Enhanced Unique Pattern of Hematopoietic Cell MobilizationInduced by the CXCR4 Antagonist 4F-Benzoyl-TN14003

MICHAL ABRAHAM,a KATIA BIYDER,a MICHAL BEGIN,a HANNA WALD,a IDO D. WEISS,a EITHAN GALUN,a

ARNON NAGLER,b AMNON PELEDa

aGoldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel; bBone MarrowTransplantation Department, Chaim Sheba Medical Center, Tel-Hashomer, Israel

Key Words. CXCR4 • Mobilization • Hematopoietic stem cells • Hematopoietic progenitors

ABSTRACT

An increase in the number of stem cells in blood followingmobilization is required to enhance engraftment after high-dose chemotherapy and improve transplantation outcome.Therefore, an approach that improves stem cell mobilizationis essential. The interaction between CXCL12 and its recep-tor, CXCR4, is involved in the retention of stem cells in thebone marrow. Therefore, blocking CXCR4 may result inmobilization of hematopoietic progenitor and stem cells. Wehave found that the CXCR4 antagonist known as 4F-benzo-yl-TN14003 (T-140) can induce mobilization of hematopoi-etic stem cells and progenitors within a few hours post-treatment in a dose-dependent manner. Furthermore,although T-140 can also increase the number of white blood

cells (WBC) in blood, including monocytes, B cells, and Tcells, it had no effect on mobilizing natural killer cells.T-140 was found to efficiently synergize with granulocytecolony-stimulating factor (G-CSF) in its ability to mobi-lize WBC and progenitors, as well as to induce a 660-foldincrease in the number of erythroblasts in peripheralblood. Comparison between the CXCR4 antagonistsT-140 and AMD3100 showed that T-140 with or withoutG-CSF was significantly more potent in its ability tomobilize hematopoietic stem cells and progenitors intoblood. These results demonstrate that different CXCR4antagonists may have different therapeutic potentials.STEM CELLS 2007;25:2158 –2166

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

All mature blood cells are derived from hematopoietic stemcells (HSCs) through intermediates that are termed hematopoi-etic progenitor cells (HPCs) [1]. Hematopoietic cells at variousstages of differentiation are localized within the bone marrow(BM), their main site of production. Their trafficking betweenBM and blood is a physiological process [2, 3], but understeady-state conditions, HPCs and HSCs circulate in peripheralblood at very low frequency. Stem cell frequencies in peripheralblood can be considerably increased both in response to variousgrowth factors and during the recovery phase following myelo-suppressive chemotherapy, a process termed mobilization. Cir-culating stem cells are capable of homing to the BM andregenerating normal hematopoiesis following myeloablativeconditioning with high doses of chemotherapy or radiation.Recently, the use of peripheral blood as a source of stem cellsfor patients undergoing autologous and allogeneic transplanta-tion has replaced BM as the preferred source of hematopoieticrescue. Under steady-state conditions, HPCs and HSCs circulatein blood at frequencies too low to allow for efficient collectionof numbers sufficient for transplantation. Therefore, an increasein the number of HSCs in blood following mobilization im-proves transplantation efficiency by shortening the duration ofcytopenia, enhances immune reconstitution, and reduces mor-bidity.

Granulocyte colony-stimulating factor (G-CSF)-mobilizedperipheral-blood mononuclear cells are routinely used as asource of HSCs for transplantation. Despite the potency ofG-CSF in mobilizing stem cells, it results in broad interindi-vidual variations in circulating progenitor and stem cell numbers[4], requiring repeated dosing, and is frequently associated withside effects. Thus, optimal and improved methods to mobilizeand collect peripheral-blood progenitor and stem cells for he-matopoietic rescue are warranted.

Over recent years, it has become apparent that the interac-tion between CXCL12 and its receptor, CXCR4, plays a pivotalrole in hematopoietic cell mobilization and engraftment [5–7].The CXCR4 receptor is widely expressed on many cell types,including HSCs and HPCs, and the interaction with its ligandwas found to be involved in chemotaxis, homing, and survival.The CXCL12/CXCR4 axis is also involved in the retention ofhematopoietic cells within the BM microenvironment [8]; con-sequently, the disruption of CXCL12/CXCR4 interactions re-sults in mobilization of hematopoietic cells. Indeed, blockingthe CXCR4 receptor with an antagonist, such as AMD3100,results in the mobilization of HPCs. Moreover, combiningAMD3100 with G-CSF has produced an additive effect [9].These approaches suggest that antagonizing the interactions ofBM-produced CXCL12 with CXCR4 that is expressed on HSCscould be an effective HSC mobilizing strategy.

