Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid ... · Furthermore, in the LP products mobilized and collected after BL-8040, there were much highernumberofCD4 þCD8 T,NKT,NK,anddendriticcells,

Cancer Therapy: Clinical

Single Dose of the CXCR4 Antagonist BL-8040Induces Rapid Mobilization for the Collectionof Human CD34þ Cells in Healthy VolunteersMichal Abraham1, Yaron Pereg2, Baruch Bulvik1, Shiri Klein3, Inbal Mishalian3,Hana Wald1, Orly Eizenberg1, Katia Beider4, Arnon Nagler4, Rottem Golan2,Abi Vainstein2, Arnon Aharon2, Eithan Galun3, Yoseph Caraco5, Reuven Or6,and Amnon Peled3,4

Abstract

Purpose: The potential of the high-affinity CXCR4 antagonistBL-8040 as a monotherapy-mobilizing agent and its derivedgraft composition and quality were evaluated in a phase I clinicalstudy in healthy volunteers (NCT02073019).

Experimental Design: The first part of the study was a ran-domized, double-blind, placebo-controlled dose escalationphase. The second part of the study was an open-label phase, inwhich 8 subjects received a single injection of BL-8040 (1 mg/kg)and approximately 4 hours later underwent a standard leukapher-esis procedure. The engraftment potential of the purified mobi-lized CD34þ cells was further evaluated by transplanting the cellsinto NSG immunodeficient mice.

Results: BL-8040 was found safe and well tolerated at alldoses tested (0.5–1mg/kg). The main treatment-related adverseevents were mild to moderate. Transient injection site and

systemic reactions were mitigated by methylprednisolone,paracetamol, and promethazine pretreatment. In the first partof the study, BL-8040 triggered rapid and substantial mobili-zation of WBCs and CD34þ cells in all tested doses. Four hourspostdose, the count rose to a mean of 8, 37, 31, and 35 cells/mL(placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysisrevealed substantial mobilization of immature dendritic, T, B,and NK cells. In the second part, the mean CD34þ cells/kgcollected were 11.6 � 106 cells/kg. The graft composition wasrich in immune cells.

Conclusions: The current data demonstrate that BL-8040is a safe and effective monotherapy strategy for the collectionof large amounts of CD34þ cells and immune cells in a one-dayprocedure for allogeneic HSPC transplantation. Clin Cancer Res;23(22); 6790–801. �2017 AACR.

IntroductionAllogenic hematopoietic stem and progenitor cell (HSPC)

transplantation (ALSPCT) has emerged as the preferred strategyin the treatment of a variety of hematologic malignancies (1, 2).Mobilization of stem cells using granulocyte colony-stimulat-ing factor (G-CSF) from healthy donors is the common clinicalpractice. G-CSF–mobilized peripheral blood mononuclearcells (PBMC) are routinely used as a source of hematopoieticstem cells (HSC) for transplantation (3, 4). Despite the potencyof G-CSF in mobilizing stem cells, it ultimately results in broad

interindividual variations in circulating progenitor and stemcell numbers (5), requiring 4–6 repeated dosing to collect asufficient number of cells. In addition, although consideredgenerally safe, G-CSF is frequently associated with a varietyof side effects. Therefore, improved methods to mobilize andcollect HSPCs for transplantation are required.

It has been proposed that G-CSF induces the mobilization ofHSPCs through an indirect mechanism by activating neutrophilsto secrete a variety of proteolytic enzymes, including elastase,cathepsinG,MMP-2, andMMP-9 that can degrade the chemokineCXCL12 and its receptor CXCR4. Over recent years, it has becomeapparent that the interaction between CXCL12 and its receptor,CXCR4, plays a pivotal role in hematopoietic stem cell mobili-zation and engraftment (6–8). Consequently, disruption ofCXCL12/CXCR4 interactions results in mobilization of hemato-poietic stem and progenitor cells from the bone marrow to theperipheral blood system.

Indeed, blockade of the CXCR4 receptor with the reversibleCXCR4 antagonist AMD3100 (Plerixafor; Mozobil) results inrapid mobilization of HSPCs (9, 10). When AMD3100 as a singleagent was compared with G-CSF as a mobilizer of CD34þ cellsin healthy volunteers, AMD3100 was inferior to G-CSF (5).However, AMD3100 increased both G-CSF–stimulated mobili-zation and the leukapheresis yield of CD34þ cells. As such,Mozobil was approved in combination with G-CSF for the mobi-lization of CD34þ cells in patients with lymphoma and multiplemyeloma that undergo stem cell mobilization (11, 12).

1Biokine Therapeutics Ltd, Ness Ziona, Israel. 2BioLineRx LTD, Modi'in, Israel.3Goldyne Savad Institute of Gene Therapy, Hebrew University Hospital, Jeru-salem, Israel. 4Hematology Division, Chaim Sheba Medical Center and Tel AvivUniversity, Tel-Hashomer, Israel. 5Clinical Pharmacology Unit, Hadassah Uni-versity Hospital, Jerusalem, Israel. 6Cancer Immunotherapy and ImmunobiologyResearch Center, Hadassah University Hospital, Jerusalem, Israel.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M. Abraham and Y. Pereg contributed equally to this article.

Corresponding Author: Amnon Peled, Hadassah University Hospital, P.O Box12000, Jerusalem, Israel. Phone: 972-2677-8780; Fax: 972-26430982; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-16-2919

�2017 American Association for Cancer Research.

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BL-8040 (BKT140) demonstrates a higher affinity andlonger receptor occupancy for CXCR4 and provides a greatereffect on the retention–mobilization balance of bone marrowSCs when compared with AMD3100 in both in vitro and in vivomice studies (13–15).

This study investigated the capacity of BL-8040 to mobi-lize and retain CD34þ cells in healthy volunteers, hypothe-sizing that a single day procedure of BL-8040 monotherapyadministration (single injection) followed by one apheresissession will provide sufficient amounts of CD34þ HSPCs fortransplantation.

Materials and MethodsClinical study

A phase I, two-part study exploring the safety, tolerability,pharmacodynamic, and pharmacokinetic effects of ascendingdoses of BL-8040 in healthy subjects (study BL-8040.02) afterinformed consent was obtained. The study was conducted at theHadassah Clinical Research Center (HCRC), Hadassah MedicalCenter, Jerusalem, Israel and approved by the Human SubjectsCommittee Institutional Review Boards of Hadassah MedicalCenter, Jerusalem, Israel. All subjects gave informed consent toparticipate in the study, whichwas approved by local InstitutionalReview Boards and conducted in accordance with the ethicalprinciples of the Declaration of Helsinki.

