JPH1080286A - Method for producing human il-3 and protein having human il-3 activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound participating in hematogenesis, etc., by constructing a DNA containing an expression cassette containing a transfer termination regulation region functioning in a host cell in the transfer direction, introducing the constructed DNA into a host cell, culturing the transformed cell and recovering the culture product. SOLUTION: Human interleukin-3(IL-3) is produced by a host cell according to the following procedures. A constructed DNA containing an expression cassette containing a transfer starting regulation region functioning in the host cell in the transfer direction, a DNA sequence coding for human IL-3 and a transfer termination regulation region functioning in the host is introduced into a host cell consisting of yeast, bacteria, true fungi or tissue cultured cell, etc., the transformed host cell containing the constructed DNA is cultured in a nutrient medium under proper culture condition to produce the human IL-3 and the produced human IL-3 is recovered to obtain the objective human IL-3 enabling the production of human IL-3 protein using a wide variety of host organisms.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a protein having human interleukin 3 (hIL-3) activity and a method for producing human interleukin 3 (hIL-3) by host cells. Also here, cDNA encoding human interleukin 3 (hIL-3) and cloning in various organisms including microorganisms, especially yeast, bacteria and fungi, tissue culture cells and transformed animals and plants. And its use in expression.

[0002]

BACKGROUND OF THE INVENTION Hematopoiesis involves the active growth and differentiation process of pluripotent progenitor cells into all types of mature blood cells and some specific tissue cells. Production of functional blood cells is a special protein, hematopoietic growth factors (HGFs)
Is adjusted by Some of the HGFs control the maturation of specific maturation systems, while others stimulate the proliferation and differentiation of precursors through a number of pathways. Much of our knowledge of the hematopoietic differentiation process has come from studies of mice in vitro and in vivo using purified growth factors. Mouse growth factor interleukin 3 (mIL-3) is also known as Multi CSF, mouse cell growth factor, stem cell activator or some other name, and developmentally early as detected by the spleen colony assay. Stimulates the proliferation of pluripotent cells (CFU-S), resulting in the production of progenitor cells through erythroblasts, megakaryocytes, granulocytes / macrophages, osteoblasts and some other systems. Furthermore, mIL-3 is involved in the replication of pluripotent stem cells,
Possibly related to interaction with other HGFs.

[0003] In recent years, a few groups have identified mIL-3cDN.
A was successfully cloned. No results have been reported on the confirmation of homologous sequences in human DNA using mIL-3 DNA as a probe. Perhaps the human gene is mI
It diverged extensively from the L-3 gene or lost its function during primate evolution. However, human leukocytes were found to produce HGF (s) that could replace mIL-3 in supporting the growth of mouse CFU-S. Therefore, the existence of human HGF, which shares biological properties with mIL-3 and can therefore be a human homolog, was postulated. Yang (Y-C) and others, Cell, (1
986) 47 : 3-10, dated October 10, discloses the retrieval of a cDNA encoding a protein having IL-3-like activity in gibbon T cells and genomic DNA encoding human equivalents. Human IL
The sequence of the cDNA encoding -3 is Yang
Can be deduced from human gene sequences published by others. However, the article does not disclose or teach a method for isolating cDNA encoding human IL-3,
Also, production of hIL-3 was not achieved. The present invention provides a cDNA comprising the entire coding sequence for human IL-3.
For the first time.

[0004]

The human IL-3 protein has not yet been prepared in purified form and its properties in certain in vitro proliferation assays and its properties other than the deduced primary structure have not been disclosed. The present invention relates to purified human I
It allows recovery of L-3 and confirmation of its properties by recombinant production from cDNA cloning.

[0005]

SUMMARY OF THE INVENTION As mentioned above, the present invention describes for the time being the isolation of a cDNA containing the entire coding sequence for human IL-3. Mouse and human IL-
The low degree of homology between the DNA sequences encoding
HIL by hybridization with 3 coding sequences
Does not allow retrieval of cDNA for -3.
Surprisingly, hIL-3 cDNA clones could be isolated by exploiting a rather high degree of homology in the 3 'non-coding portion of the cDNA. The availability of cDNA clones allows production of hIL-3 by a wide range of host organisms. After large-scale production, the protein can be purified and used for therapy.

[0006] The present invention allows for the production of recombinant human IL-3 protein in a wide range of host cells by transcription and translation of the cDNA sequence encoding the human IL-3 protein. The production of the protein is described below. E. co
li), COS cells, C127 cells, B. Sachiris (B.
subtilis) and B.I. Lisienif Olmis (B. lichen)
iformis); S. cerevisias and K. cerevisias. Illustrated in some hosts, including K. lactis. Production in other hosts using appropriate expression systems is also made possible by providing intron-free cDNA. More generally, the availability of an anti-human IL-3 antibody that allows the identification of colonies that exhibit satisfactory production of recombinant protein will assist in the production of human IL-3 from any recombinant system. Thus, in one aspect, the invention relates to human IL-3
Directs recombinant, intronless DNA encoding the protein. In another aspect, the present invention provides a hI
It is directed to an expression system in which expression of the DNA sequence encoding L-3 can be performed in a suitable host. In another aspect, the present invention relates to recombinant human IL-3 proteins in glycosylated or non-glycosylated form, human IL-3 without the substances normally associated with said proteins, and specificity for these recombinant or purified proteins. Antibodies that are highly reactive.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION

A. As used herein, "human IL-3", "hIL-3", "human multi-CSF" and "hmulti-CSF" are used interchangeably and indicate a protein preparation that exhibits the following activities: The protein stimulates colony formation by human hematopoietic progenitor cells whose colonies form include erythroblasts, granulocytes, granulocyte macrophages and mixtures. 2. The protein stimulates DNA synthesis by human acute myeloid leukemia (AML) blasts, as evidenced by, for example, labeled thymidine incorporation. Reconciling the definition of hmulti-CSF, the activity in said detection indicates that if these antibodies also do not block their activity by the exemplified hmulti-CSF described below, the immunospecificity against that generated in response to GM-CSF Must not be substantially blocked by the antibody. One exemplary form of hmulti-CSF is shown in FIG. 1 as a 133 amino acid mature protein with a 19 amino acid signal sequence. The amino acid sequence of FIG. 1 is identical to that of Yang, Y except for position 8 of the mature protein in which the Ser of the Yang protein is replaced by Pro.
-C. ) In addition, Cell (1986) 47 : 3-1
0 (supra). As shown herein, this amino acid sequence is effective in its non-glycosylated form. However, it contains two glycosylation sites, and glycosylated forms are also included within the scope of the invention. It will also be appreciated that the protein may exist in an acid addition salt form, a base salt form, or may be more neutral due to the pH around it. Derivatization to the extent that activity is not destroyed, such as by phosphorylation, acetylation, etc., also results in proteins that are included within the scope of the present invention.

It is also recognized that not all sequences may be required for activity. Portions of the amino acid sequence can be deleted or substituted while retaining biological activity. As exemplified here, if substituted by the sequence of residues of the fusion peptide sequence, the number can be as small as about the first 14 amino acid residues, so that alanine at position 1 is deleted or substituted. Can be. In addition, the mouse form of the protein appears to require only the first 79 residues for activity; this corresponds approximately to the first 83 residues of the human equivalent. Thus, fragments containing only the first 83 amino acid residues of the protein, and N-terminally substituted forms thereof, are also included within the scope of the invention. Furthermore, mature hIL-
It should also be considered that the N-terminus of 3 is formed by the residues ala-pro-met and the like (see FIG. 1). It is known that when a protein is secreted by a yeast host, it can in some cases be shortened by two amino acids (ala-pro) due to interaction with dipeptidyl aminopeptidase (72). HIL-3 without the N-terminal alanine and proline still retains its biological activity. A yeast strain having a null mutation in the X-prolyl dipeptidyl aminopeptidase gene is a complete hIL-3 (amino acids 1-13)
3) occurs. Thus, the multi-CSFs of the present invention include those with and without the X-prolyl dipeptidyl aminopeptidase mutant and the N-terminal alanine and proline produced by the wild-type host, respectively.

When produced as a mature protein in a prokaryotic host, the coding sequence for the mature protein is preceded by an ATG start codon. The resulting N-terminal methionine can then be removed or partially removed by processing in a bacterial host, depending on the nature of the subsequent amino acid sequence. Again, both forms of hIL-3 are biologically active. Thus, the hmulti-CSFs of the present invention include those with and without N-terminal methionine.
From the foregoing, it is clear that amino acid changes can be introduced into human IL-3 protein without affecting biological function. Small modifications in the amino acid sequence by chemical modification of the encoded residue, substitution of various residues, or deletion or addition of one or more, but preferably one, residue retain activity. It is found to produce protein. Thus, non-disruptive mutations, particularly naturally occurring allelic mutations and other mutations that are non-lethal to activity, are also encompassed by the present invention.

