Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
2000-02-07
2002-10-08
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C530S351000, C530S402000, C530S412000, C530S418000, C530S419000, C530S420000, C435S069520
Reexamination Certificate
active
06461604
ABSTRACT:
BACKGROUND OF THE INVENTION
IL-6 is a pleiotropic cytokine with a variety of stimulatory effects on hematopoietic cells and cells of the immune system (Hirano et al., 1986; Wong et al., 1988; Kishimoto et al., 1992). Major cellular targets include B-lymphocytes, T-lymphocytes, the enhancement of hematopoietic colony formation and the production of acute phase response proteins in the liver (Mackiewicz et al., 1992). The primary role of IL-6 appears to be as a component of the immune system, with knock out mice exhibiting an impaired IgG and IgA response. Of particular interest is the observation of the involvement of IL-6 in bone homeostasis. In Paget's disease and in multiple myeloma patients where significant bone loss occurs, a good correlation has been found with increased IL-6 levels. Interestingly, the level of IL-6 is affected by estrogen in bone marrow derived stromal cells and causes decrease in the development of osteoclasts (Girasole et al., 1992), while estrogen loss (by mouse ovariectomy) causes enhanced osteoclast development in ex vivo cultures of bone marrow and increased osteoclasts in trabecular bone. Most importantly osteoclast development was inhibited by the in vivo or in vitro administration of estrogen or neutralizing IL-6 monoclonal antibodies (Jilka et al., 1992). Mutant mice lacking IL-6 have normal amounts of trabecular bone and, ovariectomy does not cause bone loss or a change in the rate of remodeling. These studies strongly suggest that IL-6 plays an important role in post menopausal bone loss.
Interleukin-6 (IL-6) is a member of a family of cytokines/growth factors which are believed to share a common topological fold despite limited amino acid sequence homology. All of these cytokines are believed to have three dimensional structures comprised of a core bundle of 4 &agr;-helices connected by variable-lengthloops. Together with, inter alia, IL-11, IL-12, and EPO, IL-6 has been classified into a subfamily of “long chain” 4-helix bundles which share several structural features including overall polypeptide chain length, average length of helices, and characteristic packing of the antiparallel helical pairs (Sprang and Bazan, 1993). These predicted structural features for long chain 4-helix bundles have been largely confirmed by recent NMR and X-ray crystallographic studies of growth hormone, G-CSF, LIF, and CNTF (Zink et al., 1994; Hill et al., 1993; McDonald et al., 1995; Robinson et al., 1994; Ultsch et al., 1994). However, comparable experimental data on IL-6 has yet to be reported, although the sequence-specific assignments, secondary structure analysis, and overall topological fold for IL-6 have been reported (Xu et al., 1996).
The members of this cytokine family including IL-6 also share remarkably similar structural features for the receptors to which they bind. These similarities also extend to the sequential clustering events leading to transduction. The closest members of the family include LIF, CNTF, Oncostatin M, and IL-11 (Kishimoto et al., 1992; Miyajima et al., 1992; Gearing et al., 1991; Davis et al., 1991; Yamasaki et al., 1988; Kishimoto et al., 1994). The IL-6 receptor consists of two polypeptides: the &agr; chain (IL-6r), an 80 kD transmembrane glycoprotein that binds IL-6 with low affinity, and the &bgr; chain (gp130), a 130 kD transmembrane glycoprotein that binds to the IL-6/IL-6r heterodimer to form the high affinity signal transducing complex (Taga et al., 1989). gp130 is a signal transducticn component of not only the IL-6 receptor but also the LIF, CNTF, Oncostatin M, and the IL-11 receptors (Taga et al., 1992), therefore the &agr; chain distribution dictates the cellular response (Kishimoto et al., 1992). The IL-6r is a transmembrane protein composed of a cytokine binding type I domain (necessary and sufficient for binding IL-6) (Yawata et al., 1993), an Ig like domain and a short cytosolic domain (Yamasaki et al., 1988) that is not required for signalling (Taga et al., 1989). gp130 is also a transmembrane protein composed of an Ig-like domain, cytokine type I domain, a contacting-like region, a transmembrane domain, and a cytosolic domain necessary for signalling, containing a motif known as box 1, box2 (Murakami et al., 1991). Signal transduction by IL-6 follows the dimerisation of gp130, which activates a bound JAK2 (Argetsinger et al., 1993).