Patients and methodsThe study had two parts: part 1 (dose escalation) was a

randomized, double-blind, placebo-controlled study exploringthe safety, tolerability, and the pharmacodynamic and phar-macokinetic profiles of BL-8040 injected subcutaneously atdoses of 0.5, 0.75, and 1 mg/kg. Individuals who received thedose of 0.5 mg/kg (n ¼ 6) and their placebo (n ¼ 2) werenumbered 1001–1008. Individuals who received the dose of0.75 mg/kg (n ¼ 6) and their placebo (n ¼ 2) were numbered2001–2008 and individuals who received the dose of 1 mg/kg(n ¼ 6) and their placebo (n ¼ 2) were numbered 3001–3008.WBCs and CD34þ cell mobilization were measured in healthysubjects following administration of BL-8040 (once daily on

two consecutive days). Part 1 of the study served to select theoptimal safe and efficacious dose of BL-8040 to be used in part2. Part 2 (dose expansion) was an open-label study, exploringthe safety, tolerability, and pharmacodynamic effect of BL-8040in a single cohort of healthy subjects who received the selecteddose regimen of BL-8040 (1 mg/kg) based on the data collectedfrom part 1 (numbered 5001–5008). In addition, subjectsunderwent leukapheresis to examine the yield and character-istics of the mobilized cells. Each cohort in part 1 consisted of8 subjects; 6 subjects in each cohort randomly allocated toreceive BL-8040 and 2 subjects to receive placebo. Part 2involved a single cohort of 8 subjects, who received BL-8040at the selected optimal dose level.

Eligibility criteriaAs this was a dose escalation study in healthy volunteers,

men only selection was for safety reasons to exclude exposureof childbearing potential subjects. The main criteria for inclu-sion for this study were: healthy male subjects aged between18 and 45 years, with body mass index (BMI) between 18 and30 kg/m2 and weight � 60 kg. In addition, subjects had to beeither surgically sterilized (vasectomy), or if their partner was ofchildbearing potential, had to use two methods of contracep-tion, one of which had to be a barrier method, from the firstdose until 3 months after the last dose. All the subjects wereCaucasian males.

After providing an informed consent, adult male subjectsages 18–45 years old were screened for study eligibility byassessment of inclusion and exclusion criteria. Inclusion criteriaconsisted of a BMI measure between 18 and 30 kg/m2 andweight � 60 kg. The subjects were healthy as indicated by theirmedical history, physical examination, 12-lead electrocardio-gram (ECG), and laboratory safety tests. Screening proceduresincluded the collection of demographic data, medical history,physical examination [including height, weight and body massindex (BMI)], vital signs (blood pressure, pulse rate, respirationrate and oral temperature), 12-lead electrocardiogram (ECG),and safety laboratory evaluations [hematology, biochemistry,coagulation (PT/INR and aPTT)] and urinalysis.

Determination of blood counts and FACS analysisWBCs and differential counts, immunophenotyping for neu-

trophils, T, B, NK cells, CD34þ cell counts, and expression ofCXCR4 using the 12G5 mAb were assessed for part 1 and 2 byFACS analysis. Immunophenotyping of peripheral blood and ofcells collected by the leukapheresis (exploratory endpoint) wereassessed by FACS analysis for the following surface markers:CD34, CD16, CD56, CD3, CD4, CD8, CD19, CD11c, CD83,CD25, Foxp3, CXCR4 (part 2). Yields of hematopoietic progen-itor cells were tested by a methylcellulose medium with recom-binant cytokines and EPO for human cells (MethoCult H4435;StemCell Technologies Inc.)

The expression of CXCR4onmobilized CD34þ fromBL-8040–mobilized cells and from G-CSF–mobilized cells was done fol-lowing staining with two different CXCR4 antibody clones: 12G5(binds to the second extracellular loops) or 1D9 (binds to theN-terminal portion). Controls were incubated with appropriateisotype controls.

BL-8040–mobilized-CD34þ cells (n ¼ 4) and G-CSF–mobi-lized-CD34þ cells (n¼ 4) were stained with mAbs against CD34,

Translational Relevance

Allogeneic hematopoietic stem and progenitor cell (HSPC)transplantation (ALSPCT) has emerged as the preferred strat-egy in the treatment of a variety of hematologic malignancies.Improvedmethods tomobilize and collect leukapheresis (LP)products with shortened time to engraftment, immune recon-stitution, and antitumor effects are essential. In the final LPproducts mobilized and collected after BL-8040, there weremuch higher number of CD34þHPCs compared with CD34þ

HPCs collected after mobilization with granulocyte colony-stimulating factor (G-CSF). Furthermore, in the LP productsmobilized and collected after BL-8040, there were muchhigher number of CD4þCD8þ T, NKT, NK, and dendritic cells,compared with LP collected after mobilization with G-CSF.This new graft composition may have a different effect on theengraftment ability, antitumor effect, and immune reconsti-tution potential of the LP product.

Rapid and Robust Mobilization of HSPCs in Healthy Volunteers

www.aacrjournals.org Clin Cancer Res; 23(22) November 15, 2017 6791




CD38, CD45RA, Thy1 (CD90), and CD49f antigens and thedifferent hematopoietic stem and progenitors population wasanalyzed with BD LSR II flow cytometer (Becton Dickinson).

LeukapheresisFor all 8 subjects in part 2 (5001–5008), a leukapheresis

procedure was performed approximately 4 hours after a singleBL-8040 injection was administered. All mobilized products werecollected using a Cobe Spectra apheresis device (Gambro BCT).

Five subjects of G-CSF–mobilized peripheral blood cells werecollected by apheresis fromhealthy donor (Chaim ShebaMedicalCenter, Hematology Division, Tel-Hashomer, Israel) underinformed consent and Helsinki approval. Healthy donorsreceived G-CSF (Neupogen; Amgen), in a standard dose of 10mg/kg body weight subcutaneously for 4 days. On the morning ofthe fifth day, they underwent conventional leukapheresis. Stemcell harvesting was performed using Cobe Spectra ApheresisSystem (Caridian BCT, version 6.1 or 7). A 4-fold estimated bloodvolume was processed daily in 4 to 5 hours. Volume and proces-sing of apheresis products were done according to standardizedprocedures. If 1 leukapheresis was insufficient, an additional doseof G-CSF was administrated and a second leukapheresis wasperformed.

In vitro and in vivo studies in miceIsolation of CD34þ and CD3þ cells. Isolation of cells was doneusing the Human MicroBeads Isolation Kit from Miltenyi Biotec.Isolation of mobilized CD34þ cells was done from all collectedgrafts and from G-CSF–mobilized cells. Isolation of CD3þ T cellswas done from the BL-8040 collected grafts (mobilized CD3þ

cells) and from the blood of healthy donors who have notexperienced mobilization with BL-8040 (normal CD3þ cells).Purity of isolated cells was determined by FACS.

In vitro migration of mobilized cells. A migration assay wasperformed using transmigration plates of 6.5 mm/diameterand 5 mm/pore (Costar). Purified CD34þ cells (from BL-8040–mobilized cells and from G-CSF–mobilized cells) weresuspended in RPMI medium containing 1% FCS. Cells (2 � 105

cells/well) were added to the top chambers in a total volume of100 mL, and 600 mL RPMI supplemented with 100 ng/mLCXCL12 (PeproTech) was added to the bottom chambers. Thesame amount of isolated mobilized CD3þ cells from thecollected grafts and normal CD3þ cells were added to theTranswell. The cells migrating to the bottom chamber of theTranswell within 4 hours were counted using a FACSCaliburFlow Cytometer (BD Biosciences). The data were analyzedusing software from CellQuest (version 3.3; BD Biosciences).

Mice. Female NOD SCID gamma (NSG) mice (8–9 weeks old)were maintained under specific pathogen-free conditions at theHebrew University Animal Facility (Jerusalem, Israel). All experi-ments were approved by the Animal Care and Use Committee ofthe Hebrew University.

