Administration of daniplestim and granulocyte colony ... - Core

Biology of Blood and Marrow Transplantation 5:8–14 (1999) © 1999 American Society for Blood and Marrow Transplantation

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Administration of daniplestim and granulocyte colony-stimulating factor for the mobilization of hematopoietic progenitor cells in nonhuman primates William H. Fleming,1 Pamela Lankford-Turner,2 Curtis W. Turner,2 John Wong,1 Elizabeth Strobert,3 John P. McKearn4 1

Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, OR; 2Department of Medicine and 3Yerkes Regional Primate Center, Emory University, Atlanta, GA; 4Searle Research and Development, Monsanto, St. Louis, MO Offprint requests: William H. Fleming, MD, PhD, 5330 Basic Science Building (L580), Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201 (Received 10 September 1998; accepted 25 November 1998)

ABSTRACT We investigated the effects of administering a high-affinity interleukin-3 receptor agonist (daniplestim) and granulocyte colony-stimulating factor (G-CSF) on the mobilization of primitive hematopoietic progenitor cells into peripheral blood (PB). Groups of five rhesus monkeys were treated with 100 mg/kg of daniplestim for 5 days followed by 10 mg/kg of G-CSF for 5 days (D/G), daniplestim and G-CSF administered concurrently for 10 days (D1G), or G-CSF alone for 10 days. Phenotypic PB analysis indicated that the number of CD341 cells in the G-CSF group had increased to 283106/L by Day 3 and then declined. In contrast, CD341 cell counts of up to 683106/L were maintained until Day 10 in both the D/G and D1G groups. On Day 5, the total number of colonyforming cells in the PB had increased 15-fold in the D1G group and eightfold in both the D/G group and the G-CSF groups. By Day 7, the numbers of colony-forming units granulocyte/macrophage were comparable in all three groups, and 45-fold increases in the numbers of burst-forming units-erythroid and 12-fold increases in the numbers of multipotent colony-forming units were seen in both the D1G and the D/G groups. The frequency of circulating primitive progenitor cells in long-term stromal cultures was highest with D1G and lowest with G-CSF alone. These results indicate that the combination of daniplestim and G-CSF produces higher and more sustained levels of circulating stem cells than does G-CSF alone. D1G may offer advantages over D/G because it generates more long- and short-term clonogenic cells.

KEY WORDS Rhesus • IL-3 • G-CSF • Peripheral blood stem cells • Transplantation

INTRODUCTION Significant progress has recently been made in developing strategies to mobilize immature hematopoietic progenitor cells from the bone marrow into the peripheral blood (PB). The rapid, sustained engraftment of these cells seen after high-dose chemotherapy and radiotherapy has led to the widespread use of this source of stem cells in patients who undergo both autologous [1] and allogeneic [2] transplantation. The infusion of most hematopoietic growth factors leads to an increase in the peripheral blood stem cell (PBSC) population, but the magnitude of this mobilization

This work was supported by a grant from Searle Research and Development.

varies and depends on the growth factor used, the dosing schedule, and the biologic response of the recipient. The most active single agent identified to date is the myeloid growth factor granulocyte colony-stimulating factor (GCSF) [3]. Administration of this cytokine produces a significant increase in the number of circulating PBSCs as measured by in vitro assays of colony-forming cells (CFCs) and by the number of phenotypically defined (CD34 1 ) progenitor cells. The use of combinations of growth factors to mobilize PBSCs is now being evaluated in an effort to increase the number of circulating progenitor cells in the PB and thereby reduce the number of leukapheresis products required to obtain sufficient numbers of PBSCs for transplantation. An attractive approach to mobilizing greater numbers of these

