Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-20
2002-10-15
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C536S023500
Reexamination Certificate
active
06465212
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mammalian leukotriene receptors. More particularly, it relates to human leukotriene receptors, isolated nucleic acids and recombinant vectors encoding the receptors, to methods of making the receptors, to methods of making fragments or fusion proteins of the receptors using recombinant DNA methodology or chemical synthesis, and to methods of using the receptors in screening systems to identify inhibitors and activators of the receptors useful for the treatment of various diseases.
BACKGROUND OF THE INVENTION
Leukotrienes are products of eicosanoid metabolism and are implicated in a number of medical conditions, including inflammation, asthma, allergy, glomerulonephritis, neuroendocrine dysfunctions, AIDS, arthritis, bowel disease, psoriasis, diabetes, obesity, atherosclerosis, bacterial infection, etc. There are multiple classes of leukotrienes, e.g. class A, class B, class C, class D, class E and class F. Leukotrienes regulate the intensity and duration of immune responses and are involved in cell-to-cell communication. Leukotrienes are also involved in leukocyte migration and branchovasoconstriction. As established by radioligand binding as well as physiological assays, it appears that there are different types of receptors for leukotrienes. The molecular characteristics of leukotriene receptors were unknown until recently, when a leukotriene B4 receptor was cloned (Yokomizo et al.,
Nature
, 387:620-624 (1997)).
In view of the important role that leukotrienes play in many physiological processes and medical conditions, there is a need for materials and methods useful for the identification of agonists and antagonists of leukotriene receptors.
SUMMARY OF THE INVENTION
The present invention fills the foregoing need by providing such materials and methods. More particularly, this invention provides a novel mammalian leukotriene receptor, isolated nucleic acids or recombinant nucleic acids encoding the receptor, and recombinant vectors and host cells comprising such nucleic acids. The leukotriene receptor can be actively expressed in mammalian cells where it displays active ligand binding and positive intracellular signaling upon ligand activation. This novel receptor has high affinity for ligands such as leukotriene B4, leukotriene B5, 15(5)—OH—5z, 8z, 11z, 13e)-eicosatetraenoic acid, and lipoxin A4. This invention further provides methods for the discovery of selective agonists and antagonists of the receptor that may be useful in the treatment and management of a variety of diseases including, for example, inflammation, asthma, allergy, glomerulonephritis, neuroendocrine dysfunctions, alcoholic liver diseases, hepatitis, atropic liver regeneration, AIDS, arthritis, bowel disease, psoriasis, diabetes, obesity, atherosclerosis, and bacterial infection.
The isolated or recombinant nucleic acids of the present invention are selected from the group consisting of:
(a) A nucleic acid encoding a mammalian leukotriene receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof;
(b) A nucleic acid that hybridizes under moderately stringent conditions to the nucleic acid of (a) and encodes a polypeptide that (i) binds leukotriene and (ii) is at least 80% identical to a receptor encoded by the nucleic acid of (a); and
(c) A nucleic acid that, due to the degeneracy of the genetic code, encodes a mammalian leukotriene receptor encoded by a nucleic acid of (a) or (b).
This invention further provides methods of making a polypeptide comprising culturing a host cell comprising a nucleic acid encoding a mammalian leukotriene receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof, under conditions in which the nucleic acid is expressed. In some embodiments, the method further comprises isolation of the polypeptide from the culture.
This invention also provides a recombinant nucleic acid comprising a sequence having at least about 70% identity over a stretch of at least about 30 nucleotides to the nucleic acid sequence of SEQ ID NO: 1, useful, e.g., as a probe or PCR primer for a related gene. Another embodiment further includes a polypeptide comprising at least about 60% identity over a stretch of at least about 20 amino acids to the amino acid sequence of SEQ ID NO: 2.
This invention also provides polypeptides comprising a fragment of a polypeptide having an amino acid sequence corresponding to the sequence of at least about 8 contiguous residues of the amino acid sequence of SEQ ID NO: 2. Preferably, the polypeptides comprise at least about 12, more preferably at least about 20, and most preferably at least about 30 such residues.
Still further, this invention provides fusion proteins comprising a polypeptide defined by SEQ ID NO: 2 or a fragment therefrom covalently linked to a fusion partner.
The present invention also provides antibodies, both polyclonal and monoclonal, that specifically bind to one or more of the leukotriene receptors or to polypeptides therefrom, and anti-idiotypic antibodies, both monoclonal and polyclonal, which specifically bind to the foregoing antibodies.
This invention further provides a method for producing a mammalian leukotriene receptor comprising culturing a host cell comprising a nucleic acid encoding a mammalian leukotriene receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof, under conditions in which the nucleic acid is expressed. In one embodiment the receptor is isolated from the culture.
The present invention also provides a method for identifying a leukotriene agonist or antagonist comprising:
(a) Contacting a mammalian leukotriene receptor having an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof, in the presence of a known amount of labeled leukotriene or surrogate with a sample to be tested for the presence of a leukotriene agonist or antagonist; and
(b) Measuring the amount of labeled leukotriene specifically bound to the receptor;
whereby a leukotriene agonist or antagonist in the sample is identified by measuring substantially reduced binding of the labeled leukotriene to the leukotriene receptor, compared to what would be measured in the absence of such agonist or antagonist.
In a preferred embodiment, membranes isolated from mammalian cells comprising a nucleic acid encoding the leukotriene receptor are used as the source of the receptor.
The present invention also provides a method for identifying an agonist or antagonist of a mammalian leukotriene receptor comprising:
(a) contacting cells expressing a mammalian leukotriene receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or a conservative or allelic variant thereof, in the presence of a known amount of leukotriene or surrogate thereof with a sample to be tested for the presence of a mammalian leukotriene agonist or antagonist; and
(b) measuring at least one cellular function modulated by the binding of a ligand to said receptor present in the cells;
whereby a mammalian leukotriene receptor agonist or antagonist in the sample is identified by measuring its effect on said cellular function compared to what would be measured in the absence of such agonist or antagonist.
Examples of cellular functions modulated by the binding of a ligand to a mammalian leukotriene receptor include: intracellular second messenger pathways activated via the leukotriene receptors (e.g., cyclicAMP, calcium, inositol phosphate and MAP kinase), changes in cell growth rate, secretion of hormones, receptor-stimulated Ca
2++
mobilization, mitogenic effects etc.,
This invention still further provides a method for treating leukotriene-mediated medical conditions comprising administering to a mammal afflicted with a medical condition caused or mediated by leukotriene, an effective amount of an agonist or antagonist of the leukotriene receptor that specifically binds to a mammalian leukotriene receptor having an amino acid sequence defined by SEQ ID NO: 2, or a subsequence thereof, and pharmaceutical compositions comprisi
Laz Thomas M.
Pang Ling
Wang Suke
Schering Corporation
Thampoe Immac J.
Triolo Thomas A.
Ulm John
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