Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid

Published OnlineFirst August 23, 2017; DOI: 10.1158/1078-0432.CCR-16-2919

Cancer Therapy: Clinical Clinical Cancer Research Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid Mobilization for the Collection of Human CD34þ Cells in Healthy Volunteers Michal 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 antagonist systemic reactions were mitigated by methylprednisolone, BL-8040 as a monotherapy-mobilizing agent and its derived paracetamol, and promethazine pretreatment. In the first part graft composition and quality were evaluated in a phase I clinical of the study, BL-8040 triggered rapid and substantial mobili- þ study in healthy volunteers (NCT02073019). zation of WBCs and CD34 cells in all tested doses. Four hours Experimental Design: The first part of the study was a ran- postdose, the count rose to a mean of 8, 37, 31, and 35 cells/mL domized, double-blind, placebo-controlled dose escalation (placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysis phase. The second part of the study was an open-label phase, in revealed substantial mobilization of immature dendritic, T, B, þ which 8 subjects received a single injection of BL-8040 (1 mg/kg) and NK cells. In the second part, the mean CD34 cells/kg and approximately 4 hours later underwent a standard leukapher- collected were 11.6 106 cells/kg. The graft composition was esis procedure. The engraftment potential of the purified mobi- rich in immune cells. þ lized CD34 cells was further evaluated by transplanting the cells Conclusions: The current data demonstrate that BL-8040 into NSG immunodeficient mice. is a safe and effective monotherapy strategy for the collection þ Results: BL-8040 was found safe and well tolerated at all of large amounts of CD34 cells and immune cells in a one-day doses tested (0.5–1 mg/kg). The main treatment-related adverse procedure for allogeneic HSPC transplantation. Clin Cancer Res; events were mild to moderate. Transient injection site and 23(22); 1–12. 2017 AACR.

Introduction interindividual variations in circulating progenitor and stem cell numbers (5), requiring 4–6repeateddosingtocollecta Allogenic hematopoietic stem and progenitor cell (HSPC) sufﬁcient number of cells. In addition, although considered transplantation (ALSPCT) has emerged as the preferred strategy generally safe, G-CSF is frequently associated with a variety in the treatment of a variety of hematologic malignancies (1, 2). of side effects. Therefore, improved methods to mobilize and Mobilization of stem cells using granulocyte colony-stimulat- collect HSPCs for transplantation are required. ing factor (G-CSF) from healthy donors is the common clinical It has been proposed that G-CSF induces the mobilization of practice. G-CSF–mobilized peripheral blood mononuclear HSPCs through an indirect mechanism by activating neutrophils cells (PBMC) are routinely used as a source of hematopoietic to secrete a variety of proteolytic enzymes, including elastase, stem cells (HSC) for transplantation (3, 4). Despite the potency cathepsin G, MMP-2, and MMP-9 that can degrade the chemokine of G-CSF in mobilizing stem cells, it ultimately results in broad CXCL12 and its receptor CXCR4. Over recent years, it has become apparent that the interaction between CXCL12 and its receptor, CXCR4, plays a pivotal role in hematopoietic stem cell mobili- 1Biokine Therapeutics Ltd, Ness Ziona, Israel. 2BioLineRx LTD, Modi'in, Israel. zation and engraftment (6–8). Consequently, disruption of 3Goldyne Savad Institute of Gene Therapy, Hebrew University Hospital, Jeru- CXCL12/CXCR4 interactions results in mobilization of hemato- 4 salem, Israel. Hematology Division, Chaim Sheba Medical Center and Tel Aviv poietic stem and progenitor cells from the bone marrow to the University, Tel-Hashomer, Israel. 5Clinical Pharmacology Unit, Hadassah Uni- peripheral blood system. versity Hospital, Jerusalem, Israel. 6Cancer Immunotherapy and Immunobiology Research Center, Hadassah University Hospital, Jerusalem, Israel. Indeed, blockade of the CXCR4 receptor with the reversible CXCR4 antagonist AMD3100 (Plerixafor; Mozobil) results in Note: Supplementary data for this article are available at Clinical Cancer rapid mobilization of HSPCs (9, 10). When AMD3100 as a single Research Online (http://clincancerres.aacrjournals.org/). þ agent was compared with G-CSF as a mobilizer of CD34 cells M. Abraham and Y. Pereg contributed equally to this article. in healthy volunteers, AMD3100 was inferior to G-CSF (5). Corresponding Author: Amnon Peled, Hadassah University Hospital, P.O Box However, AMD3100 increased both G-CSF–stimulated mobili- þ 12000, Jerusalem, Israel. Phone: 972-2677-8780; Fax: 972-26430982; E-mail: zation and the leukapheresis yield of CD34 cells. As such, [email protected] Mozobil was approved in combination with G-CSF for the mobi- þ doi: 10.1158/1078-0432.CCR-16-2919 lization of CD34 cells in patients with lymphoma and multiple 2017 American Association for Cancer Research. myeloma that undergo stem cell mobilization (11, 12).

