EP0613498A1 - Derives de recepteurs avec sites de fixation pour rhinovirus humains - Google Patents

Derives de recepteurs avec sites de fixation pour rhinovirus humains

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Publication number
EP0613498A1
EP0613498A1 EP93915793A EP93915793A EP0613498A1 EP 0613498 A1 EP0613498 A1 EP 0613498A1 EP 93915793 A EP93915793 A EP 93915793A EP 93915793 A EP93915793 A EP 93915793A EP 0613498 A1 EP0613498 A1 EP 0613498A1
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EP
European Patent Office
Prior art keywords
gly
ser
leu
asp
arg
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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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EP93915793A
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German (de)
English (en)
Inventor
Franz Hofer
Heinrich Kowalski
Martin Gruenberger
Herwig Machat
Manfred Huettinger
Donscho Kerjaschki
Ernst Kuechler
Dieter Blaas
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Boehringer Ingelheim International GmbH
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Boehringer Ingelheim International GmbH
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Priority claimed from DE4222385A external-priority patent/DE4222385A1/de
Priority claimed from DE19924227892 external-priority patent/DE4227892A1/de
Priority claimed from DE19934305063 external-priority patent/DE4305063A1/de
Application filed by Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Publication of EP0613498A1 publication Critical patent/EP0613498A1/fr
Withdrawn legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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 present invention describes receptor derivatives with binding sites for human rhinoviruses of the "small rhinovirus receptor group", their use and DNA coding for the receptor derivatives.
  • Human rhinoviruses represent a large genus within the Picoravirus family and include about 115 different serotypes (Melnick, J.L. (1980) Prog. Med. Virol. 26, 214-232). These RNA viruses affect the respiratory tract of humans and cause acute infections which lead to colds.
  • the human rhinoviruses can be divided into two groups if the competition criteria for binding sites on the surface of human cell culture cells, such as e.g. HeLa cells. Competition experiments show that - with one exception (serotype 87) - there are two different receptors on the cell surface. So far, 91 serotypes of the "large rhinovirus receptor group” and 10 serotypes of the "small rhinovirus receptor group” have been assigned (Abraham and Colonno RJ (1984) J. Virol. 5] _, 340-345; Uncapher et al. (1991) Virology 180, 814-817).
  • the "large rhinovirus receptor group” receptor was purified and identified as ICAM-1, a protein belonging to the immunoglobulin superfamily that functions as a cell adhesion molecule (Tomassini et al. (1989) Proc. Natl. Acad. Sci. USA 86, 4907 -4911; Staunton et al. (1989) Cell 56, 849-853; Greve et al. (1989) Cell 56, 839- 847).
  • ICAM-1 was the receptor for the majority of rhinoviruses are (Greve et al. (1989) loc.
  • the receptor binding site of human rhinovirus serotype 14, a member of the "large rhinovirus receptor group”, is located in a so-called “canyon", a deepening of the virus surface (Rossmann et al. (1985) Nature 317, 145-153).
  • the amino acids in this "canyon” are relatively well preserved, while the surrounding amino acids are variable and represent binding sites for neutralizing antibodies. According to this "Canyon hypothesis", viruses can accept mutations in the hypervariable antibody binding sites and thus escape the natural immune response. In this way, a constant receptor binding site is retained which is not accessible to antibodies (Rossmann and Palmen ⁇ berg (1988) Virology 164, 373-382).
  • the receptor of the "small rhinovirus receptor group” mediates the uptake of about 10 serotypes of the human rhinoviruses in the corresponding host cells.
  • This membrane-bound receptor was isolated by various purification steps, the binding activity in the different fractions being detected by means of a filter binding assay (Mischak et al. (1988) J. Gen. Virol., 69, 2653-2656).
  • the apparent molecular weight of the native receptor in the presence of nonionic detergents corresponds to approximately 450 kD, that of the denatured form approximately 120 kD, although a number of other forms were also found (Mischak (1988) loc. Cit.).
  • the natural receptor is for inhibiting the uptake of rhinoviruses of the "small rhinovirus receptor group" due to the low solubility of this membrane protein in polar, e.g. aqueous solution systems such as aqueous buffer solutions, less suitable.
  • the identity of the receptors of the LDL receptor family with the receptors of the rhinoviruses of the "small rhinovirus receptor group” now surprisingly allows poly- To provide peptides, in particular soluble polypeptides, which have at least one binding site for rhinoviruses of the "small rhinovirus receptor group”.
  • a functional derivative is accordingly a component with the biological activity which essentially corresponds to the biological activity of the native receptor of the "small rhinovirus receptor group”. This biological activity relates to the binding capacity of the receptor for rhinoviruses of the "small rhinovirus receptor group”.
  • the term “functional derivatives” is intended to include “variants” and “chemical derivatives”.
  • the term derivative refers to any polypeptide which, measured on the native receptor protein, is a reduced form and has at least one binding site for rhinoviruses of the "small rhinovirus receptor group”.
  • a “variant” comprises the molecules which are essentially derived in function and structure from the native receptor molecule, such as allelic forms. Accordingly, the term “variant” contains molecules which can bind rhinoviruses of the "small rhinovirus receptor group", but e.g. have an altered amino acid sequence.
  • a “chemical derivative” includes additional chemical groups that are not normally part of this molecule. These groups can improve molecular solubility, absorption, biological half-life, etc., or alternatively reduce toxicity or undesirable side effects. Groups that mediate such effects are known (Remington's Pharmaceutical Sciences (1980)).
