CANCER STEM CELLS

Enhanced Unique Pattern of Hematopoietic Cell Mobilization Induced 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 Downloaded from ARNON NAGLER,b AMNON PELEDa aGoldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel; bBone Marrow Transplantation Department, Chaim Sheba Medical Center, Tel-Hashomer, Israel

Key Words. CXCR4 • Mobilization • Hematopoietic stem cells • Hematopoietic progenitors www.StemCells.com

ABSTRACT An increase in the number of stem cells in blood following cells (WBC) in blood, including monocytes, B cells, and T mobilization is required to enhance engraftment after high- cells, it had no effect on mobilizing natural killer cells. dose chemotherapy and improve transplantation outcome. T-140 was found to efficiently synergize with granulocyte Therefore, an approach that improves stem cell mobilization colony-stimulating factor (G-CSF) in its ability to mobi- is essential. The interaction between CXCL12 and its recep- lize WBC and progenitors, as well as to induce a 660-fold tor, CXCR4, is involved in the retention of stem cells in the increase in the number of erythroblasts in peripheral at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009 bone marrow. Therefore, blocking CXCR4 may result in blood. Comparison between the CXCR4 antagonists mobilization of hematopoietic progenitor and stem cells. We T-140 and AMD3100 showed that T-140 with or without have found that the CXCR4 antagonist known as 4F-benzo- G-CSF was significantly more potent in its ability to yl-TN14003 (T-140) can induce mobilization of hematopoi- mobilize hematopoietic stem cells and progenitors into etic stem cells and progenitors within a few hours post- blood. These results demonstrate that different CXCR4 treatment in a dose-dependent manner. Furthermore, antagonists may have different therapeutic potentials. although T-140 can also increase the number of white blood STEM CELLS 2007;25:2158–2166 Disclosure of potential conflicts of interest is found at the end of this article.

Granulocyte colony-stimulating factor (G-CSF)-mobilized INTRODUCTION peripheral-blood mononuclear cells are routinely used as a source of HSCs for transplantation. Despite the potency of All mature blood cells are derived from hematopoietic stem G-CSF in mobilizing stem cells, it results in broad interindi- cells (HSCs) through intermediates that are termed hematopoi- vidual variations in circulating progenitor and stem cell numbers etic progenitor cells (HPCs) [1]. Hematopoietic cells at various [4], requiring repeated dosing, and is frequently associated with stages of differentiation are localized within the bone marrow side effects. Thus, optimal and improved methods to mobilize (BM), their main site of production. Their trafficking between and collect peripheral-blood progenitor and stem cells for he- BM and blood is a physiological process [2, 3], but under matopoietic rescue are warranted. steady-state conditions, HPCs and HSCs circulate in peripheral Over recent years, it has become apparent that the interac- blood at very low frequency. Stem cell frequencies in peripheral tion between CXCL12 and its receptor, CXCR4, plays a pivotal blood can be considerably increased both in response to various role in hematopoietic cell mobilization and engraftment [5–7]. growth factors and during the recovery phase following myelo- The CXCR4 receptor is widely expressed on many cell types, suppressive chemotherapy, a process termed mobilization. Cir- including HSCs and HPCs, and the interaction with its ligand culating stem cells are capable of homing to the BM and was found to be involved in chemotaxis, homing, and survival. regenerating normal hematopoiesis following myeloablative The CXCL12/CXCR4 axis is also involved in the retention of conditioning with high doses of chemotherapy or radiation. hematopoietic cells within the BM microenvironment [8]; con- Recently, the use of peripheral blood as a source of stem cells sequently, the disruption of CXCL12/CXCR4 interactions re- for patients undergoing autologous and allogeneic transplanta- sults in mobilization of hematopoietic cells. Indeed, blocking tion has replaced BM as the preferred source of hematopoietic the CXCR4 receptor with an antagonist, such as AMD3100, rescue. Under steady-state conditions, HPCs and HSCs circulate results in the mobilization of HPCs. Moreover, combining in blood at frequencies too low to allow for efficient collection AMD3100 with G-CSF has produced an additive effect [9]. of numbers sufficient for transplantation. Therefore, an increase These approaches suggest that antagonizing the interactions of in the number of HSCs in blood following mobilization im- BM-produced CXCL12 with CXCR4 that is expressed on HSCs proves transplantation efficiency by shortening the duration of could be an effective HSC mobilizing strategy. cytopenia, enhances immune reconstitution, and reduces mor- Today, there are several known CXCR4 antagonists that bidity. have 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: [email protected] Received March 6, 2007; accepted for publication 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 STEM CELLS 2007;25:2158–2166 www.StemCells.com Abraham, Biyder, Begin et al. 2159

