JPH0380475B2 - - Google Patents

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides modified interleukin-2 in which the cysteine residue at position 125 of natural interleukin-2 is replaced by another amino acid residue.
This invention relates to a DNA encoding this DNA, a plasmid containing this DNA, and E. coli transformed with this plasmid. (Prior art and problems to be solved by the invention) Biologically active proteins produced microbiologically using recombinant DNA (rDNA) technology contain cysteine residues, Free to form undesired inter- or intra-molecular bonds, although not essential to the The present invention produces interleukin-2 that is improved so that undesirable binding does not occur by changing a part of the amino acid sequence of natural interleukin-2 that may cause such undesired binding. We provide a genetic system for this purpose. (Means for Solving the Problems) The present invention provides directed mutation (directed mutation).
mutagenesis), the parent structural analogue (parent)
Concerning the production of mutationally modified biologically active proteins that retain the activity of analogs) but lack intermolecular disulfide bonds or the ability to form undesired intramolecular disulfide bonds. ” (mutein). “Glossary of Genetics and Cytogenetics”
Genetics and Cytogenetics) 4th edition, p. 381, Springier-Berlag (1976)]. Directed mutagenesis techniques are well known and have been published by Lather, RF and Lecoq, JP Genetic Engineering, Academic Press (1983). ), pp. 31-50. Oligonucleotide-directed mutations are
Smith, M. and Gillam, S., Genetic Engineering: Principles and Methods, Plenum Press, 1981.
A detailed discussion is given in Volume 3, pp. 1-32. The DNA of the present invention can be produced, for example, as follows. (a) Single-stranded DNA consisting of a single strand of the structural gene encoding the parent protein is hybridized with a mutant oligonucleotide primer (this primer is paired with the codon for cysteine that is deleted or to be replaced, or, if necessary, with this codon). complementary to the region containing the antisense triplet that makes the combination, but mismatched with the codon or antisense triplet that defines the deletion of the codon or the triplet encoding another amino acid. ), (b) extend the primer with a DNA polymerase to form a mutant heteroduplex, and (c) replicate the mutant heteroduplex. In the present invention, cysteine residues that are not essential for the biological activity of biologically active proteins are intentionally deleted to eliminate sites for forming intermolecular crosslinks or erroneous intramolecular disulfide bonds. or muteins in which other amino acids are substituted, mutant genes encoding such muteins, and means for producing such muteins. Proteins that can be mutationally modified according to the present invention include cysteine content of biologically active proteins;
and the role that cysteine residues play in terms of activity and tertiary structure can be ascertained from available information. For proteins for which such information is not available in the literature, systematically alter each cysteine residue in the protein by the procedures described herein;
This information can be determined by testing the resulting muteins for biological activity and their tendency to form undesirable intermolecular or intramolecular disulfide bonds. Therefore, the present invention
As specifically described and exemplified below with respect to IFN-β and IL-2 muteins, functionally non-essential cysteine residues that may sensitize the protein to the formation of undesirable disulfide bonds are included. It will be appreciated that the following teachings apply to any other biologically active protein contained therein. In addition to IFN-β and IL-2, examples of proteins that are candidates for mutagenic modification according to the present invention include lymphotoxin (tumor necrosis factor) and colony-stimulating factor-
1, and IFN-α1. Candidate proteins usually have an odd number of cysteine residues. In the case of IFN-β, both glycosylated and unglycosylated IFNs show quantitatively similar specific activities; therefore, the glycosyl moiety
It was reported that it did not participate or contribute to the biological activity of However, non-glycosylated IFN-β produced by bacteria consistently exhibits quantitatively lower specific activity than glycosylated native IFN-β. IFN-β is known to have cysteine residues at positions 17, 31, and 141. Cysteine 141 was established by Siepard et al. (supra) to be essential for biological activity. In IFN-α, which contains four cysteine residues, there are two intermolecular -S-S- bonds, one with cys29.
Some are between cys138, others are between cys1 and cys98. IFN
Based on the homology between -β and IFN-α, IFN-β
cys141 has an intramolecular -S-S- bond with cys31, and cys17 can freely form an intermolecular crosslink. By deleting cys17 or substituting it with another amino acid, one can determine whether cys17 is essential for biological activity and its role in -S-S- bond formation. If cys17 is not essential for the biological activity of the protein, the resulting cys17-deficient or cys17-substituted proteins exhibit a specific activity close to that of native IFN-β;
It would also probably facilitate protein isolation and purification. An oligonucleotide-directed mutagenesis procedure using synthetic oligonucleotide primers that are complementary to the region of the cys17 codon of the IFN-β gene but contain one or more base changes in this codon. is used to create a designer gene, thus replacing cys17 with any other amino acid of choice. If a deletion is desired, use an oligonucleotide primer lacking the codon for cys17. cys17 to glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine,
Conversion to neutral amino acids such as tryptophan, serine, threonine and methionine is a preferred method. Serine and threonine are the most preferred alternatives due to their chemical similarity to cysteine. When cysteine is deleted, the mature mutein is one amino acid shorter than the natural parent protein or microbially produced IFN-β. Human IL-2 is reported to have cysteine residues at positions 58, 105, and 125 of the protein. IFN-
As in the case of β, IL-2 is in aggregated oligomeric form when isolated from bacterial cells, and in order to obtain good yields from bacterial extracts,
Must be reduced with a reducing agent. Moreover, purified reduced IL-2 is unstable and easily reoxidized to oligomeric inactive forms upon storage.