  • the biological activity of the receptor derivatives according to the invention or the chemical derivatives obtained after modification can be checked using methods known from the prior art, for example using the method described by Mischak et al. described filter binding assay (Mischak et al. (1988) J. Gen. Virol. 69, 2653-2656 and Mischak et al. (1988) Virology 163. 19-25):
  • the polypeptide can be applied to a suitable membrane, for example nitrocellulose.
  • the mixture is then saturated with a detergent mixture.
  • the membrane pretreated in this way is then incubated to check the specific binding with labeled rhinovirus, for example with HRV2 labeled with ⁇ S-methionine. After washing and drying the membrane, a specific binding can then be made visible using autoradiography.
  • receptor derivatives which are in the form of extracellular, soluble polypeptides and are removed from the receptor-carrying cells, for example, into the medium. are given. These receptor derivatives are extremely suitable for inhibiting the binding of rhinoviruses to their receptors. They can thus be used for the therapeutic or prophylactic treatment of the human body or for the production of pharmaceutical preparations. In particular, they can be used as an antiviral, preferably antirhinoviral, agent.
  • the phenomenon of the delivery of a soluble receptor derivative has been described for many receptor proteins, for example for the interleukin-4 and interleukin-7 receptor (Mosley et al. (1989) Cell 59, 335-348; Goodwin et al. (1990) Cell. 60, 941-951).
  • soluble receptor derivatives can also be formed by enzymatic, in particular proteolytic or chemical, cleavage.
  • B. receptor-carrying cell lines can be used, which are implemented with enzymes such as papain, trypsin, etc.
  • the amino acid sequence of the receptor molecule is known, the person skilled in the art can of course selectively produce extracellular derivatives by selecting suitable proteases.
  • the binding capacity of such derivatives can be checked with the filter binding assay described above, so that in this way specifically reduced receptor derivatives which can bind rhinoviruses of the "small rhinovirus receptor group" are produced.
  • Another aspect of this invention is the formation of soluble derivatives by enzymatic or chemical cleavage of native receptor molecules.
  • a native receptor protein for example by reaction with proteases or by chemical cleavage (as described above) the native receptor protein can be cleaved and the reduced, rhinovirus-binding region e.g. can be identified and isolated by the filter binding assay.
  • Suitable proteases can be derived from the respective amino acid sequence of the receptor protein.
  • cyanogen bromide or splitting of the receptor protein by reductive treatment e.g. with dithiothreitol.
  • the present invention includes the following aspects:
  • the LDL family of receptors is formed from three structurally related cell surface receptors, which manage the endocytosis of lipoproteins and other plasma proteins (Brown et al. (1991) Curr. Opin. Lipidology 2, 65-72).
  • the receptors have the following common features: cysteine-rich repeats, which are responsible for ligand binding, cysteine-rich repeats of the EGF ("epidermal growth factor") type, YWTD repeats, a single region spanning the membrane and at least an NPXY internalization signal (Willnow et al. (1992) J. Biol. Chem. 267, 26172-21180).
  • All members of this receptor family can thus be used for the formation of functional derivatives with binding properties for rhinoviruses of the "small rhinovirus receptor group".
  • the path taken in Example 3 can be followed for the isolation of soluble LDL receptor derivatives released into the medium.
  • the purification of a binding protein released into the cell culture supernatant is described here.
  • the receptor derivative is an LDL receptor derivative (Example 4).
  • the receptor derivative is purified here by means of ion exchange chromatography (anionic), affinity chromatography (lens culinaris lectin and jacalin agarose) and ammonium sulfate precipitation. Binding activity was checked using the filter binding assay (Mischak et al. (1988) 163, 19-25). This production method can also be applied to the other two proteins of the LDL receptor family.
  • the isolation of the native receptor proteins is known and has been described by Yamamoto et al. (1984) Cell 39, 27-38; Goldstein et al. (1985) Annu. Rev. Cell Biol. 1, 1-39; Mischak et al. (1988) Virology 163, 19-25; Kowal et al. (1989) Proc. Natl. Acad. Be. USA 86, 5810-5814 and Willnow et al. (1992) loc. cit.).
  • the native proteins can then be converted into the functional, soluble ones by means of enzymatic and chemical cleavages Derivatives are transferred. Since the amino acid sequence of the LDL receptor (FIG. 1), the 0C2MR / LRP (FIG. 2) and at least partially for the gp330 (FIG. 3) are known, proteolytically active enzymes or chemicals can be specifically selected, in particular to to release the respective extracellular receptor region.
  • the present invention therefore also relates to polypeptides which are derived from the amino acid sequences of the LDL receptor, o MR / LRP and gp330 and, in particular in their soluble form, are capable of rhinoviruses of the "small rhino virus" Receptor group ".
  • These polypeptides are preferably derived from the amino acid sequences which correspond to the human proteins of the LDL receptor family, although, as set out in Examples 1 and 2, corresponding receptors from mammals and amphibians are also suitable.
  • Receptor derivatives can be used in the form in which they are released from eukaryotic cells into the cell supernatant.
  • the receptor derivatives of the present invention can, however, also correspond to the membrane-bound members of the LDL receptor family in which the part of the protein which is responsible for the binding of the protein to the membrane is missing or has lost its function.
  • Receptor derivatives which consist essentially of domains 1, 2 and 3 of the receptor protein, 1 and 2 or only of domain 1 according to FIG. 4 are particularly preferred.
