A New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis And

Published OnlineFirst June 13, 2016; DOI: 10.1158/1535-7163.MCT-16-0041

Large Molecule Therapeutics Molecular Cancer Therapeutics A New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis and Mobilizes Effector Cells Matthieu Broussas1, Nicolas Boute2, Barbara Akla1, Sven Berger2, Charlotte Beau-Larvor1, Thierry Champion3, Alain Robert2, Alain Beck3, Jean-Francois¸ Haeuw4, Liliane Goetsch1, Christian Bailly5, Charles Dumontet6, Thomas Matthes7, Nathalie Corvaia8,and Christine Klinguer-Hamour3

Abstract

ThetypeIVC-X-C-motifchemokinereceptor(CXCR4)is Hz515H7 also strongly inhibits cell migration and prolifera- expressed in a large variety of human cancers, including tion and, while preserving normal blood cells, induces both hematologic malignancies, and this receptor and its ligand, antibody-dependent cellular cytotoxicity and complement- stromal cell–derived factor-1 (SDF-1), play a crucial role in dependent cytotoxicity against neoplastic cells. In mouse xeno- cancer progression. We generated a humanized immunoglob- graft models, hz515H7 displays antitumor activities with mul- ulin G1 mAb, hz515H7, which binds human CXCR4, efﬁcient- tiple hematologic tumor cell lines, with its Fc-mediated effec- ly competes for SDF-1 binding, and induces a conformational tor functions proving essential in this context. Furthermore, change in CXCR4 homodimers. Furthermore, it inhibits both hz515H7 binds to primary tumor cells from acute myeloid CXCR4 receptor–mediated G-protein activation and b-arrestin- leukemia and multiple myeloma patients. Collectively, our 2 recruitment following CXCR4 activation. The binding of the results demonstrate two major mechanisms of action, making hz515H7 antibody to CXCR4 inhibits the SDF-1–induced hz515H7 unique in this regard. Its potential as a best-in-class signaling pathway, resulting in reduced phosphorylation of molecule is currently under investigation in a phase I clinical downstream effectors, such as Akt, Erk1/2, p38, and GSK3b. trial. Mol Cancer Ther; 15(8); 1890–9. 2016 AACR.

Introduction replacement of GDP by GTP on associated G proteins (1). CXCR4 activates the Gi/o protein family, which in turn leads to the The type IV C-X-C-motif chemokine receptor (CXCR4) is a activation of phospholipase C, the PI3K pathway (2), and to the G-protein–coupled receptor (GPCR) expressed predominantly in þ regulation of gene transcription, cell migration, and cell adhesion. B and T cells, monocytes, natural killer (NK), and CD34 bone b-arrestin-1 and b-arrestin-2 have classically been assumed to shut marrow progenitor cells. The only CXCR4 ligand described to date off signal transduction following receptor activation, a process is stromal cell–derived factor-1 (SDF-1), also known as CXCL12 termed desensitization. In addition, b-arrestin-2 has been (C-X-C-motif ligand 12). SDF-1 is highly expressed in lymph reported to enhance CXCR4-mediated p38 activation and cell nodes, bone marrow, the liver and the lungs, and to a lesser extent migration following SDF-1 stimulation (3). in the kidneys, brain, and skin. In adults, the CXCR4/SDF-1 axis is Tumor tissues from at least 23 different types of human cancers involved in lymphocyte trafﬁcking and in the retention and of epithelial, mesenchymal, and hematopoietic origin express homing of hematopoietic stem cells in the bone marrow. As with CXCR4 (4). Indeed, this receptor has been found to be over- other GPCRs, the contact between SDF-1 and the CXCR4 receptor expressed in a large number of cancers, including solid tumors initiates signaling via an induced conformational change in the (4–16), leukemia (17), multiple myeloma (18), and lymphomas receptor, which is transmitted through the membrane to promote (19). The CXCR4/SDF-1 axis is involved in cancer cell proliferation, in angiogenesis, and in antiapoptotic and metastases pro- 1Department of Experimental Oncology, Centre d'Immunologie Pierre cesses (20). CXCR4 is also expressed by cancer stem cells that have Fabre (CIPF), Saint-Julien-en-Genevois, France. 2Unit of Molecular and Cellular Biology, CIPF, Saint-Julien-en-Genevois, France. 3Depart- been associated with cancer progression and treatment resistance, ment of Physico-chemistry, CIPF, Saint-Julien-en-Genevois, France. notably those that confer the resistance of residual, postche- 4Department of Biochemistry,CIPF, Saint-Julien-en-Genevois, France. motherapy acute myeloid leukemia (AML) blast to additional 5 Contract Development and Manufacturing Organization, Toulouse, chemotherapeutic agents, a major problem in the treatment France. 6INSERM 1052/CNRS 5286/University of Lyon, Lyon, France. 7Hematology Service and Service of Clinical Pathology, University of AML (21). These data constitute the rationale for cancer Hospital Geneva, Geneva, Switzerland. 8CIPF Director, Saint-Julien- treatments that target CXCR4. In the last few years, clinical trials en-Genevois, France. have been initiated on several CXCR4 antagonists, in combina- Corresponding Author: Christine Klinguer-Hamour, Centre d'Immunologie tion with anticancer drugs, in patients with hematologic malig- Pierre Fabre, 5 Avenue Napoleon III BP60497, Saint-Julien-en-Genevois nancies. In particular, plerixafor, a small-molecule CXCR4 antag- 74164, France. Phone: 334-5035-3583; Fax: 334-5035-3590; E-mail: onist registered by the FDA in combination with GCSF to enhance [email protected] the mobilization of hematopoietic stem cells to the peripheral doi: 10.1158/1535-7163.MCT-16-0041 blood for collection and subsequent autologous transplantation 2016 American Association for Cancer Research. in patients with lymphoma and multiple myeloma, is now