Today, there are several known CXCR4 antagonists thathave been described as having different levels of efficiency [10,

Correspondence: Amnon Peled, Ph.D., Hadassah Hebrew University Hospital, Gene Therapy Institute, P.O. Box 12000, Jerusalem,91120 Israel. Telephone: 972-2-677-8780; Fax: 972-2-643-0982; e-mail: peled@hadassah.org.il Received March 6, 2007; accepted forpublication May 15, 2007; first published online in STEM CELLS EXPRESS May 24, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi:10.1634/stemcells.2007-0161

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11]. 4F-benzoyl-TN14003 (T-140) is a short modified peptide(supplemental online Fig. 1), a highly selective CXCR4 antag-onist, originally designed as a human immunodeficiency virus(HIV) entry inhibitor through specific binding to CXCR4. T-140is a competitive inhibitor that hinders CXCL12-mediated cellproliferation and migration [12].

To determine the therapeutic potential of T-140, we evalu-ated the capacity of T-140 to mobilize hematopoietic cells, aloneand in combination with G-CSF. In addition, we compared theeffect of T-140 to that of another CXCR4 antagonist,AMD3100.

MATERIALS AND METHODS

ReagentsAMD3100 was purchased from Sigma-Aldrich (Rehovot, Israel,http://www.sigmaaldrich.com). G-CSF (Neupogen [filgrastim],Amgen, Thousand Oaks, CA, http://www.amgen.com) was kindlyprovided by Prof. Arnon Nagler. T-140 was kindly provided byBiokine Therapeutics, Ltd. The activity of T-140 was neutralized byincubating with Proteinase K (DAKO, Glostrup, Denmark, http://www.dako.com) for 20 minutes at 37°C followed by 10 minutes ofincubation at 95°C.

Mice and Experimental ProtocolFemale C57BL/6 mice (7–8 weeks old) were purchased from Har-lan Israel (Rehovot, Israel, http://www.harlanisrael.com) and main-tained under specific pathogen-free conditions at the Hebrew Uni-versity Animal Facility (Jerusalem, Israel). All experiments wereapproved by the Animal Care and Use Committee of the HebrewUniversity (Jerusalem, Israel). Mice were injected subcutaneouslywith various doses of T-140 or AMD3100 (1, 2.5, 5, and 10 mg/kg)in a total volume of 200 �l, 2 hours before sacrifice. In someexperiments, mice were sacrificed at different time points: 1⁄2, 1, 2,4, and 24 hours postinjection. G-CSF was subcutaneously injectedat a dose of 2.5 �g per mouse twice a day for 4 days. In thecombination experiments, 18 hours after the last injection of G-CSF, mice were injected with either T-140 or AMD3100. Controlmice were injected with phosphate-buffered saline (PBS) at theappropriate volume.

Cell Isolation and Differential CountsPeripheral blood cells were collected from mice by cardiac punctureinto tubes with heparin followed by lysis of erythrocyte populationusing a red blood cell lysis solution (0.155 M NH4Cl, 0.01 MKHCO3, 0.01 mM EDTA; pH 7.4). Cells were counted using ahemocytometer, and total number of cells per 1 ml of blood wascalculated. In some experiments, blood samples were resuspendedand processed to cytospin slides, centrifuged, air-dried, and stainedwith Giemsa. The percentage and the morphology of mobilized cellswere determined by differential counts of Giemsa-stained cytospinslides.

Flow CytometryFlow cytometry was used to assess the number of cells in blood anddistinguish between the different populations. Cells were gatedaccording to forward scatter and side scatter to exclude dead cellsand to determine granulocytes, mononuclear cells, and mature mac-rophage populations (Fig. 2E). The number of each cell populationwas counted. Cells were also stained in 0.1 ml of fluorescence-activated cell sorting (FACS) buffer with fluorescence antibodiesdirected against Gr-1, CD3, mac-1, F4/80, natural killer 1.1(NK1.1), B220, and Ter-119 molecules or matched isotype controls(all from eBioscience, San Diego, CA, http://www.ebioscience.com)for 30 minutes and washed with FACS buffer. Immunostained cellswere analyzed by flow cytometry using the FACSCaliber flow cytom-eter (BD Biosciences, San Diego, http://www.bdbiosciences.com); thedata were analyzed using software from CellQuest (version 3.3; BDBiosciences).

Migration AssayA migration assay with human Jurkat T cells was assayed by usingCostar migration buffer (6.5 mm/diameter, 5 �m/pore; CorningCostar, Cambridge, MA, http://www.corning.com/lifesciences;RPMI 1640, 1% FCS) containing 2 � 105 Jurkat cells added to theupper chamber, and 0.6 ml of chemotaxis buffer with or withoutCXCL12 (50 ng/ml) and with or without T-140 or T-140 pretreatedwith proteinase K was added to the bottom chamber. Cells migrat-ing within 4 hours to the bottom chamber of the Transwell werecounted using FACSCalibur (BD Biosciences, San Jose, CA, http://www.bdbioscience.com).

