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Letters to the Editor
Comparison of CD38 antibodies in vitro and ex vivo mechanisms of action in multiple myeloma
detected by pHrodo labeling of target cells after 4 hours (Online Supplementary Figure S2B). Daudi cells and MOLP-8 cells were phagocytosed by M2c macrophages by all three CD38 antibodies. However, LP-1 cells were relatively the most resistant to phagocytosis.
Apoptosis was assessed in the presence and absence of Fc receptor (FcR) crosslinking. Phosphatidylserine translocation to the cell surface was induced by the ISA analog in the absence of FcR crosslinking as measured by annexin V staining, similar to previously published reports (Figure 1A).9 Neither daratumumab nor the TAK- 079 analog could elicit annexin V staining in the absence of crosslinking. However, in the presence of crosslink- ing, all three CD38 mAb could induce annexin V staining in Daudi cells. In order to probe the level of cell death over time, we used a 5-day cytotoxicity assay to detect metabolically active cells (Figure 1B and C). All three CD38 mAb elicited comparably high levels of cell death in the presence of the FcR crosslinker and low levels in its absence. Minimal activation-induced cell death (AICD) was observed with LP-1 (Figure 1D) or MOLP-8 cells (Figure 1D).
Daratumumab has been shown to reduce CD38 expression levels partly by trogocytosis.14 In order to assess trogocytosis, we utilized Daudi cells as targets and THP-1 cells as effectors. Time-dependent loss of CD38 mAb staining on Daudi cells was correlated with a gain of signal on THP-1 cells (Online Supplementary Figure S3A). Daratumumab with a silent Fc did not medi- ate loss of CD38 on cell surfaces, indicating the effect is Fc dependent. Membrane dye was transferred in addi- tion to CD38 (Online Supplementary Figure S3B), and imaging showed comparable efficiency of target transfer from Daudi to THP-1 cells among all three CD38 mAb (Online Supplementary Figure S3C). In the absence of THP-1, there was a negligible loss of signal for all three mAb observed on Daudi cells. The lysosome-associated membrane protein 1 (CD107a), co-localized with CD38 in effector cells, suggesting that CD38 is degraded after trogocytosis (Online Supplementary Figure S3D). All three CD38 mAb showed comparable results, suggesting that all can mediate trogocytosis.
We aimed to compare cytotoxicity of the CD38 mAb ex vivo utilizing all mechanisms of action (MOA). The cumulative effect of the CD38 mAb was compared using europium-labeled LP-1 and MOLP-8 cells in the presence of whole blood from healthy donors containing both effector cells and complement. Within the assay, daratu- mumab demonstrated a significantly higher maximal cytotoxicity than comparator mAb in LP-1 cells (P<0.0001 for both comparisons) and MOLP-8 cells (P=0.0016 for both comparisons; Figure 2). Moreover, the EC50 was significantly lower for daratumumab ver- sus the TAK-079 analog in both cell lines (P<0.0001) and was lower than the ISA analog in MOLP-8 cells (P=0.0008). Similar trends were seen with a 24-hour assay using flow cytometry as a read-out.
Bone marrow samples from untreated newly diag- nosed patients, containing tumor cells and autologous immune effector cells, were obtained commercially to compare the cumulative impact of the mode of action (MOA) of CD38 mAb ex vivo. Depletion of the CD19–CD20–CD38+ CD138+ MM cells was measured by flow cytometry after 3 days in the presence of CD38 mAb and human complement (Figure 3A). Daratumumab elicited higher percent cytotoxicity of the CD38+CD138+ MM cells compared with ISA and TAK- 079 analogs (Figure 3B). CD38 was detected using HuMab, which was developed to not compete with
CD38, a transmembrane glycoprotein, is widely expressed on multiple immune cell populations.1,2 High expression of CD38 on myeloma cells makes it a target of choice for therapeutic antibodies targeting cell surface molecules in multiple myeloma (MM).2 CD38 functions as a receptor for CD31 and as an ectoenzyme catalyzing the reaction between NAD+ and NADP+ to generate cyclic ADP ribose (ADPR), NAADP, and ADPR.3
Daratumumab, a human IgG1κ monoclonal antibody (mAb) targeting CD38, eliminates MM cells through sev- eral direct mechanisms: antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxi- city (CDC), antibody-dependent cell-mediated cytotoxi- city (ADCC), and apoptosis,4-7 as well as immunomodu- latory mechanisms.2,8 Other CD38-targeting mAb in clin- ical development, isatuximab (ISA) and TAK-079, are reported to act similar to daratumumab.9,10 It remains unclear how the pleiotropic mechanisms of CD38-tar- geting mAb collectively impact tumor cytolysis and exhibit anti-tumor effects in a comprehensive ex vivo immune milieu. Here, we report results of mechanistic comparison studies of three CD38-targeting mAb: dara- tumumab and analogs of ISA and TAK-079 (generated based on the published antigen-binding fragment sequences for ISA11 and TAK-079,12 respectively).
In order to assess antibody binding, CD38-expressing Daudi and LP-1 tumor cells were coated with daratu- mumab, ISA analog, or TAK-079 analog antibodies at varying concentrations. Cells were washed and stained with Live/Dead® (Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 647–conjugated goat anti-human Fc (Jackson ImmunoResearch, West Grove, PA, USA), and binding was analyzed by flow cytometry on a fluorescence-acti- vated cell sorting (FACS) Celesta instrument (BD Biosciences, San Diego, CA, USA). CD38 expression was measured using CD38 (clone HIIT2) PerCp-Cy5.5 (BioLegend, San Diego, CA, USA). All three CD38 mAb (daratumumab, ISA analog, and TAK-079 analog) demonstrated similar relative binding to the target cells, which is in line with earlier findings of the binding prop- erties of the three mAb.5
CDC activity of the three CD38 mAb was tested on multiple cell lines with a range of CD38 surface expres- sion and CDC sensitivity levels (Online Supplementary Figure S1A).13 In Daudi, LP-1, and MOLP-8 cells, daratu- mumab resulted in higher levels of CDC activity com- pared with the other CD38 mAb, with a more pro- nounced difference seen in Daudi cells (Online Supplementary Figure S1B). In contrast, LP-1 and MOLP- 8 cells were susceptible to CDC activity with all three CD38 mAb. However, in LP-1 cells, daratumumab exhibited higher maximal cytotoxicity versus ISA and TAK-079 analogs and lower half maximal effective con- centration (EC50) versus TAK-079 (Online Supplementary Figure S1C). Similarly, in MOLP-8 cells, daratumumab exhibited higher maximal cytotoxicity versus ISA analog and lower EC50 versus TAK-079 analog.
In ADCC assays (E:T ratio, 50:1) using peripheral blood mononuclear cells as effectors, all three CD38 mAb induced similar levels of target cell death (Online Supplementary Figure S2A). Compared to Daudi cells, MOLP-8 and LP-1 cells were less susceptible to ADCC activity. In ADCP assays (E:T ratio 4:1) using monocyte- derived M2c macrophages as effectors, all three CD38 mAb induced similar levels of target cell phagocytosis as
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haematologica | 2021; 106(7)