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WO1997018307A1 - Proteine de fusion du ligand fas - Google Patents

Proteine de fusion du ligand fas Download PDF

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Publication number
WO1997018307A1
WO1997018307A1 PCT/EP1996/005039 EP9605039W WO9718307A1 WO 1997018307 A1 WO1997018307 A1 WO 1997018307A1 EP 9605039 W EP9605039 W EP 9605039W WO 9718307 A1 WO9718307 A1 WO 9718307A1
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WO
WIPO (PCT)
Prior art keywords
protein
hfasl
gly
glycophospholipid
gpi
Prior art date
Application number
PCT/EP1996/005039
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English (en)
Inventor
Thomas Buehler
Original Assignee
Novartis Ag
Sandoz-Patent-Gmbh
Novartis-Erfindungen Verwaltungsgesellschaft M.B.H.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Ag, Sandoz-Patent-Gmbh, Novartis-Erfindungen Verwaltungsgesellschaft M.B.H. filed Critical Novartis Ag
Priority to BR9611734A priority Critical patent/BR9611734A/pt
Priority to AU76848/96A priority patent/AU7684896A/en
Priority to JP9518596A priority patent/JP2000500336A/ja
Priority to EP96939066A priority patent/EP0879285A1/fr
Publication of WO1997018307A1 publication Critical patent/WO1997018307A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • 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 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.
  • TNF tumor necrosis factor
  • 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
  • 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
  • Fas antigen sometimes also called Fas receptor
  • 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
  • the present mvention provides in a first aspect
  • 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.
  • lipid tail mto cell membranes e.g. endothelial cell membranes
  • 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.
  • 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.
  • 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.
  • a protein is functionally equivalent to the human Fas Ligand protein, if:
  • hFasL-glycophospholipid fusion protein 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 a ⁇ variant surface glycoprotein Trypanosoma, cell adhesion molecules such as LFA-3, or complement molecules such as DAF.
  • hydrolytic enzymes such as alkaline phosphatase or acetylcholinesterase
  • mammalian antigens such as Thy-1, Thy-3, Ly-6, CD14 or CD16
  • protozoal antigens such a ⁇ variant surface glycoprotein Trypanosoma
  • cell adhesion molecules such as LFA-3
  • complement molecules such as DAF.
  • 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 ⁇ and R 2 , independently, is a fatty hydrocarbon residue, preferably C 4-24 alkyl or C 4-24 alkenyl, more preferably C 12 _ 22 alkyl or C 12-2: alkenyl , R 3 is H or 2 Man ⁇ l, R 4 is H, Gal ⁇ l-2 Gal ⁇ l- 6 Gal ⁇ l- or 4 ⁇ GalNAcl when m is 1, f
  • Gal ⁇ l or R 4 is Gal ⁇ l-6Gal ⁇ l-2 Man ⁇ l 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
  • GlcNH 2 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.
  • a linker is preferably selected to provide flexibility to the protein, particularly to the extracellular domain of the protein.
  • linkers include e.g. a sequence of non polar amino acid units, e.g. 3 to 6 non polar ⁇ -amino acid units, preferably Gly and/or Ala units, e.g. -Gly-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.
  • 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.
  • 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.
  • 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.
  • a nucleotide sequence e.g. a DNA sequence
  • 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 nucleotide sequence is a DNA sequence suitable for eukaryotic or bacterial expression.
  • 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.
  • E.coli expression a Rl or C01-E1 plasmid-derived vector may be used.
  • 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.
  • glycophospholipid e.g. GPI
  • 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.
  • ER endoplasmic reticulum
  • 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 ⁇ equence.
  • a CAS doublet cleavage/attachment site
  • 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
  • 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
  • a hFasL protem-GPI fusion may be prepared by a process which comprises:
  • 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
  • hFasL-glycophospholipid fusion protem particularly hFasL-GPI
  • Suitable methods are e.g. affinity chromatography, immunoaff ity chromatography, HPLC and FPLC.
  • the hFasL-glycophospholipid fusion prote of the invention 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 de ⁇ truction 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 result ⁇ 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 may also be useful to treat chronic transplant rejection, including both allograft and xenograft rejection.
  • a process for incorporating a hFasL prote -glycophospholipid fusion, particularly hFasL protein-GPI fusion, mto endothelial cells of a tissue or an organ comprises infusing the organ or mcubatmg a tissue with a purified hFasL protem-glycophospholipid fusion, particularly hFasL protem-GPI fusion.
  • 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.
  • 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.
  • 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 .
  • 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 .
  • the present invention also provides:
  • 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 carrier ⁇ therefor.
  • 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 pho ⁇ phorylated (using T4 Kinase) , gel purified and ligated mto PstI/EcoRI digested PXMT3 expression vector.
  • 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 ⁇ ignal 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.
  • 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)
  • FA ⁇ 4 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) .
  • 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.
  • 9x10- cells are seeded in DMEM, 10% FCS on a 60 mm plate and incubated over night at 37°C, 5% C0 2 .
  • Cells are calcium phosphate transfected with 6 ⁇ g of plasmid DNA (hFasL-GPI) using the ProFection Mammalian Transfection System (Promega) .
  • 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 depoty antibody.
  • PXMT3 mock transfected COS cells are used as negative cont r ols
  • COS cell ⁇ 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-glycopho ⁇ pholipid 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" .
  • the fusion protein may also be used in saline optionally buffered, e.g. phosphate buffered saline, or in physiologically solution.
  • concentration of hFasL-glycophospholipid may vary; it may advantageously be 10-40 mg/ml of infusion or incubation solution.
  • 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 cell ⁇ , 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.
  • 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 C C During anastomosis (10-15 mm) the graft is kept cold.
  • 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.
  • an immunosuppressive agent e.g administration of an immunosuppressive agent to the recipient after transplantation
  • an immunosuppressive agent e.g administration of an immunosuppressive agent to the recipient after transplantation
  • an immunosuppressive agent e.g administration of an immunosuppressive agent to the
  • ORGANISM Homo sapiens (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO (ill)
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA (ill)
  • HYPOTHETICAL NO (m)
  • ANTI-SENSE NO

