BG60506B2 - Human recombinant interleukin-2-mutien - Google Patents

Abstract

Muteins are used in the diagnosis and treatment of viral, bacterial, protozoal and fungal infections. They enhance cell-mediated cytotoxicity and support the recovery of immune functions, including in conditions of acquired immune deficiency. The muteins are produced by bacterial expression of mutant genes that code for muteins synthesized from the genes for the parental proteins by oligonucleotide-directed mutagenesis. Characteristic of the muteins of biologically active proteins such as ifn and il-2 is that cysteine residues, which are not essential for their biological activity, are omitted or replaced with other amino acids, thereby eliminating sites for the formation of improper disulfide bonds in the molecule. 10 claims, 17 figures

Description

1. Field of technology.

This invention is within the general field of recombinant DNA technology. In particular, it refers to mutationally altered biologically active proteins that differ from their parent analogs by one or more substituted cysteine residue elements.

2. BACKGROUND OF THE INVENTION

Biologically active proteins that are obtained microbiologically by recombinant DNA technology, hereinafter referred to as rDNA, may contain cysteine residues that are not essential for their activity but are free to form unwanted intermolecular or intramolecular bonds. Such a protein is microbially produced human beta interferon (IFN-?). During the preparation of IFN- / S by the rDNA method, dimers and oligomers of microbially produced IFN- / 3 have been observed to form E. coli extracts containing high concentrations of IFN-β. This formation of multimers makes purification and separation of IFN-? too time consuming and time consuming. Several additional operations are required in the purification and isolation process as a reduction of the protein during purification and re-oxidation to restore its initial conformation, thereby increasing the possibility of an incorrect disulfide bond. In addition, microbially produced IFN-D has been shown to exhibit persistently low specific activity, possibly due to the formation of multimers or random intramolecular iodide disulfide bridges. It is therefore desirable to modify microbially produced biologically active proteins such as 1FN-β in a way that has an undesirable effect on their activity but diminishes or eliminates their ability to cause intermolecular or intramolecular bonds, whereby the protein acquires an unwanted tertiary structure, (i.e., a conformation that reduces its activity).

The present invention relates to the preparation of methods for the direct mutagenesis of mutationally altered biologically active proteins (such proteins are called Muteins-Glossary of Genetics and Cytogenetics, 4th ed. Springer, 1976, which do not retain the activity of parental analogues but do not possess the ability to form parental analogs intermolecular bonds or unwanted intramolecular disulfide bridges In this connection, Separd et al., Nature (1981) 294: 563-565 describe an IFN-β mutein in which cysteine at position 141 of its amino acid sequence (has three cysteines in the natural human interferon-beta at positions 17, 31 and 141, Gene (1980) 10: 11-15 and Nature (1980) 285: 542-547) is replaced by tyrosine This mutein is obtained by bacterial expression of hybrid a gene derived from a partial IFN-β cDNA clone having a G> A crossing at IFN- / J gene nucleotide 485. The mutein lacks the biological activity of native IFN-β, leading to the conclusion that substituted cysteine was essential for activity.

Methods for targeted mutagenesis are well known and have been reviewed by Lather u Lecoq, Genetic Engineering Academic Press (1983) pp.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 side of the invention is a biologically active protein mutein that has at least one cysteine residue free to form a disulfide bond and is insignificant for said biological activity, with at least one of said cysteine residues removed or substituted by another amino acid in the mutein.

Another aspect of the invention relates to synthetic structural genes having DNA sequences that are specifically modeled (genes) for encoding the synthetic mutein described. Part of this aspect are expression vectors that include such structural genes, host cells, or microorganisms transformed with such vectors, and processes for producing a synthetic mutein by culturing such transformants or their progeny and restoring the mutein from culture. In the case of muteins having therapeutic use, therapeutic agents containing therapeutically active 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 in a protein having one or more cysteine residues free to form such a link, mutationally altering the protein by deletion of the cysteine residue (residues) or substituting them with other amino acids.

Another aspect of the invention is a method of producing the synthetic structural gene described by oligonucleotide-directed mutagenesis, comprising the following steps:

a) hybridization of single-stranded DNA, spanning a strand of a structural gene encoding the parent protein, with a mutant oligonucleotide primer complementary to one strand of the strand including the cysteine codon, which must be removed or substituted or in some cases meaningless triplet, the codon, except in the case of a mismatch of that codon or meaningless triplet, which determines the deletion of the codon or triplet encoding said other amino acid;

b) extension of the primer with DNA polymerase to form a mutational heteroduplex; and

c) replication of the mutational heteroduplex.

The mutant oligonucleotide primer used in this process is another aspect of the invention.

Description of the attached figures

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

Figure 2 is a diagram illustrating the production of a mutant IFN- / 5 gene by oligonucleotide-directed mutagenesis.

Figure 3 shows a diagram of a plasmid p Igr including the IFN-β gene.

Figure 4 is a diagram of the cloning vector M13tr8 phage.

Figure 5 is a restriction map of clone M13-0 1.

Figure 6 is a gel footprint of the sequence of a mutant IFN- / 9 _serl7 gene showing a single base change in the coding region.

Figure 7 is a diagram of the expression plasmid pTrP3.

Figure 8 (a) shows the Hinf1 restricted clone pSY2501, and Figure 8 (b) the resultant two 169 bp and 28 bp fragments thereof.

Figure 9 is a restriction map of clone PSY2501.

Figure 10 shows the DNA coding sequence for the mutein IFN- / 3 _{ser | 7} with the corresponding amino acid sequence thereof.

Figure 11 shows a single protein chain of 18,000 daltons corresponding to IFN- _γS ^ _d17 in the clone portion of pSY2501 and ρ β 1 trp.

Figure 12 is a diagram of the plasmid pLW1 that contains the human interleukin-2 (1L2) gene under the control of the E. coli trp promoter.

Figure 13 is a restriction map of phage clone M13-1L2.

Figure 14 is a restriction map of plasmid pLW46.

Figures 15 (a) and 15 (6) show, respectively, the nucleotide sequence of the pLW46 coding chain and the corresponding amino acid sequence of mutein IL-2, designated IL-2

16 is a diagram of the plasmid pLW32.

Figure 17 is a diagram of plasmid pLW55.

SUMMARY OF THE INVENTION

The present invention relates to muteins of biologically active proteins in which non-essential cysteine residues are intentionally deleted or substituted with other amino acids to eliminate the possibility of intermolecular cross-linking or formation of an intramolecular disulfide bond; mutant genes encoding for such muteins; and methods of producing such muteins.

Proteins that can be mutationally modified according to the invention are identified according to the available information on the cysteine content of biologically active proteins and the role of these cysteine residues in terms of activity and tertiary structure. Proteins for which no such information is available in the literature can be determined by systematically altering each of the cysteine residues of the protein by the methods described and determining the biological activity of the resultant muteins and their tendency to form unwanted intermolecular or intramolecular disulpholecular disulfide molecules. . Accordingly, although the invention specifically describes and exemplifies the preparation of IFN- and IL-2 muteins below, it should be understood that the same methods can be applied to any other biologically active protein containing functionally insignificant a cysteine residue that makes the protein sensitive to the formation of unwanted disulfide bonds. Examples of proteins other than IFN-? and IL-2, which are candidates for mutational alteration according to the invention, are lymphotoxins (tumor necrosis factor) stimulating colonies of factor-1 and 1FN-? 1. Candidate proteins typically have an odd number of cysteine residues.

