Inecalcitol Enhances Daratumumab-Induced Antibody-Dependent Cell Cytotoxicity Towards Multiple Myeloma and Acute Myeloid Leukemia Cell Lines

Daratumumab (DARA), the first monoclonal antibody directed against CD38, received accelerated approval in 2015 and regular approval in 2016 by the Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma (MM) (Bhatnagar et al., 2017); its therapeutic indications were recently extended to first-line newly diagnosed MM patients in combination with standards of care following a positive clinical trial in this setting (Mateos et al., 2018). Therefore, DARA has brought extensive evidence that targeting CD38 is a highly successful strategy against MM (Frerichs et al., 2018; Plesner & Krejcik, 2018; Touzeau & Moreau, 2017). Interestingly, the pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof et al., 2016); furthermore, low level of CD38 expression has recently been identified as an adverse prognostic factor with high significance (p < 0.001) among MM patients (Arana et al., 2018). Besides MM, there are growing preclinical hints that DARA can be efficacious against acute myeloid leukemia (AML) cell lines (Fatehchand et al., 2016; Buteyn et al., 2018) or even T-cell acute lymphoblastic leukemia cells obtained from children patients (Bride et al., 2018), provided that enough CD38 molecules are present at the surface of the leukemic cells.

Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol), a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating properties and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), has been shown to stimulate CD38 expression levels in 37 out of 38 MM cell lines (ASH 2017) and in 10 out of 11 AML cell lines (ASH 2016, AACR 2018). The next question was to know if this increase in CD38 expression induced by INE resulted in an increase in DARA-induced antibody-dependent cell cytotoxicity (ADCC).

We have tested two MM (MM1.S, L-363) and seven AML (HL-60, U-937, THP-1, Molm-13, UOC-M1, OCI-AML2, Kasumi-1) cell lines representative of various levels of spontaneous CD38 cell surface density, as measured in thousands of CD38 molecules per cell by fluorescent anti-CD38 labeling (HIT2 clone) followed by quantitative flow cytometry using Cellquant calibrator beads (BioCytex). They ranged from very high CD38-positive (MM.1S, 51.0 ± 1.6 and UOC-M1, 46.5 ± 4.9) to high (OCI-AML2, 16.6 ± 1.5), low (L-363, 7.1 ± 0.1 and THP-1, 7.0 ± 0.2), very low (Molm-13, 2.1 ± 1.0 and Kasumi-1, 1.1 ± 1.2) and CD38-negative (HL-60, U-937). After exposure to 10 nM INE or vehicle for 72h, DARA-induced ADCC was assessed using either the calcein staining of target cells exposed to healthy donors peripheral blood mononuclear cells (PBMC) as effector cells, or the ADCC Reporter Bioassays Kit from Promega (G7018).

After control vehicle incubation, DARA-induced ADCC was concentration-related in the range of 0.01 to 1 µg/mL in all cell lines and achieved a maximum ranging from 7% in THP-1, although a CD38-negative cell line, to respectively 27% and 28% in MM1.S and UOC-M1, the two highest CD38-positive cell lines. However, the correlation between maximal DARA-induced ADCC and CD38 surface density did not reach statistical significance (Spearman's rank correlation r = 0.6134, p = 0.09, n = 9).

After incubation with 10 nM INE, the initially CD38-negative U-937 and HL-60 cells were found to express low (7.9 ± 0.3) and high (25.6 ± 7.3) surface CD38 levels, respectively. All the CD38-positive cell lines have shown a marked increase in the number of surface CD38 molecules: MM1.S (228 ± 12), OCI-AML2 (76,7 ± 6.5), UOC-M1 (68.8 ± 3.1), L-363 (63.9 ± 1.6), Molm-13 (38.2 ± 8.9), THP-1 (17.9 ± 0.9) and Kasumi-1 (15.7 ± 3.3) (paired Student's t-test, p = 0.02, n = 7). DARA-induced ADCC was concentration-related in the same 0.01 to 1 µg/mL range. Maximal DARA-induced ADCC was increased by 10 nM INE in all cell lines (paired Student t-test, p = 0.0025, n = 9), ranged from 20% in the lowest CD38-expressing cell line U-937 to 42% in the highest CD38-expressing cell line MM1.S, and was significantly correlated with the number of CD38 molecules/cell (Spearman's rank correlation r = 0.8333, p < 0.01, n = 9).

Altogether, these results suggest that inecalcitol could represent a therapeutic agent of choice to enhance the clinical response of MM and AML patients to anti-CD38 antibodies such as daratumumab.