IL86077A - Receptor of the small rhinovirus receptor group its preparation and pharmaceutical compositions containing it - Google Patents

Abstract

A receptor from the small receptor group of human rhinoviruses, its purification and use. [EP0287076A1]

Description

ιιηκ ο·>ϊ»:>ηη mnpn »veoni ιη, η a—o—33 —a—a—a—a —s—s—a—a—s—a—a—o—a—a —a —a—a*>>a< Receptor of the small rhinovirus receptor group, its preparation and pharmaceutical compositions containing it BOEHRINGER INGELHEIM INTERNATIONAL GmbH C:- 74501 This invention relates to the receptor of the small receptor group of human rhinoviruses, the purification and use thereof. The invention also relates to monoclonal and. polyclonal antibodies against such receptors, to hybrid cell lines which secrete these antibodies and to processes for preparing them.

Human rhinoviruses constitute a large genus within the family of Picorna viruses and contain over 90 different serotypes (6,11) . These RNA viruses affect the respiratory · tract of humans and cause acute infections which may lead to colds, coughs, hoarseness, etc., and are generally known as colds (15) . Infections caused by rhinoviruses are among the most common diseases in man. Although the course of the diseases is generally harmless, colds do nevertheless result in general weakening of the organism. This may then give rise to secondary infections caused by other pathogens.

The large group of human rhinoviruses can be subdivided into two sub-groups if the competition for binding sites on the cell surface in human cell culture cells (generally HeLa cells) is used as the criterion for classification. This original classification of a few representatives of the rhinoviruses (10) has been extended to 88 representatives as a result of a wide range of experiments (4,1) . The result of these experiments was to indicate that in spite of the large number, there are surprisingly only two different receptors on the cell surface to which representatives of one or other group of rhinoviruses can bind. Up till now, 78 serotypes of the large "rhinovirus receptor group" and 8 of the small "rhinovirus receptor group" have been classified (RVRG) . 2 other representatives did not behave clearly so that they could not be definitively classified (1) .

In recent years, a considerable increase in rhinovirus infections has been discovered in densely populated areas. Whereas the majority of other infectious diseases result in a long-lasting or permanent immunity from the pathogen in question, infections caused by rhinoviruses may recur again and again. The reason for the absence of any lasting immunity is the large variety of strains of rhinovirus which show little or no immunological inter-reaction with one another (6,11) . After infection has occurred, antibodies against the strain of virus in question can be detected but these do not confer any protection against other rhinovirus strains. In view of the large number of strains circulating in the population, repeated infections by rhinoviruses are possible.

Therefore, the presence of only two receptors offers promising possibilities for the successful combating of rhinoviral infections.

Since receptors are generally highly specific, there is a possibility of achieving controlled influence on the receptors by means of suitable substances, for example by blocking the receptors. If substances which block the receptor are used, the penetration of receptor-specific viruses into the cell can be prevented. The same substances which can prevent infection in this way can also be -used for the treatment of a manifest rhinovirus infection. The production of such substances is made substantially easier and in some cases made possible for the first time, if the receptor in question is characterised.

The only information on rhinoviral receptors available hitherto has concerned the receptor of the large RVRG.

The purification and character sation of the receptor of the large RVRG was effected using a monoclonal antibody obtained by immunising mice with HeLa cells. This receptor is glycosylated and has a molecular weight of about 440 kD in its native state; denaturing with sodium laurylsulphate results 'in a dissociation into subunits of 90 kD, leading one to conclude that the functional receptor "is present as a penta er (17). Hitherto, the receptor for the small RVRG has neither been characterised nor purified. The only data on this receptor indicate its protein structure and also show that these or similar proteins are also present on cells in a number of other species.

This distinguishes the receptor of the small RVRG essentially from the receptor of the large group, which has only been found in human cells and, in a few cases, in monkey cells. Influencing of this receptor, for example blocking the receptor with substances which prevent penetration of the virus into the cell, would appear to be suitable as a possible prevention or even treatment for an existing infection.

The aim of this invention was therefore to provide the pre equisites for preparing substances which give protection against infections by rhinoviruses of the small receptor group.

The present invention provides a receptor with binding activity for rhinoviruses of the small receptor group in substantially pure form with a molecular weight determined by gel permeation chromatography .of about 450 kD constituted from sub-units with an apparent molecular weight determined by SDS gel electrophoresis of about 120 kD or a fragment thereof consisting from such a sub-unit.

The following description may contain subject matter exceeding the scope of the claims, which is being included for the sake of clarity and better understanding.

Thus, one aspect of the present invention provides a receptor with binding activity for rhinoviruses of the small receptor group in substantially pure form.

This aspect of the present invention was achieved by isolating the receptor from cell membranes, for example HeLa cell membranes. These cells were cultivated in suspension by methods known per se , the cells were broken up, the nuclei removed and the membranes purified. The receptors found in the membranes were then solubilised. To achieve optimum solubilisat ion of active receptors from purified HeLa cells, various detergents were tested at different concentrations.

The critical factor in choosing a specific detergent was its ability to solubilise as much membrane material as possible with the highest possible virus binding activity. 1% l-0-n-octyl->8-D-glucopyranoside proved to be the most suitable (Fig. 0).

The insoluble constituents were removed and the receptors in solution were further purified. In order to be able to monitor the virus binding activity, a sensitive filter binding test was developed which makes it 35 possible for S-labelled virus to bind to receptors which had been immobilised on nitrocellulose paper.

The viruses required for the test were cultivated and puri ied in a manner known per se (13) .

The receptors according to the invention were purified by chromatographic methods.

Thus, a further aspect of the present invention provides a process for preparing a receptor with binding activity for rhinoviruses of the small receptor group which compr ises -a. isolating membranes from cells and purifying them, b. solubilising the receptors in the membranes with detergents, c. removing the insoluble constituents and d. purifying the receptors by chromatography.

It is known that the majority of membrane proteins are glycosylated so the receptor may be purified for example on a Lens culinaris lectin column. This lectin has specificity for a-D-glucose and a-D-mannose units (16). Bound material may be eluted for example with 1M a-D-methylglucoside in phosphate-buffered NaCl solution with 1% octylglucoside .

