Deoxyribonucleic acids, recombinant deoxyribonucleic acids, hosts containing them, polypeptides and process for their production.

Field of invention

The invention relates to deoxyribonucleic acids which can be derived from human lymphoblastoid cells and which contain the code for interferon-like polypeptides, recombinant deoxyribonucleic acids (vectors) which contain corresponding deoxyribonucleic acid sequences as inserts, hosts which are transformant and polypeptides with the recombinant deoxyribonucleic acids Interferon-like activities. The invention further relates to methods of producing said deoxyribonucleic acids, said recombinant deoxyribonucleic acids, said hosts and said interferon-like polypeptides using recombinant deoxyribonucleic acid technology. The polypeptides according to the invention are useful as immunomodulators, in particular as antiviral, anti-tumor and anti-cancer agents. In accordance with this, the invention further relates to pharmaceutical preparations containing these polypeptides and methods for the treatment of viral infections, tumors and cancer.

Background of the Invention

In the following, the expression "DNA" is used in analogy to the Anglo-Saxon notation for "deoxyribonucleic acid". The term "DNA", which is common in the German language and is derived from the German word deoxyribonucleic acid, is generally not used according to general practice in the present technological field.

Interferons ("IFN") are a group of mostly glycosylated polypeptides with molecular weights in the range from 10,000 to 40,000. They are used by vertebrate cells when exposed to an IFN inducer, such as a virus, a double-stranded RNA (ribonucleic acid), intracellular microbes , Microbial products or various chemical agents (1). IFNs are generally not found in normal, healthy cells. They support the healthy cells of the vertebrate in their defense against viral infections and other attacks. IFNs were found to have immunomodulatory activities.

The nomenclature for interferon polypeptides has not yet been clearly established. According to the current recommendations (2), the classification is based on animal origin (for example "Hu" for human origin), the antigenic specificity (types a, ß,^y, etc. on the basis of their antigen-antibody reactions with a, β or Y-IFN antibodies), the structural and physiological differences (subtypes are indicated by Arabic numbers, for example a_l, a_2' etc.) and the type of the original cells (Le is derived from leukocytes, Ly is derived from lymphoblastoid cells, F is derived from fibroblasts, etc.). For example, HuIFN- &₁(Ly) or HuLyIFN- &₁ an a-type interferon of subtype 1, which is derived from human lymphoblastoid cells. In addition to these designations, it is sometimes necessary to indicate whether the interferon was obtained directly from its progenitor cell or by synthesis in a microorganism and whether the interferon belongs to a special subtype or is a mixture of two or more interferon subtypes.

The nomenclature regarding DNA sequences coding for IFN polypeptides, recombinant DNA molecules and hosts containing them has also not yet been clearly established. The nomenclature used for these DNA sequences indicates, in abbreviated form, the interferon that they encode. For example, Escherichia coli HB 101 [Z-pBr 322 (Pst) / HcIF-2h] means the bacterial strain (E. coli HB 101) which contains the recombinant plasmid DNA Z-pBR 322 (Pst) / HcIF-2h, ie the plasmid pBR 322, which at the Pst I site (insertion site of the foreign DNA) has an HcIF &_l- £ insert, which comes from Zurich (Z). The letter "H" indicates human origin, "c" means a complementary DNA and &₁ stands for the subtype [compare C. Weissmann, (3)]. At the specified nomenclature^pThe source of the IFN genes (leukocytes) is not specified. A similar nomenclature is used in the present application, however the source for the IFN genes (lymphoblastoid cells) is given.

So far, three classes of HuIFN have been identified: HulFN-a, HuIFN-ß and HuIFN-y. HulFN-a (which was formerly referred to as LeIFN, leukocyte interferon, or LyIFN, lymphoblastoid interferon) is derived from human leukocytes (fresh cells obtained from the blood of human blood donors) and by lymphoblastoid cells upon induction, for example with a virus [ EAHavell et al. (4) and AD Sagar et al. (5)]. It is stable at pH 2 (IFNe, which are stable to acid, were formerly referred to as "Type I") and contains a mixture of individual interferon polypeptides, which mainly differ in their degree of glycosylation [eg MRubinstein et al., (6)] and differ in the amino acid composition (see the following remarks). Of the two components isolated and purified so far, one with a molecular weight of 15,000 to 18,000 is not glycosylated, while the other with a molecular weight of 21,000 to 22,000 is glycosylated. WEStewart, II et al. (7) report that most or all of its HuIFN-a activity has been retained in the non-glycosylated interferon. Parts of the amino acid sequence of HuIFN-a from lymphoblastoid cells have also been described in the literature [KCZoon et al., (8)]. Various forms of HuIFN-a, which differ structurally and physiologically, are also known. Particular human individuals can form allelomorphic variations of HuIFN-a.

HuIFN-ß (formerly FIFN or FiIFN, "Type I") is induced by human fibroblasts (eg cells from the foreskin of newborns) when induced with a ds RNA and to a small extent together with HuIFN-a from human lymphoblastoid cells when induced with a Virus formed. HuIFN-ß is also stable at pH 2 (therefore it belongs to "Type I"). At least two types of HuIFN-ß have been described so far (33,48). The molecular weights are approximately 20,000 to 22,000. The amino acid sequence is partially known.

HuIFN-y [formerly referred to as IIFN (immune interferon or "type II" interferon)) is produced by T lymphocytes in response to antigens or mitogens. It is acid labile at pH 2 and differs serologically from HuIFN-a and HuIFN-ß.

HuIFNe are useful antiviral, anti-tumor and anti-cancer agents. "

As antiviral agents, they can be used for the treatment of viral infections of the respiratory tract, herpes simplex keratitis, acute hemorrhagic conjunctivitis, varicella zoster, hepatitis B, cytomegalic inclusion diseases and others.

As anti-tumor or anti-cancer agents, HuIFNe can be used to treat, for example, osteosarcoma, acute myeloid leukemia, multiple myeloma, Hodgkin's disease, melanoma, breast cancer, lymphosarcoma and papilloma, and others.

HuIFNe can be used in the form of pharmaceutical preparations for oral or parenteral administration, for example as pharmaceutical preparations for topical, intravenous, intramuscular, intranasal, intradermal or subcutaneous administration, for example in the form of tablets, ampoules, syrups, solutions or suspensions for oral administration Administration, powders, solutions for injection or infusion or suspensions, eye drops, ointments, sprays, etc.

The preparations are generally administered, for example intramuscularly, one to three times a day in dose amounts of about 10⁶ until about 10⁷ Units administered, the treatment depends on the disease, the type of application and the condition of the patient. Viral infections are generally treated daily or up to three times a day for several days to several weeks, whereas tumors and cancer are treated either one to several times a day or two or more times a week for several months or years.

To date, HuIFN-a can only be produced in insufficient amounts via induced human cells, for example human lymphoblastoid cells (for example from Burkitt's lymphoma "Namalwa" cells) or human leukocytes which are obtained from the fresh blood of blood donors. HuIFN-ß is mainly obtained from human fibroblasts. It has been described in the literature that only 2.6 x 10⁹ IU of crude HuIFN-a can be obtained from 800 l of Namalwa cells grown and that only about 10 ¹¹ IE raw HuIFN-a can be obtained annually in very large blood centers, e.g. the Finnish Red Cross Center in Helsinki. The specific activity of HuIFN-a is on the order of about 4 x 10⁸ until 10⁹_-IU / mg. The amount of HuIFN-a needed for an extensive and commercial_Aapplication would be very low compared to other pharmaceutical compounds.

100 g of pure HuIFN-a would result in 10 to 30 million dose units. However, such amounts cannot be industrially produced using human tissue culture and human leukocyte technology at an acceptable cost.

It is another disadvantage of these large scale manufacturing processes that only mixtures of interferons are obtained. It is difficult and expensive to separate these mixtures into individual subtypes. The therapeutic applications of pure, individual interferon species have therefore not yet been satisfactorily determined.

The industrial application of these methods is further limited to HuIFNe, which is of human cells that can be grown (such as human tumor cells and certain fibroblast cells), or of human cells that are available in relatively large quantities (such as leukocytes and lymphocytes). However, all of these methods are expensive and complicated.

It has been recognized that the solution to the problem of industrial synthesis of large amounts of individual interferon species could lie in the advances in molecular biology that have made it possible to express a specific, non-bacterial, eukaryotic gene in bacterial cells. Recently, SN Cohen and HW Boyer (9) described a general procedure for the replication of biologically functional DNA sequences. This method comprises the steps of cleaving circular plasmid DNA, whereby a first linear DNA segment is obtained; inserting a second linear segment of DNA with a gene for a phenotypic trait into this first segment to obtain a recombinant DNA molecule (a modified circular plasmid); transforming a unicellular microorganism with this recombinant DNA molecule; growing the transformants together with the non-transformed microorganisms under suitable nutritional conditions; and separating the transformants from the unicellular parent microorganisms. The transformants can then produce the desired protein.

The problem of producing a linear DNA sequence coding for interferon and a recombinant DNA therefrom: was not solved by Cohen and Boyer.

Description of the prior art

Various patent applications and other publications have identified DNA sequences that differ from human leukocytes and fibroblasts _Pencode olypeptides with HuIFN-a and HuIFN-B-like activity, derive recombinant DNA molecules which contain these DNA sequences, hosts which have been transformed with these recombinant DNA molecules, polypeptides or culture liquids which contain such polypeptides included, and processes for the preparation of these substances described.

Using human leukocytes induced with Sendai virus as the starting material, various polypeptides with HuIFN-a-like activity, DNA sequences, recombinant DNA molecules and hosts for their production have been described by C. Weissmann [(3); see also S. Nagata et al. (10), N. Mantei et al. (11) and M. Streuli et al. (12)].

A partially purified HuIFN-a-like polypeptide with a molecular weight of 21,000, also derived from leukocytes, in particular from the human myeloblastoid cell line KG-1 induced with Sendai virus, with the corresponding DNA and the structure of eight specific human ones LeIFN cDNAs are described by DV Goeddel et al. (13, 14).

A method for producing a polypeptide with HuIFN-a-like activity using human lymphoblastoid Namalwa cells or human blood leukocytes, both of which are induced with Newcastle disease virus, is described in general form in European Patent Application No. 34 307 ( 15), but no concrete values ​​or details are given.

In European Patent Application No. 28 033 [(16); see also Taniguchi et al. (17), Derynck et al. (18) and Goeddel et al. (19)] starting from human fibroblasts, in particular those which are obtained from the foreskin of newborns and which are induced with poly (I): poly (C), DNAs, recombinant DNA molecules which code for HuIFN-ß, and microorganisms containing them.

mRNAs, DNAs, recombinant DNA molecules and bacterial strains, the HulFN-ß_land HuIFN-B2 can be obtained by genetic engineering using human fibroblasts, especially those from FS11 or SV80 foreskin cells, which are induced with double-stranded poly (I): poly (C) [cf. GE Patent Application 2,063,882 (20)].

DNAs, recombinant DNA molecules, E. coli bacteria containing the latter, and polypeptides with the activity of HuIFN-ß, produced via mRNA, which is induced from human fibroblasts, in particular human foreskin FS-4, with poly (I) : Poly (C), and processes for their preparation are also described in BE-PS 887 397 (21).

In general form, the process for the production of a polypeptide with HuIFN-β-like activity using human fibroblasts (foreskin of newborns) which are induced with poly (I): poly (C) is described in European patent application 34 306 (22). described without concrete values ​​or details being given.

Human lymphoblastoid interferon: "HuIFN (Ly)" can be obtained in different amounts from Namalwa cells when stimulated with different inducers [MDJohnston et al. (24)] can be produced. It was shown that HuIFN (Ly) made from Namalwa cells induced by Sendai virus, HuIFN - & (Ly) (70-90%) and HuIFN-ß (Ly) (10-30 %) contains. It consists of at least seven components [KCZoon et al. (8) and G.Allen et al. (26); see also EAHavell et al. (4) and AD Sagar et al. (5)]. Although the overall structure of the lymphoblastoid interferon polypeptides is currently still unknown, it is evident that HuIFN-a (Ly) differs from HuIFN-a (Le). The main components of the lymphoblastoid interferon do not appear to be or only slightly glycosylated. The various components have not yet been separated and cleaned.

Object of the invention

Since clinical trials have been carried out with mixtures of lymphoblastoid HuIFNs, it is desirable to separate the various components and to manufacture them individually so that their therapeutic potential can be determined. None of the recombinant DNA methods described in the literature relate to the synthesis of human lymphoblastoid interferons. The object of the present invention is to solve this problem by means of recombinant DNA technology. According to the invention, the structure (amino acid sequences) of the various subtypes of HuLyIFNs is also to be clarified, and methods are to be made available which allow the internal_dindividual interferons can be produced in sufficient quantities to ensure the treatment of a large number of possible patients.

The present invention solves the problems of producing (even in large quantities) individual polypeptides that have biological activity similar to that of lymphoblastoid HuIFNs. The individual polypeptides are either the same or different from the HuIFN-a (Le) and HuIFN-ß components known to date.

The invention further relates to pharmaceutical preparations containing polypeptides which have the immunological and biological activities of HuLyIFN-a or HuLyIFN-ß and their methods of use.

Terms and abbreviations used in the present description and in the claims

Clone: ​​A population of cells that derives asexually from a single cell. Such a population is believed to be genetically identical.

Operon: A genetic unit consisting of neighboring genes that are coordinatively expressed under the control of an operator and a repressor.

Expression Control Sequence: A sequence of nucleotides that controls and regulates the expression of structural genes when operatively linked to these genes. It includes the promoter and the ribosomal binding site inter alia.

Promoter: A DNA segment that binds the RNA polymerase and initiates the transcription.

Ribosomal binding site: sequences that allow the binding of the mRNA to ribosomes, a prerequisite for translation.

Expression: A method which includes transcription and translation.

Transcription: A method which involves base pairing, wherein the genetic information contained in the DNA is used to place a complementary base sequence in an RNA chain.

-Translation: The process in which the genetic information present in the mRNA molecule determines the order of the specific amino acids during protein synthesis.

Nucleotide: The building block of nucleic acids, which contains a purine or a pyrimidine base, a ribose or a 2-deoxyribose residue and a phosphoric acid residue. The ribonucleotide bases are A, G, C and U, whereas in deoxyribonucleotides U is replaced by T.

Vector (or cloning^Gvehicle): A DNA sequence, e.g. a plasmid or a phage DNA, which can replicate autonomously in a host cell and which contains a marker which is suitable for the identification of the transformed cells, e.g. tetracycline resistance or ampicillin resistance, and on which another DNA segment can be bound experimentally, so that replication of the bound segment is possible.

Plasmid: An extrachromosomal, circular, double-stranded DNA, which can multiply in the host cell.

Recombinant (hybrid) DNA:

A DNA molecule, which consists of DNA segments derived from different genes, which have been linked outside of living cells and which have the ability to infect and maintain certain host cells.

Nucleases: Enzymes that cleave the phosphodiester bonds of nucleic acid chains.

Ribonucleases (RNAsen): Enzymes that cleave the phosphodiester bonds of RNA.

Deoxyribonucleases (DNAsen): Enzymes that cleave the phosphodiester bonds of DNA.

Restriction endonucleases: enzymes that cut polynucleotides at specific target sequences within the polymer chain. The cleavage products result in DNA fragments with "blunt" or "flushed" ends or "staggared" or "sticky" ends.

Exonucleases: Enzymes that cleave DNA from the ends of the strands.

Lysozyme: Enzymes that break down the polysaccharides found in the cell walls of certain bacteria.

Reverse transcriptase: An enzyme that is encoded by RNA tumor viruses and that is able to generate complementary single-stranded DNA chains from RNA templates and then convert these DNA chains into double-wound form.

DNA polymerases: enzymes that catalyze the formation of DNA 3'-5'-phosphodiester bonds.

DNA ligase: An enzyme that catalyzes the repair of a single-stranded DNA-phosphodiester bond break of the type introduced by an endonuclease.

Polynucleotide Kinase: An enzyme that catalyzes the phosphorylation of the 5'-hydroxyl groups of DNA.

Transformation: The introduction of an exogenous DNA, eg a plasmid or hybrid DNA, into a cell, followed by the establishment of the DNA within the cell.

