JPH04500508A - Non-glycosylated human interleukin-3 composition - 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] Non-glycosylated human interleukin-3 composition Human protein with physical activity Tarleukin-31 [similar non-glycosylated protein (rhuIL-3)] A medical composition comprising:

Granulocyte-macrophage colony weight, which has been shown to increase Another cspt, including factor (G?1-C3P), promotes selective proliferation of macrophages. Inducing macrophage C5F (?1-C3F or C5F-1) and bone marrow progenitors Contains granulocyte C3F (G-CSF) that induces the development of granulocyte stem cells from cells , and further isolated first from mouse systems and more recently from human cell sources. Another C5F is called IL-3 or multipotent C3F (-ulti-C5P). Ru.

Mouthru 3 is originally 20α-hydroxysteroid, an oxygen involved in T cells. As a factor that induces the expression of dehydrogenase, Ihle et al. al.), J.

It was shown that c DNA claw number 7, which corresponds to mouse 3, is F. Hwang et al. and Weinstein) Adv, Iwunol, 39: 1 (1986) provides a comprehensive review of publications on the physiological activity of Roux 3. Ru.

Preclinical research on Ru-3 has shown its potential as a therapeutic agent in the treatment of various cell deficiencies. This suggests that the plant has excellent nurturing properties. Therapeutic composition with human 3 activity, alone. or other lymphokines, such as JL-1, JL-2, IL-4, IL-5, I L-6, IL-7, G-C5F, G-gar C5F, ? l-C3F and other Combined with growth factors to enhance the immune response to infectious pathogens or Normal blood cell collection after hematopoietic cell suppression caused by staining, radiation, or chemotherapy Can be used to assist in rebuilding the group.

Highlights Ω! Affirmation The present invention provides at least 3.0X in human bone marrow proliferation assays obtained by expression in yeast. It exhibits a biological activity of 107 units per milligram (tl/g), and an inhibition constant (K,) The binding affinity for human monocyte IL-3 receptor is at least 2. OX IO"M-', and the endotoxin content is less than Ins per protein fig. A recombinant human IL-3 protein analog, rhuTL-3 (As p", Asp").

concave Ωdan single detachment The figure shows the cDNA salt for the wild-type human ru3 allele, designated as “Pro”. The base sequence, the nucleic acid and the corresponding amino acid sequence are less than N of the mature protein. Starting with the alanine (Ala) residue encoded by nucleotides 1-3.

Figure 2 is used in the site-directed oligonucleotide mutagenesis method described in the Examples. The oligonucleotides shown are shown below.

Founding Q Anotsuki Tomo Setomo The [1IiA segment encodes human IL-3], which encodes human peripheral blood T lymphocytes (P cDNA prepared by reverse transcription of polyadenylic acid RNA isolated from H1) isolated from a library. N-terminus and C-terminus of the human genome DNA base sequence manipulated by the East Using a synthetic oligonucleotide probe with a base sequence homologous to the terminal @ region, standard methods The library was screened using DNA hybridization technology. . [INA was isolated and IIJI]i enzyme of the clone hybridized to the probe. using fragments separated by electrophoresis, agarose gel electrophoresis, and fragments separated by electrophoresis. Analysis was performed by another hybridization experiment (2 Southern blots).

After isolating several clones that hybridized with each probe, h The hybridizing part of the ulL-3 clone was subcloned, and the The base sequence was determined. The fragment was further transcribed in vitro. , capping the resulting message and adding polyadenylic acid to the African message. was injected into frog (Xenopus) oocytes. The translation products of the live oocyte are Testing for the ability to induce proliferation of hematopoietic cells by the human bone marrow assay described below. I did it.

Sequencing of several clones isolated from the PBT library revealed that the adult ph Both suggest the presence of a proline residue at position 8 of the υIL-3 sequence. Was. Therefore, we performed site-specific oligonucleotide mutagenesis and bonded to the N-terminus. Human ratio-3 analog lacking glycosylation sites (Pro@, Aspl%) , Asp") was created. This cDNA was used to perform yeast expression. Construct a vector, use it to transform an appropriate yeast expression strain, and transform the yeast promoter. cultured under fermentation conditions that promote the derepression of the microorganisms. The resulting protein is transferred to a reversed-phase high-speed liquid Purified by a combination of chromatography and ion exchange chromatography A purified product was obtained. Using human bone marrow proliferation assay and human Rou3 receptor binding assay Testing of this product revealed that it was compared to human IL-3 protein without such molecular modification. We have confirmed the expression of analogue products suitable for human medicinal use that exhibit higher physiological activity. 1. The composition of the present invention exhibits at least 3.5% in a human bone marrow proliferation assay. Oxl Physiological activity of O'U/wg and inhibition constant or human monocyte Roux 3 receptor Characterized by a binding affinity for the host of at least 2.0 XIO"M-1 . Even better, the compositions of the present invention contain at least 64,0 x 10' U/g and and 1 are at least 4. It has a specific biological activity of OXl0111M-'.

Friend-1 "Human interleukin 3" and 'hulL-3J are a group of hematopoietic stem cells with high potential. A human endogenous drug that induces proliferation of granulocytes, macrophages and erythroid stem cells from It refers to a secreted protein. As used throughout the specification, this predicate Physiologically active proteins and amino acid sequences that are substantially the same as those shown in Figure 1. Taste. “Substantially identical” means that the specific amino acid sequence of interest is identical to the reference sequence. caused by one or more substitutions, deletions, or additions, the final effect of which Disadvantageous I! Those that do not cause functional association and the target sequence are referenced. is at least 90% identical to the sequence of Lower identity, equivalent bioactivity , and sequences with equivalent expression characteristics are considered synonymous. Determine substantive identity For purposes of definition, truncations and additions of references to arrays are ignored. 'r hulL　3j(Asp"Asp")" Means recombinant human rL-3 boribeftide with spartic acid (Asp) residues. And here, residue 1 means formation F! Refers to the first amino acid of a protein (explanation of Figure 1 above) checking).

[Mutation amino acid sequence] is a nucleotide sequence that has been intentionally changed from the original sequence. Therefore, ``mutant protein'' and ``analog protein'' refer to coated polypeptides. ” or “mutin” refers to a protein containing a mutant amino acid sequence. "Of the original "Sequence" means a sequence of nucleotides that is identical to the wild type or original form of a gene or protein. or rN-glycosylation site, which refers to a sequence of amino acids, is defined below. be done. The term "inactivate" is used to define certain aspects of the invention. As shown, the N-glycosylation site was changed to create an oligosancalide moiety. means to prevent the covalent bonding of a specific amino acid sequence.