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two consecutive days). Part 1 of the study served to select the Translational Relevance optimal safe and efficacious dose of BL-8040 to be used in part Allogeneic hematopoietic stem and progenitor cell (HSPC) 2. Part 2 (dose expansion) was an open-label study, exploring transplantation (ALSPCT) has emerged as the preferred strat- the safety, tolerability, and pharmacodynamic effect of BL-8040 egy in the treatment of a variety of hematologic malignancies. in a single cohort of healthy subjects who received the selected Improved methods to mobilize and collect leukapheresis (LP) dose regimen of BL-8040 (1 mg/kg) based on the data collected products with shortened time to engraftment, immune recon- from part 1 (numbered 5001–5008). In addition, subjects stitution, and antitumor effects are essential. In the final LP underwent leukapheresis to examine the yield and character- products mobilized and collected after BL-8040, there were istics of the mobilized cells. Each cohort in part 1 consisted of þ þ much higher number of CD34 HPCs compared with CD34 8 subjects; 6 subjects in each cohort randomly allocated to HPCs collected after mobilization with granulocyte colony- receive BL-8040 and 2 subjects to receive placebo. Part 2 stimulating factor (G-CSF). Furthermore, in the LP products involved a single cohort of 8 subjects, who received BL-8040 mobilized and collected after BL-8040, there were much at the selected optimal dose level. þ þ higher number of CD4 CD8 T, NKT, NK, and dendritic cells, compared with LP collected after mobilization with G-CSF. Eligibility criteria This new graft composition may have a different effect on the As this was a dose escalation study in healthy volunteers, engraftment ability, antitumor effect, and immune reconsti- men only selection was for safety reasons to exclude exposure tution potential of the LP product. of childbearing potential subjects. The main criteria for inclusion for this study were: healthy male subjects aged between 18 and 45 years, with body mass index (BMI) between 18 and 30 kg/m2 and weight 60 kg. In addition, subjects had to be BL-8040 (BKT140) demonstrates a higher affinity and either surgically sterilized (vasectomy), or if their partner was of longer receptor occupancy for CXCR4 and provides a greater childbearing potential, had to use two methods of contracep- effect on the retention–mobilization balance of bone marrow tion, one of which had to be a barrier method, from the first SCs when compared with AMD3100 in both in vitro and in vivo dose until 3 months after the last dose. All the subjects were mice studies (13–15). Caucasian males. This study investigated the capacity of BL-8040 to mobi- After providing an informed consent, adult male subjects þ lize and retain CD34 cells in healthy volunteers, hypothe- ages 18–45 years old were screened for study eligibility by sizing that a single day procedure of BL-8040 monotherapy assessment of inclusion and exclusion criteria. Inclusion criteria administration (single injection) followed by one apheresis consisted of a BMI measure between 18 and 30 kg/m2 and þ session will provide sufficient amounts of CD34 HSPCs for weight 60 kg. The subjects were healthy as indicated by their transplantation. medical history, physical examination, 12-lead electrocardiogram (ECG), and laboratory safety tests. Screening procedures Materials and Methods included the collection of demographic data, medical history, physical examination [including height, weight and body mass Clinical study index (BMI)], vital signs (blood pressure, pulse rate, respiration A phase I, two-part study exploring the safety, tolerability, rate and oral temperature), 12-lead electrocardiogram (ECG), pharmacodynamic, and pharmacokinetic effects of ascending and safety laboratory evaluations [hematology, biochemistry, doses of BL-8040 in healthy subjects (study BL-8040.02) after coagulation (PT/INR and aPTT)] and urinalysis. informed consent was obtained. The study was conducted at the Hadassah Clinical Research Center (HCRC), Hadassah Medical Center, Jerusalem, Israel and approved by the Human Subjects Determination of blood counts and FACS analysis Committee Institutional Review Boards of Hadassah Medical WBCs and differential counts, immunophenotyping for neu- þ Center, Jerusalem, Israel. All subjects gave informed consent to trophils, T, B, NK cells, CD34 cell counts, and expression of participate in the study, which was approved by local Institutional CXCR4 using the 12G5 mAb were assessed for part 1 and 2 by Review Boards and conducted in accordance with the ethical FACS analysis. Immunophenotyping of peripheral blood and of principles of the Declaration of Helsinki. cells collected by the leukapheresis (exploratory endpoint) were assessed by FACS analysis for the following surface markers: Patients and methods CD34, CD16, CD56, CD3, CD4, CD8, CD19, CD11c, CD83, The study had two parts: part 1 (dose escalation) was a CD25, Foxp3, CXCR4 (part 2). Yields of hematopoietic progen- randomized, double-blind, placebo-controlled study exploring itor cells were tested by a methylcellulose medium with recom- the safety, tolerability, and the pharmacodynamic and phar- binant cytokines and EPO for human cells (MethoCult H4435; macokinetic profiles of BL-8040 injected subcutaneously at StemCell Technologies Inc.) þ doses of 0.5, 0.75, and 1 mg/kg. Individuals who received the The expression of CXCR4 on mobilized CD34 from BL-8040– dose of 0.5 mg/kg (n ¼ 6) and their placebo (n ¼ 2) were mobilized cells and from G-CSF–mobilized cells was done fol- numbered 1001–1008. Individuals who received the dose of lowing staining with two different CXCR4 antibody clones: 12G5 0.75 mg/kg (n ¼ 6) and their placebo (n ¼ 2) were numbered (binds to the second extracellular loops) or 1D9 (binds to the 2001–2008 and individuals who received the dose of 1 mg/kg N-terminal portion). Controls were incubated with appropriate (n ¼ 6) and their placebo (n ¼ 2) were numbered 3001–3008. isotype controls. þ þ WBCs and CD34 cell mobilization were measured in healthy BL-8040–mobilized-CD34 cells (n ¼ 4) and G-CSF–mobi- þ subjects following administration of BL-8040 (once daily on lized-CD34 cells (n ¼ 4) were stained with mAbs against CD34,

