WO1996040041A2 - ANTICORPS DE L'ANTIGENE Fas CAPABLES D'INHIBER L'APOPTOSE - Google Patents

ANTICORPS DE L'ANTIGENE Fas CAPABLES D'INHIBER L'APOPTOSE Download PDF

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Publication number
WO1996040041A2
WO1996040041A2 PCT/US1996/009153 US9609153W WO9640041A2 WO 1996040041 A2 WO1996040041 A2 WO 1996040041A2 US 9609153 W US9609153 W US 9609153W WO 9640041 A2 WO9640041 A2 WO 9640041A2
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WIPO (PCT)
Prior art keywords
cells
fas
antibodies
antibody
fas antigen
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Application number
PCT/US1996/009153
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English (en)
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WO1996040041A3 (fr
Inventor
Thomas Gesner
Original Assignee
Chiron Corporation
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 Chiron Corporation filed Critical Chiron Corporation
Priority to AU60914/96A priority Critical patent/AU6091496A/en
Publication of WO1996040041A2 publication Critical patent/WO1996040041A2/fr
Publication of WO1996040041A3 publication Critical patent/WO1996040041A3/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the invention relates to the field of immunology. More specifically, the invention relates to antibodies capable of binding Fas antigen and preventing apoptosis, to hybndomas capable of producing the antibodies and to methods of using the antibodies to treat and prevent diseases and symptoms of diseases caused by apoptosis.
  • Background of the Invention relates to antibodies capable of binding Fas antigen and preventing apoptosis, to hybndomas capable of producing the antibodies and to methods of using the antibodies to treat and prevent diseases and symptoms of diseases caused by apoptosis.
  • Fas antigen on cell surfaces was first described by Yonehara et al, (1989) J. Exp. Med. 169:1747-1756. Fas antigen was described as a cell surface component with a molecular weight of approximately 200,000 daltons. Yonehara et al identifed Fas antigen by means a monoclonal antibody capable of binding to Fas antigen. The monoclonal antibody had an activity the authors described as "indistinguishable from the cytolytic activity of TNF.” However, it was pointed out that the molecular weight of Fas antigen is different from that of the TNF receptor. Fas antigen appears on cells that do not express the TNF receptor and on cells that do express the TNF receptor.
  • Fas antigen On those cells positive for Fas antigen and TNF receptor, Fas antigen is co-downregulated with the TNF receptor when the cells are incubated with TNF or with the anti-Fas antigen antibody. Subsequently, the Fas antigen has been detected on the cell surface of myeloid cells, T lymphoblastoid cells and diploid fibroblasts.
  • the cDNA for human Fas has been cloned and sequenced. Itoh et al, (1991) Cell 66:233-243. The sequence was also used to express Fas on the surface of murine T lymphoma WR19L and fibroblast L929 cells. Binding of an anti-Fas antibody resulted in the death of these cells by apoptosis accompanied by fragmentation of chromosomal DNA and fragmentation of nuclei.
  • the Fas Ligand has been cloned by Suda et al, (1993) Cell 75: 1169-1178 and the
  • Fas Ligand is a type ⁇ integral membrane protein with homology to tumor necrosis factor. Interaction of Fas antigen and Fas Ligand leads to apoptosis and is involved in T-cell mediated cytotoxicity.
  • Antibodies to Fas antigen are known in the art to induce apoptosis and have been administered to mice with resulting fulminating hepatitis due to apoptosis in liver cells. Ogaswara et al, (1993) Nature 364:806-809. Further, it is known that autoimmune diseases such as Sj ⁇ gren syndrome, type I diabetes and viral hepatitis are associated with T-cell migration and infiltration of the affected area. Therefore, a need exists in the art for molecules capable of binding Fas and inhibiting apoptosis onset. Brief Description of the Drawings Figure 1 shows the results of experiments utilizing the antibodies of the invention.
  • Figure 3 shows that the antibodies of the invention are capable of blocking the interaction of cells expressing Fas antigen with cells expressing Fas ligand on their respective cell surfaces.
  • SKW 6.4 cells were preincubated with and without antibody and co-cultured with SF9 cells expressing Fas ligand on the cell surface, essentially as describe in Figure 2.
