Native Granulocyte Colony-stimulating Factor Research Articles

Improving the Therapeutic Potential of G-CSF through Compact Circular PEGylation Based on Orthogonal Conjugations.

In this study, a circular conjugate of granulocyte colony-stimulating factor (G-CSF) was prepared by conjugating the two end-chains of poly(ethylene glycol) (PEG) to two different sites of the protein. For the orthogonal conjugation, a heterobifunctional PEG chain was designed and synthesized, bearing the dipeptide ZGln-Gly (ZQG) at one end-chain, for transglutaminase (TGase) enzymatic selective conjugation at Lys41 of G-CSF, and an aldehyde group at the opposite end-chain, for N-terminal selective reductive alkylation of the protein. The cPEG-Nter/K41-G-CSF circular conjugate was characterized by physicochemical methods and compared with native G-CSF and the corresponding linear monoconjugates of G-CSF, PEG-Nter-G-CSF, and PEG-K41-G-CSF. The results demonstrated that the circular conjugate had improved physicochemical and thermal stability, prolonged pharmacokinetic interaction, and retained the biological activity of G-CSF. The PEGylation strategy employed in this study has potential applications in the design of novel protein-based therapeutics.

A topological refactoring design strategy yields highly stable granulopoietic proteins

Protein therapeutics frequently face major challenges, including complicated production, instability, poor solubility, and aggregation. De novo protein design can readily address these challenges. Here, we demonstrate the utility of a topological refactoring strategy to design novel granulopoietic proteins starting from the granulocyte-colony stimulating factor (G-CSF) structure. We change a protein fold by rearranging the sequence and optimising it towards the new fold. Testing four designs, we obtain two that possess nanomolar activity, the most active of which is highly thermostable and protease-resistant, and matches its designed structure to atomic accuracy. While the designs possess starkly different sequence and structure from the native G-CSF, they show specific activity in differentiating primary human haematopoietic stem cells into mature neutrophils. The designs also show significant and specific activity in vivo. Our topological refactoring approach is largely independent of sequence or structural context, and is therefore applicable to a wide range of protein targets.

Optimizing Granulocyte Colony-Stimulating Factor Transcript for Enhanced Expression in Escherichia coli.

The human granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor used to prevent and treat neutropenia. G-CSF stimulates the bone marrow to produce infection-fighting granulocytes. Food and Drug Administration of the United States approved G-CSF in 1991 and its PEGylated version in 2002 as a prophylactic and therapeutic measure against neutropenia. Recombinant human G-CSF is produced in surrogate host Escherichia coli and is PEGylated at N-terminal. Besides neutropenia, G-CSF is also used in bone marrow transplantation for the mobilization and maturation of peripheral blood stem cells. Considering the requirement of producing G-CSF therapeutic in large quantities, construct designing for high expression is critical for the biopharmaceutical and industrial application. Earlier studies have employed approaches such as codon optimization, use of strong promoters, employment of protein tags, secretion signals, optimization of protein folding, etc., for increasing expression and yield of therapeutic proteins. In this study, it was observed that mRNA transcribed from the native human cDNA of G-CSF and the codon-optimized variant leads to low protein expression in E. coli. To understand the underlying reasons, the mRNA secondary structure of the 5′ end of the G-CSF transcript was analyzed. This analysis revealed the presence of stable secondary structures at the 5′ end of the G-CSF transcript, arising from the native human gene and even from the codon-optimized sequence. These secondary structures were disrupted through translationally silent mutations within the first 24 nucleotides of the transcript without affecting the protein sequence. Interestingly, through this approach, the G-CSF protein expression was increased 60 folds as compared to native G-CSF construct. We believe that these findings create a roadmap for optimization of G-CSF transcript for enhanced expression in E. coli and could be employed to increase the expression of other therapeutic proteins.

