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. 2018 Jul 23;7(10):e1486948.
doi: 10.1080/2162402X.2018.1486948. eCollection 2018.

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion

Jayeeta Ghose  1 , Domenico Viola  2   3 , Cesar Terrazas  4 , Enrico Caserta  2   3 , Estelle Troadec  2   3 , Jihane Khalife  2   4 , Emine Gulsen Gunes  2   5 , James Sanchez  3 , Tinisha McDonald  6 , Guido Marcucci  2   3 , Balveen Kaur  7 , Michael Rosenzweig  3 , Jonathan Keats  8 , Steven Rosen  3 , Amrita Krishnan  3 , Abhay R Satoskar  4 , Craig C Hofmeister  9 , Flavia Pichiorri  2   3
Affiliations

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion

Jayeeta Ghose et al. Oncoimmunology. .

Abstract

Daratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.

Keywords: CD38; bone marrow stromal cells; bortezomib; daratumumab; internalization; loss of adhesion; multiple myeloma; plasma cells; sensitivity.

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Figures

Figure 1.
Figure 1.
MM-PCs express higher levels of CD38 compared to other immune subsets. Flow cytometric analysis showed CD38 expression in different immune subsets of the total cellular fraction isolated from BM aspirates obtained from 2 newly diagnosed and 3 relapsed MM patients. In (A) Representative gating strategy and flow analysis of total cellular BM fraction gated in T cells (CD3+), B cells (CD19+), monocytes (CD14+) and MM-PCs (CD138+); (B) Dot plot graph showing CD38 relative abundance, expressed as median of intensity of fluorescence, in MM-PCs, compared with that in the B cells, monocytes and T cells obtained from the same patient; different points linked by the same color line represent each patient. CD138+ MM-PCs have been shown to have significantly highest expression of CD38 compared to CD19+ B cells, CD3+ T cells (14 ± 4%) and the CD14+ monocyte fraction. The t-test (2 tailed, unpaired) was used to calculate CD38 expression in the different immune subsets among patients.
Figure 2.
Figure 2.
CD38 is internalized into CD38+ MM cells. (A) Schematic representation of surface and intracellular staining of CD38 in MM cell lines incubated with mouse anti-human CD38 Ab. Cells were stained with Anti-mouse Fc Alexa Fluor 568 for surface staining, washed, fixed, and permeabilized, and then stained with Anti-mouse Fc Alexa Fluor 633 for intracellular anti-human CD38 Ab/CD38 complex staining. (B) Flowsight cytometric analysis (using intracellular localization application wizard) showed CD38 internalization into different MM cell lines; internalization score was calculated by IDEAS Amnis software for anti-human CD38 antibody treated cells. The gating strategy to define the internalized and the surface AbCD38/CD38 populations is shown in Sup. Figure1. Internalization score was observed to be 39%, 28%, and 38% for NCI-H929, MM.1S and L363 cells, respectively, whereas internalization was only 1.5% for U266 cells, which do not express CD38. For replicates, (C) Bar diagram showing mean CD38 internalization score in MM.1S, L363, NCI-H929 cells but not in U266 cells with low CD38 expression. (D) Flowsight cytometric analysis showed CD38/Dara internalization into MM.1S cells treated with Dara. Secondary antibodies for surface and intracellular staining were FITC conjugated and TRITC conjugated anti-human IgG, respectively. Internalization score was similarly evaluated and observed to be 57% for MM.1S cells treated with Dara. Anti-HER2 humanized antibody (Trastuzumab) was used as a control and not observed to be internalized into MM.1S cells (erb2 negative). (E) Representative images of treated cells; for improved visualization, green (FITC) and red (TRITC) colors were assigned in IDEAS software to indicate surface staining and intracellular staining, respectively. Brightfield (BF), internalization (Merged) indicate single cell with internalized CD38/Dara.
Figure 3.
Figure 3.
Dara is internalized into primary CD38+ MM cells in the context of the total bone marrow microenvironment. Total cellular fraction isolated from BM of a Dara naïve MM patient was treated with 100 µg/ml of Dara or Ctrl IgG and incubated at 37°C or at 4°C for 2 hrs. Cells were then washed with PBS1X and treated with FITC conjugated anti-human IgG for detecting surface Dara/CD38 complex, and TRITC conjugated anti-human IgG was used to evaluate Dara/CD38 or non-specific IgG/CD38 complex internalization. Cells were also stained with CD138 APC to label CD138+ MM-PCs among the total cellular fraction and analyzed by Flowsight cytometric analysis.(A-B) LIVE cells gated as in Sup. Fig. S4 were evaluated for CD138 expression. CD138+ and CD138+/CD38+ cells were evaluated for CD38 internalization after incubation with Dara at 4°C (A) and at 37°C (B); (C-D) LIVE cells gated as in Sup. Fig. S4 were evaluated for CD138 expression. CD138+ cells were evaluated for CD38 internalization after incubation with Ctrl IgG at 4°C (C) and at 37°C(D); (E) Representative images of cells gated in the B panels as Intern.+ showing bright field (CH01), Dara/CD38 complex on the membrane (CH02), internalized Dara/CD38 complex (CH04) and then merged images from both channels. For improved visualization, green (FITC) and red (TRITC) colors were assigned in IDEAS software to indicate surface staining and intracellular staining, respectively.