Abbreviations (according to general practice, if the Anglo-Saxon and German abbreviations differ, the Anglo-Saxon abbreviations are used)<ul id="ul0001" list-style="none"><li>A adenosine or deoxyadenosine monophosphate residue</li><li>U uridine monophosphate residue</li><li>T deoxythymidine monophosphate residue</li><li>C cytidine or deoxycytidine monophosphate residue</li><li>G guanosine or deoxyguanosine monophosphate residue</li><li>I inosine monophosphate residue</li><li>dATP deoxyadenosine triphosphate</li><li>dTTP deoxythymidine triphosphate</li><li>dCTP deoxycytidine triphosphate</li><li>dGTP deoxyguanosine triphosphate</li><li>dCMP deoxycytidine monophosphate</li><li>dGMP deoxyguanosine monophosphate</li><li>RNA ribonucleic acid</li><li>mRNA messenger ribonucleic acid</li><li>tRNA transfer ribonucleic acid</li><li>rRNA ribosomal ribonucleic acid</li><li>ds RNA double stranded ribonucleic acid</li><li>DNA deoxyribonucleic acid</li><li>cDNA complementary deoxyribonucleic acid (enzymatically synthesized from an mRNA sequence)</li><li>ds cDNA double-stranded, complementary deoxyribonucleic acid</li><li>ApPr expression control sequences of the ß-lactamase gene IFN interferon</li><li>HuLy (from) human lymphoblastoid cells.</li></ul>

Detailed description of the invention

The invention relates to a DNA, in particular a recombinant DNA, which contains a DNA sequence which can be derived from human lymphoblastoid cells, or a fragment, a variant or a mutant of this sequence and which codes for an interferon-like polypeptide, or a DNA, which hybridizes with said DNA, a host which is transformed with at least one of the recombinant DNAs mentioned, a polypeptide, which has the immunological and biological activity of human lymphoblastoid interferon, or a fragment or a derivative thereof, a pharmaceutical preparation containing said polypeptide and a method for the treatment of viral infections, cancer or tumors in humans, characterized in that humans an effective amount of the polypeptide is administered in the form of said pharmaceutical preparation.

The invention further relates to a method for producing a polypeptide which has the immunological and biological activity of a human lymphoblastoid interferon, which is characterized in that a host which is associated with at least one of the recombinant _DNAs is transformed, grown, and that the desired polypeptide is recovered, and a method for producing a transformed host microorganism.

The process for producing a transformed host microorganism comprises the steps<ul id="ul0002" list-style="none"><li>(1) isolation of human lymphoblastoid poly (A) RNA from induced human lymphoblastoid cells and their enrichment in HuLyIFN mRNA,</li><li>(2) production of a single-stranded, complementary DNA from this template and from it a double-stranded cDNA,</li><li>(3) introduction of the ds cDNA into a suitable vector DNA,</li><li>(4) transforming a suitable host microorganism with the recombinant DNA obtained,</li><li>(5) Culturing the host microorganism and selecting the clones transformed with human lymphoblastoid IFN cDNA or DNA fragments and, if necessary, isolating the recombinant D_NAs from the transformed host, optionally modification of the recombinant DNAs to increase the Asu yield of polypeptides with IFN activity and carrying out steps (4) and (5) again. The invention further relates to the individual stages and the combination of two or more of the individual stages.</li></ul>

1. Induction of human lymphoblastoid cells and isolation of human lymphoblastoid poly (A) RNA, enriched in HuLyIFN mRNA

Human lymphoblastoid poly (A) RNA enriched in HuLyIFN mRNA can be isolated using various methods known per se. The process used in the present invention comprises the following stages:<ul id="ul0003" list-style="none"><li>a. Induction of human lymphoblastoid cells for LyIFN synthesis,</li><li>b. Destruction of the induced cells,</li><li>c. Separation of the lymphoblastoid poly (A) RNA from the contaminating proteins, lipoproteins, DNAs and RNAs of other types,</li><li>d. Enrichment of LyIFN-specific mRNA.</li></ul>

a. Induction of human lymphoblastoid cells to the L

Y

IFN synthesis

As a result of exposure to an IFN inducer, human lymphoblastoid cells form LyIFN mRNA and then human LyIFN.

Suitable IFN inducers are, for example, various chemical agents, a double-stranded RNA, for example poly (I): poly (C), or specifically certain viruses, above all members of the paramyxovirus, pseudomyxovirus and reoviridial families, such as Newcastle Disease Virus (Newcastle Disease virus) (e.g. strains 110, B1, La Sota or Texas), _'Sendai virus, blue tongue virus _Masernvirus, mumpsvirus, para-influenza virus type I, II or III or Semliki Forest Virus.

Preferred human lymphoblastoid cells are those derived from Burkitt's lymphoma patients. Such a cell line, Namalwa, has been shown to produce high levels of IFN when virally stimulated (26). In addition to the Namalwa cells, other lymphoblastoid cells can be used, e.g. B. Daudic cells, Acuba cells, NC-37 cells, RN-2 cells and other cells known in the art.

Before induction, the lymphoblastoid cells can be pretreated with a lower straight chain alkanoic acid, such as butyric acid, or one of its salts, which are known to activate IFN formation by lymphoblastoid cells (27, 28). If necessary, the lymphoblastoid cells can be stimulated by treatment with a small amount of homologous IFN.

The induction of the lymphoblastoid cells takes place in a manner known per se in accordance with the analog methods described in the literature. The lymphoblastoid cells, e.g. B. Namalwa cells, are in a known nutrient medium (eg RPMI 1640 medium), which is supplemented with approximately 10% fetal calf serum, up to a sufficient cell density (eg 10th⁶ until 10⁷ Cells / ml). The cells recovered from the medium by centrifugation are resuspended in the nutrient medium and at a concentration of about 200 by adding suitable viruses, for example Newcastle Disease Virus_Haem-agglutination units / 10⁶ Cells induced for a sufficient time, for example 5 to 16 hours. As soon as the induced cells have generated IFN with sufficient titer, the cells are harvested and further, as in the following_'described, treated. The IFN activity can be determined, for example, according to the dye uptake method developed by Armstrong (29).

b. Destruction of the induced cells

The first step in isolating a nucleic acid is to dissociate from the other cellular components and involves destroying the cells and removing the contaminating proteins. Methods suitable for cell destruction include repeated freezing and thawing, mechanical destruction, e.g. homogenization with a motor-driven Teflon pestle in a glass homogenization device, breaking by osmotic shock, destruction by means of ultrasound vibration and the use of lytic, chemical agents, such as anionic detergents, for example sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium 4-aminodosalicylate, sodium 4-aminodecylate Sodium triisopropylnaphthalenesulfonate.

In certain cases, the use of anionic detergents causes a partial release of the nucleic acids from the protein complexes and a partial inhibition of the RNAse activity. The method is preferably carried out using a high concentration of detergent and a short exposure time in order to achieve a complete release of the nucleic acids, in particular RNA, with minimal degradation.

The induced cells are preferred with _Hlysed using a suitable, anionic detergent, for example sodium dodecyl sulfate, in a buffer solution known per se, for example in TNE. After a short exposure time, the suspension is treated with a deproteinizing agent as described in paragraph c.

'c. Separation of lymphoblastoid poly (A) RNA from contaminating proteins, lipoproteins, DNAs and RNAs of other types

The deproteinization of the nucleic acid mixture obtained can be carried out by the action of chemical agents, for example

Chloroform containing 1 to 4% 1-pentanol, or especially phenol. The proteins can also be removed by digestion with a protease, for example pronase or protease P, which digests almost every protein to amino acids. To ensure complete removal of the proteins, a combination method in which two chemical deproteinizing agents, e.g. Phenol and chloroform, or treatment with a protease followed by a chemical deproteinizing agent, e.g. As phenol can also be used.

In particular, the deproteinization of the nucleic acid mixture takes place by incubation with a protease, for example

Pronase, and repeated extraction of the resulting mixture with phenol and then with chloroform.

Using the phenol system, almost all denatured and digested proteins are transferred to the phenol and intermediate phases.

These and the subsequent steps for the production of poly (A) RNA can be checked with regard to the mRNA degradation by adding a small amount of a radioactively labeled marker mRNA, for example 125 I-labeled globin mRNA.

Purified deoxyribonuclease (free from RNAse) can be used to digest the contaminating DNA.

. Alternatively, the RNA can be freed of contaminating DNA by performing equilibrium centrifugation in CsCI gradients. Furthermore, the RNA can be separated from DNA by means of chromatographic methods, for example by means of hydroxyapatite column chromatography.

The mRNA molecules present in the purified solution differ from other RNA species, eg tRNA or rRNA, by a long, uninterrupted sequence of adenosine nucleotides (100 to 200 residues long) at their 3 'ends. These poly (A) chains can be used to select the mRNA in a manner known per se, for example by repeated, batchwise adsorption on oligo (dT) cellulose or poly (U) -separose. The bound poly (A) RNA is then washed through a few washes with a low ionic strength solution, e.g. B. eluted with water.

A preferred method for the isolation of poly (A) RNA comprises, for example, treating the nucleic acid mixture obtained after the lysis of the induced cells (step 1b) with a protease, extraction with phenol and then with chloroform to obtain the denatured and remove digested proteins, subject the resulting solution to oligo (dT) cellulose chromatography and elute the bound poly (A) RNA with water. If necessary or if necessary, the adsorption on oligo (dT) cellulose can be repeated several times.

At this point, the poly (A) RNA can be tested for its capacity to direct the synthesis of polypeptides that have HuIFN activity in an in vitro translation system (eg: reticulocyte translation system, Xenopus laevis). The IFN-specific polypeptides can be identified using a radio immunoassay or, in particular, a cytopathic bioassay. For this purpose, a_'Sample of the poly (A) RNA obtained dissolved in a suitable solvent, for example water, a dilute (for example 1 mM) EDTA solution or a buffer mixture known per se and in oocytes of the African claw toad (Xenopus laevis) according to Colman et al ( 30) micro-injected. The IFN formed in the oocytes can be determined by a cytopathic bioassay, e.g. using the dye binding assay according to Armstrong (29), or by reducing the cytopathic effect according to Stewart et al. (31) using a suitable challenge virus, for example vesicular stomatitis virus (VSV), on a human cell line, for example CCI-23 cells or Hep-2 cells. If desired, the HuIFN mRNA activity of an RNA which has been isolated in any desired purity state or which is derived from a corresponding DNA, for example a double-stranded cDNA, can be determined by means of one of the assays mentioned at any point in the process described.

d. Enrichment of LyIFN-specific mRNA

After removing the other contaminating _RNA-_Species, eg tRNA, rRNA, or DNA (see paragraph 1c), the solution of poly (A) RNA in 0.5 to 1 mM EDTA can be further extracted with phenol and passed through a Chelex column to remove divalent cations getting cleaned.

This poly (A) RNA fraction can be enriched for lymphoblastoid HuIFN mRNA by various methods known per se from the literature. It is essentially based on the different molecular sizes of the individual mRNA species in question.

The fractionation of the poly (A) RNA according to the size can be carried out, for example, by gel filtration on columns _'DExtra derivatives or polyacrylamide take place, the smaller RNA molecules penetrating the gel particles to different degrees, whereas the large molecules are not retained and easily pass through. Furthermore, a mixture of poly (A) RNA species can be fractionated by zone electrophoresis with polyacrylamide, starch or agarose gels. Poly (A) RNAs can also be separated according to their sedimentation rate by zone centrifugation with sucrose density gradients using sucrose solutions of about 5 to 23% as gradients.

For example, the fractionation of the poly (A) RNA mixture can be carried out as follows. The contaminating DNA and RN_ASpecies-freed poly (A) RNA solution is fractionated according to the molecular size by centrifuging through sucrose density gradients (e.g. 5 to 23%) in a buffer system known per se which contains a small amount of EDTA. The fractions are collected and can be analyzed for their IFN mRNA activity as mentioned above (section 1c). The fractions which show the highest IFN mRNA activity are pooled and applied to an oligo (dT) -cellulose or a poly (U) -sepharose column.

The bound poly (A) RNA, which is highly enriched in HuLyIFN mRNA, is eluted with water and precipitated with ethanol.

At this point, HuLyIFN mRNA activity can be determined again using the method described above (see paragraph 1c). In general, sucrose gradient centrifugation gives a 10- to 20-fold enrichment in HuLyIFN mRNA.

2nd Production of lymphoblastoid, double-stranded cDNA containing HuLvIFN ds cDNA

The poly (A) RNA, enriched in HuLyIFN mRNA and produced as described above (paragraph 1d), can be used as a template for the production of double-stranded cDNA. This conversion involves the production of a single-stranded cDNA, the synthesis of the second strand of DNA and the degradation of the terminal "hairpin" structure that was generated first.

a. Preparation of the single-stranded cDNA

A single-stranded DNA, which is complementary to the poly (A) RNA as described above (paragraph 1d), can be produced by reverse transcription of the RNA. The synthesis is catalyzed by an RNA-dependent DNA polymerase (reverse transcriptase), for example from bird myeloblastosis virus (AMV). AMV reverse transcriptase does not initiate DNA synthesis on a single stranded RNA. It is similar to DNA polymerase in that it requires a primer with a free 3'-hydroxyl group that must be base paired with the RNA template strand. Because of the poly (A) tails at the 3 'ends of the mRNAs, it is advantageous to use, for example, oligodeoxythymidylate └Oligo (dT) ┘ or poly (U) as a primer. If sequence information is available, it is also possible to program cDNA synthesis selectively for the gene of interest.

For example, the synthesis of the single-stranded cDNA is as follows. The poly (A) RNA is isolated and purified as described above and then in a buffer mixture known per se with a primer, for example oligo (dT), a magnesium salt, for example MgCl₂, a mercaptan, e.g.

Dithiothreit (DTT), dATP, dGTP, dCTP, d_TTP and ANV reverse transcriptase. High concentrations of deoxynucleoside triphosphates are preferably chosen. so that the synthesis of complete copies is favored. The subsequent purification steps of the single-stranded cDNA are facilitated if the four deoxynucleoside triphosphates used have been labeled, for example with 32p. The reaction can be terminated by adding an inhibition mixture which contains, for example, EDTA and SDS. After the reaction has ended, the product is deproteinized, for example by extracting the solution with phenol and chloroform, and then chromatographed on a Sephadex column, the salts and unincorporated deoxynucleoside triphosphates being removed. Fractions containing the synthesized cDNA (provided that one of the deoxynucleoside triphosphates has been labeled with 2p, the identification of usable fractions can be easily achieved by measuring Cerenkov radiation) are collected and the nucleic acids (RNA and cDNA) can be isolated , for example by precipitation with ethanol. The template RNA is with a ribonuclease, for example RNAse A or RNAse T1, or, preferably, by hydrolysis with alkali, for example Sodium hydroxide removed. The length of the remaining cDNA can be determined, for example, from its electrophoretic mobility in an alkaline agarose gel or in a polyacrylamide gel relative to marker DNA_s known length can be determined (see 32).

b). Production of the double strands cDNA

The single-stranded 'cDNA, prepared as described above, has a 3'-terminal "hairpin" structure. This "hairpin" structure represents a short double-stranded region, which causes the cDNA to self-"prime" in the subsequent synthesis of the second strand of DNA. This means that no additional primer is required.

The double-stranded cDNA can be RNA-dependent _DNAPolymerase, for example AMV reverse transcriptase, can be synthesized in a manner similar to that described above in the synthesis of single-stranded cDNA, except that poly (A) RNA is replaced by single-stranded cDNA and the primer is omitted becomes. Alternatively, other enzymes involved in the synthesis of DN_A catalyze from their deoxyribonucleotide precursors, are used, for example T4 DNA polymerase, E. coli - DN_APolymerase (Klenow fragment) or, preferably, E. coli DNA polymerase I.

Second strand synthesis can be performed using a buffer mixture containing the single stranded cDNA, a magnesium salt such as MgCl₂, a mercaptan, for example dithiothreitol, the four deoxynucleoside triphosphates, one of which is radiolabelled, for example with ³H, and a DNA polymerase, for example E. coli DNA polymerase I, contains. After completion of the reaction and deproteinization of the mixture (see paragraph 2a), the DNA is precipitated with ethanol.

&gt; The ds DNA obtained contains a "hairpin" loop that connects the two strands of DNA. The loop can be cut off with S1 nuclease to obtain a ds cDNA with base-paired ends. The cutting can be carried out in a buffer mixture known per se, the product from the synthesis of the second strand being treated with S1 nuclease in the presence of a zinc salt, for example zinc sulfate. The cutting can be ended by adding SDS and EDTA. After deproteinization with phenol, the solution is chromatographed on a Sephadex column. Fractions containing the ds cDNA (which can be determined, for example, by measuring the Cerenkov radiation of each fraction) are pooled and the cDNA is precipitated with ethanol.

The synthesized ds cDNA, which still contains a large number of different species, is preferably further enriched at this point in complete ds cDNA. Suitable methods for this purpose are gel filtration, electrophoresis on polyacrylamide gel or on agarose gel or zone centrifugation in a sucrose gradient. Coelectrophoresis and co-centrifugation of the DNA molecules of known molecular size in parallel experiments allow the localization of those ds cDNA molecules which have the molecular size expected for IFN ds cDNAs (700 to 1200 base pairs, according to the previously published data: 14, 16, 18, 33).

For example, the ds cDNA, which is dissolved in a suitable buffer medium, is subjected to zone centrifugation by sucrose gradients (for example 5 to 23%). DNA species that settle faster than a suitable marker DNA that is tested in parallel gradients (for example a 700 to 800 base pair marker) are isolated.

