Noncleavable Fas ligand

Details Noncleavable Fas ligand Noncleavable Fas ligand

1998-01-14

2002-09-17

Spector, Lorraine (Department: 1646)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000, C530S351000, C435S320100, C435S325000, C435S365100, C435S252300, C435S254110, C536S023500, C536S023100, C424S450000

Reexamination Certificate

active

06451759

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to the fields of molecular biology and molecular medicine and more specifically to the identification of novel polypeptides comprising a noncleavable form of a Fas ligand and having a capacity to activate a Fas receptor-mediated pathway involved in programmed cell death, and pharmaceutical compositions thereof, and therapeutic and prophylactic methods employing such polypeptides.
BACKGROUND OF THE INVENTION
Programmed cell death, or apoptosis, is a physiological process that guarantees that homeostasis is maintained between cell proliferation and cell differentiation in nearly all self-renewing tissues of multicellular organisms. Apoptosis permits the elimination of cells that are no longer necessary, are produced in excess, have developed improperly, or have sustained genetic damage. A variety of cell types appear to undergo cell death through such an apoptotic mechanism.
In addition to preserving normal tissue homeostasis, apoptosis also occurs in response to a various external stimuli, including cytotoxic lymphokines, radiation, chemotherapeutic agents, growth factor deprivation, hyperthermia, hormone withdrawal, and infection by some viruses. See. e.g., Kerr et al.,
Cancer
73:2013-2026 (1994), published erratum,
Cancer
73(12):3108 (1994). Accordingly, apoptosis is an inducible phenomenon that can be regulated by mechanisms of regulation that are similar to those involved in other metabolic pathways.
Dysregulation of apoptosis has also been observed and is implicated in the development of diseases resulting from inappropriate cell death or inhibition of cell death. For example, apoptotic dysregulation has been observed in some types of cancer cells which survive for longer periods than corresponding normal cells. The inhibition or failure of the apoptotic mechanism may permit such cells to undergo mutations leading to a transformed or cancerous state. See. e.g., Korsmeyer,
Blood
80:879-886 (1992). Inhibition or failure of the apoptotic cell death mechanism may also contribute to diseases of the immune system by allowing persistence of self-reactive B and T lymphocyte cells, thereby promoting autoimmune disorders. See. e.g., Watanabe-Fukunaga et al.,
Nature
356:314-317 (1992). Apoptotic dysregulation has also been observed in neurodegenerative diseases in which neurons die prematurely.
Apoptosis is mediated, at least in part, by a cell surface receptor protein known as the Fas antigen receptor (“Fas”) (also known as “APO-1 antigen” or “CD95”). Fas is an approximately 45-kDa (kiloDalton) type I transmembrane protein belonging to the tumor necrosis factor (“TNF”)
erve growth factor (“NGF”) receptor family of proteins. Tanaka et al.,
Nature Med
. 2:317-322 (1996); Tanaka et al.,
EMBO J
. 14:1129-1135 (1995). Fas is expressed in a variety of tissues, including the thymus, liver, lung, intestine, heart, and kidney, and in various human cell types, including, for example, lymphocytes, hepatocytes, activated B and T cells, and neutrophils, and carcinoma cells, such as breast, colon, prostate and pancreatic cancer cells. Tanaka et al.,
Nature Med
. 2:317-322 (1996); Kayagaki et al.,
J. Exp. Med
. 182:1777-1783 (1995). As a receptor protein, Fas has been found to transduce extracellular signals into a cell and, as a result, can mediate or trigger apoptosis. Itoh et al.,
Cell
66:233-243 (1991).
Because Fas is expressed on the cell surface, its mechanism of action is believed to be regulated by interacting with or binding to another cell surface protein. One such known protein is Fas ligand (“FasL”), a 40-kDa type II transmembrane protein of the TNF family. FasL has been observed to mediate and induce apoptosis by binding to Fas. Takahashi et al.,
Int'l Immunol
. 6:1567-1574 (1994); Abbas,
Cell