1 "Recombinant" as used herein, refers to the original endogenous substances and the original host. Recombinant microorganisms (e.g. Bacillus refers to a protein derived from an expression system (of Teria or fungi). “By expression in yeast, "obtained by fermentation" means produced by fermentation of a yeast strain transformed with an expression plasmid. means protein. In general, proteins expressed in yeast are It has a glycosylation pattern that is distinguishable from the expressed protein. but However, the analog protein that is the active ingredient of the pharmaceutical composition of the present invention binds to the N-terminus. Lacks carbohydrates. As shown in the illustrative example below, this modification provides high biological activity. and human monocyte Leu3 receptors.

"Open segment" means a segment that has been identified, manipulated, and The isolated fragments and larger fragments derived from the separated DNA allow for recovery and retrieval. DNA polymers in the form of elements of KinariNA constructs and e.g. the nucleic acid contained therein using standard biochemical techniques using It's called the punchline sequence. "Nucleotide sequence" means a sequence of deoxyribonucleotides. A recombinant expression vector J, which refers to a telopolymer, means (1) a promoter, for example. one or more molecules that play a regulatory role in gene expression, such as genetic factors and (2) III structure that is transcribed into wRNA and translated into protein. A plasmid that has a transcription unit containing a collection of coding sequences or a collection of coding sequences. Copy units contain leader sequences that enable extracellular secretion of translated proteins in host cells. It is more preferable to have a recombinant expression system with an expression vector and a suitable host. It means a combination of microorganisms. Sanki Romyces cere, which is often used for these The yeast expression system of Visiae 7-competition 9-tech evi = Yamako is used for protein production of the present invention. It was done.

rhu IL-2 The assay method used to measure the physiological activity of rhυ3 is described below.

1 - Upper bone marrow proliferation technique Freshly isolated human bone marrow cells were placed in a pre-incubated, pre-aerated solution of 50 units l-/d. penicillin, streptomycin at μg/d and freshly prepared 300 μg/d - Serum-free RPM11640 medium containing Noshiichi glutamine (Gibco, C Hagrin Falls, OR, USA) (hereinafter referred to as "assay medium") 5 hrs at 37°C in tissue culture flasks containing 2 x 10' cells per d. Preculture was performed under %CO□ conditions. After pre-incubation, gently spread the medium onto the flask surface. Non-adherent cells were collected by viventing.

Non-adherent cells were collected by centrifugation at 1000 rpi + 10 min at 4°C, Resuspend Triban blue in a small volume of assay medium containing % fetal bovine serum (FBS). was used to count viability and talc staining was used to count leukocyte regeneration. Cells are placed in assay plate. Stored at approximately 4°C in assay medium containing 10% FBS until added to the plate. .

50p! of assay medium in each well of a 96-well flat bottom tissue culture plate ( I put it in a hollow). 50p! diluted in assay medium in the first well of each row. of the sample and made a series of dilutions across each column using standard methods. 1.25×10′ bone marrow cells in 100 al were added to each well.

Place the plate at 37°C in a plastic box containing sterile distilled water to prevent drying. Incubated for 4 days with 5% CO□. On day 4, 5% FBS and 80 μC i/IIi [25p! containing 1ll-thymidine (80C4/■-01)] Add assay medium to each well and place the plate in a plastic box. It was incubated at 37°C for 5 hours with 5% CO2. After incubation, cells collected on a glass fiber filter, washed, and quenched radioactivity. Measured with a lacein counter. The unit of hulL-3 activity is the maximum thymidine 0 calculated by comparison with the amount of hulL3 that induces 50% of uptake, e.g. , if a 1.00 pl sample is diluted 1:20, the maximum thymidine uptake is If half is produced, one unit is 100μ! Contained in 1/20 of It is defined as the activity that Therefore, the sample is 1000 divided by 5, i.e. 2 00 units per milliliter (11/e) of bulL-3 activity. .

l-Determining the plan for the second marriage In a 96-well round bottom microtiter plate, in RPIII 1640 medium, Prepare W 2-fold dilutions of each sample starting at Q, 075 μgod. each Dispense 50μ of rbulL-3 standard (1.5μg/d) onto the assay plate. ! and a negative control (binding medium) in triplicate. In this test method, “combined "Medium" contains 2.5% (-/ν) bovine serum albumin (BSA), 0.2% (@/ ν), RPMI containing sodium azide and 20mM HEPES pH 7.2 It means 1640. 50a prepared as described below (Illustrative Example 3) i! I''I rbu IL-3 stock [IXl0-''M in binding medium , specific activity 4-8 x 101 scps/--ole) was added to each well, Next, 50 pl of human monocytes [recovered from the culture by centrifugation, RPMl 164 Wash 072 times and resuspend at 4X10' cells/I11 in binding medium]. Shake each plate vigorously on a rotary shaker. Incubated at 37°C for 1 hour. After incubation, phthalate oil centrifugation Cells and bound "'Irhu IL-3" were transferred to unbound "5Irh" by the detachment method. The following treatment was performed to separate it from u IL-3. First, each item Take two 65d aliquots from the incubage gin mixture and add 4 aliquots of each aliquot. 00.1 200, l cold lid JL4m oil mixed in polyethylene centrifuge tube Compound [1,5 dibutylphthalic acid, 1 bis((-2-ethylhexyl) -Phthalic acid (Eastman Kodak Co, Rochester, NY+ USA)] and centrifuged for 1 minute at 8°C in a microcentrifuge. cells and their “’I rhulL-3 bound to it passes through the phtano I oil mixture and precipitates. On the other hand, a low-density binding medium containing unbound "'IrhulL3" remains on the surface. be done. 1°C% IrhuIL3 bound to cells cut the tube in the middle. , count and measure the 11187 ratio of the top and tip.

The results of the IL-3 receptor binding assay are expressed as inhibition ( χ): Here, Wax represents the binding level of "'] rhu IL-3 in the presence of medium alone. Binding level in the presence of 1.5 μg/d of Rhu RT, -3 and Test is the level of binding in the presence of the diluted sample being tested. It is.

