Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-01-07
2002-12-31
Ulm, John (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C536S023500
Reexamination Certificate
active
06500922
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns novel polypeptide factors. More particularly, the invention concerns factors associated with the type 2 tumor necrosis factor receptor (TNF-R2).
BACKGROUND OF THE INVENTION
Tumor necrosis factor (TNF, also referred to as TNF-&agr;) is a potent cytokine produced mainly by activated macrophages and a few other cell types. The large number of biological effects elicited by TNF include hemorrhagic necrosis of transplanted tumors, cytotoxicity, a role in endotoxin shock, inflammatory, immunoregulatory, proliferative, and antiviral responses [reviewed in Goeddel, D. V. et al.,
Cold Spring Harbor Symposia on Quantitative Biology
51, 597-609 (1986); Beutler, B. and Cerami, A.,
Ann. Rev. Biochem.
57, 505-518 (1988); Old, L. J.,
Sci. Am.
258(5), 59-75 (1988); Fiers, W.
FEBS Lett.
285(2), 199-212 (1991)]. The literature has reported that TNF and other cytokines such as IL-1 may protect against the deleterious effects of ionizing radiation produced during the course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage [(Neta et al.,
J. Immunol.
136(7): 2483, (1987); Neta et al.,
Fed. Proc.
46: 1200 (abstract), (1987); Urbaschek,
Lymphhokine Res.
6:179 (1987); U.S. Pat. No. 4,861,587; Neta et al.,
J. Immunol.
140: 108 (1988)]. A related molecule, lymphotoxin (LT, also referred to as TNF-&bgr;), that is produced by activated lymphocytes shows a similar but not identical spectrum of biological activities as TNF (see, e.g. Goeddel, D. V. et al., supra, and Fiers, W., supra). TNF was described by Pennica et al.,
Nature
312, 721 (1984); LT was described by Gray et al.,
Nature
312. 724 (1984).
The first step in the induction of the various cellular responses mediated by TNF or LT is their binding to specific cell surface receptors. Two distinct TNF receptors of approximately 55-kDa (TNF-R1) and 75-kDa (TNF-R2) have been identified [Hohmann, H. P. et al.,
J. Biol. Chem.
264, 14927-14934 (1989); Brockhaus, M. et al.,
Proc. Natl. Acad. Sci. USA
87, 3127-3131 (1990)], and human and mouse cDNAs corresponding to both receptor types have been isolated and characterized [Loetscher, H. et al.,
Cell
61, 351 (1990); Schall, T. J. et al.,
Cell
61, 361 (1990); Smith, C. A. et al.,
Science
248, 1019 (1990); Lewis, M. et al.,
Proc. Natl. Acad. Sci. USA
88, 2830-2834 (1991); Goodwin, R. G. et al.,
Mol. Cell. Biol.
11, 3020-3026 (1991)]. Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular portions of both receptors are found naturally also as soluble TNF-binding proteins [Nophar, Y. et al.,
EMBO J.
9, 3269 (1990); and Kohno, T. et al.,
Proc. Natl. Acad. Sci. U. S. A.,
87 8331 (1990)]]. The amino acid sequence of human TNF-R1 and the underlying nucleotide sequence are disclosed in EP 417,563 (published Mar. 20, 1991), whereas EP 418,014 (published Mar. 20, 1991) discloses the amino acid and nucleotide sequences of human TNF-R2.
Although not yet systematically investigated, the majority of cell types and tissues appear to express both TNF receptors.
The individual roles of the two TNF receptors, and particularly those of TNF-R2, in cell signaling are far from entirely understood, although studies performed by poly- and monoclonal antibodies (mAbs) that are specific for either TNF-R1 or TNF-R2 have provided some very valuable insight into the functions and interactions of these receptors.
It has been observed that both polyclonal and monoclonal antibodies directed against TNF-R1 can act as specific agonists for this receptor and elicit several TNF activities such as cytotoxicity, fibroblast proliferation, resistance to chlamydiae, and synthesis of prostaglandin E
2
[Engelmann, H. et al.,
J. Biol. Chem.
265. 14497-14504 (1990); Espevik, T. et al.,
J. Exp. Med.
171, 415-426 (1990); Shalaby, M. R. et al.,
J. Exp. Med.
172, 1517-1520 (1990)]. Agonist antibodies to TNF-R1 with antiviral activity are disclosed in copending application Ser. No. 07/856,989 filed Mar. 24, 1992.