In the case of IFN-β3, it is known from the literature that both glycosylated and non-glycosylated interferon exhibit qualitatively similar specific activities and that glycosyl groups are not involved in and do not contribute to the biological activity of IFN-β. But bacterially produced IFN-? , which is non-glycosylated, exhibits consistently less specific activity than natural IFN-? which is glycosylated. For IFN- /? it is known that there are 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- / 3, which contains four cysteine residues, there are two intramolecular S-S bonds: one between cys 29 and cys 138 and the other between cys 1 and cys 98. Based on the homology between 1FN- β and IFN- as cys 141 of 1FN- / 3 could be incorporated into the intramolecular -SS- bond with cys 31, leaving cys 17 free for intramolecular cross-linking. By removing cys 17 or replacing it with a different amino acid, it can be determined whether it is essential for biological activity and its role in the formation of the -SS bond. If cys 17 is not essential to the biological activity of the protein, the resulting protein removed or substituted cys 17 may exhibit specific activity close to that of native IFN- / 3 and could also facilitate protein isolation and purification.

By using an oligonucleotide directed mutagenesis with a synthetic oligonucleotide primer that is complementary to the IFN-? the gene at the codon for evs 17, but which contains single or multiple changes of bases in that codon, one gene can be obtained, which results in the substitution of cys 17 with another amino acid of choice. When a deletion in the oligonucleotide primer is desired, a codon for cys 17 is lacking. Conversion of cys 17 to a neutral amino acid such as glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine, tryptophan, serine, threonine and threonine. Serine and threonine are the most preferred substituents 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-?

Human IL-2 has been reported to have three cysteine residues located at positions 58, 105 and 125 of the protein. As in the case of IFN-E, IL-2 is in aggregated oligomeric form when isolated from bacterial cells and must be reduced with reducing agents to obtain a good yield of bacterial extracts. In addition, the purified reduced 1L-2 protein is unstable and readily re-oxidized when stored to one oligomer inactive form. The presence of three cysteines means that after re-oxidation, the protein may accidentally form one of the three possible intramolecular disulfide bridges, with only one of them being correct, i.e. as it is in a natural molecule. As the disulfide structure of the natural IL-2 protein is unknown, it is possible to use the invention to create mutations in columns 58, 105 and 125 of the IL-2 gene and to determine which cysteine residues are essential for the activity and therefore are most likely to be involved in the natural formation of a disulfide bond (bridge). In the same connection, the cysteine residue, which is not essential for the activity, can be modified to prevent the formation of irregular intramolecular disulfide bridges and to minimize the likelihood of formation of intermolecular disulfide bridges by eliminating or replacing the free cysteine axis.

The size of the oligonucleotide primer is determined by the requirement for stable hybridization of the primer to the region of the gene in which the mutation will be induced, and by the limitations of known oligonucleotide synthesis methods. Factors to consider when designing oligonucleotides for use for oligonucleotide-directed mutagenesis (eg total size, size of the portion bordering the mutation site) have been described by Smith and Gillam. The total length of the oligonucleotide should be such as to optimize stable, unique hybridization at the mutation site, and the 5 'and 3' of the mutation site should be of sufficient size to avoid editing the mutation by the exonuclease activity of the DNA polymerase. Oligonucleotides used for mutagenesis according to the invention typically contain from 12 to 24 bases, preferably from about 14 to 20, and more preferably from 15 to 18 bases. Generally, they contain at least about three bases 3 'of the modified or missing codon.

The method of producing a modified IFN- / 5 gene generally involves inducing site-specific mutagenesis in the IFN- / 1 gene at codon 17 (TGT) using a synthetic nucleotide primer that omits the codon or alters it so that it to code for another amino acid. When threonine replaces cysteine and the primer is hybridized to the nonsense strand of the 1FN- / 1 gene, the preferred nucleotide primer is GCAATTTTCAGACTCAG (highlighting means codon altered). When cysteine removal is desired, the preferred primer is

AGCAATTTTCAGCAGAAGCTCCTG that releases the TGT codon for cys. When cysteine is replaced by a series, one 17-nucleotide primer, GCAATTTTCAGAGTCAG including the AGT codon for serine, is selected. Crossing T -> A at first base in cys 17 codon results in a change in cysteine to serine. It should be noted that when deletions are introduced, an appropriate framework for the DNA sequence for expression of the desired protein must be maintained.

The primer is hybridized to a shear phage, such as Μ13, fd, or X174, in which one strand of the IFN-β gene is cloned. It should be noted that the phage may carry the sense or meaningless strand of the gene. When the phage carries a nonsense chain, the primer is identical to that part of the sense chain that contains the codon it will mutate, except for the non-codon that determines the removal of the codon or triplet that codes for another amino acid. When the phage carries the sense strand, the primer is complementary to that portion of the sense strand containing the codon that mutates, except for a suitable mismatch, in the triplet paired with the codon to be removed. The conditions to be used for hybridization are described by Smith and Gillam. The temperature is typically from 0 to 70 ° C, in particular from 10 to 50 ° C. After hybridization, the primer is expanded to phage DNA by reaction with DNA polymerase 1, T ₄ DNA polymerase, reversible transcriptase or other suitable DNA polymerase. The resulting dsDHK is transformed into closed circular dsflHK by treatment with DNA ligase, for example T ₄ DNA ligase. DNA molecules containing single-stranded zones can be destroyed by S, endonuclease treatment.

Oligonucleotide-directed mutagenesis can be used to produce a mutant 1L-2 gene that encodes a mutein having IL-2 activity, but in which cys 125 is changed to serine 125. The preferred oligonucleotide primer is used to produce the mutant IL-2 gene. when the phage carries the sensitive strand of the gene, it is GATGATGCTTCTGAGAAAAGGTAATC. This oligonucleotide has a C-> G change in the mean base, on the triplet that is coupled to the codon 125 of the IL-2 gene.

The resulting mutational heteroduplex is then used to transform a competent host microorganism or cell.

Replicating the heteroduplex through the host gives offspring from both strands. After replication, the mutant gene can be isolated from the mutant strand progeny, incorporated into a suitable expression vector, and used to transform the appropriate microorganism or host cell. Preferred vectors are plasmids pBR322, pCR1 and variants thereof, synthetic vectors and the like. Suitable host microorganisms are E. coli, Pseudomonas, Bacillus subtilis. Bacillus thuringicnsis, various strains of yeast, Bacillus thermophius, animal cells, such as mouse, rat or Chinese Hamster (CHO) ovaries, plant cells and host plants, animal and plant hosts, and the like. When the selected host is transformed with the vector, suitable promoter operator sequences are also introduced to express the mutein. The hosts can be prokaryotic or eukaryotic cells (methods for inserting DNA into eukaryotic cells are described in PCT Application Nos. US 81/00239 and US81 / 00240, 1981). The preferred hosts are E. coli and CHO cells. The muteins produced according to the invention may be glycosylated or non-glycosylated depending on the glycosylation of the native parent protein and the host microorganism used to produce the mutein. If necessary, the non-glycosylated mutein obtained when E. coli or Bacillus are host microorganisms may possibly be glycosylated in vitro by chemical, enzymatic or other modification known to those skilled in the art.