The cloning of the HuLyIFN ds cDNA enriched ds cDNA

a. General considerations

The cloning of the (paragraph 2b) ds cDNA prepared as described above can be carried out according to methods known per se. The method comprises<ul id="ul0004" list-style="none"><li>- Linking the ds cDNA with a suitable vector DNA and</li><li>- Transfer of the resulting recombinant DNA into a suitable host cell (transformation), in which it can replicate.</li></ul>

A vector DNA is a DNA molecule that contains genetic functions that ensure that it replicates itself when it is transferred to a host cell. It is further desirable that the vector DNA contain a gene by which plasmid-bearing host cells (transformants) can be selected from a large population of cells, most of which do not contain the plasmid. Examples of vector DNAs which are generally used in genetic engineering are circular plasmid DNA and the DNA of certain bacteriophages to which the cDNA can be bound experimentally, for example derivatives of bacteriophage X and in particular the plasmid col E1 or its derivatives, for example pMB 9, pSF 2124, pBR 317 and in particular pBR 322. The plasmids mentioned contain genes for ampicillin resistance and, in part, for tetracycline resistance. Therefore, host cells containing such a plasmid will show a phenotype that allows transformants to be separated from the parent host.

So that you get a recombinant DNA molecule

for example, a suitable plasmid, for example pBR 322, is cleaved, the ds cDNA is inserted into the linearized plasmid and the ring is closed again, an enlarged recombinant plasmid molecule being formed which contains the inserted ds cDNA segment. The plasmid DNA is preferably cleaved at defined sites. A large number of restriction endonucleases are available for this purpose, which recognize specific DNA sequences. Some restriction endonucleases cleave both strands of DNA at the same site and give "blunt" ends. Others catalyze the cleavage of bonds separated by a few nucleotides and result in free, single-stranded regions at each end of the cleaved molecule ("staggered" ends).

DNA segments are generally connected via their single-stranded cohesive ends and covalently by means of a

DNA ligase, eg T4 DNA ligase, closed. Complementary ends can be formed in two different ways: either by cleavage with restriction enzymes, which form staggered cuts and cohesive termini, or by the addition of defined, single-stranded sequences (eg homopolymeric ends). Alternatively, fully base paired DNA duplexes, blunt ends, can be linked with T4 ligase. For example, a plasmid, e.g. pBR 322, cleaved by means of a suitable restriction endonuclease, for example Pst I, and the linearized plasmid and the ds cDNA to be used are each in the presence of a suitable enzyme, for example a terminal deoxynucleotidyl _Transferase, elongated with single-stranded, homopolymeric ends. For example, poly (dC) ends can be added to one DNA preparation, and poly (dG) ends can be added to another (alternatively, dA and dT ends can also be selected). The two DNAs can then be connected via their complementary ends.

Useful hosts are, for example, yeasts and in particular bacteria which are accessible for transformation and which have no restriction enzymes and modification enzymes, for example strains of E. coli, for example E. coli X 1776 or E. coli HB 101, or strains of Bacillus subtilis, Bacillus stearothermophilus, Ps6udomonas, Haemophilus, Streptococcus and other bacteria and their mutants.

The recombinant DNA molecule, prepared as described above, can be placed in a suitable host cell using standard transformation methods including a Ca²⁺- Pretreatment of the host cells are transferred. Cells which contain a recombinant plasmid DNA which transmits a phenotypic property, for example tetracycline resistance, to the host cell are selected by plating on agar plates in a selective nutrient medium, for example containing tetracycline.

In the present invention, the preferred vector DNA is the plasmid pBR 322 which, after cleavage with a suitable restriction endonuclease, in particular Pst I, is bound to the ds cDNA via complementary homopolymeric ends, in particular dG: dC ends. The resulting recombinant DNA is transferred to E. coli HB 101.

Instead of the IFN genes, which were produced via the ds cDNA synthesis path according to the previous chapters, the corresponding chromosomal DNA can also be used for the production of clones which produce polypeptides with IFN activity.

The chromosomal DNA can be obtained from human lymphoblastoid cells, such as Namalwa cells, by methods known per se, for example by partial cleavage of the entire chromosomal DNA with Alu I, and linking the fragments thus obtained with EcoR I linkers to Ä Charon 4A arms or by Cleavage of the chromosomal DNA with a restriction enzyme, for example Kpn I or Hind III, and linkage of the fragments thus obtained with a vector DNA, such as the plasmid pBR 322 or cosmid DNA. The recombinant vector DNA can be transformed into a host such as E. coli. The colonies containing chromosomal IFN-a and -ß genes are separated by colony hybridization (see Chapter 4a) using either a radiolabelled, synthetic oligodeoxynucleotide or a radiolabelled, a- and B-specific IFN cDNA Probe identified. Sub-fragments can be obtained by subjecting the identified fragments to restriction with an appropriate endonuclease or by digesting them with an appropriate exonuclease.

The invention thus also relates to a method for producing a DNA which contains a DNA sequence which can be derived from human lymphoblastoid cells, or a fragment, a variant or a mutant of this sequence which codes for an interferon-like polypeptide, or a DNA that hybridizes with this DNA. The process includes:<ul id="ul0005" list-style="none"><li>(1) the production of a single-stranded, complementary DNA from HuLyIFN mRNA and, if desired, the production of a double-stranded cDNA therefrom, or</li><li>(2) partial cleavage of chromosomal DNA from human lymphoblastoid cells and selection of fragments containing chromosomal LyIFN genes and, if a fragment of this sequence is desired, restriction of said DNA with a suitable endonuclease or partial degradation of said DNA with a suitable exonuclease or, if a recombinant DNA is to be produced, introducing said DNA into a suitable vector DNA.</li></ul>

b. Preparation of linearized, deoxynucleotide-extended pBR 322

The preferred vector of the present invention, plasmit pBR 322, is a small plasmid containing 4361 base pairs. It contains two genes (amp^r, tet^r), the _Atransfer mpicilin or tetracycline resistance to bacterial recipient cells and which can be used for selection and identification of the transformed cells. There are various restriction sites within pBR 322. A single Pst I site is within the amp^r-Gens, while the only BamHI, Hind III and _Sal I positions within the tet^r-Gene occur. A single EcoR I site is present elsewhere (34). When using one of the restriction endonucleases mentioned above, either the amp^r-Gene or that tet^r-Gene or both genes intact. Each of the enzymes listed is therefore suitable for the cleavage and linearization of pBR 322.

After the plasmid pBR 322 has been linearized, deoxynucleotide chains can be added to both 3 'ends in the presence of terminal deoxynucleotide transferase. About 20 to 50 deoxynucleotide residues are preferably added in order to ensure a stable connection to the ds cDNA, extended by chains of the complementary deoxynucleotides. For example, the plasmid pBR 322 is in a suitably buffered, aqueous medium, the MgCl₂, a mercaptan, for example 2-mercaptoethanol, and a carrier protein source, for example bovine serum albumin or gelatin, additionally treated with a restriction endonuclease, for example Pst I. After the cleavage has ended, the solution is deproteinized with, for example, phenol. The terminal addition of deoxynucleotide residues takes place in a common buffer system, the MgCl₂Contains sodium cacodylate and a carrier protein, for example bovine serum albumin, with a sufficient amount of deoxynucleoside triphosphates, for example dGTP, and terminal deoxynucleotidyl transferase.

c. Preparation of deoxvnucleotide-extended ds cDNA

The extension of the (paragraph 2b) ds cDNA obtained as described above can be carried out in the same manner as described in the synthesis of the linearized, deoxynucleotide-extended plasmid pBR 322 (see paragraph 3b), using the complementary deoxynucleoside triphosphate (e.g. dCTP instead of dGTP) and preferably using CoCl₂ instead of MgCl₂. For example, the ds cDNA is incubated in a buffer solution containing sodium cacodylate, CoCl₂, a protein, for example bovine serum albumin, which contains the corresponding deoxynucleoside triphosphate, for example dCTP, and terminal deoxynucleotidyl transferase.

d. Hybridizing ring closure of linearized, chain-extended pBR 322 and chain-extended ds cDNA

The linearized, chain-extended plasmid pBR 322 and the chain-extended ds cDNA can be hybridized and closed again to form a ring in a manner known per se, ie by base-pairing the complementary deoxynucleotide chain chains.

In order to promote ring formation and to prevent the formation of concatomers (compound of different linear hybrid plasmids), the reaction must take place at a low concentration of both the chain-extended ds cDNA and the linearized pBR 322 molecules.

For example, a mixture of deoxynucleotide-extended (e.g. dCMP-extended) ds cDNA and linearized, deoxynucleotide-extended (e.g. dGMP-extended) pBR 322 at four successive, one-hour stages at different temperatures (e.g. 65 ° C, 46 ° C, 37 ° C and 20 ° C) incubated. The hybridized, ring-closed DNA can be used directly for transformation into a compatible bacterium, for example E. coli HB 101.

It should be noted at this point that the product of the hybridization process contains recombinant DNA molecules, very few of which are related to HuLyIFN, since most of the recombinants contain a cDNA insert, which is derived from a mRNA other than LyIFN mRNA .

e

. Transformation of E. coli HB 101 with the ring-closed hybrid plasmids

The hybrid plasmids obtained can be used to transform E. coli HB 101. The plasmids are replicated within the cell and the plasmid replicas are distributed to the daughter cells when the cell divides.

E. coli HB 101 can be transformed with the ring-closed hybrid plasmids by methods known from the literature. The method comprises the Ca2 + pretreatment of the cells so that DNA uptake is possible [eg (35)] and incubation with the hybrid plasmid. The cells can then be transferred to a selective growth medium which allows the transformed cells to be separated from the stem cells. Since the hybrid plasmid is still a tet^r-Gene contains, an agar medium containing tetracycline as a growth inhibitor is preferably used.

For example, the hybrid plasmid and the E. coli HB 101 cells, pretreated with Ca, are incubated in a buffer medium containing a Ca²⁺Salt, e.g. CaCl₂, and a Mg^2+-Salt, eg MgCl₂, contains. After a sufficient incubation period (e.g. 10 to 40 min), the bacteria are subjected to a heat pulse (35 to 42 ° C), generally for a short period of time (1 to 5 min), then the cells are cooled and placed on an agar medium, e.g. Trypton agar or McConkey agar, supplemented with a sufficient amount of tetracycline, plated. Cells that survive in such a medium contain the recircularized plasmid or the hybrid plasmid DNA. The grown colonies are therefore used to screen for suitable clones.

4th Identification of the clones containing lymphoblastoid IFN cDNA

a

. Methods suitable for the identification of the clones containing LyIFN cDNA

Colonies that contain specific genes can be identified by various methods, for example by RNA selection hybridization, differential hybridization or hybridization with a synthetic probe, or clones which form a special gene product can be identified by immunological or biological assays.

While the immunological and biological methods are based on the production of the immunologically and biologically detectable gene product, the first set of methods mainly depends on the availability of a suitable probe, which is in a sufficiently purified form to prevent non-specific hybrid formation. Suitable probes are the mRNA, complementary to the desired gene, or the corresponding cDNA.

There are different ways of screening bacterial clones containing human leukocytes and fibroblast IFN. known. For example, Goeddel et al. (13) and Sugano et al. (16) IFN genes by visual comparison of two hybridization sets. The first set is hybridized with radioactive cDNA, which was synthesized by reverse transcription of an mRNA mixture, whereby the mixture was obtained from induced cells and wherein one³²P-labeled CTP as a labeling agent and oligo (dT) (Sugano) or a synthetic deoxyundecanucleotide (Goeddel) used as a primer. The second set is hybridized with a radioactive cDNA, which is similarly formed from a mRNA mixture obtained from non-induced cells. This procedure is due to the lack of_Sspecificity and reproducibility, which is evident from the values ​​given. Another possibility, which is described by Weissmann (3), makes use of the RNA selection hybridization method, which comprises a tedious and laborious, multi-stage search for the desired clone.

The methods mentioned cannot be used in the present invention for isolating both the LyIFN-a and the B genes, since the concentration of IFN-β is only about 10% of the concentration of IFN-a in human LyIFN. This small amount is below the detection limit in these methods.

Because of the unsatisfactory approaches prior to the present invention, a new screening method was developed which involves the synthesis of a 5th¹-terminally labeled oligodeoxynucleotides, complementary to both IFN-a and IFN-ß mRNAs, the reverse transcription of poly (A) RNA enriched in LyIFN mRNA, using said oligodeoxynucleotide as a primer, and the in situ colony hybridization of the filter-bound Plasmid DNAs with a labeled cDNA probe included. This approach allows the specific, rapid, and direct detection of both IFN-a and β genes that contain the base sequences of the synthesized primer oligodeoxynucleotide and does not require any other cumbersome hybridization translation assays as described in the literature to be discribed. In situ colony hybridization is based on the general method described by Grunstein and Hogness (36) or on variants thereof. In this method, colonies are grown on nitrocellulose filters or transferred to them, lysed by alkali treatment and fixed to the filters in situ. A radioactively labeled nucleic acid that is complementary to the desired gene is then hybridized as a probe with the DNA bound to the filter. Since the hybridized probe can be detected autoradiographically, the corresponding colonies that contain a hybridizable DNA can be isolated from a comparison set of nitrocellulose filters.

A comparison of the coding regions of the cDNAs of cloned human IFN-a and IFN-ß showed a region of 13 nucleotides that is common to both cDNAs (and obviously the corresponding mRNAs as well) (23). Therefore, a synthetic 13-mer oligodeoxynucleotide having the above-mentioned contiguous base sequence can be used to start cDNA synthesis from human lymphoblastoid IFN-a and β mRNA.

b. Making one

32

P-labeled, human IFN-α and IFN-B specific cDNA probe

There are various tried and tested ways of synthesizing an oligodeoxynucleotide of a given structure (37). For example, the oligodeoxynucleotide synthesis can be carried out using chemical methods, for example the diester or triester method. The basic stage of the diester procedure consists in the connection of two suitable, protected deoxynucleotides to form a dideoxynucleotide which contains a phosphodiester bond. The Triester process differs from the diester process by the presence of an additional organic protective group on the phosphate groups, which causes the solubility of the deoxynucleotides and oligodeoxynucleotides in organic solvents. Alternatively, the synthesis can be carried out enzymatically using polynucleotide phosphorylase which, under controlled conditions, mainly adds a single deoxynucleotide to the short oligodeoxynucleotide. The reactions can be carried out in solution or using solid-phase techniques, which have recently been perfected to a high degree.

For example, the synthesis of the 13-mer oligodeoxynucleotide of the formula<img file="EP0076489A2_D0001.tif" />Complementary to both human IFN-α and β mRNA according to the Triester method, as described by Itakura (38) and de Rooij (39), using protected mono-, di- and trideoxynucleotides as starting materials. A single stage of the process is illustrated in the following scheme, which illustrates the synthesis of a dinucleotide:<chemistry id="chem0001" num="0001"><img file="EP0076489A2_D0002.tif" /></chemistry>where R₁, R₂ and R₃ Protecting groups mean, dN or dN 'represent purine or pyrimidine bases and cond. means a condensing agent.

The starting materials used in the synthesis according to the invention (protected or partially protected mono-, di- or trideoxynucleotides) are known from the literature.

The protecting groups are preferably selected so that they can be successively removed under mild conditions without cleavage of the nucleotide 3'-5 'bonds. Suitable protecting groups for R₁ are, for example, monomethoxytrityl or dimethoxytrityl, for R₂ for example 2-chlorophenyl and for R₃ for example 2-cyanoethyl.

The exocyclic amino functions in the adenine, guanine and cytosine residues are protected in particular by acyl groups, for example benzoyl or isobutyryl. For example, 2,4,6-triisopropylbenzenesulfonic acid can be used as the condensing agent.

The specific removal of the individual protective groups (e.g. R₃, R₁ and R₂) in the intermediates and the complete removal of the protective groups of the fully protected 13-mer oligodeoxynucleotide can be carried out according to methods known per se. For example, a 2-cyanoethyl group R₃ removed by treatment with alkali, a monomethoxytrityl group R₁ can be removed by treatment with 80% acetic acid and a 2-chlorophenyl group R₂ is removed using tetrabutylammonium fluoride. Other known methods can also be used. The individual synthesis steps are shown in Fig. 2.

The 13-mer oligodeoxynucleotide primer produced can be purified by chromatographic methods known per se, e.g. B. by DEAE Sephadex chromatography and / or high performance liquid chromatography (HPLC). The primer is 5'-P labeled so that it is possible to detect the hybridized probe in the subsequent hybridization procedure. Labeling is done by reacting the synthesized primer with a labeled phosphorylating agent, e.g._B. [γ-³²P] -_ATP, with T4 polynucleotide kinase in a common buffer mixture. The resulting solution, which contains the 32P-labeled primer, is purified by deproteinization, for example with phenol, and by chromatographic methods, for example by means of Sephadex chromatography or polyacrylamide gel electrophoresis. If desired, the nucleotide sequence can be checked at this point in time by two-dimensional homochromatography.

