Mobilization of hematopoietic stem cells with the novel CXCR4 antagonist POL6326 (balixafortide) in healthy volunteers—results of a dose escalation trial

Volunteers were healthy male HSC donors from the German Stem Cell Donor Registry (DSSD) who had received a 5-day course of filgrastim (G-CSF, 7.5–10 µg/kg per day in 2 divided doses) for matched-unrelated stem cell donation and shown a grossly average mobilization response (121.6 ± 8.6 CD34+ cells/μL). Additional eligibility (inclusion) criteria for treatment with balixafortide were the same as for G-CSF mobilized stem cell donation [11]. Between G-CSF mobilization/HSPC donation and study participation there was a wash-out period of at least 6 weeks.

Study drug was administered on an in-patient basis in the phase I clinical trial unit of Goethe University Medical Center, the ‘Klinisches Studienzentrum Rhein-Main’. Volunteers were discharged 24 h after treatment, to return for a follow-up appointment 8–14 days thereafter.

This was a prospective Phase I open label dose escalation trial; The study design is summarized in Table 1. A total of 27 volunteers were treated with balixafortide. A treatment consisted of a single intravenous infusion of balixafortide in normal saline at doses of 500, 1000, 1500, 2000 and 2500 μg/kg, based on actual weight, followed by sequential clinical and blood analyses (see below). Initially conceived as a classical 3 + 3 dose escalation design, the volunteers were assigned to four groups defined by increasing dose levels of balixafortide (500, 1000, 1500, and 2000 μg/kg) administered by constant rate infusion at over 2 h. Subsequently, amendments were added to test additional modalities: Group 6 received 2500 μg/kg under the same conditions. Volunteers assigned to Group 5 received a dose level of 2000 μg/kg by an continuously increasing infusion rate (ramp-infusion instead of constant rate infusion) applied over 2 h. In group 7, a dose level of 1000 μg/kg was infused over 1 h at a constant rate and compared (intra-individually) to the 2 h infusion given with an interval of ≥4 weeks. A second balixafortide treatment was furthermore tested in volunteers from groups 2, 3 and 6 with groups 2 and 3 receiving 2500 μg/kg and group 6 given 1500 μg/kg as the second infusion. In as far as not all volunteers from the initial phase of the study could be recalled, they were replaced by new volunteers receiving two treatments, to have a group size of at least 3 for each cross-over modality, explaining the variable dosing group sizes between 3 and 6 (Table 1). Thus, to allow for intra-individual comparison, 12 donors received a second dose of balixafortide (2 h constant infusion rate for all) after a minimum wash-out period of 4 weeks.

Vital signs were monitored immediately prior to and in the first 24 h after the start of the infusion of balixafortide; serial blood samples were drawn for biochemical safety profiling and pharmacokinetic/pharmacodynamic analyses. Given the cationic nature of the compound [17] the risk of local or systemic symptoms of histamine release was identified and anti-histamine treatment was proposed (per protocol) in case of such symptoms. After completion of the 2000 µg/kg dosing group the protocol was amended to introduce prophylactic anti-histamine treatment in the dosage group ≥2500 µg/kg. Volunteers who received prophylactic or therapeutic anti-histamine medication are listed accordingly in Table 1.

Primary outcome parameters were safety and tolerability of balixafortide when compared to G-CSF, pharmacodynamics of mature and immature blood cell mobilization, specifically the intra-individual comparison of balixafortide- and G-CSF-induced mobilization of HSPCs. Secondary objectives included pharmacokinetic analyses and identification of a suitable window for HSPC apheresis.

Plasma samples were collected at the indicated times and kept frozen until immediately before analysis.

Blood samples were collected at the indicated times (Figs. 1, 2, 3, 4, 5 and 6) and kept at room temperature (maximum 2–3 h.) until immediately before analysis. Complete blood counts were assessed with the Sysmex XT1800 hematology analyzer (Norderstedt, Germany). CD34+ cells were quantified using the single platform flow cytometry analysis with the SCE Kit [Becton–Dickinson (BD), Heidelberg, Germany] according to the manufacturer’s instructions and ISHAGE guidelines [18]. In addition, multi-parametric flow cytometric analyses were performed to quantify co-mobilized mature cell subsets such as T (CD45+ CD3+), B (CD45+ CD19+) and NK (CD45+ CD56+ 16+) cells (Multitest, T cells, BD), T cell subpopulations (CD45+ CD3+ CD4+/CD8+, Multitest, TBNL cells, BD) and monocytes (CD45+ CD14+, all from BD). In addition, plasmacytoid dendritic cell progenitors (pro-pDCs) were identified as CD45dimCD34dimCD45RA + CD123high (all moABs from BD). Lyse-no-wash protocols were used in conjunction with BD counting beads for direct cell enumeration for CD34+ cells and T cell subsets; all other cell concentrations were calculated using frequencies relative to directly enumerated cell species, such as CD34+, CD45+ or CD3+ cells.

Circulating colony-forming units-culture (CFU-C) were quantified by plating aliquots of lysed peripheral blood in commercial cytokine-replete methylcellulose media (StemMACS HSC-CFU lite with Epo, human, Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany).

Unless otherwise mentioned, data are expressed as mean ± SEM. Descriptive statistics and Student’s t-test for paired or unpaired analysis (as appropriate) were calculated using Excel. A p < 0.05, Bonferroni-corrected for multiple testing if appropriate, was considered statistically significant.

