EP0452433A1 - Proteins inhibant la cytolyse (cti) et sequences adn codant pour lesdites proteines - Google Patents

Proteins inhibant la cytolyse (cti) et sequences adn codant pour lesdites proteines

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Publication number
EP0452433A1
EP0452433A1 EP90914723A EP90914723A EP0452433A1 EP 0452433 A1 EP0452433 A1 EP 0452433A1 EP 90914723 A EP90914723 A EP 90914723A EP 90914723 A EP90914723 A EP 90914723A EP 0452433 A1 EP0452433 A1 EP 0452433A1
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EP
European Patent Office
Prior art keywords
cli
protein
cytolysis
cytolysis inhibitor
inhibitor
Prior art date
Legal status (The legal status 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 status listed.)
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EP90914723A
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German (de)
English (en)
Inventor
Jürg TSCHOPP
Dieter Jenne
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Bayer Pharma AG
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Schering AG
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Publication of EP0452433A1 publication Critical patent/EP0452433A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • C07K14/775—Apolipopeptides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a novel blood plasma component having a strong inhibitory effect on the target cell lysis mediated by terminal complement proteins, such as, for example, by perforin secreted by killer cells or by the ⁇ -toxic of Staphylococcus aureus.
  • the component likewise plays possibly an important role in lipid metabolism.
  • Two effector systems complementing each other, ensure immunological defense against entering viruses, bacteria, virus-transformed and virus-infested cells, endogenous cells which are altered in malignant fashion or due to ageing, against sperms in the female genital tract, and against exogenous material of the environment.
  • the cellular effector system consists of specially matured immune cells, the so-called cytotoxic T-lympho- cytes and the natural killer cells. Lymphocytes and natural killer cells are capable, with the aid of inherent surface receptors, of recognizing exogenous structures on other endogenous cells indicative of abnormal states or in intersticial tissue, as well as structures on foreign materials such as, e.g., invading microorganisms, and of effectively preventing the spreading and proliferation of these exogenous materials.
  • S UBSTITUTE SHEET endogenous cells and microorganism Activated cytotoxic lymphocytes and natural killer cells enter into intimate contact with the target cells recognized as foreign and release membrane-damaging molecules in a controlled fashion, the lymphocytes and killer cells themselves being protected against these molecules.
  • the actual membrane-damaging molecule of the killer cells is the so- called perforin possessing high affinity for the lipophilic membrane of the target cell.
  • Several molecules of perforin penetrate into the double-layered membrane of the target cell and thereafter form a cylinder-like water-permeable channel transversely through the lipid double membrane of the target cell. Water molecules and electrolyte salts can freely move through the hydrophilic inner space of the transmembranous channel.
  • the humoral system of immune defense consists of a plurality of soluble plasma proteins which interact with one another and form a finely staggered cascade similar to that of the clotting system.
  • the actual terminal step in complement activation leads to membrane damage to the target cell according to the same principle as in case of perforin-caused lysis of the target cells. Grouping together of the terminal components of the complement system is initiated by the cleavage of complement protein C5 into two fragments C5a and C5b.
  • the carboxy-terminal main fragment C5b unites with C6, C7 and C8 to a macromolecular complex, the hydrophobicity of which increases considerably with the addition of C7 and C8 and which can bind to the lipid double membrane.
  • the C5b-8 complex proper does not as yet form a functional pore in the lipid membrane. Membrane-bound, it takes over the
  • Soluble perforin monomers and the stable, soluble C5b-6 complex can move away from the site of their release and/or generation and can diffuse into the surroundings so that neighboring healthy cells could be affected. Therefore, it has been supposed for a long time that effective regulatory mechanisms exist which prevent uncontrolled propagation of potentially lytic molecules.
  • the present invention provides purified natural inhibitor for the known immunological effector molecules, i.e., for perforin stemming from T cells and killer cells and for the terminal complement proteins of human plasma, as well as inhibitors produced by genetic engineering.
  • the invention further provides DNA which codes for the specific protein portion of a novel blood plasma component and to a purification process for the natural blood plasma component characterized by the CLI protein molecule and exerting an inhibitory effect on the target cell lysis mediated by complement proteins and on perforin isolated from killer cells.
  • the invention especially concerns the nucleotide base sequence of the CLI-1 clone depicted in Figure 2 and the amino acid sequence derived therefrom.
  • the invention furthermore provides the plasmid pGEM4/CLI-l shown in Figure 1; host organisms transformed with this plasmid; and cytolysis inhibitor from human plasma isolated for the first time.
  • This invention furthermore provides hybridoma cell lines, e.g., hybridoma cell line CLI-9, the monoclonal antibodies, e.g., monoclonal antibody CLI-9 produced by the hybridoma cell line CLI-9, and the use of said antibodies for the isolation and purification of CLI from
  • Figure 1 shows a scheme of the recombinant pGEM4/CLI-l plasmid containing the entire coding sequence for the cytolysis inhibitor, and several important restriction scission sites.
  • the plasmid has an ampicillin resistance gene coding for the enzyme beta- lactamase, and includes the Sp6 and, respectively, T7 promoter in the direct vicinity to the cloning scission site BamHI in the polylinker.
  • the two ends of the cDNA insertion are provided with the adapter oligomers A and B according to Haymerle, H. , Herz, J. , Bressan, G.M. , Frank, R. and Stanley, K.K., Nucleic Acids Res. 154. 8615-8624 (1986) .
