WO1997018307A1

WO1997018307A1 - Fas ligand fusion protein

Info

Publication number: WO1997018307A1
Application number: PCT/EP1996/005039
Authority: WO
Inventors: Thomas Buehler
Original assignee: Sandoz Erfindungen Verwaltungs GmbH; Novartis Erfindungen Verwaltungs GmbH; Novartis AG; Sandoz AG
Current assignee: Novartis Pharma GmbH Austria; Novartis AG; Sandoz AG
Priority date: 1995-11-16
Filing date: 1996-11-15
Publication date: 1997-05-22
Anticipated expiration: 1998-05-16
Also published as: BR9611734A; EP0879285A1; CA2232876A1; CO4520295A1; ZA969623B; AU7684896A; CN1202200A; GB9523469D0; JP2000500336A

Abstract

A protein comprising the human Fas Ligand protein (hFasL) or a truncated human Fas Ligand protein or a functionally equivalent variant thereof that retain the Fas receptor-binding and apoptosis-inducing properties of hFasL and, linked either directly or indirectly to its C-terminus, a glycophospholipid is useful in preventing or treating tissue or organ graft rejection.

Description

Fas Ligand Fusion Protein

The present invention relates to a Fas Ligand protem-glyco- phospholipid fusion and to its use, e.g. to prevent rejection of tissue or organ transplants

Fas Ligand (FasL) is a 40 kDa type II membrane protein that belongs to the tumor necrosis factor (TNF) /nerve growth factor receptor family and is expressed on immature thymocytes, activated T-cells, nonlymphoid cells in liver, ovary, heart etc. The nucleotide sequence and predicted ammo acid sequence of rat FasL cDNA is disclosed by T Suda et al. in Cell. 1993, Dec. 17, 75(6), 1169-78 SEQ ID No 1 gives the ammo acid sequences for human FasL (Takahashi et al , Inti Immunol. £, 1567-1574, 1994) . The ammo acid sequence of the intact protem is numbered from ammo acid 1 to 281

It is known that FasL interacts with the cell-surface receptor Fas expressed by certain tissue cells and induces apoptosis of these Fas antigen (sometimes also called Fas receptor) expressing cells Mark R Alderson et al J. Exp Med , 181, 71-76, January 1995 disclose that activated mature T-cells express Fas antigen on their cell surface

It is also known that endothelial cells expressing human FasL on their surface can, by interacting with the Fas antigen on cytotoxic T lymphocytes (CTL) induce apoptosis of these T-cells T-cells being involved in graft rejection, it is desirable to obtain specifically apoptosis of the T-cells which attack the transplanted organ or tissue

In accordance with the particular findings of the present m.ention, the present mvention provides in a first aspect

1. A hFasL protem-glycophospholipid fusion.

Such protem will incorporate its lipid tail mto cell membranes, e.g. endothelial cell membranes, and thus present the FasL protem on the cell surface, e.g to bind to Fas receptor present on other cells and to thereby induce apoptosis of such other cells.

As used herein, the term "hFasL protein" encompasses full length human Fas Ligand protein, including the membrane-bound protein (comprising a cytoplasmic domain, a transmembrane region and an extracellular domain) as well as truncated human Fas Ligand proteins and functionally equivalent variants thereof that retain the Fas receptor-binding and apoptosis inducing properties of human Fas Ligand.

Characteristically the hFasL protein comprises at least the extracellular domain of human Fas-Ligand or a functionally equivalent part thereof or a functionally equivalent variant of these. Thus preferably the hFasL protein comprises the polypeptide having the amino acid sequence from position 103 to position 281 inclusive, more preferably from position 106 to position 281 inclusive, or most preferably from position 136 to position 281 inclusive, of the amino acid sequence shown in SEQ ID No. 2 or a functionally equivalent variant thereof.