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undergoing phase I and phase II clinical trials for several indica- and hz515H7–IgG4 were generated by fusing the Fc region of tions, including chronic lymphocytic leukemia (CLL), small lym- mouse IgG2a and of human IgG4, to the heavy chain and light phocytic lymphoma, AML, multiple myeloma, non-Hodgkin chain variable domains of hz515H7, respectively. lymphoma (NHL), and Hodgkin disease. Similarly, phase I clinical trials are under way for the CXCR4 human immunoglobulin Flow cytometry IgG4 mAb BMS-936564, for the treatment of multiple myeloma, The specific binding of hz515H7 to human CXCR4 was AML, diffuse large B-cell leukemia, CLL, and follicular lymphoma. investigated by FACS. NIH3T3 and NIH3T3-hCXCR4–trans- Clinical trials in phase I or II are also ongoing for the BL-8040 fected cells were incubated with hz515H7 or an irrelevant hIgG1 (BKT140) peptide, as a treatment for chronic myeloid leukemia, mAb control. After incubation with a goat anti-human Alexa 488 multiple myeloma, and AML. secondary (Invitrogen) antibody and three washing steps, pro- The above-mentioned CXCR4 antagonists can all potentially pidium iodide was added to each well and viable cells were mobilize CXCR4-expressing cancer cells, facilitating their destruc- analyzed by FACS. tion by anticancer drugs. Here, we describe the generation of To determine the expression level of CXCR4 on blasts (17 hz515H7, a new humanized IgG1 mAb and CXCR4 antagonist patients for AML and 8 healthy donors, Hospices Civils de Lyon, designed to kill cancer cells via antibody- and complement- Lyon, France) and on plasmocytes (13 patients for multiple fi dependent cellular cytotoxicity (ADCC and CDC), which ef - myeloma and 10 healthy donors, Hopitaux^ Universitaires de ciently competes for SDF-1 binding and alters the conformation Geneve, Geneva, Switzerland), bone marrow samples from of CXCR4 homodimers. patients obtained after informed consent were incubated with CD45-V500 (clone HI30, BD560777), CD34-APC (clone 8G12, Materials and Methods BD345804) for blasts and CD138-PECy5 (clone BA38, Cell lines and reagents CoulterA54191), CD38-V450 (clone HB7, BD646851) for Chinese hamster ovary (CHO)-K1 and NIH3T3 cells from the plasmocytes, as per the manufacturer's recommendations, and ATCC were stably transfected to express human CXCR4. The with 5 mg/mL PE-labeled hz515H7 or a PE-labeled isotype CHO-K1 cells were cultured in DMEM-F12 medium (Cambrex) control. Two milliliters of BD (BD Biosciences) Pharm Lyse was supplemented with 5% FCS, 1% L-glutamine, and 500 mg/mL added to lyse red blood cells, followed by washing with BD Cell geneticin (all obtained from Gibco-Invitrogen) and the NIHT3 Wash and resuspended in BD Cell Wash. Samples were analyzed cells in DMEM þ GlutaMAX medium (Gibco-Invitrogen) supple- immediately on a BD FACSCanto II Flow Cytometer. Results were fl mented with 10% FCS, 1% L-glutamine, and 600 mg/mL geneticin. expressed as a CXCR4/MFI isotype mean uorescence intensity The HEK293 cells (from the ATCC) used for bioluminescence ratio. resonance energy transfer (BRET) assays were cultured in DMEM þ GlutaMAX medium and 10% FCS (Sigma). U937 [125I]SDF-1 binding assay human monocyte histiocytic lymphoma and Ramos human A radioligand binding assay was performed using membranes B lymphoblast Burkitt lymphoma tumor cell lines were obtained from CXCR4-transfected CHO-K1 cells mixed with [125I]-SDF-1 from the ATCC and KARPAS-299 anaplastic large cell lymphoma (0.1 nmol/L) and SPA-WGA beads in the presence of indicated (ALCL) human T-cell lymphoma cell line from the European concentrations of hz515H7 in 96-well plates. The plates were Collection of Cell Cultures. Cell lines were cultured according to incubated for 1 hour at room temperature, centrifuged at 1,000 the manufacturer's instructions and were authenticated using short g, and then read on a TopCount liquid scintillation counter tandem repeat (STR) DNA profiling (LGC Standards) on the dates (PerkinElmer). indicated [U937, Ramos, and KARPAS-299 (obtained, 2009; STR, 2016)]. Passages were limited to 20 for experimental procedures BRET assay of CXCR4 homodimers (<3 months) before replacing with lower passage number stocks. HEK293 cells transfected with plasmid constructs allowing the Recombinant human SDF-1 was purchased from R&D Systems, expression either of CXCR4 fused to R. reniformis luciferase 125 0 [ I]-humanSDF-1 from PerkinElmer, guanosine 5 -triphos- (CXCR4-Rluc), or of both CXCR4-Rluc and CXCR4 fused to 35 35 phate g S([ S]-GTPgS) and scintillation proximity assay (SPA) yellow fluorescent protein (CXCR4-YFP), were incubated in the wheat germ agglutinin (WGA) beads from Amersham Bios- presence of antibodies (hz515H7 or a control mAb) at 20 mg/mL ciences. The anti-CD20 chimeric mAb rituximab was obtained and 37C prior to the addition of coelenterazine H with or from Euromedex. without SDF-1 (100 nmol/L). After 5-minute incubation at 37C and 15 minutes at room temperature, light emission acquisition at Antibodies 485 and 530 nm was initiated using a Mithras LB 940 Multimode fi Anti-CXCR4 mAbs were generated by immunizing BALB/c mice Plate Reader (Berthold). BRET ratios were calculated as de ned (Charles River Laboratories) subcutaneously with NIH3T3- previously (23). For each experimental point, a mean BRET ratio hCXCR4–transfected cells. Supernatants of hybridomas were was calculated and expressed in milliBRET units (mBU). evaluated for the secretion of mAbs raised against the human CXCR4 receptor by FACS. Selected hybridomas were cloned using [35S]GTPgS binding assay a FACSVantage device. The best candidate mAb, 515H7, was The [35S]GTPgS binding assay used in this study is a radioactive humanized (yielding hz515H7) using a strategy based on CDR assay based on scintillation proximity technology (GE Health- grafting. An hz515H7 IgG1 variant with reduced Fc-mediated care). Membranes from NIH3T3-hCXCR4–transfected cells and effector functions was generated by mutating the leucines at endogenous adaptor heterotrimeric G proteins were mixed with positions 234 and 235 in the amino acid sequence to alanines unlabeled GDP, 10 nmol/L SDF-1, SPA-WGA beads, and [35S]- (22). The recombinant chimeric constructs hz515H7–mIgG2a GTPgS in the presence of indicated concentrations of hz515H7

