Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1992-07-30
2002-03-12
Kaufman, Claire M. (Department: 1646)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C536S023100, C536S023500, C530S300000, C530S324000
Reexamination Certificate
active
06355476
ABSTRACT:
FIELD OF THE PRESENT INVENTION
Lymphokines are the proteins by which the immune cells communicate with each other. Scientists produce them in sufficient quantities for therapeutic use against immunologic diseases. The present invention relates particularly to previously unknown lymphokine and receptor proteins which were isolated and identified based on specific expression of the T cell genes using a technique identified by the present inventor in a publication (
Proc. Natl. Acad. Sci. USA,
84, 2896-2900, May 1987, Immunology).
BACKGROUND OF THE PRESENT INVENTION
The immune system of humans and other species requires that white blood cells be made in the bone marrow, which white blood cells include phagocytes, lymphocytes and B cells. As presently understood, the phagocytes include macrophage cells which scavenge unwanted materials such as virus protein from the system. The lymphocytes include helper T cells and killer T cells and B cells as well as other cells, including those categorized as suppressor T cells.
The B cells produce the antibodies. The killer T cells physically pierce the cell and the helper T cells facilitate the whole process. In any event, the immune process is facilitated by lymphokines. Interleukin 1, secreted from macrophages activate the helper T cells and raise the body temperature causing fever which enhances the activity of the immune cells. The activated helper T cells produce Interleukin 2 and Interleukin stimulates the helper and killer T cells to grow and divide. The helper T cells also produce another lymphokine, B cell growth factor (BCGF), which causes B cells to multiply. As the number of B cells increases, the helper T cells produce another lymphokine known as the B cell differentiating factor (BCDF), which instructs some of the B cells to stop replicating and start producing antibodies. T cells also produce a lymphokine, gamma interferon (IF), which has multiple effects like Interleukin 2. Interferon helps activate killer T cells, enabling them to attack the invading organisms. Like BCGF, interferon increases the ability of the B cells to produce antibodies. Interferon also affects the macrophages to keep them at the site of the infection and help the macrophages to digest the cells they have engulfed. Gathering momentum with each kind of lymphokine signal between the macrophages and the T cells, the lymphokines amplify the immune system response and the virus protein or other foreign matter on the infected cells is overwhelmed. There are many other lymphokines, maybe a hundred or more, which participate in the immune process. Many lymphokines are known and many are not.
Lymphokines are sometimes called intercellular peptide signals. Among scientists there is widespread use of cloned cell lines as lymphokine producers and the isolation of lymphokine mRNA has become a common technique.
The protocol reported in the aforesaid publication can be used by scientists to detect virtually all of the lymphokines because the method is designed to detect virtually all the mRNA expressed differentially and the mRNA sequences of the immune cells are expressed differentially as they relate to the T cells and the killer T cells even though the level of expression is low and the quantity of the secreted lymphokine protein is low. The present inventor believes that the analysis described in the above identified publication can reveal biologically important molecules such as lymphokines because there are many indications that biologically important or active molecules are coded by the most scarce messages. An example is a transforming growth factor (TGF) which is present as only one of a million clones.
There are many known lymphokine proteins and they include the interferons, interleukin-1,2,3,4,5,6,7, colony-stimulating factors, lymphotoxin, tumor necrosis factor and erythropoietin, as well as others.
Most T cell factors have been classically identified by recognizing biologic activities in assays, purifying the protein information. An alternative approach is to isolate putative T cell genes based upon specific expression and then demonstrate the function of the unknown molecule. Using the aforesaid modified differential screening procedure, the present inventor has recently cloned a series of T cell subset-specific cDNAs from cloned helper T (HTL) L2 and cloned cytolytic T lymphocyte (CTL) L3.
SUMMARY OF THE PRESENT INVENTION
Apparent full length cDNAs corresponding to fourteen species of the 16 initial isolates were sequenced and were found to constitute five different species.
Three of the five were identical to previously reported cDNA sequences of proenkephalin, T cell replacing factor and HF gene (a serine esterase). The other two, represented as L2G25B and 4-1BB, were novel sequences of unknown function. The open reading frames of 4-1BB and L2G25B code for 245 and 92 amino acids, respectively. The predicted proteins of 4-1BB and L2G25B include 22 and 23 amino acid-long putative signal sequences, respectively. The protein backbones of mature proteins encoded by 4-1BB and L2G25B are composed of 234 amino acids with molecular weight of 25000 and 69 amino acids with molecular weight of 7880, respectively. 4-1BB contains two potential N-glycosylation sites while L2G25B has none. 4-1BB contains 23 cysteine residues in the putative mature protein.
The cDNA L2G25B encodes for the lymphokine, macrophage inflammatory protein-1&agr; or MIP-1&agr;. MIP-1&agr; has been described in a paper entitled, “Enhancing and Suppressing Effects of Recombinant Murine Macrophage Inflammatory Proteins on Colony Formation In Vitro by Bone Marrow Myeloid Progenitor Cells”, Hal E. Broxmeyer, Barbara Sherry, Li Lu, Scott Cooper, Kwi-Ok Oh, Patricia Tekamp-Olson, Byoung S. Kwon, and Anthony Cerami,
Blood,
76 , 111-1116, 1990 and is incorporated herein by reference. This was the first time the suppressing activity of MIP-1&agr; was characterized. Natural MIP-1 was found and characterized as an inflammatory protein in 1988 (Wolpe, S. D., G. Davatelis, B. Sherry, B. Beutler, D. G. Hesse, H. T. Hguyen, L. L. Moldawer, C. F. Nathan, S. F. Lowry, and A. Cerami. 1988. Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties.
J. Exp. Med.,
167, 570, incorporated herein by reference). The sequence for MIP-1&agr; was also published in June 1988 (Davatelis, G., P. Tekamp-Olson, S. D. Wolpe, K. Hermsen, C. Luedke Gallegos, D. Cort, J. Merryweather, and A. Cerami. 1988. Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties.
J. Exp. Med.,
167, 1939, incorporated herein by reference). The myelopoietic enhancing effects on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells was published in 1989 (Broxmeyer, H. E., B. Sherry, L. Lu, S. Cooper, C. Carow, S. D. Wolpe, and A. Cerami. 1989. Myelopoietic enhancing effects of murine macrophage inflammatory proteins and human bone marrow granulocyte/macrophage progenitor cells.
J. Exp. Med.,
170, 1583, incorporated herein by reference). In 1990, Graham and Pragnell identified and characterized an inhibitor of hematopoietic stem cell proliferation which turned out to be MIP-1&agr;. (Graham, G. J., E. G. Wright, R. Hewick, S. D. Wolpe, N. M. Wilke, D. Donaldson, S. Lorimore, and I. B. Pragnell. 1990. Identification and characterization of an inhibitor of haematopoietic stem cell proliferation.
Nature,
344, 442, incorporated herein by reference). Graham and Pragnell have also published work on in vivo experiments of the suppressing effects of MIP-1&agr; (David J. Dunlop, Eric G. Wright, Sally Lorimore, Gerald J. Graham, Tessa Holyoake, David J. Derr, Stephen D. Wolpe, and Ian B. Pragnell. 1992. Demonstration of Stem Cell Inhibition and Myeloprotective Effects of SCI/rhMIP-1&agr; In Vivo,
Blood,
79, No. 9, pp. 2221-2225, incorporated herein by reference).
The present invention includes the discovery of the cDNA sequence encoding for the protein later ref
Broxmeyer Hal E.
Kwon Byoung Se
Advanced Research and TechnologyInc
Kaufman Claire M.
Schwegman Lundberg Woessner & Kluth P.A.
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