HPC AssayTo evaluate the number of progenitor cells, a colony-forming cellassay was used. Burst-forming units-erythrocyte (BFU-E), colony-forming units granulocyte-macrophage (CFU-GM), colony-formingunits megakaryocytes (CFU-M), and colony-forming units granu-locyte-erythroblasts-macrophages-megakaryocyte (CFU-GEMM)were assayed by plating the cells in Iscove’s modified Dulbecco’smedium containing 1% methylcellulose, 15% fetal bovine serum,1% bovine serum albumin, 3 U/ml recombinant human (rh) EPO,104 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombi-nant mouse (rm)SCF, 10 ng/ml rmIL-3, 10 �g/ml rh insulin, 10ng/ml rh interleukin-6 (IL-6), and 200 �g/ml human transferrin(Methocult GF M3434; Stem Cell Technologies, Vancouver, BC,Canada, http://www.stemcell.com). The cultures were incubated at37°C in a humidified atmosphere containing 5% CO2. Seven dayslater, typical colonies were visually scored by morphologic criteriausing a light microscope, and the frequency of CFU was calculated.Staining colonies with benzidine dihydrochloride (Sigma-Aldrich)was used to localize hemoglobin-containing cells.

HSC AssayC57BL/6 mice, serving as donors, were injected with 5 mg/kg ofeither T-140 or AMD3100. Two hours later, peripheral blood cellswere collected, followed by erythrocyte lysis (as described above),and i.v. transferred into C57BL/6 recipient mice that had beenpretreated with a lethal dose of irradiation (900 cGy) 24 hoursbefore. Total cells obtained from 900 or 225 �l of blood wereinoculated into a single recipient mouse in a total volume of 200 �lof PBS. Recipient mice that were inoculated with cells obtainedfrom blood of untreated mice or with normal BM cells (5 � 106

cells per mouse) served as controls. The survival of mice wasmonitored for 4 months. Four months after the first transplantation,BM cells recovered from the first recipient mice repopulated bydonor cells were injected i.v. into lethally irradiated secondary mice.

Statistical AnalysisResults are expressed as average � SD. Statistical differences weredetermined by an analysis of two-tailed Student’s t test. Values ofp � .05 were considered to be statistically significant.

To evaluate whether the effect we observed for the combinedtreatments was synergistic, we used Student’s t-test and comparedthe sum of values obtained for each different treatment (estimatedadditive effect) and the values obtained for the combined treatment.Synergistic effect was defined as an effect that was significantlygreater (p � .05) than the sum of the individual effects.

RESULTS

T-140 Administration Induces White Blood CellMobilizationWe evaluated the effect of T-140 on mobilization of cells to theblood of intact mice and analyzed the kinetics and the dose-response of this effect. First, mice were injected with 5 mg/kgT-140, and the mobilization of white blood cells (WBC), atvarious time points postinjection, was examined. At 1⁄2, 1, 2, 4,and 24 hours after injection of T-140, mice were sacrificed and

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cells were collected from blood. A single-dose administration of5 mg/kg T-140 resulted in a rapid elevation in the total numberof WBC in blood, a threefold change from the control after 30minutes, slowly decreasing until full return to the baseline levelafter 24 hours (Fig. 1A). The characterization of the mobilizedWBC, using a flow cytometer, showed alteration in the absolutenumber of granulocytes (stained positive with Gr-1), mononu-clear cells (MNCs), and mature macrophages (stained positivewith both mac-1 and F4/80 [13]). Further characterization usingcytospin assay and Giemsa staining defined the granulocytepopulation as neutrophils. When we looked at the number ofneutrophils in blood, there was an elevation starting 30 minutespostinjection, reaching a peak after 1–2 hours (more than a12-fold change from control), and slowly decreasing to thebaseline level after 24 hours (Fig. 1B). The same pattern wasobserved for mature macrophages; these cells reached a peak 1hours postinjection of T-140 and returned to the baseline levelafter 24 hours (Fig. 1D). When we tested the effect of T-140 onthe mobilization of MNCs, we observed a twofold rapid eleva-tion change from control after 30 minutes, which rapidly de-creased until full return to the baseline level after 4 hours (Fig.1C).

To determine the effect of different doses of T-140, concen-trations of 1, 2.5, 5, and 10 mg/kg were subcutaneously injectedinto mice. Two hours postinjection, mice were sacrificed, andthe total number of WBC in 1 ml of blood was measured. T-140induced the mobilization of WBC in a dose-dependent manner.Peak mobilization of WBC occurred at a dose of 5 mg/kg ofT-140 (Fig. 2A). When we looked at the different populations,we found a similar dose-response elevation in the absolutenumber of neutrophils (8-fold increase), mature macrophages(6-fold increase), and MNCs (3.5-fold increase) in the bloodfollowing treatment with 10 mg/kg of T-140 (Fig. 2B–2D,respectively).

To further identify the different populations of MNCs thatwere mobilized by T-140, we stained the cells with specificantibodies for T cells (CD3), B cells (B220), Mac-1� cells, andNK cells (NK1.1) (Fig. 2E). As shown in Figure 2F, T-140 hadan effect on the mobilization of T cells, B cells, and Mac-1�

cells but had no effect on the mobilization of NK cells. We didnot observed any significant alteration in the number of plateletsor other coagulation factor in blood following treatment withT-140 (data not shown).

