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WO1995028427A1 - Lymphokine chimere composee de l'interleukine 3 et d'une muteine de l'interleukine 6 - Google Patents

Lymphokine chimere composee de l'interleukine 3 et d'une muteine de l'interleukine 6 Download PDF

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Publication number
WO1995028427A1
WO1995028427A1 PCT/US1994/004208 US9404208W WO9528427A1 WO 1995028427 A1 WO1995028427 A1 WO 1995028427A1 US 9404208 W US9404208 W US 9404208W WO 9528427 A1 WO9528427 A1 WO 9528427A1
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PCT/US1994/004208
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English (en)
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Charles Tackney
Colette L. Brown
Samuel D. Waksal
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Imclone Systems Incorporated
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Priority to PCT/US1994/004208 priority Critical patent/WO1995028427A1/fr
Publication of WO1995028427A1 publication Critical patent/WO1995028427A1/fr

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    • 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]
    • C07K14/5403—IL-3
    • 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]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a chimeric protein comprised of lnterleukin-3 and a mutein of lnterieukin-6.
  • the mutein of lnterleukin-6 (mlL-6) has the first two cysteine residues replaced with any other amino acid residue.
  • the chimera may be constructed according to the following formula:
  • IL-3 represents lnterleukin-3
  • mlL-6 represents the mutein of lnte ⁇ ieukin-6
  • L represents the first twenty-two amino acid residues of the lnterleukin-6 mutein.
  • SEQ. ID. NO. 1 An example of the nucleic and amino acid sequence of the chimeric IL-3/mlL-6 protein of the present invention is shown below in SEQ. ID. NO. 1.
  • the invention also includes nucleic acid sequences encoding such proteins, plasmids and vectors containing such nucleic acid sequences, cells capable of expressing the protein and methods of using the protein.
  • Figure 1 illustrates the three nucleic acid fragments used to construct the chimeric IL-3/mlL-6 protein of the present invention.
  • Line A represents nucleic acid sequences encoding human IL-3.
  • Line B represents the restriction fragment obtained from the IL-3 sequence represented by line A by endonuclease digestion with Ncc ⁇ and Dde ⁇ . The plasmid containing the fragment is designated p570.
  • Lines C and C show the 3' end of the IL-3 restriction fragment (line B), which lacks the nucleic acid sequence that encodes the eleven amino acids from the carboxy terminal end of native IL-3 (line A).
  • Lines D and D' represent an oligonucleotide pair that contains the nucleic acid sequences for the last eleven amino acids of IL-3 and the first four amino acids of mlL-6, all of which are forfeited during digestion of the nucleic acid sequences encoding IL-3 with Nco ⁇ and Dde ⁇ endonucleases and mlL-6 with EcoRH and Hind ⁇ endonucleases.
  • Line E represents the EcoRMHinc ⁇ H restriction fragment encoding mlL-6 which lacks the first four amino acid residues from the amino terminal end of the molecule.
  • Line E' represents the portion of the pKK223-2 IL-6 SSCC plasmid which contains the nucleic acid sequences that encode the mlL-6 protein which lacks the first four amino acid residues from the amino terminal end of the molecule.
  • Line E" represents the sequence from which the EcoRMHin RW restriction fragment (line E) is obtained.
  • Figure 2 illustrates the relative positions of the IL-3, L and mlL-6 portions of one embodiment of the chimera of the present invention.
  • Figure 3 illustrates the expression vector pSE420.
  • the pSE420 vector contains the lacl q gene, which allows for regulated expression in E.c ⁇ //HB101. Transcriptional control is via the trc promoter and utilizes the highly efficient translation re-initiation characteristic of mini-cistron systems. The incorporation of upstream anti-termination and g10 ribosome binding sequences ensures high level translation of inserts cloned into its polylinker. Digestion of pSE420 with Ned and Kpr ⁇ allows subsequent mobilization of the IL-3/mlL-6 chimera, by Ncci/Kpr ⁇ digestion of the IL-3/mlL-6-pKK233-2 plasmid, into this protein expression system.
  • IL-3 and IL-6 refer to human IL-3 and human IL-6, respectively.
  • the terms IL-3 and IL-6 include proteins described in the literature as having the same name as IL-3 or IL-6.
  • IL-3 is also known as multi-colony-stimulating factor (multi-CSF).
