WO1990006370A1 - Interleucine-6 synthetique - Google Patents

Interleucine-6 synthetique Download PDF

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Publication number
WO1990006370A1
WO1990006370A1 PCT/US1989/005421 US8905421W WO9006370A1 WO 1990006370 A1 WO1990006370 A1 WO 1990006370A1 US 8905421 W US8905421 W US 8905421W WO 9006370 A1 WO9006370 A1 WO 9006370A1
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peptide
protein
recombinant
gene
vector
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PCT/US1989/005421
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English (en)
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Dana M. Fowlkes
Charles T. Tackney
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The Trustees Of The University Of North Carolina
Imclone Systems, Inc.
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Priority to KR1019900701677A priority Critical patent/KR910700341A/ko
Publication of WO1990006370A1 publication Critical patent/WO1990006370A1/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]
    • C07K14/5412—IL-6
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to genes and their encoded proteins which are recombinant mature interleukin-6 (hereinafter IL-6) and a synthetic cysteine-free protein that retains IL-6 activity. These proteins are expressed in unicellular hosts as the amino or carboxy terminal peptide portion of a tri-hybrid fusion protein comprising either interleukin-6 or its modified synthetic form, together with a portion of a cleavage site and carrier DNA.
  • IL-6 recombinant mature interleukin-6
  • cysteine-free protein that retains IL-6 activity.
  • recombinant fusion protein is purified and digested in vitro with a protease that is specific for the cleavage site to liberate the peptide with IL-6 activity, which is easily separated from the large ⁇ -galactosidase protein expressed by the carrier DNA.
  • the purified peptide can be used to stimulate the production of proteins, including
  • immunoglobulins and hepatic proteins may be used to prevent viral infections.
  • the protein can also be added to vaccine preparations as an adjuvant.
  • Physiological agents that regulate metabolic activity of distant cells were given the name hormone by English scientists Bayliss and Starlinger in 1909. These agents consist of amino acid derivatives, steroids and peptides. More recently, a variety of peptides that activate and/or inhibit cell proliferation have been identified and termed stimulatory factors or growth factors. An alternative general term for such cellular factors is cytokine although more specific terminology indicates the cell of origin, i.e. lymphokines which are produced by lymphocytes. Lymphokines also belong to the interleukin class of molecules that modulate the proliferation of cells in the immune system. Other peptides produced by the immune system result in specific antiviral activities; such peptides are termed interferons. To qualify as an interferon a factor must be a protein which exerts antiviral activity through cellular metabolic processes involving the synthesis of both RNA and protein (Committee on Interferon Nonmenclature, 1980, Nature 86(2):110).
  • Interleukin-6 is the term given to a peptide described alternatively as IFNßS2A, BSF-2, HSF, G-CSF, CS- 309, HPGF, or 26 kDa protein by a multiplicity of
  • IL-6 interferon- ⁇ 2 (Zilberstein et al., 1986, EMBO J. 5:2529) or 26 kDa protein (Haegeman,et al., 1986, Eur. J. Biochem. 159:625) which was first detected in poly(rl) poly(rC) stimulated fibroblasts; 2) a potent T-cell derived lymphokine termed B-cell stimulation factor-2 (BSF-2)
  • HGF hepatocyte stimulating factor
  • IL-6 The functions which have been ascribed to the IL-6 peptide are basic to both the inflammatory and immune response in human pathology. Those functions are diverse and depend on the type of cells under examination. IL-6 is expressed in leukocytes, epithelial cells, IL-I treated fibroblasts, hepatocytes, vascular endothelial cells, cardiac myxoma tissue, certain bladder carcinomas, certain cervical cancer cells and glial cells. IL-6 is one of the peptides involved in the interaction of T cells with B cells to result in the proliferation and differentiation of antibody producing cells. IL-6 significantly enhances secretion of immunoglobulins in activated B-cells (Muraguchi et al., 1988, J. Exp. Med. 167 (2):332; Tosato et al., 1988, Science 239:502; Hirano et al., 1985, Proc. Nat:1. Acad.