T-140 Synergizes with G-CSF to Mobilize WBCRecently, the CXCR4 antagonist AMD3100 was reported topromote mobilization of stem cells and was found to synergis-tically augment G-CSF-induced mobilization of HPCs [9, 14].Thus, we evaluated the mobilizing capability of T-140 in com-parison with G-CSF, AMD3100, and the combination of bothCXCR4 antagonists with G-CSF. Similarly to T-140, injectionof AMD3100 (s.c.) induced the mobilization of WBC after 2hours at the optimal concentration of 5 mg/kg. Both T-140 andAMD3100 induced a 2.5-fold increase in the number of WBC inblood compared with control. When G-CSF was injected sub-cutaneously at a dosage of 2.5 �g per mouse twice a day for 4days, there was a 1.9-fold increase in the number of WBC inblood. To study the combination of G-CSF and either T-140 orAMD3100, the CXCR4 antagonists were injected at a concen-tration of 5 mg/ml, 18 hours after the fourth injection of G-CSF;2 hours later, mice were sacrificed, and the number and type ofcells mobilized in blood were tested.

The combination of T-140 with G-CSF induced a 5.1-foldincrease in the number of WBC in blood (Fig. 3A). This induc-tion was significantly higher than the increase seen in WBCstimulated by a combination of AMD3100 and G-CSF (3.7-foldincrease over control; p � .006; Fig. 3A).

Next, we analyzed the relative number of neutrophils,MNCs, and mature macrophages in blood of treated mice. Wefound that T-140 and AMD3100 increased the mobilization ofneutrophils by 6.1-fold and 8-fold over control, respectively(p � .05), whereas G-CSF alone stimulated no more than a4.4-fold increase in the number of neutrophils over control.Combination of T-140 or AMD3100 with G-CSF synergisticallyincreased the number of neutrophils in blood (13.3- and 11.7-fold increase over control, respectively; p � .005; Fig. 3C).

T-140 and AMD3100 induced a significant and similar foldchange in the number of MNCs in blood compared with control(2.3- and 3.4-fold increase over control, respectively; p � .005).Comparison with both CXCR4 antagonists showed that G-CSF,in our experimental model, is an inferior mobilizer of MNCs(Fig. 3B, 3D). The combination of G-CSF with T-140 induceda synergistic increase in the number of MNCs (4-fold increaseover control), whereas the combination of G-CSF withAMD3100 did not induce such an effect (Fig. 3D). T-140 andAMD3100 also induced a significant change in the number ofmature macrophages in blood compared with control (4.2- and

Figure 1. Time-dependent mobilization ofWBC induced by administration of 5 mg/kg of T-140. (A): Time-dependent mobiliza-tion of WBC in response to a single s.c.injection of 5 mg/kg T-140 into C57BL/6peripheral blood. Cells were counted using ahemocytometer, and the total number ofcells per 1 ml of blood was calculated. (B):Fluorescence-activated cell sorting analysiswas performed to distinguish between differ-ent populations of the cells. The cells weregated according to forward scatter and sidescatter, and the subpopulations of neutro-phils, MNCs, and mature macrophages weredefined (see Fig. 2E). The numbers of neu-trophils (B), MNCs (C), and mature macro-phages (D) following T-140 administrationwere analyzed. The data shown are the av-erage � SD of six mice per group from atotal of two separate experiments performed(�, p � .05). Abbreviations: MNC, mono-nuclear cell; T-140, 4F-benzoyl-TN14003;WBC, white blood cells.

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6.1-fold increase over control, respectively; p � .05). Compar-ison with both CXCR4 antagonists showed that G-CSF is also apoor mobilizer of mature macrophages (Fig. 3B, 3E). Similarlyto the effect on MNCs, the combination of G-CSF with T-140induced a synergistic increase in the number of MNCs (8.3-foldincrease over control; p � .005), whereas the combination ofG-CSF with AMD3100 did not induce such an effect (Fig. 3E).These results show that the CXCR4 antagonists are capable ofmobilizing different subtypes of cells compared with G-CSFand that T-140, but not AMD3100, synergized with G-CSF tofurther stimulate the mobilization of MNCs and mature macro-phages.

Effects of T-140 Administration on Progenitor CellMobilizationTo further study the effect of T-140 on mobilization of HPCs,we collected cells from the blood of treated and untreated miceand tested for the presence of colony-forming cells. Injection ofT-140 at a concentration between 1 and 10 mg/kg induced adose-response increase (up to 10-fold over control) in the num-ber of progenitor cells in blood (Fig. 4A; p � .005). Treatmentof mice with T-140 induced a robust dose-response mobilization

of myeloid CFU-GM colonies and the mobilization of multipo-tent progenitors CFU-M, CFU-GEMM and the erythroid pro-genitors BFU-E. Interestingly, we found that the most dramaticincrease in progenitors was observed in the number of BFU-E,reaching a significant increase of more than 110-fold overcontrol (p � .01; Fig. 4B).

