DE4008354A1 - NEW PROTEIN, ITS PRODUCTION AND USE - Google Patents

Abstract

The invention relates to soluble glycoproteins of the formula shown in sequence protocol I, their biologically active fragments, and their manufacture. The proteins can be used as diagnostic agents and as active substances in drugs.

Description

T lymphocytes play in the organism through their interaction with specific ones Target cells, or antigen-presenting cells (APC) a central Role in triggering an immune response. One of the effector functions of T cells affects cell mediating cytotoxicity, which requires that it is for the adhesion of the cytotoxic T cells to the corresponding target cells is coming. T helper cells regulate the immune response other effector cells act, recognize an antigen on the surface an APC related to major histocompatibility molecules (HLA) Class II. The interaction of the T cell / APC is also necessary here. Judge by the central position of the T lymphocytes in the immune response most attempts to pharmacologically affect the immune system in a variety of diseases on manipulation of the T lymphocytes and their effector function. T cells can either over the antigen receptor (J. Immunol. 129: 2293 (1982); Nature 303: 808 (1983); J. Exp. Med. 157: 1149 (1983)) by binding the nominal antigen in Class II molecules of the HLA complex are activated or via the CD2 molecule, a 50 KD glycoprotein that targets all human thymocytes and mature T cells is expressed (Cell 36: 897 (1984)).

In vitro, T cells can be identified by a combination of specific monoclonal Antibodies against different molecular regions of the CD2 molecule are activated. With the discovery of a natural ligand for the CD2 molecule, the CD58 (Nature 326: 298 (1987); Nature 326: 400 (1987)) and the demonstration of its stimulating effect on T cell activation could activate this mode of activation as a physiological response to be established. CD58 (see also EP 2 80 578 and PCT / - WO 88/09 820) 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 (PI) -linked form, here the molecule is directly in the cell membrane anchored and has no cytoplasmic part. The sequence of CD58 in the PI-linked form was described in 1987 (Nature 329: 840 (1987)). CD58 binds to CD2 on the T cell surface. The binding of CD58 on the target cell side plays on CD2 on the T lymphocyte side play a crucial role in intercellular adhesion, for example in the interaction of thymocytes / epithelial cells of the thymus or in the process of Cytolysis, whereby cytotoxic T cells must bind to their target cells in order to to set the so-called "lethal hit". Monoclonal antibodies specific  for CD2 and CD58 both inhibit the cytolytic activity of the T cells as well as cell-cell interactions. Both for the interaction of Endothelial cells / lymphocytes, as well as for the recirculation of the lymphocytes the bond between the CD2 and CD58 molecules is crucial.

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 genetically engineered in a known manner prepared, the DNA encoding the protein obtained according to Nature 329: 840 (1987), is used in bacculoviruses and these in one Insect cell line can be produced from Spodoptera frugiperda.

Cells of the insect species Spodoptera frugiperda are identified with Autographa californica nuclear polyhedrosis virus (AcMNPV) infected. Viral DNA is then from extracellular virus particles (ECV) from the supernatant of the cell Line won. In parallel with this DNA extraction is the bacculovirus Expression plasmid pAc436 changed by in vitro mutagenesis in such a way that Corresponding interfaces are created that make it possible for the cDNA of the PI-coupled CD58 into the mutated vector. The construct of the vector / CD58 is propagated by transformation into E. coli bacteria and purified the DNA.

The insect cells are then treated with the wild-type DNA of the AcMNPV and the Plasmid construct co-transfected to subsequently from the supernatant recombinant viruses resulting from homologous recombination, d. H. To win wild-type viruses that have incorporated the plasmid construct. Around To examine the presence of recombinant viruses is a plaque test performed the visually or by hybridization with the radioactive labeled CD58 sample is evaluated to determine whether recombinant Viruses are present.

Such recombinant viruses are then used to infect the insect cells used, which then produce viruses as well as the protein that can be detected intracellularly or in the supernatant.

The insect cell line is available from ATCC. Wild-type viruses (AcMNPV) and expression plasmid pAc436 are described in U.S. Patent 4,745,051, see also Biotechnology 6:47 (1988).  

The protein is purified by affinity chromatography with Using a CD58 specific monoclonal antibody and subsequent Gel filtration. The identity of the protein is verified in gel electrophoresis, checked in Western blot and especially in biological tests.

