Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1997-09-30
2002-05-28
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S326000, C530S327000, C530S328000, C530S350000, C530S395000
Reexamination Certificate
active
06395874
ABSTRACT:
BACKGROUND OF THE INVENTION
Diseases related to tissue inflammation associated with oxygen metabolite production is a much studied area in the fields of medicine and biology. These diseases can include for example, stroke, arthritis, pulmonary adult respiratory distress syndrome, atherosclerosis and inflammations of the eye.
During inflammation, oxygen radicals released by phagocytes can be potentially injurious to surrounding tissues. Superoxide can be one of the most injurious species through its secondary conversion to hydrogen peroxide, hydroxyl radicals and peroxynitrite.
When this sequence of events occurs in severe inflammation, it can often lead to irreversible damage. Oxygen radical-induced lipid peroxidation products can degrade cellular membranes, but also can serve to perpetuate inflammation.
Specifically in acute intraocular inflammation, neutrophils can release a variety of agents that can be potentially toxic to surrounding tissues. Amongst these agents are reactive oxygen metabolites which includes for example, superoxide. Upon release, superoxide can dismutate spontaneously to hydrogen peroxide and through the catalysis of tissue iron, can produce hydroxyl radicals. These hydroxyl radicals can cause extensive damage to tissues. Cytotoxicity of superoxide can be manifested by its reaction with phagocyte mediated nitric oxide. The combination of these two radicals can be diffusion-limited and can produce peroxynitrite which can exert its reactivity either by reacting as an anion radical or by dissociated to a hydroxyl radical-like species. Reactive oxygen species derived from superoxide are known to those skilled in the art to inflict damage on cellular macromolecules, which can include lipids and proteins.
There are several known intracellular antioxidants which are capable of scavenging these oxygen radicals. However, disadvantages with these cellular antioxidants is that they often are unable to translocate to the extracellular space in the event of inflammation, to effectively neutralize these oxygen radicals. Intracellular antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase and others, are important defenses against oxygen free radicals generated during normal cellular metabolism. These enzymes can also be found in abundant quantities in the ocular tissues. The role of reactive oxygen metabolites in the destruction of ocular tissues and amplification of the inflammatory process in uveitis, for example, has been demonstrated in animals with S-antigen induced uveo-retinitis. The extent of damage that can result from inflammation appears to be the result of an interplay between the factors that protect the ocular tissues and those that sustain the inflammation. Even though intraperitoneal injections of the antioxidant enzymes can attenuate inflammation and preserve the tissue morphology in experimental uveitis, the role of antioxidants normally found in ocular tissues in the modulation of inflammation is unclear. Antioxidants that are present intracellularly may not counter the large amounts of oxidants generated in the extracellular milieu during uveitis. The enzymes may even be inactivated by free radicals produced during the initial phases of inflammation.
For the foregoing reasons, there is a need for discovering a way to inhibit oxygen metabolite production associated with tissue inflammation related to diseases of the eye. Still further it would be advantageous to have a method for diagnosing or treating of various other diseases associated with oxygen metabolite production.
SUMMARY OF THE INVENTION
The present invention presents a protein consisting essentially of purified retinal pigment epithelial protein or purified biologically active variants thereof, or a combination of purified retinal pigment epithelial protein and biologically active variants thereof. The protein can be made from a mammal such as a rabbit, mouse, rat and human. Also disclosed are methods for purifying these proteins from retinal pigment epithelium. In particular, two proteins are disclosed, the molecular masses of these proteins are about 69-, and 75-kDa, respectively.
According to another aspect of the present invention, there is provided a high affinity monoclonal antibody which immunoreacts with retinal pigment epithelial protein or immunogenic fragments thereof. The antibody can have a Fc portion selected from the group consisting of the IgM class, the IgG class and the IgA class. There is also provided a method of making a monoclonal antibody which immunoreacts with retinal pigment epithelial protein comprising the steps of first, administering to a host retinal pigment epithelial protein in an amount sufficient to induce the production of antibodies to the retinal pigment epithelial protein form the antibody producing cells. Next, the antibody producing cells can be recovered from the host. Then, cell hybrids can be formed by fusing the antibody-producing cells to cells capable of substantially unlimited reproduction, such as myeloma cells. Then, the hybrids can be cultured and monoclonal antibodies are collected as a product of the hybrids.
There is also provided a vector containing a DNA molecule encoding retinal pigment epithelial protein and a prokaryotic or eukaryotic host cell stably transformed or transfected by the vector.
There is also provided a method of making a protein according to the present invention comprising the steps of, first culturing a microorganism transformed with DNA encoding for retinal pigment epithelial protein, and then, recovering the retinal epithelial protein or its complementary strands.
The present invention also includes a purified and isolated peptide having the Sequence I.D. No. 1: Met Gly Ile Glu Leu Tyr Gly Tyr Tyr Xaa Xaa Ser Ala Leu Gly Glu Lys, Sequence I.D. No. 2: Asn Gln Asn Lys Val Gln Lys Gln Ala Asn Met His Met Gln Xaa Tyr Leu, Sequence I.D. No. 3: Ala Phe Ser Asn Asn Glu Ala Asp Ala Val Thr Leu Asp Gly Gly Leu Val Tyr Glu Ala Gly Leu Ser Pro Asn, Sequence I.D. No. 4: Phe Leu Val Leu Asn Lys Thr Val Val Gly, Sequence I.D. No. 5: Gly Tyr Leu Ala Val Ala Val Val Ile Ser Leu Gly Ser, Sequence I.D. No. 6: Ser Ala Glu Leu Asn Lys Phe Met Gly Arg, Sequence I.D. No. 7: Glu Leu Val Glu Ser Gly Leu Val Ala Phe Val Ser Xaa Glu Ser Xaa Glu Xaa Ser Ser Pro, Sequence I.D. No. 8: Gly Tyr Leu Ala Leu Pro Ala Phe Gln Ser Leu Gly Xaa Xaa Asn. The previous sequence I.D. nos. 1-8 directly correspond to the amino acid sequences from Table II, Peak Nos. 50, 42, 52, 22, 27, 29, 34 and 24. Sequence I.D. Nos. 1-8 can be either from a natural source or from a recombinant source. Also, there is provided a high affinity monoclonal antibody which immunoreacts with this peptide. The antibody can have an Fc portion of the IgM class, the IgG class of the IgA class.
According to another aspect of the present invention, there is provided a method of detecting a protein capable of inhibiting neutrophil activation in inflammatory conditions in a sample comprising the steps of contacting the sample with a nucleic acid sequence probe capable of binding to a nucleic acid sequence encoding a retinal pigment epithelial protective protein, and detecting such binding, wherein said retinal pigment epithelial protective proteins are selected from the group consisting of retinal pigment epithelial protective proteins having molecular masses of about: 69-, and 75-kDa.
According to another aspect of the present invention, there is provided a method of detecting a protein capable of inhibiting neutrophil activation in inflammatory conditions in a sample comprising the steps of: contacting the sample with an antibody capable of binding a retinal pigment epithelial protective protein or its immunogenic fragment, and detecting such binding, wherein said retinal pigment epithelial protective protein is selected from the group consisting of retinal pigment epithelial proteins having molecular masses of about: 69-, and 75-kDa.
According to yet another aspect of the present invention, there is provided a method for detecting in a sample, an antib
Doheny Eye Institute
Fulbright & Jaworski L.L.P.
Saunders David
Tong Mary Beth
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