WO1991013981A1

WO1991013981A1 - New protein, its manufacture and use

Info

Publication number: WO1991013981A1
Application number: PCT/EP1991/000421
Authority: WO
Inventors: Anni Barbara Endler-Jobst; Stefan Meuer; Reiner Wallich; Marion Albert-Wolf
Original assignee: Basf Aktiengesellschaft
Priority date: 1990-03-15
Filing date: 1991-03-07
Publication date: 1991-09-19
Also published as: DE4008354A1

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

New protein, its production and use

Description 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). 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. Due to the central position of the T lymphocytes in the immune response, most attempts to pharmacologically influence the immune system in a variety of diseases are directed towards manipulation of the T lymphocytes and their effector function. 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)). In vitro, T cells can be activated by a combination of specific monoclonal antibodies that target different regions of the CD2 molecule. 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, this activation mode could be established as a physiological reaction become. CD58 (see also EP 280 578 and PCT / - WO 88/09820) 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 (PΙ) -linked form, here the molecule is anchored directly in the cell membrane and has no cytoplasmic part. The sequence of CD58 in the Pl-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 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

Nature 329: 840 (1987), is used in bacculoviruses and these are produced in an insect cell line from Spodoptera frugiperda. Cells of the insect species Spodoptera frugiperda are infected with Autographa californica nuclear polyhedrosis virus (AcMNPV). Viral DNA is then obtained from extracellular virus particles (ECV) from the supernatant of the cell line. In parallel to this DNA extraction, the 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.

To win wild-type viruses that have incorporated the plasmid construct. In order to examine the presence of recombinant viruses, 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. Here, 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.

Example 1) Cell line

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.

2) Autographa californica nuclear polyhedrosis virus (AcMNPV) Viral DNA was obtained from extracellular virus particles (ECV). 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 μg / 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. After ligation of the CD58 cDNA (0.9 kb Ncol / Psti fragment) into the NcoI / PstI cut vector pAcY, 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.

4) Isolation of recombinant CD58-producing Baculo viruses

Sf9 cells were mixed with the wild-type AcMNPV DNA and the pAcY-CD58

Construction co-transfected.

Sf9 cells were sown 1 h before transfection at a density of 2 × 10 6 cells per 25 cm 2 bottle. In an Eppendorf tube, 1 μg AcMNPV DNA and 2 μg pAcY-CD58 DNA were mixed with 0.75 ml transfection buffer (25 mM HEPES (pH7, 1), 140 mM NaCl, 125 mM CaCl _2. After removing the culture medium from 0.75 ml of fresh complete Grace's medium was added to the Sf9 cells and the DNA solution was then added dropwise After 4 hours of incubation at 27 ° C., the medium was 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 examined in the plaque test for the presence of recombinant viruses: 2 x 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, and the dishes were pivoted at intervals of 15 min during the one-hour incubation. 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

Hydride with 32p labeled CD58 probe, as well

visual evaluation of the virus plaques.

Homogeneous extracellular virus particles were obtained after three plaque cleaning.

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

(1988)). The representation of the glycosylation of the recombinant CD58

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

Addition of the new soluble CD58 protein inhibits rosette formation with red blood cells up to 100% depending on the dose. This can be determined microscopically by counting the rosettes, as well as with a radioimmunoassay in which ⁵¹ cr-labeled T lymphocytes are incubated with sheep erythrocytes and various concentrations of the new soluble CD58 protein, then carefully resuspended and applied to a Ficoll gradient become. If rosette formation has taken place, the T lymphocytes with the sheep erythrocytes migrate to the bottom of the centrifuge tube; if there is no rosette formation, the radioactive T cells remain on the gradient. These two compartments are harvested and measured separately in a gamma spectrometer. b) Inhibition of the Mixed Leukocyte Response (MLR)

The presence of the new soluble protein in this system blocks the mixed lymphocyte response (for

For the execution of the experiment, see B.B. Michell, S.M. Shiigi in Selected Methods in Cellular Immunology (1980)). The maximum inhibition to be achieved is of the order of 50%. This means that the interaction of CD2 and new protein is important for the MLR, but other co-activating molecules are also present. c) Inhibition of cytotoxic effector function

The new protein partially and partially inhibits the cytotoxic effector function of conventional T-lymphocytes and NK cells against their target K562. In the case of conventional T lymphocytes, up to 50% of the cytotoxic activity can be inhibited. This value is usually higher for NK cells.

To measure the inhibition, 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 γ-counter. For the execution of the experiment, see B.B. Mitchell,

SM Shiigi: Selected Methods in Cellular Immunology (1980). 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, the other represents a functionally inert binding site for CD58 / CD2. The new protein apparently has both epitopes. In analogy to cells expressing CD58, 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).