The new soluble protein can by its inhibition of E-rosetting of T lymphocytes can be characterized with sheep erythrocytes. T lymphocytes of humans spontaneously form rosettes with red blood cells from sheep and from humans. The interaction between erythrocytes and T lymphocytes is based on the binding of CD2 to CD58, which is found in the erythrocyte Membrane is expressed.

The characterization of the new soluble protein can also be done by its inhibitory property in the mixed leukocyte reaction. The mixed lymphocyte response is the in vitro equivalent to graft Rejection reaction. Here lymphocytes are irradiated with allogeneic non-lymphocytes brought together. Because the allogeneic HLA molecules are recognized as "foreign" by lymphocytes, lymphocyte activation occurs and for the generation of cytotoxic effector cells. So cytotoxic T-lymphocytes must be able to exercise their effector function i.a. via their specific receptors on CD58 molecules on the membrane of the target cells adhere.

The properties of the new soluble protein can also be seen in check what are known as cytotoxicity tests, which determine to what extent the new soluble protein is able to cytotoxic Inhibit properties of T cells because of the cytotoxic effector function lymphocytes also through adhesion and activation processes, that mediates the interaction between CD58 and CD2, is made possible. CD58 is present on all target cells and can be used with the CD2 of the effector cell interact.

The new soluble glycoprotein can due to its costimulatory effects activation with certain monoclonal CD2-specific antibodies can be characterized by T lymphocytes in vitro. It also shows surprisingly, like its fragments, the same biological activity like the membrane-bound form of the CD58 molecule. The protein and its Fragments are thus able to target appropriate receptors Bind cells and so the interaction of these cells with other cells, to prevent the CD58 from expressing on the cell membrane.  

The protein can be used in diagnostics to detect natural ligands or in the competitive ELISA of soluble circulating CD58 molecules be used. 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, disease Crohn, lupus erytematodes and autoimmune diseases and graft rejection. But it can also be used for immune stimulation in the case of immune deficiencies be used.

example 1) cell line

The insect cell line from Spodoptera frugiperda (Sf9) (ATCC CRL1711) was at 27 ° C in Grace's Medium (Gibco) with 10% FCS, and 3.3 g / l each Lactalbumin hydrolyzate and yeastolate (Difco) propagated. After reaching a cell density of approx. 9 × 10⁶ cells per 75 cm² tissue culture bottle the cells were treated with approx. 2 ml virus suspension (see under 2.) infected (<10⁸ PFU / ml). The virus suspension was after a one hour incubation removed and replaced with fresh medium. The cell supernatant, which contains both viruses and recombinant protein was collected after 2-4 days and either for more Infections (virus) or related to the purification of the new protein.

2) Autographa californica nuclear polyhedrosis virus (AcMNPV)

Viral DNA was obtained from extracellular virus particles (ECV). Spodoptera frugiperda (Sf9) cells were treated with AcMNPV (approx. 2 × 10⁶ 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 × TE.

The ECV was then cleaned using a linear one Sucrose gradient (25-56%). After centrifugation (90 min at 100,000 g) the viral band was withdrawn, the ECV pelleted again and how Resuspended above and with Proteinase K (approx. 40 µg / ml) Digested for 1-2 h at 50 ° C. After adding Sarkosyl (final concentration: 1%) the incubation was continued for 2 h. The DNA was duplicated extracted with phenol / chloroform, ethanol precipitated and in 0.1 × TE added.  

3) Plasmid construction

The baculovirus expression plasmid pAc436 was by oligonucleotide mediated in vitro mutagenesis modified around the ATG start codon, by replacing the original sequence 2 with the sequence 3 has been.

The vector (pAcX) thus obtained has restriction sites in addition to the XmaI / SmaI another NcoI interface. In the XmaI restriction site the adapter with sequence 4 was then inserted.

The resulting vector pAcY has sequence 5.

The cDNA of the PI-coupled CD58 gene (sequence 1) was digested cut out with the restriction enzymes NcoI and PstI and into the Vector pAcY ligated (figure). The NcoI interface coincides with the ATG Start codon together. After ligation of the CD58 cDNA (0.9 kb NcoI / PstI Fragment) in the NcoI / PstI cut vector pAcY, the plasmid construct pAcY-CD58 increased by transformation in E.coli HB101 and prepared the plasmid DNA. (Maniatis, Fritsch and Sambrook; Molecular Cloning - A laboratory manual (1982)). The purification of the DNA was carried out using a CsCl density gradient. Then became extensive dialyzed (TE), extracted with phenol / chloroform and with ethanol precipitated.

