HU215587B - Process for producing water-soluble polypeptides having high affinity for interferons alfa and beta and cells expressing the same and pharmaceutical preparations containing the same - Google Patents

Abstract

Field of the Invention The present invention relates to a method for the preparation of a highly agglomerated polybasic peptide peptide having a high affinity for α- and β-interferers, or in a hybrid mammal with a clotting peptide. According to the invention, the cells which are capable of expressing a peptide or hybrid expression are cultured in a suitable medium and then an isolated product is isolated. Production of the above-described peptide peptides and hybrid coding DNA sequences, cells expressing the above-mentioned peptide peptides and hybrids, and pharmaceutical compositions comprising the above-mentioned peptide peptides and hybrids, are also contemplated by the invention. ŕ

Description

The present invention relates to novel water-soluble polypeptides, to DNA sequences encoding them, to cells capable of expressing the polypeptides, and to pharmaceutical compositions containing the polypeptides. The invention further relates to a method for producing antibodies against an interferon receptor and for the diagnostic detection of the presence of such antibodies.

Interferon α and β form a group of selected proteins with different biological properties and create an antiviral and antifungal state in vertebrate cells. [I. Gresser and M. G. Tovay, Biochem. Biophys. Acta 516, 23 (1978)].

Interferon α exerts significant effects on the immune system, the cellular system and the humoral system, and primarily on the polyclonal activation of B cells [M. Peters: J. Immunol. 137, 3153 (1986)] for inhibiting T cell function [J. Knop et al., J. Immunol. 133, 2412 (1984)] and modifying the expression of histocompatibility antigens [M. Fellous et al., Eur. J. Immunol. 9, 446 (1979)]. All these processes play a role in the development of autoimmunity.

Although interferon is considered a beneficial factor in the body, its abnormal production can play a causal role in the course of some diseases and is associated with various so-called autoimmune diseases. For example, high levels of interferon can be found in the serum and tissues of patients with lupus erythematosus, rheumatoid arthritis, Behcet's syndrome, diabetes, multiple sclerosis, bone marrow aplasia, and patients with severe immune deficiency. There is a direct association between excessive interferon levels and the development of poorly predicted AIDS disease [E. Buimovivi-Klein et al., AIDS Slit. 2, 99 (1986)].

It is known that mice belonging to a specific murine strain (NZB) of the animal model of human lupus erythematosus or agonizing β-interferon exacerbate the disease [H. Heremans et al., Infect. Immun. 21, 925 (1978), C. Adam et al., Clin. Exp Immunol. 40, 373 (1980)].

In young mice, administration of high levels of interferon causes growth inhibitory syndrome, hepatic necrosis and death [I. Gresser et al., Natura 258: 76 (1973). Similarly, when female mice are infected with certain viruses (such as Pichinda lymphocyte choriomeningitis virus or reovirus) during their neonatal life, large amounts of endogenous interferon are produced, causing the same fatal syndrome. However, juvenile mice infected with such viruses at birth can be protected from the above syndrome by administration of interferon α or β. Riviere et al., Proc. Natl. Acad. Sci. USA, 74, 2135 (1977), Y. Riviere et al., J. Exp. Med. 152, 633 (1980), T. Clark et al., J. Virol. 59: 728 (1986)]. This experiment convincingly demonstrates the harmful role of interferon in the course of this disease.

It is also known that both NK cell activation and histocompatibility antigen expression modifications, which play a crucial role in the rejection of transplanted bone marrow, are regulated by [alpha] or [interferon]. Chien et al., Science 246: 66 (1989)]. Afifi et al., J. Med. Immunol. 134, 3739 (1985)] have shown that treating FI mice or allogeneic mice with anti-α or β-interferon serum from rodents allows for transplantation and reproduction of parent or allogeneic bone marrow. This fact confirms that production of interferon α or β in FI hybrid mice is a key element in the development of bone marrow transplant resistance.

The biological effects of interferons and their subtypes are known to interact with a specific receptor with high affinity for the cell surface [M. Aquet and K.E. Mogensen (Academic Press, London, 1983).

At present, there is no adequate therapy for the treatment of autoimmune diseases and related diseases such as multiple sclerosis. Known methods of treating autoimmune diseases are unsatisfactory and have toxic effects. The treatments used in "anti-rejection" therapy suppress the symptoms of the disease, but the causes are not eliminated and at the same time they are very toxic. Thus, there is a great need for drugs that are sufficiently effective in the treatment of autoimmune diseases and in preventing organ rejection, but only of low toxicity.

It would be highly desirable to block the effect of interferon alpha or β by injecting an antagonist as this would be therapeutically beneficial both in the treatment of autoimmune diseases and in the prevention of rejection of transplanted tissues. However, proven solutions based on the injection of foreign immunoglobulins have not proved effective in the practice of human medicine.

Proc. Natl. Acad. Sci. USA 80, 25-39 (1983) discloses a monoclonal antibody capable of blocking a human α-interferon species at its terminal carboxyl group. Because the N-terminal portion of the α-interferon molecule binds to the receptor, this antibody is able to recognize the terminal carboxyl group of the α-interferon even when bound to the cell surface receptor. However, this antibody does not recognize the human interferon alpha receptor and is not related to soluble forms of the alpha interferon receptor or to monoclonal antibodies to the receptor. However, the antibody described in that publication is capable of detecting soluble forms of the α-interferon receptor because of its ability to bind to the soluble receptor-bound interferon. The soluble interferon receptor / interferon / anti-interferon monoclonal antibody complex thus formed can be precipitated with protein A / Sepharose beads capable of binding to the Fc fragment of the anti-interferon monoclonal antibody and the soluble interferon receptor can be detected in the precipitate.

The present invention is based on the novel approach of using soluble forms of the specific receptor for interferon-α as antagonists to block the action of α- or β-interferon. Such soluble forms of the natural receptor produced by genetic engineering techniques

They retain their ability to bind circulating or local endogenous interferon alpha, but do not contain the moiety that binds the natural receptor to the cell surface. Therefore, soluble forms of the specific receptor for α-interferon are freely circulating in the body and, by virtue of their specificity, bind only to or interferon β.

Soluble forms of the specific receptor for α-interferon, like antibodies, are capable of blocking the effects of α or interferon in the body.

The present invention provides such soluble forms of the α-interferon specific receptor, hereinafter referred to as water-soluble polypeptides.

The present invention thus relates to a process for water soluble polypeptides

a) a polypeptide having the amino acid sequence shown in Figure 1 and its derivatives formed by deletion and / or substitution,

b) polypeptides encoded by the nucleic acid sequence shown in Figure 1, and

c) for deletion and / or substitution of the polypeptide of the amino acid sequence shown in Figure 2, alone or in the form of hybrids with another polypeptide.

According to the invention, cells capable of expressing a given polypeptide or hybrid are cultured in a suitable medium and the expressed product is isolated.

The water-soluble polypeptides of the present invention have high affinity for interferon α and β, which means that the dissociation constant of the complex is less than 10 ⁹ M.

The term "water-soluble" means that the polypeptide is able to circulate in the body (e.g., the human body) and then bind to a cell.

As used herein, the term "hybrid" refers to a fusion of a water-soluble polypeptide (a "soluble" portion of a natural receptor, a modified whole receptor, or a soluble portion of a modified natural receptor, for example) and another polypeptide (e.g., an immunoglobulin or or a conjugation product thereof.

For the sake of simplification, the term "interferon receptor" is used herein instead of "α- and β-interferon receptor".

A preferred embodiment of the water-soluble polypeptides of the present invention is a compound having the amino acid sequence shown in Figure 1, which is an extracellular soluble part of the natural native receptor for interferon alpha or.

Derivatives formed by deletion and / or substitution of this polypeptide also retain high affinity for the above interferons. It is also an object of the present invention to provide derivatives of the polypeptide shown in Figure 1 by deletions and / or substitutions.

With respect to deletions, it is noted that one or more amino acids in the polypeptide shown in Figure 1 can be deleted without adversely affecting the affinity for interferon α and β.

Deletions may also apply to the full and native receptors, particularly at the soluble level. In this way, for example, the ability to bind to the cell membrane may be lost, thereby rendering the deleted product available to the circulation.

Starting from the sequence of the native and complete interferon receptor shown in Figure 2, the transmembrane and cytoplasmic regions of the sequence may be omitted.

437-457 corresponding to the transmembrane domain. residues 458 to 557 corresponding to the cytoplasmic domain. deletion of the remainder leads, for example, to the formation of soluble (circulating) forms.

The complete sequence of the amino acids and nucleotides encoding the full Ct and β interferon receptor is shown in Figure 2.