Daniplestim and G-CSF Mobililization of Progenitor Cells

cells involves priming the bone marrow with an early-acting cytokine to increase the number of progenitor cells, followed by treatment with a later-acting factor that has significant mobilizing activity. The synergistic effect of interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on colony formation in vitro [4,5] suggests that the administration of these factors in combination may substantially increase the mobilization of PBSCs. Studies in both rhesus monkeys and humans have examined the sequential administration of IL-3 followed by either G-CSF [6–8] or GMCSF [9–14]. These studies have shown that these combinations of cytokines successfully mobilize PBSCs, but side effects, including fevers, headache, arthralgias, and myalgias are associated with the administration of IL-3 [6,7,9,10]. A similar side-effect profile has been observed in patients treated with IL-3 for bone marrow failure [15]. In addition, inflammatory reactions that are both dose-dependent and related to the duration of treatment have been described in nonhuman primates [16]. To enhance the efficacy of IL-3, a family of IL-3 receptor agonists has been engineered to increase the hematopoietic activity of this molecule without substantially increasing its inflammatory activity. Daniplestim (SC-55494, Searle Research and Development, St. Louis, MO), a novel species-specific IL-3 receptor agonist, has been shown to be 10-fold more active in proliferation assays and more than 20-fold more active in CFC assays than native IL-3 [17]. This advantage is offset by a twofold increase in the release of inflammatory response molecules such as histamine and leukotrienes, so that the overall potential therapeutic index of daniplestim over IL-3 is estimated to be five- to 10-fold [17]. When daniplestim was administered to nonhuman primates with radiation-induced myelosuppression, a more rapid recovery of both platelets and neutrophils was observed, indicating the multilineage activity of this IL-3 receptor agonist [18]. Combined therapy with daniplestim and G-CSF after radiation exposure reduces the magnitude and duration of neutropenia and thrombocytopenia to a greater degree than does daniplestim alone or G-CSF alone [19]. In the present study we have evaluated the potential of daniplestim administered concurrently or sequentially with G-CSF to mobilize PBSCs in nonhuman primates.

PATIENTS AND METHODS Animals Rhesus monkeys (Macaca mulatta) that weighed 6–8 kg were maintained at the Yerkes Regional Primate Center in Atlanta, GA. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Emory University. Growth factor administration Three groups of five animals were injected subcutaneously with 100 mg/kg daniplestim for 5 days followed by 10 mg/kg of G-CSF for 5 days (D/G), daniplestim and GCSF concurrently for 10 days (D1G), or G-CSF alone for 10 days. Injections were rotated among different sites on the abdomen and the flanks, and the injection sites were monitored daily for signs of inflammation.

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PB samples PB was obtained during ketamine hydrochloride anesthesia before the administration of the growth factors (Day 0) and on Days 3, 5, 7, 10, 12, and 14 of treatment. On each of these days, 4 mL of PB was collected; 1 mL was immediately transferred to a tube containing ethylenediaminetetraacetic acid (EDTA). The remaining 3 mL was mixed with 1 mL of Hanks’ balanced salt solution containing 3% fetal calf serum and 150 U heparin, and was used to prepare mononuclear cells. Complete blood counts Using a Coulter counter (TM, Coulter Electronics, Miami, FL) calibrated for rhesus peripheral blood cells, the total leukocyte count, serum hemoglobin level, hematocrit, and platelet count were determined. Manual differential counts were performed and the absolute number of neutrophils, lymphocytes, monocytes, basophils, and eosinophils per unit volume was obtained by counting 100 peripheral blood leukocytes. CD341 cell determination Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using ficoll-hypaque (Histopaque-1077, Sigma Chemical, St. Louis, MO). The PBMCs were washed twice in a phosphate-buffered saline solution containing 1% bovine serum albumin and were incubated for 20 minutes at 48C with a biotinylated antibody to CD34 (12.8, Cell Pro, Bothell, WA) [20]. The cells were washed, resuspended, and incubated with both a second-stage streptavidin-phycoeyrthrin and an antibody directed against CD45RA-FITC (BDIS, Sunnyvale, CA). The cells were washed and resuspended in modified Hanks’ balanced salt solution containing 1 mg/mL propidium iodide. The number of CD341, CD45RA1 mononuclear cells was determined by flow cytometry. Nonspecific binding was evaluated by using the appropriate second-stage and isotype controls. For each sample 50,000 cells were analyzed, providing a sensitivity of detection of 0.1% of the total number of PBMCs. The total number of CD341 , CD45RA1 cells per unit volume of PB was calculated by multiplying the percentage of the PBMCs that expressed CD45RA and CD34 by the number of mononuclear cells per unit volume of blood. Methylcellulose colony assays Total PBMCs were prepared as described above, then cultured in Iscoves’s Methylcellulose Ready-Mix serum supplemented with erythropoietin (3 U/mL; StemCell Technologies, Vancouver, BC), 50 mL of phytohemagglutininstimulated conditioned medium, and 1% fetal bovine serum [21]. Tissue culture plates containing 0.3–1.03105 input cells were incubated in triplicate at 378C and 5% CO2 for 14 days. The numbers of colony-forming unit granulocyte/macrophage (CFU-GM), colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and multipotent colony-forming unit (CFU-GEMM) colonies (.50 cells) were scored by using an inverted microscope. The number of colonies per unit volume of PB was determined by multiplying the number of colonies produced per input cell by the total number of mononuclear cells per unit volume of blood.

