IL-1/TNF-alpha-activated kinase (ITAK), and methods of making and using the same
AssigneeImmunex Corporation
InventorsJohn E. Sims, G. Duke Virca, Timothy A. Bird, Dirk M. Anderson
Life of this patent
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Abstract
Purified and isolated IL-1/TNF-α-activated kinase (ITAK), nucleic acids encoding ITAK, processes for production of recombinant forms of ITAK, pharmaceutical compositions containing ITAK, and use of ITAK in therapies and in various assays, including assays to detect antagonists and agonists of ITAK are provided.
Claims32
An isolated nucleic acid molecule encoding an IL-1/TNF-α-activated kinase (ITAK) or a variant thereof.
An isolated nucleic acid molecule according to claim I wherein said molecule encodes a protein having the amino acid sequence of SEQ ID:
- NO 2, from amino acid number 1 to amino acid number 979.
A recombinant vector, comprising a nucleic acid molecule according to any one of claims 14.
An isolated IL-1/TNF-α-activated kinase (ITAK) or a variant thereof.
A nucleic acid probe of at least 15 nucleotides in length which is capable of specifically hybridizing to a nucleic acid sequence encoding an IL-1/TNF-α-activated kinase (ITAK).
A method of screening for an agent that modulates the kinase activity of an IL-1/TNF-α-activated kinase (ITAK), comprising:
- (a) contacting a candidate agent with biologically active ITAK under conditions and for a time sufficient to allow the candidate agent to modulate the kinase activity of said ITAK;
- and
- (b) measuring the ability of the candidate agent to modulate the ITAK kinase activity.
A method for determining whether a selected agent is an IL-1/TNF-α-activated kinase (ITAK) agonist, comprising:
- (a) exposing the selected agent to an unstimulated ITAK response pathway under conditions and for a time sufficient to allow a stimulation of the pathway;
- and
- (b) detecting stimulation of the response pathway and therefrom determining the presence of an ITAK agonist.
A method for determining whether a selected agent is an IL-1/TNF-α-activated kinase (ITAK) agonist, comprising:
- (a) measuring the ITAK kinase activity of an ITAK response pathway;
- (b) exposing the selected agent to the measured ITAK response pathway;
- and
- (c) detecting increased ITAK kinase activity in the response pathway.
A method for determining whether a selected agent is an IL-1/TNF-α-activated kinase (ITAK) antagonist, comprising:
- (a) exposing the selected agent to an ITAK response pathway in the presence of an ITAK agonist under conditions and for a time sufficient to allow a decrease in stimulation of the pathway;
- and
- (b) detecting a decrease in the stimulation of the response pathway relative to the stimulation of the response pathway by the ITAK agonist alone, and therefrom determining the presence of an ITAK antagonist.
An ITAK agonist.
An ITAK antagonist.
An ITAK phosphorylation substrate peptide acceptor sequence that is not mammalian β-cascin and that can be phosphorylated by isolated ITAK at a rate of at least 40 nmol phosphate/mg protein/minute.
A method for detecting IL-1/TNF-α-activated kinase (ITAK) activity, comprising:
- (a) contacting ITAK with an ITAK phosphorylation substrate peptide acceptor sequence that is not mammalian β-casein in the presence of ATP under conditions and for a time sufficient to allow transfer of a γ-phosphate group from an ATP donor to the ITAK phosphorylation substrate peptide acceptor sequence;
- and
- (b) measuring the incorporation of phosphate by the ITAK phosphorylation substrate peptide acceptor sequence.
An ITAK phosphorylation substrate peptide acceptor sequence comprising the amino acid sequence:
- 9 Arg-Arg-Arg-His-Leu-Pro-Pro-Leu-Leu-Leu-Gln-Ser-Trp-Met-His-Gln-Pro-His-Gln. (SEQ ID:NO 3)
A method for treating an IL-1- or TNF-α-mediated inflammatory disorder, comprising administering to a patient a therapeutically effective amount of an ITAK antagonist.
A kit for detecting ITAK in a sample, comprising:
- an ITAK phosphorylation substrate peptide acceptor sequence that is not mammalian β-casein and that can be phosphorylated by isolated ITAK at a rate of at least 40 nmol phosphate/mg protein/minute;
- and
- a means for measuring phosphate incorporated by the ITAK phosphorylation substrate peptide acceptor sequence.
A method for identifying gene products that associate with ITAK, comprising:
- (a) introducing nucleic acid sequences encoding an ITAK polypeptide into a first expression vector such that ITAK sequences are expressed as part of a fusion protein comprising a functionally incomplete first portion of a protein that is essential to the viability of a host cell;
- (b) introducing nucleic acid sequences encoding a plurality of candidate gene products that associate with ITAK into a second expression vector such that any candidate gene products are expressed as part of a fusion protein comprising a second functionally incomplete portion of the protein that is essential to the viability of the host cell;
- (c) introducing the first and second expression vectors into a host cell under conditions and for a time sufficient such that host cell survival is dependent upon reconstitution of both the first and second functionally incomplete portions into a functionally complete protein;
- and
- (d) identifying surviving host cells, and therefrom determining the nucleic acid sequences encoding candidate gene products that associate with ITAK in the second expression vector.
Dependencies are inferred from the claim text.