The presence of three cysteines means that upon reoxidation, the protein can randomly form one of three possible intramolecular disulfide bridges, whereas only one is the correct bridge as found in natural molecules. It means. Since the disulfide structure of natural IL-2 protein is unknown, IL-2
Create mutations in codons 58, 105, and 125 of the gene to determine which cysteine residues are required for activity and are therefore also most likely involved in natural disulfide bridge formation. The present invention can be used for Similarly, the formation of erroneous intramolecular disulfide bridges can be prevented by modifying cysteine residues not required by the activity, and the opportunity for intramolecular disulfide bridges can be prevented by removing or replacing free cysteine residues. can be minimized. The size of the oligonucleotide primer is determined by the conditions necessary for stable hybridization of the primer to the gene region to be mutated and by the limitations of currently available oligonucleotide synthesis methods. When designing oligonucleotides for use in oligonucleotide-directed mutagenesis, factors to be considered (e.g., overall size, size of portion bypassing the mutation site) are described by M. Smith and Described by S. Gillam (cited above). In general, the overall length of the oligonucleotide is such that it optimizes stable and unique hybridization at the mutation site, and extensions from the mutation site to the 5' and 3' ends of the DNA It should be large enough to avoid the effects of mutations due to the exonuclease activity of the polymerase. The oligonucleotides used for mutagenesis according to the invention are:
Usually about 12 to about 24 bases, preferably about
It contains 14 to about 20 bases, more preferably about 14 to 18 bases. These usually contain at least about 3 bases 3' of the codon that is altered or deleted. The method for creating a modified IFN-β gene generally involves the use of synthetic nucleotide primers that either eliminate codon 17 or change it so that it encodes a different amino acid. (TGT) to induce site-specific mutations. When changing cysteine to threonine and hybridizing the primer to the antisense strand of the IFN-β gene, the preferred nucleotide primer is GCAATTTC
ACTCAG (underlined indicates changed codon). When it is desirable to delete cysteine,
The preferred primer is
AGCAATTTTCAGCAGAGAGCTCCTG, which has lost the TGT codon for cys. When replacing cysteine with serine, the 17-nucleotide primer GCAATTTTCAG AGT CAG containing the AGT codon for serine is selected.
A change from cysteine to serine occurs at cys17 by transposition of the first base T→A. When introducing deletions, it must be recognized that the proper reading frame of the DNA sequence must be maintained for expression of the desired protein. The primer is a primer such as M13, fd, or φ×174, in which the single strand of the IFN-β gene is cloned.
Hybridizes to double-stranded phage. It will be appreciated that phages can carry either the sense or antisense strand of a gene. If the phage carries an antisense strand, the primer is directed to a region of the sense strand that does not match this codon but defines a codon deletion or triplet encoding another amino acid, but that contains the codon to be mutated. are the same. If the phage carries a sense strand, it is appropriately mismatched in the triplet that pairs with the codon to be deleted, but complementary to the region of the sense strand that contains the codon to be mutated. The conditions used for hybrid formation are described by M. Smith and S. Gillam (supra). The temperature is usually around 0℃ or
70℃, but generally ranges from about 10℃ to 50℃. After hybridization, the primers
DNA polymerase I, T4DNM polymerase,
It is extended on the phage DNA by reaction with reverse transcriptase or other suitable DNA polymerase.
The resulting dsDNA is a T4DNA gauze-like DAN
It is converted to closed circular dsDNA by treatment with ligase. DNA molecules containing single-stranded regions can be destroyed by S1 endonuclease treatment. Oligonuclease-directed mutagenesis generates a mutant IL-2 encoding a mutein with IL-2 activity but with a cys125 to serine 125 change.
It can be similarly used to create two genes. If the phage carries the sense strand of the gene, the preferred oligonucleotide primer used to create this mutant IL-2 gene is
GATGATGCTTCTG AGA AAAGGTAATC. This oligonucleotide has a C→G change in the middle base of the triplet that pairs with codon 125 of the IL-2 gene. The resulting mutant heterodiplex is
Used to transform competent host organisms or cells. Replication of the heteroduplex by the host produces progeny strands from both strands. Following replication, the mutant gene is isolated from the progeny of the mutant strand, inserted into a suitable vector, and this vector is used to transform a suitable host organism or cell. Preferred vectors are plasmids
pBR322, pCR1 and their variants, synthetic vectors, etc. A suitable host organism is E. coli (E.
Coli), Pseudomonas, Bacillus subtilis, Bacillus thuringiensis
thuringiensis), various yeast strains, Bacillus themophilus, animal cells such as mouse, rat, and Chinese hamster ovary (CHO) cells, plant cells, and animal and plant hosts. It should be appreciated that when transforming the host of choice with the vector, appropriate promoter-operator sequences are also introduced in order for the mutein to be expressed. The host may be a prokaryote or a eukaryote. (Methods for inserting DNA into eukaryotic cells are described in PCT application numbers US81/00239 and US81/00240, published September 3, 1981). E. coli and CHO cells are preferred hosts. Muteins obtained according to the invention may or may not be glycosylated depending on the glycosylation that occurs in the natural parent protein and the host organism used for mutein preparation. As desired,
The unglycosylated muteins obtained when using E. coli and Bacillus as hosts can be produced in vivo by chemical, enzymatic and other modifications known in the art. It may optionally be glycosylated ex situ. In a preferred embodiment of the present invention regarding IFN-β, as shown in FIG. 1, the cysteine residue at position 17 in the amino acid sequence of IFN-β is
−Codons on the sense strand of the DNA sequence encoding β
The first base of 17 is converted to serine by T→A rearrangement. This site-directed mutagenesis is performed using synthetic 17-nucleotide primers.