  • This plasmid was deposited in E. coli K12 on March 28, 1989 with the DSM (German Microorganism Depository) under DSM No. 5269;
  • Figure 2 shows the nucleotide base sequence of the
  • Figure 3 shows the results of the Southern analysis: the position of the molecular weight markers is indicated in kilobases on the left-hand margin of the figure;
  • FIG. 4a shows the purified CLI after separation by
  • Figure 5 shows a Western blot identification (bars 1 to 3*) of CLI in human plasma (1), in purified C5b-9 complex (2), in seminal fluid (3 and 3').
  • Bar 4 shows the proteins of the seminal fluid after staining the SDS gel with "Coomassie" blue;
  • Figure 6 shows the inhibition of complement lysis by
  • CLI inhibits lysis of the erythrocytes by soluble C5b-6, C7, C8 and C9 in a concentration-dependent way
  • Staphylococcus-A-hemolysin is preincubated with varying
  • Figure 9 shows that ApoA-I-CLI complexes appear in the electron microscope as spherical structures having a diameter of 9.0 ⁇ 1 nm.
  • terminal complement complex contains, in addition to the already known S-protein/vitronectin, another still unknown protein.
  • This unknown protein was located by a monoclonal antibody, CLI-9, which, contrary to the expectation, did not recognize the S-protein. It was found that this protein, cytolysis inhibitor (CLI) , is not a component of the membrane-bound, lytic complement complex.
  • the amino acid sequence of part of the glycoprotein CLI was determined by sequencing the first twenty amino- terminal amino acids in accordance with the Edman degradation method. Synthetic DNA probes were then prepared with ' the aid of these sequences and used to
  • SUBSTITUTESHE screen a human liver-specific cDNA library, according to routine methods recited in Example 1.
  • the novel gene was then cloned from the human genome.
  • This gene codes for a protein molecule carrying the international name of "Cytolysis Inhibitor” (CLI or ZLI) .
  • This polypeptide is an essential and characteristic part of a novel blood plasma component with the above-mentioned properties.
  • this plasmid was analyzed with the aid of restriction enzymes. The size of the DNA insertion in the pGEM4 vector was determined.
  • Purified CLI was found to have a molecular weight of 70 kilodaltons under nonreducing conditions and a molecular weight of 35 kilodaltons under reducing conditions in sodium dodecyl sulfate gel electrophoresis.
  • the protein was also shown to be a glycoprotein. A comparison with all heretofore known sequences published in the literature showed that the complete protein structure of CLI and the corresponding complete nucleotide base sequence had not been determined heretofore.
  • CLI refers to the glycosylated and unglycosylated form of the protein, having the biological activity .of inhibiting cytolysis mediated by cytolytic proteins, such as, e.g., complement or perforin, as well as to all isoforms of CLI which may differ from the natural glycosylated form found in human serum by their carbohydrate content.
  • cytolytic proteins such as, e.g., complement or perforin
  • the term also includes therefore the CLI of seminal plasma which may have other carbohydrate structures.
  • glycoprotein CLI glycoprotein CLI
  • the isolated blood plasma component counteracts undesired cellular destruction. It neutralizes, in a way dependent on concentration, the cytolytic potential of C5b-7 complement complexes in the extracellular fluid and in the plasma. The sensitivity of endogenous cells to complement and perforin lysis is diminished, and the threshold for cellular damage is increased.
  • the glycoprotein CLI also occurs in human seminal fluid. Intensive studies of the natural CLI blood plasma component (Example 2f) yielded the following results. In human blood plasma, CLI is fixedly associated with the apolipoprotein A-I (ApoA-I) , which transports predominantly phospholipids and free as well as esterified cholesterol.
  • the ApoA-I- CLI particle migrates in the pre-beta zone. Since the ApoA-I concentrations in the plasma are about 20 times higher than the CLI concentrations, about 2.5% of the entire plasma ApoA-I is present in the form of an ApoA- I-CLI complex. High-density lipoproteins of the pre- beta fraction represent, on the average, a proportion of 4.2 ⁇ 1.8% of the entire ApoA-I in the plasma. Therefore, the predominant proportion or even the entire fraction of pre-beta HDL could consist of ApoA-I-CLI particles.
  • natural CLI blood plasma component refers to the entire apolipoprotein A-I (ApoA-I)/CLI/lipid complex found in whole human blood. It is further noted that this complex may be heterogeneous, e.g., it may bind additional components of lipid transport pathways, cholesterol- ester-transferrin protein (CETP) , lecithin cholesteryl acyltransferase (LCAT) and/or apolipoprotein A-II.
  • CETP cholesterol- ester-transferrin protein
  • LCAT lecithin cholesteryl acyltransferase
  • the lipid content of the HDL-CLI complex can vary among
  • the natural CLI blood plasma component isolated with the aid of the monoclonal antibody CLI-9 is capable, due to its special biochemical properties, of neutralizing the membrane-binding and membrane-inserting activity of the nascent complement complexes and of the monomeric perforin.
  • the biological activity of the purified blood plasma component could be mediated by the polypeptide chain, the carbohydrate proportion associated therewith, or by CLI-associated lipid proportions.
  • the complete cDNA sequence of CLI was isolated from a liver-specific gene bank and analyzed with the use of methods of genetic engineering and based on the provided partial amino acid sequence. It is noted that this sequence coincided with an already published partial sequence.
  • the clone CLI-1 which was isolated as described herein has a length of 1651 base pairs and exhibits an open reading frame starting with the 199th nucleotide base.
