BG60510B2 - STRUCTURAL GENES, PLASMIDS AND TRANSFORMED CELLS FOR OBTAINING CYSTEINE-POOR MUTEINS OF BIOLOGICALLY ACTIVE PROTEINS - Google Patents

Abstract

Muteins are used in the diagnosis and treatment of bacterial, viral, parasitic, protozoal and fungal infections, to restore immune functions and in the development of diagnostic tests. They are produced by bacterial expression of mutant genes that code for muteins synthesized from the parental protein genes by oligonucleotide-directed mutagenesis. In these muteins of biologically active proteins such as ifn and il-2, non-essential for biological activity cysteine residues are omitted or replaced with another amino acid to eliminate possible sites for the formation of unwanted intermolecular and intramolecular bonds. 17 claims, 17 figures

Description

Technical field.

The invention relates to recombinant DNA (DNA) technologies, in particular mutationally altered biologically active proteins, which differ from the parent analogs in one or more substitutions / deletions of cysteine residues.

Prior to that state of the art.

Biologically active proteins that are microbially produced, ie. obtained by recombinant DNA (rDNA) technologies may contain cysteine residues that are not realized for their activity but are free to form unwanted intermolecular and intramolecular bonds. Such a protein is the microbially derived human β-interferon (IFN-β). Upon preparation of IFN-β by rDNA methods, it has been observed that dimers and oligomers of microbially produced IFN-β are formed in E.coli extracts containing high concentrations of IFN-β. The formation of multimers makes IFN-β purification and separation very complex and time-consuming and necessitates the inclusion of several additional purification and isolation operations, such as protein reduction during purification and oxidation to restore the original conformation. thus increasing the possibility of irregular disulfide bonds. In addition, microbially derived IFN-β was found to be. exhibits persistent low specific activity, possibly due to the formation of multimers or random intramolecular bridges. Therefore, it is desirable to be able to alter microbially derived biologically active proteins such as IFN-β in a way that does not interfere with their activity undesirably but diminishes or eliminates their ability to form intermolecular cross-links or intramolecular bonds that cause the protein to acquire an unwanted tertiary structure (i.e., a conformation that reduces the activity of the protein).

The present invention relates to the preparation by targeted mutagenesis of mutationally altered biologically active proteins (such proteins are called "muteins." Glossary of Genetics and Cytogenetics, 4th ed, 381. Springer-Vcrlag (1976)), which retain the activity of parental analogs but do not have the ability to form intermolecular bonds or unwanted intramolecular disulfide bonds. In this regard, Scpard et al .. Nature, (1981) 294: 563-565 describes an IFNβ mutein. in which the cysteine at position 141 of the amino acid sequence (there are three cysteines in natural human IFN-β at positions 17, 31 and 141, Gene (1980). 10: 11-15 and Nature (1980). 285: 542-547) is substituted with tyrosine. The mutein was obtained by bacterial expression of a hybrid gene derived from a partial IFN-β cDNA clone having a G -> A transition into nucleotide 485 of the IFN-β gene. Mutein lacks the biological activity of natural IFN-β, leading to the conclusion that substituted cysteine is essential for the activity.

Methods for targeted mutagenesis are well known and are described in Lather and Lecoq, Genetic Engineering, Academic Press, 1983, 31-50. Oligonucleotide-directed mutagenesis has been specifically addressed by Smith and Gillam, Genetic Engineering: Principles and Methods, Plenum Press (1981) 3: 1-32.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a synthetic mutein of a biologically active protein, which has at least one pistin residue that is free to form a disulfide bond and is not essential for biological activity, with at least one of said cysteine residues being a mutein. removed or substituted with another amino acid.

Another aspect of the invention relates to synthetic structural genes having DNA sequences "project genes" that are specifically intended to encode the synthetic mutein described. This section also includes expression vectors that include the structures and project genes described, host cells, or rat eggs transformed with such vectors. 'act'. ' and methods for preparing a synthetic mutein by culturing the transformants described or progeny thereof and recovering the mutein from the culture. In the case of muteins having therapeutic use, therapeutic agents containing therapeutically effective amounts of muteins, and therapeutic methods are another aspect of the invention.

The invention also relates to a method of preventing the formation of an unwanted disulfide bond from a protein having one or more cysteine residues free to form such a link by mutational alteration of the protein by deletion of the cysteine residue (residues) or substitution with other amino acids.

The invention also includes a method of producing the synthetic structural gene described by oligonucleotide-directed mutagenesis, comprising the following steps:

a) hybridization of single stranded DNA, representing a strand of a structural gene encoding a parent protein with a mutant oligonucleotide primer that is complementary to that portion of the strand comprising the cysteine codon to be removed or substituted, or in some cases the meaningless triplet paired with a codon, except in the case of a mismatch of that codon or, optionally, a nonsense chain, leading to a deletion of the codon or triplet that codes for the other amino acid;

c) extending the primer with DNA polymerase to form a mutant heteroduplex;

c) replication of the mutant heteroduplex.

The mutant oligonucleotide primer used in this process is also part of the invention.

The present invention relates to muteins of biologically active proteins in which cysteine residues that are immaterial to biological activity are deliberately removed or replaced by other amino acids to eliminate sites of intramolecular cross-linking or to form an intramolecolecular intramolecular molecule; mutant genes encoding for such muteins; and methods for preparing such muteins.

Which proteins can be mutationally modified according to the invention can be determined by the available information on the cysteine content of biologically active proteins, as well as the importance of these cysteine residues for the activity and tertiary structure of the protein. If there are proteins. for which no such information is available in the literature, it can be obtained by systematically altering each of the cystine residues in the protein by the methods described herein and then measuring the biological activity of the resultant muteins and determining their ability to form unwanted intermolecular or intramolecular disulfide bonds . In fact, although the IFN-β and 1L-2 muteins are described and illustrated by the invention below, it should be known that what is described herein can also be applied to any other biologically active protein containing a functionally insignificant cysteine residue , which makes it sensitive to the formation of unwanted disulfide bonds. Examples of such proteins other than IFN-β and IL-2 that can be mutationally altered according to the invention are lymphotoxins (tumor necrosis factor), colony-stimulating factor-1 and IFN-α. Candidate proteins have an odd number of cysteine residues.

For IFN-β, both the glycosylated and non-glycosylated IFNs have been described in the literature with qualitatively similar specific activities, which means that glycosyl groups do not contribute to the biological activity of IFN-β. However, bacterially produced IFN-β, which is non-glycosylated, exhibits consistently quantitatively but low specific activity from native IFN-β, which is glycosylated.

IFN-β is known to have three cysteine residues at positions 17, 31 and 141. Cysteine 141 has been identified by Shepard, et al., (See above) as essential for biological activity. For IFN-α, which contains four cysteine residues, there are two intramolecular -S-S bonds: one between cys 29 and evs 138 and the other between cys 1 and cys 98. If the homology between IFN-β and IFN-a is based , cys 141 of IFN-β should be included in the intramolecular -SS bond with cys 31, and cys 17 should remain free of intramolecular binding. By removing cys 17 or replacing it with another amino acid, it can be determined whether it is essential for biological activity and what is important for the formation of the -SS bond. If cys 17 is not essential for biological activity, the protein in which it is removed or substituted may exhibit specific activity close to that of native IFN-β and this may facilitate the isolation and purification of the protein.

Using oligonucleotide-directed metagenesis with a synthetic oligonucleotide primer complementary to the region of the IFN-β gene in the codon for cys 17 but with a single or multiple base change in that codon, a gene can be obtained that results in the substitution of cys 17 is any other selected amino acid. If a deletion is desired, the oligonucleotide primer lacks a codon for cys 17. Preferably the conversion of cys 17 into a neutral amino acid such as glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine, tryptophan, series. The most preferred substituents are serine and threonine because of their chemical analogy to cysteine. When cysteine is removed, the mature mutein is one amino acid shorter than the native parent protein or microbially produced IFN-β.

