WO1994001553A1 - Receptor derivates with binding sites for human rhinoviruses - Google Patents

Abstract

The production of polypeptides having a binding activity of receptors of the "small rhinovirus receptor group" is disclosed, as well as a process for producing these peptides, their use and the DNA coding for these peptides.

Description

 RECEPTOR DERIVATIVES WITH BINDING POINTS FOR HUMANE RHINOVIRES

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). By demonstrating a specific binding of the purified ICAM-1 to the virus and by the ability to transfer the rhinovirus binding activity to cells which had no corresponding activity prior to the transfer, it could be clearly demonstrated that ICAM-1 was the receptor for the majority of rhinoviruses are (Greve et al. (1989) loc. cit .; Staunton et al. (1989) loc. cit). It has also been shown that monoclonal antibodies against ICAM-1 prevent the binding and infection of HeLa cells by rhinoviruses (Staunton et al. (1989), loc. Cit.). Furthermore, monoclonal antibodies which inhibit the binding of ICAM-1 to leukocytes via LFA-1 ("lymphocyte fetion associated antigen-1") - another natural ligand of ICAM-1 - can also bind to rhinovirus block the receptor. The LFA-1 and rhinovirus binding sites must therefore at least be adjacent. Experiments with chimeras and mutant ICAM-1 molecules have shown- In addition, the binding site for the rhinovirus-ICAM-1 interaction does not coincide with the binding site for LFA-1 (Staunton et al. (1990) Cell 61, 243-254).

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).

As far as is known to date, 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 (determined by gel chromatography) 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.). It was also found that a protein isolated from the cell culture supernatant of HeLa cells has the ability to bind rhinoviruses of the "small rhinovirus receptor group" (Hofer et al. (1992) J. Gen. Virol. 73, 627-632) .

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.

Surprisingly, it has now been found that the members of the LDL ("low density lipoprotein") receptor family serve as receptors for rhinoviruses of the "small rhinovirus receptor group".

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".

The polypeptides according to the invention themselves are referred to below as "functional derivatives" of the receptor proteins. 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. To block non-specific binding, 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.

One aspect of the invention is the 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).

Of course, soluble receptor derivatives can also be formed by enzymatic, in particular proteolytic or chemical, cleavage. For this, e.g. B. receptor-carrying cell lines can be used, which are implemented with enzymes such as papain, trypsin, etc. If 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. In addition to enzymatic cleavage, it is also possible to cleave extracellular receptor regions by chemical methods, for example by cleavage with cyanogen bromide.

Another aspect of this invention is the formation of soluble derivatives by enzymatic or chemical cleavage of native receptor molecules. After isolation of 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. For the chemical cleavage reactions, cyanogen bromide or splitting of the receptor protein by reductive treatment, e.g. with dithiothreitol.

In particular, the present invention includes the following aspects:

It was surprisingly found that proteins of the LDL receptor family can bind and internalize rhinoviruses of the "small rhinovirus receptor group". This means that all members of the LDL receptor family can now be used for functional purposes To produce derivatives that can bind rhinoviruses of the "small rhinovirus receptor group".

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).

Surprisingly, it could be shown that all three members of this family - the LDL receptor, the c ^ MR / LRP (012 -macroglobulin / LDL-receptor-related protein) and also the gp330 (Heymann nephritis antigen gρ330) - are able To bind and internalize rhinoviruses of the "small rhinovirus receptor group" (Examples 1 to 2).

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". For example, 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.

Surprisingly, it turned out that it is an LDL receptor derivative (Example 4). For isolation, 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 and 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. Among the proteins of the LDL receptor family, the human LDL receptor is the preferred starting compound. In particular, 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:

Domains 1 and 2 (amino acids 1 to 750, SEQ.ID.NO.l):

MGP GW LRW TVALL AAAG TAVGDRCERN EFQCQDGKCI SYKWVCDGSA ECQDGSDESQ ETCLSVTCKS GDFSCGGRVN RCIPQFWRCD GQVDCDNGSD EQGCPPKTCS QDEFRCHDGK CISRQFVCDS DRDCLDGSDE ASCPVLTCGP ASFQCNSSTC IPQL ACDND PDCEDGSDE PQRCRGLYVF QGDSSPCSAF EFHCLSGECI HSSWRCDGGP DCKDKSDEEN CAVATCRPDE FQCSDGNCIH GSRQCDREYD CKD SDEVGC VNVTLCEGPN FKCHSGECI TLDKVCNMAR DCRD SDEPI KECGTNECLD NNGGCSHVCN DLKIGYECLC PDGFQLVAQR RCEDIDECQD PDTCSQLCVN LEGGYKCQCE EGFQLDPHTK ACKAVGSIAY LFFTNRHEVR KMTLDRSEYT S IPNLRNW ALDTEVASNR IY SDLSQRM ICSTQLDRAH GVSSYDTVIS RDIQAPDG A VD IHSNIYW TDSVLGTVSV ADTKGVKRKT LFRENGSKPR AIWDPVHGF MY TD GTPA KIKKGGLNGV DIYSLVTENI Q PNGITLDL LSGRLY VDS KLHSISSIDV NGGNRKTILE DEKRLAHPFS AVFEDKVFW TDIINEAIFS ANRLTGSDVN LLAENLLSPE D VLFHNLTQ PRGVNWCERT TLSNGGCQYL CLPAPQINPH SPKFTCACPD GMLLARDMRS CLTEAEAAVA TQETSTVRLK VSSTAVRTQH TTTRPVPDTS

Domain 1 (amino acids 1 to 322, SEQ.ID.NO.2):

MGPWGWKLRW TVALLLAAAG TAVGDRCERN EFQCQDGKCI SYKWVCDGSA ECQDGSDESQ ETCLSVTCKS GDFSCGGRVN RCIPQFWRCD GQVDCDNGSD EQGCPPKTCS QDEFRCHDGK CISRQFVCDS DRDCLDGSDE ASCPVLTCGP ASFQCNSSTC IPQLWACDND PDCEDGSDEW PQRCRGLYVF QGDSSPCSAF EFHCLSGECI HSSWRCDGGP DCKDKSDEEN CAVATCRPDE FQCSDGNCIH GSRQCDREYD CKDMSDEVGC VNVTLCEGPN KFKCHSGECI TLDKVCNMAR DCRDWSDEPI KECGTNECLD NN.

The 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.). In addition, 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.

In addition, the invention includes DNA vectors that contain the DNA sequences described above. In particular, 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.

For expression in prokaryotes, other organisms known from the prior art, in particular E. coli, can be used. The 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. For example, 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-β-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 λpL promoter, the tac promoter inducible with IPTG or the T7 promoter.

Numerous plasmids with suitable promoter structures with efficient ribosome binding sites have been described, such as, for example, pKC30 (λpL; 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).

A large number of other vector systems for the expression of the DNA according to the invention in E. coli are known from the prior art and are described, for example, in Sambrook et al (1989) loc. cit.).

Suitable 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.)

The experimental implementation of the cloning experiments, the expression of the polypeptides in E. coli and the processing and purification of the polypeptides are known and are described, for example, in Sambrook et al. (1989, loc. Cit.). In addition to prokaryotes, eukaryotic microorganisms such as e.g. Yeast.

For expression in 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

(1980)) and the plasmid YEpl3 (Bwach et al, Gene 8, 121-133 (1979)) was used.

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.

Other suitable labeling genes for yeast are e.g. the URA3 and HIS3 genes. 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.

Other suitable promoter sequences for 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. 251, 2073 (1980)) or other glycolytic enzymes (Kawaski and Fraenkel, BBRC 108, 1107-1112 (1982)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose -6-phosphate isomerase, phosphoglucose isomerase and glucokinase. When constructing suitable expression plasmids, 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.

Other 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, MFαl, 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). In general, however, any vector which contains a yeast-compatible promoter, original replication and termination sequences is suitable. In this way, hybrid vectors containing sequences homologous to the yeast 2μ plasmid DNA can also be used. be applied. Such hybrid vectors are incorporated by recombination already existing 2μ plasmids or replicate autonomously.

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.).

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. 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).

For the cloning of corresponding DNA fragments in prokaryotic or eukaryotic systems, for example 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. It is advantageous to cleave the vector already containing the expression control sequence with a second restriction endonuclease within the vector DNA and to split it into the resulting vector fragment insert the DNA molecule with the correct ends. The working techniques required for this are described, for example, by Sambrook et al (1989, loc. Cit.).

In addition to the DNA molecules according to the invention, 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.

The 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. Here, for example, expression takes place in a eukaryotic system. It is clearly shown that the LDL receptor expressed expresses the binding of radioactively labeled human rhinovirus serotype 2 (HRV2) (FIG. 5). The polypeptides according to the invention can be obtained, for example, by deleting DNA sequences in the expression plasmid. For example, the method of Davis et al. (1987) Nature 326, 760-765, which describes the deletion of the entire EGF domain. Furthermore, 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).

Another aspect of the invention is the use of 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. For example, LDL (low density lipoprotein) inhibits the uptake of rhinoviruses of the "small rhinovirus receptor group" (Example 9). Other natural ligands of the LDL receptor family are e.g. by Willnow et al. (1992) J. Biol. Chem. 267, 26172-26180.

For example, the 39 kDa receptor-associated protein (RAP) 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.

Of course, the native receptors of the LDL receptor family, the LDL receptor, α2MR LRP and gp330, such as the receptor derivatives according to the invention, can also be used for inhibition.

Conversely, of course, 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). 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. In particular, the polypeptides can serve as competitive substances for inhibiting the binding of viruses, in particular rhinoviruses, to the native receptor and / or physiological LDL ligands. The polypeptides and natural ligands, in particular the extracellular, soluble form of the receptor, can be used above all as antiviral, preferably antirhinoviral, agents.

For the treatment of viral infections, 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. Examples of 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. In the case of oral use, 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. In the case of aqueous suspensions, in addition to the auxiliaries mentioned above, the active ingredients can be mixed with various flavor enhancers or colorants.

In addition, 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.

Furthermore, 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. LEGENDS TO THE FIGURES

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.

5: A) Binding and internalization of labeled HRV2 to normal human fibroblast cells or to LDL receptor-deficient FH cells (example 1). t: cultivated without the addition of cholesterol / 25-hydroxycholesterol ^• I: cultivated with the addition of cholesterol / 25-hydroxycholesterol

B) Competition of HRV2 and LDL for the receptor binding site +: with the addition of unlabeled HRV2 or LDL -: without the addition of unlabeled HRV2 or LDL.