For the purposes of the present description, a protein is functionally equivalent to the human Fas Ligand protein, if:

i. It has binding specificity for human Fas receptor similar to that of full length human Fas Ligand protein or the hFasL-GPI fusion protein as hereinafter described in the Examples, and

ii. it is capable, when present in as hFasL-glycophospholipid fusion protein, of inducing apoptosis of Fas receptor bearing cells, e.g. Lymphoma L1210-Fas cells, to a similar extent as the hFasL-GPI fusion protein as hereinafter described in the Examples, e.g. when tested in an m vitro assay as described in Example 3.

Also for the purposes of the present description, a protein is a variant of human Fas Ligand protein or of a part thereof, if the protein is at least 70%, preferably at least 80%, or more preferably at least 90% (especially at least 95%) homologous to the human Fas ligand amino acid sequence from position 1 to position 281 inclusive of SEQ ID No. 1 or the corresponding part thereof. In this context, amino acid sequences are at least 70% homologous to one another if they have at least 70% identical or conservatively replaced ammo acid residues in a like position when the sequences are aligned optimally, gaps or insertions or non conservative substitutions m the ammo acid sequences being counted as non- ldentical/non-conservatively replaced residues.

Further in this context and for the purposes of the present description conservative replacements may be made between ammo acids in the followmg groups:

(I) alanine, serine and threonine;

(II) glutamic acid and aspartic acid; (m) arginine and lysme;

(iv) asparagine and glutamine;

(v) isoleucine, leucine, valine and methionine, and

(vi) phenylalanine, tyrosine and tryptophan. It will be appreciated, however, that conservative replacements may be inappropriate withm critical regions of the sequence, such as an active site or bmdmg site, and at the methionine coded by the start codon.

Accordmg to the invention, the hFasL protem is covalently linked by its C-termmal ammo acid, either directly or indirectly to a glycophospholipid, preferably a glycosylated form of phosphatidyl- mositol, termed glycosyl-phosphatidylmositol (hereinafter GPI), e g as disclosed by M.P Lisanti et al in J Membrane Biol 117. 1-10, 1990

In particular embodiments the C-termmal ammo acid of the hFasL protem is linked by an amide bond either directly or via a linker to ethanolamine, which is turn connected through a phosphodiester linkage to an oligo-sacchande of variable composition and structure. The terminal mono-saccharide of this glycan may be non-N-acetylated glucosamme which is linked at the C-1 position to the C-6 hydroxy of the inositol ring on phosphatidylmositol The molecule may further comprise a glycerol lipid moiety which serves as the membrane-anchoring domam.

According to the mvention, the GPI may be of any structure as present m naturally occurring GPI-lmked protems, e.g. hydrolytic enzymes such as alkaline phosphatase or acetylcholinesterase, mammalian antigens such as Thy-1, Thy-3, Ly-6, CD14 or CD16, protozoal antigens such aε variant surface glycoprotein Trypanosoma, cell adhesion molecules such as LFA-3, or complement molecules such as DAF.

In particular embodiments the FasL-glycophospholipid fusion protein of the invention may be represented by the formula I

wherein is 0 or 1,

R is a direct bond or a linker,

R_a is a direct bond or one or more amino acid residues derived from the selected GPI signal each of R_λ and R₂, independently, is a fatty hydrocarbon residue, preferably C_4-24alkyl or C_4-24alkenyl, more preferably C₁₂_₂₂alkyl ^or C_12-2:alkenyl , R₃ is H or 2 Manαl, R₄ is H, Galαl-2 Galαl-⁶Galαl- or 4βGalNAcl when m is 1, f

Galαl or R₄ is Galαl-6Galαl-2 Manαl when is 0 , and

FasL being human FasL, a fragment thereof or a functionally equivalent variant thereof retaining the Fas-binding properties, Gal being galactose, Man being mannose, GalNAc being N-acetyl- galactosamine and GlcNH₂ being non-N-acetylated glucosamine.

