Purpose: The potential of the high-affinity CXCR4 antagonist BL-8040 as a monotherapy-mobilizing agent and its derived graft composition and quality were evaluated in a phase I clinical study in healthy volunteers (NCT02073019).

Experimental Design: The first part of the study was a randomized, double-blind, placebo-controlled dose escalation phase. The second part of the study was an open-label phase, in which 8 subjects received a single injection of BL-8040 (1 mg/kg) and approximately 4 hours later underwent a standard leukapheresis procedure. The engraftment potential of the purified mobilized CD34+ cells was further evaluated by transplanting the cells into NSG immunodeficient mice.

Results: BL-8040 was found safe and well tolerated at all doses tested (0.5–1 mg/kg). The main treatment-related adverse events were mild to moderate. Transient injection site and systemic reactions were mitigated by methylprednisolone, paracetamol, and promethazine pretreatment. In the first part of the study, BL-8040 triggered rapid and substantial mobilization of WBCs and CD34+ cells in all tested doses. Four hours postdose, the count rose to a mean of 8, 37, 31, and 35 cells/μL (placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysis revealed substantial mobilization of immature dendritic, T, B, and NK cells. In the second part, the mean CD34+ cells/kg collected were 11.6 × 106 cells/kg. The graft composition was rich in immune cells.

Conclusions: The current data demonstrate that BL-8040 is a safe and effective monotherapy strategy for the collection of large amounts of CD34+ cells and immune cells in a one-day procedure for allogeneic HSPC transplantation. Clin Cancer Res; 23(22); 6790–801. ©2017 AACR.

Translational Relevance

Allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation (ALSPCT) has emerged as the preferred strategy in the treatment of a variety of hematologic malignancies. Improved methods to mobilize and collect leukapheresis (LP) products with shortened time to engraftment, immune reconstitution, and antitumor effects are essential. In the final LP products mobilized and collected after BL-8040, there were much higher number of CD34+ HPCs compared with CD34+ HPCs collected after mobilization with granulocyte colony-stimulating factor (G-CSF). Furthermore, in the LP products mobilized and collected after BL-8040, there were much higher number of CD4+CD8+ 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 the engraftment ability, antitumor effect, and immune reconstitution potential of the LP product.

Allogenic hematopoietic stem and progenitor cell (HSPC) transplantation (ALSPCT) has emerged as the preferred strategy in the treatment of a variety of hematologic malignancies (1, 2). Mobilization of stem cells using granulocyte colony-stimulating factor (G-CSF) from healthy donors is the common clinical practice. G-CSF–mobilized peripheral blood mononuclear cells (PBMC) are routinely used as a source of hematopoietic stem cells (HSC) for transplantation (3, 4). Despite the potency of G-CSF in mobilizing stem cells, it ultimately results in broad interindividual variations in circulating progenitor and stem cell numbers (5), requiring 4–6 repeated dosing to collect a sufficient number of cells. In addition, although considered generally safe, G-CSF is frequently associated with a variety of side effects. Therefore, improved methods to mobilize and collect HSPCs for transplantation are required.

It has been proposed that G-CSF induces the mobilization of HSPCs through an indirect mechanism by activating neutrophils to secrete a variety of proteolytic enzymes, including elastase, cathepsin G, MMP-2, and MMP-9 that can degrade the chemokine 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 mobilization and engraftment (6–8). Consequently, disruption of CXCL12/CXCR4 interactions results in mobilization of hematopoietic stem and progenitor cells from the bone marrow to the peripheral blood system.

Indeed, blockade of the CXCR4 receptor with the reversible CXCR4 antagonist AMD3100 (Plerixafor; Mozobil) results in rapid mobilization of HSPCs (9, 10). When AMD3100 as a single agent was compared with G-CSF as a mobilizer of CD34+ cells in healthy volunteers, AMD3100 was inferior to G-CSF (5). However, AMD3100 increased both G-CSF–stimulated mobilization and the leukapheresis yield of CD34+ cells. As such, Mozobil was approved in combination with G-CSF for the mobilization of CD34+ cells in patients with lymphoma and multiple myeloma that undergo stem cell mobilization (11, 12).

BL-8040 (BKT140) demonstrates a higher affinity and longer receptor occupancy for CXCR4 and provides a greater effect on the retention–mobilization balance of bone marrow SCs when compared with AMD3100 in both in vitro and in vivo mice studies (13–15).

This study investigated the capacity of BL-8040 to mobilize and retain CD34+ cells in healthy volunteers, hypothesizing that a single day procedure of BL-8040 monotherapy administration (single injection) followed by one apheresis session will provide sufficient amounts of CD34+ HSPCs for transplantation.

Clinical study

A phase I, two-part study exploring the safety, tolerability, pharmacodynamic, and pharmacokinetic effects of ascending doses of BL-8040 in healthy subjects (study BL-8040.02) after 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 Committee Institutional Review Boards of Hadassah Medical Center, Jerusalem, Israel. All subjects gave informed consent to participate in the study, which was approved by local Institutional Review Boards and conducted in accordance with the ethical principles of the Declaration of Helsinki.

Patients and methods

The study had two parts: part 1 (dose escalation) was a randomized, double-blind, placebo-controlled study exploring the safety, tolerability, and the pharmacodynamic and pharmacokinetic profiles of BL-8040 injected subcutaneously at doses of 0.5, 0.75, and 1 mg/kg. Individuals who received the dose of 0.5 mg/kg (n = 6) and their placebo (n = 2) were numbered 1001–1008. Individuals who received the dose of 0.75 mg/kg (n = 6) and their placebo (n = 2) were numbered 2001–2008 and individuals who received the dose of 1 mg/kg (n = 6) and their placebo (n = 2) were numbered 3001–3008. WBCs and CD34+ cell mobilization were measured in healthy subjects following administration of BL-8040 (once daily on two consecutive days). Part 1 of the study served to select the optimal safe and efficacious dose of BL-8040 to be used in part 2. Part 2 (dose expansion) was an open-label study, exploring the safety, tolerability, and pharmacodynamic effect of BL-8040 in a single cohort of healthy subjects who received the selected dose regimen of BL-8040 (1 mg/kg) based on the data collected from part 1 (numbered 5001–5008). In addition, subjects underwent leukapheresis to examine the yield and characteristics of the mobilized cells. Each cohort in part 1 consisted of 8 subjects; 6 subjects in each cohort randomly allocated to receive BL-8040 and 2 subjects to receive placebo. Part 2 involved a single cohort of 8 subjects, who received BL-8040 at the selected optimal dose level.