On the other hand, it should be considered that amino acid conversions in the human IL-3 protein can be beneficial for therapeutic use of the protein. As you can see here,
The mature protein has residues 15-36, 54-61, 74-
It has four conserved domains at 91 and 107-118. Non-conserved regions 1 to 14 (they can be replaced by the sequence of the host-derived fusion protein anyway),
Proteins containing one and multiple amino acid changes within 3, 62-73, 92-106 and 119-133 are possible. However, the cysteine residues at positions 16 and 84 appear to be necessary for disulfide bridge formation as they are conserved between species. Changes in the conserved domains can affect the biological properties of the protein, such as receptor binding and signal transduction. It is contemplated that hIL-3 with altered properties may be used therapeutically. It is to be understood that derivatives of hIL-3 that can be made by known protein engineering techniques are within the scope of the present invention. The protein preparation can include the hmulti-CSF peptide in monomeric form or in aggregated form if the aggregate retains the activity.

The term "expression system", as used herein, allows for the expression of both a coding sequence desired to be expressed and an appropriate control sequence when the control sequence is compatible with the host in which the expression system is located. DNA contained within an operable linkage
Shows the sequence. As is generally understood, the term "control sequence" refers to a segment of DNA operably linked to a coding sequence that is necessary for or regulates its expression. The control sequences for all hosts include promoters that may or may not be controlled by the regulation of their environment. Typical promoters suitable for prokaryotes, for example, trp promoter (induced by tryptophan absence), (induced by galactose analog IPTG) lac promoter, beta-lactamase promoter, and the phage derived P _L promoter (a temperature change Induced by). In addition, useful promoters, especially for expression in Bacillus, are α-amylase, protease, Spo
2 and synthetic promoter sequences are included. Promoters for expression in yeast include those for the 3-phosphoglycerate kinase promoter and other glycolytic enzymes, as well as promoter regions for alcohol dehydrogenase and yeast phosphatase. Transcription elongation factor (TEF) and lactase promoters are also useful. Mammalian expression generally employs viral-derived promoters such as the adenovirus promoter and the SV40 promoter system, but they also include regulatable promoters such as the metallothionein promoter controlled by heavy metal or glucocorticoid concentrations. There are also currently available viral-based insect cell expression systems, as well as expression systems based on plant cell promoters such as the Novalin Synthetase promoter.

In addition to the promoter DNA sequence required for transcription of the gene by RNA polymerase, various regulatory sequences, including those that regulate termination (eg, resulting in a polyadenylation sequence in eukaryotic systems), are also used to control expression. Useful. Some systems also include an enhancer element that is desirable, but not necessary, to effect expression. Translational control involves a ribosome binding site (RBS) in prokaryotic systems, whereas in eukaryotic systems translation can be controlled by nucleotide sequences near the AUG codon.

[0013] As included above, recombinant protein production has been demonstrated in bacteria [mainly E. coli. E. coli, Bacill
us) and Streptomyces], and yeast and fungi (eg, Saccharomyces, Kluyveromyce).
s) and Aspergillus, and mammalian and other cell cultures such as COS cells, C127 cells, Chinese hamster ovary cells, Spodoptera.
Frugiperda (Sfoptera frugiperda) (Sf9) cells can be used. The protein can be produced as an intracellular mature or fusion protein,
Alternatively, a DNA encoding an appropriate compatibility signal
Can be secreted when included in the gene.
The invention first allows large-scale production of recombinant human IL-3, which can then be used in purified form as a therapeutic. The method described herein provides a method for producing glycosylated as well as unglycosylated forms of the protein that can be purified to substantially pure human IL-3. The term "purified" human IL-3 refers to human IL-3 as described above, usually without the other proteins associated therewith.

B. CDN encoding human IL-3
Retrieval of human A IL-3 was isolated by the following strategy: An operation was developed that allows the reproductive production of hematopoietic growth factors (HGFs) by human leukocytes. 2. mRNA was prepared from such producing cells and transcribed into double-stranded cDNA. 3. The cDNA was screened with the complete mIL-3 cDNA containing both the coding and untranslated 3 'downstream to obtain D11. 4. The hybridizing cDNA clone D11 was inserted into the expression vector pLO to obtain pLB4, which was
Expression in cells confirmed the presence of the sequence encoding human IL-3. The conditioned medium for these cells is hIL
-3 showed the expected biological activity.

Although the human cDNA had a remarkable degree of homology in the 3 'untranslated region despite significant homology with the mouse coding sequence, it was not possible to retrieve using this procedure. did it. Applicants are unaware of the prior disclosure of using 3 'untranslated sequences for retrieval against alternative species genes. More specifically, 12-O-
Tetradecanoylphorbol-13 acetate (TPA)
And conditioned medium of lymphocytes cultured in the presence of Concanavalin A (Con A) were obtained from mouse CFU-S in suspension culture.
With medium suitable for human HGFs as determined by in vitro stimulation of the medium, proliferation of mIL-3-dependent DA-1 cells, human hematopoietic precursor assay by in vitro colony formation, and in vitro stimulation of acute leukemia blasts. is there. The human lymphocyte cDNA library is λgt-10 phage (2
0) and m for the development of mIL-3-related sequences
Screening was performed using the HindIII-XbaI fragment of the IL-3 cDNA. No hybridizing clones were identified.

However, when the complete mouse IL-3 cDNA was used as a probe, 4 clones were confirmed. Restriction enzyme analysis of the largest clone (D11) revealed a 910 bp insert containing an internal EcoRI site (position 411, FIG. 1). (It was examined whether this EcoRI site was created by ligation of two independent cDNA fragments or was a naturally occurring site. Southern digestion of restriction enzyme digested human DNA using the labeled 5 'and 3' EcoRI fragments of clone D11 as a probe. The analysis shows equal DNA after digestion with Hind III (15 kb) and BamHI (4.6 kb).
Fragment indicated. Furthermore, the DNA sequence near the EcoRI site did not correspond to the linker sequence (pCCGAATTCGS) used to insert the cDNA into phage DNA, indicating that these EcoRI fragments were derived from a single mRNA. )

From hybridization and sequencing studies, it was concluded that the small clones (II, IV and VI) were equal to the 3 'nucleotide sequence of clone D11 and were derived from the same mRNA species. D11 cDNA and mIL-3c
The computer-assisted sequence of DNA (FIG. 1) is 5 '
In 00 bp, sequence homology was shown between nucleotides 236-269 and between nucleotides 598-803 in the 3 'terminal region (68%, 71% and 73, respectively).
%). In particular, the region between nucleotides 706-763 is highly conserved (93% homology) and contains repetitive AT-rich sequences.
Low homology in the 5'-terminal 600 bp of the human cDNA (52
%) Excludes detection by hybridization with the HindIII-XbaI fragment of mIL-3. Analysis of the human cDNA clone for the encoded protein shows an open reading frame to the stop codon TGA at positions 495-497 (FIG. 1). The first ATG triplet is probably the actual start codon of the encoded polypeptide. The putative encoded protein consists of a 19 amino acid hydrophobic leader protein, which is probably cleaved between glycine and alanine residues (22, 2,
3).

The sequence of the predicted amino acid residues of human and mouse IL-3 (FIG. 1) is the leader peptide (residues-26 to
+1), the mature protein (residues 1-13
Shows 28% homology to 3). There are two conserved regions of four amino acids within the leader peptide (residues -13 to -10 and -3 to +1), the second of which contains the processing site. The mature protein is 133 amino acids long and has a molecular weight of 15 kd. The mature protein has four conserved domains (residues 15-36, 54-6
1, 74-91 and 107-118) and two potential glycosylation sites (residues 15-17 and 70-7)
2). Both cysteine residues present in human proteins (positions 16 and 84) are conserved and can play an important role in protein fold formation by disulfide bridge formation.

To demonstrate that this human cDNA encodes a functional protein similar to mIL-3, the D11 cDNA was transformed into a eukaryotic expression vector (pLO, SV
40 transcription units) and the expression vector pLB4
Was transferred to COS1 cells. COS / pLB4 conditioned medium (CM) was tested for its ability to (1) stimulate colony formation by human bone marrow cells and (2) stimulate human acute myeloid leukemia (AML) blasts. Bone marrow monocytes (Vim-2 positive) and T lymphocytes (T-3 positive)
In vitro colony growth of human hematopoietic precursors depleted of accessory cells was effectively stimulated by COS / pLB4CM. The data shows stimulation of some hematopoietic differentiation lineage precursors and a subpopulation of BFU-E by COS / pLB4CM.

In another test, bone marrow was subjected to density centrifugation, T
Progenitor cells were enriched by E-rosette sedimentation except lymphocytes and adherence except mononuclear phagocytes, and cultured in a rich medium containing fetal calf serum. Under these conditions, COS / p
The majority of LB4CM-stimulated colonies contain two or more hematopoietic differentiation lines: all macrophages, about 1/2 immature blasts and / or immature blasts and / or immature It includes erythroid cells and / or neutrophil granulocytes, as well as a small number of basophils or eosinophil granulocytes. These results indicate that the human cDNA clone D11
2 shows the pluripotent stimulatory effect of the protein encoded by and its effect on developmentally fast pluripotent hematopoietic cells. For AML stimulation, AML blasts from 5 patients were
Stimulated with S / pLB4CM and responded to ³ H-TdR
Assays were made by measuring uptake and colony formation. 5
Three of the leukemia cell samples were COS / pLB4C in both assays.
M; a unique dose-response relationship to AML blast colony formation and DNA synthesis from different patients was obtained.
Response to GM-CSF is further COS / pLB4C
Phenotypic differences between leukemias responding to M were shown.