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mAb in 96-well plates. The plates were incubated for 1 hour at Cytotoxicity assays room temperature and then read on a TopCount liquid scintil- ADCC and CDC assays on cancer cells were performed as lation counter (PerkinElmer) after 10-minute room temperature described in refs. 25 and 26, respectively. CFSE-stained Ramos centrifugation at 1,000 g. cells were spiked into blood before adding buffer, human IgG1 isotype control, hz515H7, or rituximab at 10 mg/mL and then incubated for 4 hours at 37C. The percentages of both viable b-arrestin-2 recruitment BRET assay þ þ þ (CFSE ToPro3 ) and dead (CFSE ToPro3 ) Ramos cells were The antagonist potency of hz515H7 on b-arrestin-2 recruit- measured. To evaluate the effect of hz515H7 on normal blood ment following SDF-1–induced human CXCR4 activation was cells, 500 mg/mL of hz515H7, human IgG1 isotype control, or evaluated using a BRET assay. CXCR4-Rluc and CXCR4-Rluc/ rituximab was incubated overnight at 37C with blood from b-arrestin-2-YFP transfected HEK293 cells were incubated for healthy donors. The viability of B cells, T cells, monocytes, and 15 minutes at 37C in the presence of different concentrations NK cells was tracked by FACS analysis using the appropriate of hz515H7. The cells were incubated for a further 5 minutes primary antibodies (CD19, CD3, CD14, or CD56 from Invitro- at 37C after adding coelenterazine H (5 mmol/L) and SDF-1 gen) and ToPro3. The results represent the percentage of viable (100 nmol/L). Light emission acquisitions at 485 and 530 nm (ToPro3 ) cells. were then performed using a Mithras LB 940 reader.

Binding of mAbs to the mouse Fc gamma receptor IV Western blot analysis Real-time surface plasmon resonance interaction assays were Ramos cells were incubated overnight in serum-free carried out using a Biacore X device and reagents supplied by RPMI1640 medium with 1% L-glutamine, then incubated or Biacore (GE Healthcare). To study the binding of mAbs on the not with hIgG1 or hz515H7 at 10 mg/mL and stimulated or not mouse FcgRIV extracellular domain (ECD; Sino Biological Inc.), with SDF-1 (50 ng/mL). After centrifugation, lysis buffer was 1,033.5 RU of the m-FcgRIV ECD was chemically grafted on the addedtothecellsfor90minutesat4C. After immunopre- FC2 of a CM4 sensor chip (GE Healthcare) using the Amine cipitation, the immunocomplexes were suspended in sample Coupling Kit. The first flow cell (FC1) served as the reference buffer, loaded on a 4% to 12% SDS-PAGE, and transferred to surface to subtract the nonspecific interactions. Kinetic experi- nitrocellulose blot membranes. Each blot was saturated, incu- ments were carried out at 25Caflow rate of 30 mL per minute. An bated with appropriate (Cell Signaling Technology) primary injection of HBS-EP running buffer analyte was used as a double antibodies for 4 hours at room temperature, and incubated reference. The binding of mAbs to mouse FcgRIV was evaluated with horseradish peroxidase (HRP)-linked secondary antibo- using BIAevaluation 3.1 (GE Healthcare). dies (GE Healthcare). The blots were subsequently assayed using the ECL detection system (Amersham Biosciences). The Mouse models membranes were then stripped, saturated and incubated with Ten million Ramos cells were engrafted subcutaneously to anti-GAPDH antibodies for 1 hour at room temperature, and SCID mice (Charles River Laboratories). After 8 days, the mice finally washed and incubated with HRP-linked secondary anti- were randomized into groups with mean tumor sizes of 100 mm3. bodies. Optical density maps were produced using imageJ 1.46 The mice were treated weekly by intraperitoneal injections with (24) and results expressed as an optical density ratio after appropriate doses (0.15–20 mg/kg) of hz515H7 with a loading normalization with GAPDH. dose. Five million KARPAS-299 cells were engrafted subcutaneously to NOD/SCID mice (Charles River Laboratories). Six days Cell migration assays after implantation, the mice were randomized into two groups High-throughput screening 96-well 8.0-mmTranswellplates with mean tumor sizes of 130 mm3. The mice were treated by (Corning Life Sciences) were used to perform chemotaxis intraperitoneal injections of hz515H7 at 40 mg/kg (loading dose) assays. U937 cells were suspended in RPMI medium (BioWhit- and then weekly at 20 mg/kg. In both models, a histidine buffer taker) and incubated for 40 minutes at 37CwithPBS,hIgG1 group was introduced as control. Tumor volumes were measured irrelevant antibody or hz515H7 (10 mg/mL). Then, 75 mLof twice a week with an electronic caliper and calculated with the cell suspension was added to the inserts. SDF-1 was used at formula: p/6 length width height. 800 ng/mL in a 235-mL total volume of RPMI þ 2% FCS in the Ten million U937 cells were engrafted intraperitoneally on bottom wells. Transwell migration was conducted for 4 hours 7-week-old NOD/SCID or NOD/SCID g (NSG) female mice. at 37C. The cells that had migrated into the bottom wells The antibodies were administered subcutaneously, starting two were quantified using ATP measurements, expressed as relative days after cell engraftment, with a loading dose of 20 mg/kg, luminescence unit. followed by weekly injections of a 10 mg/kg dose. The efficacy of the treatment was measured by monitoring the mouse survival rate daily and by comparing the treated groups with Proliferation assay the control-untreated group (injected with histidine buffer). Ramos cells were incubated in RPMI1640 medium with 1% L-glutamine and 10% FBS, with an IgG1 isotype control or hz515H7 at 10 mg/mL for 24 hours at 37Cwithorwithouta Results cross-linking antibody (a mouse anti-human IgG1 Fc antibody hz515H7 recognizes human CXCR4, antagonizes obtained from LSBio) added at 6-fold excess. The cells were SDF-1 binding, and induces a conformational change in then suspended in culture medium and incubated for 3 days at CXCR4 homodimers 37C. Cell viability was evaluated using ATP measurements The binding specificity of hz515H7 on both NIH3T3 wild-type (CellTiter-Glo, Promega). cells and NIH3T3-hCXCR4–transfected cells was evaluated by