Eligibility criteria

As this was a dose escalation study in healthy volunteers, men only selection was for safety reasons to exclude exposure 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 either surgically sterilized (vasectomy), or if their partner was of childbearing potential, had to use two methods of contraception, one of which had to be a barrier method, from the first dose until 3 months after the last dose. All the subjects were Caucasian males.

After providing an informed consent, adult male subjects ages 18–45 years old were screened for study eligibility by assessment of inclusion and exclusion criteria. Inclusion criteria consisted of a BMI measure between 18 and 30 kg/m2 and weight ≥ 60 kg. The subjects were healthy as indicated by their medical history, physical examination, 12-lead electrocardiogram (ECG), and laboratory safety tests. Screening procedures included the collection of demographic data, medical history, physical examination [including height, weight and body mass index (BMI)], vital signs (blood pressure, pulse rate, respiration rate 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 analysis

WBCs and differential counts, immunophenotyping for neutrophils, T, B, NK cells, CD34+ cell counts, and expression of CXCR4 using the 12G5 mAb were assessed for part 1 and 2 by FACS analysis. Immunophenotyping of peripheral blood and of cells collected by the leukapheresis (exploratory endpoint) were assessed 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 progenitor cells were tested by a methylcellulose medium with recombinant cytokines and EPO for human cells (MethoCult H4435; StemCell Technologies Inc.)

The expression of CXCR4 on mobilized CD34+ from BL-8040–mobilized cells and from G-CSF–mobilized cells was done following staining with two different CXCR4 antibody clones: 12G5 (binds to the second extracellular loops) or 1D9 (binds to the N-terminal portion). Controls were incubated with appropriate isotype controls.

BL-8040–mobilized-CD34+ cells (n = 4) and G-CSF–mobilized-CD34+ cells (n = 4) were stained with mAbs against CD34, CD38, CD45RA, Thy1 (CD90), and CD49f antigens and the different hematopoietic stem and progenitors population was analyzed with BD LSR II flow cytometer (Becton Dickinson).

Leukapheresis

For all 8 subjects in part 2 (5001–5008), a leukapheresis procedure was performed approximately 4 hours after a single BL-8040 injection was administered. All mobilized products were collected using a Cobe Spectra apheresis device (Gambro BCT).

Five subjects of G-CSF–mobilized peripheral blood cells were collected by apheresis from healthy donor (Chaim Sheba Medical Center, Hematology Division, Tel-Hashomer, Israel) under informed consent and Helsinki approval. Healthy donors received G-CSF (Neupogen; Amgen), in a standard dose of 10 μg/kg body weight subcutaneously for 4 days. On the morning of the fifth day, they underwent conventional leukapheresis. Stem cell harvesting was performed using Cobe Spectra Apheresis System (Caridian BCT, version 6.1 or 7). A 4-fold estimated blood volume was processed daily in 4 to 5 hours. Volume and processing of apheresis products were done according to standardized procedures. If 1 leukapheresis was insufficient, an additional dose of G-CSF was administrated and a second leukapheresis was performed.

In vitro and in vivo studies in mice

Isolation of CD34+ and CD3+ cells.

Isolation of cells was done using the Human MicroBeads Isolation Kit from Miltenyi Biotec. Isolation of mobilized CD34+ cells was done from all collected grafts and from G-CSF–mobilized cells. Isolation of CD3+ T cells was done from the BL-8040 collected grafts (mobilized CD3+ cells) and from the blood of healthy donors who have not experienced mobilization with BL-8040 (normal CD3+ cells). Purity of isolated cells was determined by FACS.

In vitro migration of mobilized cells.

A migration assay was performed using transmigration plates of 6.5 mm/diameter and 5 μm/pore (Costar). Purified CD34+ cells (from BL-8040–mobilized cells and from G-CSF–mobilized cells) were suspended in RPMI medium containing 1% FCS. Cells (2 × 105 cells/well) were added to the top chambers in a total volume of 100 μL, and 600 μL RPMI supplemented with 100 ng/mL CXCL12 (PeproTech) was added to the bottom chambers. The same amount of isolated mobilized CD3+ cells from the collected grafts and normal CD3+ cells were added to the Transwell. The cells migrating to the bottom chamber of the Transwell within 4 hours were counted using a FACSCalibur Flow Cytometer (BD Biosciences). The data were analyzed using 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 the Hebrew University Animal Facility (Jerusalem, Israel). All experiments were approved by the Animal Care and Use Committee of the Hebrew University.

Engraftment of mobilized human CD34+ cells in mice.

NSG mice were first irradiated with 300 cGy and 24 hours later mobilized human CD34+ cells were intravenously injected (2 × 105 cells/mouse) into the mouse via the dorsal tail vein in a final volume of 200 μL. Engraftment of cells was allowed for 4, 8, and 22 weeks after transplantation.

Eight weeks following transplantation second transplantation was performed. A total of 2 × 105 bone marrow cells/mouse were transplanted as described. The engraftment of cells in the second transplantation was allowed for 14 weeks after transplantation.

Following engraftment mice were sacrificed and blood, bone marrow and spleen were taken for analysis. Cells were isolated from those organs, stained with anti-human CD45, and anti-human CD34+ antibodies and percentage of cells evaluated by FACS. For further analysis of transplanted cells, different staining was 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 Cytometer (BD Biosciences). The data were analyzed using software from CellQuest (version 3.3; BD Biosciences). To evaluate the number of human progenitor cells following transplantation of human CD34+ cells, a colony-forming cell (CFC) assay was performed. The colonies were assayed by plating 1 × 105 of cells collected from the mice bone marrow, following lysis of RBC, in a methylcellulose medium with recombinant cytokines and EPO for human cells (MethoCult H4435; StemCell Technologies Inc.). The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. The colonies that developed 10 days later were visually scored using a light microscope (employing morphologic criteria).