Aliquots were applied in duplicate to nitrocellulose and incubated both with native and with heated virus. Autoradiography of the fractions showed strong binding to the native virus in the case of the material which had been eluted, compared with the fractions from the material which had run through. The heated virus showed weak binding to the run-through material.

This indicates nonspecific interactions which are caused by the high proportion of hydrophobic proteins; heated rhinovirus has greater hydrophobicity (9) .

Since it had been established that, on being stored for a fairly long time at 4°C, purified virus gradually changes into particles which have the same antigenicity as heated virus" (C-determinants) , these contaminations were separated off by immunoprecipitation with C-determinant-specif ic monoclonal antibodies, for example mAK 2G2, immediately before the binding test was carried out. These monoclonal antibodies are obtained in a manner known per se by immunising mice or rabbits with C-determinants and subsequently cloning according to Kfthler and Milstein (18) .

In addition to the culinar is lectin column, con-canavalin A, ricin and hepar in-Sepharose columns were also used to purify the receptor. The run-through and eluted material were tested as above. Con.A-Sepharose columns were eluted with 1 a-D-methyl-mannoside, approximately 20% of the binding activity being recoverable; elution of the ricin column with 1 M galactose resulted in approximately 100% recoverable binding activity. By contrast with the receptor for the Coxsackie B virus group (7) hepar in-Sepharose did not retard, the binding activity. 5 The eluate from the culinaris column was separated on a Superose column by FPLC (Pharmacia) (gel permeation chromatography) . By comparison with marker proteins, the molecular weight of the active receptor was determined 10 as 450 kD. At the same time, a substantial proportion of contaminating protein's could be removed (Figure 1) .

The minor group receptor migrate* with an apparent molecular weight of 120 kD on the polyacrylamide gel in presence of SDS. Sometimes a barely visible band with a slightly lower molecular weight can also be observed which might represent a modification of the bulk of the receptor protein (rig. S, lane 1 ) . The molecular weight of both forms of the receptor are considerably higher than found for the major group receptor (90 kD). As both proteins exhibit a molecular weight of about 450 kD in their native state it . is not unlikely that their subunit structure is similar. The actual molecular weight might however differ from the one determined by gel permeation chromatograpy because of the small difference of retention volumes of. .proteins in this high molecular weight range. The picornavirus structure shows a deep depression (the canyon) running around the fivefold axes of icosahedral symmetry which is thought to contain the receptor binding site (./ By sucrose gradient centr ifugation, it was possible to determine the sedimentation constant of the receptor.

For this purpose, Lj_ culina is purified receptor was applied to a sucrose gradient and centrifuged.

The activity peak was found to be at the position of the gradient which corresponds to the sedimentation constant of 28.4 S (Figure 2).

Preliminary tests had shown that the receptor could no longer be eluted from an anion exchange chromatography column. Since the receptor is insensitive to neuraminidase, sialic acid was removed from the glycoprotein in ϊ* order to reduce the ionic interaction with the column material. The sample was then applied to a mono Q column (Pharmacia) and the receptor was eluted with an NaCl gradient. The binding activity could be detected as a broad peak at about 250 m NaCl (Figure 3) .

It is also possible to purify the receptors according to the invention by a combination of chromatographic purification steps on different chromatography materials.

The chemical properties and structural requirements of the receptor according to the invention for viral - 8 -binding were determined with the aid of enzymes and chemical reagents (Table 1) .

Table I Sensitivity of the small rhinovirus group receptor Pretreatment of the solubilised Binding assay small receptor group (% Bind. -act.) No pretreatment ■/ 100 10 meg trypsin 6 50 mU neuraminidase 170 10 mM dithiothreitol 15 10 mM iodacetamide 80 10 mM sodium periodate 70 10 mM EDTAa 5 All pre-incubations were carried out at 37°C for 30 minutes.

Note: (a) No pretreatment; instead, incubation was carried out with labelled HRV2 in the presence of 10 mM EDTA.

Trypsin treatment destroys the binding activity entirely. This agrees with known results from enzymatic treatment of cell surfaces (14) and indicates the protein nature of the receptor.

Treatment of the solubilised receptor with neuraminidase resulted, in reproducible manner, in a slight increase in the binding activity. This treatment may possibly lead to better accessibility of the region on the receptor molecule which is the target of the virus interaction. As a result, sialic acid is not necessary for the virus binding. - 9 - Dithiothreitol (DTT) destroys the binding activity, leading one to conclude that disulphide bridges are involved in maintaining the correct folding of the protein. The surprisingly high molecular weight, determined by gel permeation chromatography and gradient centr ifugation , indicates an oligomeric structure for the receptor molecule. The sensitivity to DTT might lead one to conclude that intermolecular disulphide bridges are necessary for the association of the hypothetical sub-units.

Treatment with iodacetamide reduces the binding activity only slightly and indicates that free sulphydryl groups are not necessary for efficient binding.

In the presence of ethylenediaminotetraacetic acid (EDTA) during incubation of the nitrocellulose filters 35 with S-labelled virus, no binding could be detected.

This agrees with earlier investigations which showed the need for the presence of divalent cations for interaction of the rhinoviruses with the cell surface (12) .

Competitive binding assays between pairs of serotypes had been used in order to classify the human rhinoviruses into the two receptor classes (10,1). In the present invention, therefore, HRV2 and HRV49 were used as representatives of the small receptor group and HRV89 as representative of the large receptor group in competitive experiments to discover the specificity of the receptors according to the invention. The nitrocellulose filters with immobilised receptors were incubated in the presence of an approximately 20-fold excess of either HRV2 or HRV89 with labelled HRV2. As shown in Table 2, the binding was massively suppressed in the presence of non-labelled HRV2 but unaffected by HRV89. In order to check these results, the tests were repeated with labelled HRV49, using - 10 - HRV2 and HRV89 as competitors. Once again, it is obvious that HRV2 reduces binding on a massive scale but HRV89 has no effect.

Table 2 Competition between various rhinovirus serotypes for the small receptor group.