In the preferred application of the invention with respect to IFN-? the cysteine residue at position 17 of the amino acid sequence of IFN-D shown in Figure 1 is altered in series by a T-> A transition at the first base of codon 17 of the sense strand of the DNA sequence that codes for mature IFN- / 7. Site-specific mutagenesis is induced using a synthetic 17-nucleotide primer GCAATTTTCAGAGTCAG, which is identical to a 17-nucleotide sequence of the IFN-? in the region of code 17, except for a single base mismatch at the first codon 17 base. The mismatch is at nucleotide 12 in the primer. The genetic code is degenerate and many of the amino acids can be encoded by more than one codon. The basic code for serine, for example, is six times degenerate, such that the codons they TCT, TCG, TCC, TCA, AGT and ACG encode for serine. The AGT codon was chosen for convenience. Similarly, threonine is encoded by each of the codons ACT, ACA, ACC and ACG. It has been stated that when a codon is specific for a particular amino acid, it includes all the degenerate codons that encode that amino acid. The 17-mer is hybridized to single-stranded DNA of phage M13, which carries the nonsense IFN gene strand. The oligonucleotide primer is then extended on the DNA using DNA polymerase I-Klenow fragment and the resulting dsflHK is converted to closed circular DNA with T ₄ ligase. Replication of the resulting mutational heteroduplex yields clones from the DNA strand containing the mismatch. Mutant clones can be identified and screened for the presence or absence of specific restriction sites, antibiotic resistance or sensitivity, or by other methods known to those skilled in the art. When cysteine is substituted with serine, the T -> A transition shown in Figure 2 results in the creation of a new Hinfi restriction site in the structural gene. The mutant clone is identified by using an oligonucleotide primer as an indicator in the hybridization screening of the mutant phage plates. The primer will have a single mismatch when it hybridizes to the parent, but there will be an exact match when it hybridizes to the mutant phage DNA, as shown in Figure 2. The conditions of hybridization may therefore be such that the oligonucleotide primer will hybridize to the mutant DNA. , not to the parent DNA. The newly acquired Hinfl site also serves as a means of confirming the single base mutation in 1FN- /? gene.

M13 phage DNA carrying the mutated gene was isolated and distributed in appropriate expression vectors such as plasmid pTrP3 and E. coli strain MM294 transformed with the vector. Suitable culture media for the transformation of transformants and their runs are known to those skilled in the art. The expressed IFN-D mutein is isolated, purified and characterized.

Embodiments of the invention illustrate the invention and illustrate it.

They do not limit its scope. Examples 1 to 11 describe the preparation of IFN-? . Examples 12 to 20 describe the production of IL-2 mutein.

Example 1. Cloning of IFN-? gene in M13 vector.

The use of the M13 phage vector as a source of single stranded DNA template is described by Temple et al, Nature (1982) 296: 537540. The plasmid ρ β ltrp (Figure 3) containing IFN-? The gene under the control of the E. coli trp promoter was digested with the Hindlll and Xholl restriction enzymes. M13mp8 (Messing, Third Cleveland Symposium on Macromolecules: Recombinant DNA 143-153, (1983) DNA replication form (RF) (Figure 4) was digested with Hindlll and Bam HI restriction enzymes and mixed with ρ β ltrp DNA, which is digested beforehand with Hind III and Xho II. The mixture was ligated with T ₄ DNA ligase and 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: 309321) Plates containing recombinant phage (white plates) are taken with a needle, inoculated into fresh culture of JM103 and are mini-preparations of RF molecules obtained from infected cells (HDBirnb oim and J. Doly, Nucleic Acid Res (1979) 7: 1513-1523) RF molecules are digested with different restriction enzymes to identify clones containing IFN-? insert. Restriction map of one such clone (M13-β 1 ) is shown in Figure

5. Single-stranded (SS) phage DNA was obtained from clone M13-β 1 to serve as template for site-specific mutagenesis using a synthetic oligonucleotide.

Example 2. Site specific mutagenesis. Forty picomoles of the synthetic oligonucleotide GCAATTTTCAGAGTCAG (primer) were treated with T ₄ kinase in the presence of 0.1 mM adenosine triphosphate (ATP), 50 mM hydroxymethylaminomethane hydrochloride (trie hydrochloric acid) pH 8.0, 10 mM magnesium pH 8.0, 10 mM magnesium (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 μξ, ss M13- /? 1 DNA in 50 μ \ mixture containing 50 mM sodium chloride, 10 mM tris hydrochloric acid with pH 8.0, 10 mM magnesium dichloride and 10 mM β-mercaptoethanol by heating at 67 ° C for 5 minutes and 42 ° C in 25 minutes. The heated mixture was cooled on ice and then 50 μl of reaction mixture containing 0.5 mM of deoxynucleotide triphosphate (dNTP), 80 mM trisolic acid with pH 7.4, 8 mM magnesium dichloride, 100 mM sodium was added. chloride, 9 units of DNA polymerase 1, Klenow fragment, 0.5 mM ATP and 2 units of T ₄ DNA ligase, incubated at 37 ° C for 3 hours and at 25 ° C for 2 hours. The reaction was terminated by phenol extraction and ethanol precipitation. The DNA was dissolved in 10 mM Tris-hydrochloric acid with 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 / wg / ml ethidium bromide. DNA bands corresponding to the RF forms of Ml3- / 3 1 are eluted from the gel sections by the perchlorate method (Davis et al., Advanced Bacterial Genetics, pp. 178-179 (1980). Eluted DNA is used to transform competent JM103 cells cultured overnight and ssHHK was isolated from the current culture fluid.This ss4HK was used as a template in the second primer extension cycle, the gel purified RF forms of DNA were transformed into competent JM103 cells, spread on agar plates and were incubated overnight to obtain phage plates.

Example 3. Site specific mutagenesis. The experiment of Example 2 was repeated, except that the gene used to modify codon 17 of the IFN- / 3 gene from one that encodes for cysteine into one that encodes a threonine synthetic oligonucleotide primer is GCAATTTTCAGACTCAG.

Example 4. Site specific deletion.

The experiment of Example 2 was repeated, except that the oligonucleotide primer used for deletion of codon 17 of the IFN- / 3 gene was AGCAATTTTCAGCAGAAGCTCCTG.

Example 5. Screening and identification of mutated plaques.

Plates containing mutated Ml3- / 3 1 plaques (Example 1) and two dishes containing non-mutated Ml3- / 3 1 phage plates were cooled to 4 ° C and plates of each plate were transferred to two nitrocellulose filter circles by placing dry filter on agar plate for five minutes for the first filter and 15 minutes for the second filter. The fil60506 was then applied to three filter papers on thick filter paper soaked in 0.2 normal sodium hydroxide, 1.5 M sodium chloride and 0.2% Triton X-100 for five minutes and neutralized by placing on filter paper impregnated with 0 , 5 M Tris-hydrochloric acid with pH 7.5 and 1.5 M sodium chloride over the next five minutes. The filters were similarly washed twice on filters soaked in 2xSSC (standard physiological citrate), dried and baked in a vacuum oven at 80 ° C for two hours. The double filters were re-hybridized at 55 ° C for four hours with 10 ml for filter DNA hybridization buffer (5xSSC) with pH 7.0, 4 x Denhardt solution (polyvinylpyrrolidine, ficol and bovine serum albumin, 1 x = 0.02% of each), 0.1% sodium dodecileulphate (SDS), 50 mM sodium phosphate buffer with pH 7.0 and 100 / rg / ml DNA from trout denatured semen. The ³² P labeled indicator was prepared by kinase of the oligonucleotide primer labeled with ³² P ATP. The filters were hybridized to 3.5x10 cpm / ml from ³² P labeled primers in 5 mi for DNA hybridization buffer filter at 55 ° C for 24 hours. The filters were washed at 55 ° C for 30 minutes, each in a wash buffer containing 0.1% SDS and reducing amounts of SSC. The filters were washed initially with a buffer containing 2 x SSC, and control filters containing unmutated M131 plates were checked for radioactivity with a Geiger counter. The concentration of SSC was reduced stepwise and the filters were washed until no radioactivity was detected on the control filters with unmutated M13-β1 plates. The lowest SSC concentration used is 0.1 x SSC. The filters are air-dried and autoradiographically at -70 ° C for 2-3 days. 480 plates of mutated M13-β1 and 100 non-mutated control plates were screened with the kinase oligonucleotide probe. None of the control plates hybridized with the sample, whereas the mutated M13-β1 plates hybridized with the indicator.