Human IL-2 is known to have three cysteine residues located at positions 58, 105 and 125 of the protein. Like IFN-β, 1L-2 is in aggregated oligomer form when isolated from bacterial cells and must be reduced with a suitable reducing agent to obtain good yield of bacterial extracts. Furthermore, the purified reduced IL-2 protein is unstable and readily re-oxidized to an oligomeric inactive form upon storage. The presence of three cysteines means that upon re-oxidation, the protein may accidentally form one of three possible intramolecular bridges, of which only one is correct, such as is present in the natural molecule. Because the disulfide structure of native IL-2 is unknown, it is possible to use the present invention to create mutations at the codons 58, 105 and 125 of the IL-2 gene and to determine which cysteine residues are required for activity and because of this is most likely to be involved in the natural formation of disulfide bridges. Similarly, cystinsinitic residues that are not essential to the activity can be modified to prevent the formation of irregular intramolecular disulfide bridges and to minimize the likelihood of intramolecular disulfide bridges by replacing the free cysteine residue.

The size of the oligonucleotide primer is determined by the requirement for stable hybridization of the primer to that region of the gene in which the mutation is to be induced, and by the limitations imposed by known oligonucleotide synthesis methods. Factors to consider in the compilation of the oligonucleotide primer The oligonucleotides used in oligonucleotide-directed mutagenesis (eg total size, size of sections bordering the mutation site) are described by Smith and Gillam (see above). In fact, the length of the oligonucleotide should be such as to optimize stable, single hybridization of the mutation site, and the 5 'and 3'-extensions of the mutation site should be of sufficient size to avoid editing the mutation by exonuclease activity of DNA polymerase. The oligonucleotides used for mutagenesis according to the invention typically contain from 12 to 24 bases, preferably from 14 to 20, and in the most preferred embodiment, from 15 to about 18. They typically comprise at least about three bases 3 'of the modified or missing codon.

The method for producing a modified IFN-β gene generally involves inducing site-specific mutagenesis in the lFN-β gene at codon 17 (TGT) using a synthetic nucleotide primer that omits the codon or alters it to encode for another amino acid . When threonine replaces cysteine and the primer is hybridized to the nonsense strand of the IFN-β gene, the preferred nucleotide primer is GCAATTTTCAGACTCAG (highlighted denotes the modified codon). When a cysteine deletion is desired, the preferred primer is AGCAATTTTCAGCAGAAGCTCCTG, which releases TGT for cys. When cysteine is replaced by serine, a 1 7-nucleotide primer is selected, GCAATTTTCAGACTCAG, which includes one AGT codon for serine. The change of TA into the first base of the cys 1 'codon results in the replacement of cysteine by serine. It should be appreciated that when deletions are induced, the correct DNA reading sequence for the expression of the desired protein must be maintained.

The primer was hybridized to single-stranded rice phage, such as Μ13, fd or ХX 174, in which a single IFN-β-rcua chain was cloned. The phage can carry the meaningful or meaningless strand of the gene. When a phage carries a nonsense strand, the primer is identical to that portion of the sense strand that contains the codon to be mutated, except if it does not coincide with that codon, resulting in codon divisions or a triplet that encodes for another amino acid. When the phage carries the sense strand, the primer is complementary to that portion of the correct strand that contains the codon to be mutated, except in the case of a mismatch with the triplet paired with that deletion codon. The conditions under which the hybridization is to be carried out are described by Smith and Gillam (see above). The temperature is generally in the range of 0 ° to 70 ° C, more often 1 ° to 50 ° C. After hybridization, the primer is expanded to phage DNA by reaction with DNA polymerase I,% DNA polymerase, reverse transcriptase or other suitable DNA polymerases. The resulting dsDHK is converted to closed circular ds DNA by treatment with DNA ligase, eg _T4 DNA legaza. DNA molecules having single-stranded regions can be destroyed by treatment with S, endotuclease.

Oligonucleotide-directed mutagenesis can similarly be used to produce a mutant IL-2 gene that encodes an eline mutein. having IL-2 activity but in which cys 125 is altered to Srp 125. The preferred oligonucleotide primer used to produce the mutant IL-2 gene when the phage carries the sense strand of the gene is GATGATG CTTCTGAGAAAAGGTAATC. This oligonucleotide has a C - G transition in the middle base of the triplet that is mated to the codon 125 of the IL2 gene.

The resulting mutant heteroduplex was then used to transform a competent microorganism or host cell. Replication of heteroduplex from the host gives progeny from both strands. After replication, the mutant gene can be isolated from mutant strands included in a suitable expression vector, and the vector can be used to transform a suitable host microorganism or cell. Preferred vectors are plasmids pBR322, pCI2 and variants thereof, synthetic vectors and the like. Suitable microorganisms are E. coli, Pseudomonas. Bacillus subtilis, Bacillus tliuringiensis, different yeast languages. Bacillus thermophilus, animal cells such as mouse, rat and Chinese hamster (CHO) ovarian cells, plant cells, animal and plant hosts, and the like. It should be noted 10 that when the selected host is transformed with the vector, suitable promoter-operator sequences for mutein expression are introduced. The hosts may be prokaryotes or sucaryotes (methods for insertion of DNA into eukaryotic cells are described in PCT Application Nos. US81 / 11239 and US81 / 00240, published September 3, 1981). Preferred hosts are E. coli and CHO cells. The muteins produced according to the invention can be glycosylated or non-glycosylated depending on the glycosylation of the native parent protein and the host used to produce the muteins. If necessary, the non-glycosylated 25 mutein obtained when the host microorganism is E. coli or Bacillus may optionally be glycosylated in vitro by chemical, enzymatic and other methods known in the art.

In the preferred application of the present invention to IFX-β, the cysteine residue at position 17 of the amino acid sequence shown in FIG. 1 is altered in series by a T -> A switch to the first base of codon 17 of the sense strand of the DNA sequence that codes for mature IFN-β. Induced site-specific mutagenesis using a single synthetic 17-nucleotide primer GCA

ATTTTCAGAGTCAG, which is identical to one sequence of seventeen nucleotides on the IFN-β sense strand in the codon 1 region. ' is the exception of a single mismatch in the first base of ko45 don 17. This mismatch is at nucleotide 12 in the primer. It should be borne in mind that the genetic code can degenerate and many of the amino acids can be encoded by more than one codon. For example, the base codon for serine is six times degenerate, so the columns TCT, TCG, TCC, TCA, AGT and ACG encode for serine. The AGT codon has been selected as the preferred convenience application. Similarly for threonine encodes each of the columns ACT, ACA, ACC and ACG. It is accepted that when a codon is specific for a particular amino acid, it includes all codon 5 degenerates that encode that amino acid. The 17-mRNA is hybridized to single-stranded DNA of M13 phage that carries the nonsense strand of the IFN-β gene. The oligonucleotide primer was then extended onto 10 DNA using the DNA polymerase 1 fragment of Klenow and the resulting dsflHK converted to closed circular DNA with T ₄ ligase. Replication of the resultant mutant heteroduplex yields clones of the DNA strand containing the mismatch. Mutant clones may be identified and screened for the presence or absence of specific restriction sites, antibiotic resistance or susceptibility, or by other methods known in the art. When cysteine is replaced by serine, the T -> A transition shown in Figure 2 results in the creation of a new Hinf1 restriction site in the structural gene. The mutant clone was identified by the oligonucleotide primer as a probe for hybridization screening of the mutated phage plates. The primer has only one mismatch when hybridized, is the parent protein, but exactly 30 when it is hybridized to the mutated phage DNA, as shown in figure 2. Hybridization conditions can be maintained such that the oligonucleotide primer is hybridized primarily to the mutant DNA, not parental DNA. The newly created Hinf1 site also serves as a means of confirming a single-base mutation in the IFN-β gene.