These data indicate that the D11 cDNA clone contains complete genetic information for the bioactive protein to be transported into the medium in transformed COS cells. Despite the apparent uncertainty (only 30%) of homology with respect to protein sequences between human proteins and mIL-3, these proteins are comparable with regard to their biological functions. Both proteins exert their effects on early developmental hematopoietic precursors of various systems. Low homology at the amino acid level is also indicated by low homology in the coding nucleotide sequence. However, quite surprisingly, homology occurred in the 3 'untranslated region, which was sufficient for retrieval of human cDNA clones. Southern analysis of human DNA indicates a single hybridizing gene, indicating that this cDNA does not belong to the group of closely related genes.

From the above results, we conclude that human cDN in D11
We conclude that the A insert encodes a human homolog of mIL-3. We use the term hmulti-CS for the protein encoded by cDNA clone D11.
It was decided to use F for its main biological effects and assays. hmulti-CSF cDNA by hybridization with the 3 'terminal region of mIL-3 cDNA
Confirmation of the clone was not expected. Homologous DNA sequences are generally found predominantly in the coding region.
-The CSF sequence is widely distributed in this part of the gene (4
5% homology). Analysis of the highly conserved domain in the 3 'terminal non-coding region all indicated the occurrence of five ATTTA repeat units conserved in the mIL-3 cDNA.

Hmulti-CSF and mIL-3 are other mouse and human growth factors or lymphokines such as GM-CSF (25), interleukin 2 (25),
Interleukin 1 (26) and interferon (27-29) show significantly less protein homology. The biological activity of mature mIL-3 appears to be contained in the first 79 amino acids, including the absolute requirement for a cysteine residue at position 17 (30). This cysteine residue is conserved in hmulti-CSF (FIG. 1, position 1).
6), can play a substantial role in protein fold formation. The generation of a potential glycosylation site near the cysteine residue can prevent the formation of disulfide bridges.

C. Production and Formulation of hmulti-CSF Applicants have provided a representative class of expression systems capable of producing human IL-3 protein in various forms, as fusion proteins, as mature intracellular proteins, and as secreted proteins. To the applicant's knowledge, anywhere in the art, recombinant forms of human IL-3 or
In fact, human IL-3 in a protein-free preparation not normally associated with this desired protein is not available.
Accordingly, the present invention provides, for the first time, human IL-3 protein in a state adaptable for therapeutic and diagnostic uses.

Human IL-3 can be produced as a fusion protein with a sequence that is heterologous to the human IL-3 amino acid sequence. The term "heterologous" refers to human IL-3
It means a sequence that is not found in itself and is an unrelated sequence. The heterologous sequence can be derived from a bacterial protein, a yeast protein, a mammalian protein, or any of a variety of incidentally encoded sequences, such as those encoded by a polylinker. From the results below,
If at least the fusion protein is further extended beyond the N-terminus, it is clear that at least the first 14 amino acids at the N-terminus of the human IL-3 sequence can be replaced by a heterologous sequence.

The protein can also be obtained as a mature intracellular protein by constructing an ATG start codon immediately upstream of the desired N-terminus. These cellular proteins, whether mature or fusion proteins, can be recovered by lysing the cells and purifying human IL-3 using standard protein purification techniques. Protein purification is simplified if human IL-3 is secreted into the medium. Once the native signal sequence has been generated in a compatible mammal, the native signal sequence can be used to effect secretion into the medium. In bacterial or yeast systems, signal sequences compatible with these hosts can be used, such as penicillinase or the α-amylase sequence in bacteria or the α-factor signal sequence in yeast.

When produced recombinantly, human IL-3 is free of the proteins normally associated with it, and can be obtained from proteins and other materials specific to the recombinant host by, for example, chromatography, gel filtration, ammonium sulfate precipitation, and the like. Can be used for purification. As described below, the proteins are useful for therapeutic and diagnostic purposes. Proteins for use in therapy can be formulated in a standard manner for pharmaceutical compositions used for administration of proteins.
Suitable excipients include, for example, saline, Ringer's solution, and the like. Alternative formulations, including solid formulations (eg, lyophilized), can also be used.

D. Preparation of Antibodies The utility of a recombinant IL-3 protein or a portion thereof, as described below, allows for the production of antibodies directed to the protein or a portion thereof. Such antibodies include, among others, hIL
Useful for in vitro detection of colonies producing -3, therapeutic applications, and purification of both native and recombinant hIL-3.

[0029]

[Statement of Utility] The nucleotide sequence of all or part of the cDNA of human IL-3, or a closely related DN
The A sequence can be used to detect genetic abnormalities, including genomic rearrangements, restriction fragment length polymorphisms, mutation and altered gene expression, analysis of chromosomal DNA using restriction enzymes, DNA and RNA
Blotting and hybridization techniques (Maniatis et al., 1982) and two-dimensional gel electrophoresis (Fisher and Lerman, 1992).
83] advantageously. The recombinant hmulti-CSF provided by the present invention facilitates a detailed analysis of its role in human hematopoiesis, in particular the potential synergy of hmulti-CSF and various other HGFs.
Furthermore, hmulti-CSF has applicability for in vitro diagnosis of human diseases including hematopoietic progenitor cells and including leukemia,
As well as for potential applications aimed at expanding hematopoiesis in vivo. The effect of hmulti-CSF on various hematopoietic malignancies associated with terminal differentiation of leukemic cells also needs to be examined. Furthermore, hmulti-CSF is m
Since stimulation with IL-3 has been shown to be necessary for successful infection of mouse stem cells with a recombinant replication-defective retrovirus, it can be required to establish the proliferative state of human stem cells in gene therapy protocols.

The IL-3 protein can also be used advantageously to detect early hematopoietic progenitor cells in standardized in vitro culture [Wagemaker and Visser 1].
980; Metcalf et al., 1982; Merchaw and Magmaker, 1984; Metcalf, 1986]. IL-3 proteins and variants can also be used for the expansion of hematopoietic stem cells in vitro, possibly for bone marrow transplantation and gene expansion of stem cells, possibly with other growth factors [Lowenberg and Diet, et al.
cke), 1977; Wagemaker an
d Petem), 1978; Lemischka et al.
986]. The IL-3 protein can be used to determine the response pattern of malignant hematopoietic cells in an in vitro test [Touw and Lowendberg, 1985; Griffin et al. 1986; Griffin et al.
d Lowenberg), 1986].

In addition, the IL-3 protein can be used to detect residual leukemia cells by an in vitro method [Touw and Lowenberg, 1986; Griffin
in) et al., 1986; Griffin et al. (Grifin and Lowe)
nberg), 1986]. In addition, the IL-3 protein
It can be used in vivo by itself or in combination for the treatment and prevention of malignant and non-malignant disorders, and the resulting specific response by the hematopoietic system can produce clinical benefits. These applications include-leukopenia and / or immunosuppression, for example by AIDS infection;-cytopenia by chemotherapy and / or radiation therapy;-bone disorders such as fractures and osteoporosis;-immunodeficiency by general anesthesia; Recovery after bone marrow transplantation,-vaccination of the infection and adjuvants to additional therapies.

[0032] The cloned human IL-3 DNA sequence or closely related DNA can be used for gene therapy in a genetic deviation from the normal IL-3 gene. Large amounts of human IL-3 are required to facilitate the analysis. The easiest way to obtain sufficient amounts of protein is to use microorganisms, especially yeasts, bacteria and fungi, such as Saccharomyce.
s), Kluyveromyces, Aspergillus, Streptomyces
s), Bacillus and E.C. Production by B. coli species. Production in mammalian and other eukaryotic systems such as C127 cells, Spodoptera cells and transformed animals and plants is also possible to one of skill in the art with the teachings of the present invention. All of these possibilities are also included within the scope of the present invention.

The expression of the human IL
As an example of how to obtain live cells that produce E-3 protein, a number of plasmids were constructed Coli (B. coli),
B. B. Subtilis, B. B. licheniformis, S. et al. S. cerevisia
e), K. Transfected into K. lactis and C127 cells. Production of recombinant human IL-3 was achieved using these host strains. The products were tested for their ability to stimulate human AML blasts as described above against COS / pLB4 conditioned media. These tests indicated that the resulting protein was biologically active.

The following examples are illustrative, but not limiting, of the present invention. The Example 1 Human multi-CSF (hmulti-CSF) encodes the cDNA of Retori Val TPA (5ng / ml) and ConA (10μg / ml) human leukocytes stimulated with the murine stem cell proliferation assay and various other colony assays Measure a significant amount of HGF
s. Cells were harvested 24 hours after stimulation, as mRNA production was often translocated following stimulation with phorbol esters and lectins. H already 24 hours later
GFs could be easily detected in CM.

The mRNA preparation cells were harvested, washed with PBS and homogenized in guanidinium isothiocyanate solution (36). RNA was pelleted with a cesium chloride cushion. Oligo (d
T) -Cellulose chromatography was used for mRNA selection (36). Substantially in accordance with cDNA synthesis Gubler and Hoffman (37), oligo (dT) as primer and A
CDNA was synthesized using MV reverse transcriptase. The second strand is RNase H and E. coli. It was synthesized with E. coli DNA polymerase I. Close the gap with T4-DNA ligase,
The ends were flushed with T4-DNA polymerase. The cDNA was methylated with EcoRI methylase to protect internal EcoRI control sites. Next, the cDNA is
The DNA was linked to a phosphorylated EcoRI linker with 4-DNA ligase. After digestion with EcoRI, excess linker was removed by Sepharose CL4B chromatography. The material recovered in the void volume of the column was greater than 250 bp and was used for library construction.

