DD251996A5 - Process for the preparation of hybrid interferons - Google Patents

Abstract

The invention relates to a process for the microbiological production of novel hybrid interferons and their treatment of viral or neoplastic diseases. The invention provides a method of producing novel hybrid interferons that exhibit specific biological activities and eliminate or reduce the undesirable side effects that limit their use. The object of the invention is to develop a process for the preparation of novel hybrid interferons which have the desired properties. According to the invention, the object is achieved by providing a process for the production of hybrid interferon polypeptide, wherein transformed host cells are cultivated with a hybrid vector which contains a DNA sequence coding for the hybrid interferon polypeptide and the hybrid Interferon is isolated. The novel hybrid interferons have valuable antiviral and proliferation-inhibiting properties.

Description

Field of application of the invention

The invention relates to a process for the microbiological production of novel hybrid interferons by means of recombinant DNA technology, as well as their use for the treatment of viral or neoplastic diseases.

Characteristic of the known technical solutions

Interferons ("IFNn") are a family of polypeptides secreted by a wide variety of eukaryotic cells after exposure to various mitogens and viruses ^ The interferons have been classified into three groups based on their chemical and biological characteristics: IFN -a, IFN-ß and IFN-γ. IFN-α and β genes are predominantly expressed in cells treated with virus, viral components and double-stranded DNA, whereas IFN-γ is synthesized in response to mitogens.

Human IFN-ct is encoded by a gene family having at least 15 non-allelomorphic genes. Human IFN-β and human IFN-γ are encoded by single genes. The primary translation product of IFN-α comprises 189 amino acids (except IFN-Cc ₂ : 188 amino acids) from which a signal peptide of 23 amino acids is removed by post-translational modification. Human IFN-β and human IFN-γ consist of 187 and 186 amino acids, of which 21 or 20 amino acids that make up the signal peptides are removed. The amino acid sequence homology among the IFN-a subspecies is 80 to 85%, while the homology between IFN-ß and IFN-a is about 30 to 40%.

IFN-γ shows only simple amino acid homologies with IFN-α corresponding to 12% identical pairs.

The IFNs show antiviral, immunomodulatory and cytotoxic activities. It has been demonstrated that IFN-a subtypes (eg, IFN-Ct ₂ , IFN-a,) show different target cell specificities. The antiviral activity of IFN-Ct ₂ and IFN-ai is about the same in bovine (MDBK) cells, but IFN-a ₂ is seven times more potent than IFN-a, in human (WISH) cells, and thirty times less effective than IFN-a in protecting murine (L929) cells. (M. Streuli, et al., Proc. Natl. Acad. Sci. USA 78, 2848 [1981]). The evidence that members of the multigene IFN-ct family lose the level and specificity of their antiviral activity was provided by the results obtained with some IFN-a "hybrids" (see EP 32 134, US Pat 4 414 150) These "hybrids" were constructed by cleaving two genes encoding interferon-a subtypes with the restriction enzyme Pvull (or in two cases with BglII) and cross ligation of the resulting fragments such that those described for the Amino terminal portion of the DNA sequence coding for an IFN gene was condensed to the coding for the carboxyl terminal portion of the other IFN gene DNA sequence. Although these hybrid interferons have changes in target cell specificity compared to the parent interferons, it could not be demonstrated that any attenuation or restriction was present in any of the three interferon activities. Such mitigation or restriction may be desirable, for example, in clinical applications where it is intended to concentrate interferon therapy on a particular problem, such as viral infection, without the possibility of complicating factors deriving from other activities of the administered interferon.

Object of the invention

The invention provides a method of producing novel hybrid interferons which exhibit specific biological activities resulting from the selective or predominant activation of only some of the interferon-induced biochemical pathways and / or some of the undesirable side effects of natural interferons · limit their use, such as eliminating or reducing influenza-like symptoms (eg fever, headache and fatigue), gastrointestinal upset, lowering blood pressure and hair loss.

Explanation of the essence of the invention

The invention has for its object to provide processes for the preparation of novel hybrid interferons, the

have desired properties.

The hybrid interferons of the invention are obtained from human lymphoblastoid interferon LylFN-a-2 having the following sequence

CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU

ILE LEU LEU ALA HLN MET ARG ARG ILE SER PRO PHE SER CYS LEU

LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP

LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU MET

ILE GLN GLN THR PHE ASN LEU PHE SER THR LYS ASP SER SER ALA

ALA LEU. ASP GLU THR LEU LEU ASP GLU PHE TYR ILE GLU LEU ASP

GLN GLN LEU ASN ASP LEU GLU SER CYS VAL MET GLN GLU VAL GLY

VAL ILE GLU SER PRO LEU MET TYR GLU ASP SER ILE LEU ALA VAL

ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU THR GLU LYS LYS

TYR SER SER CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG

SER PHE SER LEU SER ILE ASN LEU GLN LYS ARG LEU LYS SER LYS

and human lymphoblastoid interferon LylFN-a-3 having the following sequence

CYS ASP LEU PRO GLU THR HIS SER LEU ASP ASN ARG ARG THR LEU

MET LEU LEU ALA GLN MET SER ARG ILE SER PRO SER SER CYS LEU

MET ASP ARG HIS ASP PHE GLY PHE PRO GLN GLU GLU PHE ASP GLY

ASN GLN PHE GLN LYS ALA PRO ALA ILE SER VAL LEU HIS GLU LEU

ILE GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA

ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR

GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL MET GLN GLU GLU ARG

VAL GLY GLU THR PRO LEU MET ASN ALA ASP SER ILE LEU ALA VAL

LYS LYS TYR PHE ARG ARG ILE THR LEU TYR LEU THR GLU LYS LYS

TYR SER PRO CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG

SER LEU SER LEU SER THR ASN LEU GLN GLU ARG LEU ARG ARG LYS GLU

derived.

These interferons as well as processes for their preparation have been described in EU-PA No. 76,489. LylFN-ct-2 is related (but not identical) to human leukocyte interferon LelF-ctB (see U.S. Patent 4,414,150) and has a specific activity for primary calf kidney cells and human embryonic atrial cells (HEF, each infected with vesicular stomatitis viruses ) of 1.85 × 10 ⁸ IU / mg or 2.45 × 10 ⁸ IU / mg. LylFN-a-3 is related (but not identical) to human leukocyte interferon LelF-aD (see U.S. Patent 4,414,150) and has a specific activity for calf cells and HEF cells (as above) of 1.32 x 10 ⁸ IU / mg or 3.7 x 10 ^B IU / mg. In addition, LylFN-a-3 has higher thermostability and less antiproliferative activity than LylFN-cc-2.

A particular object of the invention is to provide LylFN-a-2 / a-3 hybrid polypeptides having the high antiviral titer and / or higher proliferation inhibitory activity in human LylFN-ct-2 cells and the high thermostability of LylFN -a-3 unite.

Specifically, the invention relates to a hybrid interferon polypeptide having an amino acid sequence composed of two to four subsequences that differ in amino acid identity and number of sub-sequences of human lymphoblastoid interferon LylFN-cc-2 and LylFN-a-3, wherein the hybrid interferon polypeptide is selected from the group consisting of a polypeptide having an amino acid sequence selected from amino acids 1 to 150 of LylFN-a-2 and amino acids 151 to 166 of LylFN- a-3 is a polypeptide having an amino acid sequence consisting of amino acids 1 to 92 of LylFN-a-2, amino acids 93 to 150 of LylFN-cc-3 and amino acids 151 to 166 of LylFN-a-2, and a polypeptide having an amino acid sequence selected from amino acids 1 to 60 of LylFN-a-2, amino acids 61 to 92 of LylFN-a-3, amino acids 93 to 150 of LylFN-a-2 or LylFN-a- 3 and amino acids 151 to 166 of LylFN-cc-2 or LylFN-o-3 , includes.

More specifically, the invention relates to the hybrid interferon "B ₁ B ₂ B ₃ D ₄ " having the following formula CYS ASP LEU PRO GLN THR HIS 'SER EU GLY ASN ARG ARG ALA LEU LE LEU ALA GLN MET ARG ARG ILE SER PRO PHE SER CYS LEU LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU METAL GLGL THR PHE ASN LEU PHE SER THR LYS ASP SER SER ALA ALA LEU ASP GLU THR LEU LEU ASP GLU PHE TYR ILE GLU LEU ASP GLN GLN LEU ASN ASP LEU GLU SER CYS VAL MET GLN GLU VAL GLY VAL ILE GLU SER PRO LEU MET TYR GLU ASP SER ILE LEU ALA VAL ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU THR GLU LYS LYS TYR SER SER CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER LEU SER LEU SER THR ASN LEU GLN GLU ARG LEU ARG ARG LYS

the hybrid interferon ,, BiB ₂ D ₃ B ₄ "with the following formula."

CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU, '. ¹ I

ILE LEU LEU ALA GLN MET ARG ARG ILE SER PRO PHE SER CYS LEU ^{! !} .

LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP 'ι

LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU MET 'I j

ILE GLN GLN THR PHE ASN LEU PHE SER THR LYS ASP SER SER ALA

ALA LEU ASP GLU THR LEU LEU ASP GLU PHE TYR ILE GLU LEU ASP

GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL MET GLN GLU GLU ARG

VAL GLY GLU THR PRO LEU MET ASN ALA ASP SER ILE LEU ALA VAL

LYS LYS TYR PHE ARG ARG ILE THR LEU TYR LEU THR GLU LYS LYS

TYR SER PRO CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG

SER PHE SER LEU SER ILE ASN LEU GLN LYS ARG LEU LYS SER LYS

the hybrid interferon "B, D ₂ B ₃ D ₄ " with the following formula

CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU

ILE LEU LEU ALA GLN MET ARG ILE SER PRO PHE SER CYS LEU

LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP

LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU MET

ILE GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA

ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR

GLN GLN LEU ASN ASP LEU GLU SER CYS VAL MET GLN GLU VAL GLY

VAL ILE GLU SER PRO LEU MET TYR GLU ASP SER ILE LEU ALA VAL

ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU THR GLU LYS LYS

TYR SER SER CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER LEU SER LEU SER THR ASN LEU GLN GLU ARG LEU ARG ARG LYS GLU

the hybrid interferon ,, B] D ₂ D ₃ B ₄ "with the following formula

CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU

ILE J-EU LEU _ALA_GLN__MET ARG__ARG_.ILE .. _.SER_. PER. PHE SER CYS LEU

LYS asp "~ agg ~ h1s asp phe" glu phe pro gln glu glu phe asp asp

LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU MET

ILE GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA

ALA 'TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR

GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL MET GLN GLU GLU ARG

VAL GLY GLU THR PRO LEU MET ASN ALA ASP SER ILE LEU ALA VAL

LYS LYS TYR PHE ARG ARG ILE THR LEU TYR LEU THR GLU LYS LYS

TYR SER PRO CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG

SER PHE SER LEU SER ILE ASN LEU GLN LYS ARG LEU LYS SER LYS GLU

(IV),

the hybrid interferon ,, B ₁ D ₂ D ₃ D ₄ "with the following formula

CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU

ILE LEU LEU ALA GLN MET ARG ILE SER PRO PHE SER CYS LEU

LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP

LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU MET

ILE GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA

ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR

GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL MET GLN GLU GLU ARG

VAL GLY GLU THR PRO LEU MET ASN ALA ASP SER ILE LEU ALA VAL

LYS LYS TYR PHE ARG ARG ILE THR LEU TYR LEU THR GLU LYS LYS

TYR SER PRO CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG

SER LEU SER LEU SER THR ASN LEU GLN GLU ARG LEU ARG ARG LYS GLU

and the hybrid interferon ,, B ₁ D ₂ B ₃ B ₄ "having the following formula

CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU

ILE LEU LEU ALA GLN MET ARG ILE SER PRO PHE SER CYS LEU

LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP

LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU MET

ILE GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA

ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR

GLN GLN LEU ASN ASP LEU GLU SER CYS VAL MET GLN GLU VAL GLY

VAL ILE GLU SER PRO LEU MET TYR GLU ASP SER ILE LEU ALA VAL

ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU THR GLU LYS LYS

TYR SER SER CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG

SER PHE SER LEU SER ILE ASN LEU GLN LYS ARG LEU LYS SER LYS GLU

(Vl).

The most preferred hybrid interferons of the invention are "B ₁ D ₂ D ₃ D ₄ " (V) and "B ₁ B ₂ D ₃ B ₄ " (II).

A hybrid interferon of the invention can be prepared by the recombinant DNA technique, which includes, for example, the following steps

a) Preparation of a DNA containing the structural gene coding for this hybrid interferon by in vitro recombination at the sites corresponding to amino acids 60, 92 and 150, respectively, of the interferons LylFN-o-2 and LylFN-o-3. or by chemical DNA synthesis and incorporation of the recovered DNA into an appropriate expression vector,

b) transferring the obtained hybrid vector carrying the hybrid DNA structural gene into a host,

c) selecting transformed hosts from untransformed hosts, usually by culturing under conditions where only the transformed hosts survive,

d) culturing the transformed hosts under conditions permitting expression of the hybrid IFN structural gene, and

e) Isolation of the hybrid IFN formed.

The methods used will become more apparent from the following detailed description and from the accompanying drawings. In the drawings:

Fig. 1 shows a comparison of the amino acid sequences of mature LylFN-α-2 and LylFN-α-3. Only those amino acids of LylFN-a-3 are distinguished which differ from corresponding amino acids of LylFN-a-2. The amino acid sequence of LylFN-a-3 is otherwise identical to that of LylFN-a-2;

Figure 2 shows the nucleotide sequences of the coding regions and portions of the 3 'extracistronic regions of LylFN-a-2 and LylFN-a-3. The ATG translation initiation codons, the termination triplets and the appropriate restriction sites are underlined. The coding triplets are numbered;

Figure 3 shows the restriction maps of the HindIII-PstI small segments from the plasmids pBR (AP) / LylFN-cc-2 and pBR (AP) / LylFN-oc-3. The white bar (LylFN-a-3) and the black bar (LylFN-oc-2) delimit the coding regions of the mature IFNe. Below is the subdivision of the primary structure of IFN polypeptide (166 amino acids) into four sections; Figures 4 and 5 show the radioactivity bound to the carrier bead depending on the amount of IFN-cc-2 in the test solution measured in a sandwich RIA.

1. Preparation of hybrid vectors containing a hybrid IFN-Stmktur gene The cDNAs of human interferon-a, subspecies oc-2 and a-3 were cloned in E. coli. Thus, plasmids contain pBR (AP) / LylFN-a-2 and pBR (AP) / LylFN-a-3 HindIII-PstI (or EcoRI-PstI) -DNA inserts, which comprise the coding regions of LylFN-cc-2 and LyIFN- a-3 under control of the β-lactamase promoter (see EU-PA No. 76.489). The restriction maps of the inserts are shown in FIG. It can readily be seen that the genes share some common restriction sites (located at positions 60, 92 and 150 of the IFN amino acid sequences) that can be used to advantage for the construction of novel genes that are favorable for hybrid interferons encode biological and / or physical properties. Thus, recombinants (single or multiple replacement) between LylFN-a-2 and LylFN-a-3 genes can be generated by in vitro recombination at the Pvull site (position 92) and / or at Sau3A sites (positions 60 and 150). be generated. Since the primary structure of the interferons can be divided into 4 sections in this way (first section amino acids 1 to 60, second section amino acids 61 to 92, third section amino acids 93 to 150 and fourth section amino acids 151 to 166, see FIG. 3), For example, the hybrid interferons of the invention may be identified by four letters, each letter being typical of the existing section. For example, hybrid interferon (I) _{1 is} that from sections 1 to 3 of interferon LylFN-a-2 ("B", LylFN-a-2 is related to IFN-aB, supra), and section 4 of LylFN -ct-3 ("D", LylFN-a-3 is related to IFN-aD, supra, related), designated IFN "B ₁ B ₂ B ₃ D ₄ ".

To facilitate generation of the desired hybrid interferon, the DNA inserts containing the LylFN-a-2 and LylFN-a-3 coding regions, respectively, must be modified in such a way that the restriction sites HindIII (or EcoRI), Pvull and Pstl are unique to them for religious purposes. From Fig. 3 it is apparent that there is an additional Pvull site in the 3 'extracistronic region of the LylFN-a-2 coding gene. This site can be easily removed, for example by partial cleavage of a hybrid vector containing the LylFN-a-2 coding region with Pvull, ligation of the resulting linearized DNA to a linker containing a DNA sequence defined by the restriction Endonuclease PstI, cleavage with PstI, digestion of digests at low DNA concentration and selection for plasmids (eg by restriction analysis) that have the required modification, namely a truncated 3 'extracistronic region, where the Pvull site is eliminated. Similarly, vectors used to clone IFN genes are best modified so that the restriction sites HindIII, Pvull and PstI are unique to them.