The DNAnM13phage carrying the mutated gene is isolated and separated into appropriate expression vectors, for example plasmid pTrP3 and E. coli strain MM294 transformed with the vector. Suitable culture media for the transformation of transformants and their progeny are known to those skilled in the art. The expressed IFN-β 45 mutein was isolated, purified and characterized.

Brief description of the drawings.

Figure 1 is a diagram of the amino acid sequence of IFN-β. 50

Figure 2 is a diagram showing the production of a mutant IFN-β gene by oligonucleotide-directed mutagenesis.

Figure 3 is a diagram of plasmid ρβΗτρ.

including the IFN-β gene.

Figure 4 is a diagram of the cloning vector M13 tr8 phage.

Figure 5 is a restriction map of clone M13-P 1.

Figure 6 is a footprint of a sequencing gel of the mutant IFN-β _serl7 gene, showing a single-base change in the coding region.

Figure 7 is a diagram of the expression plasmid pTrP3.

Figure 8 shows the Hinf I restriction map of clone pSY 2501, figure 8 (bi shows the resulting two 169 bp and 23 bp fragments.

Figure 9 with the PSY2501 cluster restriction map.

Figure 10 shows the mutein coding for IFN-β _h . and its corresponding amino acid sequence.

11 is a single strip of 16,000 daltons protein corresponding to lFN-β ^ in the extracts of clones pSY2501 and ρβΠτρ.

Figure 12 is a diagram of plasmid pLM 1 that contains the human interleukin -2 (1L-2) gene under the control of the E. coli trp promoter.

Figure 13 is a restriction map of a phage M13-IL-2 clone.

Figure 14 is a restriction map of plasmid pLW46.

Figures 15 (a) and 15 (c) show the nucleotide sequence of the pLW46 coding chain and the corresponding amino acid sequence of IL-2 mutein, designated IL-2 ser 1 25

Figure 16 is a diagram of the plasmid pLW32.

Figure 17 is a diagram of plasmid pLW55.

Examples of carrying out the invention.

The following examples illustrate the invention but do not limit its scope. Examples 1 to 11 describe the production of IFN-β mutein, and Examples 12 to 20-the production of IL-2 mutein.

Example 1. Cloning of IFM β gene in M13 vector.

The use of the M13 phage vector as a source of single stranded DNA template is described by GFTemple et. al, Nature (1982) 296: 537540. The plasmid ρβΠτρ (Figure 3) containing the IFN-β gene under the control of the E. coli trp promoter was digested with the restriction enzymes HindIII and Xholl. MI3inp8 (J. Massing. Third Cleveland Symposium on Macromolecules: Recombinant DNA, 1981) DNA replication form (RF) (Figure 4) was digested with HindIII and BamHI restriction enzymes and mixed with ρβΐίτρ DNA. which was pre-ground with Hindlll and Xholl. The mixture was ligated with T ₄ DNA ligase, the ligated DNA was transformed into competent cells of E.coli strain JM103 and plated onto Xgal indicator plates (Messing et. Al. Nucleic Acids Res. ( 1981) 9: 309-321). Plates containing recombinant phage (white plates) were separated, inoculated into fresh culture of JM 103 and minipreparations of RF molecules obtained from infected cells (Bimboim and Dolv, Nucleic Acid Res. (1979) 7: 1513-1523). The RF molecules were digested with various restriction enzymes to identify clones containing IFN-β insert. The restriction map of one such clone (Μ 13-β 1) is shown in Figure 5. Single-stranded (SS) phage DNA is obtained from clone Μ13-β 1 and serves as a template for site-specific mutagenesis using a synthetic oligonucleotide.

Example 2. Site-specific mutagenesis.

Forty picomoles of the synthetic oligonucleotide GCAATTTTCAGAGTCAG (primer) was treated with T ₄ kinase in the presence of 0.1 mM adenosine triphosphate (ATP), 50 mM hydroxymethylaminomethane hydrochloride (Tris-HCI) at pH 8.0, 10 mM magnesium chloride, 5 mM dithiothreitol (DTT ) and 9 units of T ₄ kinase, in 50 μΐ at 37 ° C for 1 hour. The kinase primer (12 picomoles) was hybridized to 5 pg ss Μ13-β 1 DNA in a 50 pl mixture containing 50 mM sodium chloride, 10 mM tris hydrochloric acid with pH 8.0, 10 mM magnesium dichloride and 10 mM β mercaptoethanol, at heating at 67 ° C for five minutes and at 42 ° C for 25 minutes. The heated mixture was cooled on ice and added to a 50 pl reaction mixture containing 0.5 µM of each deoxynucleoside triphosphate (dNTP), 80 mM tris hydrochloric acid with pH 7.4, 8 mM magnesium dichloride, 9 units of DNA polymerase 1 , a Klenow fragment, 0.5 mM ATP and 2 units of T ₄ DNA ligase, incubated at 37 ° C for three hours and two hours at 25 ° C. The reaction is terminated by phenolic extraction and precipitation with ethanol. The DNA was dissolved in 10 mM Tris hydrochloric acid was pH 8.0, 10 mM ethylenediaminetetraacetic acid (EDTA), 50% sucrose and 0.05% bromophenyl blue and electrophoresed on 0.8 agarose gel in the presence of 2 pg / ml ethidium bromide. DNA bands corresponding to the Μ13 βΐ RF forms. are excised from the gel sections by the perchlorate method (Davis, et al..Advanced Bacterial Genetics ”(1980) pp. 178-179. The eluted DNA is used to transform competent JM103 cells cultured overnight and bcDNA. is isolated from the supernatant This 3ZDNA was used as a template in the second cycle of iimer expansion, gel-purified RF forms of DNA were transformed into competent JM 103 cells, spread on agar plates, and incubated overnight to obtain phage plates.

Example 3. Site-specific mutagenesis.

The experiment of Example 2 was repeated, except that the synthetic oligonucleotide primer used was GCAATT TTCAGACTCAG. It changes codon 17 of the IFN-β gene from one that encodes for cysteine to one that encodes for threonine.

Example 4. Place-specific deletion.

The experiment of Example 2 was repeated, except that the synthetic oligonucleotide primer used was AGCA ATTTTCAGCAGAAGCTCCTG for deletion of codon 17 from the IFN-β gene.

Example 5. Screening and identification of mutant plaques.

Plates containing mutated Μ13-β1 plates (Example 1) and two dishes containing non-mutated Μ13-β1 phage plates were cooled to 4 ° C and plates of each plate were transferred to two nitrocellulose circular filters by placing a dry filter on the agar tile for five minutes for the first and 15 minutes for the second filter. The filters were then placed on thick filter paper soaked in 0.2 ° ₀ sodium hydroxide. 1.5 M sodium chloride and 0.2% Triton X-100. for five minutes and neutralized by loading on filter paper impregnated with 0.5 M trie hydrochloric acid pH 7.5 and 0.5 M sodium chloride for another five minutes. The filters were similarly washed twice on filters soaked in 2xSSC (standard physiological citrate), dried and then baked in a vacuum oven at 80 ° C for two hours. The double filters were pre-hybridized at 55C for four hours with 10 ml each per filter DNA hybridization buffer (5 x SSC) pH 7.0, 4 x Denhardt solution (polyvinylpyrrolidine, ficol and bovine serum albumin, 1 x = 0.02% of each), 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate buffer with pH 7.0 in 100 pg / ml of denatured trout semen. A ³² P labeled sample was prepared by kinase of the ³² P ATP labeled oligonucleotide primer. The filtrates were hybridized to 3.5x10 cpm / ml of ³² P labeled primers in 5 ml for DNA filter hybridization buffer at 55 ° C for 24 hours. Filters were washed at 55 ° C for 30 min each in wash buffer containing 0,1 ° _o SDS and decreasing amounts of SSC. First, the filters are washed with buffer containing 2xSSC, and the control filters containing the Μ13-β1 -mutated plates are checked for any radioactivity with a Geiger counter. The concentration of SSC was reduced stepwise and the filters were washed until no radioactivity was detected on control filters with н13-β1 plaque-switched. The lowest SSC concentration that has been used is O.lxSSC. The filters are air-dried and autoradiographically at -70 ° C for two to three days. Screening was performed with a kinase oligonucleotide indicator of 480 plates of mutated M13βΐ and 100 non-mutated control plates. None of the control plaques hybridized with the indicator, while five mutated Μ13-β1 plaques hybridized with it.