  • IL-6 appears to suppress the action of TNF (Kohase et al., 1987, Mol. Cell Biol. 7 : 213 ) . However, it stimulates the growth of human B-lymphoblastoid cells infected with EBV (Tosato et al., 1988, Science 239:502), and of human
  • cycloheximide and actinomycin D produced a 14S mRNA molecule which coded for a protein capable of inducing antiviral activity called IFN ⁇ 52 (Weissenbach et al., 1980, Proc. Natl. Acad. Sci. 77:7152; British Patent No. 2,063,882).
  • IFN ⁇ 52 a protein capable of inducing antiviral activity
  • Clones produced from cDNA copies of this induced mRNA fraction provided a partial sequence of the IFNß2 promoter that was clearly different from IFNß1, IFN7 ⁇ A, IFN7 ⁇ , and IFN ⁇ D
  • Natl, Acad. Sci. 83 :8957 also defined the 212 amino acid protein sequence using a full length cDNA clone rather than the partial cDNA clones of Zilberstein et al., 1986, EMBO J. 5:2529.
  • IFN ⁇ 2 was induced by TNF.
  • cycloheximide or interleukin-1 was identical to the IFNß2 of Zilberstein et al., 1986, EMBO J. 5:2529. Because the 5' terminus of the protein was missing in the cDNA clone collection, a screen of a human gene library, testing for complementarity with an internal cDNA sequence of the 26 kDa peptide, yielded genomic clones that provided the complete 212 amino acid sequence, as well as the DNA sequence of a 162 bp intron in the 5' terminus region of the human gene. A search of a protein data base containing 3309 individual peptide sequences failed to reveal any significant
  • BSF-2 protein was purified from a human T-cell line that constitutively produced the factor and was established using the HTLV-1 virus.
  • the amino acid sequence data from nine peptide fragments provided the information necessary to produce synthetic oligomers which were used to probe cDNA libraries.
  • the cDNA clones were sequenced as was the amino terminus of the purified IL-6 protein.
  • the end of the mature protein sequence was pro-val-pro-pro indicating that the 212 amino acid prepeptide that was predicted from the nucleic acid sequence contained a 28 amino acid long signal peptide which is cleaved to produce the natural mature BSF-2 (IL-6) protein.
  • BSF-2 (IL-6) genomic DNA demonstrated that the chromosomal segment contained five exons and four introns (Yasukawa et al., 1987, EMBO J. 6:2939).
  • the organization of the BSF-2 (IL-6) gene was strikingly similar to that of G-CSF when the two genes were compared.
  • cysteines of HP-1, IL-6 and G-CSF are cysteines of HP-1, IL-6 and G-CSF and noted that the
  • cysteine positions were conserved within the three peptides .
  • cysteines in a protein can cause problems in processing when the protein is being produced recombinantly in a bacterial host. Microbially produced cysteine-containing proteins may tend to form multimers which greatly complicate purification of the protein
  • cytokines such as IL-6
  • cysteines of IL-6 have been conserved (See above).
  • cysteines have been reported to be affected if cysteines are removed. For example, only one cysteine residue out of three can be removed without affecting the biological activity of the IFN- ⁇ (see U.S. Pat. No. 4,737,462). Similarly, only one of four cysteines of IFN ⁇ 1 can be removed without complete loss of activity (Mark et al., 1984, Proc. Natl. Acad. Sci. 81: 5662).
  • Recombinant DNA technology involves insertion of specific DNA sequences into a DNA vehicle (vector) to form a recombinant DNA molecule which is capable of replication in a host cell.
  • a DNA vehicle vector
  • the inserted DNA sequence is foreign to the recipient DNA vehicle, i.e., the inserted DNA sequence and the DNA vector are derived from organisms which do not exchange genetic information in nature, or the inserted DNA sequence may be wholly or partially
  • the recombinant DNA molecule must be compatible with the host cell, i.e., capable of autonomous replication in the host cell or stably integrated into one or more of the host cells chromosomes.
  • the recombinant DNA molecule should preferably also have a marker function which allows the selection of the desired recombinant DNA molecule (s).
  • s the desired recombinant DNA molecule
  • the foreign gene will be properly expressed in, e.g., the transformed bacterial cells, in the case of bacterial expression plasmids, or in permissive cell lines or hosts infected with a recombinant virus or carrying a recombinant plasmid having the appropriate origin of replication.