GCAATTTTCAG AGT CAG, this primer is identical to the 17 nucleotide sequence on the sense strand of IFN-β in the region of codon 17, except for a single base mismatch at the first base of codon 17. It is. This mismatch is at nucleotide 12 of the primer. It must be recognized that the genetic code (codons) is degenerate and many amino acids are encoded by more than one codon. For example, the base code for serine is degenerate in six ways, such that the codons TCT, TCG, TCC, TCA, AGT, and ACG all code for serine. For convenience, the AGT codon was chosen as the preferred embodiment. Similarly,
Threonine is encoded by any one of the ACT, ACA, ACC and ACG codons. When one codon is specified for a particular amino acid, it is intended that this includes all degenerate codons encoding that amino acid. 17-mer is
Hybridized to single-stranded M13 phage DNA carrying the antisense strand of the IFN-β gene.
Clones are then obtained from the DNA strand containing the mismatch by replication of the oligonucleotide heteroduplex using the DNA polymerase IKlenow fragment. Mutant clones are identified and screened for the presence or absence of specific restriction positions, antibiotic resistance or sensitivity, or other methods known in the art. When cysteine is replaced by serine, a new HinfI restriction site is created in the structural gene by the T→A rearrangement shown in FIG. Mutant clones are identified using oligonucleotide primers as probes in hybridization screens for mutated phage plaques. As shown in Figure 2, the primers will have only one mismatch when hybridized to the parent, but will have a perfect match when hybridized to the mutated phage DNA. Hybridization conditions can be devised so that the oligonucleotide primer hybridizes preferentially to the mutant DNA rather than the parent DNA. The newly generated HinfI site also serves as a means to confirm single base mutations in the IFN-β gene. M13 phages carrying mutated genes
The DNA is isolated and incorporated into a suitable expression vector, such as plasmid pTrp3, and this vector is transformed into E. coli strain MM294. Suitable growth media for culturing transformants and their progeny are known to those skilled in the art. Expressed muteins of IFN-β are isolated, purified, and characterized. The following examples are presented to aid in a further understanding of the invention and for illustrative purposes only. They should not be considered as limiting the scope of the invention in any way. Examples 1 to 9
The preparation of muteins for IFN-β is described. Examples 10-15 describe the preparation of muteins of IL-2. Example 1 (Reference example) Cloning of the IFN-β gene into the M13 vector The use of the M13 phage vector as a source of single-stranded DNA template is described by GF Temple et al., Nature (1982). Volume 296 537−
Proven on page 540. Plasmid containing the IFN-β gene under the control of the E. coli trp promoter
pβ1trp (Figure 3) was digested with restriction enzymes Hind and Xho. M13mp8〔G.Metzing (J.
Messing). "Third Cleveland Symposium on Macromolecules: Recombinant DNA," edited by A. Walton, Elsavier Press, pp. 143-153 (1981). and BamH and pre-Hind
and pβ1trpDNA that had been digested with Xho. The mixture was then ligated with T4 DNA ligase,
The ligated DNA was transformed into E. coli strain JM103 and competent cells and plated on Xgal indicator plates [J. Metzing et al.
Nucleic Acids Res (1981) Vol. 9, pp. 309-321].
Plaques containing recombinant phages (white plaques) were picked and inoculated into fresh cultures of JM103, and minipreps of RF molecules were prepared from infected cells (HDBirnboim and J.D. .
Doly), Nucleic Acid Res (1979) Vol. 7, 1513-
1523 pages). To identify clones containing IFN-β inserts, RF molecules were digested with various restriction enzymes. The restriction map of one such clone (M13-β1) is shown in FIG. Single-stranded (ss) phage DNA was generated from the M13-β1 clone and used as a template for site-directed mutagenesis using synthetic oligonucleotides. Example 2 (Reference Example) Site-directed mutagenesis 0.1mM adenosine triphosphate (ATP), 50mM hydroxymethylaminomethane hydrochloride (Tris-HCl) PH8.0, 10mM magnesium chloride, 5mM dithiothreitol (DDT) and Synthetic oligonucleotide GCAATTTTCAGAGTCAG (primer) 40 in 50μ in the presence of 9 units of T4 kinase
Picomoles were treated with T4 kinase for 1 hour at 37°C. 50mM sodium chloride, 10mM Tris-HCl, PH8.0, 10mM magnesium chloride and 10mM β
- 50μ of a mixture containing mercaptoethanol
The kinased primer (12 pmoles) was hybridized to 5 μg of single-stranded (ss) M13-β1 DNA by heating at 67° C. for 5 minutes and at 42° C. for 25 minutes. The annealed mixture was then cooled on ice and treated with 0.5mM each deoxynucleotide triphosphate (dNTP), 80mM Tris-HCl,
A 50μ reaction mixture containing PH 7.4, 8mM magnesium chloride, 100mM sodium chloride, 9 units of DNA polymerase IKlenow fragment, 0.5m MATP and 2 units of T4 DNA ligase was added and incubated for 3 hours at 37°C and 2 hours at 25°C. The reaction was then stopped by phenol extraction and ethanol precipitation. DNA in 10mM Tris-HCl, PH8.0,
10mM ethylenediaminetetraacetic acid (EDTA), 50%
It was dissolved in sucrose and 0.05% bromophenyl blue and subjected to electrophoresis on a 0.8% agarose gel in the presence of 2 μg/ml ethidium bromide. M13−β1
percolate the DNA band corresponding to the RF type of [RWDavis]
3. “Advanced Bacterial Genetics”
Genetics), Cold Spring Harbor Laboratory, NY 178-179 (1980)]. The eluted NA was used to transform competent JM103 cells, the bacteria were grown overnight, and ssDNA was isolated from the culture supernatant.
Using this ssDNA as a template in the second cycle of primer extension, the gel-purified RF type DNA is transformed into competent JM103 cells, plated on agar plates, and cultured overnight to obtain phage plaques. Example 3 (Reference example) Site-directed mutagenesis The experiment of Example 2 above is repeated. However, as a synthetic oligonucleotide primer, it is possible to change codon 17 of the IFN-β gene from one encoding cysteine to one encoding threonine.