The results were analyzed using the nonlinear least squares method that fits the equation (2) below: Equation (1) For the data generated, I'l (X) is the maximum value of inhibition and K(M-') The binding constant of LtSlrhuL3, C(M), is the concentration of “’l rhu IL-3”. , I (M) is the concentration of the test sample, and +(M-') is the inhibition (M) of the test sample combination) is a constant.

A value of 1 for the test sample is when the inhibition constant of standard rhuIL3 is Ks. , divided by the value of standard rhulL-3 (l/b) to give a relative result. Synthetic activity is obtained.

3) Murus (LIIIuluS) for Baku J Anintodoxin ” fixed Limulus amebocyte lysis assay (AL) test kit is used according to the manufacturer's instructions, Construction of the present invention assayed large quantities of products for the presence of bacterial endotoxins The product contained no more than 10 g of endotoxin per inch of protein.

Heather rice twice runi tan arrangement Nucleotides and nucleotides of hulL3 cDNA isolated as described below. and the corresponding amino acid sequences are shown in Figure 1. In Figure 1, the nucleotide 1-3 shows the GCT codon corresponding to the N-terminal alanine of the original mature protein. The original polypeptide is cleaved during secretion to give the mature protein. - Contains arrays.

The recombinant DNA segment encoding the amino acid sequence of bulL3 was DNA library screening or artificially synthesized oligonucleotides can be obtained by a set of codes. Completely synthesized human ru 3 cDNA is It has now become a product (Beckman 5speciality Bioche seals P, OBox 10015 Pa1o AIto, CA, l-shot T II; Br1tish Biotechnology Limlted, 0xfor d, England), as a matter of selection, proline or The hulL3 sequence containing the codon specifying serine was converted into a synthetic oligonucleotide. and cDN^ are collected by using a fragment.

Eh, turn to A yeast system was used to express the recombinant protein of the invention. Selection and replication in E. coli DNA sequence derived from pBR322 for production (Ap' gene and origin of replication); Alcohol dehydrogenase 2 (ADH2) protein that can be suppressed by glucose pBc102/K22 (ATCC67 , 255). ADH2 promoter is la Russell et al., J.Bio+, Chet: 25 8:2674 (1982) and Beier et al. ture 300:724 (1982). pBc1 02.K22 also contains the Ding rpl gene as a selection marker and the yeast 2μ replication. Contains the starting point. Yeast α-factor leader sequence adjacent to promoter exists and allows secretion of heterologous proteins from the yeast host. α-factor To promote the fusion of this sequence with foreign genes, the leader sequence of Asp 718 restriction yeast site nearby (Kpn I and Asp 718 are isoschizoma modified to include (is). Brake et al. , Proc, Na tl, ^cad, scj.

Secreted proteins as described by μsa 81:4642 (1984) For efficient processing of Glu-Ala-Glu-Ala, The acid-encoding sequence was removed. Further expression vectors, such as pYα) Contains α-factor promoters such as Iu GM (ATCC53157) A yeast vector, it carries the wild type human GM-C5F gene.

Other technically accomplished versions of these are known. The structure of pYαHuG- is EPA183.350, and pBC102/K22 is EPA243.153 , the disclosures of each of which are incorporated by reference in the text.

Selection of suitable yeast strains for transformation depends on the selection marker and other characteristics of the vector. will be determined. pBc102/K22 or pYα [Iu] derived from GM S. cerevisiae barrels subjected to transformation with expression vectors were loaded with yeast. Genete Inc. Stock Center C’1east Genetic 5to ck Center), Berkeley, CA, USA (see below) Available. a trpl gal adel has the his2 genotype χ218-IB, J17 (revised CC52683: a his2 adel t rysu, metM ura3) and ILI66-5B (ATCC46183; a Hisl) is included. Particularly favorable expression for use of pBC102/K22 The current strain χν2181 was created by joining two haploid cells of X2181-IB. A polyploid organism at the University of California East Genetic Stock Center Department of Physical and Medical Pharmacology, Berkeley.

CA　94702. Available from LISA, and XV 617-1-3B is available from Department of Genetics, Nton University, 5eattle, WA 98105 USA or Lwm unex Corporation, 51 Llniversity 5tre etA5eattle.

MA 98101. Available from ll5A. A suitable transformation protocol is Hinnenet al, Proc Natl cad, sci, [Description of IsA 75-1929 (1978) or Sherma et al. u et al,), Lahorator Course) Iauu I for Methods in’! eas soot@Genel, ics.

Co1d Spring Laboratory, 8th book on 1986.121 Transformants of ``Trp'' are selected using the lithium acetate method that has been developed.

Hosts transformed with vectors carrying ADH2 or α-factor promoters Main stock added 804g7111 adenine and 80ttg/I11 uracil complete medium containing 1% yeast extract, 2% peptone, and 1% glucose Grow for expression in . Derepression of ADl12 promoter is caused by glucose in the medium This occurs due to the consumption of The crude yeast supernatant was recovered by filtration until further purification. Freeze or store at 4°C.

Luni main Ω cover Recombinant human lu-3 (rbulL-3) produced from fermentation of a yeast strain was developed by Ardal et al. Urdal et al.) J, Chromatog, 296: 171 (1984) and Grabstein et al. In a manner similar to that described by Fallen Musume Nakumu Yokugi 1405 (1986), Purify by one or successive reverse phase HPLC steps on a designated HPLC column. obtain.

For example, filter yeast conditioned medium containing rbulL3 through a 0.45μ filter. 15-20 μm of reverse phase silica (Sydac, The 5eparatio s Group, He5peria, CA LISA) filled with 5cm x 3 ( km column at a flow rate of 100 af/min. Yeast conditioned medium Prior to loading, the column was equilibrated with water containing 0.1% trifluoroacetic acid (solvent A). After adding the medium, the optical absorption at 2BOnm of the effluent The solvent was allowed to flow until the baseline value was reached. Here, acetonitrile (solvent B) gradient 0.1% trifluoroacetic acid with 1 minute at appropriate times following the start of the gradient. Collect the fractions and electrolyte a portion of each fraction onto a polyacrylamide gel. Protein content is analyzed by electrophoresis and fluorescamine protein detection. If necessary Further HPLC steps can be used. Furthermore, the application to S-Sepharose The complete purification protocol, including lye and elution, is detailed in Example 2 of the Institute of Technology. .