Deletions in the transmembrane and cytoplasmic (or cellular and intracellular) regions can eliminate potentially immune epitopes. One of the advantages of the deletion of the transmembrane native α- and β-interferon receptors is that they are secreted in the supernatant of tissue culture of recombinant host cells.

As mentioned above, water-soluble polypeptides which can be derived from the polypeptides having the amino acid sequence shown in Figures 1 and 2 are also compounds of the present invention.

The invention also relates to the preparation of substitutional variants. In these cases, one or more amino acids in the interferon sequence may be removed or replaced with other amino acids so that the total number of amino acids remains unchanged.

The substitutions preferably refer to the soluble portion of the interferon receptor. To produce a significant change in the function or immunological identity of the soluble portion of the receptor, non-conservative substitutions and amino acid residues or sequences that are different from the groups originally present in the polypeptide are selected that maintain the three-dimensional structure of the polypeptide near the substitution. or is of most importance for maintaining conjugation or hydrophobicity of the majority of the side chain.

Among the substitutions modifying the properties of the receptor are the following:

- replacement of an amino acid residue (eg seryl or threonyl) with a hydrophobic residue (eg leucyl, isoleucyl, phenylalanyl, alanyl or valyl),

- replacement of the cysteine residue with any other residue,

- substitution of an electropositive residue (eg lysyl, arginyl or histidinyl) with an electronegative residue (eg glutamyl or aspartyl),

- substitution of a residue with a large volumetric chain (eg phenylalanyl) with a residue without such a moiety.

Substituted variants may also refer to the structure of the entire interferon receptor, and preferably

21 affect the transmembrane domain. Substitutions at this level reduce the affinity of the polypeptide for lipid cells or membranes and thus result in soluble forms of the α and β interferon receptors.

For example, the transmembrane moiety may be replaced by a non-unique amino acid sequence (such as a homopolinucleotide DNA sequence, or any sequence containing 5 to 50 identical amino acids, such as lysine, arginine, glutamine, and aspartic acid - or other hydrophilic amino acids) allowing soluble receptor is secreted into the culture medium when culturing recombinant host cells.

It is also an object of the present invention to provide water-soluble polypeptides which can be derived from the polypeptides shown in Figures 1 and 2.

It will be appreciated from the foregoing that substitutions or deletions, and combinations of such modifications, may occur in the polypeptides of the invention.

In general, the variants obtained by substitution and / or deletion have no functional transmembrane region and preferably no intracellular (cytoplasmic) part.

It will be appreciated that other variants of the water-soluble polypeptides of the present invention may be chemically modified. Such modifications are primarily intended to improve the intrinsic properties of the α and β interferon receptors. Chemically modified polypeptides may contain, for example, hydrophilic polymers (such as poly (ethylene glycol) grafted on free amino acid residues (such as lysine) or sulfhydryl groups). Chemical modifications can increase the plasma half-life of polypeptides, increase the solubility of polypeptides, and / or reduce the immunogenicity of the polypeptides. These modifications may be made by methods known per se, such as those described in U.S. Patent No. 4,179,337.

The invention also relates to the production of hybridized (or additionally conjugated) forms of the above-defined polypeptides.

For example, the second polypeptide involved in conjugation (hybridization) may be an immunocompetent polypeptide. Such polypeptides are, for example, those that elicit immune responses in an animal treated with the hybridized polypeptide, or that are capable of binding to any antibody to a polypeptide moiety that is not a soluble interferon receptor.

Non-receptor epitopes generally contain antigens that are recognized by antibodies already present in the body. For example, polypeptide fragments of bacteria such as β-galactosidase.

Immunoconjugation can be accomplished, for example, by crosslinking in vitro or by culturing a recombinant cell transformed with DNA encoding an immunogenic polypeptide.

Preferably, the immunogenic agent is inserted or linked to a soluble receptor or a soluble receptor-derived fragment by binding to a polypeptide to produce epitopes corresponding to the soluble receptor and at least one polypeptide chain that is foreign to the soluble receptor. These epitopes can be inserted into any other locus in the polypeptide chain that is compatible with the receptor or receptor fragment.

These hybrids are particularly useful for the production of anti-interferon receptor antibodies in warm-blooded individuals. The antibodies thus produced can be used, for example, as diagnostic agents to detect the presence of interferon receptors in biological samples or to purify the native receptor or the soluble receptor for interferon.

Another class of conjugated polypeptides are occasionally immunogenic hybrids which contain, in addition to another water soluble polypeptide conjugated to the C-terminal region of the polypeptide of the invention, a homopolymer (e.g., pentahistidine). These hybrids can be easily separated from mixtures containing impurities in addition to the hybrids by means of chelating agents (such as zinc ions) fixed on a support. The hybrids adsorbed by the carrier-bound chelating agent may be readily resolved from the support, and the hybrids thus purified may, if desired, release the soluble interferon receptor, for example by enzymatic cleavage.

Another class of hybrids are those that improve soluble receptor selection. In these hybrids, the signal polypeptide of the soluble receptor is replaced by a heterologous signal polypeptide. If the host cell recognizes this hybrid, it will use it and the receptor will be secreted.

The signal polypeptides are selected according to the characteristics of the host cell used. The signal polypeptide may be, for example, a bacterial, yeast, fungal, plant, mammalian, or viral sequence.

A preferred group of hybrids are those in which the hybrid-forming second polypeptide slows down the breakdown of the hybrid in the human body by a special structure. Highly preferred representatives of such second polypeptides are plasma proteins with a longer half-life (preferably more than 20 hours) than the soluble portion of the receptor, which, when hybridized with the soluble polypeptides of the invention, prolongs the duration of action of the soluble polypeptides. Examples of such plasma proteins are serum albumin and apolipophosphates. Particularly preferred examples of plasma proteins are immunoglobulins, in particular type G immunoglobulins, most preferably type G1 immunoglobulins.

The above hybrids are preferably non-immunogenic in the animal or human body treated with them, and the plasma proteins, by their biological activation, do not cause adverse side effects in patients.

The water-soluble polypeptides of the present invention are preferably conjugated to immunoglobulins at the constant region level. Preferably, a type G immunoglobulin is used, more preferably a G1 type.

HU 215 587 Β

Immunoglobulins and some variants thereof are known, many of which are produced by recombinant cell cultures [Kohler et al., Proc. Natl. Acad. Sci. USA 77, 2197 (Morrison, 1980), Ann. Port. Immunol. 2, 239 (1984)].

Polypeptides having the activity of the extracellular portion of the α and β interferon native receptors can be conjugated at their C-terminus to the N-terminus of the light chain or heavy chain constant region.

In this way, the variable region can be replaced and at least the transition regions CH2 and CH3 of the heavy chain constant region can be maintained in a functionally active form.

For this purpose, the appropriate DNA sequence can be constructed and expressed in recombinant cell cultures.

Similarly, the polypeptides having a longer plasma half-life at the soluble receptor for immunoglobulins and interferon α and β may also be conjugated to the above receptor and variants.

The extracellular portion of the interferon receptor contains up to 427-436 amino acids, starting from the initial methionine (see Figure 1).

Sequences containing the cell region comprising the anchoring region are generally conjugated to immunoglobulin sequences.

The exact location of the conjugation is not critical. The above binding sites are for information only, and other adjacent soluble interferon sites for conjugation can be selected to optimize the secretion or attachment characteristics of the soluble receptor for α- and β-interferon. The optimal location can be determined using standard experiments.

In general, hybrids have been found to be expressed in a cell, but the degree of secretion of recombinant host cells varies to some degree. The following table shows the different immunoglobulin interferon receptor fusions obtained.

a) RC1

(b) (RC1) 2

(c) (RCh) 2

d) (RC12) 2 (RCh) 2, wherein "R" represents a portion of the extracellular portion of the soluble receptor at the α- and β-interferon, and Cl and Ch, respectively, represent the constant region of the human immunoglobulin light chain and heavy chain. . The above structures only show the main structural features and do not, for example, show the common (J) domain or other regions of immunoglobulins or disulfide bridges. If such sequences are required for binding activity, they must be in a position that is naturally occupied by soluble receptors α- and β-interferon, immunoreceptors α-interferon or immunoglobulin.

These examples are representative of bifunctional hetero-antibodies containing different ligand / receptor sites where VhCh immunoglobulin is capable of binding to a predetermined antigen. The use of heavy chains of other classes of immunoglobulins results in more complex structures (e.g., IgM, IgG2, 3,4, IgA, IgE, IgD, preferably IgG1).