In addition, polyclonal antibodies to both murine TNF-R1 and TNF-R2 have been developed, have been shown to behave as specific receptor agonists and induce a subset of murine TNF activities. While the murine TNF-R1 was shown to be responsible for signaling cytotoxicity and the induction of several genes, the murine TNF-R2 was shown to be capable of signaling proliferation of primary thymocytes and a cytotoxic T cell line, CT6 [Tartaglia, L. A. et al.,
Proc. Natl. Acad. Sci
. USA 88, 9292-9296 (1991)]. The ability of TNF-R2 to stimulate human thymocyte proliferation has been demonstrated in experiments with monoclonal antibodies directed against the human receptor.
Monoclonal antibodies against human TNF-R1 that block the binding of TNF to TNF-R1 and antagonize several of the TNF effects have also been described [Espevik, T. et al., Supra; Shalaby, M. R. et al., Supra; Naume, B. et al.,
J. Immunol.
146, 3035-3048 (1991)].
In addition, several reports described monoclonal antibodies directed against TNF-R2 that can partially antagonize the same TNF responses (such as cytotoxicity and activation of NF-&kgr;B) that are induced by TNF-R1 agonists [Shalaby, M. R. et al., Supra; Naume, B. et al., Supra; and Hohmann, H. P. et al.,
J. Biol. Chem.
265, 22409-22417 (1990)].
It is now well established that although the two human TNF receptors are both active in signal transduction, they are able to mediate distinct cellular responses. While TNF-R1 appears to be responsible for signaling most TNF responses, the thymocyte proliferation stimulating activity of TNF is specifically mediated by TNF-R2. In addition, TNF-R2 activates the transcription factor NF-&kgr;B (Lenardo & Baltimore,
Cell
58: 227-229 [1989]) and mediates the transcriptional induction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene (Miyatake et al.,
EMBO J.
4: 2561-2568 [1985]; Stanley et al.,
EMBO J.
4: 2569-2573 [1985]) and the A20 zinc finger protein gene (Opipari et al.,
J. Biol. Chem.
265: 14705-14708 [1990]) in CT6 cells. TNF-R2 also participates as an accessory component to TNF-R1 in the signaling of responses primarily mediated by TNF-R1, like cytotoxicity ( [Tartaglia, L. A. and Goeddel, D. V.,
Immunol. Today
151-153 [1992]).
SUMMARY OF THE INVENTION
Although TNF itself, the TNF receptors and TNF activities mediated by the two receptors have been studied extensively, the post-receptor signal transduction mechanisms are unknown (see the review article by Beyaert, R. & Fiers, W., “Molecular mechanisms of tumor necrosis factor-induced cytotoxicity: what we do understand and what we do not”,
FEBS Letters
340, 9-16 (1994)). This is especially true for the very first step in the TNF receptor signal transduction cascade, i.e. for the question of how the membrane-bound receptor sends a signal into the cell after activation by the ligand, TNF.
The present invention is based on the hypothesis that polypeptide factors associated with the intracellular domain of TNF-R2 exist and participate in the TNF-R2 signal transduction cascade. More specifically, this invention is based on research directed to the identification and isolation of native polypeptide factors that are capable of association with the intracellular domain of TNF-R2 and participate in the intracellular post-receptor signaling of TNF biological activities.
It is known that the TNF induced proliferation of murine CT6 cells is mediated by TNF-R2 (Tartaglia et al., [1991], supra). To identify factors that are associated with the intracellular domain of hTNF-R2, the receptor was immunoprecipitated from lysates of [
35
S]-labeled transfected CT6 cells and from unlabeled transfected human embryonic kidney 293 cells, which were then incubated with labeled lysate from untransfected CT6 cells. Several polypeptides wi
Goeddel David V.
Rothe Mike
Genentech Inc.
Marschang Diane L.
Ulm John
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