  • IL-6 is also known as interferon- ⁇ -2 (IFN- ⁇ -2), B-cell stimulation factor-2 (BSF- 2), B-cell hybridoma/plasmacytoma growth factor (HPGF or HGF), 26 kDa protein and hepatocyte stimulating factor (HSF).
  • IFN- ⁇ -2 interferon- ⁇ -2
  • BSF- 2 B-cell stimulation factor-2
  • HPGF or HGF B-cell hybridoma/plasmacytoma growth factor
  • HGF hepatocyte stimulating factor
  • IL-6 The amino acid sequence of IL-6 has been described in the literature; see, for example, Figure 2A of Brakenhoff et al., Journal of Immunology 139. 4116-4121 (1987) and Figure 1 of Clark et al., PCT publication WO 88/00206, published 14 January 1988.
  • Thesi eferences also contain the cDNA sequence that corresponds to native IL-6 mR A.
  • mlL-6 is a mutein wherein the cysteine residues corresponding to amino acid positions 45 and 51 of native IL-6 have been replaced by other amino acids, while the cysteine residues corresponding to amino acid positions 74 and 84 have been retained.
  • the cysteine residues are replaced by neutral amino acids such as serine or alanine.
  • DNA sequences that encode native IL-3 and IL-6 include, but are not limited to, mammalian sources such as murine, pan and human sequences.
  • chimera or “chimeric protein” in this specification is understood to refer to a non-naturally occurring protein that is formed by joining one genetically distinct protein to another genetically distinct protein, end to end, in such a way that the biological activity of both proteins is retained or enhanced.
  • fusion protein in this specification is understood to refer to a protein that is produced in a system in which the desired protein is linked to a fusion partner, usually for the purpose of expediting expression or purification.
  • suitable fusion partners include t ⁇ E, b-galactosidase, Protein A, maltose binding protein, etc.
  • amino acid in this specification are understood to mean the approximately 21 naturally occurring a-amino acids or their analogs.
  • the chimeric IL-3/mlL-6 protein and fragments thereof may be prepared by methods known in the art.
  • a preferred method of preparing the chimeric protein of the present invention involves isolating DNA sequences that encode IL-3 and mlL-6, joining the IL-3 and mlL-6 encoding sequences in frame to form a single nucleic acid sequence that encodes the IL-3/mlL-6 chimera; amplifying or cloning the DNA in a suitable host; expressing the DNA in a suitable host; and harvesting the protein.
  • a chimeric IL-3/mlL-6 nucleic acid sequence may be constructed as follows:
  • an oligonucleotide is used to replace sequences from IL-3 and mlL-6 which are lost as a result of the excision of the IL-3 and mlL-6 portions of the genes from the plasmids.
  • Replacement of the missing IL-3 and mlL-6 sequences by the oligonucleotide also serves to join the IL-3 and mlL-6 sequences together to form the chimeric IL-3/mlL-6 nucleic acid sequence in such a way that both interleukins are in frame for translation;
  • the chimeric IL-3/mlL-6 nucleic acid sequence is assembled by combining the IL-3 fragment, the mlL-6 fragment, and, optionally, the oligonucleotide into a plasmid.
  • the plasmid contains a selectable marker, such as an antibiotic resistance gene.
  • the chimeric IL-3/mlL-6 sequence is amplified by, for example, PCR or cloning; 5) the amplified chimeric IL-3/mlL-6 sequence is inserted into an expression vector for expression of the chimeric IL-3/mlL-6 protein.
  • a controllable protein expression system that causes the juxtaposition of a promoter to control the amino acid coding sequence as a non-fusion process is employed.
  • the system can utilize any of several well-known, characterized and available promoters such as trp, trc, tic, tac, lac, P L , etc.
  • the chimera following expression of the chimeric IL-3/mlL-6 protein, the chimera is isolated and purified by methods known in the art.
  • the starting materials for construction of the present invention are nucleic acid sequences that encode native IL-3 and either native IL-6 or mlL-6. Nucleic acid sequences encoding native IL-3 and IL-6 may be isolated from a human cDNA or genomic DNA library.
  • the preferred method for obtaining DNA suitable as a starting material for construction of DNA encoding the chimera of the invention is to isolate DNA encoding native IL-3 and mlL-6 from an available recombinant plasmid.
  • Recombinant plasmids that encode native full length IL-3 and mlL-6 are known.
