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
2000-07-21
2003-07-08
Spector, Lorraine (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...
C435S325000, C435S006120, C514S002600, C530S350000
Reexamination Certificate
active
06589764
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to proteins that are members of the IL-1 receptor family. More particularly, the present invention relates to IL-1Rrp1 and AcPL receptor complexes that mediate high affinity IL-18 binding and activity as well as inhibit IL-18 mediated activity.
2. Description of Related Art
The type I interleukin-1 receptor (IL-1RI) mediates the biological effects of interleukin-1, a pro-inflammatory cytokine (Sims et al.,
Science
241:585-589, 1988; Curtis et al.,
Proc. Narl. Acaz Sci. USA
86:3045-3049, 1989). A second interleukin-1 receptor (designated type II IL-1R or IL-1RII) binds IL-1, but does not appear to mediate signal transduction (McMahan et al.,
EMBO J
. 10:2821, 1991; Sims et al.,
Proc. Natl. Acai Sci. USA
90:6155-6159, 1993). IL-1RI and IL-1RII each bind IL-1&agr; and IL-1&bgr;. IL-1 has been implicated in chronic inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. There is increasing evidence that IL-1 plays a role in osteoporosis. All of these activities are initiated by the signaling function of the cytoplasmic portion of the Type I IL-1R. EL-1ra inhibits the activities of IL-1 by binding to the type I IL-1 receptor, thereby blocking access to IL-1&agr; and IL-1&bgr; while eliciting no biological response of its own.
IL-1RI and IL-1RII belong to a family of proteins that exhibit significant sequence homology. One such protein is IL-1R accessory protein (IL-1R AcP), described in Greenfeder et at. (
J. Biol. Chem
. 270: 13757-13765, 1995). This protein, by itself, is not capable of binding IL-1, but does form a complex with IL-1RI and IL-1&agr; and IL-1&bgr;. When co-expressed with IL-1RI, recombinant IL-1R AcP increases the binding affinity of IL-1RI for IL-1&bgr; (Greenfeder et al., supra).
Another protein exhibiting sequence homology to the IL-1RI and IL-1RII family is the IL-1 receptor related protein 1 (IL-1Rrp1)(See Pamet et al.
J. Biol Chem
271:3967,1996, and Torigoe et al.,
J. Biol Chem
272:25737, 1997). Still another such protein is AcPL.
IL-18, is a homologue of IL-1&agr; and IL-1&bgr; and is known to activate many of the same responses activated by IL-1. For example, cells stimulated with IL-18 activate NF&kgr;B and produce known inflammatory mediators. IL-18 acts as a stimulator of Th1 cell growth and differentiation and is a potent inducer of &ggr;-interferon production from Th1 cells. The Th1 class of helper T cells are known to mediate inflammatory reactions. IL-18 enhances NK cell killing activity and has been implicated in septic shock, liver destruction, inflammatory bowel disease and diabetes.
Recently it was shown that IL-1Rrp1 binds IL-18 and mediates IL-18 signaling in transfected cells. However, the IL-1Rrp1 binding affinity for IL-18 is very low and it is likely that one or more additional receptors or receptor subunits are involved with IL-18 binding and signaling.
Thus, the identification of additional receptors of for IL-18 is desirable. Such receptor proteins can be studied to determine whether or not they bind IL-18 and, if so, whether the receptors play a role in mediating signal transduction. Furthermore, soluble forms of such receptors may be used to inhibit IL-18 activity and ameliorate any inflammatory and/or autoimmune diseases attributable to IL-18 signaling. The possible existence of additional affinity-converting subunits for IL-18 can be explored, as well.