The poly (A) RNA enriched in LyIFN mRNA (compare step 1d) is used as a template for the synthesis of an IFN-α and -B-specific cDNA probe as follows: The cDNA synthesis is carried out essentially in the same manner as above (Stage 2a), with the exception that the 5th^1-labeled, synthetic oligodeoxynucleotide is used instead of oligo (dT) as a primer. The labeled cDNA product is deproteinized with, for example, phenol and collected by precipitation with ethanol. Further purification can be carried out, for example, by polyacrylamide gel electrophoresis of the cDNA product. The product can be made visible by autoradiography.

A single DNA band that is specific for induced cell poly (A) RNA and is not present in the product obtained from non-induced cells using poly (A) RNA is extracted from the gel. Their size can be determined, for example, from the relative mobility, based on labeled marker DNAs of known length. The product represents one³²P-labeled, human, LyIFN-a and -ß specific cDNA probe.

c. Screening for clones containing lymphoblastoid IFN cDNA

Colonies that survive and grow on an agar medium supplemented with tetracycline (see paragraph 3e) are tested for clones containing lymphoblastoid IFN cDNA. For this purpose, the in situ colony hybridization method described above (paragraph 4a) is chosen. The transformant colonies are transferred to nitrocellulose filters and a comparison set of these colonies is then obtained by replica plating on additional agar plates. The colonies on the filters are lysed, their DNA is denatured and fixed in situ on the filters (36).

Before the hybridization process, the denatured DNA on the filters is preferably prehybridized with a mixture which contains an alien DNA of foreign origin in order to keep the background low and to saturate non-specific hybridization sites. The radioactively labeled cDNA probe produced as described above (paragraph 4b) is then hybridized with the DNA bound to the filter, which is covered with mineral oil. After the filter has been freed of mineral oil and other contaminants, the result of the hybridization process can be followed by autoradiographic analysis on an X-ray film. Colonies that respond positively to the X-ray film can be picked from the comparison set and used for further investigation.

Preferably only a part of the transformant colonies is transferred to the nitrocellulose filter. The remaining colonies are used for further screening procedures, which are explained below (paragraph 4d).

For example, the DNA fixed to the nitrocellulose filter is treated with a conventional prehybridization mixture which contains, inter alia, a denatured DNA, for example denatured calf thymus DNA, bovine serum albumin, Ficoll and polyvinylpyrrolidone, and then on _Pa-_raoil with the radioactively labeled cDNA probe (cf.

Paragraph 4b) hybridizes using standard hybridization methods (see 36).

After hybridization is complete, the filters are washed sequentially with chloroform and with a buffer mixture containing SDS and a low salt to remove the paraffin oil and unhybridized cDNA.

The filters are exposed to X-ray films for autoradiography. The hybridized colonies show a positive response and can be picked out on the comparison set.

The identified colonies contain recombinant DNAs with inserts complementary to those of the cDNA probe, ie DNA segments that correspond to the human lymphoblastoid genes or their fragments. Since the primary clones of the transformed cells occasionally contain more than one species of recombinant DNA molecule, the hybrid plasmid DNAs are isolated from the positively hybridized clones and used to retransform E. coli HB 101 as described above (paragraph 3e). The hybrid plasmid DNAs can be isolated, for example, by the following method. First, each identified colony is grown in a suitable nutrient medium, such as tryptone medium, in the presence of tetracycline to remove such contaminating cells that do not contain a plasmid. The surviving cells are collected in a conventional_lichen. Buffer system loosened and carefully destroyed so that the chromosomes remain inside the cell envelopes. This method includes, for example, the sequential addition of lysozyme, EDTA and a nonionic detergent, such as Triton. The cell debris and the chromosomal DNA are then removed by centrifugation. The supernatant solution is deproteinized, for example with phenol, and the RNA is broken down with an RNAse, for example RNAse A. The hybrid plasmid DNA can be separated from the RNA fragments by precipitation with polyethylene glycol and purified by reprecipitation with ethanol.

At this point, the cleavage pattern of each hybrid DNA isolated can be determined by cleaving with an appropriate restriction endonuclease, particularly that used to linearize the plasmid pBR 322, at the point where the ds cDNA has been inserted (cf. Paragraph 3b). The size of the restriction fragments can be determined, for example, from their electrophoretic mobility in an agarose gel, relative to marker DNAs of known length.

Each of the isolated hybrid DNAs is retransformed in E. coli HB 101 and grown on a suitable agar medium (see paragraph 3e) which contains tetracycline. A few clones are picked from each retransformation and the hybrid plasmid DNA from each clone is isolated as described above. The hybrid plasmid DNAs are again subjected to the restriction analysis and a complete or partial nucleotide sequence analysis of the cDNA insert is carried out in order to select hybrid DNAs which are suitable for the further methods. Furthermore, a partial sequence analysis can clarify whether the inserted IFN cDNA segments correspond to the human lymphoblastoid IFN-a or -ß genes.

Various rapid methods for sequence determination of DNA molecules are currently available. The "primed synthesis" methods, which were developed in particular by Sanger (40), use the ability of the DNA polymerases to make a complementary copy of the single-stranded_en Precisely synthesize DNA templates, using radioactively labeled DNA fragments as primers, which were generated by restriction endonuclease cleavage. Alternatively, the chemical DNA sequencing method developed by Maxam and Gilbert (41) can be used. In this method, the DNA to be subjected to sequencing is finally labeled, partially cleaved in four different reactions at each of the four bases, and the products are fractionated according to their size, for example by means of gel electrophoresis under denaturing conditions. The DNA sequence can be read from the pattern of the radioactive bands. In order to determine the complete nucleotide sequence of a certain DNA region, a restriction endonuclease is required which cleaves the DNA in this region. Using the Maxam and Gilbert method, the sequence of up to 100 to 150 bases in both directions of the cleavage site can be determined in one experiment.

For example, isolated hybrid DNAs with suitable restriction endonucleases, for example Pst I, EcoR I, Bgl II, Pvu II or Alu I, can be located at sites within the cDNA- _I.nserts occur, be split. The resulting DNA fragments are terminated with, for example, [γ-³²P] -ATP labeled in the presence of a polynucleotide kinase and cleaved with a second restriction endonuclease such that only one strand of the ds DNA remains labeled at the end. The suitable DNA fragments are isolated, for example, by polyacrylamide gel electrophoresis. The DNA fragments are then subjected to base-specific cleavage reactions, which are written by Maxam and Gilber. The products are by electrophoresis, e.g. fractionated on polyacrylamide gel under denaturing conditions, for example in 7 M urea, _'and the DNA fragments can be made autoradiographically visible.

d. Identification of additional clones containing lymphoblastoid IFN cDNA

As described above (paragraph 4c), part of the transformant colonies are transferred to nitrocellulose filters and their fixed DNA is subjected to in situ colony hybridization using a radioactively labeled cDNA as a probe, eg an IFN-specific cDNA probe. The recombinant DNAs from positively hybridizing colonies can be used as screening material for additional clones, the recombinant DNA molecules with the same or a related DNA insert (e.g. IFN-related sequences) included.

To this end, the plasmid DNAs from each of the identified colonies (corresponding to different IFN genes or gene fragments) obtained in the first screening procedure (paragraph 4c) are isolated as described above and digested with restriction endonucleases in such a way that plasmid fragments, containing the IFN cDNA insert or part thereof. After these plasmid fragments have been marked at the 5 'end, and the corresponding DNA fragments (e.g. those which contain radioactively labeled IFN inserts or a part thereof), for example by polyacrylamide gel electrophoresis, can be used either individually or alternatively as a mixture. For hybridization, transformant colonies (see above) are transferred to nitrocellulose filters and lysed. Your DNA is denatured, fixed to the filter in situ and hybridized with the IFN-specific, radioactively labeled DNA fragments according to Grunstein and Hogness (36). Hybridization colonies can be made autoradiographically visible and picked from a comparison set.

The identified colonies contain recombinant DNA molecules with an identical or similar cDNA insert as the probes used. According to this screening method, additional clones can be identified which contain lymphoblastoid IFN-a or -ß genes or their fragments. The plasmid DNA of each identified colony can be isolated and characterized by restriction analysis and (partial) sequence analysis as described above (paragraph 4c).

5. Synthesis of polypeptides with HuLyIFN-like activity by E. coli, which contain HuLyIFN-specific, recombinant DNAs

The HuLyIFN inserts according to the invention were inserted by hybridization into the Pst I site of pBR 322 (see above). Since the Pst I site of pBR 322 is within the ß-lactamase gene, a fused protein can be formed if the cDNA insert is inserted in the correct orientation for transcription and in the correct reading frame for translation. If the inserted cDNA has its own initiation signal and / or termination signal in phase with the β-lactamase sequence, initiation or / and reinitiation can also occur with the second initiation signal and an unfused protein can be formed. Moreover, even those clones that do not have IFN activity are valuable, even though a HuLyIFN cDNA is present. In this case, the cDNA insert can be isolated and linked to the expression control area in a suitable manner (see paragraph 7) in order to obtain a high degree of expression of the desired polypeptide.

Clones which contain a recombinant DNA with a HuLyIFN cDNA insert can be tested for their IFN activity by methods known per se. For example, cultures can reach a sufficient level_Z.be cultivated. The cells are harvested, resuspended and lysed (see section 1b). The cell-free extracts can be analyzed for their IFN activity using, for example, a cytopathic bioassay (eg 29).

Clones that synthesize polypeptides with HuLyIFN activity to a satisfactory extent are suitable for large-scale production. The clones can be grown and the polypeptides can be obtained according to Chapters 7 and 8.

6. Construction of recombinant plasmids that can express high levels of polypeptides with HuLyIFN activity

For sufficient expression to take place, a gene must be correctly classified with regard to the control area, including the initiator for transcription (promoter) and for translation (ribosomal binding site).

As described above (paragraph 3d), the plasmid pBR 322 was cleaved with a suitable restriction endonuclease and bound to HuLy cDNA. The resulting recombinant plasmid DNA was used for the transformation of E. coli HB 101. For example, if Pst I is used as a restriction endonuclease, HuLy cDNA is inserted within the β-lactamase gene of pBR 322. If the link continues to be in the correct orientation and reading frame, a fused protein can result that is part of the _ß-Lactamase chain followed by the HuLyIFN amino acid sequence. If the cDNA is not inserted in the correct reading frame and / or in the correct orientation, the resulting protein will have no IFN activity. In the event of an incorrect orientation, the plasmid can be reoriented by cutting out the cDNA insert with a suitable restriction endonuclease (in the present invention, all inserts can be cut out by Pst I) and reconnecting the cDNA and the linearized plasmid. The resulting hybrid plasmid can be transformed into E. coli HB 101, which in turn can be analyzed for its IFN activity, as usual.

In order to increase the expression yield of the HuLyIFN cDNA insert, it is necessary to place the HuLyIFN cDNA insert in the vicinity of the above-mentioned expression control sequence so that no additional nucleotides (and thus amino acids) precede the gene (and thus the polypeptide with HuLyIFN activity ). Furthermore, in the case of HuLyIFNs, the primary translation products are pre-interferons, which contain signal peptides that are bound to the N-terminus of "mature" interferons. The signal peptide sequences are removed post-translationally in the original progenitor cells. However, E. coli is unable to proteolytically remove the presequences. Therefore, the pre-sequences are preferred from the_C.DNA insert using a suitable _Vexperienced (see below) removed so that the primary translation product is a "mature" IFN-like polypeptide. For this purpose, the gene coding for the mature HuLyIFN is reconstituted in vitro and reinserted into a plasmid close to (operably linked to) an expression control sequence, eg the expression control sequence of the B-lactamase gene.

Other expression control sequences including inter alia the promoter and the ribosomal binding site can also be used, for example the control sequence of the lactose operon, tryptophan operon, arabinose operon and the like, the corresponding sequences from the phage λN gene and the phage fd-_Hüll- proteiningen or other sequences known per se. The expression control sequence can be inserted into a plasmid that already contains a cDNA insert, or the cDNA can be inserted into a plasmid that already contains the expression control sequence, or both DNA fragments can be inserted into the plasmid in succession.

For example, the "mature" HuLyIFN cDNA can be brought under the control of the β-lactamase expression control sequence. Since the "mature" cDNA insert, which codes for a "mature" HuLyIFN-like polypeptide, does not start with the codon ATG, which is necessary for the initiation of translation, the ATG triplet must be introduced synthetically. For example, knowing the nucleotide base sequences and, accordingly, the restriction endonuclease patterns of both pBR 322 and HuLyIFN cDNA, one can proceed as follows. The plasmid pBR 322 is digested with Pst I within the β-lactamase gene and digested with an exonuclease, eg Bal 31, in order to shorten the B-lactamase coding sequence. Alternatively, a combination of λ-exonuclease (5'-exonuclease) or 3'-exonuclease from E. coli and S1 nuclease can be used. The restricted plasmid is linked to a ds DNA linker that can be synthesized, for example according to the Triester method, as described above (paragraph 4b). The linker comprises the recognition sequence of a suitable restriction endonuclease, for example Bcl I (Sau 3A). The resulting plasmid fragment is treated with a restriction endonuclease that is suitable for the hybridized linker (e.g. Bcl I) and then digested with EcoR I (there is an EcoR I site within pBR 322 that is located near the β-lactamase expression control sequence). The resulting DNA fragment, for example the EcoR I - Bcl I DNA fragment, essentially consists of the expression control sequence of the β-lactamase (ApPr) and the hybridized linker and can be isolated by means of polyacrylamide electrophoresis.

On the other hand, the HuLyIFN cDNA insert can be cut out of the recombinant DNA molecule which contains it (paragraph 4d), for example by cleaving with the restriction endonuclease Pst.I. The isolated HuLyIFN cDNA insert is further digested with another restriction endonuclease (or, if necessary, with two other restriction endonucleases and partially relinked) to remove the DNA sequence encoding the signal peptide.

The resulting "mature" HuLyIFN cDNA has a staggered end which is complementary to that of the ApBr DNA fragment mentioned above (eg a Sau 3A end). The "mature" HuLyIFN cDNA and the ApPr DNA fragments are hybridized with ligase as usual (see paragraph 3d). Hybridization must result in the formation of an ATG codon that precedes the first codon of the "mature" cDNA to ensure the correct reading frame. The resulting hybrid DNA contains the β-lactamase expression control region, an ATG translation start codon, a DNA sequence coding for the complete HuLyIFN, and two restriction endonuclease ends (eg EcoR I and Pst I ends) which are suitable for inserting the hybrid DNA into the correspondingly cleaved plasmid pBR 322.

The resulting hybrid plasmid can be used to transform E. coli HB 101 and to control the synthesis of high levels of polypeptide with HuLyIFN activity.

7. Cultivation of HuLyJFN-specific, recombinant DN

A

s containing clones

The transformed hosts according to the invention, which do not differ morphologically from the starting strains used for the transformation, can be used for the production of polypeptides with HuLyIFN activity. The process for producing these polypeptides is characterized in that the transformed host, in particular a transformed E. coli strain is grown in a liquid nutrient medium containing assimilable sources of carbon and nitrogen and inorganic salts.

Different carbon sources can be used. Examples of preferred carbon sources are assimilable carbohydrates, such as glucose, maltose, mannitol or lactose, or an acetate, which are used either alone or in suitable mixtures. Suitable nitrogen sources are, for example, amino acids, such as casamino acids, peptides and proteins and their breakdown products, such as trypton, peptone or meat extracts; furthermore yeast extracts, malt extract,_Mai_sq_uellLiquid, as well as ammonium salts, for example ammonium chloride, sulfate or nitrate, which can be used either alone or in suitable mixtures. Inorganic salts that can be used include sulfates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium.

Furthermore, the nutrient medium can also contain substances for activating growth and / or substances which exert a selection pressure in order to prevent the loss of HuLyIFN-specific, recombinant DNA.

Substances that activate growth are, for example, trace elements such as iron, zinc, manganese and the like, or individual amino acids. In addition to the gene coding for a polypeptide with HuLyIFN activity, the recombinant DNAs according to the invention preferably contain a gene which brings about antibiotic resistance, for example resistance to ampicillin and / or tetracycline. If such an antibiotic substance is added to the culture medium, cells containing recombinant DNA will survive and grow, whereas cells which have lost the recombinant DNA or foreign antibiotic-sensitive microorganisms which will contain the culture medium _kontaminieren., will not grow.

The cultivation is carried out using methods known per se. The cultivation conditions, such as the temperature, the pH of the medium and the fermentation time, are selected in such a way that maximum contents of IFN-like polypeptides are formed. A selected E. coli strain is preferred under aerobic conditions in submerged culture with shaking or stirring at a temperature of about 20 to 40 ° C, preferably about 30 ° C, and a pH of 4 to 9, preferably at pH 7, for about 4 to 20 h, preferably 8 to 12 h. As a result of cultivation, IFN-like polypeptides accumulate intracellularly.

8th. Isolation and purification of the polypeptides with IFN activity

The human lymphoblastoid interferons according to the invention can be isolated from the culture broth by releasing the polypeptides from the cells of the transformed host and purifying them.