Competition of radioactively Binding assay labelled HRV2 with: .■···■, (% Bind. -act.) nothing 100 HRV2 13 HRV89 95 Competition of radioactively labelled HRV49 with: nothing 100 HRV2 15 HRV89 90 The filters were incubated with a 20-fold excess of non-labelled virus as described.

Although HRV2 and HRV89 bind to different receptors, their capsid proteins are surprisingly similar (5) . A detailed comparison of structure between HRV2 and HRV14, based on the X-ray structure analysis of_HRV14, was recently set up (2) . Both HRV14 and HRV89 bind to the receptor of the large RVRG. It can therefore be expected that a cluster of preserved amino acids will be found at the hypothetical receptor binding site. Up till now, however, it has not yet been possible to discover a simple pattern of conservative amino acids within the Canyon region. - 11 - The present invention makes it possible for the first time to produce receptors . for the small RVRG.

Using the receptors according to the invention it is possible for the first time to carry out controlled investigations on the virus/receptor interactions.

Of particular importance is the locating of the regions on the receptors which are finally responsible for the viral activity. Once these areas are known, it should be possible to produce substances which are directed specifically 'against these areas, and thereby possibly block the receptors for a variety of different rhinoviruses .

The present invention also relates to the receptors which can be produced from the natural receptors by methods known to those skilled in the art. By way of example, there may be mentioned the sub-units of the natural receptor, which are obtained by treating with reducing agents, and which can be purified for example by electrophoret ic methods. These sub-units may be used, for example, to produce polyclonal and/or monoclonal antibodies which can be used preparat ively , diagnost ically and/or therapeutically in a similar manner to the corresponding antibodies against the natural receptors. The receptor sub-units may also be used in a similar manner to the natural receptors.

The present invention also relates to the modifications of the natural receptors and/or the sub-units thereof which can be obtained by controlled enzymatic treatment. As has been shown in this invention, treatment with trypsin destroys the binding activity of the receptors according to the invention, whilst neuraminidase caused a slight increase in activity. Therefore it is conceivable, and anyone' skilled in the art can check this in a non-inventive manner, that specific - 12 -enzymes and/or chemical reagents result in receptors which either have an improved activity and/or are easier to apply and use and/or have better stability compared with . he natural receptors. These modifications may, for example, result in parts of the protein chain being severed or cut out and/or the protein chains being cut up, in all or some of the sub-units of the natural receptors.

In addition to these modifications it is also possible to convert the natural receptors either wholly or partially into the sub-units, for example by controlled reduction. These large or small sub-units may also be linked together, for example by controlled oxidation, to form large or small units which are rearranged compared with the natural receptors.

Suitable reducing agents for cleaving disulphide bridges include, for example, thiol compounds such as thiophenol, 4-ni trothiophenol , 1 , 4-butanedithiol and particularly 1 , 4-dithiothreitol . The reduction is advantageously carried out in an aqueous/alkaline medium, for example in the dilute aqueous solution of an alkali metal hydroxide, e.g. sodium hydroxide, alkali metal carbonate, e.g. sodium carbonate or an organic base, more particularly a tri-lower alkyl-amine, e.g. tr iethylamine , at ambient temperature.

Suitable oxidising agents for the re-linking of disulphide bonds in the reduced polypeptides include, for example, oxygen from the air, which is passed through an aqueous solution of the polypeptide to which a catalytic amount of a transition metal salt, e.g. iron(III)-sulphate, iron (III) -chloride or copper (II) -sulphate, may have been added; iodine, including iodine in the form of the potassium iodide adduct KI.-,? which is preferably used in alcoholic, e.g. methanolic, - 13 -or aqueous-alcoholic, e.g. aqueous-methanolic solution; potassium hexacyano errate (III) in aqueous solution; 1 , 2-diiodoethane or dimethyl or diethyl azodicarboxylate , which are reacted in water or in a mixture consisting of water and a water-miscible alcohol, e.g. methanol.

Oxidation is more particularly carried out at ambient temperature .

The removal of the reagents, particularly the salts and the oxidants and reducing agents and their secondary products, from the desired' compound is carried out by methods known per se , for example by molecular weight filtration, e.g. on Sephadex or Biogel.

All the modifications may be used in the same way as the natural receptors according to the invention.

The products obtainable in this way, such as the antibodies, like the modifications, fall within the scope of the present invention.

The receptor according to the invention is soluble, so that it is easy to handle and use.

The invention further relates to pharmaceutical compositions containing, in addition to one or more pharmaceutically inert carriers and/or excipients, an effective amount of a receptor according to the invention.

However, it is also possible to bind the receptors to a solid carrier and to use them in this form for diagnostic and preparative purposes. Suitable carriers include all the usual solid carriers such as polystyrene, glass, dextrans and also biological membranes and lipid vesicles.

It is also possible to bind conventional labels to the receptors and to use them in this form for diagnostic purposes. It is also possible to use the receptors - 14 -for the therapeutic treatment of viral infections.

If the receptors according to the invention are bound to a carrier, they may be used both diagnostically and also preparatively to bind the viral protein, for example by means of so-called affinity chromatography. Diagnostically, a viral protein can be detected in the usual way by a receptor bound to a carrier, e.g. by means of antibodies or labelled antibodies. The labelling used may be, for example radioactive labelling, an enzyme or a fluorescent group.

When the receptors according to the invention are used therapeutically, they may be injected in suitably highly refined form, so that they can then inhibit competitively against the natural receptor. Preferably, soluble receptors will be used for this purpose dissolved in a suitable conventional parenterally-administrable carrier. Such solutions may also be used for diagnosis and differential diagnosis.

An exceptionally important application is the use of the receptors according to the invention for producing polyclonal and/or monoclonal antibodies which act specifically against the receptors located in the cell membranes. Antibodies of this kind may be used diagnostically to show up and determine the receptors on cells or biological cell material, or may be used therapeutically to block the receptors in the cell membranes. Consequently, they open up totally new methods and possibilities. - 15 - Legend relating to the Figures Figure 0 Solubilisation of the receptors with various detergents (A: nat. virus, B: denat. virus) .