  • Domain 1 then comprises the N-terminal, cysteine-rich receptor part which binds the various ligands
  • domain 2 comprises a region with high homology to the EGF precursor protein
  • domain 3 comprises a relatively short, O-glycosylated peptide region
  • domain 4 the transmembrane region
  • domain 5 the cytoplasmic part of the receptor molecule.
  • Polypeptides consisting essentially of domains 1, 1 and 2 as well as 1, 2 and 3 can be obtained from the culture supernatant of eukaryotic cells (Example 3) or by recombinant DNA techniques known per se, such as, for example by Davis et al. (1987) Nature 326, 760-765 for the LDL receptor.
  • the human LDL receptor is the preferred starting compound.
  • the invention comprises functional receptor derivatives which essentially contain amino acids 1 to 750 (domains 1 and 2) and 1-322 (domain 1) ( Fig. 1) include.
  • the C-terminus of these polypeptides can be shortened to the extent that the binding capacity for rhinoviruses of the "small rhinovirus receptor group" is retained.
  • the preferred receptor derivatives thus essentially have the following amino acid sequences:
  • polypeptides according to the invention can be present as dimers, trimers, tetramers or multimers.
  • the methods for the production of the receptor derivatives, enzymatic or chemical treatment of the native receptor molecules, isolation of the derivatives released by cells and methods for the recombinant production are also part of the invention.
  • Another aspect of the invention is DNA molecules which code for the polypeptides according to the invention.
  • the starting molecules are accessible to the person skilled in the art by known methods.
  • the cloning of the corresponding cDNA has been described for all three members (Yamamoto et al. (1984) loc. Cit .; Goldstein et al. (1985) loc. Cit .; Pietromonaco et al. (1990) Proc. Natl. Acad. Sei USA 87, 1811-1815; Herz et al. (1988) loc. Cit.).
  • the DNA molecules with knowledge of the amino acid sequence can also be prepared synthetically (for example according to Edge et al. (1981) Nature 292, 756-762) or by means of the PCR method (Sambrook et al. (1 89) "A Laboratory Manual ", Cold Spring Harbor Laboratory Press).
  • the invention also relates to DNA sequences that include modifications that are easily obtained by mutation, deletions, rearrangement, or addition by methods known to those skilled in the art.
  • Each DNA sequence which codes for a polypeptide according to the invention and the correspondingly degenerate forms of the DNA sequences are included.
  • the invention includes DNA vectors that contain the DNA sequences described above.
  • these can be vectors in which the DNA molecules described are functionally linked to a control sequence which allows the expression of the corresponding polypeptides.
  • These are preferably plasmids which can be replicated and / or expressed in prokaryotes such as E. coli and or in eukaryotic systems such as yeasts or mammalian cell lines.
  • the invention also includes appropriately transformed host organisms.
  • DNA sequences according to the invention can be expressed as fusion polypeptides or as intact, native polypeptides.
  • Fusion proteins can advantageously be produced in large quantities. They are generally more stable than the native polypeptides and are easy to clean.
  • the expression of these fusion proteins can be controlled by normal E. coli DNA sequences.
  • the DNA sequences according to the invention can be cloned as lacZ fusion genes and brought to expression.
  • a large number of vector systems are available to the person skilled in the art, for example the pUR vector series (Rüther, U. and Müller-Hill, B. (1983), EMBO J. 2, 1791).
  • the bacteriophage promoter IpR in the form, for example, of the vectors pEX-1 to -3 can also be used for the expression of large amounts of Cro- ⁇ -galactosidase fusion protein (Stanley, KK and Luzio, JP (1984) EMBO J. 3, 1429).
  • the tac promoter inducible with IPTG can also be used in an analogous manner, for example in the form of the pROK vector series (CLONTECH Laboratories).
  • the prerequisite for the production of intact, native polypeptides by E. coli is the use of a strong, regulatable promoter and an effective ribosome binding site.
  • the promoters here can be, for example, the temperature-sensitive bacteriophage ⁇ pL promoter, the tac promoter inducible with IPTG or the T7 promoter.
  • plasmids with suitable promoter structures with efficient ribosome binding sites have been described, such as, for example, pKC30 ( ⁇ pL; Shimatake and Rosen ⁇ berg (1981) Nature 292, 128, pKK173-3 (tac, Amann and Brosius (1985) Gene 40, 183) or pET-3 (T7 promoter (Studier and Moffat (1986) J. Mol Biol 189, 113).
  • E. coli strains which are specifically tailored to the respective expression vector are known to the person skilled in the art (Sambrook et al. (1989), loc. Cit.)
  • yeast for example, the plasmid YRp7 (Stinchcomb et al. Natur 282, 39 (1979); Kingsman et al, Gene 7, 141 (1979); Tschumper et al, Gene K), 157
  • the plasmid YRp7 contains the TRPl gene, which is a selection marker for a Mutant yeast (eg ATCC No. 44076) which is unable to grow in trypthophane-free medium.
  • the presence of the TRP1 defect as a characteristic of the yeast strain used then represents an effective tool for detecting transformation if cultivation is carried out without tryptophan.
  • the situation is similar for the plasmid YEpl3, which contains the yeast gene LEU 2, which can be used to supplement a LEU-2 minus mutant.
  • yeast hybrid vectors preferably also contain a start of replication and a marker gene for a bacterial host, in particular E. coli, so that the construction and cloning of the hybrid vectors and their precursors can take place in a bacterial host.
  • Further expression control sequences suitable for expression in yeast are, for example, those of the PHO3 or PHO5 gene.