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CD38, CD45RA, Thy1 (CD90), and CD49f antigens and the Eight weeks following transplantation second transplanta- different hematopoietic stem and progenitors population was tion was performed. A total of 2 105 bone marrow cells/ analyzed with BD LSR II flow cytometer (Becton Dickinson). mouse were transplanted as described. The engraftment of cells in the second transplantation was allowed for 14 weeks after Leukapheresis transplantation. For all 8 subjects in part 2 (5001–5008), a leukapheresis Following engraftment mice were sacrificed and blood, bone procedure was performed approximately 4 hours after a single marrow and spleen were taken for analysis. Cells were isolated BL-8040 injection was administered. All mobilized products were from those organs, stained with anti-human CD45, and anti- þ collected using a Cobe Spectra apheresis device (Gambro BCT). human CD34 antibodies and percentage of cells evaluated by Five subjects of G-CSF–mobilized peripheral blood cells were FACS. For further analysis of transplanted cells, different staining collected by apheresis from healthy donor (Chaim Sheba Medical was done using specific anti-human fluorescence antibodies: Center, Hematology Division, Tel-Hashomer, Israel) under CD34/CD38, CD3/CD4/CD8, CD14/CD16, and CD19/CD56/ informed consent and Helsinki approval. Healthy donors CD3. FACS analysis was done using a FACSCalibur Flow Cyt- received G-CSF (Neupogen; Amgen), in a standard dose of 10 ometer (BD Biosciences). The data were analyzed using software mg/kg body weight subcutaneously for 4 days. On the morning of from CellQuest (version 3.3; BD Biosciences). To evaluate the the fifth day, they underwent conventional leukapheresis. Stem number of human progenitor cells following transplantation of þ cell harvesting was performed using Cobe Spectra Apheresis human CD34 cells, a colony-forming cell (CFC) assay was System (Caridian BCT, version 6.1 or 7). A 4-fold estimated blood performed. The colonies were assayed by plating 1 105 of cells volume was processed daily in 4 to 5 hours. Volume and proces- collected from the mice bone marrow, following lysis of RBC, in a sing of apheresis products were done according to standardized methylcellulose medium with recombinant cytokines and EPO procedures. If 1 leukapheresis was insufficient, an additional dose for human cells (MethoCult H4435; StemCell Technologies Inc.). of G-CSF was administrated and a second leukapheresis was The cultures were incubated at 37C in a humidified atmosphere performed. containing 5% CO2. The colonies that developed 10 days later were visually scored using a light microscope (employing mor- In vitro and in vivo studies in mice phologic criteria). þ þ Isolation of CD34 and CD3 cells. Isolation of cells was done using the Human MicroBeads Isolation Kit from Miltenyi Biotec. þ Statistical methods Isolation of mobilized CD34 cells was done from all collected þ All measured variables and derived parameters are listed indi- grafts and from G-CSF–mobilized cells. Isolation of CD3 T cells þ vidually and, where appropriate, presented using descriptive was done from the BL-8040 collected grafts (mobilized CD3 statistics. The safety parameters and changes from baseline were cells) and from the blood of healthy donors who have not þ examined and summarized for descriptive purposes. Adverse experienced mobilization with BL-8040 (normal CD3 cells). events (AE) were coded according to the MedDRA (version Purity of isolated cells was determined by FACS. 17.1) system organ class and preferred term. The individual study drug pharmacokinetic parameters and the mean, SD, and 95% In vitro migration of mobilized cells. A migration assay was confidence interval (CI) values were calculated for each dose performed using transmigration plates of 6.5 mm/diameter þ group, for all subjects. Pharmacodynamic analyses included the and 5 mm/pore (Costar). Purified CD34 cells (from BL- values, changes from predose, and fold increases of WBCs (neu- þ 8040–mobilized cells and from G-CSF–mobilized cells) were trophils, lymphocytes, monocytes, and platelets), CD34 and 5 þ suspended in RPMI medium containing 1% FCS. Cells (2 10 CD138 counts, red blood cells (RBC), and the number of stem cells/well) were added to the top chambers in a total volume of cell collections. The individual measurements and changes from 100 mL, and 600 mL RPMI supplemented with 100 ng/mL baseline by time point are presented in addition to summary CXCL12 (PeproTech) was added to the bottom chambers. The P þ tables by dose group. A value of less than 0.05 was considered same amount of isolated mobilized CD3 cells from the fi fi þ signi cant, and the signi cance of the differences between the collected grafts and normal CD3 cells were added to the groups for the stem cell collection was performed using Student t Transwell. The cells migrating to the bottom chamber of the test. A paired two-tailed Student t test was used to evaluate the Transwell within 4 hours were counted using a FACSCalibur significant differences between the groups. Flow Cytometer (BD Biosciences). The data were analyzed using software from CellQuest (version 3.3; BD Biosciences). Results Mice. Female NOD SCID gamma (NSG) mice (8–9 weeks old) Demographics and baseline characteristics were maintained under specific pathogen-free conditions at the Twenty-five subjects were enrolled into part 1 of the study; Hebrew University Animal Facility (Jerusalem, Israel). All experi- however, one subject did not receive the study drug and two ments were approved by the Animal Care and Use Committee of subjects received only one dose of the study drug. A total of the Hebrew University. 40 potential subjects were screened for part 1 of the study. Twenty-eight subjects met all inclusion and exclusion criteria þ Engraftment of mobilized human CD34 cells in mice. NSG mice and were eligible to participate in the study. Of these, 24 were were first irradiated with 300 cGy and 24 hours later mobilized initially included in the study. One subject, initially random- þ human CD34 cells were intravenously injected (2 105 cells/ ized to the BL-8040 1 mg/kg group, was withdrawn from the mouse) into the mouse via the dorsal tail vein in a final volume study due to the investigator's decision (the subject fainted of 200 mL. Engraftment of cells was allowed for 4, 8, and 22 weeks before BL-8040/placebo administration). An additional subject after transplantation. was therefore added to that group to replace this early dropout.