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Long-term stromal cell cultures and limit dilution assays The AC-6 stromal cell line was cultured in 96-well plates as previously described [22]. The PBMCs were suspended in RPMI/IMDM media with 10% fetal calf serum and stem cell factor at 0.5 ng/mL. Serial dilutions of these cells were added to confluent AC-6–containing wells (200–60,000 cells per well). These cultures were fed each week with 100 mL of RPMI/IMDM media. Those wells in which there were cobblestone-area-forming cells (.100 viable cells) after 28 days of culture were scored as positive. The number of cells that gave rise to 37% negative wells was determined [23]. This value indicates the number of early clonogenic precursors that have the potential to sustain colony formation for up to 28 days on this stromal feeder system.

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Monitoring of adverse effects associated with cytokine administration All animals were examined at least once daily for any adverse effects from the treatment. The injection sites did not show any signs of inflammation or any abrasions characteristic of a pruritic reaction. The food intake, weight, and activity levels of all animals were stable throughout the treatment period. PB samples were obtained by using ketamine anesthesia, which made it possible to assess the peripheral lymph nodes and spleen size during the treatment period. No adenopathy or splenomegaly was detected in any of the animals. In summary, all 15 animals in the three treatment groups tolerated the daily injections of these cytokines without showing any adverse effects.

Figure 1. Effects of daniplestim and granulocyte colony-stimulating factor (G-CSF) on peripheral blood cells

Effects of cytokine administration on PB counts After 5 days of treatment, the total white blood cell, neutrophil, and mononuclear cell counts in the D/G group remained unchanged (Fig. 1). In contrast, an eightfold increase in the number of circulating neutrophils and a 1.8fold increase in the number of mononuclear cells were observed in the D1G and the G-CSF groups. By Day 10 the total white blood cell count had peaked in all three treatment groups, with levels ranging from 34.53109/L (GCSF) to 60.13109/L (D/G), a four- to sixfold increase over the pretreatment levels (Fig. 1A). In all three groups the majority of these circulating cells were neutrophils (Fig. 1B); however, up to a twofold increase in the total number of circulating mononuclear cells was observed (Fig. 1C). Both the total white blood cell count and the neutrophil count returned rapidly to baseline levels after the cytokine was stopped. A similar decline in the number of mononuclear cells was observed in the G-CSF group and the D/G group; however, numbers of mononuclear cells that were 1.8-fold greater than the pretreatment levels were maintained in the D1G group for up to 4 days after the cessation of cytokine treatment. No clinically significant differences in the hemoglobin level, hematocrit, or platelet count were observed among the three treatment groups (data not shown).

Cohorts of five rhesus monkeys were treated with either daniplestim for 5 days and then G-CSF for 5 days (j), daniplestim and G-CSF concurrently for 10 days (d), or G-GSF alone for 10 days (m). Peripheral blood samples were obtained and the number of total (A) nucleated cells, (B) neutrophils, and mononuclear cells (C) was determined. The mean and SEM are shown.