One of the five mutated Μ13-β1 plaques (M13-SY2501) was taken with a needle and inoculated into JM 103 culture. From the supernatant, cbDNA was obtained and double stranded (ds) DNA was obtained from the precipitate. This ssflHK was used as a matrix for dideoxy sequencing of the clone using the M13 universal primer. The sequencing results are shown in Figure 6, confirming that the TGT cys codon has been converted to the AGT ser codon.

Example 6. Expression of Mutated lFN-β in E.coli.

The M13-SY2501 RF DNA was digested with the HindIII and XhoII restriction enzymes and the included 520 bp fragment was purified on a 1% agarose gel. Plasmid rTgrZ containing E. coli trp promoter (Figure 7) was digested with en winters Hindlll u BamHI, mixed with the purified M13-SY2501 DNA fragment, and ligated in the presence of T ₄ DNA ligase. Spleen DNA was transformed into E. coli strain MM294. Ampicillin-resistant transformants are screened for drug sensitivity tetracycline. Plasmid DNA of five tetracycline-sensitive ampicillin-resistant clones was digested with Hinfl for screening for M13-SY2501 inclusion. Figure 8 (a) shows an IllNfl restriction map of one of the clones (pSY2501) compared to the Hinfl map of the original IFN-β clone, ρβΐίτρ. As expected, there is one additional HinfI site in pSY2501 cleaving the 197 bp IFN-β internal fragment of one 169 bp fragment and one 28 bp fragment (Figure 8b). A restriction map of clone pSY2501 is shown in Figure 9. The complete DNA sequence of the mutant IFN-β gene is shown in Figure 10 together with the expected amino acid sequence.

The plasmid, designated clone pSY2501, was deposited with the Agricultural Research Culture Collections (NRRL), Fermentation Laboratory, Notrhern Regional Research Center, Science and Education Administration of the US Department of Agriculture, 1815 North University Street, Peoria Ill. 60604 on March 30, 1983 and received IASB No. 1533 and NRRL No. B-15356.

Cultures of pSY2501 and ρβΐΐτρ, which include their progeny, are grown to an optical density (OD) of 1.0. Prepare with a cell-free extract and the amount of IFN-β antiviral activity was assayed on GM2767 cells by microtiter analysis. Extracts from clone pSY2501 exhibited three to ten times higher activity than pPltrp (Table 1), indicating that clone pSY2501 synthesized more protein exhibiting IFN-β activity or that the resulting protein had higher specific activity.

Table 1

Extract Anti-virus activity (U / nil) PSY2501 6 x 10 ^s ρβΐφρ I x 10 ⁵ ptrp ³ (control) 30

To determine whether clone pSY2501 synthesized several times over the active protein, extracts from both clones were electrophoresed on an SDS polyacrylamide gel together with a control extract, and the gel was stained with coomas and blue to illustrate the proteins. As shown in Figure 11, there is only one band corresponding to one 18,000 Dalt protein contained in the extracts of clones pSY2501 and, ρβΗτρ, but not in the control extract ptrp '. This protein, which has a molecular weight of about 20,000 daltons but shows a gel migration image of 18,000 daltons, found to be IFN-β by purification of this protein from ρβΐίτρ extracts. Because the extracts of pSY2501 contain less of this protein than the extracts of ρβΐtrp, the specific activity of the protein in extracts of clone pSY250l is higher than that of clone ρβΗτρ.

Example 7.

Plasmid pSY2501 cc was transformed into a competent sub-variant of E. coli strain MM294, designated MM294-1. A sample of the transformant obtained was deposited with American Culture Collection 12301, Parklawn Drive, 10 Rockville, Md 20852 US on November 18, 1983 under ATCC number 39 517.

Example 8. Production of IFN-β serl7. IFN-β ser 17 is recovered from E. coli that have been transformed to produce

IFN-β serl7. E. coli were cultured in the following culture medium to an OD of 10-11 at 680 nm (dry weight 8.4 g / Ι).

Ingredient Concentration nh ₄ ci 20 mM K ₂ SO ₄ 16.1 tM KN RO. 7.8 tM At ₂ HPO ₄ 12.2 tM MgSO ₄ .7H ₂ O 3 tM Na ₃ citrate. 2H ₂ O 1.5 tM MnSO ₄ .4H _; 0 30 μΜ ZnSO ₄ .7H ₂ b 30 μΜ CuO ₄ .5H, O 3 μ.Μ L-tryptophan 70 mg / l FeSO ₄ .7H ₂ 0 72 μΜ Thiamine hydrochloric acid 20 mg / l Glucose 40 g / l 15 pH controlled with NH ₄ OH

The resulting 9.9 l (9.9 kg) of transformed E.coli cc were cooled to 20 ° C and concentrated by passing through a cross-flow filter at an average pressure of about 110 K and a constant filtrate rate of 260 ml / min until the weight of the filtrate reaches 8.8 kg. The concentrate (approximately 1 L) was separated into a vessel and cooled to 15 ° C. The cells in the concentrate were then destroyed by passing it through a Manton-Gaulin 45 homogenizer at 5 ° C, about 69,000 cf. The homogenizer was washed with one liter of phosphate buffered saline with pH 7.4 (PBS) and the washing liquid was added to the dierupate to obtain a final volume of two liters. This volume 50 was centrifuged continuously at 12,000 xg and a flow rate of 50 ml / min. The precipitate was separated from the supernatant and resuspended in four liters of PBS containing 2 ° w. SDS. This suspension was stirred at room temperature for 15 minutes, after which no more visibly suspended material was present. The solution was then extracted with 2-butanol in a volume ratio of butanol: solution 1: 1. This extraction is carried out in a liquid-liquid phase separator at a flow rate of 200 ml / min. The organic phase was then separated and evaporated to dryness, yielding 21.3 g of protein. It is resuspended in distilled water at a volume ratio of 1:10.

The recovered product was tested for human IFN-β activity. using analysis based on protection against viral cytopathic effect (CPE). The test is carried out in microtiter plates. A minimum of 50 μΐ of essential medium is placed in each well and 25 μΐ of the sample is placed in the first well, and serial dilutions of 1: 3 are made in subsequent wells. Each plate has a virus (Vesicular stomatitus), cells (human fibroblast GM-2767 line) and comparative IFN-β control. The IFN-β used is 100 units per milliliter. The tiles were then irradiated for 10 minutes. After irradiation, 100 µl of cell suspension (1.2 x 10 ⁵ cells / ml) was added to each well and the plates were incubated for 18-24 hours. Add a viral solution of one plaque unit per cell in each well, except for the control. The plates were incubated until viral control showed 100% CPE. This happens normally 18 to 24 hours after the addition of the viral solution. The results of the study are interpreted with respect to the location of the well with 50% CPE relative to the comparative IFN-β control. The interferon titer of all samples on the plate was determined against them. The specific activity of the recovered product is 5 x 10 ⁷ U / mg.