Figure 1 Gel permeation chromatography of the solubilised receptor on a Superose 6 HR 10/30 column. g Membranes equivalent to 10 cells were solubilised in OG (15 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 (PBS) with 1% ootyl glucoside) , applied to a 1 ml L. culinaris column and the adsorbed material was eluted with 1 M a-D-methylglucos ide in OG. The eluate was concentrated in a Centricon test tube to 0.5 ml and applied to the Superose column. The column was developed with 0.2 ml/min OG and 0.5 ml fractions were collected. The binding activity of the individual fractions, the positions of the marker proteins (determined in a separate experiment) and the extinction at 280 nm are shown.

Figure 2 Sucrose gradient centr ifugation of the solubilised receptor. Material which had been eluted from an Lj_ culinaris column was concentrated (see the legend to Figure 1) and separated on a 10 - 40% sucrose gradient in OG. The binding activity of the fractions (0.4 ml), the position of the marker proteins catalase (Cat) and aldolase (Aid) and the extinction at 280 nm are shown.

Figure 3 Mono Q anion exchange chromatography.

Fractions 14 to 16 of the gel permeation chromatography (Figure 1) were treated with neuraminidase and the material was - 16 -separated by FPLC on mono Q. The binding activity of the- individual fractions (0.5 ml), the extinction at 280 nm and the path of the ..gradient are shown.

Fig, ^ Detection of the rhinovirue minor group receptor on Western blots.

Virus binding activity obtained from the mono F anion exchange column was applied onto a Superoee 6 HR 10/30 column. Fractions of 1.2 ml were collected. Proteins were monitored as A , the 280 positions of protein markers ( thyroglobulin, 670kj apoferritin 440kj β-amylase, 200k) are also shown (a). Proteins contained in the fractione from the Superoee were concentrated and applied on a 6 % SDS-polyacrylamide gel in triplicate. The gel was stained with CoomaBBie blue (b), or the proteins were transferred onto 35 nitrocellulose paper (c,d). The blot was incubated with S labelled HRV2 in absence (c) or in presence of an excess of unlabelled HRV2 (d); the positions of marker proteins ( 2-roacroglobulin, 180k; β-galactosidase, 116kj fructose-6-phosphate kinase, 84k) and of the receptor band are indicated with arrows.

Fiqt Autoradiograph of Western blots obtained of pooled fractions containing virus binding activity from the Superoae column as described in Fig. 35 The blots were incubated with S labelled HRV2. The sample to be loaded onto the gel was incubated with SDS at room temperature, lane 1; the sample was boiled in SDS, lane 2, or incubated with 10 mM dlthiothreitol , lane 3; a blot identical to lane 1 was 35 incubated with S labelled HR 2 in presence of 10 mM EDTA, lan® 35 4, or was incubated with S labelled HRV2 which had been heated o to 56 C for 10 minutes, lane 5. - 17 - Materials l-0-n-Octyl-6-glucopyranoside , Tween 40 and 3-(3-cholamidopropyl) -dimethylammonio-l-propane sulphonate (CHAPS) were obtained from Sigma, N-tetradecyl-N,N-dimeth l-ammonio-3-propane sulphonate (Zwittergent 3 - 14) was obtained from Serva. The other detergents came from Merck, trypsin from Miles 35 and S-methionine (1350 Ci/mmol) from Amersham.

Example 1: ■■< Preparation of the viruses HRV2, HRV49 and HRV89 were cultivated essentially as described in HeLa cell suspension and then purified (13) . The cultivation, isolation and purification of HRV2 will be described here by way of example.

HeLa cells (strain HeLa Ohio, 03-147, Flow Laboratories, England) were grown in suspension at 37°C. The suspension medium (Thomas, D.C., Conant, R.M. and Hamparian, V.U., 1970, Proc. Soc. Exp. Biol. Med. 133, 62 - 65? Stott, E.J. and Heath, G.F., 1970, J. Gen. Virol, 15 - 24) consisted of a Joklik modification of MEM for suspension (Gibco 072-1300) and 7% horse serum (Seromed 0135). The inoculation density was 4 5 - 10 x 10 cells/ml and the volume was 500 ml.

The suspension was centrifuged at a cell density of 1 x 106 cells/ml under sterile conditions at 300 g for 10 minutes. The supernatant was removed by suction filtering and the cells were resuspended in 100 ml of iniection medium (Joklik modification of MEM for suspension culture with 2% horse serum and 2 mM MgCl2) . By carefully sucking up several times in a 20 ml pipette, the cells were homog-eneously distributed in the infection medium. The medium was then made - 18 -up to 500 ml. The cell suspension was brought to 34°C and infected with HRV2 (twice plaque-pu ified) at a multiplicity of 0.1 viruses per cell. The HRV2 strain was obtained from the American Type Culture Collection, (ATCC VR-482 and VR-1112) . The strain used was neutralised with antiserum against HRV2 (American Type Culture Collection, Cat. No. ATCC VR-1112 AS/GP) . The control serum used was an antiserum against HRV7 (Cat. o. ATCC VR-1117 AS/GP) which showed no neutralisation. After 60 hours at 34°C the virus was harvested. Virus was obtained both from the cells and cell fragments and also from the medium.

For this purpose, the medium was separated from infected cells and cell fragments by centrifuging for 10 minutes at 1500 g and then suction filtering. The precipitate was frozen at -70°C.

The cell precipitates from 12 litres of suspension culture were combined, resuspended in 40 ml of TM buffer (20 mM Tris/HCl, pH 7.5, 2 mM MgCl2) , put on ice for 15 minutes, then broken up in a Dounce homogeniser and the mixture was centrifuged for 30 minutes at 6000 g. The precipitate was then washed once again in 10 ml of TM buffer. The two supernatents were combined and centrifuged for 3 hours at 110,000 g in order to pellet the virus. The virus pellet was then taken up in 10 ml of KTMP buffer (50 mM KC1, 50 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 2 mM mercaptoethanol , 1 mM puromycin, 0.5 mM GTP) and after the addition of 150 meg of DNase I (Sigma, r ibonuclease-free) it was incubated for 1 hour on ice.