After the transformed E. coli cells have grown to a satisfactory cell density, the first step in obtaining the expressed polypeptide is to release it from the interior of the cell. For this purpose the cells are lysed by treatment with a detergent such as SDS or Triton. Alternatively, mechanical forces such as shear forces (e.g. Use an X press, French press) or shake with glass beads or alumina to break open the cells. The resulting polypeptide mixture can on human LyIFN by methods known per se, such as precipitation with ammonium sulfate or trichloroacetic acid, gel electrophoresis, dialysis, chromatography, e.g. B. ion exchange chromatography, size exclusion chromatography or reverse phase HPLC and the like. The final purification of the pre-cleaned product can be carried out, for example, by means of antibody affinity chromatography. In principle, the cleaning stages can be carried out using the Staehelin et al. (51), which was developed for the purification of human leukocyte interferon.

For example, isolation and purification of human LyIFN can be performed using the following steps:<ul id="ul0006" list-style="none"><li>(1) lysis of E. coli cells,</li><li>(2) removal of part of the non-proteinaceous material by treatment with polyethyleneimine,</li><li>(3) precipitation of the polypeptides by saturating the solution with ammonium sulfate,</li><li>(4) dialysis in a suitable buffer mixture,</li><li>(5) column chromatography on DEAE cellulose,</li><li>(6) affinity chromatography on a monoclonal antibody column and</li><li>(7) Separation by molecular size with a suitable Sephadex ^R -Pillar.</li></ul>

Additional cleaning stages may be required to obtain a sufficiently pure product, e.g._B. Cation or anion exchange chromatography, adsorption on hydroxyapatite, reverse phase HPLC etc. On the other hand, one or more of the above steps can be omitted if possible or the order of the steps can be changed.

The invention further relates to fragments and derivatives of the polypeptides according to the invention, for example proteolytically cleaved polypeptides, completely or partially protected, for example acylated, silylated and in particular glycosylated polypeptides, and their salts, and processes for their preparation.

The invention particularly relates to DNAs; and the polypeptides of the present invention in substantially pure form, and in particular those DNAs, transformed hosts, polypeptides and methods for their preparation, which are described in the examples.

The polypeptides according to the invention and their suitable derivatives, for example glycosylated products, are used in analogy to the known interferons for the treatment of viral infections, tumors and cancer of the human body, optionally together with other antiviral, anti-tumor or anti-cancer agents , preferably in the form of pharmaceutical _'pharmaceutical preparations containing an effective amount of the active ingredient together or in admixture with inorganic or organic, solid or liquid, pharmaceutically acceptable carriers suitable for parenteral administration.

The pharmacologically active compounds according to the invention are preferably used in the form of preparations or infusion solutions for parenteral, for example intramuscular or intravenous, administration. Such solutions are preferably isotonic, aqueous solutions or suspensions which can be prepared before use, for example from lyophilized preparations which contain the active ingredient alone or together with a pharmaceutically acceptable carrier. The pharmaceutical preparations can be sterilized or they can contain adjuvants, for example preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizing agents, salts for regulating the osmotic pressure and / or buffers. The pharmaceutical preparations according to the invention, which may contain further pharmacologically valuable substances, are produced in a manner known per se, for example by conventional dissolution or lyophilization processes, and they contain about 0.1 to 100%, in particular about 1 to about 50%, and in the case of the lyophilisates up to 100% of active ingredient.

The invention further relates to a method for producing a pharmaceutical preparation, which is characterized in that the pharmacologically active compound according to the invention is mixed with a pharmaceutically acceptable carrier.

Depending on the nature of the disease and the condition of the patient, the preparations are generally administered in dosage units of about 10 one to three times a day⁶ until about 10⁷ Units administered, eg intramuscularly.

The following examples illustrate the invention but should not be construed as a limitation.

Examples

The following abbreviations are used in the examples:<ul id="ul0007" list-style="none"><li>EtBr ethidium bromide</li><li>BSA bovine serum albumin</li><li>_DTT 1,4-dithiothreitol (1,4-dimercapto-2,3-butanediol)</li><li>EDTA ethylenediamine tetraacetic acid</li><li>SDS sodium dodecyl sulfate</li><li>_TNE solution containing 100 mM NaCl, 50 mM Tris.HCl (pH 7.5) and 5 mM EDTA</li><li>Tris (Trizma) tris (hydroxymethyl) aminomethane</li><li>Tris.HCl monohydrochloride from Tris</li></ul>

1. Isolation of poly (A) RNA enriched in HuIFN mRNA (Fig. 1)

a) Induction of Namalwa cells

Namalwa cells are grown at 37 in the RPMI 1640 culture medium containing 10% fetal calf serum⁰C bred. After a cell density of 3 x 10 cells / ml is reached, the suspension is centrifuged at 800 xg for 10 min at room temperature. The collected cells are resuspended in 200 ml of culture medium containing glutamine (0.027% by volume), penicillin (200 units / ml) and streptomycin (50_/µg / ml) contains. The cells are 90 min at 37 ° C with Newcastle Disease Virus (NDV 110) at a ratio of 190 HAU / 10⁶ Cells (HAU = hemagglutination units) incubated. By adding fresh culture medium, the cell density is reduced to 1.3 x 10⁶ Cells / ml adjusted and the cell suspension is shaken at 34 ° C and 100 U / min. After 12 hours, 6 x 10⁹ Cells were harvested and resuspended in 50 ml of phosphate-buffered saline ("PBS"; 1 1 PBS contains 80 g NaCl, 2 g KC1, 14.4 g Na₂HPO₄ and 2 g KH₂PO₄). Before harvesting the cells, a sample is taken and the interferon activity is determined according to the method of Armstrong (29) using human CCL-23 cells and the vesicular stomatitis virus (VSV) as challenge virus. 4300 IFN units / ml are found.

b. Cell destruction and deproteinization

The cell suspension (6 x 10⁹ Cells in 50 ml PBS) is added at room temperature to 800 ml lysis buffer containing 0.05 M Tris.HCl (pH 7.5), 0.1 M NaCl, 5 mM EDTA and 2% SDS (crystalline research grade, Serva) contains. The lysate is digested with 0.2 mg / ml preincubated (2 h at 37 ° C) protease (protease P, type VI, Sigma) for 1 h at room temperature while stirring the solution. The solution is deproteinized by extracting three times with 500 ml of phenol saturated with TNE and extracting five times with 500 ml of chloroform. 500 mg of nucleic acids are obtained, determined by the absorption at 260 nm.

c. Removal of the contaminating DNA and

RN

A

The somewhat viscous aqueous solution obtained in step 1b is adjusted to 0.3 M NaCl and 1 g of oligo- (dT) -cellulose (type 7, PL Biochemicals) is added. After stirring for 30 minutes at room temperature, the suspension is centrifuged in 1 1 Sorvall bottles in a Sorvall RC-3 centrifuge for 10 min at 4000 rpm and at room temperature, and the oligo (dT) -cellulose slurry is washed twice with 40 ml twice TNE containing 0.5% SDS washed. The bound poly (A) RNA is then eluted by five successive washes with 2.5 ml of water.

The yield is 720_/µg of poly (A) RNA determined by measuring the optical density. The supernatant RNA solution from the first adsorption is adsorbed a second time on 1 g of oligo (dT) cellulose and eluted as described above. You get 320_/µg of poly (A) RNA. The eluates are combined, adjusted to TNE and the poly (A) RNA is precipitated with 67% ethanol at -20 ° C. for 10 h. The RNA is collected by centrifugation at 10,000 rpm in a Sorvall RC-5B centrifuge for 10 minutes at 0 ° C. The precipitate (1 mg) is redissolved in 1 ml of 1 mM EDTA.

The HuIFN mRNA activity of the RNA is analyzed by injecting into Xenopus laevis oocytes as follows: 50 nl of the RNA solution are injected into each of 20 oocytes. The oocytes are in Barth medium (2 mM Tris, 88 mM NaCl, 1 mM KC1, 0.33 mM Ca (NO₃)₂.H₂O, 0.41 mM CaCl₂.2H₂O, ₀,₈₂ mM MgSO₄.7H₂0, 2.4 mM NaHC0₃, 0.01 mg / ml penicillin, 0.01 mg / ml streptomycin; the pH of the solution is adjusted to 7.6 with HCl) according to Gurdon (42), Barth (43) and Colman et al. (30) incubated. The injected oocytes are incubated for 42 to 48 h and the incubation medium is removed, then centrifuged in an Eppendorf centrifuge for 5 min and the supernatant is stored at -20 or -80 ° C until it is used for the analysis. The IFN activity is determined essentially according to Armstrong (29), with whom_AChange that VSV is used as a challenge virus on Hep-2 cells (Flow Laboratories). The oocyte extract has a specific activity of 600 IU interferon / µg injected RNA.

d. Enrichment of the poly (A) RNA with HuIFN mRNA

The poly (A) RNA is passed through a Chelex 100 column (200 to 400 mesh, Bio-Rad) with a bed volume of 0.5 ml. The column is rinsed with 1 ml of 1mM EDTA.

The eluate [1 mg poly (A) RNA in 2 ml EDTA] is heated for 2 min at 100 ° C. and through a sucrose density gradient [six 14 ml sucrose solutions with increasing sucrose concentration from 5 to 23% (M / V) and with a Content of 50 mM Tris.HCl (pH 7.5), 0.2 M NaCl and 1 mM EDTA] centrifuged. Centrifugation is carried out in a TST 41 rotor (Kontron AG) at 35,000 rpm for 16 h at 5 ° C. 0.3 ml fractions are collected using an ISCO gradient collector. 2 vol. Ethanol is added to each fraction and the solution is left at -20 ° C for 10 h. The precipitated mRNA is collected by centrifugation (Sorvall, HB-4 rotor at 0 ° C, 10,000 rpm for 10 min). The precipitation of each fraction is again in 25_/ul 1 mM EDTA dissolved and each fraction is analyzed for its human IFN mRNA activity as described above (step 1c) with which _AChange that only ten oocytes are injected per RNA sample instead of twenty. The results are shown in Table 1.<tables id="tabl0001" num="0001"><img file="EP0076489A2_D0003.tif" /></tables>

Fractions 23 to 29 are pooled and the poly (A) RNA is further purified as follows.

The poly (A) RNA solution is adjusted to 2 x TNE in 0.5% SDS and to a 200_/ul given oligo (dT) cellulose column. The column is filled with 2 ml of 2 x TNE in 0.5%_SDS washed and the poly (A) RNA eluted by washing five times with 0.5 ml of water. The eluate is adjusted to TNE and the solution extracted twice with an equal volume of phenol (saturated in TNE) and twice with the same volume of chloroform. The poly (A) RNA is with 2 vol. Ethanol precipitated at -20 ° C for 10 h and collected by centrifugation in an HB-4 rotor as previously described.

The poly (A) RNA is in 100_/ul of 0.5 mM EDTA dissolved. The yield is 40_/µg, determined by measuring the optical density.

Part of the poly (A) RNA is analyzed for its human IFN activity as described above using 20 oocytes / assay. The poly (A) RNA preparation has a specific activity of 8100 IU interferon / µg RNA.

2nd Preparation of the double-stranded cDNA (Fig. 1)

HuIFN mRNA-enriched poly (A) RNA (see step 1d) is used as a template for the production of double-stranded cDNA, essentially according to Efstratiadis et al. (44), Maniatis et al. (45) and Hoeijmakers et al. (46) proceeds.

a. First strand synthesis

250_/ul reaction mixture containing 40 mM Tris. HC1 (pH 7.5), 30mM NaCl, 5mM MgCl₂, 0.5 mM DTT (Calbiochem.), 1 mM dGTP, dCTP, dTTP (PL Biochemicals) and 1 mM ³²P-dATP (Amersham, specific activity 5_{0 000 c}pm / nmol), 20_/µg / ml oligo (dT)_12-18 (PL-Biochemicals), 40_/µg / ml poly (A) RNA and 100 units

Bird myeloblastosis virus (AMV) -reverse _Transcriptase (Life Sciences, Inc., St.Petersburg, Florida) are 80 min at 37⁰C incubated. The reaction is stopped by adjusting the solution to 10 mM EDTA and 0.1% SDS. The mixture is extracted once with 1 volume of phenol. The aqueous phase is extracted again with 1 vol. Chloroform and added to a 3 ml Sephadex G-50 (Pharmacia, fine) column. 0.1 ml fractions are collected. The radioactivity of each fraction is determined by measuring the Cerenkov radiation. Radioactive fractions are collected and the nucleic acids are mixed with 2 vol. Ethanol precipitated at -20 ° C for 10 h. The sample is centrifuged in an HB-4 rotor for 20 min at 10,000 rpm at 0 ° C. The precipitate appears in 95_/ul water dissolved. 5_/µl 10N NaOH are added and the mixture is incubated at 25 ° C for 40 min. After neutralization with 5 M acetic acid, 50_/ul water and 2 vol. ethanol are added and the sample is stored at -20 ° C for 10 h. The precipitate is collected by centrifugation as previously described and again in 200_/ul 0.1 mM EDTA dissolved. The yield of single-stranded cDNA is 3.7_/ug. The length of the cDNA is 700 to 1500 nucleotides, determined from its electrophoretic mobility in a 6% polyacrylamide gel in tris-borate EDTA (108 g Tris, 9.3 g disodium EDTA and 55 g boric acid per 1 1 solution at pH 8 , 3), containing 7 M urea, based on marker DNAs of known length (32).

b. Second strand synthesis and S

1

-Endonuclease cleavage

The cDNA solution obtained is heated at 100 ° C. for 90 seconds, cooled and in a 400th_/ul reaction mixture containing 0.1 M potassium phosphate buffer (pH 6.9), 10 mM MgCl₂, 10mM DTT (Calbiochem), 1mM dATP, 1mM dCTP, 1mM dTTP (PL, Biochemicals), 1mM ³H-dGTP (Amersham, specific activity 94,000 cpm / nmol) and 165 units / ml E. coli DNA polymerase I (Biolabs, New England) incubated at 15 ° C for 8 h. The reaction is stopped by adding EDTA and SDS to final concentrations of 10 mM and 0.1%, respectively. The mixture is extracted with phenol and chloroform, chromatographed on Sephadex G-50 (Pharmacia, fine, 2 ml bed volume) and precipitated with ethanol as described above (stage 2a).

The resulting DNA is 30 min at 37 ° C in a 50_/ul incubation mixture containing 0.25 M NaCl, 50 mM sodium acetate (pH 4.5) and 1 mM ZnS0₄ treated with six units of S1 endonuclease (PL Biochemicals). The reaction is stopped with 0.1% SDS and 10 mM EDTA. The reaction mixture is deproteinized with 1 vol. Phenol (saturated in 50 mM sodium acetate, pH 4.5) and chloroform. The aqueous phase is chromatographed on a 2 ml Sephadex G-50 (Pharmacia, fine) column in TNE. 100_/µl fractions are collected and the Cerenkov radiation of each fraction is determined. The excluded fractions are combined and the DNA is precipitated with 2 vol. Ethanol at -20 ° C. for 10 h as described above. The precipitate is centrifuged in an HB-4 rotor (see above) and the collected precipitate in a 100_/ul solution containing 10 mM Tris.HCl (pH 7.5) and 0.5 mM EDTA. You get 4_/µg DNA.

The DNA is fractionated over a sucrose density gradient (5 to 23%) in 50 mM Tris-HCl (pH 7.5) and 1 mM EDTA in a TST-60 rotor (Kontron AG). Centrifugation is carried out at 35,000 rpm for 5 hours at 15 ° C. The DNA, which sediments faster than an 800 base pair marker DNA, which is checked in a parallel gradient, is pooled, adjusted to TNE and precipitated with 67% ethanol at -20 ° C. for 10 h. 0.4 µg of double-stranded cDNA is obtained.

3rd Production of pBR 322-linked cDNA (

F

ig. 1)

a. Production of dCMP-extended cDNA

The 3 'termini of 0.1 μg of the ds cDNA obtained are terminated with poly (dC) ends in a 10th_/ul provided reaction volume, the 100 mM sodium cacodylate (pH 7.2), 2.5 mM CoCl₂, 50 µg BSA (Calbiochem.) Per ml, 1 mM dCTP and 10 units of terminal deoxynucleotidyl transferase (PL Biochemicals) per µg ds cDNA. After incubation (20 min at 27 ° C) EDTA is added up to a content of 10 mM and the sample is stored at -20 ° C until use.

b. Production of Pst I cleaved, dGMP extended pBR 322

10th ug pBR 322 plasmid DNA with 10 units of Pst I endonuclease (Biolabs) in a 100 ul solution containing 50 mM NaCl, 6 mM Tris.HCl (pH 7.5), 6 mM MgCl₂, 6 mM 2-mercaptoethanol and 100_/µg / ml of gelatin digested at 37 ° C for 1 h. The solution is extracted with 1 vol. Phenol and chloroform. The solution is adjusted to TNE and the linearized DNA with 2 vol.