Preferred hybrids are formed by fusion of the N-terminus of the soluble α- and β-interferon receptor, which contains a binding site for the α and β-interferon ligands in the C-terminal portion of an antibody containing immunoglobulin G1-influencing functions. Such hybrids are also illustrated in the examples.

Typically, such hybrids contain the first 436 or 427 amino acids of the soluble α- or β-interferon receptor linked to the constant region of the K chain or the G1 chain at the C-terminus. The DNA encoding the soluble α or β interferon receptor shown in Figure 1 can be synthesized by combining the PCR reaction (Polymerase Chain Reaction, see RK Saiki et al., Science 239, 487-491 (1988)) and digestion with appropriate restriction enzymes. , as detailed in the examples.

Complementary oligonucleotides are used at the predetermined sites downstream of the 5 'end of the IFN receptor cDNA sequence (a / b) as shown in Figure 2.

A complete cDNA (Uzé et al., Cell 60 (2), 225-234 (1990)) or a plasmid containing bacteriophage lambda was used as a template for the PCR reaction. Commercially available cDNA libraries may also be used. The cDNA fragment can also be directly synthesized from total RNA or from polys + RNA prepared by well known methods [see O. Ohara et al., Proc. Natl. Acad. Sci. USA 86: 5673-5677 (1989); J. Delort et al., Nucl. Acid Slit. 17, 6439-6448 (1989)]. The cDNA or RNA library can be constructed from human cells (e.g., Daudi, Namalwa) or organs (e.g., human spleen) with substantially the same result.

Although the interferon receptor appears to be a unique polypeptide, certain allelic variants of its sequence may still exist.

The DNA fragment is inserted into DNA encoding a constant region of a light or heavy chain of an immunoglobulin (preferably human immunoglobulin, if desired).

DNAs encoding immunoglobulins are known and are commercially available or synthesized (see, e.g., Adams et al., 1980, Biochemistry 19, 2711-2719, Gough et al., Biochemistry 19, 2702-2710, Dolby et al., Proc. . Natl. Acad. Sci. USA 77, 6027-6031 (1980), Rice et al., Proc. Natl. Acad. Sci. USA 79: 7862-7865 (1982); Falkner et al. (1982) Natur. 298: 286-288; Morrison et al., Ann. Port. Immunol. 2: 239-256 (1984)].

Preferably, the DNA encoding the hybrids is transfected into host cells for expression.

If the host cell produces heavy immunoglobulin chains before transfection, it is sufficient to transfect a hybrid containing light chains of soluble α and β interferon receptors to produce bifunctional hetero-antibodies. Like if the host cell already

Expression of light chains, DNA encoding a hybrid containing the heavy chains of soluble α- and β-interferon receptors can be transfected to produce a bifunctional antibody. Bifunctional immunoglobulins containing one or more chains and variable regions comprising the binding site of interferon alpha and β are doubly specific for interferon α and β and a predetermined antigen. These may be prepared by the methods described above or by in vitro methods. In the latter case, for example, the F (ab) 2 fragments of the hybrid are formed by known methods (see, for example, U.S. Patent No. 4,444,878).

Alternatively, bifunctional antibodies can be generated by fusing B cells or hybridomas that express specific antibodies to the desired antigen with cells producing immunoglobulin / soluble α-interferon receptor hybrids (e.g., myelomas). Bifunctional antibodies can be recovered from the culture supernatant of such hybridomas.

The invention further relates to the preparation of DNA sequences encoding the water-soluble polypeptides described above or their hybrids with a second polypeptide. These DNA sequences are prepared by linking the appropriate DNA fragments and / or nucleic acids in the correct order using known genetic engineering techniques and amplifying the resulting product by PCR (polymerase chain reaction).

Examples of high affinity polypeptides for mammalian α and β interferons include soluble α and β interferon receptors in primates, as well as human, rat, mouse, dog, cat, bovine, equine and porcine species. soluble interferon α and β receptors. DNA sequences encoding such polypeptides are useful in a variety of applications. In particular, these sequences or portions of the sequences and their synthetic or semisynthetic copies may be used to screen other cDNA libraries or human or animal genomic libraries by hybridization to other DNA sequences similar to soluble α and β interferon receptors. Such DNAs, fragments, and synthetic or semi-synthetic copies thereof are variants of soluble α and interferon-β receptors and can be used as starting materials for the production of other variants by mutation. These mutations are either unable to alter the sequence of the amino acids encoded by the mutated codons or alter the amino acids. Both types of mutations are advantageously utilized for the production or use of soluble α and β interferon receptors. For example, these mutations result in higher productivity, simpler purification, or higher binding capacity.

An example of a suitable DNA sequence is shown in Figs. a nucleotide sequence.

Preferably, the DNA encoding variants of the soluble forms of the natural α- and β-interferon receptor is used in a form that permits its expression in a transcription unit under the control of transcriptional and translational controls for mammals, microorganisms, or viruses compatible with the intended host cell. After transformation, transfection or infection of appropriate cells, these vectors allow expression of the recombinant polypeptide. Soluble variants of the natural α and β interferon receptor may be expressed in cells of mammals, yeast or bacteria or other cells under the control of appropriate promoters. Vectors used for the cloning and expression of bacterial, fungal, yeast or mammalian host cells have been described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant parts of which are incorporated herein by reference. Optionally, the expression vector may not contain a replication site as well as one or more selectable marker sequences for selection between transformed cells.

Variants may be inserted into the DNA encoding the polypeptides described above, but these may not alter the reading frame or create complementary sequences that produce expression-damaging secondary structures.

The polypeptides, which are soluble variants of the natural interferon receptor, have substantially the same binding activity as the natural receptor, but, as stated above, variants with other (e.g. improved) characteristics than the natural receptor may be formed.

Although the mutation site is fixed, the mutation itself is not. For example, to optimize the performance of a mutation at a particular site, the codon or target region may be randomly mutated and the resulting polypeptide variants screened for an optimal combination of secondary characteristics. Methods for inserting substitution mutations at specific sites in the DNA are well known, such as the M13 system. The DNA encoding the human receptor can be produced by any known method, and its sequence is shown in Figure 2.

Generally, prokaryotes may be used to clone the polypeptide sequences described above. E. coli strain 294 (ATCC 31446) proved to be particularly useful, but other strains such as E. coli X 1776 (ATCC 31537) may be used.

As stated above, soluble variants of the α and β interferon receptor and their hybrids can be expressed in bacteria, yeast or mammalian cells or other types of cells under appropriate promoter control. The invention also relates to the production of cells expressing such polypeptides. These cells are prepared by transfecting the host cell with the DNA sequence encoding the polypeptide.

Prokaryotic systems such as E. coli may be used to express recombinant proteins. E. coli is typically transformed using a derivative of pBR322 (ATCC 37017), a plasmid derived from an E. coli strain (Bolivar et al., Gene 2: 95 (1977)). PBR322 is an ampicillin and tetracycline resis6

HU 215,587 Β and is a simple means of identifying transformed cells. Plasmid pBR322 or other microbial plasmids must also contain or modify the expression control sequences commonly used in the recombinant DNA construct to include such control sequences.

Examples of the above controls include the lactose (lac) promoter (ATCC 37121) [J. Mole. Appl. Gene. 1, 139-147 (1989)], the β-lactamase promoter (Chang et al., Naturre 275, 615 (1987)), the tryptophan promoter (Miozzari, J. Bact. 133: 1457-1466 (1978)] and hybrid promoters such as tac (ATCC 37138) [H. de Boer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983). Also included are commercially available vectors such as trc containing the phage lambda promoter and the heat-labile c1857 repressor or pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) containing the pPLlambda promoter. However, the range of controls is not limited to those exemplified above.

Other functional promoters have also proved to be suitable. DNA sequences are generally known, so they can be conjugated to DNA encoding soluble variants of the α and β interferon receptors using appropriate binding elements or adapters. The promoters of bacterial systems also contain a so-called ShineDalgamo (SD) sequence operatively linked to DNA encoding downstream of the antigen.

After transformation of the appropriate host strain and growth of the transformed host strain to the appropriate culture density, the selected promoter is appropriately repressed (e.g., by increasing temperature or by chemical induction) and the microorganisms are further cultured. The microorganisms are then harvested, usually by centrifugation, lysed chemically or physically, and the extract is isolated for further purification.

The microorganisms are fermented under vigorous agitation under conditions of maximum growth in a fermentor, for example 10 liters. Preferably, an antifoam agent is used. The stock culture was described by Mott et al., Proc. Natl. Acad. Sci. At a culture density of ₆₀₀ = 5-6 absorbances, the promoter is depressed by raising the temperature to 42 ° C and harvesting the cells 2-20 (preferably 3-6) hours after the temperature change. The microbial mass is first compacted by filtration or otherwise, then centrifuged at 10,000 g at 4 ° C for 10 minutes, and finally the resulting pellet is solidified rapidly. The recombinant proteins produced in the bacterial culture are isolated by extraction of the pellet, followed by concentration or desalting of the extract in one or more steps, or ion exchange or exclusion chromatography.

