JPH02501827A - Human interleukin-4 mutein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

Title of the Invention Human Interleukin-4 Mutin Background of the Invention The present invention relates generally to lymphokines and, in particular, to recombinant mutants that induce the clonal expansion and maturation of activated B cells and the proliferation of cytotoxic T cells. Interloiki It is related to N-4 mutin or similar proteins. B lymphocytes, or B cells, are the precursors of antibody-secreting plasma cells. B cells pass through an intermediate cell class known as pre-B cells to hematopoietic stem cells located in the bone marrow. It is derived from cells. B cells can be distinguished from pre-B cells by the expression of surface-bound immunoglobulins that are capable of binding specific antigens. B cells have membrane B cells are activated by antigen binding to receptors, but B cells are activated by specific helper receptors. - interact with T cells, or even with certain soluble growth or differentiation factors. B cell activation is a sequential process that includes proliferation and differentiation phases. During the proliferation phase, activated B cell clones increase and participate in activation. provides a large number of cells capable of reacting with the antigen. During the differentiation phase, some activated B cells mature and secrete immunoglobulins as plasma cells in the blood circulation. Ru. Initially, different cytocans derived from T cells, named B cell growth factor J (BCGF) and B cell differentiation factor J (B CDF), are involved in the regulation of proliferation and differentiation phases. Another term applied is “B cell stimulation”. Interleukin-4J (BSF-1) and Interleukin-4J (IL-4), although the latter is currently preferred. Howard et al., Ll Tsui J He Eyu J: 914 (1982). , and Farrar et al., J.Ia+muno1.131 = 1838 (1983), stimulated by mitogens to promote proliferation of B cells. describes a B cell stimulating factor derived from murine T cells. Following this disclosure, numerous laboratories developed T-cell hybridomas, cloned T-cells, and normal T-cells. A similar murine-derived activity was reported in media conditioned by cells, e.g. Roehm et al., Noelle et al., Proc. Natl. Acad, Sci, tlsA 8:6149 (1984), Oliver et al., Proc. Natl, Acad, Sci, tlSA 82: 2465 (1985), La Rabin et al., Proc. Natl, Acad, Sci, USA 82: 2935 (1985), V. See Vitetta et al., 4L Shiwu, 1726 (1985). Purification of the four murine BSF-1/ILs to homogeneous standards was described by Grabstein et al. Li mark” (9-”) is: 1405 (1 986). Isolation of cDNA encoding a protein with murine BSF-1/IL-4 activity was recently reported by Noma et al., Nature 319:640 (1986) and Lee et al., Proc. Natl., Acad, Sci. USA 83: 2061 (1986). Yokota et al., Proc, Natl, Acad, Sci, USA Vine: 5894 (1 (986) isolated a human cDNA clone with homology to murine IL-4. Ta. This human cDNA encodes a protein consisting of 153 amino acids, including a potential signal peptide. Transfection of monkey CO5-1 cells with this cDNA induced proliferation of human B cells exposed to anti-1g1'l. I was able to guide you. This activity is due to the presence of murine IL-4 in association with murine B cells. Similar to the nature of knowledge. IL~4 also stimulates the proliferation and differentiation of factor-dependent T cell and myeloid cell classes. Grubstein et al. (supra) reported that murine IL-4 induces proliferation of murine IL-2- and IL-3-dependent progeny cell lineages. Other studies have shown that IL-4 stimulates mast cell proliferation and macrophage differentiation. It is reported that it gets worse. The availability of significant amounts of purified IL-4 has implications for B cell ontogeny and function. We have promoted research on lymphokines and clarified the therapeutic potential of this lymphokine. Current intended uses for recombinant human IL-4 include the treatment of immunodeficiency disorders characterized by B-cell depletion and as a treatment for certain B-cell-associated lymphocytic leukemias. There is induction of cell differentiation. IL-4 is also an immunoglobulin Supplying and maintaining a continuous culture of phospho-secreting B cells with a source of human monoclonal antibodies. Applicants have discovered that IL-4 induces the proliferation and differentiation of cytotoxic T cells that have been previously exposed to mitogenic stimuli. This observation indicates that IL-4 can be used as a therapeutic lymphokine in the treatment of viral infections and neoplastic conditions. Main plate emperor! The present invention relates to recombinant human Ru4 protein produced using a yeast expression system. It is something that Similar proteins, including those with inactivated asparagine-linked glycosylation sites, such as hIL-4 (Asp”, Asp”’) The present invention is directed to a DNA sequence encoding a mutin. and recombinant expression vectors containing the DNA sequences, as well as recombinant expression vectors containing the DNA sequences. How to produce mutin contained in cultured microorganisms transformed with the current vector The present invention also relates to methods of In addition, contacting T cells with a composition containing a biologically effective amount of IL-4. The present invention also provides a method for inducing proliferation and lytic activity of anti-tumor cytolytic T cell (CTL) populations comprising: In a related aspect, the present invention provides a method for proliferating anti-tumor or anti-viral cytolytic T cells in a mammal, e.g., a human, comprising administering a therapeutically effective amount of a human IL-4 therapeutic composition. Provides methods for induction and activation. Figure 1 shows the nucleotide sequence and the corresponding amino acid sequence of natural wild-type human IL-4. Showing columns. Figure 2 shows the nucleotide sequence of the DNA sequence encoding hlL-4 myutein GluA1aGluAla-hIL-4 (A sp''. AspI 29). Figure 6 schematically depicts the construction of a yeast expression vector for the production of IL-4 (Asp'', Asp''9). FIG. 7 is a graph showing the rate of increase in cytolytic T cell production by IL-2. FIG. FIG. As detailed herein, a cDNA containing the nucleotide sequence encoding native human IL-4 was inverted in polyadenylated RNA isolated from human peripheral blood T cells. It was isolated from a cDNA library prepared by photocopying. Synthetic oligonucleotides with sequences homologous to the N-terminus and C-terminus of the natural human sequence were used to search the library by Brofka, conventional DNA hybridization techniques. contains plasmid DNA that hybridizes to the probe. Clones were isolated from the library and subjected to restriction enzyme digestion and agarose gel electrophoresis. The cells were analyzed by electrophoresis and further hybridization experiments ("Southern blot") using electrophoresed fragments. After isolating a single clone that hybridized to both the N-terminal and C-terminal oligonucleotide probes, the hybridized fragment was cleaved to give a smaller restriction enzyme fragment carrying the hlL-4 gene. and this is then The vector was cloned and sequenced using conventional techniques. Coating mature hlL-4 The coding cDNA is then digested with selected restriction enzymes to extract the planned code. was reconstituted using synthetic oligonucleotides to effect conversion of the protein. The resulting mutant cDNA sequence was inserted into a yeast expression vector under the control of a specific promoter. This vector was used to transform the appropriate yeast expression strain, which was grown under conditions that promote derepression of the yeast promoter. The resulting yeast conditioned culture medium is a bioactive protein of hlL-4. protein, which was purified as described below. The terms "human interleukin-4" and rhlL-4J refer to a human endogenous secreted protein capable of inducing the maturation and proliferation of human B cells, which has the sequence shown in Figure 1. Contains amino acid sequences that are substantially homologous in whole or in significant parts. Other names for this molecule include "B cell stimulatory factor" and "B cell growth factor." ``DNA sequences'' are components of isolated fragments or large DNA structures. represents all DNA polymers, all of which are present at least once in substantially pure form. That is, it is derived from isolated DNA in amounts or concentrations such that the fragment or its constituent nucleotide sequences can be recovered by identification, manipulation, and standard biochemical techniques, such as cloning vectors. "Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. A "recombinant expression vector" is defined as (1) one or more elements that have a regulatory role in gene expression, such as a promoter or an enhancer; genetic elements, and (2) the structure that is transcribed into wa RNA and translated into protein. A plasmid containing a transcription unit, consisting of a collection of structural or coding sequences. Preferably, the transcription unit includes a leader sequence that allows the protein translated by the host cell to be secreted outside the cell. "Recombinant expression system" refers to a combination of an expression vector and a suitable host microorganism. A yeast expression system, particularly a system using Saccharomyces cerevisiae, is preferred. A "variant amino acid sequence" refers to a polypeptide encoded by a nucleotide sequence that