Mechanistic explanation of structural and functional changes induced by methionine mutation in G-CSF protein

Abstract Potency of a biotherapeutic is influenced by its binding affinity to the designated receptor., This study examines how substitution of methionine residues with Leucine/Alanine in granulocyte colony stimulating factor (G-CSF) brings about changes in its affinity to G-CSF receptor. To this effect, two mutants M1 (M122A, M127A and M138A) and M2 (M122L, M127L and M138L) were designed computationally and their stability and functional attributes were compared with the native GCSF. The simulation results show that although all atom root mean square deviations during 100 ns window were within 2–3 A for the mutants as well as the native G-CSF, yet some local conformational changes were evident. Amino acid residues in the stretch 125–145 exhibited shift in their secondary structure composition from predominantly turn form to coiled and gamma turn forms. In addition, increase in the helical content in the amino acid stretch 60–70 was also observed when compared to native G-CSF. Docking and MMPBSA studies of the GCSF variants with G-CSFR show evidence of increased number of interactions with G-CSFR owing to the observed conformational perturbations in the variants. Experimental data on the stability and the affinity of these variants are in agreement with the in-silico predictions. Thus, we show that careful substitution of key residues in G-CSF potentially presents avenues for improving the stability as well therapeutic prospects of the molecule via enhancement in their affinities to the pertinent receptors.

Determination of Critical Quality Attributes for a Biotherapeutic in the QbD Paradigm: GCSF as a Case Study.

Estimating impact of the various product-related variants and impurities on a biotherapeutic's safety and efficacy is an essential requirement in the quality by design paradigm. In view of the limited role that clinical studies offer in this regard, we demonstrate a preclinical approach to achieve this for granulocyte colony-stimulating factor (GCSF). While our repeated-dose toxicity data suggest that these variants do not elicit any adverse effects or histopathological changes, aggregated GCSF impurity caused sluggishness in animal behavior manifested by a possible muscular injury. Cell assay data revealed that the cys-64-cys74 disulfide bond in reduced GCSF imparts stabilization in absence of the cys-36-cys42 bond. PK data demonstrate variability in half lives of different species when compared to the native GCSF. PD data along with differential expression of JAK-2 and STAT5a genes show that all the tested variants triggered the required signal transduction pathways for neutrophil proliferation and activation.

Granulocyte colony-stimulating factor (GCSF) fused with Fc Domain produced from E. coli is less effective than Polyethylene Glycol-conjugated GCSF

Human granulocyte colony-stimulating factor (GCSF) is a well-known cytokine for neutropenia treatment. However, daily injections are required due to the short circulating half-life of the protein. To overcome this bottleneck, we fused GCSF with the Fc domain of IgG1 at the C terminus (GCSF-Fc) and with the maltose binding protein (MBP) tag at the N-terminus and expressed it as a soluble protein in the cytoplasm of E. coli. We also conjugated PEG aldehyde to GCSF to make PEG-GCSF. The bioactivities of GCSF-Fc and PEG-GCSF were similar to native GCSF using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50 dose-response curves for GCSF, GCSF-Fc and PEG-GCSF were 37 ± 12 pM, 75 ± 13.5 pM and 46 ± 5.5 pM, respectively. When the proteins were injected into neutropenic rats, the group injected with PEG-GCSF showed the highest and fastest recovery of neutrophils, followed by GCSF-Fc and GCSF. ELISA assay revealed the PEG-GCSF had the longest plasma circulation (>72 h), followed by GCSF-Fc (>48 h) and GCSF (~24 h), which is consistent with the in vivo activities of the proteins. In summary, the GCSF-Fc purified from E. coli was not as efficient as PEG-GCSF in treating neutropenic rats.

Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF.

Prolonged circulation and in vivo efficacy of recombinant human granulocyte colony-stimulating factor encapsulated in polymeric micelles