The microorganisms used to express soluble variants of the? And? Interferon receptor can be lysed by any suitable method (e.g., cyclic freeze-thaw, mechanical fracture, or chemical). Soluble variants of the α and β interferon receptors tend to aggregate to some extent. Extraction and purification may be carried out in the presence of a detergent such as Tween 80 or Triton XI00 to reduce aggregate formation.

For water-soluble polypeptides whose DNA sequence does not begin with methionine, the initiating signal results in an upstream addition of an additional methionine amino acid, which forms the N-terminal portion of the product. Although these additional N-terminal methionine-containing products can be used in the methods of the invention, it is more preferred to remove this methionine beforehand. Standard in vivo and ex vitro methods for the removal of N-terminal methionine are well known.

Yeast systems, such as Saccharomyces types (including readily available S. cerevisiae), may also be used to express the polypeptides or hybrids defined above.

Suitable yeast vectors generally include the following elements: an origin of replication, a selectable marker for transformation of yeast and E. coli (e.g., the E. coli ampicillin resistance gene and the S. cerevisiae trpl gene, a yeast mutant unable to grow in the presence of tryptophan). ATCC 44076) and a promoter from a gene overexpressed in yeast to induce upstream transcription of the gene. Suitable promoters for yeast hosts include the promoter of 3-phosphoglycerate kinase or other glycolysis enzymes, the promoter of acid phosphatase (e.g. PH05), and the promoter of α-type coupling factors. Further suitable promoters for yeast hosts include promoter regions such as 2-alcohol dehydrogenase [Russel et al., J. Bioi. Chem., 258, 2674 (1982)]. A signal peptide (e.g., the signal peptide of a factor enabling the selection of a heterologous yeast protein) can be inserted between the promoter and the upstream structural gene [see Bittér et al., Proc. Natl. Acad. Sci. USA 82, 5330 (1984)]. Methods for transforming yeast are well known in the art. A typical method is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75, 1929 (1978)], by means of which trp + transformants can be selected on a selection medium containing 0.67% nitrogen yeast base, 0.5% casino acid, 2% glucose and 10 pg / ml uracil. Transformed host strains containing the PH05 promoter vectors may be initially cultured in the form of a pre-culture medium in a rich medium and then repressed by reducing the concentration of inorganic phosphate in the medium. The strain is cultured by conventional methods.

The recombinant polypeptide is recovered by extracting the cell pellet lysed by glucosidase enzymatic digestion followed by detergent treatment or by mechanical means (e.g. compression), then concentrated in one or more steps, desalted, ion-exchanged or excluded by chromatography. When the polypeptide is secreted into the periplasmic space

The cells are treated with a chemical that damages the outer membrane layer and allows the release of the recombinant polypeptide (eg, EDTA) and is then swallowed by the polypeptide. If the polypeptide is excreted in the culture medium, it can be recovered directly.

The polypeptides or hybrids defined above may also be expressed in mammalian cells under the control of suitable promoters. Cell-free systems can also be used to produce soluble variants of the mammalian interferonalpha.- and .beta., Using RNAs derived from the DNAs of the present invention.

Promoters that control expression in mammalian host cells can be obtained from various sources. These include promoters from the genomes of the virus (such as polyoma, monkey SV40 virus, adenovirus, retroviral hepatitis B cytomegalovirus), some mammalian promoters (e.g., promoter with sites sensitive to the human β-globin gene DNAase I), and some insect viruses. promoters (such as the polyhedron promoter of the baculovirus system). For expression in animal cells, a control region derived from the late main promoter of adenovirus-2 can be advantageously used.

The early and late domains of SV40 are preferably isolated from the SV40 virus in the form of a restriction fragment containing the viral origin of replication (see Fiers et al., Nature272, 113 (1978)).

The early direct domain of human cytomegalovirus was isolated as a Hind IIIE restriction fragment (Greenaway, P.J. et al., Gene 18: 356-360 (1982)). The late promoter of adenovirus-2 is illustrated in Figures 8-17. isolated as a Hind III restriction fragment containing map units [S. Hu and J.L. Manley, Proc. Natl. Acad. Sci. USA 78: 820-824 (1981)]. Parent cell-derived eukaryotic promoters have also been found to be useful.

In eukaryotic expression vectors, transcription is increased by inserting sequences containing activators in addition to the promoter. Activators are DNA elements that act on the same side and contain ten to three hundred base pairs several times and enhance the promoter's ability to initiate transcription. Many activators (globin, elastase, albumin, insulin, etc.) are known to act on mammalian genes. However, it is common to use activators of viruses that infect eukaryotic cells, such as the SV40 activator of the late origin of replication (100-270 bp), the activator of the early direct promoter of cytomegalovirus, the late origin replication activator of the replication origin, side polyoma activator and adenovirus activator. In addition, expression vectors used in eukaryotic cells (yeasts, fungi, insects, plants, animals, humans) may contain sequences necessary for the transcriptional cleavage and termination of factors capable of influencing mRNA expression. Expression vectors contain a selection gene.

Examples of selection markers of mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase and neomycin. When such selection markers are transfected into a mammalian host cell, the transformed cell exposed to the selection pressure becomes capable of survival. Usually, two types of selection systems are used. One type uses mutant cell lines whose growth depends on whether or not the medium contains certain substances. These include, for example, CHO DHFR cells or murine LTK cells that cannot grow without the addition of thymidine or hypoxanthine. When transfected with these cells into a functional DHFR or TK gene, the cells become viable, whereas cells not transformed with the DHFR or TK gene remain unchanged on thymidine or hypoxanthine-supplemented medium.

The other solution is dominant selection, which does not require mutant cells. For example, a gene that renders a cell resistant to a toxic substance is transfected into a cell and expressed as described in Southern and Berg, J. Molec. Appl. Gene. 1, 327 (1982), Mulligan and Berg (1980) Science 20: 1422, Sugden et al., Mol. Cell. Biol. 5: 410-413 (1985).

Increasing or replicating certain chromosomal regions of a cell is called amplification (amplification) and is generated using a selection agent (e.g., methotrexate inactivating DHFR). As the amount of MTX increases, the amplification or copy number of the DHFR gene increases, resulting in an increase in DHFR production. The degree of amplification increases with increasing MTX concentration in the medium. Amplification of the desired gene is accomplished by co-transfection of the desired gene with the DHFR gene cointegrated in the chromosome. After co-amplification of the desired gene and the DHFR gene, the gene encoding the desired protein expresses more desired proteins.

Preferred host cells for expression of the soluble interferon receptor variants as defined above are mammalian cells, including monkey kidney cells (COS-7, ATCC CRL 1651) and Chinese hamster cells [CHO-, DHFR-, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77, 4216 (1980)].

Methods for transforming mammalian cells are well known. A preferred method for precipitating DNA with calcium phosphate is described by Graham F. and Van Der Eb (Virology 52: 456-457 (1973)). Another method involves electroporation [G. Chu et al., Nucl. Acid. Gap. 15: 1311-1326 (1987). Other transfection methods may be used, such as nuclear injection or protoplast fusion.

Expression vectors containing the desired control and coding sequences are constructed by standard methods well known in the art (see, e.g., Maniatis T. et al., Molecular Cloning, 133-134 (Cold Spring Harbor, 1982)) and Ausubel et al., Ed. Molecular Biology (Greene Publishing Associates & Wiley Interscience, 1987). Plasmids or fragments of DNA are isolated, cleaved, sized and then cut to the desired form.

HU 215 587 Β

The correct plasmid sequences were determined after transformation with ligation mixtures in E. coli HB101 or E. coli K12194 (ATCC 31446). Transformants resistant to ampicillin are selected. The plasmids are isolated from the transformants and analyzed by well-known restriction enzyme treatment or sequencing (see Messing et al., Nucl. Acid. Gap. 9, 309 (1981) and Maxam et al., Methods in Enzymology 65, 499 (1980).

Host cells are generally transformed with expression vectors and cultured in a suitable medium containing promoter expression inducers, components for selection of transformants, or genes for amplification of genes. The fermentation conditions (e.g. temperature, pH) to be used for the selected expression culture are well known to those skilled in the art.