is intentionally mutated from the natural sequence. ``Mutant protein'' or ``mutin'' refers to a protein that contains a variant amino acid sequence. ``Substantially homologous'' is used for both nucleic acid and amino acid sequences; A sequence, e.g. a mutant sequence, differs from a reference sequence by one or more substitutions, deletions or additions, the overall effect of which does not result in an undesirable functional difference between the reference sequence and the sequence in question. For the purpose of this invention Sequences having 80% or more homology and equivalent biologically specific activity are considered to be substantially homologous sequences within the scope of the present invention. Sequences with less homology and corresponding biological activity are considered equivalent. "Native sequence" refers to an amino acid or nucleic acid sequence that is identical to the wild type or native form of a gene or protein. "N-glycosylation site" is defined below. The term "inactivate" as used in defining this invention refers to modifying selected N-glycosylation sites to remove amino acids capable of forming covalent bonds with oligosaccharide moieties. It means to do. For example, IL-4 activity can be detected in cultures of human B cells derived from human tonsil cell suspensions. It is observed at The enriched B cell population is 2-aminoethyl isothiobromide T cells were prepared into rosettes using sheep red blood cells treated with Subsequently, Ficoll-Histopaque (Sigma Che+Wical Corp, St. Louis, MO, USA) was used to eliminate T cells, and monocytes, granulocytes, and active cells were removed. Sephadex GIO filtration was used to remove converted B cells. Therefore, it can be prepared. Following enrichment, B cell preparations are frozen in liquid nitrogen before use. can be done. For activity measurement, frozen B cells were thawed, washed, and mixed with 10% fetal bovine serum, 5 XlO-'M 2-mercaptoethanol, appropriately diluted test sample, 12. F(ab')z fragment purified by affinity chromatography at 5 g/d Incubate the culture in 100ml of RPM 11640 medium containing single sheep anti-human Igl'l for 68-72 hours. During the last 16 hours of this incubation period, the cells are exposed to 0.5 μci [“H]-thymidine with a specific activity of 75 C i/m 5 ole. Next, the culture is carried out until the glass fibers are collected on paper and the uptake of the radiolabel is determined. scintillation Measured by counting the number of motions. Details regarding similar assays for murine IL-4 activity can be found in the reference reviewed by Brooks et al., Methods: 372 (1985). In the bIL-4 activity measurement, the unit of activity is calculated based on the amount of hIL-4 that induces 50% of the maximum thymidine uptake.For example, after a 1:20 dilution. If the 100 m sample produced the maximum amount of 172 thymidine incorporation, one unit The position is defined as the activity contained in 1720 or 5111 of 100ul. death Therefore, this sample will contain 1000 divided by 5 or 200 units of hlL-4 activity per ld (U/d). The nucleotide sequence of the hIL-4 protein and the deduced amino acid sequence of the cDNA sequence encoding the wild-type hIL-4 protein are shown in Figure 1. The nucleotides are numbered starting with the ATG codon, which corresponds to the N-terminal methionine of the full-length natural polypeptide. Similarly, the amino acids are numbered starting from this methionine. It has been done. Natural proteins have a leading sequence of 23 or 25 amino acids in front of the histidine residue that becomes the N-terminus in the mature, secreted protein. Contains columns. Based on comparisons of hlL-4 with its murine homologue, the old st 3 is presumed to be the N-terminus. However, according to parallel expression experiments, If so, any tag with old s23 and His” as the N-terminal amino acid residue Equivalent biological activity has been shown for proteins. Figure 2 shows the nucleotide sequence of the synthetic gene encoding hlL-4 myutein. shows the amino acid sequence GluAlaGluAla-hIL-4- (Asp". 4spl!?), which represents a preferred embodiment within the scope of the present invention. Ru. 1! Numerous DNA constructs containing all or a portion of the nucleotide sequence of Figure 1 of Minnie and Minnie In can be conveniently combined with oligonucleotide cassettes containing additional useful restriction enzyme cleavage sites. It can be prepared as needed. The present invention provides one or more hlL-4 sequences that are substantially homologous to the natural hlL-4 sequence. It is related to certain similar proteins or mutins that contain the above intentional amino acid substitutions, deletions, or insertions, but do not have a negative effect on activity. Mutations are introduced at specific loci flanked by restriction enzyme sites that can be ligated to the native sequence fragment. This can be done with synthetic oligonucleotides containing mutated sequences that can be introduced into the system. After the ligation reaction, the resulting reconstructed sequence encodes a mutin with the desired amino acid substitution or deletion. This method is illustrated in Figures 3-6. It is shown. Alternatively, site-directed mutagenesis methods, identified using oligonucleotides, are used to provide modified genes with specific codons modified according to the desired substitutions, deletions, or insertions. It can be done. Bauer et al., Fallen Box 3 (1985), Craik, Biotech ni ues January 1985+12-19, Smith et al. an±Methods (Plenum Press, 1981), and U.S. Pat. No. 4,518,584, disclose techniques suitable for this and are also incorporated herein by reference. forms part of it. For either approach, conventional oligonucleotide synthesis techniques are used, such as Sood et al., Nucl. Ac1d Res, 4: 2557 (1977) and Hirose et al., Tet. Lett. 28:2449 (1978) is suitable. When performing site-directed mutagenesis, there is a sense that corresponds to the gene to be modified. The gene to be modified is cloned into M13 single-stranded phage or other suitable vector to yield single-stranded DNA containing either antisense or antisense stranded DNA. Loaned. This DNA anneals to the M13 phage fragment and is cut. This provides a double strand with an eye, which is then hybridized with an oligonucleotide primer. Make it loud. This primer is complementary to the sequence flanking the codon to be modified, but it also contains a codon that specifies a new amino acid at the site where the change is to be made (or an antisense codon that is complementary to such a codon). ). too However, if a deletion is desired, primers are used that maintain the correct reading frame and lack specific codons that determine the amino acids to be deleted. If insertion is desired If so, the primer will contain a new codon at the appropriate location in the sequence that specifies the amino acid to be inserted. Codons to be replaced with W, codons to be deleted, or codons to be inserted Preferably, the don is located at or near the center of the oligonucleotide. The size of the oligonucleotide primers used is determined to avoid influencing mutations due to the exonuclease activity of the DNA polymerase used to fill in the gaps. Considering that sufficient length is required for the The need to optimize stable and specific hybridization to multiple mutation sites Therefore, it is determined. Therefore, the oligonucleotides used according to the invention A base will usually contain about 15 to about 25 bases. Larger size o Ligonucleotides are unnecessary. The oligonucleotide primer is then attached to a single-stranded template containing the gene to be modified. hybridize to a gapped duplex with a blank region. This primer is then extended along the template strand by reaction with DNA polymerase I (Klenow fragment), T4 DNA polymerase, or other suitable NA polymerase. The resulting double-stranded DNA is then converted into closed circular DNA by treatment with a DNA ligase, such as T4 DNA ligase, and the resulting heteroduplex is isolated from E. coli JM105 (Bethesda Research). Laboratories (Bethesda Re5earch Laboratories), Gaisse suitable, such as Gaithersburg, Maryland, USA). used to transform appropriate host strains. Replication of heteroduplex by the host , the descendants below both strands are supplied. This transformed cell is then transformed into a plastic This is similar to that used for the mutagenesis method. Screening is performed using a corresponding labeled oligonucleotide. mirror strike Hybridization of primers to mutant DNA rather than land descendants Conditions are used such that this occurs preferentially. The DNA containing the mutant gene is then isolated and ligated into an appropriate expression vector, which is used to transform the host strain. This host strain then provides similar proteins. cultivated to provide food. The specific mutagenesis strategies underlying the invention are described below. After translation, many secreted proteins are covalently linked, often in the form of oligosaccharide units linked to asparagine side chains by N-glycodyl bonds. Acquire carbohydrates. The structure and number of oligosaccharides linked to specific secreted proteins are highly variable, resulting in a wide range of apparent molecular masses even when attributed to a single glycoprotein. It will be distributed over a wide range. Human IL-4 is a secreted glycoprotein of this type. It is quality. Attempts to express glycoproteins in