Mobilization of progenitor cells started after 30 minutes andreached a peak 1 to 2 hours postinjection of T-140 (Fig. 4C, 4D;9.5- and 13.6-fold increases over control, respectively; p � .01).This elevation in the number of progenitor cells in blood almostdisappeared 24 hours postinjection. The most dramatic effectwas observed in the mobilization of BFU-E, where the numberof progenitors was increased 200-fold 1 hour after injection ofT-140 (Fig. 4D). Staining mobilized cells with a marker forproerythroblasts, known as Ter119, showed that administrationof T-140 caused a 3.2-fold elevation over control in the numberof proerythroblasts in blood (supplemental online Fig. 2).

T-140 Synergizes with G-CSF to Mobilize HPCsBlocking the CXCR4 receptor with AMD3100 resulted in themobilization of HPCs [9, 15, 16]. When we compared the effectof T-140 (5 mg/kg) on the mobilization of progenitor cells tothat of AMD3100 (5 mg/kg), we found that T-140 was signif-

Figure 2. Dose-dependent mobilization of white blood cells (WBC) induced by administration of T-140. C57BL/6 mice were s.c. injected with 1,2.5, 5, or 10 mg/kg of T-140. Two hours postinjection, mice were sacrificed and peripheral blood cells were obtained. (A): Total WBC was countedusing a hemocytometer. Numbers of neutrophils (B), mature macrophages (C), and MNCs (D) were evaluated by fluorescence-activated cell sorting(FACS) according to FSC and SSC. (E): A representative FACS analysis of blood cells gated according to FSC and SSC to define maturemacrophages (R1), neutrophils (R2), and MNCs (R3) is shown. Mature macrophages were further identified by staining for MAC-1 and F4/80expression; neutrophils were also identified by staining for Gr-1. To analyze the MNC subpopulations, T cells were stained for CD3. NK cells werestained for NK1.1, but no CD3 or B cells were identified by staining for B220. (F): T-140 administration induced the mobilization of monocytes, Tcells, and B cells but not NK cells. The data are the average � SD of six mice per group from a total of two separate experiments performed (�, p �.05). Abbreviations: FSC, forward scatter; MNC, mononuclear cell; NK, natural killer; SSC, side scatter; T-140, 4F-benzoyl-TN14003.

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icantly more potent in its ability to mobilize progenitor cells intoblood. T-140 increased the number of progenitor cells in bloodby 7.1-fold over control (p � .05), whereas AMD3100 inducedthe mobilization of progenitor cells by 4.2-fold over control(p � .001). G-CSF induced a 13.6-fold increase in the numberof progenitors in blood over control, whereas in combinationwith T-140, it induced a 76.8-fold elevation in the number ofprogenitors over control. This progenitor increase induced by acombination of G-CSF and T-140 was significantly higher thanthe synergistic induction in the number of progenitors by thecombination of G-CSF with AMD3100 (76.8-compared with46.4-fold over control; p � .001; Fig. 5A).

The effect of CXCR4 antagonists alone and in combinationwith G-CSF on the mobilization of BFU-E and GEMM progen-itor cells was further characterized. T-140 and AMD3100 aloneinduced a similar increase in the number of BFU-E in blood(nearly 30-fold change from control; p � .05), whereas G-CSFalone induced a 142-fold increase in BFU-E in blood overcontrol (Fig. 5B). Coinjection of AMD3100 with G-CSF wasneither additive nor synergistic (142-fold increase with G-CSFvs. 98-fold increase with combination treatment). However,G-CSF, in combination with T-140, induced a significant ele-vation of BFU-E (142-fold increase with G-CSF vs. 660-foldincrease with combination treatment; p � .01; Fig. 5B). Similarresults were observed when we analyzed the number of CFU-

GEMM colonies mobilized with G-CSF alone or in combi-nation with the CXCR4 antagonists (Fig. 5C). T-140 wassignificantly more potent than AMD3100 in its ability tomobilize CFU-GEMM (44- vs.15.5-fold increase, respec-tively; p � .05). G-CSF alone induced a 151-fold increaseover control (p � .01), whereas its combination with T-140induced a 712-fold increase over control (p � .01). Thiselevation was significantly higher than that induced by thecombination of G-CSF with AMD3100 (a 266-fold increaseover control; p � .01; Fig. 5C). These results suggest thatT-140 synergized more efficiently with G-CSF to induce themobilization of early progenitors.

To determine whether the effects we observed were spe-cifically related to the activity of T-140, we used the neu-tralized T-140 treated with proteinase K and compared itseffect with the natural T-140. We found that treatment withproteinase K digests the peptide into smaller fragments, asdemonstrated in supplemental online Figure 3A and 3B.These fragments did not inhibit the migration of Jurkat Tcells toward CXCL12, the ligand for CXCR4, and could notinduce the mobilization of WBC or progenitor cells (supple-mental online Fig. 3C–3E, respectively). These results dem-onstrate that the mobilizing effect we observed was specifi-cally related to presence of the intact T-140 peptide capableof interacting with CXCR4.

