CA2117099A1 - Receptor derivatives having binding sites for human rhinoviruses - Google Patents

Abstract

Abstract The invention describes the preparation of polypeptides with binding activities for receptors of the "small rhinovirus receptor group", processes for preparing the peptides, the use thereof and the DNA coding for the peptides.

Description

21170.99 -FILE, I~THI`~
~F TRANSLArlC)N ~ :

RECEPTOR DERIVATIVES HAVING BINDING SITES
FOR HUMAN RHINOVIRUSES

The present invention describes receptor derivatives having binding sites for human rhinoviruses of the "small rhinovirus receptor group", the use thereof and DNA coding for the receptor derivatives.

Human rhinoviruses represent a large genus within the family of the picorna viruses and include approximately 115 different serotypes (Melnick, J.L. (1980) Prog. Med.
Virol. 26, 214-232). These RNA viruses attack the respiratory tract in humans and cause acute infections which lead to colds.

The human rhinoviruses can be subdivided into two groups if the criterion used for categorisation is their competition for binding sites on the surface of human cell culture cells such as HeLa cells. Competitive experiments show that, apart from one single exception (serotype 87), there are two different receptors on the cell surface. Hitherto, 91 serotypes have been allocated to the "large rhinovirus receptor group" and 10 serotypes to the "small rhinovirus receptor group"
(Abraham and Colonno R.J. (1984) J. Virol. 51, 340-345;
Uncapher et al. (1991) Virology 180, 814 - 817). The receptor of "large rhinovirus receptor group" was purified and identified as ICAM-1, a protein belonging to the immunoglobulin superfamily, which acts 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)-~ 2117099 By demonstrating specific binding of the purified ICAM-l to the virus and because of the possibility of transferring the rhinovirus binding activity, by gene transfer, to cells which had no such activity before the transfer, it was possible to show clearly that ICAM-l is the receptor for the majority of rhinoviruses (Greve et al. (1989) loc. cit.; Staunton et al. (1989) loc. cit).
Moreover, it has 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-l to leukocytes via LFA-l ("lymphocyte function associated antigen-l") - another natural ligand of ICAM-l - are also able to block the binding of the rhinovirus to the receptor. Thus, the ¦ LFA-l and rhinovirus binding sites must be at least I adjacent. Tests with chimeric and mutated ICAM-l molecules additionally showed that the binding site for 1 the rhinovirus-ICAM-l interaction does not coincide with the binding site for LFA-l (Staunton et al. (1990) Cell 61, 243-254).
The receptor binding site of the human rhinovirus serotype 14, an example of the "large rhinovirus receptor group", lies in a so-called "canyon", a depression in the surface of the virus (Rossmann et al.
(1985) Nature 317, 145-153). The amino acids which are located in this canyon are conserved to a relatively great extent, whilst the amino acids in the surrounding area are variable and constitute binding sites for antibodies with a neutralising effect. 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 maintained which is not accessible for antibodies (Rossmann and Palmenberg (1988) Virology 164, 373-382).

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2ll7ass As far as is known at present, the receptor of the "small rhinovirus receptor group" permits the uptake of about 10 serotypes of human rhinoviruses into the corresponding host cells. This receptor on the membrane has been isolated by various purification steps, whilst the binding activity in the various frac~ions has been demonstrated by 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 about 450 kD, that of the denatured form corresponds to about 120 kD, although a number of other forms were found (Mischak (1988) loc.
cit.). It has also been found that a protein isolated from the cell culture supernatant from HeLa cells has the capacity to bind rhinoviruses of the "small rhino-virus receptor group" (Hofer et al. (1992) J. gen.
Virol. 73, 627 - 632). ;

The natural receptor is less suitable for inhibiting the uptake of rhinoviruses of the "small rhinovirus receptor group" on the basis of the low solubility of this membrane protein in polar, e.g. aqueous solution systems such as aqueous buffer solutions.

Surprisingly, it has now been found that the members of LDL ("low density lipoprotein") receptor family act as receptors for rhinoviruses of the "small rhinovirus receptor group".
~ . :
The identical nature of the receptors of the LDL-receptor family and the receptors of rhinoviruses of the "small rhinovirus receptor group" now surprisingly makes it possible to prepare polypeptides, particularly soluble polypeptides, which have at least one bindinq -site for rhinoviruses of the "small rhinovirus receptor group".

21170~9 The polypeptides according to the invention are hereinafter referred to as ~functional derivatives" of the receptor proteins. A func~ional derivative is therefore a component with the biological activity which corresponds essentially 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 expression "functional derivatives" is intended to include "variants" and "chemical derivatives". The term derivative refers to any polypeptide which is small in size, compared with the native receptor protein, and has at least one binding site for rhinoviruses of the "small rhinovirus receptor group". A "variant" comprises the molecules which are essentially derived from the native receptor molecule in function and structure, such as the allelic forms, for example. Accordingly, the term "variant"
includes molecules which are capable of binding rhinoviruses of the "small rhinovirus receptor group"
but have a different amino acid sequence, for example.

A "chemical derivative" includes additional chemical groups which are not normally part of this molecule.
These groups may improve the molecule solubility, the absorption, the biological half-life etc. or alternatively may reduce the toxicity of undesirable side effects. Groups having such effects are known (Remington's Pharmaceutical Sciences (1980)).
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The biological activity of the receptor derivatives according to the invention or the chemical derivatives obtained after modification can be tested using methods known from the prior art, e.g. the filter binding assay described by Mischak et al. (Mischak et al. (1988) J.
Gen. Virol. 69, 2653-2656 and Mischak et al. (1988) Virology 163, 19-25): the polypeptide is applied to a ~ 2~170~9 , suitable membrane, such as nitrocellulose. Then, in order to block any non~specific binding, it is saturated with a detergent mixture. The membrane pretreated in this way is then incubated with labelled rhinovirus, e.g. with HRV2 labelled with 35S-methionine, in order to check the specific binding. After washing and drying of the membrane specific binding can then be visualised by autoradiography.

One aspect of the invention relates to the receptor derivatives which are present in the form of extracellular, soluble polypeptides and are released into the medium, for example, by receptor-carrying cells. These receptor derivatives are exceptionally well suited to inhibiting the binding of rhinoviruses to their receptors. Thus, they can be used for the therapeutic or prophylactic treatment of the human body or for producing pharmaceutical preparations. In particular, their use as antiviral and preferably antirhinoviral agents may be considered. The phenomenon of releasing a soluble receptor derivative has been ;~;
described for numerous receptor proteins, e.g. for the interleukin-4- and interleukin-7-receptor (Mosley et al.
(1989) Cell 59, 335-348; Goodwin et al. (1990) Cell. 60, 941-951). ;

Naturally, soluble receptor derivatives may also be formed by enzymatic, especially proteolytic or chemical cleaving. Receptor-carrying cell lines may be used for this purpose, which are reacted 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 deliberately prepare extracellular derivatives by a suitable choice of proteases. The binding capacity of such derivatives can be checked using the filter binding assay described above, thus making it possible to prepare deliberately smaller ~ 21~709~

receptor derivatives which are capable of binding rhinoviruses of the "small rhinovirus receptor group".
In addition to enzymatic cleaving it is also possible to cleave extracellular receptor regions by chemical methods, e.g. by cleaving with cyanogen bromide.

A further aspect of this invention consists of the formation of soluble derivatives by enzymatic or chemical cleaving of native receptor molecules. After a native receptor protein has been isolated, for example, the native receptor protein can be cleaved by reaction with proteases or by chemical cleaving (as described above) and the reduced in size, rhinovirus-binding region can be identified by the filter binding assay, for example, and isolated. Suitable proteases can be derived from the particular amino acid sequence of the receptor protein. Chemical cleaving reactions can also be carried out using cyanogen bromide or cleaving the receptor protein by a reductive treatment, e.g. with dithiothreitol.

More specifically, the present invention comprises the following aspects:

It has been found, surprisingly, that proteins of the LDL-receptor family are capable of binding and internalising rhinoviruses of the "small rhinovirus receptor group". Consequently, all the members of the LDL-receptor family can now be used to prepare functional derivatives capable of binding the rhinoviruses of the "small rhinovirus receptor group".

The LDL-receptor family is formed from three structurally related cell surface receptors which bring about the endocytosis of lipoproteins and other plasma proteins (Brown et al. (1991) Curr. Opin. Lipidology 2, 65-72). The receptors have the following common 21170~3 features: cysteine-rich repeats, which are responsible for ligand binding, cysteine-rich repeats of the EGF
("epidermal growth factor")-type, Y-W-T-D-repeats, a single region spanning the membrane and at least one NPXY-internalising signal (Willnow et al. (1992) J.
Biol. Chem. 267, 26172-21180).

Surprisingly, it has been shown that all three ~embers of this family - the LDL-receptor, the ~2MR/LRP (~2-macroglobulin/LDL-receptor-related protein) and also the gp330 (Heymann nephritis antigen gp330) - are capable of binding and internalising rhinoviruses of the "small rhinovirus receptor group" (Examples 1 to 2). All members of this receptor family can thus be used to form functional derivatives with binding properties for ~1 rhinoviruses of the "small rhinovirus receptor group".
For example, in order to isolate soluble LDL-receptor derivatives released into the medium, the method included in Example 3 can be followed. This describes the purification of a binding protein released into the cell culture supernatant. Surprisingly, it was found that this is an LDL-receptor derivative (Example 4).
For the purpose of isolation, the receptor derivative is purified by ion exchange chromatography (anionic), affinity chromatography (Lens culinaris lectin and Jacalin agarose) and ammonium sulphate precipitation.
The binding activity was checked using the filter binding assay (Mischak et al. (1988) 163, 19-25). This method of production can also be applied to the other two proteins of the LDL-receptor family.

Isolation of the native receptor proteins is known and I is described by Yamamoto et al. (1984) Cell 39, 27-38;
I Goldstein et al. (1985) Annu. Rev. Cell Biol. 1, 1-39;
I Mischak et al. (1988) Virology 163, 19-25; Kowal et al.
j (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5810-5815 and Willnow et al. (1992) loc. cit.). The native proteins ~ 2117~g.~ ' can then be converted by enzymatic and chemical cleaving into the functional soluble derivatives. Since the amino acid sequence of the LDL-receptor (Fig. 1), the MR/LRP (Fig. 2) and, at least partially, the gp330 (Fig. 3) are known, proteolytically active enzymes or chemicals can be deliberately selected in order to release, in particular, the particular extracellular receptor region. The present invention therefore also relates to polypeptides which are derived from the amino acid sequences of the LDL-receptor, ~zMR/LRP and gp330 and, in particular, in their soluble form are capable of binding rhinoviruses of the "small rhinovirus receptor group". Preferably, these polypeptides are derived from the amino acid sequences which correspond to the human proteins of the LDL-receptor family, although, as explained in Examples 1 and 2, corresponding receptors -from mammals and amphibia are also suitable.

Receptor derivatives may be used in the form in which they are released into the cell supernatant from eukaryotic cells. The receptor derivatives of the present invention may also, however, correspond to the membrane-bound members of the LDL-receptor family in which that part of the protein which is responsible for ~ -binding the protein to the membrane is missing or has lost its function.

Particularly preferred are receptor derivatives which consist essentially of domains 1, 2 and 3 of the receptor protein, or domains 1 and 2 or only domain 1 according to Figure 4. According to this, domain 1 comprises the N-terminal cysteine-rich receptor portion which binds the various ligands, domain 2 comprises a region having higher homology to the EGF-precursor protein, domain 3 comprises a relatively short, O-glycosylated peptide region, domain 4 comprises the transmembrane region and domain 5 the cytoplasmic part . - : : ' 21170~9 _ 9 -of the receptor ~olecule. Polypeptides consisting essentially of domains l, l and 2 and 1, 2 and 3 m~y be obtained from the culture supernatant of eukaryotic cells (Example 3) or by recombinant DNA techniques known ' se, such as that described by Davis et a],. (1987) Nature 326, 760-765 for the LDL-receptor. Of the proteins of the LDL-receptor family the human,LDL- :, receptor is the preferred starting compound. In particular, the invention include~ functional receptor derivatives which essentially comprise amino acids 1 to 750 tdomains l and 2) and 1-322 (domain 1) (Fig. 1). , , The C-terminus of these polypeptides can be shortened, provided that the ~inding capacity for rhinoviruses of the small rhinovirus receptor group remains intact.