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Therefore, a total of 25 subjects were randomized to participate placebo group were all below the limit of quantitation, as in part 1 of the study and 24 subjects received at least one dose expected, and were not considered in the pharmacokinetic anal- and were included in the safety analysis. A total of 22 subjects ysis. Likewise, predose plasma concentrations were below the (22/24, 91.7%) completed the study. Two subjects (8.3%) limit of quantitation in all subjects randomized to receive BL- withdrew prematurely: one subject in the BL-8040 0.5 mg/kg 8040. After subcutaneous administration of BL-8040 on day 1, the treatment group withdrew his consent for participation in the appearance of the compound in the plasma was rapid, with a study, and one subject in the BL-8040 1 mg/kg withdrew from median Tmax ranging between 0.25 and 0.5 hour. Thereafter, the study due to injection site pain, tension headaches, nausea, plasma concentrations declined monoexponentially with a short vomiting, and abdominal discomfort. half-life of approximately 1 hour. For that reason, plasma levels of AllsubjectsweremaleandCaucasian.Thedatabelowis BL-8040 were below the limit of detection in the majority of presented only for the 24 subjects who received the study drug. subjects by 8 hours and in all subjects at 23 hours after dosing. Summary tables for baseline demographic data are presented in Summary statistics of pharmacokinetic parameters of BL-8040 Supplementary Table S1A. The baseline characteristics were after day 1 (first dose and second dose) can be found in Supple- similar across groups, and no subject reported any history of mentary Table S2A. alcohol and/or drug abuse or addiction and/or active/past (up Increases in the dose of BL-8040 led to overall approximate to 2 years before screening) nicotine consumption. No subject proportional increases in plasma exposure, as measured by reported a clinically significant medical history (Supplementary Cmax,AUC0–t, AUC0–24,andAUC0–¥ (Fig. 1). Dose-normalized Table S1A). Cmax and AUC0–24 and t1/2 values were comparable across the Eight subjects were enrolled into part 2 of the study. All subjects 3 dose groups suggesting dose proportionality across the 0.5 to were male and Caucasian. All subjects received BL-8040. Sum- 1 mg/kg dose range. mary tables for baseline demographic data are presented in The plasma concentration–time profile on day 2 was con- Supplementary Table S1B. No subject reported any history of sistent with day 1 and characterized by the rapid absorption alcohol and/or drug of abuse addiction and/or active/past (up and elimination of BL-8040 from the circulation. Consistent to 2 years before screening) nicotine consumption. No subject with its short half-life, accumulation of BL-8040 upon once reported clinically significant medical history (Supplementary daily repeated administration for two days was minimal. Table S1B). The pharmacokinetic parameters on day 2 (Supplementary Table SS2B; Fig. 1) were similar to those of day 1. Pharmacokinetic analysis Pharmacokinetic samples were collected only during part 1 of Safety analysis the study. A total of 15 blood samples were collected for phar- No deaths or serious AEs occurred during the study. In part 1, macokinetic analysis from each subject. Plasma concentrations of BL-8040 was overall well-tolerated, only a single subject termi- BL-8040 in subjects that received BL-8040 were analyzed by nated early due to an AE. No AE reported during this part of the model-independent methods using Phoenix WinNonlin version study was considered life-threatening, most AEs were mild or 6.4 (Pharsight, Inc.). Nominal sampling times were used. Con- moderate and only 13% of all reported AEs were considered as centrations below the limit of quantitation (BLQ) were reported severe. The most common AE among all the subjects was injection as "0" and were used as such in the pharmacokinetic analysis and site pain, reported in 88% of subjects, including the 3 subjects summary statistics of concentration–time data. who received a placebo. No dose response was observed for the Steady-state achievement after the second dose was assessed by overall incidences of AEs. An incidence pattern among cohorts comparing predose and 23-hour concentrations on day 1, and the 0.5–1 mg/kg, possibly suggestive of a dose response was observed Cmax on days 1 and 2. Accumulation on day 2, if any, was assessed for: leukocytosis, palpitations, nausea, dizziness, headache, pru- by comparing AUC0-24 and Cmax of the second and first doses. ritus generalized, and pallor for 21 of 24 (88%) subjects who Pharmacokinetic linearity was assessed by examining the rela- received BL-8040. Seventy-nine AEs related (definitely, probably tionship between dose and the exposure parameters (Cmax and and possibly) to study treatment were reported by 6 subjects CUA) on the first and second doses. Plasma concentrations in the (100%) receiving 0.5 mg/kg BL-8040. Among all the AEs reported

C ABmax AUC0−24 1,600 2,400 Day 1 Day 1 1,400 Day 2 Day 2 2,000 1,200 1,600 1,000 Figure 1.

800 Exposure versus dose relations of (ng/mL) 1,200 (ng/mL) BL-8040 after daily subcutaneous 0–24 max 600

C administration for two days in healthy 800 400 AUC volunteers.

200 400

0 0 0.4 0.5 0.6 0.7 1.0 0.8 0.9 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Dose (mg/kg) Dose (mg/kg)