  • yeast vectors include the 5 'flanking region of the genes of ADH I (Ammerer, Methods of Enzymology 1 _ 192-201 (1983)), 3-phosphoglycerate kinase (Hitzeman et al, J. Biol. Chem.
  • glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose -6-phosphate isomerase, phosphoglucose isomerase and glucokinase.
  • the termination sequences associated with these genes can also be inserted into the expression vector at the 3 'end of the sequence to be expressed, in order to enable polyadenylation and termination of the mRNA.
  • promoters are the promoter regions of the alcohol dehydrogenase-2, isocytochrome C, acid phosphatase, and enzyme genes responsible for the metabolism of maltose and gralactose.
  • Promoters that are regulated by the yeast mating type locus for example promoters of the genes BARI, MF ⁇ l, STE2, STE3, STE5 can be used in temperature-regulated systems by using temperature-dependent sir mutations. (Rhine Ph.D. Thesis, University of Oregon, Eugene, Oregon (1979), Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, part I, 181-209 (1981), Cold Spring Harbor Labo ⁇ ratory).
  • any vector which contains a yeast-compatible promoter, original replication and termination sequences is suitable.
  • hybrid vectors containing sequences homologous to the yeast 2 ⁇ plasmid DNA can also be used. be applied.
  • Such hybrid vectors are incorporated by recombination already existing 2 ⁇ plasmids or replicate autonomously.
  • yeasts In addition to yeasts, other eukaryotic systems can of course also be used to express the polypeptides according to the invention. Since post-translational modifications such as disulfide bridge formation, glycosylation, phosphorylation and / or oligomerization are often necessary for the expression of biologically active eukaryotic proteins by means of recombinant DNA, the expression of the DNA according to the invention in mammalian cell lines but also in insect cell lines is also suitable.
  • the functional requirements of the corresponding vector systems include, in particular, suitable promoter, termination and polyadenylation signals, and elements which enable replication and selection in mammalian cell lines.
  • Vectors which can be replicated both in mammalian cells and in prokaryotes such as E. coli are particularly suitable for the expression of the DNA molecules according to the invention.
  • Vectors derived from viral systems such as SV40, Epstein-Barr virus, etc. are, for example, pTK2, pSV2-dhfv, pRSV-neo, pKO-neo, pHyg, p205, pHEBo, etc. (Sambrook et al. 1989, loc. cit.).
  • CHO cells After transformation into suitable host cells, e.g. CHO cells can be obtained with the aid of selectable markers (thymidine kinase, dehydrofolate reductase, etc.) and transformed cells and the corresponding polypeptides can be isolated after expression.
  • suitable host cells e.g. CHO cells
  • selectable markers thymidine kinase, dehydrofolate reductase, etc.
  • transformed cells and the corresponding polypeptides can be isolated after expression.
  • the host cells suitable for the vectors are known, as are the techniques for transformation (microinjection, electroporation, calcium phosphate method, etc.) (e.g. Sambrook et al., 1989).
  • the selected vector is cut with a restriction endonuclease and, optionally after modification of the linearized vector thus formed, an expression control sequence provided with corresponding restriction ends is introduced.
  • the expression control sequence contains at the 3 'end (in the direction of translation) the recognition sequence of a restriction endonuclease, so that the vector already containing the expression control sequence can be digested with said restriction enzyme and the DNA molecule according to the invention provided with suitable ends can be used.
  • the invention comprises methods for producing the vectors described, in particular expression vectors.
  • These vectors are characterized in that in a vector DNA cut with restriction endonucleases which contains the expression control sequences described by way of example, a DNA provided with corresponding ends which is suitable for a functionally new derivative of the receptor of the "small rhinovirus receptor group" encoded, so that the expression control sequences regulate the expression of the inserted DNA.
  • polypeptides according to the invention which are obtained by expression of recombinant DNA or from the native receptor molecule, can of course also be derivatized by chemical or enzymatic methods.
  • the expression of the LDL receptor is explained in Example 6.
  • expression takes place in a eukaryotic system.
  • HRV2 human rhinovirus serotype 2
  • the polypeptides according to the invention can be obtained, for example, by deleting DNA sequences in the expression plasmid.
  • the method of Davis et al. (1987) Nature 326, 760-765 which describes the deletion of the entire EGF domain.
  • soluble forms of the receptor can be formed by introducing a stop codon in front of the cytoplasmic or transmembrane domain (Yokade et al. (1992) J. Cell. Biol U7, 39).
  • the invention furthermore comprises hybrid cell lines which specifically secrete monoclonal antibodies against one of the polypeptides or functional derivatives according to the invention. These monoclonal antibodies are able to neutralize the action of the polypeptides in whole or in part or to bind specifically to one of the said polypeptides. The monoclonal antibodies can then be used for the qualitative and / or quantitative determination or for the purification of the polypeptides according to the invention.
  • the invention naturally also includes test systems which contain the monoclonal antibodies mentioned.
  • the method for producing the monoclonal antibodies is characterized in that host animals are immunized with one of the polypeptides and B-lymphocytes of these host animals are fused with myeloma cells become; the hybrid cells which secrete the corresponding monoclonal antibodies can then be subcloned and cultivated (Harlow, G. and Lane, D .: “Antibodies. A Laboratory Manual” (1988) Cold Spring Harbor Laboratory Press, USA).
  • physiological ligands of the LDL receptor family for the production of medicaments for inhibiting the binding of rhinoviruses of the "small rhinovirus receptor group".
  • the physiological ligands include the substances that are bound and / or internalized by the LDL receptor family.