The preferred receptor derivatives have essentially the following amino acid sequences:

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

401 LFFTNRHEVR KMTLDRSEYT SLIPNLRN W ALDTEVASNR IYWSDLSQRM ' 451 ICSTQLDRAH GVSSYDTVIS RDIQAPDGLA VDWIHSNIYW TDsvLGTvsv 501 ADTKGVKRKT l,FRENGSKPR AI W DPVHGF MYWTDWGTPA KIKKGGLNGv 601 DEKRLAHPFS LAVFEDKVFW TDIINEAIFS ANRLTGSDVN LLAENLLspE
651 DMVLFHNLTQ PRGVNWCERT TLSNGGCQYL CLPAPQINPH SpKFTcAcpD
701 GMLLARDMRS CLTEAEAAVA TQETSTVRLK VSSTAVRTQH TTTRpvpDTs ' -~ 211709~

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

The polypeptides according to the invention may occur as dimers, trimers, tetramers or multimers. The processes for preparing the receptor derivative, enzymatic or chemical treatment of the native receptor molecules, isolation of the derivatives released by cells and processes for recombinant preparation are also part of the invention.

A further aspect of the invention concerns DNA molecules which code for the polypeptides according to the invention.
:' The starting molecules can be obtained by the person skilled in the art using known methods. The cloning of the correspondind cDNA is described for all three members (Yamamoto et al. (1984) loc. cit.; Goldstein et al. (1985) loc. cit.; Pietromonaco et al. (1990) Proc.
Natl. Acad. Sci. U.S.A. 87, 1811-1815; Herz et al.
(1988) loc. cit.). Moreover, the DNA molecules, where ;
the amino acid sequence is known, may also be produced synthetically (e.g. according to Edge et al. (1981) 292, 756-762) or by the PCR method (Sambrook et al., loc.
cit.).

The invention relates to DNA sequences which have modifications obtained simply by methods known to those skilled in the art, by mutation, deletion, transposition ~: .

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-- 21170~9 or addition. All DNA sequences which code for a polypeptide according to the invention and the correspondingly degenerate forms of the DNA sequences are included.

In addition, the invention relates to DNA vectors which contain the DNA sequences described above. In particular, these may be vectors in which the DNA
molecules described are functionally linked to a control sequence which allows 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 relates to correspondingly transformed host organisms.

Expression in prokaryotes may be carried out using other organisms known from the prior art, especially E. coli.
The DNA sequences according to the invention may be expressed as fusion polypeptides or as intact, native polypeptides. .

Fusion proteins may advantageously be produced in large quantities. They are generally more stable than the native polypeptide and are easy to purify. The expression of these fusion proteins can be controlled by normal E. coli DNA sequences.
I
For example, the DNA sequences according to the invention can be cloned and expressed as lacZ fusion genes. The person skilled in the art has a variety of vector systems available for this purpose, e.g. the pUR-vector series (Ruther, U. and Muller-Hill, B. (1983), EMB0 J. 2, 1791). The bacteriophage promoter ~PR may also be used, in the form of the vectors pEX-1 to -3, 21170~9 for expressing large amounts of Cro-~-galactosidase fusion protein (Stanley, K.K. and Luzio, J.P. (1984) EMBO J. 3, 1429). Analogously, the tac promoter which can be induced with IPTG can also be used, for example in the form of the pROK-vector series (CLONTECH
Laboratories).

The prerequisite for producing intact native polypeptides using E. coli is the use of a strong, regulatable promoter and an effective ribosome binding site. Promoters which may be used for this purpose include the temperature sensitive bacteriophage ~pL-promoter, the tac-promoter inducible with IPTG or the T7-promoter. Numerous plasmids with suitable promoter structures and efficient ribosome binding sites have been described, such as for example pKC30 (~pL; Shimatake and Rosenberg (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 number of other vector systems for expressing 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) "A Laboratory Manual", Cold Spring Harbor Laboratory Press).

Suitable E. coli strains which are specifically tailored to the expression vector in question are known to those skilled in the art (Sambrook et al. (1989), loc. cit.).
The experimental performance of the cloning experiments, the expression of the polypeptides in E. coli and the working up 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 yeast may also be used.

-- 2117~

For expression in yeast, the plasmid YRp7 (StinchcoMb et al. Nature 282, 39 (1979); Kings~an et al., Gene 7, 141 (1979); Tschumper et al., Gene 10, 157 (1980)) and the plasmid YEpl3 (Bwach et al., Gene 8, 121-133 (1979)) are used, for example. The plasmid YP~p7 contains the TRPl-gene which provides a selection marker for a yeast mutant (e.g. ATCC No. 44076) which is incapable of growing in tryptophan-free medium. The presence of the TRPl defect as a characteristic of the yeast strain used then constitutes an effective aid to detecting transformation when cultivation is carried out without -tryptophan. The same is true with the plasmid YEpl3, -which contains the yeast gene LEU-2, which can be used to complete a LEU-2-minus mutant.
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Other suitable marker genes for yeast include, for example, the URA3- and HIS3-gene. Preferably, yeast hybrid vectors also contain a replication start and a marker gene for a bacterial host, particularly E. coli, so that the construction and cloning of the hybrid vectors and their precursors can be carried out in a bacterial host. Other expression control sequences suitable for expression in yeast include, for example, those of PHO3- or PHO5-gene.

Other suitable promoter sequences for yeast vectors contain the 5'-flanking region of the genes of ADH I
tAmmerer, Methods of Enzymology 101, 192-210 (1983)), 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.
255, 2073 (1980)) or other glycolytic enzymes (Kawaski and Fraenkel, BBRC 108, 1107-1112 (1982)) such as enolase, glycerinaldehyde-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 may also be inserted in the expression 21170~

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 genes for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase and enzymes which are responsible for the metabolism of maltose and galactose~ Promoters which are regulated by the yeast mating type locus, such as promoters of the genes BARI, MF~1, STE2, STE3, STE5 can be inserted in temperature regulated systems by the use -of 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 Laboratory). However, generally, any vector which contains a yeast-compatible promoter and origin replication and termination sequences is suitable.
Thus, hybrid vectors which contain sequences homologous to the yeast 2~ plasmid DNA may also be used. Such hybrid vectors are incorporated by recombination within the cells of existing 2~-plasmids or replicate autonomously. ~-. ~
In addition to yeasts, other eukaryotic systems may, of course, be used to express the polypeptides according to the invention. Since post-translational modifications such as disulphide bridge formation, glycosylation, phosphorylation and/or oligomerisation are frequently necessary for the expression of biologically active eukaryotic proteins by means of recombinant DNA, it may be desirable to express the DNA according to the invention not only in mammalian cell lines but also insect cell lines.
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Functional prerequisites of the corresponding vector systems comprise, in particular, suitable promoter, ~ A

21170~9 termination and polyadenylation signals as well as elements which make it possible to carry out replication and selection in mammalian cell lines. For expression -of the DNA molecules according to the invention it is particularly desirable to use vectors which are replicable both in mammalian cells and also in prokaryotes such as E. coli.

Vectors derived from viral systems such as SV40, Epstein-Barr-virus, etc., include, for example, pTK2, pSV2-dhfv, pRSV-neo, pKO-neo, pHyg, p205, pHEBo, etc.
(Sambrook et al. 1989, loc. cit.).

After transformation in suitable host cells, e.g. CHO-cells, corresponding transformed cells may be obtalned with the aid of selectable markers (thymidine-kinase, dihydrofolate-reductase etc.) and the corresponding polypeptides are isolated after expression. The host cells suitable for the vectors are known, as are the techniques for transformation (micro-injection, electroporation, calcium phosphate method, etc.) (e.g.
Sambrook et al., 1989).

For cloning corresponding DNA fragments in prokaryotic or eukaryotic systems, the selected vector is cut, for example, with a restriction endonuclease and, optionally after modification of the linearised vector thus formed, an expression control sequence equipped with corresponding restriction ends is inserted. At the 3'-end (in the direction of translation) the expression control sequence contains the recognition sequence of a restriction endonuclease, so that the vector already containing the expression control sequence is digested with the said restriction enzyme and the DNA molecule according to the invention, provided with ends which fit, can be inserted. It is a~vantageous to cleave the vector which already contains the expression control 21170~9 sequence with a second restriction endonuclease inside the vector DNA and to insert the DNA molecule provided with the correct ends into the vector fragment produced.
The techniques required are described for example by Sambrook et al. (1989, loc. cit.).

Apart from the DNA molecules specified, the invention also relates to processes for preparing the vectors described, particularly expression vectors. These vectors are characterised in that a DNA provided with corresponding ends and coding for a functional derivative of the receptor of the "small rhinovirus receptor group" is inserted into a vector DNA cut with restriction endonucleases and containing the expression control sequences described by way of example, in such a way that the expression control sequences regulate the expression of the DNA inserted. The polypeptides according to the invention which are obtained by the expression of recombinant DNA or from the native receptor molecule may, of course, also be derivatised by chemical or enzymatic processes.

The expression of the LDL-receptor is explained in Example 6. Here, expression takes place in a eukaryotic system, by way of example. It is clearly shown that the LDL-receptor expressed brings about a binding of radioactively labelled human rhinovirus serotype 2 (HRV2) (Fig. 5). The polypeptides according to the invention may be obtained, for example, by deletion of DNA sequences in the expression plasmid. This can-be done, for example, using the method of Davis et al.
(1987) Nature 326, 760-765, which describes the deletion of the entire EGF domain. Moreover, soluble forms of the receptor can be formed by inserting a stop codon in front of the cytoplasmic or transmembrane domain (Yokade et al. (1992) J. Cell. Biol. 117, 39).
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The invention further relates to hybrid cell lines which secrete monoclonal antibodies specifically against one of the polypeptides according to the invention or functional derivatives thereof. These monoclonal antibodies are capable of wholly or partially neutralising the activity of the polypeptides or specifically binding to one of the said polypeptides.
The monoclonal antibodies can then be used for qualitative and/or quantitative measurement or for purifying the polypeptides according to the invention.
The invention naturally also includes test systems which contain the monoclonal antibodies mentioned. The process for preparing the monoclonal antibodies is characterised in that host animals are immunised with one of the polypeptides and B-lymphocytes of these host animals are fused with myeloma cells; the hybrid cell lines 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).

A further aspect of the invention is the use of physioloqical ligands of the LDL-receptor family for preparing pharmaceutical compositions for inhibiting the binding of rhinoviruses of the "small rhinovirus receptor group". The physiological ligands comprise the substances which are bound and/or internalised 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 described for example by Willnow et al. (1992) J. Biol. Chem. 267, 26172-2618~.

Thus, for example, the 39 kDa receptor-associated protein (RAP) can reduce the yield of rhinoviruses of the "small rhinovirus receptor group" (Example 7). RAP

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is known ~er se. Its isolation and binding to members of the LDL-receptor family has been described for example by Kounnas et al. (1992) J. Biol. Chem. 267, 21162-21166.
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It is also, of course, possible to use the native receptors of the LDL-receptor family, the LDL-receptor, ~2MR/LRP and gp330 as the receptor derivatives according to the invention for inhibition.

Conversely, it is also, of course, possible to use rhinovirus material of the "small rhinovirus receptor group" to inhibit the binding of physiological LDL-ligands. This rhinovirus may, for example, be derived from human rhinovirus serotype 2 (HRV2). Preferably, inactivated rhinovirus, rhinovirus coat material or rhinovirus peptides with a binding activity to a receptor of the LDL-receptor family may be used as rhinovirus material. Rhinoviruses of the "small rhinovirus receptor group" can be obtained from the "American Type Culture Collection". Corresponding virus material may be prepared by 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).

Of course, the invention also includes the pharmaceutically acceptable salts of the polypeptides according to the invention and the pharmaceutically acceptable adducts and covalent compounds between the polypeptides and an inert carrier for the prophylactic and/or therapeutic treatment of the human or animal body. The adducts or covalent compounds may be formed with polyethyleneglycol, for example. The polypeptides according to the invention and the native receptor ~-proteins, the physiological ligands of the LDL-receptor family such as LDL and RAP, for example, may be used to ~ 2117Q~

produce pharmaceutical preparations for the therapeutic and/or prophylactic treatment of the human or animal body. In particular, the polypeptides can be used as competitively acting substances for inhibiting the binding of viruses, particularly rhinoviruses, to the native receptor and/or physiological LDL-ligands. The polypeptides and natural ligands, particularly the extracellular, soluble form of the receptor, may be used especially as antiviral and preferably antirhinoviral agents.

For treating viral infections the substances described may be administered nasally, for example, the quantities supplied being sufficient to suppress or competitively interact or inhibit the binding of the rhinovirus to the natural receptor. The dosage should generally be between 0.01 pg/kg of the weight of the patient up to 1 mg/kg of the weight of the patient, although larger or smaller quantities may also be used. The rhinovirus I material which may be used to inhibit the binding of physiological LDL-ligands can be used in suitable pharmaceutical compositions in the ranges of concentration specified for the polypeptides.

The receptor derivatives according to the invention and the pharmacologically acceptable salts thereof may be converted in the usual way into conventional formulations such as plain or coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert pharmaceutically acceptable carriers or solvents. The proportion of the pharmaceutically active compound or compounds should be in the range from 0.5 to 90 wt.-% of the total composition, i.e. in amounts which are sufficient to achieve the dosage range specified above.