R may be a linker as used in the art in fusion proteins to link a C-terminal carboxy group to an amino group. Such a linker is preferably selected to provide flexibility to the protein, particularly to the extracellular domain of the protein. Examples of such linkers include e.g. a sequence of non polar amino acid units, e.g. 3 to 6 non polar α-amino acid units, preferably Gly and/or Ala units, e.g. -Gly-Gly-Gly-Gly-Gly- .

Protein fusions of the invention comprising a linker between the hFasL protein and the glycophospholipid moiety are encompassed by the expressions "hFasL protein-glycophospholipid fusion" and hFasL protein-GPI fusion as used hereinafter.

The hFasL protein-glycophospholipid fusions of the invention may be prepared synthetically by chemical linking of a hFasL protein and a glycophospholipid moiety, optionally in suitably protected followed by removal of protecting groups as required.

Conveniently, however, the hFasL protein-glycophospholipid fusion may be prepared by a recombinant DNA technology process involving expression of a protein comprising the hFasL protein amino acid sequence and post-translational modification of the expressed protein to yield the hFasL protein-glycophospholipid fusion. For such a process the expressed protein characteristically comprises a signal sequence, e.g. a c-terminal sequence of amino acid residues, which act as a trigger and site for post-translational modification of the expressed protein to give the hFasL protein- glycophospholipid fusion.

Thus in a further aspect the invention also provides a nucleotide sequence, e.g. a DNA sequence, coding for a protein comprising the amino acid sequence of a hFasL protein and a signal sequence for post translational modification of the protein to give a corresponding hFasL protein-glycophospholipid fusion, particularly hFasL protein-GPI fusion.

Preferably the nucleotide sequence is a DNA sequence suitable for eukaryotic or bacterial expression. Thus the invention also provides an eukaryotic or bacterial expression vector comprising a DNA sequence coding for a protein comprising the amino acid sequence of a hFasL protein and a signal sequence for post translational modification of the protein to give a corresponding hFasL protein-glycophospholipid fusion, particularly hFasL protein-GPI fusion.

The expression vector typically contains, in addition to the protein coding sequence, appropriate expression control sequences including a suitable promoter, an operator and a ribosome binding site and other appropriate regulatory sequences. The expression vector may also contain one or more selectable markers. The promoter may be inducible by a variety of stimuli, e.g. exposure to a chemical or change in temperature. The promoter may also be cell-specific or cell cycle specific.

The promoter may be any promoter which is active in E.coli, e.g the bacteriophage T7 promoter, and the expression vector may be a plasmid. For E.coli expression a Rl or C01-E1 plasmid-derived vector may be used. For eukaryotic expression, a vector containing a viral promoter, e.g. based on pXMT2 or pXMT3 may be employed. The invention also provides bacterial or eukaryotic host cells transformed with a hFasL protein glycophospholipid fusion, particularly the hFasL fusion GPI protein, coding sequence or an expression vector as described above. Any suitable bacterial host may be used, preferably E. coli. A suitable eukaryotic host is COS cells for transient and CHO cells for stable expression.

Attachment of the glycophospholipid, e.g. GPI, moiety is a post-translational modification which conveniently occurs in the endoplasmic reticulum (ER) of the host cell. As a result of glycophospholipid (e.g. GPI) addition a hydrophobic sequence is typically removed from the carboxy terminus of the nascent protein. Such an hydrophobic sequence is often a necessary portion of the post-translational modification signal εequence. A CAS doublet (cleavage/attachment site), e.g. Ser-Ser, Ser-Gly, Ser-Ala, in conjunction with a hydrophobic carboxy terminus often provides the minimal sequence necessary for glycophospholipid, e.g. GPI, addition. A spacer of 5-20, more preferably 7-14 amino acids may be desirable between the FasL protein sequence and the post- translational modification signal sequence. The hydrophobic domain conveniently functions in the ER to slow or temporarily stop the transit of the nascent protem through the membrane of the ER so that attachment of the GPI moiety can occur