Mobilization of CD341 progenitor cells into the PB The number of phenotypically defined hematopoietic progenitor cells in the mononuclear fraction of the PB was determined during the 10-day treatment period. When

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mononuclear cell preparations were examined, it was found that almost all CD34 1 cells also expressed the CD45RA antigen. Before treatment the mean level of CD341 progenitor cells in the PB was 133106/L (Fig. 2). After 5 days of D1G administration, the number of circulating CD341 progenitor cells had increased to 543106/L. The number of CD34 1 cells in the D/G group had increased to only 203106/L by Day 5 (the last day of daniplestim treatment); however, in the G-CSF phase of the sequential-cytokine regimen, the cell count increased to 633106/L within 2 days and this value was maintained through day 10. The maximal increase in the CD34 1 cell count in the group that was given G-CSF only was 43310 6 /L by Day 3. The count returned to the baseline value by Day 5 and did not increase again during the treatment period. These results indicate that the early maximal increase in phenotypically defined progenitor cells occurs after the concurrent administration of daniplestim and G-CSF. Mobilization of the total population of colony-forming hematopoietic progenitor cells To determine the level of committed hematopoietic progenitors in the PB following cytokine treatment, we assayed PBMCs for their ability to differentiate into myeloid (CFUGM), erythroid (CFU-E and BFU-E), and mixed colonies (CFU-GEMM). The total number of colonies generated per 105 input cells was determined in each treatment group. The pretreatment numbers of total colonies per 105 input cells ranged from 3.8 to 6.4 (Fig. 3A). After 5 days of treatment, the number of colonies had increased ninefold in the D1G group (55 colonies), sevenfold in the D/G group (29 colonies), and 4.7-fold (30 colonies) in the G-CSF group. By Day 11 the number of colonies had peaked in all three treatment groups. A peak activity of 73 colonies per 105 input cells was seen in the D1G group; the corresponding values in the D/G and G-CSF groups were 61 and 41 per 105 input cells, respectively. The colony-forming activity of the mononuclear fraction of the PB was increased in all treatment groups, but the greatest increases were observed in the concurrent-treatment group. In both groups given daniplestim, the increase in activity on a per-cell basis was greater than that in the group given only G-CSF. To determine the colony-forming activity of circulating hematopoietic progenitor cells, we calculated the total number of CFCs per unit volume of PB. The number of CFCs before treatment was between 201 and 363 3103/L in all three groups (Fig. 3B). By Day 5 there was an eightfold increase in the number of CFCs in the G-CSF and D/G groups. In contrast, there was a 15-fold increase in the D1G group. By Day 10 the values were 72553103/L in the D/G group and 58223103/L in the D1G group, but only 24583103/L in the G-CSF group. The levels of circulating CFCs were higher in the D1G group than in the G-CSF group throughout the 10 days of treatment. The levels were lower in the D/G group than in the D1G and G-CSF groups during the 5 days of daniplestim treatment. When G-CSF was given in the D/G group, however, the levels increased rapidly and continued to increase throughout the period of G-CSF treatment. This finding is consistent with an increase in the number of CFCs in the bone marrow, which is induced by daniplestim and followed by the release

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Figure 2. Effects of daniplestim and G-CSF on CD341 cell counts in the peripheral blood The number of CD341 cells circulating in the peripheral blood after treatment with daniplestim followed by G-CSF (j), daniplestim plus G-CSF (d), and G-CSF alone (m) was determined. The mean and SEM for groups of five animals are shown.

