EP0162898A1 - Interleukin-2-erzeugung unter verwendung klonierter gene für interleukin-2 und hefe alpha-faktor - Google Patents

Interleukin-2-erzeugung unter verwendung klonierter gene für interleukin-2 und hefe alpha-faktor

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Publication number
EP0162898A1
EP0162898A1 EP84904300A EP84904300A EP0162898A1 EP 0162898 A1 EP0162898 A1 EP 0162898A1 EP 84904300 A EP84904300 A EP 84904300A EP 84904300 A EP84904300 A EP 84904300A EP 0162898 A1 EP0162898 A1 EP 0162898A1
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EP
European Patent Office
Prior art keywords
sequence
secretory leader
yeast
factor
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP84904300A
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English (en)
French (fr)
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EP0162898A4 (de
Inventor
Philip J. Barr
James P. Merryweather
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
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Filing date
Publication date
Application filed by Chiron Corp filed Critical Chiron Corp
Publication of EP0162898A1 publication Critical patent/EP0162898A1/de
Publication of EP0162898A4 publication Critical patent/EP0162898A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • Lymphokines are naturally-occurring polypeptides, produced by normal lymphocytes, which mediate the host's immune response to antigenic challenge.
  • a particular lymphokine, interleukin-2 appears to promote the host's immune response and has potential value in the treatment of tumors, immuno-deficiency diseases and several other clinical conditions, and as an adjuvant for vaccine administration.
  • Interleukin-2 appears to act as a potent mitogen for T lymphocytes. At present, only limited quantities of interleukin-2 are obtained by separation from human serum or from certain human cell tissue culture media. It is therefore of great scientific and clinical importance to be able to produce sufficiently large quantities of interleukin-2. Economic, efficient methods for producing a product having the properties of mature human interleukin-2 have therefore become significant goals.
  • Kurjan and Herskowitz, Cell (1982) 30:933-934 describe a putative ⁇ -factor precursor containing four tandem copies of mature ⁇ -factor, describing the sequence and postulating a processing mechanism.
  • Robb et al. (1983) Proc. Natl. Acad. Sci USA 80:5990-5994 describe a partial amino acid sequence from the N-terminal region of mature human interleukin-2 isolated from JURKAT cell tissue culture medium.
  • Novel methods and DNA constructs are provided for the production of polypeptides having biological activity analogous to interleukin-2 (IL2) .
  • IL2 interleukin-2
  • Enhanced efficiency in production of the polypeptide is achieved, in part, by the complete synthesis of the structural gene employing codons preferentially utilized by yeast, the intended host. Desirably, at least about 50%, usually, at least about 60% of the codons are modified such that most of the codons of the structural gene are those preferentially utilized by yeast.
  • the construct includes a replication system for yeast and the structural gene joined in reading frame to secretory and processing signals recognized by yeast.
  • the yeast host transformant provides for efficient and economic production of a product useful as interleukin-2.
  • Fig. 1 illustrates the order of assembly of synthetic ssDNA segments used in preparing the 5'-half of the synthetic IL2 gene.
  • Fig. 2 illustrates the scheme utilized to clone the synthetic DNA fragment of Fig. 1.
  • Fig. 3 illustrates the order of assembly of ssDNA segments used in preparing the 3'-half of the synthetic IL2 gene.
  • Fig. 4 illustrates the scheme utilized to clone the synthetic gene fragment of Fig. 3.
  • Fig. 5 illustrates the scheme utilized to join the two halves of the synthetic IL2 gene.
  • DNA constructs capable of expressing mammalian, particularly human interleukin-2 (IL2) in a eukaryotic microorganism host are provided.
  • IL2 interleukin-2
  • polypeptide (s) it is intended to include not only the naturally occurring mammalian factors, but also fragments or analogs thereof exhibiting analogous biological activity.
  • These DNA fragments can be incorporated into vectors, and the resulting plasmids used to transform susceptible hosts. Transformation of a susceptible host with such recombinant plasmids results in expression of the IL2 gene and production of a mature polypeptide product having the physiological and immunological activity of IL2. That is, it acts in the same manner as IL2 isolated from either a rat or a human host in recognized bioassays.
  • Extrachromosomal constructs having as essential elements for expression of mature polypeptides, a replication system recognized by yeast, a synthetic structural gene having a plurality of codons preferentially utilized by yeast, the structural gene being in reading frame with efficient secretory leader and processing signals to provide for the efficient secretion and processing of the polypeptide in a yeast host, and production of a product in high yield which has biological, particularly immunological and physiological activity commensurate with human interleukin-2.