84:655-657 (1996). Human FasL is a polypeptide of 281 amino acids divided into three distinct domains—an intracellular (i.e., cytoplasmic) domain, a transmembrane domain, and an extracellular domain. See, e.g., EP Patent Application, Publ. No. 0 675 200 A1 (published Oct. 4, 1995, issued to Mochida Pharmaceutical Co., Ltd.). FasL is predominantly expressed in activated T cells, but is also expressed in a number of other cell types, including Sertoli cells in the testis and the stroma cells of the retina. Tanaka et al.,
Nature Med
. 2:317-322 (1996).
The interaction between FasL and Fas has been shown to be critical to the regulation of cell number in a large number of tissue and organ systems. Nagata, Adv. in
Immunol
. 57:129-144 (1994); Nagata et al.,
Science
267:1449-1456 (1995). The Fas/FasL system has been implicated, for example, in the pathogenesis of fulminant hepatitis, GVHHD, and AIDS. Kayagaki et al.,
J. Exp. Med
. 182:1777-1783 (1995). Loss of function of the Fas/FasL system has also been observed to result in lymphoproliferative disorders and to accelerate autoimmune disorders in humans and mice. Takahashi et al.,
Cell
76:969-976 (1994).
Conversely, exaggeration of the Fas system appears to cause tissue damage. Tanaka et al.,
Nature Med
. 2:317-322 (1996). Fas expression is upregulated in hepatocytes transformed by human hepatitis C virus. Lymphocytes transformed with human immune deficiency virus (“HIV”), human T cell leukemia (“HTLV−1”) or Epstein-Barr virus (“EBV”) express a high level of Fas and appear to be sensitive to Fasmediated apoptosis. Tanaka et al.,
Nature Med
. 2:317-322 (1996).
The Fas/FasL interaction has been particularly well studied in the immune system. The activation of T cells through the T cell receptor (“TCR”) upregulates both Fas and FasL on such cells. In circumstances of low to moderate TCR stimulation, T cells proliferate. Under conditions of repetitive or high levels of TCR stimulation, T cells are driven toward apoptosis. This phenomenon has been termed “Antigen Induced Cell Death” (“AICD”). The importance of AICD in regulating the immune system has been demonstrated in the LPR mouse. Nagata et al.,
Immunol. Today
16:39-43 (1995). This mouse strain, which has a spontaneous disruption in the Fas gene from the insertion of an endogenous retroviral element, has been shown to be defective in AICD. With age, such mice develop large numbers of non-functional T lymphocytes in lymphoid and non-lymphoid tissues and develop a number of autoimmune syndromes.
The Fas/FasL system also appears to contribute to the phenomenon of immune privilege. It has been shown that in the testis and the anterior chamber of the eye, constitutive expression of FasL may serve to limit the immune response by eliminating Fas-bearing T cells and possibly other Fas-bearing inflammatory cell types. Bellgrau et al.,
Nature
377:630-632 (1995); Griffith et al.,
Science
270:1189-1192 (1995). The Fas system appears to maintain the immune privilege by preventing activated lymphocytes from infiltrating the testis or testis. In the case of the testis, it has been shown that FasL-bearing Sertoli cells can be transplanted across major histocompatibility complex (“MHC”) barriers, with constitutive FasL expression protecting against alloimmune mechanisms. Bellgrau et al., supra. This demonstration has inspired a number of investigators to study the possibility of artificially conferring immune privilege to transplanted organs via the induction of FasL expression. It has also been shown that syngeneic myoblasts expressing FasL can protect allogenic islets from alloimmune destruction. Lau et al.,
Science
273:109-112 (1996).
The mechanism of action by which FasL interacts with Fas and mediates apoptosis has been examined rather extensively. Recent studies have shown that membrane-bound FasL is converted to a soluble 26-kDa form of FasL (“sFasL”) by action of a matrix metalloproteinase-like enzyme. Kayagaki et al.,
J. Exp. Med
. 182:1777-1783 (1995); Tanaka et al.,
Nature Med
. 2:317-322 (1996); Tanaka et al.,
EMBO J
. 14:1129-1135 (1995). Soluble human FasL is believed to comprise an amino acid sequence of the extracellular domain of FasL which has been proteolytically cleaved from membrane-bound Fas

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