Provided the expressed protein comprises an appropriate post- translational modification signal sequence, addition of glycophospholipid, e.g. GPI, occurs broadly in most host cells

The DNA coding for the protem comprising the ammo acid sequence of the hFasL protem and the post-translational modification signal sequence may be prepared by appropriate ligation of hFasL protem coding sequence and DNA sequence coding for the signal sequence

In a particular embodiment, a hFasL protem-GPI fusion may be prepared by a process which comprises:

a) generating a GPI addition signal sequence e.g. usmg two overlapping oligonucleotides which are filled m using a polymerase Each of the 5 ' end and 3 ' end contams a restriction s te The filled in product is cloned mto an expression vector, e.g PXMT3

b) using a clone contammg the human Fas-ligand cDNA, e.g aε a template, to amplify the extracellular domain of human Fas-ligand, e.g the fragment comprising ammo acids 136 to 281 of the human Fas-ligand sequence The 3' oligonucleotide may be designed so that it encodes the desired lmker and a restriction site at it's 3 end The 5' oligonucleotide preferably does not contain any restriction site but overlaps the signal sequence of human Fas by several nucleotides, e.g 14 nucleotides. The human Fas signal sequence may be generated using two oligonucleotides which are filled m with a polymerase The noncoding oligonucleotide may overlap Fas-ligand by several nucleotides, e.g 10 to 22, preferably 22 nucleotides The Fas-ligand PCR product and the filled in Fas signal sequence are typically spliced, e.g. using the overlap PCR technique, digested and cloned mto the plasmid containing the GPI signal sequence The corresponding hFasL-GPI construct is indicated in Fig 1

Purification or isolation of hFasL-glycophospholipid fusion protem particularly hFasL-GPI, may be accomplished by any method known in the art that does not result in substantial degradation of the fusion prote . Suitable methods are e.g. affinity chromatography, immunoaff ity chromatography, HPLC and FPLC.

The hFasL-glycophospholipid fusion prote of the invention, particularly the hFasL-GPI fusion protem is useful for inducing Fas-mediated cell death, particularly T-lymphocyte death Where cells of a tissue for transplantation bear on their surfaces foreign histocompatibility antigens, these antigens cause cytotoxic T-lymphocyte activation m recipients, leading to donor cell deεtruction after several sequential activation steps . The hFasL protein-glycophospholipid fusion, particularly the hFasL protem-GPI fusion may be useful to treat acute graft rejection A conventional route of therapy for acute graft rejection resultε severe immunosuppression in the recipient host. Treatment with the hFasL protem-GPI fusion should provide a more specific treatment for activated T-lymphocytes, i.e. for T-lymphocytes expressing the Fas antigen which attack the transplanted tissue or organ, not all the T-lymphocytes present in the immune system.

The hFasL protem-glycophospholipid fusion, particularly the hFasL protem-GPI fusion, may also be useful to treat chronic transplant rejection, including both allograft and xenograft rejection.

In a further embodiment the invention provides:

2 A process for incorporating a hFasL prote -glycophospholipid fusion, particularly hFasL protein-GPI fusion, mto endothelial cells of a tissue or an organ, which procesε comprises infusing the organ or mcubatmg a tissue with a purified hFasL protem-glycophospholipid fusion, particularly hFasL protem-GPI fusion.

3. A method for inducing Fas-mediated death of endothelial cells at a targeted tissue or organ comprising infusing the targeted organ or mcubatmg the targeted tissue with a purified hFasL protem-glycophospholipid fusion, particularly hFasL protem-GPI fusion.

4. A method for preventing or treating tissue or organ allograft or xenograft rejection m a subject which comprises infusing the donor organ or cubatmg the donor tissue, with a purified hFasL prote -glycophospholipid fusion, particularly hFasL protein-GPI fusion prior to transplantation.