The invention relates to hybrid vectors comprising a DNA sequence coding for any of the hybrid interferons of formulas I to IV and operatively linked to an expression control sequence, and methods for their production. The vector is chosen according to the host cells intended for transformation. Examples of suitable hosts are microorganisms which lack or only have a limited amount of restriction enzymes or modification enzymes, such as yeast species, for example Saccharomyces cerevisiae, and strains of bacteria, especially strains of Escherichia coli, for example E. coli X1776, E. coli HB101, E. coli W3110, E. coli HB101 / LM1035, E. coli JA221 or E. coli K-12 strain 294, Bacillus subtilis, Bacillus stearothermophilus, Pseudomonas, Haemophilus, Streptococcus and others, and also cells of higher organisms all introduced human and animal cell lines. The above strains of E. coli, for example, E. coli HB101 and E. coli Ja221 and, moreover, Saccharomyces cerevisiae are preferred as host microorganisms. In principle, all vectors that replicate and print the hybrid IFN genes of the invention in the chosen host are suitable. Examples of suitable vectors for the expression of the hybrid interferon genes in an E. coli strain are bacteriophages, for example derivatives of λ bacterial phage, or plasmids, in particular the plasmid Co1 E1 and its derivatives, for example pMB9 , pSF2124, pBR317 and pBR322. The preferred vectors of the invention are derived from plasmid pBR322. Suitable vectors contain a complete replicon and a marker gene, whereby the hosts transformed with the expression plasmids can be selected and identified on the basis of a phenotypic property. Suitable marker genes confer upon the host, for example, resistance to heavy metals, antibiotics and the like. In addition, preferred vectors of the invention outside of the replicon and marker gene regions contain restriction endonuclease recognition sequences such that the hybrid IFN gene and, if appropriate, the expression control sequence are inserted at these sites can. The preferred vector, plasmid pBR322, contains an intact replicon, marker genes conferring resistance to tetracycline and ampicillin (tet ^R and amp "), and a number of unique restriction endonuclease recognition sites, for example, PstI (splits into the amp ^R gene, the tet ^R gene remains intact), BamHi, HindIII, and Sail (all split into the tet ^R gene, the amp ^R gene remains intact), Nrul, and EcoRI Various expression control sequences can be used In particular, expression control sequences of highly expressed genes of the host to be transformed are used, for example, in the case of pBR322 as the hybrid vector and E. coli as the host microorganism, the expression controls are Sequences (containing, inter alia, the promoter and ribosome binding site) of the lactose operon, tryptophan operon, arabinose operon, and the like, the β-lactamase gene, which is complementary suitable sequences of the phage λΝ gene or the phage fd coat protein gene and others. While the plasmid pBR322 already contains the promoter of the β-lactamase gene (β-lac gene), the other expression control sequences must be incorporated into the plasmid. Vectors suitable for yeast replication and expression contain a yeast replication start and a yeast selective genetic marker. Hybrid vectors containing a yeast replication start, for example, a chromosomal autonomously replicating segment (ars) are extrachromosomally contained within the yeast cells after transformation and are replicated autonomously. In addition, hybrid vectors containing yeast 2μ-Ρΐ35ηηία-ΟΝ5 homologous sequences can be used. Such hybrid vectors are integrated by recombination into the 2μ-Γ33Γηίαβ, which are already present in the cell, or replicate autonomously. 2μ sequences are particularly suited for high-frequency-transforming plasmids and allow high copy numbers. The preferred yeast vector of the invention is the plasmid pJDB207.

Suitable marker genes for yeasts are, in particular, those which confer antibiotic resistance to the host, or in the case of auxotrophic yeast mutants, genes which complement the host lesions. Corresponding genes, for example, confer resistance to the antibiotic cycloheximide or provide protrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, HIS3 or especially the TRP1 gene. In addition, yeast hybrid vectors preferably contain an origin of replication and a marker gene for a bacterial host, especially E. coli, so that the construction and cloning of the hybrid vectors and their intermediates can occur in a bacterial host.

Expression control sequences that are suitable for expression in yeast are, for example, those of highly expressed yeast genes. Thus, the promoters of the TRP1 gene, the ADHI or ADHII gene, acid phosphatase (PHO3 or PHO5) gene, isocytochrome gene, or a promoter involved in the glycolytic pathway, such as the promoter of the enolase, glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, tropic phosphate isomerase, phosphoglucose isomerase and glucokinase genes. Preferred vectors of the invention contain promoters with transcriptional control, e.g. For example, the promoters of the PHO5, ADHII and GAPDH genes, which can be switched on or off by changing the growth conditions. For example, the PHO5 promoter can be suppressed or relaxed solely by increasing or decreasing the concentration of the organic phosphate in the medium. Promoters for use in mammalian cells are, for example, virus promoters such as HTLV, SV40, vaccinia promoter and the like.

The promoter is operatively linked to the coding region so as to ensure efficient expression of the hybrid interferon. In one embodiment of the invention, the promoter is directly linked to the coding region of mature hybrid IFN with a translation start signal (ATG) inserted at the junction. A preferred region for combining the promoter with the hybrid IFN coding region is between the major mRNA start and the ATG of the gene naturally attached to the promoter.

In another embodiment of the invention, especially when yeast is used as the host microorganism, a signal sequence is included in the design. Suitable signal sequences are those naturally linked to the promoter used. For example, the PHO5 signal sequence, as well as the signal sequences of the yeast invertase or a-factor genes can be used. Preferred are those combinations which permit accurate cleavage between the signal sequence and the amino acid sequence of mature hybrid IFN. Additional sequences, such as pro and spacer sequences, which may or may not carry specific processing signals, may also be included in the design for easier accurate processing of precursor molecules. Alternatively, condensed proteins can be generated that contain internal processing signals that allow for proper maturation in vivo or in vitro. Preferably, the hybrid vectors of the invention also have the 3 'flanking sequence of a gene containing the correct signals for transcription termination and polyadenylation. Suitable 3-flanking sequences are, for example, those of the gene that are naturally linked to the promoter used. If yeast is used as the host microorganism and the PHO5 promoter is used as the expression control sequence, then the 3 'flanking sequence is preferably that of the PHO5 gene.

In a preferred embodiment, the invention relates to hybrid vectors having the ability to replicate and phenotypically select in a host strain comprising a promoter and a DNA sequence encoding each of the novel hybrid interferons, wherein the DNA sequence is co-ordinated with Transcriptional start and termination signals and translation start and stop signals are housed in the hybrid vectors under such a control of the promoter that they are expressed in a transformed host to produce a hybrid interferon according to the invention. The hybrid vectors of the invention may be prepared by in vitro ligation of appropriate segments of the LylFN-ct-2 and LylFN-cc-3 coding regions and inserting the resulting hybrid IFN gene into an appropriate vector or by ligation of a particular IFN DNA segment to a linearized vector DNA containing the remaining sections already in such a way that the gene coding for the desired hybrid interferon is generated. It is recommended that the amino terminal DNA sequence-donating DNA segment is already fused to the expression control sequence.

Corresponding segments of the LylFN-a-2 and LylFN-a-3 coding regions are mainly taken from the preferred starting vectors according to the invention, namely the pBR322-derived plasmids pAM2 and pAM21. Plasmid pAM2 contains a HindIII PstI insert with the LylFN a-3 coding region under the control of the / J lactamase promoter. The vector portion of plasmid pAM2 is resistant to Pvull. The plasmid pAM21 contains a HindIII PstI insert wherein the LylFN-a-2 coding region has a unique Pvull site at position 92 and is under the control of the / i lactamase promoter. The vector portion of plasmid p21 is also resistant to Pvull.

Specifically, the gene encoding hybrid interferon ,, B ₁ B ₂ B ₃ D ₄ "is amplified by ligation of the large PstI-Pvull fragment from plasmid pAM21, the small San3A-Pvuli fragment from plasmid pAM21, and the small Sau3A-PstI fragment. This construction results in a plasmid (pJC344) containing the hybrid interferon ,, B ₁ B ₂ B ₃ D ₄ "coding region under the control of the β-lactamase promoter. In a similar manner, the gene encoding hybrid interferon ,, B ₁ B ₂ D ₃ D ₄ "is ligated by ligation of the large Pvull-PstI fragment from plasmid pAM21, the small Sau3A-Pvull fragment from plasmid pAM2 and the small Sau3A fragment. This construct results in a plasmid (pJC342) containing the hybrid interferon ,, B ₁ B ₂ D ₃ B ₄ "coding region under the control of the β-lactamase promoter. The gene encoding hybrid interferon ,, B ₁ D ₂ D ₃ D ₄ "can be obtained by ligation of the small HindIII Sau3A fragment from plasmid pAM21, the small Sau3A Pvull fragment from plasmid pAM2 and the large Pvull HindIII fragment This construction results in a plasmid (pAM94) containing the hybrid interferon ,, B ₁ D ₂ D ₃ D ₄ "coding region under the control of the β-lactamase promoter. In an analogous manner, the gene coding for hybrid interferon ,, B ₁ D ₂ B ₃ B ₄ "by ligation of the small HindIII Sau3A fragment of plasmid pAM21, the small Sau3A Pvull fragment of plasmid pAM2 and the large Pvull This construct results in a plasmid (pAM90) containing the hybrid interferon ,, B ₁ D ₂ B ₃ B ₄ "coding region under control of the / S-lactamase promoter. Plasmids containing the hybrid interferon ,, B ₁ D ₂ B ₃ D ₄ "coding region (eg, plasmid pDM1) or the hybrid interferon ,, B ₁ D ₂ D ₃ B ₄ " coding region (e.g. Plasmid pDM2) can be prepared in a similar manner. These plasmids are useful for replication and expression of hybrid IFN genes in E. coli. If desired, the DNA insert encoding any of the hybrid interferons may be excised from the corresponding plasmid and inserted into another vector DNA

be inserted. In this way, plasmids containing the hybrid interferon coding region under the control of a promoter other than the 6-lactamase promoter can be obtained, and / or the gene can be expressed in a host other than E. coli.

The production of DNA containing the structural gene for each of the said hybrid interferons can also be carried out by means of chemical synthesis. Suitable methods for the synthesis of DNA have been summarized by SA Narang [Tetrahedron 39, 3 (1983)]. Known synthetic techniques allow the production of polynucleotides of up to 20 bases in good yield, high purity and in a relatively short time. Suitable protected nucleotides are prepared by the phosphodiester method [K. L Agarwal, et al., Angew. Chem. 84, 489 (1972)] or by the more effective phosphotriester method [CB Reese, Tetrahedron 34, 3143 (1972)] or the phosphite triester method [RL Letsinger et al., J. Am. Chem. Soc. 98, 3655 (1976)]. Simplification of the synthesis of the oligonucleotides and polynucleotides is made possible by the solid-phase approach of attaching the nucleotide chains to a suitable polymer. Itakura, et al., [J. At the. Chem. Soc. 103, 706 (1981)] use trinucleotides linked by the phosphotriester method in solid-phase synthesis, rather than single nucleotides, and these can be condensed in a short time and in good yields to give, for example, a 31-base polynucleotide. The actual . Double-stranded DNA can be constructed enzymatically from chemically prepared short segments. Khorana, et al., [J. Biol. Chem. 251, 565 (1976)] overlapping polynucleotide sequences from both DNA strands, which are held together in the correct arrangement by base pairing and then chemically linked by the enzyme DNA ligase. Another possibility comprises in each case, the incubation of a polynucleotide sequence from the two DNA strands with a short overlapping segment in the presence of the four deoxynucleoside triphosphates required with a DNA polymerase, for example DNA polymerase I, a Klenow fragment of polymerase I or T ₄ DNA polymerase, or with AMV (avian myoblastosis virus) reverse transcriptase. The two polynucleotide sequences are held together in the correct arrangement by base pairing and are supplemented with the necessary nucleotides by the enzyme to give a complete double-stranded DNA [SA Narang, et al., Anal. Biochem. 121, 356 (1982)]. Itakura, et al., [J. Biol. Chem. 257, 9226 (1982)], as based on this principle, a 132 base pair segment of the human leucocyte interferon a ₂ gene in the presence of DNA polymerase I (Klenow fragment) of four chemically synthesized Fragments with a length of 39 to 42 bases can be built, resulting in a 40% saving in chemical synthesis compared to the method using only ligase. A suitable procedure for preparing the DNSn according to the invention has been described, for example, in EU-PA No. 146,785, the findings of which here. to be incorporated by reference.

The chemically synthesized DNA having the structural gene for each of the hybrid interferons of the present invention can be inserted into a vector DNA containing an expression control sequence in such a manner that the expression control sequence expresses regulates this gene.

2. Transformation of the host cells

The invention also relates to a method of producing a transformed host comprising transforming a host with an expression vector containing a DNA sequence encoding each of the hybrid interferons of the invention and regulated by an expression control sequence becomes.

Examples of suitable hosts are the above-mentioned microorganisms such as strains of Sachharomyces cerevisiae. Bacillus subtilis and Escherichia coli. The transformation with the expression plasmids according to the invention is carried out, for example, as described in the literature, as for S. cerevisiae [A. Hinnen, u. a., Proc. Nati. Acad, Sei. USA 75, 1929 (1978)], B. subtilis Anagnostopoulos, et al. ., J. Bacteriol. 81, 741 (1961)] and E. coli M. Mandel, u. J. Mol. Biol. 53: 159 (1970)].

Thereafter, the transformation process of E. coli cells involves Ca ²⁺ pre-treatment of the cells to allow for DNA uptake and incubation with the hybrid vector. The cells are transferred to a selective nutrient medium which allows separation of the transformed cells from the parent cells. Cells that do not contain the Vector will not survive in such a medium. The transformation of yeast includes, for example, the steps of (1) enzymatically removing the yeast cell wall by means of glucosidases, (2) treating the recovered spheroplasts with the vector in the presence of polyethylene glycol and Ca ²⁺ ions, and (3) regenerating the cell wall by embedding the spheroplasts in agar. The regeneration agar is preferably prepared in a manner that permits the simultaneous regeneration and selection of the transformed cells

 The invention also relates to the transformed hosts to be obtained in the manner described.

3. Breeding of the transformed host cells and isolation of the expressed hybrid interferon

The invention further relates to a method for producing the hybrid interferons of the formulas I to IV, which is characterized in that host cells which have been transformed with a hybrid vector containing a DNA sequence coding for each of the hybrid interferons, are grown and the hybrid interferon is isolated.

The transformed host cells are grown by methods known in the art in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts.

Various carbon sources can be used to grow the transformed hosts of the present invention. Examples of preferred carbon sources are absorbable hydrocarbons, such as glucose, maltose, mannitol or lactose, or an acetate, which can be used either as such or in suitable mixtures. Examples of suitable nitrogen sources are amino acids such as casamino acids, reptides and proteins and their degradation products such as tryptone, peptone, or meat extracta; and furthermore yeast extracts, malt extract and also ammonium salts, for example ammonium chloride, sulphate or nitrate, which can be used either as such or in suitable mixtures. Inorganic salts which can also be used are, for example, sulfates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium.

The medium also contains, for example, growth-promoting substances such as trace elements, for example iron, zinc, manganese and the like, and preferably substances which exert selective pressure and prevent the growth of cells which have lost the expression plasmid. For example, ampicillin is added to the medium when the expression plasmid contains an amp ^R gene. Such addition of antibiotic substances also has the effect of destroying contaminating, antibiotic-sensitive microorganisms. When a yeast strain which is auxotrophic in, for example, an essential amino acid is used as the host organism, the plasmid preferably contains a gene coding for an enzyme supplementing the host defect. The cultivation of the yeast strain is carried out in a minimal medium in which said amino acid is insufficient.

The cultivation is carried out according to methods known in the art. Culturing conditions such as temperature, pH of the medium and fermentation time are chosen so that a maximum titer of hybrid interferon is obtained. Thus, an E. coli or yeast strain is preferably under aerobic conditions by submerged culture with shaking or stirring at a temperature of about 20 to 40 "C, preferably about 30 ⁰ C, and a pH value of 4 to 8, preferably at a pH Value of about 7, for about 4 to 30 hours, preferably until maximum yields of hybrid interferon are achieved It has been surprisingly found that the maximum titers of the hybrid interferons according to the invention, for example the hybrid interferons "BiD ₂ D ₃ B ₁ ", "B ₁ D ₂ B ₃ D ₄ ", "BiD ₂ D ₃ D ₄ " and "B ₁ D ₂ B ₃ B ₄ ", which are prepared in crude extracts of For example, under standard conditions of cultured transformed yeast, they are significantly higher than those of interferon-a-2 and hybrid interferons, "B ₁ B ₂ D ₃ D ₄ " and "D, D ₂ B ₃ B ₄ ", labeled with the hybrid interferons "BD" and "DB" described in U.S. Patent 4,414,150 are closely related.

When the cell density has a sufficient value, the cultivation is stopped and the hybrid interferon is released from the cells of the host. For this purpose, the cells are destroyed, for example by treatment with a detergent such as SDS or Triton, or lysed with lysozyme or a similarly acting enzyme. When yeast is used as a host microorganism, the cell wall can be removed by enzymatic digestion with a glucosidase. Alternatively or additionally, mechanical forces such as shear forces (for example X-press, French press, Dyno mill) or shaking with glass beads or alumina, or alternately freezing, for example in liquid nitrogen, and thawing, for example from 30 to 40 ⁰ C, as well as ultrasound are used to break up the cells. The resulting mixture, which contains proteins, nucleic acids and other cell components, is enriched with proteins, including hybrid interferon, in a manner known per se after centrifugation. For example, most of the non-protein constituents are removed by polyethylenimine treatment, and the proteins, including hybrid interferon, are precipitated, for example, by saturation of the solution with ammonium sulfate or with other salts. Bacterial proteins can also be precipitated by acidification with acetic acid (for example, 0.1%, pH 4 to 5). Further purification steps include, for example, ultrafiltration, diafiltration, gel electrophoresis, chromatographic techniques such as ion exchange chromatography, size exclusion chromatography, HPLC, reverse phase HPLC and the like, separation of the components of the mixture by molecular size using a suitable Sephadex column, dialysis , Affinity chromatography, for example antibody, especially monoclonal antibody affinity chromatography and other known methods, especially those known in the art.

Surprisingly, the hybrid interferons according to the invention have higher thermal stabilities than interferon LylFN-a-2 and hybrid interferon ,, B ₁ B ₂ D ₃ D ₄ "Thus, the temperature at which 50% of the antiviral activity is lost (temperature increase of IFN solutions of 1 ° C / min) 62.8 ⁰ C in the case of LylFN-a-2 and 63 ° C in the case of hybrid interferon ,, B ₁ B ₂ D ₃ D ₄ ", while the corresponding values of for example, the interferons "B ₁ D ₂ D ₃ D ₄ ", "B ₁ D ₂ B ₃ B ₄ ", "B ₁ D ₂ B ₃ D ₄ " and "B ₁ D ₂ D ₃ B ₄ "65 ° C, 64.7 ° C, 65.3 ⁰ C and 64.2 ° C, respectively.

The invention further relates to the hybrid interferons of the formulas I to IV, if they are each prepared by the process according to the invention.

The invention also relates to the hybrid interferons which can be obtained by the method according to the invention. The invention specifically relates to the hybrid vectors, the transformed host cells, the hybrid interferon polypeptides and the methods of making the same, as described in the Examples.

Biological properties of hybrid interferons and pharmaceutical formulations

The hybrid interferons according to the invention show interesting biological properties which qualify them as valuable pharmaceuticals.

