Granulocyte-macrophage Colony-stimulating Factor Fusion Protein Research Articles

Development of an extended half-life GM-CSF fusion protein for Parkinson's disease.

Transformation of CD4+ T cell effector to regulatory (Teff to Treg) cells have been shown to attenuate disease progression by restoring immunological balance during the onset and progression of neurodegenerative diseases. In our prior studies, we defined a safe and effective pathway to restore this balance by restoring Treg numbers and function through the daily administration of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These studies were conducted as a proof-of-concept testing in Parkinson's disease (PD) preclinical models and early phase I clinical investigations. In both instances, they served to ameliorate disease associated signs and symptoms. However, despite the recorded efficacy, the cytokine's short half-life, low bioavailability, and injection site reactions proved to be limitations for any broader use. To overcome these limitations, mRNA lipid nanoparticles encoding an extended half-life albumin-GM-CSF fusion protein were developed for both mouse (Msa-GM-CSF) and rat (Rsa-GM-CSF). These formulations were tested for immunomodulatory and neuroprotective efficacy using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and human wild-type alpha-synuclein (αSyn) overexpression preclinical models of PD. A single dose of the extended half-life mouse and rat mRNA lipid nanoparticles generated measurable GM-CSF plasma cytokine levels up to four days. Increased Treg frequency and function were associated with a resting microglial phenotype, nigrostriatal neuroprotection, and restoration of brain tissue immune homeostasis. These findings were substantively beyond the recorded efficacy of daily recombinant wild-type GM-CSF with a recorded half-life of six hours. Mechanistic evaluation of neuropathological transcriptional profiles performed in the disease-affected nigral brain region demonstrated an upregulation of neuroprotective CREB and synaptogenesis signaling and neurovascular coupling pathways. These findings highlight the mRNA-encoded albumin GM-CSF fusion protein modification linked to improvements in therapeutic efficacy. The improvements achieved were associated with the medicine's increased bioavailability. Taken together, the data demonstrate that mRNA LNP encoding the extended half-life albumin-GM-CSF fusion protein can serve as a benchmark for PD immune-based therapeutics. This is especially notable for improving adherence of drug regimens in a disease-affected patient population with known tremors and gait abnormalities.

Malignant Progenitors From Patients With Acute Myelogenous Leukemia Are Sensitive to a Diphtheria Toxin–Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 × 10−12 mol/L (range, 5 to >4,000 × 10−12 mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing ≥35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 × 10−9 mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.

Monoclonal Anti-idiotype Antibody 6G6.C4 Fused to GM-CSF Is Capable of Breaking Tolerance to Carcinoembryonic Antigen (CEA) in CEA–Transgenic Mice

Internal image anti-idiotypic antibodies are capable of mimicking tumor-associated antigens and thus may serve as surrogate for vaccination strategies in cancer patients. The monoclonal antibody (mAb) 6G6.C4 mimics an epitope specific for the human carcinoembryonic antigen (CEA) and generates a CEA-specific response (Ab3) in various experimental animals. In humans, however, 6G6.C4 only yields a very limited humoral anti-CEA reaction presumably due to tolerance against the CEA autoantigen. In this study, we investigated the CEA-specific Ab3 response in mice transgenic for the human CEA and tested whether the antigen tolerance could be overcome by fusing a recombinant single-chain variable fragment of 6G6.C4 (scFv6G6.C4) to the murine granulocyte macrophage colony-stimulating factor (GM-CSF). Like mAb 6G6.C4, the fusion protein (scFv6G6.C4/GM-CSF) retained binding to the CEA-specific idiotype mAb T84.66. Also, scFv6G6.C4/GM-CSF was biologically active as measured by proliferation of the GM-CSF-dependent murine FDC-P1 cells in vitro. After immunization with the scFv6G6.C4/GM-CSF fusion protein, CEA-transgenic animals showed significantly enhanced Ab3 antibody responses to scFv6G6.C4 (P=0.005) and to CEA (P=0.012) compared with the scFV6G6.C4 alone. Sera from mice immunized with the fusion protein specifically recognized CEA in Western blot analyses with no cross-reaction to CEA-related antigens. Finally, the Ab3 antisera detected single CEA-expressing tumor cells in suspension as shown by flow cytometry. Taken together, these data show in a model antigenically related to the human system that vaccination with scFv6G6.C4/GM-CSF improves vaccination against an endogenous tumor-associated antigen resulting in a highly specific humoral Ab3 response in vivo that is capable of bind single circulating CEA-positive tumor cells.