11]. 4F-benzoyl-TN14003 (T-140) is a short modified peptide Migration Assay (supplemental online Fig. 1), a highly selective CXCR4 antag- A migration assay with human Jurkat T cells was assayed by using onist, originally designed as a human immunodeficiency virus Costar migration buffer (6.5 mm/diameter, 5 ␮m/pore; Corning (HIV) entry inhibitor through specific binding to CXCR4. T-140 Costar, Cambridge, MA, http://www.corning.com/lifesciences; is a competitive inhibitor that hinders CXCL12-mediated cell RPMI 1640, 1% FCS) containing 2 ϫ 105 Jurkat cells added to the proliferation and migration [12]. upper chamber, and 0.6 ml of chemotaxis buffer with or without To determine the therapeutic potential of T-140, we evalu- CXCL12 (50 ng/ml) and with or without T-140 or T-140 pretreated ated the capacity of T-140 to mobilize hematopoietic cells, alone with proteinase K was added to the bottom chamber. Cells migrat- Downloaded from and in combination with G-CSF. In addition, we compared the ing within 4 hours to the bottom chamber of the Transwell were effect of T-140 to that of another CXCR4 antagonist, counted using FACSCalibur (BD Biosciences, San Jose, CA, http:// AMD3100. www.bdbioscience.com). HPC Assay MATERIALS AND METHODS To evaluate the number of progenitor cells, a colony-forming cell assay was used. Burst-forming units-erythrocyte (BFU-E), colony- forming units granulocyte-macrophage (CFU-GM), colony-forming www.StemCells.com Reagents units megakaryocytes (CFU-M), and colony-forming units granu- AMD3100 was purchased from Sigma-Aldrich (Rehovot, Israel, locyte-erythroblasts-macrophages-megakaryocyte (CFU-GEMM) http://www.sigmaaldrich.com). G-CSF (Neupogen [filgrastim], were assayed by plating the cells in Iscove’s modified Dulbecco’s Amgen, Thousand Oaks, CA, http://www.amgen.com) was kindly medium containing 1% methylcellulose, 15% fetal bovine serum, provided by Prof. Arnon Nagler. T-140 was kindly provided by 1% bovine serum albumin, 3 U/ml recombinant human (rh) EPO, Biokine Therapeutics, Ltd. The activity of T-140 was neutralized by 104 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombi- incubating with Proteinase K (DAKO, Glostrup, Denmark, http:// nant mouse (rm)SCF, 10 ng/ml rmIL-3, 10 ␮g/ml rh insulin, 10 www.dako.com) for 20 minutes at 37°C followed by 10 minutes of ng/ml rh interleukin-6 (IL-6), and 200 ␮g/ml human transferrin at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009 incubation at 95°C. (Methocult GF M3434; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). The cultures were incubated at Mice and Experimental Protocol 37°C in a humidified atmosphere containing 5% CO2. Seven days later, typical colonies were visually scored by morphologic criteria Female C57BL/6 mice (7–8 weeks old) were purchased from Har- using a light microscope, and the frequency of CFU was calculated. lan Israel (Rehovot, Israel, http://www.harlanisrael.com) and main- Staining colonies with benzidine dihydrochloride (Sigma-Aldrich) tained under specific pathogen-free conditions at the Hebrew Uni- was used to localize hemoglobin-containing cells. versity Animal Facility (Jerusalem, Israel). All experiments were approved by the Animal Care and Use Committee of the Hebrew University (Jerusalem, Israel). Mice were injected subcutaneously HSC Assay with various doses of T-140 or AMD3100 (1, 2.5, 5, and 10 mg/kg) C57BL/6 mice, serving as donors, were injected with 5 mg/kg of in a total volume of 200 ␮l, 2 hours before sacrifice. In some either T-140 or AMD3100. Two hours later, peripheral blood cells experiments, mice were sacrificed at different time points: 1⁄2,1,2, were collected, followed by erythrocyte lysis (as described above), 4, and 24 hours postinjection. G-CSF was subcutaneously injected and i.v. transferred into C57BL/6 recipient mice that had been at a dose of 2.5 ␮g per mouse twice a day for 4 days. In the pretreated with a lethal dose of irradiation (900 cGy) 24 hours combination experiments, 18 hours after the last injection of G- before. Total cells obtained from 900 or 225 ␮l of blood were CSF, mice were injected with either T-140 or AMD3100. Control inoculated into a single recipient mouse in a total volume of 200 ␮l mice were injected with phosphate-buffered saline (PBS) at the of PBS. Recipient mice that were inoculated with cells obtained appropriate volume. from blood of untreated mice or with normal BM cells (5 ϫ 106 cells per mouse) served as controls. The survival of mice was Cell Isolation and Differential Counts monitored for 4 months. Four months after the first transplantation, Peripheral blood cells were collected from mice by cardiac puncture BM cells recovered from the first recipient mice repopulated by into tubes with heparin followed by lysis of erythrocyte population donor cells were injected i.v. into lethally irradiated secondary mice. using a red blood cell lysis solution (0.155 M NH Cl, 0.01 M 4 Statistical Analysis KHCO3, 0.01 mM EDTA; pH 7.4). Cells were counted using a hemocytometer, and total number of cells per 1 ml of blood was Results are expressed as average Ϯ SD. Statistical differences were calculated. In some experiments, blood samples were resuspended determined by an analysis of two-tailed Student’s t test. Values of and processed to cytospin slides, centrifuged, air-dried, and stained p Ͻ .05 were considered to be statistically significant. with Giemsa. The percentage and the morphology of mobilized cells To evaluate whether the effect we observed for the combined were determined by differential counts of Giemsa-stained cytospin treatments was synergistic, we used Student’s t-test and compared slides. the sum of values obtained for each different treatment (estimated additive effect) and the values obtained for the combined treatment. Flow Cytometry Synergistic effect was defined as an effect that was significantly Ͻ Flow cytometry was used to assess the number of cells in blood and greater (p .05) than the sum of the individual effects. distinguish between the different populations. Cells were gated according to forward scatter and side scatter to exclude dead cells and to determine granulocytes, mononuclear cells, and mature mac- RESULTS rophage populations (Fig. 2E). The number of each cell population was counted. Cells were also stained in 0.1 ml of fluorescence- activated cell sorting (FACS) buffer with fluorescence antibodies T-140 Administration Induces White Blood Cell directed against Gr-1, CD3, mac-1, F4/80, natural killer 1.1 Mobilization (NK1.1), B220, and Ter-119 molecules or matched isotype controls We evaluated the effect of T-140 on mobilization of cells to the (all from eBioscience, San Diego, CA, http://www.ebioscience.com) blood of intact mice and analyzed the kinetics and the dose- for 30 minutes and washed with FACS buffer. Immunostained cells were analyzed by flow cytometry using the FACSCaliber flow cytom- response of this effect. First, mice were injected with 5 mg/kg eter (BD Biosciences, San Diego, http://www.bdbiosciences.com); the T-140, and the mobilization of white blood cells (WBC), at data were analyzed using software from CellQuest (version 3.3; BD various time points postinjection, was examined. At 1⁄2,1,2,4, Biosciences). and 24 hours after injection of T-140, mice were sacrificed and www.StemCells.com 2160 Unique CXCR4-Dependent Mobilization

