Hematopoietic properties of granulocyte colony-stimulating factor/immunoglobulin (G-CSF/IgG-Fc) fusion proteins in normal and neutropenic rodents.

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

ADH IB expression, but not ADH III, is decreased in human lung cancer.

Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer

Schematic diagram of (A) G-CSF/IgG-FcL (linkered) and (B) G-CSF/IgG-FcD (direct) fusion proteins.

<p>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.</p

SDS-PAGE analysis of purified G-CSF/IgG-Fc direct fusion proteins.

<p>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.</p

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.

<p>Mice received subcutaneous injections every day (ED; on Days 1–5) or every other day (EOD; on Days 1, 3 and 5) with vehicle solution, or the indicated doses of G-CSF or G-CSF/IgG1-FcL. On day 6 blood samples were obtained from the mice and the number of neutrophils (PMN), white blood cells (WBC), red blood cells (RBC), platelets, and lymphocytes (Lymphs) was determined. On day 6 the animals were sacrificed, their spleens weighed and the ratio of myeloid to erythroid cells (M: E ratio) in sections of the bone marrow determined. Blood and tissue samples were obtained from untreated mice on Day 1. Data are means ± SE for 5 mice per group. ^ap≤0.05 versus untreated controls; ^bp≤0.05 versus G-CSF.</p

Changes in neutrophil and white blood cell counts in neutropenic rats treated daily with G-CSF/IgG1-FcL.

<p>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 (<b>Panel A</b>) and white blood cell (<b>Panel B</b>) levels were measured. Data are means ± SE for 5 rats/group.</p

Changes in neutrophil and white blood cell counts in neutropenic rats treated once with G-CSF/IgG-Fc proteins.

<p>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 (<b>Panel A</b>) and white blood cell (<b>Panel B</b>) levels measured. Data are means ± SE for 5 rats/group.</p

Effect of every day (days 1-5) dosing of G-CSF and G-CSF/IgG1-FcL on day 6 blood cell counts in mice.

<p>Mice received a subcutaneous injection every day for 5 days with vehicle solution, or the indicated doses of G-CSF or G-CSF/IgG1-FcL. On day 6 blood samples were obtained from the mice and the number of neutrophils (PMN), white blood cells (WBC), red blood cells (RBC), platelets, and lymphocytes (Lymphs) was determined. On day 6 the animals were sacrificed, their spleens weighed and the ratio of myeloid to erythroid cells (M: E ratio) in sections of the bone marrow determined. Blood and tissue samples were obtained from untreated mice on Day 1. Data are means ± SE for 5 mice per group. ^ap≤0.05 versus untreated controls; ^bp≤0.05 versus G-CSF.</p