The virus was precipitated from the infection medium with stirring at 4°C with polyethylene glycol 6000 (PEG 6000; Merck) at a' concentration of 7% and 450 mM NaCl (Korant, B.D. , Lonberg-Holm, K. , Noble, J. and - 19 - Stasny, J.T., 1972, Virology 48, 71 - 86). After 4 hours in the cold, the virus was centrifuged off for 30 minutes at 1500 g, the precipitate was resuspended in 10 ml of KTMP buffer containing 75 meg of DNase I, the mixture was incubated for 1 hour on ice and then frozen at -70°C.

The virus suspensions obtained from the cells and from the medium were combined, incubated for 5 min. at 37°C, cooled by the addition of 60 ml of cold TE buffer (10 mM Tris/HCl,' pH 7.4, 1 mM EDTA) and then sonicated for 5 min in an ice bath.

The suspension- was then centrifuged for 30 minutes at 6000 g. 920 ml of TE buffer containing 7% PEG 6000 and 450 mM NaCl were added to the supernatant, this was stirred carefully for 4 hours at 4°C and the precipitate formed was pelleted for 30 minutes at 6000 g. The precipitate was once again taken up in 100 ml of TM buffer, the virus was precipitated as above by the addition of PEG 6000 and NaCl and pelleted. The precipitate was resuspended in 40 ml of TM buffer, the suspension was centrifuged for 30 minutes at 6000 g and the virus was pelleted for 3 hours at 110,000 g. The precipitate was dissolved in 1 ml TM buffer, incubated for 1 hour at 4°C after the addition of 50 meg of DNase I and then 1 ml of TE buffer was added. For further purification, the virus suspension was centrifuged on sucrose gradients (10 - 30% w/w in TE buffer) for 4 hours at 4°C and at 110,000 g. From the extinction at 260 nm, the fractions containing the virus were discovered and diluted with TM buffer so that the final sucrose concentration was 10%. Then centrifuging was carried out for 8 hours at 85,000 g.

The virus pellet was taken up in 1 ml TM buffer and - 20 -stored at -70°C. To check the purity of the virus preparation, electrophoresis was carried out on a 12.5% polyacrylamide gel in the presence of 0.1% sodium dodecyisulphate (Laemmli, U.K., 1970 Nature (London) 277 , 680-685) and the protein bands were stained with Coomassie Brilliant Blue.

Example 2: 35 Preparation of S-methionine-labelled human rhinovirus serotype 2 (HRV2) - 2 2 HeLa cell mono layers in 165 cm Petri dishes were infected with an MOI (Multiplicity of Infection) of 40 with HRV2 for 1 hour at 34°C in methionine-free MEM medium (Gibco) with 2% dialysed foetal calf serum (Flow) . The cells were washed twice with PBS and incubation was continued at 34°C in fresh medium. 35 After 3 hours, 1 mCi S-methionine (1350 Ci/mmol, Amersham) was added to each mono layer and incubation was continued for a total of 24 hours. The medium of infected cells and cell fragments was separated by 10 minutes' centrifuging at 1500 g and suction filtered. The precipitate was frozen at -70°C in 5 ml of 10 mM Tris, 10 mM EDTA, pH 7.5 (Tris/EDTA) and thawed again. The supernatant and frozen/thawed precipitate were combined and centrifuged for 30 min. at 45,000 x g. The supernatant from this centrifug-ation was centrifuged for 2 hours at 140,000 x g.

The virus pellet was resuspended in 300 mcl Tris/EDTA and the virus was purified over a 10-30% sucrose gradient as above. The individual fractions of the gradient were analysed by SDS electrophoresis in 12% polyacrylamide gels. The pure virus fractions were combined and stored in the presence of 1% BSA (bovine serum albumin) at 4°C for at most 4 weeks. - 21 - In order to remove virus with an altered capsid structure from the preparations from Examples 1 and 2, 20 mcl of immunoadsorbant , containing monoclonal antibodies against the C-determinant , for example mAK 2G2, were incubated with the purified virus for 30 min. and pelleted before the viral probes were removed.

Preparation of the immunoadsorbant Staphylococcus aureus (BRL) cells were suspended as a 10% w/w suspension in' water. The cells were washed twice with PBS , 1/5 vol rabbit-anti-mouse IgG serum (Behring) was added; the suspension was incubated for 1 hour at ambient temperature. The cells were washed twice with PBS and incubated again for 1 hour with 1/5 vol mouse ascites fluid, which contained monoclonal antibodies against the C-determinants (e.g. mAK 2G2) . After washing twice, it was pelleted, the pellet was resuspended in PBS (10% w/w) and the preparations of the radioactive viruses were added.

Example 3: Solubilisation of the receptor from HeLa cells HeLa cells (strain HeLa-Ohio, 03-147, Flow Laboratories, England) were cultivated in suspension at 37°C.

The suspension medium (Thomas, D.C., Conant, R.M. and Hamparian, V.U., 1970, Proc. Soc. Exp. Biol.

Med. 133, 62 - 65? Stott, E.J. and Heath, G.F., 1970, J. Gen. Virol. 6, 15 - 24) consisted of a Joklik modification of MEM for suspension (Gibco 072-1300) and 7% horse serum- (Seromed 0135) . The inoculation 4 density was 5 - 10 x 10 cells/ml and the volume was 500 ml. The suspension was centrifuged at a cell density of 1 x 10 cells/ml under sterile conditions at 300 g for 10 min. The supernatant was removed - 22 -by suction filtering and the cells were washed twice 9 with phosphate-buffered saline solution (PBS) . 10 cells were suspended in 20 ml of isotonic buffer (10 mM HEPES-KOH, pH 7.9, 140 mM KC1, 1.5 mM MgCl2, 0.5 mM EDTA, 0.2 mM phenylmethylsulphonylfluor ide) and broken up in the cold with 200 pulses of a 50 ml Dounce homogeniser. Cell nuclei were removed by centrifuging for 3 minutes at 1000 x g. The membranes were then further purified by the two-phase method o (3) . Membranes corresponding to 2x10 cells per ml were taken up in PBS and stored in liquid nitrogen. g To solubilise them, 2 x 10 cell equivalents were suspended in 1 ml of 1% octylglucos ide in PBS (OG) and any insoluble material was removed by centrifuging at 80,000 x g for 1 hour. The supernatant was used for column chromatography.