  • RNA transcription and processing events control levels of gene expression such as DNA transcription and messenger RNA (mRNA) translation.
  • Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and
  • procaryotic promoters are not recognized and do not function in eucaryotic cells.
  • ribosomal RNA complementary to the 3' end of the 16S rRNA (ribosomal RNA), and probably promote binding of mRNA to ribosomes by
  • Expression vectors have been used to express genes under the control of an active promoter in a suitable host, and to increase protein production.
  • the entire IFN/32A cDNA sequence and some adjacent nucleotides of the original cloning vector followed the SV40 early promoter and the first 60
  • T antigen RNA nucleotides of the T antigen RNA.
  • the IFN ⁇ 2A sequence was followed by the T antigen splicing region and
  • plasmid constructs wherein the entire IFN ⁇ 2A cDNA sequence was fused to the T7 RNA polymerase promoter, also were transcribed in vitro to produce mRNA that was subsequently translated in rabbit reticulocyte lysates. Following immunoprecipitation of the lysate, the proper sized IFN ⁇ 2 (IL-6) protein could be detected on SDS-polyacrylamide gels.
  • BSF-2 (IL-6) was functionally expressed in COS7 cells following transfection of a plasmid containing the entire coding region of BSF-2 adjacent to the SV40 early promoter (Hirano et al., 1986, Nature 324:73). BSF-2 activity could be detected after purification and concentration of the media from the transfected cells using immunoaffinity gel methods. Recombinant IL-6 produced using this method was used to analyze the regulation of fibrinogen and albumin mRNA in FAO cells (Andus et al., 1987, FEBS Letts. 221: 18).
  • IL-6 CDNA sequence was ligated into the p91023B plasmid which contains the SV40 enhancer, major adenovirus late promoter, DHFR coding sequence, SV40 late message poly-A addition site and the VA I gene.
  • IL-6 hematopoietic stimulating activity could be recovered at a 10-4 dilution of conditioned tissue culture cell medium.
  • interleukin-2 interleukin-2 (Takai et al., 1988, J. Immunol. 140:508).
  • Alternative methods of higher cell production of recombinant IL-6 include in vitro synthesis following injection of IL-6 mRNA into Xenopus oocytes (Weissenbach et al., 1980, Proc. Natl. Acad. Sci. 77 : 7152 ; Coulie et al., 1987, Eur. J.
  • pAL- Sec-IL6-181 was constructed to produce IL-6 by coupling the cDNA sequence of IL-6 to a synthetic signal peptide leader sequence under P L promoter control.
  • IL-6 was isolated from the periplasm of the transformed cells as a homogeneous protein from which the signal peptide had been removed. No yield of purified protein was reported.
  • Recombinant BSF-2 (IL-6) was produced in E . coli using pTBCDF-12 (Hirano et al . , 1988 , Immunol . Letters 17:41). Induction of this plasmid resulted in the production of a fusion protein in which the recombinant IL-6 peptide was fused to the IL-2 peptide. Protease digestions using
  • Kallikrein and amino peptidase-P were required to obtain mature IL-6 protein. However, the details of the procedure other than this nonenabling description were to be published elsewhere (see their manuscript in preparation citation).
  • the recombinant fused IL-6 protein was injected into rabbits to produce a polyclonal antiserum.
  • the polyclonal antiserum was used to characterize the natural IL-6 protein in fibroblastic FS4 cells following TNF and cycloheximide induction of IL-6 or in human monocytes.Incubation of the antiserum with the FS4 cell medium
  • IL-6 in bacteria exist (see Section 2.5.3), production of high yields of the natural protein in bacteria has not been successful even using recombinant DNA bioengineering methods
  • the unsuccessful Asagoe expression plasmid contained the mature processed BSF-2 (IL-6) protein coding sequence adjacent to and in frame with the P TAC promoter and ATG start sequence. Although the plasmid was analyzed in a variety of strain backgrounds, IL-6 protein was not detected following
  • oligonucleotide sequence producing a Xa factor peptide recognition site (ile-glu-gly-arg); and 3) the-Xa factor recognition site was then fused to either a glu-phe-met- BSF-2 sequence or an ala-BSF-2 coding sequence.