Figure 1. Time-dependent mobilization of WBC 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/6 peripheral blood. Cells were counted using a hemocytometer, and the total number of

cells per 1 ml of blood was calculated. (B): Downloaded from Fluorescence-activated cell sorting analysis was performed to distinguish between differ- ent populations of the cells. The cells were gated according to forward scatter and side scatter, and the subpopulations of neutro- phils, MNCs, and mature macrophages were defined (see Fig. 2E). The numbers of neu- trophils (B), MNCs (C), and mature macro- www.StemCells.com phages (D) following T-140 administration were analyzed. The data shown are the av- erage Ϯ SD of six mice per group from a total of two separate experiments performed -p Ͻ .05). Abbreviations: MNC, mono ,ء) nuclear cell; T-140, 4F-benzoyl-TN14003; WBC, white blood cells. at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009 cells were collected from blood. A single-dose administration of T-140 Synergizes with G-CSF to Mobilize WBC 5 mg/kg T-140 resulted in a rapid elevation in the total number Recently, the CXCR4 antagonist AMD3100 was reported to of WBC in blood, a threefold change from the control after 30 promote mobilization of stem cells and was found to synergis- minutes, slowly decreasing until full return to the baseline level tically augment G-CSF-induced mobilization of HPCs [9, 14]. after 24 hours (Fig. 1A). The characterization of the mobilized Thus, we evaluated the mobilizing capability of T-140 in com- WBC, using a flow cytometer, showed alteration in the absolute parison with G-CSF, AMD3100, and the combination of both number of granulocytes (stained positive with Gr-1), mononu- CXCR4 antagonists with G-CSF. Similarly to T-140, injection clear cells (MNCs), and mature macrophages (stained positive of AMD3100 (s.c.) induced the mobilization of WBC after 2 with both mac-1 and F4/80 [13]). Further characterization using hours at the optimal concentration of 5 mg/kg. Both T-140 and cytospin assay and Giemsa staining defined the granulocyte AMD3100 induced a 2.5-fold increase in the number of WBC in population as neutrophils. When we looked at the number of blood compared with control. When G-CSF was injected sub- neutrophils in blood, there was an elevation starting 30 minutes cutaneously at a dosage of 2.5 ␮g per mouse twice a day for 4 postinjection, reaching a peak after 1–2 hours (more than a days, there was a 1.9-fold increase in the number of WBC in 12-fold change from control), and slowly decreasing to the blood. To study the combination of G-CSF and either T-140 or baseline level after 24 hours (Fig. 1B). The same pattern was AMD3100, the CXCR4 antagonists were injected at a concen- observed for mature macrophages; these cells reached a peak 1 tration of 5 mg/ml, 18 hours after the fourth injection of G-CSF; hours postinjection of T-140 and returned to the baseline level 2 hours later, mice were sacrificed, and the number and type of after 24 hours (Fig. 1D). When we tested the effect of T-140 on cells mobilized in blood were tested. the mobilization of MNCs, we observed a twofold rapid eleva- The combination of T-140 with G-CSF induced a 5.1-fold tion change from control after 30 minutes, which rapidly de- increase in the number of WBC in blood (Fig. 3A). This induc- creased until full return to the baseline level after 4 hours (Fig. tion was significantly higher than the increase seen in WBC 1C). stimulated by a combination of AMD3100 and G-CSF (3.7-fold To determine the effect of different doses of T-140, concen- increase over control; p ϭ .006; Fig. 3A). trations of 1, 2.5, 5, and 10 mg/kg were subcutaneously injected Next, we analyzed the relative number of neutrophils, into mice. Two hours postinjection, mice were sacrificed, and MNCs, and mature macrophages in blood of treated mice. We the total number of WBC in 1 ml of blood was measured. T-140 found that T-140 and AMD3100 increased the mobilization of induced the mobilization of WBC in a dose-dependent manner. neutrophils by 6.1-fold and 8-fold over control, respectively Peak mobilization of WBC occurred at a dose of 5 mg/kg of (p Ͻ .05), whereas G-CSF alone stimulated no more than a T-140 (Fig. 2A). When we looked at the different populations, 4.4-fold increase in the number of neutrophils over control. we found a similar dose-response elevation in the absolute Combination of T-140 or AMD3100 with G-CSF synergistically number of neutrophils (8-fold increase), mature macrophages increased the number of neutrophils in blood (13.3- and 11.7- (6-fold increase), and MNCs (3.5-fold increase) in the blood fold increase over control, respectively; p Ͻ .005; Fig. 3C). following treatment with 10 mg/kg of T-140 (Fig. 2B–2D, T-140 and AMD3100 induced a significant and similar fold respectively). change in the number of MNCs in blood compared with control To further identify the different populations of MNCs that (2.3- and 3.4-fold increase over control, respectively; p Ͻ .005). were mobilized by T-140, we stained the cells with specific Comparison with both CXCR4 antagonists showed that G-CSF, antibodies for T cells (CD3), B cells (B220), Mac-1ϩ cells, and in our experimental model, is an inferior mobilizer of MNCs NK cells (NK1.1) (Fig. 2E). As shown in Figure 2F, T-140 had (Fig. 3B, 3D). The combination of G-CSF with T-140 induced an effect on the mobilization of T cells, B cells, and Mac-1ϩ a synergistic increase in the number of MNCs (4-fold increase cells but had no effect on the mobilization of NK cells. We did over control), whereas the combination of G-CSF with not observed any significant alteration in the number of platelets AMD3100 did not induce such an effect (Fig. 3D). T-140 and or other coagulation factor in blood following treatment with AMD3100 also induced a significant change in the number of T-140 (data not shown). mature macrophages in blood compared with control (4.2- and Abraham, Biyder, Begin et al. 2161 Downloaded from www.StemCells.com at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009