4) Isolation of recombinant CD58-producing Baculo viruses

Sf9 cells were analyzed with the wild-type AcMNPV DNA and the pAcY-CD58 Construction co-transfected.

Sf9 cells were 1 h before transfection at a density of 2 × 10⁶ Seeded cells per 25 cm² bottle. In an Eppendorf tube 1 µg AcMNPV DNA and 2 µg pAcY-CD58 DNA with 0.75 ml transfection Buffer (25 mM HEPES (pH 7.1), 140 mM NaCl, 125 mM CaCl₂ mixed. After Removal of the culture medium from the Sf9 cells became 0.75 ml of fresh complete Grace's medium was added and then the DNA Solution added dropwise. After 4 h of incubation at 27 ° C. the medium is removed from the cells and replaced with 5 ml of fresh medium. The transfected Sf9 cells were incubated for 4-6 days at 27 ° C. The supernatant (ECV) was removed and tested for the plaque Existence of recombinant viruses examined:  

2 × 10⁶ Sf9 cells were seeded in 60 mm tissue culture dishes (Nunc) in 5 ml Grace's medium. After the cells had been attached and the medium had been suctioned off, 1 ml of a dilution (10 ^-2 to 10 ^-5 ) of the recombinant virus supernatant was added. During the one hour incubation, the dishes were swiveled at 15 minute intervals. The virus supernatant was then removed and the cells were covered with a layer of 4 ml of liquid (40 ° C.) soft agarose (50 ml of 3% agarose solution, 40 ml of 2 × Grace's medium, 10 ml of FCS). After incubation at room temperature for one hour, the tissue culture dishes were incubated in a moist chamber at 27 ° C. for 4-6 days.

Recombinant viruses have been detected by

• - Hydridization with ³²P labeled CD58 probe, as well
• - visual evaluation of the virus plaques.

Homogeneous extracellular virus particles were found after three plaque Cleaning won.

The recombinant human CD58 glycoprotein was isolated from the supernatant of infected Sf9 cells cleaned. Monoclonal anti-CD58 antibody TS2 / 9 (ATCC HB205) were activated on ®Sepharose 4B (Pharmacia), respectively the manufacturer's protocol, covalently bound. The TS2 / 9 ®Sepharose 4B Column was used for affinity-chromatographic purification of the recombinant CD58 used, according to the method described in: "Current Protocols In Molecular Biology; F.M. Ausubel et al .; John Wiley &Sons; ISBN 0-471-62594-9 ". The glycine HCl eluted recombinant CD58 glycoprotein was then passed through a gel filtration column Bio-Gel P100 (BIO-RAD) further purified (method: Current Protocols In Molecular Biology, so.). Immunoprecipitations were used to determine protein size and modifications and Western blotting using monoclonal anti-CD58 Antibodies carried out (method: Current Protocols In Molecular Biology, so.). Radiolabeled recombinant CD58 glycoproteins were generated by the metabolic incorporation of ³⁵S-methionine produced (Summers, A manual of methods for Baculovirus Vectors and Insect Cell Culture Procedures (1988)). The representation of the glycolization of the recombinant CD58 Protein was made using endoglycosidases (Endo F and Endo H) or glycoprotein biosynthesis inhibitor tunicamycin (Boehringer Mannheim) according to the manufacturer's instructions. That determined Molecular weight of the protein portion of the recombinant CD58 is approx. 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 determine as follows:

a) Inhibition of E-rosetting of T lymphocytes with sheep erythrocytes

Addition of the new soluble CD58 protein inhibits rosette formation red blood cells up to 100% depending on the dose. This can be done both determine microscopically by counting the rosettes, as well as with a Radioimmunoassay in which ⁵¹Cr-labeled T lymphocytes with sheep erythrocytes and various concentrations of the new soluble CD58 Protein incubated, then carefully resuspended and opened be given a Ficoll gradient. Has rosette formation occurred the T lymphocytes migrate to the sheep erythrocytes Bottom of the centrifuge tube; there is no rosette formation, the radioactive T cells remain on the gradient. These Both compartments are harvested and separated from each other in one gamma spectrometer measured.

