Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-03-08
2001-08-28
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C424S278100, C424S810000, C530S324000
Reexamination Certificate
active
06281193
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns compounds such as proteins, peptides and organic compounds which are characterized by their ability to block the interaction between Raf-1 protein and/or 14-3-3 proteins with the intracellular domain of the &bgr; chain of the interleukin-2 receptor molecule (IL-2R&bgr;), and thereby block the intracellular signaling process mediated by IL-2R&bgr;. The compounds of the invention are intended to inhibit the activity of IL-2 or IL-15 where desired, for example in autoimmune diseases in general, or graft-versus-host reactions in particular. The present invention also concerns in vitro assays for the isolation, identification and characterization of the above compounds, as well as pharmaceutical compositions containing as active ingredient one or more compounds of the invention.
BACKGROUND OF THE INVENTION
Interleukin-2 (IL-2) is a T-cell derived factor that amplifies the response of T cells to any antigen by stimulating the growth of the T cells. Thus, IL-2 is a critical T-cell growth factor which plays a major role in the proliferation of T cells that occurs subsequent to antigen activation, this proliferation resulting in the amplification of the number of T cells responsive to any particular antigen. IL-15 can generally substitute for IL-2 to exert most, if not all, of these activities (Bamford et al., 1994).
The high affinity (Kd:10
−11
M) IL-2 receptor (IL-2R) is composed of at least three non-covalently associated IL-2 binding proteins: the low affinity (Kd:10
−8
M) p55 (&agr; chain) and the intermediate affinity subunits (Kd:10
−9
M) p75 (&bgr; chain) and p64 (&ggr; chain) (Smith, K. A., 1988; Waldmann, T. A., 1993). Proliferative signals for the T cells are delivered through high affinity IL-2 receptors consisting of all three subunits, but not via the low affinity site (Robb, R. J. et al., 1984; Siegal, J. P. et al., 1987; Hatakeyama, M. et al., 1989). IL-2R&agr;, IL-2R&bgr;, and IL-2R&ggr; chains have 13, 286 and 86 amino acid intracytoplasmic domains, respectively.
IL-15, a cytokine with many IL-2-like activities, also utilizes the IL-2R&bgr; as a part of its receptor complex (Giri et al., 1994). This IL-2R&bgr; dependent signaling process is fundamental to the cellular effects induced by the binding of IL-2 to its receptor (IL-2R) as well as the effects induced by the binding of IL-15 to its receptor. The IL-2R&bgr; and &ggr; chains, but not the &agr; chain, are essential for IL-2- as well as IL-15-mediated signal transduction (Nakamura, Y. et al., 1994). The 64 kDa IL-2R&ggr; chain protein is rapidly phosphorylated on tyrosine residues after stimulation with IL-2. The &ggr; chain has also been shown to be a part of other receptor complexes such as the receptor for IL-4 and IL-7 (Noguchi, M. et al., 1993; Russell, S. M. et al., 1993). Absence of the &ggr; chain leads to a severe combined immunodeficiency disease in humans (Noguchi, M. et al., 1993). IL-2R&ggr; contains sequences from positions 288 to 321 homologous to the Src homology region 2 (SH2) that can bind to phosphotyrosine residues of some phosphoproteins. Another molecule, designated pp97, has been suggested to be the tyrosine kinase physically associated with the IL-2R&ggr; chain (Michiel, D. F. et al., 1991).
