Ulocuplumab (BMS-936564 / MDX1338): a fully human anti-CXCR4 antibody induces cell death in chronic lymphocytic leukemia mediated through a reactive oxygen species-dependent pathway

The CXCR4 receptor (Chemokine C-X-C motif receptor 4) is highly expressed in different hematological malignancies including chronic lymphocytic leukemia (CLL). The CXCR4 ligand (CXCL12) stimulates CXCR4 promoting cell survival and proliferation, and may contribute to the tropism of leukemia cells towards lymphoid tissues. Therefore, strategies targeting CXCR4 may constitute an effective therapeutic approach for CLL. To address that question, we studied the effect of Ulocuplumab (BMS-936564), a fully human IgG4 anti-CXCR4 antibody, using a stroma – CLL cells co-culture model. We found that Ulocuplumab (BMS-936564) inhibited CXCL12 mediated CXCR4 activation-migration of CLL cells at nanomolar concentrations. This effect was comparable to AMD3100 (Plerixafor - Mozobil), a small molecule CXCR4 inhibitor. However, Ulocuplumab (BMS-936564) but not AMD3100 induced apoptosis in CLL at nanomolar concentrations in the presence or absence of stromal cell support. This pro-apoptotic effect was independent of CLL high-risk prognostic markers, was associated with production of reactive oxygen species and did not require caspase activation. Overall, these findings are evidence that Ulocuplumab (BMS-936564) has biological activity in CLL, highlight the relevance of the CXCR4-CXCL12 pathway as a therapeutic target in CLL, and provide biological rationale for ongoing clinical trials in CLL and other hematological malignancies.


CXCR4 phenotyping in primary CLL samples and cell lines
The phenotyping of CLL cells for CXCR4 expression was carried out by flow cytometry using a 1:200 dilution of PE mouse anti-human CD184 Antibody (Catalog # 555974, BD Biosciences).
Overnight buffer-coated (1% milk/PBS) glass fiber plates (Catalog # MAFBNOB50, Millipore) were washed 3 times with 0.2 mL of binding buffer. Fifty microliters of buffer alone was added to the maximum binding wells (total binding) and control wells. 25 µL of varying concentrations of 125 I-Ulocuplumab (BMS-936564) were added to all wells. 25 µL of varying concentrations of unlabeled antibody at 100-fold excess was added to control wells to determine nonspecific binding (NSB). 25 µL of cells suspended in binding buffer was added to all wells to bring the total volume to 50 µL. The plates were incubated for 2 hours at 4°C followed by three washes with 0.2 mL cold wash buffer (24 mM Tris pH 7.2, 500 mM NaCl, 2.7 mM KCl, 2 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 0.1% BSA). Filters were removed and radioactivity was measured on a Wizard® gamma counter (PerkinElmer).

Flow Cytometry
The flow cytometric data collection and analysis was carried out using FACScalibur (BD Biosciences, San Jose, CA) and FlowJo software (version 9, TreeStar Inc, Ashland, OR).

Detection of apoptosis
Apoptotic and viable cells were discriminated by staining the cells with a 1:1000 dilution of

Inhibition of actin polymerization
The Ulocuplumab (BMS-936564) antibody was assessed for its ability to inhibit actin polymerization in primary leukemia cells from patients with CLL. Actin polymerization is a surrogate marker of cancer cell migration, metastatic potential and stromal cell dependency induced by the interaction of CXCR4 with CXCL12. 6

Inhibition of migration of cells in a transwell assay
Ulocuplumab (BMS-936564) was assessed for its ability to inhibit CXCL12 induced chemotaxis in primary leukemia cells from CLL patients using a transwell migration assay.

Detection of reactive oxygen species (ROS) by flow cytometry
CLL cells were seeded at 2.5x10 5 /ml in RPMI media and treated with 10 g/ml of Ulocuplumab (BMS-936564) for 4 hours at 37°C and 5% CO2 in 24-well plates. The generation of ROS was detected using dihydroethidium (HE) staining (Catalog # D1168, Sigma-Aldrich, St. Louis, MO). HE is a cell-permeable fluorogenic probe that reacts with ROS to form ethidium, which intercalates within double-stranded DNA in the nucleus and emits red fluorescence. Cells were re-suspended in phosphate buffered saline (PBS) containing 2.5 M of HE dye and incubated for 30 minutes in the dark at room temperature.
Cells were then washed with annexin V binding buffer (AVB) (catalog # 51-66121E, BD Bioscience) twice and stained with a 1:100 dilution of annexin V (catalog # 556421, BD Biosciences) in AVB at room temperature for 15 minutes. The samples were then analyzed by flow cytometry followed by data analysis using FlowJo software. Further, to test whether ROS specificity, we used ROS inhibitor Tiron (Catalog # 172553, Sigma-Aldrich) at 30 mM concentration. The CLL cells were incubated with BMS-36564, rituximab, GA101, and F-ara-A in absence or presence of Tiron for 6 hrs. The CLL cells were assessed for ROS production by CD19/CD5/HE staining and cell death by CD19/CD5/Annexin-V staining followed by flow cytometry analysis.