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 counted using 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 mature macrophages (R1), neutrophils (R2), and MNCs (R3) is shown. Mature macrophages were further identified by staining for MAC-1 and F4/80 expression; neutrophils were also identified by staining for Gr-1. To analyze the MNC subpopulations, T cells were stained for CD3. NK cells were stained for NK1.1, but no CD3 or B cells were identified by staining for B220. (F): T-140 administration induced the mobilization of monocytes, T p Ͻ ,ء) cells, and B cells but not NK cells. The data are the average Ϯ SD of six mice per group from a total of two separate experiments performed .05). Abbreviations: FSC, forward scatter; MNC, mononuclear cell; NK, natural killer; SSC, side scatter; T-140, 4F-benzoyl-TN14003.

6.1-fold increase over control, respectively; p Ͻ .05). Compar- of myeloid CFU-GM colonies and the mobilization of multipo- ison with both CXCR4 antagonists showed that G-CSF is also a tent progenitors CFU-M, CFU-GEMM and the erythroid pro- poor mobilizer of mature macrophages (Fig. 3B, 3E). Similarly genitors BFU-E. Interestingly, we found that the most dramatic to the effect on MNCs, the combination of G-CSF with T-140 increase in progenitors was observed in the number of BFU-E, induced a synergistic increase in the number of MNCs (8.3-fold reaching a significant increase of more than 110-fold over increase over control; p Ͻ .005), whereas the combination of control (p Ͻ .01; Fig. 4B). G-CSF with AMD3100 did not induce such an effect (Fig. 3E). Mobilization of progenitor cells started after 30 minutes and These results show that the CXCR4 antagonists are capable of reached a peak 1 to 2 hours postinjection of T-140 (Fig. 4C, 4D; Ͻ mobilizing different subtypes of cells compared with G-CSF 9.5- and 13.6-fold increases over control, respectively; p .01). and that T-140, but not AMD3100, synergized with G-CSF to This elevation in the number of progenitor cells in blood almost disappeared 24 hours postinjection. The most dramatic effect further stimulate the mobilization of MNCs and mature macro- was observed in the mobilization of BFU-E, where the number phages. of progenitors was increased 200-fold 1 hour after injection of T-140 (Fig. 4D). Staining mobilized cells with a marker for Effects of T-140 Administration on Progenitor Cell proerythroblasts, known as Ter119, showed that administration Mobilization of T-140 caused a 3.2-fold elevation over control in the number To further study the effect of T-140 on mobilization of HPCs, of proerythroblasts in blood (supplemental online Fig. 2). we collected cells from the blood of treated and untreated mice and tested for the presence of colony-forming cells. Injection of T-140 Synergizes with G-CSF to Mobilize HPCs T-140 at a concentration between 1 and 10 mg/kg induced a Blocking the CXCR4 receptor with AMD3100 resulted in the dose-response increase (up to 10-fold over control) in the num- mobilization of HPCs [9, 15, 16]. When we compared the effect ber of progenitor cells in blood (Fig. 4A; p Ͻ .005). Treatment of T-140 (5 mg/kg) on the mobilization of progenitor cells to of mice with T-140 induced a robust dose-response mobilization that of AMD3100 (5 mg/kg), we found that T-140 was signif- www.StemCells.com 2162 Unique CXCR4-Dependent Mobilization Downloaded from www.StemCells.com at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009