N-glycodyl'no Many secreted proteins acquire covalently linked carbohydrate units post-translationally, which are often It takes the form of an oligosancalide linked to the asparagine side chain via an N-glycodyl bond. Ru. The structure and number of oligosaccharide units added to specific secreted proteins are highly variable. are diverse, resulting in a wide range of precise molecular weights that can be attributed to a single glycoprotein. 6 Recombinant hulL3 is a glycoprotein of this type. Glycoproteins are produced using recombinant systems. Attempts to clarify this are complicated by the heterogeneity of this diverse carbohydrate component. , human or mouse granulocyte-macrophage colony stimulating factor (GM-C3F) The purified mixture of &Il recombinant proteins, such as It can be done. Miyaj i*a et al, EMBOJ , 5: 1193 (1986), the N-glycosylation site was mutated and the glycodylation Recombinant mouse GM-C that prevents cell synthesis and reduces heterogeneity of yeast-expressed products. reported the expression of 5F.

The presence of variable amounts of carbohydrates attached to recombinant secreted glycoproteins complicates purification procedures and This reduces the yield. In addition, if the glycoprotein is used as a therapeutic agent, If so, there is a possibility that Fool could develop an allergy to the carbohydrate part of yeast. However, for these reasons, discontinuation of treatment would be required. homogeneous analogues of glycoproteins involved in immunomodulation with reduced carbohydrates The rhu of the present invention, which is desirable for therapeutic use and further lacks carbohydrates attached to the N-terminus. The rL-3 (Asp", Asp") analog protein is expressed in a recombinant yeast system. 2-4 times higher than controls containing unmodified N-glycosylation sites. The specific activity and human monocyte Roux 3 receptor binding affinity are shown.

The N-glycosylation site in eukaryotic proteins is the amino acid Asn-A'-Z. Characterized by an acid triplet, where ^1 is any amino acid except Pro, Z is S er or Thr. In this sequence, asparagine is on the side to which the carbohydrate is covalently bonded. Provides chain amino groups. Such sites include Asn or two residues to other amino acids. Substitution, deletion of Asn or Z, or insertion of an amino acid other than Z between ^1 and Z. can be removed by insertion or insertion of a non-Asn amino acid between Asn and AI. 0 The analog protein of the present invention has N-glyphsylation present in the original human ratio-3 sequence. Asparagine # (Asp) residue in place of each asparagine side chain group at the site. have

Alog and Myein The mutated sequence is flanked by restriction enzyme sites that allow binding to fragments with the native sequence. Mutations can be introduced at specific positions by synthetic oligonucleotides containing . In addition, The reassembled sequence resulting from subsequent conjugation contains the desired amino acid insertions, substitutions or Encodes a mutin with a deletion.

In addition, using oligonucleotide-induced site-directed mutagenesis, Altered genes with specific codons changed according to desired substitutions, deletions, or insertions Can provide a child. f) Lt Dar and Walder (1+1 alder and W alder)% Gene 42:133 (1986); Bauer et al. auer et al.), Gene 37: 73 (1985); Clay Craik Biotechniques% January 1985, 12-1 9; Smith et al. Eclipse ■ L embryo - Nijie L: Pr1 nci Ies and Methods (PIenu* Press, 19 81); and U.S. Patent 4,518.58S discloses suitable techniques and is incorporated by reference in the text.

In either method, for example, Sood et al., Nu cl, Acjds, Res.

i: 2557 (1977) and Hirose et al. Tet, Lett, 2B: 2449 (1978). Conventional oligonucleotide synthesis techniques such as Stell synthesis are suitable.

In site-directed mutagenesis, the strand of the gene to be changed is M13 single-stranded phage or other suitable phage containing either the sense or antisense strand. This D NA is complementary to the sequence surrounding the codon to be changed, but the point affected by the substitution is A codon that specifies a new amino acid (or a complementary amino acid to such a codon) hybridized with an oligonucleotide primer containing an antisense codon) Ru. If a deletion is desired, the primer specifies the amino acid to be deleted. lacking certain codons while maintaining the correct reading frame. If inserted If insertion is desired, the primer is placed at the appropriate position in the sequence to identify the amino acid to be inserted. Contains a new codon specifying a no acid. Substituted, deleted, or inserted codons is preferably placed at or near the center of the oligonucleotide.

The size of the oligonucleotide primer used depends on the extension in the 5' and 3' directions. The long portion is long enough to avoid editing by exonucleases. However, it is necessary to optimize stable and unique hybridization at the mutation site. Determined from the point of view. Thus 9 oligonucleotides used according to the invention Usually contains about 15 to 2 mollusks.

In a more preferred method (see Walder and Walder, supra), the resulting oligo The nucleotide/ss vector hybrid is directly transformed into yeast. another person In this method, mutagenic primers target a single-stranded template portion containing the gene to be changed. hybridized with a gapped duplex. In the latter case, the primer is DNA polymerase I (Klenow fragment) along the template strand, DNA Extend by reaction with a polymerase or other suitable NA polymerase. Generates double-stranded DNA that is circular and used for transfecting a suitable host strain. stomach Even in the case of misalignment, replication of the heteroduplex by the host produces progeny of both strands. L Once the transfected cells are plated, they form colonies. , selected using the same labeled oligonucleotide used for mutagenesis. Ru. If yeast was transformed directly, transformants were pooled and DNA isolated. and transform the cells. Select the colonies that have grown by hybridization. Ru. The primer will hybridize to the changed DNA, but no offspring from the original strand will hybridize. Zero-order change using conditions that yield results that will not hybridize to grandchildren The DNA containing the gene is isolated and recombined into an appropriate expression vector.

Company planning program iΦ structure connection In one embodiment of the invention, mature hu11 31F white matter N, is attached to the end. Octapeptide Asp -Tyr-Lys-Asp -Asp -Asp - hulL3 protein as a fusion construct containing Asp-Lys (DYKDDDDK) quality was expressed. Octapeptide has high antigenicity and can be used with specific monoclonal antibodies. Provides an epitope that the body reproducibly binds to, allowing rapid production of expressed recombinant proteins. Enables accelerated detection and purification. This sequence can also be used in bovine mucosal tissues if desired. It can be specifically cleaved by telokinase at the residue immediately after the Asp-Lys bearing. Ru.