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within the BL-8040 cohorts only 3 were assessed as related and number of WBCs reached its peak between 8 and 12 hours severe. Of the severe AEs (SAE), 2 subjects in the 1 mg/kg treat- following BL-8040 injection (4.54, 5.16, 5.81-fold increase, ment group had severe asthenia and 1 subject in the 0.75 mg/kg respectively) and started to decline after 12 hours (Fig. 2D). treatment group had severe syncope. The event resolved within Interestingly, the number of WBCs in the blood following the the same day it was reported and no supportive care required. In second injection did not increase above the maximal number the 02, no SAE were reported. The most common AE among all the achieved after the first injection, and neither did the mean fold subjects was injection site pain, reported in 88% of subjects, increase of WBCs. Forty-eight hours after the last injection of including the 3 subjects who received a placebo. Other common BL-8040 the number of WBCs in the blood was significantly AEs reported in more than 10% of all subjects included injection reduced (50% or less relative to peak accumulation of WBCs in site erythema (75%), injection site edema (75%), flushing (67%), the blood). In most of the subjects, the number of WBCs pruritus (63%), hot flush (46%), nausea (42%), injection site normalized 72 hours following the last injection. The increase pruritus (38%), urticaria (33%), headache (29%), asthenia in the number of WBCs could be attributed mainly to increase (25%), chills (25%), dizziness (25%), parasthesia (25%), hyper- in the number of neutrophils and lymphocytes subpopula- hidrosis (25%), pruritus generalized (25%), rash macular (21%), tions (70% and 20%, respectively;Fig.2EandF).Withinthe pallor (21%), vomiting (17%), injection site hematoma (17%), lymphocyte subpopulation, an increased mobilization of B, T, injection site induration (17%), edema peripheral (17%), leuko- and NK cells was noted (Fig. 2G). It is worth mentioning that a þ cytosis (13%), tachycardia (13%), diarrhea (13%), peripheral mobilization of dendritic cells (DC) (lin /CD11c cells) was swelling (13%), and pruritus genital (13%). Detailed drug-related observed following administration of BL-8040 (1mg/kg) AEs by preferred term and treatment group are shown in Supple- reaching a peak at 3.5 hours posttreatment and by 24 hours mentary Table S3A and S3B. postdose, the DC levels completely returned to the baseline After reviewing the data gathered from cohorts 1–3ofpart1, level (Fig. 2H). Mobilization of WBCs to the periphery and it was decided for part 2 to use a single dose of 1 mg/kg SC. In the recovery of normal numbers of WBCs correlate with the part 2, based on the safety data of part 1, premedication with ability of the CXCR4 12G5 antibody to bind to its epitope on steroids and antihistaminic drugs was implemented prior to the the CXCR4 receptor (Fig. 2H). þ BL-8040 injection. Premedication with paracetamol (1 g, oral- After the first dose administration of BL-8040, CD34 HSPCs ly), methylprednisolone 100 mg (i.v.), and promethazine counts increased rapidly reaching maximal levels between 4 and 25 mg (orally) were given 1 hour prior to the BL-8040 injection, 8 hours postdose (Fig. 3A). The maximal counts increased with which was given in the second part of the study. BL-8040 was dose averaging 9.3, 38.2, 43.7, and 45.5 IU/mL for placebo, 0.5, overall well tolerated and all subjects completed the study. No 0.75, and 1 mg/kg groups, respectively. By 24 hours postdose, þ AE reported during this part of the study was considered life- the CD34 levels had declined slightly but were still 5- to 7-fold threatening, all the AEs were mild or moderate and no AEs were higher than the baseline. In contrast to WBCs, the number of þ considered severe. The most common AEs reported in all CD34 cells after the second dose increased in a dose-depen- subjects were injection site pain, hot flush, and injection site dent manner (Fig. 3A). Upon administration of the second þ erythema. Overall, 100% of the subjects who received 1 dose of dose, an increase in CD34 was observed 4 to 8 hours after þ 1 mg/kg of BL-8040 reported AEs. The majority of these AEs dosing. In correlation with the expression of CXCR4, the CD34 (88%) were assessed as related (definitely, probably, and pos- counts declined thereafter reaching baseline level approximate- sibly) and were considered mild and moderate by the principal ly 48 hours after the second dose (Fig. 3A and E). At doses of investigator. The most common AE reported in 100% of the 0.5 and 0.75 mg/kg, 4 of 12 injected individuals failed to þ subjects was injection site erythema, injection site pain, and hot achieve 20 CD34 cells per mL4–8 hours following the first flush. Other common AEs reported in more than 10% of the administration of BL-8040, whereas all 6 individuals treated þ subjects were injection site edema (88%), macular rash (88%), with 1mg/kg achieved 20 CD34 cells per mL(Fig.3B–D). We flushing (75%), palpitations (50%), nausea (50%), asthenia therefore selected the 1 mg/kg as the dose for the second part of (50%), paresthesia (50%), injection site hematoma (38%), the study and performed aphaeresis 3–4 hours following the dizziness (38%), tachycardia (25%), dry mouth (25%), vomit- BL-8040 treatment. In part 2, similar to the results of part 1 of ing (25%), pallor (25%), injection site induration (25%), the study, injection of 1 mg/kg of BL-8040 induced a rapid þ abdominal discomfort (13%), abdominal pain (13%), dyspha- increase in the number of WBCs and CD34 cells in the gia (13%), injection site inflammation (13%), injection site periphery that declined between 24 and 48 hours (Fig. 4A and ischemia (13%), injection site necrosis (13%), injection site B) posttreatment. All 8 subjects (no placebo was administered þ paraesthesia (13%), injection site pruritus (13%), injection site in part 2 of the study) increased their CD34 count above 20 per scab (13%), peripheral swelling (13%), headache (13%), pre- mL approximately 3.5 hours after injection of BL-8040 before syncope (13%), tension headache (13%), epistaxis (13%), leukapheresis (Fig. 4C). The average blood volume processed tachypnoea (13%), and throat irritation (13%). during leukapheresis was 15.8 liters (range 9.8–17.5 liters). þ CD34 cell counts were assessed by FACS analysis and the þ Hematopoietic cell mobilization and collection amount of CD34 cells collected per kg was calculated for each þ In part 1 of the study a single administration of BL-8040 subject. The mean CD34 cells/kg collected was 10.3 106 (0.5, 0.75, 1 mg/kg) induced a rapid and dose-dependent cells/kg and the median was 10.5 106 cells/kg (range of 5.1 mobilization of WBCs into the blood in all subjects tested 106 to 15.2 106) when calculated on the basis of a 79 kg (Fig. 2A–C). The maximal number of WBCs in the different average recipient weight. Table 1A summarizes the individual þ cohorts was achieved after the first injection, and was 35.5 data and the amount of CD34 cells collected when calculated 103, 49.4 103,and44.3 103, respectively, for cohorts on the basis of actual donor weight as well as based on 79 kg receiving 0.5, 0.75, and 1 mg/kg. The mean fold increase in the average recipient weight.

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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 in H. 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.

þ Lifelong blood cell production is dependent on rare CD49f HSCs were found as the population with the highest þ þ hematopoietic stem cells (HSCs). The bulk of HSCs are CD34 long-term and secondary repopulating activity (17). CD34 þ cells; however, most CD34 cells are lineage-restricted pro- cells were purified from BL-8040 and G-CSF–mobilized grafts genitors and HSCs remain rare. HSCs can be enriched further and stained for CD38, CD45RA, CD90, and CD49f. The þ based on CD45RA, Thy1 (CD90), and CD38 expression. Loss percentage of CD34 CD38 hematopoietic stem and progeni- þ of CD90 expression in the CD34 CD38 CD45RA compart- tors was similar in both grafts (Fig. 4D). However, whereas þ ment of lineage depleted cord blood (CB) was proposed 23.2 % of BL-8040–mobilized CD34 CD38 cells did not þ þ þ to be sufficient to separate CD34 CD38 CD45RA CD90 express CD45RA, only 1.6% of G-CSF–mobilized CD34 þ HSCs from CD34 CD38 CD45RA CD90 multipotent prog- CD38 cells did not express CD45RA (Fig. 4D). The per- þ enitors (MPP; ref. 16). Recently, it was demonstrated that centage and absolute number of CD34 CD38 CD45RA þ þ þ þ þ þ CD49f is a specific HSC marker. Single CD49f cells were CD49f CD90 / ,CD34 CD38 CD45RA CD49f CD90 , and þ þ highly efficient in generating long-term multilineage grafts, CD34 CD38 CD45RA CD90 HPCS were increased signifi- and the loss of CD49f expression identified transiently engraft- cantly by 45, 25, 12 (% of cells) -fold and by 61.7, 36.6, 59.7 þ þ ing MPPs. Furthermore, CD34 CD38 CD45RA CD90 (absolute number of cells per kg) in the BL-8040 graft compare

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Figure 3. Mean BL-8040 dose-dependent (0.5, 0.75, 1 mg/kg) mobilization of CD34þ cells/mL of blood is shown in A. Red arrows indicate the times of the two injections of BL-8040. Patients (1001–1008), placebo, and BL-8040 dose-dependent (0.5, 0.75, 1 mg/kg) mobilization of CD34þ cells/mL of blood is shown in B–D. CXCR4 12g5 Ab binding to its epitope on the CXCR4 receptor is partially blocked on CD34þ cells by treatment with BL-8040 as shown in E.