  • the construct provides for the initial formation of "pre”-IL2.
  • pre-IL2 it is meant that the DNA sequence encoding the mature polypeptide is joined to and in reading frame with a leader sequence including processing signals efficiently recognized by the yeast host.
  • pre denotes the inclusion of secretory leader and processing signals sequences (on the precursor polypeptide) recognized by yeast and does not refer to any processing signals associated with the natural human IL2 gene.
  • the IL2 structural gene of this invention is synthetic DNA, which is prepared using codons preferred by yeast as evidenced by the codon frequency in structural genes encoding for yeast glycolytic enzymes.
  • the secretory leader and processing signals are conveniently derived from naturally-occurring DNA sequences in yeast, which sequences provide for the secretion and processing of polypeptide (s).
  • polypeptides which are naturally secreted by yeast include ⁇ -factor, a-factor, acid phosphatase, and the like.
  • the remaining sequences in the construct, including the replication system, promoter, and terminator, are well known and amply described in the literature.
  • DNA constructs of the present invention it is necessary to bring the individual DNA sequences embodying the structural gene, the secretory leader and processing signals, replication system, promoter, and terminator together in a predetermined order to assure that they are able to properly function in the resulting plasmid. Since the various DNA sequences which are joined to form the DNA construct of the present invention will be derived from diverse sources, in some instances it will be convenient or necessary to join the sequences by means of connecting or adaptor molecules, which in the subject invention are incorporated into the synthetic gene.
  • a pre-existing vector which includes a multicopy number yeast replication system, a bacterial replication system, appropriate markers for selection, as well as a promoter, transcriptional terminator and modified leader sequence associated with the secretion of ⁇ -factor. See copending application Serial No. 522,909, filed August 12, 1983.
  • the structural gene encoding for IL2 is prepared by first preparing a series of single stranded fragments ranging in number of from about 10 to 40, conveniently 14 to 34 bases, which provide for overlapping of other fragments and overhangs, so that upon bringing together the fragments under ligating conditions, dsDNA is produced having appropriate cohesive ends.
  • a 5'- and 3'-fragment were prepared and then combined to provide for a structural gene encoding the entire amino acid sequence of IL2 and providing for appropriate termini for linking to restriction sites in the vector, which provide for the structural gene being in frame with the secretory leader sequence.
  • Each of the fragments encoding for the gene - 5 '-fragment and 3 '-fragment with flanking regions as appropriate - may be cloned and amplified in an appropriate vector to expand the amount of the fragment and to ensure its integrity.
  • the 5' -fragment is inserted downstream from transcriptional regulatory signals for transcription initiation and a secretory leader sequence which includes a convenient restriction site at or about the processing signal.
  • the termini of the fragment can be tailored to fulfill the requirements necessary for subsequent processing steps.
  • its 5' -terminus is designed to join to the secretory leader sequence and processing signal in reading frame and to replace any nucleotides which have been lost due to restriction or other processing of the nucleotide sequence encoding for the secretory leader and processing signal.
  • a restriction site can be chosen which is internal to the coding region for the secretory leader and processing signal.
  • the 5'-fragment in the 3'-direction beyond the site where the 5' -fragment and 3 '-fragment are to be linked. In this way, a convenient 3' -terminus of the 5' -fragment is present for joining to the vector 5' -terminus. The extension is then removed by restriction to provide a 3' -terminus of the 5 '-fragment which is complementary to the 5 '-terminus of the 3 '-fragment.
  • Parallel manipulations may be employed with the 3' -fragment to provide for appropriate restriction sites and termini for joining to a vector and to the 5' -fragment, as well as supplying nucleotides in the 3 ' -non-coding region associated with termination of transcription and translation.
  • this new fragment After cloning the synthetic 5' -fragment in the vector containing the secretory leader sequence, a new extended 5' -fragment is excised.
  • the new 5 ' -fragment has its 5 ' -end beginning with the transcriptional regulatory signals controlling the transcription of the secretory leader sequence and processing signal, followed by the synthetic 5 '-fragment in reading frame with the secretory leader sequence.
  • this new fragment now includes the promoter and other associated transcriptional regulatory sequences, such as the TATA box and capping sequence, as well as any other sequences involved in the efficient transcription of ⁇ -factor.
  • the fragment is obtained which has all the necessary transcriptional regulatory functions, the secretory leader and processing signal, the 5 '-end of the IL2 structural gene and a cohesive end or blunt end for ligating to the 5 '-terminus of the 3 '-fragment.