The hybrid interferons according to the invention are characterized by a very strong antiviral activity towards mammalian cells such as bovine and especially human cells. Thus, antivirus titers, which are considered to reduce the cytopathic effect according to the method of S. Rubinstein, et al. a., [J. Virol. 37, 755 (1981) using vesicular stomatitis virus (VSV) as an immunity test virus in bovine (MDBK) and human (WISH) cells], as follows:

Table 1 Anti-virus titers of hybrid interferons using VSV as the immunity test virus

Interferon specific activity / mg protein

Cattle Human

,, B ₁ D ₂ D ₃ D ₄ "1.25 x 10 ⁸ 1.3 x 10 ⁸

"B, D ₂ B ₃ B ₄ " 1.25x10 ⁸ 1.3 X10 ⁸

,, B ₁ B ₂ D ₃ B ₄ "9.5 X10 ⁷ > 2x10 ⁸

,, B ₁ D ₂ B ₃ D ₄ "'1.9 x 10 ⁸ 7.7 x 10 ⁷

,, B ₁ D ₂ D ₃ B ₄ "· 7.2 x 10 ⁷ 3.8 X 10 ⁷

,, B ₁ B ₂ B ₃ D ₄ "1.05x10 ⁸ 8x10 '

"D ₁ D ₂ B ₃ B ₄ " (DB) 8.5 x 10 ⁷ 3.3 x 10 ⁵

"B ₁ B ₂ D ₃ D ₄ " (BD) 1.15 x 10 ⁸ 2.33 X 10 ⁸

a-2 ("B") 1.05x10 ⁸ 2.13x10 ⁸

a-3 ("D") 5.5 x 10 ⁷ x 6.7 x 10 ⁶

Thus, the antiviral activity of the hybrid interferons of the present invention is not limited to human cells, but is consistently interspecific. Thus, the antiviral activities of the hybrid interferon "B ₁ D ₂ D ₃ D ₄ ", "B ₁ D ₂ B ₃ B ₄ ", "B ₁ B ₂ D ₃ B ₄ ", "B ₁ D ₂ B ₃ D ₄ "and" B ₁ B ₂ B ₃ D ₄ "which they show in human cells, in degree with those of parent interferon a-2 and hybrid interferon ,, B ₁ B ₂ D ₃ D ₄ "closely related to the hybrid interferon" BD "as presented in U.S. Patent 4,414,150 but the parent interferon a-3 (by a factor of 10 to 30) and the reference hybrid Interferon ,, D ₁ D ₂ B ₃ B ₄ "(by a factor of 230 to 600) is clearly superior.

Apart from the general antiviral activity in human cells, the hybrid interferons according to the invention lead to a more specific reaction of target cells. Thus, various intracellular enzymes are involved in the interferon effect. These enzymes include (2'-5 ') oligoisoadenylate synthetase ("2-5 A synthetase"), which generates (2'5 ^; ) linked oligonucleotides, which in turn activate a latent endoribonuclease that cleaves viral mRNA and has surprisingly been found in that the hybrid interferons according to the invention, such as "B ₁ D ₂ D ₃ D ₄ ", "B ₁ D ₂ B ₃ B ₄ ", "B ₁ B ₂ B ₃ D ₄ " and "B ₁ B ₂ D ₃ B ₄ "are particularly active stimulators of 2-5 A synthetase activity in human cells, resulting in hybrid interferons," B ₁ D ₂ D ₃ D ₄ "," B ₁ D ₂ B ₃ B ₄ ", ,, B ₁ B ₂ B ₃ D ₄ "and" B ₁ B ₂ D ₃ B ₄ "is a 17.5, 11.4, 8.5 and 9.6-fold increase in 2-5 A synthetase activity in Daudi cells at units / ml versus 5.6, 3.9, 4.0 or 4.3 times increase in effect on parent interferons a-2 and a-3 and reference hybrid interferons ,, B ₁ B ₂ D ₃ D ₄ "or" D ₁ D ₂ B ₃ B ₄ ".

In addition, the hybrid interferons according to the invention have proliferation-inhibiting activities. The antiproliferative activity can be determined as follows: Interferon-containing solutions are incubated with 5 x 10 ⁴ Daudi cells per ml in 2 ml of RPMI medium containing 10% fetal calf serum. Cell multiplication is determined after an incubation period of 3 days by counting the viable cells in a hemocytometer using the trypan blue exclusion test. Thus, the concentration of hybrid interferon required to induce 50% inhibition of Daudi cell multiplication is "B ₁ D ₂ D ₃ D ₄ ", "B ₁ D ₂ B ₃ B ₄ ", "B ₁ B ₂ B ₃ D ₄ "," B ₁ B ₂ D ₃ B ₄ "," B ₁ D ₂ B ₃ D ₁ "and" B ₁ D ₂ D ₃ B ₄ "are approximately 4, 4, 1, 3, 1,3, 4 or

4 units / ml, and is thus approximately equal to the parent interferon a-2 and the reference hybrid interferon ,, B ₁ B ₂ D ₃ D ₄ "(approx

1.3 units / ml) and superior to parent interferon a-3 (about 20 units / ml) and the reference hybrid interferon, "D ₁ D ₂ B ₃ B ₄ " (about 400 units / ml).

The antiproliferative activity against human cancer can also be demonstrated in vivo. Thus, various cancers of human origin such as cancer of the breast, colon and lung, ovarian cancer and melanoma were excited in nude mice. Small fragments of the tumors generated are isolated and implanted through a hollow needle under the venal capsule of (Balb / c χ DBA / 2) F ₁ (CDF ₁ ) mice. IFN is given intramuscularly in two daily doses of 5 χ 10 ⁵ to

5 x 10 ⁷ lU / kg / lnj. on days 1 to 5 (day of administration: day 0). On day 6, animals are sacrificed, final tumor sizes are measured and compared to initial tumor sizes (prior to IFN treatment) and tumor sizes in untreated control mice. After treatment with the hybrid interferons according to the invention, for example "B ₁ D ₂ D ₃ D ₄ " and "B ₁ B ₂ D ₃ B ₄ ", a significant and significant inhibition of tumor growth and partial regression of tumors was observed become. Surprisingly, the antiproliferative effect of the hybrid interferons is that of the parent interferons a-2.and a-3 as well as that of the hybrid interferons ,, B ₁ B ₂ D ₃ D ₄ "and" D ₁ D ₂ B ₃ B ₄ " think.

The anti-viral and anti-proliferative properties of the hybrid interferons of the present invention make these polypeptides useful for the treatment of viral infections such as influenza and other viral infections of the respiratory tract, herpes virus infections, rabies and hepatitis infection, and neoplastic diseases such as melanoma, renal cancer and hair cells. Leukemia of the human or animal body, possibly in combination with other antiviral or antitumor agents, preferably in the form of pharmaceutical preparations, which preferably mix together a therapeutically effective amount of the active ingredient together with inorganic or organic, solid or liquid, pharmaceutically acceptable carriers suitable for parenteral administration.

Parenteral formulations are, above all, injectable fluids which are effective in various ways, such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal or subcutaneous. Such liquids are preferably isotonic aqueous solutions or suspensions which may be formulated before use, for example from lyophilized preparations containing the active ingredient alone or together with a pharmaceutically acceptable carrier. The pharmaceutical preparations can be sterilized and / or contain adjuvants, for example preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, salts for regulating the osmotic pressure and / or buffers. The pharmaceutical compositions according to the invention, which may contain further pharmacologically valuable substances if desired, are prepared in a manner known per se, for example by means of conventional dissolution of lyophilization processes, and contain approximately 0.1% to 100%, especially approximately 1 % to approximately 50%, and in the case of lyophilisates up to 100% active ingredient. It should not go unmentioned that the inventive

Hybrid interferons such as "BiD ₂ D ₃ D ₄ " can be lyophilized and redissolved without loss of activity, whereas lyophilized preparations of the parent interferons a-'2 and a-3 give cloudy solutions with a marked decrease in Activity The latter interferons can therefore only be stored in buffered solutions.

The invention also relates to a process for the preparation of a pharmaceutical composition which comprises mixing a pharmaceutically active compound of the invention with a pharmaceutically acceptable carrier

The mode of administration and dosage will be chosen by the physician, who will take into account the peculiarities of the patient, the disease and the condition of the disease. For example, viral infections are usually treated by doses administered daily or twice daily for several days to a few weeks, while the treatment of neoplasms generally requires daily or repeated daily doses over weeks or months. The same daily dosages, about 10 ⁶ to 10 ⁷ units, as used in conventional interferon therapy may be used.

Monoclonal antibodies to the interferon hybrids

It is an object of the invention to provide a novel monoclonal antibody for IFNα having high affinity for, various subtypes of IFNα, including IFN-ct hybrids, and low affinity for other subtypes of IFNα, and hybridoma cells leave them out.

The novel monoclonal antibody is characterized in that it has a high affinity for IFN α / Β, D, F and related subtypes (see DV Goeddel, et al., Nature 290, 20 [1981]) and hybrids of these subtypes, especially the hybrid according to the invention Interferons, but has a low affinity to, for example, IFN cc / A subspecies.

The monoclonal antibody is referred to as 144 BS. This monoclonal antibody is secreted by the hybridoma cell series designated 144 BS 22-6-19. The invention also relates to derivatives of this monoclonal antibody, for. B.

Antibody fragments, radioactively labeled monoclonal antibodies and conjugates of the monoclonal antibody with enzymes or the like.

Fragments of the monoclonal antibody of the invention are e.g. Fab, Fab 'or F (ab') ₂ fragments which retain their specificity for the antigenic determinants, ie, retain the characteristic binding pattern of the monoclonal parent antibody to human IFN-a and IFN-o hybrid subtypes.

Radiolabeled monoclonal antibodies contain, for. Radioactive iodine ( ¹²³ I, ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H) or the like. Preference is given to monoclonal antibodies which are labeled with radioactive iodine.

Antibody conjugates according to the invention are e.g. B. conjugates of the monoclonal antibody or fragments thereof with enzymes such as horseradish peroxidase, alkaline phosphatase, ß-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenase, or with avidin or biotin. In such conjugates, the antibody is linked to the enzyme directly or through a spacer or linker group. Preference is given to conjugates of the monoclonal antibody with the enzymes horseradish peroxidase or alkaline phosphatase.

The monoclonal antibody of the present invention is characterized by its binding ability to polypeptides belonging to various subtypes of human IFNα. The binding ability is z. B. in the so-called combined immunoprecipitation bioanalysis, as described by S. S. Alkan, u. a, in "Protides of the biological fluid", publisher H. Peeters, Pergamon Press, Vol 30, pages 495 to 498 (1983) In this analysis, a solution containing IFN.alpha incubated the monoclonal antibody to be tested, the bound and unbound monoclonal antibodies precipitated by the addition of a polyclonal serum that binds all the antibodies tested, the immunoprecipitate separated and redissolved in acidic solution, and released thereby the IFN α in a classical bioanalysis the basis of the antiviral activity of IFN α determined.

It is surprising that the monoclonal antibody according to the invention binds strongly to the IFN-cc subtypes B, D, F and hybrids thereof, likewise to the IFN-α-uneararts C and J, but has a low affinity to the IFN-ct- Subspecies A shows a subspecies bound by any other known monoclonal antibody recognizing more than three IFN-ct subtypes,

d. H. Antibody with broad subtype recognition such as the monoclonal antibody NK2 (D. Secher, et al., Nature 285, 446 [1980]), the monoclonal antibodies EBI-1, 2 and 3, which are disclosed in EU-PA 119 476, or the monoclonal Antibody LO-22, which is presented in PA-WO 84/03106.

Compared to the monoclonal antibody YOK, which is specific for the IFN-cc subtype D, presented in the PA-WO 84/03105, the monoclonal antibody according to the invention surprisingly exhibits a broad specificity, above all with respect to the IFN-ct subtypes B and F, which are not bound by the antibody YOK.

The monoclonal antibody of the invention, designated 144 BS, was found to belong to the immunoglobulin class IgG ₁ K (kappa) as determined by well-known methods, e.g. For example, the immuno-diffusion Ouchterlony technique using class-specific second antibody.

The monoclonal antibody of the present invention and derivatives thereof are obtained by methods known per se, which are characterized in that the hybridoma cell series 144 BS 22-6-19

a) is grown in vitro and the monoclonal antibody is isolated from the supernatant liquid, or

b) is propagated in vivo in a suitable mammal and the monoclonal antibody is recovered from the bodily fluids of the mammal concerned and, if required

c) the recovered monoclonal antibody is converted into a derivative thereof.

Suitable culture media for the in vitro cultivation according to the invention according to method a) are standard nutrient media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally by a mammalian serum, ζ. As fetal calf serum, supplemented. The isolation of the monoclonal antibody is carried out by precipitation of the protein present in the supernatant liquid by ammonium sulfate or the like, followed by purification of the immunoglobulin by conventional chromatographic methods such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose or immunoaffinity chromatography.

Large amounts of the intended monoclonal antibody can be obtained by propagating the hybridoma cell line according to method b). Cell clones are injected into syngeneic mammals to grow antibody-producing tumors. After one to three weeks, the intended monoclonal antibody is recovered from body fluids of the mammal in question. As an example, the hybridoma cell line obtained from Balb / c mice is intraperitoneally transduced into Balb / c mice

ascites which have been optionally pretreated with a hydrocarbon such as pristane, and after one to two weeks, ascites fluid is collected from these mice. The monoclonal antibody is isolated by known methods, e.g. B. by precipitation of the protein with ammonium sulfate or the like, isolated from the body fluids, followed by the purification of the immunoglobulin by means of conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose or immunoaffinity chromatography is made.

Fragments of the monoclonal antibody, for example, Fab, Fab 'or F (ab') ₂ fragments, which retain their specificity to the antigenic determinants of the IFN-cc subtypes, may be derived from the monoclonal antibody obtained by methods a) and b) per se known methods are obtained, for. B. by digestion with enzymes such as pepsin or papain and / or cleavage of disulfide bonds by chemical reduction.

Radioactive iodine-labeled monoclonal antibodies can be prepared by iodination methods known in the art, for example, by labeling the monoclonal antibody with radioactive sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidizing agent such as lactoperoxidase, glucose oxidase and glucose. Radioactively labeled monoclonal antibodies according to the invention are also produced by the addition of radiolabeled nutrients to the nutrient media of the in vitro culture of step a). Such labeled nutrients include, for example, radioactive carbon ( ¹⁴ C), tritium ( ³ H), sulfur ( ³⁵ S) or the like, and include, for example, L- (' ⁴ C) -leucine, L- ( ³ H) -leucine or L-l ( ³⁵ S) -methionine.

Conjugates of the monoclonal antibody of the invention are prepared by methods known in the art, e.g. Example, by the reaction of the monoclonal antibody prepared according to method a) or b) or a fragment prepared as described above with an enzyme in the presence of a coupling agent, for. Glutaraldehyde, periodate, Ν, Ν'-o-phenylenedimaleimide, N- (m-maleimidobenzyloxy) succinimide, N- (3- (2'-pyridyldithio) -propionoxy) -succinimide, N-ethyl-N '- (3 dimethyiaminopropyl) carbodiimide or the like. Conjugates with avidin are also prepared. Conjugates with biotin are z. B. by the reaction of the monoclonal antibody with an activated ester of biotin, such as the biotin N-hydroxysuccinimidester produced.

The invention also relates to the hybridoma cell line designated 144 BS 22-6-19 deposited on March 14, 1985 in the Collection National de Cultures de Microorganisms, Institut Pasteur, Paris, under the number 1-424. This hybridoma cell line is a hybrid of the mouse myeloma cell line Sp2 / 0-Ag14 and a spleen B lymphocyte of a human IFN α-immunized Balb / c mouse monoclonal antibody designated as 144 BS The cell line may be maintained in culture or may be frozen in liquid nitrogen and reactivated by thawing.

The invention also relates to a method for producing said hybridoma cell series which secretes the monoclonal antibody 144 BS, characterized in that Balb / c mice are immunized with a natural human IFN α, antibody-producing spleen cells of the mice with cells of the Myolom cell series Sp2 / 0-Ag14 are fused, the hybrid cells obtained by the fusion are cloned and a cell clone leaving the desired antibody is selected. The immunization of the mice is made so that z. For example, natural human IFNα is injected two to four times parenterally, such as intraperitoneally and / or subcutaneously, at 10 to 40 day intervals in amounts of about 10 ⁵ IU to about 10 ⁶ IU. The injections optionally contain a lymphocyte production stimulating adjuvant such as complete or incomplete Freund's adjuvant.

Antibody-producing spleen cells taken two to five days after the last repeat injection are fused in the presence of a fusion promoter to cells of the myeloma cell line Sp2 / 0-Ag14. As fusion promoters z. B. Sendai virus or other paramyxo viruses, optionally in UV-inactivated form, Claciumionen, surface-active lipids such as lysolecithin, or polyethylene glycol. Preferably, the myoloma cells are fused with a 3 to 20 fold excess of spleen cells from immunized mice in a solution containing about 30% to about 60% polyethylene glycol having a molecular weight of between 1,000 and 4,000.

After fusion, the cells are resuspended and grown in selective HAT medium. Only Habridoma cells will survive because they combine the ability to grow and replicate in vitro, and this ability derives from the myeloma cells, with the lack of HGPRT or TK genes that indicate survival in the HAT -Medium are important, these genes are derived from the antibody-producing spleen cells of the immunized mice.

Suitable culture media for the propagation of hybridoma cells are the standard nutrient media, such as Dulbecco's Modified Eagle Medium, Minimal Essential Medium, RPMI 1640 medium and the like, optionally supplemented by serum, e.g. B. 10 to 15% fetal calf serum. Preferably, at the beginning of cell growth, feeder cells are added, e.g. Normal mouse peritoneal exudate cells, spleen cells, marrow bone macrophages or the like. The nutrient media are supplemented at regular intervals by selective HAT medium to prevent overgrowth of the hybridoma cells by normal myeloma cells.