Use GCAATTTTCAGACTCAG. Example 4 (Reference Example) Site-Specific Deletion The experiment of Example 2 above is repeated. However, as a synthetic oligonucleotide primer, it is possible to delete codon 17 of the IFN-β gene.
Use AGCAATTTTCAGCGAAGCTCCTG. Example 5 (Reference Example) Screening and identification of mutagenized plaques Plates with mutated M13-β1 plaques (Example 1) and unmutated M-13
- Cool the two plates containing β1 phage plaques to 4°C and remove 2 plaques from each plate.
For the first filter, place the dried filter on the agar plate for 5 minutes; for the second filter, for 15 minutes.
Transferred in layers for a minute. The filters were then placed on thick filter paper soaked in 0.2N NaOH, 1.5M sodium chloride, and 0.2% Triton X-100 for 5 minutes;
Neutralization was achieved by layering on filter paper soaked in 0.5M Tris-HCl, pH 7.5, and 1.5M sodium chloride for an additional 5 minutes. The filters were washed in a similar manner twice on filters soaked in 2×SSC (standard citrate), dried, and dried in a vacuum drying oven at 80° C. for 2 hours. 10ml per filter with duplicate filters
DNA hybridization buffer (5x SSC), PH
7.0, 4x Denhard solution (polyvinylpyrrolidone,
Ficoll and bovine serum albumin, 1× = 0.02 each
%), 0.1% Sodium Dodecyl Sulfate (SDS), 50
mM sodium phosphate buffer, PH7.0 and 100μg/
ml of denatured sperm DNA at 55°C for 4 hours.
Prehybridization was performed. A ^32P -labeled probe was created by underkinase treatment of the oligonucleotide primer with ^32P -labeled ATP. The filters were hybridized to ³² P-labeled primer 3.5 x ¹⁰⁵ cpm/ml in 5 ml of DNA hybridization buffer per filter for 24 hours at 55°C. 30% each in wash buffer containing 0.1% SDS and decreasing amounts of SSC.
Filters were washed at 55°C for minutes. Filters were first washed with buffer containing 2x SSC, and control filters containing non-mutated M13-β1 plaques were examined for the presence of radioactivity using a Geiger counter. The SSC concentration was decreased stepwise and the filters were washed until no detectable radioactivity remained on the control filter with unmutated M13-β1 plaques. The minimum concentration of SSC used was 0.1×SSC. Filters were air dried and autoradiographed at -70°C for 2-3 days. 480 mutated M13-β1 plaques and 100 nonmutated control plaques were screened with a kinased oligonucleotide probe. None of the control plaques hybridized with the probe, while five mutated M13-β1 plaques hybridized with the probe. One of the five mutant M13-β1 plaques (M13-SY2501) was picked and inoculated onto JM103 culture medium. ssDNA was prepared from the supernatant and double-stranded (ds) DNA from the cell pellet.
ssDNA as a template for dideoxy sequencing of clones using the M13 universal primer
It was used. The results of the sequencing analysis are shown in FIG. This result confirms that the TGTcys codon was converted to an AGTser codon. . Example 6 (Reference example) Expression of mutant IFN-β in Escherichia coli RF DNA from M13-SY2501 was extracted with restriction enzymes.
Digested with Hind and Xho and the 520 bp insert was purified on a 1% agarose gel. Plasmid pTrp3 containing the E. coli trp promoter (Figure 7)
was digested with the enzymes Hind and BamH and purified.
It was mixed with the M13-SY2501 fragment and ligated in the presence of T4 DNA ligase. Concatenate DNA with E. coli MM294
transformed into a strain. Ampicillin-resistant transformants were screened for sensitivity to the drug tetracycline. Plasmids from 5 ampicillin-resistant and tetracycline-sensitive clones
DNA was digested with Hinf to screen for the presence of the M13-SY2501 insert. 8th
Figure a shows Hinf of one of the clones (pSY2501).
The restriction pattern is shown in the figure, which is compared to the original IFN-
The Hind patterns of the β clone, pβ1trp, are compared. As expected, pSY2501 has additional
There is a Hinf site, and the 197 bp IFN-β internal fragment is cut into a 169 bp fragment and a 28 bp fragment (Figure 8b). The restriction map of clone pSY2501 is shown in FIG. Complete DNA of mutant IFN-β gene
sequence along with the predicted amino acid sequence.
As shown in the figure. The plasmid, designated clone pSY2501, has been deposited at the Agricultural Research Culture Collection (NRRL), Fermentation Research Institute, Northern Research Center, U.S. Department of Agriculture, Service of Science and Education, 1815 North University Street, Peoria, Illinois 60604. has been done. The designated deposit numbers are CMCC No. 1533 and NRRLB-15356. Cultures of pSY2501 and pβ1trp (including progeny)
The cells were grown to an optical density of 1.0 (0D ₆₀₀ ). Cell-free extracts were prepared and the amount of antiviral activity of IFN-β was assayed using GM2767 cells using a microtiter assay. The extract of clone pSY2501 is from pβ1trp to
The clone showed 10 times higher activity (Table 1).
It is shown that either pSY2501 synthesizes a higher amount of a protein exhibiting IFN-β activity, or that the protein produced has a higher specific activity. Table 1 Extract antiviral activity (units/ml) pSY2501 6×10 ⁵ pβ1tpr 1×10 ⁵ ptrp3 (control) 30 Extraction of both clones to determine whether clone pSY2501 was synthesizing several times more active protein. The samples were subjected to electrophoresis on SDS polyacrylamide gels along with control extracts, and the gels were stained with Coomassie blue to develop the proteins. As shown in Figure 11, approximately
There was only one protein band corresponding to a protein of 18,000 daltons. Although this protein has a molecular weight of approximately 20,000 daltons, it exhibits a gel migration pattern of a protein of 18,000 daltons, and purification of this protein from extracts of pβ1trp revealed that IFN-β
I knew in advance that this would be the case. Since the amount of this protein is lower in the extract of pSY2501 than in the extract of pβ1trp, the specific activity of the protein in the extract of clone pSY2501 is higher than that of clone pβ1trp. Example 7 (Reference Example) Purification of IFN-βser17 E.
