Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-01-08
2004-10-19
Allen, Marianne P. (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000
Reexamination Certificate
active
06806254
ABSTRACT:
2. BACKGROUND
2.1 Technical Field
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods. In particular, the invention relates to novel alpha-2-macroglobulin-like polypeptides.
2.2 Background Art
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences. Proteins are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity. It is to these polypeptides and the polynucleotides encoding them that the present invention is directed. In particular, this invention is directed to novel alpha-2-macroglobulin-like polypeptides and polynucleotides.
Alpha-2-macroglobulin is a highly conserved proteinase inhibitor present in plasma at relatively high concentrations (2-4 mg/ml). It is unique in its ability to inhibit all the major classes of proteinases (Bhattacharjee et al (2000) J. Biol. Chem. 275, 26806-26811). Alpha-2-macroglobulin is a tetramer of four identical 180 kDa subunits that forms a hollow cylinder-like structure. It presents multiple target peptide bonds to attacking proteinases in its central “bait” domain. Binding of the proteinase and subsequent cleavage of the bait domain leads to a conformational change trapping the proteinase in the central cavity. The “activated” alpha-2-macroglobulin is now recognizable by its receptor. The receptor bound activated alpha-2-macroglobulin is then endocytosed, thus removing the potentially harmful proteinases from the circulation. Alpha-2-macroglobulin is the major proteinase inhibitor acting on foreign proteinases like snake venoms.
However, there are many other proteinase inhibitors in the circulation and it is proposed that alpha-2-macroglobulin may have other functions including binding to and regulation of cytokine and growth factor activity, promotion of tumoricidal capabilities of macrophages, and enhancement of antigen presentation. In accordance with above findings, numerous cytokines, growth factors and other plasma proteins have been shown to bind to alpha-2-macroglobulin including transforming growth factor-beta, platelet-derived growth factor, nerve growth factor, tumor necrosis factor-alpha, basic fibroblast growth factor, interleukin-6, beta-amyloid peptide, apolipoprotein E, myelin basic protein, leptin, beta-microglobulin and vascular endothelial growth factor (VEGF). Most of these proteins do not bind to the bait domain. Also, the native and activated alpha-2-macroglobulins show different binding specificities and kinetics. Binding of these proteins to alpha-2-macroglobulin could sequester them from proteinases and help maintain effective concentrations.
Epidemiological and genetic studies have indicated that alpha-2-macroglobulin deficiency and/or polymorphism may be associated with Alzheimer's disease and cerebral amyloid angiopathy, multiple sclerosis, and congenital antithrombin deficiency. Alpha-2-macroglobulin and VEGF are also implicated in rheumatoid arthritis and growth of various tumors, coronary or limb ischemia and retinopathies. Binding of alpha-2-macroglobulin to beta-microglobulin could be involved in regulation of immune response to tumors and viral infections. It is conceivable that alpha-2-macroglobulin exerts its effects by binding to various biologically important molecules and regulating their activities.
Thus, the alpha-2-macroglobulin-like polypeptides and polynucleotides of the invention may be used in the treatment of Alzheimer's disease and cerebral amyloid angiopathy, multiple sclerosis, congenital antithrombin deficiency, rheumatoid arthritis and growth of various tumors, coronary or limb ischemia, retinopathies, and regulation of immune response to tumors and viral infections.
3. SUMMARY OF THE INVENTION
This invention is based on the discovery of novel alpha-2-macroglobulin-like polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies. Specifically, the polynucleotides of the present invention are based on an alpha-2-macroglobulin-like polynucleotide isolated from a cDNA library prepared from fetal brain (Clontech) (SEQ ID NO: 1) and from uterus (Clontech) (SEQ ID NO: 26).
The compositions of the present invention additionally include vectors such as expression vectors containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The compositions of the invention provide isolated polynucleotides that include, but are not limited to, a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1-3, 5, 26-28, or 30; or a fragment of SEQ ID NO: 1-3, 5, 26-28, or 30; a polynucleotide comprising the full length protein coding sequence of SEQ ID NO: 1-3, 5, 26-28, or 30 (for example, SEQ ID NO: 4 or 29); and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of any of SEQ ID NO: 1-3, 5, 26-28, or 30. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any of the nucleotide sequences set forth in SEQ ID NO: 1-3, 5, 26-28, or 30; (b) a nucleotide sequence encoding any of SEQ ID NO: 4, 6-22, 25, 29, or 31-40; a polynucleotide which is an allelic variant of any polynucleotides recited above having at least 70% polynucleotide sequence identity to the polynucleotides; a polynucleotide which encodes a species homolog (e.g. orthologs) of any of the peptides recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide comprising SEQ ID NO: 4 or 29.
A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.
This invention further provides cloning or expression vectors comprising at least a fragment of the polynucleotides set forth above and host cells or organisms transformed with these expression vectors. Useful vectors include plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
The compositions of the present invention include polypeptides com
Arterburn Matthew C.
Boyle Bryan J.
Deng Cenhua
Drmanac Radoje T.
Godbole Shubhada D.
Allen Marianne P.
Nuvelo, Inc.
Polizotto Renée S.
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