Statistical methods

All measured variables and derived parameters are listed individually and, where appropriate, presented using descriptive statistics. The safety parameters and changes from baseline were examined and summarized for descriptive purposes. Adverse events (AE) were coded according to the MedDRA (version 17.1) system organ class and preferred term. The individual study drug pharmacokinetic parameters and the mean, SD, and 95% confidence interval (CI) values were calculated for each dose group, for all subjects. Pharmacodynamic analyses included the values, changes from predose, and fold increases of WBCs (neutrophils, lymphocytes, monocytes, and platelets), CD34+ and CD138+ counts, red blood cells (RBC), and the number of stem cell collections. The individual measurements and changes from baseline by time point are presented in addition to summary tables by dose group. A P value of less than 0.05 was considered significant, and the significance of the differences between the groups for the stem cell collection was performed using Student t test. A paired two-tailed Student t test was used to evaluate the significant differences between the groups.

Demographics and baseline characteristics

Twenty-five subjects were enrolled into part 1 of the study; however, one subject did not receive the study drug and two subjects received only one dose of the study drug. A total of 40 potential subjects were screened for part 1 of the study. Twenty-eight subjects met all inclusion and exclusion criteria and were eligible to participate in the study. Of these, 24 were initially included in the study. One subject, initially randomized to the BL-8040 1 mg/kg group, was withdrawn from the study due to the investigator's decision (the subject fainted before BL-8040/placebo administration). An additional subject was therefore added to that group to replace this early dropout. Therefore, a total of 25 subjects were randomized to participate in part 1 of the study and 24 subjects received at least one dose and 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/kg treatment group withdrew his consent for participation in the study, and one subject in the BL-8040 1 mg/kg withdrew from the study due to injection site pain, tension headaches, nausea, vomiting, and abdominal discomfort.

All subjects were male and Caucasian. The data below is presented only for the 24 subjects who received the study drug. Summary tables for baseline demographic data are presented in Supplementary Table S1A. The baseline characteristics were similar across groups, and no subject reported any history of alcohol and/or drug abuse or addiction and/or active/past (up to 2 years before screening) nicotine consumption. No subject reported a clinically significant medical history (Supplementary Table S1A).

Eight subjects were enrolled into part 2 of the study. All subjects were male and Caucasian. All subjects received BL-8040. Summary tables for baseline demographic data are presented in Supplementary Table S1B. No subject reported any history of alcohol and/or drug of abuse addiction and/or active/past (up to 2 years before screening) nicotine consumption. No subject reported clinically significant medical history (Supplementary Table S1B).

Pharmacokinetic analysis

Pharmacokinetic samples were collected only during part 1 of the study. A total of 15 blood samples were collected for pharmacokinetic analysis from each subject. Plasma concentrations of BL-8040 in subjects that received BL-8040 were analyzed by model-independent methods using Phoenix WinNonlin version 6.4 (Pharsight, Inc.). Nominal sampling times were used. Concentrations below the limit of quantitation (BLQ) were reported as “0” and were used as such in the pharmacokinetic analysis and summary statistics of concentration–time data.

Steady-state achievement after the second dose was assessed by comparing predose and 23-hour concentrations on day 1, and the Cmax on days 1 and 2. Accumulation on day 2, if any, was assessed by comparing AUC0-24 and Cmax of the second and first doses. Pharmacokinetic linearity was assessed by examining the relationship between dose and the exposure parameters (Cmax and CUA) on the first and second doses. Plasma concentrations in the placebo group were all below the limit of quantitation, as expected, and were not considered in the pharmacokinetic analysis. Likewise, predose plasma concentrations were below the limit of quantitation in all subjects randomized to receive BL-8040. After subcutaneous administration of BL-8040 on day 1, the appearance of the compound in the plasma was rapid, with a median Tmax ranging between 0.25 and 0.5 hour. Thereafter, plasma concentrations declined monoexponentially with a short half-life of approximately 1 hour. For that reason, plasma levels of BL-8040 were below the limit of detection in the majority of subjects by 8 hours and in all subjects at 23 hours after dosing. Summary statistics of pharmacokinetic parameters of BL-8040 after day 1 (first dose and second dose) can be found in Supplementary Table S2A.

Increases in the dose of BL-8040 led to overall approximate proportional increases in plasma exposure, as measured by Cmax, AUC0–t, AUC0–24, and AUC0–∞ (Fig. 1). Dose-normalized Cmax and AUC0–24 and t1/2 values were comparable across the 3 dose groups suggesting dose proportionality across the 0.5 to 1 mg/kg dose range.

Figure 1.

Exposure versus dose relations of BL-8040 after daily subcutaneous administration for two days in healthy volunteers.

Figure 1.

Exposure versus dose relations of BL-8040 after daily subcutaneous administration for two days in healthy volunteers.

Close modal

The plasma concentration–time profile on day 2 was consistent with day 1 and characterized by the rapid absorption and elimination of BL-8040 from the circulation. Consistent with its short half-life, accumulation of BL-8040 upon once daily repeated administration for two days was minimal. The pharmacokinetic parameters on day 2 (Supplementary Table SS2B; Fig. 1) were similar to those of day 1.