Engraftment of mobilized human CD34þ cells in mice. NSG micewere first irradiated with 300 cGy and 24 hours later mobilizedhuman CD34þ cells were intravenously injected (2 � 105 cells/mouse) into the mouse via the dorsal tail vein in a final volumeof 200 mL. Engraftment of cells was allowed for 4, 8, and 22 weeksafter transplantation.

Eight weeks following transplantation second transplanta-tion was performed. A total of 2 � 105 bone marrow cells/mouse were transplanted as described. The engraftment of cellsin the second transplantation was allowed for 14 weeks aftertransplantation.

Following engraftment mice were sacrificed and blood, bonemarrow and spleen were taken for analysis. Cells were isolatedfrom those organs, stained with anti-human CD45, and anti-human CD34þ antibodies and percentage of cells evaluated byFACS. For further analysis of transplanted cells, different stainingwas done using specific anti-human fluorescence antibodies:CD34/CD38, CD3/CD4/CD8, CD14/CD16, and CD19/CD56/CD3. FACS analysis was done using a FACSCalibur Flow Cyt-ometer (BD Biosciences). The data were analyzed using softwarefrom CellQuest (version 3.3; BD Biosciences). To evaluate thenumber of human progenitor cells following transplantation ofhuman CD34þ cells, a colony-forming cell (CFC) assay wasperformed. The colonies were assayed by plating 1 � 105 of cellscollected from themice bonemarrow, following lysis of RBC, in amethylcellulose medium with recombinant cytokines and EPOfor human cells (MethoCult H4435; StemCell Technologies Inc.).The cultures were incubated at 37�C in a humidified atmospherecontaining 5% CO2. The colonies that developed 10 days laterwere visually scored using a light microscope (employing mor-phologic criteria).

Statistical methodsAll measured variables and derived parameters are listed indi-

vidually and, where appropriate, presented using descriptivestatistics. The safety parameters and changes from baseline wereexamined and summarized for descriptive purposes. Adverseevents (AE) were coded according to the MedDRA (version17.1) system organ class and preferred term. The individual studydrug pharmacokinetic parameters and the mean, SD, and 95%confidence interval (CI) values were calculated for each dosegroup, for all subjects. Pharmacodynamic analyses included thevalues, changes from predose, and fold increases of WBCs (neu-trophils, lymphocytes, monocytes, and platelets), CD34þ andCD138þ counts, red blood cells (RBC), and the number of stemcell collections. The individual measurements and changes frombaseline by time point are presented in addition to summarytables by dose group. A P value of less than 0.05 was consideredsignificant, and the significance of the differences between thegroups for the stem cell collection was performed using Student ttest. A paired two-tailed Student t test was used to evaluate thesignificant differences between the groups.

ResultsDemographics and baseline characteristics

Twenty-five subjects were enrolled into part 1 of the study;however, one subject did not receive the study drug and twosubjects received only one dose of the study drug. A total of40 potential subjects were screened for part 1 of the study.Twenty-eight subjects met all inclusion and exclusion criteriaand were eligible to participate in the study. Of these, 24 wereinitially included in the study. One subject, initially random-ized to the BL-8040 1 mg/kg group, was withdrawn from thestudy due to the investigator's decision (the subject faintedbefore BL-8040/placebo administration). An additional subjectwas therefore added to that group to replace this early dropout.

Abraham et al.

Clin Cancer Res; 23(22) November 15, 2017 Clinical Cancer Research6792




Therefore, a total of 25 subjects were randomized to participatein part 1 of the study and 24 subjects received at least one doseand were included in the safety analysis. A total of 22 subjects(22/24, 91.7%) completed the study. Two subjects (8.3%)withdrew prematurely: one subject in the BL-8040 0.5 mg/kgtreatment group withdrew his consent for participation in thestudy, and one subject in the BL-8040 1 mg/kg withdrew fromthe study due to injection site pain, tension headaches, nausea,vomiting, and abdominal discomfort.

All subjects were male and Caucasian. The data below ispresented only for the 24 subjects who received the study drug.Summary tables for baseline demographic data are presented inSupplementary Table S1A. The baseline characteristics weresimilar across groups, and no subject reported any history ofalcohol and/or drug abuse or addiction and/or active/past (upto 2 years before screening) nicotine consumption. No subjectreported a clinically significant medical history (SupplementaryTable S1A).

Eight subjects were enrolled into part 2 of the study. All subjectswere male and Caucasian. All subjects received BL-8040. Sum-mary tables for baseline demographic data are presented inSupplementary Table S1B. No subject reported any history ofalcohol and/or drug of abuse addiction and/or active/past (upto 2 years before screening) nicotine consumption. No subjectreported clinically significant medical history (SupplementaryTable S1B).

Pharmacokinetic analysisPharmacokinetic samples were collected only during part 1 of

the study. A total of 15 blood samples were collected for phar-macokinetic analysis from each subject. Plasma concentrations ofBL-8040 in subjects that received BL-8040 were analyzed bymodel-independent methods using Phoenix WinNonlin version6.4 (Pharsight, Inc.). Nominal sampling times were used. Con-centrations below the limit of quantitation (BLQ) were reportedas "0" and were used as such in the pharmacokinetic analysis andsummary statistics of concentration–time data.

Steady-state achievement after the second dose was assessed bycomparing predose and 23-hour concentrations on day 1, and theCmax on days 1 and 2. Accumulation on day 2, if any, was assessedby comparing AUC0-24 and Cmax of the second and first doses.Pharmacokinetic linearity was assessed by examining the rela-tionship between dose and the exposure parameters (Cmax andCUA) on the first and second doses. Plasma concentrations in the

placebo group were all below the limit of quantitation, asexpected, and were not considered in the pharmacokinetic anal-ysis. Likewise, predose plasma concentrations were below thelimit of quantitation in all subjects randomized to receive BL-8040.After subcutaneous administrationof BL-8040onday1, theappearance of the compound in the plasma was rapid, with amedian Tmax ranging between 0.25 and 0.5 hour. Thereafter,plasma concentrations declined monoexponentially with a shorthalf-life of approximately 1 hour. For that reason, plasma levels ofBL-8040 were below the limit of detection in the majority ofsubjects by 8 hours and in all subjects at 23 hours after dosing.Summary statistics of pharmacokinetic parameters of BL-8040after day 1 (first dose and second dose) can be found in Supple-mentary Table S2A.

Increases in the dose of BL-8040 led to overall approximateproportional increases in plasma exposure, as measured byCmax, AUC0–t, AUC0–24, and AUC0–¥ (Fig. 1). Dose-normalizedCmax and AUC0–24 and t1/2 values were comparable across the3 dose groups suggesting dose proportionality across the 0.5 to1 mg/kg dose range.

The plasma concentration–time profile on day 2 was con-sistent with day 1 and characterized by the rapid absorptionand elimination of BL-8040 from the circulation. Consistentwith its short half-life, accumulation of BL-8040 upon oncedaily repeated administration for two days was minimal.The pharmacokinetic parameters on day 2 (SupplementaryTable SS2B; Fig. 1) were similar to those of day 1.