recombinant systems have Recombinant glycoprotein mixtures, e.g., purified human or murine granulocyte-macrophage-colony stimulating factor (GM-CSF), may be complicated by heterogeneity due to the hydrate moiety. may contain up to 50% carbohydrates by weight. Miyajima et al., EIIBO Journal 5: 1193 (1986), introduced a mutation in the N-glycosylation site to prevent N-glycosylation and reduce the heterogeneity of the expression product in yeast. reported the expression of murine GM-C5F. The presence of diversity in the amount of carbohydrate attached in recombinant secreted glycoproteins complicates purification methods and therefore reduces yields. In addition, if this sugar protein If the yeast is used as a therapeutic agent, the recipient will have to react to the carbohydrate moiety of the yeast. Allergic reactions may occur and treatment may have to be discontinued. For these reasons, we have developed a biologically active, homogeneous immune system regulatory glycoprotein with reduced carbohydrate content. Quality analogs are being considered for use in therapy. Functional mutant analogs of human IL-4 with inactivated N-glycosylation sites can be generated by oligonucleotide synthesis and ligation or by site-directed mutagenesis methods, as described above. can be produced by These analogue proteins can be produced in high yields, homogeneously, and with carbohydrates using yeast expression systems. The present invention provides human IL-4 containing at least one amino acid substitution, deletion or insertion so as to inactivate the N-glycosylation site. It is related to the indigestion type. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-A'-Z, where AI is any amino acid and Z is serine or represents threonine. Within this sequence, asparagine (Asn) is a carbohydrate provides an amino group on the side chain for covalent bonding. Such sites include substitution of Asn or residue Z with another amino acid, deletion of Asn or Z, insertion of an amino acid other than Z between A1 and Z, or insertion of an amino acid between Asn and A10. It can be removed by inserting an amino acid other than Asn. Substitutions are preferably carried out conservatively; in other words, it is most preferable that the amino acid to be substituted has similar physicochemical characteristics to the residue to be replaced. Delicious. Similarly, when deletion or insertion methods are applied, the effect of the deletion or insertion on biological activity should be considered. Thus, the analog hIL-4 according to the invention has a variant amino acid sequence substantially homologous to the natural sequence of hIL-4, in which case at least one Asn-A' position in the natural sequence is present. -Z is replaced in the variant sequence by Asn-A2-Y or X-A''-A3, where AI, Am and A3 are the same or different any amino acid, X is any amino acid other than Asn, Y is any amino acid other than Z, and Z represents Set or Thr. All Asn-A'-Z in the natural sequence are Asn in the mutant sequence. -A"-Y or The protein contains two putative N-glycosylation sites, the first being a triad of AsnThrThr starting at residue 62 and the second starting at residue 129. AsnG1n5er, Asn Appropriately conserved substituted amino acids include Asp, G1r++G1u, Ala, Gly, Set, and Thr; and Glu are preferred; when Z is Set, suitable substituents are Met, Leu, He, Val, Asp, Gin, Glu or Asn, of which Net, Leu, lie and Val are preferred. When Z is Thr, conservative substituents are Val, Glu, Asp, Gln, Gly and Ala, with Val, Glu, 8sp or Gin being preferred. - Preferred substitutions to inactivate the glycosylation site are the substitutions of vals for Thr", Asp for Asn" and Asp for AsnI29. Other conservative amino acid substitutions can also be made to provide proteins lacking N-glycosylation sites. Mutins containing these substitutions are It is considered equivalent to that specifically disclosed and claimed herein. these places The following table 1 is given by equimarginalization according to the order of substitution effects at positions. 1: hIL-4 amino 1 Most preferred Asp Val Asp MetAsp Val Thr Thr Asn Cis-in

[Replacement] In order to avoid the formation of intermolecular cross-links or incorrect intramolecular disphite bonds, the present invention provides hlL-4 in which cysteine residues that are not essential for biological activity are deleted or substituted with other amino acids. He also has some thoughts on mutin. The native hlL-4 sequence contains six cysteine residues at positions 27.48.70.89.122 and 151 (see Figure 1), the first five cysteine residues. Stin has a counterpart among its rat analogues, while the last Sistine There is no match for the last residue, so the last residue cannot be replaced or deleted. This is a reasonable candidate. Site-directed mutagenesis or oligonucleotide substitution methods can be used to delete specific cysteine residues or to make conservative substitutions. Preferred amino acids for making substitutions are neutral amino acids such as Gly, Ala, Val, Leu, Ile, Tyr+Phe, His, Trp, Ser+Thr or Met. Among the above, Ser and Thr are preferred. Deletion, addition, or substitution of residues to alter the absence of adjacent pairs of basic residues, such as the pairs Arg-Arg, Arg-Lys, or Lys-Arg, of KEX2 Broase Kuniba-1. Appropriate mutagenesis methods can be used to inactivate sites that undergo processing by KEχ2 protease by altering the sequence. In particular, the Lys-Lys pair is considerably less susceptible to cleavage by KEX2, and conversion from Arg-Lys or Lys-Arg to Lys-Lys inactivates the KEX2 site. This represents a conservative approach. The resulting mutin targets the KEX2 protease at a site other than the α-factor leader sequence that is scheduled for cleavage upon secretion. becomes less susceptible to cleavage by Looking at FIG. 1, the Lys-Arg pair is present at position 123 of the natural sequence of hlL-4. If LysIt3 or ArgIz4 is replaced with an amino acid other than Arg, By substituting the KEX2 processing site of Arg-Arg, Arg-Lys, or Lys-Arg, mutant hlL-4 can be obtained. available. Comparison with the mouse IL-4 sequence indicates that the deletion of Lys123 is a conservative change. It is a heterogeneous method and therefore suggests that it is preferable. with other methods Arg+24 can also be replaced with Lys. KEX2po-a-1,a+(7)1 A preferred expression system for the IL-4 proteins of the present invention uses the yeast α-factor leader sequence to induce secretion of the recombinant protein by the yeast host. is also used. Ideally, this system would be compatible with the yeast KEX2 protein. From the N-terminus of the protein where rotease is required, α-factor is first released during secretion. and configured to cut the conductive array. The configuration of the α-factor leader sequence-hlL-4 protein with the Lys-ArgKEX2 protease site immediately adjacent to the N-terminal former S residue of the wild type does not necessarily result in cleavage upon secretion by recombinant yeast. Lys-Arg KEX2 recognition site and N-terminus of hIL-4 Insertion of the four peptide sequence Glu-Ala-Glu-Ala between the ends results in more efficient cleavage at the KEX2 site. The resulting product is the hlL-4 protein with the four peptides Glu-Ala-Glu-Ala at the N-terminus. Probably the yeast 5TE13 gene product, i.e. the N-terminal Overproduction of dipeptidyl aminopeptidase A that cleaves the Glu-Ala pair at the end It would be possible to remove these residues in vivo using a yeast strain that is capable of producing a protein. However, due to the presence of Glu-Ala-Glu-Ala sequence at the N-terminus, This suggests that there is no significant difference in the biological activity of this analog compared to the wild-type protein. has been observed. As mentioned above, yeast is preferred for the expression of analogs and native recombinant human IL-4. A typical expression vector is designed for selection and replication in E. coli. The DNA sequence (Ap' gene and origin of replication) derived from pBR322 of the female Alcohol dehydrogenase inhibited by yeast pBc104 (ATCC 67, 232), which contains yeast DNA sequences. The DH2 promoter was described by Russell et al., J. et al. Biol. Chem. 258:2674 (1982) and Beier et al., Nature 300:724 (1982). Contains the starting point. There is an α-factor leader sequence next to the promoter, and yeast hosts Enables secretion of foreign proteins from the main body. The α-factor leader sequence has been modified to include an Asp''% I) restriction enzyme cleavage site near its 3' end to facilitate fusion of this sequence to foreign genes. pBc104 also contains a cDNA insert encoding wild type hIL-4. Regarding the construction of this plasmid Details are given below. Other expression vectors that can be used include yeast vectors containing the α-factor promoter. vectors, such as pY a fHuGM (ATCC 53.157) 7: Yes, which carries the wild-type human GM-CSF gene. Other vectors are known to those skilled in the art. The construction of pYαfHuGM is described in previously published European patent application BP-A-183,350. The method for selecting an appropriate yeast strain for transformation will be determined by the nature of the selectable marker and other properties of the vector. S. cerevisiae suitable for transformation with various constructs derived from pBc104 or pyαfHuGM and their vectors. S. cerevisjae strains were obtained from the East Genetic Stock Center, Berkeley, California, USA [ It is available from [see below] and includes -iro' 工 1 - v↓ 1 adel his 2 gaki IV shi 19 - and so on. A particularly preferred expression strain, χν2181, Genetic Stock Center, Department of Biophysics and Medical Physics, California χ2181-IB available from Runia University, Berkeley, CA 94702, USA, and Department of Genetics, University of Washington, Seattle, WA 98 105, USA, or Immunex Corporation, 51 University Street, Seattle, WA 98101, USA, by combining two haploid strains, XV617-1-3B. It is a diploid. A suitable transformation method is that described by Hinnen et al., Proc, Natl., Acad, Sci, USA 75:1929 (197B), using a 0.67% yeast nitrogen source base, 0. Transformation of pBc104 or other constructs containing AIIH2 or α-factor promoters is selected on a selection medium consisting of 5% Kazaamino acids, 2% glucose, 10 μg/ld anidene and 20 μg/−uracil. The host strain containing the construct was injected with 80 μg/In1 adenine and 80 μg/d horse for expression. Composed of 1% yeast extract, 2% polypeptone, 1.1% glucose, supplemented with Rasil. and grown in a rich nutrient medium. Derepression of the ADH2 promoter is performed by removing glucose from the medium. cormorant. Recombinant human IL-4 protein produced by fermentation of a yeast strain of rhlL-4 protein has been described by Urdal et al., J. Chromato, 296: 17H1984) and Grabstein et al. 63:1405 (1986) by reverse phase HPLC steps using single or sequential preparative HPLC columns. For example, yeast conditioned medium containing hlL-4 is filtered through a 0.45μ filter. First, batch adsorption with a cation exchange carrier such as S-Sepharose was performed. and purification by elution. The pooled fractions from the batch adsorption/elution steps were then coated on 1°-20μ reversed-phase silica (Vydac, The Cell). The 5eparation Group, Hess A flow rate of 100 d/win was injected into a 5c1oX30cmO column packed with 100 d/win (Berea, CA, USA). The column was equilibrated with 0.1% aqueous trifluoroacetic acid before passing through the yeast-conditioned medium, and then, after the medium was loaded into the column, this column was equilibrated until the optical absorption of the eluate at 280n+* approached the baseline value. Wash with solvent. In this case, a gradient of 0.1% trifluoroacetic acid derived from 0 to 60-100% solvent B in acetonitrile at a flow rate of 100 d/min and a rate of change of 1-2% per minute may be used. At an appropriate time (10-20 min) after starting the gradient, a small aliquot of both aliquots is collected and a portion of both aliquots is analyzed for protein analysis by polyacrylamide gel electrophoresis and fluorescence (fluorescamine) protein assay. Analyzed for content. If necessary, further use HPLC or cation exchange chromatography. Sometimes. The HL-4 protein is a promising therapeutic agent for the treatment of immunodeficiency or neoplastic conditions. In such therapies, physiologically acceptable carriers or The hlL-4 protein in the form of a purified composition comprising the hlL-4 protein in combination with a parenteral injection, at a dosage effective to induce proliferation of B cells and T cells. Administered by subcutaneous injection or other suitable method. Suitable doses for IL-4 therapy are from 0.1 to 1100 p/kg body weight per day, as shown by animal studies. Another method is to In the form of adoptive immunotherapy, where certain immune cell classes are isolated, expanded in vitro in the presence of hlL-4 protein, and re-administered with additional doses of hlL-4 to cause tumor regression. Can be used. hlL-4 protein can also be used with human interleukin-2. These cancer treatments demonstrate that purified murine IL-4 stimulates the production of cytolytic T leukocytes in primary mixed leukocyte cultures, which induce the production of mixed lymphocytes that have been previously exposed to antigens from allogeneic cells. demonstrated by the observation that it induces cytolytic activity in the population. being suggested. Cytolytic T lymphocytes (CTLs) are cytotoxic or effector T cells. It is also known as a receptor-containing, antigen-specific lymphocyte. Alloreactive CTL are target cells that carry major histocompatibility (M)Ic) antigens that are the same as the allogeneic cell antigen used to stimulate or induce cytolytic cells. Lyse the vesicles. Virus- or tumor-specific CTLs are ``restricted'' in recognizing antigens, meaning that target cells bearing the antigen are It must have the same MBC antigen as the body. CTLs control viral replication by killing cells expressing virus-associated membrane antigens. It has also been indirectly linked to immune system surveillance and destruction of certain neoplastic cell types. Ru. CTL generation has been most extensively studied in mixed leukocyte cultures (MLC). Here, lymphocytes from genetically different (allogeneic) individuals are cultured together to induce T cell proliferation. These T cells are antigen-specific (an antigen present on the cells of one individual but not on another); Therefore, they are called alloreactive T cells. CTL activation and differentiation requires the participation of CTL progenitors, T "helper" cells, and accessory cells of the monocyte/macrophage lineage. CTL responses are initiated by antigen recognition by specific T cell populations. The activation of CTL precursors is triggered by exposure to appropriate antigens. expression of lymphokine receptors and secretion of lymphokines by helper T cells. Begins. Binding of lymphokines by CTL precursors induces antigen-activated CTL precursor proliferation and possibly differentiation into a cytolytic state. However, CTL precursors do not necessarily need to proliferate to reach their lytic state. However, the ability to kill is apparently a separate function. The lytic cycle carried out by T cells is initiated by cell-cell contact between live effector cells and target cells bearing appropriate antigenic determinants. A natural enzyme that exhibits cytolytic activity against a wide range of target cells without the need for activation by overt antigens. Unlike normal (NX) cells, CTLs perform lysis with distinguishable specificity, following contact and adhesion of effector and target cells, the so-called "lethal virus" A lethal bit is produced, thereby destroying the membrane permeability of the target. It will be done. This leads to increased osmotic pressure and eventual loss of cytoplasm. This effect Target cells also retain the ability to recognize and lyse other target cells. CTL proliferation and differentiation are controlled by soluble growth hormones, of which interleukin-2 (IL-2) is considered to be the most important. This time, it has been shown that IL-4 is also deeply involved in the generation of functionally active CTL. In particular, IL-4 acts as a potent helper factor for the generation of CTLs in primary mixed leukocyte cultures (MLCs) and induces lytic activity in MLC memory populations induced in vitro. Purified recombinant IL-4 and A direct comparison of IL-4 and IL-2 showed that IL-4 was more potent than IL-2 in increasing CTL generation in primary MLC. These two lymphokines differ from IL-4 in IL-2 and do not cause obvious antigen stimulation. Even if the It's different. t! by IL-4! The specificity of cell lysis induced could provide important therapeutic approaches, if by introduction of non-specific lymphokine-activated killer (LAK) cells (e.g. IL-2LAK therapy). Reducing side effects would enhance the effectiveness of adoptive immunotherapy. In a related observation, recombinant IL-4 has been linked to alloreactive T cell clones of various functional subtypes, including mitogen-activated T cells, thymocytes, memory T cells, and CTLs. has been shown to effectively induce the proliferation of . IL-4 stimulates the expansion of mitogen-activated murine spleen cells with Lyt2+ surface antigens. Regarding proliferation induction, it has been shown to have a stimulating effect comparable to that of IL-2. It is therefore clear that IL-4 is an important regulator of T cell proliferation and function. The following discussion is intended to provide further explanation regarding certain embodiments of the invention. In the experiments described below, restriction enzyme digestion of DNA, purification of DNA fragments by gel electrophoresis, ligation of DNA fragments, transformation into E. coli (RRI strain was used throughout), and restriction enzyme digestion were performed. Standard molecular biology techniques, such as construct analysis and confirmation by Maniatis et al., Molecular Cloning Near Laboratory Manual The method described by Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, 19B2) was followed. ! -One-zeta engineering of the cDNA encoding hIL-4 A synthetic oligonucleotide was constructed to be complementary to human IL-4ON and C-terminal coding region sequences. The N-terminal probe has the sequence 5'-CAGTTG GGAGGTGAGACCCAT-3', while the C-terminal probe has the sequence 5'-TCAGCTCGAACACTTTGAATA-3'. The synthesis method is described by Soot et al., Nucleic Acids Res, 4:255? (1977) and Hirose et al., Tet, Lett, 28:2449 (1978). Following synthetic synthesis, the oligonucleotides are deprotected and purified by Sephadex G-50 chromatography followed by preparative gel electrophoresis. This oligonucleotide is used as a screening probe. For use as a library, Maniatis et al. Laboratory Manual (Cold Spring Harbor Laboratory, 1) 982) by standard techniques such as those disclosed in ``P-ATP and T4 polynucleotides'' Using cleotide kinase! ! The end is radiolabeled by p. Human peripheral blood T lymphocytes (PBT) stimulated with phytohemagglutinin (PI (A) and phorbol 12-myristate 13-acetate (TMA)) A cDNA library was constructed by reverse transcription of polyadenylated mRNA isolated from total aiRNA extracted from a. The EcoRI cleavage site in the cDNA is methylated with EcoRI methylase to protect it from subsequent EcoR1 cleavage, and ligated with an EcoRI linker, making one copy of the linker at both ends of the cDNA. The bacteriophage λgtlo-Z was cut with coRI and then dephosphorylated, and the resulting arm was injected with ligage gin. (Huynh et al., DNA Cloning: A Practical Approach, Glover, IRL Publishing pp. 49-78), La. The igated DNA is packaged into phage particles to create a library of 2.5×10′ recombinants. 