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 α ^ MR / LRP antiserum (lane 3 or with gp330 antiserum (lane 4).

Lane 1 LM extracts, lane 2 rat kidney microvilli extracts, lane 3 protein extracts like lane 1, lane 4 protein extracts like lane 2. Fig. 7 Gel electrophoretic analysis of the purified HRV2 binding protein. a) 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).

8 shows the column chromatographic separation of the tryptic peptides of the soluble form of the obtained from the HeLa cell supernatant

"Small rhinovirus receptor group" receptor. The peptides were grown on a µBondapak C 18,250-4 column under the following

Conditions separated:

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,

37.5% B to 75% B from 60 to 90 min, 75% B to 98% B from 90 to 105 min; Temperature: room temperature; Detection: photometric at 214 nm,

0.08 AUFS (paper feed: 0.25 cm / min).

9: Chromatographic separation of fractions 23 to 27 under the following conditions:

Column: Merck Superspher 4 μm, C18, 125-H; Buffer A: dest. Water / 0.1% TFA; Buffer B: acetonitrile / 0.1% TFA; Flow rate: 1 ml / min, linear gradient from 0% B to 70% B in 70 min; Temperature: 30 ° C; Detection: photometric at 214 nm, 0.1 AUFS, paper feed 1 cm / 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

X = amino acid not identifiable; subscript = amino acid not clearly identifiable.

*: The sequencing of fraction 33 (Fig. 9) resulted in 2 amino acids per degradation step; peptides B and E could, however, because of the different

Quantities can be assigned.

13: Inhibition of the binding of [ ³ ^ S] -labeled HRV2 to the LDL receptor bound to Immobilon by Jacalin. A) Filter binding test in the absence of

Jacalin

B) as A, but in the presence of 0.1 mg / ml jacalin.

14: Expression of the human LDL receptor in COS-7 cells (Example 6). Detection of bound [ ³ - ^ S] -HRV2 on u: untransfected COS-7 cells +: transformed with the "sense" (pSVL-LDLR +) - vector -: transformed with the "antisense" (pSVL-LDLR -) - vector

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 ΔHRV2H48P-G

• HRV2 wild type * HRV2 _{3 182R:} τ

19: Competition of the binding of HRV2u4 p _; G and HRV23 ig2R _: τ through HRV14 (■) or HRV2 (.). EXAMPLES

Examples 1: Binding and internalization of [ ³ ^ S] - labeled HRV2 by human fibroblast cells and competition of HRV2 and LDL for the receptor binding site

a) Binding and internalization of HRV2 Normal human fibroblast cells or LDL receptor deficient cells (FH cells; NTH Collection No. GM 00486A) were on "6 well plates" (Nunc) in MEM, the 10% delipidated fetal calf serum (Gibco), either with (1) or without (t) addition of 12 μg / ml cholesterol and 2 μg / ml 25-hydroxycholesterol, for 24 h. 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 μg / 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.

b) Competition of HRV2 and LDL for the receptor binding site

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.. cpm [- ^ S] - labeled HRV2 with (+) or without (-) 80 mg / ml unlabeled LDL for 60 min at 37 ° C. The cell-associated radioactivity was determined with a g or ß counter. The radioactivity values for the high affinity binding of 12 ^ 1-LDL were obtained by subtracting the radioactivity in the presence of a 20-fold excess of unlabelled LDL obtained (approx. 40,000 cpm / mg of total cell protein) was determined from the total LDL binding (150,000 cpm / mg). Without a competitor, a radioactivity value of 1900 cpm was normally determined for the HRV2 binding. The maximum binding values were set to 100% in each case. The data obtained (FIG. 5b) show the result of two individual experiments.

Example 2: Binding of [ ³⁵ S] -labeled HRV2 by o- ^ MR / LRP and gp 330

To detect the binding of rhinoviruses of the "small rhinovirus receptor group" to other members of the LDL receptor family, plasma membrane preparations were checked for HRV2 binding.

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.

Incubation of the separated LM extracts with [ ³ ^ S] -labeled HRV2 showed binding to a protein with an apparent molecular weight of approximately 500 kDa (FIG. 6, lane 1). This gang emigrates with cc2MR / LRP. This could be shown by an identical blot, which was developed with α2MR / LRP antiserum (Moestrup and Gliemann (1991) J. Biol. Chem. 266, 14011-14017) (FIG. 6; lane 3).

In extracts from microvilli rat kidneys, a protein with an apparent molecular weight of also about 500 kDa could be detected with radioactively labeled HRV2 (FIG. 6; lane 2). After analysis with a gp330 antiserum, the band could be identified as Heymann nephritis antigen gp330 (FIG. 6, lane 4).

Example 3: Purification of a binding protein for the rhinoviruses of the "small rhino virus receptor group"

200 l of 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 α- [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 α- [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. 0.5 ml fractions were collected and checked for binding activity by means of a filter binding assay (Mischak et al, Virology (1988) 163, 19-25). The active fractions from all 5 chromatographies were concentrated to 1.5 ml in a Centricon 30 (Amicon) and by means of preparative gel electrophoresis under non-reducing conditions on a 7.5% polyacrylamide gel (Laemmli, UK (1970) Nature 227,680). 685) separated. The gel was stained with copper chloride (Lee et al. (1987) Anal Biochem. 166. 308-312), the band corresponding to the active protein was localized and cut out. 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 μl 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. Then 84 μl water and 80 μl 0.1 M ammonium hydrogen carbonate, pH 7.6, were added, the protein solution with 800 ng trypsin (in 5 μl) among the Conditions of the manufacturer (Promega) added and incubated at 37 ° C for 18 h. The solution was acidified with 1/10 volume of 10% trifluoroacetic acid (TFA), centrifuged for 5 min and the peptides on a C-18 "reversed-phase" column (Baker), which was treated with a 0.06% aqueous TFA Solution had been equilibrated, with a gradient up to 80% acetonitrile, 20% water, 0.052% TFA (Fig. 8). 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).

The sequencing of 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.

1 shows the overall sequence of the human LDL receptor (Yamamoto et al. (1984) Cell 31, 27-38). 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.

By partially filling in both the vector and the LDL receptor DNA, the restriction sites were made compatible and LDL receptor coding and vector DNA were ligated using T4 ligase. Competent E. coli bacteria were transformed as described. Several colonies were examined by restriction digestion with Xhol for orientation of the insert with respect to the SV40 late promoter. One colony each with positive (sense, pSVL-LDLR +) and negative (antisense, pSVL-LDLR-) orientation of the insert were grown and plasmids were obtained in large amounts. The transfection of COS-7 cells (ATCC CRL 1651) was carried out by means of lipofection (lipofectin reagent, BRL) in 9 cm petri dishes according to the manufacturer's instructions. After transfection, the cells were sown in 6-well dishes and cultivated for a further 24 hours in RPMI / 10% HiFCS and 12 μg / ml cholesterol and 2 μg / 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. As shown in FIG. 14, 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

Two aliquots of the protein purified to jacalin agarose chromatography (see Example 3, each corresponding to an initial amount of cell supernatant of approximately 50 ml) were placed on a 7.5% SDS polyacrylamide gel (Laemmli, UK 1970, Nature 227, 680-685) under non-reducing conditions separated and transferred electrophoretically to a Immobilon membrane (Millipore) (Mischak et al, 1988, loc. cit., Hofer et al, 1992, loc. cit.). A trace was in the absence (Fig. 13, 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.

Example 7: Reduction of virus yield by RAP (receptor associated protein)

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 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / 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 μg 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).

Example 9: Mutations of the HRV2 receptor binding site

Different receptor binding sites of the rhinoviruses of the "small" and the "large rhinovirus receptor group" should also be reflected in the "canyon structure" of the viral capsids responsible for the interaction with the corresponding receptor. A depression around the 5-fold axis of symmetry of the viral capsid with an extension of approximately 30 A is referred to as a canyon. A hypothesis regarding the Canyon structure states that the amino acid side groups in this region are inaccessible to immunoglobins and are therefore not exposed to any immunological pressure (Rossmann et al. (1985) Nature 317, 145-154). In this way, the different rhinoviral serotypes of a receptor group could conserve structures which are important for the interaction with the corresponding receptor and at the same time develop a broad serotypic diversity (Rossmann (1989) Viral Immunology 2, 143-161). Furthermore, the hypothesis implies that differences in the Canyon structure between "large" and "small rhinovirus receptor groups" are responsible for the use of two different receptors. This would include one set of amino acid residues preserved in certain positions of the "large rhinovirus receptor group" rhinoviruses and a second set conserved in certain positions of the "small rhinovirus receptor group" rhinoviruses. 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).

The following 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).

The preparation of the cDNA required for the mutagenesis, the in vitro production of corresponding infectious RNA and the transfection of corresponding cells are described (Duechler et al. (1989) Virology 168, 159-161; Maniatis et al. (1982) " Molecular Cloning: A laboratory manual ". Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Taylor et al (1989) Nucl. Acids. Res. 13, 8764-8785; Ho et al. (1989) Gene 77, 51- 59 and Herlitze & Koenen (1990) Gene £ 1, 143-147.

Transfection with RNAs which corresponded to the wild-type HRV2 and the mutants HRV2j Hgp.G and HRV23i 2 _: τ in Heia cells gave yields of approx. 300 pu ml. Plaque size and morphology of the wild type and of the two mutants were identical. 18 also shows that there was no significant difference in the binding behavior of these viruses to Heia cells (HRV2wt (•), HRV2ι i4 p _: G (Δ) and HRV23 i82R: T (♦); Neubauer et al. (1987) Virology 158.255-258). No viable mutants were obtained from the 1081K: E, 3229L: T or the mutant with the exchanged TEK motif at position 1222.

Competition experiments were carried out to show whether the mutants can bind to the receptor of the small rhinovirus group (FIG. 19). As soon as increasing amounts of unlabeled HRV2 (•) were present during the incubation of HeLa cells, the binding of [ ³ ^ S] labeled virus material of the mutants was reduced. Addition of HRV14 (■) had no effect on binding. Obviously, the affinity of the viruses for the "small rhinovirus receptor group" receptor does not change due to the mutations. These data show that the Pro 148 from VPl (equivalent to Pro 155 in HRV14) is not involved in the interaction of HRV2 with its receptor. - In contrast to HRV14, which indicates an important function of this amino acid in the interaction of HRV14 with ICAM-1 (Colonno et al (1988) Proc. Natl. Acad. Be. USA 85, 5449-5453). No conserved oligopeptide sequence corresponding to the TEK element could be detected in the large group of rhinoviruses.