Abrogation of the Hematological and Biological Activities of the Interleukin-3/Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein PIXY321 by Neutralizing Anti-PIXY321 Antibodies in Cancer Patients Receiving High-Dose Carboplatin

This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 micrograms/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 micrograms/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/microL but was able to speed the time of recovery of platelet counts to 100,000/microL (15 v 20 days; P =.01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/microL by an average of at least 8 days as compared with cycle A-1 (without PIXY321; P </=.004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The immunogenicity of this fusion protein provides a cautionary warning that clinical development of bioengineered human molecules requires thorough testing for immune neutralization.

Modulation of the Apoptotic Response of Human Myeloid Leukemia Cells to a Diphtheria Toxin Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractIt has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor–bearing cells. We now report that DT388–GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF–receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL ). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I–GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388–GM-CSF at 4 × 10−9 mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 × 10−9 mol/L to 3 × 10−13 mol/L. GM-CSF–receptor occupancy at 0.7 nmol/L GM-CSF–ligand concentration correlated with the protein synthesis IC50 . Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL , in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388–GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.

Developments in cytokine therapy for haematological toxicity

Will thrombopoietin*join the successes or the failures that have arisen from research into the use of cytokines to treat haematological toxicity? The clinical development of the thrombopoietic factors interleukin (IL)-1 and IL-6 has been halted due to dose-limiting systemic toxicity. However, the growth factors granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and epoetin are now enjoying successful use in the clinical setting, having received approval for attenuating chemotherapy-induced neutropenia. Still under investigation for preventing thrombocytopenia are IL-3, IL-11, PIXY-321 (an IL-3/GM-CSF fusion protein), and thrombopoietin.**The current status of these agents was reviewed at the 15th Annual Conference on Clinical Haematology and Oncology [San Diego, US; February 1997].

Effect of flt3 Ligand on the Ex Vivo Expansion of Human CD34+ Hematopoietic Progenitor Cells

A ligand for the tyrosine kinase receptor flt3/flk-2, referred to here as flt3 ligand (flt3L), was recently cloned. The effect of flt3L on purified human CD34+ progenitor cells was examined. flt3 receptor (flt3R) was detected on the surface of human bone marrow cells that were enriched for CD34 expression. The effects of flt3L and the c-kit ligand Steel factor (SLF) on hematopoietic progenitors were compared in clonal colony assays. Both factors synergized with Pixy321 (interleukin-3 [IL-3]-granulocyte-macrophage colony-stimulating factor fusion protein) to induce granulocytic-monocytic (GM) and high proliferative potential (HPP) colonies and synergized with Pixy321 + erythropoietin (EPO) to induce multipotent granulocytic-erythroid-monocytic-megakaryocytic colonies. Although SLF had a potent effect on colony formation of erythroid restricted progenitor cells (burst-forming unit-erythroid), no effect by flt3L was observed. The addition of flt3L to irradiated long-term marrow cultures seeded with CD34+ cells augmented both total and progenitor cell production. Ex vivo expansion studies with isolated CD34+ bone marrow cells from normal donors showed that flt3L alone supported maintenance of both GM and HPP progenitors for 3 to 4 weeks in vitro. The addition of flt3L to a growth factor combination of IL-1 alpha + IL-3 + IL-6 + EPO resulted in a synergistic effect on progenitor cell expansion comparable to that observed with the addition of SLF to IL-1 alpha + IL-3 + IL-6 + EPO. These data show a function for flt3L in the regulation of both primitive multipotent and lineage-committed hematopoietic progenitor cells.

New Cytokines with Thrombopoietic Activity

The use of growth factors to stimulate haemopoietic proliferation in various clinical conditions such as renal failure-associated anaemia, post-chemotherapy cytopenia and enhancement of bone marrow engraftment has been well described. Of the 3 haemopoietic lineages, the use of growth factors has been most beneficial in the erythroid and myeloid compartments. Currently, there are no cytokines available specifically for the purpose of stimulating thrombopoiesis. However, expanding knowledge of haemopoiesis in general and megakaryocytopoiesis in particular has led to an appreciation of the fact that multiple growth factors with overlapping or specific activities are involved in various stages of megakaryocyte proliferation and differentiation. Although application of early acting, multiline-age cytokines such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) has resulted in significant augmentation of thrombopoiesis in vitro, little such activity has been seen in vivo. The IL-3/GM-CSF fusion protein PIXY-321 (pixykine), also active in vitro, similarly has limited thrombopoietic effect in vivo. On the other hand, interleukin-6 (IL-6) and, in early studies, interleukin-11 (IL-11), which both augment IL-3-dependent progenitor proliferation and have significant megakaryocytopoietic activity in vitro, produce a more pronounced thrombopoietic effect. Further analyses and trials will be required to establish proper cytokine dosages, regimens and combinations in order to achieve maximal platelet enhancement.

Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

Long-Term Generation and Expansion of Human Primitive Hematopoietic Progenitor Cells In Vitro

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP-CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.