  • IL-3 see, for example, PCT publication WO 88/00598, published 28
  • mlL-6 is produced by mutating the native sequence.
  • muteins may be introduced into native IL-6 by site-directed mutagenesis, in order to encode amino acid residues other than cysteine at amino acid positions 45 and 51.
  • Site-directed mutagenesis is carried out by methods known in the art. See, for example, Zoller and Smith, Nucl. Acids Res. 10, 6487-6500 (1982); Methods in Enzymology 100, 468-500 (1983); and DNA 3, 479-488 (1984).
  • codons for the cysteine residues at positions corresponding to positions 45 and 51 of native IL-6 are replaced by codons for other amino acids, preferably by codons for any other neutral amino acids, and more preferably by codons for serine or alanine residues.
  • IL-6 in which all four cysteine residues have been replaced by serine residues may be obtained as described in Fowlkes et al., PCT application US89/05421.
  • the codons for the serine residues at positions corresponding to positions 74 and 84 of native IL-6 are replaced by cysteine residues by, for example, site- directed mutagenesis.
  • the codons for the serine residues at positions corresponding to 45 and 51 may be retained or replaced by other amino acid residues, such as by alanine, in the same way.
  • DNA encoding IL-3, IL-6, mlL-6 or the IL-3/mlL-6 chimera may be synthesized from individual nucleotides. Chemical synthesis of DNA from the four nucleotides may be accomplished in whole or in part by methods known in the art. Such methods include those described by Caruthers in Science 230, 281-285 (1985). DNA may also be synthesized by preparing overlapping double-stranded oligonucleotides, filling in the gaps, and ligating the ends together.
  • the DNA obtained may be amplified by methods known in the art.
  • One suitable method is the polymerase chain reaction (PCR) method described by Saiki et al. in Science 239, 487 (1988), Mullis et al in U.S. Patent 4,683,195 and by Sambrook, Fritsch and Maniatis (eds) in Molecular Cloning. A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press (1989). It is convenient to amplify the clones in the Iambda-gt10 or Iambda-gt11 vectors using Iambda-gt10 or Iambda-gt11 -specific oligomers as the amplimers (available from Clontech, Palo Alto, California).
  • PCR polymerase chain reaction
  • the DNA fragments encoding the protein of the invention may be assembled in the proper order and replicated following insertion into a wide variety of host cells in a wide variety of cloning vectors.
  • the host may be prokaryotic or eukaryotic.
  • Cloning vectors may comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Some suitable prokaryotic cloning vectors include plasmids from E.coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4.
  • Prokaryotic vectors also include derivatives of phage DNA such as M13 fd, and other filamentous single-stranded DNA phages.
  • Vectors for expressing proteins in bacteria are also known.
  • Such vectors include the pK233 (or any of the tec family of plasmids), T7, and lambda P .
  • Examples of vectors that express fusion proteins are PATH vectors described by Dieckmann and Tzagoloff in J. Biol. Chem. 260. 1513- 1520 (1985). These vectors contain DNA sequences that encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy terminus.
  • TrpE anthranilate synthetase
  • Vectors useful for cloning and expression in yeast are available.
  • a suitable example is the 2m circle plasmid.
  • Suitable cloning/expression vectors for use in mammalian cells are also known.
  • Such vectors include well-known derivatives of SV-40, adenovirus, cytomegalovirus (CMV) retrovirus-derived DNA sequences.
  • CMV cytomegalovirus
  • the expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed.
  • the control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence.
  • useful expression control sequences are the lac system, the t ⁇ system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses
  • Useful expression hosts include well-known prokaryotic and eukaryotic cells.
  • Some suitable prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli ⁇ B 101, E. co// ' W3110, E. CO// X1776, E. coli X2282, E. coli DHI, and E. coli MRCI, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.
  • Suitable eukaryotic cells include yeasts and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture.
  • the chimeric protein of the invention may be expressed in the form of a fusion protein with an appropriate fusion partner.
  • the fusion partner preferably facilitates purification and identification. Increased yields may be achieved when the fusion partner is expressed naturally in the host cell.
  • Some useful fusion partners include beta-galactosidase (Gray, et al., Proc. Natl. Acad. Sci. USA 79, 6598 (1982)); trpE (Itakura et al., Science 198, 1056 (1977)); protein A (Uhlen et al., Gene 23369 (1983)); glutathione S-transferase (Johnson, Nature 338.
