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. 2014 Mar 17;9(3):e91990.
doi: 10.1371/journal.pone.0091990. eCollection 2014.

Hematopoietic Properties of Granulocyte Colony-Stimulating Factor/Immunoglobulin (G-CSF/IgG-Fc) Fusion Proteins in Normal and Neutropenic Rodents

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Free PMC article

Hematopoietic Properties of Granulocyte Colony-Stimulating Factor/Immunoglobulin (G-CSF/IgG-Fc) Fusion Proteins in Normal and Neutropenic Rodents

George N Cox et al. PLoS One. .
Free PMC article

Abstract

Previously we showed that granulocyte colony-stimulating factor (G-CSF) in vitro bioactivity is preserved when the protein is joined via a flexible 7 amino acid linker to an immunoglobulin-1 (IgG1)-Fc domain and that the G-CSF/IgG1-Fc fusion protein possessed a longer circulating half-life and improved hematopoietic properties compared to G-CSF in normal rats. We have extended this analysis by comparing the relative hematopoietic potencies of G-CSF/IgG1-Fc to G-CSF in normal mice and to G-CSF and polyethylene glycol (PEG) -modified G-CSF in neutropenic rats. Mice were treated for 5 days using different doses and dosing regimens of G-CSF/IgG1-Fc or G-CSF and circulating neutrophil levels in the animals measured on Day 6. G-CSF/IgG1-Fc stimulated greater increases in blood neutrophils than comparable doses of G-CSF when administered using daily, every other day or every third day dosing regimens. In rats made neutropenic with cyclophosphamide, G-CSF/IgG1-Fc accelerated recovery of blood neutrophils to normal levels (from Day 9 to Day 5) when administered as 5 daily injections or as a single injection on Day 1. By contrast, G-CSF accelerated neutrophil recovery when administered as 5 daily injections, but not when administered as a single injection. G-CSF/IgG1-Fc was as effective as PEG-G-CSF at accelerating neutrophil recovery following a single injection in neutropenic rats. G-CSF/IgG1-Fc and G-CSF/IgG4-Fc fusion proteins in which the 7 amino acid linker was deleted also were effective at accelerating neutrophil recovery following a single injection in neutropenic rats. These studies confirm the enhanced in vivo hematopoietic properties of G-CSF/IgG-Fc fusion proteins.

Conflict of interest statement

Competing Interests: GNC, DJS, SJC, SJB and DHD are employees or former employees of Bolder BioTechnology, Inc. EAC has a financial interest in BolderPATH, Inc., which received compensation from Bolder BioTechnology, Inc. to perform the animal studies. GC and DD are inventors on patents related to G-CSF/IgG-Fc fusion proteins.

Figures

Figure 1
Figure 1. Schematic diagram of (A) G-CSF/IgG-FcL (linkered) and (B) G-CSF/IgG-FcD (direct) fusion proteins.
In the linkered constructs, the carboxy-terminus of G-CSF is joined via a seven amino acid linker (L) to the amino terminus of IgG1-Fc and IgG4-Fc domains. In the D (direct) constructs, the carboxy-terminus of G-CSF is joined directly to the amino terminus of IgG1-Fc and IgG4-Fc domains. The hinge (H), CH2 and CH3 regions of the IgG-Fc fragments are indicated. The fusion proteins are dimeric due to disulfide bonds (S-S) that form between cysteine residues located in the IgG Hinge region.
Figure 2
Figure 2. SDS-PAGE analysis of purified G-CSF/IgG-Fc direct fusion proteins.
Lanes 1-3 are reducing SDS-PAGE and lanes 4-6 are non-reducing SDS-PAGE. Lanes 1 and 4 are molecular weight markers; lanes 2 and 5 are G-CSF/IgG1-FcD; and lanes 3 and 6 are G-CSF/IgG4-FcD.
Figure 3
Figure 3. Changes in neutrophil and white blood cell counts in neutropenic rats treated daily with G-CSF/IgG1-FcL.
Rats were made neutropenic by injection of cyclophosphamide (CPA) on Day 0. Beginning on Day 1 and continuing through Day 5 different groups of rats received daily injections of G-CSF (100 μg/kg), G-CSF/IgG1-FcL (100 μg/kg) or vehicle solution. The No CPA control group did not receive CPA but did receive injections of vehicle solution on Days 1 through 5. Blood samples were obtained from the rats on the days indicated and neutrophil (Panel A) and white blood cell (Panel B) levels were measured. Data are means ± SE for 5 rats/group.
Figure 4
Figure 4. Changes in neutrophil and white blood cell counts in neutropenic rats treated once with G-CSF/IgG-Fc proteins.
Rats were made neutropenic by injection of CPA on Day 0. On Day 1 different groups of rats received injections of G-CSF (100 μg/kg), PEG-G-CSF (100 μg/kg), G-CSF/IgG1-FcL (100 μg/kg), G-CSF/IgG1-FcD (100 μg/kg), G-CSF/IgG4-FcD (100 μg/kg), or vehicle solution. The No CPA control group did not receive CPA but did receive an injection of vehicle solution on Day 1. Blood samples were obtained from the rats on the days indicated and neutrophil (Panel A) and white blood cell (Panel B) levels measured. Data are means ± SE for 5 rats/group.

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