  • the amino acid sequence derived from the nucleotide base sequence has 448 amino acid residues.
  • the first 21 amino acids form a typical hydrophobic signal peptide which is removed during translocation into the rough endoplasmic reticulum.
  • the mature secreted protein thus begins with the sequence D-Q-T-V-S-D-N-E (aspartic acid, D, is number 1) .
  • the double-chain form of CLI is due to the fact that under reducing conditions, even prior to secretion of CLI into the human plasma, the peptide bond between arginine-205 and serine-206 is hydrolyzed by a still unknown cellular protease.
  • CLI The structural properties of CLI are typical of apolipoproteins.
  • the predicted ⁇ -helix content of CLI is 41%.
  • There are several helical segments of amphiphilic nature (residues 1-76, 150-170, 215-240, 300-350 and 406- 420) which are conserved across human, rat and quail species.
  • Similar amphiphilic structures are shared by all apolipoproteins, e.g., ApoA-I and A-II. These predicted secondary structural elements are likely to interact with lipid bilayers and the lipids of the CLI-
  • SUBSTITUTE SHEET HDL complex The hydrophobic surface of these protein segments are expected to mediate contact with the hydrophobic leaflet of lipid bilayers, the lipid micelles of CLI-HDL complexes and the hydrophobic, membrane- inserting domains of channel-forming, cytolytic proteins of the terminal complement system, the killer cell- derived lytic protein perforin and cytolytic proteins of pathogenic microorganisms.
  • the hydrophilic surfaces are exposed to the surrounding hydrophilic fluid (plasma, interstitial fluid) and determine the water solubility of the CLI-HDL complexes.
  • corresponding to sequences that are identical to the DNA and amino acid sequences particularly identified herein or are different due to natural variations which produce proteins having a biological activity of CLI.
  • this invention also encompasses DNA sequences coding for the same protein but differing due to degeneracy of the DNA code, as well as sequences coding for variants, e.g., allelic variants, of the CLI protein which have the biological activity of CLI, i.e., of inhibiting cytolysis mediated by cytolytic proteins.
  • This invention also encompasses DNA sequences coding for synthetic mutant CLI proteins (muteins) having a biological activity of CLI.
  • mutants mutant CLI proteins
  • this invention also encompasses synthetic variants of the CLI protein which have the biological activity of inhibiting cytolysis mediated by cytolytic proteins, as well as such variants having such activity or which are capable of binding to antibodies that bind specifically to CLI. Suitable variants can be recognized, identified and made by the skilled worker according to the particular use required by reference to the physical characteristics of
  • SHEET the natural CLI protein as outlined above, including, e.g., the helical conserved regions.
  • the stability and lipid affinity of the CLI-HDL complex can be increased; conversely, by introducing highly hydrophilic residues and generating additional asparagine-1inked glycosylation sites (Asn-X-Ser/Thr) at the water interface, the water solubility and half-life of CLI and CLI-HDL complexes could be increased.
  • DNA sequences, plasmids, cytolytic proteins, antibodies and cell lines of this invention can be produced by any and all means conventional in the art, based upon the disclosure of this invention. Specific and general methods for making these products are described above and below.
  • Natural CLI can be purified from human bodily fluids by any means routine to one of ordinary skill in the art, given the disclosure of this invention.
  • the monoclonal antibodies to CLI of this invention are utilized for this purpose by attaching them to an activated solid support material, e.g., Sepharose, and contacting a solution containing the bodily fluid, e.g., plasma or seminal fluid, with the antibody-solid support matrix.
  • the CLI will bind to the antibodies, while the rest of the constituents of the bodily fluid do not.
  • the CLI is then obtained in highly purified form upon release from the antibodies, e.g., by use of a glycine/NaCl buffer.
  • CLI can also be produced by recombinant DNA technology according to methods well-known to those of ordinary skill in the art. Given the amino acid sequence disclosed herein, as well as the DNA sequence also disclosed, probes for the CLI gene can be routinely synthesized which are complementary to DNA sequences
  • DNA sequences for the gene encoding the CLI gene e.g., sequences contained in the cloning plasmid pGEM4/CLI-l, can be isolated from, e.g., endogenous plasmid sequences in genomic libraries, and cloned into an expression vector suitable for the expression of the gene, according to techniques which are well-known to one of ordinary skill in the art.
  • the DNA sequence can be expressed in either non-glycosylating procaryotic or eucaryotic organisms.
  • the DNA sequence may be expressed in organisms, e.g., eucaryotic cells, which endogenously perform the required glycosylation.
  • non-glycosylated protein may be glycosylated in vitro according to well-known methods, e.g., by use of the appropriate glycosylating enzymes.
  • the protein can then be isolated and purified from the cells by means appropriate to the cell type in which it is expressed; i.e., if produced in a cell type which secretes the protein, it may be purified from the cell- free medium separated from the expressing cells. If the protein remains intracellular, then it can be isolated by any one of numerous means known in the art, e.g., the cells are mechanically or osmotically ruptured, insoluble cellular debris is removed from the soluble fraction, and the desired protein is purified by chromatographic means, ionic precipitation, HPLC, etc. , including the affinity chromatography method also described herein using an antibody specific for the CLI protein bound to a solid support.
  • Antibodies specific for CLI are made by methods well known to one of ordinary skill in the art, e.g. , by multiply injecting purified CLI into mice or rabbits and
  • SUBSTITUTE SHEET purifying the thus-elicited antibodies from the animals' serum, e.g., by affinity chromatography using CLI affixed to a support medium.