The mutants HRV2] ogιK: E, HRV23229L: T ^{un (} ^ the TEK mutant were not viable. 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. In addition, 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.

The change from Proi 14g to Gly in HRV2 had no effect on the ability of the virus to bind to its receptor. With HRV14, a corresponding exchange leads to a considerably stronger binding to the receptor. Pro \ 155 forms a kind of base at the foot of the canyon and seems to prevent the receptor from penetrating further into the viral capsid. The increase in binding affinity by replacing Pro with Gly can therefore be interpreted as a reduction in steric hindrance. Since such an effect was not observed in HRV2, it is likely that the rhinoviruses in the small group will have a virus / receptor interaction at a different location than that used by the rhinoviruses in the large group.

SEQUENCE LOG

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Boehringer Ingelheim

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(C) LOCATION: Ingelheim (D) FEDERAL STATE: Rhineland-Palatinate

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(ii) REGISTRATION TITLE: RECEPTOR DERIVATIVES (iii) NUMBER OF SEQUENCES: 5

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(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPA)

(2) INFORMATION ABOUT SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 750 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu 1 5 10 15 Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe

20 25 30

Gin Cys Gin Asp Gly Lys Cys Ile Ser Tyr Lys Trp Val Cys Asp Gly 35 40 45

Ser Ala Glu Cys Gin Asp Gly Ser Asp Glu Ser Gin Glu Thr Cys Leu 50 55 60

Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn 65 70 75 80

Arg Cys Ile Pro Gin Phe Trp Arg Cys Asp Gly Gin Val Asp Cys Aεp 85 90 95

Asn Gly Ser Asp Glu Gin Gly Cys Pro Pro Lys Thr Cys Ser Gin Asp 100 105 110

Glu Phe Arg Cyε Hiε Aεp Gly Lyε Cys Ile Ser Arg Gin Phe Val Cyε 115 120 125

Asp Ser Asp Arg Asp Cyε Leu Asp Gly Ser Aεp Glu Ala Ser Cyε Pro 130 135 140

REPLACEMENT LEAF Val Leu Thr Cys Gly Pro Ala Ser Phe Gin Cys Asn Ser Ser Thr Cyε 145 150 155 160

Ile Pro Gin Leu Trp Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly 165 170 175

Ser Aεp Glu Trp Pro Gin Arg Cys Arg Gly Leu Tyr Val Phe Gin Gly 180 185 190

10

Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cyε Leu Ser Gly Glu 195 200 205

Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp

15 210 215 220

Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cyε Arg Pro Aεp Glu 225 230 235 240

20 Phe Gin Cys Ser Asp Gly Asn Cys Ile Hiε Gly Ser Arg Gin Cys Aεp 245 250 255

Arg Glu Tyr Asp Cys Lys Asp Met Ser Asp Glu Val Gly Cyε Val Asn 260 265 270

25

Val Thr Leu Cyε Glu Gly Pro Aεn Lys Phe Lys Cys His Ser Gly Glu 275 280 285

Cyε Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cyε Arg Aεp 30. 290 295 300

Trp Ser Aεp Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp

305 310 315 320

35 Aεn Asn Gly Gly Cys Ser Hiε Val Cyε Asn Asp Leu Lys Ile Gly Tyr

325 330 335

Glu Cys Leu Cys Pro Asp Gly Phe Gin Leu Val Ala Gin Arg Arg Cys 340 345 350

40

Glu Asp Ile Asp Glu Cyε Gin Asp Pro Asp Thr Cys Ser Gin Leu Cys 355 360 365

Val Asn Leu Glu Gly Gly Tyr Lys Cyε Gin Cys Glu Glu Gly Phe Gin 45 370 375 380

Leu Asp Pro His Thr Lys Ala Cys Lyε Ala Val Gly 'Ser Ile Ala Tyr 385 390 395 400

50 Leu Phe Phe Thr Asn Arg His Glu Val Arg Lys Met Thr Leu Asp Arg

405 410 415

Ser Glu Tyr Thr Ser Leu Ile Pro Asn Leu Arg Asn Val Val Ala Leu 420 425 430

55

Asp Thr Glu Val Ala Ser Asn Arg Ile Tyr Trp Ser Asp Leu Ser Gin 435 440 445

Arg Met Ile Cys Ser Thr Gin Leu Asp Arg Ala His Gly Val Ser Ser 60 450 455 460

Tyr Asp Thr Val Ile Ser Arg Asp Ile Gin Ala Pro Asp Gly Leu Ala 465 470 475 480

65 Val Asp Trp Ile His Ser Asn Ile Tyr Trp Thr Aεp Ser Val Leu Gly

485 490 495

HE Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe 500 505 510

Arg Glu Asn Gly Ser Lys Pro Arg Ala Ile Val Val Asp Pro Val His 515 520 525

Gly Phe Met Tyr Trp Thr Asp Trp Gly Thr Pro Ala Lys Ile Lys Lys 530 535 540

Gly Gly Leu Asn Gly Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile 545 550 555 560

Gin Trp Pro Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr 565 570 575

Trp Val Asp Ser Lyε Leu Hiε Ser Ile Ser Ser Ile Aεp Val Aεn Gly 580 585 590

Gly Asn Arg Lys Thr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro 595 600 605

Phe Ser Leu Ala Val Phe Glu Asp Lys Val Phe Trp Thr Asp Ile Ile 610 615 620

Asn Glu Ala Ile Phe Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn 625 630 635 640

Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Met Val Leu Phe Hiε 645 650 655

Aεn Leu Thr Gin Pro Arg Gly Val Aεn Trp Cyε Glu Arg Thr Thr Leu 660 665 670 Ser Asn Gly Gly Cys Gin Tyr Leu Cyε Leu Pro Ala Pro Gin Ile Asn 675 680 685

Pro His Ser Pro Lys Phe Thr Cys Ala Cyε Pro Aεp Gly Met Leu Leu 690 695 700

Ala Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala 705 710 715 720

Thr Gin Glu Thr Ser Thr Val Arg Leu Lyε Val Ser Ser Thr Ala Val 725 730 735

Arg Thr Gin Hiε Thr Thr Thr Arg Pro Val Pro Asp Thr Ser 740 745 750

(2) INFORMATION ABOUT SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 322 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu 1 5 10 15

REPLACEMENT LEAF Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cyε Glu Arg Aεn Glu Phe 20 25 30

Gin Cys Gin Asp Gly Lyε Cys Ile Ser Tyr Lyε Trp Val Cyε Aεp Gly 35 40 45

Ser Ala Glu Cys Gin Aεp Gly Ser Aεp Glu Ser Gin Glu Thr Cys Leu 50 55 60

Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cyε Gly Gly Arg Val Aεn 65 70 75 80

Arg Cyε Ile Pro Gin Phe Trp Arg Cyε Asp Gly Gin Val Aεp Cys Aεp 85 90 95

Aεn Gly Ser Aεp Glu Gin Gly Cyε Pro Pro Lyε Thr Cys Ser Gin Aεp 100 105 110

Glu Phe Arg Cys His Asp Gly Lyε Cyε Ile Ser Arg Gin Phe Val Cyε 115 120 125

Asp Ser Asp Arg Aεp Cyε Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro 130 135 140

Val Leu Thr Cys Gly Pro Ala Ser Phe Gin Cys Asn Ser Ser Thr Cys 145 150 155 160

Ile Pro Gin Leu Trp Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly 165 170 175

Ser Asp Glu Trp Pro Gin Arg Cys Arg Gly Leu Tyr Val Phe Gin Gly 180 185 190 Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cyε Leu Ser Gly Glu 195 200 205

Cys Ile His Ser Ser Trp Arg Cyε Asp Gly Gly Pro Asp Cys Lys Asp 210 215 220

Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu 225 230 235 240

Phe Gin Cyε Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gin Cys Asp 245 250 255

Arg Glu Tyr Asp Cys Lys Asp Met Ser Asp Glu Val Gly Cyε Val Asn 260 265 270 Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cyε His Ser Gly Glu 275 280 285

Cys Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp 290 295 300

Trp Ser Asp Glu Pro Ile Lyε Glu Cyε Gly Thr Aεn Glu Cyε Leu Aεp 305 310 315 320

Aεn Asn

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 860 amino acids

ERS (B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu 1 5 10 15

Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cyε Glu Arg Aεn Glu Phe 20 25 30

Gin Cyε Gin Asp Gly Lys Cyε Ile Ser Tyr Lyε Trp Val Cyε Asp Gly 35 40 45

Ser Ala Glu Cys Gin Asp Gly Ser Asp Glu Ser Gin Glu Thr Cyε Leu 50 55 60

Ser Val Thr Cyε Lys Ser Gly Asp Phe Ser Cyε Gly Gly Arg Val Aεn 65 70 75 80

Arg Cyε Ile Pro Gin Phe Trp Arg Cys Asp Gly Gin Val Aεp Cyε Aεp 85 90 95

Aεn Gly Ser Aεp Glu Gin Gly Cyε Pro Pro Lyε Thr Cyε Ser Gin Aεp 100 105 110

Glu Phe Arg Cys His Asp Gly Lyε Cyε Ile Ser Arg Gin Phe Val Cyε 115 120 125

Asp Ser Asp Arg Asp Cys Leu Aεp Gly Ser Asp Glu Ala Ser Cyε Pro 130 135 140

Val Leu Thr Cyε Gly Pro Ala Ser Phe Gin Cys Asn Ser Ser Thr Cys

145 150 155 160

Ile Pro Gin Leu Trp Ala Cys Asp Aεn Asp Pro Asp Cyε Glu Aεp Gly 165 170 175

Ser Asp Glu Trp Pro Gin Arg Cys Arg Gly Leu Tyr Val Phe Gin Gly 180 185 190

Aεp Ser Ser Pro Cyε Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu 195 200 205 Cyε Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Aεp Cys Lys Asp 210 215 220

Lyε Ser Aεp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu

225 230 235 240

Phe Gin Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gin Cyε Aεp 245 250 255

Arg Glu Tyr Asp Cys Lyε Aεp Met Ser Aεp Glu Val Gly Cys Val Asn 260 265 270

Val Thr Leu Cys Glu Gly Pro Aεn Lyε Phe Lys Cyε His Ser Gly Glu 275 280 285 Cys Ile Thr Leu Asp Lyε Val Cyε Asn Met Ala Arg Asp Cys Arg Asp 290 295 300 Trp Ser Asp Glu Pro Ile Lys Glu Cys Gly Thr Aεn Glu Cys Leu Asp 305 310 315 320