  • This recombinant IL-6 (or BSF-2) peptide was thus the carboxy terminal portion of the human growth hormone/Xa factor sequence/BSF-2 hybrid fusion product in this
  • the recombinant IL-6 activity existing either as glu-phe-met-BSF-2 peptide in one plasmid construct or ala- BSF-2 peptide in the other, could be purified only after solubilization of the fusion protein in 8M urea.
  • the Xa factor cleavage produced a heterogeneous mixture containing intact fusion protein, recombinant IL-6 peptides, and human growth hormone peptide, in addition to other partial cleavage products.
  • the Xa factor/cleavage mixture first had to be denatured with 6M guanidinium hydrochloride. After chromatographical purification of the recombinant IL-6 peptide, it was subjected to an additional round of extensive dialysis in order to recover a refolded active peptide.
  • the resultant recombinant peptides either glu-phe-met-BSF-2 or ala-BSF-2, were examined only for their activity in a B-cell stimulatory factor assay.
  • the present invention provides DNA
  • the invention also provides a cysteine-free form of IL-6 which retains the biological activity of the native IL-6 molecule.
  • the present invention is directed to the economical production of recombinant synthetic cysteine-free IL-6 proteins produced by bacteria as well as active peptide fragments of each of the proteins.
  • the proteins produced are either cysteine-containing or cysteine-free, and retain IL-6 biological activity.
  • Bacterial cultures express either recombinant protein as a high percentage, generally at least 20%, of total soluble cytosol protein.
  • the recombinant IL-6 protein does not require treatment with harsh denaturing agents like 8M urea, or ⁇ -mercaptoethanol to solubilize the protein from a pellet.
  • the phrase "substantially soluble in water” refers to this property.
  • the IL-6 is produced
  • the fusion protein comprises first, second and third peptide portions.
  • the first peptide portion has IL-6 activity;
  • the second peptide portion is a chemically or enzymatically cleavable sequence which links the first peptide portion to the third peptide portion;
  • the third peptide portion is a protein or portion thereof which is capable of being expressed by the unicellular host, and which preferably has a detectable function.
  • the three peptide portions are referred to as "first", “second” or “third” only for convenience, and should not be read as requiring a specific order, relative to the promoter
  • the positions of the first and third peptide portions are interchangeable .
  • the third peptide portion provides a protein or portion thereof which the unicellular host cell can make.
  • the presence of the carrier DNA that expresses the third peptide facilitates production of the eucaryotic IL-6 protein.
  • the third peptide portion also provides a means by which transformed clones producing the fusion protein' can be readily identified and isolated.
  • the first peptide portion, which possesses the desired biological activity, is
  • the recombinant IL-6 peptide is then separated and purified from the protein mixture by methods known in the art such as by high
  • HPLC performance liquid chromatography
  • inventions also -contemplates nucleotide seuuences encoding the fusion protein, recombinant vectors comprising these nucleotide sequences, as well as unicellular hosts
  • a synthetic peptide having IL-6 activity is produced with all four cysteines of the native IL-6 sequence replaced by serine residues.
  • synthetic cysteine-free IL-6 or recombinant cysteine-free IL-6 is used in the
  • the cysteine-free form of IL-6 has been shown to exhibit hepatocyte activation and B-cell stimulation.
  • the proteins of the present invention may have antiviral activity and may prevent viral infection of cells.
  • the cysteine-free synthetic protein may be nonpyrogenic or at least
  • the proteins of the present invention also include different cysteine-free and cysteine-containing.
  • IL-6 peptides which have deleted up to 27 amino acid residues from the IL-6 amino terminus, and/or which add up to 50 amino acid residues on the amino terminus and/or up to 350 amino acids on the carboxy terminus of the IL-6 sequence, and retain the
  • the basic IL-6 sequence can be modified substantially without any
  • the invention therefore encompasses the gene sequences encoding the cysteine-free peptides, as well as truncated and extended cysteine-free or cysteine-containing peptides which retain IL-6 activity.
  • the present method represents an improvement over known recombinant methods for producing IL-6 in that it provides means for producing IL-6 in commercial quantities in a recombinant/vector system.