An analysis of cells transformed with a series of IL-2R&bgr; chain deletion mutants identified a 46 amino acid serine and proline rich intracytoplasmic region of the IL-2R&bgr; chain (a.a. 267-312), which is crucial for growth promoting signal transduction (Hatakeyama, M. et al., 1989). This same region is crucial for promoting IL-15 mediated effects. Upon stimulation with IL-2, enzymatically active protein tyrosine kinases and, as the laboratory of the present inventors has previously shown (Remillard, B. et al., 1991), the novel lipid kinase, phosphatidyinositol-3-kinase activity blocks proliferation. Cells that express wild-type IL-2R&agr; and &ggr; chains and mutant IL-2R&bgr; chains lacking this 46 a.a. region bind and internalize IL-2, but fail to proliferate in response to IL-2 (Hatakeyama, M. et al., 1989). An identical set of circumstances pertains to IL-15 responses. Although the intracytoplasmic domain of the IL-2R&bgr; and &ggr; chains lacks a protein tyrosine kinase consensus sequence, several cellular proteins are phosphorylated upon tyrosine residues following IL-2 stimulation (Benedict, S. H. et al., 1987; Ferris, D. K. et al., 1989; Saltzmann, E. M. et al., 1988; Asao, H. et al., 1990; Mills, G. B. et al., 1990; Merida, I. and Gaulton, G. N., 1990). IL-2 induced protein tyrosine kinase activity is due, at least in part, to activation of the p56
lck
(lck), a src-family protein tyrosine kinase. Controversy exists as to whether the serine/proline rich (Fung, M. R. et al., 1991) or an adjacent tyrosine rich “acidic” region (Hatakeyama, M. et al., 1991) of the IL-2R&bgr; chain is the lck binding site.
IL-2 also stimulates phosphorylation on serine residues of several proteins (Turner, B. et al., 1991; Valentine, M. V. et al., 1991). Raf-1, a serine/threonine kinase, has been identified as a likely signal transducing element for several growth factor receptors (Carroll, M. P. et al., 1990; Morrison, D. K. et al., 1988; Baccarini, M. et al., 1991; Kovacina, K. S. et al., 1990; Blackshear, P. J. et al., 1990; App, H. et al., 1991). The Raf-1 molecule has a molecular weight of 74 kD and can be divided into 2 functional domains, the amino-terminal regulatory half and the carboxy-terminal kinase domains (for review see Heidecker, G. et al., 1991). Raf-1 has been identified as a crucial signal transducing element for ligand activated EPO receptors (Carroll, M. P. et al., 1991). The IL-2R&bgr; chain and EPO receptors belong to the same family of receptors and share homologies within their cytoplasmic domains (D'Andrea, A. D. et al., 1989). Stimulation of the IL-2R results in the phosphorylation and activation of cytosolic Raf-1 serine/threonine kinase. IL-2R stimulation leads to a 5 to 10 fold immediate/early induction of the c-raf-1 mRNA expression on freshly isolated, resting T cells (Zmuidzinas, A. et al., 1991) and results in up to a 12-fold increase in Raf-1 protein expression. In addition, a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increases through G1.
Enzymatically active Raf-1 appears in the cytosol of IL-2 stimulated CTLL-2 cells (Hatakeyama, M. et al., 1991) and human T blasts (Zmuidzinas, A. et al., 1991). Following IL-2 stimulation, cytosolic Raf-1 molecules are phosphorylated on tyrosine and serine residues (Turner, B. et al., 1991). The laboratory of the present inventors have studied the signaling pathway by which IL-2 signals T cells to begin dividing. In these studies Raf-1 was identified in immunoprecipitates of the IL-2R&bgr; chain, suggesting that Raf-1 may be involved as an important element in IL-2 signaling. Further, it was determined that prior to IL-2 stimulation, enzymatically active Raf-1 molecules are physically associated with the IL-2R&bgr; chain and that following stimulation with IL-2, a protein tyrosine kinase phosphorylates Raf-1 thereby leading to translocation of Raf-1 from the IL-2 receptor into the cytosol (Maslinski, W. et al., 1992). Moreover, dissociation of enzymatically active Raf-1 from the IL-2R&bgr; chain, but not maintenance of IL-2R associated kinase activity, is completely abolished by genistein, a potent tyrosine kinase inhibitor (Maslinski, W. et al., 1992). The above-noted suggested requirement of Raf-1 for IL-2 signaling has been supported by evidence showing that by blocking Raf-1 expression, IL-2 could not induce T cell proliferation in the absence of Raf-1. Thus, from the afore-mentioned, it is widely accepted that activation of the Raf-1 serine/theonine kinase is critical for IL-2-mediated T-cell proliferation (see also Riedel et al., 1993).
Prior to IL-2 stimulation, several serine, but not tyrosine nor threonine, residues of the IL-2R&bgr; chain are phosphorylated (Asao, H. et al., 1990). IL-2 induces rapid
Maslinski Wlodzimierz
Strom Terry
Applied Research Systems ARS Holding N.V.
Browdy and Neimark
DeCloux Amy
Saunders David
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