Safety analysis

No deaths or serious AEs occurred during the study. In part 1, BL-8040 was overall well-tolerated, only a single subject terminated early due to an AE. No AE reported during this part of the study was considered life-threatening, most AEs were mild or moderate and only 13% of all reported AEs were considered as severe. The most common AE among all the subjects was injection site pain, reported in 88% of subjects, including the 3 subjects who received a placebo. No dose response was observed for the overall incidences of AEs. An incidence pattern among cohorts 0.5–1 mg/kg, possibly suggestive of a dose response was observed for: leukocytosis, palpitations, nausea, dizziness, headache, pruritus generalized, and pallor for 21 of 24 (88%) subjects who received BL-8040. Seventy-nine AEs related (definitely, probably and possibly) to study treatment were reported by 6 subjects (100%) receiving 0.5 mg/kg BL-8040. Among all the AEs reported within the BL-8040 cohorts only 3 were assessed as related and severe. Of the severe AEs (SAE), 2 subjects in the 1 mg/kg treatment group had severe asthenia and 1 subject in the 0.75 mg/kg treatment group had severe syncope. The event resolved within the same day it was reported and no supportive care required. In the 02, no SAE were reported. The most common AE among all the subjects was injection site pain, reported in 88% of subjects, including the 3 subjects who received a placebo. Other common AEs reported in more than 10% of all subjects included injection site erythema (75%), injection site edema (75%), flushing (67%), pruritus (63%), hot flush (46%), nausea (42%), injection site pruritus (38%), urticaria (33%), headache (29%), asthenia (25%), chills (25%), dizziness (25%), parasthesia (25%), hyperhidrosis (25%), pruritus generalized (25%), rash macular (21%), pallor (21%), vomiting (17%), injection site hematoma (17%), injection site induration (17%), edema peripheral (17%), leukocytosis (13%), tachycardia (13%), diarrhea (13%), peripheral swelling (13%), and pruritus genital (13%). Detailed drug-related AEs by preferred term and treatment group are shown in Supplementary 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. In part 2, based on the safety data of part 1, premedication with steroids and antihistaminic drugs was implemented prior to the BL-8040 injection. Premedication with paracetamol (1 g, orally), methylprednisolone 100 mg (i.v.), and promethazine 25 mg (orally) were given 1 hour prior to the BL-8040 injection, which was given in the second part of the study. BL-8040 was overall well tolerated and all subjects completed the study. No AE reported during this part of the study was considered life-threatening, all the AEs were mild or moderate and no AEs were considered severe. The most common AEs reported in all subjects were injection site pain, hot flush, and injection site erythema. Overall, 100% of the subjects who received 1 dose of 1 mg/kg of BL-8040 reported AEs. The majority of these AEs (88%) were assessed as related (definitely, probably, and possibly) and were considered mild and moderate by the principal investigator. The most common AE reported in 100% of the subjects was injection site erythema, injection site pain, and hot flush. Other common AEs reported in more than 10% of the subjects 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%), vomiting (25%), pallor (25%), injection site induration (25%), abdominal discomfort (13%), abdominal pain (13%), dysphagia (13%), injection site inflammation (13%), injection site ischemia (13%), injection site necrosis (13%), injection site paraesthesia (13%), injection site pruritus (13%), injection site scab (13%), peripheral swelling (13%), headache (13%), presyncope (13%), tension headache (13%), epistaxis (13%), tachypnoea (13%), and throat irritation (13%).

Hematopoietic cell mobilization and collection

In part 1 of the study a single administration of BL-8040 (0.5, 0.75, 1 mg/kg) induced a rapid and dose-dependent mobilization of WBCs into the blood in all subjects tested (Fig. 2A–C). The maximal number of WBCs in the different cohorts was achieved after the first injection, and was 35.5 × 103, 49.4 × 103, and 44.3 × 103, respectively, for cohorts receiving 0.5, 0.75, and 1 mg/kg. The mean fold increase in the number of WBCs reached its peak between 8 and 12 hours following 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 the second injection did not increase above the maximal number achieved after the first injection, and neither did the mean fold increase of WBCs. Forty-eight hours after the last injection of BL-8040 the number of WBCs in the blood was significantly reduced (50% or less relative to peak accumulation of WBCs in the blood). In most of the subjects, the number of WBCs normalized 72 hours following the last injection. The increase in the number of WBCs could be attributed mainly to increase in the number of neutrophils and lymphocytes subpopulations (70% and 20%, respectively; Fig. 2E and F). Within the lymphocyte subpopulation, an increased mobilization of B, T, and NK cells was noted (Fig. 2G). It is worth mentioning that a mobilization of dendritic cells (DC) (lin/CD11c+ cells) was observed following administration of BL-8040 (1mg/kg) reaching a peak at 3.5 hours posttreatment and by 24 hours postdose, the DC levels completely returned to the baseline level (Fig. 2H). Mobilization of WBCs to the periphery and the recovery of normal numbers of WBCs correlate with the ability of the CXCR4 12G5 antibody to bind to its epitope on the CXCR4 receptor (Fig. 2H).

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.

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.

Close modal

After the first dose administration of BL-8040, CD34+ HSPCs counts increased rapidly reaching maximal levels between 4 and 8 hours postdose (Fig. 3A). The maximal counts increased with dose 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 CD34+ levels had declined slightly but were still 5- to 7-fold higher than the baseline. In contrast to WBCs, the number of CD34+ cells after the second dose increased in a dose-dependent manner (Fig. 3A). Upon administration of the second dose, an increase in CD34+ was observed 4 to 8 hours after dosing. In correlation with the expression of CXCR4, the CD34+ counts declined thereafter reaching baseline level approximately 48 hours after the second dose (Fig. 3A and E). At doses of 0.5 and 0.75 mg/kg, 4 of 12 injected individuals failed to achieve 20 CD34+ cells per μL 4–8 hours following the first administration of BL-8040, whereas all 6 individuals treated with 1mg/kg achieved 20 CD34+ cells per μL (Fig. 3B–D). We therefore selected the 1 mg/kg as the dose for the second part of the study and performed aphaeresis 3–4 hours following the BL-8040 treatment. In part 2, similar to the results of part 1 of the study, injection of 1 mg/kg of BL-8040 induced a rapid increase in the number of WBCs and CD34+ cells in the periphery that declined between 24 and 48 hours (Fig. 4A and B) posttreatment. All 8 subjects (no placebo was administered in part 2 of the study) increased their CD34+ count above 20 per μL approximately 3.5 hours after injection of BL-8040 before leukapheresis (Fig. 4C). The average blood volume processed during leukapheresis was 15.8 liters (range 9.8–17.5 liters). CD34+ cell counts were assessed by FACS analysis and the amount of CD34+ cells collected per kg was calculated for each subject. The mean CD34+ 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 kg average recipient weight. Table 1A summarizes the individual data and the amount of CD34+ cells collected when calculated on the basis of actual donor weight as well as based on 79 kg average recipient weight.

Figure 3.