IFN-βser17 was recovered from E. coli. E. coli was grown in the following growth medium to an optical density of 10-11 at 680 nm (8.4 g/dry weight). Ingredient concentration Ammonium chloride 20mM Potassium sulfate 16.1mM Monopotassium phosphate 7.8mM Disodium phosphate 12.2mM Magnesium sulfate heptahydrate 3mM Trisodium citrate dihydrate 1.5mM Manganese sulfate tetrahydrate 30μM Zinc sulfate - Heptahydrate 30μM Copper sulfate pentahydrate 3μM L-tryptophan 70mg / Ferrous sulfate heptahydrate 72μM Thiamine hydrochloride 20mg / Glucose 40g / PH adjusted with ammonia Harvest of transformed E. coli 9.9 (9.9Kg)
Cool to 20℃ until the filtrate weight is 8.8Kg.
The harvest was concentrated by passing through a cross-flow filter with an average pressure drop of -100 kpa and a constant filtrate flow rate of 260 ml per minute. The concentrate (approximately 1) was poured into a container and cooled to 15°C. Heat the concentrate at 5℃, approx.
The bacterial cells in the concentrate were disrupted by passing it through a Manton-Gorlin homogenizer at 69,000 kpa.
Phosphate buffered saline, PH7.4 (PBS) 1
The homogenizer was washed with water and the wash was added to the crush to give a final volume of 2. This volume was subjected to continuous centrifugation at 12000xg with a flow rate of 50ml per minute. The solids were separated from the supernatant and resuspended in PBS4 containing 2% by weight SDS. After stirring this suspension for 15 minutes at room temperature, there was no visible suspension. The solution was then extracted with 2-butanol at a 1:1 volume ratio of 2-butanol:solution. The extraction was carried out in a liquid-liquid phase separator using a flow rate of 200 ml per minute. The organic phase was then separated and evaporated to dryness, yielding 21.3 g of protein. This was resuspended in distilled water at a volume ratio of 1:10. The recovered product was assayed for human IFN-β activity using a protection against viral cytopathic effects (CPE)-based assay. This assay was performed in microtiter plates. Fill each container with a minimum of 50μ of the basic doubler, and put the materials in the first container.
25μ was added, and subsequent dilutions of 1:3 were carried out continuously. Virus (vesicular stomatitis), cells (human fibroblast GM-2767 line) and standard IFN-β control group were loaded onto each plate. Standard IFN-β at 100 units per ml was used.
The plate was then irradiated with ultraviolet light for 10 minutes. After irradiation, 100μ of cell suspension (1.2 × ¹⁰ cells per ml)
was added to each container and the trays were incubated for 18-24 hours. Virus solution was added at 1 plaque forming unit per cell to each vessel except the cell control plate. Trays were incubated until the virus control showed 100% CPE. This usually occurred 18-24 hours after adding the virus solution. Standard IFN-
The assay results were interpreted in terms of the location of the β control 50% CPE container. From this point, interferon titers were determined for all samples on the plate. The specific activity of the recovered product was determined to be 5×10 ⁷ U/ml. Example 8 (Reference example) Purification by acid precipitation and chromatography The method of Example 7 was repeated, except that the extraction,
After separation of the aqueous and organic phases and mixing of the organic phase with PBS in a volume ratio of 3:1, the PH of the mixture was lowered to about 5 by addition of glacial acetic acid. The resulting precipitate
Separate by centrifugation at 10,000-17,000xg for 15 minutes and pellet in 10% W/V SDS, 10mM
DTT was redissolved in 50mM sodium acetate buffer, PH5.5 and heated to 80°C for 5 minutes. The solution is then converted using a Beckman gradient system to
Brownley RP-300, 10 μM, was applied to an “Aquabore” column. Buffer A was 0.1% trifluoroacetic acid (TFA) in water and buffer B was 0.1% TFA in acetonitrile. Detection was by UV absorbance at 280 nm. Solvent program is 0 in 3 hours
The gradient was linear from % Buffer B to 100% Buffer B. The fractions containing the highest interferon activity were pooled, and the specific activity of this pooled interferon preparation was approximately 2 x 10 ⁸ international units per mg of protein in native IFN-β.
It was determined to be between 9.0×10 ⁷ and 3.8×10 ⁸ U/ml. Example 9 (Reference Example) Biochemical characterization of IFN-βser17 Amino acid composition was determined after 40 μg of sample was subjected to hydrolysis in 200 μ of 5.7N HCl, 0.1% phenol at 108° C. for 24-72 hours. Proline and cysteine were determined in the same manner after performic acid oxidation. In this case, phenol was omitted from the hydrolysis. Tryptophan was sampled 400μ in 5.7N hydrochloric acid, 10% mercaptoacetic acid (no phenol).
was analyzed after 24 hours of hydrolysis. The analysis is
Beckmann using a single column of AA10 resin
The analysis was carried out using a 121MB amino acid analyzer. Purified IFN
-The amino acid composition calculated from representative 24-, 48-, and 72-h acid hydrolysis of βser17 is predicted by the DNA sequence of the cloned IFN gene, except that the N-terminal methionine is missing. It matches well. The amino acid sequence of the first 58 residues from the amino acid terminus of purified IFN was determined from 0.7 mg of sample using a Beckman 890C sequencer using 0.1 M Quadrol buffer. PTH amino acids were purified by reverse-phase high-pressure liquid chromatography (HPLC) on an Artex Ultrasphere 0DS column (4.6 x 250 mm) at 45°C for 1.3 min with 40% buffer B and 40-70% buffer B.