The supernatant fluid of the hybridoma cell culture is screened for the required monoclonal antibody, preferably with combined immunoprecipitation bioanalysis or radioimmunoassay. Positive hybridoma cells are cloned, e.g. B. by limiting the dilution, preferably twice or more. The cloned cell lines can be frozen in a conventional manner.

The monoclonal antibody and / or its derivatives according to the invention are prepared for the qualitative and quantitative determination and / or purification of human IFN-a subsets from natural sources or by recombinant DNA methods and of human hybrid IFNs, especially the according to the invention, useful.

For example, the monoclonal antibody or its derivative, such as an enzyme conjugate or a radioactive derivative, can be used in any of the known immunoassays that rely on the binding interaction between the antigenic determinant, e.g. A polypeptide having IFN-α activity, and the monoclonal antibody. Examples of such analyzes are radioimmunoanalyses, enzyme immunoanalyses, immunofluorescence, latex agglutination and hemagglutination. Such immunoanalyses are valuable, e.g. In the monitoring of the production and purification of IFNα from natural sources or genetically engineered microorganisms and of hybrid IFN-α proteins and for the qualitative and quantitative determination of IFNα and hybrid IFNα in biological fluids, e.g. of patients with IFN-a therapy or those in need of such therapy.

The monoclonal antibody according to the invention can be used as such or in the form of a radiolabeled derivative in a radioimmunoassay (RIA). Any of the known modifications of an RIA can be applied, for example

RIA in homogeneous phase, solid phase RIA or heterogeneous RIA, single RIA or double (sandwich) RIA with direct or indirect (comparative) determination of IFN o. A sandwich RIA is preferred in which a suitable carrier, for example the plastic surface of a microtiter plate or a test tube, for example of polystyrene, polypropylene or polyvinyl chloride, glass or plastic beads, filter paper or dextran cellulose acetate or nitrocellulose sheets or the like with a monoclonal antibody specific for an IFN-cc subtype simply by adsorption or optionally after the activation of the carrier, for example with glutaraldehyde or cyanogen bromide, and incubated with the test solution and a solution of a ¹²⁵ I radiolabelled monoclonal antibody, the dissolved monoclonal antibody being an epitope of the IFN-ct subtype other than the carrier recognizes bound monoclonal antibodies, and the amount de IFNα or hybrid IFNα is determined by measuring the radioactivity bound to the support. In such preferable radioimmunoassay, either the carrier-bound antibody or the radiolabelled antibody is the monoclonal antibody of the present invention or a derivative thereof as described above, wherein the other antibody is a monoclonal antibody for IFNα out of the scope of the invention, or a polyclonal antibody , optionally in the form of a radiolabeled derivative.

Particularly preferred is a sandwich radioimmunoanalysis as described above in which the monoclonal antibody of the invention is bound to a bead, for example a polystyrene bead, in a test of standard solution containing IFNα or hybrid IFNα, in coated test beads. is finally incubated with a radiolabelled monoclonal antibody that recognizes another IFN-ct epitope.

Figures 4 and 5 show the radioactivity bound to the carrier beads as a function of the amount of IFN-α / B subspecies IFN-α-2 polypeptide in the test solution, as measured by sandwich RIA can be described in detail in the experimental part. The sensitivity of the RIA allows rapid, reliable and quantitative determination of 150 to 5000 IU / ml IFN-a-2 subspecies or even only one IU / ml of the IFN-ct-2 subtype, using a more time-consuming procedure becomes. Similar results can be seen in a sandwich RIA for determination of other IFN-ct subtypes, e.g. As the IFN-a / D subspecies or the IFN-ct / F subspecies, belonging polypeptides and especially the hybrid interferons according to the invention achieve.

The monoclonal antibody of the present invention may be used as such or in the form of an enzyme-conjugated derivative in an enzyme immunoassay. Such immunoassays include test methods employing an enzyme-labeled monoclonal antibody derivative of the invention, enzyme-labeled antibodies known per se which recognize and bind the epitopes of the anti-IFN-α antibody of the invention, or other anti-IFN-ct antibodies. An ELISA (enzyme-linked immunoadsorbant analysis) is preferred in which a carrier as described above for RIA is coated with the monoclonal antibody according to the invention, with a test solution containing IFNα and then with a polyclonal serum for IFNα, for example Sheep serum, incubated, and finally the bound antibodies of the polyclonal serum are developed by enzyme-labeled antibodies that recognize and bind them, and the amount of bound IFN a is determined by an enzyme-substrate reaction. Such an enzyme-labeled antibody is, for example, a phosphatase-labeled goat anti-sheep immunoglobulin. An ELISA is also preferred in which a carrier coated with a monoclonal antibody specific for an IFN-a subtype is incubated with a test solution containing IFNα and a solution of a monoclonal antibody conjugated to an enzyme wherein the dissolved monoclonal antibody recognizes a different epitope of the IFN-a subtype than the carrier-bound monoclonal antibody. By means of an enzyme-substrate reaction which results, for example, in a color change and can be followed by eye or by optical measuring instruments, the amount of bound enzyme corresponding to the amount of IFNα or IFN-α hybrid subspecies in the Test solution is proportional, measured.

Particularly preferred is an enzyme immunoanalysis, called immunodot analysis, of the test or standard solutions containing IFNα or hybrid IFNα on a microporous support having high intrinsic affinity for polypeptides, for example nitrocellulose. spotted, the carrier carrying one or more drops of the IFN-cc-containing probes in a solution of the monoclonal antibody of the invention, then in a solution of an enzyme-labeled second antibody which recognizes and binds the monoclonal antibody of the invention, and finally in a solution an enzyme substrate which results in a detectable signal, for example a colored substance, is incubated. Such an enzyme-labeled second antibody is z. Rabbit anti-mouse immunoglobulin which has been conjugated to horseradish peroxidase and can be developed with suitable enzyme substrates such as 4-chloro-1-naphthol or the like. The use according to the invention of the monoclonal antibody and its derivatives as described above for the qualitative and quantitative determination of IFN-a subtypes or hybrid IFNα also encompasses other immunoanalyses known per se, for example immunofluorescence tests involving antibody-conjugates or antigen-conjugates can be used with fluorescent substances, latex agglutination with antibody-coated or antigen-coated latex particles or haemagglutination with antibody-coated or antigen-coated red blood cells or the like.

The invention also relates to test material for the qualitative and quantitative determination of IFN-a subtypes and hybrid IFN α according to the invention, comprising the monoclonal antibody 144 BS and / or a derivative thereof and optionally other monoclonal or polyclonal antibodies to human IFN α and / or adjuncts.

For example, radioimmunoassay assay kits of the present invention include a suitable carrier, uncoated or coated with a monoclonal antibody to IFNα, optionally freeze-dried or concentrated solutions of a monoclonal or polyclonal antibody to IFNα and / or a radiolabelled derivative thereof, wherein at least one of the Carrier in solution or in radiolabeled monoclonal antibody is a monoclonal antibody of the invention, standard solutions of human IFNα, buffer solutions and optionally polypeptides and detergents for preventing non-specific adsorption and aggregation, pipettes, reaction vessels, calibration curves and the like. The test equipment according to the invention for an enzyme immunoassay contain, for example, a suitable carrier, optionally freeze-dried or concentrated solutions of a monocionalen and / or polyclonal antibody to IFN α and an enzyme-labeled monocionalen or polyclonal antibody to IFN α or for a first IFN α antibody recognizing wherein at least either the monoclonal antibody for IFN α or the enzyme-labeled antibody derivative for IFNα is the monoclonal antibody or its derivative according to the invention, enzyme substrates in solid or dissolved form, standard solutions of human IFN a, buffer solutions and optionally polypeptides and detergents, pipettes , Reaction vessels, calibration curves, color scale plates and the like.

Natural origin IFNα or IFN-α subtypes or IFNα-related polypeptides such as the hybrid interferons of the present invention can be purified by immunoaffinity chromatography using the monoclonal antibody of the present invention. The monoclonal antibody or a fragment thereof is bound to a suitable carrier of organic or inorganic origin, such as cross-linked agarose, dextran or polyacrylamide in appropriately functionalized form, optionally after activation, using techniques well known in the art. For example, an activated ester bearing carrier is suspended in an aqueous buffer solution, mixed with a solution of the monoclonal antibody or a fragment thereof, filtered, resuspended and washed to remove excess monoclonal antibody and treated with a solution of irrelevant proteins to give free block reactive positions of the carrier. This antibody-coated carrier is used to target human IFN-a subtypes, e.g. B. IFN α / Β, IFN α / D and IFN a / F subtypes and related polypeptides, such as the hybrid interferons according to the invention to bind selectively in glycosylated or unglycosylierter form. For this purpose, support material is suspended in a suitable aqueous solvent, for example a saline solution such as NaCl solution or a buffer solution such as a phosphate-buffered NaCl solution, NaHCO _r solution or 3- (N-morpholino) propanesulfonic acid solution and mixed with the IFN α containing solution, for example, by pouring it into a chromatography column and adding the IFN-containing solution and pumping it through the support material, if desired under pressure. Unbound proteins and other contaminants are washed out with aqueous solutions, for example buffer solutions having a pH range of approximately pH 5 to approximately pH 9 and / or salt solutions, for example NaCl solutions. The IFNa bound to the antibody on the support material is eluted with suitable aqueous solutions, for example buffer solutions having a pH ranging from approximately pH 2 to approximately pH 5, such as glycine buffer or citric acid, or pH gradients of different composition or salt solutions. for example, concentrated HN ₄ SCN solution. The IFNα-containing eluate is optionally neutralized and the purified IFNα is isolated therefrom by known methods. The following examples serve to illustrate the invention, but should not be construed as limiting it.

Experimental part

Abbreviations used in the examples

DTT 1,4-dithiothreitol

TNE solution containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl 2 mM EDTA

Tris tris (hydroxymethyl) aminomethane

EDTA ethylenediaminetetraacetic acid

TE solution containing 10 mM Tris, 50 μM EDTA

N medium contains Bacto-tryptone (10 g, Difco), yeast extract (1 g, Difco), glucose (1 g), NaCl (8 g), CaCl ₂ H ₂ O

(249 mg) per 1 L solution

TBE solution containing 89 mM Tris, 89 mM boric acid, 2 mM EDTA

BSA bovine serum albumin

Has hypoxanthine / aminopterin / thymidine

Ig immunoglobulin

IU International Units (Interferon Activity)

Mab. monoclonal antibody

PBS phosphate buffered saline

SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis

Part I: Construction and Cloning of Interferon a-2 / a-3 Hybrids in Escherichia coli All the hybrid interferon protein coding sequences described below were operably linked to the β-lactamase promoter, cloned into a pBR322-derived expression plasmid and cloned in Transformed E. coli HB101.

embodiments

Example 1: Construction of Pvull resistant vectors carrying DNA sequences of lymphoblastoid IFNe a-2 and a-3 pBR322 is cut with Pvull (Biolabs) and the linear DNA is ligated to a ( ³² P) Bcll linker (i.e. [ATGTGTGATCACACAT], which was synthesized by the solid-phase phosphotriester method, see H. Rink, et al., Nucleic Acids Research 12, 6369 [1984]), as follows:

The restricted plasmid DNA (1 μg, about 0.7 pmol ends) is ligated to ³² P-labeled BclI linker DNA (about 40 pmol ends) in 50 μl reaction volume containing 20 mM Tris-HCl, pH 7, 8, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM rATP and 40 U / μΙ T ₄ DNA ligase (Biolabs). After an incubation period of 2 to 3 h at 15 ° C, the aqueous phase is extracted with an equal volume of phenol / chloroform and the DNA is purified by the addition of 1/9 volume 1OxTNE, 2.5 volumes of ethanol and at a constant temperature precipitated from -20 ⁰ C overnight. The DNA pellet is resuspended in 50 μΙ TE, pH 8.0 and by a 5 to 23% sucrose gradient (50 mM Tris-HCl, pH 8.0, / 1 mM EDTA) in a Kontron rotor TST 60 centrifuged for 4 hours at 58,000 rpm and at 15 ° C. The gradient is fractionated from top to bottom, and the radioactivity in each fraction is monitored by Cerenkov counting. Interesting fractions are taken together and the DNA is precipitated with TNE and ethanol. The DNA pellet is again precipitated in restriction buffer and digested with BC1I and reprecipitated after extraction of the aqueous phase with phenol / chloroform. The linear DNA is then recirculated at a concentration of 5 μg / ml under conditions identical to those described for linker ligation.

The ligated DNA is cut with the enzymes HindiIi, Pvull (to eliminate residual intact pBR322) and PstI, and the HindIII-PstI fragment (3600 BP + linker) containing the origin of replication and the amp ^R gene isolated by sucrose gradient centrifugation.

HindIII-PstI inserts derived from sucrose gradient centrifugation of CG-pBR (AP) / Lylfn-a-2 (1160 bp) and CG-pBR (AP) / LylFN-a-3 (1020 bp) (EU -PA 76.489) are each incorporated into the DNA prepared above, resulting in the plasmids pAM4 and pAM2, which contain the LylFN-a-2 and LylFN-a-3 coding regions under the control of the β-lactamase promoter.

Ligated DNA is used directly to transform efficient E. coli as follows: The DNA ligation mixture (or

Fractions thereof) is transferred to 150μl of 15mM Tris-HCl, pH 7.5, 10mM CaCl ₂ , 10mM MgCl ₂ , 10mM NaCl, 0.5mM EDTA, and 50μl of CaCl ₂ -treated recipient E. coli HB101 are added. The mixture is held at 0 ° C for 20 minutes, then brought to 42 ° C for 2 minutes, and after cooling to room temperature, 1 ml of N-medium is added. Incubation is continued at 37 ° C for 1 h (shaking platform, 250 rpm), followed by 600 μΙ on McConkey agar / (Difco) containing tetracycline (10 μg / ml). The Petri dishes are incubated at 37 ⁰ C to 18 hours, with bacterial colonies are observed. '·

Plasmid DNA is recovered from the transformants by the following procedure. 10 ml of N medium containing tetracycline (10 μg / ml) are each inoculated with a transformant colony and the cultures are agitated at 37 ° C (shaking platform, 150-300 rpm) until an optical density from 0.9 to 1.1 (650 nm). The cells are harvested and resuspended in an equal volume of N medium containing tetracycline (10 μg / ml) and chloramphenicol (80 μς / μl). Incubation is continued at 37 ^° C. for an additional 18 hours to increase the number of plasmid copies per cell. The bacteria are harvested and the bacterial pellet resuspended in 50 mM Tris-HCl, pH 8 (10 ml per gram wet cell weight), and lysozyme (Sigma) is added to a final concentration of 2 mg / ml. After 10 min at 0 ° C the solution to 50 mM EDTA is set, after 10 min at 0 ° C Triton X 100 to a final concentration of 0.8% was added, and the preparation is for 1 hour at ⁰ ° C. followed by centrifugation in a Sorvall SS-34 rotor for 30 minutes at 18000 rpm. After extraction of the clear supernatant liquid with phenol and two subsequent extractions with chloroform, RNase A (Sigma) is added to a final concentration of 25 pg / ml and the solution is incubated for 1 h at 37 ° C. To remove RNA, NaCl (1 M final concentration) and polyethylene glycol 6000 (7.5% final concentration) are added and the preparation is kept at -10 ° C for 2 hours, followed by centrifugation at 10,000 rpm for 10 minutes and at 0 ⁰ C in a Sorvall rotor SS-34 follows. The pellets are resuspended in 200 μΙ TNE and again extracted with phenol / chloroform (1: 1) before reprecipitation (by addition of 500 μΙ ethanol, 5 min at -80 ⁰ C, 10 min Eppendorf centrifuge). The DNA pellets are resuspended in 20 to 40 μΙ TE. Typical yields obtained in this procedure are about 20 μg of plasmid DNA. The prepared plasmid DNAs (pAM4 and pAM2) are subjected to restriction enzyme analysis. The structure of pAM4 and pAM2 was confirmed.

Example 2: Elimination of the S'-extracistronic Pvull site of pAM4

In contrast to pAM2 (oc-3), pAM4 (a-2) still contains two Pvull sites (see Figure 3). To facilitate the construction of hybrid IFN genes at the Pvull site within the IFN coding region, the second Pvull site within the extracistronic region is eliminated.

This modification of plasmid pMA4 is essentially a deletion of the 3 'extracistronic IFNct-2 cDNA sequence between PstI and the 3' extracistronic Pvull site (see Figure 3). pAM4 is partially digested with Pvull (5 μg pAM4 in 50 mM NaCl, 6 mM MgCl ₂ , 6 mM 2-mercaptoethanol, 10 mM Tris-HCl, pH 7.5, 1 unit / μg Pvull (Biolabs) added, 5 min termination digested at 37 ⁰ C, by phenol extraction) and ligating described ⁽³² P) -Pstl linker (Collaborative Research, procedure as in example 1). The DNA mixture is transferred to E. coli HB 101 as above, and the plasmids from 16 colonies are screened by restriction enzyme analysis using PstI, Pvull and HindIII for the presence of a correct size deletion (246 bp) become. Of the 16 colonies screened, three are unmodified pAM4, one is a multimer, five have a large deletion (620 bp), and seven carry the small desired deletion (246 bp). A clone with a truncated IFNa-2 insert (HindIII PstI small fragment now 907 bp) is selected and designated pAM21.

Example 3: Construction of Pvu Hybrids Crossing at Amino Acid 92 Plasmids pAM2 (cc-3) and PaM21 (a-2) are each cut with HindIII and Pvull resulting in the excision of DNA fragments (521 bp) which contain the β-lactamase promoter sequences and the N-terminal half of the IFN genes (which code for amino acids 1 to 92). The large and small fragments (about 4 Kb and 521 BP, respectively) are separated by sucrose gradient centrifugation, and the large fragments are prepared by treating 1 μg DNA fragments in 50 μM 50 mM Tris, pH 8, with 1 unit Alkaline vesicant phosphatase per 10 pmol 5 'ends, dephosphorylated at 37 ° C for 30 min. Hybrid IFn ,, B] B ₂ D ₃ D ₄ "and" D _t D ₂ B ₃ B ,, "genes are constructed by ligation of the corresponding large and small DNA fragments described above (large HindIII Pvull fragment [~ 4 Kb] of PAM2 ligated to small HindIII-Pvull fragment [521 bp] of pAM21 resulted in B ₁ B ₂ D ₃ D ₄ -IFN, etc.). Ligaton is carried out in ΙΟ-μΙ volumes containing 20 mM Tris-HCl, pH 7.8, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM rATP, 40 units of μΙ T ₄ -DNS ligase (Biolabs). and contain approximately 500 ng DNA fragments and 20 to 40 ng small DNA fragments. After 5 h at 15 ° C, 5 μΙ of the ligation mixture are used to transform E. coli HB101 as above, yielding about 1,000 transformed colonies each. Three colonies are selected, their plasmid DNA is isolated and analyzed with the enzymes HindIII, Pvull, EcoRI, Taql and Pstl. Two colonies containing plasmid DNAs with the predicted structure (pAm 27 [,, B ₁ B ₂ D ₃ D ₄ "] and pAM 33 [,, D ₁ D ₂ B ₃ B ₄ "]) are selected.