5X10S recombinant is in large intestine strain C600hfl- and radiolabeled the above oligonucleotide protein. Screen using standard plaque hybridization techniques using will be processed. Three clones showing positive hybridization were selected for PBT labeling. isolated from the library. These clones were plaque purified and bactericidal. It was used to prepare lyophage DNA and was digested with EcoRI. This digest was electrophoresed on an agarose gel, plotted on a nylon filter, and Hybridization of the fragments to the two types of oligonucleotide probes described above was re-assayed. One clone contained fragments that showed positive hybridization to both probes. This DNA fragment containing an internal EcoRI cleavage site was isolated by preparative agarose gel electrophoresis following partial digestion with EcoRI, and then isolated with a unique EcoRI site, a Baai HI site and a number of other unique restrictions. A standard polymerization base containing a polylinker with an enzymatic cleavage site. subcloning into the EcoRl cleavage of vector pBR322 (pGemb l ). Googled. The resulting plasmid was named pGe+++bl:hTL-4. An example of a typical vector that is substantially the same as pGe+++bl is Described by Dente et al., Nucleic Acids Research 11:1645 (1983). After transformation of the appropriate E. coli host strain, the plasmid DNA is transformed by standard methods. It was purified using EcoRV and cleaved with EcoRV. The resulting fragment was inserted into Hi18 and BamHI-digested pBC(C5F-1) or and ligated to the linker fragment below which provides the KEX2α-factor processing site and the first four amino acids of hIL-4 with the old sts amino acid terminus. Leu Asp Lys Arg His Lys Cys AspKEχ2 Rotease cleaves the peptide immediately after the Arg residue. This construct was called pBC104. pBc103 was prepared in substantially the same way, with the shadow of hlL-4, the 118- and β-I-cleaved pBC (CSF-1) immediately after the MHX2 cleavage site, and the old were prepared by ligation with the following oligonucleotide fragments encoding an additional old 5-Gly located before szs. The resulting expression vectors were designated pBc103 and pBc104 and were amplified in S. enterica and transformed into yeast strain XV2181 by the method previously described. It was used to The transformed yeast has AI)) derepression of the 12 promoter. cultured in a nutrient medium under conditions that promote regulation, and the resulting conditioned medium Then, the activity of hI L-4 was measured using a sheep anti-human IgM A(ab)z fragment as a coactivator. This activity measurement showed a medium activity of 43.4270/d for the medium conditioned by pBc104-transformed XV2181 and 46.4 for the medium conditioned by pBc103-transformed A medium activity of 149 U/ail was demonstrated. Thrust difference 3Ii" stop (fuchi 1Jλ2" maj1 force)

[Two asparagine-linked glycosylation sites (Asn62 and Asn129) present in the natural hlL-4 protein require the codon at this position to be replaced with a codon encoding aspartic acid. removed by adding This prevents N-linked glycosylation or even hyperglycosylation of proteins secreted by yeast cells, thereby allowing the production of a more homogeneous product. The N-glycosylation site of the hlL-4 eDNA (pBc104) described above was inactivated by substituting a portion of the cDNA with a synthetic oligonucleotide containing the desired nucleotide change as described below. Cloning vector containing the wild type hlL-4 cDNA sequence shown in Figure 1 - (pGemb I: hIL-4) is the 12th nucleotide of mature hIL-4. It was cleaved with the restriction enzyme EcoRV, which cuts after the octide, and Ba+a H1, which cuts downstream from the hIL-4 cDNA within the polylinker region of the vector. An approximately 550 base pair hlL-4 cDNA fragment was subcloned into the pBR322-derived vector pPL-3 by digesting this vector with EcoRV and BamHI (see Figure 3). The resulting plasmid was named L225. The L225-derived IINA fragment containing the hIL-4 cDNA was then obtained by digesting the plasmid L225 with Cla I (5' of the hIL-4 cDNA) and T4 DNA polynucleotide. The pBR322-derived vector pGEM- was treated with merase to make blunt ends and digested with Mutan1 (3' to the hlL-4 cDNA within the polylinker region) to remove the cDNA-containing fragment. 3 (Promega Biotec, Madison, WI, USA). Vector pGEM-3 was digested with Hindnl, treated with T4 DNA polymerase to create blunt ends, and then digested with SstI. Living The resulting plasmid was named L257. This plasmid contains the oligonucleotides listed below. It was used to perform the cleotide substitution mutagenesis method. Amino acid residue or null All references to cleotide numbering should be made according to the numbering in Figure 1. Residues and nucleotides are counted from the N-terminus of the full-length translation product, including the putative natural signal peptide. The codon encoding asparagine at position 62 is converted to asparagine as follows. The codon encoding paragic acid was changed. Plasmid L257 contains Hinc II that cuts at nucleotide 152 and Digested with Pst I cutting at cleotide 211. The vector break that occurred here The piece was isolated and ligated to oligonucleotide A described below. Thr Thr Glu Lys Glu Thr Phe Cys. The underlined nucleotides above indicate changes from the wild-type cDNA sequence. are doing. A single A/T to G/C change at nucleotide 184 alone The other 5 base changes do not alter the amino acid sequence but create a restriction enzyme cleavage site (Ha) to facilitate identification of the modified sequence. Similarly, the codon encoding asparagine at position 129 is introduced using the following synthetic oligonucleotide: The EcoR1 site (nucleonucleotide) in hIL-4 cDNA was Substituting a DNA fragment from Rsa 1 site (nucleotide 393) from nucleotide 360) The codon was changed to one that encodes aspartic acid. Underlined nucleotides indicate changes from the wild type cDNA sequence. The change from A/T to G/C at nucleotide 385 alone results in one amino acid Generates a codon that specifies a noic acid change (Asn129 to Asn129). the Other base changes were made without altering the amino acid sequence (introducing a Sal 1 restriction enzyme site). The medium was named pBC132. To prepare a yeast expression vector for mutin, a DNA fragment encoding hIL-4 (85p6z, Asp+29) was engineered as shown below. It was removed from the pBc132 vector by digestion with EcoRV and 5stl and inserted into the yeast expression vector plXY120. pIXY120 is virtually identical to pBc104 except for its heterologous insert. As described later, pBC104 is also used for expression of the mutin of the present invention. Therefore, it can be used instead of plXY120. Yeast expression vector pIXY120 (Fig. 4) contains the DNA sequence derived from the following. 1. Derived from E. coli vector pBR322, used for replication origin and selection within the large intestine. A long restriction fragment from vomer I (nucleotide 562) to Eco Rl (nucleotide 4361) containing an ampicillin resistance marker. 2. DNA fragment derived from yeast S. cerevisiae. This DNA fragment is selected in yeast. The TRP-1 gene as a selective marker, the yeast 2-replication origin, S. cerevisiae MDI (2 promoter), and the secreted peptide α-factor (Brake et al., Proc. Natl., Acad, Sci. USA 81: 4642 (1984), Kurjan and Haas Herskowitz, Ce1l 30:933 (1982, and see U.S. Pat. No. 4,546,082). It contains the original 85 amino acid signal peptide. The 18 restriction enzyme sites are located within the α-factor signal peptide to facilitate fusion to a heterologous gene. Introduced into leotide 237. The T residue at nucleotide 241 is an oligonucleotide. C residue was changed by a do-targeted in vitro mutagenesis method. 3. A synthetic oligonucleotide containing a multiple cloning site was inserted from the box 18 site (amino acid 79) near the 3' end of the α-factor signal conjugate into the 4-sided 1 site contained in the 2μ sequence:. 4. A 514 base pair DNA fragment derived from single-stranded bacteriophage f1 containing the origin of replication and the gene open region. This fragment was inserted into the Nru 1 site of the pBR322 DNA sequence. Because of the presence of the fl replication initiation site, it is possible to transform into an appropriate (male) E. coli strain and receive multiple infections with bacteriophage f1. A single-stranded copy of the vector is produced. This allows the DNA sequence of the vector to be determined. This facilitates identification and allows for in vitro mutagenesis methods. The yeast expression vector plXY120 was digested with the restriction enzyme Ha18, which cuts near the 3' end (nucleotide 237) of the α-factor leading peptide, and with BamHI to cut within the polylinker. Amputated. this big The vector fragment was purified and ligated to one piece of the following two types of DNA as shown in FIG. 1. Plasmid Gembl: Obtained from hlL-4, from the MutaV site (nucleotide 136 of mature hlL-4) to the BamH1 site (Gembl: 3' side to the hlL-4 cDNA in the hlL-4 polylinker). hlL-4 cDNA fragment. 