WO1991013981A1 - Nouvelle proteine, sa fabrication et son utilisation - Google Patents

Nouvelle proteine, sa fabrication et son utilisation Download PDF

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Publication number
WO1991013981A1
WO1991013981A1 PCT/EP1991/000421 EP9100421W WO9113981A1 WO 1991013981 A1 WO1991013981 A1 WO 1991013981A1 EP 9100421 W EP9100421 W EP 9100421W WO 9113981 A1 WO9113981 A1 WO 9113981A1
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WO
WIPO (PCT)
Prior art keywords
cells
protein
lymphocytes
ser
leu
Prior art date
Application number
PCT/EP1991/000421
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German (de)
English (en)
Inventor
Anni Barbara Endler-Jobst
Stefan Meuer
Reiner Wallich
Marion Albert-Wolf
Original Assignee
Basf Aktiengesellschaft
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.)
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Publication date
Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1991013981A1 publication Critical patent/WO1991013981A1/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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70528—CD58
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2806—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 immunoglobulin superfamily against CD2
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2824—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 immunoglobulin superfamily against CD58
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • T-lymphocytes play a central role in triggering an immune response in the organism through their interaction with specific target cells or antigen-presenting cells (APC).
  • APC antigen-presenting cells
  • One of the effector functions of T cells relates to cell-mediating cytotoxicity, which presupposes that the cytotoxic T cells adhere to the corresponding target cells.
  • T helper cells which have a regulatory effect on other effector cells in the immune response, recognize an antigen on the surface of an APC in connection with major histocompatibility molecules (HLA) of class II. The interaction of the T cell / APC is also necessary here.
  • HLA major histocompatibility molecules
  • T cells can be linked either via the antigen receptor (J. Immunol. 129: 2293 (1982); Nature 303: 808 (1983); J. Exp. Med. 157: 1149 (1983)) by binding the nominal antigen activated with class II molecules of the HLA complex or via the CD2 molecule, a 50 KD glycoprotein which is expressed on all human thymocytes and mature T cells (Cell 36: 897 (1984)).
  • T cells can be activated by a combination of specific monoclonal antibodies that target different regions of the CD2 molecule.
  • CD58 is a glycoprotein with a molecular weight of 60-70 KD and is expressed on a number of body cells.
  • Two forms of the CD58 molecule are known: a transmembrane and a so-called phosphatidyl-inositol (P ⁇ ) -linked form, here the molecule is anchored directly in the cell membrane and has no cytoplasmic part.
  • CD58 binds to CD2 on the T cell surface.
  • the binding of CD58 on the part of the target cell to CD2 on the part of the T lymphocytes plays a decisive role in the intercellular adhesion, for example in the interaction of thymocytes / epithelial cells of the thymus or in the process of cytolysis, with cytotoxic T cells binding to their target cells to set the so-called "lethal hit”.
  • Monoclonal antibodies specific for CD2 and CD58 both inhibit the cytolytic activity of
  • T-cell and cell-cell interactions The binding between the molecules CD2 and CD58 is crucial both for the interaction of endothelial cells / lymphocytes and for the recirculation of the lymphocytes.
  • the invention relates to a glycosylated protein with the
  • Sequence listing No. 1 given amino acid sequence and its biologically active fragments.
  • the protein and its fragments are produced in a known manner by genetic engineering, the DNA coding for the protein being obtained in accordance with
  • bacculovirus expression plasmid pAc436 is changed by in vitro mutagenesis in such a way that corresponding interfaces arise which make it possible to ligate the cDNA of the Pl-coupled CD58 into the mutated vector.
  • the construct of the vector / CD58 is propagated by transformation into E. coli bacteria and the DNA is purified.
  • the insect cells are then co-transfected with the wild-type DNA of the AcMNPV and the plasmid construct in order to subsequently use the supernatant to produce recombinant viruses which arise from homologous recombination, i.e.
  • a plaque test is carried out, which is evaluated visually or by hybridization with the radioactively labeled CD58 sample to determine whether recombinant viruses are present.
