Mutant forms of Fas ligand and uses thereof

Details Mutant forms of Fas ligand and uses thereof Mutant forms of Fas ligand and uses thereof

1997-11-12

2003-04-08

Spector, Lorraine (Department: 1647)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Fusion protein or fusion polypeptide

C424S198100, C435S069700, C435S069800, C435S325000, C435S252300, C435S320100, C530S350000, C536S023100, C536S023400, C536S023500, C514S012200

Reexamination Certificate

active

06544523

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to Fas ligand muteins and chimeric proteins that act as Fas ligand agonists, and the DNA encoding the same. More particularly, the present invention relates to novel forms of Fas ligand, which when expressed in a transformed host cell, are surface bound in a conventional type II prohormone form but non-cleavable therefrom. These non-cleavable forms of Fas ligand and the transformed host cells expressing them are useful in diagnostic assays and in reducing populations of Fas expressing cells (e.g., activated T cells and activated B cells) both in vitro and in vivo. The Fas ligand and transformed host cells of the present invention are particularly useful as pharmaceutical agents in the treatment of transplant rejection and the various autoinmmune diseases that are well known in the art.
BACKGROUND OF THE INVENTION
The tumor necrosis factor superfamily includes ligands that bind the corresponding members of the tumor necrosis factor receptor superfamily of receptors. Such members of the receptor superfamily include, for example, tumor necrosis factor receptors (TNFR) (type I or 55K or TNFR60 and type II or 75K or TNFR80), CD30, nerve growth factor receptor, CD27, CD40, CD95/APO-1 or Fas, CD120a, CD120b, lymphtoxin beta receptor (LT beta R), and a TRAIL receptor. The receptors of this family are membrane bound and recognize soluble or membrane bound ligands that mediate diverse cellular responses. The corresponding ligands for these receptors include in some cases membrane or soluble forms and include, for example, tumor necrosis factor alpha (TNF&agr;), CD30 ligand, nerve growth factor, CD70/CD27 ligand, CD40 ligand, Fas ligand, and TNF-related apoptosis-inducing ligand TRAIL (as described in Wiley et al, Immunity 3:673-82 (1995)). The ligands of the superfamily, including Fas ligand, are type II transmembrane glycoproteins with beta strands that form a jelly-roll beta-sandwich as described in Lotz et al,
J. of Leukocyte Biol
60: 1-7 (1996), which is hereby incorporated by reference. Treatment of autoimmune diseases present a unique challenge to molecular biology and medical research. Autoimmune diseases affect between 5 and 7% of the human population, often causing chronic debilitating illnesses, as described in Kuby, IMMUNOLOGY, (W.H. Freeman, NY 1992).
Although precise details of an autoimmune response are incompletely understood, the outcome of antigenic stimulation, whether antibody formation or activated T-cells, or tolerance, seems to depend on the same factors whether a reaction to auto-antigen or exogenous antigen, as described in T
HE
M
ERCK
M
ANUAL
, 16th edition (Merck & Co. Inc. 1992). Also described in T
HE
M
ERCK
M
ANUAL
are four classes of auto-antigens. Class 1 is antigens from intracellular regions of the cells of the body that, by virtue of their sequestration from the immune system, are not recognized as “self” in the body once secreted. For example, sympathetic ophthalmia causes the release of eye antigens, and a subsequent self reaction to the antigen. Class 2 is represented by self antigens that may become immunogenic by chemical, physical, or biological alteration, for example, when a chemical couples to a self antigen and produces a “foreign” reaction, for example in contact dermatitis and hypersensitivity to drugs. Class 3 is represented by foreign antigen that cross reacts with self antigen, and induces a self reaction to the self antigen, for example, as shown with the development of encephalitis after rabies vaccination. Class 4 is represented by a mutation in immunoincompetent cells, such as the autoimmune phenomena seen with mammals having lymphoma. Finally, an autoimmune reaction may be epiphenomena, developing secondarily after an immune response to an obscure antigen, for example, a virus. All autoimmune diseases have in common an involvement of the immune system, and many involve either activated B-cells, activated T-cells, or both.
Although various ameliorative and palliative therapies exist for some autoimmune diseases, and while the autoimmune diseases can spontaneously regress in a remission, effective treatment has yet to be developed for treating autoimmune diseases. It would be desirable to advance the capacity of medical and clinical research to develop effective treatments of autoimmune diseases by discovery of new methods and new therapeutic agents targeting the molecular biology of autoimmune diseases.
SUMMARY OF THE INVENTION
The present invention has multiple aspects. In a first aspect, the present invention is directed to non-cleavable forms of Fas ligand, including Fas ligand muteins and Fas ligand chimeras, and to the polynucleotides encoding them. Preferably, the Fas ligand deletion mutein comprises an effective length of the transmembrane region of Fas ligand up to residue 129 of SEQ ID NO: 12 linked by a substantially non-cleavable peptide linkage to the Fas binding domain of human Fas ligand begining from about residue 139 to residue 146 of SEQ ID NO: 12, and the Fas ligand chimera comprises the transmembrane region of a cell surface protein linked by a substantially non-cleavable peptide linkage to the Fas binding domain of human Fas ligand begining from about residue 139 to residue 146 of SEQ ID NO: 12.
A second aspect is directed to a vector (DNA or RNA) comprising a promoter operably linked to a gene encoding a Fas ligand mutein or chimera that when membrane bound is substantially non-cleavable. Preferably, the vector comprises a promoter operably linked to a recombinant gene encoding a Fas ligand deletion mutein or a Fas ligand chimera, the Fas ligand deletion mutein comprising an effective length of the transmembrane region of Fas ligand up to residue 129 of SEQ ID NO: 12 linked by a substantially non-cleavable peptide linkage to the Fas binding domain of human Fas ligand begining from about residue 139 to residue 146 of SEQ ID NO: 12, said Fas ligand chimera comprising the transmembrane region of a cell surface protein linked by a substantially non-cleavable peptide linkage to the Fas binding domain of human Fas ligand begining from about residue 139 to residue 146 of SEQ ID NO: 12.
A third aspect is directed to a transformed host cell, wherein the host cell is transformed with vector (DNA or RNA) encoding a Fas ligand mutein or chimeric protein that when expressed in the cell remains membrane bound.
A fourth aspect of the present invention is directed to compositions comprising the above described host cell and a carrier. In a preferred embodiment, the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.
Another aspect of the present invention is directed to a method for determining the presence in a sample of cells expressing Fas, the method comprising the steps of:
a) providing a transformed cell having a recombinant Fas ligand surface bound thereto, said surface bound, recombinant Fas ligand being resistant to cleavage;
b) contacting said transformed cell with a sample containing cells suspected of having surface bound Fas thereon to obtain a cell mixture;
c) allowing said cell bound Fas to bind to said surface bound recombinant Fas ligand; and
d) observing said cell mixture for rosette formation or clumping, whereby the binding of cells from said sample to said transformed cell indicates the presence of Fas on the surface of cells in said sample.
In another aspect, the present invention is directed to a method for reducing a population of Fas expressing cells, the method comprising the steps of:
a) providing a transformed cell having a recombinant Fas ligand surface bound thereto, said surface bound, recombinant Fas ligand being resistant to cleavage; and
b) contacting a population of Fas expressing cells with said transformed cell, whereby said Fas ligand binds to Fas on said Fas expressing cells, causing said Fas expressing cells that are so bound to undergo apoptosis such that said population is reduced.
In a preferred embodiment of the above method, the Fas expressing cells are activated T cells and/or B cell

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