Highly purified cytokine activating factor and methods of use

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai

Reexamination Certificate

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C424S581000, C530S324000, C530S412000, C530S853000

Reexamination Certificate

active

06420337

ABSTRACT:

BACKGROUND OF THE INVENTION
Biological factors, such as coenzymes, cofactors, vitamins and others, play important roles in biological conversion processes. They are usually produced in small amounts in a whole cell (1×10
−5
-
1
×
10
−6
). Isolation and purification of such biological factors from normal cells, however, is a difficult project.
Hyperimmunized eggs have been developed and have been shown to over-produce antibodies and certain biological factors. Because hyperimmunization is performed by injection of polyvalent bacterial antigens into the target animal, the amount of biological factors found in eggs from these hyperimmunized animals can not be increased more than one order of magnitude. Therefore, the small amount of biological factors in hyperimmunized egg makes purification of biological factors difficult, and, as such, there is a need for an efficient process for isolating, purifying or otherwise producing such biological factors.
The normal immune system is under a balance in which proinflammatory and anti-inflammatory cells and molecules are carefully regulated to promote normal host immune defense without the destruction of host's tissues. Once this careful regulatory balance is disturbed, nonspecific stimulation and activation can lead to increased amounts of potent destructive immunological and inflammatory molecules being produced and released. Thus, excess production of proinflammatory cytokines or production of cytokines in the wrong biological context, are associated with mortality and pathology in a wide range of diseases, such as malaria, sepsis, rheumatoid arthritis, inflammatory bowel disease, cancer and AIDS, among others.
Cytokines are pluripotent polypeptides that act in autocrine/paracrine fashions by binding to specific cellular receptors. Their secretion is important in determining the duration and intensity of an immune response. For example, in mice, distinct subsets of CD4+ T helper (Th) clones secrete what have classically been described as Th1 and Th2 cytokines. Th1 cells produce interleukin-2 (IL-2) and interferon-&ggr; (IFN-&ggr;) and facilitate the cellular immune response. Th2 cells produce IL-4, IL-5, IL-6 and IL-10 and support the activation of immunoglobulin secreting cells. During the process of inflammation, cytokines such as IL-1&bgr;, IL-6 and tumor necrosis factor-&agr; (TNF&agr;) are released at the site of inflammation. These cytokines have pleiotropic effects and mediate a wide range of symptoms associated with inflammation.
A key cytokine, TNF-&agr;, also known as cachectin, is a 17 kiloDalton protein composed of 157 amino acids and produced mainly by monocytes and activated macrophages. TNF-&agr; has been shown to possess tumoricidal activity as well as a variety of physiological effects with most major organ systems. In the central nervous system, TNF-&agr; is involved in fever, anorexia, and alterations in pituitary hormone release. In the cardiovascular system, TNF-&agr; plays a role in shock, acute respiratory distress and capillary leakage syndrome (procoagulation). TNF-&agr; is instrumental in the process of acute tubular necrosis and nephritis in the kidney and ischemia, colitis, and hepatic necrosis in the gastrointestinal system. It is also a key cytokine involved in the process of inflammation.
Various genera of the class Aves, such as chickens (gallus domesticus), turkeys, and ducks, produce antibodies in blood and eggs against immunogens that cause avian diseases, as well as against other immunogens. For example, LeBacq-Verheyden et al. (
Immunology
27:683 (974)) and Leslie, G. A., et al. (
J. Med.
130:1337 (1969)), have quantitatively analyzed immunoglobulins of the chicken. Polson et al. (
Immunological Communications
9:495-514 (1980)) immunized hens against several proteins and natural mixtures of proteins, and detected IgY antibodies in the yolks of the eggs. Fertel et al. (
Biochemical and Biophysical Research Communications
102:1028:1033 (1981)) immunized hens against prostaglandins and detected antibodies in the egg yolk. Jensenius et al. (
Journal of Immunological Methods
46:63-68 (1981)) provide a method of isolating egg yolk IgG for use in immunodiagnostics. Polson et al. (Immunological Communications 9:475-493 (1980)) describe antibodies isolated from the yolk of hens that were immunized with a variety of plant viruses.
U.S. Pat. No. 4,357,272 discloses the isolation of antibodies from the yolks of eggs derived from hyperimmunized hens. The antibody response was elicited by repetitive injections of immunogens derived from plant viruses, human IgG, tetanus antitoxin, snake antivenins, and Serameba.
U.S. Pat. No. 4,550,019 discloses the isolation from egg yolks of antibodies raised in the hen by hyperimmunization with immunogens having a molecular or particle weight of at least 30,000. The immunogens used to hyperimmunize the chickens were selected from among plant viruses, human immunoglobulins, tetanus toxin, and snake venoms.
U.S. Pat. No. 4,748,018 discloses a method of passive immunization of a mammal that comprises parenterally administering purified antibody obtained from the eggs of an avian that has been immunized against the corresponding antigen, and wherein the mammal has acquired immunity to the eggs.
U.S. Pat. No. 5,772,999, assigned to DCV-Biologics, discloses a method of preventing, countering or reducing chronic gastrointestinal disorders or Non-Steroidal Anti-Inflammatory Drug-induced (NSAID-induced) gastrointestinal damage in a subject by administering hyperimmunized egg and/or milk or fractions thereof to the subject.
SUMMARY OF THE INVENTION
The present invention is based on the present inventors' discovery that there is specific immunoregulatory activity in egg, and particularly in egg obtained from hyperimmunized animals, which when administered to a subject animal, and particularly, to mammals, activates cytokine production in that subject animal.
More specifically, the present invention is directed to a highly purified Cytokine Activating Factor which can be obtained from the eggs of an avian. The Cytokine Activating Factor can be highly purified from fractions isolated from both egg yolk and egg white. The Cytokine Activating Factor can also be produced recombinantly or by chemical synthesis.
One embodiment of the present invention relates to an isolated Cytokine Activating Factor (CAF) protein. Such protein comprises an amino acid sequence selected from the group of: (a) an amino acid sequence selected from the group of SEQ ID NO:1 and SEQ ID NO:6; and, (b) an amino acid sequence comprising at least 9 consecutive amino acid residues of either of the amino acid sequences of (a). The isolated CAF protein of the present invention upregulates expression of tumor necrosis factor &agr; (TNF&agr;), interleukin-1&bgr; (IL-1&bgr;) and/or interleukin-6 (IL-6), and/or downregulates expression of transforming growth factor &bgr; (TGF&bgr;). In a preferred embodiment, the isolated protein of the present invention comprises an amino acid sequence having at least about 15 consecutive amino acid residues of either of the amino acid sequences of (a), and more preferably, at least about 20 consecutive amino acid residues, and even more preferably, at least about 25 consecutive amino acid residues of either of the amino acid sequences of (a). In another embodiment, the isolated protein of the present invention comprises an amino acid sequence having at least about 50 consecutive amino acid residues of SEQ ID NO:6.
In one embodiment of the present invention, an isolated protein of the present invention comprises an amino acid sequence that is at least about 65% identical to an amino acid sequence of (a) over at least 15 amino acids of the amino acid sequence of (a). Preferably, the protein comprises an amino acid sequence that is at least about 75% identical to an amino acid sequence of (a) over at least 15 amino acids of the amino acid sequence of (a). In another embodiment, the protein comprises an amino acid sequence that is at least about 60% id

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