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
1996-11-08
2001-08-14
Mertz, Prema (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...
C435S069700, C435S006120, C435S071100, C435S071200, C435S471000, C435S325000, C435S252300, C435S254110, C536S023100, C536S023500, C536S024300, C536S023400, C530S351000
Reexamination Certificate
active
06274342
ABSTRACT:
1. BACKGROUND OF THE INVENTION
Chemokines are proteins involved in the activation of chemotaxis of leukocytes. They are believed to be important mediators of inflammation. (Baggiolini et al.,
Immunology Today
15:127, 1994).
Chemokines have been divided into three families. In chemokines of the C-X-C family, one amino acid separates the first two cysteines. Chemokines in this family are thought to be involved in the chemotaxis of neutrophils, induction of changes in cell shape, transient increase of intracellular calcium, granule exocytosis, and respiratory burst. Interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and granulocyte chemotactic protein (GCP) belong to this class. All known CXC chemokines have been mapped to human chromosome 4 and mouse chromosome 5.
In the C-C family, the first two cysteines are adjacent to one another. Members of this family are chemotactic for monocytes, but not neutrophils. Recent studies have shown that they are capable of activating basophils and eosinphils. Proteins belonging to the C-C class of chemokines include monocyte chemotactic proteins 1, 2, and 3 (MCP-1, MCP-2, and MCP-3), RANTES, and macrophage inflammatory proteins &agr; and &bgr; (MIP-1&agr; and MIP-1&bgr;). Recently, MIP-3, MIP-4, and MIP-1&ggr; have also been described (WO 95/17092). All known C-C chemokines have been mapped to human chromosome 17 and mouse chromosome 11.
An example of a third class of chemokine has also been identified. This chemokine, lymphotactin, was isolated from progenitor T lymphocytes. Lymphotactin is chemotactic to lymphocytes (Kelner et al.,
Science
266:1395, 1994).
Unlike the chemokines of the CC and CXC families in which two disulfide bonds stabilize the protein, lymphotactin has only one disulfide bond. Lymphotactin was mapped to human and mouse chromosome 1.
A variety of cell types are involved in the various inflammatory states. For example, acute infiltrates found after bacterial infection are mainly neutrophilic, while mononuclear cells predominate after infection by an intracellular pathogen. Basophils and easinophils dominate in both immediate-type allergic response and autoimmune diseases. Increased understanding of the regulation of these various cell types by chemokines will facilitate the development of more effective therapies for disorders related to inflammation.
2. SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel C-C chemokine molecules, referred to herein as “Monocyte Chemotactic Protein 5” (MCP-5) nucleic acid and polypeptide molecules. An exemplary MCP-5 molecule has been deposited with the American Type Culture Collection (ATCC)® on Sep. 19, 1996 and has been assigned ATCC® designation number 98172. The murine MCP-5 gene transcript is approximately 540 base pairs in length, including 5′ and 3′ untranslated regions and a 341 base open reading frame encoding a 104 amino acid polypetide. The mature protein (minus the 22 amino acid signal sequence) is comprised of about 82 amino acids, containing 5 cysteine residues, four of which create the characteristic motif of the CC chemokine family. MCP-5 is mainly expressed by alveolar macrophages and smooth muscle cells of the eosinophilic lung. The presence of lymphocytes was found to be critical for its expression during allergic inflammation. During non-inflammatory situations, expression of MCP-5 in the lymph nodes and thymus is constitutive and restricted to stromal clls (macrophages and dendritic cells) present in the germinal centers of the lymphoid follicles. The murine MCP-5 polypeptide is approximately 11-12 kD.
In one aspect, the invention features isolated vertebrate MCP-5 nucleic acid molecules. The disclosed molecules can be non-coding, (e.g. probe, antisense or ribozyme molecules) or can encode a functional MCP-5 polypeptide (e.g. a polypeptide which specifically modulates, e.g., by acting as either an agonist or antagonist, at least one bioactivity of the human MCP-5 polypeptide). In one embodiment, the nucleic acid molecules can hybridize to the MCP-5 gene contained in ATCC® designation number 98172 or to the complement of the MCP-5 gene contained in ATCC® designation number 98172. In another embodiment, the nucleic acids of the present invention can hybridize to a vertebrate MCP-5 gene or to the complement of a vertebrate MCP-5 gene. In a further embodiment, the claimed nucleic acid can hybridize with the nucleic acid sequence shown in
FIG. 1
(SEQ ID NOs. 1 and 3). In a preferred embodiment, the hybridization is conducted under mildly stringent or stringent conditions.
In further embodiments, the nucleic acid molecule is a MCP-5 nucleic acid that is at least 70%, preferably 80%, more preferably 85%, and even more preferably at least 90% or 95% homologous in sequence to any of the nucleic acids shown as SEQ ID Nos: 1 or 3 or to the complement of the nucleic acid shown as SEQ ID Nos: 1 or 3. In a further embodiment, the nucleic acid molecule is a MCP-5 nucleic acid that is at least 70%, preferably 80%, more preferably 85% and even more preferably at least 90% or 95% similar in sequence to the MCP-5 gene contained in ATCC® designation number 98172 or to the complement of the MCP-5 gene contained in ATCC® designation number 98172.
The invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least 6 consecutive nucleotides of any of the sequences set forth as SEQ ID Nos: 1 or 3 or complements of any of the sequences set forth as SEQ ID Nos 1 or 3 or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject nucleic acids can include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer sequence, which regulatory sequence is operably linked to the gene sequence. Such regulatory sequences in conjunction with a MCP-5 nucleic acid molecule can provide a usefuil vector for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing MCP-5 proteins by employing said expression vectors.
In another aspect, the invention features isolated MCP-5 polypeptides, preferably substantially pure preparations, e.g. of plasma purified or recombinantly produced polypeptides. In a preferred embodiment, the polypeptide is approximately 11 kD. In particularly preferred embodiments, the subject polypeptides have a MCP-5 bioactivity, for example, they are capable of inducing eosinophil; monocyte- and lymphocyte- (e.g. B-lymphocyte) mediated inflammation.
In a preferred embodiment, the MCP-5 polypeptide is encoded by a nucleic acid which hybridizes with the nucleic acid sequences represented in SEQ ID Nos. 1 or 3 or with the gene or gene fragment contained in ATCC® Designation No. 98172. In a further preferred embodiment, the MCP-5 polypeptide is comprised of the amino acid sequence set forth in SEQ ID No. 2. The subject MCP-5 proteins also include modified proteins, which are resistant to post-translational modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The MCP-5 polypeptides can comprise a full length protein or they can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or 200 amino acids in length. In preferred embodiments, the polypeptide is capable of inducing eosinophil-, monocyte- and lymphocyte- (e.g., B-lymphocyte) mediated inflammation.
Another aspect of the invention feature
Gonzalo Jose-Angel
Gutierrez-Ramos Jose-Carlos
Jia Gui-Quan
Center For Blood Research, Inc.
Laccotripe Maria C.
Lahive & Cockfield LLP
Mandragouras Amy E.
Mertz Prema
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