  • Such fusion proteins may be purified by affinity chromatography using reagents that bind to the fusion partner.
  • the reagent may be a specific ligand of the fusion partner or an antibody, preferably a monoclonal antibody.
  • fusion proteins containing beta-galactosidase may be purified by l o affinity chromatography using an anti-beta-galactosidase antibody column
  • fusion proteins containing maltose binding protein may be purified by affinity chromatography using a column containing cross-linked amylose; see Guan, European Patent Application 286,239.
  • the fusion protein may occur at the amino-terminal or the carboxy- terminal side of the cleavage site.
  • the DNA that encodes the fusion protein is engineered so that the fusion protein contains a cleavable site between the protein and the fusion partner. Both chemical and enzymatic
  • cleavable sites are known in the art. Suitable examples of sites that are cleavable enzymatically include sites that are specifically recognized and cleaved by collagenase (Keil et al., FEBS Letters 56, 292-296 (1975)); enterokinase (Hopp et al., Biotechnology 6, 1204-1210 (1988)); factor Xa (Nagai et al., Methods Enzymol. 153.461-481 (1987)); and thrombin (Eaton et al., 5 Biochemistry 25, 505 (1986)). Collagenase cleaves between proline and X in the sequence Pro-X-Gly-Pro wherein X is a neutral amino acid.
  • Enterkinase cleaves after lysine in the sequence Asp-Asp-Asp-Asp-Lys.
  • Factor Xa cleaves after arginine in the sequence lle-Glu-Gly-Arg.
  • Thrombin cleaves between arginine and glycine in the sequence Arg-Gly-Ser-Pro.
  • Specific chemical cleavage agents are also known. For example, cyanogen bromide cleaves at methionine residues in proteins.
  • the chimeric protein is purified by methods known in the art. Such methods include affinity chromatography using specific antibodies. Alternatively, the recombinant protein may be purified using a combination of ion-exchange, size-exclusion, and hydrophobic interaction chromatography using methods known in the art. These and other suitable methods are described by Marston, "The Purification of Eukaryotic Proteins Expressed in E. coli" in DNA Cloning. D. M. Glover, Ed., Volume III, IRL Press Ltd., England, 1987.
  • SEQ. ID. NOS. 1-2 show the amino acid sequence of one chimeric IL- 3/mlL-6 protein of the invention. This sequence shows an embodiment in which the carboxy terminal end of IL-3 is attached to the amino terminal end of mlL-6.
  • a nucleotide sequence that expresses the chimer is also shown in SEQ. ID. NO. 1.
  • the invention also includes equivalent variants of the IL-3 and mlL-6 portions of the chimeric protein described above and the nucleic acid molecules that encode such variants.
  • Equivalent variants include proteins comprising substitutions and additions in the amino acid and nucleotide sequences of the chimeras of the invention and the corresponding nucleic acid molecules.
  • Variants are included in the invention as long as the resulting chimeras and nucleic acid molecules continue to satisfy the structural and functional criteria described above, i.e., retain activity at least comparable to that of native IL-3 and mlL-6 and lack cysteine residues at positions 45 and 51 of the IL-6 portion.
  • amino acid or nucleotide sequence that is substantially the same as another sequence, but that differs from the other sequence by means of one or more substitutions or additions is considered to be an equivalent sequence. Except for the substitutions of cysteine residues at positions corresponding to positions 45 and 51 of native, mature IL-6, preferably less than 25%, more preferably less than 10%, and most preferably less than 5% of the total number of amino acids or nucleotides in the chimeras of the invention are substituted for or added to in the equivalent sequences.
  • Additions to the IL-3/mlL-6 muteins may be made at the C-terminal or N- terminal ends by adding the corresponding codons at the 5' or 3' ends of the nucleic acid sequences and expressing the nucleic acid molecules.
  • Examples of internal additions to the nucleic acid molecules include the introns present in genomic DNA. The introns are not expressed in a suitable eukaryotic host cell.
  • Equivalents of the nucleic acid molecules encoding the chimeric IL- 3/mlL-6 protein also include silent mutations at sites that do not alter the amino acid sequence expressed. Preferably, the silent mutation results in increased expression in a particular host.
  • the chimera may contain the entire IL-3 and mlL-6 proteins, or a biologically active fragment of either or both whole proteins.