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 hours 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 blood cells were obtained. (A): Total number of WBC in blood was counted by fluorescence-activated cell sorting (FACS). (B): A representative FACS analysis of 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 were p Ͻ .05 of GCSFϩT-140 compared with GCSFϩAMD3100; ⌬, p Ͻ .05 of T-140 compared ,ءء ;p Ͻ .05 compared with control ,ء .analyzed with AMD3100. The data are the average Ϯ 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 blood cells. icantly more potent in its ability to mobilize progenitor cells into GEMM colonies mobilized with G-CSF alone or in combi- blood. T-140 increased the number of progenitor cells in blood nation with the CXCR4 antagonists (Fig. 5C). T-140 was by 7.1-fold over control (p Ͻ .05), whereas AMD3100 induced significantly more potent than AMD3100 in its ability to the mobilization of progenitor cells by 4.2-fold over control mobilize CFU-GEMM (44- vs.15.5-fold increase, respec- (p ϭ .001). G-CSF induced a 13.6-fold increase in the number tively; p Ͻ .05). G-CSF alone induced a 151-fold increase of progenitors in blood over control, whereas in combination over control (p Ͻ .01), whereas its combination with T-140 with T-140, it induced a 76.8-fold elevation in the number of induced a 712-fold increase over control (p Ͻ .01). This progenitors over control. This progenitor increase induced by a elevation was significantly higher than that induced by the combination of G-CSF and T-140 was significantly higher than combination of G-CSF with AMD3100 (a 266-fold increase the synergistic induction in the number of progenitors by the over control; p Ͻ .01; Fig. 5C). These results suggest that combination of G-CSF with AMD3100 (76.8-compared with T-140 synergized more efficiently with G-CSF to induce the 46.4-fold over control; p ϭ .001; Fig. 5A). mobilization of early progenitors. The effect of CXCR4 antagonists alone and in combination To determine whether the effects we observed were spe- with G-CSF on the mobilization of BFU-E and GEMM progen- cifically related to the activity of T-140, we used the neu- itor cells was further characterized. T-140 and AMD3100 alone tralized T-140 treated with proteinase K and compared its induced a similar increase in the number of BFU-E in blood effect with the natural T-140. We found that treatment with (nearly 30-fold change from control; p Ͻ .05), whereas G-CSF proteinase K digests the peptide into smaller fragments, as alone induced a 142-fold increase in BFU-E in blood over demonstrated in supplemental online Figure 3A and 3B. control (Fig. 5B). Coinjection of AMD3100 with G-CSF was These fragments did not inhibit the migration of Jurkat T neither additive nor synergistic (142-fold increase with G-CSF cells toward CXCL12, the ligand for CXCR4, and could not vs. 98-fold increase with combination treatment). However, induce the mobilization of WBC or progenitor cells (supple- G-CSF, in combination with T-140, induced a significant ele- mental online Fig. 3C–3E, respectively). These results dem- vation of BFU-E (142-fold increase with G-CSF vs. 660-fold onstrate that the mobilizing effect we observed was specifi- increase with combination treatment; p Ͻ .01; Fig. 5B). Similar cally related to presence of the intact T-140 peptide capable results were observed when we analyzed the number of CFU- of interacting with CXCR4. Abraham, Biyder, Begin et al. 2163 Downloaded from www.StemCells.com at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009

Figure 4. Time- and dose-response effect of T-140 on mobilization of hematopoietic progenitor cells into blood. (A, B): Dose-response analysis at 2 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 of colony-forming units (CFU), CFU-M (colony-forming cells granulocyte-macrophage [CFC-GM]), CFU-GEMM, or BFU-E in blood was evaluated by 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 as ,p Ͻ .05). Abbreviations: BFU-E ,ء) control. The data are the average Ϯ SD of six mice per group from a total of two separate experiments performed 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. T-140 Mobilizes HSCs with Long-Term T-140 is a powerful mobilizer of long-term repopulating stem Repopulating Capacity cells. Although mobilization of HPCs may be of use for short-term repopulation in a transplant setting, HSCs are required for DISCUSSION long-term repopulation. To assess the T-140 effect on mobi- lization of murine HSCs, we used a transplantation model in which donor C57Bl/6 mice were treated with T-140 or Autologous and allogeneic stem cell transplantation has AMD3100. After 2 hours, blood was collected, and cells that emerged as a preferred strategy in the treatment of a variety of hematological malignancies [17, 18]. In recent years, the use of were isolated from an equal blood volume (900 or 225 ␮l) peripheral blood as a source of HSCs for transplantation fol- were transferred into lethally irradiated recipients C57Bl/6 lowed by a high dose of chemotherapy has emerged into com- mice. Survival of recipient mice, as indicated by long-term mon clinical practice [19]. Successful blood and marrow trans- repopulating activity and presence of HSCs, was monitored. plants require the infusion of a sufficient number of HSCs As shown in Figure 6A, 100% of mice transplanted with ␮ capable of homing to the marrow cavity and regenerating a full cells mobilized by T-140 (900 l of blood) survived the array of hematopoietic cell lineages. Thus, an increase in the treatment. In contrast, only 73% of mice transplanted with number of hematopoietic cells in blood will improve the yield of cells that were mobilized by AMD3100 survived the treat- cell collection for transplantation and will also have the poten- ϭ ment (p .06; Fig. 6A). Transplantation of cells mobilized tial to shorten recovery from cytopenia and reduce morbidity ␮ by T-140 using a blood volume of 225 l resulted in the and mortality. survival of 70.6% of the treated mice, compared with the G-CSF was first identified as a colony-stimulating factor for survival of only 37.5% of the treated mice when mobilization granulocyte precursors and as a stimulator of granulocyte dif- was performed by AMD3100 (p Ͻ .05). Although all un- ferentiation and activation [20]. Due to its activity, G-CSF is treated mice died after irradiation, 100% of mice survived used to reduce neutropenia and increase the production of neu- irradiation when normal BM cells had been transplanted (Fig. trophils [21, 22]. In addition to inducing the production and 6A, 6B). To test for self-renewal and long-term repopulating mobilization of neutrophils, G-CSF is also a powerful mobilizer activity, the transplanted mice were maintained for more than of HSCs. Although several cytokines, such as GM-CSF, IL-3, 4 months, and then their BM cells were injected into a second stem cell factor, and flt-3 ligand, and chemokines, such as IL-8, recipient. Stem cells mobilized by T-140 maintained 100% of were also shown to mobilize HPCs and HSCs [23–25], the the transplanted mice for longer than 4 months and were cytokine G-CSF remains the major growth factor used for capable of producing long-term rescue in lethally irradiated mobilization of stem cells for transplantation. The current pro- secondary transplanted mice. These results indicate that tocols for stem cell mobilization necessitate 4–5 days of 1–2 s.c. www.StemCells.com 2164 Unique CXCR4-Dependent Mobilization Downloaded from www.StemCells.com at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009

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. Eighteen hours 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 blood cells 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-GEMM p Ͻ ,ءء ;p Ͻ .05 compared with control ,ء .was tested by staining the colonies with benzidine dihydrochloride to detect hemoglobin-containing cells .05 of GCSFϩT-140 compared with GCSFϩAMD3100; ⌬, p Ͻ .05 of T-140 compared with AMD3100. The data are the average Ϯ SD of four mice per 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.