Mean BL-8040 dose-dependent (0.5, 0.75, 1 mg/kg) mobilization of CD34+ cells/μL 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 CD34+ cells/μL of blood is shown in B–D. CXCR4 12g5 Ab binding to its epitope on the CXCR4 receptor is partially blocked on CD34+ cells by treatment with BL-8040 as shown in E.

Figure 3.

Mean BL-8040 dose-dependent (0.5, 0.75, 1 mg/kg) mobilization of CD34+ cells/μL 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 CD34+ cells/μL of blood is shown in B–D. CXCR4 12g5 Ab binding to its epitope on the CXCR4 receptor is partially blocked on CD34+ cells by treatment with BL-8040 as shown in E.

Close modal
Figure 4.

Number of WBCs and CD34+ cells/μL of blood is shown in patients 5001–5008 (n = 8) that were administrated with BL-8040 and 4 hours later underwent apheresis to collect their CD34+ cells is shown in A and B. Mean BL-8040 time-dependent mobilization of CD34+ cells/μL of blood is shown in C. Percentage of CD34+CD38 cells from total of CD34+ cells and percentage of CD45RA cells out of CD34+CD38 from cells mobilized by BL-8040 or G-CSF is shown in D. Percentage of CD34+CD38 CD45RA CD49f+ from a total of CD34+CD38 cells and percent of CD34+CD38CD45RACD90+CD49f+ from a total of CD34+CD38 CD45RA CD49f+ of cells mobilized by BL-8040 or G-CSF is shown in E. SDF-1 migration of BL-8040–mobilized CD34+ cell and G-CSF–mobilized-CD34+ cells is shown in F. Migration of CD3+ cells which mobilized by BL-8040 and normal CD3+ cells served as controls. The expression of CXCR4 on mobilized CD34+ cells are shown in G. CD34+ cells were separated from BL-8040–mobilized cells (n = 8) and from G-CSF–mobilized cells (n = 3). CD34+ 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.

Figure 4.

Number of WBCs and CD34+ cells/μL of blood is shown in patients 5001–5008 (n = 8) that were administrated with BL-8040 and 4 hours later underwent apheresis to collect their CD34+ cells is shown in A and B. Mean BL-8040 time-dependent mobilization of CD34+ cells/μL of blood is shown in C. Percentage of CD34+CD38 cells from total of CD34+ cells and percentage of CD45RA cells out of CD34+CD38 from cells mobilized by BL-8040 or G-CSF is shown in D. Percentage of CD34+CD38 CD45RA CD49f+ from a total of CD34+CD38 cells and percent of CD34+CD38CD45RACD90+CD49f+ from a total of CD34+CD38 CD45RA CD49f+ of cells mobilized by BL-8040 or G-CSF is shown in E. SDF-1 migration of BL-8040–mobilized CD34+ cell and G-CSF–mobilized-CD34+ cells is shown in F. Migration of CD3+ cells which mobilized by BL-8040 and normal CD3+ cells served as controls. The expression of CXCR4 on mobilized CD34+ cells are shown in G. CD34+ cells were separated from BL-8040–mobilized cells (n = 8) and from G-CSF–mobilized cells (n = 3). CD34+ 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.

Close modal
Table 1A.

Summary of the individual data and the amount of CD34+ 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)CD34+ cells (%)CD34+/kg × 106 (donor weight)CD34+/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 × 10610.3 × 106 (±2.8 × 106
Median 16.5 ± 2.3 L 0.77 ± 0.13% 11.2 × 106 (±2.8 × 10610.5 × 106 (±2.8 × 106
Subject no.Whole blood processed (L)CD34+ cells (%)CD34+/kg × 106 (donor weight)CD34+/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 × 10610.3 × 106 (±2.8 × 106
Median 16.5 ± 2.3 L 0.77 ± 0.13% 11.2 × 106 (±2.8 × 10610.5 × 106 (±2.8 × 106

Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs). The bulk of HSCs are CD34+ cells; however, most CD34+ cells are lineage-restricted progenitors and HSCs remain rare. HSCs can be enriched further based on CD45RA, Thy1 (CD90), and CD38 expression. Loss of CD90 expression in the CD34+CD38CD45RA compartment of lineage depleted cord blood (CB) was proposed to be sufficient to separate CD34+CD38CD45RA CD90+ HSCs from CD34+CD38CD45RA CD90 multipotent progenitors (MPP; ref. 16). Recently, it was demonstrated that CD49f is a specific HSC marker. Single CD49f+ cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting MPPs. Furthermore, CD34+CD38CD45RACD90+ CD49f+ HSCs were found as the population with the highest long-term and secondary repopulating activity (17). CD34+ cells were purified from BL-8040 and G-CSF–mobilized grafts and stained for CD38, CD45RA, CD90, and CD49f. The percentage of CD34+CD38 hematopoietic stem and progenitors was similar in both grafts (Fig. 4D). However, whereas 23.2 % of BL-8040–mobilized CD34+ CD38 cells did not express CD45RA, only 1.6% of G-CSF–mobilized CD34+ CD38 cells did not express CD45RA (Fig. 4D). The percentage and absolute number of CD34+CD38CD45RACD49f+CD90+/−, CD34+CD38CD45RACD49f+CD90+, and CD34+CD38CD45RA CD90+ HPCS were increased significantly 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 to G-CSF graft–derived CD34+CD38 cells (Fig. 4E; Supplementary Table S4A).

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

It was already shown before that G-CSF mobilizes CD34+ cells collected from the blood migrate less in response to CXCL12 (18). Interestingly, we found that CD34+ cells mobilized with BL-8040 migrated better than G-CSF–mobilized cells in response to CXCL12 (Fig. 4F).

We stained CD34+ cells mobilized cells from BL-8040 and G-CSF–mobilized CD34 cells with 12g5 mAb, which binds the second extracellular loop of CXCR4, and 1D9 mAb, which binds the N-terminal portion of CXCR4. When cells were stained with 1D9 antibodies similar expression levels of CXCR4 were observed while the level of CXCR4 following staining with 12g5 antibodies was significantly reduced on BL-8040 mobilized cells but not on G-CSF mobilized cells (Fig. 4G). This suggests a specific block of the 12g5 with no internalization of the CXCR4 receptor.