The formulations are prepared, for example, by admixing .1 ' ~
~ `,:

.~.. ,......... - ~ . . ~ . , . ~ ~ ............................ ..
. - ~ .. . .. ., . .. . . ,.. .,. ..... , ., ., ~ ... . . ... .

211709~ ~ ~

the active substances with solvents and/or carriers, optionally using emulsifiers and/or dispersants, whilst if water is used as the diluent, organic solvents may be used as solubilising agents or auxiliary solvents.

The excipients used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins, vegetable oils, mono- or polyfunctional alcohols, carriers such as natural mineral powders, synthetic mineral powders, sugars, emulsifiers and lubricants.

The substances are administered in the usual way, preferably nasally. In the case of oral administration the tablets may, of course, contain in addition to the above-mentioned carriers, additives such as sodium citrate, calcium carbonate and dicalcium phosphate together with various additives such as starch, preferably potato starch, gelatine and the like.
Furthermore, lubricants such as magnesium stearate, sodium laurylsulphate and talc can be used to form tablets. In the case of aqueous suspensions, the active substances may contain, in addition to the above-mentioned excipients, various flavour improvers or colourings.

The invention also relates to processes for isolating substances which inhibit the binding of ligands to the LDL-receptor. These processes comprise incubating the LDL-receptor protein or an LDL-receptor derivative with a potentially inhibiting substance. This process can be carried out in the presence of labelled rhinovirus material. The extent of the binding of labelled rhinovirus material then allows conclusions to be drawn as to the activity of the test substance. The preparation of rhinovirus material with various binding activities is described in Example 9. ~ -~

r~ 21~7099 The invention also relates to processes for detecting LDL-receptors, in which a substance derived from virus material of the "small rhinovirus receptor group" with a binding activity for the LDL-receptor is labelled, incubated with a suitable sample and the extent of binding is detected. Another process serves to supply therapeutically active substances in which virus material of the "small rhinovirus receptor" group with a binding activity for the LDL-receptor is coupled with the therapeutic substance and the said conjugate is added to cell material carrying LDL-receptor and the therapeutically actlve substance is inserted into the j cell by binding and internalisation.

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'1 :

,~ 21~7~

- 2~ -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 Lipopro-tein Receptor Related Proteins" (LRP, Herz et al. (1988) EMB0 J. 7, 4119-4127).

Fig. 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: Diagrammatic representation of a receptor of the LDL-receptor family (according to Yamamoto et al. (loc. cit.). The receptor has five domains: domain 1 comprises the N-terminal cysteine-rich receptor part which is presumably responsible for the ligand binding.
Domain 2 with homology for the EGF-precursor protein adjoins domain 3, the amino acids of which are partially 0-glycosylated. Domain 4 forms the part of the receptor situated at the membrane whilst domain 5 forms the cytoplasmic part of the receptor.

Fig. 5: A) Binding and internalisation of labelled HRV2 on normal human fibroblast cells and on LDL-receptor deficient FH cells, respectively (Example 1).

t: cultivated without the addition of cholesterol/25-hydroxycholesterol 1: cultivated with the addition of cholesterol/25-hydroxycholesterol D, 21~70~9 B) Competition of HRV2 and LDL for the receptor binding site +: with the addition of unlabelled HRV2 or LDL
-: without the addition of unlabelled HRV2 or LDL.
ig. 6: Binding of [35S]-labelled HRV2 to ~2MR/LRP and gp330. Membrane extracts have been electrophoretically separated and transferred to nitrocellulose. Detection was carried out with [35S]-labelled HRV2 (trace 1 and 2) with ~2MR/LRP antiserum (trace 3) or with gp330 antiserum (trace 4).
Trace 1: LM-extracts, Trace 2: Rat kidney microvilli-extracts, Trace 3: Protein extracts as in trace 1, Trace 4: Protein extracts as in trace 2.
ig. 7: Gel electrophoretic analysis of the purified HRV2 binding protein.
a) The purified H~V2 binding protein was subjected to electrophoresis in a 7.5 SDS gel under reducing (trace 1) and non-reducing conditions ~trace 2) and made -~
visible by silver staining. Under non-reducing conditions a molecular weight of about 120 kDa is obtained whilst under reducing conditions the molecular weight is 160 kDa.
b) Ligand blots of a gel as described under a (trace 2), developed with [3sS]-HRV2 (trace 1) according to Mischak et al.
(1988) Virology 163, 19-25. Trace 2 shows the development with an antibody specific for the human LDL-receptor (IgG-C7, Beisiegel et al. (1982) J. Biol.

~ 21170~

Chem. 257, 13150-13156).

,, .
Fig. 8: Shows the column chromatographic separation of the tryptic peptides of the soluble form of the receptor of the "small rhinovirus receptor group", obtained from the HeLa cell supernatant. The peptides were separated on a ~Bondapak C18,250-4 column under the following conditions:
~ Buffer A: distilled 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% 8 from 60 to 90 min, 75% B to 98% B from 90 to 105 min; ~-temperature: ambient temperature; detection:
photometrically at 214 nm, 0.08 AUFS (paper advance: 0.25 cm/min).

Fig. 9: Chromatographic separation of fractions 23 to 27 under the following conditions:
Column: Merck Superspher 4 ~m, C18, 125-H;
buffer A: distilled 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: 30C; detection: photometrically ' at 214 nm, 0.1 AUFS, paper advance 1 cm/min.

Fig. 10: Rechromatography of fraction 29. The experimental conditions are described in the legend to Fig. 9.

Fig. 11: Rechromatography of fraction 38. The experimental conditions are described in the legend to Fig. 9.

Fig. 12: Sequences of the peptide analysed X = amino acid not identifiable; subscript = amino acid .`
' :
~ 5~

~11709~

not clearly identifiable.
*: The sequencing of fraction 33 (Fi~. g) yielded 2 amino acids for each breakdown step;
however, peptides B and E were able to be classified because of the different quantities.
ig. 13: Inhibition of binding of [35S]-labelled HRV2 to the immobilon-bound LDL-receptor by jacalin.
A) filter binding test in the absence of jacalin B) as in ~, but in the presence of 0.1 mg/ml of jacalin.
ig. 14: Expression of the human LDL-receptor in COS-7 cells (Example 6).
Detection of bound[3sS]-HRV2 on u: untransfected COS-7 cells +: transformed with the "sense" (pSVL-LDLR+)-vector -: transformed with the "antisense"
(pSVL-LDLR-)-vector ig. 15: Reduction of the virus yield by RAP, given in p.f.u./ml (infectious particles per millilitre).
ig. 16: Inhibition of HRV2-infection of HeLa cells by human LDL.
ig. 17: Comparison of sequences for determining the positions in or on the edge of the canyon which are conserved in rhinoviruses of the small group.
ig. 18: Binding characteristics of HRV2ll48pG and HRV23lB2RT to HeLa cells ^` 2117099 ~:

AHRv2 1 148P:G
HRV2-Wild type _HRV23l8ZR r Fig. l9: Competition of binding of HRV2~148pG and .
HEV2~zpl by HRVl4 (-) or HRV2 (~

:~;

~ 2117~9~

EXAMPLES

Example 1: sinding and internalisation of [35S]-labelled HRV2 by human fibroblast cells and competition between HRV2 and LDL for the receptor binding site -~

a~ Binding and internalisation of HRV2 Normal human fibroblast cells or LDL receptor-deficient cells (FH cells; NIH Collection No. GM 00486A) were grown for 24 hours on 6-well plates (Nunc) in MEM, containing 10% delipidated foetal calf serum (Gibco), either with (1) or without (t) the addition of 12 ~g/ml of cholesterol and 2 ~g/ml of 25-hydroxycholesterol.
Then the cells were washed twice with PBS, 10,000 cpm [35S]-labelled HRV2 in 0.5 ml of PBS, containing 2% ~SA
and 30 mM MgCl2 were added and the mixture was incubated for 60 minutes at 34C (Mischak et al. (1988) Virology 163, 19-25). After the removal of superficially bound HRV2 using 10 ~g/ml of trypsin and 25 mM EDTA in PBS the cells were washed once more and then the bound radioactivity was measured. The data provided are the averages from four experiments in each case. The radioactivity levels of the cell pellets from normal fibroblasts (normally about 1900 cpm) grown without steroids, minus background radioactivity, was set at 100%. The background activity was determined either with HRV2 which had been heated to 56'C for 30 minutes (Mischak et al. (1988) loc. cit.) or by incubation with a 1000-fold excess of unlabelled HRV2. For both methods it was between 40 and 50 cpm. The data obtained from ~f~
four separate experiments are shown in Fig. Sa. ~
:.
b) Competition of HRV2 and LDL for the receptor bindinq ;;~
site :~
Normal fibroblast cells were grown as described under a) (without the addition of cholesterol and 25-hydroxy-. ~

~ 21170~9 cholesterol). The cells were incubated at 37C for 60 minutes with approximately 1.4 x 106 cpm lZsI-labelled LDL
(250 cpm/ng; Huettinger et al. (1992) J. Biol. Chem., 267, 18551-7) with (+) and without (-) the addition of 100 Pfu ("plaque forming units"; corresponding to about 2400-24000 virus particles; Abraham & Colonno (1984) J.
Virol. 51, 340-345) per cell on purified, unlabelled HRV2 or with about 10000 cpm of [35S]-labelled HRV2 with (+) or without (-) 80 ~g/ml of unlabelled LDL. The cell-associated radioactivity was measured with a y- or ~-counter. The radioactivity levels for the high affinity binding of l2sI-LDL were determined by subtracting the radioactivity, obtained in the presence of a 20-fold excess of unlabelled LDL (approximately 40000 cpm/mg of whole cell protein), from the entire LDL-bond (150,000 cpm/mg). Without a competitor, a radioactivity level of 1900 cpm was normally found for HRV2 binding. The maximum binding levels wère put at 100% in each case. The data obtained (Fig. 5b) show the results of two separate experiments in each case.

Example 2: Binding of [35S]-labelled HRV2 by ~2MR/LRP
and gp 330 Plasma membrane preparations were tested for HRV2-binding in order to demonstrate the binding of rhinoviruses of the small rhinovirus receptor group to other members of the LDL receptor family. Plasma membranes were isolated from murine LM fibroblasts and kidney epithelial microvilli (Malathi et al. (1979) Biochem. Biophys. Acta, 554, 259-263; Fornistal et al.
(1991) Infect. Immun. 59, 2880-2884 and Kerjaschki and Farquhar (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5557-5561). Proteins from the membrane extracts were separated by SDS-qradient-polyacrylamide-electrophoresis and transferred on to nitrocellulose.

^` 211709~ ~

The incubation of the separated ~-extracts with [35S]-labelled HRV2 showed a binding to a protein with an apparent molecular weight of about 500 kDa (Fig. 6, trace 1). This band comigrates with ~2MR/LRP. This was demonstrated by an identical blot which was developed with ~2MR/LRP antiserum (Moestrup and Gliemann (1991) J.
Biol. Chem. 266, 14011-14017) (Fig. 6; trace 3).

A protein with an apparent molecular weight of again about 500 kDa could be detected with radioactively labelled HRV2 in extracts from rat kidney microvilli (Fig. 6; trace 2). After analysis with a gp330 antiserum the band was identified as Heymann Nephritis ' Antigen gp330 (Fig. 6, trace 4~.

:i l Example 3: Purification of a binding protein for the i rhinoviruses of the "small rhinovirus ¦ receptor group"

200 1 of HeLa-cell culture supernatant (made by Computer Cell Culture Center, Mons, Belgium) were concentrated down to 20 1 by ultrafiltration and dialysed against `~ 250 1 of distilled water (with 0.02% NaN3). Then the bùffer concentration was adjusted to 20 mM N-methylpipe~azine, pH ~.5, the mixture was centrifuged at 4000 rpm in a Beckman J6B centrifuge, filtered through a 0.8 ~m preliminary filter and the filtrate was transferred on to an anion exchanger 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 using 1 M tris-HCl, pH 8.0, and transferred to a Lens culinaris lectin column (100 ml; Pharmacia); bound protein was eluted -~
with 0.5 M ~-[D]-methylglucose in PBS, the eluted protein was precipitated at 50% saturation with ammonium sulphate, pH 7.2, the precipitate was washed with 50% ~ ;

:~

~ 211709~

saturated ammonium sulphate solution, pH 7.2, and taken up in 200 ml of PBS. The protein solution was transferred to a Jacalin agarose column (40 ml; Vector-Labs) and eluted with 120 ml of 100 mM ~-tD]-methyl-galactopyranoside in PBS. The eluted protein was precipitated with ammonium sulphate as described above, washed, taken up in 20 mM methylpiperazine, pH 4.5, and desalinated using a PD10-column (Pharmacia). The desalinated material was added to a Mono Q anionic exchanger column (HR5/5; Pharmacia) in 5 aliquots per ml and eluted with a gradient from o to 0.5 M NaCl, 20 mM
methylpiperazine, pH 4.5. 0.5 ml fractions were collected and tested for binding activity using a filter binding assay (Mischak et al., Virology (1988) 163, 19-25). The active fractions from all five chromatographies were concentrated down to 1.5 ml in a Centricon 30 (Amicon) and resolved by preparative gel electrophoresis under non-reducing conditions on a 7.5%
polyacrylamide gel (Laemmli, U.K. (1970) Nature 227, 680 - 685). The gel was stained with copper chloride (Lee et al. (1987) Anal. Biochem. 166, 308 - 312), the band corresponding to the active protein was located and excised. The gel fragments were decolorised in 0.25 M
Tris-HCl, 0.25 M EDTA, pH 9.0, and the protein was eluted in 50 mM N-ethylmorpholinoacetate, pH 8.5, by electrophoresis. One aliquot was again tested for activity using the filter binding assay. The protein was then separated by gel electrophoresis under reducing conditions, eluted and lyophilised.