þ to G-CSF graft–derived CD34 CD38 cells (Fig. 4E; Supplemen- NK cells (Supplementary Table S4B). Furthermore, in the þ tary Table S4A). CD4 T-cell population, we found that the graft contained mostly na€ve CD4 T cells (35%), effector memory CD4 T cells (21%), These results suggest the BL8040 is a better mobilizer of central memory CD4 T cells (38%), and almost no effector CD4 T þ HSCs than G-CSF cells (6%). Whereas in the CD8 T-cell population, we found that þ It was already shown before that G-CSF mobilizes CD34 the graft contained mostly na€ve CD8 T cells (64%), and central cells collected from the blood migrate less in response to memory CD8 T cells (33%), and almost no effector memory CD8 þ CXCL12 (18). Interestingly, we found that CD34 cells mobi- T cells (1%), and effector CD8 T cells (2%; Fig. 5). lized with BL-8040 migrated better than G-CSF–mobilized cells The different immune subpopulation in the BL-8040 com- in response to CXCL12 (Fig. 4F). pared with G-CSF–mobilized graft were determined (Supple- þ We stained CD34 cells mobilized cells from BL-8040 and mented Table S4B). Significant increase in the percentage of þ þ þ þ þ G-CSF–mobilized CD34 cells with 12g5 mAb, which binds the CD3 CD4 ,CD3 CD8 ,DC,na€ve, and effector CD4 T cells, second extracellular loop of CXCR4, and 1D9 mAb, which binds central memory CD4 T cells, na€ve CD8 T cells, and effector the N-terminal portion of CXCR4. When cells were stained memory CD8 cells was observed (Fig. 5). with 1D9 antibodies similar expression levels of CXCR4 were The SCID-reconstituting capacity of fresh or frozen purified þ observed while the level of CXCR4 following staining with 12g5 CD34 cells (>90%) was evaluated in transplant experiments antibodies was significantly reduced on BL-8040 mobilized cells in NSG mice using human anti-CD45 antibody at a threshold but not on G-CSF mobilized cells (Fig. 4G). This suggests a specific level of 0.5% of marrow and spleen cellularity and blood. block of the 12g5 with no internalization of the CXCR4 receptor. Significant engraftment was observed in the bone marrow, The graft collected was further tested for its immune compo- spleen,andbloodofalltransplantedmice(Fig.6A).Thebone þ þ þ þ sition. It was found that the graft was enriched for immune cells, was engrafted with CD45 /CD19 B cells, CD45 /CD56 / þ þ þ þ including immature dendritic cells (ImDC), T cells, B cells, and CD3 NK cells, CD45 /CD14 /CD16 myelomonocytic

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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 underwent apheresis 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.Percentage of CD34þCD38 cells from total of CD34þ cells and percentage of CD45RA cells out of CD34þCD38 from cells mobilized by BL-8040 or G-CSF is shown in D.PercentageofCD34þCD38 CD45RA CD49fþ from a total of CD34þCD38 cells and percent of CD34þCD38CD45RACD90þCD49fþ from atotalofCD34þCD38 CD45RA CD49fþ of cells mobilized by BL-8040 or G-CSF is shown in E. SDF-1 migration of BL-8040–mobilized CD34þ cell and 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. The expression 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–mobilized cells (n ¼ 3). CD34þ cells were stained with two different CXCR4 antibody clones 12g5 (binding to extracellular loops) or 1D9 (binding to the N-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 kg average 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 4.5 5002 16.0 1.01 11.9 10.6 5003 16.6 0.85 13.7 13.2 5004 16.2 0.76 10.2 10.5 5005 16.6 0.78 11.4 13.5 5006 16.5 0.87 13.7 13.0 5007 17.5 0.64 11.1 8.6 5008 16.7 0.61 9.6 8.9 Average 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 BL-8040 (n ¼ 8) AMD3100a (n ¼ 24) G-CSFa (n ¼ 8) Cell type Median (range) Median (range) 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/Kg aDevine and colleagues (5).

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transplanted mice and re-transplanted them in to a secondary recipient. Successful and robust long-term human engraftment of secondary recipient was observed 14 weeks following the second transplantation (Fig. 6E and F). In correlation with the engraftment potential we observed þ in NSG mice, the ability of thawed puriﬁed CD34 HPCs þ and CD3 cells to migrate in response to the CXCR4 ligand CXCL12 was found to be intact (Fig. 4F).