The polypeptides of the invention are isolated and purified from supernatants of recombinant host cells. The supernatants are usually concentrated by ultrafiltration and then purified by ion exchange chromatography or immunoaffinity to adsorb the expected polypeptides and subsequently elute. It is highly prone to formation of antigenic aggregates. Therefore, the cleaning operations are preferably carried out in the presence of a detergent such as Tween 80, Triton X100 or CHAPS. The final product is stabilized with proteins (e.g., albumin). Optionally, these proteins may contain a detergent.

Note that all vectors or sequences for expression control and all host cells do not function equally in all expression systems. The selection of appropriate vectors, expression control sequences and host cells within the scope of the present invention is within the skill of the art. When selecting suitable single-cell hosts, for example, the following properties must be considered: compatibility with the vector selected, toxicity of the product encoded by the DNA sequences of the invention, secretion characteristics, protein folding properties, fermentation requirements, DNA sequences of the invention The ease of purification of the recombinant products obtained after.

Given the above parameters, one skilled in the art will readily select those vector system / expression control system / host cell combinations that express the DNA sequences of the invention during large-scale fermentation or cultivation of animal cells (e.g., CHO or COS-7 cells).

As stated above, the polypeptides of the invention are useful as immunomodulators (particularly immunosuppressants) and are useful in medicine, for example, for the treatment of autoimmune diseases and for the prevention of transplant rejection. For these purposes, the polypeptides of the invention are preferably used in the form of pharmaceutical compositions.

The present invention further relates to a process for the preparation of a pharmaceutical composition by converting a water-soluble polypeptide of the invention into pharmaceutically acceptable carriers, diluents and / or other excipients using conventional pharmaceutical technology.

The pharmaceutical compositions may be, for example, solid, semi-solid and liquid compositions such as tablets, pills, powders or solutions for injection or infusion. The preferred form of preparation will depend upon the mode of administration and the therapeutic use. The pharmaceutical compositions of the present invention may be used in a manner similar to the use of other pharmaceutically significant polypeptides, and in the form of compositions containing the other polypeptides. Thus, for example, the polypeptide may be stored in lyophilized form, from which the ready-to-use drug is formulated prior to use by the addition of sterile water, and administered by conventional means such as parenterally (e.g., subcutaneously, intravenously, intramuscularly or intralesionally). An effective dose of the active compounds may be in the range of 1-5 mg / kg / day, but it is noted that doses below or twice the upper limit can be administered without adverse effects.

The pharmaceutical compositions of the present invention can be administered to patients suffering from abnormal production of α and β interferons for the treatment of, for example, lupus erythematosus, Bechet syndrome, aplastic anemia, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, severe immune deficiency, AIDS. The pharmaceutical compositions of the present invention are also useful in treating patients at risk of rejection of transplanted organs or tissues by virtue of their immune-modifying properties (e.g., activation of NK cells or expression of histocompatible antigens).

Because of their nature and mechanism of action, the pharmaceutical compositions of the present invention are devoid of the general toxicity of previously known agents (e.g., chemical immunosuppressants, such as glucocorticoids) and therefore represent a significant improvement over drugs used in current clinical practice. Examples of currently used drugs include immunosuppressors and their derivatives (such as adrenal corticosteroids) that inhibit cell division and cytokine synthesis of all elements of the immune system, as well as the cyclic undecapeptide cyclosporin. it also has non-immunological toxic effects, see N. Engl. J. Med. 321 (25): 1725-1738 (1989)].

The polypeptides of the invention may also be used to regulate the function of agonists or antagonists other than α and β interferons.

HU 215 587 Β

The polypeptides of the present invention, when purified, can also be used to determine the tertiary structure of the binding sites for soluble α and β interferon receptors. Knowledge of the tertiary structure is a prerequisite for the development of a molecular model from which structural conclusions can be drawn for the synthesis of antagonists for use as therapeutic immunosuppressants or agonists for use as antiviral or antitumor agents.

The polypeptides of the invention may also be used for diagnostic purposes.

One example of diagnostic uses is the generation of antibodies against the interferon receptor by administering to a warm-blooded animal the polypeptide of the invention and a pharmaceutically acceptable substrate, and after the immune response has been completed, the animals are bled and the antibodies are isolated from the blood. These operations are carried out using standard immunobiological techniques. The antibodies thus formed can be used, for example, for various diagnostic assays or pharmacological model tests.

Another example of diagnostic applications is the detection of antibodies to interferon α and β in biological samples. Antibodies are detected by an immunobiological method known per se, which involves contacting the biological sample to be tested with a substance complexed with the antibody under conditions which allow the formation of the complex and detecting the presence of the complex. In accordance with the present invention, the polypeptides of the invention are used as complexing agents with the antibody.

It is noted that, unless expressly stated otherwise, the term "(water soluble) polypeptide produced by the present invention" refers to both free and hybridised forms of the polypeptides listed in the introductory part of the specification.

The following examples further illustrate the invention without limiting the scope of the invention to the examples.

/. example

Construction of Vectors for Expression of Water Soluble Receptors and Variants of Native α and β Interferons Selected by Mammalian Cells

Section 1

Construction of plasmids for expression of native interferon receptor

DNA fragments containing complete cDNA and encoding the receptor for native α and β interferons were synthesized by PCR. The template is a lambda ZAP bacterial phage containing the entire length of the receptor cDNA for the α and β interferons and specific oligonucleotides are used as primers for the reaction. Specifically, the lambda bacterial phage ZAP is incubated with a synthetic oligonucleotide pair complementary to the antisense at the 5 'end of the cDNA, the oligonucleotide encoding the oligo 0: 5' -CCGGCTGCAGGGATCTGCGGCG with the codon gCTCCCAG-3 ', and the oligo-1: 3 '-C consists of an oligonucleotide having the sequence AAAAAGTCGTCCTCAATGTG ACCATGGCC-5'.

The oligonucleotides are constructed such that, after completion of the PCR reaction, the resulting double-stranded DNA fragments contain a restriction site at the 5 'end for PstI and at the 3' end for KpnI.

The PCR reaction was performed in 100 µl reaction volume in PCR buffer containing 1-1 mM primers, 100 µg lambda ZAP bacterophage and 1 unit Taq polymerase. The reaction conditions are as follows: 25 cycles; one cycle consists of incubation at 95 ° C for one hour (denaturation), treatment at 37-40 ° C for 3 minutes (hybridization) and then heating at 72 ° C for 4 minutes (polymerization). After the final cycle, the reaction was continued at 72 ° C for 10 minutes and samples were stored at 4 ° C. The reaction products were extracted with chloroform and after precipitation with ethanol, the products were successively digested with KpnI and PstI. The 1.7 kb fragment (fragment 1) was collected after electrophoresis on low melting point agarose and ligated to the expression vector pSVAdpAl.

The pSVAdpAl expression vector is constructed as follows:

SV40 sequences were obtained from plasmid pSV2DHFR [Subrami et al., Molec. Cell. Biol. 1, 854-864 (1981)]. They contain the origin of replication, the activator, the early and late promoters (Pwull-HindIII fragments) and the intron of the antigen surrounded by its cleavage sites (donor and acceptor) and the early domain polyadenylation site (Bgl II-Eco RI fragment).

The 2-adeno late major promoter region was obtained from plasmid pAdD26SVpA (EcoRI-PstI fragment) [Kaufman and Shimke, P. A., J. Mol. Biol. 159: 601-621 (1982)]. This promoter, the tripartite leader [Zain et al., Cell. 16, 851 (1979)] and comprises a hybrid cleavage site consisting of the 2-adeno-donor cleavage site and the acceptor cleavage site of the variable region of the immunoglobulin.

The multiple cloning site is identical to that found in pUC18. It contains restriction sites for Pst I, Scal I, Acc I, Hinc II, Xba I, Bam HI, Xma I, Sma I, Kpn I, Sac I, Ban II, Eco RI.

The plasmid contains the replication origin of E. coli and the ampicillin-resistant gene and was derived from the plasmid pML (Lusky and Botchan, Natura 293: 79-81 (1981)).

Plasmid pSVAd1 was digested sequentially with KpnI and PstI, treated with phosphatase, and the fragment containing the major portion of the plasmid (fragment 2) was isolated by low melting point agarose electrophoresis. Fragment 2 is linked to fragment 1 and

The coupled mixture was transfected into competent E. coli HB 101 cells. The transformed culture is poured into petri dishes containing ampicillin-containing medium and the ampicillin-resistant colonies are selected. From these transformants, the DNA plasmid was prepared and analyzed; during the analysis, the presence of the appropriate insert is examined by restriction enzymes and the sequence of the insert at the 5 'and 3' ends is confirmed by sequence analysis to confirm the correctness of the sequence.

The plasmid pSVAdIFRpA was used to express the native cell receptor in mammalian cells.