Ethanol precipitated at -20 ° C for 5 h.

The linearized plasmid DNA is extended with dGMP in a 200 ul reaction volume containing 100 mM sodium cacodylate (pH 7.2), 2 mM MgCl₂, 20 mM NaH₂PO₄, 50_/µg of BSA per ml, 1 mM dGTP and 100 units of terminal deoxynucleotidyl transferase (PL Biochemicals).

After 2₀After incubation at 37 ° C. for a minute, EDTA is added to a content of 10 mM and the reaction mixture is frozen at -20 ° C. until use.

c. Hybridization of dGMP-extended pBR 322 with dCMP-extended ds cDNA

A mixture of dCMP-extended, double-stranded cDNA (0.1 µg) and dGMP-extended, linearized pBR 322 (0.5 µg) in 500 µl TNE buffer is 1 h at 65 ° C, 1 h at 46 ° C, Incubated for 1 h at 37 ° C and 1 h at 20 ° C. The solution containing the cDNA linked to pBR 322 is placed on ice and used immediately for the transformation.

4th Transformation of E. coli HB 101 with the hybrid plasmid

Calcium-treated E. coli HB 101 is made according to the method of Mandel et al. (35) made for the transformation.

10th µl of the reaction mixture containing the pBR 322 hybrid plasmid DNAs prepared as described above (stage 3c) are added to a mixture containing 150 µl of calcium-treated E. coli HB 101 in 10 mM MgCl₂, 10 mM CaCl₂and contains 10 mM Tris.HCl (pH 7.5) in a total volume of 200 µl.

The mixture is cooled in ice for 20 min, heated to 42 ° C. for 1 min and incubated at 20 ° C. for 10 min. 1 ml trypton medium [trypton medium contains 10 g bacto-trypton (Difco); 1 g yeast extract (Difco); 1 g glucose; 8 g NaCl and 294 mg CaCl₂.2H₂O in 1 l of distilled water] is added and the mixture is incubated at 37 ° C. for 30 min with shaking at 300 rpm. The mixture is plated on two agar plates (Mc Conkey Agar, Difco; 0.6 ml / plate), supplemented with 10 μg / ml tetracycline (Sigma). The plates are incubated at 37 ° C for 12 to 17 h. About 5600 TetracycLin-resistant colonies of transformed E. coli HB 101 are produced.

5. Identification of the clones containing HuIFN cDNA

a. Synthesis of the 13-mer oligodeoxynucleotide primer (Fig. 2)

An oligodeoxynucleotide that is complementary to a 13 nucleotide range that contains both HuIFN-a_l as well as HuIFN-ß mRNA is common, is chemically according to the phosphotriester method [cf. Itakura et al. (38), de Rooij et al. (39)] synthesized. The individual stages of the synthesis are shown in Fig. 2.

The starting materials shown in line 1 of FIG. 2 (protective groups carrying mono- and dideoxynucleotides) are known from the literature. The protecting groups are designed according to the methods described by Itakura et al. split off the described process: The deblocking of 5'-monomethoxytrityl (M) or dimethoxytrityl (D) -substituted hydroxyl groups is carried out with acetic acid (80%) at room temperature, and the β-cyanoethylphosphate groups are mixed with 0.1N sodium hydroxide in dioxane Split off water (4: 1) at room temperature. The building blocks are condensed using triisopropylbenzenesulfonyl chloride as the activating agent, giving oligodeoxynucleotides up to the fully protected 13-mer primer shown in line 7 of FIG. 2. The last stage (complete removal of all protective groups) is carried out as follows:

A solution containing 64.6 mg of the completely protected 13-mer oligodeoxynucleotide in 3 ml of dioxane and 1 ml of acetonitrile is mixed with 200 mg of syn-p-nitrobenzaldoxime and 124 mg of N¹, N¹, N³, N³-Tetramethylguanidin treated and left for 27 h. 10 ml of ammonia (25%) are added and the solution is stored at 50 ° C. for 24 hours. After evaporation of the solvent in vacuo, the residue is dissolved in water, adjusted to pH 4 with acetic acid and the solution extracted 20 times with chloroform. The aqueous solution is evaporated in vacuo and the

'Residue dissolved in 1 ml acetic acid (80%). The solution is left to stand for 1 h, diluted with 6 ml of water, extracted three times with chloroform and lyophilized.

A third of the product obtained is purified by chromatography on DEAE-Sephadex A 25 (column size: 10 × 1.3 cm) using a 200 ml 0.2-1.2 M triethylammonium bicarbonate gradient. The elution of the main fraction occurs at a gradient concentration of 0.87 M. The main fraction, which contains the pure product, which can be recognized by an HPLC test, is evaporated three times with water, through 10 ml of Dowex 50 W (NH₄Salt) filtered and lyophilized. HPLC (Permaphase AAX, column size 90 x 0.3 cm, 60 ° C, 2 ml / min; gradient: A = 0.005 M KH₂PO₄, B = 0.5 M KH₂PO₄, 0.5 M KCl, pH 4.5; 20% A → 100% B in 30 min): t_R 11.8 min.

b. Making one

32

P-labeled, human IFN-a and IFN-ß specific cDNA probe (Fig. 3)

40 pmol of the synthetic 13-mer oligodeoxynucleotide primer (compare step 5a) and 40 pmol [γ-³²P] -ATP (5700 Ci · mmol^-1, Amersham) will be in 100_/ul 50mM Tris.HCl (pH 9.5), 10mM MgCl₂ and 5 mM DTT combined. 50 units of T.₄ Polynucleotide kinase (PL biochemicals) are added and after 30 min at 37 ° C. a further 20 units of the enzyme are added. Incubation is continued at 37 ° for a further 15 min_C.. The the³²Aqueous solution containing P-labeled primer is purified by phenol extraction. A further purification is carried out by chromatography on a 4 ml Sephadex G-50 (Pharmacia, fine) column in 1 mM Tris.HCl (pH 8.0). 0.1 ml fractions are collected. The radioactivity of each fraction is determined by measuring the Cerenkov radiation. A specific activity of 4 x 10⁶Cerenkov cpm / pmol oligodeoxynucleotide is obtained. Of the³²P-labeled primer (40 pmol) is lyophilized, again in 91 ul water containing 14_/µg of poly (A) RNA (from induced Namalwa cells, produced according to step 1), suspended and heated at 100 ° C. for 60 seconds. 9 μl of 4 M KC1 are added and the mixture is incubated at 25 ° C. for 60 min. 450 ul of a reverse transcriptase mixture are added so that the reaction volume 40 mM Tris.HCl (pH 8), 4 mM MgCl₂, 1mM DTT (Calbiochem., Inc.), 74mM KCl, 1mM each of dATP, dGTP, dCTP, dTTP (PL-Biochemicals) and 90 units of bird myeloblastosis virus (AMV) -reverse transcriptase. The incubation is continued for 1 h at 37 ° C. The solution is extracted with 1 vol. Phenol (saturated in TNE) and the nucleic acids are precipitated with 2 vol. Ethanol for 10 h at -20 ° C. The precipitate is centrifuged (HB-4 rotor, 20 min, 10,000 rpm, 0^OC) collected and dissolved in 20 ul dye mixture containing 90% (v / v) formamide (Merck, per analysis), 1mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyano blue. The sample is heated at 90 ° C. for 2 min and placed on a 5% polyacrylamide gel in tris borate EDTA [cf. Peacock et al. (32)] is given. A single band can be seen on the autoradiogram, which is between the 267 bp and 435 bp³²P-marked _Marker DNA fragments obtained from the Hae III digest of the plasmid pBR 322 migrated. The³²P-labeled cDNA fragment is extracted from the gel and according to Mueller et al. (47) cleaned. 20,000 Cerenkov cpm are obtained³²P-labeled human IFN-a and IFN-ß specific cDNA probe.

c. Screening for colonies containing HuIFN cDNA (Fig. 3)

1650 Transformant colonies, prepared in accordance with stage 4, are transferred to nitrocellulose filters BA 85 (Schleicher &amp; Schuell, 8 cm diameter). The cells are lysed, their DNA denatured and fixed in situ to the filters according to Grunstein and Hogness (36). The colony-bearing filters are placed in 4 x SET [a solution containing 0.15 M NaCl, 30 mM Tris.HCl (pH 8.0), 1 mM EDTA], 0.1% (w / v) Ficoll 400 (Pharmacia), 0.1% (w / v) polyvinyl pyrrolidone (PVP-360, Sigma), 0.1% (vol / vol) BSA, 0.5% SDS, 50_/µg / ml denatured veal_Hymus DNA [prepared as follows: 5 mg calf thymus DNA (type I, Sigma) are boiled for 10 min in 0.5 M NaOH to shear the DNA, neutralized with 5M acetic acid and precipitated with 2 vol. ethanol at -20 ° C. ] pre-hybridized. The precipitate is centrifuged in an HB-4 rotor for 10 min at 0⁰C collected and again in 500_/ul 0.5 mM EDTA at 65 ° C for 4 h using 20 ml mixtures / filters and dissolved with 10³ Cerenkov _cp_m of the 32P-labeled sample per nitrocellulose filter in 5 x SET, 0.02% (w / v) Ficoll, 0.01% polyvinylpyrrolidone, 0.02% (vol / vol) BSA, 0.2% SDS and 50_/µg / ml denatured calf thymus DNA hybridizes. The hybridization is carried out at 65 ° C. for 36 h.

The filters are rinsed once in chloroform, twice in SET, 0.5% SDS at room temperature and twice in SET, 0.5% SDS for 1 h at 60 ° C and once with 3 mM Trizma base for 1 h at room temperature. The filters are dried by placing them on 3MM paper (Whatman) and an X-ray film (Fuji) is placed under the filters_Vexposed to the use of a film filter (Ilford amplification filter) at 80 ° C for 72 h.

Nine positive colonies are identified on the autoradiogram and these are used for further investigation.

Since the primary clones of transformed cells sometimes contain more than one species of recombinant DNA molecule, the hybrid plasmid DNAs are isolated from the nine positively hybridized clones and used to retransform E. coli HB 101 as described above.

The hybrid plasmid DNA is isolated as follows: 1 colony is used to inoculate 10 ml tryptone medium, supplemented with 10_/µg / ml tetracycline as above used in a 25 ml Erlenmeyer flask. The culture is 15 to 18 h at 37 ° C and 300 rpm. shaken. The cells are harvested by centrifugation (Sorvall, HS-4 rotor, 10 min at 4000 rpm, 4 ° C). About 0.1 g of cells are obtained and these are resuspended in 1 ml of 50 mM Tris.HCl (pH 8.0). 0.25 ml lysozyme solution [10 mg / ml in 50 mM Tris.HCl (pH 8.0); Lysozyme is sold by Sigma] are added and, after 10 minutes of incubation at 0 ° C., 0.15 ml of 0.5 M EDTA (pH 7.5) are added. After a further 10 min at 0 ° C, 60_/ul 2% Triton X-100 (Merck) added. After 30 minutes at 0 ° C, the sample is centrifuged for 30 minutes at 15,000 rpm and 4 ° C in a Sorvall SA-600 rotor. The supernatant is deproteinized with 1 vol. Phenol (saturated in TNE). The phases are separated by centrifugation (Sorvall HB-4 rotor) for 10 min at 5000 rpm and 4 ° C. The upper phase is extracted twice with 1 vol chloroform. Pancreatic RNAse A (Sigma; 10 mg / ml in TNE preheated at 85 ° C for 10 min) is up to a final concentration of 25_/µg / ml added and the mixture incubated at 37 ° C for 40 min.

The solution is then adjusted to 1 M NaCl and 10% polyethylene glycol 6000 (Fluka, treated in an autoclave for 20 min at 120 ° C.) and incubated at -10 ° C. for 2 h.

The precipitate is collected in a Sorvall HB-4 rotor (20 min at 10,000 rpm, 0 ° C) and again in 100_/ul TNE solved. The DNA solution is extracted with 1 vol. Phenol and the DNA is precipitated with 2 vol. Ethanol for 10 min at -80 ° C. The precipitate is collected by centrifugation in an Eppendorf centrifuge and the DNA again in 20_/ul 10 mM Tris.HCl (pH 7.5) and 0.5 mM EDTA dissolved. 8 to 10 are obtained from a 10 ml culture_/µg of hybrid plasmid DNA.

E. coli HB 101 is transformed with each of the nine hybrid DNAs isolated and the transformed cells are plated on agar plates containing tetracycline as previously described (step 4). Three tetracycline-resistant clones are picked from each transformation; 10 ml cultures are prepared and the hybrid DNAs are isolated from the cultures as previously described.

All DNA samples before and after the retransformation are analyzed by cleavage with Pst I endonuclease and electrophoresis through a 1% agarose gel in 50 mM Tris acetate (pH 7.8) and 1 mM EDTA. All samples show identical cleavage patterns before and after retransformation.

One of the cloned recombinant DNA molecules gives two bands, one of which shows the mobility of Pst I-cleaved pBR 322 and the other shows the mobility corresponding to about 1000 bp. It is referred to as CG-pBR 322 / HLycIFN-1'b.

Another recombinant DNA gives three bands, one of which corresponds to the mobility of Pst I-cleaved pBR 322, one to the mobility of approximately 600 bp and one to the mobility of approximately 150 bp. The recombinant DNA molecules in this clone are called CG-pBR 322 / HLycIFN-ß₁ designated.

d. Characterization of the clones CG-pBR 322 / HLycIFN-1'b and CG-pBR 322 / HLycIFN-ß

1

The recombinant plasmid DNAs of the clones CG-pBR 322 / HLycTFN-1'b and CG-pBR 322 / HLycIFN-ß₁ are isolated and characterized from the cultures as described above (step 5c) by using the nucleotide sequence of the cDNA- _I.nserts using the method described by Maxam and Gilbert (41). Basically, the following procedure is used.

The isolated, recombinant plasmid DNA is digested with various restriction endonucleases. The enzymes are used essentially according to the manufacturer's instructions (New England Biolabs), with the change that BSA is replaced by gelatin in the enzyme buffers. The solution containing the restricted DNA is deproteinized with phenol (saturated with TNE). The DNA is precipitated with ethanol, redissolved in 50 mM Tris-HCl (pH 8.0) at a DNA concentration of 50 µg / ml and with 0.1 units of intestinal alkaline calf phosphatase (Boehringer) per pmol of DNA-5 Incubated at 37 ° C for 30 min. The enzyme is inactivated by heating the solution at 65 ° C for 60 min. The DNA is by DEAE cellulose chromatography according to Mueller et al. (47) purified and precipitated with ethanol. The DNA will then be 5^!-terminal with [γ-³²P] -ATP (&gt; 5000 Ci / mmol, Amersham) and T4 polynucleotide kinase (PL Biochemicals) were marked essentially according to the method described by Maxam and Gilbert (41), with the change that the DNA was not denatured before the kinase reaction . In general, the specific activities are 1 to 3 x 10⁶ cpm / pmol 5 'ends.

The labeled DNA fragments are digested with a second restriction endonuclease and the products are separated by electrophoresis through a 6%, 8% or 10% polyacrylamide gel in Tris-Borate-EDTA buffer. The DNA fragments are extracted from the gel and according to Mueller et al. (47) cleaned. For the determination of the nucleotide sequences, the DNA fragments are broken down chemically and the products are separated by polyacrylamide gel electrophoresis according to Maxam and Gilbert (41).

In particular, the isolated plasmid DNAs of the clone CG-pBR 322 / HLycIFN-1'b are treated as follows. On the one hand, 5_/µg of the plasmid DNA digested with Bgl II, 5'-terminally labeled and digested with Pvu II. The Pvu II-Bgl II⁺- (+ denotes the marked position) and Bgl II-Pvu II DNA fragments are isolated on a 6% polyacrylamide gel. On the other hand, 5th_/µg of the plasmid digested with Alu I, 5'-terminally labeled and cleaved with Pst I. The Pst I-Alu I⁺ DNA fragment is isolated on an 8% polyacrylamide gel. The individual fragments are broken down one after the other and then the sequence is determined according to Maxam and Gilbert. The nucleotide sequence obtained is shown in FIG. 4. A range of about 25 to 35 deoxyguanosine residues precedes the 5 'end of the cDNA insert. The nucleotide sequence shown is similar to that of IFN-a (type F) cDNA described by Goeddel et al. [(14); compare also Weissmann (3)], but shows many clear deviations (point mutations), some of which influence the resulting amino acids (see FIG. 4).