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Abstract

Cette protéine comprend, d'une part, la protéine ligand humaine Fas ou une protéine ligand humaine Fas tronquée ou une variante de celle-ci, fonctionnellement équivalente, retenant les propriétés de hFasL, lesquelles consistent à fixer le récepteur de Fas et à induire l'apoptose, et liée soit directement soit indirectement à sa partie C-terminale, et d'autre part, un glycophospholipide. Cette protéine est utile pour prévenir ou traiter le rejet de greffe d'organe ou de tissu.
PCT/EP1996/005039 1995-11-16 1996-11-15 Proteine de fusion du ligand fas WO1997018307A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
BR9611734A BR9611734A (pt) 1995-11-16 1996-11-15 Proteina de fusão de ligante fas
AU76848/96A AU7684896A (en) 1995-11-16 1996-11-15 Fas ligand fusion protein
JP9518596A JP2000500336A (ja) 1995-11-16 1996-11-15 Ｆａｓリガンド融合タンパク質
EP96939066A EP0879285A1 (fr) 1995-11-16 1996-11-15 Proteine de fusion du ligand fas

Applications Claiming Priority (2)

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GBGB9523469.6A GB9523469D0 (en) 1995-11-16 1995-11-16 Organic compounds
GB9523469.6 1995-11-16

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WO1997018307A1 true WO1997018307A1 (fr) 1997-05-22

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JP (1) JP2000500336A (fr)
CN (1) CN1202200A (fr)
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BR (1) BR9611734A (fr)
CA (1) CA2232876A1 (fr)
CO (1) CO4520295A1 (fr)
GB (1) GB9523469D0 (fr)
WO (1) WO1997018307A1 (fr)
ZA (1) ZA969623B (fr)