  • the 3 '-half of the IL2 is inserted into an appropriate vector for cloning.
  • the resulting plasmid has a unique restriction site at the 5 '-end of the 3 '-fragment resulting in cohesive termini when cut, and appropriate transcriptional termination sequences downstream, such as a terminator and polyadenylation signal.
  • the fragment to be inserted conveniently has the same cohesive termini.
  • the plasmid is treated with phosphatase.
  • the fragment containing the transcriptional regulatory signals and 5 ' -end of the IL2 structural gene is then inserted into the phosphatase-treated plasmid for bacterial transformation and subsequently excised and ligated to provide for a plasmid capable of transforming a yeast host efficiently, being multicopy, and providing for the efficient secretion of the polypeptide encoded by the IL2 gene.
  • homologous promoter associated with the secretory leader sequence may be used, it may also be replaced with other promoters or may be used in tandem with other promoters.
  • Promoters of particular interest include acid phosphatase and those promoters involved with enzymes in the glycolytic pathway, such as promoters for alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, triose phosphate isomerase, phosphoglucoisomerase, phosphofructokinase, etc.
  • promoters By employing these promoters with regulatory sequences, such as enhancers, operators, etc., and using a host having an intact regulatory system, one can regulate the expression of the "pre"-IL2, and various small organic molecules, e.g., glucose, may be employed for the regulation of production of the desired polypeptide.
  • regulatory sequences such as enhancers, operators, etc.
  • small organic molecules e.g., glucose
  • temperature controlled sytems e.g., temperature-sensitive regulatory mutants which allow for modulation of transcription by varying the temperature.
  • genes provide for amplification, where upon stress to the host, not only is the gene which responds to the stress amplified, but also flanking regions.
  • plasmids may be obtained which have a plurality of repeating sequences, which sequences include the "pre"-polypeptide gene with its regulatory sequences.
  • Illustrative genes include metallothioneins and dihydrofolate reductase.
  • the construct may include, in addition to the secretory leader and processing signal sequence, other DNA homologous to the host genome. If it is desired that there be integration of the IL2 gene into the chromosome (s) , integration can be enhanced by providing for sequences flanking the IL2 gene construct which are homologous to host chromosomal DNA.
  • the replication system which is employed will be recognized by the yeast host. Therefore, it is desirable that the replication system be native to the yeast host.
  • yeast vectors are reported by Botstein et al. , Gene (1979) 8:17-24.
  • YEp plasmids which contain the 2 ⁇ m plasmid replication system. These plasmids are stably maintained at multiple copy number.
  • ARSa and CEN4 may be used to provide for stable maintenance.
  • the plasmids may be introduced into the yeast host by any convenient means, employing yeast host cells or spheroplasts and using DNA for transformation, or liposomes, or calcium precipitated DNA or other conventional technique.
  • the modified hosts may be selected in accordance with the genetic markers which are usually provided in a vector used to construct the expression plasmid.
  • An auxotrophic host may be employed, where the plasmid has a gene which complements the host and provides prototrophy.
  • resistance to an appropriate biocide e.g., antibiotic, heavy metal, toxin, or the like, may be included as a marker in the plasmid.
  • Selection may then be achieved by employing a nutrient medium which stresses the parent cells, so as to select for the cells containing the plasmid.
  • the plasmid containing cells may then be grown in an appropriate nutrient medium, and the desired secreted polypeptide isolated in accordance with conventional techniques.
  • the polypeptide may be purified by chromatography, filtration, extraction, etc. Since the polypeptide will be present in mature form in the nutrient medium, one can cycle the nutrient medium, continuously removing the desired polypeptide.
  • the following examples are offered by way of example and not by way of limitation .
  • a nucleotide sequence for interleukin-2 comprising preferentially utilized yeast codons was devised. The sequence included a portion of a modified ⁇ -factor secretory leader and processing signal at its
  • the sequence is provided with a Kpnl cohesive end at the 5 ' -end and a Sail cohesive end at the
  • the coding sequence for the mature polypeptide begins after the processing site.
  • a synthetic DNA fragment for interleukin-2 having the above sequence was prepared by synthesizing and cloning two halves of the fragment separately. Each half fragment was prepared by synthesizing overlapping ssDNA segments using the phosphoramidite method, as described by Beaucage and Caruthers (1981) Tetrahedron Lett. 22:1859-1862.