  • LDL low density lipoprotein
  • Other natural ligands of the LDL receptor family are e.g. by Willnow et al. (1992) J. Biol. Chem. 267, 26172-26180.
  • the 39 kDa receptor-associated protein can reduce the yield of rhinoviruses of the "small rhinovirus receptor group" (Example 7).
  • RAP is known per se. Its isolation and binding to members of the LDL receptor family is described, for example, by Kounnas et al. (1992) J. Biol. Chem. 267, 21162-21166.
  • the native receptors of the LDL receptor family, the LDL receptor, ⁇ 2MR LRP and gp330, such as the receptor derivatives according to the invention, can also be used for inhibition.
  • rhinovirus material of the "small rhinovirus receptor group” can also be used to inhibit the binding of physiological LDL ligands.
  • This rhinovirus material can e.g. be derived from human rhinovirus serotype 2 (HRV2).
  • HRV2 human rhinovirus serotype 2
  • Inactivated rhinovirus, rhinovirus covering material or rhinovirus peptides with binding activity to a receptor of the LDL receptor family can preferably be used as rhinovirus material.
  • Rhinoviruses of the "small rhinovirus receptor group” are available from the "American Type Culture Collection”.
  • Corresponding virus material can be provided using known methods (e.g. Putnak and Phillips (1981) Microbiol Reviews 45, 287-315 and Palmenberg (1990) Annu. Rev. Microbiol 44, 603-623 and the literature cited therein).
  • the invention naturally also includes the pharmaceutically tolerable salts of the polypeptides according to the invention and the pharmaceutically tolerable adducts and covalent compounds between the polypeptides and an inert carrier for prophy- lactic and / or therapeutic treatment of the human or animal body.
  • the adducts or covalent compounds can be formed, for example, with polyethylene glycol.
  • the polypeptides according to the invention and the native receptor proteins, the physiological ligands of the LDL receptor family, such as, for example, LDL and the RAP, can be used for the production of pharmaceutical preparations for the therapeutic and / or prophylactic treatment of the human or animal body.
  • polypeptides can serve as competitive substances for inhibiting the binding of viruses, in particular rhinoviruses, to the native receptor and / or physiological LDL ligands.
  • viruses in particular rhinoviruses
  • the polypeptides and natural ligands, in particular the extracellular, soluble form of the receptor, can be used above all as antiviral, preferably antirhinoviral, agents.
  • the substances described can e.g. are administered nasally, providing a quantity which is sufficient for suppression or competitive interaction or for inhibition of the rhinovirus binding to the natural receptor.
  • the dose should generally be between 0.01 pg / kg patient weight and 1 mg / kg patient weight, although larger or smaller amounts can also be used.
  • the rhinovirus material which can be used to inhibit the binding of physiological LDL ligands can be used in suitable pharmaceutical compositions in the concentration ranges given for the polypeptides.
  • the receptor derivatives according to the invention and their pharmacologically acceptable salts can be converted in a known manner into the customary formulations - such as tablets, tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert pharmaceutically suitable excipients or solvents.
  • the proportion of the pharmaceutically active compound (s) should in each case be in the range from 0.5 to 90% by weight of the total composition, i.e. in amounts sufficient to reach the dosage range indicated above.
  • the formulations are prepared, for example, by stretching the active ingredients with solvents and / or carriers, if appropriate using emulsifiers and / or dispersants, where, for example, if water is used as the diluent, organic solvents may optionally be used as solubilizers or auxiliary solvents can be used.
  • auxiliaries include water, pharmaceutically acceptable organic solvents such as paraffins, oils of vegetable origin, monofunctional or polyfunctional alcohols, carriers, such as natural rock powders, synthetic rock powders, sugar, emulsifiers and lubricants.
  • the application is carried out in the usual way, preferably nasally.
  • the tablets can of course also contain additives, such as e.g. Contain sodium citrate, calcium carbonate and dicalcium phosphate together with various additives such as starch, preferably potato starch, gelatin and the like.
  • Lubricants such as magnesium stearate, sodium lauryl sulfate and talc can also be used for tableting.
  • the active ingredients can be mixed with various flavor enhancers or colorants.
  • the invention also includes methods for isolating substances which inhibit the binding of ligands to the LDL receptor.
  • these methods include incubating the LDL receptor protein or an LDL receptor derivative with a potentially inhibitory substance. This procedure can be done in the presence of labeled rhinovirus material. The extent of the binding of labeled rhinovirus material then provides information about the effect of the tested substance. The provision of rhinovirus material with different binding activities is shown in Example 9.
  • the invention comprises methods for the determination of LDL receptors, in that a substance derived from virus material of the "small rhinovirus receptor group” with binding activity to the LDL receptor is labeled, incubated with a corresponding sample and the extent of the binding is detected.
  • Another method is used to deliver therapeutically active substances, in which virus material of the "small rhinovirus receptor group” with binding activity to the LDL receptor is coupled with the therapeutic substance and the said conjugate is added to the LDL receptor-carrying cell material and by binding and internalization the therapeutically active substance is introduced into the cell.
  • Fig. 1 Amino acid sequence of the "Low Density Lipoprotein Receptors" (LDL, Yamamoto et al (1984) Cell 31, 27-38).
  • Fig. 2 Amino acid sequence of the "Low Density Lipoprotein Receptor Related Protein” (LRP, Herz et al. (1988) EMBO J. 7, 4119-4127).