Asn Asn Gly Gly Cys Ser Hiε Val Cys Asn Aεp Leu Lyε Ile Gly Tyr 325 330 335

Glu Cyε Leu Cys Pro Asp Gly Phe Gin Leu Val Ala Gin Arg Arg Cys 340 345 350

Glu Asp Ile Asp Glu Cys Gin Asp Pro Asp Thr Cys Ser Gin Leu Cyε 355 360 365

Val Asn Leu Glu Gly Gly Tyr Lyε Cyε Gin Cyε Glu Glu Gly Phe Gin 370 375 380

Leu Aεp Pro His Thr Lys Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr 385 390 395 400

Leu Phe Phe Thr Aεn Arg Hiε Glu Val Arg Lyε Met Thr Leu Aεp Arg 405 410 415

Ser Glu Tyr Thr Ser Leu Ile Pro Aεn Leu Arg Asn Val Val Ala Leu 420 425 430

Asp Thr Glu Val Ala Ser Asn Arg Ile Tyr Trp Ser Aεp Leu Ser Gin 435 440 445

Arg Met Ile Cys Ser Thr Gin Leu Asp Arg Ala His Gly Val Ser Ser 450 455 460

Tyr Aεp Thr Val Ile Ser Arg Asp Ile Gin Ala Pro Asp Gly Leu Ala 465 470 475 480

Val Asp Trp Ile His Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly 485 490 495 Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg Lyε Thr Leu Phe

500 505 510

Arg Glu Aεn Gly Ser Lyε Pro Arg Ala Ile Val Val Asp Pro Val His 515 520 525

Gly Phe Met Tyr Trp Thr Aεp Trp Gly Thr Pro Ala Lyε Ile Lyε Lyε 530 535 540

Gly Gly Leu Asn Gly Val Aεp Ile Tyr Ser Leu Val Thr Glu Asn Ile 545 550 555 560

Gin Trp Pro Aεn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr 565 570 575 Trp Val Asp Ser Lys Leu His Ser Ile Ser Ser Ile Asp Val Aεn Gly

580 585 590

Gly Asn Arg Lys Thr Ile Leu Glu Asp Glu Lys Arg Leu Ala Hiε Pro 595 600 605

Phe Ser Leu Ala Val Phe Glu Aεp Lyε Val Phe Trp Thr Asp Ile Ile 610 615 620

Asn Glu Ala Ile Phe Ser Ala Aεn Arg Leu Thr Gly Ser Aεp Val Aεn 625 630 635 640

Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Met Val Leu Phe Hiε 645 650 655 Asn Leu Thr Gin Pro Arg Gly Val Asn Trp Cys Glu Arg Thr Thr Leu 660 665 670

Ser Asn Gly Gly Cys Gin Tyr Leu Cys Leu Pro Ala Pro Gin Ile Asn 675 680 685

Pro His Ser Pro Lys Phe Thr Cys Ala Cys Pro Asp Gly Met Leu Leu 690 695 700

Ala Arg Asp Met Arg Ser Cyε Leu Thr Glu Ala Glu Ala Ala Val Ala 705 710 715 720

Thr Gin Glu Thr Ser Thr Val Arg Leu Lyε Val Ser Ser Thr Ala Val 725 730 735

Arg Thr Gin Hiε Thr Thr Thr Arg Pro Val Pro Aεp Thr Ser Arg Leu 740 745 750

Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu Ile Val Thr Met Ser 755 760 765

His Gin Ala Leu Gly Asp Val Ala Gly Arg Gly Asn Glu Lys Lyε Pro 770 775 780

Ser Ser Val Arg Ala Leu Ser Ile Val Leu Pro Ile Val Leu Leu Val 785 790 795 800

Phe Leu Cys Leu Gly Val Phe Leu Leu Trp Lyε Asn Trp Arg Leu Lyε 805 810 815

Asn Ile Aεn Ser Ile Asn Phe Asp Asn Pro Val Tyr Gin Lys Thr Thr 820 825 830

Glu Asp Glu Val Hiε Ile Cyε Hiε Asn Gin Asp Gly Tyr Ser Tyr Pro 835 840 845

Ser Arg Gin Met Val Ser Leu Glu Aεp Aεp Val Ala 850 855 860

(2) INFORMATION ABOUT SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4492 amino acids

(B) TYPE: Amino Acid (C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Leu Thr Pro Pro Leu Leu Leu Leu Leu Pro Leu Leu Ser Ala Leu 1 5 10 15

Val Ala Ala Ala Ile Asp Ala Pro Lys Thr Cys Ser Pro Lys Gin Phe 20 25 30 Ala Cyε Arg Aεp Gin Ile Thr Cyε Ile Ser Lyε Gly Trp Arg Cyε Asp 35 40 45 Gly Glu Arg Asp Cys Pro Asp Gly Ser Asp Glu Ala Pro Glu Ile Cys 50 55 60

Pro Gin Ser Lys Ala Gin Arg Cys Gin Pro Asn Glu His Asn Cys Leu 65 70 75 80

Gly Thr Glu Leu Cys Val Pro Met Ser Arg Leu Cyε Aεn Gly Val Gin 85 90 95

Asp Cys Met Asp Gly Ser Asp Glu Gly Pro Hiε Cyε Arg Glu Leu Gin 100 105 110

Gly Aεn Cys Ser Arg Leu Gly Cyε Gin His His Cys Val Pro Thr Leu 115 120 125

Asp Gly Pro Thr Cys Tyr Cyε Asn Ser Ser Phe Gin Leu Gin Ala Asp 130 135 140

Gly Lyε Thr Cys Lyε Asp Phe Asp Glu Cys Ser Val Tyr Gly Thr Cyε 145 150 155 160

Ser Gin Leu Cys Thr Asn Thr Asp Gly Ser Phe Ile Cyε Gly Cys Val 165 170 175

Glu Gly Tyr Leu Leu Gin Pro Asp Asn Arg Ser Cys Lys Ala Lyε Asn 180 185 190

Glu Pro Val Asp Arg Pro Pro Val Leu Leu Ile Ala Asn Ser Gin Asn 195 200 205

Ile Leu Met Pro Gly Leu Lys Gly Phe Val Asp Glu His Thr Ile Asn 210 215 220

Ile Ser Leu Ser Leu His His Val Glu Gin Met Ala Ile Asp Trp Leu 225 230 235 240

Thr Gly Asn Phe Tyr Phe Val Aεp Aεp Ile Aεp Asp Arg Ile Phe Val 245 250 255

Cys Asn Arg Asn Gly Aεp Thr Cys Val Thr Leu Leu Asp Leu Glu Leu 260 265 270 Tyr Asn Pro Lys Gly Ile Ala Leu Asp Pro Ala Met Gly Lys Val Phe 275 280 285

Phe Thr Asp Tyr Gly Gin Ile Pro Lys Val Glu Arg Cys Asp Met Asp 290 295 300

Gly Gin Asn Arg Thr Lys Leu Val Asp Ser Lys Ile Val Phe Pro Hiε 305 310 315 320

Gly Ile Thr Leu Asp Leu Val Ser Arg Leu Val Tyr Trp Ala Asp Ala 325 330 335

Tyr Leu Asp Tyr Ile Glu Val Val Asp Tyr Glu Gly Lys Gly Arg Gin 340 345 350 Thr Ile Ile Gin Gly Ile Leu Ile Glu His Leu Tyr Gly Leu Thr Val 355 360 365

Phe Glu Aεn Tyr Leu Tyr Ala Thr Asn Ser Asp Aεn Ala Aεn Ala Gin 370 375 380

Gin Lys Thr Ser Val Ile Arg Val Asn Arg Phe Asn Ser Thr Glu Tyr 385 390 395 400 Gin Val Val Thr Arg Val Asp Lys Gly Gly Ala Leu Hiε Ile Tyr His 405 410 415

Gin Arg Arg Gin Pro Arg Val Arg Ser Hiε Ala Cyε Glu Aεn Asp Gin 420 425 430

Tyr Gly Lyε Pro Gly Gly Cyε Ser Asp Ile Cys Leu Leu Ala Asn Ser 435 440 445 His Lys Ala Arg Thr Cyε Arg Cys Arg Ser Gly Phe Ser Leu Gly Ser 450 455 460

Asp Gly Lys Ser Cys Lyε Lyε Pro Glu His Glu Leu Phe Leu Val Tyr 465 470 475 480

Gly Lys Gly Arg Pro Gly Ile Ile Arg Gly Met Asp Met Gly Ala Lyε 485 490 495

Val Pro Aεp Glu Hiε Met Ile Pro Ile Glu Aεn Leu Met Aεn Pro Arg 500 505 510

Ala Leu Aεp Phe His Ala Glu Thr Gly Phe Ile Tyr Phe Ala Asp Thr 515 520 525

Thr Ser Tyr Leu Ile Gly Arg Gin Lys Ile Asp Gly Thr Glu Arg Glu 530 535 540

Thr Ile Leu Lyε Asp Gly Ile His Asn Val Glu Gly Val Ala Val Asp 545 550 555 560

Trp Met Gly Aεp Asn Leu Tyr Trp Thr Asp Asp Gly Pro Lys Lys Thr 565 570 575

Ile Ser Val Ala Arg Leu Glu Lys Ala Ala Gin Thr Arg Lys Thr Leu 580 585 590

Ile Glu Gly Lys Met Thr Hiε Pro Arg Ala Ile Val Val Asp Pro Leu 595 600 605

Asn Gly Trp Met Tyr Trp Thr Asp Trp Glu Glu Asp Pro Lyε Aεp Ser 610 615 620 Arg Arg Gly Arg Leu Glu Arg Ala Trp Met Asp Gly Ser His Arg Asp 625 630 635 640

Ile Phe Val Thr Ser Lys Thr Val Leu Trp Pro Asn Gly Leu Ser Leu 645 650 655

Asp Ile Pro Ala Gly Arg Leu Tyr Trp Val Asp Ala Phe Tyr Asp Arg 660 665 670

Ile Glu Thr Ile Leu Leu Asn Gly Thr Asp Arg Lys Ile Val Tyr Glu 675 680 685

Gly Pro Glu Leu Asn His Ala Phe Gly Leu Cyε His His Gly Asn Tyr 690 695 700 Leu Phe Trp Thr Glu Tyr Arg Ser Gly Ser Val Tyr Arg Leu Glu Arg 705 710 715 720