Determined by elution at 8.4 minutes. Here buffer A was 0.0115M sodium acetate, 5% tetrahydrofuran (THF), pH 5.11 and buffer B was 10% THF in acetonitrile. The determined N-terminal amino acid sequence of IFN-βser17 corresponds to the predicted sequence predicted from the DNA sequence, except for the absence of the N-terminal methionine. As shown above, IFN-βser17 preparations exhibit activity very close to or even superior to the specific activity level of native IFN-β. IFN-βser17 has no free sulfhydryl groups, but exhibits one -S-S- bond between the remaining cysteines only at positions 31 and 141. Proteins do not easily form oligomers and are considered to be essentially in monomeric form. The IFN-βser17 obtained according to the invention, as a single product or as a mixture of various types, is inert, non-toxic, non-allergenic and can be prepared into pharmaceutically acceptable formulations in a physiologically acceptable carrier medium. Can be prescribed for clinical and therapeutic use in cancer therapy or in conditions where interferon therapy is indicated.
It can also be used for viral infections. Such vehicles 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 such as dextrose, HSA (human serum albumin), etc. may also be optimally included. Therapeutic formulations can be administered orally or parenterally, such as by intravenous, intramuscular, intraperitoneal and subcutaneous administration. The modified IFN-β formulations of the present invention can also be used for topical application in suitable vehicles commonly utilized for topical applications. The main advantage of the above-mentioned IFN-β muteins is that IFN-β muteins
The free sulfhydryl group (-SH group) at position 17 of β is eliminated, which allows the mutein to form the correct disulfide bond between cys31 and cys141, clearly resulting in full biological activity. I am getting the necessary conformation. IFN-
Due to the increased specific activity of βser17, lower doses can be used for treatment. By deleting the cysteine at position 17 and eliminating the free -SH group,
IFN-βser17 protein is an IFN-β protein produced by microorganisms.
It does not form dimers or oligomers as easily as β. This facilitates purification of the protein and enhances its stability. Example 10 The nucleotide sequence of a cDNA clone encoding human IL-2, the procedure for constructing an IL-2 cDNA library, and the procedure for screening it for IL-2 were described by Taniguchi.
T.) Nature (1983) Vol. 24, pp. 305 onwards. A cDNA library enriched for potential IL-2 cDNA clones was created from enriched IL-2 mRNA fractions obtained from induced peripheral blood lymphocytes (PBL) and Jakarta cells by conventional procedures. To enrich mRNA for IL-2, the mRNA was fractionated and the fraction was injected into immature oocytes of Xenopus laevis, and the IL-2 activity of the oocyte lysate was measured in HT-2 cells. This was done by confirming the fraction with IL-2 mRNA activity [J. Watson, J. Exp. Med.
(1979) Vol. 150, pp. 1570-1519 and S. Gillis et al., Immun (1978) Vol. 120, pp. 2027-
2032 pages]. Example 11 Screening and identification of IL-2 cDNA clones IL-2 cDNA clones were isolated using colony hybridization method.
2 cDNA libraries were screened. Each microtiter plate was copied by a replica method onto a layered nitrocellulose filter paper (S&S type BA-85), and grown on L agar containing 50 μg/ml ampicillin at 37° C. for 14 to 16 hours. Colonies were lysed and treated with 500mM sodium hydroxide, 1.5M sodium chloride sequentially for 5 minutes.
DNA was fixed on the filter and washed twice with 5x standard citrate solution (SSC) for 5 minutes each. The filter was air dried and oven dried at 80°C for 2 hours. Add 10 ml of DNA hybridization buffer (50% formamide, 5x
SSC, PH7.0, 5x Denhard's solution (polyvinylpyrrolidine, Ficoll and bovine serum albumin; 1
x = 0.2% each), 50mM sodium phosphate buffer of pH 7.0, 0.2% SDS, 20μg/ml polyU and 50μg/ml
ml of modified salmon sperm DNA for 6-8 hours at 42°C. A 20-mer oligonucleotide probe labeled with ³² P was created based on the IL-2 gene sequence reported by Taniguchi (supra). The nucleotide sequence of the probe is
It was GTGGCCTTCTTGGGCATGTA. Samples were hybridized for 24-36 hours at 42°C with 5 ml of DNA hybridization buffer per filter containing the ³² PcDNA probe. The filters were washed twice for 30 min each at 50°C with 2x SSC, 0.1% SDS, then washed with 1x SSC and 0.1% SDS at 90°C.
Wash at 50°C for 2 min each, air dry, and dry at -70°C for 2 min.
Autoradiographed for ~3 days. Positive clones were identified and rescreened with the probe. Clones of sufficient length were identified by restriction enzyme mapping and IL-
This was confirmed by comparison with the sequence of 2 cDNA clones. Example 12 Cloning of the IL-2 gene into the M13 vector The IL-2 gene was cloned in the same manner as described in Example 1 using the plasmid pLW1 (Figure 12) containing the IL-2 gene under the control of the E. coli trp promoter. Two genes were cloned into M13mp9. The pLW1 sample was dated August 4, 1983, 12301 Park Lawn Drive, Lockeville, MD 20852;
It was deposited with the American Type Culture Collection and designated ATCC No. 39 and 405. IL−
One clone containing two inserts (M13-IL-2
Figure 13 shows the restriction map of Single-stranded phage DNA was generated from clone M13-IL-2 and used as a template for oligonucleotide-directed mutagenesis. Example 13 Oligonucleotide-Directed Mutagenesis As noted above, IL-2 contains cysteine residues at amino acid positions 58105 and 125. Based on the nucleotide sequence of the portion containing codons for these three cysteine residues in the IL-2 gene,
Designed three oligonucleotide primers,
The codons for these residues were synthesized to mutate them into codons for serine. These oligonucleotides have the following sequences. Change cys58 CTTCTAGAGACTGC AGA
TGTTTC (DM27) Change cys105 CATCAGCATACTC AGA
CATGAATG (DM28) Change cys125 GATGATGCTCTG AGA
AAAGGTAATC (DM29) 0.1mM ATD, 50mM Tris-HCl, PH8.0,
10mM magnesium chloride, 5mM DTT and T4
Forty pmoles of each oligonucleotide were separately kinased in 50μ for 1 hour at 37°C in the presence of 9 units of kinase. Each of the kinased primers (10 pmoles) was incubated at 67°C for 5 min and 42 min in 15 μM of a mixture containing 100mM sodium chloride, 20mM Tris-HCl, PH 7.9, 20mM magnesium chloride and 20mM β-mercaptoethanol.