  • Such recombinant viruses are then used to infect the insect cells, which then produce viruses as well as the protein, which can be detected intracellularly or in the supernatant.
  • the insect cell line is available from ATCC. Wild-type viruses (AcMNPV) and the expression plasmid pAc436 are described in US Pat. No. 4,745,051, see also Biotechnology 6:47 (1988).
  • the protein is purified by affinity chromatography using a CD58-specific monoclonal antibody and then gel filtration. The identity of the protein is checked in gel electrophoresis, in Western blot and above all in biological tests.
  • the new soluble protein can be characterized by its inhibition of E-rosetting of T lymphocytes with sheep erythrocytes.
  • Human T-lymphocytes spontaneously form rosettes with red blood cells from sheep and humans.
  • the interaction between erythrocytes and T-lymphocytes is based on the binding of CD2 to CD58, which is expressed in the erythrocyte membrane.
  • the new soluble protein can also be characterized by its inhibitory properties in the mixed leukocyte reaction.
  • the mixed lymphocyte reaction is the in vitro equivalent of the graft rejection reaction.
  • lymphocytes are brought together with irradiated allogeneic non-lymphocytes. Since the allogeneic HLA molecules are recognized as "foreign" by lymphocytes, lymphocyte activation and the generation of cytotoxic effector cells occur. In order for cytotoxic T lymphocytes to perform their effector function, they must, among other things. Adhere to CD58 molecules on the membrane of the target cells via their specific receptors.
  • the properties of the new soluble protein can also be checked in so-called cytotoxicity tests, in which it is determined to what extent the new soluble protein is able to inhibit the cytotoxic properties of T cells, since the cytotoxic effector function of lymphocytes is also determined by adhesion and Activation processes that mediate the interaction between CD58 and CD2.
  • CD58 is present on all target cells and can interact with the CD2 of the effector cell.
  • the new soluble glycoprotein can be characterized by its costimulatory effects with certain monoclonal CD2-specific antibodies on the activation of T-lymphocytes in vitro. Like its fragments, it surprisingly shows the same biological activity as the membrane-bound form of the CD58 molecule. The protein and its fragments are thus able to bind to corresponding receptors on cells and thus prevent the interaction of these cells with other cells that express CD58 on the cell membrane.
  • the protein can be used in diagnostics for the detection of natural ligands or in the competitive ELISA of soluble circulating CD58 molecules. It is also suitable for the detection and separation of CD2 + cells.
  • the protein can also be used to treat diseases. It is particularly suitable as an immunosuppressant for combating chronic inflammation, such as rheumatoid arthritis, multiple sclerosis, Crohn's disease, lupus erytematodes, and autoimmune diseases and transplant rejections. However, it can also be used for immune stimulation in the case of immune defects.
  • the insect cell line from Spodoptera frugiperda (Sf9) (ATCC CRL1711) was propagated at 27 ° C. in Grace's Medium (Gibco) with 10% FCS, as well as 3.3 g / l lactalbumin hydrolyzate and yeastolate (Difco). After reaching a cell density of approx. 9 x 10 6 cells per 75 cm2 tissue culture bottle, the cells were infected with approx. 2 ml virus suspension (see under 2.) (> 10 8 PFU / ml). The virus suspension was removed after an hour's incubation and replaced with fresh medium. The cell supernatant, which contained both viruses and recombinant protein, was collected after 2-4 days and used either for further infections (virus) or to purify the new protein.
  • AcMNPV Autographa californica nuclear polyhedrosis virus
  • ECV extracellular virus particles
  • Spodoptera frugiperda (Sf9) cells were infected with AcMNPV (approx. 2x10 6 cells / ml) and the supernatant was removed after 48 h.
  • the ECV were pelleted by centrifugation (30 min at 100,000 g) and resuspended in 0.1 x TE.
  • the ECV was then purified using a linear sucrose gradient (25-56%). After centrifugation (90 min at 100,000 g), the viral band was drawn off, the ECV pelleted again and resuspended as described above and digested with proteinase K (approx. 40 ⁇ g / ml) at 50 ° C. for 1-2 h. After adding Sarkosyl (final concentration: 1%), the incubation was continued for 2 h. The DNA was extracted twice with phenol / chloroform, ethanol precipitated and taken up in 0.1 x TE. 3) Plasmid construction