Figure 4.
Figure 4.
Dara impairs MM cell adhesion to BMSCs. Luciferase assays showing (A) Percent increase in luciferase activity of Dara treated MM.1S GFP+/Luc+ cells in suspension at 3 and 24 hours compared to control cells treated with control human IgG (IgG) or with a clinically irrelevant antibody not targeting MM cells (Trastuzumab, Trast.). Similarly, luciferase assays showing (B) percent decrease in luciferase activity of adherent fraction of Dara treated MM.1S GFP+/Luc+ cells at 3 and 24 hours compared to control treated cells. These data indicated an increase in suspension; i.e., loss of adhesion of Dara treated MM.1S GFP+/Luc+ cells over time compared to control. (C) IF showing changes in LAMP-1 cellular localization and colocalization with CD38 in the membrane area of MM.1S cells after Dara treatment compared to control IgG. IgG and Dara treated MM.1S cells were co-stained with multi epitope anti-CD38 (green) and anti-LAMP-1 (red), and nuclei were stained with DAPI (blue); (D) Colocalization was visualized by confocal microscopy at × 40 magnification with 4X (zone1) and 6X (zone2) optical zoom. Representative co-localization signals were shown in the merged image as yellow. Scale bars, 10 μm; (E) Flow cytometric analysis showing surface expression of CD38 in MM.1S cells treated for 24 hours with Dynasore (80μM) in presence of control IgG or Dara (100μg/ml). (F) Luciferase assay showing percent decrease in luciferase activity of MM.1S GFP+/Luc+ cells adhered to HS-5 monolayer when treated for 24 hours with Dara alone compared to those treated with Dynasore+IgG or Dynasore+Dara. The t-test (2 tailed, unpaired) was used to calculate percentage of adhesion upon Dara treatment. Each experiment was performed at least in triplicate ±SD.
Figure 5.
Figure 5.
Dara sensitizes MM cells to bortezomib in vitro and in vivo. (A) Annexin-V/PI staining of CD138+ cells in a co-culture of MM.1S cells and HS-5. MM.1S cells were seeded alone or on top of HS-5 monolayer and treated successively with 100 µg Dara and 5 nM BTZ. The cells were then stained with CD138 BUV 737 to specifically label CD138 expressing MM.1S cells from HS-5 cells in the co-culture (top panel). In the lower panel [CD138+] PI staining shows percentage of MM.1S cell death to be 17% in the presence of BTZ, which was lowered to 7% in the presence of HS-5 monolayer. Similarly, (B) Propidium iodide (PI) staining of CD138+ gated MM.1S cells under similar conditions in an independent experiment shows 20% MM.1S cell death in the presence of 5 nM BTZ, which was reduced to 14% in the presence of HS-5, indicating HS-5 mediated protection of MM.1S to BTZ. However, the addition of Dara to the co-culture potentiated MM cell sensitivity to BTZ, as indicated by increased (25%) MM.1S cell death in the presence of both Dara and BTZ. (C) Bar diagram showing percentages of PI positive MM.1S cells under different conditions as indicated. (D) 20 × 106 MM.1S GFP+/Luc+ cells were subcutaneously injected into the right flank of athymic nude mice. Once tumors were palpable, mice were divided into 4 treatment groups: a) PBS-1+Control IgG (n = 3, Ctrl); b) PBS + Mouse Anti-Human CD38 antibody (n = 4, clone HB7, Ab-CD38); c) BTZ + Control IgG (n = 4); d) Ab-CD38 + BTZ (n = 4). Mice were treated three times a week (M-W-F) by subcutaneous injection (adjacent to the tumor site) with AbCD38 or control IgG (1 mg/kg). BTZ (0.8 mg/kg) or control vehicle (PBS) were intraperitoneally (IP) injected three times a week. Tumor volumes were measured three times weekly with calipers. Images of mice treated as indicated bearing subcutaneous tumor on their right flank (top panel) and images of the tumors isolated from respective mice (lower panel); (E) Graphical representation of sub-cutaneous tumor volume in xenograft mouse model of MM.1S GFP+/Luc+ in athymic nude mice in the different treatment groups. Data for the tumor growth between the different treatment groups were analyzed by 1 way analysis of variance (ANOVA).
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