  • the ssDNA segments were as follows:
  • IL2-3 GCAAATGATCTTGAACGGTATCAACAACTACA
  • IL2-6 GAAGCACCTGCAGTGTCTAGAGGAAGAGTTG IL2-7 AAGCCATTGGAAGAAGTCCTGAACTTGGCTCAAT
  • IL2-16 GTTCAAGATCATTTGCAAGTCCAACAACAAGT IL2-17 GTCAACTTTGGGTTCTTGTAGTTGTTGATACC
  • IL2-23 GTTTCAGAACCCTTCAATTCCAAAACGATAAC IL2-24 GGTTTCGTCAGCGTATTCACACATGAAGGTG
  • the 5'-half of the sequence was assembled as illustrated in Fig. 1.
  • Fifty pmoles of each ssDNA segment (except Linker and IL2-20') were 5 ' -phosphorylated with T4 polynucleotide kinase.
  • the segments were then annealed in a single step by combining and cooling from 95°C to 25°C over 1.5 hours.
  • Ligation was performed in a reaction volume of 30 ⁇ l containing 1mM ATP, 10mM DTT, 100mM tris-HCl, pH 7.8, 10mM MgCl 2 , 1 ⁇ g/ml spermidine and T4 ligase.
  • the resulting double stranded fragment was purified on a 7% native polyacrylamide electrophoresis gel.
  • the dsDNA fragment included a Kpnl cohesive end at the 5 ' -end and a Sail cohesive end at the 3 '-end.
  • EGF human epidermal growth factor
  • the sequence was inserted into the EcoRI site of pBR328 to produce a plasmid p328EGF-1 and cloned. Approximately 30 ⁇ g of p328EGF-1 was digested with EcoRI and approximately 1 ⁇ g of the expected 190 base pair EcoRI fragment was isolated. This was followed by digestion with the restriction enzyme Hgal. Two synthetic oligonucleotide connectors Hindlll-Hgal and Hgal-Sall were then ligated to the 159 base pair Hgal fragment.
  • the Hgal-Hindlll linker had the following sequence:
  • CTTCGATTGAG This linker restores the ⁇ -factor processing signals interrupted by the Hindlll digestion and joins the Hgal end at the 5 '-end of the EGF gene to the Hindlll end of pAB112.
  • the Hgal-Sall linker had the following sequence: TGAGATGATAAG
  • ACTATTCAGCT This linker has two stop codons and joins the Hgal end at the 3' -end of the EGF gene to the Sail end of pAB112.
  • the resulting 181 base pair fragment was purified by preparative gel electrophoresis and ligated to 100ng of pAB112 which had been previously completely digested with the enzymes Hindlll and Sail. Surprisingly, a deletion occurred where the codon for the 3rd and 4th amino acids of EGF, asp and ser, were deleted, with the remainder of the EGF being retained.
  • pAB112 is a plasmid containing a 1.75 kb EcoRI fragment with the yeast ⁇ -factor gene cloned in the EcoRI site of pBR322 in which the Hindlll and Sail sites had been deleted (pAB11) .
  • pAB112 was derived from plasmid pAB101 which contains the yeast ⁇ -factor gene as a partial Sau3A fragment cloned in the BamHI site of plasmid YEp24.
  • pAB101 was obtained by screening a yeast genomic library in YEp24 using a synthetic 20-mer oligonucleotide probe
  • Plasmid pAB201 (5 ⁇ g) was digested to completion with the enzyme EcoRI and the resulting fragments were: a) filled in with DNA polymerase I Klenow fragment; b) ligated to an excess of BamHI linkers; and c) digested with BamHI.
  • the 1.75 kbp EcoRI fragment was isolated by preparative gel electrophoresis and approximately 100ng of the fragment was ligated to 100ng of pCl/1, which had been previously digested to completion with the restriction enzyme BamHI and treated with alkaline phosphatase.
  • Plasmid pCl/1 is a derivative of pJDB219,
  • f. shows the sequences at the C-terminal region of hEGF, which is the same for all constructions. Synthetic oligonucleotide linkers used in these constructions are boxed. These fusions were made as follows.