Example 4: Tryptic digestion and sequence analysis of the binding protein for the rhinoviruses of the "small rhinovirus receptor group"

The purified and lyophilised protein (Example 3) was taken up in 30 ~1 of 6 M guanidine-HCl, 0.4 M ammonium '-` 2117~99 hydrogen carbonate, pH 7.6, and mixed with 3 ~1 of 45 mM
dithiothreitol and incubated for 15 minutes at 56C.
After cooling to ambient temperature, 3 ~1 of 100 mM
iodoacetamide were added and the mixture was incubated for a further 15 minutes at ambient temperature. Then 84 ~1 of water and 80 ~1 of 0.1 M ammonium hydro~en carbonate, pH 7.6, were added, the protein solution was mixed with 800 ng of trypsin (in 5 ~1) under the conditions specified by the manufacturer (Promega) and incubated for 18 hours at 37~C. The solution was acidified with 1/10 volume of 10% trifluoroacetic acid (TFA), centrifuged for 5 minutes and the peptides were eluted on a C-18 "reversed phase" column (Baker) which had been equilibrated with a 0.06~ aqueous TFA solution, with a gradient up to 80~ acetonitrile, 20~ water, 0.052% TFA (Fig. 8). ;~

Fractions 20 and 33 were sequenced directly using a gas ¦ phase sequenator whilst fractions 23 to 27 and 29 and 38 I were re-chromatographed under the conditions specified j in the Figures (C18 "reversed-phase" column, Merck;
I Figs. 9, 10 and 11). The peptides designated "A", "D" and "F" in the Figures and fractions 33 and 20 were selected for sequencing in the gas phase sequenator.
The results are assembled in Fig. 12. The sequences obtained were compared with the protein sequences available in the "SwissProt" databank. Comparison showed total 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).

~ .~

2~17ass Peptide Position Sequence The sequencing of fraction 33 yielded two amino acids per breakdown step. Taking as the basis the LDL-sequence and the ratio of amino acid quantities present in each breakdown step, from about 40% to 60~, fraction 33 was identified as a mixture of two peptides. The sequences of these two peptides also correspond to sequences of the human LDL-receptor.

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

~, j 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 introduced into competent E. coli SK and amplified using known methods (Sambrook et al., loc. cit.). After extraction and purification of the plasmid DNA the latter was digested with the restriction enzyme HindIII and the fragments were separated in a 0.8% agarose gel. After elution of the fragment coding for the LDL-receptor the latter was precipitated with ethanol, taken up in TE-buffer and partially filled with Klenow fragment using dATP and dGTP.

21170~9 The eukaryotic expression vector pSVL (Pharmacia) was replicated in E. coli 5K, purified and cut with XbaI.
After partial filling with dCTP and dTTP, phenol-chloroform extraction and ethanol precipitation, the plasmid was dephosphorylated with alkaline phosphatase.

By partial filling both of the vector and of the LDL-receptor DNA the restriction cutting sites were made compatible and LDL-receptor coding and vector DNA were ligated using T4-ligase. Competent E. coli bacteria were transformed as described. A number of colonies were investigated, by restriction digestion with XhoI, for orientation of the insert with regard to the SV40 "late promoter". One colony with positive (sense, pSVL-LDLR+) orientation and one colony with negative (antisense, pSVL-LDLR-) orientation of the insert were cultivated and plasmids were obtained in large quantities.

The transfection of COS-7 cells (ATCC CRL 1651) was carried out by lipofection (lipofectin reagent, BRL) in 9 cm petri dishes in accordance with 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 ~g/ml of cholesterol as well as -2 ~g/ml of 25-hydroxycholesterol. The cells were washed with PBS/2% BSA and then incubated for 1 hour at 34C
with about 10,000 cpm/well of [35S ] -HRV2 in PBS/2~ BSA.
After washing several times, the cells were lysed in PBS/2% SDS and the quantity of bound [3sS]-HRV2 was determined by counting in a liquid scintillation counter. The addition of foetal calf serum, cholesterol and 25-hydroxycholesterol brings about a suppression of the endogenous LDL-receptors (Davis et al., 1987, Nature 326, 760), so that in the subsequent binding test only the LDL-receptors expressed by transfection are detected. As shown in Fig. 14, the quantity of bound -' 21~70~9 HRV2, compared with untransfected control cells, is twice as great if the cells are transfected with the sense construct pSVL-LDLR+. Transfection with pSVL-LDLR- shows no difference in binding compared with the control cells.

Example 6: Inhibition of binding of [35S]-labelled rhinovirus serotype 2 (HRV2) by Jacalin Two aliquots of the protein purified by all but Jacalin agarose chromatography (see Example 3, corresponding to a starting quantity of cell supernatant of about 50 ml) were separated on a 7.5% SDS polyacrylamide gel (Laemmli, U.K. 1970, Nature 227, 680 - 685) under non-reducing conditions and transferred by electrophoresis on to an immobilon membrane (Millipore) (Mischak et al., 1988, loc. cit., Hofer et al., 1992, loc. cit.). One trace was incubated in the absence (Fig. 13, trace A) or in the presence (trace B) of 0.1 mg/ml of jacalin (Vector Labs) with radioactively labelled rhinovirus (Mischak et al., 1988, loc. cit.), washed, dried and exposed on X-ray film (Hofer et al.~ 1992, loc.cit.).
As shown in Figure 13 the binding of the virus to the LDL-receptor is totally inhibited under the conditions specified.

Example 7: ~eduction of the virus yield by RAP
(receptor associated protein) FH cells (cf. Example 1) were sown in 24-well dishes (Nunc) in RPMI with 10% foetal calf serum and cultivated overnight to a cell density of about sx104 cells per well. The cells were washed once with PBS and mixed with RPMI/2% foetal calf serum/30 mM MgC12. Human ~ -recombinant RAP was obtained as described in Kunnas et ~

~ ' :

21170~

al., loc. cit., then purified and added to the mediu~ in -concentrations of 0.5 ~g/ml, 5 ~g/ml, 10 ~g/ml and 20 ~g/ml, and the cells were incubated for 2 hours at 4C. HRV2 was added to each test batch in an m.o.i of lO0 and incubation was continued for a further 2 hours at 4C. Then the cells were washed 3x with PBS, mixed with RPMI/2% foetal calf serum/30 mM MgC12 and incubated overnight at 34C. The next day the cells were broken up by freezing and thawing three times. Cell fragments were removed by centrifuging at lO,OOOxg and the number of infectious virus particles in the supernatant was determined by the plaque test (Neubauer et al., loc.
cit.). Fig. 15 shows that the yield of HRV2 decreases as the concentration of RAP rises and at an RAP
concentration of 20 ~g/ml it is reduced to about 5% of the comparison value without RAP.

xample 8: Inhibition of ~RV2-infection of HeLa cells by human LDL

HeLa cells were sown in 24-well dishes (Nunc) in MEM
with 10% foetal calf serum and cultivated overnight to a cell density of about 2xlO5 cells per well. The cells were washed once with PBS and mixed with RPMI/2% foetal calf serum/30 mM MgCl2. Purified LDL (Huttinger 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 were incubated for 30 minutes at 34C. HRV2 or HRV14 (a virus of the large receptor group, used as a control) were added to each test batch in an m.o.i of 100 and incubation was continued for 45 minutes at 34C. Then the cells were washed three times with PBS, mixed with RPMI/2% foetal calf serum/30 mM M~Cl2 and incubated for 60 hours at 34C. The medium was suction filtered and intact cells were stained with crystal violet. Fig. 16 shows that in the presence Oe LDL at a concentration Oe 21170~9 1 mg/ml the infection of HeLa cells by HRV2 is prevented (all the cells are intact). In the case of HRV14, no effect was observed (cells fully lysed).

Example 9: Mutations of the HRV2-receptor binding site Different receptor binding sites of the rhinoviruses of the "small" and "large rhinovirus receptor group" should also be reflected in the "canyon structure" of the viral capsids which is responsible for the interaction with the corresponding receptor. The term "canyon" is used to denote the 5-numbered axis of symmetry of the viral capsid with an extent of roughly 30 ~. One hypothesis regarding the canyon structure claims that the amino acid side groups in this region are inaccessible to immunoglobulins and are therefore not exposed to any immunological pressure (Rossmann et al. (1985) Nature 317, 145-154). The different rhinoviral serotypes of a receptor group might in this way conserve structures which are important for the interaction with the corresponding receptor and at the same time develop a broad serotypical diversity (Rossmann (1989) Viral Immunology 2, 143-161). The hypothesis goes on to say that differences in the canyon structure between the "large" and the "small rhinovirus receptor group" are responsible for the use of two different receptors.
This would contain one set of amino acid groups which is conserved in specific pOsitiolls of the rhinoviruses of the "large rhinovirus receptor group" and a second set which is conserved in specific positions of the rhinoviruses of the "small rhinovirus receptor group".

Fig. 17 shows a sequence comparison for determining the positions in or on the edge of the canyon which are preserved in the rhinoviruses of the small group, by contrast with the rhinoviruses of the large group. They include the basic groups 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 VPl-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 cDNA required for mutagenesis, the in vitro production of corresponding infectious RNA and the transfection of corresponding cells have been described Duechler et al. (1989) Virology 168, 159-161; Maniatis et al. (1982) "Molecular Cloning: A laboratory manual".
I Cold Spring Harbour Laboratory, Cold Spring Harbour, 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 91, 143-147.
i~ .
The transfection with RNAs which corresponded to the j Wild-type HRV2 and the mutants HRV2l148pG and HRV23182RT, in HeLa cells gave yields of about 300 Pfu/ml. The plaque size and morphology of the Wild type and the two mutants were identical. Fig. 18 also shows that there was no significant difference in the binding characteristics of these two viruses to HeLa cells (HRV2wt ( ), HRV21148pG
( ) and HRV23182RT (_); Neubauer et al. (1987) Virology 158, 255-258). No viable mutants were obtained Erom the 1081K:E, 3229L:T or the mutant with the exchange TEK-motif at position 1222.

To demonstrate whether the mutants are capable of binding to the receptor of the small rhinovirus group, ::

21170~

competitive experiments were carried out (Fig. 19). As soon as increasing quantities of unlabelled HRV2 (-) were present in the incubation of HeLa cells, the binding of [35S]-labelled virus material of the mutants was reduced. The addition of HRV14 (O) had no effect on the binding. Obviously the affinity of the viruses for the receptor of the small rhinovirus receptor group is not affected by the mutations. These data show that the Pro 148 of VPl (equivalent to Pro 155 in HRV14) is not involved in the interaction of HRV2 with its receptor.
- Unlike 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. Sci. USA 85, 5449-5453).

No conserved oligopeptide sequence corresponding to the TEK element could be detected in rhinoviruses of the large group.

mutantS HRV2 1081~:~, HRV23229~r and the TEK mutant were not viable. Analysis of the three-dimensional structure of HRVlA - a serotype closely related to ~RV2 - provided no indications of sterical or electrostatic disorders caused by the exchanged amino acid side groups. All the ~-side groups are located on the surface and are accessible for the solvent. For this reason it is certainly possible that they are involved in the interaction with the receptor of the small group and that their change results in a loss of binding capacity and infectiousness.
.

The change from Pro114~ to Gly in HRV2 had no effect on the ability of the virus to bind to its receptor. In HRV14, a corresponding exchange leads to a substantially firmer bonding to the receptor. Prollss forms a kind of base at the foot of the canyon and appears to prevent the receptor from penetrating further into the viral ~ 2~170~

capsid. The increase in the binding affinity caused by replacing Pro with Gly can therefore be interpreted as a reduction in the sterical hindrance. Since no such effect was observed in HRV2 it is probable that the virus/receptor interaction occurs at a different place in rhinoviruses of the small group than that used by the rhinoviruses of the large group.