  • Monoclonal antibodies are also made by well-known methods, e.g., according to the well-known " method of Kohler and Milstein.
  • immunoassay tests for the detection and quantitation of CLI, CLI-fragments, and CLI- containing complexes, e.g., CLI-ApoA-I and/or CLI-HDL complexes, is a routine matter for one of ordinary skill in the art.
  • Test development protocols are well-known and can be modified by routine experimentation to customize the assay for the detection of CLI.
  • Such immunoassay tests can be used to detect and establish levels of CLI present in any normal and disease state of interest in animals, including humans, according to methods well known to one of ordinary skill in the art.
  • serum can then be tested for the presence of normal/abnormal levels of CLI, either alone or as a complex with various other materials, e.g., with ApoA-I, or with HDL.
  • seminal fluid can be routinely tested for the presence of normal/abnormal levels of CLI.
  • tests can be routinely performed and quantitated, e.g., according to well-known methods.
  • antibodies according to this invention can be used to perform immunohistological differential diagnoses and in vivo diagnoses, e.g., according to the usual methods known in the art.
  • an antibody of this invention can be labelled in a way detectable by external detecting means, e.g. , radiolabelled for X-ray imaging or labelled with a paramagnetic species for magnetic resonance imaging, the labelled antibody administered to the patient, and the imaging procedure performed.
  • SUBSTITUTESHEET CLI-containing complexes e.g., CLI-ApoA-I and CLI- HDL
  • CLI-ApoA-I and CLI- HDL can be isolated from bodily fluids using routine methods known to one of ordinary skill in the art. For example, they can be isolated electrophoretically, as described above, or the anti-CLI-antibody/support material affinity chromatography method can be employed, with our without concurrent use of anti-ApoA-I antibodies.
  • reconstituted complexes comprising isolated CLI can be routinely made in the presence or absence of lipids, e.g., phospholipids, and optionally also in the presence of cholesterol, e.g., by mixing the components in the presences of detergents to totally solubilize the proteins and lipids (Ann. Rev. Biophys.
  • the detergent is then removed either by dialysis or molecular sieve chromatography over a gel filtration column that includes the detergent and excludes the final reconstituted product.
  • Suitable detergents include, e.g. , sodium cholate, sodium taurocholate or octylglucoside.
  • Egg phosphatidylcholine and free cholesterol are commercially available, and lipid-free ApoA-I can be isolated from human plasma by the method of Peitsch et al. Analyt. Biochem. 178. 301- 305 (1989).
  • the cytolytic inhibitor protein of this invention can be used as a therapy for conditions which are mediated by cytolytic protein-mediated cell lysis.
  • increasing the CLI plasma level e.g., by administration of exogenous CLI, represents a therapeutic approach for counteracting increased systemic complement activity in the body caused by various disease and traumatic states, as well as the activity of perforin-induced cell lysis, and/or activity of other membrane-active cytolytic proteins.
  • SUBSTITUTESHEET Further approaches made possible by this invention include control of plasma levels of the cytolysis inhibitor by pharmaceutical or biologically active compounds altering the gene expression of the cytolysis inhibitor in the liver or in other tissues, e.g., the
  • CLI has been postulated to have a role in human fertility, in that it is involved in preventing premature lysis of the cell membrane of the sperm prior to fertilization of the egg. Therefore, congenital or acquired lack of CLI may be a cause of male infertility. Similarly, the presence of CLI-specific antibodies in intravaginal and/or intrauterine fluid may also be a cause of infertility. Therefore, CLI or fragments thereof, including the carbohydrate portion of the glycoprotein, are used to develop a vaccine against CLI, thereby producing in a male immunized therewith a contraceptive effect. Such a vaccine is produced and administered according to well-known methods-.
  • the invention additionally includes the systemic or local administration of CLI for the treatment of tissue- destroying pathological conditions evoked by complement and killer cells, and the local or systemic administration of CLI for detoxification of membrane- active peptides and cytolytic proteins secreted by pathogenic organisms, for example by bacteria (inter alia Staphylococcus aureus, Escherichia coli) , fungi, or insects (for example, in the poison of honeybees, wasps, hornets, bumblebees) .
  • bacteria inter alia Staphylococcus aureus, Escherichia coli
  • fungi for example, in the poison of honeybees, wasps, hornets, bumblebees
  • CLI-protein, glycoprotein, protein fragments and/or complexes are administered for the various treatment protocols analogously to other inhibitory proteins, e.g., protease
  • UBSTITUTESHEET inhibitor which are used in animal, especially human, medicine. Particular dosages for a given indication, patient and compound or complex can be routinely determined, e.g., by using standard pharmaceutical procedures well known to practitioners.
  • CLI can be involved in tissue damage resulting from, e.g., chemical, metabolic, hormonal, ischemic, mechanical, and immunological causes, as well as in response to involution of normal human tissues.
  • CLI localization during cytolytic tumor therapy is useful to detect the progress and/or regression of such tumors.
  • the antibodies to CLI have a particular utility for the immunological detection, quantitation, immunohistological differential diagnosis, and localization of CLI and/or CLI-containing complexes in patients, including in bodily fluids, tissue samples, histological specimens, and, in the case of in vivo imaging as discussed above, in a whole patient, by use of appropriately labelled antibodies, which can be labelled according to any of the many suitable ways known to one of ordinary skill in the art for labelling proteins in general and immunological molecules in particular.