Example 9. Acid precipitation and chromatographic purification.

The process of Example 8 was repeated with the exception that after extracting and separating the aqueous and organic phases and mixing the organic phase with PBS in a volume ratio of 3: 1, the pH of the mixture was lowered to about 5 by the addition of glacial acetic acid. acid. The resulting precipitate was separated by centrifugation at 10,000 to 17,000 g for 15 minutes and the residue was redissolved in 10% (w / v) SI) S, 10 ml DTT, 50 ml sodium acetate buffer pH 5.5 and heated to 8 () In five minutes.

The solution was then placed on a Brownlee RP-300, 10μΜ, “Aquaporc” column using a Beckman gradient system. Buffer A is 0.1% trifluoroacetic acid (TFA) in H ₂ O, buffer B is 0.1% TFA in acetonitrile. Detection is by UV absorption at 280 ps. The solvent program is a linear gradient of 0% buffer B to 100% buffer B for three hours. The fractions exhibiting the highest interferon activity were collected in one place and it was determined that the specific activity of the collected interferon preparation was 9.0 x 10 'to 3.8 x 10 ^s international units per mg of protene compared to this of about 2x10 'U / mg of native IFN-β.

Example 10. Biochemical characterization of 1ΕΝ-β _{κΓ 17} .

The amino acid composition was determined after hydrolysis of a sample of 40 µg in 200 μΐ 5.7-hydrochloric acid for 24 to 72 hours. 0.1% phenol at 108 ° C. Proline and cysteine are determined in the same way after oxidation with permaic acid, in which case phenol is omitted during hydrolysis. Tryptophan was analyzed after 24 hours hydrolysis of 400 μΐ sample samples in 5.7-hydrochloric acid, 10% mercaptoacetic acid (without phenol). The assay was performed on a Beckman 121 MB amino acid analyzer using a single column of AAO resin.

The amino acid composition calculated from representative 24-, 48-, and 72-hour hydrolyses of purified IFN-β4 _{] 7} matches the expected DNA sequence of the cloned IFN-β gene, minus the missing N-kpa methionine.

The amino acid sequence of the first 58 residues of the amino acid end of purified IFN was determined on a 0.7 mg sample in a Beckman 890 ° C sequencer with 0.1 m Quadrol buffer. PTH amino acids were determined by reverse phase HPLC on an Altex ultraspheric ODS column (4.6 x 250 mm) at 45 ° C, eluting 1.3 minutes with 40% buffer B and 8.4 minutes with 40-70% buffer B, where buffer A is 0.0115 moles of sodium acetate, 5% tetrahydrofuran (THF), pH 5.11, and buffer B is 10% THF in acetonitrile.

The N-terminal amino acid sequence10 of the IFN-β _{icr? 7} sequence, after determination, coincides with the expected DNA-based sequence except for the N-terminal mystinin.

Example 11. Alternative Method for Preparation and Purification of 1ΓΝ-β

E. coli transformed pSY2501 were cultured in the following medium.

Ingredient Approximate initial concentration l, citrate 2H, 0 1 mM KN ₂ PO ₄ 30 tM (NH ₄ ) ₂ SO ₄ 74 tM MgSO ₄ .7H, O 3 tM MnSO ₄ .H ₂ 0 46 μΜ ZnSO ₄ .7H ₂ O 46 μΜ CuSO ₄ .5H, O 1-2 μ Μ L-tryptophan 350 μΜ FeSO ₄ .7H O 74 μΜ Thiamine hydrochloric acid 0,002% Glucose 1.5%

If necessary, add a Dow Corning defoamer-25 percent solution of polypropylene glycol, 50 percent glucose solution and 5-nor.-potassium hydroxide.

The temperature was maintained at 37 ± 1 ° C, pH 6.5 ± 0.1 with sodium hydroxide and dissolved oxygen at 30% air saturation. The optical density and the amount of residual glucose are measured after 14 hours and at intervals of approximately 1 hour thereafter. The material is collected when glucose consumption reaches 40 ± 6 g / l / OD at 680 nm = 10-1I).

The collected material was concentrated about three times by passing through a microporous cross-flow filter. The concentrated cells were diafiltered with deionized ox while the collected material was concentrated 4-5 times. The cells were then cleaved by passage through a Manton-Gaulin homogenizer at about 4.1 to 5.5x10 ⁴ feet. After the first pass, SDS sodium phosphate buffer was added to a final concentration of 2% SDS, 0.08 M sodium phosphate and homogenization continued for another hour. Then solid DTT was added to a final concentration of 50 mM and the homogenate heated to 90 ± 5 ° C for 10 minutes. The resulting cell suspension was extracted with 2-butanol at a volume ratio of 1: 1 butanol: suspension in a static mixer.

The mixture was centrifuged and the 2-butanol-rich phase was separated.

The butanol-rich phase was mixed with 2.5 volumes of 0.1% SDS in phosphate-buffered saline (PBS). A solid 35 DTT was added to a final concentration of 2 mM. The pH of the mixture was adjusted to 6.2 ± 0.1 with glacial acetic acid and the mixture was centrifuged. The resulting precipitate was separated and resuspended in PBS + 10% SDS at a pH of 40 8.5 ± 0.1 with 1-Ν sodium hydroxide base. Add solid DTT to a final concentration of 100 mM and heat the suspension to 90 ± 5 ° C for 10 minutes. The suspension was then cooled to about 25C. The pH was adjusted to 5.5 ± 0.1 with ice45 acetic acid and the solution filtered.

The solution was then placed in a Sephacryl S-200 column and the fractions exhibiting the highest activities of intraferon were separated and then concentrated by ultrafiltration with 10 Kdal molecular weight separation. The concentrate is oxidized by the addition of equimolar amounts of protein and iodine-acid in the reaction vessel. containing 2mM sodium pyrophosphate, 0.1% SDS and 1mM EDTA. The pH was maintained during oxidation to 9.0 ± 0.1 with 0.5-normal sodium hydroxide and adjusted to 5.5 ± 0.2 when 5 oxidation was complete. After oxidation, the concentrate is again passed through the ultrafiltration unit at 10 Kdal molecular weight separation.

The concentrate was placed on a major Sephac-10 ryl S-200 column and fractions were analyzed by SDS-PAGE to detect those containing no high molecular weight pollutants. These fractions are collected in one place and passed through an ultrafiltration device. The filtered concentrate was fractionated on a Sephadex G-75 column. SDS-PAGE analysis of fractions is performed to determine those that do not contain low or high molecular weight pollutants. These fractions were collected 20 at a single desalination site.

A Sephadex G-25 column calibrated with 1 mM sodium hydroxide was charged to the collected fractions of the Sephadex G-75 column using distilled water set pH 25 to 10.8-11 with 50% sodium hydroxide. The purified product is collected to a blank. From this desalinated, purified IFN-β mutein, formulations for therapeutic use can be prepared by known methods. 30

IFN-β biological assays

Antigenic comparison.

IFN-β was compared antigenically to IFNβ produced by diploid fibroblasts using the 35 virus neutralization assay. Polyvalent diploid fibroblast IFN-β antiserum is obtained in rabbits. This antiserum blocks the antiviral activity of both diploid fibroblast IFN-β and ΙΡΝ-β _κΓ17 in virus neutralization assays, indicating that the two proteins are indistinguishably antigenic.

Antivirus activity.

Purified iFN-β ^ ₁₇ was compared by antiviral activity with native IFN-β. Inhibition of vesicular stomatitis virus replication in diploid skin fibroblasts (HS27F) is indistinguishable from that of the natural molecule. Similarly, the inhibition of herpes simplex virus type I in HS27F fibroblasts of natural and mutant protein is comparable.

Antiproliferative activity.