  • Figure 3 A is a photomicrograph of a co-culture without antibody. Multiple rosettes of SF9 (larger) and SKW 6.4 (smaller) cells may be seen indicating cell-cell interactions.
  • Figure 3B is a photomicrograph of a co-culture with antibody and no rosettes are seen.
  • Figure 4 shows the inhibition of Fas ligand killing of SKW 6.4 cells cells were preincubated with C42 or ZB.4 (Immunotech, Westbrook ME) and co-cultured with Sf9 cells expressing Fas ligand on the cell surface essentially as described above. Thymidine incorporation is shown for C42 (open squares) and ZB.4 (closed squares). Incorporation of thymidine for SKW 6.4 cells alone (upper arrow) and for SKW 6.4 cells co-cultured with Sf9-Ligand cells (lower arrow) are shown on the right vertical axis.
  • C42 or ZB.4 Immunotech, Westbrook ME
  • the invention relates to hybridomas capable of producing antibodies capable of recognizing and bind Fas antigen and to inhibit apoptosis upon binding.
  • the invention also relates to the antibodies produced by the hybridomas.
  • the invention relates to methods of treatment using the antibodies.
  • Fas antigen refers to the cell surface antigen cloned by Itoh et al, (1991) Cell 66:233-243.
  • the Fas antigen is approximately 200,000 MW and can mediate cell death by apoptosis.
  • Fas Ligand refers to the cell surface protein cloned by
  • Fas Ligand interacts with Fas antigen and induces apoptosis in the cell having Fas antigen on its surface.
  • antibody encompasses monoclonal antibodies and fragments thereof. Such fragments will include Fab, Fab2, Fv.
  • Antibodies should also be understood to include humanized antibodies. Such antibodies are designed to reduce immunogenicity of the antibodies in human patients and therefore mitigate any HAMA response. Humanization may be accomplished, for example, by a CDR grafting approach in which CDR regions are inserted within a set of human framework regions, or by a veneering approach in which amino acids exposed at the surface of the folded antibody protein are replaced with consensus amino acids from the human framework regions at the same positions (see EP 519,596, published 23 December 1992).
  • Fas + cells e.g. U937 cells
  • RNA pellet may then be solubilized in water and the total RNA preparation incubated with reverse transcriptase to synthesize cDNA.
  • cDNA encoding the extracellular region of Fas with a C-terminus epitope tag (glu-glu) (sFas) may be constructed.
  • the 5' primer may utilize a sequence 5' of the initiating ATG codon preceded by an insertion of a sequence encoding for a Pst I
  • the 3 1 primer may consist of a coding region 5' of ASN ⁇ ,
  • sequence of the 5' and 3' primers may be: 5'- GTACCTGCAGGGAAGCTCTTTCACTTCGGAGG-3 and 5'-
  • Both the baculovirus expression plasmid pAcC13 and PCR product are digested with Pst I and Not I.
  • the plasmid may be further treated with calf intestine alkaline phosphatase.
  • the digested plasmid and PCR product are purified (Glassmilk, BIO-101), and subsequently ligated with T4 ligase.
  • Escherichia coli (E. coli) are transformed with the products of the ligase reaction. Positive colonies (i.e. those colonies containing plasmid with insert) are further screened by PCR using the cloning primers.
  • the cDNA for sFas may be incorporated into baculovirus for expression of sFas in Sf9 cells (Spodopterafrugiperda). Expression in insect cells has also been described by Summers and Smith ( 1987) Texas Agricultural Experiment Station Bulletin No. 1555.
  • kits include the MaxBac kit (Invitrogen, San Diego, CA). The baculovirus infected Sf9 cells lead to the expression and secretion of sFas into culture medium.
  • Affinity purification of sFas sFas contained in Sf9 medium is concentrated over a YM10 membrane (Amicon), then passed over an anti-glu-glu antibody column (anti-glu-glu monoclonal antibody coupled to protein G Sepharose). Bound sFas is eluted with the glu-glu hexapeptide. After separation of the hexapeptide from sFas, the homogeneous sFas is used as antigen for monoclonal antibody production. 6. Production of Sf9 cells expressing Fas-Ligand on the cell surface
  • Sf9 cells capable of expressing full length Fas-Ligand on the cell surface may be developed essentially as described above, using the published sequence of Fas-Ligand (Suda et al, (1993) Cell 75:1169-1178). It will be appreciated that the addition of a glu- glu epitope tag is unnecessary.
  • Fas antigen produced by expression in SF9 cells was used as an immunogen in mice to produce hybridomas capable of producing antibodies which recognize and bind Fas antigen.