Construction of the phage cDNA library The cDNA was ligated to the λgt10 phage arm (20) and a commercially available packaging extract [Gigapack, Vector Cloning Systems (Vector Cloning Systems)] was prepared.
stems)]. The recombinant phage was transformed into E. coli. It was propagated in E. coli C600hfl. Screening of the phage library Two nitrocellulose filter replicas of each plate containing 1-500 plaques were made according to standard procedures. The filter was then washed with HindIII-Xba of the mIL-3 cDNA.
Radioactively labeled mIL-3 prop of I fragment or complete mIL-3cDN labeled with random primers
Hybridized with the A clone. mIL-3cD
NA clone (pL101) was isolated from the WEHI-3B cDNA library. WEHI-3B mRNA was isolated using the guanidium isothiocyanate CsCl method, size fractionated on a sucrose gradient, and injected into Xenopus laevis oocytes. Using an RNA fraction that induces oocytes to produce a factor capable of supporting mouse stem cell proliferation for the synthesis of the cDNA,
At the end and inserted into the PstI site of pUC9. The mIL-3 clone was confirmed using a synthetic oligonucleotide (mIL-3 sequence published, 11). The insert of pL101 was purified on a polyacrylamide gel and used for screening a human cDNA library. Probe DNA was labeled using the random primer method (38). Latent positive plaques were screened again and plaques were purified. By this method, four clones containing phage D11 were confirmed.

Sequencing of cDNA clones Recombinant phage were grown and purified on a large scale, and the cDNA insert was removed from the phage arm by digestion with EcoRI and purified on polyacrylamide gel. The purified fragment was purified using M13mp18 and pTZ18R DN.
A (digested with EcoRI). E. coli
It was used for transformation of JM109. Single-stranded DNA was prepared and sequenced by established procedures (39). Sequence data can be obtained from various computer programs (40-4).
Analyzed using 3). The sequence obtained for the insert in phage D11 is shown in FIG. This 910 bp
The sequence includes the entire coding region for hmulti-CSF and its signal sequence and shows high homology to the mouse clone pL101 in the 3 'untranslated region. The homology upstream of the coding sequence is relatively limited. As described above, the protein is a 133 amino acid mature protein containing a putative 19 amino acid signal sequence, followed by two glycosylation sites (15-17 and 70-72) and two cysteine residues at 16 and 84. Having. The deduced amino acid sequence is Yang (YC)
Et al., Supra, except for those encoded by the genomic DNA disclosed above and one amino acid [position 8 of the putative mature protein; Yang DNA encodes Ser and this cDNA encodes Pro]. The same is true. The intronless sequence obtained in phage D11 can be used for prokaryotic expression as well as expression in eukaryotic systems as described below.

Example 2 Expression in Mammalian Cells Construction of Eukaryotic Expression Vector pLB4 Phage D11 (including the longest cDNA insert) was inserted into Hind.
III and Bgl II and digested with plasmid pT1 (pT1
A derivative of Z18R, subcloned in a multilinker containing some additional restriction sites (see Example 3A). Clones containing phage fragments containing the cDNA insert were confirmed by restriction analysis. The cDNA insert was removed from this plasmid by partial digestion with EcoRI and purified by polyacrylamide gel electrophoresis. The appropriate fragment was inserted into a eukaryotic expression vector (pLO) in the SV40 transcription unit.

PLO is EcoRI (filled) -pBR32
2 PstI (1-755), pBR329 PstI-A
vaI (756-1894), AvaI-PvuII adapter (1850-1868), SV40 (promoter)
Pvu II-Hind III (filled) (1869-1221)
1), Pvu II-Bam HI adapter containing unique EcoRI site (2211-2251), MboI "splice fragment" of SV40 (2252-2861), SV
40 BclI-BamHI (filled) “poly A fragment” (2862-3098), SV40 Pvu II-Hi
nd III promoter fragment (3099-344)
0), HindIII-BamHIEco gpt gene (3441-
4501), SV40 MboI "splicing fragment" (4502-5111) and SV40 Bcl
I-BamHI (filled) "Poly A fragment" (511
2-5348). The Eco gpt transcription unit is not critical for translocation expression of the protein in COS1 cells. The resulting hmul
The expression plasmid for ti-CSF was named pLB4 and was purified on CSCl. E. FIG. This plasmid in E. coli is a central bureau of
Shimmer Cultures (CBS) (Centraal Bureau of S
1986 in Chimmel-culture, Baarn, the Netherlands)
CBS on 12 December 2012 in accordance with the terms of the Budapest Treaty
Deposited under 568.86. The construct is shown in FIG.

B. Generation of hmulti-CSF in COS1 cells
Current and bioassay pLB4 DNA was transferred to COS1 cells using the calcium phosphate co-precipitation method (45). Cells were cultured for 48-72 hours in alpha medium containing 10% fetal calf serum. The medium was collected, filtered and used in assays to confirm biological activity. Human bone marrow precursor colony assay and acute myeloblast colony and proliferation assay were performed as follows. Bone marrow was obtained by iliac crest puncture from a hematologically normal adult volunteer after consent was given. Mononuclear cells were separated by density gradient centrifugation on a Ficoll gradient (from Nijegaard and Co., Oslo, Norway), washed and resuspended in Hanks' balanced salt solution (HBSS). . Then bone marrow cells and T lymphocytes were removed. For this purpose, bone marrow cells were purified by monoclonal antibody OKT-3 [CD3; Ortho, Ravitan, N .;
Y.) and Vim2 (bone marrow monocytic cells, 46) in the presence of rabbit complement at saturating concentrations (40%, 30 min, 2
(5 ° C) and then dissolved. HBSS cells
Twice, and resuspended in Iscove's modified Dulbecco's medium (IMDM).
and Messner) in the presence of autologous plasma according to (16)
1.5-3 × 10 ⁴ / ml as previously described (48)
At a concentration of 1. Erythropoietin 1 U / ml (sheep, stage III, Connaught, Willowdale, Ca
nada) and COS / pLB4CM were added as growth stimulating activities. In a direct comparison with COS / pLB4CM, phytohemagglutinin-stimulated leukocyte CM (PH-L
The results of a standard culture with CM) are also given. 60% of the colonies were picked and confirmed by microscopic analysis. CM in COS cells transferred the vector without insert (pLO) and did not stimulate colony formation alone.

The results are shown in FIG. As shown in the figure, erythroid (BFU-E), granulocyte-macrophage (CFU-GM), granulocyte (CFU-G), eosinophil (C
FU-E), macrophage (CFU-M) and mixed (CFU-MIX) colony variable COS / pLB
The average number of duplicate cultures stimulated with 4CM (± SD) is shown.

Induction of AML proliferation (see FIG. 4) AML blasts were purified using bovine albumin (BSA) density gradient. The remaining T lymphocytes were transformed into E. coli. Rosette sedimentation (1
7, 49, 50) from the AML sample. AML
(Patient 1) Colony formation was measured not only in the established PHA leukocyte feeder (PHA lf) system, but also in a modified form in which leukocytes were replaced by COS / pLB4CM, as shown in FIG. 4A. It was possible to evaluate the colony stimulating activity (17, 18, 49, 50). A
DNA synthesis of ML blasts (patient 2) was assayed by thymidine incorporation as described in reference (51) and the results are shown in FIG. 4B. Both tests showed a dose dependence on the added COS / pLB4CM. Addition of control COS medium did not affect AML growth in either assay.

C. Construction of eukaryotic expression vector pLB / BPV
Built human IL-3, C127 cells (ATCC CRL161
Derivatives of pLB4 were transferred to establish stable cell lines expressing 6). This derivative can be used for all BP
The V-1 genome (69) was constructed by inserting it into pLB4. The BPV-1 BamHI fragment was excised from the vector pdBPV-MMTneo (342-12) (70). The BamHI sticky ends were filled with Klenow polymerase. Then the vector pLB4 was
Cleavage at a unique EcoRV site within the gpt gene. Then, at the plant end, the BPV-1 fragment was cloned into EcoRV cut pLB4, resulting in the vector pLB4 / BPV capable of replicating in C127 cells. pLB4 / BP
V was converted to C12 using the calcium phosphate precipitation method (45).
Transferred to 7 cells. The transferred cells were cultured for 16 days, after which the focus was collected from the culture dish. Some independent cell lines were identified. The pLB4 / BPV vector appears to be stably maintained intracellularly as judged by Southern blotting of the Hirt extract (71) of some cell lines. The conditioned medium was purified using the AML proliferation assay for IL-
Three activities were tested. The stable cell line is an active human IL
-3.