~ 21170~9 Cas~ IV140, 141, 149-PCT 40 SEQUENCE LISTING
(1) GENERAL INFORMATION:
S (i) APPLICANT:
(A) NAME: Boehringer Ingelheim (B) STREET: Binger Strasse (C) CITY: Ingelheim (D) STATE: Rheinland-Pfalz (E) COUNTRY: Deutschland (F) POSTAL CODE: W-6507 (G) TELEPHONE: 06132-77-0 (H) TEL8FAX: 06132-77-3000 - (I) TELEX: 418791-0 bi d : 15 (ii) TITLE OF INVENTION: RECEPTOR DERIVATIVES HAVING BINDING SITES
WITH RECEPTOR BINDING SITES
(iii) N~MBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/NS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPA) I (2) INFORMATION ZU SEQ ID NO: 1:

3 30 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 750 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: :
Met Gly Pro Trp Gly Trp Lyg Leu Arg Trp Thr Val Ala Leu Leu Leu Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cys Glu Axg Asn Glu Phe Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lya Trp Val Cy8 Asp Gly :
Ser Ala Glu Cys Gln A8p Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu ~ :
50 S5 60 ~:
Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp A8n Gly Ser ABP Glu Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys : Asp Ser Asp Arg Agp Cy8 Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro 2117~
CaseIV140,141,149-PCT 41 Val Leu Thr Cy8 Gly Pro Ala Ser Phe Gln Cy8 Asn Ser Ser Thr Cys Ile Pro Gln Leu Trp Ala Cy5 Asp Asn A6p Pro Asp Cys Glu Asp Gly Ser Asp Glu Trp Pro Gln Arg Cy5 Arg Gly Leu Tyr Val Phe Gln Gly laO 185 190 Asp Ser Ser Pro Cys Ser Ala Phe GlU Phe His Cys Leu Ser Gly Glu Cys Ile His Ser Ser Trp Arg Cys Agp Gly Gly Pro Asp Cys Lys Asp lS 210 215 220 Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp Arg Glu Tyr A~p Cys Lys Asp Met Ser Asp Glu Val Gly Cys Val Asn Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cy~ His Ser Gly Glu Cys Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp Trp Ser A~p Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cy8 Leu A6p Asn A~n Gly Gly Cys Ser Hig Val Cy~ Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Ile Asp Glu Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys Gln Cys Glu 51u Gly Phe Gln Leu Asp Pro His Thr Ly8 Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr Leu Phe Phe Thr Asn Arg Hi~ Glu Val Arg Lys Met Thr Leu Asp Arg Ser Glu Tyr Thr Ser Leu Ile Pro Asn Leu Arg Asn Val Val- Ala Leu Asp Thr Glu Val Ala Ser Asn Arg Ile Tyr Trp Ser Asp Leu Ser Gln Arg Met Ile Cys Ser Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser Tyr Asp Thr Val Ile Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala Val Asp Trp Ile His Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly 211709~
Casc IV140,141,149-PCT 42 Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe Arg Glu Agn Gly Ser Lys Pro Arg Ala Ile Val Val AEP Pro Val His Gly Phe Met Tyr Trp Thr Asp Trp Gly Thr Pro Ala Lys Ile Lys Lys Gly Gly Leu Asn Gly Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile Gln Trp Pro Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr Trp Val Asp Ser Lys Leu Hig Ser Ile Ser Ser Ile Asp Val Asn Gly Gly Asn Arg Lys Thr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro Phe Ser Leu Ala Val Phe Glu A~p Lys Val Phe Trp Thr A&p Ile Ile Asn Glu Ala Ile Phe Ser Ala Agn Arg Leu Thr Gly Ser Asp Val Asn Leu Leu Ala Glu A8n Leu Leu Ser Pro Glu Asp Met Val Leu Phe His Asn Leu Thr Gln Pro Arg Gly Val Agn Trp Cys Glu Arg Thr Thr Leu 1 660 665 670 :.
! 35 Ser Asn Gly Gly Cys Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn - ~-Pro Hi8 Ser Pro Ly8 Phe Thr Cya Ala Cys Pro Asp Gly Net Leu Leu 1 690 695 700 ; -~
¦ Ala Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala.Ala Val Ala ' 705 710 715 720 Thr Gln Glu Thr Ser Thr Val Arg Leu Lys Val Ser Ser Thr Ala Val Arg Thr Gln His Thr Thr Thr Arg Pro Val Pro Asp Thr Ser 740 7~5 750 (2) INFORMATION ZU SEQ ID NO: 2: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) NOLEC~LE 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 .- 2ll7n~.~
Caso IV140,141,149-PCI 43 Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cy8 Glu Arg Asn Glu Phe Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lys Trp Val Cy8 Aap Gly Ser Ala Glu Cys Gln Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu Ser Val Thr Cys Lys Ser Gly Agp Phe Ser Cys Gly Gly Arg Val Asn Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp Asn Gly Ser Asp G1U Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys Asp Ser Asp Arg Asp Cys Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro Val Leu Thr Cys Gly Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys Ile Pro Gln Leu Trp Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly Ser Asp Glu Trp Pro Gln Arg cyg Arg Gly Leu Tyr Val Phe Gln Gly Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cy5 Lys Asp Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp Arg Glu Tyr Asp Cys Lys Asp DIet Ser Asp Glu Val Gly Cys Val Asn Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cys ~lis Ser Gly Glu Cys Ile Thr Leu Asp Lys Val Cy8 Asn Met Ala Arg Asp Cys. Arg Asp Trp Ser Asp Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp Asn Asn 65 (2) INFORMATION ZU SEQ ID NO: 3:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 860 amino acids ~` ~117~9 Cl~se 12/140,141,149-K~ 44 (B) TYPE: amino ac~id (C) STR~NDEDNESS: single (D) TOPOLOGY: linear :
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTIO~: SEQ ID NO: 3:
Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lys Trp Val Cys Asp Gly 35 40 45 . :
20 Ser Ala Glu Cys Gln Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu Ser Val Thr Cy8 Lys Ser Gly Asp Phe Ser Cy8 Gly Gly Arg Val Asn Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp Asn Gly Ser Asp Glu Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys 35 Asp Ser ABP Arg Asp Cys Leu Asp Gly Ser Agp Glu Ala Ser Cys Pro Val Leu Thr Cys Gly Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys 145 150 155 1~0 Ile Pro Gln Leu Trp Ala Cyg A~p Asn Asp Pro A~3p Cys Glu Asp Gly 165 170 175 ~;
Ser Asp Glu Trp Pro Gln Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly : -Asp Ser Ser Pro Cy8 Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu 50 Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp Lys Ser ABP Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro- Asp Glu Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp 245 250 255 :
Arg Glu Tyr Asp Cys Lys Asp Met Ser A p Glu Val Gly cyg Val Asn Val Thr Leu Cyg Glu Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu 65 Cys Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg A6p cyg Arg Asp '~
-~: ::::::

- 21170.~9 Ca~e 12/140,141,149-PCT 45 Trp Ser Asp Glu Pro Ile Lys Glu Cy8 Gly Thr Asn Glu Cys Leu Asp 5 Asn Asn Gly Gly Cy8 Ser His Val Cys Asn Asp Leu Lys Ile Gly Tyr Glu Cy8 Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys , 10 :: :
Glu Asp Ile Asp Glu Cy8 Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys Val Asn Leu Glu Gly Gly Tyr Lyg Cys Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr 385 390 395 400 ::
' 20 Leu Phe Phe Thr Asn Arg His Glu Val Arg Lys Met mr Leu Asp Arg 25 Ser Glu Tyr Thr Ser Leu Ile Pro Asn Leu Arg Asn Val Val Ala Leu , 420 425 430 Asp Thr Glu Val Ala Ser A8n Arg Ile ~yr Trp Ser Asp Leu Ser Gln ::

~¦ Arg Met Ile Cys Ser Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser Tyr Asp Thr Val Ile Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala Val Asp Trp Ile Hig Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly ~¦ 40 Thr Val Ser Val Ala A8p Thr 1,y8 Gly Val l.ys Arg Lys Thr Leu Phe Arg Glu Asn Gly Ser Lys Pro brg Ala Ile Val Val Asp Pro Val His . 45 Gly Phe Met Tyr Trp Thr A6p Trp Gly Thr Pro Ala Ly8 Ile Ly6 Ly5 Gly Gly Leu Asn Gly Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile Gln Trp Pro Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr : 565 570 575 . 55 Trp Val A8p Ser Lys Leu His Ser Ile Ser Ser Ile A8p Val A8n Gly , 580 5a5 590 Gly Asn Arg Lys mr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro Phe Ser Leu Ala Val Phe Glu A6p Ly8 Val Phe Trp Thr Asp Ile Ile Asn Glu Ala Ile Phe Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Me~ Val Leu Phe His 645 650 655 ;.

:
. ' , , ,, ~ ~.. ,... , . . , ,~, , , j. .. . .. ..

~ 2117099 12/140,141,149-PCI' 46 Asn Leu Thr Gln Pro Arg Gly Val Asn Trp Cys Glu Arg Thr Thr Leu 660 ~ 665 670 - :
Ser Asn Gly Gly Cys Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn Pro His Ser Pro Lys Phe Thr CYB Ala Cy6 Pro A~p Gly Met Leu Leu Ala AIg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala Thr Gln Glu Thr Ser Thr Val Arg Leu Ly~ Val Ser Ser Thr Ala Val Arg Thr Gln His Thr Thr Thr Arg Pro Val Pro Asp Thr Ser Arg Leu Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu Ile Val Thr Met Ser His Gln Ala Leu Gly Asp Val Ala Gly Arg Gly Asn Glu Lys Lys Pro 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 Lys Asn Trp Arg Leu Lys Asn Ile A~n Ser Ile Asn Phe Asp A6n Pro Val Tyr Gln Lys Thr Thr Glu Asp Glu Val His Ile Cy6 His A6n Gln Asp Gly Tyr Ser Tyr Pro Ser Arg Gln Met Val Ser Leu Glu Asp Asp Val Ala (2) INFORMATION ZU SEQ ID NO: 4~
(i) SEQUENCE CHARACTERISTICS: - :
(A) LENGTH: 4492 amino acid6 (B) TYPE: amino acid (C) STRANDEDNESS: ~ingle (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 Val Ala Ala Ala Ile Asp Ala Pro Lys Thr Cys Ser Pro Lys Gln Phe 20 25 30 .: ~ :
Ala Cys Arg Asp Gln Ile Thr Cyg Ile Ser Lys Gly Trp Arg Cy6 Asp r~
2117~
C~e IV140.141,149-PCr 47 .
Gly Glu Arg Asp CYE; Pro Asp Gly Ser Asp Glu Ala Pro Glu Ile Cys Pro Gln Ser Lys Ala Gln Arg Cys Gln Pro Asn Glu His A~n Cy5 Leu Gly Thr Glu Leu Cys Val Pro Met Ser Arg Leu Cys Asn Gly Val Gln Asp Cys Met Asp Gly Ser Asp Glu Gly Pro His Cy8 Arg Glu Leu Gln Gly Asn Cys Ser Arg Leu Gly Cy8 Gln His His Cys Val Pro Thr Leu Asp Gly Pro Thr Cys Tyr Cy8 Asn Ser Ser Phe Gln Leu Gln Ala Asp Gly Lys Thr Cy8 Lys Asp Phe Asp Glu Cys Ser Val Tyr Gly Thr Cys Ser Gln Leu Cys Thr Asn Thr Asp Gly Ser Phe Ile Cys Gly Cys Val Glu Gly Tyr Leu Leu Gln Pro Asp Aan Arg Ser Cys Lys Ala Lys Asn Glu Pro Val Asp Arg Pro Pro Val Leu Leu Ile Ala Asn Ser Gln Asn Ile Leu Met Pro Gly Leu Lys Gly Phe Val A~p Glu His Thr Ile Asn Ile Ser I:-eu Ser Leu His His Val Glu Gln ~et Ala Ile Asp Trp Leu Thr Gly Asn Phe Tyr Phe Val A8p A8p Ile Asp A8p Arg Ile Phe Val Cys Asn Arg A~n Gly Asp Thr CYB Val Thr Leu Leu Asp Leu Glu Leu Tyr Asn Pro Lys Gly Ile Ala Leu Asp Pro Ala Met Gly Lys Val Phe Phe Thr Asp Tyr Gly Gln Ile Pro Lys Val Glu Arg Cys Asp Met Asp Gly Gln A8n Arg Thr Lys Leu Val Asp Ser Lys Ile Val Phe Pro His Gly Ile Thr Leu Asp Leu Val Ser Arg Leu Val Tyr Trp Ala Asp Ala Tyr Leu Asp Tyr Ile Glu Val Val A~p Tyr Glu Gly Lys Gly Arg Gln Thr Ile Ile Gln Gly Ile Leu Ile Glu His Leu Tyr Gly Leu Thr Val Phe Glu A8n Tyr Leu Tyr Ala Thr Asn Ser A8p Asn Ala Asn Ala Gln Cln Lys Thr Ser Val Ile Arg Val Asn Arg Phe Asn Ser Thr Glu Tyr 2117(1~
Ca~e Ivl40,141,149-PCr 48 Gln Val Val Thr Arg Val Asp Lys Gly Gly Ala Leu Hia Ile Tyr His Gln Arg Arg Gln Pro Arg Val Arg Ser Hia Ala Cys Glu Asn Asp Gln Tyr Gly Ly8 Pro Gly Gly Cys Ser Asp Ile Cys Leu Leu Ala Asn Ser His Lys Ala Arg Thr Cys Arg Cys Arg Ser Gly Phe Ser Leu Gly Ser Asp Gly Lys Ser Cys Lys LYB Pro Glu His Glu Leu Ph~ Leu Val Tyr 465 470 475 ~80 Gly Lys Gly Arg Pro Gly Ile Ile Arg Gly Met Asp Met Gly Ala Lys Val Pro Agp Glu His Met Ile Pro Ile Glu Asn Leu Net Asn Pro Arg Ala Leu Asp Phe His Ala Glu Thr Gly Phe Ile Tyr Phe Ala Asp Thr Thr Ser Tyr Leu Ile Gly Arg Gln Lys Ile Asp Gly Thr Glu Arg Glu Thr Ile Leu Lys Asp Gly Ile His Agn Val Glu Gly Val Ala Val Asp :: ~
545 550 555 560 ~ ::

Trp Met Gly Asp Asn Leu Tyr Trp Thr Asp Asp Gly Pro Lys Lys Thr : -Ile Ser Val Ala Arg Leu Glu Lys Ala Ala Gln Thr Arg Lys Thr Leu :~
580 5a5 590 .. ::
Ile Glu Gly Lys Met Thr His 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 Lys Asp Ser 610 615 620 ~ :
Arg Arg Gly Arg Leu Glu Arg Ala Trp Met Asp Gly Ser His Arg Asp p Ile Phe Val Thr Ser Lys Thr Val Leu Trp Pro Asn Gly Leu Ser Leu ~ -~

ABP Ile Pro Ala Gly Arg Leu Tyr Trp Val Asp Ala Phe Tyr Asp Arg .