In the methods of the invention as defined under 3 to 4 above, the protein fusion of the invention is particularly useful in preventing symptoms associated with acute or chronic organ or tissue allo- or xenograft transplant rejection, e.g. heart, lung, combined heart-lung, liver, kidney, pancreatic (complete or partial, e.g. Langerhans islets), skin, corneal transplants or bone marrow, particularly transplant vasculopathies, e.g. graft atherosclerosis .

As alternatives to the above, the present invention also provides:

5. A hFasL protein-glycophospholipid, particularly hFasL protein-GPI fusion for use in any method as defined under 2 to 4 above; or

6. A hFasL protein-glycophospholipid, particularly hFasL protein-GPI fusion for use in the preparation of a pharmaceutical composition for use in any method as defined under 2 to 4 above; or

7. A composition for use in any method as defined under 2 to 4 above comprising a hFasL protein-glycophospholipid, particularly hFasL protein-GPI fusion, together with one or more pharmaceutically acceptable diluents or carrierε therefor.

The following examples are given by way of illustration and are not to be construed as limiting the invention in any way inasmuch as many variations of the invention are possible within the spirit of the invention.

Example 1: Plasmid Construction and Cloning of the DNA encoding hFasL-GPI fusion protein

The GPI addition signal sequence derived from human CD16 is generated using two overlapping oligonucleotides which are filled in using Klenow polymerase. The 5' end contains a PstI and a Spel site, the 3' end an EcoRI site. The filled in product is phoεphorylated (using T4 Kinase) , gel purified and ligated mto PstI/EcoRI digested PXMT3 expression vector.

Oligonucleotides :

Pst Spel GPI5' : GTC ACT AGT TTG GCA GTG TCA ACC ATC TCA TCA TTC TCT

CCA CCT GGG TAC CAA GTC TCT TTC TGC TTG GTG ATG GTA

(SEQ ID No. 3)

EcoRI

GPI3": GTC GAA TTC TCA AAT GTT TGT CTT CAC AGA GAA ATA TAG TCC TGT GTC CAC TGC AAA AAG GAG TAC CAT CAC CAA GCA GAA (SEQ ID No. 4)

A clone contammg the human Fas-ligand cDNA is used as a template to amplify the extracellular domain of human Fas-ligand comprising ammo acids 136 to 281 of the published human Fas-ligand sequence. The 3' oligonucleotide (Fas4) is designed so that it encodes for additional 6 glycme residues and a Spel site at it's 3' end. The 5' oligonucleotide (Fas3) does not contain any restriction site but overlaps the signal sequence of human Fas by 14 nucleotides. The human Fas εignal sequence is generated using two oligonucleotides (Fasl, Fas2) which are filled in with Klenow polymerase. The noncoding oligonucleotide overlaps Fas-ligand by 22 nucleotides. The Fas-ligand PCR product and the filled m Fas signal sequence are spliced using the overlap PCR technique, gel purified, digested with PstI and Spel and cloned into similarly digested above described plasmid containing the GPI signal sequence. Oligonucleotides:

PstI

FAS1: TCT CTG CAG ATG CTG GGG ATC TGG (SEQ ID No. 5)

FAS2 : GGG TGG AGC AAC AGA CGT AAG AAC CAG AGG TAG GAG GGT CCA GAT GCC CAG CAT CTG CAG AGA (SEQ ID No. 6)

FAS3 : TTA CGT CTG TTG CTC CAC CCC CTG AAA AAA AGG AG (SEQ ID No. 7)

Spel

FAΞ4: CAA ACT AGT GCC ACC ACC GCC TCC ACC GAG CTT ATA TAA GCC GAA AAA CG (SEQ ID No. 8)

The entire construct is sequenced using the T7 sequencing kit from Pharmacia Biotech (Cat. No. 27-168201) .

Example 2; Purification

The recombinant fusion protein is purified using a Fas-Fc affinity column (or an anti FasL antibody affinity column) as described by T. Suda and S. Nagata in J. Exp. Med. 179: 873-879, 1994. An endotoxin free fusion protein is obtained.