  • CLI and variants thereof and muteins, as well as the DNA sequences, plasmids and antibodies coding therefor or binding thereto, are similarly useful.
  • muteins which may or may not have a particular biological activity of CLI, can be used to map the binding sites of monoclonal antibodies and to study the domain interaction
  • Nucleotide acid sequences are derived via the genetic code from the partial amino acid sequence of the two chains of CLI, and the corresponding oligonucleotides are synthetized as hybridization probes. Since the genetic code is degenerated, i.e. in most cases several codons can code for the same amino acid, two long oligonucleotides are synthetized and, in accordance with the rules by Lathe, J. Mol. Biol. 183 : 1-12 / 1985, only those codons are selected which, based on statistic- al empirical values, can be found with maximum probability in human genese for the respective amino acids.
  • the partial sequence DNELQEMSNQG is selected for probe 1, and the partial sequence PYEPLNFHAMFQPFLEM is chosen for probe 2.
  • These protein sequences are determined in accordance with Example 2 and/or according to Murphy et al., J. Clin, Invest. 81 : 18589-1864, 1988.
  • the resultant synthetic hybridization probes have the fol ⁇ lowing nucleotide base sequences for probe 1: 5'-GAC AAT GAG CTG CAG GAG ATG TCC AAC CAG GG-3'; for probe 2: 5'-CCC TAT GAG CCC CTG AAC TTC CAC GCC ATG TTC CAG CCC TTC CTG GAG ATG-3 1 .
  • the probes are labeled radioactively on the 5'-terminus with the use of [gamma- 32 P] dATP (A ersham) and T4 polynucleotide kinase (Pharmacia-LKB, Sweden) .
  • ⁇ ,he 20 ⁇ l reaction solution contains 70 mmol of tris- . hydrochloride, pH 7.6, 10 mmol of MgCl- and 5 mmol of DTT, 3 pmol of the respective oligomer, 9 pmol of [gamma- 3 --V]dATP, and 6 units of T4 DNA polynucleotide kinase, and is incubated for 30 minutes at 37° C.
  • the free, nonused [gamma- 3 --P]dATP is separated from the oligonucleotides by gel filtration (NAP 25, Pharmacia- LKB, Sweden) .
  • the roundish colonies of a size of about 1 mm are transferred by simple kiss method from the agar plates to nitrocellulose filters (Schleicher und Schull) .
  • the bacterial colonies are further cultivated on the original agar substrates for 7 hours and in this way a replicate capable of growth is provided for each colony on the nitrocellulose filters.
  • the bacterial colonies on the nitrocellulose filters are solubilized by means of sodium dodecyl sulfate and sodium hydroxide solution.
  • the bacterial DNA is released, denatured, and bound as single-strand DNA to the filter membrane. After several washing steps with neutral buffers, the DNA is firmly fixed to the nitrocellulose by heating
  • the filters are pre- hybridized for four hours at 50° C.
  • the solution consists of 6 x SSC (1 x SSC contains 0.15 moles NaCl, 0.015 moles sodium citrate) , 5 x Denhardt's solution (according to Maniatis, T. , et al. in Molecular Cloning, Cold
  • hybridization solution DNA subjected to ultrasound and denatured by boiling for five minutes. Hybridization with the 51 oligomer probe takes place at 50° C in fresh hybridization solution over a period of four hours.
  • 4 hybridization solution is 5 x 10 cpm/ml.
  • the nitro- cellulose filters are washed at 45° C in 1 x SSC and 0.1% SDS (weight/volume) for 60 minutes and exposed overnight to an X-ray film at -70° C.
  • the 51 probe is removed by boiling for 10 minutes in a 10 mmol EDTA and 1% aqueous glycerol solution.
  • the filters are subjected to a second screening procedure with the 32 probe. A strong unequivocal signal with both probes is observed only on three places on the 20 filters. Bacterial clones from these sites are examined a second time by streaking and renewed cultiva ⁇ tion on small agar plates.
  • the recombinant plasmid of the CLI-1 clone is isolated according to a routine method and analyzed by restriction enzymes (L.G. Davies et al., Basic Methods in Molecular Biology, Elsevier, New York, 1986) .
  • the DNA is cleaved with the restriction enzymes BamHI and Kpnl.
  • the resultant DNA fragments are separated by agarose gel electrophoresis, and the size of the DNA insertion in the pGEM4 vector is determined.
  • the length of the cDNA amounts to 1.7 kb; there are no internal BamHI and Kpnl scission sites ( Figure 1) .
  • Approximately 20 ⁇ g of the cDNA is separated from the pGEM4 vector plasmid by agarose gel electrophoresis and purified for DNA sequence determination.
  • the cDNA of the CLI-1 clone is separated, after spontaneous ligation, by ultrasonic treatment in an ultrasonic water bath into randomly distributed subfrag- ments and fractionated according to size by electro ⁇ phoresis in 1.3% strength NA-agarose (Pharmacia) . Those fragments which fall within the molecular weight range from 300 to 600 bp are isolated, and the ends are repaired with the aid of T4 DNA polymerase so that smooth termini are produced.
  • the cDNA fragments produced in this way are inserted into the Smal restriction scission site of M13mp8 vector with the use of T4 DNA ligase.
  • the single-stranded phages of the M13 vector are prepared according to standard methods (L.G. Davies et al. , Basic Methods in Molecular Biology, Elsevier, New York, 1986) , and the nucleotide sequence of about 50 subfragments is determined with the aid of the
  • the nucleotide base sequence of the CLI-1 cDNA is determined at least once on both strands.