  • the baculovirus expression plasmid pAc436 was modified by oligonucleotide-mediated in vitro mutagenesis around the ATG start codon by replacing the original sequence 2 with sequence 3.
  • the vector thus obtained (pAcX) has an Ncol site in addition to the Xmal / Smal restriction sites.
  • the adapter with the sequence 4 was then inserted into the X mal restriction site.
  • the resulting vector pAcY has sequence 5.
  • the cDNA of the Pl-coupled CD58 gene (sequence 1) was cut out by digestion with the restriction enzymes Ncol and PstI and ligated into the vector pAcY ( Figure).
  • the Ncol interface coincides with the ATG start codon.
  • the plasmid construct pAcY-CD58 was expanded by transformation in E.coli HB101 and the plasmid DNA was prepared. (Maniatis, Fritsch and Sambrook; Molecular Cloning - A laboratory manual (1982)).
  • the DNA was purified using a CsCl density gradient. Subsequently, dialysis was carried out extensively (TE), extracted with phenol / chloroform and precipitated with ethanol.
  • Sf9 cells were mixed with the wild-type AcMNPV DNA and the pAcY-CD58
  • Sf9 cells were sown 1 h before transfection at a density of 2 ⁇ 10 6 cells per 25 cm 2 bottle.
  • 1 ⁇ g AcMNPV DNA and 2 ⁇ g pAcY-CD58 DNA were mixed with 0.75 ml transfection buffer (25 mM HEPES (pH7, 1), 140 mM NaCl, 125 mM CaCl 2.
  • the recombinant human CD58 glycoprotein was purified from the supernatant from infected Sf9 cells.
  • Monoclonal anti-CD58 antibodies TS2 / 9 (ATCC HB205) were covalently bound to activated ® Sepharose 4B (Pharmacia) according to the manufacturer's protocol.
  • the TS2 / 9- ® Sepharose 4B column was used for affinity-chromatographic purification of the recombinant CD58, according to the method described in: "Current Protocols In Molecular Biology; FM Ausubel et al .; John Wiley &Sons; ISBN 0- 471-62594-9 ".
  • the glycine-HCl eluted recombinant CD58 glycoprotein was then further purified via a gel filtration column Bio-Gel P100 (BIO-RAD) (method: Current Protocols In Molecular Biology, see above). To determine the protein size and modifications, immunoprecipitations and Western blotting were carried out using monoclonal anti-CD58 antibodies (method: Current Protocols In Molecular Biology, see above). Radiolabeled recombinant CD58 glycoproteins were produced by the metabolic incorporation of 35s-methionine (Summers, A manual of methods for Baculovirus Vectors and Insect Cell Culture Procedures
  • Proteins were carried out using endoglycosidases (Endo F and Endo H) or glycoprotein biosynthesis inhibitor tunicamycin (Boehringer Mannheim) according to the manufacturer's instructions.
  • the determined molecular weight of the protein portion of the recombinant CD58 is approximately 25 kDa; the glycated precursor molecules have a molecular weight of 37-41 kDa and the molecular weight of the mature form is in the range of 65 kDa.
  • the functional effects of the recombinant new protein can be determined as follows: a) Inhibition of E-rosetting of T-lymphocytes with sheep erythrocytes
  • the new protein partially and partially inhibits the cytotoxic effector function of conventional T-lymphocytes and NK cells against their target K562.
  • conventional T lymphocytes up to 50% of the cytotoxic activity can be inhibited. This value is usually higher for NK cells.
  • the target cells are marked with siChrom, then cultivated with T lymphocytes and after 5 hours the 5ichrome released by the lysed target cells is measured in a ⁇ -counter.
  • CD2 and CD58 come into contact via two different epitopes. One of these can cause activation processes via CD2, the other represents a functionally inert binding site for CD58 / CD2.
  • the new protein apparently has both epitopes.
  • this protein when combined with certain monoclonal CD2 antibodies, can have costimulatory effects on the activation of human T lymphocytes in vitro. T lymphocytes are incubated with monoclonal antibodies (eg Tll 2 + Tll 3 ) in the presence of CD58.
  • the monoclonal antibodies are used in submitogenic doses so that proliferation only occurs in the presence of a costimulus. This can be mediated by the new protein and measured as 3H-thymidine uptake (d3 / 4).
  • CAA TGT AAA CGT AAC TCA ACC AGT ATA TAT TTT AAG ATG GAA AAT GAT CTT 623 Gin Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys Met Glu Asn Asp Leu
  • Sequence type nucleotide sequence with an amino acid sequence derived therefrom. Nucleotide sequence: 862 base pairs
  • Type of molecule synthetic oligonucleotide
  • Type of molecule vector
  • the upper strand has 4 protruding nucleotides at its 5 'end
  • the lower strand has 4 protruding nucleotides at its 5 ' end