2. Synthetic oligonucleotide linker 1 below: This revamps the 3' end of the α-factor leader peptide and attaches it to the 5' 4 amino acids of hIL-4 in reading frame. It is something that fuses with sea urchins. This oligonucleotide also encodes an 8 amino acid identification peptide fused to the N-terminus of hlL-4. hlL-4tan By fusing this sequence to protein, detection using specific antibodies becomes possible. This plasmid, called plXY118 (Figure 5), was initially used to monitor the expression and purification of hIL-4, and was designed to contain the ADH2 protein, which is repressed by glucose. Contains the wild type hlL-4 gene under control of the motor. α-factor leading vector Butide allows secretion of hlL-4 from yeast cells. Proteolytic processing of the alpha-factor leader sequence occurs after the Lys-Arg residues (amino acids 83 and 84) of the alpha-factor leader sequence. Contains the blL-4 gene excluding consensus sites that undergo N-linked glycosylation. To create a yeast expression vector, plasmid pIXY118 was digested with EcoRl (which cuts the 5' side of the ADH2 promoter) and 5stl (which cuts the 3' side of the hIL-4 gene). Figure 5). this big The vector fragment was purified and ligated to the following DNA fragment. 1, ADH2 promoter, α-factor leader sequence and first 4 amino acids of hlL-4 2. EcoRl to EcoRV DNA fragment from plXY118 carrying the amino acid (this was required due to the presence of a shadow I site within this fragment); 2. EcoRV site (mature) contained in plasmid pBc132; hlL-4 cDNA insert from nucleotide 13 of type hlL-4 to the 5st1 site (3' side of hlL-4 cDNA). The plasmid generated here was named plXY133. It contains the hIL-4 gene in a yeast expression vector with codon changes of Asp62 and Asp129 and a fusion peptide consisting of 8 amino acids at the N-terminus. The final yeast expression plasmid is identical to plasmid pIXY133, except for the oligonucleotide linker sequence used to fuse the hlL-4 cDNA to the alpha-factor leader sequence (Oligonucleotide 2, Figure 6). This yeast expression plasmid was constructed as described below and shown in FIG. Yeast expression vector plXY120 was cut with restriction enzymes box 18 and B ag HI as described above. This large vector fragment is then ligated with the next DNA fragment. was applied. (1) EcoRV (nuclease) derived from plasmid pxxy133 hlL-4 (Asp"Asp") cDNA from the Ban HI site (3' side of the hlL-4 cDNA) (position 13), (2) the 3' end of the α-factor leading peptide to the Ban HI site (3' side of the hlL-4 cDNA), -Leu-Asp-Lys-Arg-Glu-Ala-Glu-Ala) site, and this Synthetic oligonucleotide fused in reading frame to a amino acid at the EcoRV site (oligonucleotide 2, Figure 6). The sequence of this oligonucleotide is shown below. The resulting plasmid was named plXY157 (Figure 6). child The vector, when present in yeast, allows glucose-controlled expression and secretion of non-glycosylated mutant hlL-4. Since the recovered hlL-4 is not processed by the yeast protease dipeptidyl-amino-peptidase A, it contains the four amino acids Glu-Ala-Glu-Ala at its N-terminus. I'm there. The majority of the α-factor leader sequence is proteolytically removed by the KEX2 gene product after the Lys-Arg residues (amino acids 83 and 84 of the leader sequence). The somewhat redundant route described above involves excising the fragment containing the IL-4 cDNA of EC0RV BJυ)(I) from pBC104, digesting this fragment, and asperating the Aspergillus aspergillus as described above. Using synthetic oligonucleotides to modify lagin-linked glycosylation sites It can be shortened by reconstitution as an EcoRV-5st I fragment. pBc104 contains vector fragments that can be cut with EcoRI and Sst I and EcoRI and EcoRV and ligated with the reconstituted Eco RV-5st I fragment of mutant IL-4, as described above for pIXY118. Next, this construct, which can generate 1-EcoRV, was digested with Ha118 and Bag HI, and the resulting vector fragment was used to generate (1) EcoRV derived from the same plasmid containing the IL-4-like gene; - Bas+HI fragment, and and (2) can be ligated to the synthetic oligonucleotide 2 described above to generate a yeast expression vector for GluAlaGluAla-hlL-4- (Asp”. Aspl!?) identical to plXY157. 4: and Yeast containing an expression plasmid encoding the protein GluAlaG1uA1a-hIL-4-(Aspz, Asp'''), which is similar to protein GluAlaG1uA1a-hIL-4 (Asphz, Asp'''), was maintained on YNB-trp agar that was stored at 4°C. Fresh agar plates were prepared from frozen glycerol storage (-70°C) every -week. Preculture was carried out by adding several monomers to 1 liter of YNB-trp medium (6,7 g/f yeast nitrogen source, 5 g/l casamino acids, 40 wg/l adenine, 160+ ag/j! uracil, and 200 mg/f tyrosine). Isolated transformant yeast colonies and cultured overnight in two 2-liter flasks at 30°C with vigorous shaking. fed. By the next morning, the culture had entered stationary phase and reached saturation, going from 2 to 7 00600. After cleaning and sterilizing two 10-liter fermenters, fill them to 80% of their capacity with 12150 YEP medium (12 g/ffi yeast extract, 50 g/i! peptone) and store at 30 °C. , stirring at 500 to 600 rpm, and maintaining aeration conditions at 10-1 to 2 LPM. Inoculum was added. After 2 hours of growth, 50% glucose nutrients were added at a rate of 50 g/Q during 10-12 hours. The nutrient source was then transferred to 50% ethanol and added for a total of 10 d//2 over 6 hours. The total elapsed time of fermentation was approximately 20 hours. The final optical density (600 nm) ranged from 30 to 45. The fermentor was cooled to 20"C and the bacterial collection process was started. First, 5M NaOH was added to adjust the pH to 8.0. The contents were collected in a large clean glass bottle. The yeast beer was then filtered through a Milliporee Pelicon filter system using a 0.45μ filter cassette and collected into sterile 102 large glass bottles. GluAlaGluAla-hlL-4-(Aspbt, Asp''') mutin (IL-4 mutin) in the filtered yeast supernatant is S-Sepharo Luhatch adsorption on Sgel, washing with 50+eM B-7 ranin pH 4,0 and 50mM HEPES pH7,4, elution with 0.5M NaCl and 50mM HEPES pH7,4, high performance liquid chromatography (HPLC), MONO-S column. chromatography, and 100mM) Purified by dialysis. In the first step, IL-4 mini-tin contained in yeast beer is It was bound to an S-Sepharose gel by a binding method. In a typical operation, a gel slurry of 400d of Sepharose (1 volume of gel: 1 volume of 0.5M β-alanine pH 4.0) is added to 10f of yeast beer. added to the file. The pH of this solution was adjusted to pH 3.6 by adding 2N H(J).The solution was then stirred for 10 minutes and the gel was allowed to precipitate for 30 minutes. The supernatant was decanted through a sintered glass funnel and the recombinant hlL-4 mute A gel slurry containing the quinine remained in the funnel. The gel was washed with 500 μM of 50 M β-alanine pH 4.0, then twice with 12 μM of 50 mM HEPBS pH 7.4. In is 0.5M NaCj of 200d! , was eluted from the gel by washing five times with 50 mM HEPES pH 7.4 solution. The highest concentration of IL-4 Miute The S-Sepharose eluates 1 to 3 containing the protein were pooled, oversterilized, and stored at 4°C until subjected to IPLC. The fourth and fifth eluates containing less than 10% IL-4 myutein were separated, filter sterilized, and stored at 4°C. rough fermentation Mother beer, unbound fraction, 3 washes, 1-3 and 4-5 stored Samples obtained from each eluate were examined for the presence of IL-4 by immunodot plot and 5DA-PAGE. Protein concentration in the pooled eluates was determined by BCA assay. The S-Sepharose fraction is 100i! The yeast of the beer was collected until it was processed. At this point, pool all eluates from 1-3 washes (as described above) and equilibrate with 0.1% trifluoroacetic acid (TFA)/pyrogen-free water. The reaction mixture was applied to a 5C11X30C11 column packed with 15-20μ C-4 reverse phase silica using a Waters LC-500 HPLC. C-4 column is 1! The fraction containing 0Mi recombinant hlL-4 washed with 0.1% TFA/pyrogen-free aqueous solution was applied to the column at a change rate of 2% per minute and a flow rate of 100-min per minute. It was eluted with a gradient of acetonitrile solution. Save the peak fractions from C-J RPC columns and add 1/10 volume of 0.5M β-alcohol. Lanin pH4 was added. Samples were collected and pooled onto a 20Id MONO-S column (1,6em x 10CI11.Pharmacia) at a flow rate of 7d/min. I called fast. After injecting the sample, fill the column with 100al! IL-4 myutein was washed with 50a+M Tris pH 7,4 and washed with IM NaCl 1,100w+M Tris pH 7,4. The peak fraction of IL-4 eluted with a linear gradient of pH 8 was then saved, dialyzed against 100 mM) pH 8 overnight at 4°C, and filter sterilized. Once production and purification were complete, the entire highly active product was stored at 4°C in sterile polyethylene tubes. The specific activity of purified hlL-4 (Asp", Asp"") was 3.1 ± 107 units per 1 according to the BCGF activity measurement method. IL-4 stimulates