  • Bioactive fragments of bioactive proteins may be identified by methods known in the art. For example, IL-6 fragments lacking amino acids 1-28 are known to be active. See, for example, Brakenhoff, J.P.J., et al., J. Immunol. 143. 1175-1182 (1989).
  • Fragments containing bioactive sequences may be selected on the basis of generally accepted criteria of potential bioactivity. Such criteria include analysis of which region(s) of a protein is required for bioactivity.
  • the present invention includes nucleic acid molecules that encode the chimera of the present invention. Any nucleic acid sequence that encodes the amino acid sequence of SEQ. ID. NOS. 1-2 can be used to express the chimeric protein of the present invention. For example, nucleic acid sequences that are found in nature or can be selected that will maximize expression in bacteria.
  • the nucleic acid molecule may be DNA or RNA.
  • the nucleic acid molecules may be used as probes for detecting DNA encoding IL-3, IL-6, mlL-6 or chimeric IL-3/mlL-6 as explained below, or to produce a protein of the invention, as explained above.
  • the chimeric protein and DNA can be used to prepare probes that detect the presence of IL-3, IL-6, mlL-6 or the chimeric IL-3/mlL-6 protein or DNA in a sample.
  • the method involves use of a labelled probe that recognizes IL-3, IL-6, mlL-6 or the chimeric IL-3/IL-6 protein or DNA present in biological samples, including, but not limited to, lymphatic fluid, synovial fluid, cerebral-spinal fluid, blood, tissue and cell samples.
  • the probe may be an antibody raised against the chimeric IL-3/mlL-6 protein, or a fragment thereof, or an oligonucleotide that hybridizes to DNA encoding IL-3, IL-6, mlL-6 or the chimeric IL-3/mlL-6 protein.
  • the antibody may be polyclonal or monoclonal.
  • Polyclonal antibodies are isolated from mammals that have been innoculated with the chimeric protein or a functional analog in accordance with methods known in the art. Briefly, polyclonal antibodies may be produced by injecting a host mammal, such as a rabbit, mouse, rat, or goat, with the chimeric protein or a fragment thereof. Sera from the mammal are extracted and screened to obtain polyclonal antibodies that are specific to the chimeric protein or protein fragment. The antibodies are preferably monoclonal. Monoclonal antibodies may be produced by methods known in the art.
  • the probes described above are labelled in accordance with methods known in the art.
  • the label may be a radioactive atom, an enzyme, or a chromophoric moiety.
  • the label may be radioactive.
  • useful radioactive labels include *P, 125 l, 131 l, and 3 H. Use of radioactive labels have been described in U.K.2,034,323, U.S.4,358,535, and U.S.4,302,204.
  • non-radioactive labels include enzymes, chromophors, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties.
  • the probe may be an antibody, preferably a monoclonal antibody.
  • the antibodies may be prepared as described above.
  • Assays for detecting the presence of proteins with antibodies have been previously described, and follow known formats, such as standard blot and ELISA formats. These formats are normally based on incubating an antibody with a sample suspected of containing the protein and detecting the presence of a complex between the antibody and the protein. The antibody is labelled either before, during, or after the incubation step.
  • the protein is preferably immobilized prior to detection. Immobilization may be accomplished by directly binding the protein to a solid surface, such as a microtiter well, or by binding the protein to immobilized antibodies.
  • a protein is immobilized on a solid support through an immobilized first antibody specific for the protein.
  • the immobilized first antibody is incubated with a sample suspected of containing the protein. If present, the protein binds to the first antibody.
  • a second antibody also specific for the protein, binds to the immobilized protein.
  • the second antibody may be labelled by methods known in the art. Non-immobilized materials are washed away, and the presence of immobilized label indicates the presence of the protein. This and other immunoassays are described by David, et al. in U.S. Patent 4,376,110 assigned to Hybritech, Inc., LaJolla, California.
  • the chimeric protein may be labelled and used as probes in standard immunoassays to detect antibodies against IL-3, IL-6, mlL-6 or chimeric IL- 3/mlL-6 proteins in samples, such as in the sera or other bodily fluids of patients.
  • a protein in accordance with the invention is incubated with the sample suspected of containing antibodies to the protein.
  • the protein is labelled either before, during, or after incubation.
  • the detection of labelled protein bound to an antibody in the sample indicates the presence of the antibody.
  • the antibody is preferably immobilized.