Section 2

Construction of plasmids for expression of selected variants of? And? Interferon receptors

DNA fragments containing different deletions of the re5 receptor containing different carboxy terminals and encoding selected forms of the receptor were synthesized by PCR. Specifically, the lambda ZAP bacterial phage containing the complete cDNA of the receptor is incubated with various synthetic oligonucleotide pairs 10 which are complementary to the 5 'end of the antisense,

5 'Ligigo: an oligonucleotide having the sequence 5'-CCGGCTGCAGGGATCTGCGGCGGCTCCCAG-3 and a coding strand at various sites on the cDNA

3 'end oligo 1: 3'-CGTCCTTTATGGAGATTTACTCCATGGCC-5' 3 'end oligo 2: 3'-TCACTGCGACATACACTCACTCCATGGCC-5' 3 'end oligo 3: 3'-GTCAGACCTTTGTGCGGAACTCCATGGCC-5' 3'AGGG 4ACG-5 '3' end The '3' end contains one of the oligonucleotides having the sequence oligo 5: 3'-CTCTGATGAATAACAGATACTCCATGGCC-5 '.

The oligonucleotides are constructed such that, after completion of the PCR reaction, the resulting double-stranded DNA fragments contain a restriction site at the 5 'end of the PstI enzyme and at the 3' end of the KpnI.

The PCR reaction was performed in 100 µΐ reaction volume in PCR buffer containing 1–1 µΜ primers, 100 µg bacterial phage Lambda and 1 unit Taq polymerase. The reaction conditions are as follows: 25 cycles; each cycle consists of incubation at 95 ° C for one minute (denaturation), treatment at 37-40 ° C for one minute (hybridization) and heating at 72 ° C for one minute (polymerization). After the final cycle, the reaction was continued at 72 ° C for 10 minutes and samples were stored at 4 ° C. The reaction products were extracted with chloroform and after precipitation with ethanol, the products were successively digested with KpnI and PstI. The desired fragments were collected after electrophoresis on low melting point agarose. The length of the fragments collected is approximately the following:

Reaction 1 (5 'end oligo fragment 3

Reaction 2 (5 'end oligo fragment 4

Reaction 3 (5 'end oligo + 3' end oligo 3): 1.1 kb fragment 5

Reaction 4 (5 'end oligo + 3' end oligo 4): 0.9 kb fragment 6

Reaction 5 (5 'end oligo + 3' end oligo 5): 0.6 kb fragment 7

The DNA fragments of 3-7 are separately linked to the expression vector pSVAdpAl.

Plasmid pSVAd1 was sequentially digested with KpnI and PstI, then treated with phosphatase and

3 'end oligo 1): 1.3 kb

3 'end oligo 2): The 1.3 kb fragment (fragment 2) was isolated by low melting point agarose electrophoresis. Fragment 2 is linked separately to the sequence of Figures 3-7. fragments and the coupled mixture was transfected into competent E. coli HB 101 cells. The transformed cultures contain medium containing ampicillin

Pour into petri dishes and select resistant colonies. From these transformants, the DNA plasmid was prepared and analyzed; during the analysis, the presence of the appropriate insert is examined by restriction enzymes and the sequence of the insert at the 5 'and 3' ends is confirmed by sequence analysis to confirm the correctness of the sequence.

The plasmids are named pSVAdsIFlpA, pSVAdsIFR2pA, pSVAdsIFR3pA, pSVAdsIFR4pA, pSVAdsIFR5pA. These plasmids are used to express the soluble receptor and its fragments in mammalian cells.

Example 2

Expression in CHO Cells After testing for the expression of the soluble receptor, a stable cell line continuously expressing the soluble receptor is constructed. To accomplish this, a dihydrofolate reductase-deficient line was used as the host cell, using the CHO DUK-X line [F. Kao et al., 1969, PNAS USA 64: 1284-1291; Chasin L. and Urlaub G., Proc. Natl. Acad. Sci, 77, 4216-4280 (1980)]. Using this system, the soluble interferon α and β receptor are co-transfected with the pSV2DHFR vector containing the murine DHFR gene [Subrami et al., Molec. Cell. Biol. 1, 854-864 (1981)]. Prior to this co-transfection, all plasmids were linearized by restriction with a restriction enzyme, and prior to transfection, each plasmid was separately mixed with plasmid pSV2DHFR so that the SV2DHFR11

The ratio of the plasmid to the IFR plasmid should be 1:10. Thus, the number of copies of IFR genes per transfectant is maximized. Plasmids are linked in the cell to form polymers that can be integrated into the host cell chromosome by recombination [Haynes and Weismann; Nucl. Acids. Gap. 11: 687-706 (1983); Scahill, S.J., et al., Proc. Natl. Acad. Sci. USA 80: 4654-4658 (1983). Transfectants expressing rat murine DHFT in an α-medium were selected; α-medium is a nucleoside-free medium. Methotrexate (MTX; a toxic folate analogue associated with DHFR) is then added to select cells expressing high levels of DHFR. Resistance to MTX is a consequence of the high expression of DHFR and is often the result of amplification of the DHFR gene, which may contain long chromosome segments called "amplified units" [Kaufman and Sharp, Molec. Cell. Biol. I, 1069-1076 (1981)]. Thus, co-integration of DHFR sequences with soluble α- and β-interferon receptors allows amplification of soluble α- and β-interferon receptor genes.

Stably transfected cell lines were isolated by cloning in selective medium supplemented with 10% fetal bovine serum [DMEM / HAM FI2 1: 1, Gibco]. Clones are then screened for in vivo immunoprecipitation after labeling with ³⁵ C-cysteine to find a clone expressing the most soluble α- and β-β-interferon receptors. Specifically, pSVAdIFRpA or pSVAdsIFRlpA or pSVAdsIFR2pA or pSVAdsIFR3pA or pSVAdpA or pSV2dhfr plasmid transfected about 10 ⁷ CHO cells for 5 hours at 37 ° C with 4 ml RPMI cys medium (Gibco) supplemented with 100 mCi / ml ³⁵ S-cysteine (Amersham) were incubated . After labeling the cells, 1 ml of filtered conditioned medium is adjusted with 0.5 mM phenylmethylsulfonyl fluoride and subjected to immuno-precipitation as described below. The precipitate was fractionated by electrophoresis under reductive conditions on a 7.5% PAGE gel (UK Laemmli, Nature 227: 680-685 (1980)).

Example 3

Purification of soluble α and β interferon receptors

A clone of pSVAdsIFR3pA-structured CHO cells expressing recombinant soluble α- and β-interferon receptors was selected and treated with 1% fetal calf serum (FCS), 1 g / l glucose and antibiotics [e.g. streptomycin and penicillin (100 g / ml)] and DMEM / HAM F12 (1: 1) supplemented with 0.5 mM MTX in 4 bottles (Becton and Dickinson) for 8 days. The supernatant was collected, filtered through a 0.2 µm maxi dish (Gelman Sciences), and the selected protein was concentrated on a FPLC mono Q anion exchange column (Pharmacia). The column was rinsed with a buffer containing 100 mM Heps pH 7, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMFS) and 400 inhibitory units aprotinin kallikrein / ml, followed by a continuous gradient of sodium chloride (0.2). M (1.0 M to 1.0 M). The soluble receptor is capable of binding interferon and subsequently binding the ¹²⁵ iodine-labeled interferon is used to monitor the purity of the receptor.

The receptor-containing material was eluted on a dialyzed mono Q column and concentrated in a Micro-Prodicon. A protein retaining membrane with a molecular weight greater than 10,000 daltons is used. The concentrated material was precipitated on a 3 mm gel containing 7.5% polyacrylamide and 0.1% SDS. Simultaneously, a sample (about 10 pg) is precipitated on the other side of the same gel. Following electrophoresis, this portion of the gel was electrophoresed on a polyvinyl bifluoride membrane (Millipore) and hybridized with ¹²⁵ iodine-labeled recombinant human interferon alpha-8. In this way, the position of the soluble receptor can be identified after electrophoresis. The soluble receptor portion of the gel was excised, concentrated by molecular ultrafiltration, and dialyzed to remove SDS.

Example 4

Construction of plasmids fused to human kappa immunoglobulin constant region and human gl immunoglobulin constant region to express soluble receptor variants of α and β interferons

Light chain fusions

Plasmids for the expression of soluble receptors for interferons α and β are constructed which contain extracellular domains of various lengths fused to the immunoglobulin constant region of kappa. These plasmids are hereinafter referred to as pSVAdsIFRIK, pSVAdsIFR2K and pSVAdsIFR3K.

