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.
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.
Table 1.
Effect of every day (days 1-5) dosing of G-CSF and G-CSF/IgG1-FcL on day 6 blood cell counts in mice.
Table 2.
Effect of every other day (days 1, 3 and 5) dosing of G-CSF and G-CSF/IgG1-FcL on day 6 blood cell counts in mice.
Table 3.
Effect of every third day (dosing on days 1 and 4) dosing of G-CSF and G-CSF/IgG1-FcL on day 6 blood cell counts in mice.
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.
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.