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

Abstract

Glycoprotéines solubles de la formule dans le protocole de séquence I ainsi que leurs fragments biologiquement actifs et leur fabrication. Lesdites protéines peuvent être utilisées comme agents de diagnostique ou comme substances actives dans des médicaments.
PCT/EP1991/000421 1990-03-15 1991-03-07 Nouvelle proteine, sa fabrication et son utilisation WO1991013981A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19904008354 DE4008354A1 (de) 1990-03-15 1990-03-15 Neues protein, seine herstellung und verwendung
DEP4008354.3 1990-03-15

Publications (1)

Publication Number Publication Date
WO1991013981A1 true WO1991013981A1 (fr) 1991-09-19

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WO (1) WO1991013981A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0517174A2 (fr) * 1991-06-06 1992-12-09 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha LFA-3 protéine et dérivés
WO1994001547A2 (fr) * 1992-07-09 1994-01-20 Cetus Oncology Corporation Procede permettant de produire des anticorps contre des molecules de surface des cellules
US6001651A (en) * 1998-03-20 1999-12-14 Isis Pharmaceuticals Inc. Antisense modulation of LFA-3

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009820A1 (fr) * 1987-06-03 1988-12-15 Biogen N.V. Sequences adn, molecules adn recombinant, et procedes de production de l'antigene-3 associe a la fonction lymphocyte
WO1989010938A1 (fr) * 1988-05-04 1989-11-16 Dana-Farber Cancer Institute Micelles de proteine
WO1990012099A1 (fr) * 1989-04-10 1990-10-18 Biogen, Inc. Liaison de proteines par phosphatidylinositole

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009820A1 (fr) * 1987-06-03 1988-12-15 Biogen N.V. Sequences adn, molecules adn recombinant, et procedes de production de l'antigene-3 associe a la fonction lymphocyte
WO1989010938A1 (fr) * 1988-05-04 1989-11-16 Dana-Farber Cancer Institute Micelles de proteine
WO1990012099A1 (fr) * 1989-04-10 1990-10-18 Biogen, Inc. Liaison de proteines par phosphatidylinositole

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NATURE, Band 329, Nr. 6142, 29 Oktober 1987 (London), B. Seed "An LFA-3 cDNA en- codes a phospholipid-linked membrane protein homo *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0517174A2 (fr) * 1991-06-06 1992-12-09 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha LFA-3 protéine et dérivés
EP0517174A3 (en) * 1991-06-06 1993-05-12 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Lfa-3 protein and derivatives
US5556943A (en) * 1991-06-06 1996-09-17 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Cell adhesion protein, gene coding for the same, process for preparing the same and carrier onto which the same is immobilized
WO1994001547A2 (fr) * 1992-07-09 1994-01-20 Cetus Oncology Corporation Procede permettant de produire des anticorps contre des molecules de surface des cellules
WO1994001547A3 (fr) * 1992-07-09 1994-03-31 Cetus Oncology Corp Procede permettant de produire des anticorps contre des molecules de surface des cellules
US6001651A (en) * 1998-03-20 1999-12-14 Isis Pharmaceuticals Inc. Antisense modulation of LFA-3

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DE4008354A1 (de) 1991-09-19

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