  • Construction (a) was made as described above. Construction (b) was made in a similar way except that linker 2 was used instead of linker 1. Linker 2 modifies the ⁇ -factor processing signal by inserting an additional processing site (ser-leu-asp-lys-arg) immediately preceding the hEGF gene. The resulting yeast plasmid is named pY ⁇ EGF-22. Construction (c) , in which the dipeptidyl aminopeptidase maturation site (glu-ala) has been removed, was obtained by in vitro mutagenesis of construction (a) . A Pstl-Sall fragment containing the ⁇ -factor leader-hEGF fusion was cloned in phage M13 and isolated in a single-stranded form. A synthetic 31-mer of sequence
  • 5'-TCTTTGGATAAAAGAAACTCCGACTCCCG-3 ' was synthesized and 70 picomoles were used as a primer for the synthesis of the second strand from 1 picomole of the above template by the Klenow fragment of DNA polymerase. After fill-in and ligation at 14° for 18 hrs., the mixture was treated with S, nuclease (5 units for 15 minutes) and used to transfect E . coli JM101 cells. Bacteriophage containing DNA sequences in which the region coding for (glu-ala) was removed were located by filter plaque hybridization using the
  • plasmid pAB114 was derived as follows: plasmid pABH2 was digested to completion with Hindlll and then religated at low (4 ⁇ g/ml) DNA concentration and plasmid pAB113 was obtained in which three 63bp Hindlll fragments have been deleted from the ⁇ -factor structural gene, leaving only a single copy of mature ⁇ -factor coding region.
  • a BamHI site was added to plasmid pAB11 by cleavage with EcoRI, filling in of the overhanging ends by the Klenow fragment of DNA polymerase, ligation of BamHI linkers, cleavage with BamHI and religation to obtain pAB12.
  • Plasmid pAB113 was digested with EcoRI, the overhanging ends filled in, and ligated to BamHI linkers. After digestion with BamHI the 1500bp fragment was gel-purified and ligated to pAB12 which had been digested with BamHI and treated with alkaline phosphatase.
  • Plasmid pAB114 which contains a 1500bp BamHI fragment carrying the ⁇ -factor gene, was obtained. The resulting plasmid (pAB114 containing the above described construct) is then digested with BamHI and ligated into plasmid pCl/1.
  • the resulting yeast plasmid is named pY ⁇ EGF-23.
  • Construction (d) in which a new Kpnl site was generated, was made as described for construction (c) except that the 36-mer oligonucleotide primer of sequence 5' -GGGTACCTTTGGATAAAAGAAACTCCGACTCCGAAT-3 ' was used.
  • the resulting yeast plasmid is named pY ⁇ EGF-24.
  • Construction (e) was derived by digestion of the plasmid containing construction (d) with Kpnl and Sail instead of linker 1 and 2.
  • the resulting yeast plasmid is named pY ⁇ EGF25.
  • the 5' -half of the IL2 sequence was inserted according to the scheme illustrated in Fig. 2. Plasmid p ⁇ EGF-24 was restricted with a mixture of restriction endonucleases Kpnl and Sail to remove a KpnI/Sall fragment. The 5 '-synthetic fragment of IL2 was inserted into the resulting cut vector to produce plasmid p ⁇ IL2-5' which was then cloned in E . coli HB101.
  • the 3 '-half of the sequence was assembled as illustrated in Fig. 3. Fifty pmoles of each ssDNA segment (except IL2-6 and IL2-27) were 5'-phosphorylated with T4 polynucleotide kinase. The segments were then annealed in a single step by combining and cooling from 95°C to 25°C over 1.5 hours. Ligation was performed in a reaction volume of 30 ⁇ l containing 1mM ATP, 10mM DTT, 100mM tris-HCl, pH 7.8, 10mM MgCl 2 , 1 ⁇ g/ml spermidine and T4 ligase. The resulting double stranded fragment was purified on a 7% native polyaerylamide electrophoresis gel. The dsDNA fragment included a Xbal cohesive end at the 5 ' -end and a Sail cohesive end at the 3 '-end.
  • Plasmid pAB114 is described in application serial no. 522,909 which has in part been reproduced above.
  • Plasmid pAB114 was restricted with a mixture of restriction endonucleases Xbal and Sail to remove a Xbal/Sall fragment. The 3 '-synthetic fragment was inserted into the resulting cut vector to produce p ⁇ IL2-3' which was then cloned in E. coli HB101.
  • Plasmid p ⁇ IL2-5' was restricted with restriction endonuclease Xbal to generate an Xbal/Xbal fragment carrying the ⁇ -factor transcriptional regulatory sequences and modified secretory leader and processing signals derived from plasmid p ⁇ EGF-24 and the 5 '-half of the IL2 sequence.
  • the 3 '-proximal Xbal site in the 5' -segment is located interior of the synthetic sequence so that a 15 bp segment at the 3 '-end is removed.