  • Figure 3 Part of the amino acid sequence of the "Heymann Nephritis Antigen gp330 (Pietromonaco et al (1990) Proc. Natl. Acad. Sci. U.S.A 87, 1811-1815).
  • Fig. 4 Schematic representation of a receptor of the LDL receptor family (according to Yamamoto et al. (Loc. Cit.).
  • the receptor comprises five domains: Domain 1 comprises the N-terminal, cysteine-rich receptor part, which is presumably responsible for ligand binding Domain 2 with homology to EGF-
  • Precursor protein joins domain 3, some of whose amino acids are O-glycosylated. Domain 4 forms the membrane-bound part of the receptor, domain 5 the cytoplasmic part.
  • Figure 6 Binding of [ 35 S] -labeled HRV2 to c ⁇ MR / LRP and gp330.
  • Membrane extracts were separated electrophoretically and transferred to nitrocellulose. Detection was performed with [ 3 ⁇ S] -labeled HRV2 (lanes 1 and 2) with ⁇ ⁇ MR / LRP antiserum (lane 3 or with gp330 antiserum (lane 4).
  • Fig. 7 Gel electrophoretic analysis of the purified HRV2 binding protein.
  • the purified HRV2 binding protein was electrophoresed in a 7.5% SDS gel under reducing (lane 1) and under non-reducing conditions (lane 2) and visualized by silver staining. A molecular weight of approx. 120 kDa, a molecular weight of 160 kDa under reducing conditions.
  • b) Ligand blots of a gel as described under a (lane 2), developed with [ 35 S] -HRV2 (lane 1) according to Mischak et al. (1988) Virology 163, 19-25.
  • Lane 2 shows the development with an antibody specific for the human LDL receptor (IgG-C7, Beisiegel et al. (1982) J. Biol. Chem. 257, 13150-13156).
  • Buffer A dest. Water / 0.06% TFA; Buffer B: 80% acetonitrile 0.052% TFA; Flow rate: 0.5 ml / min; Gradient: 2% B to 37.5% B from 0 to 60 min,
  • Fig. 10 Rechromatography of fraction 29. The experimental conditions are listed in the legend to Fig. 9.
  • Fig. 11 Rechromatography of fraction 38. The experimental conditions are listed in the legend to Fig. 9.
  • Fig. 12 Sequences of the peptides analyzed
  • Fig. 15 Reduction of virus yield by RAP, given in p.f.u./ml (infectious particles per milliliter).
  • Fig. 16 Inhibition of HRV2 infection of HeLa cells by human LDL.
  • Fig. 17 Sequence comparison for determining the positions in or on the edge of the canyon, which are conserved in the rhinoviruses of the small group.
  • Fig. 18 Binding behavior of HRV2j i4gp : G and ⁇ HRV23 i82R: T on HeLa cells ⁇ HRV2H48P-G
  • the cells were then washed twice with PBS, 10000 cpm [- ⁇ Sj- labeled HRV2 in 0.5 ml PBS containing 2% BSA and 30 mM MgCl2, added and incubated for 60 min at 34 ° C (Mischak et al. (1988) Virology 163, 19-25). After removal of HRV2 bound to the surface with 10 ⁇ g / ml trypsin, 25 mM EDTA in PBS, the cells were washed again and then the bound radioactivity was determined. The data shown are mean values from four experiments each. The radioactivity values of the cell pellets from normal fibroblasts (normally around 1900 cpm) grown without steroids minus background radioactivity were set equal to 100%.
  • the background activity was determined either with HRV2 which had been heated to 56 ° C. for 30 min (Mischak et al. (1988) loc. Cit.) Or by incubation with a 1000-fold excess of unlabelled HRV2. It was between 40 and 50 cpm for both methods.
  • the data obtained from four individual experiments are shown in FIG. 5a.
  • Normal fibroblast cells were grown as described under a) (without the addition of cholesterol and 25-hydroxycholesterol). The cells were cultured with approximately 1.4 x 10 ⁇ cpm 125 I-labeled LDL (250 cpm / ng; Huettinger et al. (1992) J. Biol Chem., 267, 18551-7) with (+) and without (- ) addition of 100 Pfu ( "plaque forming units, etc., equivalent to about 2400 to 24,000 virus particles; Abraham & Colonno (1984) J. Virol 5, 340-345) per cell of purified unlabeled HRV2 or about 10,000..
  • Plasma membranes were isolated from mouse LM fibroblasts and renal epithelial microvilli (Malathi et al. (1979) Biochem. Biophys. Acta, 554, 259-263; Fomistal et al. (1991) Infect. Immun. 59, 2880-2884 and Kerjaschki and Farquhar (1982) Proc. Natl. Acad. Sci. USA 79, 5557-5561). Proteins from the membrane extracts were separated using an SDS gradient polyacrylamide electrophoresis and transferred to nitrocellulose.
  • Example 3 Purification of a binding protein for the rhinoviruses of the "small rhino virus receptor group"
  • HeLa cell culture supernatant (Computer Cell Culture Center, Mons, Belgium) was concentrated to 20 l by means of ultrafiltration and dialyzed against 250 l of distilled water (with 0.02% NaN 3 ). The buffer concentration was then increased to 20 mM N-methylpiperazine, pH 4.5, adjusted, centrifuged at 4000 rpm in the Beckman J6B centrifuge, filtered through a 0.8 mm prefilter and the filtrate loaded onto an anion exchange column (0.5 1 Makroprep 50 Q; biorad). Bound material was eluted with 20 mM N-methylpiperazine, pH 4.5, 0.5 M NaCl.