ssM13−IL− by heating for 25 min at °C.
2DNA was hybridized to 2.6 μg. The annealed mixture was cooled on ice, then 0.5mM each
dNTP, 17mM Tris-HCl (PH7.9), 17mM magnesium chloride, 83mM sodium chloride, 17mM
Mβ-mercaptoethanol, 5 units of DNA polymerase IKlenow fragment, 0.5 mMATP, and
Reaction mixture containing 2 units of T4 DNA ligase
A final volume of 25μ was prepared and incubated at 37°C for 5 hours. The reaction was stopped by heating to 80°C, and the reaction mixture was used to transform competent JM103 cells, plated on agar plates and cultured overnight to obtain phage plaques. Example 14 Screening and identification of mutagenized phage plaques Plates containing mutagenized M13-IL-2 plaques and non-mutagenized M13-
Two plates containing IL-2 phage plaques were cooled to 4°C and the phage plaques from each plate were transferred onto two nitrocellulose circular filters, and in the case of the first filter, the dried filter was transferred to an agar plate. Transfer for 5 minutes on top and 15 minutes in the case of the second filter. The filter was then placed on thick filter paper soaked in 0.2N sodium hydroxide, I5M sodium chloride and 0.2% Triton for 5 minutes, then 0.5M Tris-HCl (PH7.5) and 1.5M
Neutralization was achieved by layering on filter paper soaked in NaCl for an additional 5 minutes. The filters were washed twice in a similar manner on filters soaked in 2×SSC, dried and then dried in a vacuum drying oven at 80° C. for 2 hours. A multilayer filter was added with 10 ml per filter of DNA hybridization buffer (5x SSC), pH 7.0, 4x Denhard's solution (polyvinylpyrrolidine, Ficoll, and bovine serum albumin, 1x = 0.02% each), 0.1% SDS,
50mM sodium phosphate buffer (PH7.0) and 100μ
Prehybridization was performed with g/ml denatured salmon sperm DNA for 4 hours at 42°C. labeled
A ³² P-labeled probe was created by kinase treatment of the oligonucleotide primer with ATP. 5ml per filter
8 hours at 42°C in DNA hybridization buffer.
The filter was hybridized to 0.1×10 ⁵ cpm/ml of ³² P-labeled primer. 0.1% SDS
and 2 x 30 min each at 50 °C in a washing and drying solution containing 0.1x SDS and 0.2x SSC.
Filters were washed twice in SSC at 50°C for 30 minutes each. Air dry the filter and store at -70℃ for 2~
It was subjected to autoradiography for 3 days. Oligonucleotide primer DM28 and
DM29 is new to the mutagenized clone
RF-DNA from several clones hybridized with these kinased primers was digested with the restriction enzyme Dde, as it was designed to create a Dde restriction site (Table 14). hybridized with primer DM28 and mutagenized with a new Dde restriction site.
One of the M13-IL-2 plaques (M13-LW44) was picked up and inoculated into a JM103 culture, ssDNA was prepared from the culture supernatant, and dsRF-IL-2 was extracted from the cell pellet.
DNA was prepared. Similarly, plaques hybridized with primer DM29 (M13-
LW46) will be taken up, and from now on, ssDNA and RF−
DNA was prepared. Oligonucleotide primer DM27 is designed to create a new Pst restriction site in place of the Dde position. Plaques hybridized to this primer were therefore screened for the presence of new Pst sites. One such phage plaque was identified (M13-LW42) and ssDNA and RF-
DNA was prepared from this. For target cysteine
To confirm that the TGT codon was converted to a TCT codon for serine, the DNA obtained from all three clones was sequenced. Example 15 Mutagenic IL for expression in E. coli
- Recloning of 2 genes M13-LW42, M13-LW44, and M13-
Restriction enzyme Hind of RF-DNA from LW46
and Ban and the insert was purified from a 1% agarose gel. Similarly, plasmid pTrp3
(Fig. 7) was digested with Hind and Ban, and the large plasmid fragment containing the trp promoter was purified on an agarose gel.
Each of the inserts isolated from LW44 and M13-LW46 were ligated. The ligated plasmid was transformed into competent E. coli strain K-12MM294.
Plasmid DNA from these transformants was analyzed by restriction enzyme mapping and plasmid
The existence of pLW42, pLW44, and pLW46 was confirmed. Figure 14 is a restriction map of pLW46. To induce the trp promoter, each of these individual clones was grown in the absence of tryptophan, the cell-free extracts were analyzed on an SDS-polyacrylamide gel, and three clones pLW42, pLW44 and
All of pLW46 was shown to synthesize a 14.5 Kd protein similar to that found in the positive control pLW21, which was demonstrated to synthesize a 14.4 Kd IL-2 protein. When these same extracts were assayed for IL-2 activity in mouse HT-2 cells, clones
Only pLW21 (positive control) and pLW46 were found in significant amounts.