Plasmid pSVAdsIFRIK contains the N-terminal portion from the methionine initiation codon to the fusion point downstream of lysine 436, followed immediately by the kappa immunoglobulin constant region starting at codon 109 of the human kappa immunoglobulin [Kabat et al., Hieter PA et al. : Cell 22: 197-207 (1980)].

Similarly, plasmid pSVAdsIFR2K contains the N-terminal portion from the methionine initiation codon to the fusion point after glutamate codon 427, which is immediately followed by the kappa immunoglobulin constant region starting at codon 109 of human kappa immunoglobulin [Kabat et al. Hieter, P. A., et al., Cell 22: 197-207 (1980).

These plasmids are constructed as follows.

The kappa immunoglobulin DNA fragment was synthesized from a human spleen cDNA library (Clontech Laboratories, Inc.). For the synthesis of the desired cDNA, oligonucleotides containing sequences complementary to predetermined regions based on the published DNA sequence are used as primers for the PCR reaction (Hieter, P. A., et al., Cell 22: 197-207 (1980)). The 5 'end of the oligonucleotide has the sequence:

'- ACTGTGGCTGCACC ATCTGTCTTCA-3'

EN 215 587 Β while the 3 'end has the sequence:

3 '-CCCTCTCACAATCTCCCTCCATGGCCAG-5' The PCR reaction was performed as described in Example 1 with the exception that 1 μg of plasmid was used as a template and after the reaction the mixture was reconstituted with Klenow polymerase in the presence of 4 triphosphate nucleosides. treated.

The DNA was digested with the restriction enzyme Kpn1 and the desired fragment was isolated by low melting point agarose electrophoresis.

DNA fragments encoding soluble α and β interferon receptor fragments were synthesized in the same manner. The 5 'end of the 5' oligonucleotide is the same as that described in Example 1, i.e., '-CCGGCTGC AGGGATCTGCGGCGGCTCCCAG-3', while the 3 'end of the oligonucleotide is as follows:

3'-CTCTTTTGTTTTGGTCCTTTATGGAGATTT-5 'oligo 1 3'-TCGTCACAAAAATCACAGCGACATACACTC-5' oligo 2 3'-CCACGAGGTTTTGTCAGACCTTTGTGCGGA-5 'oligo 3

The PCR reactions were performed as described in Example 1, except that after the final reaction cycle, the reaction products were treated with Klenow polymerase to repair the ends of the two strands, and then with PstL to generate IFR1, IFR2 and IFR3 fragments. treated.

The pSVAdAl expression vector was sequentially digested with KpnI and PstI followed by phosphatase, and the P-fragment containing most of the plasmid was isolated by low melting point agarose electrophoresis.

In a three-component reaction, the Pffagments are coupled to the immunoglobulin fragment and the sIFR1 fragment. Similarly, the P fragment is linked to the immunoglobulin fragment and the sIFR2 fragment, and finally the P fragment is linked to the immunoglobulin fragment and the sIFR3 fragment.

Each ligation mixture is transformed individually into competent E. coli HB101, the transformed cultures are plated in Petri dishes containing ampicillin medium and the ampicillin resistant colonies are selected. The plasmids were isolated from the transformants and analyzed; in the assay, the insert is determined by restriction enzymes, or sequencing is performed to confirm the DNA sequence of the insert. These plasmids are used to express the soluble receptor fusion of α- and β-interferons.

Heavy chain fusions

Plasmids containing different lengths of extracellular domains fused to the constant region of immunoglobulin gl are fused to express soluble α and β interferons. These plasmids are hereinafter referred to as pSVAdsIFR1gl, pSVAdsIFR2gl and pSVAdsIFR3gl.

Plasmid pSVAdsIFR1g1 contains the N-terminal portion from the methionine initiation codon to the fusion point after codon 436 of lysine, which is immediately followed by the constant region of immunoglobulin gl, beginning at codon 113 of human gl immunoglobulin [Kabat et al; Ellison J.W. et al., Nucl. Acids Slit. 10: 4071-4079 (1982)].

Similarly, plasmid pSVAdsIFR2gl contains the N-terminal portion from the methionine initiation codon to the fusion point after codon 427 of glutamate, which is immediately followed by the constant region of the immunoglobulin gl, starting at codon 113 of human gl immunoglobulin [Kabab et al; Ellison J.W. et al., Nucl. Acids Slit. 10: 4071-4079 (1982)].

Plasmid pSVAdsIFR3gl contains the N-terminal portion from the methionine initiation codon to the fusion point downstream of the proline codon 360, which is immediately followed by the constant region of immunoglobulin gl, beginning at codon 113 of human gl immunoglobulin [Kabat et al; Ellison J.W. et al., Nucl. Acids Slit. 10: 4071-4079 (1982)].

These plasmids are constructed as follows:

The immunoglobulin coding sequence for gl was synthesized from a human spleen cDNA library (Clontech Laboratories Inc.). For the synthesis of the desired cDNA, oligonucleotides are used as primers for the PCR reaction, based on the published DNA sequence [Ellison et al., Nucl. Acids Slit. 10: 4071-4079 (1982)] contain a sequence complementary to predetermined domains.

The 5 'end of the oligonucleotide has the sequence:

5 '-GCCTCCACCAAGGGCCCATCGGTCTTC CCC-3', while the 3 'end has the sequence:

'-GACAGAGGCCCATTTACTCACCATGGC CAG-5'

The PCR reaction is performed as described below.

The IFR1, sIFR2 and sIFR3 fag fragments are the same as described above and the expression vectors are constructed as described above for light chain fusion.

Example 5

Construction of vectors for expression of soluble interferon α and β in E. coli

The expression vector pHR148 was used to express sIFR in E. coli. The pHR148 vector has been described by Rink et al., Nucl. Acid. Gap. 12: 6369-6387 (1984)]. Plasmid HR148 was sequenced with NcoI restriction enzymes and then the ends of the two strands

For the recovery of HU 215 587 Β, Klenow polymerase and KpnI were digested in the presence of 4 triphosphate nucleosides. After treatment with phosphatase, the DNA fragment (fragment 1) containing the major part of the plasmid was isolated by electrophoresis on low melting point agarose.

A DNA fragment encoding the mature soluble receptor for α- and β-interferons (i.e., containing no peptide signal) is a template containing the complete cDNA as a template (UZE et al., Cell 60 (2): 225-234 (1990)). and synthesized using a pair of oligonucleotides as primer.

The 5 'end of the oligonucleotide (derived from the N-terminal region of interferon) has the following sequence:

'-GGTGGAAAAAATCTAAAATCTCCTCAA AAAG-3' (oligo 1), while the 3 'end of the oligonucleotide (derived from the region just prior to the transmembrane region of interferon) is as follows:

'-TC ACTGCGACATACACTCATCCCATGG CC-5' (oligo 2).

The oligonucleotides are selected such that, after PCR fusion, the synthesized fragment contains a Kpn I restriction site at the 3 'end and a "blunt" end at the 5' end.

The PCR reaction was performed as described in Example 1 except that after the reaction, the products were treated with Klenow polymerase followed by KpnI to repair the ends of the two strands. The 1.3 kb fragment was isolated by low melting point agarose electrophoresis and ligated to fragment 1.

A second DNA fragment is similarly synthesized by the replacement of the 5 'end oligonucleotide with the sequence AG-3' - GG A AA AATCTA A AATCTCCTC AAA.

The ligation products are transformed into E. coli strain HB101 and the transformed cultures are poured into a petri dish containing ampicillin medium. Ampicillin-resistant colonies were selected, plasmids were isolated from the transformants and analyzed; digestion with restriction enzymes and sequencing to confirm the fusion point of the insert.

The plasmids ptrpIFR1 and ptrpIFR2 are used to express the soluble receptor in E. coli. To express the recombinant protein, transformants were obtained in 20-50 ml of culture medium in M9 medium to obtain a 1.0 absorbance (A650) value [Rink et al., Nucl. Acid. Gap. 12: 6369-6387 (1984)]. The cells are then centrifuged, washed, and the soluble interferon receptor extracted from the disrupted cells and purified. Soluble interferon-α and β-receptor were determined by Western biot or biot analysis as described below.

Example 6

Methods for determining the soluble receptor for human interferon

Methods for determining the soluble receptor are based on the ability of the receptor to bind specifically to human interferon alpha.