of these cells into the periphery by G-CSF. By Day 10 the number of circulating CFCs in the D/G group was 36-fold higher than the pretreatment level, but only eightfold higher in the G-CSF group. In all three treatment groups, there was a decrease in the concentration of CFCs when the growth factor was discontinued. These findings indicate that both the progenitor activity per mononuclear cell and the progenitor activity per unit volume of PB are highest in the D1G group. Mobilization of lineage-specific progenitor cells When the effects of cytokine mobilization on the number of lineage-specific progenitor cells were evaluated, clear differences were observed after 7 days of treatment (Fig. 4). The number of CFU-GM was similar in each group, ranging from a mean of 130 colonies per 106 input cells to 281 colonies per 106 cells. The number of BFU-E colonies was markedly different in the treatment groups, with a mean of 0.8 colonies per 106 input mononuclear cells in the G-CSF group and means of 36 and 43 colonies per 106 input cells in the D/G and D1G groups, respectively. This difference in the number of erythroid precursors was more pronounced when the CFU-E were examined. A mean of 20 CFU-E per 106 input cells was seen in both the D/G group and the D1G group. In contrast, no CFU-E were detected in the G-CSF group. Mixed myeloid and erythroid colonies (CFU-GEMM) are believed to arise from more primitive bipotential progenitor cells. Both the D/G group and the D1G group had a mean of 16 colonies per 106 input cells, but the G-CSF group had a mean of only 1.4 colonies per 106 input cells. These results indicate that both D1G and D/G produce higher levels of

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Figure 3. Mobilization of hematopoietic progenitor cells into the peripheral blood (A) The number of colony-forming cells (CFCs) per 105 input mononuclear cells in cohorts of five rhesus monkeys after treatment with daniplestim for 5 days and then G-CSF for 5 days (j), daniplestim and G-CSF concurrently for 10 days (d), or G-GSF alone for 10 days (m) are shown. (B) The number of CFCs per unit volume of peripheral blood in each of these treatment groups was determined. The mean and SEM are shown.

circulating erythroid progenitor cells and early mixed-lineage progenitor cells than does G-CSF alone. Detection of early hematopoietic progenitor cells To determine the presence of primitive hematopoietic cells with the ability to generate colonies in long-term culture, we performed a limit dilution assay. The number of clonogenic precursors was determined by using Poisson analysis [23,24]. The number of these progenitor cells was highest in the D1G group, in which there was a fourfold increase in the number of circulating progenitor cells by Day 7. In the D/G group there was a twofold increase and in the G-CSF group a 1.5-fold increase within the same time period (Fig. 5). The levels of progenitor cells peaked by Day 7 in all three treatment groups and then declined to levels below baseline by Day 10. These results indicate that an increase in the levels of circulating early progenitor cells occurs earlier and is less sustained than the mobilization of the more differentiated methylcellulose colony-forming progenitor cells.

DISCUSSION Early studies of IL-3 found that this pleiotropic speciesspecific growth factor [25] interacts with early progenitor cells [26] and produces a marked increase in the activity of later-acting lineage-specific cytokines. In vitro studies of murine progenitor cells indicate that the combination of homologous IL-3 and G-CSF increases the formation of multipotential blast colonies [4]. When human bone marrow cells were evaluated in culture, a synergistic effect on colony growth was observed when IL-3 was combined with either G-CSF or GM-CSF [5]. The administration of IL-3 in nonhuman primates was found to have a modest effect on hematopoiesis; however, pretreatment with IL-3 greatly potentiated the hematopoietic response to GM-CSF