Safety analysisNo deaths or serious AEs occurred during the study. In part 1,

BL-8040 was overall well-tolerated, only a single subject termi-nated early due to an AE. No AE reported during this part of thestudy was considered life-threatening, most AEs were mild ormoderate and only 13% of all reported AEs were considered assevere. Themost commonAE among all the subjects was injectionsite pain, reported in 88% of subjects, including the 3 subjectswho received a placebo. No dose response was observed for theoverall incidences of AEs. An incidence pattern among cohorts0.5–1mg/kg, possibly suggestive of a dose response was observedfor: leukocytosis, palpitations, nausea, dizziness, headache, pru-ritus generalized, and pallor for 21 of 24 (88%) subjects whoreceived BL-8040. Seventy-nine AEs related (definitely, probablyand possibly) to study treatment were reported by 6 subjects(100%) receiving 0.5mg/kg BL-8040. Among all the AEs reported

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Figure 1.

Exposure versus dose relations ofBL-8040 after daily subcutaneousadministration for two days in healthyvolunteers.






within the BL-8040 cohorts only 3 were assessed as related andsevere. Of the severe AEs (SAE), 2 subjects in the 1 mg/kg treat-ment group had severe asthenia and 1 subject in the 0.75 mg/kgtreatment group had severe syncope. The event resolved withinthe same day it was reported and no supportive care required. Inthe02, no SAEwere reported. Themost commonAEamong all thesubjects was injection site pain, reported in 88% of subjects,including the 3 subjects who received a placebo. Other commonAEs reported in more than 10% of all subjects included injectionsite erythema (75%), injection site edema (75%),flushing (67%),pruritus (63%), hot flush (46%), nausea (42%), injection sitepruritus (38%), urticaria (33%), headache (29%), asthenia(25%), chills (25%), dizziness (25%), parasthesia (25%), hyper-hidrosis (25%), pruritus generalized (25%), rashmacular (21%),pallor (21%), vomiting (17%), injection site hematoma (17%),injection site induration (17%), edema peripheral (17%), leuko-cytosis (13%), tachycardia (13%), diarrhea (13%), peripheralswelling (13%), andpruritus genital (13%).Detailed drug-relatedAEs by preferred term and treatment group are shown in Supple-mentary Table S3A and S3B.

After reviewing the data gathered from cohorts 1–3 of part 1,it was decided for part 2 to use a single dose of 1 mg/kg SC. Inpart 2, based on the safety data of part 1, premedication withsteroids and antihistaminic drugs was implemented prior to theBL-8040 injection. Premedication with paracetamol (1 g, oral-ly), methylprednisolone 100 mg (i.v.), and promethazine25 mg (orally) were given 1 hour prior to the BL-8040 injection,which was given in the second part of the study. BL-8040 wasoverall well tolerated and all subjects completed the study. NoAE reported during this part of the study was considered life-threatening, all the AEs were mild or moderate and no AEs wereconsidered severe. The most common AEs reported in allsubjects were injection site pain, hot flush, and injection siteerythema. Overall, 100% of the subjects who received 1 dose of1 mg/kg of BL-8040 reported AEs. The majority of these AEs(88%) were assessed as related (definitely, probably, and pos-sibly) and were considered mild and moderate by the principalinvestigator. The most common AE reported in 100% of thesubjects was injection site erythema, injection site pain, and hotflush. Other common AEs reported in more than 10% of thesubjects were injection site edema (88%), macular rash (88%),flushing (75%), palpitations (50%), nausea (50%), asthenia(50%), paresthesia (50%), injection site hematoma (38%),dizziness (38%), tachycardia (25%), dry mouth (25%), vomit-ing (25%), pallor (25%), injection site induration (25%),abdominal discomfort (13%), abdominal pain (13%), dyspha-gia (13%), injection site inflammation (13%), injection siteischemia (13%), injection site necrosis (13%), injection siteparaesthesia (13%), injection site pruritus (13%), injection sitescab (13%), peripheral swelling (13%), headache (13%), pre-syncope (13%), tension headache (13%), epistaxis (13%),tachypnoea (13%), and throat irritation (13%).

Hematopoietic cell mobilization and collectionIn part 1 of the study a single administration of BL-8040

(0.5, 0.75, 1 mg/kg) induced a rapid and dose-dependentmobilization of WBCs into the blood in all subjects tested(Fig. 2A–C). The maximal number of WBCs in the differentcohorts was achieved after the first injection, and was 35.5 �103, 49.4 � 103, and 44.3 � 103, respectively, for cohortsreceiving 0.5, 0.75, and 1 mg/kg. The mean fold increase in the

number of WBCs reached its peak between 8 and 12 hoursfollowing BL-8040 injection (4.54, 5.16, 5.81-fold increase,respectively) and started to decline after 12 hours (Fig. 2D).Interestingly, the number of WBCs in the blood following thesecond injection did not increase above the maximal numberachieved after the first injection, and neither did the mean foldincrease of WBCs. Forty-eight hours after the last injection ofBL-8040 the number of WBCs in the blood was significantlyreduced (50% or less relative to peak accumulation of WBCs inthe blood). In most of the subjects, the number of WBCsnormalized 72 hours following the last injection. The increasein the number of WBCs could be attributed mainly to increasein the number of neutrophils and lymphocytes subpopula-tions (70% and 20%, respectively; Fig. 2E and F). Within thelymphocyte subpopulation, an increased mobilization of B, T,and NK cells was noted (Fig. 2G). It is worth mentioning that amobilization of dendritic cells (DC) (lin�/CD11cþ cells) wasobserved following administration of BL-8040 (1mg/kg)reaching a peak at 3.5 hours posttreatment and by 24 hourspostdose, the DC levels completely returned to the baselinelevel (Fig. 2H). Mobilization of WBCs to the periphery andthe recovery of normal numbers of WBCs correlate with theability of the CXCR4 12G5 antibody to bind to its epitope onthe CXCR4 receptor (Fig. 2H).

After the first dose administration of BL-8040, CD34þ HSPCscounts increased rapidly reaching maximal levels between 4 and8 hours postdose (Fig. 3A). The maximal counts increased withdose averaging 9.3, 38.2, 43.7, and 45.5 IU/mL for placebo, 0.5,0.75, and 1 mg/kg groups, respectively. By 24 hours postdose,the CD34þ levels had declined slightly but were still 5- to 7-foldhigher than the baseline. In contrast to WBCs, the number ofCD34þ cells after the second dose increased in a dose-depen-dent manner (Fig. 3A). Upon administration of the seconddose, an increase in CD34þ was observed 4 to 8 hours afterdosing. In correlation with the expression of CXCR4, the CD34þ

counts declined thereafter reaching baseline level approximate-ly 48 hours after the second dose (Fig. 3A and E). At doses of0.5 and 0.75 mg/kg, 4 of 12 injected individuals failed toachieve 20 CD34þ cells per mL 4–8 hours following the firstadministration of BL-8040, whereas all 6 individuals treatedwith 1mg/kg achieved 20 CD34þ cells per mL (Fig. 3B–D). Wetherefore selected the 1 mg/kg as the dose for the second part ofthe study and performed aphaeresis 3–4 hours following theBL-8040 treatment. In part 2, similar to the results of part 1 ofthe study, injection of 1 mg/kg of BL-8040 induced a rapidincrease in the number of WBCs and CD34þ cells in theperiphery that declined between 24 and 48 hours (Fig. 4A andB) posttreatment. All 8 subjects (no placebo was administeredin part 2 of the study) increased their CD34þ count above 20 permL approximately 3.5 hours after injection of BL-8040 beforeleukapheresis (Fig. 4C). The average blood volume processedduring leukapheresis was 15.8 liters (range 9.8–17.5 liters).CD34þ cell counts were assessed by FACS analysis and theamount of CD34þ cells collected per kg was calculated for eachsubject. The mean CD34þ cells/kg collected was 10.3 � 106

cells/kg and the median was 10.5 � 106 cells/kg (range of 5.1 �106 to 15.2 � 106) when calculated on the basis of a 79 kgaverage recipient weight. Table 1A summarizes the individualdata and the amount of CD34þ cells collected when calculatedon the basis of actual donor weight as well as based on 79 kgaverage recipient weight.