  • the over ⁇ lapping partial sequences are compared with the aid of a microcomputer and composed into the total sequence ( Figure 2) .
  • the alternative route of complement cascade is activated in 1 liter of human serum by the addition of insulin (10 mg/ml, Merck) .
  • the thus-produced soluble terminal complement complex is isolated according to Bhakdi and Roth (J. Immunol., 127 : 576-582, 1981) .
  • this complex is produced by the uniting of the complement proteins C5b, C6, C7, C8, C9, S-protein, and CLI.
  • anionic detergents S-protein and CLI will dissociate from the complex.
  • DOC deoxycholate
  • SC5b-9 complex 250 mmol of deoxycholate (DOC) in the solid form is added to a solution of 0.6 mg to 1.2 mg/ml of SC5b-9 complex. This solution is incubated at 37° C for one hour in the presence of 2 mmol of PMSF. Respectively 4 ml is loaded onto a linear sucrose gradient (40 ml total volume, 10% by weight to 40% by weight of sucrose in 6.25 mmol of DOC, 10 mmol of tris-hydrochloride, pH 8.1, 50 mmol of NaCl and 7.5 mmol of NaN_) , and centrifuged for 3 hours at 4° C and 250,000 g's in a Beckman vertical rotor (model Vto-50) .
  • a linear sucrose gradient 40 ml total volume, 10% by weight to 40% by weight of sucrose in 6.25 mmol of DOC, 10 mmol of tris-hydrochloride, pH 8.1, 50 mmol of NaC
  • fractions are collected from the bottom of the centrifuge tube.
  • the fractions containing S-protein and CLI are pooled and concentrated approximately fivefold (Amicon PM10 membranes) .
  • the proteins are then separated with the aid of a gel filtration column ("Sephacryl" S-300,. 1 cm x 60 cm column, Pharmacia) .
  • the equilibration buffer is 10 mmol of tris-hydrochloride, 50 mmol of NaCl, pH 8.1. CLI and S-protein elute in a peak.
  • the fractions are concentrated (protein concentration between 0.4 mg/ml and 0.6 mg/ml) and stored at -20° C.
  • 500 ⁇ l of the mixture of S-protein and CLI are injected subcutaneously into female Balb/c mice in complete Freund's adjuvant (1:1; vol./vol.). After 5 weeks, the injection of 500 ⁇ l of the antigen is repeated, this time in incomplete Freund's adjuvant. After another 5 weeks, 500 ⁇ l of antigen without adjuvant is administered intraperitoneally, and the mice are then sacrificed after 3-4 days.
  • Myeloma cells are cultivated in RPMI 1640 medium containing additionally 10% inactivated fetal calf serum, 1% glutamine, 5,000 U/ml of penicillin, 5 ⁇ g/ml of streptomycin, and 0.02 mmol of 2-mercapt ⁇ - ethanol (myeloma medium) .
  • the myeloma cells are cultivat-ed at 37° C in 12 Petri dishes having a dia ⁇ meter of 9 cm until they begin to become confluent.
  • the myeloma cells and the cells from the spleen of an immunized mouse are mixed with each other in a serum-free medium and pelletized at 1,200 rpm.
  • the cell sediment is gently resuspended in 2 ml of the fusion solution (50% PEG 4000 in RPMI 1640) and mixed for 2 minutes at 37° C gradually by means of a pipette.
  • the fusion solution is then diluted with 20 ml of RPMI 1640. Thereafter the cells are sedimented and distributed in 20 ml of fresh myeloma medium on
  • the culture chambers contain macrophages obtained by flushing the peritoneal cavity of 2 mice. On each subsequent second day, the myeloma medium which, for the purpose of selection,
  • _2 then contains additionally 0.1 x 10 mmol of hypo- xanthine, 4 x 10 -4 mmol of aminopterin, and 1.6 x 10 - 2 mmol of thymidine (-HAT medium) , is changed.
  • Hybridoma culture supernatants are examined with the aid of an
  • the monoclonal antibody CLI-9 is purified from 1 ml of ascites fluid.
  • the ascites fluid is passed over a 2 ml protein A "Sepharose” column equilibrated in 10 mmol of tris-hydrochloride, pH 7.4, 150 mmol NaCl (subsequently called TBS) .
  • the antibodies are eluted with 10 ml of 0.2-molar glycine, pH 2.8, 0.5-molar NaCl. 4 mg of the purified antibody is subsequently coupled to 1.5 ml of cyanogen bromide- activated "Sepharose" (in accordance with the directions by the manufacturer, Pharmacia) .
  • Pure CLI is eluted with 10 ml of 0.2-moles glycine buffer, 0.5 moles NaCl, pH 2.8 ( Figure 4) . Approximately 0.5 mg of CLI can thus be obtained in a buffer volume of 5 ml. Purified CLI is dialyzed against TBS (16 hours) and either stored at 4° C for several days or deep-frozen (-20° C) in small portions (100 ⁇ l) .
  • the electrophoresis buffer consists of 25 mmol tris- hydrochloride, 192 mmol glycine and 20 vol-% methanol. The proteins are transferred for 3 hours at 60 V and
  • saturation buffer 1% gelatin [Merck], 0.1% bovine albumin [Bohringer] , 20 mmol tris-hydrochloride, pH 8.4, 150 mmol NaCl, 5 mmol EDTA and 0.02% sodium azide
  • a protein A peroxidase conjugate (dilution 1:1000, Sigma) or an anti-mouse IgG peroxidase conjugate (dilution 1:1000, Dakopatis) is added thereto.