The isolated plasmid DNA of the clone CG-pBR 322 / HLycIFN-ß₁ is treated in a similar way. 5 µg of the plasmid are digested with Pvu II and 5^1-marked terminal. Half of the mixture is cleaved with Pst I and the rest with Bgl II. The Pst I-Pvu II^+- and Bgl II-Pvu II^+-Fragments are isolated by electrophoresis on a 6% polyacrylamide gel and broken down as described above. The nucleotide sequence (N-terminal sequence) can be seen from FIG. 5 and shows that the cDNA insert at the nucleotide number 102 is IFN-β₁ cDNA as described by Taniguchi et al. (17) begins. Therefore, the cDNA insert has the capacity for human IFN-ß₁, which lacks 11 amino acids at the N-terminus. Of the_cDNA-Insert is flanked at its 5 'end by a range of about 20 to 25 deoxyguanosine residues and shows a point mutation in position 153 where a C- has been converted to a T residue without affecting the corresponding amino acid.

e. Identification of the clones which contain recombinant DNA molecules which contain the inserts from CG-pBR 322 / HLycIFN-l'b and CG-pBR 322 / HLycIFN-ß

1

cross hybridize

The recombinant plasmid DNAs of the clones CG-pBR 322 / HLycIFN-1'b and CG-pBR 322 / HLycIFN-ß₁ are isolated from the cultures according to level 5C. The CG-pBR 322 / HLycIFN-1'b plasmid DNA (5th_/ug) is digested with Bgl II, 5^1-marked terminally and cleaved with Pvu II. On the other hand, the isolated CG-pBR 322 / HLycIFN-ß₁ Plasmid DNA (5th_/ug) digested with Pvu II, 5^1-marked terminally and cleaved with Bgl II. The Pvu II-Bgl II⁺(351 bp) DNA fragment (probe A) and the Pvu II^+-Bgl II (368 bp) DNA fragments (probe B) are isolated from an 8% polyacrylamide gel according to step 5d and used for in situ colony hybridization (cf. below). The restriction of the plasmid DNAs, the labeling and the purification of the DNA fragments are carried out in the same way as described in step 5d above.

4000 the transformant colonies produced in accordance with stage 4 above are transferred to nitrocellulose filters BA 85 (Schleicher &amp; Schuell, 8 cm diameter). The cells are lysed, their DNA is denatured and fixed to the filters in situ according to Grunstein and Hogness (36). Hybridization of probes A and B (both samples are mixed) is carried out according to step 5c. Autoradiography identifies six positive colonies, three of which are referred to as<ul id="ul0008" list-style="none"><li>E. coli HB 101 CG-pBR 322 / HLycIFN-4₁,</li><li>E. coli HB 101 CG-pBR 32ZhILycIFN-5₁ and</li><li>E. coli HB 101 CG-pBR 322 / HLycIFN-8 '₁</li></ul>

be used for further investigation. The plasmid DNAs from these clones are isolated, retransformed and re-isolated as described in steps 5c and 5d above.

To establish the nature of the inserts of the recombinant DNAs, the nucleotide sequences of the cDNA inserts (partially or completely) are determined using the method described in step 5d above.

In particular, 5_/µg of the isolated plasmid DNAs CG-pBR 322 / HLycIFN-4₁ and CG-pBR 322 / HLycIFN-81 digested with Pvu II, 5'-terminally labeled and digested with Pst I. The DNA fragments are fractionated on an 8% polyacrylamide gel and Pst I-Pvu II⁺ (~ 120 bp) from 8 '₁ DNA and Pst I-Pvu II⁺ (82 bp) from 4₁ DNA isolated in the usual way.

The isolated plasmid DNA CG-pBR 322 / HLycIFN-5₁ is treated as follows. On the one hand, 5_/µg of the plasmid DNA digested with Hae III, 5^1-marked terminally and cleaved with Pst I. The Pst I-Hae III⁺ (57 bp) DNA fragment is isolated on 10% polyacrylamide gel. On the other hand, 5th_/µg of the plasmid digested with EcoR I, 5'-terminally labeled and cleaved with Pst I. The Pst I-EcoR I⁺ (235 _bp) and EcoR I⁺-Pst 1 (~ 700 bp) DNA fragments are isolated on 8% polyacrylamide gel. The various DNA fragments are subjected to sequence analysis according to Maxam and Gilbert (41).

The nucleotide sequences of the cDNA inserts can be seen in FIGS. 6 to 8. In Figure 6 is the nucleotide sequence of the cDNA insert from CG-pBR322 / HLycIFN-4₁ shown. The insert is flanked at the 5 'end by a region of 23 deoxyguanosine residues and comprises part of the IFN-α₂(Le) cDNA as described by Streuli et al. (12). In the 3'-extra-cistronic region there are some minor deviations (point mutations) and an additional 318 nucleotides. The nucleotide sequence of the cDNA insert of CG-pBR 322 / HLycIFN-8 '₁ is shown in Fig. 7. The insert is flanked at the 5 'end by a range of 20 to 23 deoxyguanosine residues and is similar but not identical to that described by Goeddel et al. [(14); see also Mantei et al. (11)] described IFN-a (type D) cDNA. In addition to the differences in the cDNA regions that precede and follow the IFN coding sequence, the IFN gene contains a GCC triplet at positions 28 to 30 and a GCG triplet, coding for alanine, at positions 409 to 411 from GTC or GTG and coding for valine. Finally, the nucleotide sequence of the cDNA insert from CG-pBR 322 / HLycIFN-5 shows₁ (see Figure 8) a range of 17 deoxyguanosine residues at the 5 'end. The nucleotide sequence is related to that of IFN-a (type B) cDNA as described by Goeddel et al. (14) is described. However, additional nucleotides appear at the 5 'end of the HLycIFN-5 cDNA insert₁, Point mutations, excisions and insertions in the extracistronic region and in the IFN coding sequence, in particular in positions 22 and 361 to 372.

6. Synthesis of human interferons by E. coli, containing human IFN-specific recombinant DNA molecules

The five clones that were shown to contain human IFN-specific recombinant DNA molecules, namely<ul id="ul0009" list-style="none"><li>E. coli 11B 101 CG-pBR 322 / HLycIFN-l'b</li><li>E. coli IIB 101 CG-pBR 322 / HLycIFN-4₁,</li><li>E. coli HB 101 CG-pBR 322 / HLycIFN-5₁,</li><li>E. coli HB 101 CG-pBR 322 / HLycIFN-8 '₁, and</li><li>E. coli HB 101 CG-pBR 322 / HLycIFN-ß_{l '}</li></ul>

are checked for IFN activity, whereby in each case proceed as follows:

Cultures of the corresponding E. coli clone (30 ml suspensions) are grown in tryptone medium to an optical density _(OD650) about ₁ bred. The cells are harvested and resuspended in 0.5 ml of an aqueous solution containing 30 mM NaCl and 50 mM Tris-HCl (pH 8.0). Lysozyme (Sigma) is added up to 1 mg / ml. After 30 min at 0 ° C, the suspensions are frozen (liquid nitrogen) and thawed at 37 ° C (five times) and 20 min at 20,000 rpm in an SS 34 Sorvall rotor centrifuged at 4 ° C. The supernatants are analyzed for IFN activity using the Armstrong (29) cythopathic bioassay as described in step 1c. The following activities are noted:<tables id="tabl0002" num="0002"><img file="EP0076489A2_D0004.tif" /></tables>

Clones that show no measurable IFN activities may contain recombinant DNAs in which the HuLyIFN cDNA insert has the wrong orientation with respect to the direction of transcription. Therefore, the recombinant DNA of such a clone (CG-pBR 322 / HLycIFN-l'b), which contains a complete cDNA insert, is reoriented as follows:

The plasmid DNA of the clone E. coli HB 101 CG-pBR 322 / HLycIFN-1'b is isolated according to step 5c and digested with Pst I. 0.5_/µg of the cleaved DNA in 20_/µl of a buffer mixture containing 20 mM Tris-HCl (pH 7.8), 10 mM NgCl₂, 10 mM DTT, 25 mM NaCl and 50_/µg / ml gelatin are treated with 0.2 units of T4 DNA ligase (Biolabs) and 0.5 mM ATP for 2 h at 15 ° C. E. coli HB 101 is transformed with the cDNA mixture according to step 4. Transformed colonies are selected on Mc Conkey agar plates supplemented with tetracycline and then replica-plated on nitrocellulose filters. Four colonies of bacteria, which with the³²P-marked Pvu II-Bgl II^+- Hybridize fragments (351 bp) of the recombinant DNA CG-pBR 322 / HLycIFN-1'b (cf. step 5e) are called E. coli HB 101 CG-pBR 322 / HLycIFN-1'b₁ to -1'b₄ designated. Extracts of the four clones are prepared and tested for IFN activity as described above.

The following activities are found:

<tables id="tabl0003" num="0003"><img file="EP0076489A2_D0005.tif" /></tables>The plasmid CG-pBR 322 / HLycIFN-1'b₄ contains a cDNA insert that can direct the synthesis of a polypeptide with IFN activity.

7. Construction of recombinant plasmids that can produce high levels of polypeptides with IFN activity and transformation of E. coli HB 101 with these plasmids

A. Construction of the recombinant plasmid CG-pBR (AP) / LyIFN-a-1

In order to improve the IFN-specific protein yield of the clone E. coli HB 101 CG-pBR 322 / HLycIFN-1'b, the following construction is carried out, as indicated schematically in FIG. 9.

a. Preparation of the cDNA insert

The recombinant plasmid DNA (150_/µg) of the clone E. coli HB 101 CG-pBR 322 / HLycIFN-1'b is cleaved with Pst I (Biolabs) using standard methods (compare step 5d). Following the phenol extraction and the ethanol precipitation, the excised insert is isolated by means of sucrose density gradient centrifugation (5 to 23%) in 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA. Centrifugation takes place at 35,000 rpm in a TST 41 rotor (Kontron AG) at 15 ° C for 16 h. 0.3 ml fractions are made with an ISCO gradient collector. collected at 1 ml / min. The fractions containing the small fragment (ie the insert) are pooled. The DNA is precipitated in a conventional manner with ethanol and the precipitate is collected by centrifugation in an HB-4 rotor (Sorvall) at 10,000 rpm and 0 ° C. for 10 minutes. The precipitate turns 60 again_/ul 10 mM Tris-HCl (pH 7.5) and 0.05 mM EDTA dissolved. You win 30_/µg of DNA determined by measuring the optical density.

The insert DNA (10_/µg) is digested with Hae III (Biolabs) and the fragments are on 2% agarose gel in a solution containing 50 mM Tris, 50 mM boric acid, 1 mM EDTA and 0.5_/µg / ml ethidium bromide fractionated. The largest DNA fragments, Hae III-Pst I (869 bp) and Hae III-Hae III (82 bp, see FIG. 9, fragments 3 and 4) are each excised from the gel by a thin needle with a Syringe in 5 ml 0.15 M NaCl, 50 mM Tris.HCl (pH 8.0), 1 mM EDTA and eluted overnight by shaking. The eluate is separated by a 100_/ul DE-52 (Whatman) Pasteur pipette column for adsorption of DNA. The column is washed with 2 ml of the same buffer and the DNA with 400_/µl of a solution eluted containing 1.5 M NaCl, 50 mM Tris (pH 8.0) and 1 mM EDTA. The DNA is precipitated with 2 vol. Ethanol at -20 ° C. overnight. The precipitate is collected by centrifugation in an Eppendorf centrifuge.

The HaeIII-Hae III DNA fragment (82 bp) is redissolved and digested with Sau 3A (Biolabs). The enzyme is at 65 for 30 min⁰C in_dit inactivates heat. 1_/µg of the Hae III-Pst I DNA fragment (869 bp) is added, the solution is made up to 10 mM MgCl₂, 10 mM DTT and 0.5 mM ATP and T4 DNA ligase (Biolabs) becomes 30 units / _/ul reaction volume added. The solution is 10 h at 15⁰C incubated. Following extraction with phenol and chloroform, the mixture is fractionated on a 2% agarose gel in tris-borate EDTA in the presence of ethidium bromide. The Sau 3A-Pst I DNA fragment (cf. FIG. 9, fragment 5) is extracted as described above, precipitated with ethanol and again in 10_/ul of a solution containing 10 mM Tris.HCl (pH 7.5) and 0.05 mM EDTA.

b. Preparation of the DNA fragment which contains the regulatory region of the β-lactamase gene (ApPr) from pBR 322

The plasmid pBR 322 is cleaved with Pst I (see step 3b) and treated with 4 U / ml exonuclease Bal 31 (Bethesda Research Lab.) For 4 to 10 min at 30 ° C. to remove the β-lactamase coding segment.

A chemical DNA linker of the formula

<img file="EP0076489A2_D0006.tif" />

is synthesized using the method described in step 5a. The linker is added to the Bal 31 treated pBR 322 DNA by means of a known link. The resulting hybrid molecule is cleaved with the restriction endonucleases Bcl I (Biolabs) and EcoR I. The digestion products are fractionated on 8% polyacrylamide gel in tris-borate EDTA according to stage 2a. DNA fragments (ApPr DNA fragments) that migrate between 184 bp and 234 bp marker DNAs are isolated according to step 7a and precipitated in a conventional manner with ethanol. The precipitate is redissolved in a solution containing 10 mM Tris.HCl (pH 7.5) and 0.05 mM EDTA.

c. Linking the ApPr-DNA fragment to the cDNA insert and producing the plasmid CG-pBR (AP) / LyIFN-a-1

The solutions containing the ApPr DNA fragments and the cDNA insert are combined. The mixture is made up to 10 mM MgCl₂, 10 mM DTT and 0.5 mM ATP and 12 h at 15 ° C with 30 U /_/ul T4 DNA ligase (Biolabs) incubated. Following the extraction with phenol and chloroform, the mixture is fractionated on a 1% low-melting agarose gel (organic wheel). The ApPr cDNA fragment obtained is linked to the large fragment of pBR 322 which has been digested with both Pst I (Biolabs) and EcoR I (Biolabs) as follows. 'That the ApPr cDNA fragment (about 20_/The gel piece containing ul) is mixed with the Pst I-EcoRI fragment from pBr 322, melted at 65 ° C. for 2 min, cooled to 37 ° C., to 0.5 mM ATP, 10 mM DTT and 10 mM MgCl₂ adjusted and incubated for 12 h at 15 ° C with 30 U / ul T4 DNA ligase (Biolabs), whereby a solution is obtained which contains the recombinant plasmid, designated CG-pBR (AP) / LyIFN-a-1.

d. Transformation of E. coli HB 101 with the plasmid CG-pBR (AP) / LyIFN-α-1

_1/10th Volume of a solution containing 100 mM Tris.HCl (pH 7.5), 100 mM CaCl₂ and 100 mM MgCl₂ is added to the solution containing the plasmid CG-pBR (AP) / LyIFN-α-1. The combined solutions are heated at 65 ° C for 10 min to inactivate the ligase and cooled to 37 ° C. The solution is then used to approx^2+-transformed E. coli HB 101 according to step 4, and on Mc Conkey agar plates, supplemented with 10_/µg / ml tetracycline, plated. The transformed colonies are checked for their IFN activity (see step 6). The clone that synthesizes the highest level of IFN activity is selected and designated E. coli HB 101 CG-pBR (AP) / LyIFN-α-1. An activity of 40,000 (IU / ml) is determined, which corresponds to a 1300-fold stimulation compared to the original clone E. coli HB 101 CG-pBR 322 / HLycIFN-1'b.

The recombinant plasmid DNA of the clone CG-pBR (A) / LyIFN-a-1 is isolated from the culture according to step 3c and characterized by the nucleotide sequence of the cDNA- _I.nserts (IFN gene) and the ß-lactamase regulatory region determined. The result is shown in FIG. 10.

B. Construction of the recombinant plasmid CG-pBR (AP) / LyIFN-α-3.

The IFN-specific protein yields of the clone E. coli HB 101 CG-pBR 322 / HLycIFN-8 '₁ is improved as follows (see FIG. 11):

a. Preparation of the DNA fragment containing the β-lactamase regulatory region from CG-pBR (AP) / LyIFN-a-1

CG-pBR (AP) / LyIFN-α-1 DNA (100 µg) is cleaved with Hind III (Biolabs) and Bgl II (Biolabs). Following the phenol extraction and the ethanol precipitation, the excised DNA fragment is isolated by sucrose density gradient centrifugation (5 to 23%) in 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The centrifugation is carried out for 4 hours at 15 ° C. at 58,000 rpm in a TST 60 rotor (Kontron AG). 0.2 ml fractions are collected as previously described. The fractions containing the small fragment (Hind III-Bgl II) are combined and the DNA is precipitated with ethanol as usual. The precipitate turns 80 again_/ul 10 mM Tris-HCl (pH 7.5) and 0.05 mM EDTA dissolved. 16_/µg of DNA are isolated, determined by measuring the optical density.

The DNA fragment (Hind III-Bgl II) (4th_/ug) with sow 3_A (Biolabs) cleaved and the digestion products are fractionated on 6% polyacrylamide gel in Tris-Borate-EDTA, as previously described. The DNA fragments are in EtBr (0.5_/µg / ml) stained. The Hind III-Sau 3A DNA fragment (239 bp) is extracted and isolated as previously described. The DNA is precipitated with ethanol in the usual way. The precipitate appears again in 20_/ul 10 mM Tris-HCl (pH 7.5) and 0.05 mM EDTA dissolved.

b. Preparation of the cDNA insert

The cDNA insert is made from the recombinant plasmid CG-pBR 322 / HLycIFN-8 '₁ with Pst I as described above (Section 7a).