  • Plasmid p ⁇ IL2-3' was also restricted with Xbal and treated with alkaline phosphatase to prevent recircularization.
  • the Xbal fragment from p ⁇ IL2-5' was then inserted into the Xbal site on p ⁇ IL2-3' to form plasmid p ⁇ IL2 which had the two fragments in frame in the correct orientation. The latter was determined by restriction analysis after plasmid p ⁇ IL2 was cloned in E. coli HB101.
  • Plasmid p ⁇ IL2 was then digested to completion with BamHI, and the resulting fragment carrying the IL2 construct was isolated by preparative gel electrophoresis. Approximately 100 ng of the fragment was inserted into the BamHI site of pCl/1, which had been previously digested to completion with BamHI and treated with alkaline phosphatase. The resulting plasmids were designated pY ⁇ IL-2/3.6 and pY ⁇ IL-2/18.7, representing the opposite orientations of the inserted sequence. Plasmid pCl/1 was described above.
  • Microtiter dishes (96-well) were prepared with 2x10 4 HT-2 cells in 100 ⁇ l of RPMI-1640 mammalian cell growth medium with L-glutamine (300 ⁇ g/ml) supplemented with 10% fetal calf serum, 50 ⁇ M
  • HT-2 cells are a subclone of the HTL-1 line of murine T lymphocytes described by Watson et al. (1979) J. Exp. Med. 150:849 and 1510, dependent upon the presence of interleukin-2 for viability and/or growth. Since these mouse cells respond to human material, their use provides a bioassay for human interleukin-2.
  • a semi-quantative assay was employed by visually estimating cell survival and growth employing a microscope and comparing test results with standards employing known amounts of Serial 2-fold dilutions of the yeast me i um dialysate above were then prepared in mammalian cell growth medium (RPMI-1640 with supplements, as above) and 100 ⁇ l of each diluted sample added to individual wells containing HT-2 cells.
  • mammalian cell growth medium RPMI-1640 with supplements, as above
  • Reference standards known to contain interleukin-2 (concanavalin A-free, conditioned rat splenocyte medium) were either obtained commercially (Monoclone ® , Collaborative Research, Inc.) or prepared by stimulation of rat spleen cell cultures (in RPMI-1640 medium with supplements, as above) with concanavalin A (1 ⁇ g/10 cells) for 48 hours at 37°C in
  • the HT-2 cell microplate cultures were incubated at 37°C in 7% CO 2 /air for 48 hours, scored for viability and/or growth and the approximate interleukin-2 content of the yeast preparation determined by reference to the standards. This comparison indicated activities equivalent to or greater than the commercial material, i.e., estimated to be in the range of 20-100 ng/ml, probably about 50 ng/ml.
  • novel DNA constructs are provided which may be inserted into vectors to provide for expression of "pre"-interleukin-2 and intracellular processing and secretion of the mature polypeptide in good yield to promote a polypeptide product having high IL2 biological activity in a recognized bioassay based on murine cellular growth.
  • pre interleukin-2
  • intracellular processing and secretion of the mature polypeptide in good yield to promote a polypeptide product having high IL2 biological activity in a recognized bioassay based on murine cellular growth.

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EP19840904300 1983-11-14 1984-11-09 Interleukin-2-erzeugung unter verwendung klonierter gene für interleukin-2 und hefe alpha-faktor. Withdrawn EP0162898A4 (de)

Applications Claiming Priority (2)

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US55144383A 1983-11-14 1983-11-14
US551443 1983-11-14

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EP0162898A1 true EP0162898A1 (de) 1985-12-04
EP0162898A4 EP0162898A4 (de) 1987-07-23

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2304586C1 (ru) * 2006-03-27 2007-08-20 Михаил Николаевич Смирнов Препарат интерлейкина-2 и способ его получения

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JPS60115528A (ja) * 1983-11-28 1985-06-22 Takeda Chem Ind Ltd ヒトインタ―ロイキン―２蛋白質を含有する抗腫瘍用または免疫機能低下疾患治療用組成物
US4925791A (en) * 1984-07-20 1990-05-15 Celltech Limited Eucaryotic expression vectors
US5496924A (en) * 1985-11-27 1996-03-05 Hoechst Aktiengesellschaft Fusion protein comprising an interleukin-2 fragment ballast portion
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WO2007111534A1 (en) 2006-03-27 2007-10-04 Mikhail Nikolaevich Smirnov Method for producing an interleukin-2 preparation and the thus obtainable preparation

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