Figure 3. T-140 synergizes with GCSF to mobilize WBC. C57BL/6 mice were s.c. injected with GCSF 2.5 �g twice a day for 4 days. Eighteen hoursafter the last injection, mice were injected with 5 mg/kg of either T-140 or AMD3100; 2 hours later, mice were sacrificed and peripheral blood cellswere obtained. (A): Total number of WBC in blood was counted by fluorescence-activated cell sorting (FACS). (B): A representative FACS analysisof blood cells is shown. The cells were gated according to FSC and side scatter, and subpopulations of mature macrophages (R1), neutrophils (R2),and MNCs (R3) were defined. The numbers of neutrophils (C), MNCs (D), and mature macrophages (E) following the different treatments wereanalyzed. �, p � .05 compared with control; ��, p � .05 of GCSF�T-140 compared with GCSF�AMD3100; �, p � .05 of T-140 comparedwith AMD3100. The data are the average � SD of four mice per group from a total of four separate experiments performed. Abbreviations: AMD,AMD3100; FSC, side scatter; GCSF, granulocyte colony-stimulating factor; MNC, mononuclear cell; T140, 4F-benzoyl-TN14003; WBC, white bloodcells.

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T-140 Mobilizes HSCs with Long-TermRepopulating CapacityAlthough mobilization of HPCs may be of use for short-termrepopulation in a transplant setting, HSCs are required forlong-term repopulation. To assess the T-140 effect on mobi-lization of murine HSCs, we used a transplantation model inwhich donor C57Bl/6 mice were treated with T-140 orAMD3100. After 2 hours, blood was collected, and cells thatwere isolated from an equal blood volume (900 or 225 �l)were transferred into lethally irradiated recipients C57Bl/6mice. Survival of recipient mice, as indicated by long-termrepopulating activity and presence of HSCs, was monitored.

As shown in Figure 6A, 100% of mice transplanted withcells mobilized by T-140 (900 �l of blood) survived thetreatment. In contrast, only 73% of mice transplanted withcells that were mobilized by AMD3100 survived the treat-ment (p � .06; Fig. 6A). Transplantation of cells mobilizedby T-140 using a blood volume of 225 �l resulted in thesurvival of 70.6% of the treated mice, compared with thesurvival of only 37.5% of the treated mice when mobilizationwas performed by AMD3100 (p � .05). Although all un-treated mice died after irradiation, 100% of mice survivedirradiation when normal BM cells had been transplanted (Fig.6A, 6B). To test for self-renewal and long-term repopulatingactivity, the transplanted mice were maintained for more than4 months, and then their BM cells were injected into a secondrecipient. Stem cells mobilized by T-140 maintained 100% ofthe transplanted mice for longer than 4 months and werecapable of producing long-term rescue in lethally irradiatedsecondary transplanted mice. These results indicate that

T-140 is a powerful mobilizer of long-term repopulating stemcells.

DISCUSSION

Autologous and allogeneic stem cell transplantation hasemerged as a preferred strategy in the treatment of a variety ofhematological malignancies [17, 18]. In recent years, the use ofperipheral blood as a source of HSCs for transplantation fol-lowed by a high dose of chemotherapy has emerged into com-mon clinical practice [19]. Successful blood and marrow trans-plants require the infusion of a sufficient number of HSCscapable of homing to the marrow cavity and regenerating a fullarray of hematopoietic cell lineages. Thus, an increase in thenumber of hematopoietic cells in blood will improve the yield ofcell collection for transplantation and will also have the poten-tial to shorten recovery from cytopenia and reduce morbidityand mortality.

G-CSF was first identified as a colony-stimulating factor forgranulocyte precursors and as a stimulator of granulocyte dif-ferentiation and activation [20]. Due to its activity, G-CSF isused to reduce neutropenia and increase the production of neu-trophils [21, 22]. In addition to inducing the production andmobilization of neutrophils, G-CSF is also a powerful mobilizerof HSCs. Although several cytokines, such as GM-CSF, IL-3,stem cell factor, and flt-3 ligand, and chemokines, such as IL-8,were also shown to mobilize HPCs and HSCs [23–25], thecytokine G-CSF remains the major growth factor used formobilization of stem cells for transplantation. The current pro-tocols for stem cell mobilization necessitate 4–5 days of 1–2 s.c.

Figure 4. Time- and dose-response effect of T-140 on mobilization of hematopoietic progenitor cells into blood. (A, B): Dose-response analysis at2 hours postinjection (s.c.) using 1, 2.5, 5, or 10 mg/kg T-140 into C57BL/6 mice. Peripheral blood cells were obtained, and the total number ofcolony-forming units (CFU), CFU-M (colony-forming cells granulocyte-macrophage [CFC-GM]), CFU-GEMM, or BFU-E in blood was evaluatedby CFU assay. (C, D): Time-dependent mobilization of CFC in response to a single s.c. injection of 5 mg/kg of T-140. Untreated mice served ascontrol. The data are the average � SD of six mice per group from a total of two separate experiments performed (�, p � .05). Abbreviations: BFU-E,burst-forming units-erythroblasts; CFU-GEMM, colony-forming units granulocyte-erythroblasts-macrophages-megakaryocyte; CFU-GM, colony-forming units granulocyte-macrophage; CFU-M, colony-forming units macrophages; T-140, 4F-benzoyl-TN14003.

