Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-10-01
2004-09-28
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S358000, C530S350000
Reexamination Certificate
active
06797493
ABSTRACT:
BACKGROUND
Granulocyte colony-stimulating factor (G-CSF) is a 20 kilodalton (kDa) glycoprotein that promotes the proliferation of progenitor cells and induces their differentiation into neutrophils. In addition, G-CSF prolongs the survival of mature neutrophils and activates their functions Human G-CSF (hG-CSF) is produced by monocytes, macrophages, fibroblasts and endothelial cells (see, for example, Moore,
Annu. Rev. Immunol
, 9:159-191, 1991; Nicola,
Annu. Rev. Biochem
., 58:45-77, 1991). The biological effects of G-CSF are mediated through its interaction with the G-CSF receptor (G-CSF-Rc) expressed on the surface of bone marrow hematopoietic progenitors and cells of the myeloid lineage. Upon binding G-CSF, the receptor is activated and undergoes homodimerization, followed by phosphorylation of Janus family of tyrosine kinases. Subsequently, a series of intracellular signal transduction events take place, leading to the increase of the number of progenitor cells, their maturation into neutrophils, and further activation of effector functions in mature neutrophils (see, for example, Demetri et al.,
Blood
, 78:2791-2808, 1991). Therefore, G-CSF plays an essential role not only in the regulation and maintenance of hematopoiesis, but also in host defense against infection and inflammation.
Recombinant human G-CSF (rhG-CSF) is widely used in the treatment of patients with neutropenia as a result of receiving chemotherapy. Administration of rhG-CSF is effective in restoring functioning neutrophils to these patients, leading to a decrease of infection-related events. Use of rhG-CSF allows intensified dosing or scheduling of chemotherapeutic agents that may be of benefit to cancer patients. Besides chemotherapy-induced neutropenia, rhG-CSF has been used for the treatment of myelosuppression after bone marrow transplantation, acute leukemia, aplastic anemia, myelodysplastic syndrome, severe chronic neutropenias, and mobilization of peripheral blood progenitor cells for transplantation (see, for example, Welte et al.,
Blood
, 88:1907-1929, 1996).
The elimination half-life of the serum concentration of rhG-CSF is approximately 3 to 4 h for intravenous or subcutaneous administration. The safety profile and patient tolerance of rhG-CSF are good with medullary bone pain being the only frequent and significant side effect. The relatively low toxicity of rhG-CSF has made it feasible to develop longer-acting derivatives to decrease the inconvenience of the daily or twice-daily injection schedule. Attachment of polyethylene glycol (PEG) to various proteins, including G-CSF, has been reported to yield derivatives with higher in vivo potency due to their longer half-lives (see, for example, Zalipsky et al., in “
PEG chemistry: biotechnical and biomedical applications
”, pp. 347-370, 1992). PEG-conjugated proteins usually have considerably lower in vitro biological activity than their unmodified parent proteins (Eliason et al.,
Stem Cells
, 18:40-45, 2000). The increased in vivo potency of these modified proteins is, at least in part, due to decreased removal by the kidney in a manner proportional to their molecular weight (Yamaoda et al.,
J. Pharmaceut. Sci
., 83:601-606, 1994). We unexpectedly discover that it is possible to increase the potency of hG-CSF through prolonging its half-life as well as enhancing its biological activity is to attach the Fc region derived from human IgG at the C-terminus of hG-CSF, as described in this invention.
