Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1999-11-10
2002-12-17
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
C530S324000, C536S023500, C435S069500, C435S071100, C435S071200, C435S471000, C435S325000, C435S254300, C435S254110, C435S320100, C435S069700
Reexamination Certificate
active
06495128
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to deletion and substitution mutant polypeptides of human chemokine &bgr;-7 (Ck&bgr;-7), as well as nucleic acid molecules encoding such polypeptides and processes for producing such polypeptides using recombinant techniques. In one aspect, the invention also relates to uses of the full-length and mature forms of Ck&bgr;-7, as well as deletion and substitution mutants, in medical treatment regimens. In particular, the Ck&bgr;-7 polypeptides described herein may be employed to treat a variety of conditions, including rheumatoid arthritis, inflammation, respiratory diseases, allergy, IgE-mediated allergic reactions, kidney diseases and may be employed for transplantation therapy. &bgr;-7 is also known as MIP-4, PARC, AMAC1 and DCCK1.
2. Related Art
Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and fimctionally related cytokines. These molecules are 8-14 kd in size. In general chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two major subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the “C—X—C” subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the —C—C—C subfamily. Thus far, at least eight different members of this family have been identified in humans. More recently, two additional chemokine families, the C and CX
3
C families, have been described.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, eosinophils, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence oftarget immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein I (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis. An example of a hematopoietic lineage regulator is MIP-1. MIP-1 was originally identified as an endotoxin-induced proinflammatory cytokine produced from macrophages. Subsequent studies have shown that MIP-1 is composed of two different, but related, proteins MIP-1&agr; and MIP-1&bgr;. Both MIP-1&agr; and MIP1&bgr; are chemo-attractants for macrophages, monocytes and T lymphocytes. Interestingly, biochemical purification and subsequent sequence analysis of a multipotent stem cell inhibitor (SCI) revealed that SCI is identical to MIP-1&bgr;. Furthermore, it has been shown that MIP-1&bgr; can counteract the ability of MIP-1&agr; to suppress hematopoietic stem cell proliferation. This finding leads to the hypothesis that the primary physiological role of MIP-1 is to regulate hematopoiesis in bone marrow, and that the proposed inflammatory function is secondary. The mode of action of MIP-1&agr; as a stem cell inhibitor relates to its ability to block the cell cycle at the G
2
S interphase. Furthermore, the inhibitory effect of MIP-1&agr; seems to be restricted to immature progenitor cells and it is actually stimulatory to late progenitors in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF).
Murine MIP-1 is a major secreted protein from lipopolysaccharide stimulated RAW 264.7, a murine macrophage tumor cell line. It has been purified and found to consist of two related proteins, MIP-1&agr; and MIP-1&bgr;.
Several groups have cloned what are likely to be the human homologs of MIP-1&agr; and MIP-1&bgr;. In all cases, cDNAs were isolated from libraries prepared against activated T-cell RNA.
MIP-1 proteins can be detected in early wound inflammation cells and have been shown to induce production of IL-1 and IL-6 from wound fibroblast cells. In addition, purified native MIP-1 (comprising MIP-1, MIP-1&agr; and MIP-1&bgr; polypeptides) causes acute inflammation when injected either subcutaneously into the footpads of mice or intracistemally into the cerebrospinal fluid of rabbits (Wolpe and Cerami,
FASEB J
. 3:2565-73 (1989)). In addition to these proinflammatory properties of MIP-1, which can be direct or indirect, MIP-1 has been recovered during the early inflammatory phases of wound healing in an experimental mouse model employing sterile wound chambers (Fahey, et al.
Cytokine
, 2:92 (1990)). For example, International Patent Application Serial No. PCT/US92/05198 filed by Chiron Corporation, discloses a DNA molecule which is active as a template for producing mammalian macrophage inflammatory proteins (MIPs) in yeast.
The murine MIP-1&agr; and MIP-1&bgr; are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil finction and cause local inflammation and fever. MIP-1&agr; has been expressed in yeast cells and purified to homogeneity. Structural analysis confirmed that MIP-1&agr; has a very similar secondary and tertiary structure to platelet factor 4 (PF-4) and interleukin 8 (IL-8) with which it shares limited sequence homology. It has also been demonstrated that MIP-1&agr; is active in vivo to protect mouse stem cells from subsequent in vitro killing by tritiated thymidine. MIP-1&agr; was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage colony-forming cells in response to granulocyte macrophage colony-stimulating factor. (Clemens, J. M. et al.,
Cytokine
4:76-82 (1992)).
There are four forms of monocyte chemotactic protein, namely, MCP-1, MCP-2, MCP-3 and MCP-4. All of these proteins have been structurally and fimctionally characterized and have also been cloned and expressed. MCP-1 and MCP-2 have the ability to attract leukocytes (monocytes, and leukocytes), while MCP-3 also attracts eosinophils and T lymphocytes (Dahinderi, E., et al.,
J. Exp. Med
. 179:751-756(1994)). MCP-4 attracts eosinophils and monocytes (Garcia-Zapeda, E. A., et al.,
J. Immunol
. 157:5613 (1996); Uguccioni, M., et al.,
J. Exp. Med
. 183:2379 (1996); Forssmann, U., et al.,
J. Exp. Med
. 185:2171 (1997)).
Human MCP-1 is a basic peptide of 76 amino acids with a predicted molecular mass of 8,700 daltons. MCP-1 is inducibly expressed mainly in monocytes, endothelial cells and fibroblasts. Leonard, E. J. and Yoshimura, T.,
Immunol. Today
11:97-101 (1990). The factors which induce this expression is IL-1, TNF or lipopolysaccharide treatment.
Other properties of MCP-1 include the ability to strongly activate mature human basophils in a pertussis toxin-sensitive manner. MCP-1 is a cytokine capable of directly inducing histamine release by basophils, (Bischoff, S. C., et al.,
J. Exp. Med
. 175:1271-1275 (1992)). Furthermore, MCP-1 promotes the formation of leukotriene C4 by basophils pretreated with Interleukin 3, Interleukin 5, or granulocyte/macrophage colony-stimulating factor. MCP-1 induced basophil mediator release may play an important role in allergic inflammation and other pathologies expressing MCP-1.
Clones havi
Graham Gerard John
Nibbs Robert John Benjamin
Patel Vikram P.
Salcedo Theodora W.
Human Genome Sciences Inc.
Mertz Prema
Sterne Kessler Goldstein & Fox P.L.L.C.
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