Experimental considerations A, hulL 3 encoding cDN^ Ficoll Hypaque (Fic o + i bypaque) Whole blood (Botland Red Cloth) was collected by density centrifugation. Peripheral blood phosphorus was obtained from buffy coat prepared from Pa bulbs were isolated. T cells were treated with 2-aminoethylthiouronium bromide Isolated by rosetting with sheep red blood cells .

Cells were mixed with 100 d RPMI and 10% fetal calf serum. serum), 50 μM β-mercaptoethanol, 1% phytohemagglutinin (PHA) and 10 ng/d phorbol 12-myristic acid 13-acetic acid (PM A) Cultured for 18 hours at 5X10h cells/I11 in a 175 d flask. Ta. RNA was extracted by the guadinium CsCl method, and then extracted using 16-cell cellulose chromatography. Poly A' RNA was prepared by matography (Maniatis Molecular Cloning: A Labor atory Manual, Cold Spring Harbor, 1982 j, cDNA is Essentially by Gubler and Hoffman Following what was described (Gene25: 263-269 (1983)) Prepared from poly A'' RNA. cDNA was digested with DNA polymerase I. Make it a full strand, make it blunt-ended with DNA polymerase, and add EcoR in the cDNA. To protect the I cleavage site, it was methylated with EcoRI methylase and then Ligated to RI linker. These compositions were digested with EcoRI to generate cDNA. Remove all but one copy of the linker at each end of A and cut with EcoRI. It was ligated to the arm of a batataeriophage Igt 10 that had been cut and dephosphorylated. Follow the manufacturer's instructions and use L. Daphage Extract (Stratagene, Sun Diego, California, TLSA).　500,000 The glue recombinant is a big one! Strain C600hfl- Standard plaque hybridization using the following probes: Screening was carried out using the technique.

Has complementary base sequences to the selection 5' and 3' base sequences of the bulL3 gene Two oligonucleotides were synthesized. The 5' probe is the buIL-3 reader 5'-GAGTTGGAGCAG It had a base sequence of GAGGAGGAC-3'. The 3' probe is the mature protein. Corresponds to the region encoding amino acids 123 to 130 of the 5' -GATCGCGAGGCTCAAAGTCG-3' base sequence 0 The synthesis method is described by Sood et al., Nucl, Ac1ds Res, 4: 2577 (1977)) h and Hirose et al. Published by Ding et, Lett, 28: 2449 (197B)) Following the 0 synthesis, which was an automated triester method substantially similar to (deblocked the oligonucleotide), a spare gel cell Purified by pneumophoresis.

When used as a screening probe, oligonucleotides are “P-ATP and T4 polypropylene” using a technique similar to that published by Niatis et al. The ends were radiolabeled with nucleotide kinase. For library screening The E. coli strain used was C600hfl- (Huynh et al., 1985 ．． 5upra).

Thirteen positive plaques were purified, two hybridization probes It was re-examined separately in the 11 colonies reacted to both oligonucleotides. Hybridized. of cDNA from several positive recombinant phages. inserts with unique EcoR1 sites, Bam81 sites, and numerous other A standard PCR clone containing a polylinker with unique restriction enzyme cleavage sites. subtracted into the EcoRI-cleaved derivative (pGEMBLlB) of pHR322, which is a vector. I cloned it. pEMBL, an exemplary vector of this type, is nte) et al. (Nucl, Aeids Res, 11:1 645 1655 (1983)), in which the SF3 and T7 polymerase blocks were The Cl motor is attached to the 9 selected clones near the multi-cloning site. The nucleotide sequence of the loan was processed using the chain termination method (chain te+vina). tion method). In particular, λGTIO: IL-3 black 2. 3. EcoR1 partial digestion of 4 and 5 was performed from 850 bp to 1, o Obtain fragments in the oobp size range and convert them to pGE? 1BL18 Eco Separately subcloned into the R1 site. pGEMBLlB:rhu IL 3 sub Place the clone insert next to the multi-cloning site of pGEMBL18. A universal primer that binds to and a synthetic oligo derived from the hulL-3 base sequence Sea fenced using nucleotide primer. Restricted map is h) IL-3 showed the presence of restriction enzyme sites corresponding to those previously reported. nine sea fencing of loan 5, the proline at the 8th position of the mature protein The existence of a codon (CCC) encoding (Pro) was revealed. 3 more Sea fencing on cDNA isolated from three clones (2, 3, 4) was reported by Yang et al. (Cell 47: 3 (198 6)) This mutation was confirmed from the base sequence. 22nd of formation=+t-3 This only T→C change in the nucleotide is the 8th 1y of formation = n, -3! i Some people have suggested that (1) the 8th hulL3 containing proline represents the naturally occurring hulL3 gene. In order to determine whether the To confirm the amount, we screened the Pro' and Ser'1L-3 genes. Ta. (Cold Spring H) described by Mullis et al. arbor Symposiaor + Quantitatjve Biolo gy, Vol. LI: 263 (1986)) Polymerase Chain Rear PCR ) is included in part of the IL-3 gene from 13 people (from peripheral blood lymphocytes) Used to amplify DNA. Contains the site encoding the 8th amino acid The amplified DNA base sequence is set specific to Set”@ or Pro” DNA base sequence. was hybridized to an oligonucleotide probe.

This analysis revealed that of the 13 people tested, all 3 were positive for hulL-3 with the 8th amino acid. was also positive (and therefore heterozygous) and was therefore isolated (P ro') hu IL-3 cDNA is the naturally occurring hurL-3 gene, 2 The more predominant of the two alleles.

Souse 1. For the rhulL3 protein as an N-terminal peptide solid pBc102-K22 (ATCC67,255) with Asp71B and 5pel The fragment constituting the mature sequence of human G-C5P was removed and the following oligonucleotide was digested with A yeast expression vector is created by ligating a leotide polylinker to the vector fragment. Configured vector: Asp718 ↓Ncol ↓5tul↑Ba@al 5p el The resulting vector was named pBc115. c encoding hulL-3 I) GEM [IL1B cloning vector (see Example A, above)] From the 14th amino acid of adult Pht+IL 3, downstream of the coding sequence By digesting with L and HasHl to provide fragments spanning the site of I cut it out.