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Figure 1. Hz515H7 recognizes human CXCR4, antagonizes SDF-1 binding, and induces a conformational change in CXCR4 homodimers. A, FACS analysis of viable NIH3T3 (NIH) and NIH3T3- hCXCR4–transfected cells incubated with hz515H7 or an irrelevant hIgG1 mAb. Ab, antibody; MFI, mean ﬂuorescence intensity; WT, wild-type. B, membranes from CXCR4- transfected CHO-K1 cells in the presence of hz515H7 using SDF-1 as a competitor ligand. The data points represent the means of three independent experiments, two sets of which were performed for each point. C, BRET analysis (% of the basal BRET signal) of HEK293 cells expressing CXCR4-Rluc or both CXCR4-Rluc and CXCR4 fused to YFP, incubated in the presence of antibodies (hz515H7 or a control mAb) with or without SDF-1 (100 nmol/L). Ctrl, control. The data shown are means of at least ﬁve repeat measurements, and the bars represent the associated SEs.

FACS analysis. Neither hz515H7 nor hIgG1 bound wild-type observed on the surface of NIH3T3-hCXCR4–transfected cells NIH3T3 parent cells (see insert in Fig. 1A). In contrast, concen- (Fig. 1A). The Kd value of the mAb measured on NIH3T3- tration-dependent binding of hz515H7 (but not of hIgG1) was hCXCR4–transfected cells was 0.290 0.009 nmol/L.

Table 1. CXCR4 expression on blasts (AML patients) and on plasmocytes (MM patients) AML patient blast cells MM patient plasma cells Patient MFI MFI MFI Patient MFI MFI MFI number isotype CXCR4 ratioa number isotype CXCR4 ratiob 1 61 4,334 71 149722 2 106 360 3 2 47 78 2 3 108 334 3 3 32 32 1 4 119 478 4 4 3 33 11 5 778 1,559 2 5 3 64 20 69781786177 7 83 3,815 46 722915 8 86 3,291 38 8232 9 123 2,410 20 96498 10 196 4,407 22 10 8 44 6 11 74 927 13 11 2 16 8 12 126 1,900 15 12 2 26 13 13 48 435 9 13 3 39 13 14 93 239 3 15 65 1,094 17 16 116 1,428 12 17 165 4,419 27 NOTE: Positive cases of the subjects tested are in bold. Abbreviations: MFI, mean ﬂuorescence intensity; MM, multiple myeloma. aMFI ratio is 10 6 for blast cells from healthy donors. bMFI ratio is 2.6 0.9 for CD34þ cells from healthy donors.

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Furthermore, to confirm CXCR4 specificity, hz515H7-1 was eval- change. In contrast, the binding of hz515H7 (20 mg/mL) to uated for its ability to recognize human cell lines by FACS analysis. CXCR4 homodimers led to a decrease in the BRET signal both This profile of cell recognition was compared with the one in the absence and presence of SDF-1. These results suggest that obtained by qPCR-quantifying mRNA encoding for hCXCR4. the binding of hz515H7 to a CXCR4 homodimer induces a hz515H7-1 was able to recognize cells for which mRNA encoding conformational change in the receptor that prevents any further for hCXCR4 was quantified but not cells for which mRNA encod- changes induced by SDF-1. ing for hCXCR4 was not detected (data not shown). To evaluate the ability of hz515H7 to compete with SDF-1 hz515H7 recognizes CXCR4 on primary blasts and plasmocytes for CXCR4 binding, saturation-binding experiments were con- from AML and multiple myeloma patients 125 ducted using radio-labeled [ I]-SDF-1 and membranes of To evaluate CXCR4 expression on primary leukemic blasts CHO-K1 cells stably expressing human CXCR4. Hz515H7 and malignant plasmocytes, we stained CXCR4 in fresh bone 125 inhibited [ I]SDF-1 binding concentration dependently with marrow samples from AML and multiple myeloma patients an IC50 of approximately 1.5 nmol/L (Fig. 1B). As reported with PE-labeled hz515H7 or PE-isotype control (18 healthy previously (27, 28), we used a BRET assay to monitor for donors). For 6 of 17 (35%) samples from AML patients and ligand-induced conformational changes in CXCR4 homodi- for 9 of 13 (69%) from multiple myeloma patients, the mers (Fig. 1C). The addition of SDF-1 (100 nmol/L) induced surface expression levels of CXCR4 of blast cells (CD45Dim, þ þ þ an increase in the BRET signal, reflectingaconformational CD34 ) and of plasmocytes (CD38 ,CD138)werehigher

Figure 2. Hz515H7 inhibits the G-protein–dependent and b-arrestin-2 signaling pathways induced by SDF-1. A, inhibition of SDF-1–induced [35S]GTPgS binding to membranes of NIH3T3-hCXCR4–transfected cells as a function of hz515H7 concentration. Each data point is the mean of three experiments, and measurements at each concentration were performed in duplicate. B, BRET analysis, following the addition of coelenterazine H (5 mmol/L), of CXCR4-Rluc and CXCR4-Rluc/b-arrestin-2- YFP–expressing HEK293 cells incubated in the presence of hz515H7 with or without SDF-1 (100 nmol/L). The data are means of seven experimental values, and the bars represent the associated SEs. C and D, immunoblot analysis of Ramos cells incubated or not with hIgG1 or hz515H7 at 10 mg/mL and stimulated or not with SDF-1 (50 ng/mL) for the time periods indicated in C. D, optical density (OD) ratios, using GAPDH and the corresponding protein to normalize, obtained from OD maps produced using imageJ software. ctl, control.