  • Suitable assays are known in the art, such as the standard ELISA protocol described by R.H. Kenneth, “Enzyme-Linked Antibody Assay with Cells Attached to Polyvinyl Chloride Plates” in Kenneth et al, Monoclonal Antibodies. Plenum Press, N.Y., page 376 (1981).
  • the probe may also be an oligonucleotide complementary to a target nucleic acid molecule.
  • the nucleic acid molecules may be RNA or DNA.
  • the length of the oligonucleotide probe is not critical, as long as it is capable of hybridizing to the target molecule.
  • the oligonucleotide should contain at least 6 nucleotides, preferably at least 10 nucleotides, and, more preferably, at least 15 nucleotides. There is no upper limit to the length of the oligonucleotide probes. Longer probes are more difficult to prepare and require longer hybridization times. Therefore, the probe should not be longer than necessary. Normally, the oligonucleotide probe will not contain more than 50 nucleotides, preferably not more than 40 nucleotides, and, more preferably, not more than 30 nucleotides.
  • the chimeric IL-3/mlL-6 protein of the present invention possesses in vitro and in vivo biological activity at least comparable to that of a mixture of IL-3 and IL-6 or IL-3 and mlL-6. Accordingly, the chimeric IL-3/mlL-6 protein is useful in the in vitro and in vivo stimulation of the formation, proliferation and differentiation of a broad range of hematopoietic cells, including granulocytes, macrophages, eosinophils, mast cells, erythroid cells, B cells, T cells, megakaryocytes, and multi-potential hematopoietic progenitor cells. The stimulation of proliferation of megakaryocytes leads to the production of platelets.
  • the mlL-6 portion of the chimeric IL-3/mlL-6 protein induces various acute phase proteins in liver cells.
  • the chimeric IL-3/mlL-6 protein is useful in immunotherapeutic and anti-inflammation compositions.
  • the chimera may also be used for the treatment of patients suffering from thrombocytopenia and patients undergoing chemotherapy or bone marrow transfers.
  • the starting material for the construction of the chimeric IL-3/mlL-6 nucleic acid sequence is a plasmid, designated p570 (ATCC 69242).
  • the p570 plasmid contains the cloned mature human IL-3 gene.
  • An analogous plasmid containing sequences that encode mature human IL-3 can be obtained from R&D Systems Inc., Minneapolis, Mn., catalog No. BBG 14. Mature human IL-3 contains 133 amino acids. (See line A in Figure 1 and SEQ. ID. NO. 3-4)
  • the p570 plasmid is digested with the restriction endonucleases ⁇ /col and Dde ⁇ . (New England Bio Labs, Beverly, Ma.) Digestion of the plasmid with these enzymes liberates a 0.375 kbp fragment (Line B in Figure 1 ) which encodes the natural amino terminus of human IL-3 and extends toward the carboxy terminus of the protein to the codon encoding alanine at amino acid position number 121. (See SEQ. ID. NO. 5)
  • the mlL-6 nucleic acid sequences are obtained from a plasmid designated pKK233-2 IL-6 SSCC. (See SEQ. ID. NO. 6-7 for the portion of the plasmid the encodes the sequence of mlL-6) Construction of the plasmid is described by Skelly et al., in example 5 of co-pending U.S. application 07/907,710, which is incorporated herein by reference and in Dagan et al., Protein Expression and Purification s, 290-294 (1992).
  • the pKK233-2 IL-6 SSCC plasmid contains a 0.6 kbp Ncol/HindlW restriction fragment that encodes mature mlL-6.
  • the Nco ⁇ restriction site of this plasmid places an ATG codon immediately upstream of the initial mlL-6 amino acid residue, alanine.
  • the Nco ⁇ site is followed 12 bp downstream by a unique EcoRtt recognition sequence.
  • Figure 1 when pKK233-2 IL-6 SSCC is digested with EcoRU and HindlU restriction enzymes (New England Bio Labs, Beverly, Ma.), a 0.59 kbp fragment is generated. (See line E and SEQ. ID. NO. 8) This fragment encodes the complete mlL-6 product minus the alanine-proline-valine-proline amino terminal residues and is followed by a Kpn ⁇ restriction site and three random in- frame stop codons.
  • an oligonucleotide pair (lines D and D' in Figure 1) encoding the lost amino acids is used to replace the lost nucleic acid sequences.