  • the nitrocellulose mem ⁇ branes are incubated for another two hours.
  • the peroxidase activity is determined with 4-chloro-l- naphthol (Merck, 0.075%) in the presence of 0.01%
  • 28 kilodalton component are Asp-Glu-Pro-Pro-Gln-Ser-Pro- Trp-Asp-Arg and exactly coincide with the amino-terminal sequence of the apolipoprotein A-I (ApoA-I) .
  • the ApoA-I of the CLI complex shows the same mobility in the SDS-PAGE as ApoA-I of purified HDL and reacts with commerically available ApoA-I-specific antisera (Behringwerke, Marburg). From this, the inventors have drawn the conclusion that the 28 kilodalton com ⁇ ponent represents the mature form of the intact ApoA-I.
  • ApoA-I-CLI complex Since the same molecular weight form of ApoA-I is considered to be the specific protein marker of the high-density lipoproteins (HDL) , the supposition presented itself that the ApoA-I-CLI complex could involve a new special HDL subfraction. Therefore, the inventors analyzed the lipid content and the lipid composition of the ApoA-I-CLI complex.
  • the ApoA-I-CLI particle consists of 78% by weight of protein and 22% by weight of lipid.
  • the lipids were separated by thin-layer chromatography into individual lipid fractions and quantified by comparison with commercially available lipid standards in situ on the thin-layer plate. The lipid proportion in mol-% is broken down as follows: 54% total cholesterol (unesterified to esterified cholesterol, 0.58)
  • a further indication that the purified ApoA-I- CLI complex involves an HDL particle is constituted by the results of investigations with the electron micro ⁇ scope.
  • ApoA-I-CLI complexes are presented in the electron microscope as spherical, homogeneous-appearing structures of uniform size ( Figure 9) .
  • the average particle dia ⁇ meter amounts to 9.0 ⁇ 1.0 nm and thus is similar to the HDL particles of size category 3 (8.5 nm to 9.6 n ) , the smallest of the three size categories.
  • C7 Podack, E.R., Kolb, W.P., Esse ' r, A.F., and Muller-Eberhard, H.-J., J. Immunol. 123 : 1071-1077, 1979
  • C8 Kolb, W.P. and Muller- Eberhard, H.-J., J. Exp. Med. 1_3 : 1131-1139, 1976
  • C9 Podack, E.R., Tschopp, J. and Muller-Eberhard, H.-J., J. Exp. Med. 156 : 268-282 are purified in accordance with standard methods.
  • C5b-6 which lyses, in the absence of CLI, about 80% of the erythrocytes under the conditions set forth below, is mixed with 30 ⁇ l of sheep erythrocytes in 10 mmol veronal buffer, pH 7.4, 142 mmol NaCl, 0.1% gelatin and 10 mmol EDTA (abbreviated GVBE) , and incubated for 20 minutes at 25° C.
  • GVBE mmol EDTA
  • C7 (1 ⁇ g/ml final concentra ⁇ tion)
  • C8 0.2 ⁇ g/ml final concentration
  • C9 (1 ⁇ g/ml final concentration)
  • CLI 1 ⁇ g/ml final concentration
  • the four proteins had been pre- incubated for 5 minutes at room temperature. After an incubation of 30 minutes at 37° C, the intact erythrocytes are removed by centrifuging (1500 rpm, 2 minutes) , and the amount of hemoglobin released into the supernatant is determined at a wavelength of 412 nm in a spectrophotometer.
  • Perforin is purified according to the method by Masson, D., and Tschopp, J. , J. Biol. Chem. 260 :
  • Staphylococcus aureus is among the most frequent bacterial pathogens in man.
  • the ⁇ -hemolysin formed by almost all human-pathogenic Staphylococcus aureus strains is presently considered to be an important pathogenicity factor.
  • S. aureus secretes ⁇ -hemolysin as a water-soluble monomeric protein with a molecular weight of 34,000.
  • 6 toxin monomers group together into a hydrophilic, transmembranous channel having a diameter of 1-2 mm.
  • the arachidonic acid cascade can be activated with release of highly active prostaglandins and leukotrienes in endothelial cells.
  • Human blood platelets are particularly sensitive with respect to subcytolytic doses of staphylococcus- ⁇ -hemolysin. Due to the action of the toxin on platelets, platelet aggregation occurs, along with reduction of the clotting time in the platelet-rich plasma (Bhakdi et al., J. Exp. Med., _168_ : 527-542, 1988) .
  • plasma LDL ⁇ -lipoproteins
  • Staphylococcus- ⁇ - hemolysin staphylolysin reagent, 5 units per bottle, Behringwerke, Marburg
  • GVB buffer GVB buffer without EDTA, see above
  • 50 ⁇ l of a rabbit o erythrocyte suspension (10 /ml) is admixed to 200 ⁇ l of this solution.
  • the specimens After incubating for one-half hour at 37° C, the specimens are centrifuged (2 minutes at 12,000 g's) , and the released hemoglobin in the super ⁇ natant is measured by spectrophotometry at 412 nm. Total hemolysis is measured after adding sodium dodecyl -3k-
  • SUBSTITUTE SHEET Figure 1 shows a scheme of the recombinant pGEM4/CLI-l plasmid containing the entire coding sequence for the cytolysis inhibitor, and several important restriction scission sites.