Abraham et al.





Lifelong blood cell production is dependent on rarehematopoietic stem cells (HSCs). The bulk of HSCs are CD34þ

cells; however, most CD34þ cells are lineage-restricted pro-genitors and HSCs remain rare. HSCs can be enriched furtherbased on CD45RA, Thy1 (CD90), and CD38 expression. Lossof CD90 expression in the CD34þCD38�CD45RA� compart-ment of lineage depleted cord blood (CB) was proposedto be sufficient to separate CD34þCD38�CD45RA� CD90þ

HSCs from CD34þCD38�CD45RA� CD90� multipotent prog-enitors (MPP; ref. 16). Recently, it was demonstrated thatCD49f is a specific HSC marker. Single CD49fþ cells werehighly efficient in generating long-term multilineage grafts,and the loss of CD49f expression identified transiently engraft-ing MPPs. Furthermore, CD34þCD38�CD45RA�CD90þ

CD49fþ HSCs were found as the population with the highestlong-term and secondary repopulating activity (17). CD34þ

cells were purified from BL-8040 and G-CSF–mobilized graftsand stained for CD38, CD45RA, CD90, and CD49f. Thepercentage of CD34þCD38� hematopoietic stem and progeni-tors was similar in both grafts (Fig. 4D). However, whereas23.2 % of BL-8040–mobilized CD34þ CD38� cells did notexpress CD45RA, only 1.6% of G-CSF–mobilized CD34þ

CD38� cells did not express CD45RA (Fig. 4D). The per-centage and absolute number of CD34þCD38�CD45RA�

CD49fþCD90þ/�, CD34þCD38�CD45RA�CD49fþCD90þ, andCD34þCD38�CD45RA� CD90þ HPCS were increased signifi-cantly by 45, 25, 12 (% of cells) -fold and by 61.7, 36.6, 59.7(absolute number of cells per kg) in the BL-8040 graft compare

Figure 2.

Patients (1001–1008), placebo, and BL-8040 dose-dependent (0.5, 0.75, 1 mg/kg) mobilization of WBCs are shown in A–C. Mean fold change of WBCs,neutrophils, and lymphocytes in the treated and placebo patients are shown in D–F. Red arrows indicate the time of the two injections of BL-8040.Mobilization of B, T, and NK cells lymphocyte subpopulation following 1 mg/kg of BL-8040 is shown in G. Mobilization of dendritic cells (DC) is shown inH. CXCR4 12g5 antibody binding to its epitope on the CXCR4 receptor is completely blocked on WBCs by treatment with BL-8040 as shown in I.






to G-CSF graft–derived CD34þCD38� cells (Fig. 4E; Supplemen-tary Table S4A).

These results suggest the BL8040 is a better mobilizer ofHSCs than G-CSF

It was already shown before that G-CSF mobilizes CD34þ

cells collected from the blood migrate less in response toCXCL12 (18). Interestingly, we found that CD34þ cells mobi-lized with BL-8040 migrated better than G-CSF–mobilized cellsin response to CXCL12 (Fig. 4F).

We stained CD34þ cells mobilized cells from BL-8040 andG-CSF–mobilized CD34 cells with 12g5 mAb, which binds thesecond extracellular loop of CXCR4, and 1D9 mAb, which bindsthe N-terminal portion of CXCR4. When cells were stainedwith 1D9 antibodies similar expression levels of CXCR4 wereobserved while the level of CXCR4 following staining with 12g5antibodies was significantly reduced on BL-8040 mobilized cellsbut not onG-CSFmobilized cells (Fig. 4G). This suggests a specificblock of the 12g5 with no internalization of the CXCR4 receptor.

The graft collected was further tested for its immune compo-sition. It was found that the graft was enriched for immune cells,including immature dendritic cells (ImDC), T cells, B cells, and

NK cells (Supplementary Table S4B). Furthermore, in theCD4þ T-cell population, we found that the graft containedmostlyna€�ve CD4 T cells (35%), effector memory CD4 T cells (21%),central memory CD4 T cells (38%), and almost no effector CD4 Tcells (6%).Whereas in the CD8þ T-cell population, we found thatthe graft contained mostly na€�ve CD8 T cells (64%), and centralmemory CD8 T cells (33%), and almost no effector memory CD8T cells (1%), and effector CD8 T cells (2%; Fig. 5).

The different immune subpopulation in the BL-8040 com-pared with G-CSF–mobilized graft were determined (Supple-mented Table S4B). Significant increase in the percentage ofCD3þCD4þ, CD3þCD8þ, DC, na€�ve, and effector CD4þ T cells,central memory CD4 T cells, na€�ve CD8 T cells, and effectormemory CD8 cells was observed (Fig. 5).

The SCID-reconstituting capacity of fresh or frozen purifiedCD34þ cells (>90%) was evaluated in transplant experimentsin NSG mice using human anti-CD45 antibody at a thresholdlevel of 0.5% of marrow and spleen cellularity and blood.Significant engraftment was observed in the bone marrow,spleen, and blood of all transplanted mice (Fig. 6A). The bonewas engrafted with CD45þ/CD19þ B cells, CD45þ/CD56þ/CD3þ NK cells, CD45þ/CD14þ/CD16þ myelomonocytic

Figure 3.

Mean BL-8040 dose-dependent(0.5, 0.75, 1 mg/kg) mobilization ofCD34þ cells/mL of blood is shown inA. Red arrows indicate the times ofthe two injections of BL-8040.Patients (1001–1008), placebo, andBL-8040 dose-dependent (0.5,0.75, 1 mg/kg) mobilization ofCD34þ cells/mL of blood is shown inB–D. CXCR4 12g5 Ab binding to itsepitope on the CXCR4 receptor ispartially blocked on CD34þ cells bytreatment with BL-8040 as shownin E.

Abraham et al.





Figure 4.

Number of WBCs and CD34þ cells/mL of blood is shown in patients 5001–5008 (n ¼ 8) that were administrated with BL-8040 and 4 hours later underwentapheresis to collect their CD34þ cells is shown in A and B. Mean BL-8040 time-dependent mobilization of CD34þ cells/mL of blood is shown in C. Percentageof CD34þCD38� cells from total of CD34þ cells and percentage of CD45RA� cells out of CD34þCD38� from cells mobilized by BL-8040 or G-CSF isshown in D. Percentage of CD34þCD38� CD45RA� CD49fþ from a total of CD34þCD38� cells and percent of CD34þCD38�CD45RA�CD90þCD49fþ froma total of CD34þCD38� CD45RA� CD49fþ of cells mobilized by BL-8040 or G-CSF is shown in E. SDF-1 migration of BL-8040–mobilized CD34þ celland G-CSF–mobilized-CD34þ cells is shown in F. Migration of CD3þ cells which mobilized by BL-8040 and normal CD3þ cells served as controls. Theexpression of CXCR4 on mobilized CD34þ cells are shown in G. CD34þ cells were separated from BL-8040–mobilized cells (n ¼ 8) and from G-CSF–mobilizedcells (n ¼ 3). CD34þ cells were stained with two different CXCR4 antibody clones 12g5 (binding to extracellular loops) or 1D9 (binding to theN-terminus). Controls were incubated with appropriate isotype controls.

