Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Fas T~ Fusion ProtQin
The present invention relates to a Fas Ligand protein~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
~o the tumor necrosis factor (TNF)/nerve growth factor receptor
~amily and is expressed on immature thymocytes, activated T-cells,
nonlymphoid cells in liver, ovary, heart etc. The nucleotide
sequence and predicted amino acid se~uence 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 amino acid sequences for human FasL
(Tak~h~hi et al., Intl. Tm~llnol~ 6, 1567-1574, 1994). The amino
acid sequence of the intact protein is numbered from amino 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. in 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 f; n~i ngS of the present
ir.-~ention, the present invention provides in a ~irst aspect:
1. A hFasL protein-glycophospholipid fusion.
Such protein will incorporate its lipid tail into cell membranes,
e.g. endothelial cell membranes, and thus present the FasL protein
on the cell surface, e.g. to bind to Fas receptor present on other
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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 ~m~in, a trAn~m~mhrane region and an
extracellular ~om~in) 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 ~m~; n of human Fas-Ligand or a functionally
e~uivalent part thereof or a functionally e~uivalent 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. ~ or a
functionally e~uivalent variant thereof.
For the purposes of the present description, a protein is
functionally equivalent to the human Fas Ligand protein, if:
i. It has bin~;ng 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 n 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 ~EQ ID No. 1 or the corresponding part thereof. In
this context, amino acid seguences are at least 70% homologous to
one another if they have at least 70% identical or conservatively
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replaced amino acid residues in a like position when the sequences
are aligned optimally, gaps or insertions or non conservative
substitutions in the amino acid se~uences being counted as non-
identical/non-conservatively replaced residues.
Further in this context and ~or the purposes of the present
description conservative replacements may be made between amino
acids in the ~ollowing groups:
(i) alanine, serine and threonine;
(ii) glutamic acid and aspartic acid;
~iii) arginine and lysine;
(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 within critical regions of the sequence, such as
an active site or binding site, and at the methionine coded by the
start codon.
According to the invention, the hFasL protein is covalently linked
by its C-terminal amino acid, either directly or indirectly to a
glycophospholipid, preferably a glycosylated form of phosphatidyl-
inositol, termed glycosyl-phosphatidylinositol (hereina~ter GPI),
e.g. as disclosed by M.P. Lisanti et al. in J. Membrane Biol. 117,
1-10, 1990.
In particular em~odiments the C-tPrmi n~ 1 amino acid o~ the hFasL
protein is linked by an amide bond either directly or via a linker
to ethanolamine, which is in turn connected through a
phosphodiester linkage to an oligo-saccharide of variable
composition and structure. The t~rm; n~l mono-saccharide of this
glycan may be non-N-acetylated glucosamine which is linked at the
C-l position to the C-6 hydroxy of the inositol ring on
phosphatidylinositol. The molecule may further comprise a glycerol
lipid moiety which serves as the membrane-anchoring ~m~; n .
According to the invention, the GPI may be of any structure as
present in naturally occurring GPI-linked proteins, e.g. hydrolytic
enzymes such as alkaline phosphatase or acetylcholinesterase,
m~mm~lian antigens ~uch as Thy-l, Thy-3, Ly-6, CD14 or CD16,
protozoal antigens such as variant surface glycoprotein
Trypanosoma, cell adhesion molecules such as ~FA-3, or complement
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W097/l8307 _4_ PCT~P96/05039
molecules such as DAF.
In particular embodiments the FasL-glycophospholipid fusion protein
of the invention may be represented by the formula I
NH2~ \--~
I a
NH
CH2
~CH2
o
O--P=O
o
R3--2 Mano~1t2 Man D~1~ 6 H~ OH ~
i O--C--(CH ) --CH
Man~1-- 4GlcNH2a1--O--~--OH 2 ,~ 3 n
~ O~ P=O
0--P = O O
O CH2--CH--ICH2
C IH2 0 0
CH2
C=O C=O
NH2 l l
Rt R2
wherein
m is 0 or 1,
n is 0 or 1,
R is a direct bond or a linker,
R~ is a direct bond or one or more amino acid residues derived from
the selected GPI signal
each of Rl and R2, independently, is a fatty hydrocarbon residue,
preferably C424alkyl or C424alkenyl, more preferably Cl222alkyl
or C1222alkenyl,
R3 is H or 2 Manal,
R4 is H, Galal-2 Galal-6Galal- or 4~GalNAcl when m is 1,
Gal~1
or R4 is Galal-6Gal~1-2 Man~l when m is 0, and
FasL being human FasL, a fragment thereof or a functionally
e~uivalent variant thereof ret~;n;n~ the Fas-binding properties,
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Gal being galactose, Man being mannose, GalNAc being N-acetyl-
galactosamine and GlcNH2 belng non-N-acetylated glucosamine.
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 ~nm~; n 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 a-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 'IhFasL 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-t~rm; n~ 1 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 se~uence is a DNA sequence suitable for
eukaryotic or bacterial expression.