The cDNA insert (2µg) is digested with 2.5 units of Sau 3A (Biolabs) in 10µg / ml EtBr and incubated at 37 ° C for 60 min. The cleavage pieces are extracted with phenol and the DNA is precipitated in ethanol as above. The D_{N / A}Fragments are on 1.2% agarose gel in a solution containing 50 mM Tris, 50 mM boric acid, 1 mM EDTA and 0.5_/µg / ml ethidium bromide fractionated.

The second largest DNA (Sau 3A-Pst I: 693 bp) is extracted from the gel and purified according to section 7a. The DNA is again in 20_/ul 10 mM Tris-HCl (pH 7.5) and 0.05 mM EDTA dissolved.

c. Linking the Hind III-Sau 3A DNA fragment with the cDNA insert (Sau 3A-Pst I)

Equal amounts of both DNA fragments (-50 ng) are incubated in a solution containing 10 mM MgGl₂, 10 mM DTT, 0.5 mM ATP and 30 U /_/ul T4 DNA ligase (Biolabs) contains, for 3 h at 15 ° C. The mixture is incubated at 80 ° C. for 15 min and adjusted to 50 mM NaCl. The DNA mixture is digested with 0.5 units of Pst I (Biolabs) and 1 unit of Hind III (Biolabs) for 20 min at 37 ° C. The DNA is extracted with phenol, precipitated with ethanol and again in 10_/µl 10mM Tris-HCl (pH 7.5) and 0.05mM EDTA dissolved.

Half of the resulting mixture is with the large Hind III-Pst I DNA fragment of the plasmid pBR 322 (-100 ng) in 10 mM MgCl₂, 10 mM DTT, 0.5 mM ATP, containing 30 U / µl T4 DNA ligase (Biolabs), linked for 2 h at 15 ° C. A solution is obtained which contains the recombinant plasmid CG-pBR (AP) / LyIFN-α-3.

d. Transformation of E. coli HB 101 with the plasmid CG-pBR (AP) / LYIFN-α-3

1/10 volume of the above solution is used to transform E. coli HB 101 as described in step 4. The transformed colonies are used to test level 6 IFN activity.

The clone that synthesizes the highest level of IFN activity is selected and designated E. coli HB 101 CG-pBR (AP) / LyIFN-a-3.

IFN activity is determined according to step 6 above. An activity of 70,000 (IU / ml) is determined, which is a 700-fold stimulation in comparison with the original clone E. coli HB 101 CG-pBR 322 / HLycIFId-8 '₁ corresponds.

The recombinant plasmid DNA of the clone CG-pBR (AP) / LyIFN-a-3 is isolated from the culture according to step 3c and characterized by the nucleotide sequence of the cDNA insert (IFN gene) and the ß-lactamase regulatory region certainly. The result is summarized in Fig. 12.

The construction protocol for the plasmid CG-pBR (AP) / LyIFN-a-3 can generally be used for all α-IFN cDNA genes or appropriately cut chromosomal a-IFN genes.

For example, starting from the plasmid CG-pBR 322 / HLycIFN-5₁obtained the plasmid CG-pBR (AP) / LyIFN-a-2 in an identical manner as described for the plasmid CG-pBR (AP) / LyIFN-a-3. This new plasmid contains the DNA insert from CG-pBR 322 / HLycIFN-5₁ and the β-lactamase regulatory region of CG-pBR (AP) / LyIFN-a-1. A clone designated E. coli HB 101 CG-pBR (AP) / LyIFN-a-2 is selected as described above. An IFN activity of 50,000 (IU / ml) is found, which is a 5-fold stimulation in comparison with the original E. coli HB 101 CG-pBR 322 / HLycIFN-5₁ corresponds. The nucleotide sequence of the cDNA insert and the B-lactamase regulatory region of the plasmid CG-p_BR (AP) / LyIFN-a-2 is determined as described above and is shown in Fig. 13.

8th. Cultivation of the strain E. coli HB 101 CG-pBR (AP) / LyIFN-α-3 on the scale of a fermentation direction

The strain E. coli HB 101 CG-pBR (AP) / LyIFN-a-3 is grown in medium No. X, which contains the following components per 1 1 solution:

<tables id="tabl0004" num="0004"><img file="EP0076489A2_D0007.tif" /></tables>Separated from the mass of the medium, cerelose and tetracycline are sterilized by heat sterilization or sterile filtration. Three 2 1 shake flasks containing 500 ml of medium No. X are each inoculated with the cells from a slant agar tube. The shake flasks with four indentations are incubated for 11 hours at 120 rpm in a rotary shaker. 1.5 l of this preculture are transferred to a 500 l fermenter which contains 300 l medium no. X contains, and then cultivated under the following conditions: stirring at 350 to 500 rpm with a disc stirrer, aeration rate 0.3 to 1.0 1/1 · min, fermenter pressure 0.3 bar, temperature 30 ° C. The content of dissolved oxygen is prevented from falling below 50% saturation by increasing the aeration rate and, if appropriate, the stirring speed to the maximum values. The pH is kept above 6.8 by the controlled addition of NaOH. After about 10 hours of culture, the culture reaches a maximum interferon titer [determined according to Armstrong (29)], and then harvesting.

9. Isolation and purification of HLyIFN-a-3

a. Preparation of the polypeptide solution for the monoclonal antibody column

280 1 culture broth of pH 7.2 is cooled to 10 ° C. and the cells are separated using an Alfa-Laval BRPX-207 sludge separator. The clear supernatant contains no IFN activity. Before the cell mass which has collected in the sludge chamber of the separator is obtained, the supernatant is washed with 20 l of lysis buffer A [50 mM Tris. HCl, 50mM EDTA, 0.2M NaCl, 10µM PMSF (phenylmethylsulfonyl fluoride); 1 mM L-cysteine, adjusted with HC1 to pH 8.2], and the contents of the separator drum (7 l) are expelled by means of a total sludge removal. The separator drum is rinsed three times with 2 l of lysis buffer A. The cell mass obtained is adjusted to 20 l with buffer A and has a pH of 6.9. After cooling to 5 to 10 ° C, the suspension is replaced by a DYNO^(R) -Mill (_Typ KDL-Pilot, 1.4 1), which is equipped with polyurethane stirring disks and with 1170 ml glass beads with a diameter of 0.5 to 0.75 mm. The stirring speed is 3350 rpm and the flow rate is 5 l / h. The cells are unlocked. 8th₀₀ ml of lysis buffer A (with an additional content of 100 g of polyethyleneimine, adjusted to pH 8.2 with HC1) are added to the resulting suspension of the digested cells at 2 ° C. with moderate stirring. The suspension with a pH of about 7.6 is cooled to -2 ° C. for 3 h and centrifuged. 3028 g of ammonium sulfate are added to the supernatant (17.2 l). The slightly cloudy solution is centrifuged after standing for 1 hour at 6 ° C. The supernatant is treated with 4324 g of ammonium sulfate and, after standing overnight, centrifuged at 3000 rpm. The wet sediment (about 1224 g) is dissolved in buffer B (25 mM Tris. HCl, 10 μM PMSF, adjusted to pH 8.5 with HCl), giving 2800 ml of a solution which contains the desired polypeptide.

An aliquot of 700 ml of this polypeptide solution is diafiltered at room temperature through an H1P10 ultrafilter cartridge using an Amicon DC-2 Hollow Fiber System using 7 1 B buffer system. The filter cartridge is washed with buffer B, the diafiltered solution and the washing solutions are combined (1440 ml) and at a flow rate of 200 ml / h on a DEAE column (Trisacryl® M DEAE, LKB 2205-300) with a bed volume of 450 ml, the column having been pre-equilibrated with buffer B beforehand. The first polypeptide fraction with UV absorption at 280 nm wavelength is discarded. The column is further washed with Buffer B until at least five bed volumes of wash solution are the bases_sline absorption of 280 nm. The adsorbed polypeptides are then eluted with 2.8 l of buffer C (0.2 M NaCl, 25 mM Tris-HCl, pH 8.5). Column chromatography is carried out at 4 ° C. In the analysis according to Armstrong (29), the eluate shows an IFN activity of 1.4 × 10⁵ IU / mg polypeptide. The eluate is adjusted to a pH of 7.4 with 2M HGl and frozen in 100 ml aliquots at -20 ° C. until they are used for the monoclonal antibody column.

b. Purification of human LyIFN-a-3 on a monoclonal antibody column

The monoclonal antibody column 1K2-20 (bed volume 0.8 ml, see below) is washed with PBS (phosphate-buffered saline: 0.137 M _NaCl, 0.0027 M KCl, ₀,₀₀₇₇ M Well₂H^P0₄.1²H₂^{0, 0,}0015 M KH₂PO₄, ^pH 7.4) equilibrated, and 10 ml portions of the above polypeptide solution are applied to the column at room temperature at a flow rate of 10 ml / h. The first fractions containing the non-adsorbed polypeptides and 3 ml PBS wash solutions are discarded. Furthermore, non-specifically bound polypeptides are eluted with 3 ml PBS with an additional content of 0.5 M NaCl and 0.2% Triton X 100. The column is washed with 3 ml PBS, after which the specifically adsorbed polypeptides are eluted with 3 ml buffer D (0.1 M citric acid, 0.3 M NaCl, pH 2). This fraction and 4 ml of a subsequent PBS wash solution are combined, the pH is adjusted to 6.3 with 2N NaOH and then concentrated 10-fold at 4 ° C. using an immersible CX ™ molecular separator (Millipore®). The concentrate is applied to a Sephadex G-25 column (2.6 x 34 cm, 200 ml bed volume), equilibrated with 0.025 M histidine.HCl (pH 6.3). At 4 ° C and with a flow rate of 42 ml / h, the column is eluted with the same histidine.HCl of pH 6.3. 20 fractions are collected (10.5 ml each). The fractions containing the polypeptide are determined by their optical absorption at 280 nm. Fractions 7 and 8 contain the polypeptide with IFN activity, which was determined by the analysis according to Armstrong (29). The active fractions containing LyIFN-a-3 are stored at -20 ° C or in an ice bath until further use. The IFN activity of the fractions is 1.8 x 10⁸ IU / mg polypeptide (29).

Lyophilization of the above fractions from 1 ml of solution gives 20 to 40_/µg of the polypeptide.

SDS-polyacrylamide gel electrophoresis [comp. (49)] shows a molecular weight of the LyIFN-a-3 obtained of about 18 kDalton.

Ultrathin isoelectric focusing on polyacrylamide gel (100th_/uM), carried out according to BJRadola (50), within a pH range of 4.5 to 6.5, the pure active human LyIFN-α-3 indicates at the isoelectric point of 5.3 to 5.4 pH units.

c. Preparation of the 1K2-20 A. monoclonal antibody column. Immunization of mice

Balb / c mice (8 weeks old, obtained from the Sisseln animal farm, Switzerland) are given 3 x 10⁵ Units of human leukocyte IFN-α (purity 1%) in Freund's complete adjuvant (Difco), distributed into the four paws, injected. On day 30, the same amount of IFN is injected in Freund's incomplete adjuvant in the same way. The third injection is on day 85 when 4 x 10⁵ Units of human leukocyte IFN are administered intraperitoneally in saline. 4 days later, the spleen is removed for the fusion.

B. Preparation of Hybridomas

All fusion experiments using the X63-Ag8-653 myeloma line (52) are carried out essentially in accordance with the method of Köhler and Milstein (53), using 10⁸ Spleen cells with 10⁷ Myeloma cells mixed using 1 ml of 50% polyethylene glycol (PEG 1500, Serva) (54). After washing, the cells are resuspended in 48 ml of standard Dulbecco's minimum essential medium (Gibco). 15% fetal calf serum and 3 x 10⁶ normal peritoneal mouse exudate cells / fusion are added as feeder cells. The cells are distributed in 48 x 1 ml costar wells. The cultures are fed twice daily with standard selective medium (53) for 3 to 6 weeks. After the growth of the hybrids, they are frozen and the supernatants are put on the anti_-IFN activity as described below was examined. The hybridoma cells are cloned by limiting the dilution in microtiter plates.

C. Antibody Assays

To check the anti-IFN activity of the supernatant, 50_/ul IFN-α (at the end 10 to 20 units IFN / ml) incubated with 50 µl culture supernatant at room temperature, and 30 to 60 min later the residual activity on IFN is tested in a standard IFN assay. This method, which is suitable for the conventional antibodies, either fails or gives no reproducible results in the analysis of the hybridoma supernatants. The following combined immunoprecipitation bioassay was therefore developed for this purpose. 50 µl crude IFN-α (₁0⁴ U / ml) are mixed (in microtubes 3810, Eppendorf) with equal amounts of culture supernatant and the mixture is incubated at 37 ° C. for 2 to 4 h. Then 50_/ul of previously titrated rabbit anti-mouse Ig antibody (Nordic) is added and the mixture is first stirred at 37 ° C. for 1 h and then at +4 for 16 h⁰C incubated for the formation of immune complexes. The tubes are centrifuged at 12,000 rpm in a cold room for 5 minutes. The supernatant is collected and the precipitate is washed once with 1 ml of buffered saline (pH 7.2). After washing, the precipitate becomes 200_/ul saline solution (pH 2.2) dissolved. According to Armstrong (29), the IFN activity is checked.

D. Purification of the anti-IFN antibody isolated from ascitic fluid

Balb / c mice are pretreated intraperitoneally with 0.4 ml Pristan (Carl Roth). 1 week later the mice are ip with 2 to 5 x 10⁶ Hybridoma cells injected.

Ascitic fluid is repeatedly collected from each mouse. The liquid materials are collected and frozen at -80 ° C. After thawing, the collected material is centrifuged at 16,000 rpm for 30 minutes. The fat on the upper part is suctioned off and the debris-free supernatant is collected. If necessary, the centrifugation is repeated. A crude immunoglobulin fraction is extracted from the ascitic liquid by 18% Na₂SO₄ Obtain precipitation at room temperature. This fraction is then passed through Sephacryl G 200 (Pharmacia) according to the manufacturer's instructions using 0.1 M Tris-HCl buffer (pH 8.2). The active fractions are collected and concentrated with Amicon XM 50 filters (Amicon). The protein is determined by OD₂₈₀-Measurement, assuming that 1 mg protein gives an absorption of 1.2 at 280 nm in a 1 cm cuvette.

E. 1K2-20 Immunoadsorbent Column

1 ml of discarded Affi-Gel (Bio-Rad) is mixed with 15 mg of immune _' noglobulin of the anti-IFN monoclonal antibody is coupled according to the manufacturer's instructions: Affi-Gel 10 is placed on a glass frit funnel first with cold distilled water and then with 0.1 M NaHC0₃Solution (pH 8.0) (coupling buffer). 50% of the gel in the coupling buffer is transferred to a plastic tube, mixed with the same amounts of purified antibody solution and rotated for 4 hours at room temperature. After coupling, the gel is washed with coupling buffer. To block the unreacted areas, it is treated with 0.1 ml of 1M ethanolamine HCl (pH 8.0) / ml of gel for 1 to 2 hours at room temperature. The gel is washed with phosphate buffered saline in the presence of 10 mM NaN₃ washed and kept at 4 ° C. 0.8 ml of the resulting gel is used to prepare the monoclonal antibody column (designated 1K2-20), which is used to prepare human LyIFN (see above).

10th Pharmaceutical preparations (parenteral administration)

2nd mg of lymphoblastoid interferon, for example LyIFN-a-3, isolated from clone E. coli HB 101 CG-pBR (AP) / LyIFN-α-3 (see Example 9), with a specific activity of 1.8 x 108 U / mg are dissolved in 30 ml of 5N human serum albumin. The resulting solution is passed through a bacteriological filter and the filtered solution under aseptic conditions in 100 ampoules, each 3.6 x 10⁶ Units containing pure lymphoblastoid interferon divided. The ampoules suitable for parenteral administration are preferably stored in the cold, for example at -20 ° C.

In the same way, ampoules containing ₇, 2 x ₁₀⁶ or 1.08 x 10⁷ Units are made using 4 and 6 mg of the above lymphoblastoid interferon, respectively.

Deposit of the produced microorganisms

As examples of the microorganisms and recombinant DNA molecules which were produced by the processes according to the invention, cultures were deposited at the collection point of the Agricultural Research Culture Collection (NRRL) on September 14, 1981, to which the following deposit numbers were assigned:<tables id="tabl0005" num="0005"><img file="EP0076489A2_D0008.tif" /></tables>

References

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ATTACHMENT

The symbols used in FIGS. 4 to 5 have the following meanings:<tables id="tabl0006" num="0006"><img file="EP0076489A2_D0009.tif" /></tables>

In the corresponding figures, the symbols given relate to the closest prior art listed in the examples.