  • the eluate was adjusted to a pH of 7.2 with 1 M Tris-HC1, pH 8.0 and loaded onto a lens culinaris lectin column (100 ml; Pharmacia); bound protein was eluted with 0.5 M ⁇ - [D] methylglucose in PBS, the eluted protein was precipitated at 50% saturation with ammonium sulfate, pH 7.2, the precipitate with 50% saturated ammonium sulfate solution, pH 7.2 , washed and taken up in 200 ml of PBS.
  • the protein solution was loaded onto a jacalin agarose column (40 ml; Vector-Labs) and eluted with 120 ml of 100 mM ⁇ - [D] methyl-galactopyranoside in PBS.
  • the eluted protein was precipitated with ammonium sulfate as described above, washed, taken up in 20 mM methylpiperazine, pH 4.5 and desalted using a PDIO column (Pharmacia).
  • the desalted material was loaded in 5 1 ml aliquots onto a Mono Q anion exchange column (HR5 / 5; Pharmacia) and eluted with a gradient of 0 to 0.5 M NaCl, 20 mM methylpiperazine, pH 4.5.
  • the gel pieces were decolorized in 0.25 M Tris-HCl, 0.25 M EDTA, pH 9.0 and the protein was electrophoresed in 50 mM N-ethylmorpholine acetate, pH 8.5. An aliquot was again checked for activity using the filter binding assay. The protein was then gel electrophoresed under reducing conditions, eluted and lyophilized.
  • Example 4 Tryptic digestion and sequence analysis of the binding protein for the rhinoviruses of the "small rhinovirus receptor group"
  • the purified and lyophilized protein (Example 3) was taken up in 30 ml of 6 M guanidine-HCl, 0.4 M ammonium hydrogen carbonate, pH 7.6 and mixed with 3 ⁇ l of 45 mM dithiothreitol and incubated at 56 ° C. for 15 min. After cooling to room temperature, 3 ml of 100 mM iodoacetamide were added and incubated for a further 15 minutes at room temperature.
  • Fractions 20 and 33 were sequenced directly with the aid of a gas phase sequencer, while fractions 23 to 27 and fractions 29 and 38 were re-chromatographed under the conditions specified in the figures (C18 "reversed-phase” column, Merck; FIG. 9 , 10 and 11).
  • the peptides designated in the figures with "A”, “D” and “F” or the fractions 33 and 20 were selected for sequencing in the gas phase sequencer.
  • the result is summarized in Fig. 12.
  • the sequences obtained were compared with the protein sequences available in the "SwissProt" database. The comparison showed complete agreement with corresponding peptide sequences of the human LDL receptor:
  • the following table shows the sequences of the isolated tryptic peptides and the position in the sequence of the human LDL receptor (FIG. 1).
  • fraction 33 gave two amino acids per degradation step. On the basis of the LDL sequence and the ratio of the amounts of amino acids present in each degradation step from approximately 40% to 60%, fraction 33 could be identified as a mixture of two peptides. The sequences of these two peptides also correspond to sequences of the human LDL receptor.
  • Example 5 Expression of the human LDL receptor in COS-7 cells
  • the plasmid pTZl which contains the entire coding sequence of the human LDL receptor from the plasmid pLDLR2 (Yamamoto et al., Loc. Cit.) was converted into competent E. coli 5K using known methods (Sambrook et al., Loc. Cit.) introduced and amplified. After extraction and purification of the plasmid DNA, it was digested with the restriction enzyme Hind TU and the fragments separated in a 0.8% agarose gel. After elution of the fragment coding for the LDL receptor, it was precipitated with ethanol, taken up in TE buffer and partially filled in with Klenow fragment using dATP and dGTP.
  • the eukaryotic expression vector pSVL (Pharmacia) was propagated in E. coli 5K, purified and cut with Xbal. After partial filling with dCTP and dTTP, phenol-chloroform extraction and ethanol precipitation, the plasmid was dephosphorylated with alkaline phosphatase.
  • the cells were sown in 6-well dishes and cultivated for a further 24 hours in RPMI / 10% HiFCS and 12 ⁇ g / ml cholesterol and 2 ⁇ g / ml 25-hydroxycholesterol.
  • the cells were washed with PBS / 2% BSA and then incubated for 1 hour at 34 ° C. with about 10000 cpm / well [ 35 S] -labeled HRV2 in PBS / 2% BSA. After washing several times, the cells were lysed in PBS / 2% SDS and the amount of bound [ 3 ⁇ S] -HRV2 was determined by counting in a liquid scintillation counter.
  • the addition of fetal calf serum, cholesterol and 25-hydroxycholesterol suppresses the endogenous LDL receptors (Davis et al, 1987, Nature 326, 760), so that only the LDL receptors expressed by transfection are detected in the subsequent binding test.
  • the amount of bound HRV2 is twice as high compared to untransfected control cells if the cells with the sense construct pSVL- LDLR + were transfected. Transfection with pSVL-LDLR- shows no difference in binding compared to control cells.
  • Example 6 Inhibition of [35s] -labeled rhinovirus serotype 2 (HRV2) by Jacalin
  • trace A or in the presence (trace B) of 0.1 mg / ml Jacalin (Vector Labs) with radioactively labeled rhinovirus (Mischak et al, 1988, loc. Cit.) incubated, washed, dried and exposed on X-ray film (Hofer et al, 1992, loc.cit.). As shown in FIG. 13, the binding of the virus to the LDL receptor is completely inhibited under the specified conditions.
