Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1998-08-17
2001-07-17
Kunz, Gary L. (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C435S007100, C435S007200, C530S324000, C530S326000, C530S327000, C530S328000, C530S329000, C530S350000, C530S351000
Reexamination Certificate
active
06262228
ABSTRACT:
FIELD OF THE INVENTION
The field of this invention is enzymes involved in signal transduction.
BACKGROUND
Interleukin 1 (IL-1) receptor associated kinase (IRAK) functions as an intracellular signal transducer for the pro-inflammatory cytokine IL-1. IL-1 treatment of cells induces the complex formation of the two IL-1 receptor chains, IL-1R1 and IL-1RAcP, which recruits an adaptor molecule designated as MyD88 which binds to IRAK. IRAK is subsequently phosphorylated, released from the receptor complex to interact with TRAF6. TRAF6 triggers either the NIK/IKK kinase cascade to activate the transcription factor NF-&kgr;B or an undefined kinase cascade to activate the transcription factor AP-1. Both transcription factors regulate large numbers of genes that regulate immune and inflammatory responses.
The genome project has facilitated the identification of a large number of membrane bound receptor-like molecules that are related to IL-1RI and IL-1RAcP by sequence homology. One member of this family, IL-1RrP, has been recently shown to function as a receptor of an IL-1 related cytokine, IL-1 8, that regulates immune response by promoting the production of interferon&ggr;. Like IL-1RI and IL-1RAcP, IL-1RrP signals NF-&kgr;B activation. Gene disruption experiments and biochemical analyzes indicate that IL-1RrP also utilizes MyD88 and IRAK and TRAF6 as intracellular signal transducers.
Although MyD88 deficient mice failed to respond to IL-1 and IL-18, IRAK deficient mice still have a residual response to IL-1. This observation indicates that other molecules in the cells can partially substitute for the function of IRAK in IL-1 signaling. Recently, an IRAK-related molecule designated IRAK2, was described. Although upon over expression, IRAK2 could interact with IL-1R and IRAK, it has not been shown to be recruited to the receptor complex after IL-1 treatment like IRAK. Therefore, we searched for molecules that can substitute for IRAK in an IL-1 response.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to isolated IRAK3 polypeptides and related polynucleotides having IRAK3-specific structure and activity. The subject IRAK3 polypeptides and polynucleotides can regulate cellular responsiveness to cytokine activation and hence provide important regulators of cell function. The polypeptides may be produced recombinantly from transformed host cells from the subject IRAK3 polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated IRAK3 hybridization probes and primers capable of specifically hybridizing with the disclosed IRAK3 gene, IRAK3-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for IRAK3 transcripts), therapy (e.g. IRAK3 kinase inhibitors to inhibit IL-1 induced signal transduction) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).
DETAILED DESCRIPTION OF THE INVENTION
The nucleotide sequence of a natural human cDNA encoding a human IRAK3 polypeptide is shown as SEQ ID NO:1, and the full conceptual translate is shown as SEQ ID NO:2. To clone this novel IRAK3 cDNA, we used a the cDNA insert from a mouse EST clone (AA840598, Genome Systems) as a hybridization probe to screen a lambda cDNA library constructed with Phytohemagglutinin-L (PHA-L) activated human peripheral blood leukocytes under low stringent conditions. We isolated a 2.2 kb cDNA clone with an open reading frame encoding 617 amino acids, and determined that the methionine at position 22 of the open reading frame is the first amino acid of the full-length IRAK3 protein, which consists of 596 amino acids with a calculated molecular weight of 67675 daltons. We determined that IRAK3 can function as a signaling molecule for either IL-1 or cytokines that signal through IL-1R related receptors, can bind MyD88 in coexpression and in vitro binding assays, and plays a role in inflammatory responses and/or immune regulation and thus provides a drug target for treatment of inflammatory diseases and immune disorders.
The IRAK3 polypeptides of the invention include incomplete translates of SEQ ID NO:1 which translates and fragments of SEQ ID NO:2 have human IRAK3-specific amino acid sequence, binding specificity or function. Preferred translates/deletion mutants comprise at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, most preferably at least 50 consecutive residues of SEQ ID NO:2, preferably of at least one of SEQ ID NO:2, residues 1-99, residues 100-199, residues 200-299, residues 300-399, residues 400-499 and residues 500-596. The subject domains provide IRAK3 domain specific activity or function, such as IRAK3-specific kinase or kinase inhibitory activity, IRAK-3 specific MyD88-binding or binding inhibitory activity, IRAK3 specific antibody binding or binding inhibitory activity.
IRAK3-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an IRAK3 polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as an IRAK3 substrate, an IRAK3 regulating protein or other regulator that directly modulates IRAK3 activity or its localization such as MyD88; or non-natural binding target such a specific immune protein such as an antibody, or an IRAK3 specific agent such as those identified in screening assays such as described below. IRAK3-binding specificity may be assayed by kinase activity or binding equilibrium constants (usually at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
), by the ability of the subject polypeptide to function as negative mutants in IRAK3-expressing cells, to elicit IRAK3 specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In any event, the IRAK3 binding specificity of the subject IRAK3 polypeptides distinguishes any discernable translation product of EST clone AA840598.
In a particular embodiment, the subject domains provide IRAK3-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to IRAK3-specific domains are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of IRAK3-specific antibodies is assayed by solid phase immunosorbant assays using immobilized IRAK3 polypeptides of SEQ ID NO:2, see, e.g. Table 2.
TABLE 2
Immunogenic IRAK3 polypeptides eliciting IRAK3-specific rabbit
polyclonal antibody: IRAK3 polypeptide-KLH conjugates immunized
per protocol described above.
IRAK3 Polypeptide Sequence
Immunogenicity
SEQ ID NO:2, residues 1-10
+++
SEQ ID NO:2, residues 12-21
+++
SEQ ID NO:2, residues 25-37
+++
SEQ ID NO:2, residues 42-59
+++
SEQ ID NO:2, residues 62-71
+++
SEQ ID NO:2, residues 72-85
+++
SEQ ID NO:2, residues 88-89
+++
SEQ ID NO:2, residues 105-112
+++
SEQ ID NO:2, residues 116-122
+++
SEQ ID NO:2, residues 120-128
+++
SEQ ID NO:2, residues 175-182
+++
SEQ ID NO:2, residues 180-195
+++
SEQ ID NO:2, residues 201-208
+++
SEQ ID NO:2, residues 213-222
+++
SEQ ID NO:2, residues 222-230
+++
SEQ ID NO:2, residues 228-237
+++
SEQ ID NO:2, residues 230-338
+++
SEQ ID NO:2, residues 237-245
+++
SEQ ID NO:2, residues 440-450
+++
SEQ ID NO:2, residues 442-451
++&plu
Kunz Gary L.
Landsman Robert S.
Osman Richard Aron
Tularik Inc.
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