Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

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Prevention of Thrombocytopenia and Neutropenia in a Nonhuman Primate Model of Marrow Suppressive Chemotherapy by Combining Pegylated Recombinant Human Megakaryocyte Growth and Development Factor and Recombinant Human Granulocyte Colony-Stimulating Factor

Blood ◽

10.1182/blood.v89.1.155 ◽

1997 ◽

Vol 89 (1) ◽

pp. 155-165 ◽

Cited By ~ 39

Author(s):

Laurence A. Harker ◽

Ulla M. Marzec ◽

Andrew B. Kelly ◽

Ellen Cheung ◽

Aaron Tomer ◽

...

Keyword(s):

Platelet Count ◽

Neutrophil Count ◽

Growth And Development ◽

Granulocyte Colony Stimulating Factor ◽

Serum Concentrations ◽

Colony Stimulating Factor ◽

Platelet Counts ◽

Development Factor ◽

Trough Serum ◽

Stimulating Factor

Abstract This report examines the effects on hematopoietic regeneration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF ) (2.5 μg/kg/d) alone and in combination with recombinant human granulocyte colony stimulating factor (rHu-GCSF ) (10 μg/kg/d) for 21 days in rhesus macaques receiving intense marrow suppression produced by single bolus injections of hepsulfam (1.5 g/m2). In six hepsulfam-only control animals thrombocytopenia (platelet count <100 × 109/L) was observed between days 12 and 25 (nadir 39 ± 20 × 109/L on day 17), and neutropenia (absolute neutrophil count <1 × 109/L) occurred between days 8 and 30 (nadir 0.167 ± 0.120 × 109/L on day 15). PEG-rHuMGDF (2.5 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.9 ± 0.2 ng/mL and increased the platelet count twofold over basal prechemotherapy levels (856 ± 594 × 109/L v baseline of 416 ± 88 × 109/L; P = .01). PEG-rHuMGDF alone also shortened the period of posthepsulfam neutropenia from 22 days to 12 days (P = .01), although the neutropenic nadir was not significantly altered (neutrophil count 0.224 ± 0.112 × 109/L v 0.167 ± 0.120 × 109/L; P < .3). rHu-GCSF (10 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.4 ± 1.1 ng/mL, and reduced the time for the postchemotherapy neutrophil count to attain 1 × 109/L from 22 days to 4 days (P = .005). The postchemotherapy neutropenic nadir was 0.554 ± 0.490 × 109neutrophils/L (P = .3 v hepsulfam-only control of 0.167 ± 0.120 × 109/L). However, thrombocytopenia of <100 × 109 platelets/L was not shortened (persisted from day 12 to day 25), or less severe (nadir of 56 ± 32 × 109 platelets/L on day 14; P = .7 compared with untreated hepsulfam animals). The concurrent administration of rHu-GCSF (10 μg/kg/d) and PEG-rHuMGDF (2.5 μg/kg/d) in four animals resulted in postchemotherapy peripheral platelet counts of 127 ± 85 × 109/L (P = .03 compared with 39 ± 20 × 109/L for untreated hepsulfam alone, and P = .02 compared with 856 ± 594 × 109/L for PEG-rHuMGDF alone), and shortened the period of neutropenia <1 × 109/L from 22 days to 4 days (P = .8 compared with rHu-GCSF alone). Increasing PEG-rHuMGDF to 10 μg/kg/d and maintaining the 21-day schedule of coadministration with rHu-GCSF (10 μg/kg/d) in another four animals produced postchemotherapy platelet counts of 509 ± 459 × 109/L (P < 10−4compared with untreated hepsulfam alone, and P = .04 compared with 2.5 μg/kg/d PEG-rHuMGDF alone), and 4 days of neutropenia. Coadministration of rHu-GCSF and PEG-rHuMGDF did not significantly alter the pharmacokinetics of either agent. The administration of PEG-rHuMGDF (2.5 μg/kg/d) from day 1 through day 22 and rHu-GCSF (10 μg/kg/d) from day 8 through day 22 in six animals produced peak postchemotherapy platelet counts of 747 ± 317 × 109/L (P < 10−4 compared with untreated hepsulfam alone, and P = .7 compared with PEG-rHuMGDF alone), and maintained the neutrophil count < 3.5 × 109/L (P = .008 v rHu-GCSF therapy alone). Thus, both thrombocytopenia and neutropenia are eliminated by initiating daily PEG-rHuMGDF therapy on day 1 and subsequently adding daily rHu-GCSF after 1 week in the rhesus model of hepsulfam marrow suppression. This improvement in platelet and neutrophil responses by delaying the addition of rHu-GCSF to PEG-rHuMGDF therapy demonstrates the importance of optimizing the dose and schedule of cytokine combinations after severe myelosuppressive chemotherapy.