Synthesize and assemble oligonucleotides, (1) KE starting from ^5p718 the C-terminal 5 amino acids of the yeast α-factor leader peptide ending at the X2 recognition site; (2) Synthetic N-terminal “flag” identification Mibutide (DYKDDDDK) ; see above); and (3) an 8-codon sequence encoding the adult phulL-3 protein. encodes the N-terminal #A14 amino acids of the protein up to the blunt end. provided a fragment encoding a short sequence; Approximately 40 nuclei each The salt of this 84 base pair Kpnl-Hpal fragment composed of 4 oligomers of The base sequence is shown below: 5'-CTA CCT TTG GAT AA A　AGA　GACTACAAG　GACGACGAT　GACAAG　-GA AACCTA Ding T Ding TCT CTG A Ding G TTCCTG CTG C TA CTG TTC-Vol Pro Leu Asp Lys Arg A sp ding yr Lys Asp Asp Asp Asp Lys〈-・・-・ α factor →−−・・〉1・−・−1−−−Identification vebutide・−・−・− ・-> 1-CCT CCCA Ding G ACCCAG ACG ACG CCC Ding Child G AAG ACCAGCTG G clove -3'-CGA GGG TAC TGG GTCTGCTGCAGG AACTTCTGG TCG ACCCA AAla Pro Met Dinghr Gln Dinghr Thr Pro Leu Lys Thr Ser Trp Vall-・・・・・・−・・−−−１・ ・−・−−−1−−・・−・・hulL−3−・−・・−・−・Song・−・・ -...Group-...〉The above fragment, Asp718 Ba5al vector derived from pBcI15. Ligate the HpaT-Bag)tl hulL-3 fragment and the HpaT-Bag)tl hulL-3 fragment together. and provided an expression vector (referred to as pBC) 25).

Plasmid DNA from an E. coli clone containing the pBC125 vector was isolated. , transfection of yeast strain XV 2181 to express the rhuIL-3 product. Used for home machine. AD) Conditions that promote release of repression of the 12 promoter Below, transformed yeast strains were grown in shake flask cultures. yeast strip The processed supernatant was collected by centrifugation and assayed for bone marrow proliferation-inducing activity. child experiments showed high level expression of rhu IL-3 protein.

Example 1: hulL 3 cDNA coded Σ-g1 cosillage 5-ohmnu 1-11 natural vector of rho TL-(Pro@As 1sAs”) Two asparagines (As) involved in glycosylation exist in proteins. nl′ and Asr+′. ), the codon at this position also encodes aspartic acid. In contrast, lycosylation (sometimes hyperglycosylation) is inhibited, A more uniform product is obtained. These changes were observed in the hulL3 cDNA yeast expression vector. Subcloning to vector plXY 120 l! ! , as described below The 0m mother expression vector p1λY 120 created in Identical to the described pBC115 but containing the multiple cloning site: The synthetic oligonucleotide described above is located near the 3' end of the α-factor signal peptide. 5pe included in the base sequence around 2μ from the ASp 718 site (amino acid position 79) Two Asp718j Ncol inserted up to l site l;TACCTTTGGATAAAAGAGACTACAAGGACGACGA TGACAAGAGGCCTCCAdingG6^TCCC,CCfGG^CA GAAACCTATTTCTCTGATGTTCCTGCTGC-ACTGT TCTCCGGAGII;TACCTA[;GGGGCCCCsGTG^engineering 5 ↑BamHI 5pel Furthermore, replication origins and intergenic regions (interεenie r 5140p I′lN derived from single-stranded batateriophage f1 containing The A fragment was inserted into the Nru1 site of the pBR322 DNA base sequence. reproduction of fl The presence of a starting point allows for transformation into a suitable (male) E. coli strain and When superinfected with phage f1, a single-stranded copy of the vector can be produced. can. This ability facilitates vector DNA sequencing and inv Enables itro mutagenesis.

The yeast expression vector plXY 120 was added to the 3' end of the α-factor leader peptide. Asp7]8, which is cut near the end (nucleotide 237), and the polylinker Digested with Ram old restriction enzyme that cuts within -. Purify large vector fragments, Ligated with the following DNA fragment = (1) Plasmid GEM BL18: Claal site derived from hulL-3 (nucleotide 58 of mature huIL-3 ) to the Ba++l11 site (within the polylinker, 3′ of huiL 3 cDNA side) hulL 3 cDNA fragment; (2) α-factor leader peptide We reproduced the base sequence encoding the C-terminus of RhuJL-3 in frame with the octapeptide DYKDDDD fused to the N-terminus of ( in-fra lipid e) fused synthetic oligonucleotide linker (oligonucleo (Fig. 2), this fusion to the rhu IL-3 protein is an octapeptide. Enables detection with specific antibodies and inhibits expression and purification of rhulL-3 It was first used in This oligonucleotide also has this N-linked glycosyl -Amino M change at position 15 that changes the binding site (Asn”→Aspl 6) The underlined nucleotides in Figure 2 are wild-type cDNA@ID. It represents the NA base sequence. (Equivalent to the N-terminal alanine of three molecules of adult PhulL) (counting from the 1st nucleotide) A to G and C to the 43rd and 4th nucleotide, respectively. Simply changing to T causes an amino acid change (AsplS). Other base changes are Useful restriction enzyme sites (AhaII and PvuII) without changing the amino acid sequence will be introduced.

The resulting plasmid is p! XY 139, N-linked glycostrain, Contains 3 cDNAs with one common sequence (Asn") rhull.

Using plasmid plXY 139, Asn16 was converted to Asρ'. By changing to , an oligonucleotide aligned with the second N-linked glycosylated silane consensus sequence. Performed directional mutagenesis. in vitro mutagenesis is described by Walder and Walder (supra). A similar method was used. Yeast vector plXY 139 is a single-stranded bacterium Contains a reproduction starting point for phage f1 and is adapted (of a) large! ! strain helper phage was stained with Ram. Time-end strand DNA can be produced.