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þ than those of normal CD34 cells in healthy bone marrow stimulation with an IC50 of 3 nmol/L. Furthermore, Fig. 2C samples (Table 1). clearly shows that within 5 minutes, hz515H7 dramatically reduces the SDF-1–induced phosphorylation of Akt, Erk1/2, hz515H7 inhibits both G-protein–dependent and b-arrestin-2– p38, and GSK3b. This inhibition is maintained for at least 60 dependent SDF-1–mediated signaling pathways minutes (Fig. 2D). Altogether, our results indicate that – Following SDF-1 binding, the conformation adopted by hz515H7 inhibits both G-protein- and b-arrestin-2 dependent – CXCR4 leads to the activation of heterotrimeric G proteins SDF-1 mediated intracellular signaling pathways. Finally, Fig. – associated with an exchange between bound GDP and GTP 3 demonstrates that hz515H7 inhibits SDF-1 induced U937 from the Ga subunit (29). In a ﬁrst assay, the incorporation of cell migration and proliferation in the presence of a cross- radio-labeled [35S]GTPgSwasmonitoredasaproxyforthe linking antibody by 80.0 12.0% (Fig. 3A) and approximately activation level of CXCR4. The incubation of hz515H7 with 50% (Fig. 3B), respectively. SDF-1 (10 nmol/L) activated membranes of NIH3T3-CXCR4 inhibited [35S]GTPgS binding by up to 89% in a concentration- hz515H7 induces both ADCC and CDC of tumor cells but has dependent manner (IC50 3.5 nmol/L; Fig. 2A). no impact on normal blood cells in vitro The binding of SDF-1 to CXCR4 induces its internalization ADCC and CDC are important mechanisms in mAb cancer through the recruitment of b-arrestin and clathrin-mediated therapy (31). The ability of hz515H7 to trigger ADCC was endocytosis (30). The BRET assay in Fig. 2B demonstrates that assessed on Ramos cells using puriﬁed NK cells from healthy hz515H7 inhibits b-arrestin-2 recruitment in response to SDF-1 donors. As shown in Fig. 3C, 10 mg/mL of the hz515H7 IgG1 mAb

Figure 3. Hz515H7 inhibits U937 cancer cell migration and Ramos cell proliferation. A, U937 cell migration after incubation in PBS, 10 mg/mL hIgG1 isotype control, or hz515H7 in the presence or absence of SDF-1 (800 ng/mL). RLU, relative luminescence unit. The data shown are the mean and SDs of three independent measurements, each performed in triplicate. B, percentage inhibition of Ramos cell proliferation after incubation for 72 hours at 37 C either in medium (RPMI1640 with 1% L-glutamine and 10% FBS) by itself or with an IgG1 isotype control or hz515H7 at 10 mg/mL, with or without cross-linking antibodies (viz. anti-human IgG1 Fc antibodies). The data shown are the mean of two independent experiments, each performed in duplicate, and the associated SEs. C–F, Hz515H7-induced ADCC and CDC in tumor cells. C, mean cytotoxicity percentage (SDs) of Ramos cells preincubated with 10 mg/mL hIgG1 isotype control (Ctl), hz515H7 IgG1, or hz515H7 IgG4. Puriﬁed NK cells from healthy donors were used as effector cells. D, FACS analysis of CXCR4 expression in Ramos cells preincubated with increasing concentrations of hz515H7 IgG1 before human sera were added. E, mean cytotoxicity percentage (SDs) of Ramos cells preincubated with 10 mg/mL hIgG1 isotype control or hz515H7 IgG1 in heated or nonheated human or baby rabbit sera (10% of the ﬁnal concentration) to evaluate CDC. F, mean cytotoxicity percentage (SDs) of Ramos cells preincubated with buffer, 10 mg/mL hIgG1 isotype control, hz515H7 IgG1, or hz515H7 IgG4 before adding human serum.

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Figure 4. Hz515H7 does not affect normal blood cells in vitro. A and B, viability (as measured by FACS; A) of CFSE-stained Ramos cells spiked into blood before adding buffer, a human IgG1 isotype control (Ctl) mAb, hz515H7, or rituximab (RTX) at 10 mg/mL and then incubated for 4 hours at 37C; and of B cells, T cells, monocytes, and NK cells (B) in blood from healthy donors (n ¼ 6) incubated overnight at 37C in the presence of a human IgG1 isotype control mAb, hz515H7, or rituximab at 500 mg/mL.

induced 50 10% cell cytotoxicity, whereas the IgG1 control respectively). The absence of cell lysis in the presence of heated mAb and the hz515H7 IgG4 mAb, which binds tumor cells in a serum (Fig. 3E) conﬁrms the involvement of the complement in manner similar to that of the corresponding IgG1 antibody, did the CDC assay. As expected, the hz515H7 IgG4 mAb did not not lead to ADCC. trigger any CDC on Ramos cells (Fig. 3F). Next, we investigated CDC at different culture times in Ramos Regarding the elimination of tumor cells in blood, Fig. 4A cells expressing different levels of CXCR4. Hz515H7 induced shows that more than 90% of the Ramos cells expressing both concentration-dependent cell lysis (Fig. 3D). Interestingly, there CXCR4 and CD20 are killed in the presence of either hz515H7 or is a strong correlation between the concentration response curves rituximab, but not in the presence of a control isotype. In of CDC and of hz515H7 binding to Ramos cells. The EC50s for addition, Fig. 4B reveals that whereas rituximab (a positive both assays are similar (1.4 10 10 and 1.6 10 10 mol/L, control) causes B-cell depletion, hz515H7 has no major impact

Figure 5. Efﬁcacy of hz515H7 in xenograft mice tumor models. A and B, evolution of tumor volumes in 7-week-old female SCID mice (A) engrafted subcutaneously with 107 Ramos cells and treated intraperitoneally (n ¼ 6) with appropriate doses of hz515H7 or a histidine buffer after 8 days and then once a week for 5 weeks; and 7-week-old NOD/SCID mice (B) engrafted subcutaneously with 5 106 KARPAS-299 cells and treated intraperitoneally (n ¼ 6) with a histidine buffer or hz515H7 at 40 mg/kg after 6 days and then once a week at 20 mg/kg. A Mann–Whitney statistical test was performed after each measurement. The data from one experiment representative of at least two are shown.