the proliferation of certain factor-dependent, non-B-cell lineages that normally respond to IL-2 or the myeloid growth factor IL-3. See Grubstein et al., J. Exl. L163: 1405 (1986) and Lee et al., K. Natl. Acad, Sci. USA 83: 2061 (1986). IL- To demonstrate that 4 also influences the initial T cell population, particularly the generation of functionally active T cells, we demonstrated the growth of CTLs in mixed leukocyte cultures (Ml, C). We investigated the effects on growth. MLC was established using suboptimal concentrations of C57BL/6 spleen responsive cells and allogeneic, irradiated DBA/2 lung stimulating cells. Five days after the start of culture, the lytic activity against 5ICr di-labeled P815 murine (DBA/2 origin) tumor target cells was measured. The murine IL-4 cDNA was derived from the cDNA sequence described by Lee et al., Proc. Natl., Acad, Sci. LISA 83:2061 (1986). EL4 thymoma stimulated by phorbol myristate acetate using column It was cloned from a library prepared from size-sorted mRNA from tumor cells. The full-length cDNA contains the PBR322 sequence, the yeast TRPI gene for tryptophan selection, the yeast 2μ origin of replication and the yeast alcohol dehydrogenase 2 (ADH2) promoter, and sufficient α-factor to direct synthesis and secretion. Subcloned into a yeast expression vector containing the leader sequence. The expression plasmid was transformed into yeast strain 79 (CR, TR and U, 1EU2-2) and Trp” transformants were selected. For purification, 1 liter of rich medium was Culture by inoculating the soil (1% yeast extract, 2% peptone, 1% glucose). The cells were cultivated and grown at 30°C until reaching the stationary phase. PMSF and Pepsta Chin A was added during bacterial collection. Cells were removed by centrifugation and filtration through a 0.45-cellulose acetate filter. rlL-4 was described by Ardal et al. The yeast supernatant was purified to homogeneity by performing 5 cycles of high performance liquid chromatography (IPLc) using a solvent system previously described by Shidan + Ro + Su1 Ho-296: 17H1984). Homogeneous recombinant and native murine IL-4 was measured in the B cell proliferation assay described below at 2. Same as OX 105U/μg They showed similar specific activities. MLC was prepared by mixing 5 x 10 s of C57BL/6 murine spleen cells and stimulated cells of DBA/2 murine spleen cells that had undergone 5 x 10' of gamma irradiation (2,50 Or). The cells were started in 16 mm diameter culture wells. The culture medium was Dulbecco's modified Eagle's medium containing 5% fetal bovine serum (FBS), 5x10'. Dulbecco's Modified Eagle's Medium, which is substantially the same as that disclosed by Cerottini et al., J. Ex. Med, 140:703 (1974). Cultures were initially supplemented with 2 ng/- of homogeneous native murine IL-4 (nIL-4), Long/d of recombinant human IL-2, or medium. Culture development Five days after initiation, the effector was cultured in a 96-well V-bottom microtiter plate. The lytic activity was tested by incubating in duplicate at 2001 Jl volume at various ratios of S + Cr-labeled 1P815 target cells (2 x 10' cells/well). After incubation for 3.5 hours, the plate was centrifuged and 150 At! of supernatant was removed from each well. was collected and counted using a gamma counter. The obtained results are shown in Figure 6. It is. In Figure 6, the reported percentage 5ICr release is determined by 100X [cpm (experimental) - cp - (spontaneous)] / [cp - (maximum) - cps (spontaneous)], Here, spontaneous release (118 cpm) was determined by incubating P815 in culture and maximum release (801 cp+++) was determined by incubating P815 in INHCJ. One lytic unit (Ltl) is required for 50% lysis of 2X10' P815 target cells. It is defined as the number of cells present in a given dose and determined from a dose-response curve. Percent recovery equals the number of cells recovered on day 5 as a percentage of the initial number of reacting cells cultured. Cultures initially supplemented with 2 ng/- of homogeneous, native IL-4 exhibited 50-fold higher cytolytic activity on a cell-by-cell basis than control cultures in the absence of exogenous IL-4; , showed an additional 100-fold increase in activity. Cultures supplemented with 10 ng/im rlL-2 showed higher CTL activity compared to control cultures, but lytic activity was sevenfold lower than that seen in cultures supplemented with IL-4. . Responsiveness The lytic activity against cells and syngeneic target cells is directed against specific allogeneic targets. The cytolytic 1923 cells generated in MLC supplemented with IL-4 or IL-2 were specific for alloantigens, as less than 2% of the cells (data not shown) were present. This data shows that IL-4 has a strong effect on the generation of allogeneic cytolytic 1923 spheres. This shows that it is a powerful helper factor. 6: t T ・L-sickness: MLC populations cultured over a certain period gradually lose CTL activity, but when exposed to allogeneic cells or culture supernatant, MLC populations cultured over a certain period gradually lose CTL activity. By this, high levels of cytolytic activity can be induced again. To examine the effect of IL-4 on such MLC memory populations, cells obtained from day 14 C57BL/6 anti-DBA/2 primary MLC were incubated with recombinant IL-2 or IL-4. After 3 days, the cytolytic activity was measured. Mixed leukocyte culture was carried out in a total volume of 20 m in a 25 d flask with 25 X 10'C 57BL/6 splenic cells and 25 X 10' irradiated (250 Or) DBA/2 splenic stimulated cells. It was composed of 14 days after starting, the primary culture Cells were harvested from cultured cells and 5×10′ cells were added to 2 d volumes containing lng/In1 recombinant murine IL-4, 0,5 ng/d recombinant human IL-2 or medium. Quantities were recultured in Costar 16 mm culture wells. After 3 days, the culture was! +(r-labeled P815 target cells were tested for lytic activity. The spontaneous release in the experiment was 204 cp+m, while the maximum release was 1.829 cpm. As shown in Figure 7, exposure of cells to either lymphokine results in cell proliferation and induction of high levels of cytolytic activity. Keeps you warm with IL-4 The lytic activity that occurs in incubated cultures is similar to that observed in control cultures incubated in medium. (Figure 7) and 80 times higher than the activity of the 14-day population before exposure to exogenous lymphokines (data not shown). IL-4-induced CTL activity during these cultures was antigen-specific, as was the case with primary MLCs (data not shown). Therefore, like Roux 2, Roux 4 induces the expression of antigen-specific cytolytic activity in the dormant memory T cell population once activated, without the need for further antigen stimulation. Induce. 17: Recombination in the ability of IL-4 and IL-2 to increase CTL production To directly assay the relative effects of recombinant IL-4 and IL-2, various concentrations of homogeneous recombinant IL-4 or IL-2 were added to allogeneic primary mixed leukocyte cultures, thereby The resulting lytic activity was measured after 5 days. In this experiment, the mixed leukocyte culture (MLC) consisted of 2 x 10'' C57BL/6 spleen cells and 5 x 10' irradiated (2,50 Or) DBA/2 (allogeneic) or C57BL/6 (syngeneic) cells. ) consists of murine tileable cells, and the species Various amounts of recombinant IL-4 or IL-2 were added. Lytic activity against 5lCr-labeled P81 5 was measured after 5 days as described above. Spontaneous release averaged 125 cpI11, but a maximum release of 886 cpm was observed. Although both lymphokines increase cell proliferation and CTL activity, the level of lytic activity that appears in cultures containing the optimal amount of IL-4 is greater than that observed in cultures with the lowest amount of IL-2. It was 3-4 times higher. Furthermore, at suboptimal lymphokine doses, approximately 171° of IL-4 was required to obtain similar lytic activity. These data are based on mixed leukemia consisting of a combination of allogeneic strains. In sphere cultures, IL-4 has been shown to be a more potent helper than IL-2 for the generation of CTLs from unstimulated precursors. Because approximately the same number of cells are recovered in allogeneic CTLs supplemented with IL-2 or IL-4, this data suggests that CTLs are present at a higher frequency or that individual CTLs have a greater solubility. This probably reflects the fact that it has deactivation. Medium 55 4 21 <1 The data presented above and in Examples 6 and 7 demonstrate that IL-4 induces both proliferation and cytolytic activity in primary and memory MLC populations, providing novel information regarding CTL generation. The mechanism of control is revealed. 8:2 Chi1 thymocytes were obtained from 6-10 week old C57BL/6J female mice, containing 5% FBS, culture supplements as described above, and as shown in Table 3 below. 200 I! with or without 0.25% PHA-M (Gibco Laboratories, Grand Island, NY) and with recombinant human IL-2 or murine Lu-4. ! (7) Culture at 1.5 X 10' cells/well in RPfll 1640. nurtured. Cultures were pulse labeled with 2.0 g Ci of [3H]thymidine (75Ci/mole) for 72 hours in lllJlrel & for 18 hours afterward. The bacteria were collected on a glass fiber filter, and the incorporated radioactive label was measured. rIL-2 and rIL-4 were used at 10 ng/d. The results are expressed in Table 3 below as the mean cps of duplicate cultures. 