One l1iDNA was transformed into E. coli strain non-107 to generate a helper gene. 1it1. -Isolate and mature the terminal strand DNA Codon switch that replaces Asp at position 700 of hulL-3 Annealed to the mutadiene oligonucleotide l’B shown in Figure 2 to provide I let it go. Annealing and yeast transformation conditions were as described by Walder. Yeast transformation was done as described (supra) by Wargoo and -mants were selected by growth on medium lacking tryptophan and pooled. , DNA was described by Holm et al. (Gene 42: 1 69 (1986)). This DNA is a wild type and mutant plasmid. It contains a mixture of DNA and is made by transforming E. coli R111 into ampicillin. Used to provide resistance to phosphorus. The resulting colonies were removed using standard techniques. For hybridization to radiolabeled oligonucleotide B using I screened more. Consists of DNA encoding hulL 3Asp” Plasmids were incubated with radiolabeled oligonucleotides under stringent conditions. Identified by hybridization to deB and nucleotide sea fencing It was confirmed by.

The resulting yeast expression plasmid was designated plXY138 and was designated as Asp The hulL~3 gene that encodes the amino acid change of “Asp” and the N-terminal It contained the octapeptide DYKDD [lOK. Final yeast expression plasmid is the same as plXY138, but the base sequence encoding the octapeptide is It lacks a column and therefore produces adult rhu IL-3 as a product.

The final yeast expression plasmid was constructed as described below. Yeast expression vector vector plXY 120 as described above, restriction enzymes ^sp 718 and Ba1I Cut at H1. The large vector fragment was inserted into (1) (nucleus of adult pjhulL 3). Cut at position 19) from Aha■ site to Ram) 11 parts on cDNA 3' side hulL3 cDNA fragment derived from pTX'l13&, (2) As 3' end of α-factor leader peptide from p718 site @ (amino acid Pro -Leu-Asp-Lys-Arg) and the Ahan site of hulL3 A synthetic oligonucleotide that regenerates the N-terminal 7 amino acids up to It was ligated together with C1 (Fig. 2).

The resulting plasmid was designated as plXY 1.51. This vector is , present in yeast, rhu IL-3 (Pro", Asp", Asp") Allows glucose-controlled expression and secretion.

Example 2; 7t<0 rhu TL-3 (Pro”, As’f host strain Xν2181 (S , cereν1siae diploid strain) was obtained from the Genetics Institute, University of Washington. East Strain Bank Department, Seattle, Washington, USAClhe 1 lnjνersity of Washjn5ton, Dept, of Gen etics Yeast 5train Bank, 5eattle, W`, U SA)? Utori XV-617-13B (a, his6.1eu2-1. V animals", ura3.5te5) and East Genetic Stock Center, Califo Runia University, Berkeley, California, USA (the Yeast Ge netics 5tock Center, University of Ca 1iforn 奄=B χ21B1 1B Cry, obtained from Berkeley, CA, [l5A) k iyu, 1. It is formed by joining adel, his2). Ta. The host strain was derived from Sherman et al.

The expression plasmid was transformed by the method of 5urpa).

The yeast containing the expression plasmid p1χY151 (see above, Example 1) was Preculture maintained on YNB-trp N top plates stored at °C. - Transfer some isolated or combined yeast colonies to YNB-trp medium. (6.1g/L Yeast Nitrogen Hese, 5g Casamino Acid, 40■ /L adenine, 16 to luuracil, 200■/L tyrosine) to inoculate 1 liter. Start by heating in two 2 liter flasks at 30'C with vigorous shaking. Propagate with Burnite. By morning, the culture is saturated and ready for stage 5. - Hit with stationary phase. It became 2-7 ing.

The fermenters (3 machines with a working volume of 10 liters) were pre-cleaned and sterilized. 5D-2 medium (4.0 g/L ammonium sulfate) until 80% of working capacity. Nitrium, 3.2g/L - Potassium Phosphate Base, 3.0g/L Yeast, Extract Lacto, 1. Og citric acid, 0.1g/L sodium chloride, 5d/L 2% chloride Calcium, 2.5d Rubitami:/101 solution, 0.5d Rutrace element Solution, 0.5 d/L 20% magnesium sulfate, 2. .

d/L glucose) ”?’ Fill, 30°C, 500,600 rpm, 7 settings Maintain at 10-161 pm airage. Add inoculum. proliferation After 2 hours, feed with 50% glucose at 50R/L for 10-12 hours. Start with a ratio such that . Nutrient supply then takes 30-40 minutes until collection. Changed to 50% ethanol added at 11/hr.

The total elapsed time of fermentation was approximately 20 hours, after which the optical density (600 nm) was 3o It is in the range of 45. The fermenter is then chilled to 20°C and the pH of the yeast beer is adjusted to 20°C. was adjusted to 8.0 by adding 5M Nao)I, and the resultant was adjusted to 0.45μ Miliboa Bellicon (Mr I) 1por with garden filter cassette attached e　Pel　I　1con) Filter the filter system with a sterilized 10L Collect in a boxed large carboy.

RhulL-3 contained in yeast broth was transferred to 15-215-20tI reverse phase silica. (Bydak.

Separation! Hesveria, California, LISA) The yeast broth filtered into the 51X 30Cl column was injected directly into the column. More common.

The column was soaked with 0.1% aqueous trifluoroacetic acid (solvent A) before application of yeast broth. Following injection filling of the column with broth, the absorbance of the effluent reaches the baseline. This f4 medium is used to wash out the liquid until it approaches the value of . At this point, 0.1% trifle The gradient of the oloacetic acid acetonitrile solution (solvent B) was created from 0% B to 100% B. Set at a flow rate of 100 m/min with a rate of change of 2% B/min. The gradient reaches 30%B Program the slope controller to interrupt the slope for 5 minutes when the slope starts. Starting from 0 minutes, collect fractions every minute and add bovine serum albumin (BSA) to the 0 fraction solution. Fluorescamine assay used as standard The protein content was analyzed by assay). rhu IL - 3 (Asp", Aspffl') was found to be eluted in fraction 8.9.10. It was.

The fraction containing rhuJL-3 from the first step contained approximately 1 gram of total protein. Stored at 4°C in polyethylene bottles until obtained. Double the amount of 0. A 1% aqueous trifluoroacetic acid solution is added. This solution is then mixed with solvent A (0 15-20 μC-18 silica (Vydac, 1% TFA/water) equilibrated with Severe Research Group, Hesveria, California, LISA) The sample is injected onto a second 5CIX3 column. After injecting the material, remove the column from the solvent. A, then run the same gradient as above for 10 minutes with a rate of change of 1% solvent B per minute. Set at a flow rate of 0 me/sin.

Run the gradient for 24 minutes, collecting fractions every 0.4 minutes. The fractionated solution was prepared using BSA as standard. Analyze protein content using a fluorescamine assay.