Gly Val Gly Gly Ala Pro Pro Thr Val Thr Leu Leu Arg Ser Glu Arg 725 730 735

Pro Pro Ile Phe Glu Ile Arg Met Tyr Aεp Ala Gin Gin Gin Gin Val 740 745 750

REPLACEMENT LEAF Gly Thr Asn Lys Cys Arg Val Asn Aεn Gly Gly Cys Ser Ser Leu Cyε 755 760 765

Leu Ala Thr Pro Gly Ser Arg Gin Cys Ala Cys Ala Glu Aεp Gin Val 770 775 780

Leu Aεp Ala Aεp Gly Val Thr Cyε Leu Ala Aεn Pro Ser Tyr Val Pro 785 790 795 800 Pro Pro Gin Cyε Gin Pro Gly Glu Phe Ala Cys Ala Asn Ser Arg Cys

805 810 815

Ile Gin Glu Arg Trp Lys Cys Asp Gly Asp Asn Asp Cys Leu Asp Asn 820 825 830

Ser Asp Glu Ala Pro Ala Leu Cyε His Gin Hiε Thr Cys Pro Ser Asp 835 840 845

Arg Phe Lys Cys Glu Asn Asn Arg Cyε Ile Pro Asn Arg Trp Leu Cys 850 855 860

Asp Gly Asp Asn Asp Cys Gly Asn Ser Glu Asp Glu Ser Asn Ala Thr 865 870 875 880

Cyε Ser Ala Arg Thr Cyε Pro Pro Asn Gin Phe Ser Cyε Ala Ser Gly 885 890 895

Arg Cyε Ile Pro Ile Ser Trp Thr Cys Asp Leu Asp Asp Asp Cyε Gly 900 905 910

Asp Arg Ser Asp Glu Ser Ala Ser Cys Ala Tyr Pro Thr Cys Phe Pro 915 920 925

Leu Thr Gin Phe Thr Cys Asn Asn Gly Arg Cyε Ile Asn Ile Aεn Trp 930 935 940

Arg Cyε Aεp Asn Asp Aεn Aεp Cyε Gly Asp Asn Ser Aεp Glu Ala Gly 945 950 955 960

Cys Ser His Ser Cys Ser Ser Thr Gin Phe Lys Cys Asn Ser Gly Arg 965 970 975

Cys Ile Pro Glu Hiε Trp Thr Cyε Aεp Gly Aεp Aεn Aεp Cys Gly Asp 980 985 990

Tyr Ser Asp Glu Thr His Ala Asn Cys Thr Asn Gin Ala Thr Arg Pro 995 1000 1005

Pro Gly Gly Cyε His Thr Asp Glu Phe Gin Cys Arg Leu Asp Gly Leu 1010 1015 1020

Cys Ile Pro Leu Arg Trp Arg Cys Asp Gly Asp Thr Asp Cys Met Asp 1025 1030 1035 1040

Ser Ser Asp Glu Lys Ser Cys Glu Gly Val Thr His Val Cys Asp Pro 1045 1050 1055 Ser Val Lys Phe Gly Cys Lys Asp Ser Ala Arg Cyε Ile Ser Lyε Ala

1060 1065 1070

Trp Val Cyε Aεp Gly Aεp Aεn Asp Cys Glu Asp Asn Ser Asp Glu Glu 1075 1080 1085

Asn Cyε Glu Ser Leu Ala Cyε Arg Pro Pro Ser His Pro Cys Ala Asn 1090 1095 1100

ATZBLATT Asn Thr Ser Val Cys Leu Pro Pro Aεp Lys Leu Cyε Aεp Gly Asn Aεp 1105 1110 1115 1120

Aεp Cyε Gly Asp Gly Ser Asp Glu Gly Glu Leu Cyε Asp Gin Cys Ser 1125 1130 1135

Leu Asn Aεn Gly Gly Cyε Ser His Asn Cys Ser Val Ala Pro Gly Glu 1140 1145 1150 Gly Ile Val Cyε Ser Cyε Pro Leu Gly Met Glu Leu Gly Pro Aεp Aεn 1155 1160 1165

His Thr Cys Gin Ile Gin Ser Tyr Cyε Ala Lyε His Leu Lys Cys Ser 1170 1175 1180

Gin Lys Cys Asp Gin Aεn Lys Phe Ser Val Lyε Cyε Ser Cyε Tyr Glu 1185 1190 1195 1200

Gly Trp Val Leu Glu Pro Aεp Gly Glu Ser Cyε Arg Ser Leu Asp Pro

1205 1210 1215

Phe Lys Pro Phe Ile Ile Phe Ser Asn Arg His Glu Ile Arg Arg Ile 1220 1225 1230

Asp Leu His Lys Gly Asp Tyr Ser Val Leu Val Pro Gly Leu Arg Aεn 1235 1240 1245

Thr Ile Ala Leu Aεp Phe His Leu Ser Gin Ser Ala Leu Tyr Trp Thr 1250 1255 1260

Aεp Val Val Glu Asp Lys Ile Tyr Arg Gly Lyε Leu Leu Aεp Aεn Gly 1265 1270 1275 1280 Ala Leu Thr Ser Phe Glu Val Val Ile Gin Tyr Gly Leu Ala Thr Pro

1285 1290 1295

Glu Gly Leu Ala Val Aεp Trp Ile Ala Gly Aεn Ile Tyr Trp Val Glu 1300 1305 1310

Ser Aεn Leu Asp Gin Ile Glu Val Ala Lys Leu Asp Gly Thr Leu Arg 1315 1320 1325 Thr Thr Leu Leu Ala Gly Asp Ile Glu His Pro Arg Ala Ile Ala Leu 1330 1335 1340

Asp Pro Arg Asp Gly Ile Leu Phe Trp Thr Asp Trp Asp Ala Ser Leu 1345 1350 1355 1360

Pro Arg Ile Glu Ala Ala Ser Met Ser Gly Ala Gly Arg Arg Thr Val 1365 1370 1375

His Arg Glu Thr Gly Ser Gly Gly Trp Pro Aεn Gly Leu Thr Val Asp 1380 1385 1390

Tyr Leu Glu Lys Arg Ile Leu Trp Ile Aεp Ala Arg Ser Aεp Ala Ile 1395 1400 1405 Tyr Ser Ala Arg Tyr Aεp Gly Ser Gly His Met Glu Val Leu Arg Gly 1410 1415 1420

Hiε Glu Phe Leu Ser Hiε Pro Phe Ala Val Thr Leu Tyr Gly Gly Glu 1425 1430 1435 1440

Val Tyr Trp Thr Asp Trp Arg Thr Asn Thr Leu Ala Lys Ala Asn Lys 1445 1450 1455 Trp Thr Gly His Asn Val Thr Val Val Gin Arg Thr Asn Thr Gin Pro 1460 1465 1470

Phe Asp Leu Gin Val Tyr Hiε Pro Ser Arg Gin Pro Met Ala Pro Aεn 1475 1480 1485

Pro Cys Glu Ala Asn Gly Gly Gin Gly Pro Cys Ser Hiε Leu Cys Leu 1490 1495 1500 Ile Asn Tyr Asn Arg Thr Val Ser Cys Ala Cys Pro His Leu Met Lyε 1505 1510 1515 1520

Leu His Lyε Aεp Aεn Thr Thr Cyε Tyr Glu Phe Lyε Lyε Phe Leu Leu 1525 1530 1535

Tyr Ala Arg Gin Met Glu Ile Arg Gly Val Asp Leu Asp Ala Pro Tyr 1540 1545 1550

Tyr Asn Tyr Ile Ile Ser Phe Thr Val Pro Asp Ile Asp Asn Val Thr 1555 1560 1565

Val Leu Aεp Tyr Aεp Ala Arg Glu Gin Arg Val Tyr Trp Ser Aεp Val 1570 1575 1580

Arg Thr Gin Ala Ile Lyε Arg Ala Phe Ile Asn Gly Thr Gly Val Glu 1585 1590 1595 1600

Thr Val Val Ser Ala Aεp Leu Pro Asn Ala His Gly Leu Ala Val Asp 1605 1610 1615

Trp Val Ser Arg Asn Leu Phe Trp Thr Ser Tyr Asp Thr Asn Lys Lys 1620 1625 1630 Gin Ile Asn Val Ala Arg Leu Asp Gly Ser Phe Lyε Asn Ala Val Val 1635 1640 1645

Gin Gly Leu Glu Gin Pro Hiε Gly Leu Val Val His Pro Leu Arg Gly 1650 1655 1660

Lys Leu Tyr Trp Thr Asp Gly Asp Asn Ile Ser Met Ala Asn Met Asp 1665 1670 1675 1680 Gly Ser Asn Arg Thr Leu Leu Phe Ser Gly Gin Lys Gly Pro Val Gly

1685 1690 1695

Leu Ala Ile Asp Phe Pro Glu Ser Lys Leu Tyr Trp Ile Ser Ser Gly 1700 1705 1710

Asn His Thr Ile Asn Arg Cyε Asn Leu Asp Gly Ser Gly Leu Glu Val 1715 1720 1725

Ile Asp Ala Met Arg Ser Gin Leu Gly Lys Ala Thr Ala Leu Ala Ile 1730 1735 1740

Met Gly Aεp Lys Leu Trp Trp Ala Asp Gin Val Ser Glu Lys Met Gly 1745 1750 1755 1760 Thr Cys Ser Lyε Ala Aεp Gly Ser Gly Ser Val Val Leu Arg Aεn Ser

1765 1770 1775

Thr Thr Leu Val Met Hiε Met Lys Val Tyr Asp Glu Ser Ile Gin Leu 1780 1785 1790

Asp His Lys Gly Thr Aεn Pro Cyε Ser Val Aεn Aεn Gly Aεp Cyε Ser 1795 1800 1805

REPLACEMENT Gin Leu Cys Leu Pro Thr Ser Glu Thr Thr Arg Ser Cys Met Cys Thr 1810 1815 1820

Ala Gly Tyr Ser Leu Arg Ser Gly Gin Gin Ala Cys Glu Gly Val Gly 1825 1830 1835 1840

Ser Phe Leu Leu Tyr Ser Val Hiε Glu Gly Ile Arg Gly Ile Pro Leu 1845 1850 1855 Aεp Pro Asn Asp Lyε Ser Asp Ala Leu Val Pro Val Ser Gly Thr Ser

1860 1865 1870

Leu Ala Val Gly Ile Asp Phe His Ala Glu Asn Asp Thr Ile Tyr Trp 1875 1880 1885

Val Asp Met Gly Leu Ser Thr Ile Ser Arg Ala Lys Arg Asp Gin Thr 1890 1895 1900

Trp Arg Glu Asp Val Val Thr Asn Gly Ile Gly Arg Val Glu Gly Ile 1905 1910 1915 1920