SUMMARY OF THE INVENTION
The present invention provides receptor polypeptides designated herein as IL-18 receptor complexes. More particularly, the present invention provides multimeric receptor polypeptides that include an AcPL polypeptide, or fragments thereof, and an IL-1Rrp1 polypeptide, or fragments thereof. The AcPL polypeptide may be covalently linked or noncovalently to the IL-1Rrp1 polypeptide by any suitable means. Such means include via a cross-linking reagent, a polypeptide linker, and associations such as via disulfide bonds or by use of leucine zippers. In one embodiment of the invention, the receptor is a fusion protein produced by recombinant DNA technology. This multimeric receptor of the present invention binds IL-18 with an affinity greater than that of IL-1Rrp1 alone. Disorders mediated by IL-18 may be treated by administering a therapeutically effective amount of this inventive receptor to a patient afflicted with such a disorder.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based upon the discovery that the coexpression of AcPL and IL-1Rrp1 results in a dramatic enhancement of NF&kgr;B activity in cells stimulated with IL-18. Because IL-1Rrp1 alone binds IL-18 only weakly and AcPL does not bind IL-18, the enhancement of NF&kgr;B activity by coexpressed AcPL and IL-1Rrp1 indicates that these polypeptides are subunits of an IL-18 receptor complex. In accordance with the present invention novel polypeptides, designated IL-18 receptor complexes, are provided. Advantageously, such dimeric IL-18 receptor complexes comprising IL-1Rrp1 and AcPL, or fragments thereof, are useful for inhibiting IL-18 activity, including the proinflammatory effects of IL-18, and can include IL-1Rrp1 and AcPL as proteins coexpressed in the same cell, or as IL-1Rrp1 linked to an AcPL as receptor subunits. Preferably the subunits are linked via covalent linkages. The subunits may be covalently linked by any suitable means, such as via a cross-linking reagent or a polypeptide linker.
In one embodiment of the present invention, the receptor is a fusion protein produced by recombinant DNA technology. Such fusion proteins can be prepared by transfecting cells with DNA encoding IL-1Rrp1:Fc fusion protein and DNA encoding AcPL:Fc fusion protein and coexpressing the dimers in the same cells.
Alternatively, AcPL/IL-1Rrp1 dimers can be prepared by fusing one of the receptor subunits to the constant region of an immunoglobulin heavy chain and fusing the other receptor subunit to the constant region of an immunoglobulin light chain. For example, an AcPL protein can be fused to the CH
1
-hinge-CH
2
—CH
3
region of human IgG1 and an IL-1Rrp1 protein can be fused to the C kappa region of the Ig kappa light chain, or vice versa. Cells transfected with DNA encoding the immunoglobulin light chain fusion protein and the immunoglobulin heavy chain fusion protein express heavy chain/light chain heterodimers containing the AcPL fusion protein and the IL-1Rrp1 fusion protein. Via disulfide linkages between the heavy chains, the heterodimers further combine to provide multimers, largely tetramers. Advantageously, in the event homodimers of two heavy or two light chain fusions are expressed, such homodimers can be separated easily from the heterodimers.
In addition to IL-18 receptor protein complexes, the present invention includes isolated DNA encoding heteromer polypeptides, expression vectors containing DNA encoding the heteromer polypeptides, and host cells transformed with such expression vectors. Methods for production of recombinant IL-18 receptor, including soluble forms of the protein, are also disclosed. Antibodies immunoreactive with the novel polypeptide are provided herein as well.
In one embodiment of the present invention, the polypeptide subunits of the heteromer IL-18 receptors include at least one ACPL subunit as described in SEQ ID NO:2 or SEQ ID NO: 6, and at least one IL-1Rrp1 subunit as described in SEQ ID NO:4 or SEQ ID NO:8. DNA encoding these polypeptide subunits are presented in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:3 and SEQ ID NO:7, respectively. The AcPL subunit protein encoded by SEQ ID NO:1 includes an extracellular domain of 356 amino acids (residues 1-356 from N- to C-terminus of SEQ ID NO:2) that includes a signal peptide of 14 amino acids (residues 1-14 of SEQ ID NO:2); a transmembrane region of 25 amino acids (residues 357-381) and a cytoplasmic domain of 218 amino acids (residues 382-599). The AcPL subunit protein encoded by SEQ ID NO:5 includes an extracellular domain of 356 amino acids (residues 1-356 of SEQ ID NO:6) that includes a signal
Born Teresa L.
Sims John E.
Henry Janis C.
Immunex Corporation
Jiang Dong
Perkins Patricia Anne
Spector Lorraine
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