(a) Immunoassay

The determination of the soluble receptor for human α- and β-interferons is based on the formation of a complex between the soluble receptor and ¹²⁵ iodine-labeled human interferon α-8, which is directed against the ¹²⁵ I-IFN monoclonal terminal portion of interferon α-2. inhibits its binding to an antibody [H. Amheiter et al., Proc. Natl. Acad. Sci. (USA) 80, 2539 (1983). Once bound to the interferon receptor, the terminal amino portion of the molecule is not accessible to the antibody [H. Amheiter et al., 1983]. The remaining ¹²⁵ I-IFN is complexed with the anti-IFN antibody and precipitated by the addition of A-sepharose protein (Pharmacia). The anti-IFN antibody is conjugated to protein A according to the manufacturer's instructions.

Serial dilutions of soluble receptors to be determined in 50 μΐ borate buffer (pH 8.3; 0.1 MH ₃ BO ₃ , 0.025 M Na ₂ B ₄ O ₇ , 0.075 M NaCl, 0.1% NP40) with ¹²⁵ Iodine with specific activity of 81 Bq per ffnol (75 pmol) of labeled α-2 interferon (KE Mogensen and G. Uze, Methods in Enzymol. 119C, 267 (1986)] and incubated for 1 hour at 4 ° C. A constant volume of A-anti-IFN protein was added in 50 μΐ volume with 50 μΐ borate buffer pH 8.3; 0.2 MH ₃ BO ₃ , 0.05 M Na ₂ B ₄ O ₇ , 0.15 M NaCl , 0.1% NP40). After stirring for one hour at 4 ° C, the mixture is centrifuged in an Eppendorf microcentrifuge at 10,000 g for one minute and the supernatant discarded. The precipitate was borne six times with borate buffer, pH 8.3; 0.1 MH ₃ BO ₃ , 0.025 M Na ₂ B ₄ O ₇ , 1 M NaCl, 0.5% NP40, and 2% Wash twice with glycerol, once with 40 mM HEPES (pH 8) and 1 M urea, and finally twice with 40 mM HEPES (pH 8). Counting the radioactivity of the immune complex in the precipitate, as compared to the inhibition curve of the interferon-anti-interferon complex, allows the amount of soluble α- and β-interferon receptor to be determined in the sample so tested.

b) Determination of soluble receptor for human α and β interferons by inhibiting the binding of labeled interferon to the cell receptor

The a- and β-interferons, soluble receptors of serial dilutions for 30 min at 4 ° C with labeled recombinant ^l25 iodine (specific radioactivity 25 Ci per fmol) human α-interferon 8 Varying concentrations of the quantities of (0.50, 100, 125, 500, 1000 and 2000 IU / ml). Daudi human lymphoid cells are then added to various ¹²⁵ I-IFN soluble receptor mixtures at a final concentration of 10 ⁷ cells / ml and incubated for 2 hours at 4 ° C. The cells were centrifuged (800 g for 10 minutes) and the supernatant discarded. Then washed three times with RPMI 1640 medium containing 0.5% fetal bovine serum (Flow Laboratories) and added to the cells.

Bound radioactivity is determined by counting on a LKB 1270 gamma counter.

The amount of soluble receptor for α and β interferons is determined from the inhibition curve of specific binding of interferon to the cell receptor. Specific binding is the difference between the binding curves of ¹²⁵ I-IFN alone and 100 times unlabelled excess interferon.

c) determination of the soluble receptor for α and β interferons by the Western biot method

The sample to be tested for the presence of the α-interferon receptor is concentrated using an Amicon Centricon 30 membrane retaining protein with a molecular weight greater than 30,000 daltons. Samples were taken up in 60 mM Tris-HCl buffer (pH 6.8; 1.25% SDS, 1 mM PMFS and 400 inhibitory units kallikrein / ml aprotinin / ml) and 7.5% polyacrylamide gel containing 0.1% SDS. After electrophoresis under standard conditions, the samples were polyvinidyl in a buffer containing 39 mM glycine, 48 mM tris base (pH 8.3) and 18% methanol in an "semi-solid" type electrotransfer (Nova Biot) at 200 mA for one hour. Bifluoride membrane (Millipora) After transfer, the membrane was agitated at 25 ° C with 20 mM tris base, 0.2% Tween 20 (pH 7.8) and 10% skimmed milk (Regilait, France). Stir at C for 4 hours, then hybridize with 10 ¹⁰ M ¹²⁵ Ia-8-interferon (specific activity 50 Bq per Phenol) for 2 hours at 25 DEG C. with stirring and wash the membrane five times with aliquots of the same buffer. then air-dried and exposed to X-rays y film.

Recombinant human α-8- (α-8) -interferon is labeled with iodine, the chloramine method described above [K. E. Mogensen and G. Uze, Methods in Enzym. 119C, 267 (1986)]. This corresponds to a specific radioactivity of 25 Bq per fmol or 6 Bq per international interferon unit.

(d) Determination of the soluble receptor for human α and β interferons by the 'dot biot' method

Serial dilutions of the soluble receptor preparations to be assayed were made in 50 μΐ buffer containing 39 mM glycine and 48 mM tris base (pH 8.3) and sedimented in wells of a Bio-Biot device (Biorad) filled with polyvinyldilifluoride membrane (Millipore). forget it.

The membrane was treated with a buffer containing 20 mM tris base, 0.2% Tween 20 (pH 7.8) and 10% skimmed milk (Regilait, France) for 4 hours at 25 ° C. Then, in the same ^{buffer, 25} I-IFN α-8 10 with ¹⁰ M (specific activity 25 Ci per fmol) at 25 ° C for 2 hours and hybridized with stirring, and the above buffer and aliquots were washed five times and parts of stirring exposed to X-ray sensitive film. By autoradiographic analysis and comparison with a standard curve, the amount of ¹²⁵ I-IFN bound to the membrane can be determined and the concentration of soluble receptor in the test sample can be determined.

Labeling of recombinant human α-8- (α-8) -interferon-1-iodine was performed using the chloramine T method [K. E. Mogensen and G. Uze, Methods in Enzymol. 119C. 267 (1986)]. This corresponds to a specific radioactivity of 25 Bq per fmol or 6 Bq per international interferon unit.

Example 7

Medical preparation

An injection composition is prepared as follows:

Component Quantity

Example 3 100 mg

Excipients for injection can be used in 2 ml

Claims (10)

PATENT CLAIMS A process for water soluble polypeptides, namely a) a polypeptide having the amino acid sequence shown in Figure 1 and its derivatives formed by deletion and / or substitution, b) polypeptides encoded by the nucleic acid sequence shown in Figure 1, and c) for producing derivatives of the polypeptide deletion and / or substitution of the amino acid sequence shown in Figure 2, either alone or in the form of hybrids with a second polypeptide, characterized in that the cells expressing the polypeptide or hybrid are cultured in a suitable medium; . The method of claim 1 for preparing a water-soluble polypeptide as defined in claim 1 in the form of a hybrid with a plasma protein such as a second polypeptide having a plasma half-life greater than the first polypeptide, characterized in that cells capable of expressing said hybrid are cultured in a suitable medium. and isolating the express product. 3. The method of claim 2 for producing hybrids with an immunoglobulin as a second polypeptide, wherein cells capable of expressing said hybrid are cultured in a suitable medium and the expressed product is isolated. 4. The method of claim 3, wherein said second polypeptide is a hybrid of type G, preferably G1, immunoglobulin, comprising culturing cells capable of expressing said hybrid in a suitable medium and isolating said expressed product. 5. Procedure 1-4. DNA sequences for encoding polypeptides and hybrids as defined in any one of claims 1 to 3, characterized in that the appropriate DNA fragments and / or nucleic acids are linked in the correct order by known genetic engineering techniques and the resulting product amplified by polymerase chain reaction (PCR). 6. Procedure 1-4. A method for producing a cell for the production of polypeptides and hybrids as defined in any one of claims 1 to 3, characterized in that a cell Is transferred with a DNA sequence as defined in claim 5. A process for the preparation of a pharmaceutical composition comprising the steps of: 1-4. The polypeptide or hybrid of any one of claims 1 to 6 is formulated into a pharmaceutical composition using pharmaceutically acceptable carriers, diluents and / or other excipients, using known pharmaceutical technology. 8. A method of producing antibodies to an interferon receptor comprising the steps of: 1-4 in warm-blooded animals. The polypeptide or hiride produced according to any one of claims 1 to 6 and a pharmaceutically acceptable substrate is administered, and after the immune response has been completed, the animal is bled and the antibodies are isolated from the blood. 9. A method of detecting the presence of antibodies to the α and β interferon receptor in biological samples, comprising contacting the biological sample with a substance that forms an immunological complex with the antibody under conditions that allow the formation of the complex, followed by the immunological complex formed. 10, characterized in that as a complexing agent according to any one of claims 1-4. The polypeptide or hybrid of any one of claims 1 to 6 used.