Example 4; Filter binding test Fractions from column chromatography to be tested for activity were applied to a nitrocellulose membrane (BA85, Schleicher and Schtlll) in a dot-blot apparatus (Bio-Rad) . The probes were left to seep in at ambient temperature. Then liquid residues were suction filtered under a gentle water jet vacuum and non-specific protein binding sites were saturated with 2% bovine serum albumin (BSA) in PBS at 4°C overnight. The 5 35 filters- were then incubated with 10 cpm S-methionine labelled HRV2 in 1% Tween 40, 0.5% sodium deoxycholate and 10 mM ( 3- ( 3-cholamidopropyl) -dimethyla monio-1-propane sulphonate) in PBS for 1 hour. The membranes were washed twice with 2% BSA in PBS, then dried and the round areas corresponding to the probes were stamped out; the radioactivity was measured in a liquid scintillation counter. - 23 - As a specificity control, HRV2 was heated to 56°C for 10 minutes before the . incubation of the nitrocellulose filters (8) . After this treatment, no binding to any of the probes could be detected (Fig. IB) . From this it was concluded that the binding of native HRV2 to the immobilised material can actually be ascribed to a specific interaction of the virus with the receptor.

Example 5; Affinity chromatography on Lens culinaris lectin columns 10 cell equivalents were solubilised as described and applied to an L. culinaris column (1 ml) equilibrated with OG. The column was washed with 5 ml of OG and bound material was eluted with 2 ml of 1 M a-D-methyl-glucoside in OG. The binding test showed that almost 100% of the binding activity could be recovered, whereas about 90% of the total protein had been removed.

Example 6; Gel permeation chromatography The eluate from the culinar is column was concentrated down to 0.5 ml with a Centricon tube (exclusion 30 kD) and separated on a Superose 6 HR 10/30 column (equilibrated with OG-buffer) by FPLC (Pharmacia) . By comparison with marker proteins the molecular weight of the active receptor could be determined as 450 kD. At the same time a large proportion of contaminating proteins could be removed (Figure 1) . - 24 - Example 7: Sucrose gradient centr ifuqation. culinar is purified receptor (as above) was applied to a sucrose gradient (10 - 40% in OG) and centrifuged for 8 hours at 38 krpm at 4°C. The activity peak was found at the position on the gradient corresponding to the sedimentation constant of 28.4 S. The positions of the marker proteins were determined in another gradient (Figure 2) . As- a' esult of the presence of detergents, the markers sedimented at calculated sedimentation coefficients of 15.0 S and 21.9 S (7.3 S and 11.3 S in the absence of detergents) .

Example 8: Anion exchange chromatography It had been found in preliminary tests that the receptor could no longer be eluted by mono Q HR 5/5 columns (Pharmacia) . Therefore, receptor which had been subjected to preliminary purification using culinar is and gel permeation was treated with 1 U neuraminidase/mg of protein for 60 min at 37°C in order to remove the strongly acidic neuramino acid groups. The sample was then diluted with 10 mM sodium phosphate buffer, pH 7, 1% octylglucoside , to twice the quantity and applied to a mono Q column. The column was developed with a gradient of 0 to 1 M NaCl in the same buffer. The binding activity could be detected as a broad peak at about 250 mM NaCl (Figure 3) .

Isolation and purification of the receptor - 9 flasma membranes of 2x10 HeLa calls were solubilized in 5 ml PBS containing Ϊ % w/v 1 -O-n-octyl-B-D-glucopyranoside and 0.01% w/v oaah of L«#-p- tosyl-L-li/einechl nrnmcf-hyl · Ve nnft (TLCK) L~l-tosylamide-2-phenylethylchloromethyl ketone (TPC ), and phenylmethyl-eulphonylfluoride (PMSF ) (all from Sigma) for 10 minutes at room temperature. Insoluble material was removed by centrifugation at 30 krpm for 30 minutes in the Beckman 65 fixed angle rotor. The supernatant was applied onto a 25 ml ^ cullnaris lectin column equilibrated with OG (PBS, 1% w/v octyl-glucoeide) and bound material was eluted with 5 ml of OG containing 1 M ^- e h glucose. The eluate was brought to 50 % saturation by addition of the same volume of saturated ammonium sulfate. The precipitated material was dissolved in 2 ml of buffer A (10 mM Tris-HCl (pH 7.5), 5 fliM EDTA, 1 %w/v octyl-glucoside) and injected onto a Mono P anion exchange column connected to a Pharmacia FPLC system. The proteins were separated using a gradient from 0 to 100 % buffer B (as A but containing 1.5 M NaCl). Fractions containing Lh« virus binding activity woro pooled, concentrated with a centricon tube to 0.5 ml and run on a Superoee 6 column equilibrated with OG containing 5 mM EDTA. The protein concentration was monitored by the absorbance at 280 n The fractions from this column were concentrated' to 50 JAX, made 0.1 % w/v SDS and run in triplicate on a 6% polyacryl-amide gel containing 5 mM EDTA The proteins separated in the gel were then either stained with Coomassie blue (Fig.^b) or electrophoretically transferred to a nitrocellulose 5 35 sheet (22) which was incubated with 4x10 cpm S -labelled HRV2 under conditions as described for the dot blots /(Mioohak—e£—aim ΊΟ&β- The nitrocellulose was then dried and autoradiography (Fly.^ ). λ& & control for opeoifio binding an identical blot was incubated in presence of a 20 fold excess of unlabelled HRV2 (Fig.^d). It can be seen that fractions 6 and 7 from the Superose column contained material which was able to bind HRV2 when transferred to the nitrocellulose. The autoradiograph shows several bands with an apparent molecular weight greater than 300 kD in addition to one band at a position corresponding to approximately 120 kD when compared to protein markers run on the same gel (Fig, 4c). Only this 120 kD band disappears in the control containing excess unlabelled virus (Fig.^d) demonstrating specific interaction of this protein with HRV2. The polyacrylamide gel containing identical samples and stained with Coomassie blue shows a very faint band at a position corresponding to the radioactive band on the Western blot. This band is only found in the samples obtained from fractions 6 and 7 which exhibit virus binding activity (Fig.^b).