[0044] Example 3 E. Construction of E. coli expression vector Construction of pGB / IL-301 (see FIG. 5, FIG. 6, FIG. 7, and FIG. 8 to FIG. 9) For the construction of an E. coli expression vector, the following modifications were made according to standard procedures (36). 1. The 3 'non-coding sequence between the AvaI site (position 541) and the XhoI site (position 856) in pLB4 was deleted by fusing the DNA fragment after filling the sticky ends with Klenow enzyme (FIG. 5). ).

2. Hmulti-CSF into bacterial expression vector
The following steps were performed to introduce the insert. pLH
One vector was digested with Ava II and the recessed ends were filled with Klenow polymerase. Bgl II linker (CAGAT
After ligation of CTG), the DNA was digested with BglII and BamHI. The BglII-BamHIhmulti-CSF fragment was purified on a polyacrylamide gel, and the derivative of pTZ18R (Pharmacia) modified in the multiple cloning site was subcloned into the BglII site of pT1 (Pharmacia). See FIG. 6). Two clones were obtained, which had the insert in the reverse orientation with respect to the lacZ promoter (see FIG. 5). The inserts of these two clones were digested with Bgl II and EcoRV, separated on a polyacrylamide gel, and subcloned into Bgl II and Hind II digested pT1. The binding of the Bgl II linker and hmulti-CSF DNA was confirmed by sequence analysis and A
The fusion of the linker to the va II site was shown (this Ava
The II site was created by ligation of an EcoRI linker to the cDNA molecule). This construct (pGB / IL-30)
0) is not in phase with the lac Z protein, so Bgl I
The I-EcoRV insert was digested with BamHI and HindII.
Subcloned into UC8 (52). The resulting construct (pGB / IL-301, FIG. 5, FIG. 7 and FIG.
) Was tested for production of the lac Z / hmulti-CSF fusion protein.

B. pGB / IL-302, pGB / IL
-303, pGB / IL-304 and pGB / IL-
Construction of 305 (FIGS. 5, 7 and 8-9) Introduction of the synthetic oligonucleotide into pGB / IL-300 introduced some base conversions into the coding sequence for the N-terminal part of the fusion protein. pGB
/ IL-302, pGB / IL-303 and pGB /
A new expression vector, designated IL-304, was constructed as follows: the HindII-HindIII fragment of pGB / IL-300 was separated on an agarose gel and nucleotides 99-137 and the 5 'terminal SalI recognition sequence of hmulti-CSF. And SalI and Hind III
Into pTZ18R digested with. The sequence of some clones has been established. In fact, some base conversion was observed,
Modification of the hmulti-CSF protein has occurred. Some cloned inserts were transferred to pUC8 for expression of the lacZ fusion protein (pGB / IL-302, pGB
/ IL-303). Clone pGB / IL-304 was created in the lacZ bearing phase by ligation of the SalI site after filling the recessed ends with Klenow. The construct was verified by Pvu I digestion. Some clones lacked synthetic nucleotides and were found to fuse in frame to the lac Z protein. One example of these clones is pGB / IL-
305.

C. Construction of pGB / IL-306 (FIG. 5,
(See FIGS. 7 and 8-9) An expression vector encoding a protein lacking the lacZ N-terminal amino acid was constructed from pGB / IL-300 by deletion looping as described in reference (53). Synthetic oligonucleotides include pTZ containing the ATG start codon.
The 22 nucleotides upstream of the lac Z gene and the first 24 nucleotides encoding mature IL-3 were included. This plasmid was named pGB / IL-306 (FIGS. 5, 7 and 8-9). Plasmids pGB / IL-300, pGB / IL-301 and p
E. coli containing GB / IL-302. The E. coli strain is 19
July 13, 1987, CBS 377.87, CBS 379.8
7 and has been deposited with the CBS under CBS 378.87. 8-9 show the sequences of the fusion regions for the various plasmids constructed. The sequence of the clone is shown from the start of the lac Z protein coding region (lower letter) and the hmulti-CSF coding region (upper letter) in pUC8 or pTZ18R to the ClaI site at position 158. Mutations of hmulti-CSF DNA sequence are ^{underlined, trp 13 → arg 13 (pGB} / IL-30
2); leu ⁹ → pro ⁹ and trp ¹³ → arg ¹³ (pGB / IL
−303); met ³ → thr ³ and silent change (pGB
/ IL-304).

In the priority application EP 87201322.2 filed on Jul. 13, 1987, other names for these plasmids were used: pGB / IL-300 = pT-hIL3; pGB / IL-301 = pUC / hmulti; pGB / IL-302 = pUC / hmultiΔ1A; pGB / IL-303 = pUC / hmultiΔ1B; pGB / IL-304 = pUC / hmultiΔ1C; pGB / IL-305 = pUC / hmultiΔ2; / IL-306 = pTZ / hmulti;

D. E. FIG. Lac Z / hmul in E. coli
ti-CSF fusion protein and mature hmulti-CSF
Expression E. coli with various expression vectors E. coli strain (JM
109) were grown in LB medium containing 50 μg / ml ampicillin at 37 ° C. until an optical density of 0.5 at 550 nm was reached. IPTG (isopropyl β-D-thiogalactoside, Pharmacia) was then added to the culture to a final concentration of 1 mM and incubation was performed for 3-4 minutes.
Continued for hours. Plasmids pGB / IL-306 and p
GB / IL-302 is also E.I. E. coli DH1
(Wild-type lac Z operon). These strains were grown for 16 hours at 37 ° C. in LB or 2 × TY medium containing 50 μg / ml ampicillin. Bacteria are collected by centrifugation and 0.1 M Tris / HCl, pH 8.
0: 5 mM-EDTA 0.2% Nonidet P40
Sonicated in a buffer containing (NP-40) and 1 mM phenylmethylsulfonyl fluoride (PMSF).
Centrifuged at 000 × g for 30 minutes. The pellet was subjected to polyacrylamide gel electrophoresis.
Most of the CSF protein was shown to be stored in bacteria in an insoluble form.

The pellet was re-extracted with 0.5% NP-40 buffer and finally solubilized with 8 M urea, 0.1 M Tris / HCl, pH 8.0 and 5 mM dithiothreitol. Thus, extensive purification of the fusion protein was achieved (FIG. 10). As shown, pGB / IL-301 and pGB / IL-301
Bacteria containing IL-302 [E. E. coli] were isolated as described above. Lane 1 is 0.2% NP4
0 supernatant (sample corresponding to 0.1 ml of original bacterial culture).
Lane 2 shows 0.5% NP40 supernatant (0.2 ml), and lane 3 shows 8M urea buffer (A: 0.05 ml; B: 0.2 ml).
Is shown. Proteins were separated on a 13.5% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. Molecular weight (kd) of labeled protein (lane M)
Is shown on the right. The human hmulti-CSF fusion protein is indicated by the arrow. The fusion protein encoded by pGB / IL-301 is approximately 20 kd, pGB / IL-301.
Those generated from IL-302 have an expected MJ of about 16 kd.

E. Biological activity of bacterial hmulti-CSF preparation
The bacterial protein preparation was diluted in α-medium containing 1% bovine serum albumin, sterilized by filtration and assayed in the AML blast proliferation assay. The diluted sample was added to purified AML blasts and cultured for 4 days. DNA synthesis was measured using ³ H thymidine as described in reference (51). AML per unit per ml
Defined as the amount of hmulti-CSF required for half maximal blast proliferation. FIG. 11 shows this titration. Plasmid pGB /
Bacteria containing the IL-302, were assayed for stimulation of AML blast proliferation using various dilutions of the urea extracted protein preparation of ³ H-thymidine. The fusion protein concentration of this protein preparation was 33 μg / ml. Based on a given titration curve, the activity of this preparation was 16,000 units /
ml. The amount of bacterial fusion protein in the preparation was assessed from polyacrylamide gel electrophoresis and used to calculate relative activity. The results are shown in the following table.

[0052]

TABLE 1 Biological activity of bacterial hmulti-CSF preparation ────────────────────────────────── Mr (X10 ^-3 ) Protein unit relative activity lacZ / hmulti μg / ml per ml unit (1) (2) (3) per mg IL-3 ───────────────────── ───────────── pGB / IL−301 20 20 45 4,500 pGB / IL−302 / 303 16 5 2400 480,000 pGB / IL−304 18 ND (4) 18 − pGB / IL−305 16 1 300 300,000 pGB / IL−306 15 ND 70 ND ──────────────────────────────────

(1) The approximate molecular weight is the DNA of the fusion protein
Evaluated from the sequence (FIGS. 8-9). (2) IL-3 concentration was evaluated on SDS-polyacrylamide gel and calculated per ml of starting culture. (3) The activity of urea solubilized protein is measured by AML proliferation assay and is expressed in ml of starting culture. (4) Not measured. It was concluded that human multi-CSF expressed as a fusion protein in E. coli was obtained in a biologically active form. The results show that changes introduced into the N-terminus of the fusion proteins can affect the relative activities of these proteins.

Example 4 Preparation of antibody capable of immunospecific reaction with human IL-3 protein
Preparation of A. A gel for preparing a polyclonal rabbit anti-human IL-3 antiserum was prepared using E. coli containing plasmid pGB / IC-301. It was made from a lysate of E. coli. The 20 kd band with the IL-3 fusion protein is sliced, minced in saline in a mortar and 1 mg of Mycobacterium tuberculosis H37RA
Emulsified in a 1: 1 ratio in Freund's adjuvant containing every ml. New Zealand White
Rabbit (spf) was added to 1 ml of emulsion (± 100 μg IL
5 injection sites (2 × im
(3 × sc on the back of the thigh). Booster injections of the same antigen in incomplete Freund's adjuvant were given at weeks 2, 4 and 6. Serum was collected from ears by venipuncture at week 8.