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WO1998021232A3 (fr) * 1996-11-13 1998-07-09 Chiron Corp Formes mutantes du ligand du fas et leurs utilisations
WO1998042824A3 (fr) * 1997-03-20 1999-01-07 Cellfactors Plc Procedes permettant de selectionner des cellules et utilisations de ces procedes
EP0909816A1 (fr) * 1997-04-01 1999-04-21 Sankyo Company Limited Anticorps contre Fas
WO2000047740A3 (fr) * 1999-02-12 2000-12-07 Amgen Inc Proteines associees au facteur de necrose des tumeurs
WO2001052664A1 (fr) * 2000-01-24 2001-07-26 University Of Louisville Research Foundation, Inc. Modulation immunitaire avec apoptose induite par recepteur de mort cellulaire
US6451759B1 (en) * 1998-01-14 2002-09-17 The Regents Of The University Of California Noncleavable Fas ligand
EP1248645A1 (fr) * 2000-01-03 2002-10-16 Tr Associates, L.L.C. Proteines chimeres et procedes d'utilisation
US6972323B1 (en) 1997-04-01 2005-12-06 Sankyo Company, Limited Anti-Fas antibodies
US7238360B2 (en) 2000-06-30 2007-07-03 Unversity Of Louisville Research Foundation, Inc. Alteration of cell membrane with B7
US7888318B1 (en) 1999-03-10 2011-02-15 Adprotech Limited Method of preparing an organ by perfusion
US7927602B2 (en) 2002-07-23 2011-04-19 University Of Louisville Research Foundation, Inc. Fas ligand-avidin/streptavidin fusion proteins
US9388230B2 (en) 2010-09-28 2016-07-12 Kahr Medical(2005) Ltd Compositions and methods for treatment of hematological malignancies
US20200054676A1 (en) * 2017-02-17 2020-02-20 Purdue Research Foundation Targeted Ligand-Payload Based Drug Delivery for Cell Therapy

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US6544523B1 (en) 1996-11-13 2003-04-08 Chiron Corporation Mutant forms of Fas ligand and uses thereof
WO1998021232A3 (fr) * 1996-11-13 1998-07-09 Chiron Corp Formes mutantes du ligand du fas et leurs utilisations
WO1998042824A3 (fr) * 1997-03-20 1999-01-07 Cellfactors Plc Procedes permettant de selectionner des cellules et utilisations de ces procedes
US6972323B1 (en) 1997-04-01 2005-12-06 Sankyo Company, Limited Anti-Fas antibodies
EP0909816A1 (fr) * 1997-04-01 1999-04-21 Sankyo Company Limited Anticorps contre Fas
US6451759B1 (en) * 1998-01-14 2002-09-17 The Regents Of The University Of California Noncleavable Fas ligand
WO2000047740A3 (fr) * 1999-02-12 2000-12-07 Amgen Inc Proteines associees au facteur de necrose des tumeurs
US7888318B1 (en) 1999-03-10 2011-02-15 Adprotech Limited Method of preparing an organ by perfusion
EP1908780A1 (fr) * 2000-01-03 2008-04-09 Mark L. Tykocinski Nouvelles protéines chimiques et leurs procédés d'utilisation
JP4723782B2 (ja) * 2000-01-03 2011-07-13 ティーアール アソシエイツ，エル．エル．シー． 新規なキメラ蛋白質及び該蛋白質の使用方法
EP1248645A4 (fr) * 2000-01-03 2003-08-06 Tr Associates L L C Proteines chimeres et procedes d'utilisation
JP2003521485A (ja) * 2000-01-03 2003-07-15 ティーアール アソシエイツ，エル．エル．シー． 新規なキメラ蛋白質及び該蛋白質の使用方法
EP1248645A1 (fr) * 2000-01-03 2002-10-16 Tr Associates, L.L.C. Proteines chimeres et procedes d'utilisation
WO2001052664A1 (fr) * 2000-01-24 2001-07-26 University Of Louisville Research Foundation, Inc. Modulation immunitaire avec apoptose induite par recepteur de mort cellulaire
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US8728747B2 (en) 2000-06-30 2014-05-20 University Of Louisville Research Foundation, Inc. Alteration of cell membrane for new functions
US9255133B2 (en) 2000-06-30 2016-02-09 University Of Louisville Research Foundation, Inc. Alteration of cell membrane for new functions using IL-2 and streptavidin
US7927602B2 (en) 2002-07-23 2011-04-19 University Of Louisville Research Foundation, Inc. Fas ligand-avidin/streptavidin fusion proteins
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US10000549B2 (en) 2010-09-28 2018-06-19 Kahr Medical Ltd. Compositions and methods for treatment of hematological malignancies
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JP2000500336A (ja) 2000-01-18
CN1202200A (zh) 1998-12-16
EP0879285A1 (fr) 1998-11-25
GB9523469D0 (en) 1996-01-17
ZA969623B (en) 1998-05-15
CO4520295A1 (es) 1997-10-15
CA2232876A1 (fr) 1997-05-22
BR9611734A (pt) 1999-02-23

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