Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-06-26
2001-03-27
Romeo, David (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000
Reexamination Certificate
active
06207642
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to extracellular signal molecules, and more particularly, relates to a member of the tumor necrosis factor (TNF) family of molecules designated as TREPA, reagents and methods for its detection as well as its use in therapeutics.
The TNF (tumor necrosis factor) family is an expanding set of extracellular signaling molecules (ligands) with biological activities that are intimately associated with a variety of disease conditions. The prototypic member of this family, TNF, is well known as a mediator of septic shock, inflammation, and graft versus host disease. See, for example, A. Cerami,
Immunol Today,
9:28-31 (1988); M. Revel,
Ciba Found Symp,
129:223-233 (1987); J. Cohen,
J. Bone Marrow Transplant
3(3):193-197 (1988). Also, because of its beneficial effects on vasculature of solid tumors, isolated perfusion of TNF is being evaluated as a therapeutic agent for cancer patients. M. W. Boehme,
Eur. J. Clin. Invest.
26:404-410 (1996).
The important role of the TNF family in immune regulation has been demonstrated by mutations both in mice and humans. For instance, mice with a loss of function mutation in the TNF family member known as Fas ligand present a variety of disorders including lymphadenopathy and autoimmune disease. See, for example, R. Watanabe-Fukunaga,
Nature
356:314-317 (1992); Takahishi et al.,
Cell
76:969-966, (1994); Adiachi et al.,
PNAS,
90:1756 (1993); Fisher et al.,
Cell
81:953-946 (1995); F. Rieux-Laucat,
Science
268:1347 (1995). Another example of the immune regulation role is mutation of the TNF family member CD40 ligand in humans. Spontaneous CD40 ligand mutations in human patients result in hyper IgM syndrome, demonstrating the requirement of CD40 ligand for B-cell maturation and isotype switching. Also, targeted disruption of TNF family member LTa in mice results in failure to develop peripheral lymph nodes. P. De Togni,
Science
264:703-707 (1994).
Another property of this family of ligands which is potentially clinically useful is the ability to selectively induce apoptosis (programmed cell death) in a variety of cancer cells, but not in most normal cells. The two known members of the TNF family which induce apoptosis in the widest variety of cell lines are TRAIL (TNF related apoptosis inducing ligand) and Fas ligand. See, for example, T. Suda et al.,
Cell
75:1169-1178 (1993); Wiley et al.,
Immunity
3:673-682, (1995). This property is unique to the TNF family of ligands.
Members of the TNF family of ligands can be identified by a region of amino acid conservation, which is approximately restricted to the N-terminal 150 amino acids. This region forms a b-pleated sheet structure, which trimerizes and interacts with the cognate receptors.
Specifically, with the exception of lymphotoxin-alpha, members of the TNF family of ligands are type II membrane spanning proteins whole extracellular C-terminal domains combine to form these trimeric complexes. These ligand trimers, either presented on cell surfaces or shed to produce soluble molecules, initiate a variety of biological activities by cross-linking congnate members of the parallel family of TNF receptors (Smith, C. A. et al.,
Cell
76:959-962). These activities include T-cell co-stimulation (Goodwin, R. G. et al.,
Eur. J. Immunol,
23:2631-2641; Smith, C. A. et al.,
Cell
73:1349-1360; and Goodwin, R. G. et al.,
Cell
73:447-456.), apoptosis (Daniel, P. T. et al.,
J. Immunol.
152:5624-5632 and Wiley, S. R. et al.,
Immunity.
3:673-682), B-cell proliferation and isotype switching (Allen, R. C. et al.,
Science
259:990-993 and Alderson, M. R. et al.,
J. Exp. Med.
181:71-77), and development of peripheral lymph nodes (De Tongi, P. et al.,
Science
264:703-707).
Further, within the aforementioned N-terminal domain there are isolated regions of homology which correspond to the strands of the b-pleated sheet. Therefore, the total amino acid sequence identity between TNF family members is not high, but can be recognized by those skilled in the art.
Given the crucial roles of members of this family of molecules in immune regulation, investigation into the existence and identity of other members of the TNF family is desirable. The identification and characterization of these molecules provide means to identify and treat a variety of immune disorders.
SUMMARY OF THE INVENTION
The present invention provides a novel member of the TNF family of ligands, as well as cDNA encoding this novel member, and expression vectors encoding a soluble form of this novel member. Since this novel member of the TNF family is able to induce proliferation in human umbilical endothelium cells, it is called TREPA for TNF Related Endothelium Proliferative Agent.
A method for producing TREPA polypeptides involves expression in host cells transformed with a recombinant expression vector that contains an engineered soluble version of TREPA, as well as a cell surface expressed form of TREPA.
The present invention also provides a method of detecting target polynucleotides of TREPA in a test sample which comprises contacting a target polynucleotide specific for TREPA with at least one TREPA specific polynucleotide and fragment or complement thereof provided herein and detecting the presence of the target in the test sample. The polynucleotide comprises SEQUENCE ID NO 1 and fragments or complements thereof. Also, the TREPA polynucleotide may be attached to a solid phase prior to performing the assay.
The present invention also provides a method for amplifying 5′ end cDNA of TREPA gene in a test sample, which comprises performing reverse transcription with random primers, amplifying the cDNA obtained by using other oligonucleotide primer(s) of TREPA as sense and antisense primer(s) in a first-stage PCR to obtain amplified cDNA and detecting the presence of TREPA amplicon in the test sample. Amplification can be performed by the polymerase chain reaction. Also, the test sample can be attached to a solid phase prior to performing the method. Further, the detection step can comprise utilizing a detectable label capable of generating a measurable signal. The detectable label can be attached to a solid phase.
The present invention further provides a method of detecting TREPA in a test sample suspected of containing TREPA, which comprises contacting said test sample with at least one polynucleotide as a sense primer and with at least one polynucleotide as an anti-sense primer and amplifying same to obtain a first stage reaction product; contacting said first stage reaction product with at least one of said polynucleotides of the contacting step and a second polynucleotide, with the proviso that the second oligonucleotide is located 3′ to the first oligonucleotide utilized and is of opposite sense to said first oligonucleotide, and detecting said TREPA as an indication of the presence of disease. The amplification may be performed by the polymerase chain reaction. The test sample can be attached to a solid phase prior to performing the method. The detection step also comprises utilizing a detectable label capable of generating a measurable signal, and the detectable label can be attached to a solid phase. Test kits useful for detecting TREPA target in a test sample further are provided which comprise a container containing at least one polynucleotide selected from the group consisting of SEQUENCE ID NO 1, and fragments and complements thereof. These test kits further comprising containers containing tools useful for collecting test samples such as blood, urine, saliva, and stool. Such tools include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; cups for collecting and stabilizing urine or stool samples. Collection materials, papers, cloths, swabs, cups and the like, may optionally be treated to avoid denaturation or irreversible adsorption of the sample. They may also be treated with or contain preservatives, stabilizers or antimicrobial agents to help maintain the integrity of t
Abbott Laboratories
Becker Cheryl L.
Goller Mimi C.
Romeo David
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