mechanism by activating neutrophils within the BM. Indeed, it was recently demonstrated that G-CSF stimulates neutrophils to secrete 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 the chemokine receptor CXCR4, a key molecule that is expressed by the BM stroma and plays an important role in the retention of stem cells expressing CXCR4 within the BM [7, 26]. An important role for the interaction between CXCL12 and its receptor CXCR4 has been described for the mobilization of hematopoietic progenitors and stem cells [5–7]. More specifi- cally, AMD3100, a selective antagonist of CXCR4, was recently shown to rapidly mobilize CD34ϩ HPCs from the human marrow into the peripheral blood. Furthermore, AMD3100 sig- nificantly increased both G-CSF-stimulated (10 ␮g/kg per day) Figure 6. T-140 mobilizes hematopoietic stem cells with long-term ϩ repopulating capacity that can rescue mice following lethal irradiation. mobilization of CD34 cells (3.8-fold) and leukapheresis yield C57BL/6 mice serving as donors were injected with 5 mg/kg of either of CD34ϩ cells. Moreover, AMD3100-mobilized leukapheresis T-140 or AMD3100. Two hours later, peripheral blood cells were products contained significantly greater numbers of T and B collected and transplanted into C57BL/6 recipient mice that were le- cells compared with G-CSF-stimulated leukapheresis products thally irradiated (900 cGy) 24 hours before i.v. injection of the cells. [27]. AMD3100 was also reported to mobilize HSCs with long- Cells obtained from 900 (A) or 225 (B) ␮l of blood were transplanted term repopulating capacity in humans [28] and nonhuman pri- into single recipient mice. As a control, recipient mice were also mates [29]. In addition, AMD3100 was reported to be a safe and transplanted with cells obtained from blood of untreated mice or with effective agent for the mobilization of CD34ϩ cells in patients p Ͻ .06 of T-140 compared with AMD3100 when ,ء .normal BM cells -p Ͻ .05 of T-140 compared with with multiple myeloma or lymphoma receiving prior chemo ,ءء ;␮l was transplanted 900 AMD3100 when 225 ␮l was transplanted. Data are the average of 17 therapy [30, 31]. On the other hand, a clinical trial with mice per group from a total of four separate experiments performed. AMD3100 in HIV-infected individuals produced premature Abbreviations: AMD, AMD3100; BM, bone marrow; T-140, 4F-ben- ventricular contraction side effects, resulting in the discontinu- zoyl-TN14003. ation of this trial [27]. On the basis of this knowledge, we evaluated the ability of injections of G-CSF; moreover, one or more pheresis procedures a novel specific CXCR4 antagonist, known as T-140, to mobi- are required because of failure to collect the number of CD34ϩ lize hematopoietic cells. T-140 and its analogues were first cells necessary for successful transplantation. It is suggested that developed initially as anti-HIV agents that inhibit HIV-1 infec- G-CSF induces the mobilization of HSCs through an indirect tion through their specific binding to CXCR4 [32, 33]. Unlike Abraham, Biyder, Begin et al. 2165

AMD3100, T-140 is a short, modified peptide. Comparative progenitor cells in blood that was superior to G-CSF alone or to studies between T-140 and AMD3100 found that each of these a combination of G-CSF with AMD3100. This superiority was agents inhibited CXCR4 via different mechanisms [34, 35]. highly significant when we witnessed the number of CFU- Whereas T-140 binds residues in extracellular domains of the GEMM and BFU-E in the blood of treated mice. CXCR4 receptor and regions of the hydrophobic core proximal Although improved mobilization of HPCs in response to to the cell surface, amino acids in the central hydrophobic core T-140 is of interest, proof that T-140 also mobilizes HSCs is of are critical to binding of AMD3100 [34]. Analysis of antago- paramount importance. A comparison of the abilities of T-140 and AMD3100 to mobilize HSCs into blood was made using a nists revealed that exposure to AMD3100 induced G-protein Downloaded from activation by CXCR4, whereas T-140 decreased autonomous transplantation model. We found that T-140 is more potent in its signaling [35]. Therefore, AMD3100 was defined to have a ability to mobilize HSCs with long-term repopulating activity weak partial agonist activity, whereas T-140 functions as an compared with AMD3100. inverse agonist. Mobilization of WBC and progenitor cells was synergisti- Our results indicate that T-140 can efficiently induce mobi- cally enhanced when T-140 was used in combination with lization of mature white blood cells, progenitors, and stem cells G-CSF, and this effect was significantly superior to that ob- into blood within a few hours after treatment in a dose-depen- served for the combination of G-CSF with AMD3100. Future www.StemCells.com dent manner and can efficiently synergize with G-CSF for this studies should address the questions about the mechanism(s) by effect. A variety of cell types express the CXCR4 receptor, which T-140 induces cell mobilization, including studies regard- including monocytes/macrophages, B and T lymphocytes, pro- ing the blocking of the CXCR4 receptor and the immunogenic- genitors, and stem cells. Indeed, we found that T-140 can induce ity properties of T-140. mobilization of all of these cell subsets. Although CXCR4 is In summary, our results indicate that the ability of the also expressed by NK cells [36] and platelets [37], we could not CXCR4 antagonist T-140 to mobilize WBC, as well as progen- demonstrate the effect of T-140 on their mobilization. Similarly, itor and stem cells, differs both qualitatively and quantitatively CXCR4/CXCL12 interaction was not found to be involved in from that of the well-characterized CXCR4 antagonist at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009 platelet activation, as demonstrated by the inability of CXCL12 AMD3100. Furthermore, T-140 differs from AMD3100 in its to augment aggregation or stimulate calcium mobilization of ability to efficiently synergize with G-CSF. It is therefore pos- platelets [37]. sible that an improved CXCR4 antagonist will provide better Administration of T-140, AMD3100, and G-CSF can induce methods of collecting mobilized stem cells for transplantation. an effective mobilization of WBC into blood. However, whereas Future studies should address these issues and define the poten- G-CSF induces mainly the exit of neutrophils, both CXCR4 tial clinical advantage and usefulness of T-140 in clinical stem antagonists induced a robust mobilization of MNCs and mature cell mobilization and transplantation. macrophages. T-140 or AMD3100 alone induced the mobilization of a similar number of MNCs and activated macrophages. However, ACKNOWLEDGMENTS the combination of T-140 with G-CSF induced a significant increase in the mobilization of MNCs and mature macrophages We thank Nobutaka Fujii from Kyoto University (Sakyo-ku, into blood compared with AMD3100. Kyoto, Japan) and Mary Clausen from the Gene Therapy Insti- Even though mobilization of WBC is an important factor for tute, Hadassah University Hospital (Jerusalem, Israel), for tech- transplantation, the major factor contributing to transplantation nical assistance. This work supported by a grant from the Lady efficiency is the number of HPCs and HSCs in blood. When we Davis Fellowship Trust. looked at the effect of T-140 on mobilization of progenitor cells, we observed a significant elevation in the number of both DISCLOSURE OF POTENTIAL CONFLICTS myeloid pluripotent and erythroid progenitors, that is, CFU- GM, CFU-M, CFU-GEMM, and BFU-E. A comparative study OF INTEREST demonstrated that administration of a single dose of T-140 resulted in a significant increase of progenitor cell mobilization A.P. owns stock in, has acted as a consultant to, has performed compared with AMD3100. Moreover, the combination of T-140 contract work for, is a Board member for, and has a financial with G-CSF resulted in a dramatic elevation in the number of interest in Biokine Therapeutics.