Recently studies utilising size exclusion columns and equilibrium centrifugation have shown that IL-6 binds to sIL-6r to form a heterodimer (Ward et al., 1994). However, in the presence of sgp130 a hexameric complex is formed that is composed of IL-6, sIL-6r, sgp130 in a 2:2:2 stoichiometry (Ward et al., 1994). These studies combined with the evidence from structural, biochemical, and mutagenesis studies of the human growth hormone (hGH), human growth hormone receptor (hGHr), human prolactin receptor (hPRLr) complexes (De Vos et al., 1992; Somers et al., 1994) provide evidence that assembly of the IL-6 signalling complex is an ordered and sequential process.
Analysis of IL-6 site-directed mutagenesis data provides further support for such a structural model The first class of IL-6 mutants (site 1) show reduced binding to IL-6r (Savino et al., 1993). Two additional, distinct classes of IL-6 mutants (sites 2 and 3) have been isolated which bind to IL-6r and yet fail to transduce (Brakenhoff et al., 1994; Ehlers et al., 1994). IL-6 with both site 2 and site 3 mutations not only fails to transduce signal but functions as an antagonist in an IL-6 dependant proliferation assay (Brakenhoff et al., 1994). IL-6r point mutants have also been identified which result in normal IL-6 binding but no signal transduction (Yawata et al., 1993). It has been speculated that these mutations are in a region of IL-6r that is involved in low affinity binding to gp130.
However, the study of IL-6 and its interaction with its receptor components has been hindered by the lack of detailed information concerning the structure of IL-6. Therefore, it would be desirable to determine the structure of IL-6 in order to better enable the study of its interactions with its receptor and to identify possible inhibitors of the IL-6/IL-6r interaction.
SUMMARY OF THE INVENTION
The present invention provides for the first time crystalline IL-6. Preferably, the crystalline IL-6 human IL-6; however, crystalline IL-6 from non-mammalian species is also encompasssed by the invention. The crystalline IL-6 may be recombinant IL-6 or IL-6 purified from naturally occurring or other non-recombinant sources; however, crystalline recombinant IL-6 is preferred. In certain preferred embodiments,the IL-6 may be glycosylated, although non-glycosylated forms are also contemplated by the present invention. In other embodiments, the crystalline IL-6 may comprise the mature sequence of naturally-occurring IL-6, although other forms (such as, for example, IL-6 comprising an additional N-terminal methionine residue) are also encompassed by the invention.
The present invention also provides for crystallization of IL-6 in association with a second chemical species, including without limitation potential inhibitors of IL-6 activity, potential inhibitors of IL-6 binding and all or a portion of the IL-6 receptor (IL-6R).
Other aspects of the invention provide for a model of the structure of IL-6 comprising a data set embodying the structure of IL-6. The data set embodying such structure can be derived from any available means for obtaining such information, including without limitation by crystallographicanalysis of IL-6 and by NMR analysis of IL-6. Such model can embody the entire structure of IL-6 or a portion of such structure. Preferably, the portion of the IL-6 structure embodied by such model comprises the active binding site of IL-6 and/or another epitope or binding domain of IL-6.
Any available method may be used to construct such model from the crystallographic and/or NMR data disclosed herein or obtained from independent analysis of crystalline IL-6. Such a model can be constructed from available analytical data points using known s
Hong Jin
McDonagh Thomas E.
Seehra Jasbir S.
Somers William S.
Stahl Mark L.
Genetics Institute LLC
Mertz Prema
Wyeth Rebecca Barrett
LandOfFree
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