Table 1A. Summary of the individual data and the amount of CD34þ cells collected when calculated on the basis of actual donor weight as well as based on 79 kgaverage recipient weight

Subject no. Whole blood processed (L) CD34þ cells (%) CD34þ/kg � 106 (donor weight) CD34þ/kg � 106 (79 kg recipient weight)

5001 9.8 0.75 4.1 4.55002 16.0 1.01 11.9 10.65003 16.6 0.85 13.7 13.25004 16.2 0.76 10.2 10.55005 16.6 0.78 11.4 13.55006 16.5 0.87 13.7 13.05007 17.5 0.64 11.1 8.65008 16.7 0.61 9.6 8.9Average 15.8 � 2.3 L 0.78 � 0.13% 10.6 � 106 (�2.8 � 106) 10.3 � 106 (�2.8 � 106)Median 16.5 � 2.3 L 0.77 � 0.13% 11.2 � 106 (�2.8 � 106) 10.5 � 106 (�2.8 � 106)

Table 1B. CD34þ cells collected after mobilization with BL-8040, AMD3100, or G-CSF

Cell typeBL-8040 (n ¼ 8)Median (range)

AMD3100a (n ¼ 24)Median (range)

G-CSFa (n ¼ 8)Median (range)

CD34þ � 106/Kg stem cells 11.9 (5.1–15.0) � 106/Kg 2.9 (1.2–6.3) � 106/Kg 4.2 (2.5–18.7) � 106/KgaDevine and colleagues (5).






cells, and CD45þ/CD34þ/CD38þ progenitors and CD45þ/CD34þ/CD38� stem cells as well as human colony-formingcells (Fig. 6B–D).

To assess the long-term engraftment potential of the BL-8040–mobilized CD34þ cells, engraftment was allowed for 4,8, and 22 weeks after transplantation. Successful and robustlong-term human engraftment of CD45þ and CD45þCD34þ

cells was observed at week 22 (Fig. 6E and F). The percent ofCD45 cells in the bone marrow remained stable in the bonemarrow, whereas the percentage of CD45 cells in the blood andspleen increased at week 22 (Fig. 6G). At 4 weeks, humanCD3þCD4þ T cells were only observed at a low percentage inthe spleen but not in the bone marrow, whereas no significantpercentage of CD3þCD8þ cells were found neither in the bonemarrow nor in the spleen (Fig. 6H and I). Following 22 weeks oftransplantation, the percentage of human CD3þCD4þ andCD3þCD8þ T cells was significantly increased in the spleen(30% vs. 5%, respectively). The percentage of humanCD3þCD4þ and CD3þCD8þ T cells was increased at muchlower levels in the bone marrow (Fig. 6H and I).

To further assess the long-term engraftment potential of theBL-8040, we collected the bone marrow cells from 8-week-old

transplanted mice and re-transplanted them in to a secondaryrecipient. Successful and robust long-term human engraftment ofsecondary recipient was observed 14 weeks following the secondtransplantation (Fig. 6E and F).

In correlation with the engraftment potential we observedin NSG mice, the ability of thawed purified CD34þ HPCsand CD3þ cells to migrate in response to the CXCR4 ligandCXCL12 was found to be intact (Fig. 4F).

DiscussionThe most frequently used graft of hematopoietic stem and

progenitor cells for allogeneic transplantation is peripheralblood stem cells (PBSC) that are collected after mobilizationwith G-CSF. Both in vitro and in vivo experiments have recentlysuggested that G-CSF induces HSPC mobilization by indirectlydisrupting the interaction between CXCR4 and SDF-1 (6–8).Furthermore, G-CSF activates neutrophils leading to the releaseof proteases, neutrophil elastase, and cathepsin G, which candirectly cleave the adhesive interactions between HSPCs andthe bone marrow microenvironment. Mobilization of CD34þ

cells peaked in the peripheral blood between days 4 and 5 ofG-CSF dosing. A dose between 10 and 16 mg/kg split into twodoses per day was used for PBSC mobilization (5). Pulsipherand colleagues reported that among 2,408 unrelated donorsof PBSCs, two-thirds (66%) underwent two apheresis proce-dures, whereas the remaining donors completed their dona-tions in a single day (19). Ings and colleagues reported thatamong 400 unrelated donors of PBSCs target recipient doseswere reached with one aphaeresis in 63% of donors and withtwo apheresis in 81% of donors; 19% of the donors neededmore than two apheresis (20).

AMD3100 (plerixafor, Mozobil) is a reversible direct antag-onist of the interaction between the chemokine SDF-1 and itsreceptor CXCR4 and is used for HSPC mobilization. Plerixaforwas approved for use in combination with G-CSF to mobilizeHSPC for autologous HSCT (12, 21). Studies conducted inhealthy donors reported that Plerixafor induces a transientincrease in CD34þ that peaks 9 hours after subcutaneousinjection with 80–240 mg/kg of plerixafor and declines at 24hours (22). In another study reported by Devine and collea-gues (5), plerixafor (n ¼ 24) was compared with G-CSF (n ¼ 8)in mobilization and collection from healthy sibling donors. At4 hours after a single dose of AMD3100 (240 mg/kg), theCD34þ count rose to a median of 16/mL (range, 4–54/mL).One out of the 25 donors could not undergo large volume (27liters) leukapheresis following AMD3100 due to repeatedvasovagal episodes during peripheral line placement prior tocommencing leukapheresis. A total of 8 (33%) of the remain-ing 24 donors who commenced leukapheresis followingAMD3100 administration on day 1 did not collect the min-imum required CD34þ cell dose in one day (2 � 106/kg) andwere thus eligible for a second day of drug administration andleukapheresis on day 3. The median number of CD34þ cellscollected from all 24 donors was 2.9� 106/kg (range, 1.2–6.3).In the G-CSF group, the CD34þ count rose to a median of46/mL on day 5 (range, 4–54/mL). The median number ofCD34þ cells collected from all 8 donors was 4.2 � 106/kg(range, 2.5–18.7). The increase in CD34þ cells after 5 days ofG-CSF was significantly greater than the rise 4 hours afterAMD3100 administration (P < 0.001; ref. 5).

Figure 5.

Percentages of different subpopulations of BL-8040–mobilized cells (n ¼ 8) ascompared with G-CSF–mobilized cells (n¼ 5) was analyzed by flow cytometry.Pie charts show the cells distribution within the CD4þ or CD8þ populations.CD3þ/CD4þ/CD45RAþ/CCR7þ (na€�ve CD4þ cells); CD3þ/CD4þ/CD45RAþ/CCR7� (effector CD4 T cells); CD3þ/CD4þ/CD45RA�/CCR7� (effector memoryCD4 T cells); CD3þ/CD4þ/CD45RA�/CCR7þ (central memory CD4 T cells).CD3þ/CD8þ/CD45RAþ/CCR7þ (na€�ve CD8þ cells); CD3þ/CD8þ/CD45RAþ/CCR7� (effector CD8 T cells); CD3þ/CD8þ/CD45RA�/CCR7� (effector memoryCD8 T cells); CD3þ/CD4þ/CD45RA�/CCR7þ (central memory CD4 T cells).