It shows IL-2 activity (Table 2 below), cys58 and
cys105 is required for biological activity, and converting it to serine (pLW42 and
This means that biological activity is lost due to pLW44). while cys125 uses this as ser125
(pLW46) does not affect biological activity, so it must be unnecessary for activity. Table 2 Clone IL-2 Activity (μ/ml) PIL2-7 (negative control) 1 PLW21 (positive control) 113000 PLW42 660 PLW44 1990 PLW46 123000 Figure 15a shows the nucleotide sequence of the coding strand of clone pLW46. Compared to the coding strand of the natural human IL-2 gene, clone pLW46 has a single base change from G to C at nucleotide 374.
Figure 15b shows the corresponding amino acid sequence of the IL-2 mutein encoded by pLW46. This mutein is called IL-2ser125. Compared to natural IL-2,
Mutein has serine instead of cysteine at position 125. E. coli K-12MM294 transformed with pLW46
Strain samples were sent to the American Type Culture Collection, 12301 Drive, Park Lawn, Lockeville, MD 20852, September 26, 1983.
Collection) and designated ATCC No. 39452. IL-2 muteins in which cysteine at position 125 is deleted or replaced with another amino acid, such as mutein IL-2ser125, retain IL-2 activity. Therefore, they can be formulated and used in the same way as natural IL-2. Therefore, such muteins are useful in the diagnosis and treatment of bacterial, viral, parasitic, protozoan, and fungal infections, in the development of lymphokines or immunodeficiency, and in the reconstitution of normal immune function in older humans and animals. In addition, the development of diagnostic assays, such as enzyme amplification, radiolabeling, radioimaging, and other methods known in the art to monitor IL-2 levels in pathological conditions, will utilize receptor sites for lymphokines. It is useful for the promotion of T cell growth in vitro for therapeutic and diagnostic purposes, as well as for various other therapeutic, diagnostic, and research applications. human IL
-2 for various therapeutic and diagnostic applications, see S.A. Rosenberg,
E.A. Grimm et al., A. Mazumder et al., and E.A. Grimm and S.A. Rosenberg report on the study. IL-2 can be used by itself or in combination with other immunologically relevant B or T cells or other therapeutic agents. For therapeutic or diagnostic use, they can be formulated in non-toxic, non-allergenic, physiologically acceptable carrier media such as distilled water, Ringer's solution, Hank's solution, physiological saline and the like. Administration of IL-2 muteins to humans or animals can be oral, or intraperitoneal or intramuscular or subcutaneous, as deemed appropriate by the physician. Examples of relevant cells are B or T cells, natural killer cells, etc. Examples of therapeutic reagents that can be used in combination with the polypeptides of the invention are various interferons,
Especially gamma interferon, B cell growth factor,
IL-1 etc.

[Brief explanation of drawings]

FIG. 1 is a diagram of the amino acid sequence of IFN-β.
FIG. 2 is a schematic diagram showing the preparation of mutant IFN-β genes by oligonuclease-directed mutagenesis. FIG. 3 is a diagram of plasmid pβ1trp containing the IFN-β gene. FIG. 4 is a diagram of the cloning vector M13mp8 phage.
Figure 5 shows the restriction map of clone M13-β1.
FIG. 6 shows a sequencing gel pattern of a mutant IFN-βser17 gene showing a single base change in the coding region. Figure 7 shows the expression plasmid
FIG. 3 is a diagram of pTrp3. Figure 8a shows the clone
Showing the Hinf restriction pattern of pSY2501, section 8b
The figure shows the two resulting 169bp and 28bp fragments. FIG. 9 is a restriction map of clone pSY2501. FIG. 10 shows the DNA sequence encoding the mutein IFN-βser17 and the corresponding amino acid sequence. Figure 11 shows clones pSY2501 and
A single 18000 Dalton protein band corresponding to IFN-βser17 in the extract of pβ1trp is shown. FIG. 12 is a diagram of plasmid pLW1 containing the human interleukin-2 (IL-2) gene under the control of the E. coli trp promoter. Figure 13 shows
Restriction map of Phage clone M13-IL2.
Figure 14 is a restriction map of plasmid pLW46. Figures 15a and 15b show clone pLW46
The nucleotide sequence of the coding strand of and IL-2ser125
The corresponding amino acid sequences of the IL-2 muteis, respectively, are shown.

Claims (1)

[Scope of Claims] 1. A DNA encoding a modified interleukin-2 in which the cysteine residue at position 125 of natural interleukin-2 is replaced with another amino acid residue. 2 The cysteine residue is serine, threonine,
The DNA of claim 1 encoding a modified interleukin-2 substituted with glycine, alanine, valine, leucine, isoleucine, histidine, tyrosine, phenylalanine, tryptophan or methionine. 3. A plasmid containing DNA encoding modified interleukin-2 in which the cysteine residue at position 125 of natural interleukin-2 is replaced with another amino acid residue. 4 The cysteine residue is serine, threonine,
Claim 3 containing DNA encoding a modified interleukin-2 substituted with glycine, alanine, valine, leucine, isoleucine, histidine, tyrosine, phenylalanine, tryptophan or methionine. Plasmid. 5. Escherichia coli transformed with a plasmid containing DNA encoding a modified interleukin-2 in which the cysteine residue at position 125 of natural interleukin-2 is replaced by another amino acid residue. 6 The cysteine residue is serine, threonine,
Claims transformed with a plasmid containing DNA encoding a modified interleukin-2 substituted with glycine, alanine, valine, leucine, isoleucine, histidine, tyrosine, phenylalanine, tryptophan or methionine E. coli according to Section 6. 7. E. coli according to claim 5 or 6, which produces interleukin-2 in which the cysteine residue is replaced by a serine residue and is not glycosylated.