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A New Anti-CXCR4 Antibody

on normal B cells, T cells, monocytes, or NK cells. These results were compared on one hand with chimeric mouse/human indicate that in vitro, hz515H7 triggers Ramos cancer cell lysis in mIgG2a hz515H7, designed to have optimal effector functions whole blood while preserving normal cells. in mice, and on the other with an hz515H7 IgG1 Ala/Ala mutant designed to have reduced effector function (32). Figure 6A clearly Efficacy of hz515H7 in xenograft mouse tumor models and shows that mIgG2a hz515H7 and hz515H7 IgG1 bind very the role of effector functions in the activity of this mAb efficiently to mouse FcgRIV, whereas the Ala/Ala mutant does The activity of hz515H7 against tumor growth was evalu- not. The survival rates of mice treated with the two former atedinRamoshumanBBurkittlymphoma and KARPAS-299 constructs are much higher than those of mice treated with the ALCL xenograft models. In the Ramos model (Fig. 5A), Ala/Ala mutant or in the control group (Fig. 6B). Furthermore, as hz515H7 inhibited tumor growth in a dose-dependent man- displayed in Fig. 6C, hz515H7 has virtually no antitumor activity ner, by as much as 98% at 20 mg/kg at day 23. Tumor growth in NSG mice lacking the IL2 receptor g chain. These results was also significantly inhibited (by as much as 68%) in the therefore demonstrate that the effector functions of hz515H7 are KARPAS-299 model (Fig. 5B). It has to be noted that as essential for its antitumor activity. hz515H7 does not recognize the murine CXCR4, it does not allow in these mouse models the measurement of the activity of mAb on endothelial cells in the process of angiogenesis and Discussion the potential toxicity on the different normal tissues expres- Despite huge progress in the treatment of cancer patients, sing CXCR4. residual disease still leads to cancer relapse and mortality. CXCR4 Finally, the importance of effector functions in hz515H7's is known to contribute to microenvironment-mediated chemore- mechanism of action was investigated with a U937 AML xenograft sistance, an important barrier to the eradication of residual disease tumor model in NOD/SCID mice. The effects of hz515H7 IgG1 that has yet to be overcome, notably when bone marrow niches

Figure 6. The role of effector functions in the in vivo activity of hz515H7. A, binding, as a function of time, of chimeric mouse/human mIgG2a hz515H7, hz515H7 hIgG1, and hz515H7 hIgG1 Ala/Ala-mutant mAbs to the mouse FcgRIV. Resp. Diff. RU, response differential resonance units. B and C, survival rates of 7-week-old NOD/SCID (B) and NSG female mice (C) engrafted intraperitoneally with 107 U937 cells and injected subcutaneously with histidine buffer, mIgG2a hz515H7, hz515H7 hIgG1, or hz515H7 hIgG1 Ala/Ala mutant at 20 mg/kg (loading dose), then weekly at 10 mg/kg. At the end of the experiment, the data were analyzed using a survival log-rank test. The data from one experiment representative of two are shown.

www.aacrjournals.org Mol Cancer Ther; 15(8) August 2016 1897

Broussas et al.

offer protection to cancer cells (33, 34).The value of targeting that CXCR4 is overexpressed in cancer cells, in respectively 35% CXCR4 with IgG4 mAbs (35), peptides (36), and small molecules and approximately 69% of samples from AML and multiple (37) has already been demonstrated in pharmacologic models. myeloma patients, with expression levels of CXCR4 on blast cells þ Indeed, these agents mobilize cancer cells, thereby facilitating and of plasmocytes higher than those of CD34 cells in healthy their elimination by chemotherapeutic agents. These CXCR4 bone marrow samples, suggesting that hz515H7 should prefer- antagonists are now under investigation in clinical trials. None- entially kill the neoplastic cells while preserving normal hemato- theless, it may be possible to further increase CXCR4 antagonist poietic cells. Indeed, our in vitro results show that hz515H7 does efficacy. For example, Kularatne and colleagues (38) recently not decrease the viability of B cells, monocytes, T cells, or NK cells, described an antibody–drug conjugate strategy to selectively whereas rituximab, used as a positive control for ADCC (44) and þ deliver the cytotoxic agent to cancer cells using a CXCR4 mAb CDC (40), leads to CD20 B-cell depletion as described previ- coupled to monomethyl auristatin F, which allowed the selective ously (44, 45). þ elimination of CXCR4 metastatic cancer cells both in vitro and The two major mechanisms of action demonstrated here for in vivo. Importantly, this approach was also shown to spare hz515H7, namely interference with the CXCR4/SDF-1 axis and þ CXCR4 hematopoietic cells in vivo. The new humanized IgG1 the triggering of effector functions (ADCC and CDC), make it mAb reported here, hz515H7, binds the human chemokine unique among mAbs. A phase I clinical trial is under way on this receptor CXCR4, competes for SDF-1 binding, and induces a potentially best-in-class molecule for the treatment of hemato- conformational change in CXCR4 homodimers. It inhibits both logic malignancies. Furthermore, as CXCR4 is expressed in solid CXCR4 receptor–mediated G-protein activation and b-arrestin-2 tumors, hz515H7 is also being investigated for its antitumor recruitment following CXCR4 activation. The binding of activity in solid xenograft models. hz515H7 to CXCR4 inhibits SDF-1–induced signaling pathways and the migration and proliferation of cancer cells. These prop- Disclosure of Potential Conflicts of Interest erties are similar to those of other CXCR4/SDF1 antagonists C. Dumontet reports receiving other commercial research support from currently under clinical trial. In addition, however, this study Pierre Fabre. T. Matthes is a consultant/advisory board member for Phi Pharma fl demonstrates that hz515H7 induces cell lysis in cancer cells in SA. No potential con icts were disclosed by the other authors. vitro, via ADCC and CDC. Moreover, our findings for multiple hematologic tumor xenograft models suggest that these in vitro Authors' Contributions properties translate into a strong antitumor activity in vivo. We also Conception and design: M. Broussas, N. Boute, B. Akla, C. Beau-Larvor, T. Champion, A. Robert, L. Goetsch, C. Dumontet, N. Corvaia, C. Klinguer- evaluated the in vivo antitumor activity of three different isotypes Hamour of hz515H7: the IgG1 wild-type, an Ala/Ala IgG1 mutant with Development of methodology: M. Broussas, S. Berger, A. Robert, T. Matthes, reduced effector functions, and a chimeric mouse/human con- N. Corvaia struct, mIgG2a hz515H7, designed to have optimal effector Acquisition of data (provided animals, acquired and managed patients, functions in mice. The dramatic improvement in survival rates provided facilities, etc.): N. Boute, B. Akla, S. Berger, C. Beau-Larvor, observed here in a U937 survival model for mice treated T. Champion, A. Robert, C. Dumontet, T. Matthes Analysis and interpretation of data (e.g., statistical analysis, biostatistics, with hz515H7 IgG1 and mIgG2a hz515H7 demonstrates the computational analysis): M. Broussas, N. Boute, B. Akla, C. Beau-Larvor, importance of effector functions for the antitumor activity of T. Champion, A. Robert, J.-F. Haeuw, L. Goetsch, C. Dumontet, T. Matthes, hz515H7, the Ala/Ala mutant losing most of its in vivo activity C. Klinguer-Hamour and hz515H7 also proving significantly less efficient in NSG Writing, review, and/or revision of the manuscript: M. Broussas, N. Boute, mice lacking NK cell activity. A. Robert, A. Beck, C. Bailly, C. Dumontet, T. Matthes, N. Corvaia, C. Klinguer- Several other studies have demonstrated the importance of Hamour Administrative, technical, or material support (i.e., reporting or organizing CDC and ADCC for the therapeutic efficacy of mAbs, particularly data, constructing databases): A. Robert for rituximab, a CD20 mAb largely used in the treatment of NHL Study supervision: A. Beck, C. Bailly, C. Dumontet, N. Corvaia and CLL (39, 40). Daratumumab, a CD38 mAb, kills multiple myeloma tumor cells by inducing CDC and ADCC (41) and is a Acknowledgments therapeutic mAb with high potential for the treatment of multiple The authors thank C. Tardy, A. Blaecke, M. Fournier, S Bernois, C. Bertaux, myeloma patients. Finally, elotuzumab, a CS1 mAb recently K. Leveque, V. Bonnet, L. Revy, C. Daviet, L. Troncy, P. Aspinion, M. Excoffier, approved by the FDA for the treatment of multiple myeloma and M.C. Janin-Bussat for their technical assistance and C. Catry and A. Dorison patients, has been shown to inhibit the binding of multiple for their clerical support. The costs of publication of this article were defrayed in part by the payment of myeloma cells to bone marrow stromal cells and to induce ADCC page charges. This article must therefore be hereby marked advertisement in in multiple myeloma cells (42). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Our results also show that the CDC induced by hz515H7 is correlated with the expression of CXCR4 on the cell membrane of Received January 26, 2016; revised May 24, 2016; accepted May 29, 2016; Ramos cells (Fig. 3). Similar to previous reports (18, 43), we found published OnlineFirst June 13, 2016.