Binding tests The restoration of an active receptor is dependent on mild conditions as boiling in SDS irreversibly destroys its activity (compare Fig..5, lane 1 and 2). When the receptor preparation was incubated with 10 mM dithiothreitol prior to loading onto the polyacrylamide gel, no binding was observed (Fig. S, lane 3). As the specific interaction of rhinoviruses with their receptors is dependent on the presence of divalent cations (.12) Kiochak ct gin 1968) a blot obtained from a sample identical to the one applied onto lane 1 (Fig. S~) was incubated with virus in presence of EDTA. Under these conditions no binding could be observed (Fig. , lane 4). As further control an incubation of the nitrocellulose sheet with HRV2 which had o been heated to 56 C was carried out. This treatment leads to a structural change of the viral capsid which precludes recognition of the virus on the HeLa cell surface (23).

As seen on Fig. , lane.5, no binding occurred under these conditions. - 25 - Bibliography Abraham, G. , and Colonno, R.J. (1984). Many rhinoviru's serotypes share the same cellular receptor. J. Virol. 51, 340 - 345.

Blaas, D. , Kuechler, E., Vriend. G., Arnold, E..

Griffith, J. P., Luo, . , Rossmann, M.G. (1987).

Comparison of three-dimensional structure of two human rhinoviruses (H V2 and HRV14). Proteins (in p ess ) .

Brunette. D.M. , and Till. J.E. (1971). A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. embr. Biol. 5.. 215 - 224.

Colonno. R.J.. Callahan, P.L., and Long, W.J. (1986). solation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses. J. Virol. 57., 7 - 12.

Duechler. .. Skern, T.. Sommergruber , W. , Neubauer. Ch.. Gruendler. P., Fogy, I., Blaas, D. and Kuechler. E. (1987). Evolutionary relationships within the human rhinovirus genus; comparison of serotypes 89. 2 and 14. Proc. Natl. Acad. Sci.

U.S.A. (in press).

Fox, J. P. (1976). Is a rhinovirus vaccine poss ible? American J.Epidemiol. 103 , 345 - 354.

Krah. D.L.. and Crowell. R.L. (1985). Properties of the desoxycholate-solubilized HeLa cell plasma membrane receptor for binding group B coxsackieviruses. J. Virol. 53: 867 - 870. - 26 - Lonberg-Holm, K. , and Yin, F.H. (1973). Antigenic determinants of infective and inactivated human rhinovirus type 2. J. Virol. 12, 114 - 123.

Lonberg-Holm, K. , and hiteley, N.M. (1976).

Physical and metabolic reguirements for early interaction of poliovirus and human rhinovirus with HeLa cells. J. Virol. 19., 857 - 870.

Lonberg-Holm, ., Crowell, R.L., and Philipson, L. (1976). Unrelated animal viruses share receptors. Nature 259 , 679 - 681.

Melnick, J.L. ( 1980 ). Taxonomy o£ Viruses. Proc . Med. Virol. 2_6, 214 - 232.

Noble-Harvey, J., and Lonberg-Holm, . (1974).

Seguential steps in attachment of human rhinovirus type 2 to HeLa cells. J. Gen. Virol. .25, 83 - 91.

Skern, T., Sommergruber , W. , Blaas, D. , Pieler, Ch., and Kuechler, E (1984). The seguence of the polymerase gene of human rhinovirus type 2.

Virology 136, 125 - 132.

Stott, E.J., and Heath, G.F. (1970). Factors affecting the growth of rhinovirus 2 in suspension cultures of L132 cells. J. gen. Virol. 6_, 15 - 24.

Stott, E.J., Killington, R.A. ( 1.972) Rhi novi ruses . Ann. Rev. Microbiol. 26_, 503 - 525.

Young, N.M., Leon, M.A., Takahashi, T. , Howard, I.K., and Sage, H.J. (1971). Studies on a phytohemagglut i nin from the lentil. III. Reaction of Lens cul^naris hemagglutinin with polysaccharides, glycoproteins, and lymphocytes. .1. Biol. Chem. 271. 1596 - 1601. - 27 - Tomassini, J'.E. and Colonno, R.J. (1986). Isolation of a receptor protein involved in attachment of human r inoviruses . J. Virol. 58, 290 - 295.

Kohler, G. and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256 , 495 497. - ' S9. Rosemann, M. G., Arnold, E., Erickeon, J. W., Frankenberger , E. A. / Griffith, J. p., Hecht, H-J./ Johnson, J . E., Kamer, G. , Luo, M., Moaser, A, M. , Rueckert, R. R. , Sherry, B. A. & Vriend, G. (1985). Structure of a common cold virus, human rhinovirus 14 (HRV14). Nature (London) 317, 145-154. 0· Mapoles, J. E. , Krah, D. L. & Crowell, R. L. (1985). Purification of a HeLa cell receptor protein for group B coxsackieviruses. Journal of Virology 55, 560-566.

Laemmli, U. K, (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277, 680-66$.

Burnette, w. N. (1981). Western blotting: electrophoretic transfer of proteins from sodium dodecyl eulfate - polyacrylamide gels to unmodified nitrocelluloee and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry . 112, 195-203.

Lonberg-Holm, , Yin, Γ. H. (1973). Antigenic determinants infective and inactivated human rhinovirus type 2. Journal Virology 9, 29-40.

Claims (35)

1. 86077/5 - 28 - CLAIMS : 1. A receptor with binding activity for rhihoviruses of the small receptor group in substantially pure form with a molecular weight determined by gel permeation chroma- 5 tography of about 450 kD constituted from sub-units with an apparent molecular weight determined by SDS gel elec- 14. IV.88 — — - trophoresis of about 120 kD or a fragment thereof consisting from such a sub-unit.

2. A receptor as claimed in claim 1 being, the total receptor with a molecular weight, determined by gel permeation chromatography, of about 450 kD.

3. A receptor as claime-d in claim 1 or 2 with a 15 sedimentation constant, determined by sucrose gradient centrifugation in the presence of detergents, corresponding to about 28.4 S.