Abraham et al.





The effect of BL-8040 as compared with AMD3100 wasstudied in in vitro and in vivo experimental settings. BL-8040binds and inhibits the CXCR4 chemokine receptor with highaffinity, showing an IC50 of approximately 1 nmol/L (23–25)compared with the values obtained with AMD3100 (IC50 ¼651 � 37 nmol/L; ref. 26). Moreover, BKT140 hinders thecell migration stimulated by CXCL12 within IC50 values of0.5–2.5 nmol/L (24, 27) compared with the IC50 value of51 � 17 nmol/L for Mozobil (26). Furthermore, BL-8040 haslong receptor occupancy (>24 hours) compared withAMD3100, resulting in the extended inhibition of CXCR4(15). In animal studies, comparison between the BL-8040and AMD3100 showed that BL-8040 with or without G-CSFwas significantly more potent in its ability to mobilize hemato-poietic stem cells and progenitors into blood (14, 15).

In this study, we found that at 4–8 hours after a single doseof BL-8040 (0.5, 0.75, 1 mg/kg), the CD34þ count rose to amedian of 38.2, 43.7 and 45.5 mL (respectively). In contrast totreatment with AMD3100, by 24 hours, CD34þ levels had

declined only slightly. Furthermore, the mean CD34þ cells/kgcollected from the individuals treated with 1mg/kg BL-8040was 11.6 � 106 cells/kg and the median was 11.9 � 106 cells/kg(range of 5.1 � 106 to 15.2 � 106). The results of this trialsupport the hypothesis that in healthy humans the numberof CD34þ cells that can be rapidly mobilized and collectedis greater than observed with either G-CSF or plerixafor. Fur-thermore, we have found that the percentage of CD34þCD38�

CD45RA�CD49fþCD90þ cells, and CD34þCD38�CD45RA�

CD90þ cells were significantly higher in the BL-8040 graftcompared with G-CSF graft (Fig. 4E). In association withthe high percentage of HPCs in the BL-8040 graft, we founda robust myeloid and lymphoid long-term engraftment(week 22) of BL-8040–mobilized human CD34þ cells in NSGmice (Fig. 6).

In Devine and colleagues' study (5), there were notabledifferences in the final leukapheresis products mobilized andcollected after AMD3100 or G-CSF were administered. Therewere significantly fewer CD34þ cells/kg (2.9 � 106 vs. 4.2 �

Figure 6.

The SCID reconstituting capacity of fresh or frozen purified CD34þ cells (>90% purity) was evaluated in transplant experiments in NSG mice following4 weeks, using human anti-CD45 antibodies is shown in A. The number of human colony-forming cells HPCs in the bone marrow of mice transplantedwith fresh or frozen purified CD34þ cells is shown in B. The bars represent the number of human colonies from total of 100,000 bone marrow cellsfollowing 4 weeks of transplantation. Engraftment of CD45þCD34þCD38þ and CD45þ CD34þ CD38� cells demonstrated in C. The number of humanHPCs in the bone marrow of mice following short transplantation (4 weeks) and long transplantation (22 weeks) is shown in D. Mice weretransplanted with purified CD34þ and the number of colonies from total of 100,000 bone marrow was measured. Engraftment percentage of hCD45þ

and hCD45þ/CD34þ cells in the bone marrow following 4, 8, and 22 weeks of transplantation as well as following 14 weeks of second transplantationis shown in E and F. Engraftment percentage of hCD45þ cells in the bone marrow, spleen, and blood following 4 and 22 weeks of transplantation isshown in G. Engraftment percentage of human CD3þ, CD3þCD4þ, and CD3þCD8þ T cells in the bone marrow and spleen following 4 and 22 weeks oftransplantation are shown in H and I. Engraftment percentage of human CD45þCD19þ B cells, CD45þCD56þCD3� NK cells, CD45þCD14þCD16þ

myelomonocytic cells following 4 and 22 weeks of transplantation is shown in J.






106; P < 0.006), but greater numbers of CD3þ cells/kg (4.7 �108 vs. 1.5 � 108; P < 0.006) and CD4þ cells/kg (3.1 � 108 vs.1.1 � 108; P < 0.002) in the AMD3100-mobilized productscompared with the G-CSF–mobilized allografts. Therefore,more CD3þ and CD4þ cells were mobilized per unit of bloodfollowing AMD3100 compared with G-CSF treatment. How-ever, the differences in CD8þ, CD19þ, and CD56þ cell contentwere not significant. Interestingly, the rates of acute and chronicGVHD observed in the 20 transplanted patients were notsignificantly different compared with historical control patientsat Washington University receiving G-CSF–mobilized allograftstogether with the exact same conditioning and GVHD prophy-laxis. There was no difference in the median time to neutrophilor platelet engraftment between recipients in the AMD3100 groupor the historical group receiving G-CSF–mobilized allograftsin this study and all surviving patients in remission continue tohave robust and durable trilineage hematopoiesis.

In the final leukapheresis products mobilized and collectedafter BL-8040 there were much higher number of CD34þ

progenitor and stem cells compared with the leukapheresiscollected after mobilization with AMD3100 or G-CSF (5,Table 1B). Furthermore, in the leukapheresis products mobi-lized and collected after BL-8040 there was a significant in-crease in the percentage of CD3þCD4þ, CD3þCD8þ, DC, na€�veand effector CD4þ T cells, central memory CD4 T cells, na€�veCD8 T cells, and effector memory CD8 cells compared withG-CSF–mobilized graft (Fig. 5). This new composition mayhave an effect on the engraft ability, GVHD, GVL, and immunereconstitution potential of the leukapheresis product.

In summary, our results suggest that BL-8040 is a rapid androbust mobilizer of a of CD34þ progenitors and stem cells withan intact long-term engraftment potential, as assessed byengraftment of these human cells in a mouse model. Further-more, these results suggest that in the bone marrow of humans,the number of CD34þwith the potential to exit rapidly into theblood upon a strong and sustained inhibition of CXCR4 is

clearly greater than seen with the first generation of CXCR4antagonists. A phase II study assessing safety and efficacy ofBL-8040 for the mobilization of donor hematopoietic stemcells and allogeneic transplantation in patients with advancedhematologic malignancies is currently being conducted at theWashington University School of Medicine, Division of Onco-logy and Hematology, St. Louis, MO (NCT02639559).

Disclosure of Potential Conflicts of InterestM. Abraham, H. Wald, B. Bulvik, and O. Eizenberg are employees and

shareholders of Biokine Therapeutics Ltd.; A. Peled serves as consultant forBiokine Therapeutics and is also a shareholder. Y. Pereg, R. Golan, A. Vainstein,and A. Aharon are employees of BioLineRx Ltd. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: M. Abraham, Y. Pereg, O. Eizenberg, A. Aharon,A. PeledDevelopment of methodology: M. Abraham, Y. Pereg, B. Bulvik, H. Wald,A. Aharon, R. Or, A. PeledAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):B. Bulvik, S. Klein, I.Mishalian, A.Nagler, A. Vainstein,Y. Caraco, R. Or, A. PeledAnalysis and interpretation of data (e.g., statistical analysis, biostati-stics, computational analysis): M. Abraham, Y. Pereg, B. Bulvik, R. Golan,A. Vainstein, Y. Caraco, A. PeledWriting, review, and/or revision of the manuscript: M. Abraham, Y. Pereg,O. Eizenberg, A. Nagler, R. Golan, A. Vainstein, A. Aharon, E. Galun, Y. Caraco,A. PeledAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Pereg, O. Eizenberg, K. Beider, R. GolanStudy supervision: A. Vainstein, A. Aharon, Y. Caraco

The costs of publication of this articlewere defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 22, 2016; revised June 22, 2017; accepted August 16,2017; published OnlineFirst August 23, 2017.