References 1. Li JH, Hamdan FF, Kim SK, Jacobson KA, Zhang X, Han SJ, et al. Ligand- 3. Sun Y, Cheng Z, Ma L, Pei G. b-arrestin2 is critically involved specific changes in M3 muscarinic acetylcholine receptor structure in CXCR4-mediated chemotaxis, and this is mediated by its detected by a disulfide scanning strategy. Biochemistry 2008;47: enhancement of p38 MAPK activation. J Biol Chem 2002; 2776–88. 277:49212–9. 2. Busillo JM, Benovic JL. Regulation of CXCR4 signaling. Biochim Biophys 4. Balkwill F. The significance of cancer cell expression of the chemokine Acta 2007;1768:952–63. receptor CXCR4. Semin Cancer Biol 2004;14:171–9.

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A New Anti-CXCR4 Antibody

5. Ottaiano A, Franco R, Aiello Talamanca A, Liguori G, Tatangelo F, Delrio P, 25. Broussas M, Broyer L, Goetsch L. Evaluation of antibody-dependent cell et al. Overexpression of both CXC chemokine receptor 4 and vascular cytotoxicity using lactate dehydrogenase (LDH) measurement. Methods endothelial growth factor proteins predicts early distant relapse in stage Mol Biol 2013;988:305–17. II-III colorectal cancer patients. Clin Cancer Res 2006;12:2795–803. 26. Broyer L, Goetsch L, Broussas M. Evaluation of complement-dependent 6. Kato M, Kitayama J, Kazama S, Nagawa H. Expression pattern of CXC cytotoxicity using ATP measurement and C1q/C4b binding. Methods Mol chemokine receptor-4 is correlated with lymph node metastasis in human Biol 2013;988:319–29. invasive ductal carcinoma. Breast Cancer Res 2003;5:144–50. 27. Toth PT, Ren D, Miller RJ. Regulation of CXCR4 receptor dimerization by 7. Sun YX, Wang J, Shelburne CE, Lopatin DE, Chinnaiyan AM, Rubin MA, the chemokine SDF-1alpha and the HIV-1 coat protein gp120: a fluores- et al. Expression of CXCR4 and CXCL12 (SDF-1) in human prostate cancers cence resonance energy transfer (FRET) study. J Pharmacol Exp Ther (PCa) in vivo. J Cell Biochem 2003;89:462–73. 2004;310:8–17. 8. Phillips RJ, Burdick MD, Lutz M, Belperio JA, Keane MP, Strieter RM. The 28. Percherancier Y, Berchiche YA, Slight I, Volkmer-Engert R, Tamamura H, stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological Fujii N, et al. Bioluminescence resonance energy transfer reveals ligand- axis in non-small cell lung cancer metastases. Am J Respir Crit Care Med induced conformational changes in CXCR4 homo- and heterodimers. 2003;167:1676–86. J Biol Chem 2005;280:9895–903. 9. Scotton CJ, Wilson JL, Scott K, Stamp G, Wilbanks GD, Fricker S, et al. 29. Radhika V, Dhanasekaran N. Transforming G proteins. Oncogene Multiple actions of the chemokine CXCL12 on epithelial tumor cells in 2001;20:1607–14. human ovarian cancer. Cancer Res 2002;62:5930–8. 30. McCormick PJ, Segarra M, Gasperini P, Gulino AV, Tosato G. Impaired 10. Koshiba T, Hosotani R, Miyamoto Y, Ida J, Tsuji S, Nakajima S, et al. recruitment of Grk6 and beta-arrestin 2 causes delayed internalization and Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor desensitization of a WHIM syndrome-associated CXCR4 mutant receptor. system in pancreatic cancer: a possible role for tumor progression. Clin PLoS One 2009;4:e8102. Cancer Res 2000;6:3530–5. 31. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer 11. Wang L, Wang L, Yang B, Yang Q, Qiao S, Wang Y, et al. Strong expression of 2012;12:278–87. chemokine receptor CXCR4 by renal cell carcinoma cells correlates with 32. Xu D, Alegre ML, Varga SS, Rothermel AL, Collins AM, Pulito VL, et al. In metastasis. Clin Exp Metastasis 2009;26:1049–54. vitro characterization of five humanized OKT3 effector function variant 12. Barbero S, Bajetto A, Bonavia R, Porcile C, Piccioli P, Pirani P, et al. antibodies. Cell Immunol 2000;200:16–26. Expression of chemokine receptor CXCR4 and its ligand stromal cell- 33. Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between tumor derived factor-1 in human brain tumors and their involvement in glial cells and their microenvironment. Blood 2006;107:1761–7. proliferation in vitro. Ann N Y Acad Sci 2002;973:60–9. 34. Zeng Z, Shi YX, Samudio IJ, Wang RY, Ling X, Frolova O, et al. Targeting the 13. Bian XW, Yang SX, Chen JH, Ping YF, Zhou XD, Wang QL, et al. Preferential leukemia microenvironment by CXCR4 inhibition overcomes resistance to expression of chemokine receptor CXCR4 by highly malignant human kinase inhibitors and chemotherapy in AML. Blood 2009;113:6215–24. gliomas and its association with poor patient survival. Neurosurgery 35. Kuhne RM, Mulvey T, Belanger B, Chen S, Pan C, Chong C, et al. BMS- 2007;61:570–9. 