One of the five M13- / J1 mutant plates (M13-SY2501) was separated and inoculated into JM103 culture. From the current liquid was obtained cbDNA and from the precipitate double-stranded (ds) flHK. SSflHK was used as a matrix for dideoxy sequencing of the M13 universal primer clone. The result of the sequence analysis is shown in Figure 6 and confirms that the TGT evs codon has been converted to an ATG ser codon.

Example 6. Expression of Mutated IFN-?

in E. coli.

PF DNA of Μ13-SY2501 was digested with Hind III and XhoII restriction enzymes and the 520 bp insertion fragment was purified on a 1% agarose gel. Plasmid pTrP3 containing the E. coli trp promoter (Figure 7) was digested with the Hind III and BamHI enzymes, mixed with the purified M13-SY2501 DNA fragment and ligated in the presence of T1 ligase. The ligated DNA was transformed into E. coli strain MM294. Ampicillin-resistant transformants are screened for tetracycline sensitivity. Plasmid DNAs from five ampicillin-resistant [tetracycline] tetracycline-sensitive clones were digested with Hinfl for screening for M13-SY2501 insertion. Figure 8a shows the Hinf1 restriction map of one of the clones (pSY2501) and compares it with the Hinf I map of the original IFN-β clone p β trp. Is there an additional Hinfl site in pSY2501 separating 197 bp IFN- /? the inner fragment of the 169 bp fragment and the 28 bp fragment (Figure 86). The restriction map of clone pSY2501 is shown in Figure 9. The entire DNA sequence of the mutant 1FN-β gene is shown in Figure 10, together with the expected amino acid sequence.

The plasmid, designated clone pSY2501, has been deposited with the Agricultural Research Collection (NRRL), Fermentation Laboratory, North Regional Research Center, Administration of Science and Training, Agricultural Ministry, 1815 North University St., Peoria, 111.60604 on March 30, 1983 and is designated by IASB No. 1533 and NRRL No. B-15356.

Cultures of pSY2501 and p / ltrp, which include their progeny, were cultured to optical density (OD.J of 1.0. Cell-free extract obtained and tested IFNβ antiviral activity on GM2767 cells by microtiter assay. pSY2501 exhibited three to four times more activity than ρβ ltrp (Table 1), indicating that the pSY2501 clone either synthesized more protein exhibiting IFN-? activity, or that the resulting protein had a higher specificity

Table A

activity. Table I Anti-viral activity extract (one / ml) pSY2501 6 x 10 ' p / 91 trp 1 x 10 ' ptrp3 (control) 30

In order to determine whether clone pSY2501 synthesized several times more active protein, extracts from both clones were electrophoresed on an SDS polyacrylamide gel together with a control extract, and the gel was stained with goat blue to express the proteins. As shown in Figure 11, there is only one protein band corresponding to about 18,000 daltons of protein, which is present in the extracts of clones pSY2501 and p / Jltrp but not in the control extract of ptrp3. This protein, which has a molecular weight of about 20,000 daltons but shows a gel migration pattern of 18,000 daltons, has previously been shown to be IFN- / 3 by purification of this protein from ru31 trp extracts. As the p /? 1 trp extracts contain less of this protein, the specific activity of the protein in the extracts of clone pSY2501 is higher than that of the clone p / Jltrp.

Example 7. The plasmid pSY2501 was transformed into a competent sub-variant of E. coli strain MM294, designated MM294-1. A sample of the transformant obtained was deposited with the American Type Culture Collection 12301 Parklawn Dr., Rockville, Md. 20852 in the United States on November 18, 1983 under ATCC No. 39 517.

Example 8. Production of IFN-β _{scr | 7}

IFN-β is recovered from E. coli that have been transformed to produce 1FN-. E. coli are cultured in the following culture medium (see table) up to an OD of ΙΟΙ! at 600 nm (dry weight 8.4 g / l).

Ingredient Concentration nh ₄ cl 20 mM K, SO ₄ 16.1 mM kn ₂ ro ₄ 7.9 mM Na, HPO ₄ 12.2 mM MgSO ₄ .7H ₂ O 3 mM Na ₃ citrate. 2H ₂ O 1.5 mM MnSO, .4H Ο 30 μΜ ZnSO ₄ .7H ₂ O 30 μΜ CuSO, .5H Ο 3 μΜ L-tryptophan 70 mg / L FeSO..7H, O 72 μΜ Thiamine HCI 20 mg / L Glucose 40 μg / L

The yield of 9.91 (9.9 kg) of transformed E. coli was cooled to 20 ° C and concentrated by passing through a filter with a transverse flow at an average pressure of about 110 K and a constant flow rate of the filtrate of 260 ml / min at filtrate weight 8.8 kg. The concentrate (approximately 1 I) was drained into a vessel and cooled to 15 ° C. Cells in the concentrate were destroyed by passing the concentrate through a MantonGaulin homogenizer at 5 ° C and about 69,000 Kg. The homogenizer is washed with one liter of phosphate buffered saline, pH

7.4 (PBS) and the washing liquid was added to the homogenization to obtain a final volume of two liters. This amount is subjected to continuous centrifugation at 12,000 x g and a flow rate of 50 ml / min. The precipitate was separated from the current liquid and resuspended in four liters of PBS containing 2% SDS. This suspension was stirred at room temperature for 15 minutes, after which no more suspended material was visible. The solution was then extracted with 2-butanol at a volume ratio of butanol to a solution of 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 to give 21.3 g of protein. It is resuspended in distilled water at a volume ratio of 1:10.

The reconstituted product was tested for human IFN- / 3 activity by a viral cytopathic effect (CPE) assay. The test is carried out in microtiter plates. Fifty microlitres of minimum essential medium was placed in each well, placing 25 μΐ of the sample in the first well and serial 1: 3 volume dilutions in the next wells. Virus (Vesicular stomatitus), cells (human fibroblast GM-2767 line) and comparative IFN-β controls were included in each plate. Used for comparison 1FN- /? is 100 units per ml. The tiles are then irradiated for 10 minutes. After irradiation, ΙΟΟμΙ of the cell suspension (1.2 x 10 ^s cells / ml) was added to each well and the plates incubated for 18-24 hours. A viral suspension in the amount of a plate-forming unit per cell was added to each well except the control. The plates were then incubated until the viral control showed 100% CPE. This happens normally 18 to 24 hours after the addition of the viral solution. The test results are interpreted with respect to the location of the well with 50% CPE relative to the comparative IFN- / J control. From this point the interferon titer is determined for all the samples on the plate. The specific activity of the recovered product was determined to be 5 x 10 'units / mg.

Example 9. Acid precipitation and chromatographic purification.