References1. Cashen AF, Lazarus HM, Devine SM. Mobilizing stem cells from normal

donors: is it possible to improve upon G-CSF? Bone Marrow Transplant2007;39:577–88.

2. Ozkan MC, Sahin F, Saydam G. Peripheral blood stem cell mobilizationfrom healthy donors. Transfus Apher Sci 2015;53:13–6.

3. Arslan O, Moog R. Mobilization of peripheral blood stem cells. TransfusApher Sci 2007;37:179–85.

4. Deotare U, Al-Dawsari G, Couban S, Lipton JH. G-CSF-primed bonemarrow as a source of stem cells for allografting: revisiting the concept.Bone Marrow Transplant 2015;50:1150–6.

5. Devine SM, Vij R, RettigM,McGlauchlen K, Fisher N,DevineH, et al. Rapidmobilization of functional donor hematopoietic cells withoutG-CSF usingAMD3100, an antagonist of the CXCR4/SDF-1 interaction. Blood 2008;112:990–8.

6. Peled A, Petit I, KolletO,MagidM, Ponomaryov T, Byk T, et al. Dependenceof human stem cell engraftment and repopulation of NOD/SCID mice onCXCR4. Science 1999;283:845–8.

7. Lapidot T, Petit I. Current understanding of stem cell mobilization: theroles of chemokines, proteolytic enzymes, adhesion molecules, cytokines,and stromal cells. Exp Hematol 2002;30:973–81.

8. Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, et al. G-CSFinduces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 2002;3:687–94.

9. Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA,et al. Rapid mobilization of murine and human hematopoietic stem and

progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 2005;201:1307–18.

10. Steinberg M, Silva M. Plerixafor: A chemokine receptor-4 antagonist formobilization of hematopoietic stem cells for transplantation after high-dose chemotherapy for non-Hodgkin's lymphoma or multiple myeloma.Clin Ther 2010;32:821–43.

11. DiPersio JF, Stadtmauer EA, Nademanee A, Micallef IN, Stiff PJ, KaufmanJL, et al. Plerixafor and G-CSF versus placebo and G-CSF to mobilizehematopoietic stem cells for autologous stem cell transplantation inpatients with multiple myeloma. Blood 2009;113:5720–6.

12. DiPersio JF, Micallef IN, Stiff PJ, Bolwell BJ, Maziarz RT, Jacobsen E, et al.Phase III prospective randomized double-blind placebo-controlled trial ofplerixafor plus granulocyte colony-stimulating factor compared with pla-cebo plus granulocyte colony-stimulating factor for autologous stem-cellmobilization and transplantation for patients with non-Hodgkin's lym-phoma. J Clin Oncol 2009;27:4767–73.

13. Abraham M, Beider K, Wald H, Weiss ID, Zipori D, Galun E, et al. TheCXCR4 antagonist 4F-benzoyl-TN14003 stimulates the recovery of thebone marrow after transplantation. Leukemia 2009;23:1378–88.

14. Abraham M, Biyder K, Begin M, Wald H, Weiss ID, Galun E, et al.Enhanced unique pattern of hematopoietic cell mobilization inducedby the CXCR4 antagonist 4F-benzoyl-TN14003. Stem Cells 2007;25:2158–66.

15. Peled A, Abraham M, Avivi I, Rowe JM, Beider K, Wald H, et al. The high-affinity CXCR4 antagonist BKT140 is safe and induces a robust

Abraham et al.





mobilization of human CD34þ cells in patients with multiple myeloma.Clin Cancer Res 2014;20:469–79.

16. Majeti R, Park CY, Weissman IL. Identification of a hierarchy of multi-potent hematopoietic progenitors in human cord blood. Cell Stem Cell2007;1:635–45.

17. Notta F, Doulatov S, Laurenti E, Poeppl A, Jurisica I, Dick JE. Isolation ofsingle human hematopoietic stem cells capable of long-term multilineageengraftment. Science 2011;333:218–21.

18. Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC. The chemokineSDF-1 is a chemoattractant for human CD34þ hematopoietic progeni-tor cells and provides a new mechanism to explain the mobilization ofCD34þ progenitors to peripheral blood. J Exp Med 1997;185:111–20.

19. Pulsipher MA, Chitphakdithai P, Miller JP, Logan BR, King RJ, Rizzo JD,et al. Adverse events among 2408 unrelated donors of peripheral bloodstem cells: results of a prospective trial from the national marrow donorprogram. Blood 2009;113:3604–11.

20. Ings SJ, Balsa C, Leverett D, Mackinnon S, Linch DC, Watts MJ. Peripheralblood stem cell yield in 400 normal donors mobilised with granulocytecolony-stimulating factor (G-CSF): impact of age, sex, donor weight andtype of G-CSF used. Br J Haematol 2006;134:517–25.

21. DiPersio JF, Schuster MW, Abboud CN, Winter JN, Santos VR, CollinsDM, et al. Mobilization of peripheral-blood stem cells by concurrent

administration of daniplestim and granulocyte colony-stimulating fac-tor in patients with breast cancer or lymphoma. J Clin Oncol 2000;18:2762–71.

22. Liles WC, Broxmeyer HE, Rodger E, Wood B, H€ubel K, Cooper S, et al.Mobilization of hematopoietic progenitor cells in healthy volunteers byAMD3100, a CXCR4 antagonist. Blood 2003;102:2728–30.

23. Tamamura H, Fujisawa M, Hiramatsu K, Mizumoto M, Nakashima H,YamamotoN, et al. Identification of a CXCR4 antagonist, a T140 analog, asan anti-rheumatoid arthritis agent. FEBS Lett 2004;569:99–104.

24. Jacobson O, Weiss ID, Kiesewetter DO, Farber JM, Chen X. PET of tumorCXCR4 expression with 4-18F-T140. J Nucl Med 2010;51:1796–804.

25. Jacobson O, Weiss ID, Szajek LP, Niu G, Ma Y, Kiesewetter DO, et al.PET imaging of CXCR4 using copper-64 labeled peptide antagonist.Theranostics 2011;1:251–62.

26. Fricker SP, Anastassov V, Cox J, Darkes MC, Grujic O, Idzan SR, et al.Characterization of the molecular pharmacology of AMD3100: a specificantagonist of theG-protein coupled chemokine receptor, CXCR4. BiochemPharmacol 2006;72:588–96.

27. Tamamura H, Hori A, Kanzaki N, Hiramatsu K, Mizumoto M, Naka-shima H, et al. T140 analogs as CXCR4 antagonists identified as anti-metastatic agents in the treatment of breast cancer. FEBS Lett 2003;550:79–83.






2017;23:6790-6801. Published OnlineFirst August 23, 2017.Clin Cancer Res Michal Abraham, Yaron Pereg, Baruch Bulvik, et al. Volunteers

Cells in Healthy+Mobilization for the Collection of Human CD34Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid

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