One volume of serum was treated with sonicated pUC8-containing E. coli. Absorbed with 9 volumes of E. coli (overnight at 4 ° C.) to remove non-specific antibodies. E. FIG. E. coli, B. B. licheniformis, B. licheniformis; Sachiris (B. s
ubtilis); Cerevisiae and K. cerevisiae.
Immunoblotting of all IL-3 constructs made in K. lactis showed an immunospecific reaction with the absorbed serum at a dilution of 1 in 6500. Some of these results are shown in FIG. Proteins were separated from the recombinant host, separated on a 13.5% polyacrylamide gel, and plotted on a nitrocellulose membrane. Lane 1: pTZ1
8R-containing E.C. E. coli (control); Lane 2: pG
B / IL-301; Lane 3: pGB / IL-301;
Lane 4: pGB / IL-302; Lane 5: pUC1
9 (control); Lane 6: pGB / IL-301; Lane 7: pGB / IL-302. Lanes 6 and 7 show the proteins present in the pellet after sonication of the bacteria.
Lanes 3, 4 and 5 show the proteins present in the pellet after the first washing step, and lanes 1 and 2 show the final urea solubilized protein fraction. Arrows indicate pGB / IL-
Shown are fusion proteins (of expected size) expressed from 301 and pGB / IL-302.

FIG. 13A shows AML with anti-IL-3 antiserum.
4 shows inhibition of IL-3-dependent proliferation of blast cells. FIG. 13B shows that pre-immune serum does not affect the effect of IL-3 on AML blast cell proliferation. ▲ = IL in both panels
-3, 10 U / ml; ■ = IL-3, 1 U / ml; ● = control, no addition. FIG. 13A shows the AML blast proliferation assay (5
FIG. 13B shows that IL-3 dependent growth in 1) was blocked by serum in a dose-dependent manner, and FIG. 13B shows that pre-immune serum did not have this effect. GM-CSF as control
Dependent growth was not affected by these sera in the same assay (FIG. 13A, ◆ = GM-CSF, 100 U
/ Ml).

B. Monoclonal mouse anti-human IL-3 anti
Body Balb / c mice were used in the same emulsion 3 × as used for rabbits
Immunized with 0.1 ml (sc). A booster (0.1 ml, ip) of the antigen in incomplete Freund's adjuvant was given in the second week and three days later splenic lymphocytes were fused with SP2 / 0 bone marrow cells according to standard procedures (65). The hybridoma supernatant was used for E. coli. E. coli pGB / IL-302 (1
Lysate (including the 7 kdIL-3 fusion product) as a positive control; A lysate of E. coli pUC8 was used as a negative control and screened in an enzyme-linked immunosorbent assay. A total of 29 IL-hybridoma cultures secreting antibodies specific for IL-3 were selected and stabilized.

Example 5 Construction of Bacillus Expression Vector A general cloning technique was used (36). A. Construction of pGB / IL-307 (FIG. 14) For construction of pGB / IL-307, hmulti-CSF was used.
The SmaI fragment of pLB4 carrying the gene was transferred to Pvu II
Ligation into digested pUB110 (54). DB105
[The spo ^- derivative of the protease-deficient strain DB104 (5
5)], two clones were obtained as expected and the fragments were cloned in both orientations. The so-called "Hpa II promoter"
The plasmid harboring the fragment in the correct orientation for (57) was named pGB / IL-307. In this case, a fusion protein is made (see FIG. 14).

B. Construction of pGB / IL-310 An hmulti-CSF expression plasmid was prepared as follows. 1. Promoter cloning (FIG. 15) For expression in Bacillus, the synthetic σ ⁴³ promoter described in reference (58) was used (the promoter is commonly referred to as σ ⁵⁵ ). Plasmid pPROM55s (5
8), a promoter-containing plasmid, and pGPA1
4 (59) was digested with EcoRI and XbaI. The promoter fragment was ligated into the vector fragment purified on an agarose gel. E. FIG. E. coli
After transformation into (JM101), the correct plasmid was obtained and named pGB / IL-308 (FIG. 15).

2. PGB / I of synthetic oligonucleotides
Introduction into L-308 (FIG. 16) Nucleotide sequences 39-158 and 484-546 of hmulti-CSF and a synthetic nucleotide containing a 5 'terminal SalI recognition sequence and a 3' terminal XmaIII site were converted to SalI-Xma.
Ligation into III digested pGB / IL-308. The ligation mixture was introduced into JM101. After analysis of many transformants, the correct plasmid pGB / IL-309 was found.

3. Introduction of hIL-3 (FIG. 17) Subtilis DB105,
After separation from it, plasmid pGB / IL-309
Was digested with Xma III. The recessed ends were filled with Klenow polymerase and the plasmid was cut with ClaI. Plasmid pGB / IL-307 was digested with AvaI, filled in with Klenow, and then cut with ClaI. Next, hmulti-
The CSF containing fragment was ligated into the pGB / IL-309 fragment and transformed into JM101. The resulting plasmid was named pGB / IL-310 (FIG. 1).
7). This plasmid harbored the hIL-3 gene with its own signal sequence. After isolation of the correct plasmid, it was also Subtilis DB10
5 was introduced.

C. pGB / IL-311 and pGB /
Construction of IL-312 (FIGS. 18 to 20, 21) pGB / IL-310 was partially digested with HindIII and completely digested with PvuII. Pvu II ^- Hin two hmulti-CSF containing
Digested with dIII and SmaI. 18 to 20 show the nucleotide sequence of plasmid pBHA1. The plasmids are located at positions 11-105 and 121-125; bacteriophage FD terminator (duplicate): positions 221-2
307; part of plasmid pBR (ie position 206
9-2153): positions 313-768; bacteriophage F1, origin of replication (i.e. positions 5482-594).
3); positions 772-2571; plasmid pBR322
Part; the origin of replication and the β-lactamase gene: positions 2572-2684; transposon Tn9
03, complete genome: positions 2719-2772; tryptophan terminator (duplicate): positions 2773-372
9; consists of transposon Tn9, a chrome nifenicol acetyltransferase gene. Position 3005
(A), 3038 (C), 3302 (A) and 340
The nucleotide in 9 (A) differs from the wild-type cat coding sequence. These mutations are NcoI, BalI, E
coRI and Pvu II sites: positions 3730-3804;
Multicloning site: positions 3807-7264; plasmid pUB110, a replication function and kanamycin resistance gene (EcoRI-Pvu II fragment) (6
6, 67): Positions 7267-7331, introduced to eliminate multicloning sites. Fragments were constructed by known cloning techniques such as filling of sticky ends with Klenow, adapter cloning, and the like. All data are Genbank (Genbank ^R , National Nucleic
Acid Sequence Data Bank, NIH, USA).

After transformation into JM101 and analysis of many ampicillin resistant colonies, two different plasmids were used: pGB / IL-312 (contains the complete gene with complete control sequences) and pGB / IL-311 (complete gene and- A promoter lacking the 35 region in other orientations was found (see FIG. 21). pGB / IL-3
11 to B. B. subtilis DB105 and B. subtilis B. licheniformis strain T3
99 (Δamy, spo ^-, exo protease negative, ri
f ^r , ref. 68).

D. Construction of pGB / IL-313 (FIG. 2)
2) To obtain a small plasmid with the hmulti-CSF gene after the "Hpa II promoter", pGB / IL-3
12 was digested with BamHI and ligated again. The ligation mixture was transformed into DB105 qualified cells. Many neomansin resistant colonies were analyzed and the correct plasmid was obtained. The plasmid was named pGB / IL-313.

E. Construction of pGB / IL-317 (FIG. 2)
3) B. One of the pOL5-delta vectors (68) described above, for cloning the hmulti-CSF gene after the B. licheniformis α-amylase transcription and translation initiation region and signal sequence,
That is, pOL5-2delta was used. This plasmid contains, in addition to the α-amylase signal sequence (29 amino acids in length), one amino acid (Ala) of the α-amylase mature sequence,
Next, a large amount of cloning site: EcoRI-XmaIII-XmaI
-SalI-HindIII is contained (68). hmulti-C
Sal of plasmid pGB / IL-310 containing SF gene
SalI-PvuII digested pOL of I-PvuII fragment
Ligation into the 5-2 delta vector and transformation into DB105. The resulting plasmid was named pGB / IL-317 (FIG. 23). The hIL-3 gene contains its own signal sequence on this plasmid. The plasmid can also be Licheniformis (B. licheniform
is) Introduced into T399.