The graft collected was further tested for its immune composition. 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 the CD4+ T-cell population, we found that the graft contained mostly naïve CD4 T cells (35%), effector memory CD4 T cells (21%), central memory CD4 T cells (38%), and almost no effector CD4 T cells (6%). Whereas in the CD8+ T-cell population, we found that the graft contained mostly naïve CD8 T cells (64%), and central memory CD8 T cells (33%), and almost no effector memory CD8 T cells (1%), and effector CD8 T cells (2%; Fig. 5).

Figure 5.

Percentages of different subpopulations of BL-8040–mobilized cells (n = 8) as compared with G-CSF–mobilized cells (n = 5) was analyzed by flow cytometry. Pie charts show the cells distribution within the CD4+ or CD8+ populations. CD3+/CD4+/CD45RA+/CCR7+ (naïve CD4+ cells); CD3+/CD4+/CD45RA+/CCR7 (effector CD4 T cells); CD3+/CD4+/CD45RA/CCR7 (effector memory CD4 T cells); CD3+/CD4+/CD45RA/CCR7+ (central memory CD4 T cells). CD3+/CD8+/CD45RA+/CCR7+ (naïve CD8+ cells); CD3+/CD8+/CD45RA+/CCR7 (effector CD8 T cells); CD3+/CD8+/CD45RA/CCR7 (effector memory CD8 T cells); CD3+/CD4+/CD45RA/CCR7+ (central memory CD4 T cells).

Figure 5.

Percentages of different subpopulations of BL-8040–mobilized cells (n = 8) as compared with G-CSF–mobilized cells (n = 5) was analyzed by flow cytometry. Pie charts show the cells distribution within the CD4+ or CD8+ populations. CD3+/CD4+/CD45RA+/CCR7+ (naïve CD4+ cells); CD3+/CD4+/CD45RA+/CCR7 (effector CD4 T cells); CD3+/CD4+/CD45RA/CCR7 (effector memory CD4 T cells); CD3+/CD4+/CD45RA/CCR7+ (central memory CD4 T cells). CD3+/CD8+/CD45RA+/CCR7+ (naïve CD8+ cells); CD3+/CD8+/CD45RA+/CCR7 (effector CD8 T cells); CD3+/CD8+/CD45RA/CCR7 (effector memory CD8 T cells); CD3+/CD4+/CD45RA/CCR7+ (central memory CD4 T cells).

Close modal

The different immune subpopulation in the BL-8040 compared with G-CSF–mobilized graft were determined (Supplemented Table S4B). Significant increase in the percentage of CD3+CD4+, CD3+CD8+, DC, naïve, and effector CD4+ T cells, central memory CD4 T cells, naïve CD8 T cells, and effector memory CD8 cells was observed (Fig. 5).

The SCID-reconstituting capacity of fresh or frozen purified CD34+ cells (>90%) was evaluated in transplant experiments in NSG mice using human anti-CD45 antibody at a threshold level 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 bone was engrafted with CD45+/CD19+ B cells, CD45+/CD56+/CD3+ NK cells, CD45+/CD14+/CD16+ myelomonocytic cells, and CD45+/CD34+/CD38+ progenitors and CD45+/CD34+/CD38 stem cells as well as human colony-forming cells (Fig. 6B–D).

Figure 6.

The SCID reconstituting capacity of fresh or frozen purified CD34+ 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 CD34+ 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 CD45+CD34+CD38+ and CD45+ CD34+ 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. Mice were transplanted with purified CD34+ and the number of colonies from total of 100,000 bone marrow was measured. Engraftment percentage of hCD45+ and hCD45+/CD34+ 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 hCD45+ cells in the bone marrow, spleen, and blood following 4 and 22 weeks of transplantation is shown in G. Engraftment percentage of human CD3+, CD3+CD4+, and CD3+CD8+ 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 CD45+CD19+ B cells, CD45+CD56+CD3 NK cells, CD45+CD14+CD16+ myelomonocytic cells following 4 and 22 weeks of transplantation is shown in J.

Figure 6.

The SCID reconstituting capacity of fresh or frozen purified CD34+ 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 CD34+ 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 CD45+CD34+CD38+ and CD45+ CD34+ 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. Mice were transplanted with purified CD34+ and the number of colonies from total of 100,000 bone marrow was measured. Engraftment percentage of hCD45+ and hCD45+/CD34+ 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 hCD45+ cells in the bone marrow, spleen, and blood following 4 and 22 weeks of transplantation is shown in G. Engraftment percentage of human CD3+, CD3+CD4+, and CD3+CD8+ 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 CD45+CD19+ B cells, CD45+CD56+CD3 NK cells, CD45+CD14+CD16+ myelomonocytic cells following 4 and 22 weeks of transplantation is shown in J.

Close modal

To assess the long-term engraftment potential of the BL-8040–mobilized CD34+ cells, engraftment was allowed for 4, 8, and 22 weeks after transplantation. Successful and robust long-term human engraftment of CD45+ and CD45+CD34+ cells was observed at week 22 (Fig. 6E and F). The percent of CD45 cells in the bone marrow remained stable in the bone marrow, whereas the percentage of CD45 cells in the blood and spleen increased at week 22 (Fig. 6G). At 4 weeks, human CD3+CD4+ T cells were only observed at a low percentage in the spleen but not in the bone marrow, whereas no significant percentage of CD3+CD8+ cells were found neither in the bone marrow nor in the spleen (Fig. 6H and I). Following 22 weeks of transplantation, the percentage of human CD3+CD4+ and CD3+CD8+ T cells was significantly increased in the spleen (30% vs. 5%, respectively). The percentage of human CD3+CD4+ and CD3+CD8+ T cells was increased at much lower levels in the bone marrow (Fig. 6H and I).

To further assess the long-term engraftment potential of the BL-8040, we collected the bone marrow cells from 8-week-old 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 in NSG mice, the ability of thawed purified CD34+ HPCs and CD3+ cells to migrate in response to the CXCR4 ligand CXCL12 was found to be intact (Fig. 4F).