3: Effect of rIL-4. These results indicate that IL-4 induces thymocyte proliferation in the presence or absence of comitogenic stimulation. In the presence of added mitogens, proliferation was significantly (7x) promoted. 9: IL-4 induced 1' memory T cells were generated in 14 day old primary MLCs. The first generation! 1LC was composed of 25 x 10' C57BL/6 splenic cells and DBA/2 splenic stimulated cells that had been irradiated (2500r) for 25 x 10 h in 20 m (7) culture medium in a 25c1iI flask. Ta. The culture medium was Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 5 x 10-'M 2-mercaptoethanol, and additional amino acids, which was essentially the same as described in Serroti et al. 1. It is the same as that disclosed by "Shishishi" Tank9-1 Edition: 703 (1974). First time on the 13th for the second generation MLC. 5X10' cells recovered from the MLC were cultured with 5X10' irradiated (250 Or) DBA/2 splenic cells in 1611II 11 culture wells containing 2-cell culture medium. These cells were then treated with 200 Il! Before or after restimulation with allogeneic cells by incubating at 10' cells/well in 96-well flat-bottomed plates containing 100 cells/well of culture medium and the indicated additives. 3 days later, it reacted with rlL-4 (4 ng/d) or rlL-2 (4 ng/d). The cells were subjected to tests for corresponding proliferation. Cultures were grown in 1.0 p Ci of [3H]thymidine. Cells were pulse-labeled for the last 18 hours of a 72-hour incubation period with microorganisms (75 Ci/mmole, New England Nuclear, Boston, MA) and harvested onto glass fiber filters. . Radiolabel uptake was determined by liquid scintillation spectroscopy. Results are shown in Table 4 below as mean cps (with standard deviation) for duplicate cultures. Table 4 and the above results show the difference in proliferation-inducing effects between IL-4 and IL-2. Unlike IL-4, IL-2 can induce proliferation of cells late in the culture cycle even in the absence of antigenic stimulation. However, when stimulated again with alloantigen, memory cells show a clearly stronger response to the addition of IL-4 than to IL-2. Engraving (no change in content) 8TG GGT CTCACCTC Leu 1sTTA CAG GAG ATCATCAAA ACT TTG AAG AGCCTCACA GAG CAC input AG 135Leu Gin Glu lie Ile Lys Thr Leu Asn Ser Leu Thr Glu Gin LYII 458CT CTG TGCAc e GAG TTG ACCGTA ACA GACATCTTT GCT GC CTCC180Thr Leu CYS Thr Glu Leu Thr Val Thr Asp Ile Phe Ala Ala Ser 60GG T GCG ACT GCA CM ; CAG TTCCACAGG CACA AG CAG CTG ATCCG 315Gly 1a Thr 8la Gin Gin The Hls Arg His Lys Gin Leu Ile Arg 105 2nd concave GluAloGILN a-hTL-4 (Asp 62.Asp12 Tan's [F I 4th concave S Concave V76 Figure 0 舅 92 <1.5 Submission of translation of written amendment (Article 184-8 of the Patent Law) June 1989 TytW Station Director General of the Japan Patent Office Yoshi 1) Moon Takeshi 1. Indication of patent application per /Us87103114 2. Title of the invention: Human interleukin-4 mutin 3. Patent applicant Address: 51 University Street, Seattle, Washington, D.C. 98101, United States of America. Immunex Building Name: Immunex Corporation 4, Agent Address: Tokyo, Japan 206-ku, Shin-Otemachi Building, No. 1, Otemachi 2-chome, Chiyoda-ku has a mutant amino acid sequence substantially homologous to the wild-type amino acid sequence of hIL-4, and at least one Asn-A'-Z in the wild-type sequence is replaced by Asn, -A''l-Y or -A"-A", where AI, AX and A3 are any same or different amino acids, X is any amino acid other than Asn, and Y is Z Any amino acid other than , Z is Ser or Dhr; Interleukin-4 (hlL-4) Similar proteins. 2. hlL-4 (Asp", 85p129), which is a hIL-4-like protein according to claim 1. 3. GluAlaGluAla-hlL, which is hIL-4-like protein according to claim 2. -4 (Asp”, AspI29). 4. hIL-4 analogous protein according to any one of claims 1 to 3 DNA sequence encoding protein. 5. A recombinant expression vector comprising the DNA sequence according to claim 4. 6. The recombinant expression vector according to claim 5 under conditions that promote expression. A method for producing an hlL-41-like protein, comprising culturing a transformed microorganism. 7. A biologically effective amount of IL-4 or an analogue according to any one of claims 1 to 3 together with a physiologically acceptable carrier or diluent. inducing the proliferation and lytic activity of antitumor cytolytic T-lymphoid populations, including Pharmaceutical composition. 8. A biologically effective amount of IL-4 or an IL-4 analog according to any one of claims 1 to 3, together with a physiologically acceptable carrier or diluent. A method of inducing proliferation of a cytolytic T lymphocyte population and lytic activity in the population, the method comprising contacting T cells with a composition comprising the composition. 9. The method according to claim 8, wherein the lymphocytes are activated by prior exposure to virus-related antigens. 10. The method according to claim 8, wherein the lymphocytes are activated by prior exposure to a tumor antigen. 11. The method of claim 10, wherein the lymphocytes are induced and expanded ex vivo and readministered to the patient in adoptive immunotherapy. 12. @Comprising administering to mammals a therapeutically effective amount of IL-4 or similar hIL-4 as described in any one of claims 1 to 3, Induces proliferation and activation of antitumor or antiviral cytolytic T lymphocytes. How to do it. 13. Antibiotics comprising a biologically effective amount of IL-4 or similar hIL-4 as defined in any one of claims 1 to 3 and a physiologically acceptable carrier or diluent. Viral agent composition. 14. Antibiotics consisting of a biologically effective amount of IL-4 or similar hIL-4 as defined in any one of claims 1 to 3 and a physiologically acceptable carrier or diluent. Tumor agent composition. 15. The antitumor according to claim 14, additionally comprising a therapeutically effective amount of T lymphocytes expanded ex vivo in the presence of IL-4 or IL-2. Tumor composition. Written amendment (method) 1. Indication of the case PCT/US87103114 2. Title of the invention Human-interleukin-4 myutein 3. Person making the amendment Relationship to the case Patent applicant Address Name Immunex Corporation 4, Agent Address: Shin-Otemachi Building, No. 1, 2-2-2 Otemachi, Chiyoda-ku, Tokyo 206th Ward International Investigation Report: 1°″″abe+wl A″1sl-eabee H@, p (7/U397/ Q3114

Claims (16)

[Claims] 1. Contains at least one amino acid substitution, deletion or insertion, resulting in an aspa Inactivation of lagin-linked glycosylation sites, deletion or substitution of cysteine residues human interlocutors in which the yeast XEX2 protease recognition site has been modified. Ikin-4 (hIL-4)-like protein. 2. A mutant amino acid sequence that is substantially homologous to the wild-type amino acid sequence of hIL-4. However, at least one Asn-A1-Z in the wild type sequence is Asn-A2- is substituted with a mutant sequence of Y or X-A2-A3, where: A1, A2 and A3 are any same or different amino acids, X is any amino acid other than Asn, and Y is any amino acid other than Z. , Z is Ser or Thr; hIL-4 analogue according to claim 1. 3. hIL-4 (As p62, Asp129). 4. GluAlaGl, which is a hIL-4-like protein according to claim 3; uAla-hIL-4 (Asp62, Asp129). 5. hIL-4 analogous protein according to any one of claims 1 to 4. DNA sequence encoding protein. 6. A recombinant expression vector comprising the DNA sequence according to claim 5. 7. into the recombinant expression vector according to claim 6 under conditions that promote expression. Therefore, the preparation of hIL-4-like proteins, including culturing the transformed microorganisms, Production method. 8. A biologically effective drug in combination with a physiologically acceptable carrier or diluent. IL-4 in the amount described in any one of claims 1 to 4. The anti-tumor cytolytic T lymphocyte population is generated by contacting with T cells. A pharmaceutical composition for inducing population proliferation and lytic activity in the population. 9. A biologically effective amount of I, together with a physiologically acceptable carrier or diluent. L-4 or IL-4 according to any one of claims 1 to 4 A cytolytic T lymphocyte collection comprising contacting a T cell with a composition comprising an analog. A method of inducing population proliferation and lytic activity in the population. 10. Lymphocytes are activated by prior exposure to virus-related antigens 10. The method according to claim 9, wherein: 11. Lymphocytes are activated by prior exposure to tumor antigens , the method according to claim 9. 12. Inducing and expanding lymphocytes ex vivo and in adoptive immunotherapy 10. The method of claim 9, wherein the method is readministered to a patient. 13. A therapeutically effective amount of IL-4 or claim 1 for mammals. comprising administering a similar hIL-4 as described in any one of paragraphs to paragraphs 4. , induces proliferation and activation of antitumor or antiviral cytolytic T lymphocytes. How to do it. 14. A biologically effective amount of IL-4 or claims 1 to 4. A similar hIL-4 as described in any one above and a physiologically acceptable carrier or An antiviral composition comprising a diluent. 15. A biologically effective amount of IL-4 or claims 1 to 4. A similar hIL-4 as described in any one of the preceding paragraphs and a physiologically acceptable carrier, or An antitumor agent composition comprising a diluent. 16. Additionally, in the presence of IL-4 or IL-2, increased ex vivo 16. The antitumor according to claim 15, comprising a therapeutically effective amount of T lymphocytes. Tumor composition.