F. 5 expression vectors in Bacillus strains
Expression of Rasmid B. with the following expression plasmid: Subtilis (B. subtilis)
And B. The strain B. licheniformis was transformed with neomycin 20 μg / ml or erythromycin 1
(16 to 2) in a TSB medium containing 0 μg / ml at 37 ° C.
4 h) and grown, 300 μg / ml culture was centrifuged. The pellet was resuspended in sample buffer and analyzed using polyacrylamide gel electrophoresis followed by Western blotting. The supernatant was TCA precipitated and the pellet was resuspended in sample buffer. Both supernatant and pellet were analyzed for IL-3 protein (Table 2).
reference). The following steps were performed to determine the biological activity of the resulting protein: The cell pellet was washed with 0.1 M Tris /
HCl, and resuspended in buffer containing pH 8.0 and 10 mM-MgCl _2. Lysozyme was added to a final concentration of 1 mg / ml and PMSF to a final concentration of 1 mM. The solution was incubated at 37 ° C. for 30 minutes. Next, DNase
(Final concentration 20 μg / ml) and the solution
Incubated for minutes. Finally, the biological activity of the supernatant of this preparation and of the cultured cells was determined as described. The results are shown in Table 2.

[0067]

Table 2 Expression of Bacillus vector─────────────────────────────────── MW IL-3 Biologically active plasmid strain Pellet supernatant Pellet supernatant (kd) (kd) ─────────────────────────────────── pGB / IL−307 DB105 21 − + − pGB / IL−310 DB105 15; 17 15; 17 − − pGB / IL−311 DB105 12.5; 15 − + − T399 − − + − pGB / IL−313 DB105 15; 17 12.5 ; 15 +-T399--+-pGB / IL-317 DB105 12.5; 15 12.5; 15 + +17; 20 17 T399 12.5; 15 12.5; 15 + +17; 20 17 ────────── ─────────────────────────

B. PGB in subtilis (B. subtilis)
It can be concluded that a fusion protein with IL-3 activity is made using / IL-307. When the human IL-3 gene contains only its own signal sequence
No significant secretion of L-3 is obtained. All IL-3 activity is found intracellularly. In these cases, the precursor IL-
In addition to 3, mature IL-3 (15 kd) appears to have formed in the cells. Thus, some transport may have occurred across the membrane, but the protein is not transported across the cell wall. However, using α-amylase regulation and secretion signals (pGB / IL-317),
Most of the L-3 activity appeared to be secreted into the medium.
In addition to the decomposition products, about 15 kd and about 17 kd
Two proteins, possibly mature IL-3 and precursor IL-3, respectively, are detected in the supernatant. These data indicate that both processing sites are used, an α-amylase and an hmulti-CSF processing site. The most abundant product in the cells is the precursor IL-3 (20 kd protein) containing the α-amylase signal sequence, as shown by Western blotting.
It is. Occasionally decomposition products are detected.

Example 6 Kluyverromyces lactis
Construction of expression vector Construction of pGB / IL-316 Similar to that described by Bennetzen and Hall (62), A DNA fragment containing the Tn5 gene (61) conferring gentamicin G418 resistance under the direction of the alcohol dehydrogenase I (ADHI) promoter of S. cervisiae was cloned into pUC19.
(63) was inserted into the SmaI site. E. coli containing the resulting plasmid E. coli strain, pUC-G418 is 1
CBS under CBS 872.87 on December 4, 987
Has been deposited. XbaI-HindIII cleaved pUC-G41
8 into K. Plasmid p containing the K. lactis lactase promoter and calf prochymosin DNA
Insertion of the XbaI-HindIII fragment of GB903 (64) resulted in plasmid pGB / IL-314. The SalI-HindIII fragment of this plasmid was replaced by a synthetic DNA fragment containing a small multiple cloning site and a lactase terminator (see FIGS. 24 and 25). The resulting plasmid was pGB /
Named IL-315.

SacII-XhoI digested pGB / IL-31
The following fragments were ligated into the 5 vector: The 3 'part of the lactase promoter and the S. lactase promoter. PKS105 having the 5 'portion of the S. cerevisiae α-factor signal sequence (U.S. patent application Ser. No. 078,539,6)
1) the SacII-XbaI fragment of 4); 2. a synthetic oligonucleotide comprising the 3 'part of the alpha factor signal sequence starting at the XbaI site and the 5' part of the mature hIL-3 cDNA sequence up to the 5 'half (aa-residue 14) of the HpaI site; Most of the hIL-3 cDNA sequence (residues 15-13
HpaI-Xho with 3 plus 3 'non-coding region)
I fragment.

The resulting plasmid (pGB / IL-316)
) Are schematically shown in FIG. The complete vector sequence from the SacII site in the lactase promoter sequence to the HindIII site at the end of the synthetic terminator is shown in FIG. FIG. 25 shows a unique Sac II site in the lactase promoter and Hind III after the terminator.
Plasmid pGB / IL-316 (residue 4)
457 to 7204). Residue 4
457-6100 contain a lactase promoter sequence. Residues 6101-6355 contain the alpha factor signal sequence. Residues 6356-7115 contain the sequence for mature IL-3 plus the 3 'non-coding cDNA sequence. Residues 7116-7204 contain the synthetic terminator sequence.

B. Construction of pGB / IL-318 Coding information for the α-factor signal sequence of S. cerevisiae Lactis (K. lactis)
PGB substituted by the α-factor signal sequence of (64)
An expression vector similar to / IL-316 was constructed. The rest of the plasmid is equal to pGB / IL-316.
PGB / IL-31 between the SacII site in the lactase promoter and the HindIII site after the terminator
The sequence of 8 (residues 4457-7190) is shown in FIG. Residues 4457-6087 contain the lactase promoter sequence and the small linker sequence. Residues 6088-6
342 is K. Includes K. lactis α-factor signal sequence. Residues 6343-7102 contain the sequence for mature human IL-3 plus the 3 'non-coding cDNA sequence. Residues 7103-7190 contain the synthetic terminator sequence.

C. Kluyve
romyces lactis) and secreted hIL-3
Analysis of plasmids pGB / IL-316 and pGB / IL-
318 to the unique Bac II in the lactase promoter region
Digested at the site; K. lactis strain CBS23
60 (see 64). Thus, integration of the plasmid is targeted to the chromosomal lactase gene promoter region. The resulting G418 resistant transformant was transformed into liquid Y
The culture supernatant and cell lysate were grown to saturation in EPD medium and IL-3 was analyzed using the AML cell DNA synthesis assay.
Assayed for activity. Virtually all IL-3 was secreted into the medium and appeared to be active. The culture supernatant proteins were precipitated using ethanol and analyzed using denaturing polyacrylamide gel electrophoresis followed by Western blotting. Main product is about 21kd apparent M
W and a clear band at about 15 kd. The latter product is probably mature non-glycosylated IL-
Corresponding to 3, the 21 kd product is a product with core glycosylation at two potential glycosylation sites. Incubation with endoglycosidase H resulted in migration of the protein to the 15 kd range, suggesting that all IL-3 was correctly processed during the secretory process and that most of the protein was glycosylated.

Example 7 Saccharomyces cerevisi
ae) structure of the expression vector Built A. Construction of pGB / IL-319 First, an expression vector called pGB / TEFact was constructed. This pTZ18R [Pharmacia (Pharmacia)
Manufactured). S. cerevisia
e) The translation elongation factor (EF-1α) promoter sequence (cloned and sequenced as described in refs. 73, 74) was applied to Applied Biosystems (Appl.
ied Biosystems).
The S. cerevisiae actin transcription terminator sequence (75) is linked by a small SalI-BglII-XhoI linker. The sequence of the expression cassette is shown in FIG. Residues 1-949 contain the EF-1α promoter. Residues 950-967 contain the sequence of the SalI-BglII-XhoI linker. Residues 968-1113 contain the actin terminator sequence. The unique SmaI site in pGB / TEFact was used to introduce into the G418 resistance cassette described in Example 6. The resulting plasmid is called pGB / TE
Fact G418.

Finally, SalI-XhoI digested pGB / TEF
By introducing the following DNA sequence into the act G418 plasmid, the IL-3 expression vector pGB / IL-318
Was constructed: A SalI-NruI fragment of pGB / IL-316 with the S. cerevisiae α-factor signal sequence and the hIL-3 coding sequence up to the NruI site; the remaining nucleotides encoding hIL-3 and T
Synthetic NruI containing XhoI recognition sequence immediately after GA stop codon
-XhoI DNA fragment.

B. Sacchamyces cerevisiae (Saccha
romyces cervisiae) and of secreted hIL-3
The analysis plasmid pGB / IL-319 was cut at the unique EcoRI site in the EF-1α promoter. Thus, integration of the plasmid targets the chromosomal EF-1α region. S.
S. cerevisiae wild type strain D273-103
[Alpha (ATCC25657)] by K.K. Transformations were performed as described for K. lactis (64). G418 resistant colonies were picked and transformants were saturated in liquid YEPD medium. The culture supernatant is A
HIL-3 activity was assayed using the ML assay.
S. The protein produced by S. cerevisiae was found to be biologically active. Supernatant proteins were precipitated with ethanol and then analyzed by polyacrylamide gel electrophoresis followed by Western blotting. Two prominent products, a 21 kd glycosylated product and an approximately 15 kd non-glycosylated product, could be distinguished on Western blots.