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 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 μg/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 donations in a single day (19). Ings and colleagues reported that among 400 unrelated donors of PBSCs target recipient doses were reached with one aphaeresis in 63% of donors and with two apheresis in 81% of donors; 19% of the donors needed more than two apheresis (20).

AMD3100 (plerixafor, Mozobil) is a reversible direct antagonist of the interaction between the chemokine SDF-1 and its receptor CXCR4 and is used for HSPC mobilization. Plerixafor was approved for use in combination with G-CSF to mobilize HSPC for autologous HSCT (12, 21). Studies conducted in healthy donors reported that Plerixafor induces a transient increase in CD34+ that peaks 9 hours after subcutaneous injection with 80–240 μg/kg of plerixafor and declines at 24 hours (22). In another study reported by Devine and colleagues (5), plerixafor (n = 24) was compared with G-CSF (n = 8) in mobilization and collection from healthy sibling donors. At 4 hours after a single dose of AMD3100 (240 μg/kg), the CD34+ count rose to a median of 16/μL (range, 4–54/μL). One out of the 25 donors could not undergo large volume (27 liters) leukapheresis following AMD3100 due to repeated vasovagal episodes during peripheral line placement prior to commencing leukapheresis. A total of 8 (33%) of the remaining 24 donors who commenced leukapheresis following AMD3100 administration on day 1 did not collect the minimum required CD34+ cell dose in one day (2 × 106/kg) and were thus eligible for a second day of drug administration and leukapheresis on day 3. The median number of CD34+ cells collected from all 24 donors was 2.9 × 106/kg (range, 1.2–6.3). In the G-CSF group, the CD34+ count rose to a median of 46/μL on day 5 (range, 4–54/μL). The median number of CD34+ cells collected from all 8 donors was 4.2 × 106/kg (range, 2.5–18.7). The increase in CD34+ cells after 5 days of G-CSF was significantly greater than the rise 4 hours after AMD3100 administration (P < 0.001; ref. 5).

The effect of BL-8040 as compared with AMD3100 was studied in in vitro and in vivo experimental settings. BL-8040 binds and inhibits the CXCR4 chemokine receptor with high affinity, showing an IC50 of approximately 1 nmol/L (23–25) compared with the values obtained with AMD3100 (IC50 = 651 ± 37 nmol/L; ref. 26). Moreover, BKT140 hinders the cell migration stimulated by CXCL12 within IC50 values of 0.5–2.5 nmol/L (24, 27) compared with the IC50 value of 51 ± 17 nmol/L for Mozobil (26). Furthermore, BL-8040 has long receptor occupancy (>24 hours) compared with AMD3100, resulting in the extended inhibition of CXCR4 (15). In animal studies, comparison between the BL-8040 and AMD3100 showed that BL-8040 with or without G-CSF was significantly more potent in its ability to mobilize hematopoietic stem cells and progenitors into blood (14, 15).

In this study, we found that at 4–8 hours after a single dose of BL-8040 (0.5, 0.75, 1 mg/kg), the CD34+ count rose to a median of 38.2, 43.7 and 45.5 μL (respectively). In contrast to treatment with AMD3100, by 24 hours, CD34+ levels had declined only slightly. Furthermore, the mean CD34+ cells/kg collected from the individuals treated with 1mg/kg BL-8040 was 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 trial support the hypothesis that in healthy humans the number of CD34+ cells that can be rapidly mobilized and collected is greater than observed with either G-CSF or plerixafor. Furthermore, we have found that the percentage of CD34+CD38CD45RACD49f+CD90+ cells, and CD34+CD38CD45RACD90+ cells were significantly higher in the BL-8040 graft compared with G-CSF graft (Fig. 4E). In association with the high percentage of HPCs in the BL-8040 graft, we found a robust myeloid and lymphoid long-term engraftment (week 22) of BL-8040–mobilized human CD34+ cells in NSG mice (Fig. 6).

In Devine and colleagues' study (5), there were notable differences in the final leukapheresis products mobilized and collected after AMD3100 or G-CSF were administered. There were significantly fewer CD34+ cells/kg (2.9 × 106 vs. 4.2 × 106; P < 0.006), but greater numbers of CD3+ cells/kg (4.7 × 108 vs. 1.5 × 108; P < 0.006) and CD4+ cells/kg (3.1 × 108 vs. 1.1 × 108; P < 0.002) in the AMD3100-mobilized products compared with the G-CSF–mobilized allografts. Therefore, more CD3+ and CD4+ cells were mobilized per unit of blood following AMD3100 compared with G-CSF treatment. However, the differences in CD8+, CD19+, and CD56+ cell content were not significant. Interestingly, the rates of acute and chronic GVHD observed in the 20 transplanted patients were not significantly different compared with historical control patients at Washington University receiving G-CSF–mobilized allografts together with the exact same conditioning and GVHD prophylaxis. There was no difference in the median time to neutrophil or platelet engraftment between recipients in the AMD3100 group or the historical group receiving G-CSF–mobilized allografts in this study and all surviving patients in remission continue to have robust and durable trilineage hematopoiesis.

In the final leukapheresis products mobilized and collected after BL-8040 there were much higher number of CD34+ progenitor and stem cells compared with the leukapheresis collected after mobilization with AMD3100 or G-CSF (5, Table 1B). Furthermore, in the leukapheresis products mobilized and collected after BL-8040 there was a significant increase in the percentage of CD3+CD4+, CD3+CD8+, DC, naïve and effector CD4+ T cells, central memory CD4 T cells, naïve CD8 T cells, and effector memory CD8 cells compared with G-CSF–mobilized graft (Fig. 5). This new composition may have an effect on the engraft ability, GVHD, GVL, and immune reconstitution potential of the leukapheresis product.

Table 1B.

CD34+ cells collected after mobilization with BL-8040, AMD3100, or G-CSF

Cell typeBL-8040 (n = 8) Median (range)AMD3100a (n = 24) Median (range)G-CSFa (n = 8) Median (range)
CD34+ × 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 
Cell typeBL-8040 (n = 8) Median (range)AMD3100a (n = 24) Median (range)G-CSFa (n = 8) Median (range)
CD34+ × 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).