Exam le 3: Transient expression of hFasL-GPI in COS cells

9x10- cells are seeded in DMEM, 10% FCS on a 60 mm plate and incubated over night at 37°C, 5% C0₂. Cells are calcium phosphate transfected with 6 μg of plasmid DNA (hFasL-GPI) using the ProFection Mammalian Transfection System (Promega) . As shown in Fig. 2, cells are analyzed by FACS 36 h after transfection using an anti-human Fas-ligand primary monoclonal antibody (clone NOK-1; Pharmingen) and a phycoerythrin labeled anti mouse IgG secundary antibody. PXMT3 mock transfected COS cells are used as negative cont^rols, COS cellε transfected with a construct containing human Fas-ligand cDNA are used as positive controls (Fig. 3) .

Fig. 2: FACS analysis of COS cells transiently transfected with hFasL-GPI expression construct Fig. 3: Positive and negative controls The infusion or the incubation with a hFasL-glycophospholipid fusion protein according to the invention may advantageously be performed at a temperature of about 4° to 37° C. Preferably it is carried out for a duration period of about 2 to 20 hours. The hFasL-glycophoεpholipid fusion protein of the invention may be added to a solution or preparation as usually used for storing donor tissue or a donor organ prior to transplantation, e.g. a so-called "University of Wisconsin solution" . Alternatively, the fusion protein may also be used in saline optionally buffered, e.g. phosphate buffered saline, or in physiologically solution. The concentration of hFasL-glycophospholipid may vary; it may advantageously be 10-40 mg/ml of infusion or incubation solution.

Utility of the hFasL-glycophospholipid fusion protein of the invention may be demonstrated for example in accordance with the methodε hereinafter described.

In vitro Assay

COS cells are transiently transfected with the construct of Example 1 or with a control construct encoding the entire hFasL protein (without glycophospholipid moiety) . This leads to incorporation into the cell membrane via transmembrane domain and expression on the cell surface. The apoptotic effect of above mentioned cellε, native COS cells and COS cells incubated with purified hFasL-GPI on Cr labeled lymphoma L1210 and lymphoma L1210-FAS are compared (Lymphoma cell lines: Schulz M. et al . Eur. J. Immunol. 25: 474-480, 1995) . Cell death of Lymphoma L1210-Fas is significantly higher with COS cells transfected with the construct of Example 1 due to Fas-L induced apoptosis.

In vivo Assays

A. Mouse heart infusion

Mouse hearts are infused with a University of Wisconsin solution (Eurocollinε εolution) comprising 25 mg/ml of hFasL-GPI for 8 hours at 37°C. Paraffin sections of the hearts are probed with biotinylated hFasL antibodies for assessment of the hFasL coating. B. Vascularized heterotopic heart transplantation

Following heparmization and simultaneous exsangumation of the donor animal, the thorax is opened and packed with ice The donor heart is prepared by ligation and division of the superior vena cava inferior vena cava, left pulmonary artery and right pulmonary veins The aorta is ligated and divided distal to the branchiocephalic trunk which is divided at the first bifurcation (right common carotid and subclavian arteries) . Additional cold heparinized salme is infused via the brachiocephalic stump. The organ is infused with recombinant hFasL-GPI fusion protem of Example 2 Remaining pulmonary veins are ligated in one ligature and the heart removed into cold salme.

The hearts are implanted onto the recipients abdominal vessels brachiocephalic trunk to aorta and right pulmonary artery to inferior vena cava with end-to-side anastomoses using 11/0 Ethilon (Ethicon, Norderstedt, Germany) continuous sutures. Animals are closed in two layers with 6/0 Vicryl (Ethicon) and kept warm until fully recovered Total lschaemia times are m the range of 40-50 mm of which 25-35 mm are at 4^CC During anastomosis (10-15 mm) the graft is kept cold.