A summary of adverse events that were documented throughout the trial is shown in Table 2. No severe adverse events (SAEs) were observed. Mild skin reactions such as flushing, urticaria or local itching were reported by 1/3, 4/10, 2/7, 8/9 and 7/10 volunteers receiving 500, 1000, 1500, 2000 and 2500 µg/kg of balixafortide respectively. Upon treatment with a combination of H1 and H2 blockers symptoms rapidly abated. These reactions were rated likely related to study drug.

Three adverse events (AEs) were considered possibly related to study drug: mild bone pain (1 subject), an unexplained elevation in serum creatinine kinase (2 subjects), and a systolic blood pressure reading of >150 mmHg (2 subjects).

Constant-slope infusion, tested at the 2000 µg/kg dose level in three volunteers (group 6), as well as increased infusion rates (1 vs. 2 h, group 3), tested in paired analyses in three volunteers at the 1000 µg/kg dose level, did not influence the tolerability of the agent (Table 1).

At the time of follow-up, volunteers were questioned about their subjective rating of G-CSF vs. balixafortide as mobilizing agents; there was an overwhelming preference for balixafortide. See also Table 3 for the questionnaire and volunteer responses.

Serial plasma samples were assayed for balixafortide concentrations and pharmacokinetic parameters were calculated using Phoenix WinNonlin 6.4. We observed dose linearity for both Cmax and AUC (Fig. 1a). The volume of distribution was approximately 500–600 mL/kg. Balixafortide was cleared from plasma with a terminal half-life of approximately 5 h over all application schemes and doses of 5:45 ± 0:35 h (mean ± SD; Fig. 1b). The clearance of balixafortide appeared to be almost equal to the glomerular filtration rate suggesting that balixafortide is mainly cleared through the kidney. Different infusion durations (1 vs. 2 h) did not notably influence the PK profile except for an earlier C_max (Fig. 1c), and the same applied to ‘constant-slope’ vs. ‘ramp’ infusion (Fig. 1d).

At all doses tested, balixafortide infusions quickly resulted in an increase in circulating HSPCs, as measured phenotypically (CD34+ cells, Fig. 2a) or functionally in colony assays (Fig. 2b). Clonogenicity of balixafortide vs. G-CSF mobilized CD34+ cells was lower with 1 CFU-C out of 5.9 ± 0.5 balixafortide mobilized CD34+ cells vs. 1 CFU-C out of 3.2 ± 0.2 CD34+ cells mobilized with G-CSF (Fig. 2c). At lower doses (500 vs. 1000 vs. 1500 µg/kg), dose-dependent mobilization was clearly observed, while the later dose increments to 2000 and 2500 µg/kg did not result in a commensurate increase in the number of mobilized HSPC compared to 1500 µg/kg (Fig. 3a, b). This was confirmed in paired analyses in small cohorts (Fig. 3c). Therefore, for some analyses all mobilization data for doses ≥1500 µg/kg are analyzed together. As such, mean peak mobilization in response to doses of 1500–2500 µg/kg was 38.2 ± 2.8 CD34+ cells/µL (Fig. 3d). Thus at these doses intra-individual comparison of balixafortide vs. G-CSF induced mobilization revealed that—on average—the G-CSF regimen was about three times as effective as the CXCR4 antagonist. There appeared to be a good correlation between the two mobilizing agents (Fig. 3d), suggesting that—as had been shown in mice [16, 19]—good mobilizers mobilize efficiently with either agent and poor mobilizers are refractory to both.

Peak mobilization at the 500 µg/kg dose was observed 1 h after the end of the balixafortide infusion/after reaching C_max (Figs. 1b, 2a). At higher doses, the observed mobilization peak appeared later, approximately 4 h after the end of the infusion. Thereafter, the number of circulating CD34+ cells slowly decreased but remained elevated beyond baseline at the 24 h time point for all except the lowest dose (Fig. 2a, b). Constant-slope (ramp) vs. constant-rate infusions (at 2000 µg/kg only) had no discernible effect on stem cell mobilization efficiency, and the same applied to infusion rate (1 vs. 2 h) (Fig. 4).

A population of “stem cells” co-expressing CD45RA and CD123, previously described in blood of plerixafor-mobilized donors and identified as plasmacytoid dendritic cell progenitors (pro-pDCs) [20], was detected at high frequencies (22.4 ± 2.3% of SSCdim/FSCmid-hi/CD45dim/CD34+ cells) after balixafortide-treatment, but was rare after G-CSF (Fig. 5).

Stem cell mobilization was accompanied with mixed leukocytosis affecting all cell lineages. It followed the same kinetics as stem cell mobilization and was dose-dependent as well as short-lived. At balixafortide doses of 1500–2500 µg/kg white blood counts (WBCs) of 25.3 ± 1.4 × 10*3 WBC/µL were reached, i.e. balixafortide mobilized approximately half as many mature cells as G-CSF (Fig. 6a). The lineage distribution of mature leukocytes differed markedly between both agents, in that balixafortide mobilized higher relative and absolute numbers of B-cells and fewer myeloid cells (Fig. 6b, c). The ratio between T-lymphocytes and CD34+ cells was 26.2 ± 1.98:1 in G-CSF mobilized blood, vs. 95.7 ± 8.9:1 in balixafortide mobilized blood, predicting that apheresis products from balixafortide mobilized donors will contain more T-cells than from G-CSF treated donors. Within the T cell population the proportion of T helper (CD4+) and cytotoxic T cells (CD8+) was very similar between the differently mobilized blood specimens as well as compared to steady state (baseline) (Fig. 6d).