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In vitro and in vivo effects of recombinant human granulocyte colony-stimulating factor on neutrophil functions.

Journal of the Japan Society of Blood Transfusion ◽

10.3925/jjtc1958.35.647 ◽

1989 ◽

Vol 35 (6) ◽

pp. 647-652

Author(s):

Akimichi Ohsaka ◽

Seiichi Kitagawa ◽

Akira Yuo ◽

Takashi Obata ◽

Youichi Amemiya ◽

...

Keyword(s):

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

In Vivo Effects ◽

Stimulating Factor

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Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates

Blood ◽

10.1182/blood.v86.1.54.bloodjournal86154 ◽

1995 ◽

Vol 86 (1) ◽

pp. 54-59 ◽

Cited By ~ 145

Author(s):

AM Farese ◽

P Hunt ◽

T Boone ◽

TJ MacVittie

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Growth And Development ◽

Nonhuman Primates ◽

Normal Male ◽

Platelet Counts ◽

Development Factor ◽

Development In Vitro

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.

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Platelets Exposed to Elevated Levels of Endogenous Thrombopoietin In Vivo Have a Reduced Response to Megakaryocyte Growth and Development Factor In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612918 ◽

2001 ◽

Vol 85 (01) ◽

pp. 152-159 ◽

Cited By ~ 5

Author(s):

Uichi Nishiyama ◽

Haruhiko Morita ◽

Yoshifumi Torii ◽

Tomoaki Kuwaki ◽

Eiko Shimizu ◽

...

Keyword(s):

Growth And Development ◽

Platelet Reactivity ◽

Biochemical Properties ◽

Human Platelets ◽

Janus Kinase ◽

Intravenous Injections ◽

Development Factor ◽

Lower Reactivity

SummaryThrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

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Neutrophils express the high affinity receptor for IgG (Fc gamma RI, CD64) after in vivo application of recombinant human granulocyte colony- stimulating factor

Blood ◽

10.1182/blood.v78.4.885.885 ◽

1991 ◽

Vol 78 (4) ◽

pp. 885-889 ◽

Cited By ~ 134

Author(s):

R Repp ◽

T Valerius ◽

A Sendler ◽

M Gramatzki ◽

H Iro ◽

...

Keyword(s):