Ala Val Asp Trp Ile Ala Gly Aεn Ile Tyr Trp Thr Asp Gin Gly Phe 1925 1930 1935

Asp Val Ile Val Ala Arg Leu Asn Gly Ser Phe Arg Tyr Val Val Ile 1940 1945 1950

Ser Gin Gly Leu Aεp Lyε Pro Arg Ala Ile Thr Val His Pro Glu Lyε 1955 1960 1965

Gly Tyr Leu Phe Trp Thr Glu Trp Gly Gin Tyr Pro Arg Ile Glu Arg 1970 1975 1980 Ser Arg Leu Aεp Gly Thr Glu Arg Val Val Leu Val Asn Val Ser Ile 1985 1990 1995 2000

Ser Trp Pro Aεn Gly Ile Ser Val Aεp Tyr Gin Aεp Gly Lys Leu Tyr 2005 2010 2015

Trp Cys Asp Ala Arg Thr Asp Lyε Ile Glu Arg Ile Asp Leu Glu Thr 2020 2025 2030

Gly Glu Asn Arg Glu Val Val Leu Ser Ser Asn Aεn Met Asp Met Phe 2035 2040 2045

Ser Val Ser Val Phe Glu Asp Phe Ile Tyr Trp Ser Asp Arg Thr His 2050 2055 2060

Ala Asn Gly Ser Ile Lys Arg Gly Ser Lys Asp Asn Ala Thr Aεp Ser 2065 2070 2075 2080

Val Pro Leu Arg Thr Gly Ile Gly Val Gin Leu Lys Asp Ile Lys Val 2085 2090 2095

Phe Asn Arg Asp Arg Gin Lyε Gly Thr Aεn Val Cyε Ala Val Ala Asn 2100 2105 2110 Gly Gly Cys Gin Gin Leu Cys Leu Tyr Arg Gly Arg Gly Gin Arg Ala 2115 2120 2125

Cyε Ala Cys Ala His Gly Met Leu Ala Glu Asp Gly Ala Ser Cys Arg 2130 2135 2140

Glu Tyr Ala Gly Tyr Leu Leu Tyr Ser Glu Arg Thr Ile Leu Lyε Ser 2145 2150 2155 2160

REPLACEMENT LEAF Ile Hiε Leu Ser Asp Glu Arg Asn Leu Asn Ala Pro Val Gin Pro Phe 2165 2170 2175

Glu Asp Pro Glu His Met Lys Asn Val Ile Ala Leu Ala Phe Asp Tyr 2180 2185 2190

Arg Ala Gly Thr Ser Pro Gly Thr Pro Asn Arg Ile Phe Phe Ser Aεp 2195 2200 2205 Ile His Phe Gly Asn Ile Gin Gin Ile Asn Asp Asp Gly Ser Arg Arg 2210 2215 2220

Ile Thr Ile Val Glu Asn Val Gly Ser Val Glu Gly Leu Ala Tyr His 2225 2230 2235 2240

Arg Gly Trp Asp Thr Leu Tyr Trp Thr Ser Tyr Thr Thr Ser Thr Ile 2245 2250 2255

Thr Arg His Thr Val Aεp Gin Thr Arg Pro Gly Ala Phe Glu Arg Glu

2260 2265 2270

Thr Val Ile Thr Met Ser Gly Aεp Aεp Hiε Pro Arg Ala Phe Val Leu 2275 2280 2285

Aεp Glu Cys Gin Asn Leu Met Phe Trp Thr Asn Trp Asn Glu Gin His 2290 2295 2300

Pro Ser Ile Met Arg Ala Ala Leu Ser Gly Ala Asn Val Leu Thr Leu 2305 2310 2315 2320

Ile Glu Lys Asp Ile Arg Thr Pro Asn Gly Leu Ala Ile Asp Hiε Arg 2325 2330 2335 Ala Glu Lys Leu Tyr Phe Ser Asp Ala Thr Leu Asp Lys Ile Glu Arg

2340 2345 2350

Cys Glu Tyr Asp Gly Ser His Arg Tyr Val Ile Leu Lys Ser Glu Pro 2355 2360 2365

Val Hiε Pro Phe Gly Leu Ala Val Tyr Gly Glu Hiε Ile Phe Trp Thr 2370 2375 2380

Asp Trp Val Arg Arg Ala Val Gin Arg Ala Asn Lys Hiε Val Gly Ser 2385 2390 2395 2400

Asn Met Lyε Leu Leu Arg Val Asp Ile Pro Gin Gin Pro Met Gly Ile 2405 2410 2415

Ile Ala Val Ala Asn Aεp Thr Aεn Ser Cys Glu Leu Ser Pro Cys Arg 2420 2425 2430

Ile Asn Asn Gly Gly Cys Gin Aεp Leu Cys Leu Leu Thr Hiε Gin Gly 2435 2440 2445

His Val Asn Cys Ser Cys Arg Gly Gly Arg Ile Leu Gin Aεp Asp Leu 2450 2455 2460 Thr Cyε Arg Ala Val Asn Ser Ser Cyε Arg Ala Gin Asp Glu Phe Glu 2465 2470 2475 2480

Cys Ala Asn Gly Glu Cys Ile Asn Phe Ser Leu Thr Cyε Asp Gly Val 2485 2490 2495

Pro Hiε Cyε Lys Asp Lys Ser Asp Glu Lys Pro Ser Tyr Cys Asn Ser 2500 2505 2510

REPLACEMENT LEAF Arg Arg Cys Lys Lyε Thr Phe Arg Gin Cys Ser Asn Gly Arg Cyε Val 2515 2520 2525

Ser Aεn Met Leu Trp Cys Aεn Gly Ala Asp Asp Cyε Gly Aεp Gly Ser 2530 2535 2540

Asp Glu Ile Pro Cys Asn Lys Thr Ala Cyε Gly Val Gly Glu Phe Arg 2545 2550 2555 2560 Cys Arg Asp Gly Thr Cys Ile Gly Asn Ser Ser Arg Cys Asn Gin Phe

2565 2570 2575

Val Asp Cys Glu Asp Ala Ser Asp Glu Met Asn Cyε Ser Ala Thr Aεp 2580 2585 2590

Cyε Ser Ser Tyr Phe Arg Leu Gly Val Lyε Gly Val Leu Phe Gin Pro 2595 2600 2605

Cys Glu Arg Thr Ser Leu Cys Tyr Ala Pro Ser Trp Val Cyε Aεp Gly 2610 2615 2620

Ala Aεn Aεp Cys Gly Asp Tyr Ser Aεp Glu Arg Aεp Cyε Pro Gly Val 2625 2630 2635 2640

Lyε Arg Pro Arg Cys Pro Leu Asn Tyr Phe Ala Cyε Pro Ser Gly Arg 2645 2650 2655

Cyε Ile Pro Met Ser Trp Thr Cys Asp Lys Glu Asp Asp Cys Glu His 2660 2665 2670

Gly Glu Asp Glu Thr His Cys Asn Lys Phe Cys Ser Glu Ala Gin Phe 2675 2680 2685

Glu Cyε Gin Asn Hiε Arg Cyε Ile Ser Lyε Gin Trp Leu Cys Asp Gly 2690 2695 2700

Ser Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Ala His Cys Glu Gly 2705 2710 2715 2720

Lys Thr Cyε Gly Pro Ser Ser Phe Ser Cys Pro Gly Thr His Val Cyε 2725 2730 2735

Val Pro Glu Arg Trp Leu Cys Asp Gly Asp Lys Asp Cyε Ala Aεp Gly 2740 2745 2750

Ala Asp Glu Ser Ile Ala Ala Gly Cys Leu Tyr Aεn Ser Thr Cyε Asp 2755 2760 2765

Asp Arg Glu Phe Met Cyε Gin Asn Arg Gin Cys Ile Pro Lys His Phe 2770 2775 2780

Val Cys Asp His Asp Arg Asp Cys Ala Asp Gly Ser Asp Glu Ser Pro 2785 2790 2795 2800

Glu Cys Glu Tyr Pro Thr Cys Gly Pro Ser Glu Phe Arg Cys Ala Asn 2805 2810 2815 Gly Arg Cyε Leu Ser Ser Arg Gin Trp Glu Cys Asp Gly Glu Aεn Aεp

2820 2825 2830

Cyε His Aεp Gin Ser Aεp Glu Ala Pro Lyε Asn Pro His Cys Thr Ser 2835 2840 2845

Pro Glu His Lys Cys Aεn Ala Ser Ser Gin Phe Leu Cyε Ser Ser Gly 2850 2855 2860

REPLACEMENT BLADE Arg Cys Val Ala Glu Ala Leu Leu Cyε Aεn Gly Gin Aεp Aεp Cyε Gly 2865 2870 2875 2880

Asp Ser Ser Asp Glu Arg Gly Cyε His Ile Asn Glu Cyε Leu Ser Arg 2885 2890 2895

Lyε Leu Ser Gly Cys Ser Gin Asp Cyε Glu Asp Leu Lys Ile Gly Phe 2900 2905 2910 Lyε Cyε Arg Cyε Arg Pro Gly Phe Arg Leu Lyε Aεp Aεp Gly Arg Thr 2915 2920 2925

Cys Ala Asp Val Asp Glu Cyε Ser Thr Thr Phe Pro Cyε Ser Gin Arg 2930 2935 2940

Cys Ile Asn Thr Hiε Gly Ser Tyr Lyε Cyε Leu Cyε Val Glu Gly Tyr 2945 2950 2955 2960

Ala Pro Arg Gly Gly Asp Pro His Ser Cyε Lyε Ala Val Thr Asp Glu 2965 2970 2975

Glu Pro Phe Leu Ile Phe Ala Aεn Arg Tyr Tyr Leu Arg Lyε Leu Aεn 2980 2985 2990

Leu Aεp Gly Ser Aεn Tyr Leu Leu Lyε Gin Gly Leu Asn Asn Ala Val 2995 3000 3005

Ala Leu Asp Phe Asp Tyr Arg Glu Gin Met Ile Tyr Trp Thr Asp Val 3010 3015 3020

Thr Thr Gin Gly Ser Met Ile Arg Arg Met Hiε Leu Asn Gly Ser Asn 3025 3030 3035 3040