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[11,12]. When IL-3 was administered to patients with bone marrow failure [15], an increase in the numbers of reticulocytes, leukocytes, and platelets was observed, confirming the multilineage activity of this hematopoietic growth factor. Analysis of human hematopoietic progenitor cells after the administration of IL-3 showed an increase in DNA synthesis [27]. This proliferation of progenitor cells was accompanied by an increase in bone marrow cellularity and an increase in the myeloid-to-erythroid cell ratio. These observations suggested that IL-3 could be used to expand early hematopoietic progenitor cells within the bone marrow. The subsequent addition of GM-CSF or G-CSF would be expected to mobilize these expanded bone marrow–derived progenitor cells into the PB. When this hypothesis was tested in rhesus monkeys, a 12-fold increase in the number of circulating CFU-GM was observed with IL-3 alone and a 63-fold increase with IL-3 followed by GM-CSF [13]. A subsequent phase I study in humans found that the sequential administration of IL-3 and GM-CSF was more effective in increasing the number of circulating CFU-GM than was IL-3 alone [9]. In another study, the mobilization of progenitor cells after chemotherapy, IL-3, and GM-CSF was greater than that with chemotherapy and IL-3 alone [28]. Successful mobilization in patients treated with IL-3 followed by G-CSF with or without prior chemotherapy has resulted in levels of circulating progenitor cells sufficient to enable autografting [6,7]. When autologous PBSCs mobilized with IL-3 followed by GM-CSF were infused into patients who had been treated with high-dose chemotherapy, hematopoietic engraftment was observed [10,14]. In a prospective pilot trial to evaluate the relative mobilizing potential of these cytokine combinations, leukapheresis products obtained from the sequential administration of IL3 followed by GM-CSF resulted in slower engraftment than did leukapheresis products obtained with IL-3 priming followed by G-CSF treatment (22 vs. 13 days) [8]. These find-


Figure 4. Colony-forming cell activity in cohorts of five treated rhesus monkeys Treatments included daniplestim for 5 days and then G-CSF for 5 days (h), daniplestim and G-CSF concurrently for 10 days ( ), or G-CSF alone for 10 days ( ). The number of CFU-GM, BFU-GM, CFU-E, and GFU-GEMM per 106 PBMCs was determined. The mean and SEM are shown. *Below the limit of detection of the assay.

ings underscore the importance of comparing the mobilizing ability of different cytokines in the same clinical trial. The goal of our study was to determine whether the sequential administration of the early-acting IL-3 receptor agonist daniplestim followed by G-CSF would produce higher numbers of circulating progenitor cells than would concurrent administration of these cytokines. For direct comparison we also treated a group of animals with G-CSF only. We found that both phenotypically identified and functionally defined hematopoietic progenitor cells were mobilized into the PB of nonhuman primates by all three treatment regimens. The magnitude of the mobilization of progenitor cells was greater in both the D/G and the D1G groups than in the G-CSF group. Essentially, all progenitor cell measurements indicated equal or better results with concurrent treatment than with sequential treatment. These findings are consistent with those of a recent study that evaluated the effects of Flt3/Flt2 ligand on the mobilization of hematopoietic progenitor cells in nonhuman primates [29]. This ligand slowly mobilized hematopoietic progenitor cells, with the peak activity occurring after 2 weeks of treatment. The administration of G-CSF during the last 5 days of a 12-day course of treatment with the ligand produced levels of circulating progenitor cells that were 10-fold higher than those observed with the ligand alone. When a short course of combined treatment with both agents was given, similar increases in the number of circulating progenitor cells were observed. Both this study and our present study clearly show that a period of pretreatment with an early-acting multipotent cytokine does not produce higher levels of circulating progenitor cells than does a shorter course of concurrent therapy with the same cytokines.

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Figure 5. Limit dilution analysis of clonogenic hematopoietic progenitor cells Peripheral blood samples were obtained after cytokine treatment and were analyzed by limit dilution. The frequency of clonogenic precursors per 105 peripheral blood mononuclear cells is shown for two representative animals per treatment group (D/G, [j]; D1G, [d]; G-CSF, [m]).

The early increase in the levels of circulating progenitor cells seen in the D1G group in our study indicates that this schedule would likely be more efficacious for collecting PBSCs and that a shorter treatment period would be required for mobilization. Further studies to evaluate the engraftment potential of progenitor cells mobilized by these treatment schedules appear to be warranted. In addition, these data suggest that a chimeric molecule consisting of a bifunctional IL-3 receptor agonist and a G-CSF receptor agonist may prove useful for mobilizing hematopoietic progenitor cells.

ACKNOWLEDGMENTS We thank Dr. Harold McClure and Ellen Lockwood of the Yerkes Regional Primate Center for their assistance with this project.

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