Immunoglobulins of IgG class are among the most abundant proteins in human blood. Their circulation half-lives can reach as long as 21 days. Fusion proteins have been reported to combine the Fc regions of IgG with the domains of another protein, such as various cytokines and soluble receptors (see, for example, Capon et al.,
Nature
, 337:525-531, 1989; Chamow et al.,
Trends Biotechnol
., 14:52-60, 1996); U.S. Pat. Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein linked through cysteine residues in the hinge region of IgG Fc, resulting in a molecule similar to an IgG molecule without the CHI domains and light chains. Due to the structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profile comparable to that of human IgG with a similar isotype. This approach has been applied to several therapeutically important cytokines, such as IL-2 and IFN-&agr;
2a
, and soluble receptors, such as TNF-Rc and IL-5-Rc (see, for example, U.S. Pat. Nos. 5,349,053 and 6,224,867). It is desirable to extend the circulating half-life of G-CSF and/or to increase its biological activity by making fusion proteins containing G-CSF linked to the Fc portion of the human IgG protein as disclosed and/or described in this invention.
Erythropoietin (EPO) derivatives, such as dimers, have been reported. Relative to the EPO monomer, a fusion protein consisting of two complete EPO domains separated by a 3- to 7-amino acid peptide linker exhibited reduced activity (Qiu et al.,
J. Biol. Chem
., 273:11173-11176, 1998). However, when the peptide linker between the two EPO domains was 17 amino acids in length, the dimeric EPO molecule exhibited considerably enhanced in vitro and in vivo activities (see, for example, Sytkowski et al.,
J. Biol. Chem
., 274:24773-24778, 1999; U.S. Pat. No. 6,187,564). The length of the peptide linker between the two hematopoietic growth factors is important, while not bound by this theory, presumably due to its effect on the flexibility of such molecular forms. We find that this approach is generally applicable to other therapeutic proteins, including G-CSF. We'll also refer this to this as a flexible peptide linker.
In most of the reported Fc fusion protein molecules, a hinge region serves as a spacer between the Fc region and the cytokine or soluble receptor at the amino-terminus, allowing these two parts of the molecule to function separately (see, for example, Ashkenazi et al.,
Current Opinion in Immunology
, 9:195-200, 1997). A human G-CSF fusion protein with an appropriate peptide linker between the hG-CSF and Fc moieties (hG-CSF-L-Fc) is more active than rhG-CSF, with in vitro activity at least 2-fold as that of rhG-CSF on a molar basis. It is discovered according to this invention that an added peptide linker present between hG-CSF and a human IgG Fc variant enhances the in vitro biological activity of the hG-CSF-L-Fc molecule in two ways: (1) keeping the Fc region away from the G-CSF-Rc binding sites on G-CSF, and (2) keeping one G-CSF from the other G-CSF domain, so both G-CSF domains can interact with G-CSF-Rc on the granulocyte precursor cells independently. For the present invention, a flexible peptide linker of about 20 or fewer amino acids in length is preferred. More preferably, the peptide linker should have at least two amino acids in length. Furthermore, it is even more preferable to use a peptide linker comprising two or more of the following amino acids: glycine, serine, alanine, and threonine.
The Fc region of human immunoglobulins plays a significant role in immune defense for the elimination of pathogens. Effector functions of IgG are mediated by the Fc region through two major mechanisms: (1) binding to the cell surface Fc receptors (Fc
&ggr;
Rs) can lead to ingestion of pathogens by phagocytosis or lysis by killer cells via the antibody-dependent cellular cytotoxicity (ADCC) pathway, or (2) binding to the C1q part of the first complement component C1 initiates the complement-dependent cytotoxicity (CDC) pathway, resulting in the lysis of pathogens. Among the four human IgG isotypes, IgG1 and IgG3 are effective in binding to Fc
&ggr;
R. The binding affinity of IgG4 to Fc
&ggr;
R is an order of magnitude lower than that of IgG1 or IgG3, while binding of IgG2 to Fc
&ggr;
R is below detection. Human IgG1 and IgG3 are also effective in binding to C1q and activating the complement cascade. Human IgG2 fixes complement poorly, and IgG4 appears quite deficient in the ability to activate the complement cascade (see, for example, Jefferis et al.,
Immunol. Rev
., 163:59-76, 1998).
Sun Bill N. C.
Sun Cecily R. Y.
Sun Lee-Hwei K.
Kemmerer Elizabeth
Sun Hsiang-ning
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