Pool the peak fractions from the C-18 column and add 0.5M β-alanine ptl 3 ．． Add 111O of 6. After collecting the sample, the pool was divided into 1. (7!S-Sefa Inject into a loin column (1cm x 10cm, manufactured by Pharmacia) at 5IIi/min. Ru. After sample injection, wash the column with 10mM) I Tris pH 7,4 to 20011M) Lis pH 7,4 to (200-total Gradient amount) Elute Roux 3 with a linear gradient. The peak fraction of rbulL3 was then Pool and dialyze overnight at 4°C against 1100II) Lis pH 7.4. and then filter sterilized.

Example 3: 93 rhu-Rhu-3 proteins was prepared for analysis as follows.

First, rhu IL 3 (Pr The vector was expressed using the yeast expression plasmid pB0125 described above. Yeast culture Purification of this fusion protein from loin was performed using the published PCT ls87103113 as described in ls87103113. in a single affinity chromatography step using noclonal antibodies achieved.

Second, rhu IL 3 (Pro', AspIS, Asp'') was fermented with yeast. prepared using a reversed-phase high performance liquid chromatograph as described in Example 2 above. Purified from yeast broth by roughy. Third, octapeptide and number 15 The rbulL3 containing both the th and 700th change is described in Example 1 above. was prepared by fermentation of yeast containing the expression plasmid plXY13B and as described above. Purified by sea urchin affinity chromatography. Purified rbulL-3 Protein concentration was determined by amino acid analysis.

Purified rbulL3 (octapeptide fusion protein) was Enzymo beads as previously described for MiGM-CSF (25) radioiodine reagent (erz) + 5obad radioiodi nation reagent) (Biorad Laboratories, Richsend . Radioactive using California, LISA)! I got 83.

Simply put, 0. 5 μg of IL-32 in glycine HCI p) 17.4 , 0.2H sodium phosphate pH 7.2, purest amount 50p! Make it. Enzymobi Reagent 50JZ,'! ’+(2■C1)20 pl.

2.5% β-D-glucose 10p! and keep the mixture at 25°C for 10 minutes. did. Sodium azide (50m to 10p7) and sodium metabisulfite (1 g/d at 1 opZ at 5° C.) were added one after another. After 5 minutes at 25°C, -Nylated rbulL3 was mixed with 2% bovine serum albumin, 2(b+M Hepes) RPM containing buffer, 0.2% sodium azide pl'17.2 (binding medium) 2i Sephadex G-25 color equilibrated with I-1640 It was separated from free M1tS1 by chromatography on a chromatographic column. rbulL The fractions containing 3 were pooled, diluted with the same buffer to a concentration of 0.5 μg/d, and incubated at 4°C. For samples that ran out of sodium azide during the 0 separation step, they were stored in In the human bone marrow proliferation assay described above! Determining the physiological activity of 5I-rhulL3 Established. The specific activity of the radioactive 1a-identified preparation is 36 Xl0Iscps/m5o It was calculated that le. The N-terminal octapeptide of the fusion protein contains one tyrosine residues, which in the case of rbulL3, the native IL-3 molecule Since it contains only one syn residue, this molecule is labeled with 1181 with high specific activity. It is useful for

Biological activity (units/μg) and binding affinity of the various rhulL3 compositions described above. and then human myeloproliferation and human monocyte IL-3 receptor binding assays as described above. It was decided by. The results are shown in Table I below.3 In Table I, all values are 2 The average value and standard deviation of samples run three times in two separate assays. +S, D,).

It is expressed as

Table ■: Ehito TL-3 8.2 and 4 Set-8AspIsAsp” L/No record 5.00:t3.4 XIO "Pro-8Asp" Asp" None 4.36±1.25xlO' 4.7 4±1.30xlO”Pro-8Asp”Asp” 4.93±1.9 5xlO' 2.23±, 42 mowing 0■ No Pro-8 Yes 1.10 soil 0.1 5xlO' 8.02+2.94 cut 09Ser -8 None Yes 2.1 company 1. 1 axis 10' 1.12±, 02 cutting 0'' As shown in the data summarized in Table 1 Both the biological activity and the inhibition constant (K,) for the human ratio-3 molecular form The addition of the octapeptide YXDDDDK to the amine terminus is as follows: The fusion protein analog is used for receptor binding research without essentially changing either of the parameters It is a form of human IL-3 that is reasonable for use as a research reagent and as a medicine. provides evidence that Furthermore, the 15.700th asparagine is added to asparagus. Inhibiting the addition of N-linked carbohydrates by replacing ginate can disrupt the biology of human IL-3. showing a significant (2-4 fold) increase in both biological activity and binding affinity.

Figure 1 (Figure 2 Go to engineering rhiji 2 ken second 2 Nihiro Chojo] Kogo Emi sp Procedural amendment □ October 2, 1991! ; day [distribution]

Claims (7)

[Claims] 1. Recombinant human IL-3 protein analog derived by yeast expression, rhu A pharmaceutical composition containing a pharmaceutically effective amount of IL-3 (Asp15, Asp70) Therefore, the pharmaceutical composition is (a) at least 3.0 x 107 units/mg of human bone marrow proliferation assay; physical specific activity, (b) at least for the human monocyte IL-3 receptor, expressed as an inhibition constant. avidity of 2.0 x 1010 M-1, and (c) less than 1 ng/mg protein. The above pharmaceutical composition characterized by an endotoxin content of. 2. A recombinant human IL-3 protein analog has essentially the same sequence as The composition according to claim 1, having the amino acid sequence. [There is an array] 3. The recombinant human IL-3 protein contains an amino acid sequence identical to such a sequence. The composition according to claim 2, comprising: 4. Biological ratio of at least 4.0 x 107 U/ng in human bone marrow proliferation assay activity and at least 4.0 x 1010 M-1 towards the human monocyte IL-3 receptor. A composition according to claim 1 characterized by binding affinity. 5. Claim 1, comprising the N-terminal octapeptide DYKDDDDK. Composition. 6. formulated in a physiologically acceptable dilution for injection or continuous infusion; A composition according to claim 1. 7. IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL -7, consisting of GM-CSF, G-CSF, M-CSF, EPO or TNF one or more biologically responsive modifying genes selected from the group 6. A composition according to claim 5, additionally comprising in a clinically effective amount.