planted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice. REFERENCES Leukemia 2002;16:1992–2003. 7 Levesque JP, Hendy J, Takamatsu Y et al. Disruption of the CXCR4/ CXCL12 chemotactic interaction during hematopoietic stem cell mobi- 1 Kondo M, Wagers AJ, Manz MG et al. Biology of hematopoietic stem lization induced by GCSF or cyclophosphamide. J Clin Invest 2003;111: cells and progenitors: Implications for clinical application. Annu Rev 187–196. Immunol 2003;21:759–806. 8 Kim CH, Broxmeyer HE. In vitro behavior of hematopoietic progenitor 2 Wright DE, Wagers AJ, Gulati AP et al. Physiological migration of hema- cells under the influence of chemoattractants: Stromal cell-derived fac- topoietic stem and progenitor cells. Science 2001;294:1933–1936. tor-1, steel factor, and the bone marrow environment. Blood 1998;91: 3 Abkowitz JL, Robinson AE, Kale S et al. Mobilization of hematopoietic 100–110. stem cells during homeostasis and after cytokine exposure. Blood 2003; 9 Broxmeyer HE, Orschell CM, Clapp DW et al. Rapid mobilization of 102:1249–1253. murine and human hematopoietic stem and progenitor cells with 4 Roberts AW, DeLuca E, Begley CG et al. Broad inter-individual AMD3100, a CXCR4 antagonist. J Exp Med 2005;201:1307–1318. variations in circulating progenitor cell numbers induced by granu- 10 Princen K, Schols D. HIV chemokine receptor inhibitors as novel anti- locyte colony-stimulating factor therapy. STEM CELLS 1995;13: HIV drugs. Cytokine Growth Factor Rev 2005;16:659–677. 512–516. 11 Tamamura H, Fujii N. The therapeutic potential of CXCR4 antagonists 5 Kollet O, Petit I, Kahn J et al. Human CD34(ϩ)CXCR4(-) sorted cells in the treatment of HIV infection, cancer metastasis and rheumatoid harbor intracellular CXCR4, which can be functionally expressed and arthritis. Expert Opin Ther Targets 2005;9:1267–1282. provide NOD/SCID repopulation. Blood 2002;100:2778–2786. 12 Juarez J, Bradstock KF, Gottlieb DJ et al. Effects of inhibitors of the 6 Lapidot T, Kollet O. The essential roles of the chemokine SDF-1 and its chemokine receptor CXCR4 on acute lymphoblastic leukemia cells in receptor CXCR4 in human stem cell homing and repopulation of trans- vitro. Leukemia 2003;17:1294–1300. www.StemCells.com 2166 Unique CXCR4-Dependent Mobilization

13 Leenen PJ, de Bruijn MF, Voerman JS et al. Markers of mouse macro- 26 Petit I, Szyper-Kravitz M, Nagler A et al. G-CSF induces stem cell phage development detected by monoclonal antibodies. J Immunol mobilization by decreasing bone marrow SDF-1 and up-regulating Methods 1994;174:5–19. CXCR4. Nat Immunol 2002;3:687–694. 14 Flomenberg N, Devine SM, Dipersio JF et al. The use of AMD3100 plus 27 Hendrix CW, Collier AC, Lederman MM et al. Safety, pharmaco- G-CSF for autologous hematopoietic progenitor cell mobilization is kinetics, and antiviral activity of AMD3100, a selective CXCR4 receptor superior to G-CSF alone. Blood 2005;106:1867–1874. inhibitor, in HIV-1 infection. J Acquir Immune Defic Syndr 2004;37: 15 Flomenberg N, DiPersio J, Calandra G. Role of CXCR4 chemokine 1253–1262. receptor blockade using AMD3100 for mobilization of autologous he- 28 Burroughs L, Mielcarek M, Little MT et al. Durable engraftment matopoietic progenitor cells. Acta Haematol 2005;114:198–205. of AMD3100-mobilized autologous and allogeneic peripheral-blood