b) Inhibition of the Mixed Leukocyte Response (MLR)

The presence of the new soluble protein in this system blocks the mixed lymphocyte response (depending on dose) For the execution of the experiment, see B.B. Michell, S.M. Shiigi in Selected Methods in Cellular Immunology (1980)). The maximum to achieve Inhibition is on the order of 50%. This means that the interaction of CD2 and new protein of importance for the MLR but other co-activating molecules are also present.

c) inhibition of cytotoxic effector function

The new protein inhibits the cytotoxic effector function more conventionally T lymphocytes and the NK cells against their target K562 dose-dependent and partial. In the case of conventional T lymphocytes can achieve up to 50% inhibition of cytotoxic activity will. This value is usually higher for NK cells.

To measure the inhibition, the target cells are marked with ⁵¹ chromium, then cultivated with T-lymphocytes and after 4 hours that of the lysed target cells released ⁵¹chrom in a γ-counter measured. For the execution of the experiment, see B.B. Mitchell, S.M. Shiigi: Selected Methods in Cellular Immunology (1980).  

d) Costimulation of T cell activation via CD2

CD2 and CD58 come into contact via two different epitopes. One of these can cause activation processes via CD2 that another represents a functionally inert binding site for CD58 / CD2. The new protein apparently has both epitopes. Analogous to CD58- expressing cells can namely this protein if with certain monoclonal CD2 antibodies combined, costimulatory effects exercise on the activation of human T lymphocytes in vitro. T- Lymphocytes with monoclonal antibodies (e.g. T11₂ + T11₃) in Incubated in the presence of CD58. The monoclonal antibodies are in submitogenic doses are used, so that only in the presence of a costimulus proliferation occurs. This can be mediated by the new protein and measured as ³H-thymidine uptake (d3 / 4).  

Sequence listing Sequence No. 1 Human LFA-3 (CD58) cloned in the Baculo expression vector (Underlined sequences = Baculo Virus Genome)

Sequence type: nucleotide sequence with an amino acid sequence derived therefrom
Nucleotide sequence: 862 base pairs
Strand shape: double strand
Topology: linear
Type of molecule: cDNA
original origin: human
Features: human CD58

Sequence No. 2

Sequence type: nucleotide
Sequence length: 30 base pairs
Strand form: single strand
Topology: linear
Type of molecule: synthetic oligonucleotide
Original origin: Baculovirus expression plasmid pAc436
Immediate experimental origin: fission product
Characteristics:
between 24 and 25 XmaI cleavage sites
between 27 and 28 SmaI cleavage sites

Sequence No. 3

Sequence type: nucleotide
Sequence length: 30 base pairs
Strand form: single strand
Topology: linear
Type of molecule: vector
Original origin: Baculovirus expression plasmid pAc46
Immediate experimental origin: modified fission product
Characteristics:
between 16 and 17 NcoI cleavage sites
between 24 and 25 XmaI cleavage sites
between 27 and 28 SmaI cleavage sites

Sequence No. 4

Sequence type: nucleotide
Sequence length: 14 + 14 base pairs
Strand shape: mostly double strand
Topology: linear
Type of molecule: synthetic oligonucleotides
Original origin: Baculovirus expression plasmid pAc46
Features: Insert in the XmaI interface
the upper strand has 4 protruding nucleotides at its 5'-end
the lower strand has 4 protruding nucleotides at its 5'-end

Sequence No. 5

Sequence type: nucleotide
Sequence length: 44
Strand shape: double strand
Topology: linear
Type of molecule: plasmid (pAcY)
Characteristics:
between 16 and 17 NcoI cleavage sites
between 34 and 35 PstI cleavage sites
between 41 and 42 SmaI cleft

Claims (6)

1. Protein of the formula according to sequence listing No. 1 and its biological active fragments. 2. A method for producing the proteins according to claim 1 in itself known manner, characterized in that the protein encoding DNA is used in bacculoviruses and this in an insect cell line be produced from Spodoptera frugiperda. 3. Proteins obtainable according to claim 2. 4. Diagnostics containing a protein according to claim 1 or 3. 5. Proteins according to claim 1 or 3 for use in combating Diseases. 6. Use of the proteins according to claim 1 or 3 for the production of Medicines to treat diseases caused by an influence of the immune system.