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injections of G-CSF; moreover, one or more pheresis proceduresare required because of failure to collect the number of CD34�cells necessary for successful transplantation. It is suggested thatG-CSF induces the mobilization of HSCs through an indirect

mechanism by activating neutrophils within the BM. Indeed, itwas recently demonstrated that G-CSF stimulates neutrophils tosecrete a variety of proteolytic enzymes, including elastase,cathepsin G, matrix metalloproteinase (MMP)-2, and MMP-9,that are capable of degrading CXCL12, the ligand for thechemokine receptor CXCR4, a key molecule that is expressedby the BM stroma and plays an important role in the retention ofstem cells expressing CXCR4 within the BM [7, 26].

An important role for the interaction between CXCL12 andits receptor CXCR4 has been described for the mobilization ofhematopoietic progenitors and stem cells [5–7]. More specifi-cally, AMD3100, a selective antagonist of CXCR4, was recentlyshown to rapidly mobilize CD34� HPCs from the humanmarrow into the peripheral blood. Furthermore, AMD3100 sig-nificantly increased both G-CSF-stimulated (10 �g/kg per day)mobilization of CD34� cells (3.8-fold) and leukapheresis yieldof CD34� cells. Moreover, AMD3100-mobilized leukapheresisproducts contained significantly greater numbers of T and Bcells compared with G-CSF-stimulated leukapheresis products[27]. AMD3100 was also reported to mobilize HSCs with long-term repopulating capacity in humans [28] and nonhuman pri-mates [29]. In addition, AMD3100 was reported to be a safe andeffective agent for the mobilization of CD34� cells in patientswith multiple myeloma or lymphoma receiving prior chemo-therapy [30, 31]. On the other hand, a clinical trial withAMD3100 in HIV-infected individuals produced prematureventricular contraction side effects, resulting in the discontinu-ation of this trial [27].

On the basis of this knowledge, we evaluated the ability ofa novel specific CXCR4 antagonist, known as T-140, to mobi-lize hematopoietic cells. T-140 and its analogues were firstdeveloped initially as anti-HIV agents that inhibit HIV-1 infec-tion through their specific binding to CXCR4 [32, 33]. Unlike

Figure 5. T-140 synergizes with GCSF to mobilize HPCs. C57BL/6 mice were s.c. injected with 2.5 �g of GCSF twice a day for 4 days. Eighteenhours after the last injection, mice were injected with 5 mg/kg of either T-140 or AMD3100; 2 hours later, mice were sacrificed and peripheral bloodcells were collected. (A): Number of total CFU cells in the blood was evaluated by colony assay. (B, C): The number of BFU-E and CFU-GEMMwas tested by staining the colonies with benzidine dihydrochloride to detect hemoglobin-containing cells. �, p � .05 compared with control; ��, p �.05 of GCSF�T-140 compared with GCSF�AMD3100; �, p � .05 of T-140 compared with AMD3100. The data are the average � SD of four miceper group from a total of four separate experiments performed. Abbreviations: AMD, AMD3100; BFU-E, burst-forming units-erythroblasts;CFU-GEMM, colony-forming units granulocyte-erythrocyte-monocyte-macrophage; GCSF, granulocyte colony-stimulating factor; T-140, 4F-benzoyl-TN14003.

Figure 6. T-140 mobilizes hematopoietic stem cells with long-termrepopulating capacity that can rescue mice following lethal irradiation.C57BL/6 mice serving as donors were injected with 5 mg/kg of eitherT-140 or AMD3100. Two hours later, peripheral blood cells werecollected and transplanted into C57BL/6 recipient mice that were le-thally irradiated (900 cGy) 24 hours before i.v. injection of the cells.Cells obtained from 900 (A) or 225 (B) �l of blood were transplantedinto single recipient mice. As a control, recipient mice were alsotransplanted with cells obtained from blood of untreated mice or withnormal BM cells. �, p � .06 of T-140 compared with AMD3100 when900 �l was transplanted; ��, p � .05 of T-140 compared withAMD3100 when 225 �l was transplanted. Data are the average of 17mice per group from a total of four separate experiments performed.Abbreviations: AMD, AMD3100; BM, bone marrow; T-140, 4F-ben-zoyl-TN14003.