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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 seq~ences
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 COl-El plasmid-derived
vector may be used. For eukaryotic expression, a vector cont~; n; ng
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 t~rm;nl]c of the nascent protein.
Such an hydrophobic sequence is often a necessary portion of the
post-translational modification signal se~uence. A CAS doublet
(cleavage/attachment site), e.g. Ser-Ser, Ser-Gly, Ser-Ala, in
conjunction with a hydrophobic carboxy t~rm;nl1~ often provides the
m;n;mAl 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 ~mA;n
conveniently functions in the ER to slow or temporarily stop the
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transit of the nascent protein 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 protein comprising the amino acid sequence
of the hFasL protein and the post-translational modification signal
se~uence may be prepared by appropriate ligation of hFasL protein
coding sequence and DNA sequence coding for the signal sequence.
In a particular embodiment, a hFasL protein-GPI fusion may be
prepared by a process which comprises:
a) generating a GPI addition signal sequence e.g. using two
overlapping oligonucleotides which are filled in using a
polymerase. Each of the 5' end and 3' end contains a
restriction site. The filled in product is cloned into an
expression vector, e.g. PXMT3.
b) using a clone cont~;n;ng the human Fas-ligand cDNA, e.g. as
a template, to amplify the extracellular ~mA; n of human
Fas-ligand, e.g. the fragment comprising amino acids 136 to 281
of the human Fas-ligand sequence. The 3' oligonucleotide may be
designed so that it encodes the desired linker 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 in with a polymerase. The
noncoding oligonucleotide may overlap Fas-ligand by
several nucleotides, e.g. lO 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 techni~ue,
digested and cloned into the plasmid cont~;n;n~ the GPI signal
sequence. The corresponding hFasL-GPI construct is indicated in
- Fig. l.
Purification or isolation of hFasL-glycophospholipid fusion protein
particularly hFasL-GPI, may be accomplished by any method known in
the art that does not result in substantial degradation of the
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fusion protein. Suitable methods are e.g. affinity chromatography,
immunoaffinity chromatography, HPLC and FPLC.
The hFasL-glycophospholipid fusion protein of the invention,
particularly the hFasL-GPI fusion protein 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 in recipients, leading to donor cell
destruction after several sequential activation steps. The hFasL
protein-glycophospholipid fusion, particularly the hFasL
protein-GPI fusion may be useful to treat acute graft rejection. A
conventional route of therapy for acute graft rejection results in
severe immunosuppression in the recipient host. Treatment with the
hFasL protein-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 protein-glycophospholipid fusion, particularly the hFasL
protein-GPI fusion, may also be useful to treat chronic transplant
rejection, including both allograft and xenograft re~ection.
In a further embodiment the invention provides:
2. A process for incorporating a hFasL protein-glycophospholipid
fusion, particularly hFasL protein-GPI fusion, into endothelial
cells of a tissue or an organ, which process comprises infusing
the organ or incubating a tissue with a purified hFasL
protein-glycophospholipid fusion, particularly hFasL
protein-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 incubating the targeted tissue with a purified hFasL
protein-glycophospholipid fusion, particularly hFasL
protein-GPI fusion.
4. A method for preventing or treating tissue or organ allograft
or xenograft rejection in a subject which comprises infusing
the donor organ or incubating the donor tissue, with a purified
hFasL protein-glycophospholipid fusion, particularly hFasL
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protein-GPI fusion prior to transplantation.
n 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
ph~rm~ceutical 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 carriers
therefor.
The following examples are given by way of illustration and are
not to be construed as limiting the invention in any way ;nA.cml-rh
as many variations of the invention are possible within the spirit
of the invention.
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E8ample 1: Plasmid Construction and Cloning of the DNA encoding
hFasL-GPI fusion protein
The GPI addition signal sequence derived from human CD1~ is
generated using two overlapping oligonucleotides which are filled
in using Klenow polymerase. The 5' end contains a PstI and a Spe
site, the 3' end an EcoRI site. The filled in product is
phosphorylated (using T4 Kinase), gel purified and ligated into
PstI/~coRI digested PXMT3 expression vector.
Oligonucleotides:
Pst Spel
GPI5': GTC ACT AGT TT~ 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 containing the human Fas-ligand cDNA is used as a template
to amplify the extracellular ~om~in of human Fas-ligand comprising
amino acids 136 to 281 of the published human Fas-ligand sequence.
The 3 oligonucleotide (Fas4) is designed so that it encodes for
additional 6 glycine residues and a SpeI site at it's 3' end. The
5' oligonucleotide (Fas3) does not contain any restriction site
but overlaps the signal sequence o~ human Fas by 14 nucleotides.
The human Fas signal 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 in Fas
signal sequence are spliced using the overlap PCR technique, gel
purified, digested with PstI and SpeI and cloned into similarly
digested above described plasmid cont~in;ng the GPI signal
sequence.
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WO97tl8307 ~ PCT~P96/05039
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)
SpeI
FAS4: 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).
