BD, were used to estimate the number of cell-surface CD38 molecules by flow cytometry. For daratumumab binding studies, we incubated the cells with 2.5 µg/mL daratumumab or human IgG1 kappa isotype control, and then stained with mouse anti-human IgG Fc or control and analyzed them by flow cytometry.
Complement-dependent cytotoxicity (CDC)
Complement-rich human serum (CRHS) was from Innovative Research (Novi, MI), was aliquoted, cryopreserved and thawed for immediate use. For CDC studies, cells were aliquoted at 4 × 10
5 per well, incubated in 10% complement-rich serum with daratumumab or isotype control at 1 µg/mL for 15 min at room temperature, then for 1 h at 37° C in 5% CO
2, and then were washed, resuspended with 5 μg/mL propridium iodide (PI, Sigma-Aldrich) and analyzed by flow cytometry [
13]. In these and other studies the doses of daratumumab used in vitro were based on the activity defined for daratumumab in assays against human myeloma cells [
14]. For C1q binding studies, we used the same steps of incubation and washing, then stained with either the FITC-conjugated rabbit polyclonal anti-human C1q or isotype control. For BRIC 229 and GB24 studies we incubated with BRIC 229 or GB24, washed and resuspended, and then CDC in response to daratumumab was analyzed as above.
Progenitor cell assays
Progenitor cell assays (PCA) (Stem Cell Technologies, Vancouver, CA; Cat #04435) were performed according to manufacturer’s instructions. Fresh or thawed unselected cells at 5 × 104/mL and CD34-selected cells at 500/mL medium were plated in triplicate. In some experimental situations we incubated prior to plating with daratumumab, BRIC229, isotype control or combinations for 1 h at 37° C in 5% CO2 and then added CRHS and incubated for another hour before plating directly into methylcellulose medium without washing the cells. Results of at least three experiments with specimens from different patients are reported. PCA were coded and counted on day 14 by a blinded investigator.
Caspase 3/7 activity
Luciferin-based caspase 3/7 activity (Promega; Madison, WI) was evaluated following manufacturer’s instructions on a Promega GloMax microplate luminometer with 5 × 103 cells per well, reported as relative luminescence units (RLU).
Statistics
Descriptive statistics, analyses and plots were performed with MedCalc Statistical Software version 17.9.2 (MedCalc Software, Ostend, Belgium;
http://www.medcalc.org; 2017) and with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA,
http://www.graphpad.com). All assays were performed at least three times unless otherwise noted.
Discussion
Antibody-mediated complement-dependent cytotoxicity (CDC) depends on target antigen levels, antibody affinity and isotype, complement membrane regulatory proteins (cMRP; CD46, CD55, CD59), and the localization of sufficient bound antibody to permit docking of C1q [
13,
16]. Therefore, after confirming that approximately 75% of CD34+ mobilized peripheral blood progenitor cells from myeloma patients en route to SCT co-expressed CD38 (Fig.
1a, b), we sought to determine the number of CD38 molecules per CD34+ cell. Although CD38 was expressed on many CD34+ mobilized cells (Fig.
1b), the number of C38 molecules per CD34+ (or IM-9) cell was significantly lower than the number per Daudi or KG-1 cell respectively (Fig.
1c).
We then asked whether daratumumab bound to these cells, whether C1q docked on daratumumab coated cells, and whether CDC using complement-rich human serum (CRHS) occurred. After incubation with daratumumab and control, daratumumab could be found on Daudi, KG-1 and CD34+ cells but not on IM-9 cells (Fig.
2a). Moreover, 55% of daratumumab-coated Daudi cells permitted C1q docking, while very few IM-9 or KG-1 cells did (Fig.
2b). CDC occurred with two-thirds of daratumumab-coated Daudi cells but with less than 5% of each of the others (Fig.
2c), likely because antigen density was not adequate for sufficient antibody binding and C1q docking. Although the differences we describe are related primarily to CD38 expression on the surface of Daudi, IM-9 and KG-1 cells, the cells we used were not synchronous prior to testing, and therefore cell-cycle related variables creating subpopulations may have contributed to the activity levels we observed.
Daratumumab then does not significantly impact the viability or colony-forming capacity of CD34+ progenitor cells from myeloma patients in vitro. We show this for cryopreserved and fresh unselected as well CD34+ cells (Fig.
5), and also show that in vitro caspase 3/7 activity is not increased in CD34+ cell suspensions with increasing doses of daratumumab in complement-rich human serum (Fig.
5d). Nevertheless, our claims are limited in this report because not all mechanisms of daratumumab activity were extensively examined and the number of observations is small. Our claims also require further study in part because additional possibilities exist. Just as daratumumab treatment can modulate CD38 and cMRP expression on myeloma cells, it may be that long-term exposure to daratumumab leads to attenuation of CD38 expression on CD34+ cells, perhaps affecting mobilization kinetics and lineage-specific progenitor cell frequencies or proliferative capacity, particularly when daratumumab is combined with other agents [
13]. Hypothetically, such effects may be linked not only to altered CD38 expression on CD34+ cells but also to changes in migration signaling and in the hematopoietic niche. Nevertheless, it is important to note that we and others recently reported that six myeloma patients induced with lenalidomide, carfilzomib and daratumumab, who proceeded to post-induction mobilization and SCT, had a median of 20 × 10
6 CD34+ cells per kg (range 6.5–38) collected with cytokine mobilization after a median of 4.5 cycles of induction (4–9), and that these 6 patients then underwent SCT with clinical outcomes and hematopoietic recoveries typical for myeloma SCT [
17].
Authors’ contributions
XM, SWW, PZ, CPC, AK, DT, MW, LL and RLC designed the protocol and the experimental work and acquired data; AKK, KS, KBM assisted with accrual; PD assisted with experimental design and edited the paper; RLC wrote the manuscript. All authors played important roles in interpreting the results and approved the final version. All authors read and approved the final manuscript.
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