Ile Glu Thr Ile Leu Leu Asn Gly Thr Asp Arg Lys Ile Val Tyr Glu 675 680 685 :.
Gly Pro Glu Leu Asn Hi8 Ala Phe Gly Leu Cys His His Gly Asn Tyr Leu Phe Trp Thr Glu Tyr Arg Ser Gly Ser Val Tyr Arg Leu Glu Arg .:

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 Asp Ala Gln Gln Gln Gln Val :~
: :

ca~e IVI40 141 149-PCl-Gly Thr Asn Lys Cy5 Arg Val Asn Asn Gly Gly Cy9 Ser Ser Leu Cy5 Leu Ala Thr Pro Gly Ser Arg Gln Cy8 Ala Cys Ala Glu Asp Gln Val Leu Asp Ala Asp Gly Val Thr Cys Leu Ala Asn Pro Ser Tyr Val Pro Pro Pro Gln Cys Gln Pro Gly Glu Phe Ala cy5 Ala Asn Ser Arg Cy8 Ile Gln Glu Arg Trp Lys Cys A~p Gly Asp Asn Asp Cys Leu Asp Asn Ser A~p Glu Ala Pro Ala Leu Cys His Gln His Thr Cys Pro Ser Asp Arg Phe Lys Cy8 Glu Asn Asn Arg Cys Ile Pro Asn Arg Trp Leu Cys 850 855 860 :' Asp Gly Asp Asn Asp Cys Gly AF:n Ser Glu Asp Glu Ser Asn Ala Thr ~.
Cys Ser Ala Arg Thr Cys Pro Pro Asn Gln Phe Ser Cys Ala Ser Gly 885 890 895 .
Arg Cys Ile Pro Ile Ser Trp Thr Cys Asp Leu Asp Asp Asp Cys Gly Asp Arg Ser Asp Glu Ser Ala Ser Cys Ala Tyr Pro Thr Cys Phe Pro ! 35 Leu Thr Gln Phe Thr Cys Asn Asn Gly Arg Cys Ile Asn Ile Asn Trp Arg Cys Asp Asn Asp Asn Asp Cys Gly Asp Asn Ser Asp Glu Ala Gly Cys Ser His Ser Cy9 Ser Ser Thr Gln Phe Lys Cys Asn Ser Gly Arg Cys Ile Pro Glu His Trp Thr Cys Asp Gly ~sp Asn Asp Cys Gly Asp Tyr Ser Asp Glu Thr His Ala Asn Cys Thr Asn Gln Ala Thr Arg Pro Pro Gly Gly Cys His Thr Asp Glu Phe Gln Cys Arg Leu Asp Gly Leu Cys Ile Pro Leu Arg Trp Arg Cys Asp Gly Asp Thr Agp Cys Net Asp Ser Ser Asp Glu Lys Ser Cy9 Glu Gly Val Thr His Val Cys Asp Pro 1045 1050 lOS5 Ser Val Lys Phe Gly Cys Lys Asp Ser Ala Arg Cys Ile Ser Lys Ala Trp Val Cys Asp Gly Asp Asn Asp Cys Glu Asp Asn Ser Asp Glu Glu . 1075 1080 1085 Asn Cys Glu Ser Leu Ala Cys Arg Pro Pro Ser His Pro Cys Ala Asn f' 21~709~
Caso IV140,141,149-PC~ 50 Asn Thr Ser Val Cy8 Leu Pro Pro AE:p Lys Leu Cys A3p Gly Asn Asp Asp Cys Gly Asp Gly Ser Asp Glu Gly Glu Leu Cys Asp Gln Cy8 Ser Leu Asn Agn Gly Gly Cy~ Ser His Asn Cys Ser Val Ala Pro Gly Glu Gly Ile Val Cys Ser Cys Pro Leu Gly Met Glu Leu Gly Pro Asp Asn His Thr Cys Gln Ile Gln Ser Tyr Cys Ala Lys His Leu Lys Cy8 Ser Gln LYB Cys Asp Gln Asn Lys Phe Ser Val Lys Cys Ser Cy8 Tyr Glu : ~:
1185 1190 1195 1200 :
Gly Trp Val Leu Glu Pro Asp Gly Glu Ser Cys Arg Ser Leu Asp Pro 1205 1210 1215 -.;
Phe Lys Pro Phe Ile Ile Phe Ser Asn Arg His Glu Ile Arg Arg Ile Asp Leu His Lys Gly Asp Tyr Ser Val Leu Val Pro Gly Leu Arg Asn Thr Ile Ala Leu Asp Phe His Leu Ser Gln Ser Ala Leu Tyr Trp Thr Asp Val Val Glu Asp Lys Ile Tyr Arg Gly Lys Leu Leu Asp Asn Gly 1265 1270 1275 1280 : ~:
Ala Leu Thr Ser Phe Glu Val Val Ile Gln Tyr Gly Leu Ala Thr Pro ;:
Glu Gly Leu Ala Val Asp Trp Ile Ala Gly Asn Ile Tyr Trp Val Glu 1300 1305 1310 ~ :.
Ser Asn Leu Asp Gln Ile Glu Val Ala Lys Leu Asp Gly Thr Leu Arg Thr Thr Leu Leu Ala Gly Asp Ile Glu His Pro Arg Ala Ile Ala Leu 1330 1335 1340 :
Aap Pro Arg A8p Gly Ile Leu Phe Trp Thr A~p Trp Aap Ala Ser Leu ., 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 Aan Gly Leu Thr Val Asp Tyr Leu Glu Lys Arg Ile Leu Trp Ile Asp Ala Arg Ser Asp Ala Ile Tyr Ser Ala Arg Tyr Asp Gly Ser Gly His Met Glu Val Leu Arg Gly His Glu Phe Leu Ser Hia Pro Phe Ala Val Thr Leu Tyr Gly Gly Glu 1425 1430 1435 1~40 Val Tyr Trp Thr Asp Trp P,rg Thr Agn Thr Leu Ala Lys Ala Agn Lys ~ 211709~
C~o 121140,141.149-PCr 5 l Trp Thr Gly His A~n Val Thr Val Val Gln Arg Thr Asn Thr Gln Pro lg60 1465 1470 Phe Asp Leu Gln Val Tyr His Pro Ser Arg Gln Pro Met Ala 2ro Asn Pro Cys Glu Ala Asn Gly Gly Gln Gly Pro Cy~ Ser His Leu Cy8 Leu Ile Asn Tyr Asn Arg Thr Val Ser Cys Ala Cy5 Pro His Leu Met Lys Leu Hi~ Lys Asp Asn Thr Thr Cys Tyr Glu Phe Lys Ly8 Phe Leu Leu Tyr Ala Arg Gln Met Glu Ile Arg Gly Val Asp Leu Asp Ala Pro Tyr Tyr Asn Tyr Ile Ile Ser Phe Thr Val Pro Asp Ile A~p Asn Val Thr 1555 1560 1565 .
Val Leu Agp Tyr Asp Ala Arg Glu Gln Arg Val Tyr Trp Ser Asp Val Arg Thr Gln Ala Ile Lys Arg Ala Phe Ile Asn Gly Thr Gly Val Glu Thr Val Val Ser Ala Asp Leu Pro Asn Ala His Gly Leu Ala Val Asp Trp Val Ser Arg Asn Leu Phe Trp Thr Ser Tyr Asp Thr Asn Lys Lys Gln Ile Aan Val Ala Arg Leu Asp Gly Ser Phe Lys Asn Ala Val Val Gln Gly Leu Glu Gln Pro His Gly Leu Val Val His Pro Leu Arg Gly Lys Leu Tyr Trp Thr Asp Gly Asp Aan Ile Ser Met Ala Asn Met Asp Gly Ser Asn Arg Thr Leu Leu Phe Ser Gly Gln Lys Gly Pro Val Gly Leu Ala Ile Asp Phe Pro Glu Ser Lys Leu Tyr Trp Ile Ser Ser Gly Asn Hi8 Thr Ile Asn Arg Cys Asn Leu Asp Gly Ser Gly Leu Glu Val Ile Asp Ala Met Arg Ser Gln Leu Gly Lys Ala Thr Ala Leu Ala Ile Met Gly Asp Lys Leu Trp Trp Ala Asp Gln Val Ser Glu Lys Met Gly Thr Cys Ser Lys Ala Asp Gly Ser Gly Ser Val Val Leu Arg Asn Ser Thr Thr Leu Val Met His Met Lys Val Tyr Asp Glu Ser ~le Gln Leu Asp His Lys Gly Thr Agn Pro Cyg Ser Val Asn Asn Gly Asp Cys Ser ,~ 211709~
C~se IV140,141,149-PCI` 52 Gln Leu Cy8 Leu Pro Thr Ser Glu Thr Thr Arg Ser Cyæ Met Cys Thr ~:
1810 1815 1820 :
Ala Gly Tyr Ser Leu Arg Ser Gly Gln Gln Ala Cya Glu Gly Val Gly 1825 1830 la35 1840 Ser Phe Leu Leu Tyr Ser Val ~i8 Glu Gly Ile Arg Gly Ile Pro Leu ~:
1845 1850 1855 :
Asp Pro Asn Asp Lys Ser Aap Ala Leu Val Pro Val Ser Gly Thr Ser .