Example 4 Construction of hybrid IFN genes crossed at amino acid 150 Plasmids pAM2 (cc-3) and pAM21 (a-2) are each digested with Pvull and PstI. The DNA fragments are separated by sucrose gradient centrifugation into Pvull-PstI-sized fragments (approximately 4 Kb encoding the N-terminal 92 amino acids of IFNn a-2 and a-3). In a second step, the purified Pvull-PstI small fragments of pAM2 and pAM21 are each ³² P-labeled as follows:

The DNA fragments (~ 1 μg) are each dissolved in 50 μl 50 mM Tris-HCl, pH 8.0, and dephosphorylated by treatment with 1 U / 10 pmol 5 'ends of alkaline phosphatase at 37 ° C. for 30 min. The enzyme is inactivated by 1 h incubation at 65 ⁰ C, and the DNA is purified by adsorption and elution of DEAE-cellulose and precipitated in ethanol. The DNA is phosphorylated in a 20 μΙ reaction volume containing 50 mM Tris-HCl, pH 9.5 / 10 mM MgCl ₂ / (5 mM DTT, 30 to 40 μL lyophilized Y- [ ³² P] -ATP (Amersham, 6000 includes Ci / mmol, 1 mCi / ml) and 0.5 U / μΙ T ₄ kinase (PL Biochemicals). After 20 min at 37 ⁰ C, the reaction by the addition of EDTA to 10 mM is terminated Then, an excess (. ~ 4 μg) of appropriate untreated DNA fragment and the solution is extracted with phenol and chloroform The DNA is separated from the remaining Y- [ ³² P] -ATP by chromatography through Sephadex G-50 in TNE Fractions determined by Cerenkov counting are pooled and precipitated with ethanol.

The ³² P-labeled Pvull-PstI small fragments are each completely digested with Sau3A, and the restricted DNA is separated by electrophoresis in a 6% polyacrylamide gel using TBE as running buffer. 4 DNA fragments, the Pvull Sau3A (172 bp each, which encode amino acids 93 to 150 of IFNe α-2 and α-3), and Sau3A Psti (214 bp and 324 bp, respectively, amino acids 151 to 166 IFNn α-2 and α-3) are isolated from the gel.

Religation of 3 matched fragments (see Table 2) produces cross-hybridized 4-hybrid IFN species at amino acid 150. The corresponding plasmids are reconstructed in a series of steps; In a first ligation reaction, equimolar amounts of Pvuil-Sau3A and Sau3A-PstI DNA fragments in 10 μM ligation buffer containing 20 mM Tris-HCl, pH 7.8, 10 mM MgCl ₂ , 10 mM DTT, 0.5 mM rATP and 40 units / μΙ of T4 DNA ligase ligated for 3 to 6 hours at 15 ° C (from which concatemeric [linked?] DNA resuspended). After digestion with Pvull-PstI and extraction with phenol / chloroform (1: 1), an excess of the corresponding Pvull-PstI-large fragment is dephosphorylated: in general, 1 μg of DNA in 50 μl of 50 mM Tris-HCl, pH 8, ( about 0.6 pmol 5 'ends) was treated with 0.06 units calf alkaline phosphatase kidney 30 min added at 37 ⁰ C] and the DNA religated. E. coli HB101 is transformed; and 4 clones carrying DNA with the predicted structure determined by restriction analysis (Pvull, Sau3A, Taql and EcoRI) are selected. The clones are designated E. coli HB101 pJC334, pJC37, pJC342 and pJ344 (see Table 2).

Table 2: Origin of DNA fragments used to construct the hybrid IFNe crossed at amino acid 150. In parentheses the IFN type and the a IFN-section encoded on the fragments are given.

resulting

Hybrid iFN Type Clone and Plasmid Ligated DNA Fragments (Length)

Designation (Pvull-Pstl Pvull-Sau3A Sau3A-Pstl

(about 4 Kb) (170 BP) (210BP, α-2)

(320 BP, α-3)

AM2 (α-3, .92) Am2 (α-3 ₉₃ _ ₁₅₀₎ AM21 (a-2, _5, _ ₁₆₆₎

AM2 (a-3 _ ₉₂₎ AM21 (a-3 _{9 15} 3_ o) Am2 _{(a-3151 _! 66)}

AM21 (a-2, _ ₉₂₎ Am2 (a-3 _M _, 5o) Am2.1 (a-2 ₁₅ _ _1M)

AM21 (a-2, _s ₂ ) Am21 (a-2 _{93, 15} o) Am21 (α-3 _{151, 166} )

D ₁ D ₂ D ₃ B ₄ " pJC334 D ₁ D ₂ B ₃ D ₄ " pJC337 B ₁ B ₂ D ₃ B ₄ " pJC342 B ₁ B ₂ B ₃ D ₄ " pJC344

Example 5: Construction of Hybrid IFN Genes Crossed at Amino Acid 60 The construction of these hybrid IFN genes is performed essentially by the same procedure as described in Example 4

has been described.

The plasmids pAM2 (α-3) and pAm21 (α-2) are each cut with HindIII and Pvull, and the fragments are separated by sucrose gradient centrifugation. The Hindll-Pvull small fragments (521 bp containing the β-lac promoter region and encoding the N-terminal 92 amino acids of Ifne α-2 and α-3) are ³² P-labeled and partially digested with Sau3A (0.5 units enzyme per ug DNA for 5 minutes at 37 ⁰ C, digested termination of the reaction by phenol extraction. the resulting mixture of fragments of polyacrylamide gels is redissolved by electrophoresis in 6%, and 4 fragments (two 424 BP HindIII-Sau3A fragments and two 97 BP Sau3A-Pvull fragments) are extracted from the gel Religation of 3 corresponding fragments by the stepwise procedure described in Example 4 (see Table 3) and transformation into E. coli HB101 results in transformants with only 2 of the expected 4 constructs: pAM65 (,, D ₁ B ₂ D ₃ D ₄ ") and pAM90 (,, B ₁ D ₂ B ₃ B ₄ ").

Table 3: Origin of DNA fragments used to construct two hybrid IFN genes crossed at amino acid 61. In parentheses, the IFN type and the fragments-encoded section are designated by alFN.

Resulting hybrid IFN type

Clone and plasmid name

DNS fragment type (length)

HindIII Sau3A Sau3A Pvull HindIII Pvull

(424 BP) (97 BP) (about 4 Kb)

D ₁ B ₂ D ₃ D ₄ B ₁ D ₂ B ₃ B ₄

pAm65 pAM90

Am21 (a2 ₆₂ _ ₉₂₎ Am2 (a-3 ₆₂ _ ₉₂ )

Am2 (a-393_, 66) AM 21 (a-2 ₉₃ _ ₁₆₆₎

For the construction of the two other hybrid IFN genes cross-over at amino acid 61, plasmids pAM65 and pAM90 are each cut with HindIII and Pvull, and the small HindIII-Pvull DNA fragments (521 bp) are isolated in agarose gel after electrophoresis. Ligation of the Hind III Pvull small fragment of pAM90 with the Hind III Pvull-sized fragment of pAM2 results in pAM94 (B ₁ D ₂ D ₃ D ₄ ), and ligation of the small Hind III Pvull fragment of pAM65 the HindIII-Pvull-sized fragment of pAM21 yields plasmid pAM76 (D ₁ B ₂ B ₃ B ₄ ). The structure of the above four plasmid DNSn is determined by

Restriction enzyme analysis confirmed.

The corresponding clones are referred to as E. coli HB101 pAM65, pAM90, pAM94 and PEM76.

Part II: Construction and Cloning of Interferon a-2 / a-3 Hybrids in Saccharomyces cerevisiae All the hybrid interferon protein coding sequences described below were linked to the PHO5 promoter and the PHOS transcription termination signals, into the yeast 2 μ Vector pJDB207 cloned and into the yeast strain

S. cerevisiae GRF18.

Example 6: Construction of expression plasmid p31 RT (Δ 72)

Plasmid pJDB207 / IF2 (5,) A72 (EU-PA No. 100,561) is digested with HindIII and EcoRI, and a 470-BP fragment which has the carboxy terminal end of interferon α-2 and PHO5 transcription termination signals , is isolated by agarose gel electrophoresis and electroelution (R. Yang, et al., Methods Enzym., 68, 176 [1979]) using Micro-Collodion Bags (Sartorius, Gottingen, FRG). Plasmid p31R (see EP 100 561) is digested with HindIII and EcoRI, and

the 4.1 Kb vector part is isolated using the same technique. After ligation of 200 ng of vector and insert DNA using E. coli T4 DNA ligase and transformation of E. coli HB101 to ampicillin resistance, a plasmid called p31 RT (A 72) becomes the PH05 promoter , which contains the C-terminal end of interferon ce-2 and PHO5 transcription terminating signals, and confirms its structure by restriction analysis.

Example 7: Construction of plasmids pJDB207 PHO5 / IFN AM104, AM114 and AM119 PHO5 expression plasmid p31 RT (A 72) (see Example 6) is digested with EcoRI and Xhol, and the recessed 3 'ends are digested with dCTP, dGTP, dATP and dTTP were filled in using the Klenow fragment of DNA polymerase I from E. coli. The blunt ends are then dephosphorylated with bacterial alkaline phosphatase. Plasmids pJC334, pAM90 and pJC337 (Examples 4 and 5) are digested with TaqI (pJC334 and pAM90) or with TaqI and EcoRI (pJC337) and the 3'-recessed ends are filled in with the appropriate deoxynucleotides as described above. DNA fragments of approximately 560 base pairs are isolated by agarose gel electrophoresis and electroelution using Micro-Collodion Bags as above. Ligation of 200 ng of prepared vector portion of plasmid p31 RT (A 72) and 200 ng of purified DNA fragments using T4 DNA ligase and transformation of E. coli HB101 to ampicillin resistance yield plasmids p31 R / IFN AM104 (containing the hybrid interferon ,, D ₁ D ₂ D ₃ B ₄ "coding sequence), p31R IFN AM119 (containing the hybrid interferon ,, B ₁ D ₂ B ₃ B ₄ " coding sequence) or p31R IFN AM114 ( D ₁ D ₂ B ₃ B ₄ "containing the hybrid interferon ,, coding sequence by cutting these plasmids with BamHI and HindIII a DNA fragment of approximately 1 300 bp with the configuration PHO5 promoter -. IFN protein coding sequence - PHO5 transcriptional Termination signal is isolated by agarose gel electrophoresis and electroelution This fragment is cloned between the HindIII and BamHI sites of plasmid pJDB207 The plasmids are transformed into yeast strain GRF18 to Leu ⁺ (see EP 100 561, see also Example 14) result the clones are designated S. cerevisiae GRF 18 / pJDB207 PHO5 / IFN AM104, GRF18 / pJDB207 PHO5 / IFN AM114 and GRF18 / pJDB207 PHO5 / IFN AM119.

Example 8: Construction of plasmid pJDB207 PHO5 / IFN AM129 Plasmid pAM94 (Example 5) is completely digested with TaqI and partially (in the presence of 10 ng / ml ethidium bromide) with EcoRI. A DNA fragment of approximately 580 bp is isolated by agarose gel electrophoresis and electroelution and ligated to the EcoRI and Xhol-cut gel-purified vector portion of plasmid p31RT (A 72) (see Example 6). After transformation of E. coli HB101 to ampicillin resistance, the plasmid p31R PHO5 / IFN AM129 is isolated. This plasmid containing the interferon ,, B ₁ D ₂ D ₃ D ₄ "coding region is digested with Hind III and Bam HI and the 1 300 BP DNA fragment is ligated into pJDB207 as described in Example 7. The plasmid is amplified as described in supra Yeast strain GRF 18. The resulting clone is designated S. cerevisiae GRF18 / pJDB 207 PHO5 / AM129.

Example 9: Construction of plasmids pJDB207 PHO5 / IFN AM106, AM110 and AM112 Plasmids pJDB207 PHO5 IFN / AM104, AM114 and AM129 (see Examples 7 and 8) are digested with BamHI and Pvull and the 6.8 Kb vector parts containing the respective C-terminal ends of the interferon coding regions are isolated by agarose gel electrophoresis and electroelution. In the same way, plasmid pJDB207R / (a-2) A 72 (EU-PA 100 561) is digested with BamHI and Pvull and a DNA fragment of 800 BP isolated. Ligation of the respective fragments and transformation of E. coli HB101 results in plasmids pJDB207 PHO5 / IFN AM106 (containing the hybrid interferon ,, B ₁ B ₂ B ₃ D ₄ "coding region), AM112 (containing the hybrid interferon "B, B ₂ D ₃ B ₄ " coding region contains) and AM110 (containing the hybrid interferon "B, B2D ₃ D ₄ " coding region) The yeast strain GRF18 is transformed as usual The resulting cions are identified as S. cerevisiae (GRF18 pJDB207 PHO5 / IFN AM106, pJDB207 PHO5 / 1FN AM110.

Example 10: Construction of plasmids pJDB207 PHO5 / IFN DM1 and DM2

Plasmid pJDB207 PHO5 / IFN AM119 (Example 7) is digested with HindIII and BglII, and the 7.5 Kb vector portion containing the N-terminal end of the interferon coding region is isolated by agarose gel electrophoresis and electroelution. Similarly, plasmids pJDB207 PHO5 / IFN AM114 and pJDB207 PHO5 / IFN AM104 (see Example 7) are each digested with the same enzymes, and DNA fragments of about 600 bp containing the C-terminal end of the interferon coding region, be isolated. The corresponding fragments are ligated with T4 DNA ligase. Following transformation of E. coli HB101, plasmids pJDB207 PHO5 / IFN DM1 containing the hybrid interferon "B, D ₂ D ₃ D ₄ " coding region and pJDB207 PHO5IFN DM2 containing the hybrid interferon ,, B ₁ D ₂ D ₃ B ₄ "coding region contains, recovered. The yeast strain GRF18 is transformed as described above. The resulting clones are termed S. cerevisiae GRF18 pJDB207 PHO5 / IFN DM1 and S. cerevisiae GRF18 pJDB207 PHO5 / IFN DM2.

Example 11 construction of plasmid pJDB207 PHO5 / IFN HRi43

Plasmid pJDB207R / IF (cc-2) A 72 (EU-PA No. 100 561) is digested with BamHI and Pvull and a DNA fragment of 6.8 Kb is isolated. Similarly, plasmid pJDB207R / IF (cc-3) (EU-PA 100 561) is digested with the same enzymes and a DNA fragment of 800 bp is isolated. The ligation of the two DNA fragments and the transformation of E. coli HB101 to ampicillin resistance results in plasmid pJDB207 PHO5 / IFN HRi43 containing the hybrid interferon ,, D ₁ D ₂ B ₃ B ₄ "coding region The yeast strain GRF18 is transformed as above The resulting clones are designated S. cerevisiae GRF18 pJDB207 PHO5 / IFN HRi43.

Example 12: Transformation of Saccharomyces cerevisiae GRF18 and Induction of Interferon Production Plasmid pJDB207 PHO5 / IFN AM129 (,, B ₁ D ₂ D ₃ D ₄ ") is prepared analogously to the description (Hinnen et al., Proc. Natl. Acad. Sci., USA 75, 1929 [1978]) into the Saccharomyces cerevisiae strain GRF18 (a, his 3-11, his 3-15, leu 2-3, leu 2-112, can®). One μg of plasmid DNA is added to 100 μΙ of a Spheroplast suspension, and the mixture is treated with polyethylene glycol.The spheroplasts are mixed with 10 ml of regeneration agar and streaked on yeast minimal medium plates without leucine.After an incubation period of 3 days at 30 ° C A single yeast colony from the yeast transformation plates (called Saccharomyces cerevisiae (GRF18 / pJDB207 PHO5 / IFN AM129) is added to 10 ml of yeast minimal medium in a 100 ml Erlenmeyer flask and extracted at 30 ° C ⁰ C at 200 rpm for 24 hours to a density of about 2 to 3 x 10 ⁷ ts bred / ml. The cells are supplemented once with 20 ml of low-P, minimal medium (such as "Difco yeast minimal medium without amino acids" supplemented with 20 g / l glucose, but from the ingredients according to the formulation of Difco

[Difco Handbook, Difco Laboratories, Detroit, USA] except that 0.03 g / L KH ₂ PO ₄ plus 1 g / L KCl is used instead of 1 g / L KH ₂ PO ₄ ). Three ml of resuspended cells are used to inoculate 300 ml of low-Pi minimal medium or 300 ml of normal minimal medium in 1,000 ml Erlenmeyer flasks. The incubation is carried out at 30 ° C and 160 U / min. Induction of the PHO5 promoter is followed by measurement of the appearance of acid phosphatase activity in whole cells as described (Toh-e, et al., J. Bacteriol., 113, 727 [1973]). The cells are grown to about 1 to 2 × 10 ⁷ cells / ml (incubation time 26 to 30 hours).