16 Liles WC, Broxmeyer HE, Rodger E et al. Mobilization of hematopoietic mononuclear cells in a canine transplantation model. Blood 2005;106: Downloaded from progenitor cells in healthy volunteers by AMD3100, a CXCR4 antago- 4002–4008. nist. Blood 2003;102:2728–2730. 29 Larochelle A, Krouse A, Metzger M et al. AMD3100 mobilizes hema- 17 Hartmann O, Le Corroller AG, Blaise D et al. Peripheral blood stem cell topoietic stem cells with long-term repopulating capacity in nonhuman and bone marrow transplantation for solid tumors and lymphomas: primates. Blood 2006;107:3772–3778. Hematologic recovery and costs. A randomized, controlled trial. Ann 30 Devine SM, Flomenberg N, Vesole DH et al. Rapid mobilization of Intern Med 1997;126:600–607. CD34ϩ cells following administration of the CXCR4 antagonist 18 Schmitz N, Linch DC, Dreger P et al. Randomised trial of filgrastim- AMD3100 to patients with multiple myeloma and non-Hodgkin’s lym- mobilised peripheral blood progenitor cell transplantation versus autol-

phoma. J Clin Oncol 2004;22:1095–1102. www.StemCells.com ogous bone-marrow transplantation in lymphoma patients. Lancet 1996; 31 Grignani G, Perissinotto E, Cavalloni G et al. Clinical use of AMD3100 347:353–357. to mobilize CD34ϩ cells in patients affected by non-Hodgkin’s lym- 19 Beyer J, Schwella N, Zingsem J et al. Hematopoietic rescue after phoma or multiple myeloma. J Clin Oncol 2005;23:3871–3872; com- high-dose chemotherapy using autologous peripheral-blood progenitor ment 3872–3873. cells or bone marrow: A randomized comparison. J Clin Oncol 1995;13: 1328–1335. 32 Tamamura H, Fujii N. Two orthogonal approaches to overcome multi- 20 Zarco MA, Ribera JM, Urbano-Ispizua A et al. Phenotypic changes in drug resistant HIV-1s: Development of protease inhibitors and entry neutrophil granulocytes of healthy donors after G-CSF administration. inhibitors based on CXCR4 antagonists. Curr Drug Targets Infect Disord Haematologica 1999;84:874–878. 2004;4:103–110. 33 Fujii N, Tamamura H. Peptide-lead CXCR4 antagonists with high anti-

21 Dale DC. Colony-stimulating factors for the management of neutropenia at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009 in cancer patients. Drugs 2002;62(suppl 1):1–15. HIV activity. Curr Opin Investig Drugs 2001;2:1198–1202. 22 Bedell C. Pegfilgrastim for chemotherapy-induced neutropenia. Clin J 34 Trent JO, Wang ZX, Murray JL et al. Lipid bilayer simulations of Oncol Nurs 2003;7:55–56, 63–64. CXCR4 with inverse agonists and weak partial agonists. J Biol Chem 23 Glaspy JA, Shpall EJ, LeMaistre CF et al. Peripheral blood progenitor 2003;278:47136–47144. cell mobilization using stem cell factor in combination with filgrastim in 35 Zhang WB, Navenot JM, Haribabu B et al. A point mutation that confers breast cancer patients. Blood 1997;90:2939–2951. constitutive activity to CXCR4 reveals that T140 is an inverse agonist 24 Huhn RD, Yurkow EJ, Tushinski R et al. Recombinant human interleu- and that AMD3100 and ALX40-4C are weak partial agonists. J Biol kin-3 (rhIL-3) enhances the mobilization of peripheral blood progenitor Chem 2002;277:24515–24521. cells by recombinant human granulocyte colony-stimulating factor (rhG- 36 Maghazachi AA. G protein-coupled receptors in natural killer cells. CSF) in normal volunteers. Exp Hematol 1996;24:839–847. J Leukoc Biol 2003;74:16–24. 25 Molineux G, McCrea C, Yan XQ et al. Flt-3 ligand synergizes with 37 Kowalska MA, Ratajczak J, Hoxie J et al. Megakaryocyte precursors, granulocyte colony-stimulating factor to increase neutrophil numbers and megakaryocytes and platelets express the HIV co-receptor CXCR4 on to mobilize peripheral blood stem cells with long-term repopulating their surface: Determination of response to stromal-derived factor-1 by potential. Blood 1997;89:3998–4004. megakaryocytes and platelets. Br J Haematol 1999;104:220–229.

See www.StemCells.com for supplemental material available online. Enhanced Unique Pattern of Hematopoietic Cell Mobilization Induced by the CXCR4 Antagonist 4F-Benzoyl-TN14003 Michal Abraham, Katia Biyder, Michal Begin, Hanna Wald, Ido D. Weiss, Eithan Galun, Arnon Nagler and Amnon Peled Stem Cells 2007;25;2158-2166; originally published online May 24, 2007; DOI: 10.1634/stemcells.2007-0161 This information is current as of April 18, 2009 Downloaded from

Updated Information including high-resolution figures, can be found at: & Services http://www.StemCells.com/cgi/content/full/25/9/2158 Supplementary Material Supplementary material can be found at: http://www.StemCells.com/cgi/content/full/2007-0161/DC1 www.StemCells.com at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009