In summary, our results suggest that BL-8040 is a rapid and robust mobilizer of a of CD34+ progenitors and stem cells with an intact long-term engraftment potential, as assessed by engraftment of these human cells in a mouse model. Furthermore, these results suggest that in the bone marrow of humans, the number of CD34+ with the potential to exit rapidly into the blood upon a strong and sustained inhibition of CXCR4 is clearly greater than seen with the first generation of CXCR4 antagonists. A phase II study assessing safety and efficacy of BL-8040 for the mobilization of donor hematopoietic stem cells and allogeneic transplantation in patients with advanced hematologic malignancies is currently being conducted at the Washington University School of Medicine, Division of Oncology and Hematology, St. Louis, MO (NCT02639559).

M. Abraham, H. Wald, B. Bulvik, and O. Eizenberg are employees and shareholders of Biokine Therapeutics Ltd.; A. Peled serves as consultant for Biokine Therapeutics and is also a shareholder. Y. Pereg, R. Golan, A. Vainstein, and A. Aharon are employees of BioLineRx Ltd. No potential conflicts of interest were disclosed by the other authors.

Conception and design: M. Abraham, Y. Pereg, O. Eizenberg, A. Aharon, A. Peled

Development of methodology: M. Abraham, Y. Pereg, B. Bulvik, H. Wald, A. Aharon, R. Or, A. Peled

Acquisition 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. Peled

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Abraham, Y. Pereg, B. Bulvik, R. Golan, A. Vainstein, Y. Caraco, A. Peled

Writing, 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. Peled

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Pereg, O. Eizenberg, K. Beider, R. Golan

Study supervision:A. Vainstein, A. Aharon, Y. Caraco

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

1.
Cashen
AF
,
Lazarus
HM
,
Devine
SM
. 
Mobilizing stem cells from normal donors: is it possible to improve upon G-CSF?
Bone Marrow Transplant
2007
;
39
:
577
88
.
2.
Ozkan
MC
,
Sahin
F
,
Saydam
G
. 
Peripheral blood stem cell mobilization from healthy donors
.
Transfus Apher Sci
2015
;
53
:
13
6
.
3.
Arslan
O
,
Moog
R
. 
Mobilization of peripheral blood stem cells
.
Transfus Apher Sci
2007
;
37
:
179
85
.
4.
Deotare
U
,
Al-Dawsari
G
,
Couban
S
,
Lipton
JH
. 
G-CSF-primed bone marrow as a source of stem cells for allografting: revisiting the concept
.
Bone Marrow Transplant
2015
;
50
:
1150
6
.
5.
Devine
SM
,
Vij
R
,
Rettig
M
,
McGlauchlen
K
,
Fisher
N
,
Devine
H
, et al
Rapid mobilization of functional donor hematopoietic cells without G-CSF using AMD3100, an antagonist of the CXCR4/SDF-1 interaction
.
Blood
2008
;
112
:
990
8
.
6.
Peled
A
,
Petit
I
,
Kollet
O
,
Magid
M
,
Ponomaryov
T
,
Byk
T
, et al
Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4
.
Science
1999
;
283
:
845
8
.
7.
Lapidot
T
,
Petit
I
. 
Current understanding of stem cell mobilization: the roles 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-CSF induces 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 for mobilization 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
,
Kaufman
JL
, et al
Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients 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 of plerixafor plus granulocyte colony-stimulating factor compared with placebo plus granulocyte colony-stimulating factor for autologous stem-cell mobilization and transplantation for patients with non-Hodgkin's lymphoma
.
J Clin Oncol
2009
;
27
:
4767
73
.
13.
Abraham
M
,
Beider
K
,
Wald
H
,
Weiss
ID
,
Zipori
D
,
Galun
E
, et al
The CXCR4 antagonist 4F-benzoyl-TN14003 stimulates the recovery of the bone 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 induced by 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 mobilization of human CD34+ 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 multipotent hematopoietic progenitors in human cord blood
.
Cell Stem Cell
2007
;
1
:
635
45
.
17.
Notta
F
,
Doulatov
S
,
Laurenti
E
,
Poeppl
A
,
Jurisica
I
,
Dick
JE
. 
Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment
.
Science
2011
;
333
:
218
21
.
18.
Aiuti
A
,
Webb
IJ
,
Bleul
C
,
Springer
T
,
Gutierrez-Ramos
JC
. 
The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ 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 blood stem cells: results of a prospective trial from the national marrow donor program
.
Blood
2009
;
113
:
3604
11
.
20.
Ings
SJ
,
Balsa
C
,
Leverett
D
,
Mackinnon
S
,
Linch
DC
,
Watts
MJ
. 
Peripheral blood stem cell yield in 400 normal donors mobilised with granulocyte colony-stimulating factor (G-CSF): impact of age, sex, donor weight and type of G-CSF used
.
Br J Haematol
2006
;
134
:
517
25
.
21.
DiPersio
JF
,
Schuster
MW
,
Abboud
CN
,
Winter
JN
,
Santos
VR
,
Collins
DM
, et al
Mobilization of peripheral-blood stem cells by concurrent administration of daniplestim and granulocyte colony-stimulating factor in patients with breast cancer or lymphoma
.
J Clin Oncol
2000
;
18
:
2762
71
.
22.
Liles
WC
,
Broxmeyer
HE
,
Rodger
E
,
Wood
B
,
Hübel
K
,
Cooper
S
, et al
Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist
.
Blood
2003
;
102
:
2728
30
.
23.
Tamamura
H
,
Fujisawa
M
,
Hiramatsu
K
,
Mizumoto
M
,
Nakashima
H
,
Yamamoto
N
, et al
Identification of a CXCR4 antagonist, a T140 analog, as an anti-rheumatoid arthritis agent
.
FEBS Lett
2004
;
569
:
99
104
.
24.
Jacobson
O
,
Weiss
ID
,
Kiesewetter
DO
,
Farber
JM
,
Chen
X
. 
PET of tumor CXCR4 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 specific antagonist of the G-protein coupled chemokine receptor, CXCR4
.
Biochem Pharmacol
2006
;
72
:
588
96
.
27.
Tamamura
H
,
Hori
A
,
Kanzaki
N
,
Hiramatsu
K
,
Mizumoto
M
,
Nakashima
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
.