Example 13: Preparation of Yeast Cell Extracts and Determination of Interferon Titer Cells from the 300 ml nutrient medium (see Example 12) are centrifuged in a Sorvall GSA rotor at a density of 1 to 2 χΊθ '/ ml over a period of 5 min collected at 8000 rpm and 4 ° C. The cells are washed once with 100 ml H ₂ O, again in 6 ml ice-cold lysis mixture [0.1 M potassium phosphate buffer, pH 7.4, 1% [V / V] Triton X-100, 0.0001 IV PMSF [Merck] ) and transferred to a 30 ml Corex tube. The suspension is again centrifuged for 5 minutes in a Sorvall-Roor SS-34 at 8000 rpm at 4 ° C and resuspended in 3 ml lysing mixture at ⁰ ° C. Four grams of glass beads (0.4 mm diameter) are added to the cells and the suspension is shaken on a vortex mixer (Scientific Instruments Inc., USA) for 30 seconds at full speed and then cooled in an ice bath for 1 minute. This shaking is repeated 5 to 10 times until more than 90% of the cells are broken (checked under the light microscope). Cell pieces and glass beads are removed by centrifuging at 8,000 rpm for 10 minutes at 4 ° C in a Sorvall HB-4 rotor. The supernatant is transferred to Eppendorf tubes, frozen in liquid nitrogen and stored at -6O ⁰ C. Interferon activity is determined according to S. Rubinstein, et al., (J. Virol. 37, 755 [1981]) as 7 × 10 ⁹ units / ml cell extract. ι, *

In a manner analogous to that described in Examples 12 and 13, S. cerevisiae strain GRF18 is supplemented with plasmids pJDB207 PHO5 / IFN AM119 (,, B ₁ D ₂ B ₃ B ₄ "), AM106 (,, B ₁ B ₂ B ₃ D ₄ "), AM112 (,, B ₁ B ₂ D ₃ B ₄ "), DM ₁ (,, B ₁ D ₂ B ₃ D ₄ "), DM ₂ (,, B ₁ D ₂ D ₃ B ₄ ") , AM110 ("B ₁ B ₂ D ₃ D ₄ ") and HR443 ("D ₁ D ₂ B ₃ B ₄ ") The resulting strains are cultured, the cells are harvested and the interferon titers are as determined above The IFN titers are as follows: S. cerevisiae GRF18 / pJDB207 PHO5 / IFN AM119: 5 x 10 ⁹ units / ml

S. cerevisiae GRF18 / pJDB207 PHO5 / IFN AM106: 7 10 ⁷ units / ml

S. cerevisiae GRF18 / pJDB207 PHO5 / IFN AM112: 2 10 units / ml

S. cerevisiae GRF18 / pJDB207 PHO5 / IFN DM1: 3 10 ⁹ units / ml

S. cerevisiae GRF18 / pJDB207 PHO5 / IFN DM2: 5 x 10 ⁹ units / ml

S. cerevisiae GRF18 / pJDB207 PHO5 / IFN AMHO: 3 x 10 ⁸ units / ml

S. cerevisiae GRF18 / pJDB207 PHO5 / IFN HRi43: 1 x 10 ⁹ units / ml

Under the same conditions, S. cerevisiae strains GRF18 / pJDB207R / IF (a-2) and / IF (a-3) (EU-PA 100 561) give titers of 1 x 10 ⁸ and 3 x 10 ⁹ units / ml, respectively ,

Example 14: Production of hybrid interferon ,, B ₁ D ₂ D ₃ D ₄ "by transformed yeast cells on a 30 ml scale yeast strain GRF 18 which carries the plasmid pJDB207 PHO5 / IFN AM129, which is the gene for hybrid interferon "B ₁ D ₂ D ₃ D ₄ " encoded below the acid phosphatase promoter PHO5 is streaked onto the surface of an agar culture of YNB medium. The plate is incubated at 30 ^° C. until confluent growth is observed. A sterile loop is used to transfer a portion of the surface culture to a shake flask containing the preculture medium IFN / 21, consisting of: yeast extract (Difco) 10.0 g

L-asparagine H ₂ O 6.6 g

KH ₂ PO ₄ 1.0 g

MgSO ₄ -7H ₂ O 1.9 g

L-histidine 0.02 g

D-glucose (monohydrate) 33.0 g per 1 l deionized water

The 500 ml flask has a single divider and contains 100 ml of medium. The medium is prepared using deionised water and has a pH value of approximately "6.0. The glucose is sterilized separately. After inoculation the first pre-culture is 24 hours at 3O ⁰ C with on an orbital shaker The flask with the first pre-culture delivers the inoculum to the second pre-culture flasks, which receive an inoculum of 1% v / v. The medium and incubation conditions are identical to those for the first pre-culture Sufficient flasks from the second pre-culture stage are pooled to give 1% v / v inoculum for the 30 L fermentor (3 flask / fermentor) Operating volume of 30 l, contains four partitions and a single blade turbine agitator with six blades 115 mm in diameter, the speed of movement is 600 rpm, air is blown in at 1 vol / vol / min and the overpressure becomes a kept at 0.3 bar. The fermentation temperature is 30 ⁰ C. The fermenter is sterilized with medium IFN / 23 that includes:>

Yeast extract (Difco) 2.0 G L-asparagine H ₂ O 6.6 G MgSO ₄ - 7H ₂ O 1.0 G L-histidine 0.02 G D-glucose (monohydrate) 33.0 s 5 each 1

I deionized water

wherein glucose is separately sterilized and added to obtain a final volume of 30 l.

After inoculation, the pH of the fermentation is adjusted by the addition of sodium hydroxide so that it does not fall below 6.0. The fermentation lasts about 18 hours or until the maximum yield of interferon is reached.

The optical density, which is a general measure of the growth of the yeast, reaches a value between 6 and 7 OD units. However, glucose is not fully consumed for the most part, and induction of acid phosphatase activity occurs concurrently with generation of interferon.

The interferon titer can be measured in crude extracts prepared by mechanical cell destruction in the laboratory and is 7 x 10 ⁹ units / l cell extract. The protein content of the crude extract is approximately 1 mg / ml.

At the end of the fermentation, the nutrient broth may be cooled to 10 ° C before harvesting and recovery of the hybrid interferon if necessary.

30 l of nutrient broth with a pH of 6.0 are cooled to 10 ° C and the cells are separated using a Sharpies® centrifuge. The clear supernatant contains no IFN activity. The cell mass obtained is filled up to 1400 ml with Buffer x (1 mM Tris, pH 8.0, 5 mM EDTA, 0.5 M NaCl, 20 μM phenylmethylsulfonyl fluoride) and has a pH of 8.0. After cooling to 5 to 10 ⁰ C, the suspension is passed through a DYNO®-Mill (type KDL Pilot, 0.6 l), which with polyurethane stirring blades and 500 ml glass beads with a diameter of 0.5 to 0.75 mm and has a stirring speed of 3000 rpm and a feeding speed of 10 l / h, whereby the cells are torn. The suspension has a pH of 8. The suspension containing the broken cells is clarified by centrifugation. The centrifugation is carried out in a Sorvall Rotor 6SA at 12000 rpm, at 4 ° C for 20 minutes.

The plasmid pJDB207 PHO5 / IFN AM119, AM1.06, AM112, DM1 and DM2 carrying strain S. cerevisiae GRF18 can be cultured, and a clarified solution containing the corresponding hybrid interferon can be prepared in an analogous manner.

Example 15: Preparation of extracts of E. coli strain HB101 / pAM94 and determination of IFN activity

E. coli strain HB101 / pAM94 is grown to an optical density of 8 at 650 nm in 1 liter of M9 medium containing 0.4% casamino acids and 2% glucose. The cells are sedimented and resuspended in buffer (16 g cells in 160 ml buffer) containing 0.1 M Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM EDTA and 100 μM PMSF. Lysozyme is added to a final concentration of 1 mg / ml. The solution is then kept on ice for 30 minutes. The cells in this suspension are opened with the aid of a Sorvall Omnimix at a setting of three times for 60 seconds. The suspension containing the broken cells is clarified by centrifugation for 20 minutes in a Sorvall 6SA rotor with 120Ö0 U / min at 4 ⁰ C. The supernatant liquid is analyzed for IFN activity using the method of S. Rubinstein et al. (Supra).

An IFN activity of 5 x 10 ⁷ units / ml cell extract was found.

In an analogous manner, the E. coli strains HB101 / pAM90, pJC344 and pJC342 are cultured and the interferon titres ^β as above than 5 χ 10, 5 χ 10 ⁷ and 5 x 10 ⁶ determines units / ml cell extract.

Example 16: Preparation of hybridoma cells

a) Source of Immunogen: A sample of semi-purified human leukocyte IFN (IFN α) obtained from Dr. med. K. Cantell (Helsinki), with a specific activity of 13 x 10 ⁶ IU per mg of protein, is used for immunization.

b) Immunization protocol: Seven 10 to 14 week old female Balb / c mice (Tierfarm, Sisseln, Switzerland) are emulsified by injection of 4 x 10 ⁵ IU of IFN α (Example 16) emulsified in Freund's complete adjuvant (Difco), immunized into the four balls of foot. A further injection of 4 χ 10 ⁵ IU of IFN α in incomplete Freund's adjuvant is administered on days 30 and a booster injection (ip) of 6 χ 10 ⁵ IU of IFNa in PBS at day 60. Three days later each of the spleen for the Used fusion.

c) Cell fusion: All fusion experiments are performed according to the procedure of G. Köhler and C. Milstein [Nature 256, 495 (1975)] using the non-exuding Sp 2/0-Ag14 myeloma series [M. Shulman, CD Wilde and G. Köhler, Nature 276, 269 (1978)]. 10 ^{s of} spleen cells are mixed with 10 'myeloma cells in the presence of 1 ml of 50% polyethylene glycol (PEG 1500, Serva). After washing, cells are resuspended in 48 ml of standard Dulbecco's Minimum Essential Medium (Gibco # 0422501). 3 × 10 ⁶ normal mouse peritoneal exudate cells are added per feeding as feeding cells. The cells are distributed in 48 x 1 ml growth wells and fed with standard HAT selection medium three to three weeks a week. When the growth of hybridoma cells becomes apparent, the supernatant fluids are screened by a combined immunoprecipitation bioassay (Example 19). Of the total of 221 hybridomas, 10 hybridomas were found to produce anti-IFNd antibodies. The hybridoma cells are cloned by limiting dilution at least once in microtiter plates. Hybridoma 144 BS is selected for further study because it is particularly stable and excretes large quantities of immunoglobulin.

Example 17: Isolation and purification of monoclonal antibody

8-10 weeks old Balb / c mice (Tierfarm, Sisseln, Switzerland) are pretreated intraperitoneally with 0.3 ml pristane (Aldrich).

After 2 to 3 weeks, 2 to 5 × 10 ⁶ hybridoma cloned cells and 0.2 ml pristane are intraperitoneally inoculated. After 8 to 10 days, ascites fluid is collected, centrifuged at 800 χ g and stored at -20 ° C.

Thawed ascites fluid is centrifuged at 50,000 xg for 60 min. A layer of fat floating on the surface is carefully removed and the protein concentration is adjusted to a concentration of 10 to 12 mg / ml. Crude immunoglobulin is precipitated by dropwise addition of 0.9 volume equivalents of saturated ammonium sulphate at 0 ⁰ C, then in 20 mM Tris-HCI / 50 mM NaCl (pH 7.9) and dialyzed against the same buffer. An immunoglobulin fraction is recovered by DEAE-D52 cellulose (Whatman) chromatography using a buffer gradient system of 20 mM Tris-HCl / 25-400 mM NaCl, pH 7.9. The immunoglobulin is again precipitated with ammonium sulfate and dissolved in PBS at a concentration of 10 mg / ml.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates greater than 95 percent purity for monoclonal antibodies 144 BS.

Example 18: Determination of Class and Subclass of Monoclonal Antibodies The class and subclass of monoclonal antibodies produced by hybridoma cloned cells are determined by Ouchterlony's well known agar gel immunodiffusion technique using class and subclass specific rabbit antibodies (Bionetics). certainly. The results are confirmed by an enzyme immunoassay (ELISA) as follows: Microtiter plates are coated with 1 μg of a rabbit immunoglobulin preparation of a class or subclass specific serum (Bionetics) in 50 μΙ PBS per well.

The free binding capacity of the plate is saturated with a buffer of 1% bovine serum albumin in PBS containing 0.2% NaN ₃ (M / V), pH 7.4. Containing ΙΟΟ-μΙ probes, the monoclonal antibodies are incubated for 1 h at 37 ⁰ C in the wells. The plates are washed with PBS, then incubated for 1 h at 37 ^° C. with a phosphatase-conjugated rabbit immunoglobulin preparation with the same specificity used for coating the plates. The fixed enzyme is determined by incubating (37 ⁰ C, 30 min) (with a solution of the enzyme substrate p-nitrophenylphosphate 1 mg / ml in diethanolamine buffer 10% containing 0.5 mW! MgCl ₂ and 0.02% (M / V) contains NaN ₃ , pH 9.8) and measuring the optical density at 405 nm. The monoclonal antibodies 144 BS belong to the class IgG ₁ K (kappa).

Example 19: Combined Immunoprecipitation Bioanalysis

Fifty μΙ crude natural human leukocyte IFN or recombinant IFN-α polypeptides (Example 20a) (10 ⁴ IU / ml) are mixed with equal amounts of test solution, e.g. B. the supernatants of hybridomas -, or PBS solutions of purified monoclonal antibodies, and incubated for 2 hours at 37 ° C in microtubes (Eppendorf 3810). Subsequently, 50 μΙ of previously titrated rabbit anti-mouse Ig antibody (Nordic) is added and the mixture is then incubated for 1 hour at 37 ° C and 16 hours at 4 ⁰ C, whereby free and IFN-bound monoclonal Make antibody immune complexes. The tubes are centrifuged at 12000 rpm at 4 ° C for 5 min. The immune precipitate is washed once with 1 ml cold buffered saline (pH 7.2), then dissolved in 200 μM buffered saline, pH 2.2. The IFN activity released thereby is determined in a standard bioassay according to JA Armstrong, Appl. Microbiol. 21, 723 (1971). The inhibition of the cytopathic effect of Mengo virus (400 plaque forming units per well) is determined using Hep-2 cells (3 x 10 ⁴ cells per well) in microtiter plates.

Example 20: Determination of interferon specificity.

a) Source of Recombinant IFNa Polypeptides: LylFN-a-1, LylFN-a-2 and LylFN-a-3 polypeptides related to IFN-aF, IFN-aB and IFN-aD, respectively, become EU -PA No. 76489. IFN-a-A and IFNo-J are J. Davies (Biogen, Geneva) and from Prof. C. Weissmann (University of Zurich, Switzerland), IFN-cc-C from the Weissmann Institute of Sciences, Virology Department, Rehovot, Israel.

b) Source of monoclonal antibody used for comparison for IFN

Monoclonal antibodies 1K2 and 2K2 are prepared as described in British Pat. No. 2,108,510. MAb NK2 and YOK are manufactured by Celltech (Berkshire), IY-1 and IY-2 of Inter-Yeda (Israel), S-1, S-2, S-3 and S-4 by Dr. Ing. C. Favre, UNICET, Dardilly (France) and MAb I / 24 by M. Aguet, University of Zurich (Switzerland).

c) The determination of the reactivity of various monoclonal antibodies to the IFN-a subtypes is made using the combined immunoprecipitation bioanalysis of Example 19 and the tandem radioimmunoanalysis described by D.S. Secher, Nature 290, 501 (1981). The results are summarized in Table 4.

The monoclonal antibodies 144 BS, 1K2 and 2K2 do not react with human IFNβ (Rentschier, Laupheim, West Germany) or human IFNγ (Bio Science Emmenbrücke, Switzerland).

Table 4: Immunoprecipitation by MAb's of recombinant IFN-a subspecies

Monoclonal IFN-a subspecies

Antibody A a-2 C a-3 a-1

1K2 + ++ '-

 100% bond + + + 75% binding

50% bond + 25% bond

+ 10% binding - 0% binding

Blank test: binding was not determined (approximate values)

The results of Table 4 show that the monoclonal antibody 144 BS has a unique binding pattern.

Particularly noteworthy are the low binding to IFN-α-A and the effective binding to IFN-α-2, IFN-α-3 and IFN-α-1 polypeptides.

In addition, the results of Table 4 allow the epitope analysis, which is shown schematically in Table 5.

Table 5: Epitope analysis of recombinant IFN-ct subtypes

Recombinant epitope recognized by monoclonal antibody

IFN-a sub-type 144Bs "1K2 NK2, S-3 YOK S-1S-2S-4,

2K2 IY-I, 1/24 IY-2

AV VV VV V Y Y VV Λ ΛΛ '' "''" "" '"' ΛΛ / \ Λ / \ / \ y \

_a _2 xx XX χ

_a _3 xx xx XX

et-1 XXX

The analysis in Table 5 reveals the fact that the epitope combination of each IFN-a subtype is unique. In addition, the analysis allows the prediction of appropriate pairs of monoclonal antibodies to determine specific subtypes of IFN α with the exclusion of other subtypes in a tandem immunoassay. For example, the combination of monoclonal antibody 144 BS with either MAb's NK2, S-3, IY-1 or I / 24 will be useful for the detection of IFN'-aB without IFN-aD interference, and the combination of MAb 144BS with MAb YOK for the detection of IFN-aD in the presence of IFN-aB.

Example 21: Preparation of an Immunoadsorbent Column

One ml of detached Äffi-Gel® 10 (Bio-Rad) is coupled to 17 mg of monoclonal antibody 144BS according to the manufacturer's instructions: Äff i-Gel® is placed on a sintered glass filter first with cold distilled water and then with a 0.1 M NaHCO3 ₃ solution, pH 8.0 (coupling buffer). A suspension of 50% gel in coupling buffer is transferred to a plaster tube, mixed with an equal volume of purified monoclonal antibody solution and rotated for 4 hours at room temperature. After coupling, the gel is washed with coupling buffer and treated with 0.1 ml of 1 M etharolamine HCl (pH 8.0) for 1 to 2 hours at room temperature to block unreacted sites. The gel is washed with PBS in the presence of 10 mM NaN ₃ and stored therein at 4 ° C.