The process of Example 8 was repeated, except that after extraction and separation of the aqueous and organic phases and mixing of the organic phase with PBS at a volume ratio of 3: 1 the pH of the mixture was reduced to about 5 by the addition of glacial acetic acid. The resulting precipitate was separated by centrifugation at 10,000 to 17,000 xg for 15 minutes and the residue was redissolved in 10 wt% / volume SDS and 10 mM sodium acetate buffer, pH 5.5, and heated at 80 ° C for five minutes.

The solution was then placed on a Brownlee RP-300, 10 μΜ Aquapore 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 was performed by ultraviolet absorption at 280 nm. Program for dissolving a linear gradient of 0 ° _o buffer B to 100% buffer B for three hours. The fractions containing the highest interferon activity were separated and the specific activity of the interferon collected was reported to be 9.0 x 10 ⁷ to 3.8 x 10 'international units per milligram protein compared to 2 x 10' units / mg for natural IFN- /? .

EXAMPLE 10 Biochemical Characteristics of IFN-J 'ser 17

The amino acid composition was determined after 24-72 hours hydrolysis of 40 / g samples of IFN- / 1 in 200 μΐ of 5.7 N hydrochloric acid, 0.1% phenol at 108 ° C. Proline and cystine are determined in the same way after oxidation with perfluoric acid, in which case phenol is removed upon hydrolysis. Tryptophan was analyzed after 24 hours hydrolysis of 400 µg sample in 5.7 N hydrochloric acid, 10% mercaptoacetic acid (without phenol). The analysis was performed in a Beckman 121 MB amino acid analyzer using a single AA10 resin column.

The amino acid composition calculated from representative 24-, 48- and 72-hour hydrolyses of purified 1FN- _{mg 17} matches the expected DNA-based sequence of the cloned 1FN gene, except for the missing N-terminal 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 ultrasphere ODS column (4.6 x 250 mm) at 45 ° C, eluting for 1.3 min with 40% buffer A and 8.4 min with 40 to 70% buffer B, wherein buffer A is 0.0115 M sodium acetate, 5% tetrahydrofuran (THF) pH 5.11, and buffer B is 10% THF in acetonitrile.

The N-terminal amino acid sequence of IFN- / 3 and ₁₇ coincides with the expected DNA-based sequence, except in the absence of the N-terminal methionine.

Example 11. Alternative production of IFN-β _{ig 17} and purification method.

E. coli transformed with pSY2501 are cultured in the following medium:

Table B

Ingredient Approx. starting concentration Na ₃ citrate. 2H ₂ O 3 mM KN ₂ PO ₄ 30 tM (NH ₄ ) ₂ SO ₄ 14 tM MgSO ₄ .7H ₂ O 46 μΜ ZnSO ₄ .7H ₂ O 46 μ Μ CuSO..5H, O 1-2 μΜ L-tryptophan 351 μΜ FeSO ₄ .7H ₂ O 74 Μ Thiamine 0,002% Glucose 0.5%

If necessary, add Dow Corning defoaming agent - 25% glycol solution, 50% glucose solution and 5-N 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. Optical density and residual glucose measurements are taken at 14 hours and at approximately 1 hour intervals thereafter. Yield is collected when glucose consumption reaches 40 ± 6 g / l (OD at 680 nm = 10 - 11).

The collected material is concentrated about three times by circulating through a microporous cross-flow filter. The concentrated cells were diafiltered against deionized water, while the collected material was concentrated 4-5 times. The cells are then destroyed by passing through a Manton Gaulin homogenizer at about 4.1 to 5.5 wt. After the initial passage, SDS sodium phosphate buffer was added to a final concentration of 2% SDS, 0.08 m sodium phosphate and homogenization continued for one hour. Solid DTT was then 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 2-butanol to a 1: 1 suspension in a static mixer. The mixture was then centrifuged and the 2-butanol-rich phase was collected.

The 2-butanol-rich phase is mixed with

2.5 volumes of 0.1% SDS in phosphate buffered saline (PBS). Solid 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 collected and resuspended in PBS + 10% SDS with a pH set to 8.5 ± 0.1 with 1-Ν sodium hydroxide. Solid DTT was added to a final concentration of 100 mM and the suspension was heated to 90 ± 5 ° C for 10 minutes. The suspension was then cooled to -25 ° C, the pH adjusted to 5.5 ± 0.1 with glacial acetic acid, and the solution filtered.

The solution was then placed in Sephacryl

The S-200 precolumn and the fractions exhibiting the highest interferon activity were collected in one place and concentrated by ultrafiltration. The concentrate was oxidized by adding equimolar amounts of protein and iodobenzoic acid to a reaction vessel containing 2 µM sodium pyrophosphate, 0.1% SDS, and 1 mM EDTA. During oxidation, the pH was maintained at 9.0 ± 0.1 with 0.5-1 sodium hydroxide and adjusted to 5.5 ± 0.2 when oxidation was complete. After oxidation, the concentrate is again subjected to ultrafiltration by separation at 10 Kdal molecular weight.

The concentrate was placed in the main Sephacryl S-200 column and fractions were analyzed by SDS-PAGE to determine those containing no high molecular weight contaminants. These fractions are separated and passed through the ultrafiltration device. The filtered concentrate was fractionated on a Sephadex G-75 column. SDSPAGE analysis of fractions was performed to determine those that did not contain low or high molecular weight pollutants. These fractions are separated for desalination.

A Sephadex G-25 column calibrated with 1 mM sodium hydroxide was charged to the fractions collected by the Sephadex G-25 column using distilled water, adjusted to pH 10.8-11 with 50% sodium hydroxide. The purified product is collected to a blank. This desalinated, purified 1FN-β mutein can be prepared by known routes for therapeutic use.

IFN-J * ser P Biological Tests

Antigene comparison.

1FN- / J is compared antigenically with IFN- /? produced by diploid fibroblasts using viral neutralization tests. A polyvalent IFN- / 3 antiserum from diploid fibroblasts is obtained in rabbits. This antiserum blocks the antiviral activity of both dibloid fibroblast IFN-D and 1FN- / J in viral neutralization assays, indicating that the two proteins are antigenically indistinguishable.

Antivirus activity

Purified IFN-? _Kr17 is compared in antiviral activity with naturally produced IFN- ?. 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 proteins is comparable.

Anti-proliferative activity The anti-proliferative activity of IFN-? _{ser | 7} for continuous cell lines compares to that of naturally produced 1FN-? .

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 5 proteins.

Stimulation of natural killer (NK) cells

IFN- /? _{scr 17} to stimulate an NK cell (spontaneous cell cytotoxicity) was tested. Separated with Ficoll-hypaque human peripheral mononuclear cells (PMC) or NK enriched lymphocytic preparations (devoid of monocytes by plastic adhesion and OKT3-positive 15 T cells by treatment with OKT3 antibody plus complement) were incubated overnight in culture medium, containing different concentrations of IFN-? _{ser p} . ^{* * * * * * * * * * * * * 51} Cr target cells are incubated with effector cells (at effector cells to target cells ratio = 50: 1) for 2 to 4 hours. NK cell cytotoxicity is determined by measuring the amount of labeled substance in the medium. The results of this test are shown in the following table:

Table C

Cell Effective NK cell cytotoxicity by interferon (specifically target cell percentage release of ⁵¹ Cr-SFM) 1FN (units / ml)

0 10 30 100 300 T24 PMC 7.23 ± 5.1 23.1 ± 4.4 24.4 ± 1.1 34.1 ± 2.5 50.0 ± 2.0 Chang PMC 4.7 ± 0.5 7.2 ± 0.8 9.5 ± 1.7 15.9 ± 1.3 21.9 ± 1.4 Chang NK Enr 19.2 ± 4.6 39.4 ± 4.1 ND 54.2 ± 6.1 ND K562 NK Enr 41.0 ± 4.6 48.4 ± 3.6 ND 62.2 ± 3.5 ND

As shown in target cell killing more efficiently than treated with IFN _{| 7} cells than in untreated cells.