936564/MDX-1338: a fully human anti-CXCR4 antibody induces apopto- 14. De Falco V, Guarino V, Avilla E, Castellone MD, Salerno P, Salvatore G, sis in vitro and shows antitumor activity in vivo in hematologic malignan- et al. Biological role and potential therapeutic targeting of the chemokine cies. Clin Cancer Res 2013;19:357–66. receptor CXCR4 in undifferentiated thyroid cancer. Cancer Res 36. Beider K, Begin M, Abraham M, Wald H, Weiss ID, Wald O, et al. CXCR4 2007;67:11821–9. antagonist 4F-benzoyl-TN14003 inhibits leukemia and multiple myeloma 15. Scala S, Giuliano P, Ascierto PA, Ierano C, Franco R, Napolitano M, et al. tumor growth. Exp Hematol 2011;39:282–92. Human melanoma metastases express functional CXCR4. Clin Cancer Res 37. Azab AK, Runnels JM, Pitsillides C, Moreau AS, Azab F, Leleu X, et al. 2006;12:2427–33. CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma 16. Meng X, Wuyi L, Yuhong X, Xinming C. Expression of CXCR4 in oral cells with the bone marrow microenvironment and enhances their sensi- squamous cell carcinoma: correlations with clinicopathology and pivotal tivity to therapy. Blood 2009;113:4341–51. role of proliferation. J Oral Pathol Med 2010;39:63–8. 38. Kularatne SA, Deshmukh V, Ma J, Tardif V, Lim RKV, Pugh HM, et al. 17. Burger JA, Burkle€ A. The CXCR4 chemokine receptor in acute and chronic A CXCR4-targeted site-specific antibody-drug conjugate. Angew Chem Int leukaemia: a marrow homing receptor and potential therapeutic target. Br J Ed Engl 2014;53:11863–7. Haematol 2007;137:288–96. 39. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors 18. Trentin L, Miorin M, Facco M, Baesso I, Carraro S, Cabrelle A, et al. Multiple modulate in vivo cytoxicity against tumor targets. Nat Med 2000;6:443–6. myeloma plasma cells show different chemokine receptor profiles at sites 40. Di Gaetano N, Cittera E, Nota R, Vecchi A, Grieco V, Scanziani E, et al. of disease activity. Br J Haematol 2007;138:594–602. Complement activation determines the therapeutic activity of rituximab in 19. Trentin L, Cabrelle A, Facco M, Carollo D, Miorin M, Tosoni A, et al. vivo. J Immunol 2003;171:1581–7. Homeostatic chemokines drive migration of malignant B cells in patients 41. De Weers M, Tai YT, van der Veer MS, Bakker JM, Vink T, Jacobs CDH, et al. with non-Hodgkin lymphomas. Blood 2004;104:502–8. Daratumumab, a novel therapeutic human CD38 monoclonal antibody, 20. Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin induces killing of multiple myeloma and other hematological tumors. Cancer Res 2010;16:2927–31. J Immunol 2011;186:1840–8. 21. Calandra G, Bridger G, Fricker S. CXCR4 in clinical hematology. Curr Top 42. Tai YT, Dillon M, Song W, Leiba M, Li XF, Burger P. Anti-CS1 humanized Microbiol Immunol 2010;341:173–91. monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and 22. Hezareh M, Hessell AJ, Jensen RC, Van De Winkel JGJ, Parren PWHI. induces antibody-dependent cellular cytotoxicity in the bone marrow Effector function activities of a panel of mutants of a broadly neutralizing milieu. Blood 2008;112:1329–37. antibody against human immunodeficiency virus type 1. J Virol 43. Spoo AC, Lubbert€ M, Wierda WG, Burger JA. CXCR4 is a prognostic marker 2001;75:12161–8. in acute myelogenous leukemia. Blood 2007;109:786–91. 23. Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, et al. 44. Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R, et al. Detection of beta 2-adrenergic receptor dimerization in living cells using Depletion of B cells in vivo by a chimeric mouse human monoclonal bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci U S antibody to CD20. Blood 1994;83:435–45. A 2000;97:3684–9. 45. Vugmeyster Y, Howell K, Bakshl A, Flores C, Canova-Davis E. Effect of anti- 24. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of CD20 monoclonal antibody, Rituxan, on cynomolgus monkey and human image analysis. Nat Methods 2012;9:671–5. B cells in a whole blood matrix. Cytometry A 2003;52:101–9.

www.aacrjournals.org Mol Cancer Ther; 15(8) August 2016 1899

A New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis and Mobilizes Effector Cells

Matthieu Broussas, Nicolas Boute, Barbara Akla, et al.

Mol Cancer Ther 2016;15:1890-1899. Published OnlineFirst June 13, 2016.

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