Exam~le 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 ~ree fusion protein is obtained.
Ex~m~le 3: Transient expression of hFasL-GPI in COS cells
9x105 cells are seeded in DMEM, 10~ FCS on a 60 mm plate and
incubated over night at 37~C, 5% CO2. Cells are calcium phosphate
transfected with 6 ,ug of plasmid DNA (hFasL-GPI) using the
ProFection M~mm~l ian 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;
ph~rm;ngen) and a phycoerythrin labeled anti mouse IgG secundary
antibody. PXMT3 mock transfected COS cells are used as negative
controls, COS cells transfected with a construct cont~;n;ng 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
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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-glycophospholipid 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
methods hereinafter described.
In vitro Assav
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 tr~n~m~mhrane ~m~jn and
expression on the cell surface. The apoptotic effect of above
mentioned cells, native COS cells and COS cells incubated with
purified hFasL-GPI on Cr labeled lymphoma L1210 and lymphom~
L1210-FAS are compared (Lymphoma cell lines: Schulz M. et al. Eur.
J. Immunol. 2s: 474-480, 199~). Cell death of Lym~hom~ L1210-Fas
is significantly higher with CoS cells transfected with the
construct of Example 1 due to Fas-L induced apoptosis.
In vivo Assavs
A. Mouse heart infusion
Mouse hearts are infused with a University of Wisconsin solution
(Eurocollins solution) 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.
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B. Vascularized heterotopic heart transplantation
Following heparinization and slmultaneous exsanguination o~ the
donor Anim~l ~ the thorax is opened and packed with ice. The donor
heart is prepared by ligation and division of the superior vena
cava, inferior ve~a cava, left pl~1m~n~ry artery and right
pll1m~nAry 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 saline is infused via the brachiocephalic stump. The
organ is infused with recombinant hFasL-GPI fusion protein of
Example 2. R~m~n;ng pl~1m~n~ry veins are ligated in one ligature
and the heart removed into cold saline.
The hearts are implanted onto the recipients ab~m; n~ 1 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 ischaemia times are in the range of
40-50 min of which 25-35 min are at 4~C. During anastomosis (10-15
min) 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 protein prior to transplantation compared with
control An;m~ls (no hFasL-GPI infusion prior to transplantation).
The use of the fusion protein of the invention to prevent or treat
graft rejection may be combined 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,
mizoribine, 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.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) 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
lF) POSTAL CODE (ZIP): D-79539
(A) NAME: Sandoz-Erfindungen Verwaltungsgesellschaft
MBH
(B) STREET: Brunner Strasse 59
(C) CITY: Vienna
(E) COUNTRY: Austria
(F) POSTAL CODE (ZIP): A-1230
(ii) TITLE OF INVENTION: Fas Ligand Fusion Protein
(iii) 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:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 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: 1:
Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro C.ln 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
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W O 97/18307 -15- PCT~EP96/05039
Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro
Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro
Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly
4 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 Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110
Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu
115 120 125
Lys Gln 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 Gln Ser Cys Asn Asn Leu Pro Leu Ser
195 200 205
His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met
210 215 220
Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala
225 230 235 240
Ar~ Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His
245 250 255
Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser
260 265 270
Gln Thr Phe Phe Gly Leu Tyr Lys Leu
275 280
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids
(B) TYPE: amino acid
(C) STRA~h~SS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
CA 02232876 l998-03-24
W O 97/18307 -16- PCT~EP96/05039
(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
Lys Ser Asn Ser Arg Ser Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly
Ile Val Leu Leu Ser Gly Val Lys Tyr Lys Lys Gly Gly Leu Val Ile
Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser Lys Val Tyr Phe Arg Gly
Gln Ser Cys Asn Asn Leu Pro Leu Ser His Lys Val Tyr Met Arg A~n
Ser Lys Tyr Pro Gln Asp Leu Val Met Met Glu Gly Lys Met Met Ser
Tyr Cys Thr Thr Gly Gln 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 Gln Thr Phe Phe Gly Leu Tyr
130 135 140
Lys Leu
145
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 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: 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
CA 02232876 1998-03-24
.
W O 97/18307 -17- PCTAEP96/05039
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GTCGAATTCT CAAATGTTTG TCTTCACAGA GAAATATAGT CCTGTGTCCA CTGCAAAAAG 60
GAGTACCATC ACCAAGCAGA A 8l
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 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: 5:
TCTCTGCAGA TGCTGGGGAT CTGG 24
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 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: 6:
GGGTGGAGCA ACAGACGTAA GAACCAGAGG TAGGAGGGTC CAGATGCCCA GCATCTGCAG 60
AGA 63
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STR~ :ss: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02232876 1998-03-24
W O 97/18307 -18- PCT~EP96/05039
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
TTACGTCTGT TGCTCCACCC CCTGAAAAAA AGGAG 35
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linea~
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAD: NO
(iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CAAACTAGTG CCACCACCGC CTCCACCGAG CTTATATAAG CCGAAAAACG 50