Leu Ala Val Gly Ile Asp Phe His Ala Glu Asn ABP Thr Ile Tyr Trp 1875 1880 1885 . :
Val Asp Met Gly Leu Ser Thr Ile Ser Arg Ala Lys Arg Asp Gln Thr .:
1890 la95 1900 : .
Trp Arg Glu Asp Val Val Thr A8n Gly Ile Gly Arg Val Glu Gly Ile Ala Val Asp Trp Ile Ala Gly A6n Ile Tyr Trp Thr Asp Gln Gly Phe ~:
1925 1930 1935 ~ :
::
Asp Val Ile Val Ala Arg Leu A8n Gly Ser Phe Arg Tyr Val Val Ile : :
1940 1945 1950 ~.
Ser Gln Gly Leu Asp Lys Pro Arg Ala Ile Thr Val His Pro Glu Lys 1955 1960 1965 .
Gly Tyr Leu Phe Trp Thr Glu Trp Gly Gln Tyr Pro Arg Ile Glu Arg ~ :

Ser Arg Leu Asp Gly Thr Glu Arg Val Val Leu Val A~n Val Ser Ile 1985 1990 1995 2000 - :
Ser Trp Pro Asn Gly Ile Ser Val Asp Tyr Gln ABP Gly Lys Leu Tyr Trp Cys Asp Ala Arg Thr Asp Lys Ile Glu Arg Ile Asp Leu Glu Thr Gly Glu Asn Arg Glu Val Val Leu Ser Ser Asn Asn Met Asp Met Phe 2035 2040 2045 ~:
Ser Val Ser Val Phe Glu Asp Phe Ile Tyr Trp Ser Asp Arg Thr His Ala A8n Gly Ser Ile Ly8 Arg Gly Ser Lys ABP Asn Ala Thr A8p Ser 2065 2070 2075 2080 . :
Val Pro Leu Arg Thr Gly Ile Gly Val Gln Leu Lys Asp Ile Lys Val Phe Asn Arg Asp Arg Gln Lys Gly Thr Agn Val Cys Ala Val Ala Asn Gly Gly Cys Gln Gln Leu Cys Leu Tyr Arg Gly Arg Gly Gln Arg Ala CYB Ala Cys Ala His Gly Net Leu Ala Glu Asp Gly Ala Ser Cys Arg Glu Tyr Ala Gly Tyr Leu Leu Tyr Ser Glu Arg Thr Ile Leu Lys Ser ~ 21170~ -C~e 12/140,141,149-P~r 53 Ile His Leu Ser Asp Glu Arg Asn Leu Asn Ala Pro Val Gln Pro Phe Glu Asp Pro Glu His Met Lys Asn Val Ile Ala Leu Ala Phe Asp Tyr Arg Ala Gly Thr Ser Pro Gly Thr Pro Asn Arg Ile Phe Phe Ser Asp Ile His Phe Gly Asn Ile Gln Gln Ile Agn Asp Asp Gly Ser Arg Arg Ile Thr Ile Val Glu Aan Val Gly Ser Val Glu Gly Leu Ala Tyr His ', Arg Gly Trp Asp Thr Leu Tyr Trp Thr Ser Tyr Thr Thr Ser Thr Ile Thr Arg Hia Thr Val Asp Gln Thr Arg Pro Gly Ala Phe Glu Arg Glu I

Thr Val Ile Thr Met Ser Gly Asp Asp His Pro Arg Ala Phe Val Leu Asp Glu Cys Gln Asn Leu Met Phe Trp Thr Asn Trp Asn Glu Gln Hi s Pro Ser Ile Met Arg Ala Ala Leu Ser Gly Ala Aan Val Leu Thr Leu Ile Glu Lys Asp Ile Arg Thr Pro Asn Gly Leu Ala Ile Asp His Arg Ala Glu Lys Leu Tyr Phe Ser Asp Ala Thr Leu Asp Lys Ile Glu Arg Cys Glu Tyr Asp Gly Ser His Arg Tyr Val Ile Leu Lys Ser Glu Pro Val His Pro Phe Gly Leu Ala Val Tyr Gly Glu His Ile Phe Trp Thr Asp Trp Val Arg Arg Ala Val Gln Arg Ala Asn Lys His Val Gly Ser Asn Met Ly~ Leu Leu Arg Val Asp Ile Pro Gln Gln Pro Met Gly Ile Ile Ala Val Ala Asn Asp Thr Asn Ser Cys Glu Leu Ser Pro Cys Arg Ile Asn Asn Gly Gly Cy8 Gln Asp Leu Cys Leu Leu Thr His Gln Gly His Val Asn Cys Ser Cys Arg Gly Gly Arg Ile Leu Gln Asp Asp Leu Thr Cys Arg Ala Val Asn Ser Ser Cys Arg Ala Gln Asp Glu Phe Glu Cys Ala Asn Gly Glu Cy8 Ile Asn Phe Ser Leu Thr Cys Asp Gly Val Pro His Cya Lys Asp Lys Ser Agp Glu Lys Pro Ser Tyr Cyg Agn Ser ~ 211709~
Case IV140,141.149-PCT 54 Arg Arg Cy8 Ly8 Lys Thr Phe Arg Gln Cy5 Ser Asn Gly Arg Cys Val Ser Asn Met Leu Trp Cys Asn Gly Ala Asp Asp Cys Gly Asp Gly Ser Asp Glu Ile Pro Cyg A~n Ly8 Thr Ala Cys Gly Val Gly Glu Phe Arg Cys Arg Asp Gly Thr Cy8 Ile Gly Asn Ser Ser Arg Cys Asn Gln Phe ~ ~
2565 2S70 2575 ~: :
Val Asp Cys Glu Agp Ala Ser Asp Glu Met Asn Cyg Ser Ala Thr Asp ::
2580 2585 2590 :
Cys Ser Ser Tyr Phe Arg Leu Gly Val Lys Gly Val Leu Phe Gln Pro Cys Glu Arg Thr Ser Leu Cys l'yr Ala Pro Ser Trp Val Cys Asp Gly 2610 2615 2620 ~ :
Ala Asn Asp Cys Gly Asp Tyr Ser Asp Glu Arg Asp Cys Pro Gly Val Lys Arg Pro Arg Cys Pro Leu Asn Tyr Phe Ala Cys Pro Ser Gly Arg Cys Ile Pro Net Ser Trp ~r Cys Asp Lys Glu Asp Asp Cys Glu His 2660 2665 2670 ~ -Gly Glu ABP Glu Thr Hi5 Cya Asn Lys Phe Cys Ser Glu Ala Gln Phe ~ -Glu Cys Gln Asn His Arg Cys Ile Ser Lys Gln Trp Leu Cys Asp Gly 2~90 2695 2700 Ser Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Ala His Cys Glu Gly Lys Thr Cy8 Gly Pro Ser Ser Phe Ser Cys Pro Gly Thr His Val Cys Val Pro Glu Arg Trp Leu Cys Asp Gly Asp Lys Asp Cys Ala Aæp Gly Ala Asp Glu Ser Ile Ala Ala Gly Cys Leu Tyr Asn Ser Thr Cys Asp Asp Arg Glu Phe Met Cys Gln Asn Arg Gln Cys Ile Pro Lys His Phe Val Cys Asp Hi8 Asp Arg Agp Cys Ala Asp Gly Ser Asp Glu Ser Pro Glu Cys Glu Tyr Pro Thr Cys Gly Pro Ser Glu Phe Arg Cys Ala Asn Gly Arg Cys Leu Ser Ser Arg Gln Trp Glu Cys ABP Gly Glu Agn Asp CYB Hi8 Asp Gln Ser Asp Glu Ala Pro Lys Asn Pro His Cys Thr Ser 2835 2840 2845 ~ .
6~ :
Pro Glu HiS Lys Cys Asn Ala Ser Ser Gln Phe Leu Cys Ser Ser Gly ~ ,"-'"',""',:"~

- 21171~9 Casc IV140.141,14!~-PCI 55 Arg Cy8 Val Ala Glu Ala Leu Leu Cys Asn Gly Gln Asp Asp Cys Gly Asp Ser Ser Asp Glu Arg Gly Cys Hig Ile A8n Glu Cys Leu Ser Arg Lys Leu Ser Gly Cys Ser Gln Agp Cys Glu A~p Leu Lys Ile Gly Phe Lys Cys Arg Cyg Arg Pro Gly Phe Arg Leu Lys Asp Asp Gly Arg Thr Cys Ala Asp Val Asp Glu Cys Ser Thr Thr Phe Pro Cys Ser Gln Arg Cy8 Ile Asn Thr His Gly Ser Tyr Lys Cy8 Leu Cys Val Glu Gly Tyr Ala Pro Arg Gly Gly Asp Pro His Ser Cys Lys Ala Val Thr Asp Glu Glu Pro Phe Leu Ile Phe Ala Asn Arg Tyr Tyr Leu Arg Lys Leu Asn Leu Asp Gly Ser Asn Tyr Leu Leu Lys Gln Gly Leu Asn Asn Ala Val Ala Leu Asp Phe A~p Tyr Arg Glu Gln Met Ile Tyr Trp Thr Asp Val Thr Thr Gln Gly Ser Met Ile Arg Arg Met His Leu Asn Gly Ser Asn Val Gln Val Leu His Arg Thr Gly Leu Ser Asn Pro Asp Gly Leu Ala Val Asp Trp Val Gly Gly Asn Leu Tyr Trp Cys Asp Lys Gly Arg Asp Thr Ile Glu Val Ser Lys Leu A8n Gly Ala Tyr Arg Thr Val Leu Val Ser Ser Gly Leu Arg Glu Pro Arg Ala Leu Val Val Asp Val Gln Asn Gly Tyr Leu Tyr Trp Thr Asp Trp Gly Asp Hi8 Ser Leu Ile Gly Arg Ile Gly Met Asp Gly Ser Ser Arg Ser Val Ile Val Asp Thr Lys Ile 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 Asp Tyr Ile Glu Phe Ala Ser Leu Asp Gly Ser Asn Arg His Val Val Leu Ser Gln A8p Ile Pro His Ile Phe Ala Leu Thr Leu Phe Glu Asp Tyr Val Tyr Trp Thr Asp Trp Glu Thr Lys Ser Ile Aan Arg Ala His Lys Thr Thr Gly Thr Asn Lys Thr Leu Leu 2~ ~ ~ ~ 2 ~

~ 2~17099 C.~s~ 12/140,141.149-PCI' 56 Ile Ser Thr Leu His Arg Pro Met ABP Leu His Val Phe His Ala Leu Arg Gln Pro Asp Val Pro Asn His Pro Cys Ly~; Val Asn Asn Gly Gly Cys Ser Asn Leu Cys I.eu Leu Ser Pro Gly Gly Gly His Lys Cys Ala Cys Pro Thr Asn Phe Tyr Leu Gly Ser Asp Gly Arg Thr Cys Val Ser Asn Cys Thr Ala Ser Gln Phe Val Cys Lys Asn Asp Lys Cys Ile Pro 32a5 3290 3295 Phe Trp Trp Lys Cys Asp Thr Glu Asp Asp Cys Gly Aap Hi s Ser Asp Glu Pro Pro A8p Cys Pro Glu Phe Lys Cys Arg Pro Gly Gln Phe Gln Cys Ser Thr Gly Ile Cys Thr Asn Pro Ala Phe Ile Cys Asp Gly Asp Asn Asp CYB Gln Asp Asn Ser Asp Glu Ala Asn Cys Asp Ile His Val Cys Leu Pro Ser Gln Phe Lys Cys Thr Asn Thr Asn Arg Cys Ile Pro Gly Ile Phe Arg Cys Asn Gly Gln Asp A~n Cy8 Gly Asp Gly Glu Asp Glu Arg A8p Cys Pro Glu Val Thr Cys Ala Pro Asn Gln Phe Gln Cys Ser Ile Thr Lys Arg Cys Ile Pro Arg Val Trp Val Cys Asp Arg Asp Asn Asp Cys Val Asp Gly Ser Asp Glu Pro Ala Asn Cy6 Thr Gln Met 3425 3430 3435 3440 : :
Thr Cy~ Gly Val Asp Glu Phe Arg Cys Lys Asp Ser Gly Arg Cys Ile Pro Ala Arg Trp Lys Cys Asp Gly Glu Agp Asp Cys Gly Asp Gly Ser A8p Glu Pro Ly8 Glu G1U Cy~ Agp Glu Arg Thr Cy8 Glu Pro Tyr Gln Phe Arg Cy~ Lys Asn Asn Arg Cy~ Val Pro Gly Arg Trp Gln.Cys Asp Tyr Asp Asn Asp Cys Gly Asp Asn Ser Asp Glu Glu Ser Cys Thr Pro 3505 3510 3515 3520 ; :
Arg Pro Cys Ser Glu Ser Glu Phe Ser Cys Ala Asn Gly Arg Cys Ile Ala Gly Arg Trp Lys Cys Asp Gly Agp Hi8 Agp Cys Ala Agp Gly Ser 3540 3545 3550 - :
~5 ~:
Asp Glu Lys Asp Cys Thr Pro Arg Cys Asp Met Asp Gln Phe Gln Cys , ,, :

- 2~170~.~
Case 12/140,141,149-PC1 57 Lys Ser Gly His Cy8 Ile Pro Leu Arg Trp Arg Cy8 Asp Ala Asp Ala Asp Cys Net Asp Gly Ser Asp Glu Glu Ala Cys Gly Thr Gly Val Arg Thr Cys Pro Leu Asp Glu Phe Gln Cys Asn Asn Thr Leu Cys Lys Pro O Leu Ala Trp Lys Cys Asp Gly Glu Asp Asp Cy8 Gly Asp Asn Ser Asp Glu Asn Pro Glu Glu Cy8 Ala Arg Phe Val Cys Pro Pr~ Asn Arg Pro Phe Arg Cys Lys Asn Asp Arg Val Cys Leu Trp Ile Gly Arg Gln Cys Asp Gly Thr Asp Asn Cys Gly Asp Gly Thr Asp Glu Glu Asp Cys Glu Pro Pro Thr Ala His Thr Thr His Cy6 Lys Asp Lys Lys Glu Phe Leu Cys Arg Asn Gln Arg Cys Leu Ser Ser Ser Leu Arg Cys Asn Met Phe Asp Asp Cys Gly Asp Gly Ser Asp Glu Glu Asp Cys Ser Ile Asp Pro Lys Leu Thr Ser Cys Ala Thr Asn Ala Ser Ile Cys Gly Asp Glu Ala Arg Cys Val Arg Thr Glu Lys Ala Ala Tyr Cys Ala Cys Arg Ser Gly : :
3745 3750 3755 3760 :
Phe His Thr Val Pro Gly Gln Pro Gly Cys Gln Asp Ile Asn Glu Cys Leu Arg Phe Gly Thr Cys Ser Gln Leu Cys Asn Asn Thr Lys Gly Gly His Leu Cys Ser Cys Ala Arg Asn Phe Met Lys Thr His Asn Thr Cys 3795 3aoo 3805 Lys Ala Glu Gly Ser Glu Tyr Gln Val Leu Tyr Ile Ala Asp Asp Asn Glu Ile Arg Ser Leu Phe Pro Gly His Pro His Ser Ala Tyr Glu Gln Ala Phe Gl~ Gly Asp Glu Ser Val Arg Ile Asp Ala Met Asp.Val His 3845 3850 385~5 Val Lys Ala Gly Arg Val Tyr Trp Thr Asn Trp His Thr Gly Thr Ile : ~
Ser Tyr Arg Ser Leu Pro Pro Ala Ala Pro Pro Thr Thr Ser Asn Arg , ~:

His Arg Arg Gln Ile Asp Arg Gly Val Thr His Leu Asn Ile Ser Gly -Leu Lys Met Pro Arg Gly Ile Ala Ile Asp Trp Val Ala Gly Asn Val ~-~ 2~1709~
Case 121140.141,149-PCl' 58 Tyr Trp Thr Asp Ser Gly Arg Asp Val Ile Glu Val Ala Gln Met Lys Gly Glu A6n Arg Lys Thr Leu Ile Ser Gly 2iet Ile Asp Glu Pro His Ala Ile Val Val Asp Pro Leu Arg Gly Thr Met Tyr Trp Ser A~p Trp Gly Asn His Pro Lys Ile Glu l~hr Ala Ala Met Asp Gly Thr Leu Arg Glu Thr Leu Val Gln A~p Asn Ile Gln Trp Pro Thr Gly Leu Ala Val 39a5 3990 3995 4000 Asp Tyr His Asn Glu Arg Leu Tyr Trp Ala Asp Ala Lys Leu Ser Val Ile Gly Ser Ile Arg Leu Asn Gly Thr Asp Pro Ile Val Ala Ala Asp Ser Lys Arg Gly Leu Ser His Pro Phe Ser Ile Asp Val Phe Glu Asp Tyr Ile Tyr Gly Val Thr Tyr Ile Asn Asn Arg Val Phe Lys Ile His Lys Phe Gly Hi8 Ser Pro Leu Val Asn Leu Thr Gly Gly Leu Ser His Ala Ser A8p Val Val Leu Tyr His Gln His Lys Gln Pro Glu Val Thr 4085 4090 409~
Asn Pro Cy~ Asp Arg Lys Lys Cys Glu Trp Leu Cys Leu Leu Ser Pro Ser Gly Pro Val Cys Thr Cys Pro Asn Gly Lys Arg Leu Asp Asn Gly 4115 4120 4125 :
Thr Cys Val Pro Val Pro Ser Pro Thr Pro Pro Pro Asp Ala Pro Arg Pro Gly Thr Cys Asn Leu Gln Cys Phe Asn Gly Gly Ser Cys Phe Leu Asn Ala Arg Arg Gln Pro Lys Cys Arg Cys Gln Pro Arg Tyr Thr Gly 4165 4170 4175 . ~
Asp Ly~ Cys Glu Leu Asp Gln Cys Trp Glu His Cys Arg Asn Gly Gly ~3 4180 41a5 4190 ~:
Thr Cy5 Ala Ala Ser Pro Ser Gly Met Pro Thr Cys Arg Cys . Pro Thr Gly Phe Thr Gly Pro Lys Cys Thr Gln Gln Val Cys Ala Gly Tyr Cys Ala Asn Asn Ser Thr Cys Thr Val Asn Gln Gly Asn Gln Pro Gln Cys Arg Cys Leu Pro Gly Phe Leu Gly Asp Arg Cy8 Gln Tyr Arg Gln Cys ~-Ser Gly Tyr Cys Glu Asn Phe Gly Thr Cys Gln Met Ala Ala Asp Gly r~ 2117(3~

Cas; ~V140.141.149.PCI 59 : Ser Arg Gln Cy8 Arg Cys Thr Ala Tyr Phe Glu Gly Ser Arg Cys Glu Val Asn Lys Cys Ser Arg Cy8 Leu Glu Gly Ala Cys Val Val Aan Lya Gln Ser Gly Asp Val Thr Cys Asn Cy9 Thr Asp Gly Arg Val Ala Pro Ser Cys Leu Thr Cys Val Gly His Cys Ser Asn Gly Gly Ser Cys Thr ' Met Asn Ser Lys Met Met Pro Glu Cy8 Gln Cys Pro Pro His Net Thr ; 4340 4345 4350 IS
Gly Pro Arg Cys Glu Glu His Val Phe Ser Gln Gln Gln Pro Gly His Ile Ala Ser Ile Leu Ile Pro Leu Leu Leu Leu Leu Leu Leu Val Leu 4370 4375 43ao Val Ala Gly Val Val Phe Trp Tyr Lys Arg Arg Val Gln Gly Ala Lys Gly Phe Gln His Gln Arg Met Thr Asn Gly Ala Met Asn Val Glu Ile Gly Asn Pro Thr Tyr Lys Met Tyr Glu Gly Gly Glu Pro Asp Asp Val Gly Gly Leu Leu Asp Ala Asp Phe Ala Leu Asp Pro Asp Lys Pro Thr Asn Phe Thr Asn Pro Val Tyr Ala Thr Leu Tyr Met Gly Gly His Gly Ser Arg His Ser Leu Ala Ser Thr Asp Glu Lys Arg Glu Leu Leu Gly Arg Gly Pro Glu Asp Glu Ile Gly Asp Pro Leu Ala ' , ' (2) INFORMATION ZU SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 319 amino acids (P) TYPE: amino acid (C) STRANDEDNESS: 8ingle (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: peptide (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO: 5:
Arg Ser Ala Glu Lys Asn Glu Pro Glu Met Ala Ala Lys Arg Glu Ser Gly Glu Glu Phe Arg Met Glu Lyg Leu ~sn Gln Leu Trp Glu Lys Ala : :

2 1 1 7 0 ~r`~ 9 CA~C 12/14~),141,149-PC~ 60 Lys Arg Leu His Leu Ser Pro Val Arg Leu Ala Glu Leu His Ser A~p Leu Lys Ile Gln Glu Arg Asp Glu Leu Asn Trp Lys Ly~ Leu Lys Val Glu Gly Leu Asp Gly Asp Gly Glu Lys Glu Ala Lys Leu Val His Asn .:
Leu Asn Val Ile Leu Ala Arg Tyr Gly Leu Asp Gly Arg Lys Asp Thr Gln Thr Val His Ser Asn Ala Leu Asn Glu Asp Thr Gln Asp Glu Leu Gly Asp Pro Arg Leu Glu Lys Leu Trp His Lys Ala Lys Thr Ser Gly Ile Ser Val Arg Leu Thr Ser Cys Ala Arg Val Leu His Tyr Lys Glu Lys Ile His Glu Tyr Asn Val Leu Leu Asp Thr Leu Ser Arg Ala Glu Glu Gly Tyr Glu Asn Leu Leu Ser Pro Ser Asp Net Thr His Ile Lys . .
165 170 175 ~ .
Ser Asp Thr Leu Ala Ser Lys His Ser Glu Leu Lys Asp Arg Leu Arg Ser Ile A8n Gln Gly Leu Asp Arg Leu Arg Lys Val Ser His Gln Leu -~
195 200 205 . ~:
Arg Pro Ala Thr Glu Phe Glu Glu Prc Arg Val Ile Asp Leu Trp Asp Leu Ala Gln Ser Ala Asn Phe Thr Glu Lys Glu Leu Glu Ser Phe Arg :::
Glu Glu Leu Lys His Phe Glu Ala Lys Ile Glu Lys His Asn His Tyr 245 250 2~5 ~.
Gln Lys Gln Leu Glu Ile Ser His Gln Lys Leu Lys His Val Glu Ser Ile Gly Asp Pro Glu His Ile Ser Arg Asn Lys Glu Lys Tyr Val Leu Leu Glu Glu Lys Thr Lys Glu Leu Gly Tyr Lys Val Lys Lys His Leu Gln Asp Leu Ser Ser Arg Val Ser Arg Ala Arg His Asn Glu-Leu

Claims (29)

1. Polypeptide, characterised in that it is a functional derivative of a receptor for rhinoviruses of the "small rhinovirus receptor group".

2. Polypeptide according to claim 1, characterised in that it is a soluble derivative.

3. Polypeptide according to claims 1 to 2, characterised in that it is the soluble extracellular form of a receptor protein.

4. Polypeptide according to one of claims 1 to 3, characterised in that it is derived from the LDL-receptor family.

5. Polypeptide according to claim 4, characterised in that it is derived from the amino acid sequence of the human LDL-receptor according to Fig. 1 or from that of .alpha.2MR/LRP according to Fig. 2 or that of gp330 according to Fig. 3.

6. Polypeptide according to claim 5, characterised in that it essentially comprises the domain 1, the domains 1 and 2 or the domains 1, 2 and 3 of a receptor of the LDL-receptor family according to Fig. 4.

7. Polypeptide according to claim 6, characterised in that it comprises the amino acid sequence according to SEQ.ID.NO.1 or SEQ.ID.NO.2.

8. Polypeptide according to claim 6, characterised 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 about 120 kDa, the molecular weight being determined by SDS-gel electrophoresis under non-reducing conditions.

9. Polypeptide according to one of claims 1 to 8, characterised in that it occurs as a dimer, trimer, tetramer or multimer.

10. DNA coding for a polypeptide according to one of claims 1 to 9.

11. DNA according to claim 10, characterised in that it is inserted in a vector.

12. DNA according to claim 11, characterised in that the DNA according to one of the preceding claims is functionally linked with an expression control sequence with a vector and is replicable in microorganisms and/or mammalian cells.

13. Host organism, characterised in that it is transformed with a DNA according to one of claims 11 or 12.

14. Process for preparing a DNA molecule according to claim 12, characterised in that a DNA provided with suitable ends which codes for a functional derivative of the receptor of the small rhinovirus receptor group according to claim 10 is inserted into a vector DNA
which contains expression control sequences and is cut with restriction endonucleases, in such a way that the expression control sequences regulate the expression of the inserted DNA.

15. Process for preparing a functional derivative of a receptor of the "small rhinovirus receptor group", characterised in that the polypeptide is taken from the native receptor molecule by enzymatic, preferably proteolytic or chemical, preferably reductive, treatment.

16. Process for preparing a functional derivative of a receptor of the "small rhinovirus receptor group", characterised in that it is obtained by expression of a DNA according to one of claims 10 to 12.

17. Hybrid cell line, characterised in that it secretes monoclonal antibodies against one of the polypeptides according to one of claims 1 to 9.

18. Monoclonal antibodies, characterised in that they specifically neutralise the activity of the polypeptides according to one of claims 1 to 9 or specifically bind to one of said polypeptides.

19. Use of the monoclonal antibodies according to claim 18 for qualitatively and/or quantitatively determining or purifying one of the polypeptides according to one of claims 1 to 9.

20. Test kit for determining polypeptides according to one of claims 1 to 9, characterised in that it contains monoclonal antibodies according to claim 18.

21. Process for preparing monoclonal antibodies according to claim 18, characterised in that host animals are immunised with one of the polypeptides according to one of claims 1 to 9, .beta.-lymphocytes of these host animals are fused with myeloma cells, the hybrid cell lines secreting the monoclonal antibodies are subcloned and cultivated.

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 an antiviral, more particularly antirhinoviral agent.

24. Agent for therapeutic treatment, characterised 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 molecule of the LDL-receptor family.

25. Pharmaceutical composition containing one or more polypeptides according to one of claims 1 to 9 and a suitable carrier material.

26. Use of rhinovirus of the "small rhinovirus receptor group" for inhibiting the binding of physiological LDL-ligands.

27. Process for identifying substances which inhibit the binding of ligands to "receptors of the LDL-receptor family", characterised in that a. the receptor or a soluble form of the receptor which can be isolated from culture supernatants is incubated in the presence of a potential inhibitor substance with b. labelled rhinovirus material of the "small rhinovirus receptor group" and c. the extent of binding is determined.

28. Process for detecting receptors of the LDL-receptor family, characterised in that a. a substance derived from virus material of the "small rhinovirus receptor group" with a binding activity for the receptor is labelled, b. incubated with the probe in question and c. the extent of binding of the labelled virus material is detected.

29. Process for supplying a therapeutically active substance into a carrying cell, characterised in that a. virus material of the "small rhinovirus receptor group" with a binding activity on the LDL-receptor is coupled with the therapeutic substance and b. the said material is added to the corresponding cell material, bound to the receptor and in this way the therapeutically active substance is introduced into the cell.