  • the oligonucleotide pair (lines D and D' in Figure 1) join the IL-3 fragment (line B in Figure 1) to the mlL-6 fragment (line E in Figure 1) to form a chimeric IL-3/mlL-6 cassette with Nco ⁇ and HindlH termini. (See SEQ. ID. NO. 9) Synthesis of the oligonucleotides is described below in Section B. (See SEQ. ID. NOS. 10-11)
  • the chimeric IL-3/mlL-6 cassette is assembled by simultaneously combining the IL-3 fragment (component 1; line B in Figure 1), the mlL-6 fragment (component 2; line E in Figure 1) and the oligonucleotide pair (component 3; lines D and D' in Figure 1) with a plasmid (component 4) that has been pre-digested and purified by standard methods to remove a Ncd/HindW restriction fragment from its sequence.
  • the plasmid used in this example is designated pKK233-2 (Pharmacia LKB, Piscataway, N.J.).
  • the chimeric IL-3/mlL-6 cassette which has Nco and HindlU termini, replaces the original Ncd/HindW restriction fragment in the plasmid.
  • the pKK233-2 plasmid contains an ampicillin resistance gene that is rendered functional if the four components of the reaction correctly assemble themselves to form the chimeric IL-3/mlL-6-pKK233-2 plasmid.
  • the plasmid is transfected into E.coli.
  • E.coli containing the chimeric IL-3/mlL-6 nucleic acid in the plasmid are selected for by growing the bacteria on agar containing ampicillin.
  • the IL-3/mlL-6-pKK233-2 plasmid is amplified to desired levels by growing the bacteria in a standard culture.
  • the ampicillin-resistant clone is verified as having the IL-3/mlL-6 gene by restriction enzyme analysis, sequencing data (Sanger, et al., 1977 Proc. Nat. Acad. of Sci., 74:5463) and expression of the IL-3/mlL-6 protein.
  • Expression of the IL-3/mlL-6 chimeric protein in E.coli is accomplished by inserting the chimeric IL-3/mlL-6 nucleic acid sequence into an expression vector.
  • the expression vector pSE420 (In Vitrogen, San Diego, Ca.) contains the lacl q gene which allows for regulated expression in E.co//HB101. Transcriptional control is via the trc promoter and utilizes the highly efficient translation re-initiation characteristic of mini-cistron systems. The incorporation of upstream anti-termination and g10 ribosome binding sequences ensures high level translation of inserts cloned into its polylinker.
  • Oligonucleotide chains are specifically synthesized on a Model 392 Applied Biosystems apparatus utilizing beta-cyanoethyl phosphoramidites as substrate. Synthesized nucleotide oligomers are deprotected and cleaved from resin supports using standard procedures as recommended by the manufacturer. One may utilize any of a variety of oligonucleotide purification cartridges or proceed with HPLC purification and isolation.
  • chimeric IL-3/mlL-6 protein Following expression of chimeric IL-3/mlL-6 protein in E.coli, the bacteria are harvested by centrifugation at 4°C and washed once in cold PBS. Bacterial pellets are suspended in 5ml/gm of cold 50mM Tris-HCI (pH 8.0), 100 mM NaCl, 1mM EDTA. Protease inhibitors PMSF (0.5mM), leupeptin (5mg/ml), aprotinin (5mg/ml) are included. Lysozyme, 50mg, is added and the suspension held on ice for 30 minutes.
  • lysis buffer 50mm Tris-HCI, pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate
  • MgS0 4 is added to a final concentration of 50mM followed by 25mg DNAasel (New England Bio Labs, Beverly, Ma.).
  • the mixture is incubated at room temperature until viscosity is minimal.
  • This solution is then centrifuged at 10k ⁇ m in a Beckman JS 13.1 swing-bucket rotor at 4°C. The pellet is washed once in Tris-HCI (pH 8.0), 100 mM NaCl and resuspended in this solution for protein determination by BioRad (Richmond, Ca.) assay.
  • E. coli cell pellets (10g) are suspended in 50mM Tris-HCI pH 8.5, 5mM EDTA, 1mM AEBSF (buffer A). Lysozyme is added to a final concentration of 300mg/ml and the lysate is incubated on ice for 30 minutes. The lysate is homogenized on ice and then centrifuged at 10,000Xg for 30 minutes. The resulting pellet is washed 2X by centrifugation with buffer A containing 0.5% Triton X-100 and the supernatants discarded.