Discussion The most frequently used graft of hematopoietic stem and progenitor cells for allogeneic transplantation is peripheral blood stem cells (PBSC) that are collected after mobilization with G-CSF. Both in vitro and in vivo experiments have recently suggested that G-CSF induces HSPC mobilization by indirectly disrupting the interaction between CXCR4 and SDF-1 (6–8). Furthermore, G-CSF activates neutrophils leading to the release of proteases, neutrophil elastase, and cathepsin G, which can directly cleave the adhesive interactions between HSPCs and þ the bone marrow microenvironment. Mobilization of CD34 cells peaked in the peripheral blood between days 4 and 5 of G-CSF dosing. A dose between 10 and 16 mg/kg split into two doses per day was used for PBSC mobilization (5). Pulsipher and colleagues reported that among 2,408 unrelated donors of PBSCs, two-thirds (66%) underwent two apheresis procedures, whereas the remaining donors completed their dona- tions in a single day (19). Ings and colleagues reported that among 400 unrelated donors of PBSCs target recipient doses Figure 5. were reached with one aphaeresis in 63% of donors and with Percentages of different subpopulations of BL-8040–mobilized cells (n ¼ 8) as compared with G-CSF–mobilized cells (n ¼ 5) was analyzed by flow cytometry. two apheresis in 81% of donors; 19% of the donors needed Pie charts show the cells distribution within the CD4þ or CD8þ populations. more than two apheresis (20). CD3þ/CD4þ/CD45RAþ/CCR7þ (na€ve CD4þ cells); CD3þ/CD4þ/CD45RAþ/ AMD3100 (plerixafor, Mozobil) is a reversible direct antag- CCR7 (effector CD4 T cells); CD3þ/CD4þ/CD45RA/CCR7 (effector memory onist of the interaction between the chemokine SDF-1 and its þ þ þ CD4 T cells); CD3 /CD4 /CD45RA /CCR7 (central memory CD4 T cells). receptor CXCR4 and is used for HSPC mobilization. Plerixafor CD3þ/CD8þ/CD45RAþ/CCR7þ (na€ve CD8þ cells); CD3þ/CD8þ/CD45RAþ/ þ þ was approved for use in combination with G-CSF to mobilize CCR7 (effector CD8 T cells); CD3 /CD8 /CD45RA /CCR7 (effector memory CD8 T cells); CD3þ/CD4þ/CD45RA/CCR7þ (central memory CD4 T cells). HSPC for autologous HSCT (12, 21). Studies conducted in healthy donors reported that Plerixafor induces a transient þ increase in CD34 that peaks 9 hours after subcutaneous þ þ þ þ cells, and CD45 /CD34 /CD38 progenitors and CD45 / injection with 80–240 mg/kg of plerixafor and declines at 24 þ CD34 /CD38 stem cells as well as human colony-forming hours (22). In another study reported by Devine and collea- cells (Fig. 6B–D). gues (5), plerixafor (n ¼ 24) was compared with G-CSF (n ¼ 8) To assess the long-term engraftment potential of the BL- in mobilization and collection from healthy sibling donors. At þ 8040–mobilized CD34 cells, engraftment was allowed for 4, 4 hours after a single dose of AMD3100 (240 mg/kg), the þ 8, and 22 weeks after transplantation. Successful and robust CD34 count rose to a median of 16/mL(range,4–54/mL). þ þ þ long-term human engraftment of CD45 and CD45 CD34 One out of the 25 donors could not undergo large volume (27 cells was observed at week 22 (Fig. 6E and F). The percent of liters) leukapheresis following AMD3100 due to repeated CD45 cells in the bone marrow remained stable in the bone vasovagal episodes during peripheral line placement prior to marrow, whereas the percentage of CD45 cells in the blood and commencing leukapheresis. A total of 8 (33%) of the remain- spleen increased at week 22 (Fig. 6G). At 4 weeks, human ing 24 donors who commenced leukapheresis following þ þ CD3 CD4 T cells were only observed at a low percentage in AMD3100 administration on day 1 did not collect the min- þ the spleen but not in the bone marrow, whereas no significant imum required CD34 cell dose in one day (2 106/kg) and þ þ percentage of CD3 CD8 cells were found neither in the bone were thus eligible for a second day of drug administration and þ marrow nor in the spleen (Fig. 6H and I). Following 22 weeks of leukapheresis on day 3. The median number of CD34 cells þ þ transplantation, the percentage of human CD3 CD4 and collected from all 24 donors was 2.9 106/kg (range, 1.2–6.3). þ þ þ CD3 CD8 T cells was significantly increased in the spleen In the G-CSF group, the CD34 count rose to a median of (30% vs. 5%, respectively). The percentage of human 46/mLonday5(range,4–54/mL). The median number of þ þ þ þ þ CD3 CD4 and CD3 CD8 T cells was increased at much CD34 cells collected from all 8 donors was 4.2 106/kg þ lower levels in the bone marrow (Fig. 6H and I). (range, 2.5–18.7). The increase in CD34 cells after 5 days of To further assess the long-term engraftment potential of the G-CSF was significantly greater than the rise 4 hours after BL-8040, we collected the bone marrow cells from 8-week-old AMD3100 administration (P < 0.001; ref. 5).

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Figure 6. The SCID reconstituting capacity of fresh or frozen purified CD34þ cells (>90% purity) was evaluated in transplant experiments in NSG mice following 4 weeks, using human anti-CD45 antibodies is shown in A. The number of human colony-forming cells HPCs in the bone marrow of mice transplanted with 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 cells following 4 weeks of transplantation. Engraftment of CD45þCD34þCD38þ and CD45þ CD34þ CD38 cells demonstrated in C. The number of human HPCs in the bone marrow of mice following short transplantation (4 weeks) and long transplantation (22 weeks) is shown in D.Micewere transplanted 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 transplantation is shown in E and F. Engraftment percentage of hCD45þ cells in the bone marrow, spleen, and blood following 4 and 22 weeks of transplantation is shown in G. Engraftment percentage of human CD3þ,CD3þCD4þ,andCD3þCD8þ T cells in the bone marrow and spleen following 4 and 22 weeks of transplantation 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.