Val Gin Val Leu His Arg Thr Gly Leu Ser Aεn Pro Asp Gly Leu Ala 3045 3050 3055

Val Aεp Trp Val Gly Gly Aεn Leu Tyr Trp Cys Asp Lys Gly Arg Asp 3060 3065 3070

Thr Ile Glu Val Ser Lyε Leu Asn Gly Ala Tyr Arg Thr Val Leu Val 3075 3080 3085

Ser Ser Gly Leu Arg Glu Pro Arg Ala Leu Val Val Asp Val Gin Asn 3090 3095 3100

Gly Tyr Leu Tyr Trp Thr Aεp Trp Gly Asp His Ser Leu Ile Gly Arg 3105 3110 3115 3120 He Gly Met Asp Gly Ser Ser Arg Ser Val Ile Val Asp Thr Lyε Ile

3125 3130 3135

Thr Trp Pro Asn Gly Leu Thr Leu Asp Tyr Val Thr Glu Arg Ile Tyr 3140 3145 3150

Trp Ala Asp Ala Arg Glu Aεp Tyr Ile Glu Phe Ala Ser Leu Aεp Gly 3155 3160 3165 Ser Asn Arg His Val Val Leu Ser Gin Aεp Ile Pro Hiε Ile Phe Ala 3170 3175 3180

Leu Thr Leu Phe Glu Asp Tyr Val Tyr Trp Thr Asp Trp Glu Thr Lys 3185 3190 3195 3200

Ser Ile Asn Arg Ala Hiε Lyε Thr Thr Gly Thr Asn Lys Thr Leu Leu 3205 3210 3215 Ile Ser Thr Leu Hiε Arg Pro Met Asp Leu Hiε Val Phe His Ala Leu 3220 3225 3230

Arg Gin Pro Asp Val Pro Asn His Pro Cys Lyε Val Aεn Asn Gly Gly 3235 3240 3245

Cys Ser Asn Leu Cyε Leu Leu Ser Pro Gly Gly Gly His Lyε Cys Ala 3250 3255 3260 Cys Pro Thr Asn Phe Tyr Leu Gly Ser Aεp Gly Arg Thr Cyε Val Ser 3265 3270 3275 3280

Aεn Cys Thr Ala Ser Gin Phe Val Cyε Lyε Aεn Aεp Lys Cyε Ile Pro 3285 3290 3295

Phe Trp Trp Lyε Cyε Aεp Thr Glu Aεp Aεp Cyε Gly Aεp Hiε Ser Aεp 3300 3305 3310

Glu Pro Pro Asp Cyε Pro Glu Phe Lyε Cys Arg Pro Gly Gin Phe Gin 3315 3320 3325

Cyε Ser Thr Gly Ile Cyε Thr Asn Pro Ala Phe Ile Cyε Asp Gly Asp 3330 3335 3340

Asn Asp Cys Gin Asp Asn Ser Asp Glu Ala Asn Cys Aεp Ile His Val 3345 3350 3355 3360

Cyε Leu Pro Ser Gin Phe Lyε Cyε Thr Asn Thr Asn Arg Cyε Ile Pro 3365 3370 3375

Gly Ile Phe Arg Cyε Aεn Gly Gin Asp Asn Cys Gly Asp Gly Glu Aεp 3380 3385 3390

Glu Arg Asp Cys Pro Glu Val Thr Cyε Ala Pro Aεn Gin Phe Gin Cyε 3395 3400 3405

Ser Ile Thr Lyε Arg Cyε Ile Pro Arg Val Trp Val Cyε Aεp Arg Asp 3410 3415 3420

Asn Asp Cys Val Asp Gly Ser Asp Glu Pro Ala Asn Cys Thr Gin Met 3425 3430 3435 3440

Thr Cyε Gly Val Asp Glu Phe Arg Cyε Lyε Asp Ser Gly Arg Cys Ile 3445 3450 3455

Pro Ala Arg Trp Lys Cys Asp Gly Glu Asp Asp Cys Gly Asp Gly Ser 3460 3465 3470 Asp Glu Pro Lys Glu Glu Cys Aεp Glu Arg Thr Cys Glu Pro Tyr Gin 3475 3480 3485

Phe Arg Cys Lys Asn Asn Arg Cys Val Pro Gly Arg Trp Gin Cys Asp 3490 3495 3500

Tyr Asp Asn Asp Cys Gly Aεp Aεn Ser Asp Glu Glu Ser Cys Thr Pro 3505 3510 3515 3520 Arg Pro Cyε Ser Glu Ser Glu Phe Ser Cys Ala Asn Gly Arg Cys Ile

3525 3530 3535

Ala Gly Arg Trp Lys Cyε Asp Gly Aεp Hiε Aεp Cyε Ala Aεp Gly Ser 3540 3545 3550

Aεp Glu Lyε Aεp Cyε Thr Pro Arg Cys Asp Met Asp Gin Phe Gin Cyε 3555 3560 3565 Lys Ser Gly Hiε Cyε Ile Pro Leu Arg Trp Arg Cyε Aεp Ala Aεp Ala 3570 3575 3580

Aεp Cyε Met Asp Gly Ser Asp Glu Glu Ala Cys Gly Thr Gly Val Arg 5 3585 3590 3595 3600

Thr Cyε Pro Leu Aεp Glu Phe Gin Cyε Aεn Aεn Thr Leu Cyε Lyε Pro 3605 3610 3615

10. Leu Ala Trp Lyε Cyε Aεp Gly Glu Aεp Aεp Cyε Gly Aεp Asn Ser Asp

3620 3625 3630

Glu Asn Pro Glu Glu Cys Ala Arg Phe Val Cys Pro Pro Asn Arg Pro 3635 3640 3645

15

Phe Arg Cys Lys Aεn Asp Arg Val Cys Leu Trp Ile Gly Arg Gin Cyε 3650 3655 3660

0 Asp Gly Thr Asp Asn Cyε Gly Aεp Gly Thr Aεp Glu Glu Aεp Cyε Glu 3665 3670 3675 3680

Pro Pro Thr Ala Hiε Thr Thr Hiε Cyε Lyε Asp Lys Lys Glu Phe Leu 3685 3690 3695 5

Cys Arg Asn Gin Arg Cys Leu Ser Ser Ser Leu Arg Cys Aεn Met Phe 3700 3705 3710

Aεp Asp Cys Gly Asp Gly Ser Asp Glu Glu Asp Cyε Ser Ile Aεp Pro 0 3715 3720 3725

Lyε Leu Thr Ser Cys Ala Thr Asn Ala Ser Ile Cyε Gly Aεp Glu Ala 3730 3735 3740 5 Arg Cys Val Arg Thr Glu Lys Ala Ala Tyr Cyε Ala Cyε Arg Ser Gly 3745 3750 3755 3760

Phe His Thr Val Pro Gly Gin Pro Gly Cys Gin Asp Ile Asn Glu Cyε 3765 3770 3775 0

Leu Arg Phe Gly Thr Cyε Ser Gin Leu Cys Asn Asn Thr Lys Gly Gly 3780 3785 3790

His Leu Cys Ser Cys Ala Arg Asn Phe Met Lys Thr His Asn Thr Cys 5 3795 3800 3805

Lyε Ala Glu Gly Ser Glu Tyr Gin Val Leu Tyr Ile Ala Asp Aεp Aεn 3810 3815 3820 0 Glu Ile Arg Ser Leu Phe Pro Gly Hiε Pro Hiε Ser Ala Tyr Glu Gin 3825 3830 3835 3840

Ala Phe Gin Gly Asp Glu Ser Val Arg Ile Asp Ala Met Aεp Val Hiε 3845 3850 3855 5

Val Lyε Ala Gly Arg Val Tyr Trp Thr Aεn Trp His Thr Gly Thr Ile 3860 3865 3870

0 Ser Tyr Arg Ser Leu Pro Pro Ala Ala Pro Pro Thr Thr Ser Asn Arg 3875 3880 3885

Hiε Arg Arg Gin Ile Aεp Arg Gly Val Thr His Leu Asn Ile Ser Gly 3890 3895 3900 5

Leu Lys Met Pro Arg Gly Ile Ala Ile Aεp Trp Val Ala Gly Aεn Val 3905 3910 3915 3920 Tyr Trp Thr Aεp Ser Gly Arg Aεp Val Ile Glu Val Ala Gin Met Lyε 3925 3930 3935

Gly Glu Asn Arg Lyε Thr Leu Ile Ser Gly Met Ile Aεp Glu Pro Hiε 3940 3945 3950

Ala Ile Val Val Asp Pro Leu Arg Gly Thr Met Tyr Trp Ser Asp Trp 3955 3960 3965 Gly Asn His Pro Lys Ile Glu Thr Ala Ala Met Asp Gly Thr Leu Arg 3970 3975 3980

Glu Thr Leu Val Gin Asp Asn Ile Gin Trp Pro Thr Gly Leu Ala Val 3985 3990 3995 4000

Asp Tyr His Asn Glu Arg Leu Tyr Trp Ala Asp Ala Lys Leu Ser Val 4005 4010 4015

Ile Gly Ser Ile Arg Leu Asn Gly Thr Aεp Pro Ile Val Ala Ala Aεp 4020 4025 4030

Ser Lyε Arg Gly Leu Ser Hiε Pro Phe Ser Ile Asp Val Phe Glu Aεp 4035 4040 4045

Tyr Ile Tyr Gly Val Thr Tyr Ile Asn Asn Arg Val Phe Lys Ile Hiε 4050 4055 4060

Lyε Phe Gly His Ser Pro Leu Val Asn Leu Thr Gly Gly Leu Ser His 4065 4070 4075 4080

Ala Ser Asp Val Val Leu Tyr Hiε Gin Hiε Lyε Gin Pro Glu Val Thr 4085 4090 4095 Aεn Pro Cyε Aεp Arg Lyε Lyε Cys Glu Trp Leu Cys Leu Leu Ser Pro

4100 4105 4110

Ser Gly Pro Val Cys Thr Cys Pro Asn Gly Lys Arg Leu Asp Asn Gly 4115 4120 4125

Thr Cyε Val Pro Val Pro Ser Pro Thr Pro Pro Pro Aεp Ala Pro Arg 4130 4135 4140

Pro Gly Thr Cyε Asn Leu Gin Cyε Phe Asn Gly Gly Ser Cys Phe Leu 4145 4150 4155 4160

Asn Ala Arg Arg Gin Pro Lys Cys Arg Cys Gin Pro Arg Tyr Thr Gly 4165 4170 4175 Asp Lys Cys Glu Leu Asp Gin Cyε Trp Glu Hiε Cyε Arg Aεn Gly Gly