After transplantation, graft function is monitored by daily assessment of graft beat (palpation) Rejection is considered to be complete when heart beat stopps In all experiments rejection is confirmed by histological examination of the grafts. Significant improvements are obtained with hearts infused with hFasL-GPI fusion protem prior to transplantation compared with control animals (no hFasL-GPI infusion prior to transplantation)

The use of the fusion protem of the mvention to prevent or treat graft rejection may be combmed with an immunosuppressive treatment, e.g administration of an immunosuppressive agent to the recipient after transplantation such as cyclosporin A, cyclosporin G, FK 506, leflunomide or an analogue thereof, mizoribme, mycophenolic acid, mycophenolate mofetil, immunosuppressive monoclonal antibodies, e.g. monoclonal antibodies to leucocyte receptors, e.g. MHC, CD2 , CD3 , CD4, CD7 , CD25, CD28, CTLA4 , B7 , CD45 or CD58 or their ligands. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT:

(A) NAME: Sandoz Ltd

(B) STREET: Lichtstrasse 35

(C) CITY: Basel

(D) STATE: BS

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP) : CH-4002

(G) TELEPHONE: 061-324-2327 (H) TELEFAX: 061-322-7532

(A) NAME: Sandoz Patent GMBH

(B) STREET: Humboldtstrasse 3

(C) CITY: Loerrach

(E) COUNTRY: Germany

(F) POSTAL CODE (ZIP) : D-79539

(A) NAME: Sandoz-Erfindungen Verwaltungsgesellschaf

MBH

(B) STREET: Brunner Strasse 59

(C) CITY: Vienna

(E) COUNTRY: Austria

(F) POSTAL CODE (ZIP) : A-1230

(n) TITLE OF INVENTION: Fas Ligand Fusion Protein (ill) NUMBER OF SEQUENCES: 8

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY- unknown

(ii) MOLECULE TYPE: protein

(in) HYPOTHETICAL: NO

(in) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Gin Gin Pro Phe Asn Tyr Pro Tyr Pro Gin Ile Tyr Trp Val Asp 1 5 10 15

Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys 20 25 30 Pro Thr Ser Val Pro Arg Arg Pro Gly Gin Arg Arg Pro Pro Pro Pro 35 40 45

Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro 50 55 60

Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly 65 70 75 80

Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly 85 90 95

Leu Gly Leu Gly Met Phe Gin Leu Phe His Leu Gin Lys Glu Leu Ala 100 105 110

Glu Leu Arg Glu Ser Thr Ser Gin Met His Thr Ala Ser Ser Leu Glu 115 120 125

Lys Gin Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg 130 135 140

Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu 145 150 155 160

Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr 165 170 175

Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr 180 185 190

Ser Lys Val Tyr Phe Arg Gly Gin Ser Cys Asn Asn Leu Pro Leu Ser 195 200 205

His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gin Asp Leu Val Met 210 215 220

Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gin Met Trp Ala 225 230 235 240

Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Aεp His 245 250 255

Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser 260 265 270

Gin Thr Phe Phe Gly Leu Tyr Lys Leu 275 280

(2) INFORMATION FOR SEQ ID NO: 2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

Pro Pro Pro Glu Lys Lys Glu Leu Arg Lys Val Ala His Leu Thr Gly

1 5 10 15

Lyε Ser Asn Ser Arg Ser Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly 20 25 30

Ile Val Leu Leu Ser Gly Val Lys Tyr Lys Lys Gly Gly Leu Val Ile 35 40 45

Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser Lyε Val Tyr Phe Arg Gly 50 55 60

Gin Ser Cys Asn Asn Leu Pro Leu Ser His Lys Val Tyr Met Arg Asn 65 70 75 80

Ser Lys Tyr Pro Gin Asp Leu Val Met Met Glu Gly Lys Met Met Ser 85 90 95

Tyr Cys Thr Thr Gly Gin Met Trp Ala Arg Ser Ser Tyr Leu Gly Ala 100 105 110

Val Phe Asn Leu Thr Ser Ala Asp His Leu Tyr Val Asn Val Ser Glu 115 120 125

Leu Ser Leu Val Asn Phe Glu Glu Ser Gin Thr Phe Phe Gly Leu Tyr 130 135 140

Lys Leu 145

(2) INFORMATION FOR SEQ ID NO: 3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (m) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTCACTAGTT TGGCAGTGTC AACCATCTCA TCATTCTCTC CACCTGGGTA CCAAGTCTCT 60 TTCTGCTTGG TGATGGTA 78