  • FH cells (see Example 1) were seeded in 24-well plates (Nunc) in RPMI with 10% fetal calf serum and grown overnight to a cell density of about 5x10 ⁇ cells per well. The cells were washed once with PBS and treated with RPMI / 2% fetal calf serum / 30mM MgCl2. Human recombinant RAP was as described in Kunnas et al., Loc. cit. given and cleaned and added to the medium in concentrations of 0.5 ⁇ g / ml, 5 ⁇ g / ml, 10 ⁇ g / ml and 20 ⁇ g / ml and the cells incubated at 4 ° C. for 2 hours.
  • HRV2 was added in a moi of 100 to each test batch and incubated for a further 2 hours at 4 ° C.
  • the cells were then washed 3 times with PBS, treated with RPMI / 2% fetal calf serum / 30 mM MgCl2 and incubated at 34 ° C. overnight.
  • the next day the cells were broken up by freezing / thawing three times. Cell fragments removed by centrifugation at 100,000 g and the number of infectious virus particles in the supernatant determined using a plaque test (Neubauer et al, loc. Cit.). 15 shows that the yield of HRV2 decreases with increasing concentration of RAP and is reduced to approx. 5% of the comparison value without RAP at a RAP concentration of 20 ⁇ g ml.
  • Example 8 Inhibition of HRV2 infection of HeLa cells by human LDL
  • HeLa cells were seeded in 24-well plates (Nunc) in MEM with 10% fetal calf serum and grown overnight to a cell density of approximately 2x10 ⁇ cells per well. The cells were washed once with PBS and treated with RPMI / 2% fetal calf serum / 30mM MgCl2. Purified LDL (Huettinger et al, loc. Cit.) was added in concentrations of 0.1 mg / ml, 0.3 mg / ml, 0.5 mg / ml and 1 mg / ml and the cells at 34 ° for 30 min C incubated.
  • HRV2 or HRV14 (a virus from the large group of receptors, used as a control) were added in a m.o.i of 100 to each test batch and incubated at 34 ° C. for a further 45 min. The cells were then washed 3 times with PBS, mixed with RPMI / 2% fetal calf serum / 30 mM MgCl2 and incubated at 34 ° C. for 60 hours. The medium was aspirated and intact cells stained with crystal violet.
  • Fig. 16 shows that in the presence of LDL at a concentration of 1 mg / ml the infection of HeLa cells by HRV2 is prevented (all cells are intact). No effect was observed in the case of HRV14 (cells completely lysed).
  • 17 shows a sequence comparison for determining the positions in or at the edge of the canyon which are conserved in the rhinoviruses of the small group in contrast to the rhinoviruses in the large group. They include the basic residues at position 1081 (HRV2 numbering: Blaas et al. (1987) Proteins 2, 263-272) and 3182, Ile or Leu at position 3229 and the sequence Thr-Glu-Lys (TEK at position 1222- 1224).
  • HRV2 mutants were constructed: at position 1081 (1081K: E) and at 1222-1224 (replacement of TEK by the corresponding sequence derived from HRV14, HRV39, HRV89) in the VP1 protein and the mutants 3182R: T and 3229L: T in VP3.
  • Another mutant (1148P: G) was constructed analogously to HRV14 (1155P: G) (Colonno et al. (1988) Proc. Natl Acad. Sci. U.S.A. 85, 5449-5453).
  • Analysis of the three-dimensional structure of HRVIA - a serotype closely related to HRV2 - showed no signs of steric or electrostatic disturbances from the exchanged amino acid side groups.
  • all side groups are on the surface and accessible to the solvent, so it is quite possible that they are involved in the interaction with the receptor of the small group and that their change leads to a loss of binding capacity and infectivity.
  • Trp Met Gly A ⁇ p Asn Leu Tyr Trp Thr Asp Asp Gly Pro Lys Lys Thr 565 570 575

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Abstract

L'invention concerne la préparation de polypeptides ayant une activité de liaison des récepteurs appartenant au "petit groupe de récepteurs des rhinovirus", leur procédé de préparation, leur utilisation et l'ADN de codage de ces peptides.
EP93915793A 1992-07-08 1993-07-05 Derives de recepteurs avec sites de fixation pour rhinovirus humains Withdrawn EP0613498A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
DE4222385 1992-07-08
DE4222385A DE4222385A1 (de) 1992-07-08 1992-07-08 Verfahren zur Isolierung von Inhibitoren des Rhinovirus-Rezeptors
DE19924227892 DE4227892A1 (de) 1992-08-22 1992-08-22 Derivate des Rezeptors der "kleinen Rhinovirus-Rezeptorgruppe" sowie die dafür codierenden DNA-Moleküle
DE4227892 1992-08-22
DE4305063 1993-02-19
DE19934305063 DE4305063A1 (de) 1993-02-19 1993-02-19 Rezeptorderivate
PCT/EP1993/001728 WO1994001553A1 (fr) 1992-07-08 1993-07-05 Derives de recepteurs avec sites de fixation pour rhinovirus humains

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US5496926A (en) 1992-01-19 1996-03-05 Yeda Research And Development Co. Ltd. Process of preparing a soluble LDL receptor
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DE3712678A1 (de) * 1987-04-14 1988-10-27 Boehringer Ingelheim Int Rezeptor der kleinen rhinovirus rezeptor gruppe
EP0358977A1 (fr) * 1988-08-23 1990-03-21 The General Hospital Corporation Antigène de nephritis cloné

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