Example 22 Purification of Recombinant IFNa by Immunoaffinity Chromatography A suspension of recombinant IFNa-producing disrupted E. coli cells, eg, E. coil HB101 / pAM94 (see Example 15), is centrifuged in a Sorvall 6SA rotor at 12,000rpm for 20min clarified at 4 ⁰ C. Polyethyleneimine (Polyimine P®, Fluka, pH 8.0) is added to the supernatant liquid until a final concentration of 0.25% (M / V) is achieved. The solution is stirred for 1 h and then centrifuged. The pellet is discarded and the supernatant liquid is brought to 65% saturation with solid ammonium sulfate. This mixture is stirred for 1 h and then centrifuged. The supernatant is discarded and the pellet is suspended in one-tenth volume (15 to 20 ml) of PBS (pH7.2). The solution is dialyzed overnight against PBS containing 0.02% NaN ₃ .

The dialyzed solution is mixed with 1 ml of the immunoaffinity gel of Example 21. This mixture is gently agitated for 1 hour, then poured into a column and washed sequentially with 5 column volumes of PBS, 5 column volumes of 0.5M NaCl, and 0.2% Triton X-100® containing PBS and 5 column volumes of PBS. Alternatively, the immunoaffinity gel is first filled into the column and the dialyzed solution is added at the top, then washed as described. The recombinant hybrid IFNα ("B ₁ D ₂ D ₃ D ₄ ") is eluted with an acid buffer, pH 2.5, containing 0.1 M citric acid and 0.3 M NaCl. Fractions are neutralized with 1 M Tris, dialysed against PBS, and concentrated to about 0.1 mg / ml protein using XM 10 ultrafiltration flatbed membranes (Amicon Corp.).

The maximum capacity of the immunoaffinity column of Example 21 containing 17mg monoclonal antibody 144BS is approximately 1.2 mg of each IFN-α-2 and IFN-α-3 polypeptides, at least 0.8 mg of polypeptide type IFN-α-1, 1.15mg hybrid interferon ,, B ₁ B ₂ D ₃ D ₄ ", 1.25mg hybrid interferon ,, B ₁ D ₂ B ₃ B ₄ ", 1.25mg hybrid interferon ,, B ₁ D ₂ D ₃ D ₄ ", 0.44 mg hybrid interferon ,, B ₁ B ₂ B ₃ D ₄ " 1.6 mg hybrid interferon ,, B ₁ D ₂ B ₃ D ₄ ", 1.6 mg hybrid interferon ,, B ₁ D ₂ D ₃ B ₄ ", 1.15 mg hybrid interferon ,, B ₁ B ₂ D ₃ D ₄ " and 1.25 mg hybrid interferon ,, D ₁ D ₂ B ₃ B ₄ ". The immunoadsorbent gel can be used for up to 50 separations without significant loss of capacity. IFN-a-2, IFN-a-3, IFN-a-1 recombinant polypeptides and hybrid interferons purified by this method are homogeneous on SDS-PAGE.

Example 23: Immunodot analysis

The immunodot analysis is according to R. Hawkes, u. a., anal. Biochem. 119, 142 (1982). Natural or recombinant interferons-containing probes are spotted on nitrocellulose leaves at various concentrations. The spots are air-dried and the remaining binding capacity of the nitrocellulose is blocked by incubation in a Tris buffer (0.15 M NaCl, 0.01% Tris-HCl, pH 7.6) containing 10% horse serum. The nitrocellulose strips are then incubated with a solution of monoclonal antibody 144 BS (23 μg / ml) and similarly prepared strips are probed either with a solution of monoclonal antibody 2K2 (30 μg / ml), a solution of a normal mouse immunoglobulin (Dilution 1: 100) as a negative control, or a solution of polyclonal anti-human interferon a mouse serum (Enzo, dilution 1: 1000) as a positive control. After washing, all strips are treated with rabbit anti-mouse immunoglobulin labeled with peroxidase (Nordic, 1: 400 dilution) and the bound second antibody is developed with a peroxidase substrate, 4-chloro-1-naphthol.

The monoclonal antibody stains IFN-aD type polypeptides (such as IFN-a-3) at concentrations of only 0.4 ng per spot and IFN-aB type polypeptides (such as IFN-a-2) at 10 ng spots, while MAb 2K2 recognizes only polypeptides of the IFN-aD type (4 ng per spot).

Example 24: Kegelohen radioimmuno-tandem analysis

a) Radiolabelling of monoclonal antibody 144 BS with ¹²⁵ J: 0.1 mg monoclonal antibody 144 BS is treated with ¹²⁵ J sodium iodide (1 mC) and chloramine T according to a standard method of FC Greenwood, et al., Biochem. J. 89,114 (1963). The reaction product is purified with an ion exchange column Bio-Rad® AG 1 × 8 and the activity is standardized to 4 × 10 ⁵ cpm / ml by dilution with PBS.

The iodination of monoclonal antibody 2K2 is also done. Monoclonal antibodies YOK and NK2 are commercially available in radiolabelled form from Celltech.

b) Coupling of monoclonal antibody 144 BS to macrobeads

Polystyrene beads (diameter 6.3 mm, Spherotech AG, Zurich) for 16 hours at 4 ⁰ C by gentle rocking in a solution of 1 mg / ml MAb 144 BS in a coupling buffer of PBS containing 5 mM EDTA and 0.1% NaN ₃ (pH 8), incubated in a ratio of 20 beads per 10 ml of solution. The coated beads are then transferred to a blocking buffer of PBS containing 10% horse serum and 0.1% NaN ₃ and held therein at 4 ° C until use, for at least 24 hours

 The coupling of the other MAb is done in the same way.

c) Analysis method: Plaster tubes (Falcon, 5 ml) are coated overnight with 0.5 ml blocking buffer of PBS containing 10% horse serum and 0.1% NaN ₃ . The buffer is removed by aspiration and 0.2 ml of an IFN-containing test or standard solution in blocking buffer is added directly below in the tube. A MAb-coupled and blocked bead of Example 24b is briefly dried on a clean cloth, gently transferred to the tube containing the IFN solution without foaming. The bead is incubated for 4 to 6 hours at room temperature with gentle rocking. The IFN solution is removed by aspiration and each bead is washed three times with 2 ml of blocking buffer. The bead is then incubated in 0.2 ml of a second ¹²⁵ I-labeled antibody (Example 24a) corresponding to 80 x 10 ³ cpm for 16 hours at 4 ⁰ C, washed three times with PBS and transferred to a new tube for counting.

Table 6 contains the results for standard solutions of IFN-a-3 and IFN-a-2 polypeptides obtained with various combinations of solid phase (bead-coupled) first antibody and ^12S- labeled second antibody.

Table 6: Selection of a suitable combination of monoclonal antibody for IFN-a-2 and IFN-a-3 polypeptide analysis

solid phase radiolabeled cpm per 10 ³ IU / ml found antibody second antibody IFN-a-3 IFN-a-2 by 144BS 2K2 113 105 144BS 144BS 103 58 144BS YOK 16,793 313 144BS NK2 78 3 733. 2K2 144BS 52 2 polyclonal 144BS 1 572 546

Polyclonal: bead-coupled polyclonal sheep anti-IFN-a antibody purchased from Celltech.

The best results are achieved with a combination of MAb 144 BS with MAb YOK for the detection of IFN-a-3 and with the combination of MAb 144 BS with MAb NK2 for the detection of IFN-a-2.

Figure 4 shows the result of quantitative titration experiments with the combination of MAb 144 BS coupled to the solid phase and MAb NK2 in radiolabelled form. The analysis results in linear results of 1 to 5000 IU / ml (2 pg / ml to 10 ng / ml) of polypeptides of the IFN-a-B type. Differences in the titration curves of IFN-a-2 and "standard B" type polypeptide (from Celltech) are due to deviations in the measurements of the two IFN preparations in bioanalysis.

Similar results are obtained in quantitative experiments to measure polypeptides of the IFN-a-D type with the combination of MAb 144 BS coupled to the solid phase and MAb YOK in radiolabelled form. The analysis reliably detects up to 1 IU / ml (2 pg / ml) of IFN-a-D-type polypeptides. The combination of MAb 144 BS and MAb NK2 coupled to the solid phase can also be used for the determination of polypeptides of the IFN-a-F type.

d) Abbreviated analysis method (simultaneous analysis):

Using the general method described in Example 24c), the 'MAb-coupled bead is incubated for 3 hours at room temperature with the test solution (0.1 ml) and the radiolabelled second antibody (0.1 ml), eliminating the intervening washes ,

Figure 5 shows that the abbreviated simultaneous analysis can be reliably applied to the rapid screening of IFN-aB type polypeptide samples (such as IFN-a-2) containing 150 IU / ml or more, but it is considerably less sensitive than the analysis of Example 24c).

bg = background

e) Test equipment for bead radio-immuno-tandem analysis:

Test equipment for the analysis described in Example 24c) contains: Plaster Tube, Falcon 5 ml

Polystyrene beads, 6.3 mm, coated with monoclonal antibody 144 BS in PBS containing 10% horse serum and 0.1% NaN ₃

²⁵ J labeled monoclonal antibody YOK with a specific activity of 4 x 10 ⁵ cpm / ml ¹²⁵ I-labeled monoclonal antibody NK2 with a specific activity of 4 x 10 ⁵ cpm / ml standard solution IFN-a-1 containing 10 ⁴ IU / ml Standard solution IFN-a-2 containing 10 * IU / ml contains standard solution IFN-a-3 containing 10 ⁴ IU / ml 'PBS containing 10% horse serum and 0.1% NaN ₃

Example 25: Enzyme Immunoanalysis (EUSA)

a) Labeling of monoclonal antibody 144 BS with alkaline phosphatase:

1.4 mg MAb 144 BS in 1.4 ml PBS are treated with a solution containing 5 mg alkaline phosphatase (SIGMA P6774, type VII-T) for 2 hours according to the standard procedure of Voller, et al. a., Bull. World Health Organization. 53, 55 (1976) using glutaraldehyde (0.2% v / v) coupled. The conjugate is transferred to 5 ml Tris buffer 0.05 M, pH 8.0, containing 1 mmol MgCl ₂ , 1% BSA and 0.02% NaN ₃ . The solution is stored in the dark at 4 ° C.

b) Method of analysis: Polypropylene microtiter plates (Dynatech Labs Inc.) are incubated in a period of 2 hours at 37 ° C and overnight at 4 ° C with 150 μM of a solution of the monoclonal antibody NK2 (Celltech, 10 μg / ml) a buffer, pH 8.6, (carbonate-buffered 0.9% saline containing 0.02% sodium azide). The plates are washed 5 times with PBS, and remaining protein responsive sites are incubated for 1 hour at 37 ° C with 250 μM buffer pH 7.4 (0.2% gelatin and 0.2% NaN ₃ in PBS ) saturated. The plates coated in this manner can be stored at 4 ° C for several days in this buffer.

50 μΙ of a dilution series of a test solution or a standard solution containing IFN-ct subtypes, 50 μΙ buffer, pH 7.4, and 50 μΙ of a solution of the phosphatase-labeled antibody 144 BS (Example 25a) containing 1: 100 with buffer pH 7.4 was mixed and incubated in the wells of the microtiter plates for 2 hours at 37 ⁰ C and for 30 minutes at 4 ° C. The plates are washed five times with PBS, then incubated for 30 minutes at 37 ° C with 150 μΙ of a solution of p-nitrophenyl phosphate (1 mg / ml in 10% diethanolamine buffer, 0.5 mM MgCl ₂ , pH 9.8). By measuring the optical density at 405 nm, the amount of p-nitrophenol released is determined, which is proportional to the amount of bound enzyme phosphatase and thus proportional to the amount of IFN-a subtype in the test solution.

This analysis can be used to determine the amount of IFN-a-B type polypeptides (such as IFN-a-2) or IFN-o-F. To determine polypeptides of the type IFN-cc-D (such as IFN-a-3), the microtiter plate is coated with the monoclonal antibody YOK (Celltech) instead of NK2. Similar results can be obtained when the microtiter plates are coated with MAb 144 BS and the phosphatase-coupled MAbs NK2 and YOK are used as second antibodies.

c) Test Equipment for ELISA: A test kit for the analysis described in Example 25b) contains: polypropylene microtiter plates,

20 ml monoclonal antibody NK2 (10 μg / ml) in carbonate buffered saline (0.9% NaCl, 0.42% NaHCO ₃ , 0.0072% Na ₂ CO ₃ , 0.02% NaN ₃ )

1 ml of alkaline phosphatase-coupled MAb 144 BS (Example 10.1, 0.3 mg antibody per ml) in Tris buffer (0.05 M, 1 mM

MgCl ₂ , 1% BSA, 0.02% NaN ₃ , pH 8.0)

2 ml standard IFN-a-1 solution containing 10 ⁴ IU / ml 2 ml standard IFN-a-2 solution containing 10 "IU / ml 2 ml standard IFN-a-3 solution containing 10" IU / ml

300 ml of PBS

300 ml buffer pH 7.4 (0.2 gelatin and 0.2% NaN ₃ in PBS) 50 ml p-nitrophenyl phosphate (1 mg / ml) in diethanolamine buffer (10%, 0.5 mM MgCl ₂ , 0.02% NaN ₃ , set with HCI on one

pH value of 8.9) calibration curve color intensity scale.

Example 26: Purification of Hybrid Interferons Using Precipitation and Chromatography Methods

1 l interferon ,, B ₁ D ₂ D ₃ D ₄ "purified by centrifugation (see Example 13 to 15) is using

2 M HCl acidified until the solutions reach a pH of 2.2. The solution is stirred for 1 h at 4 ° C and the precipitated protein is separated by centrifugation (GSA Sorvall rotor at 12000 rpm, for 20 min at 4 ⁰ C). The pellet is discarded and the supernatant liquid is brought to 30% saturation with solid ammonium sulfate. This solution is stirred for 1 h at 4 ⁰ C and then centrifuged. The supernatant containing the interferon is then dialyzed against 0.1 M NH ₁ HCO ₃ at pH 8.2. An interferon activity of about 10 ⁸ IU / ml is found.

A column containing 50 ml of gelling Sepharose 6B (Pharmacia) supplemented with Cu ²⁺ ions according to the manufacturer's instructions is prepared using 250 ml of 0.05 M sodium acetate, pH 7.0, and 0.5 M NaCl equilibrated. 200 ml of dialyzed solution (see above) are added to the column at 4 ° C. The column is washed with 150 ml of equilibration buffer. Elution of bound interferon is performed using a linear gradient of

250 ml of 0.05 M sodium acetate, pH 7.0, and 0.5 M NaCl

250 ml of 0.05 M sodium acetate, pH 2.8, and 0.5 M NaCl

and then through a wash of 150 ml of 0.05 M sodium acetate, pH 2.8, and 0.5 M NaCl.

The active interferon fractions are pooled and dialyzed against 0.05 M Tris-HCl buffer, pH 8.0. The interferon activity in the dialyzed solution is approximately 10 ⁸ IU / ml. The maximum capacity of the column is about 75 mg interferon ,, B ₁ D ₂ D ₃ D ₄ ".

A column containing 50 ml of Q-Sepharose (Pharmacia) ion exchanger is equilibrated using 250 ml of 0.05 M Tris-HCl buffer, pH 8.0. 100 ml of the above dialyzed, interferon-containing solution are applied to the column at room temperature. The column is then washed with 50 ml of equilibration buffer. Bound interferon is eluted using a linear gradient of 0 to 0.7 M NaCl in 200 ml of Tris-HCl, pH 8.0.

The pure interferon elutes at about 0.3 M NaCl. The active fractions show a single band with an apparent molecular mass of 19,000 when analyzed on SDS-polyacrylamide gels. The maximum capacity of the anion exchange column is about 50 mg of hybrid interferon ,, BiD ₂ D ₃ D ₄ ".

In an analogous manner, the hybrid interferons "BiD ₂ B ₃ B ₄ ", "B, D ₂ B ₃ D ₄ ", "B, D ₂ D ₃ B ₄ ", "B ₁ B ₂ B ₃ D ₄ "and" B ₁ B ₂ D ₃ B ₄ "are cleaned.

Example 27 Lyophilization of Hybrid Interferon ,, B ₁ D ₂ D ₃ D ₄ "Interfero ,, BiD ₂ D ₃ D ₄ " by either immunoaffinity chromatography using monoclonal antibody 144 BS or by conventional chromatographic methods (see Example 26) was dialyzed against 0.5 M NH ₄ HCO ₃ and then lyophilized. Small volumes of water are added twice to the lyophilized sample to completely remove NH ₄ HCO ₃ . The solution is then lyophilized again. The reconstitution of the interferon "BiD ₂ D ₃ D ₄ " solution is carried out as follows: 0.5 M NH ₄ HCO ₃ , pH 8.2, is added to the lyophilized sample to give an interferon concentration of about 0.1 mg / ml The reconstituted interferon has a specific antiviral activity of 2 × 10 ⁸ IU / mg, this activity being identical to the corresponding value before lyophilization.

Example 28: Physico-chemical characterization of hybrid interferons The hybrid interferons according to the invention and the parent interferons are subjected to polyacrylamide SDS gel electrophoresis according to the method described by Laemmli (Nature 227, 680 [1970]). The apparent molecular weights of the hybrid interferons expressed in kilo daltons (kD) are summarized in Table 7. Table 7 also contains the isoelectric points (pI) of the hybrid interferons as determined using LKB polyacrylamide gels containing Ampholine®.

Table 7: Obvious molecular weight and isoelectric points of hybrid interferons

α-2 27,000 α-3 20,000 BiB ₂ D ₃ D ₄ 27,000 D ₁ D ₂ B ₃ B ₄ 20,000 B1B2B3D4 27,000 B, B ₂ D ₃ B ₄ 26,000 B ₁ D ₂ D ₃ D ₄ 19,000 B ₁ D ₂ B ₃ D ₄ 20,000 BiD ₂ D ₃ B ₄ 20,000 B ₁ D ₂ B ₃ B ₄ 20,000

IFN offens. molecular weight pl

5.2

5.2

5-6 (scattered)

5.0

5.2

5-6 (scattered)

5.4

5.6

5.5

5.4

The amino acid sequence of the hybrid interferons of the invention is determined using an Applied Biosystems 470A protein sequencer or using an 890C Beckman sequencer. In both cases, automated Edman degradation is done. The found structure agrees perfectly with the assumed one. The N-terminal amino acid is cysteine.