The antiproliferative activity of 1FNβ ^ ₁₇ for continuous cell lines is comparable to that of native IFN-β. T24 cells derived from transitional cell carcinoma were treated with 200 units / ml of protein. Cell growth was significantly inhibited (p <0.02) by both proteins.

Stimulation of natural killer cells (ΝΚ>.

The ability of IFN-β ^ ₁₇ to stimulate cellular (spontaneous cell-mediated cytotoxicity) activity is tested. Separated with Ficoll-hvpaque peripheral mononuclear cells (PMC) or NK enriched lymphocyte preparations (devoid of monocytes by plastic adhesion and OKT3-positive T cells by treatment with OKT3 antibody plus complement) were incubated overnight in nutrient medium β-IFN _ in different concentrations. There are ⁵ marked ^; Target cells are incubated with effector cells (at effector cell to cell ratio = 50: 1) for 2-4 hours. NK cell cytotoxicity is determined by measuring the amount of labeled cells in the medium. The results of these tests are shown in Table 1 below

Table 1

NK cell cytotoxicity with interferon (specific release rate ⁵ Cr SEM) IFN u / ml

Cage Effective cell 0 10 30 100 300 1000 T24 RMS 7.23 ± 5.1 23.1 ± 4.4 24.4 ± 1.1 34.1 ± 2.5 50.0 ± 2.0 40.4 ± 4.4 Chang RMS 4.7 ± 0.5 7.2 ± 0.8 9.5 ± 1.7 15.9 ± 1.3 21.9 ± 1.4 26.9 ± 1.8 Chang NK Epg 19.2 ± 4.6 39.4 ± 4.1 ND 54.2 ± 6.1 ND 41.7 ± 5.5 K562 NK Epg. 41.0 ± 4.6 48.4 ± 1.6 ND 62.2 ± 3.5 ND 63.2 ± 3.5

As indicated target cells cc kill more effectively than treatment with IFN-β ^ ₇ cells than the untreated.

Clinical trials.

Phase I of clinical trials to confirm the safety of IFN-β _h in humans has been initiated. These experiments include administration of the protein to patients intramuscularly and intravenously at doses ranging from 1 x 10 ^s units (1 pg protein) to 400 x 10 ⁶ units. No unexpected side effects were observed in the initial phase I clinical trials.

As indicated, the IFN-β preparation exhibits a specific activity too close to or greater than that of native IFN-β. 1FNβ _ιΓ has no free sulfhydryl groups, but a single -SS- bond between the only remaining cysteines at positions 31 and 141. The protein does not easily form oligomers and exists mainly in monomers. The IFN-β ^ ₁₇ obtained according to the invention can be administered either alone or in various mixtures, in pharmaceutically acceptable formulations in inert, non-toxic, non-invasive allergies, physiologically acceptable carriers with clinical and therapeutic applications in cancer therapy or in conditions requiring interferon therapy and viral infections such as viral herpes simplex I and II. viral hepatitis B, viral colds and rhinovirus. Carriers include, but are not limited to, distilled water, saline, Ringer's solution, Hank's solution, and the like. Other non-toxic stabilizing and solubilizing additives may be included, such as dextrose, HS A (human serum albumin) and the like. Therapeutic agents may be administered orally or parenterally, for example, intravenously, intramuscularly, intraperitoneally and subcutaneously. The modified IFN-β formulations of the invention may also be used for topical administration in suitable carriers normally used for such pellets. IFN-β mutein may be administered topically or systemically, alone or in combination with other therapeutic agents, such as acyclovir for therapeutic and prophylactic purposes according to US Patent No. 4,355,032. The dose of mutein administered to human patients will depend on this. whether it is administered continuously (including intermittent) or as a bolus. The amount administered continuously is usually less than the amount of bolus applied. This amount is typically in the range of 1 x 5 ⁴ to 4 x 10 ^s units, more often from about I x 10'' to I x 10 '.

The advantages of the IFN-β mutein described are based on the elimination of a free sulfhydryl group at IFN-β position 17, thereby forcing the protein to form the correct disulfide bonds between cys31 and cvsl 41 and to accept the conformation required for complete biological activity. The higher specific activity of IFN-β ^ ₁₇ allows the use of smaller doses for therapeutic use. Thanks to the deletion of the free -SH group. 1FN-β ^, the protein does not form dimers and oligomers as easily as the microbially produced IF.N-β. This facilitates the purification of the protein and increases its stability.

Example 12.

The nucleotide sequence of the cDNA clone encoding for human 1L-2, methods for producing the IL-2 cDNA bank and screening for the same for 1L-2 are described by Taniguchi et al., Nature (1983) Volume 24. P. 305 et seq .

cDNA banks enriched with potential IL-2 cDNA clones were obtained from the IL-2 enriched mRNA fraction obtained from induced peripheral blood lymphocytes PBL and Jurkat cells by conventional methods. Enrichment of mRNA with 1L-2 information is performed by fractionating the mRNA and identifying the fraction having IL-2 mRNA activity by injecting the fractions into oocytes of Xenopus laevis and analyzing the oocyte lysates for IL-2 activity on HT-2 cells ( Watson J. Exp.Med. (1979) 150: 1570-1519 in Gillis et al., J Immun (1978) 120: 2027-2032).

Example 13. Screening and identification of 1L-2 cDNA clones.

IL-2 cDNA banks were screened using a colony hybridization method. Each microtiter plate was replicated on double nitrocellulose filter paper (S <kS type BA-85) and the colonies were cultured at 37 ° C for 14-16 hours on L agar containing 50 pg / ml ampicillin. Colonies were lysed and DNA was fixed to the filter by successive treatment for five minutes with 500 mM sodium hydroxide, 1.5 M sodium chloride, washing twice for 5 minutes each time with 5 x standard physiological citrate (SSC). The filters are air-dried and baked at 80 ° C for two hours. The double filters were pre-hybridized at 42C for 6-8 hours with 10 ml for filter DNA hybridization buffer (50% formamide, 5 x SSC with pH 7.0, 5 x Denhardl solution (polyvinylpyrrolidine, and ficol and bovine serum albumin; 1 x = -0.2% of each), 50 mM sodium phosphate buffer with pH 7.0, 0.2% SDS, 20gg / ml Poly U, and 50 pg / ml trout denatured DNA.

A ² P-labeled oligonucleotide probe was prepared based on the sequence of the IL-2 gene described by Taniguchi et al. (See above). The nucleotide sequence of the sample is GTGGCCTTCTTGGGCATGTA.

The sample samples were hybridized at 42 ° C for 24 to 36 hours with 5 ml / filter DNA hybridization buffer containing ³² P nucleotide sample. The filters were washed twice for 30 min each time at 50 ° C with 2 x SSC, 0.1% SDS, then washed twice with 1 x SSC and 0.1% SDS at 50 ° C for 90 minutes, dried air and autoradiograph at -70 ° C for 2 to 3 days. Positive clones are identified and screened again. Full-length clones were identified and confirmed by restriction enzyme mapping and compared to the sequence of the 1L-2 cDNA clone established by Taniguchi et al. (See above).

Example 14. Cloning of an IL-2 gene into an M13 vector.

The IL-2 gene was cloned into I13mp9 as in Example 1 using plasmid pLW1 (Figure 12) containing the IL-2 gene under the control of the E. coli trp promoter. One sample of pLWl was deposited at American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md, 20852, US on August 4, 1983 and was given ATCC number 39 405. The restriction map of one branch (designated M13-IL2) containing 1L The -2 insert is shown in Figure 13. Single-stranded phage DNA is derived from clone M13-1L2 and serves as a template for oligonucleotide-directed mutagenesis.

Example 15. Oligonucleotide-directed mutagenesis.