  • buffer A containing 0.5% Triton X-100
  • the final pellet containing chimeric IL-3/mlL-6 inclusion bodies is resuspended in 50mM Tris- HCI pH 8.5, 6M guanidine-HCI, 1mM EDTA, 5mM DTT, 0.1 mM AEBSF and incubated at room temperature for 2 hours.
  • the extract is then clarified by centrifugation at 15,000Xg for 1hr.
  • the solubilized IL-3/mlL-6 is refolded by diluting the extract ten fold with 50mM Tris-HCI pH 8.5, 100mM NaCl, 1mM EDTA, 0.1 mM AEBSF and incubating for 36hrs at 4°C.
  • the protein concentration during refolding is ⁇ 0.2mg/ml.
  • Insoluble material is removed by centrifugation and the supernatant dialyzed against 20mM Tris-HCI pH 8.5, 1mM EDTA, 0.1 mM DTT.
  • Dialyzed IL-3/mlL-6 is applied to a Q-Sepharose HP (Pharmacia LKB, Piscataway, N.J.) anion exchange column (1.6 X 10cm) equilibrated in 20mM Tris-HCI pH 8.5 and eluted with a linear gradient of 500mM NaCl.
  • Fractions containing the chimeric IL-3/mlL-6 are identified by ELISA, pooled and loaded onto a C4 reverse-phase column (Vydac C4, 4.6mm X 250mm) equilibrated in 100mM ammonium acetate (pH 6.0):isopropanol(85:15).
  • the IL-3/mlL-6 is eluted with a linear gradient of 100mM ammonium acetate (pH 6.0):isopropanol (18:82) over 80 minutes at a flow rate of 0.7mi/min. Fractions containing purified IL-3/mlL-6 are pooled and stored at -70°C.
  • ATC GAT AAA CAA ATT CGG TAC
  • AGA 527 lie Asp Lys Gin He Arg Tyr He Leu Asp Gly He Ser Ala Leu Arg
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • ATC CTG ATG GAA AAC AAC CTG CGT CGA CCG AAC CTG GAA GCA TTC AAC 191 lie Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn 50 55 60
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:11 :

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Abstract

Cette invention se rapporte à une protéine chimère comprenant une portion amino possédant la séquence d'acides aminés de l'interleukine 3 et une portion carboxy possédant la séquence d'acides aminés d'une mutéine de l'interleukine 6.
PCT/US1994/004208 1994-04-15 1994-04-15 Lymphokine chimere composee de l'interleukine 3 et d'une muteine de l'interleukine 6 WO1995028427A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US1994/004208 WO1995028427A1 (fr) 1994-04-15 1994-04-15 Lymphokine chimere composee de l'interleukine 3 et d'une muteine de l'interleukine 6

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1994/004208 WO1995028427A1 (fr) 1994-04-15 1994-04-15 Lymphokine chimere composee de l'interleukine 3 et d'une muteine de l'interleukine 6

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WO1995028427A1 true WO1995028427A1 (fr) 1995-10-26

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Publication number Priority date Publication date Assignee Title
US6617135B1 (en) 1999-08-09 2003-09-09 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents

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JOURNAL OF EXPERIMENTAL MEDICINE, Volume 170, issued August 1989, BERGUI et al., "Interleukin 3 and Interleukin 6 Synergistically Promote the Proliferation and Differentiation of Malignant Plasma Cell Precursors in Multiple Myeloma", pages 613-618. *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7576193B2 (en) 1997-12-08 2009-08-18 Merck Patent Gmbh Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7226998B2 (en) 1997-12-08 2007-06-05 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7582288B2 (en) 1999-08-09 2009-09-01 Merck Patent Gmbh Methods of targeting multiple cytokines
US6617135B1 (en) 1999-08-09 2003-09-09 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7141651B2 (en) 1999-08-09 2006-11-28 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7507406B2 (en) 2000-02-11 2009-03-24 Emd Serono Research Center, Inc. Enhancing the circulating half-life of antibody-based fusion proteins
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7601814B2 (en) 2001-03-30 2009-10-13 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US8926973B2 (en) 2001-03-30 2015-01-06 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US7459538B2 (en) 2001-05-03 2008-12-02 Merck Patent Gmbh Recombinant tumor specific antibody and use thereof
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7462350B2 (en) 2001-12-04 2008-12-09 Emd Serono Research Center, Inc. Cancer treatments including administering IL-2 fusion proteins with modulated selectivity
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof

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