Clinical trials

Phase I of the clinical trials is to confirm the safety of IFN- _{kg | 7} in humans.

These results include administration of the protein to patients intramuscularly and intravenously at doses ranging from 1 x 10 ⁵ units (1 g protein) to 400 x 10 'units. No adverse effects occurred in the initial phase I of the clinical trials.

As indicated, the preparations of β _{κί 17} exhibit specific activity at a level very close to or better than that of natural IFN-β. 1FN- / 1, there are no free sulfhydryl groups, however, shows an -SS bond between the only remaining cysteines at positions 31 and 141. The protein does not easily form oligomers and exists mainly in the monomeric form. IFN- /? _jag17 obtained according to the invention can be used alone or as a mixture of various forms for the preparation of pharmaceutically acceptable preparations in inert, non-toxic, non-invasive allergies, physiologically acceptable carriers for clinical and therapeutic use in cancer therapy or in conditions in which it is required interferon therapy and viral infections such as herpes simplex virus I and II, viral hepatitis B, viral colds and viral rhinitis. Carriers include, but are not limited to, distilled water, saline, Ringer's solution, Hank's solution, and the like. Non-toxic stabilizers and solubilizers such as dextrose, HSA (human serum albumin) and the like may be optionally included. The therapeutic compositions may be administered orally or parenterally, for example intravenously, intramuscularly, intraperitoneally and subcutaneously. The modified IFN- / 3 formulations of the invention can also be used for topical administration in suitable carriers commonly used for such purposes. The 1FN- / 3 mutein can be administered either topically or systemically, alone, mixed or in combination with other therapeutic agents such as acyclovir for prophylactic or curative purposes. The dose at which mutein is administered to humans will depend on whether it is administered continuously (including intermittently) or as a bolus. When administered continuously, the amounts are usually lower than those administered as a bolus. The dose is typically from about 1 x 10 ^s to 1 x 10 'units, preferably about 1 x 10 ⁶ to 1 x 10'.

The principal advantages of the IFN- / 3 mutein described are the elimination of a free sulfhydryl group at IFN- / 3 position 17, thereby forcing the protein to form the correct disulfide bonds between cys 31 r cys 141 and to adopt the conformation required for full biological activity. The increased specific activity of 1FN- / 3 allows the use of smaller doses for therapeutic use. Due to the deletion of cysteine at position 17 and the elimination of the free SH group, the 1FN- [3 _{ser | 7} protein does not form dimers and oligomers as easily as the microbially produced IFN- ?. This facilitates the purification of the protein and increases its stability.

Example 12. The nucleotide sequence for a cDNA clone encoding for a human

1L.-2, methods for producing II.-2 cDNA banks and screening for IL-2 are described by Taniguchi et al., Nature, (1983) Volume 24.

page 308.

cDNA banks enriched with potential 1L-2 cDNA clones were obtained from 1L-2 enriched and RNA [fraction] obtained from induced peripheral blood lymphocytes (PBL) and from Jurkat cells by conventional methods. Enrichment of mRNA with IL2 information is performed by fractionating the mRNA and identifying the fraction having IL-2 mRNA activity, injecting fractions into oocytes of Xenopus laevis and examining oocyte lysates for IL-2 activity on HT-2 cells ( Watson, J. Exp. Med, (1979) 150: 1570-1519 in Gillis et al., Immun. (1978) 120: 2027-2032.

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

1L-2 cDNA banks are screened by colony hybridization method. Each microtiter plate was replicated in double nitrocellulose filter paper (S S type BA85) and colonies were cultured at 37 ° C for 14-16 hours on L agar containing / rg / ml ampicillin. The colonies were lysed and DNA was fixed to the filter by successive treatment for 5 minutes with 500 mM sodium hydroxide,

1.5 M sodium chloride and washed twice for 5 minutes with 5 x standard physiological citrate (SSC). The filters were air-dried and baked at 80 ° C for 2 hours. The double filters were re-hybridized at 42 ° C for 6-8 hours with 10 ml for filter DNA hybridization buffer (50% formamide, 5 x SSC. PH 7.0, 5 x Denhardt solution (polyvinylpyrrolidine, ficol and bovine serum albumin, 1 x = 0.2% of each) 50 mM sodium phosphate buffer with pH 7.0, 0.2% SDS, 20 µg / ml Poly U and 50 µg of denatured trout semen.

A labeled ³² P 20-dimensional oligonucleotide probe was prepared based on the 1L2 gene sequence described by Taniguchi et al. (See above). The nucleotide sequence of the sample is

GTGGCCTTCTTGGGCATGTA.

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

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

The 1L-2 gene was cloned into M13tr9 as described in Example 1 using plasmid pLWl (Figure 12) containing the 1L-2 gene under the control of the E. coli trp promoter. One sample of pLW1 was deposited with the American Type Culture Collection in August 1983 and is designated ATCC No. 39 405. The restriction map of one clone (designated M13-IL 2) containing IL-2 insert is shown in Figure 13. End-chain phage DNA is derived from clone M13-1L 2 to serve as an template for oligonucleotide-directed mutagenesis.

Example 15. Oligonucleotide-directed mutagenesis

As indicated, IL-2 contains a cysteine residue at amino acid positions 58, 105 and 125. Based on the nucleotide sequence of the portions of the IL-2 gene that contains the codons for these three cysteine residues, three oligonucleotide primers are identified and synthesized for mutation of codons for these residues to codons for series. The oligonucleotides are as follows: CTTCTAGAGACTGCAGATGTTTC (DM27) for cys 58 change;

CATCAGCATACTCAGACATGAATG (DM28) for modification of cys 105 and GATGATGCTCTGAAAAGGTAATC (DM29) for modification of cys 125.

Forty picomoles of each oligonucleotide were kinased individually in the presence of 0.1 mM ATP, 50 mM trisolic acid, pH 8.0, 10 mM magnesium dichloride, 5 mM DTT, and 9 units of T ₄ kinase in 50 μ 1 at 37 ° C. Each of the kinase primers (10 picomoles) was hybridized to 2.6µg of ssM13IL-2 DNA in a 15µl mixture containing 100 mM sodium chloride, 20 mM trisolic acid, pH 7.9, 20 mM magnesium dichloride and 20mM / Tmercaptostanol by heating at 67C for 5 minutes and 42C for 25 minutes. The heated mixtures were cooled on ice and then adjusted to a final volume of 25μ 1 of the 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 / l - mercaptoethanol, 5 units of Klenow polymorphase I fragment, 0.5 mM ATP and 2 units of T ₄ DNA ligase incubated at 37 ° C for 5 hours. The reactions were interrupted by heating to 80 ° C and the reaction mixtures used to transform competent JM103 cells spread on agar plates and incubated overnight to obtain phage plates.