  • the method used was essentially that of Kohler and Milstein, (1975) Nature 256:495-497 with a standard PEG fusion modification.
  • Fas antigen produced by in SF9 cells was used as an immunogen, Fas antigen from other sources may also be used. Such sources include recombinant Fas antigen produced in E. coli and yeast such as Saccharomyces cerevisiae.
  • Cells expressing Fas antigen on their surface may also be used as immunogen, including SKW 6.4 and U937 cells.
  • SF9 cells capable of expressing Fas antigen on the cell surface may also be prepared as described in U.S. 5,397,703, the disclosure of which is herein incorporated by reference.
  • Fas ELIS A Primary screens of hybridomas for antibodies capable of binding Fas antigen may be conducted by Fas ELIS A.
  • the Fas ELIS A consists essentially of coating Fas antigen (100 ng or more/well) onto a 96 well plate, blocking said wells with an irrelevant protein (e.g. bovine serum albumin) and incubating with hybridoma supernatants.
  • the wells are then incubated with an enzyme conjugated anti-mouse antibody (e.g. alkaline phosphatase or horse radish peroxidase - coupled to goat, rabbit, donkey or sheep anti mouse immunoglobulin) and develeoped with an appropriate substrate.
  • the wells are read on an ELISA reader (e.g. Dynatech MRX plate reader).
  • Figure 1 shows the results of experiments utilizing four antibodies of the invention.
  • SKW 6.4 cells (2 x 10 4 cells) were preincubated with C33-1.6.1 (C33) (open triangle); C40-1.3.3 (C40) (closed square); C28-3.7.1 (C28) (closed triangle); or C42- 7.4.5 (C42) (closed circle) for 1 hour at 37°C.
  • CHI 1 apoptosis - inducing antibody
  • the results show that C28 and C42 antibodies are potent inhibitors of CHI 1 induced cell death across the range of concentrations tested.
  • C33 and C40 were more effective at higher concentrations of antibody.
  • Their respective isotypes of the four antibodies are C28:IgG ⁇ , C33:IgG ⁇ , C40:IgG2b and C42:IgG ⁇
  • FIG. 2 shows the inhibition of Fas ligand killing of SKW 6.4 cells by the monoclonal antibodies of the invention.
  • SKW 6.4 cells (2.86 x 10 4 cells) were preincubated with C33 (open square); C40 (open diamond); C28 (closed diamond); or C42 (closed square) for 1 hour at 37°C.
  • the cells were then co-cultured for 6 hours at 37°C. The last 2.5 hours in the presence of 1 ⁇ Ci 3 H- thymidine.
  • the results show that that all of the antibodies tested are capable of inhibiting apoptosis.
  • Figure 4 shows the inhibition of Fas ligand killing of SKW 6.4 cells cells were preincubated with C42 or ZB.4 (Immunotech, Westbrook ME) and co-cultured with Sf9 cells expressing Fas ligand on the cell surface essentially as described above. Thymidine incorporation is shown for C42 (open squares) and ZB.4 (closed squares). Incorporation of thymidine for SKW 6.4 cells alone (upper arrow) and for SKW 6.4 cells co-cultured with Sf9-Ligand cells (lower arrow) are shown on the right vertical axis.
  • Figure 3 shows that the antibodies of the invention are capable of blocking the interaction of cells expressing Fas antigen with cells expressing Fas ligand on their respective cell surfaces.
  • SKW 6.4 cells were preincubated with and without antibody and co-cultured with SF9 cells expressing Fas ligand on the cell surface, essentially as describe in Figure 2.
  • Sf9/Fas-L expressing cells were added and the mixture was centrifuged (1500 rpm, 2 minutes) and incubated 1-3 hrs at 25°C-27°C. The cell pellet was gently resuspended and resolved by photomicroscopy.
  • Figure 3 A is a photomicrograph of a co- culture without antibody. Multiple rosettes of SF9 (larger) and SKW 6.4 (smaller) cells
  • Figure 3B is a photomicrograph of a co-
  • a license may be required to make, use, or sell the deposited materials, and no such license is granted hereby.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des anticorps capables de reconnaître et de fixer l'antigène Fas et d'autre part d'inhiber l'apoptose, ainsi que des hybridomes capables de produire ces anticorps. Sont également décrits des procédés pour l'utilisation de ces anticorps afin de traiter des maladies où l'apoptose est impliquée.
PCT/US1996/009153 1995-06-07 1996-06-05 ANTICORPS DE L'ANTIGENE Fas CAPABLES D'INHIBER L'APOPTOSE WO1996040041A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60914/96A AU6091496A (en) 1995-06-07 1996-06-05 Antibodies to fas antigen capable of inhibiting apoptosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US48346195A 1995-06-07 1995-06-07
US08/483,461 1995-06-07