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GTCGAATTCT CAAATGTTTG TCTTCACAGA GAAATATAGT CCTGTGTCCA CTGCAAAAAG 60 GAGTACCATC ACCAAGCAGA A 81

(2) INFORMATION FOR SEQ ID NO: 5:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (in) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCTCTGCAGA TGCTGGGGAT CTGG 24

(2) INFORMATION FOR SEQ ID NO: 6:

(l ) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GGGTGGAGCA ACAGACGTAA GAACCAGAGG TAGGAGGGTC CAGATGCCCA GCATCTGCAG 60

AGA 63 (2) INFORMATION FOR SEQ ID NO: 7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: cDNA (lii) HYPOTHETICAL: NO ( ll) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TTACGTCTGT TGCTCCACCC CCTGAAAAAA AGGAG 35

(2) INFORMATION FOR SEQ ID NO^■ 8:

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH- 50 base pairs

(B) TYPE, nucleic ac d

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (m) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8. CAAACTAGTG CCACCACCGC CTCCACCGAG CTTATATAAG CCGAAAAACG 50

Claims

A protein comprising the human Fas Ligand protein (hFasL) or a truncated human

Fas Ligand protein or a functionally equivalent variant thereof that retain the Fas receptor-binding and apoptosiε inducing propertieε of hFaεL and, linked either directly or indirectly to itε C-terminuε, a glycophospholipid.

A protein according to claim 1 wherein the glyco-phospholipid is glycosyl-phosphatidyl inositol (GPI) .

A protein according to claim 1 of formula I

NH

I

CH,

O

I ^"O- P = O

R₃- 2 Man c- 1 — f : 2 Mar>

wherein m is 0 or 1 , n is 0 or 1 ,

R is a direct bond or a linker, R_a is a direct bond or an amino acid residue deriving from the selected GPI εignal each of R_x and R₂, independently, is a fatty hydrocarbon residue, R₃ is H or 2 Manαl,

R₄ is H, Galol-2 Galαl-⁶Galαl- or 4βGalNAcl when m iε 1,

or R₄ iε Galαl-6Galαl-2 Manαl when iε 0, and

FaεL being hFasL as defined in claim 1 , Gal being galactose, Man being mannose, GalNAc being N-acetyl- galactosamine and Glc H₂ being non-N-acetylated glucosamine.

4. A protein according to any one of the preceding claims wherein hFasL iε the human εoluble polypeptide having the amino acid sequence between amino acid 136 to 281 of hFasL.

5. A protein according to any one of the preceding claims wherein the glycophospholipid is attached by a linker to the C-terminus of hFasL.

6. A protein according to claim 5 wherein the linker is -Gly-Gly-Gly-Gly-Gly- .

7. A nucleotide sequence coding for a protein compriεing the amino acid εequence of a hFaεL protein and a signal sequence for post- translational modification of the protein to give a protein according to claim 1.

8. A process for producing a protein according to claim 1 which comprises

a) chemically linking a hFasL protein and a glycophospholipid moiety, optionally in protected form and, where required, removing the protecting groups, or

b) a recombinant DNA process involving expression of a protein comprising the hFasL protein amino acid sequence and post- translational modification of the expressed protein. 9. A protein according to claim 1 for use m the preparation of a pharmaceutical composition for use in preventing or treating tissue or organ allo- or xeno-graft rejection.

10. A pharmaceutical composition comprising a protein according to claim 1 together with one or more pharmaceutically acceptable diluents or carriers thereof.