Example 16. Screening and identification of mutant phage plates

Plates containing mutated M13-IL 2 plates and two plates containing non-mutated M13-IL 2 phage plates were cooled to 4 ° and phage plates from each plate were transferred to two nitrocellulose filter circles by spreading a dry filter on agar plate five minutes for the first filter and 15 minutes for the second filter. The filters were then placed on thick filter paper soaked in 0.2-N sodium hydroxide, 1.5 M sodium chloride and 0.2% Triton for five minutes and neutralized by spreading on filter paper soaked in 0.5 M Tris-hydrochloric acid pH 7.5, 1.5 M sodium chloride over the next five minutes. The filters were washed in the same way twice on filters soaked in 2 x SSC, dried and baked in a vacuum oven at 80 ° C for 2 hours. The double filters were re-hybridized at 42 ° C for 4 hours with 10 ml for filter DNA hybridization buffer (5 x SSC, pH 7.0, 4 x Denhardt [polyvinylpyrrolidine] polyvinylpyrrolidone solution, ficol and bovine serum albumin, 1x = 0.02 % of each), 0.1% SDS, 50 mM sodium phosphate buffer, pH 7.0 and 100 µg of denatured trout semen DNA. ³² P-labeled indicators were prepared by kinating the oligonucleotide primers into labeled ATP. The filters were hybridized to 0.1 x 10 ⁵ cpm / ml of the labeled ³² P primers in five milliliters per liter of DNA hybridization buffer at 42 ° C for 8 hours. The filters were washed twice at 50 ° C for 30 min each in wash buffer containing 0,1% SDS and 2 x SSC, and twice at 50C for 30 minutes each with 0,1 ° _v SDS and 0.2 x SSC. The filters are air-dried and autoradiographically at -70C for two to three days.

As the oligonucleotide primers DM28 and DB29 are designed to create a new Ddel restriction site in the mutant clones (Figure 14), RF-DNA from a number of clones that hybridize to each of the three kinase primers is digested with the Ddel restriction enzyme. One of the mutant M13-IL 2 plaques, which hybridizes to the DM28 primer and has a new Ddel restriction site (M13-LW44), is taken with a needle and inoculated into JM103 culture, with ssflHK obtained from the culture supernatant, dsRF- DNA is derived from the cell mass. Similarly, a plate that hybridizes to the DM29 primer is taken with a needle (M13-LW46) to produce ssflHK and RF-DNA. The DM27 oligonucleotide primer was milled to create a new PstI restriction site instead of the Ddel site. Therefore, the plates that hybridize to this primer are screened for availability on a new PstI site. One such phage plaque is identified (M13-LW42) to produce ssflHK and RF-DNA. The DNAs from these three clones were sequenced to confirm that the target TGT codon for cysteine was converted to the TST codon for serine.

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

The RF DNAs of M13-LW42, M13-LW44 and M13-LW46 were digested, each with Hind III and Ban II restriction enzymes and the insertion fragments purified from a 1% agarose gel. Similarly, the plasmid pTrP3 (Figure 7) was digested with HindIII and Banll, the large plasmid fragment containing the trp promoter purified on an agarose gel and then ligated with each of the insertion fragments isolated otM13-LW42, M13-LW44u LW46. The ligated plasmids were transformed into competent E. coli strain K _{| 2} MM294. Plasmid DNAs from these transformants were analyzed by preparation of restriction enzyme maps to confirm the availability of plasmids pLW42, pLW44, and pLW46. Figure 14 is a restriction map of pLW46. When each of these individual clones was cultured in the absence of tryptophan to induce the trp promoter and cells free of the extract were analyzed on SDS polyacrylamide gels, all three clones pLW42, pLW44 and pLW46 were found to synthesize 14.5 kd protein , similar to that established in the positive control, pLW21, which was found to produce one 14.4 kd IL-2 protein. When the same extracts were tested for IL-2 activity on murine HT-2 cells, only the pLW21 (positive control) and pLW46 clones displayed significant IL-2 activity (Table II), indicating that cys 58 and cys 105 were significant for biological activity and their change in serines (pLW42 and pLW44, respectively) leads to a loss of biological activity. Cys 125, on the other hand, is not significant for biological activity because its change to ser 125 (pLW46) has no effect on biological activity.

Table 2

Clones of IL-2 activity (μ / ml) pIL2-7 (negative control) I pLW21 (positive control) 113 000 pLW42 660 pLW44 1 990 pLW46 123 000

Figure 15a shows the nucleotide sequence of the pLW46 clone coding chain. Compared to the coding chain of the natural human IL-2 gene, clone pLW46 has a single G-> C base change at nucleotide 374. Figure 156 shows the corresponding amino acid sequence of the 1L-2 mutein encoded by pLW46. The mutein is designated desalanin (ala) IL-2 _serl2J . When compared to native IL-2, the mutein contains serine instead of cysteine at position 125, the initial N-terminal methionine (which is altered) and lacks the native N-terminal alanine of the natural molecule.

One sample of E. coli K _{| 2} strain MM294 transformed with pLW46 was deposited with the American Culture Type Collection on September 26, 1983 and designated ATCC No. 39 452.

Examples 18 and 19 describe the preparation of an alternative and preferred vector for the expression of alanyl (ala) IL-2 ^r ser125

Example 18. Preparation of AIa-IL-2 Expression Vector pLW32

One codon (GCG) for alanine is inserted (insertion) immediately after the start codon of the IL-2 gene of pLW1 by oligonucleotide-directed mutagenesis as follows. The 5GAAGTAGGCGCCATAAG-3 'oligonucleotide primer is kinase, hybridized to ssM13-lL-2 DNA and expanded by the general method described in Example 15 to form a mutant heteroduplex. In addition to the insertion of the GCG codon, mutagenesis gives rise to a new Narl restriction site in the gene. The heteroduplex was converted into a closed circular heteroduplex and the circular heteroduplexes were used to transform competent JM103 cells, spread on agar plates and incubated as in Example 15. The plates were screened to identify the mutant M13-IL 2 by the method of Example 16. E a mutant phage identified as M13-LW32 was selected for further cloning to produce RF DNA. Figure 16 is a diagram of the plasmid pLW32.

Example 19. Preparation of Ala-IL-2 _serl2J expression clone pLW55

The RF DNA of M13-LW46 (Examples 16 and 17) was digested with Xbal and PstI and a 530 bp fragment containing the carboxy-terminal coding region of the IL-2 _scrl2j gene was purified from agarose gel. Similarly, pLW32 was digested with Xbal and PstI and the large fragment consisting of the plasmid vector and the ala-IL-2 N-terminal coding sequence was purified. The two purified DNA fragments are collected in one place and ligated with T ₄ DNA ligase. The ligated DNA was transformed into competent E. coli K-12 strain MM294. Tetracycline resistant transformants were analyzed by preparation of a restriction enzyme cartridge for the presence of a plasmid containing alalL-2 _{scr | 2} , a gene identified as pLW55, which has a new Ddel site not detected in pLW32. Figure 17 is a diagram of pLW55. Cell-free bacterial extracts containing pLW55 were found to exhibit more than 10 'units of IL-2 activity per milliliter in HT-2 cell assay. J. Watson (see above) and S. Gillis (see above). The Ala-IL-2 _wrl2J protein is identical to the IL2 _ser i25 molecule shown in Figure 15 (6), except that the first includes the native N-terminal alanine of the natural molecule.

A sample of E. coli K-12 strain MM294 transformed with pLW55 was deposited with the American Culture Type Collection on November 18, 1983 and is designated ATCC No. 39 516.