Sequence listing

Sequence No. 1

Human LFA-3 (CD58) cloned in the Baculo expression vector

(Underlined sequences = Baculo Virus Genome)

ATAΑAΑTGA TAACGATCTC GCAAATAAAT AAGTATTTTA CTG.TTTTCGT AACAGTTTTG 60

TAATAAAAAA ACCTATAACC ATG GTT GCT GGG AGC GAC GCG GGG CGG GCC CTG 112

Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu

-28 -20

GGG GTC CTC AGC GTG GTC TGC CTG CTG CAC TGG TTT GGT TTC ATC AGC TGT 164 Gly Val Leu Ser Val Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys

-10 -1

TTT TCC CAA CAA ATA TAT GGT GTT GTG TAT GGG AAT GTA ACT TTC CAT GTA 215 Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val

+1 5 10 15

CCA AGC AAT GTG CCT TTA AAA GAG GTC CTA TGG AAA AAA CAA AAG GAT AAA 266 Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gin Lys ASD Lys

20 25 30

GTT GCA GAA CTG GAAAAT TCT GAA TTC AGA GCT TTC TCA TCT TTT AAA AAT 317

Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn

35 40 45 50

AGG GTT TAT TTA GAC ACT GTG TCA GGT AGC CTC ACT ATC TAC AAC TTA ACA 368 Arg Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tvr Asn Leu Thr

55 60 65

TCA TCA GAT GAA GAT GAG TAT GAA ATG GAA TGG CCA AAT ATT ACT GAT ACC 413 Ser Ser ASD Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Aso Thr

70 75 80 85

ATG AAG TTC TTT CTT TAT GTG CTT GAG TCT CTT CCA TCT CCC ACA CTA ACT 470 Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr Leu Thr

90 95 100

TGT GCA TTG ACT AAT GGA AGC ATT GAA GTC CAA TGC ATG ATA CCA GAG CAT 521 Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gin Cys Met Ile Pro Glu His

105 110 115

TAC AAC AGC CAT CGA GGA CTT ATA ATG TAC TCA TGG GAT TGT CCT ATG GAG 572 Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp Cys Pro Met Glu

120 125 130 135

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

140 145 150

CCA CAA AAA ATA CAG TGT ACT CTT AGC AAT CCA TTA TTT AAT ACA ACA TCA 674

Pro Gln Lys Ile Gin Cys Thr Leu Ser Asn Pro Leu Phe Asn Thr Thr Ser

155 160 165 170

TCA ATC ATT TTG ACA ACC TGT ATC CCA AGC AGC GGT CAT TCA AGA CAC AGA 725 Ser Ile Ile Leu Thr Thr Cys Ile Pro Ser Ser Gly His Ser Arg His Arg

175 180 185 TAT GGA CTT ATA CCC ATA CGA TTA GGA GTA ATT ACA ACA TGT ATT GTG CTG 776 Tyr Ala Leu Ile Pro Ile Pro Leu Ala Val Ile Thr Thr Cys Ile Val Leu

190 195 200

TAT ATG AAT GTT CTT TAA TTGA GAAGACAATT TCTTCATTTT TAGGTATTCT 828 Tyr Met Asn Val Leu Stop

205

GAAATGTGAC AGAAAACCAG ACAGAACCAA CTCCAATTGA TTGGTAACAG AAGATGAAGA 888

CAACAGCATA ACTAAATTAT TTTAAAAACT AAAAAGCCAT CTGATTTCTC ATTT Poly- 942 left baculose sequences

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

Properties: 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 Xmal cleft

between 27 and 28 smal fission points

TAAAAAAACC TATAAATATG CCGGCCCGGG 30

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 Ncol cleavage sites

between 24 and 25 Xmal cleft

between 27 and 28 smal fission points

TAAAAAAACC TATAACCATG GCGGCCCGGG 30

Sequence No. 4:

Sequence type: nucleotide

Sequence length: 14 + 14 base pairs

Strand shape: largest i l s double strand

Topology: linear

Type of molecule: synthetic oligonucleotides

Original origin: Baculovirus expression plasmid pAc46

Features: Insert in the Xmal interface

the upper strand has 4 protruding nucleotides at its 5 'end, the lower strand has 4 protruding nucleotides at its 5 ^' end

CCGGCTGCAG TAC 13

GACGTC ATGGGCC 13

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 Ncol cleavage sites

between 34 and 35 Pstl splitting point

between 41 and 42 smal splitting points

TAAAAAAACC TATAACCATG GCGGCCCGGC TGCAGGTACC CGGG 44

Claims

1. Protein of the formula according to sequence listing No. 1 and its biologically active fragments.

2. A method for producing the proteins according to claim 1 in a manner known per se, characterized in that the DNA coding for the protein is used in bacculoviruses and these are produced in an insect cell line 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 manufacture of medicaments for the treatment of diseases which are caused by an influence on the immune system.