As indicated previously, 1L-2 contains cysteine residues at amino acid positions 58, 105, and 125. Based on the nucleotide sequences of portions of 1L-2 genes that contain codonitis for these three cysteine residues, three oligonucleotide primers were designed and synthesized for a codoniga mutation for these residues to code for serine. These oligonucleotides have the following sequences:

CTTCTAGAGACTGCAGATGГТТС (DM27) for modification of cys 58;

CATCAGCATACTCAGACATGAATG (DM28) for modifying cys 105 and

GATGATGCTCTGAGAAAAGGTAATC (DM29) for cys 125 change.

Forty picomoles of each oligonucleotide were kinase individually in the presence of 0.1 mM ATP, 50 mM trisolic acid with pH 8.0, 10 mM magnesium dichloride, 5 mMDTT, and 9 units of T ₄ kinase in 50 μΙ at 37 ° C. 1 hour. Each of the kinase primers (10 picomoles) was hybridized with 2.6 gg of M13-IL2 DNA in 15 μΙ of one cmc. containing 100 mM sodium chloride, 20 mM trisolic acid at pH 7.9, 20 mM magnesium dichloride and 20 mM β-mercaptoethanol by heating to 67 ° C for five minutes and 42 ° C for 25 minutes. The warmed mixtures were cooled on ice and adjusted to a final volume with 25 μΐ reaction mixture containing 0.5 mM of each dNTP, 17 mM trisolic acid, pH 7.9, 17 mM magnesium dichloride, 83 mM sodium chloride. 17 mM β mercaptoethanol, 5 units of DNA polymerase 1 fragment of Klenow, 0.5 mM adenosine triphosphate (ATP) and 2 units of T ₄ DNA ligase incubated at 37 ° C for five hours .. Reactions were terminated by heating to 80 ° C and the reaction mixtures are then used to transform competent JM103 cells spread on agar plates and incubated overnight to obtain phage plates.

Example 16, Screening and Identification of Mutated Phage Plates.

Plates containing mutated M13-1L2 plates and two plates containing non-mutated M13-IL2 phage plates were cooled to 4 ^P C and phage plates from each plate were transferred to two nitrocellulose filter circles by pressing a dry filter on the agar plate for 5 minutes for the first filter and 15 minutes for the second filter. The filters were then placed on thick filter paper impregnated with 0.2-N sodium hydroxide, 1.5 M sodium chloride and 0.2% Triton for five minutes and neutralized by spreading on filter 14 paper impregnated with 0.5 M Tris-hydrochloric acid with pI 7.5 and 1.5 M sodium chloride for another five minutes. The filters were similarly washed twice on filters soaked in 2 x SSC, air-dried, and then vacuum-baked at 80C for two hours. The double filters were re-hybridized at 42 ° C for four hours with 10 ml each to filter the DNA hybridization buffer (5 x SSCrH 7.0, 4 x Denhardt solution (polyvinylpyrrolidone, ficol and bovine serum albumin, 1 x 0.02% each) ), 0.1% SDS, 50 mM sodium phosphate buffer pH 7.0 and 100 pg / ml trout denatured DNA Indicators ³² P labeled by kinase of ATP-labeled oligonucleotide primers were prepared. with 0.1 x 10 ⁵ cpm / ml of ³² P labeled primers in five ml for filtering DNA hybridization buffer at 42 ° C for eight hours. and ^50! C for 30 min each in wash buffer containing 0,1% SDS and 2 x SSC and twice at 50 ° C for 30 minutes each with 0,1% SDS and 0.2 ° ₀ SSC. The filters are dried on air and autoradiograph at -70 ° C for two to three days.

As the oligonucleotide primers DM28 and DM29 are designed to create a new Ddel restriction site in the mutated clones (Figure 14), RF-DNA from a number of clones that are hybridized to each of these kinase primers is digested with the restriction enzyme Ddel. One of the M13-1L2 mutant plates, which is hybridized with primer DM28 and has a new Ddel restriction site (M13-LW44), is separated and inoculated into JM103 culture to obtain ssJHK from the culture supernatant and cell Maca- dsRF-DNA. Similarly, a plate that is hybridized with primer DM29 (Μ 13 LW46) is separated and ss4HK and RF-DNA are obtained. The DM27 oligonucleotide primer is designed to create a new PstI restriction site instead of the Ddel site. Therefore, plaques that hybridize with the primer are screened for identity on a new PstI site. One such phage plaque cc identifies (M I3-IW42) and it produces both xDNA and RF-DNA. DNA from each of these three clones was sequenced to confirm that the cysteine target TGT codons were converted to TST columns for series.

Example 17. Reclamation of a mutant IL2 gene for expression in E. coli.

Each RF-DNA of M13-LW42, M13-LW44 and M13-LW46 was digested with Hindlll and Banll restriction enzymes and the included fragments were purified from a 1% agarose gel. Similarly, the plasmid pTrP3 (Figure 7) was digested with HindIII and Bdnll, the large plasmid fragment containing the trp promoter was purified on an agarose gel and then ligated with each of the included fragments isolated from M13-LW42, Μ13-LW44 and M13-LW44. . The ligated plasmids were transformed into competent E. coli K12 strain MM294. The plasmid DNA of these transformants was analyzed by producing a restriction enzyme map to confirm the availability of plasmids pLW42, pLW44 and pLW46. Figure 14 is a restriction map of pI.W46.

When each of these individual clones was cultured in the absence of tryptophan to induce the trp promoter and cell-free extracts were analyzed on an SDS polyacrylamide gel, all three clones pLW42, pLW44 and pLW46 were found to synthesize 14.5 kd protein similar of that found in the positive control pLW21 known to synthesize 14.4 kd IL-2 protein. When these extracts were tested for IL-2 activity on murine HT-2 cells, only the pLW21 (positive control) and pLW46 clones displayed significant IL2 activity (Table II below), indicating that cys 58 and cys 105 were significant for biological activity and their change in serines (pLW42 and pLW44, respectively) will lead to loss of biological activity. Cys 125, on the other hand, must be essential for biological activity because its change to series 125 (pL \ V46) does not affect biological activity.

Table 2

Clones IL-2 activity (μ / nil) pIL2-7 (negative control) 1 pLW21 (positive control) 113,000 pLW42 660 pLW44 1,990 th most common pLW46 123,000

Figure 15 (a) shows the nucleotide sequence of the coding chain of clone pI.W46. Compared to the coding strand of the natural human 1L-2 gene, clone pLW46 has one single change based on G -> C at nucleotide 374. Figure 15 (c) shows the corresponding amino acid sequence of 1L-2 mutein encoded by pLW46. The mutein is designated des-alanyl (ala) IL-2 ^^. When compared to native IL-2, the mutein has serine instead of cysteine at position 125, has an initial N-terminal methionine (which is altered), and lacks the native N-terminal alanine of the natural molecule.

A sample of E. coli K12 strain MM294 transformed with pLW46 was deposited at American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md 20852, US on September 26, 1983 and is designated ATCC number 39 452.

Examples 18 and 19 describe the preparation of an alternative and preferred vector for the expression of alanyl (ala) IL-2 _jerl2J .

Example 18. Preparation of the Ala-IL-2 Expression Vector pLW32.

One codon (GCG) for alanine was inserted immediately after the start codon of the 1L-2 gene from pLW1 by oligonucleotide targeted mutagenesis as follows. The oligonucleotide primer, 5'-GAAGTAGGCGCCATAAG-3 ', is kininated. hybridized to ssM13-IL2 DNA and expanded using the general method of Example 15 to form a mutational heteroduplex. In addition to the insertion of the GCG codon, mutagenesis generates a new Nar! restriction site in the gene. The heteroduplex is transformed into a closed circular heteroduplex and is used to transform competent JM103 cells that are spread on agar plates and incubated as in Example 15. Screening of plates for identification of M13-IL2 mutant is performed by the example procedure 16. A mutant phage identified as M13LW32 is selected for use in additional cloning to produce RF DNA. Figure 16 is a diagram of the plasmid pLW32.