Example 20. Preparation and purification of Ala-IL-2 ...

ser125

E. coli transformed with pLW55 is se

cultured in a fermenter containing the following environment: Table D (NK ₄ ) ₂ SO ₄ 150 tM KN ₂ PO ₄ 21.6 tM At ₃ citrate 155 tM ZnSO ₄ .7H ₂ O 30 μΜ MnSO ₄ .H ₂ O 30 μΜ CuSO ₄ .5H ₂ O ΙμΜ pH maintained 6.50 with sterile 2.5 N sodium hydroxide. Sterile additives (after autoclaving) MgSO ₄ .7H ₂ O 3 tM FeSO ₄ 100 μΜ L-tryptophan 14 mg / l Thiamensolic acid 20 mg / l Glucose 5 g / l Tetracycline 5 mg / l Ethanol 2% Casino acids 2%

Dow Corning defoamer, 20% polypropylene glycol solution, 50% glucose and 5 N potassium hydroxide are added as needed.

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. Casino acids (20% stock solution) to a concentration of 2% are added when OD _6g0 is about 10. Material collection is done three hours after OD has reached 20.

The collected material was concentrated and homogenized as in Example 11. After heat treatment, DTT material was centrifuged and the resulting paste residue was extracted with urea to a final concentration of 4M. The suspension is centrifuged and SDS is added to the solid precipitate to a concentration of 5 O ·

The solution was applied to a Sephacryl S-200 column and the fractions containing 1L-2 separated (according to SDS-PAGE). The separated fractions were applied to a Whatman M-40 column filled with 18 micron Vydac C ₄ silica gel with a pore size of 300 A calibrated with 0.1% TFA. The IL-2 mutein was eluted with a gradient of 40% to 60% 2-propanol containing 0.1% TFA for 160 minutes. The fractions exhibiting the highest 1L-2 activities were pooled and found to have specific activities comparable to those of native 1L-2.

1L-2 muteins, in which the cysteine at position 125 is replaced by another amino acid, while the mutein 1L-2 _serl2J retains its activity. They may therefore be incorporated into compositions and used in the same way as natural IL-2. Accordingly, such 1L-2 muteins are used to diagnose and treat (locally or systemically) bacterial, viral, parasitic, protozoal and fungal infections, to enhance cell-mediated cytotoxicity, to stimulate lymphokinase-activated active killer cells, to mediate the restoration of the immune function of lymphocytes, to enhance alloantigen reactivity, to facilitate the restoration of immune function in conditions of acquired immune deficiency, to restore normal immunofunction in the elderly and animals to perform diagnostic studies such as those using enzymatic amplification, radiolabeling, radiography and other methods known to those skilled in the art, to monitor IL-2 levels in disease states, to initiate growth of In vitro T cells for therapeutic and diagnostic purposes for blocking receptor sites for lymphokines and in various other applications in therapy, diagnosis and research. Various therapeutic and diagnostic uses of human IL-2 have been studied and described by Rosenberg et al., Mazumder et al., Grimm et al. IL-2 muteins can be used alone or in combination with other immunologically appropriate B or T cells or other therapeutic agents. Examples of suitable cells are B or T cells, natural killer cells and the like, and exemplary therapeutic reagents that can be used in combination are the polypeptides of the invention are various interferons, in particular gamma interferon. growth factor for B cells, IL- and the like. For therapeutic or diagnostic use, they may be prepared with non-toxic, non-allergenic, physiologically acceptable carriers such as distilled water, Ringer's solution, Hank's solution, saline and the like. Administration of the 1L-2 muteins to humans or animals may be orally or intraperitoneally, intramuscularly or subcutaneously, as appropriate by the physician. The amount of 1L-2 mutein to be administered is usually between 1 x 10 ⁴ and 2 x 10 'units.

Modifications to the disclosed embodiments of the invention known to those of skill in the field of genetic engineering, protein chemistry, medicine, and related fields are also encompassed within the scope of the following claims.

Claims (10)

Claims 1. Human recombinant interleukin- 2 mutein is omitted, optionally substituted by a neutral amino acid, cysteine at position 125, numbered according to natural human interleukin-2, exhibiting the biological activity of natural human interleukin- 2. The mutein of claim I, wherein the neutral amino acid is serine. 3. Human recombinant alanine-interleukin-2- _{serine 12J} mutein. 4. Human recombinant des-alanylinterleukin-2- _{serine 2J} mutein. 5. Human recombinant interleukin- 2- mutein, with the biological activity of natural human interleukin-2, which has the amino acid sequence shown in Figure 15 (b) with or without the N-terminal methionine. A mutein according to claims 1, 2, 3, 4 or 5 which is non-glycosylated. 7. Composition for the diagnosis or therapeutic treatment (local or systemic) of bacterial, viral, parasitic, protozoal and fungal infections, to enhance cell-mediated cytotoxicity, to stimulate lymphokine-activated killer cell activity, to mediate the restoration of function and lymphocytes, to enhance alloantigen reactivity, to facilitate the restoration of immune function in conditions of acquired immune deficiency, to restore normal immune function in the elderly, and animals to develop diagnostic studies such as those using enzyme 15 amplification, radiolabeling, radiography to monitor interleukin-2 levels in disease states, to initiate T-cell growth in vitro for diagnostic and therapeutic purposes to block receptor sites 20 for lymphokines, characterized in that it comprises (a) an effective amount of recombinant human interleukin-2 mutein in which the cysteine residue at position 125, numbered 25 wound according to natural human interleukin-2, is [removed] substituted tral amino acid, said mutein exhibits the biological activity of native human interleukin-2; and (b) an inert, non-allergenic, pharmaceutically acceptable carrier. Composition according to claim 7, characterized in that the mutein is alanylinterleukin-2- ₍ . _{With [) and || 2} , or des-alanine-interleukin-2-. _The carrier is from the group consisting of distilled water, Ringer's solution, Hank's solution and saline. A composition comprising (a) a recombinant human interleukin-2 mutein in which the cysteine residue at position 125 numbered according to natural human interleukin-2 is [removed or] substituted with a neutral amino acid and exhibits biological activity of natural human interleukin-2 and (b) a polypeptide selected from the group consisting of interferon gamma, a growth factor for B cells and IL-1. Composition according to claim 9, characterized in that the mutein is alanine-interleukin-2 or des alanine-in ^g series 115 terleukin-2 ^g series 125 Attachment: 17 figures. 5 10 1520 MetSerTyrAsnLeu LeuGlyPheLeuGln ArgSerSerAsnPhe GlnCysGlnLysLeu 25 30 3540 LeuTrpGlnLeuAsn GlyArgLeuGluTyr CysLeulysAspArg MetAsnPheAspIle 45 50 5560 ProGluGluIleLys GlnteuGlnGInPhe GlnLysGluAspAla AlaLeuThrlleTyr 65 70 7580 GluMetLeuGlnAsn IlePheAl allePhe ArgGlnAspSerSer SerThrGlyTrpAsn 85 90 95100 GluThrlleValGlu AsnLeuLeuAlaAsn YalTyrHIsGlnlle AsnHIsLeuLysThr 105 110 115120 ValLeuGluGluLys LeuGluLysGluAsp PheThrArgGlyLys LeuMetSerSerLeu 125 130 135140 HIsleulysArgTyr TyrGlyArglleLeu HisTyrLeuLysAla LysGluTyrSerHIs 145 150 155160 CysAlaTrpThrlle Vai ArgValGluIle LeuAgAsnPheTyr PhelleAsnArgLeu 165 170 175180 ThrGlyTyrLeuArg Asn—