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AMD3100, T-140 is a short, modified peptide. Comparativestudies between T-140 and AMD3100 found that each of theseagents inhibited CXCR4 via different mechanisms [34, 35].Whereas T-140 binds residues in extracellular domains of theCXCR4 receptor and regions of the hydrophobic core proximalto the cell surface, amino acids in the central hydrophobic coreare critical to binding of AMD3100 [34]. Analysis of antago-nists revealed that exposure to AMD3100 induced G-proteinactivation by CXCR4, whereas T-140 decreased autonomoussignaling [35]. Therefore, AMD3100 was defined to have aweak partial agonist activity, whereas T-140 functions as aninverse agonist.

Our results indicate that T-140 can efficiently induce mobi-lization of mature white blood cells, progenitors, and stem cellsinto blood within a few hours after treatment in a dose-depen-dent manner and can efficiently synergize with G-CSF for thiseffect. A variety of cell types express the CXCR4 receptor,including monocytes/macrophages, B and T lymphocytes, pro-genitors, and stem cells. Indeed, we found that T-140 can inducemobilization of all of these cell subsets. Although CXCR4 isalso expressed by NK cells [36] and platelets [37], we could notdemonstrate the effect of T-140 on their mobilization. Similarly,CXCR4/CXCL12 interaction was not found to be involved inplatelet activation, as demonstrated by the inability of CXCL12to augment aggregation or stimulate calcium mobilization ofplatelets [37].

Administration of T-140, AMD3100, and G-CSF can inducean effective mobilization of WBC into blood. However, whereasG-CSF induces mainly the exit of neutrophils, both CXCR4antagonists induced a robust mobilization of MNCs and maturemacrophages.

T-140 or AMD3100 alone induced the mobilization of asimilar number of MNCs and activated macrophages. However,the combination of T-140 with G-CSF induced a significantincrease in the mobilization of MNCs and mature macrophagesinto blood compared with AMD3100.

Even though mobilization of WBC is an important factor fortransplantation, the major factor contributing to transplantationefficiency is the number of HPCs and HSCs in blood. When welooked at the effect of T-140 on mobilization of progenitor cells,we observed a significant elevation in the number of bothmyeloid pluripotent and erythroid progenitors, that is, CFU-GM, CFU-M, CFU-GEMM, and BFU-E. A comparative studydemonstrated that administration of a single dose of T-140resulted in a significant increase of progenitor cell mobilizationcompared with AMD3100. Moreover, the combination of T-140with G-CSF resulted in a dramatic elevation in the number of

progenitor cells in blood that was superior to G-CSF alone or toa combination of G-CSF with AMD3100. This superiority washighly significant when we witnessed the number of CFU-GEMM and BFU-E in the blood of treated mice.

Although improved mobilization of HPCs in response toT-140 is of interest, proof that T-140 also mobilizes HSCs is ofparamount importance. A comparison of the abilities of T-140and AMD3100 to mobilize HSCs into blood was made using atransplantation model. We found that T-140 is more potent in itsability to mobilize HSCs with long-term repopulating activitycompared with AMD3100.

Mobilization of WBC and progenitor cells was synergisti-cally enhanced when T-140 was used in combination withG-CSF, and this effect was significantly superior to that ob-served for the combination of G-CSF with AMD3100. Futurestudies should address the questions about the mechanism(s) bywhich T-140 induces cell mobilization, including studies regard-ing the blocking of the CXCR4 receptor and the immunogenic-ity properties of T-140.

In summary, our results indicate that the ability of theCXCR4 antagonist T-140 to mobilize WBC, as well as progen-itor and stem cells, differs both qualitatively and quantitativelyfrom that of the well-characterized CXCR4 antagonistAMD3100. Furthermore, T-140 differs from AMD3100 in itsability to efficiently synergize with G-CSF. It is therefore pos-sible that an improved CXCR4 antagonist will provide bettermethods of collecting mobilized stem cells for transplantation.Future studies should address these issues and define the poten-tial clinical advantage and usefulness of T-140 in clinical stemcell mobilization and transplantation.

ACKNOWLEDGMENTS

We thank Nobutaka Fujii from Kyoto University (Sakyo-ku,Kyoto, Japan) and Mary Clausen from the Gene Therapy Insti-tute, Hadassah University Hospital (Jerusalem, Israel), for tech-nical assistance. This work supported by a grant from the LadyDavis Fellowship Trust.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

A.P. owns stock in, has acted as a consultant to, has performedcontract work for, is a Board member for, and has a financialinterest in Biokine Therapeutics.

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DOI: 10.1634/stemcells.2007-0161 2007;25;2158-2166; originally published online May 24, 2007; Stem Cells

Galun, Arnon Nagler and Amnon Peled Michal Abraham, Katia Biyder, Michal Begin, Hanna Wald, Ido D. Weiss, Eithan

CXCR4 Antagonist 4F-Benzoyl-TN14003Enhanced Unique Pattern of Hematopoietic Cell Mobilization Induced by the

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& ServicesUpdated Information

http://www.StemCells.com/cgi/content/full/25/9/2158including high-resolution figures, can be found at:

Supplementary Material http://www.StemCells.com/cgi/content/full/2007-0161/DC1

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