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WO1996040041A2 true WO1996040041A2 (fr) 1996-12-19
WO1996040041A3 WO1996040041A3 (fr) 1997-03-13

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0799891A1 (fr) * 1996-04-01 1997-10-08 Sankyo Company Limited Anticorps recombinant contre l'antigène Fas et son ADN complémentaire
WO1998018487A1 (fr) * 1996-10-31 1998-05-07 Mochida Pharmaceutical Co., Ltd. Agent prophylactique/therapeutique
EP0909816A1 (fr) * 1997-04-01 1999-04-21 Sankyo Company Limited Anticorps contre Fas
EP1176965A1 (fr) * 1999-04-12 2002-02-06 Isis Pharmaceuticals, Inc. Modulation antisens de la transduction de signaux induite par fas
WO2003022299A1 (fr) * 2001-08-01 2003-03-20 Genset S.A. Agonistes et antagonistes de genobix utiles dans le traitement des troubles metaboliques
US6972323B1 (en) 1997-04-01 2005-12-06 Sankyo Company, Limited Anti-Fas antibodies
WO2010102792A3 (fr) * 2009-03-12 2010-11-18 Imed Ab Anticorps humains dirigés contre fas humain et leur utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991010448A1 (fr) * 1990-01-19 1991-07-25 German Cancer Research Center Antigene de surface cellulaire associe a l'apoptose cellulaire
WO1995010540A1 (fr) * 1993-10-14 1995-04-20 Immunex Corporation Antagonistes de fas et leurs applications
DE4447484A1 (de) * 1994-04-08 1995-10-26 Deutsches Krebsforsch Hemmer von Apoptose

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991010448A1 (fr) * 1990-01-19 1991-07-25 German Cancer Research Center Antigene de surface cellulaire associe a l'apoptose cellulaire
WO1995010540A1 (fr) * 1993-10-14 1995-04-20 Immunex Corporation Antagonistes de fas et leurs applications
DE4447484A1 (de) * 1994-04-08 1995-10-26 Deutsches Krebsforsch Hemmer von Apoptose

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
INTERNATIONAL IMMUNOLOGY, vol. 6, no. 11, November 1994, OXFORD, GB, pages 1799-1806, XP000616652 M. ALDERSON ET AL.: "Regulation of apoptosis and T cell activation by Fas-specific mAb." *
INTERNATIONAL IMMUNOLOGY, vol. 6, no. 12, December 1994, OXFORD, GB, pages 1849-1856, XP000616653 S. YONEHARA ET AL.: "Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes." *
THE JOURNAL OF IMMUNOLOGY, vol. 149, no. 10, 15 November 1992, BALTIMORE, MD, USA, pages 3166-3173, XP002024490 J. DHEIN ET AL.: "Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of Apo-1 cell surface antigens." *
VIROLOGY, vol. 209, no. 2, 1 June 1995, SAN DIEGO, CA, USA, pages 288-296, XP002024489 T. TAKIZAWA ET AL.: "Activation of the apoptotic Fas antigen-encoding gene upon influenza virus infection involving spontaneously produced beta-interferon." *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0799891A1 (fr) * 1996-04-01 1997-10-08 Sankyo Company Limited Anticorps recombinant contre l'antigène Fas et son ADN complémentaire
WO1998018487A1 (fr) * 1996-10-31 1998-05-07 Mochida Pharmaceutical Co., Ltd. Agent prophylactique/therapeutique
US7128905B2 (en) 1996-10-31 2006-10-31 Mochida Pharmaceutical Co., Ltd. Method of treating graft versus host disease by administration of a Fas antagonist
EP0909816A1 (fr) * 1997-04-01 1999-04-21 Sankyo Company Limited Anticorps contre Fas
US6972323B1 (en) 1997-04-01 2005-12-06 Sankyo Company, Limited Anti-Fas antibodies
EP1176965A1 (fr) * 1999-04-12 2002-02-06 Isis Pharmaceuticals, Inc. Modulation antisens de la transduction de signaux induite par fas
EP1176965A4 (fr) * 1999-04-12 2005-01-26 Isis Pharmaceuticals Inc Modulation antisens de la transduction de signaux induite par fas
WO2003022299A1 (fr) * 2001-08-01 2003-03-20 Genset S.A. Agonistes et antagonistes de genobix utiles dans le traitement des troubles metaboliques
WO2010102792A3 (fr) * 2009-03-12 2010-11-18 Imed Ab Anticorps humains dirigés contre fas humain et leur utilisation

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WO1996040041A3 (fr) 1997-03-13
AU6091496A (en) 1996-12-30

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