Fc Receptors ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

High Affinity ◽

Healthy Donors ◽

High Affinity Receptor ◽

Affinity Receptor ◽

Stimulating Factor

Abstract Fc receptors are important effector molecules of neutrophilic granulocytes (polymorphonuclear neutrophils [PMN]), connecting phagocytic cells and the specific immune response. Neutrophils from healthy donors express the low-affinity receptors for IgG Fc gamma RII (CD32) and Fc gamma RIII (CD16), but not the high-affinity receptor Fc gamma RI (CD64). The latter has been found on neutrophils from patients with certain bacterial infections and can be induced in vitro after incubation with interferon-gamma. We show here that neutrophils strongly express Fc gamma RI after in vivo application of recombinant human granulocyte colony-stimulating factor (rhG-CSF). PMN from patients receiving rhG-CSF displayed higher cytotoxicity against Daudi lymphoma cells in vitro compared with control patients and with healthy donors. Fab fragments against Fc gamma RII (monoclonal antibody [MoAb] IV.3) inhibited neutrophil-mediated cytotoxicity of healthy donors but not of patients during rhG-CSF therapy. Therefore, expression of Fc receptors by PMN was investigated by flow cytometry and the mean fluorescence intensity (MFI) was compared. After staining with MoAb 32.2 against Fc gamma RL, the median MFI of neutrophils from G-CSF patients (median, 4.78; range, 2.40 to 8.50; n = 5) was significantly higher (P = .002 and P = .001, respectively) than the median MFI of patients not receiving G-CSF (median, 1.23; range, 1.01 to 1.58; n = 6) and the median MFI of healthy donors (median, 1.04; range, 0.67 to 1.12; n = 6). Fc gamma RI disappeared after the discontinuing of the G- CSF injections, but was reinduced during the next treatment cycle with rhG-CSF. The high expression of Fc gamma RI during rhG-CSF therapy correlated with enhanced cytotoxicity. In vitro incubation with rhG-CSF also enhances cytotoxicity, but only minor increments in Fc gamma RI expression were observed. Thus, during in vivo application of rhG-CSF neutrophils acquire an additional potent receptor for mediating tumor cell killing in vitro by induction of the high-affinity receptor for IgG (Fc gamma RI, CD64).

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In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody

Blood ◽

10.1182/blood.v86.3.1124.1124 ◽

1995 ◽

Vol 86 (3) ◽

pp. 1124-1130 ◽

Cited By ~ 24

Author(s):

J Michon ◽

S Moutel ◽

J Barbet ◽

JL Romet-Lemonne ◽

YM Deo ◽

...

Keyword(s):

Binding Site ◽

Cancer Patients ◽

Bispecific Antibody ◽

Neuroblastoma Cells ◽

Bispecific Antibodies ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Stimulating Factor

Abstract Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.

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Granulocyte colony-stimulating factor administration to healthy volunteers: analysis of the immediate activating effects on circulating neutrophils

Blood ◽

10.1182/blood.v84.11.3885.bloodjournal84113885 ◽

1994 ◽

Vol 84 (11) ◽

pp. 3885-3894 ◽

Cited By ~ 129

Author(s):

M de Haas ◽

JM Kerst ◽

CE van der Schoot ◽

J Calafat ◽

CE Hack ◽

...

Keyword(s):

Healthy Volunteers ◽

Plasma Levels ◽

Subcutaneous Administration ◽

Secretory Vesicles ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Specific Granules ◽

Stimulating Factor

In four healthy volunteers, we analyzed in detail the immediate in vivo effects on circulating neutrophils of subcutaneous administration of 300 micrograms of granulocyte colony-stimulating factor (G-CSF). Neutrophil activation was assessed by measurement of degranulation. Mobilization of secretory vesicles was shown by a decrease in leukocyte alkaline phosphatase content of the circulating neutrophils. Furthermore, shortly postinjection, Fc gamma RIII was found to be upregulated from an intracellular pool that we identified by immunoelectron microscopy as secretory vesicles. Intravascular release of specific granules was shown by increased plasma levels of lactoferrin and by upregulation of the expression of CD66b and CD11b on circulating neutrophils. Moreover, measurement of fourfold elevated plasma levels of elastase, bound to its physiologic inhibitor alpha 1- antitrypsin, indicated mobilization of azurophil granules. However, no expression of CD63, a marker of azurophil granules, was observed on circulating neutrophils. G-CSF--induced mobilization of secretory vesicles and specific granules could be mimicked in whole blood cultures in vitro, in contrast to release of azurophil granules. Therefore, we postulate that the most activated neutrophils leave the circulation, as observed shortly postinjection, and undergo subsequent stimulation in the endothelial microenvironment, resulting in mobilization of azurophil granules. Our data demonstrate that G-CSF should be regarded as a potent immediate activator of neutrophils in vivo.

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