Example 19. Construction of Ala- _iL -2 _serl2 , expression clone pLW55.

The PF-DNA of MI3-LW46 (Examples 16 and 17) was digested with Xbal and PstI and the 530 bp fragment containing carboxy, the final coding region of the IL-2 gene was purified from agarose gel. pLW42 was digested with Xbal and PstI and the large fragment containing the plasmid vector and the alaIL-2 N-terminal coding sequence were purified. The two purified DNA fragments were combined together and ligated for using T ₄ DNA ligase. The quoted DNA was transformed into competent cells of E. coli K-12 strain M.M294. Tetracycline-resistant transformants were analyzed by preparation of a restriction enzyme map for the presence of a plasmid containing ala-lL-2 _{and | 2}} identified as pLW55, which has a new Ddel site not detected in pLW32. Figure 17 is a diagram of pLW55. Cell-free extracts of bacterial cultures containing pLW55 were found to exhibit more than 10 'units of II.-2 activity per milliliter by HT-2 cell assay, see Watson (above) and GilliS (see above). . The Ala-IL-2 _, ₅ protein is identical to the IL-2 _{Kr | 25} molecule shown in Figure 18 (c), except that the first includes the native N-terminal alanine of the natural molecule.

One specimen of E. coli K-12 strain MM294 transformed with pLW55 was deposited with the American Collection of Culture Types on 18 November 16, 1983 and is designated ATCC No. 39 516. E. coli transformed with pl.W55 is cultured. 20. Preparation and purification tivated in a fermenter containing the following medium of Ala-11L:

scr 12.5 '

(N11) 150 m.M kn ₂ ro ₄ 21.6 mM At ₃ citrate 1.5 mMol ZnSO ₄ .7H ₂ O 30 μΜ MnSO ₄ .5H, O 30 μΜ CuSO ₄ .5H ₂ O 1 μΜ

The pH set at 6.50 is 2.5-N sodium hydroxide autoclaved.

Sterile additives (after autoclaving)

MgSO ₄ .7H ₂ O 1 tM FeSO ₄ 100 μΜ L-tryptophan 14 mg / ml Thiamine hydrochloric acid 20 mg / ml Glucose 5 g / l Tetracycline 5 mg / l Ethanol 2% Casino acids 2%

If necessary, add Dow Corning defoamer polypropylene glycol, 20 percent solution, 50 percent glucose solution and 5N potassium hydroxide.

The pH of the fermenter was maintained at 6.8 with 5-N potassium hydroxide. The residual glucose was maintained between 5-10 g / Ι, the dissolved oxygen at 40% and the temperature at 37 ± 1 ° C. The amino acids (20 percent stock solution) are added to a concentration of 2% when OD _6SO is about 10. Material is collected three hours after OD reaches about 20.

The collected material was concentrated and homogenized as in Example 11. After DTT heat treatment, the material was centrifuged and the resulting precipitate was extracted with ursa to a final concentration of 4 M. The suspension was centrifuged and SDS was added to the solid phase to a concentration of 5%.

The solution was introduced into a Sephacryl S-200 column and fractions containing IL-2 (by SDS-PAGE) were separated. The separated fractions were placed on a Whatman M-40 column filled with 18 m Vvdac C ₄ with a pore size of 300 A and a coupled silica gel calibrated with 0.1% TFA. The 1L-2 mutein was eluted with a gradient of 40% to 60% 2-propanol containing 0.1% TFA for 160 minutes. The fractions having the highest $ 1L-2 activity were pooled together and their specific activities were found to be comparable to those of native IL-2.

IL-2 muteins in which the cysteine at position 125 is replaced by another amino acid, for example mutein IL-2 ^^ retain IL-2 activity. Therefore, they can be prepared and used in the same way as native IL-2. Accordingly, such IL2 muteins are used to diagnose and treat (locally or systemically) bacterial, viral, parasitic, protozoal, and fungal infections, to enhance cell-mediated cytotoxicity; for stimulating lymphokine-activated activity of killer cells;

5θ to mediate the restoration of lymphocyte immune function; to enhance alloantigen reactivity; to facilitate the recovery of immune functions in conditions of acquired immune deficiency; for the restoration of normal immunofunction in the elderly and animals; in the development of diagnostic studies, such as those using enzymatic amplification, isotope radiographic marking, and other methods known to those skilled in the art for monitoring levels of

II.-2 in disease states; for inducing growth of T cells in vitro for diagnostic and therapeutic purposes for blocking receptor sites for lymphokinases and various other therapeutic, diagnostic and research applications. Various therapeutic and diagnostic uses of human IL-2 have been studied and described by Rosenberg, Grimm et al., Ma / iinider et al., Grimm and Rosenberg. IL-2 muteins can be used alone or in combination with other immunologically appropriate B and T cells or with other therapeutic agents. Examples of suitable cells are B or T cells, natural natural killer (NK) cells and the like, and the therapeutic agents that can be used in combination with the polypeptides of the present invention are the various interferons, especially interferon gamma, cell growth factor B, IL-1 and the like. For therapeutic or diagnostic applications, they may be formulated in non-toxic, non-allergenic, physiologically acceptable carriers such as distilled water, Ringer's solution, Hank's solution, saline and the like. Administration of 1L-2 muteins to humans and animals may be orally, intraperitoneally, intramuscularly, or subcutaneously, as may appear most favorable and convenient to the physician. The amount of IL-2 mutein administered is typically in the range of about 1 x 10 ⁴ to 2 x 10 ⁸ units.

Modifications to the described uses of the invention that are known to those skilled in the art of genetic engineering, protein chemistry, medicine, and related fields are also encompassed within the scope of the following claims.

Claims (17)

Claims A structural gene with a DNA sequence that encodes a synthetic interleukin-2 mutein in which the cysteine residue at position 125 numbered according to natural human interleukin-2 is substituted with a neutral amino acid. The structural gene of claim I, wherein the neutral amino acid is selected from the group consisting of serine, threonine, glycine, alanine, valine. leucine. isoleucine, histidine, tyrosine, phenylalanine, tryptophan and methionine. The structural gene of claim 1, wherein the neutral amino acid is serine or threonine. The structural gene of claim 1, wherein the neutral amino acid is a series. A structural gene according to claim 1, 2, 3, 4 with or without an ATG start codon. 6. A structural gene as presented in Figure 15 (a), with or without the starting ATG codon. An expression vector comprising the structural gene of claim 1, 2, 3, 4 and 6. The expression vector of claim 7, wherein the vector is pBR322 or pCR1. 9. Plasmid pLW46 with ATCC number 39 452. 10. Plasmid pLW55 with ATCC number 39 516. A host cell transformed with an expression vector comprising a structural gene with a DNA sequence that encodes a synthetic interleukin-2 mutein, in which the cysteine residue at position 125 numbered according to natural interleukin-2 is replaced by a neutral amino acid and the mutein is the biological activity of natural human interleukin-2. wherein the host cell is selected from the group consisting of bacteria, yeast, animals and plants. The host cell of claim 11, wherein the host cell is a bacterium. The host cell of claim 12, wherein the bacterium is E. coli. The host cell of claim 11.12 or 13. wherein the neutral amino acid is from the group consisting of serine, threonine. glycine, alanine, valine, leucine, isoleucine, histidine, tyrosine, phenylalanine, tryptophan and methionine. The host cell of claim 11,12 or 13, wherein the neutral amino acid is serine. 16. E. coli transformed with an expression vector comprising the structural gene shown in Figure 15 (a) with or without the starting ATG codon. 17. E.coli. transformed with a plasmid from the group consisting of pLW46 and pLW55 and their progeny.