4. A receptor as claimed in any one of claims 20 1 to 3 which is bound by Le s culinar is lectin.

5. A receptor as claimed in any one of claims 1 to 4 which is not bound by hepa in-Sepharose .

6. A receptor as' claimed in any one of claims 1 to 5 which binds irreversibly to an anion exchanger.

7. A receptor as claimed in any one of claims 1 to 6 which has a binding activity which is insensitive 30 to neuraminidase.

8. A receptor as claimed in any one of claims 1 to 7 which consists of sub-units associated by inter olecular disulphide bridges. 35

9. A receptor as claimed in any one of claims 1 to 8 which shows no binding to rhinoviruses in the presence of EDTA.

10. A receptor as claimed in any one of claims 1 to 9 which has a binding activity to rhinoviruses which i-s only slightly influenced by iodacetamice .

11. A receptor sub-unit produced by controlled or complete reduction of a receptor as claimed in any one of claims 1 to 10.

12. A receptor comprising at least two of the receptor sub-units according to claim 11 and which is not the natural receptor.

13. A receptor produced by controlled reduction of a receptor as claimed in any one of claims 1 to 10.

14. A receptor prepared from a receptor as claimed in any one of claims 1 to 10 by controlled treatment with one or more enzymes and/or chemicals.

15. A receptor produced by controlled ' ox idation of at least two of the receptor sub-units as claimed in claim 11.

16. A receptor as claimed in claim 1 substantially as described herein.

17. A process for preparing the receptor of claim 1 which comprises a. . isolating membranes from cells capable of binding HRV2 ana/or HRV49 and purifying them, b. solubilising the receptors in the membranes with detergents, c. removing the insoluble constituents and d. purifying the receptors' by chromatography. - 30 -

18. A process as claimed in claim 17 wherein the cells used are HeLa cells.

19. A proces.s as claimed in claim 17 or claim 18 wherein Triton-X100, CHAPS, Zwittergent, octylglucoside or DOC is used as the detergent.

20. A process as claimed in claim 19 wherein octylglucoside is used as the detergent.

21. A process as claimed' in any one of claims 17 to 20 wherein the receptors are chromatographed on a concanavalin-A, r icin-Sepharose or Lens culinar is lectin column.-

22. A process as claimed in any one of claims 17 to 21 wherein the receptors are chromatographed on an anion exchange column after treatment with neuraminidase.

23. A process as claimed in claim 22 wherein the anion exchange column used is a mono Q column.

24. A process as claimed in any one of claims 17 to 23 wherein a second chromatographic purification is carried out on a Superose column after the first chromatographic purification.

25. A process for preparing a receptor unit as claimed in claim 11 wherein the receptor as claimed in any one of claims 1 to 10 is reduced in controlled manner .

26. A process for preparing a receptor as cTaimed in claim 15 wherein at least two receptor sub-units as claimed in claim 11 are oxidised in controlled manner .

27. A process for preparing a receptor as claimed 86077/2. in claim 14 wherein a receptor as claimed in any one of claims 1 to 10 is treated with one or more enzymes -and/or chemicals in a controlled manner.

28. A process as claimed in claim 17 substantially as described herein.

29. A pharmaceutical composition containing in addition to an effective amount of a receptor as claimed in any one of claims 1 to 16 a pharmaceutically inert carrier and/or exciplent.

30. A pharmaceutical composition as claimed in claim 29 comprising a solution of said receptor in a parenterally administrable pharmaceutical carrier.

31. A hybrid cell line which secretes a monoclonal antibody against a receptor as claimed in any one of claims 1 to 16.

32. A monoclonal antibody which specifically binds to and/or wholly or partially neutralises a receptor as claimed in any one of claims 1 to 16.

33. A polyclonal antibody which specifically binds to and/or wholly or partially neutralises a receptor as claimed in any one of claims 1 to 16.

34. A process for preparing a monoclonal antibody as claimed in claim 32 wherein a host animal is immunised with a receptor as claimed in any one of claims 1 to 16, B-lymphocytes from said host animal aire fused with myeloma cells, the hybrid cell lines secreting said monoclonal antibody are subcloned and cultivated iri vitro or in vivo and said monoclonal antibody is isolated. " I- - 32 - 86077/ 3

35. An antibody-affinity carrier wherein as claimed in claim 32 or . claim 33 is bound suitable carrier material. 3 - A test kit for detecting rhinoviruses which contains at least one receptor as claimed in any one of claims 1 to 16..· · ·' : A process for the preparation of a polyclonal .or a monoclonal antibody which utilizes a receptor as claimed in any one of claims 1 to 16. 38. A process for purifying a receptor as claimed in any one of claims 1 to 16 which utilizes a polyclonal or monoclonal antibody as claimed in claim 32 or 33. 39. A method for qualitative and/or quantitative detection of rhinoviruses. comprising incubating a sample of body fluid with an analyte and detecting binding of an agent to said analyte, wherein said analyte is the receptor of any one of claims- l*i to '16. · 40. A method according to claim 39 wherein the receptor is bound to a solid carrier. 41. A method for analytical characterization or purification of rhinoviruses comprising incubating a sample of body fluid with an analyte and detecting binding of an agent to said analyte, wherein said analyte is the receptor of any one of claims 1 to 16. 42. A method for diagnosis comprising incubating a sample of body fluid with an analyte and detecting binding of an agent to said analyte, wherein said analyte is the receptor of any one of claims 1 to 16. - 33 43. A method according to claim 40 wherein the polyclonal or monoclonal antibody as claimed in claim 32 or 33 is used. 44. A receptor according to claim 1 as herein defined. 45. A process according to claim 17 as herein defined. 46. A pharmaceutical composition according to claim 29 as herein defined. 47. A hybrid cell line according to claim 31 as herein defined. 48. A monoclonal antibody according to claim 32 as herein defined. 49. A polyclonal antibody according to claim 33 as herein defined. _50. A process according to claim 34 as herein defined. _51. An antibody-affinity carrier according to claim 35 as herein defined. 52. A test kit according to claim 36 as herein defined. .53. A process according to claim 37 as herein defined. 54. A process according to claim 38 as herein defined. 55. A process according to claim 40 as herein defined. 56. A process according to claim 42 as herein defined. 57. A process according to claim 43 as herein defined. 58. A process according to claim 44 as herein defined.