þ The effect of BL-8040 as compared with AMD3100 was declined only slightly. Furthermore, the mean CD34 cells/kg studied in in vitro and in vivo experimental settings. BL-8040 collected from the individuals treated with 1mg/kg BL-8040 binds and inhibits the CXCR4 chemokine receptor with high was 11.6 106 cells/kg and the median was 11.9 106 cells/kg 6 6 affinity, showing an IC50 of approximately 1 nmol/L (23–25) (range of 5.1 10 to 15.2 10 ). The results of this trial compared with the values obtained with AMD3100 (IC50 ¼ support the hypothesis that in healthy humans the number þ 651 37 nmol/L; ref. 26). Moreover, BKT140 hinders the of CD34 cells that can be rapidly mobilized and collected cell migration stimulated by CXCL12 within IC50 values of is greater than observed with either G-CSF or plerixafor. Fur- þ 0.5–2.5 nmol/L (24, 27) compared with the IC50 value of thermore, we have found that the percentage of CD34 CD38 þ þ þ 51 17 nmol/L for Mozobil (26). Furthermore, BL-8040 has CD45RA CD49f CD90 cells, and CD34 CD38 CD45RA þ long receptor occupancy (>24 hours) compared with CD90 cells were significantly higher in the BL-8040 graft AMD3100, resulting in the extended inhibition of CXCR4 compared with G-CSF graft (Fig. 4E). In association with (15). In animal studies, comparison between the BL-8040 the high percentage of HPCs in the BL-8040 graft, we found and AMD3100 showed that BL-8040 with or without G-CSF a robust myeloid and lymphoid long-term engraftment þ was significantly more potent in its ability to mobilize hemato- (week 22) of BL-8040–mobilized human CD34 cells in NSG poietic stem cells and progenitors into blood (14, 15). mice (Fig. 6). Inthisstudy,wefoundthatat4–8 hours after a single dose In Devine and colleagues' study (5), there were notable þ of BL-8040 (0.5, 0.75, 1 mg/kg), the CD34 count rose to a differences in the final leukapheresis products mobilized and median of 38.2, 43.7 and 45.5 mL (respectively). In contrast to collected after AMD3100 or G-CSF were administered. There þ þ treatment with AMD3100, by 24 hours, CD34 levels had were significantly fewer CD34 cells/kg (2.9 106 vs. 4.2

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Rapid and Robust Mobilization of HSPCs in Healthy Volunteers

þ 106; P < 0.006), but greater numbers of CD3 cells/kg (4.7 clearly greater than seen with the first generation of CXCR4 8 10 vs. 1.5 108; P < 0.006) and CD4þ cells/kg (3.1 108 vs. antagonists. A phase II study assessing safety and efficacy of 1.1 108; P < 0.002) in the AMD3100-mobilized products BL-8040 for the mobilization of donor hematopoietic stem compared with the G-CSF–mobilized allografts. Therefore, cells and allogeneic transplantation in patients with advanced þ þ more CD3 and CD4 cells were mobilized per unit of blood hematologic malignancies is currently being conducted at the following AMD3100 compared with G-CSF treatment. How- Washington University School of Medicine, Division of Onco- þ þ þ ever, the differences in CD8 ,CD19 ,andCD56 cell content logy and Hematology, St. Louis, MO (NCT02639559). were not significant. Interestingly, the rates of acute and chronic GVHD observed in the 20 transplanted patients were not Disclosure of Potential Conflicts of Interest fi signi cantly different compared with historical control patients M. Abraham, H. Wald, B. Bulvik, and O. Eizenberg are employees and at Washington University receiving G-CSF–mobilized allografts shareholders of Biokine Therapeutics Ltd.; A. Peled serves as consultant for together with the exact same conditioning and GVHD prophy- Biokine Therapeutics and is also a shareholder. Y. Pereg, R. Golan, A. Vainstein, laxis. There was no difference in the median time to neutrophil and A. Aharon are employees of BioLineRx Ltd. No potential conflicts of interest or platelet engraftment between recipients in the AMD3100 group were disclosed by the other authors. or the historical group receiving G-CSF–mobilized allografts in this study and all surviving patients in remission continue to Authors' Contributions have robust and durable trilineage hematopoiesis. Conception and design: M. Abraham, Y. Pereg, O. Eizenberg, A. Aharon, In the final leukapheresis products mobilized and collected A. Peled þ after BL-8040 there were much higher number of CD34 Development of methodology: M. Abraham, Y. Pereg, B. Bulvik, H. Wald, A. Aharon, R. Or, A. Peled progenitor and stem cells compared with the leukapheresis Acquisition of data (provided animals, acquired and managed patients, collected after mobilization with AMD3100 or G-CSF (5, provided facilities, etc.): B. Bulvik, S. Klein, I. Mishalian, A. Nagler, A. Vainstein, Table 1B). Furthermore, in the leukapheresis products mobi- Y. Caraco, R. Or, A. Peled lized and collected after BL-8040 there was a significant in- Analysis and interpretation of data (e.g., statistical analysis, biostati- þ þ þ þ crease in the percentage of CD3 CD4 ,CD3 CD8 ,DC,na€ve stics, computational analysis): M. Abraham, Y. Pereg, B. Bulvik, R. Golan, þ and effector CD4 T cells, central memory CD4 T cells, na€ve A. Vainstein, Y. Caraco, A. Peled Writing, review, and/or revision of the manuscript: M. Abraham, Y. Pereg, CD8 T cells, and effector memory CD8 cells compared with O. Eizenberg, A. Nagler, R. Golan, A. Vainstein, A. Aharon, E. Galun, Y. Caraco, G-CSF–mobilized graft (Fig. 5). This new composition may A. Peled have an effect on the engraft ability, GVHD, GVL, and immune Administrative, technical, or material support (i.e., reporting or organizing reconstitution potential of the leukapheresis product. data, constructing databases): Y. Pereg, O. Eizenberg, K. Beider, R. Golan In summary, our results suggest that BL-8040 is a rapid and Study supervision: A. Vainstein, A. Aharon, Y. Caraco þ robust mobilizer of a of CD34 progenitors and stem cells with The costs of publication of this article were defrayed in part by the payment of an intact long-term engraftment potential, as assessed by page charges. This article must therefore be hereby marked advertisement in engraftment of these human cells in a mouse model. Further- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. more, these results suggest that in the bone marrow of humans, þ the number of CD34 with the potential to exit rapidly into the Received November 22, 2016; revised June 22, 2017; accepted August 16, blood upon a strong and sustained inhibition of CXCR4 is 2017; published OnlineFirst August 23, 2017.

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OF12 Clin Cancer Res; 23(22) November 15, 2017 Clinical Cancer Research

Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid Mobilization for the Collection of Human CD34 + Cells in Healthy Volunteers

Michal Abraham, Yaron Pereg, Baruch Bulvik, et al.

Clin Cancer Res Published OnlineFirst August 23, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-2919

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