To improve the pharmacokinetics of granulocyte colony-stimulating factor (G-CSF) and decrease dosing frequency, polyethylene glycol polyglutamate block copolymers were used as delivery carriers. The block copolymers are partially substituted with hydrophobic octyl or benzyl groups to form a micellar structure in aqueous media and encapsulate the protein. G-CSF is encapsulated in the polymeric micelles with a diameter of 60–70 nm. The present study was designed to evaluate the plasma pharmacokinetics, G-CSF release and in vivo efficacy of G-CSF-encapsulating micelles. Pharmacokinetic studies in rats showed highly enhanced plasma retention of the micelles compared with native G-CSF. The AUC (area under the curve) of the octyl-based polymer formulation showed a 5-fold increase, compared with native G-CSF. Size-exclusion chromatography of the blood from rats injected with the micelles demonstrated the release of G-CSF from the micelles in the blood circulation. The pharmacokinetic behavior supports the in vivo studies showing that the micelles display a comparable efficacy to PEGylated G-CSF. Simultaneous pharmacokinetic analysis of released and encapsulated G-CSF plasma levels showed that the G-CSF release occurs with the first-order kinetics and the half-life is 4.8 h. In conclusion, G-CSF is endowed by the polymeric micelles with prolonged half-life and increased efficacy without any chemical modification.

Surgical wound healing using hemostatic gauze scaffold loaded with nanoparticles containing sustained-release granulocyte colony-stimulating factor

BackgroundThe therapeutic strategies for malignant melanoma are still cancer chemotherapy, radiotherapy, and tumor resection. However, these therapeutic strategies often lead to a reduced neutrophilic granulocyte count or loss of more blood after surgical tumor resection. In this study, we developed a formulation of hemostatic gauze impregnated with sustained-release granulocyte colony-stimulating factor (G-CSF) with increasing of the neutrophilic granulocyte count in the blood following chemotherapy and decreasing blood loss after surgical tumor resection.MethodsWe designed a formulation with both hemostatic properties and increased neutrophil content to be used in cancer chemotherapy, radiotherapy, and tumor resection, comprising a hemostatic gauze as a scaffold and (G-CSF)-loaded dextran nanoparticles coated with polylactic-co- glycolic acid (PLGA) solution fabricated by direct spray-painting onto the scaffold and then vacuum-dried at room temperature. The performance of this system was evaluated in vitro and in vivo.ResultsNearly zero-order release of G-CSF was recorded for 12–14 days, and the cumulative release of G-CSF retained over 90% of its bioactivity in a NFS-60 cell line proliferation assay when the scaffold was incubated in phosphate-buffered saline (pH 7.4) at 37°C. The in vivo hemostatic efficacy of this formulation was greater than that of native G-CSF, the scaffold directly spray-painted with G-CSF solution or PLGA organic solution as a coating, or when a blank scaffold was covered with the coating.ConclusionOur results suggest that this formulation has both hemostatic properties and increased neutrophil activity.

Does granulocyte colony-stimulating factor ameliorate the proinflammatory response in human meningococcal septic shock?

To test the hypothesis that granulocyte colony-stimulating factor acts cooperatively with interleukin-10 in down-regulating monocyte function in severe meningococcal septic shock. 1) We quantified the plasma levels of granulocyte colony-stimulating factor, interleukin-10, Neisseria meningitidis lipopolysaccharide and the number of N. meningitidis DNA copies in 28 patients with systemic meningococcal disease. 2) We studied the inhibitory effect of recombinant human granulocyte colony-stimulating factor on normal human monocytes stimulated with purified meningococcal lipopolysaccaride. 3) We monitored the inhibitory effects of endogenously produced granulocyte colony-stimulating factor and interleukin-10 in meningococcal shock plasmas on monocytes. Comparative, experimental study. University Hospital and laboratory. Twenty-eight patients with systemic meningococcal disease, 13 with persistent shock, 7 died, and 15 without shock. The median levels of granulocyte colony-stimulating factor in shock and nonshock patients were 1.7 x 10(6) and 8.1 x 10(2) pg/mL; interleukin-10, 2.1 x 10(4) and 4 x 10(1) pg/mL; number of N. meningitidis DNA copies, 2.9 x 10(7) and <10(3)/mL; and lipopolysaccharide, 105 and <0.04 endotoxin units/mL, respectively. The plasma levels of granulocyte colony-stimulating factor were reduced by 50% within 4 to 6 hrs after initiation of antibiotic treatment. In model experiments with lipopolysaccharide-stimulated human monocytes, recombinant human granulocyte colony-stimulating factor and interleukin-10 reduced the release of tumor necrosis factor-alpha by mean 30% and 92%, respectively. When plasmas from three shock patients were depleted of native granulocyte colony-stimulating factor or interleukin-10 by immunoprecipitation, no increase in tumor necrosis factor-alpha release occurred after removal of granulocyte colony-stimulating factor, whereas removal of interleukin-10 increased the tumor necrosis factor-alpha release eight-fold. Although granulocyte colony-stimulating factor in plasma increases by five orders of magnitude in patients with meningococcal shock, the anti-inflammatory effect on patients' monocytes is uncertain.

Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state.

Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37 degrees C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1-2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.

Effect of human G-CSF on clonogenic cells in acute myeloblastic leukemia

The effects of purified human native granulocyte colony-stimulating factor (G-CSF) on the growth of clonogenic leukemic blast cells from 10 Japanese patients with acute myeloblastic leukemia (AML) were studied, using an in vitro leukemic blast colony assay. The clonogenic leukemic blast cells from six patients with AML were stimulated to form colonies in viscous medium in vitro by the addition of 100 ng/ml G-CSF. The possibility that G-CSF may be a leukemic blast growth factor warrants further attention.

Effects of purified human native granulocyte colony-stimulating factor (G-CSF) on proliferation of blast progenitors in acute myeloblastic leukemia.

Blast progenitors in acute myeloblastic leukemia (AML) grow in methylcellulose and suspension cultures. Blast colony formation in methylcellulose culture reflects the terminal divisions of blast progenitors, while secondary colony formation, by replating in methylcellulose and recovering clonogenic cells in suspension culture, reflects the self-renewal of blast progenitors. To analyze the regulatory mechanisms of the proliferation of leukemic blast progenitors, the effects of highly purified native granulocyte colony-stimulating factor (G-CSF) obtained from human squamous cell carcinoma line (CHU-2) on blast progenitors in AML patients were studied in methylcellulose and suspension cultures. Purified G-CSF stimulated the growth of blast progenitors in both culture systems, although sensitivity to G-CSF varied from patient to patient. No obvious maturation of leukemic blasts was noted in suspension culture in the presence of G-CSF. The data suggest that a normal hemopoietic regulator may play a role in the growth of blast progenitors in AML patients.

Characterization of recombinant human granulocyte-colony-stimulating factor produced in mouse cells.

Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus DNA and human granulocyte-colony-stimulating factor (G-CSF) cDNA placed under the control of the SV40 early promoter. The transformed cells secreted constitutively a high level of human G-CSF, 10-20 micrograms/ml in a low-serum medium. The secreted G-CSF has been purified to homogeneity by a two-step procedure including gel filtration and hydrophobic column chromatography. The purified recombinant G-CSF runs as a single band with an apparent Mr of 19,000 on a polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value corresponds to that of the native human G-CSF purified from the medium conditioned by human carcinoma CHU-2 cells. The recombinant human G-CSF was as active as native G-CSF in vitro in supporting proliferation of mouse NFS-60 cells and stimulating colony formation from human as well as mouse bone marrow cells. When the recombinant human G-CSF was subcutaneously administrated into mice, a remarkable stimulation of granulopoiesis and splenomegaly was observed.

Induction of neutrophilic granulocytosis in mice by administration of purified human native granulocyte colony-stimulating factor (G-CSF)

Mice were subcutaneously (sc) injected once a day for up to 15 days with a purified human native G-CSF sample at a dose of 2.5 μg/injection or with control samples with or without added endotoxin. In the G-CSF-treated mice, blood neutrophil counts began to rise as early as 2 hours after the first injection, reached a level 8 times above the preinjection level after 15 days of injections with marked elevation of all progenitor cell levels in spleen, and returned to normal within 48 hours after cessation of the injections. Such neutrophilia was observed even when endotoxin-resistant C3H HeJ mice were used, but not in control mice. It is possible that repeated G-CSF injections after administration of cyclophosphamide (CY) in mice could accelerate recovery of granulopoiesis with a rather transient rise in blood neutrophil counts.