Example 29: Influence of hybrid interferons on the degree of (2'-5 ') oligoisoadenylate synthetase activity of Daudi cells Daudi cells are incubated with 2 x 10 ⁵ cells / ml in RPMI 1640 medium with 10% fetal calf serum, the indicated concentrations (see Table 8) of the hybrid interferons ,, B ₁ B ₂ B ₃ D ₄ "," B ₁ D ₂ B ₃ B ₄ "," BiD ₂ D ₃ D ₄ "and" B ₁ B ₂ D ₃ B ₄ ", bred. After a treatment time of 24 hours, 5 × 10 ⁶ cells per sample are centrifuged (9,000 rpm) and again in 500 μl cold lysis buffer (20 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid buffer, pH 7.5 , 5 mM MgCl ₂ , 120 mM KCl, 7 mM dithiothreitol, 10% glycerol, 0.5% Nonidet P40) and incubated on ice for 2 min. The samples are then centrifuged (9000 rpm, 8 min, 4 ° C) and the supernatant fluid is recovered and analyzed for (2'-5 ') oligoisoadenylate synthetase activity as follows:

The supernatant is mixed with 50 μM poly (rl). (RC) agarose (P.L. Biochemicals) for 15 min at 30 ° C, and the non-adsorbed material is removed by centrifugation. The poly (rl). (RC) -adsorbierte material (2.5 mM (X incubated ³² P) ATP (400 Ci / mmol Amersham International) for 20 h at 3O ⁰ C, treated with bacterial alkaline phosphatase (Sigma) and then from a column of acidic alumina (Sigma) (300 μΙ) with 3.0 ml of 1 M glycine-HCl buffer, pH 2.0, as described (Merlin, et al., Analyt. Biochem. The results in Table 8 are reported as a ... fold increase in (2'-5 ') oligoisoadenylate synthetase activity in interferon-treated cells relative to the level of enzyme activity of untreated control cells (mean Activity 51.6 ± 8.7 pmol / h / 10 ⁵ cells).

Table 8

... -fold increase in (2'-5 ') (A)

Synthetase activity

[pMol ATP added / h / 10 ⁵ cells]

IFN concentration 1 10 100 1 000 [Units / ml] IFN a-2 ("B") 3.6 4.0 5.7 5.6 α-3 ("D") 2.2 3.2 3.2 3.9 ,, B ₁ B ₂ D ₃ D ₄ " 3.4 3.2 4.6 4.0 ,, D ₁ D ₂ B ₃ B ₄ " 2.6 2.1 4.8 4.3 ,, B ₁ B ₂ B ₃ D ₄ " 9.4 8.4 10.0 8.5 ,, B ₁ D ₂ B ₃ B ₄ " 5.6 5.7 7.8 11.4 ,, B ₁ D ₂ D ₃ D ₄ " 5.8 8.9 12.1 17.5 ,, B ₁ B ₂ D ₃ B ₄ " 5.6 7.0 9.1 9.6

Example 30: Pharmaceutical preparation (parenteral administration)

2 mg hybrid interferon ,, B ₁ D ₂ D ₃ D ₄ "with a specific activity of 1.3 × 10 ⁸ units / mg in human WILSH cells are dissolved in 30 ml of 5 N human serum albumin Solution is passed through a bacteria-proof filter and the filtered solution is dispensed under aseptic conditions into 100 vials, each containing 2.6 x 10 ⁶ units of pure hybrid interferon The vials suitable for parenteral administration are preferably refrigerated, e.g. Example at -20 ° C, stored.

In the same way, vials containing 5.2 × 10 ⁶ or 1.04 × 10 ⁷ units can be prepared by using 4 or 8 mg of hybrid interferon, respectively.

In an analogous manner, hybrid interferon, "B ₁ D ₂ B ₃ B ₄ ", "B ₁ B ₂ D ₃ B ₄ ", "B ₁ D ₂ B ₃ D ₄ ", "B ₁ D ₂ D ₃ B ₄ "or" B ₁ B ₂ B ₃ D ₄ "(specific activities 1.28 × 10 ⁸ , 2 × 10 ⁸ , 7.7 × 10 ⁷ , 3.8 × 10 ⁷ or 8 × 10 ⁷ ) containing vials are prepared.

Drawings:

Figure 1 Comparison of the amino acid sequences of LylFN-a-2 and LylFN-a-3

I ASP LEU PER GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU 16 a-2 CYS GLU ASP ILE a-3 MET LEU ALA GLN MET ARG ARG ILE SER PER PHE SER CYS LEU LYS 32 a-2 LEU SER SER mead ASP a-3 HIS ASP PHE GLU PHE PER GLN GLU GLU PHE ASP ASP LYS GLN 48 a-2 ARG GLY GLY ASN PHE a-3 60 LYS ALA GLN ALA ILE SER VAL LEU HIS GLU MET ILE GLN GLN 64 a-2 GLN PER LEU THR '· I a-3 ILe ' ASN LEU PHE SER THR LYS ASP SER SER ALA ALA LEU ASP GLU 80 ": a-2 PHE THR TRP THR. a-3 92 ^{> ASP} -. I ι LEU ASP GLU PHE TYR ILE GLU LEU ASP GLN GLN LEU ASN ASP 96: I ^! a-2 LEU LYS CYS THR TYR LEU ^! I j a-3 SER CYS VAL MET GLN GLU VAL GLY VAL ILE GLU SER PER LEU 112 a-2 GLU ALA GLU ARG GLY THR MET a-3 GLU ASP SER ILE LEU ALA VAL ARG LYS TYR PHE GLN ARG ILE 128 a-2 ' TYR ALA LYS ARG THR a-3 ANS TYR LEU THR GLU LYS LYS TYR SER SER CYS ALA TRP GLU VAL 144 a-2 LEU PER VAL a-3 150 ALA GLU ILE MET ARG SER PHE SER LEU SER ILE ASN LEU GLN 160 a-2 ARG LEU THR LYS a-3 166 GLU LEU LYS SER LYS GLU a-2 ARG ARG ARG a-3

Fig. 2: Nucleotide sequence of the coding regions of LylFN-a-2 and LylFN-a-3 {coding triplets are numbered) a-2 ... GAG ACAATAACCCTG ATAAATG CTTCAATAATATG AAAAAG G AAG AGTAATG

a-3 ... GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAATG

1 Sau3A 16

a-2 TGT GAT CTG CCT CAG ACT CAC AGC CTG GGT AAC AGG AGG GCC TTG ATA 'a-3 TGT GAT CTC CCT GAG ACC CAC AGC CTG GAT AAC AGG AGG ACC TTG ATG

32

a-2 CTC CTG GCA CAA ATG CGA AGA ATC TCT CCT TTC TCC TGC CTG AAG GAC a-3 CTC CTG GCA CAA ATG AGC AGA ATC TCT CCT TCC TCC TGT CTG ATG GAC

48

A-2 AGA CAT GAC TTT GAA TTC CCC CAG GAG GAG TTT GAT GAT AAA CAG TTC A-3 AGA CAT GAC TTT GGA TTT CCC CAG GAG GAG TTT GAT GGC AAC CAG TTC

Sau3A 64

a-2 CAG AAG GCT CAA GCC ATC TCT GTC CTC CAT GAG ATG ATC CAG CAG ACC a-3 CAG AAG GCT CCA GCC ATC TCT GTC CTC CAT GAG CTG ATC CAG CAG ATC

80

a-2 TTC AAC CTC TTC AGC ACA AAG GAC TCA GCT GCT TTG GAT GAG ACC a-3 TTC AAC CTC TTT ACC ACA AAA GAT TCA TCT GCT GCT TGG GAT GAG GAC

Pvull 96

a-2 CTT CTA GAT GAA TTC TAC ATC GAA CTT GAC CAG CAG CTG AAT GAC CTG a-3 CTC CTA GAC AAA TTC TGC ACC GAA CTC TAC CAG CAG CTG AAT GAC TTG

112

a-2 GAG TCC TGT GTG ATG CAG GAA GTG GGG GTG ATA GAG TCT CCC CTG ATG -3 GAA GCC TGT GTG ATG CAG GAG GAG AGG GTG GGA GAA ACT CCC CTG ATG

128

a-2 TAC GAG GAC TCC ATC CTG GCT GTG AGG AAA TAC TTC CAA AGA ATC ACT a-3 AAT GCG. GAC TCC ATC TTG GCT GTG AAG AAA TAC TTC CGA AGA ATC ACT

144

a-2 CTA TAT CTG ACA GAG AAG AAA TAC AGC TCT TGT GCC TGG GAG GTT GTC a-3 CTC TAT CTG ACA GAG AAG AAA TAC AGC CCT TGT GCC TGG GAG GTT GTC

160

a-2 AGA GCA GAA ATC ATG AGA TCC TTC TCT TTA TCA ATC AAC TTG CAA AAA a-3 AGA GCA GAA ATC ATG AGA TCC CTC TCT TTA TCA ACA AAC TTG CAA GAA a-2 AGA TTG AAG AGT AAG GAA TGA GACCTG GTACAACACG GAAATGATTCTTATAGACT a-3 AGA TTA AGG AGG AAG GAA TAA CACCTGGTCCAACATGAAACAATTCTTATTGACTC a-2 a-3 AATACAGCAGCTCACACTTCGACAAGTTGTGCTCTTTCAAAGACCCTTGTTTCTGCCAAAACC ATATACCAGGTCACGCTTTCATGAATTCTGCCATTTCAAAGACTCTCACTTCTGCTATAACTA a-2 a-3 ATGCTATGmTGAATCAAATGTGTCAAGTGTTTTCAGGAGTGTTAAGCAACATCCTGTT TGACCATGCTGATAAACTGATTTATCTATTTAAATATTTATTTAGCTATTCATAAGATTT

Pvull Sau3A

a-2 a-3 CAGCTGTATGGGCACTAGTCCCTTACAGATGACCATGCTGATGGATCTATTCATCTATTTATT AAATTAI I I I IGTTCATATAACATCATGTGCATCTTTACACTGTGGTTAGTGTAATAAAACAT a-2 a-3 TAAATCTTTATTTAGTTAACTATCTATAGGGCTTAAATTAGTTTTGTTCATATTATATTATGT GTTCCTTATATTTACTCAAATTCATTATTTT ...

a-2 GAACTTTTACATTGTGAATTGTGTAACAAAACATGTTCTnATATTTATTATTTTGCCTTGT a-2 TTATTAAATTTTACTATAG ...

FIG. 3: Restriction maps of HindIII-Pst small fragments of the plasmids CG-pBR (AP) / LylFN-a-2 and CG-pBR (AP) / LylFN-a-3 The white bars (IFN-a-3) and the black bars (IFN-a-2) delimit the coding regions of the mature IFNe. Among them is the IFN polypeptide (166 amino acids). In brackets, the number of amino acid residues that distinguish IFNe-a-2 and a-3 in each section is given.

Legend of restriction sites:

FIG. 4: 1 standard IFN-a-B-2 IFN type B concentration (IU / ml)

5: IFN-a-2 concentration (IU / ml)

Claims (8)

A method of producing a hybrid interferon polypeptide having an amino acid sequence composed of two to four subsequences corresponding in amino acid identity and number of subsequences to human lymphoblastoid interferons LylFN-a-2 and LylFN-a-3, wherein the hybrid interferon polypeptide is selected from the group consisting of a polypeptide having an amino acid sequence consisting of amino acids 1 to 150 of LylFN-a-2 and amino acids 151 to 166 of LylFN-a-3, a polypeptide having one of Amino acids 1 to 92 of LylFN-a-2, amino acids 93 to 150 of LylFN-a-3 and amino acids 151 to 166 of LylFN-a-2 existing amino acid sequence, and a polypeptide having one of the amino acids 1 to 60 of LylFN -a-2, amino acids 61 to 92 of LylFN-a-3, amino acids 93 to 150 of LylFN-a-2 or LylFN-a-3 and amino acids 151 to 166 of LylFN-a-2 or LylFN-a -3 existing amino acid sequence, characterized by that host cells transformed with a hybrid vector containing a DNA sequence coding for the hybrid interferon polypeptide are grown and the hybrid interferon is isolated. 2. Method according to item 1, characterized in that the hybrid interferon polypeptide is selected from the group consisting of hybrid interferon polypeptide ,, B ₁ B ₂ B ₃ D ₄ "having the formula CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU I LE LEU LEU ALA GLN MET ARG ILE SER PRO PHE SER CYS LEU LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU METAL GLN GLN THR PHE ASN LEU PHE SER THR LYS ASP SER SER ALA ALA LEU ASP GLU THR LEU LEU ASP GLU PHE TYR ILE GLU LEU ASP GLN GLN LEU ASN ASP LEU GLU SER CYS VAL MET GLN GLU VAL GLY VAL ILE GLU SER PRO LEU METTYR GLU ASP SER ILE LEU ALA VAL ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU THR GLU LYS LYS TYR SER SER CYS ALA TRP GLU VAL VAL ARGALA GLU ILE MET ARG SER LEU SER LEU SER THR ASN LEU GLN GLU ARG LEU ARG ARG LYS GLU, Hybrid interferon polypeptide ,, B ₁ B ₂ D ₃ B ₄ "of the formula CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU LE LEU ALU GLN MET ARG ARG ILE SER PRO PHE SER CYS LEU LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU METAL GLN GLN THR PHE ASN LEU PHE SER THR LYS ASP SER SER ALA ALA LEU ASP GLU THR LEU LEU ASP GLU PHE TYR ILE GLU LEU ASP GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL MET GLN GLU GLU ARG VAL GLY GLU THR PRO LEU MET ASN ALA ASP SER ILE LEU ALA VAL LYS LYS TYR PHE ARG ARG ILE THR LEU TYR LEU THR GLU LYS LYS TYR SER PRO CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER PHE SER LEU SER ILE ASN LEU GLN LYS ARG LEU LYS SER LYS GLU, Hybrid interferon polypeptide ,, B ₁ D ₂ B ₃ B ₄ "having the formula CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU LE LEU ALU GLN MET ARG ARG ILE SER PRO PHE SER CYS LEU LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU METAL GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR GLN GLN LEU ASN ASP LEU GLU SER CYS VAL MET GLN GLU VAL GLY VALJLE GLU SER PRO LEU METTYR GLU ASP SER ILE LEU ALAVAL "ARG LYS TYR> HE ~ GLN ARG ILE 'THRLEU'TYR LEUTHR GLU LYS LYS" TYR SER SER CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER LEU SER LEU SER THR ASN LEU GLN GLU ARG LEU ARG ARG LYS GLU, Hybrid interferon polypeptide ,, B ₁ D ₂ D ₃ B ₄ "having the formula CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU LE LEU ALU GLN MET ARG ARG ILE SER PRO PHE SER CYS LEU LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU METAL GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR GtN GLN LEU ASN ASP LEU GLU ALA CYS VAL MET GLN GLU GLU ARG VAL GLY GLU THR PRO LEU METASNALA ASP SER ILE LEU ALA VAL LYS LYS TYR PHE ARG ARG ILE THR LEU TYR LEU THR GLU LYS LYS TYR SER PRO CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER PHE SER LEU SER ILE ASN LEU GLN LYS ARG LEU LYS SER LYS GLU, Hybrid interferon polypeptide ,, B ₁ D ₂ D ₃ D ₄ "having the formula CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU LE LEU ALU GLN MET ARG ARG ILE SER PRO PHE SER CYS LEU LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU METAL GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL MET GLN GLU GLU ARG VAL GLY GLU THR PRO LEU MET ASN ALA ASP SER ILE LEU ALA VAL LYS LYS TYR PHE ARG ARG ILE THR LEU TYR LEU THR GLU LYS LYS TYR SER PRO CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER LEU SER LEU SER THR ASN LEU GLN GLU ARG LEU ARG ARG LYS GLU, Hybrid interferon polypeptide ,, B ₁ D ₂ B ₃ B ₄ "having the formula CYS ASP LEU PRO GLN THR HIS SER LEU GLY ASN ARG ARG ALA LEU LE LEU ALU GLN MET ARG ARG ILE SER PRO PHE SER CYS LEU LYS ASP ARG HIS ASP PHE GLU PHE PRO GLN GLU GLU PHE ASP ASP LYS GLN PHE GLN LYS ALA GLN ALA ILE SER VAL LEU HIS GLU METAL GLN GLN ILE PHE ASN LEU PHE THR THR LYS ASP SER SER ALA ALA TRP ASP GLU ASP LEU LEU ASP LYS PHE CYS THR GLU LEU TYR GLN GLN LEU ASN ASP LEU GLU SER CYS VAL MET GLN GLU VAL GLY VAL ILE GLU SER PRO LEU METTYR GLU ASP SER ILE LEU ALA VAL ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU THR GLU LYS LYS TYR SER SER CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER PHE SER LEU SER ILE ASN LEU GLN LYS ARG LEU LYS SER LYS GLU. > 3. The method according to item 1 for the preparation of hybrid interferon ,, B ₁ B ₂ B ₃ D ₄ ", characterized in that the hybrid vector contains a coding for the hybrid interferon DNA sequence. 4. The method according to item 1 for the preparation of hybrid interferon ,, B ₁ B ₂ D ₃ B ₄ ", characterized in that the hybrid vector contains a coding for the hybrid interferon DNA sequence. 5. The method according to item 1 for the preparation of hybrid interferon ,, B ₁ D ₂ B ₃ D ₄ ", characterized in that the hybrid vector contains a coding for the hybrid interferon DNA sequence. 6. The method according to item 1 for the preparation of hybrid interferon ,, B ₁ D ₂ D ₃ B ₄ ", characterized in that the hybrid vector contains a coding for the hybrid interferon DNA sequence. 7. The method according to item 1 for the preparation of hybrid interferon ,, B ₁ D ₂ D ₃ D ₄ ", characterized in that the hybrid vector contains a coding for the hybrid interferon DNA sequence. 8. The method according to item 1 for the preparation of hybrid interferon ,, B ₁ D ₂ B ₃ B ₄ ", characterized in that the hybrid vector contains a coding for the hybrid interferon DNA sequence.