4180 4185 4190

Thr Cys Ala Ala Ser Pro Ser Gly Met Pro Thr Cys Arg Cys Pro Thr 4195 4200 4205

Gly Phe Thr Gly Pro Lys Cyε Thr Gin Gin Val Cyε Ala Gly Tyr Cys 4210 4215 4220

Ala Asn Asn Ser Thr Cys Thr Val Asn Gin Gly Asn Gin Pro Gin Cyε 4225 4230 4235 4240

Arg Cys Leu Pro Gly Phe Leu Gly Asp Arg Cys Gin Tyr Arg Gin Cys 4245 4250 4255

Ser Gly Tyr Cys Glu Asn Phe Gly Thr Cys Gin Met Ala Ala Asp Gly 4260 4265 4270

REPLACEMENT LEAF Ser Arg Gin Cys Arg Cys Thr Ala Tyr Phe Glu Gly Ser Arg Cys Glu 4275 4280 4285

Val Aεn Lyε Cyε Ser Arg Cyε Leu Glu Gly Ala Cyε Val Val Aεn Lys 4290 4295 4300

Gin Ser Gly Asp Val Thr Cyε Asn Cys Thr Asp Gly Arg Val Ala Pro 4305 4310 4315 4320 Ser Cys Leu Thr Cyε Val Gly His Cys Ser Asn Gly Gly Ser Cys Thr

4325 4330 4335

Met Asn Ser Lys Met Met Pro Glu Cys Gin Cyε Pro Pro Hiε Met Thr 4340 4345 4350

Gly Pro Arg Cyε Glu Glu His Val Phe Ser Gin Gin Gin Pro Gly His 4355 4360 4365

Ile Ala Ser Ile Leu Ile Pro Leu Leu Leu Leu Leu Leu Leu Val Leu 4370 4375 4380

Val Ala Gly Val Val Phe Trp Tyr Lys Arg Arg Val Gin Gly Ala Lys 4385 4390 4395 4400

Gly Phe Gin His Gin Arg Met Thr Asn Gly Ala Met Asn Val Glu Ile 4405 4410 4415

Gly Asn Pro Thr Tyr Lyε Met Tyr Glu Gly Gly Glu Pro Aεp Aεp Val 4420 4425 4430

Gly Gly Leu Leu Asp Ala Asp Phe Ala Leu Asp Pro Asp Lyε Pro Thr 4435 4440 4445 Asn Phe Thr Asn Pro Val Tyr Ala Thr Leu Tyr Met Gly Gly His Gly 4450 4455 4460

Ser Arg His Ser Leu Ala Ser Thr Aεp Glu Lyε Arg Glu Leu Leu Gly 4465 4470 4475 4480

Arg Gly Pro Glu Asp Glu Ile Gly Asp Pro Leu Ala 4485 4490

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 319 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Arg Ser Ala Glu Lys Aεn Glu Pro Glu Met Ala Ala Lys Arg Glu Ser 1 5 10 15

Gly Glu Glu Phe Arg Met Glu Lys Leu Asn Gin Leu Trp Glu Lys Ala 20 25 30

REPLACEMENT LEAF Lys Arg Leu Hiε Leu Ser Pro Val Arg Leu Ala Glu Leu His Ser Asp 35 40 45

Leu Lys Ile Gin Glu Arg Asp Glu Leu Asn Trp Lys Lyε Leu Lyε Val 50 55 60

Glu Gly Leu Asp Gly Asp Gly Glu Lys Glu Ala Lys Leu Val His Asn 65 70 75 80

Leu Asn Val Ile Leu Ala Arg Tyr Gly Leu Asp Gly Arg Lys Asp Thr 85 90 95

Gin Thr Val His Ser Asn Ala Leu Asn Glu Asp Thr Gin Aεp Glu Leu 100 105 110

Gly Aεp Pro Arg Leu Glu Lyε Leu Trp His Lys Ala Lys Thr Ser Gly 115 120 125

Ile Ser Val Arg Leu Thr Ser Cys Ala Arg Val Leu Hiε Tyr Lyε Glu 130 135 140

Lyε Ile Hiε Glu Tyr Aεn Val Leu Leu Aεp Thr Leu Ser Arg Ala Glu 145 150 155 160

Glu Gly Tyr Glu Asn Leu Leu Ser Pro Ser Asp Met Thr His Ile Lyε 165 170 175

Ser Asp Thr Leu Ala Ser Lys His Ser Glu Leu Lys Aεp Arg Leu Arg 180 185 190

Ser Ile Aεn Gin Gly Leu Aεp Arg Leu Arg Lyε Val Ser Hiε Gin Leu 195 200 205 Arg Pro Ala Thr Glu Phe Glu Glu Pro Arg Val Ile Aεp Leu Trp Asp 210 215 220

Leu Ala Gin Ser Ala Asn Phe Thr Glu Lyε Glu Leu Glu Ser Phe Arg 225 230 235 240

Glu Glu Leu Lyε Hiε Phe Glu Ala Lys Ile Glu Lys His Asn His Tyr 245 250 255

Gin Lys Gin Leu Glu Ile Ser His Gin Lys Leu Lys His Val Glu Ser 260 265 270

Ile Gly Asp Pro Glu Hiε Ile Ser Arg Aεn Lyε Glu Lyε Tyr Val Leu 275 280 285 Leu Glu Glu Lyε Thr Lyε Glu Leu Gly Tyr Lyε Val Lyε Lyε Hiε Leu 290 295 300

Gin Asp Leu Ser Ser Arg Val Ser Arg Ala Arg His Asn Glu Leu 305 310 315

REPLACEMENT LEAF

Claims

PATENT CLAIMS 1. polypeptide, characterized in that it is a functional derivative of a receptor for rhinoviruses of the "small rhinovirus receptor group". 2. Polypeptide according to claim 1, characterized in that it is a soluble derivative. 3. Polypeptide according to claim 1-2, characterized in that it is the soluble extracellular form of a receptor protein. 4. Polypeptide according to any one of claims 1-3, characterized in that it is derived from the LDL receptor family. 5. Polypeptide according to claim 4, characterized in that it is derived from the amino acid sequence of the human LDL receptor according to FIG. 1 or from that of the CX2-V-R / LRP according to FIG. 2 or from that of the gp330 according to FIG. 3. 6. Polypeptide according to claim 5, characterized in that it essentially comprises domain 1, domains 1 and 2 or domains 1, 2 and 3 of a receptor from the LDL receptor family according to FIG. 4. 7. Polypeptide according to claim 6, characterized in that it comprises the amino acid sequence according to SEQ.ID.NO.l or SEQ.ED.NO.2. 8. Polypeptide according to claim 6, characterized in that it consists essentially of domains 1 and 2 of the LDL receptor, is released by eukaryotic cells and has a molecular weight of approximately 120 kDa, the Molecular weight is determined by SDS gel electrophoresis under non-reducing conditions. 9. Polypeptide according to any one of claims 1-8, characterized in that it is present as a dimer, trimer, tetramer or multimer. 10. DNA coding for a polypeptide according to any one of claims 1-9. 11. DNA according to claim 10, characterized in that it is inserted into a vector. 12. DNA according to claim 11, characterized in that the DNA according to one of the preceding claims in a vector in functional connection with a Expression control sequence is linked and is replicable in microorganisms and / or mammalian cells. 13. Host organism, characterized in that it is transformed with a DNA according to one of claims 11 or 12. Process for the preparation of a DNA molecule according to claim 12, characterized in that in a vector DNA which has been cut with restriction endonucleases and which contains expression control sequences, a DNA provided with corresponding ends which is suitable for a functional derivative of the "small" receptor Rhinovirus receptor group "encoded according to claim 10, so that the expression control sequences regulate the expression of the inserted DNA. 15. A method for producing a functional derivative of a receptor of the "small rhinovirus receptor group", characterized in that the Polypeptide is taken from the native receptor molecules by enzymatic, preferably proteolytic or chemical, preferably reductive, treatment. 16. A method for producing a functional derivative of a receptor of the "small rhinovirus receptor group", characterized in that it is obtained by expression of a DNA according to any one of claims 10-12. 17. Hybrid cell line, characterized in that it secretes monoclonal antibodies against one of the polypeptides according to one of claims 1 to 9. 18. Monoclonal antibodies, characterized in that they specifically neutralize the action of the polypeptides according to one of claims 1 to 9 or bind specifically to one of said polypeptides. 19. Use of the monoclonal antibodies according to claim 18 for the qualitative and / or quantitative determination or for the purification of one of the polypeptides according to one of claims 1 to 9. 20. Test kit for the determination of polypeptides according to one of claims 1 to 9, characterized in that it contains monoclonal antibodies according to claim 18. 21. A method for producing monoclonal antibodies according to claim 18, characterized in that host animals are immunized with one of the polypeptides according to one of claims 1 to 9, B-lymphocytes of these host animals are fused with myeloma cells, which subclones and cultivates the monoclonal antibody-secreting hybrid cell lines become. 22. Use of the polypeptides according to one of claims 1 to 9 and the native receptor molecules of the LDL receptor family or corresponding pharmaceutically suitable salts for the therapeutic or prophylactic treatment of the human body. 23. Use of the polypeptides according to one of claims 1 to 9 and the native receptor molecules of the LDL receptor family as antiviral, in particular antirhinoviral agents. 24. Agent for therapeutic treatment, characterized in that it contains, in addition to pharmaceutically inert carriers, an effective amount of a polypeptide according to one of claims 1 to 9 or the native receptor molecules of the LDL receptor family. 25. Pharmaceutical composition containing one or more polypeptides according to one of claims 1-9 and a suitable carrier material. 26. Use of "small rhinovirus receptor group" rhinovirus material for inhibiting the binding of physiological LDL ligands. 27. A method for identifying substances which inhibit the binding of ligands to "receptors of the LDL receptor family", characterized in that a. the receptor or a soluble form of the receptor that can be isolated from culture supernatants in the presence of a potential inhibitor substance with b. labeled rhinovirus material of the "small rhinovirus receptor group" and c. the extent of binding is determined. 28. A method for determining receptors from the LDL receptor family, characterized in that a. a substance derived from virus material from the "small rhinovirus receptor group" is labeled with binding activity to the receptor, b. incubated with the appropriate sample and c. the extent of the binding of the labeled virus material is detected. 29. A method for delivering a therapeutically active substance into a supporting cell, characterized in that a. Virus material of the "small rhinovirus receptor group" with binding activity to the LDL receptor is coupled with the therapeutic substance and b. said material to the corresponding one Given cell material, bound to the receptor and so the therapeutically active substance is introduced into the cell.