  • the plasmid has an ampicillin resistance gene coding for the enzyme 'beta-lactamase, and includes the Sp6 and, respectively, T7 promoter in the direct vicinity to the cloning scission site BamHI in the polylinker.
  • the two ends of the cDNA insertion are provided with the adapter oligo- mers A and B according to Haymerle, H. , Herz. , J., Bressan, G.M., Frank, R. and Stanley, K.K. , Nucleic Acids Res.
  • Figure 2 shows the nucleotide base sequence of the CLI-1 clone and the amino acid sequence derived therefrom in the line therebelow.
  • the amino- erminal sequences of the a-chain (amino acids 1 to 205) and the b-chain (amino acids 206 to 427) are underlined.
  • the numbering is indicated for the nucleotide bases, and on the right-hand margin of the figure the numbering of the amino acids is set forth.
  • Figure 3 shows the results of the Southern analysis: the position of the molecular weight markers is indicated in kilobases on the left-hand margin of the figure.
  • Figure 4a shows the purified CLI after sep ⁇ aration by SDS polyacrylamide gel electrophoresis (10% polyacrylamide proportion) under nonreducing (left) and reducing conditions (right) .
  • Figure 4b shows the purified CLI after sep ⁇ aration by SDS polyacrylamide gel electrophoresis (12% polyacrylamide proportion) .
  • Figure 5 Western Blot identification (bars 1 to 3 ' ) of CLI in human plasma (1) , in purified SC5b-9 complex (2) , in seminal fluid (3 and 3'). Bar 4 shows the proteins of the seminal fluid after staining the SDS gel with "Commassie" blue.
  • Figure 6 Inhibition of complement lysis by CLIi CLI inhibits lysis of the erythrocytes by soluble C5b-6, C7, C8 and C9 in concentration-dependent way (bottom curve) , but does not inhibit lysis of C5b-7 erythrocyte intermediate stages by C8 and C9 (top curve) .
  • Figure 7 Inhibition of perforin-mediated erythrocyte lysis at various CLI concentrations.
  • Figure 8 Inhibition of staphylococcus- ⁇ - hemolysin-mediated erythrocyte lysis by CLI.
  • Staphylococcus- ⁇ -hemolysin is preincubated with varying CLI concentrations at 37° C in GVB buffer and thereafter mixed with a rabbit erythrocyte suspension in GVB buffer.
  • Subphysiological CLI con ⁇ centrations (15 ⁇ g/ml) almost completely suppress the toxin-caused lysis of the erythrocytes.

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Abstract

On prévoit une protéine inhibant la cytolyse, des codes ADN qui lui sont destinés, des plasmides contenant ledit ADN, des cellules hôtes contenant lesdits plasmides, et des anticorps monoclonaux à ladite protéine, ainsi qu'un procédé d'isolation de l'inhibiteur de cytolyse dans les fluides de l'organisme, un procédé de fabrication de l'inhibiteur de cytolyse au moyen de la technique de recombinaison, des procédés immunologiques d'utilisation desdits anticorps, et des procédés de traitement de diverses maladies.
EP90914723A 1989-10-06 1990-10-08 Proteins inhibant la cytolyse (cti) et sequences adn codant pour lesdites proteines Withdrawn EP0452433A1 (fr)

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DE3933850 1989-10-06
DE3933850A DE3933850A1 (de) 1989-10-06 1989-10-06 Zytolyse-inhibitor (zli), eine diese blutplasmaprotein kodierende dna-sequenz, sowie ein plasmid, ein wirtsorganismus und ein verfahren zur gewinnung dieses proteins

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EP0452433A1 true EP0452433A1 (fr) 1991-10-23

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Publication number Priority date Publication date Assignee Title
WO1994002719A1 (fr) * 1992-07-15 1994-02-03 Linde Aktiengesellschaft Procede et dispositif d'elimination de particules dans des gaz d'echappement de moteurs a combustion interne

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WO1998006752A1 (fr) * 1996-08-16 1998-02-19 The Rockefeller University Proteine de fixation de leptine et son utilisation dans des procedes de diagnostic et de traitement des anomalies du chemin de leptine endogene
WO2001032837A1 (fr) * 1999-11-02 2001-05-10 Human Genome Sciences, Inc. 19 proteines humaines secretees
US6930085B2 (en) 2002-04-05 2005-08-16 The Regents Of The University Of California G-type peptides to ameliorate atherosclerosis
US20080107639A1 (en) * 2004-01-20 2008-05-08 Develogen Aktiengesellschaft Use of a Dg147 Protein Product for Preventing and Treating Metabolic Disorders
WO2007030930A1 (fr) * 2005-09-13 2007-03-22 National Research Council Of Canada Methodes et compositions permettant de moduler l'activite des cellules tumorales
CN102666585B (zh) 2009-11-24 2015-02-18 阿莱斯亚生物疗法股份有限公司 抗簇蛋白抗体和抗原结合片段及其减小肿瘤体积的用途
CA2862739A1 (fr) 2012-02-22 2013-08-29 Alethia Biotherapeutics Inc. Utilisation conjointe d'un inhibiteur de clusterine et d'un inhibiteur d'egfr pour traiter le cancer

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Title
See references of WO9105043A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002719A1 (fr) * 1992-07-15 1994-02-03 Linde Aktiengesellschaft Procede et dispositif d'elimination de particules dans des gaz d'echappement de moteurs a combustion interne

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CA2042605A1 (fr) 1991-04-07

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