[0077]

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[Brief description of the drawings]

FIG. 1: D of human multi-CSF and mouse IL-3
4 shows a comparison of NA and protein sequences. hmulti-CS
The F protein and DNA sequence (clone D11, top row) was sequenced along with the mIL-3 cDNA (11, 35) and protein sequence (30). Equal nucleotides are indicated by vertical lines and equal amino acids are indicated in boxes. Black dots indicate polyadenylation signal sequence, horizontal bars indicate ATTTA repeat units.

FIG. 2: Plasmid pLB containing human IL-3 cDNA
4 shows the construction of 4. E = EcoRI, Sm = SmaI, B = B
amHI, S = SstI, K = KpnI.

FIG. 3 shows the biological activity of COS / pLB4 on human bone marrow precursors. Erythroid (BFU-E), granulocyte-macrophage (CFU-GM), granulocyte (CFU-G),
Eosinophils (CFU-E.), Macrophages (CFU-
M) and mean number of mixed (CFU-MIX) colonies (± SD) are shown for duplicate cultures stimulated with variable amounts of COS / pLB4CM.

FIG. 4 shows the induction of AML proliferation assessed in a colony culture assay (panel A) and a DNA synthesis ( ³ H-TdR incorporation) assay (panel B).

FIG. E. coli expression vector pGB / IL
-301, pGB / IL-302, pGB / IL-30
3, the construction diagram of pGB / IL-304, pGB / IL-305 and pGB / IL-306 is shown. In this figure, X is XhoI, E is EcoRI, B is BamHI and A
Represents the AvaI site.

FIG. 6: pTZ18R (Pharmacia)
And the sequence of the multicloning site in its derivative pT1.

FIG. 7 shows a schematic diagram of hmulti-CSF expressing clones. Only the hmulti-CSF cDNA insert is shown for the eukaryotic expression plasmids pLB4 and pLH1. Leader peptide (shaded area A) and mature hmulti
-CSF protein (solid B) coding region is indicated in the box. The bacterial expression clone of hmulti-CSF (derived from pLH1) contains lacZ and the multilinker protein coding region (shaded area C), hmul
Includes the 5 'terminal non-coding region of ti-CSF (empty box part D) and the hmulti-CSF coding region. Arrows indicate the ATG start codon used in each vector.

FIG. 8. Lac Z / hmul against various bacterial expression vectors.
2 shows the sequence of the fusion region of ti-CSF DNA. It continues to FIG. The sequence of the clone is from the starting point of the lac Z protein in pUC8 or pTZ18R (lower letter) and hmul
The ClaI site at position 158 of the ti-CSF DNA sequence is shown. Mutations in the hmulti-CSF DNA sequence are underlined and trp ¹³ → arg ¹³ (pGB / IL-30
2); leu ⁹ → pro ⁹ and trp ¹³ → arg ¹³ (pGB / IL
−303); met ³ → thr ³ and silent change (pGB
/ IL-304). The superscript indicates the amino acid residue number of the mature protein.

FIG. 9 is a continuation of FIG. 8;

FIG. 10 shows pGB / IL-301 and pGB / IL-
Bacterial hmulti-CSF produced from bacteria containing S.302
1 shows a polyacrylamide gel electrophoresis.

FIG. 11 shows titration of hmulti-CSF fusion protein in AML blast cells.

FIG. 12. E. coli transformed with pGB / IL-301. 2 shows a Western blot showing the IL-3 specific response of rabbit antiserum raised against a 21 kd protein isolated from a lysate of E. coli.

FIG. 13 shows the effect of the antiserum of FIG. 12 on IL-3 activity.

FIG. 14 shows a schematic representation of the plasmid pGB / IL-307. The hatched box indicates the human IL-3 coding sequence. The N-terminal amino acids of the fusion protein are shown at the bottom of the figure.

FIG. 15 shows a schematic of the plasmid pGB / IL-308. The nucleotide sequence of the promoter region is shown at the bottom of the figure.

FIG. 16 shows the construction of plasmid pGB / IL-309. The first box A shows part of the human IL-3 sequence, ie the signal sequence of the mature protein plus 20 amino acids. The other box B is 3 'of the IL-3 cDNA sequence.
The portion of the non-coding region is shown.

FIG. 17 is a schematic representation of the plasmid pGB / IL-310.

FIG. 18 shows the nucleotide sequence of plasmid pBHA1. FIG. 19 and further to FIG.

FIG. 19 shows the nucleotide sequence of plasmid pBHA1. It is a continuation of FIG.

FIG. 20 shows the nucleotide sequence of plasmid pBHA1. It is a continuation of FIG.

FIG. 21. Plasmids pGB / IL-311 and pG
4 shows the construction of B / IL-312. The hatched box indicates the precursor human IL-3 coding region.

FIG. 22 shows the construction of plasmid pGB / IL-313. The sequence at the 5 'side of the IL-3 sequence is shown at the bottom of the figure.

FIG. 23 shows a schematic of the plasmid pGB / IL-317.

FIG. 24 shows a schematic of the plasmid pGB / IL-316.

FIG. 25 shows the nucleotide sequence of the plasmid pGB / IL-316 (residues 4457-7204) between the unique Sac II site in the lactase promoter and the Hind III site behind the terminator.

FIG. 26 shows the nucleotide sequence (residues 4457-7190) of plasmid pGB / IL-318 between the unique SacII site in the lactase promoter and the HindIII site behind the terminator.

FIG. 27 shows the nucleotide sequence of the EF-1α promoter, SalI-BglII-XhoI linker and actin terminator present on plasmid pGB / TEFact.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location C12P 21/02 C12N 5/00 B // (C12N 1/19 C12R 1: 865) (C12N 1 / (21 C12R 1: 125) (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1: 865) (C12P 21/02 C12R 1: 125) (C12P 21/02 C12R 1:19) (72) Invention Wahmer Marker Herald Nuel, Netherlands 2564 Höthe Den Haag Ananasstraat 146 (72) Inventor Fös Yvonne Johanna Netherland 2907 Embe Kapel Ardeis Eisel Zatkinerade 1 (72) Inventor Van Lane Robert Weerem Nölle Nehää Gewewe, Netherlands Sackel 23-23

Claims (20)

[Claims] 1. A method for producing human IL-3 by a host cell, comprising: a transcription initiation regulatory region functioning in the host cell in the direction of transcription;
A DNA construct containing a DNA sequence encoding human IL-3 and an expression cassette containing a transcription termination regulatory region that functions in the host is introduced into a host cell, and the host cell containing the DNA construct is appropriately placed in a nutrient medium. Growing the cells under different culture conditions, thereby producing human IL-3, and recovering the human IL-3 product. 2. The method of claim 1, wherein the host cell is a live transformed host cell containing genetic material encoding human IL-3 derived from a recombinant DNA material. 3. The transformed live host cell is selected from the group consisting of yeast, bacteria, fungi and tissue culture cells.
The described method. 4. The transformed live host cell is Saccharomyces (S)
accharomyces) and Kluyveromyce
4. The method of claim 3, wherein the method is selected from the group of s). 5. The transformed live host cell is E. coli. E.coli
And Bacillus selected from the group consisting of:
The method described in. 6. The transformed host cell is COS, C127.
4. The method according to claim 3, wherein the method is selected from the group of insect cells. 7. A host cell comprising a D-encoding human IL-3 operably linked to effective regulatory sequences in the host.
7. The method of any one of claims 2 to 6, wherein the method is transformed with an expression system operable in a recombinant host consisting essentially of the NA sequence. 8. The method of claim 7, wherein the DNA encoding human IL-3 is free of introns. 9. The control sequence includes a promoter selected from the group consisting of a lac promoter, an HpaII promoter, a σ43 promoter, an α-amylase promoter, an EF-1α promoter and an SV40 promoter.
9. A method according to claim 7 or claim 8. 10. A purified protein having human IL-3 activity, which is substantially free of other substances associated with the protein. 11. Amino acids 1 to 3 in the sequence "H" in FIG.
13. The protein of claim 10, produced by recombinant expression of a DNA sequence shown as encoding 133 or a naturally occurring variant thereof. 12. The protein of claim 10 or 11 in glycosylated or non-glycosylated form. 13. Human I derived from recombinant DNA material.
The protein according to any one of claims 10 to 12, which is produced in a transformed live host cell containing genetic material encoding L-3. 14. The protein of claim 13, wherein the transformed live host cell is selected from the group consisting of yeast, bacteria, fungi and tissue culture cells. 15. The transformed live host cell is Saccharomyces.
(Saccharomyces) and Kluyveromyc
The protein according to claim 14, which is selected from the group of es). 16. The transformed live host cell is E. coli. E.col
15. The protein according to claim 14, which is selected from the group of i) and Bacillus. 17. The transformed live host cell is COS, C12.
15. The protein of claim 14, wherein the protein is selected from the group of 7 and insect cells. 18. A transformed product transformed with an expression system operable in a recombinant host consisting essentially of a DNA sequence encoding human IL-3 operably linked to effective regulatory sequences in the host. 2. The cell of claim 1, which is produced in a host cell.
18. The protein according to any one of 3 to 17. 19. A DNA encoding human IL-3
19. The protein of claim 18, wherein does not contain an intron. 20. A control sequence comprising a lac promoter and HpaII.
20. The protein according to claim 18, comprising a promoter selected from the group consisting of a promoter, a σ43 promoter, an α-amylase promoter, an EF-1α promoter, and an SV40 promoter.