  • Previous IL-6 fusion protein constructs such as the one described by Asagoe, supra, have not been successful in obtaining expression of the protein in large quantities, and also require extensive harsh conditions
  • the peptides produced by culturing these recombinant hosts retain characteristic IL-6 activity in their
  • the present invention also contemplates methods of treatment of viral disease, immunodeficiencies and hepatic disorders, as well as overall modulation of immune response, by administration of the claimed synthetic IL-6 peptide.
  • BSF-2 B-cell Stimulation Factor
  • G-CSF Granulocyte Colony Stimulating
  • HGF Hepatocyte Growth Factor
  • HPGF Hybridoma Plamacytoma Growth Factor
  • HSF Hepatocyte Stimulating Factor
  • IGF Insulin-Like Growth Factor IgG
  • IgM Immunoglobulin G, M
  • IL-6 Interleukin 1 , 3 , or 6
  • mRNA messenger RNA
  • PBS Phosphate buffered saline
  • PDGF Platelet Derived Growth Factor
  • TN-F Tumor Necrosis factor
  • YT Yeast-tryptone growth media
  • FIG. 1 Diagram of the construction of p360.
  • the plasmid p360 is a plasmid expression vector constructed to express the cysteine free synthetic IL-6 like peptide as a protein of approximately 22-23 KDa in E. coli.
  • Oligonucleotide 737 [AGC TGA TTA AAT AAG GAG GAA TAA CCA TGG CTG CA 3'] and 738 [GCC ATG GTT ATT CCT CCT TAT TTA ATC 3'] were fused and replaced the Hind III-Pst I fragment of pBS (+), a phagemid purchased from Stratagene Systems, to form p350.
  • inducible lac promoter through the minicistron sequence and through the synthetic cysteine free IL-6 like sequence fails to produce a peptide of the size of IL-6 in extracts of E. coli (see Figure 3 for gel of expressed proteins).
  • FIG. 2 Diagram of the construction of p369.
  • the fragment containing the sequence for the synthetic cysteine free IL-6 like peptide of plasmid p365, which contains no peptide terminating codon is this construction, replaced the Nco I-Bam HI fragment of p350 (described in Figure 1) to form plasmid p369 wherein the synthetic cysteine free IL-6 like peptide sequence was fused in frame to the alpha- complementing fragment of ⁇ -galactosidase.
  • FIG. 3 Expression and nonexpression of plasmid constructs containing sequences for the synthetic cysteine free IL-6 peptide.
  • Cultures of E. coli cells carrying different plasmids were induced with IPTG. Samples of cell cultures were lysed and electrophoresed in SDS-polyacrylamide gels. The gels were stained with coomasie blue to visualize proteins. Lanes a and b are duplicate aliquots from
  • Lanes 1a and b are from cells bearing plasmid p369.
  • the expected induction protein should be a fusion protein of the alpha-complementing portion of ⁇ -galactosidase and the synthetic IL-6 like peptide. No large molecular weight protein representing the fusion is present.
  • Lanes 2a and b are from cells bearing plasmid p367, the successful expression vector for the fusion product of the synthetic IL-6 like peptide/collagen/ ⁇ -galactosidase. Note the large amount of high molecular weight fusion protein in the approximately 130 kDA position of the gel.
  • Lanes: 3a and b are from cells bearing plasmid p360 which were expected to produce a peptide of 22-23 ⁇ Da, the size of deglycosylated IL-6.
  • the arrowhead marks the position of the expected small peptide.
  • FIG. 4 Diagram of the construction of p340-1. The steps in the construction of p340-1 are described in Section 5.2.
  • Oligonucleotide 237 was [AAT TCT AAT ACG ACT CAC TAT AGG GTA AGG AGG TTT AAC CAT GGA GAT CTG 3'].
  • Oligonucleotide 238 was [GAT CCA GAT CTC CAT GGT TAA ACC TCC TTA CCC TAT AGT GAG TCG TAT TAG 3']
  • Oligonucleotide 703 was [CAT GTA TCG ATT AAA TAA GGA GGA ATA ACC 3'].
  • Oligonucleotide 704 was [CAT GTA TCG ATT AAA TAA GGA GGA ATA ACC 3'].
  • Oligonucleotide 704 was [CAT
  • P T RC is the hybrid promoter tryptophan/lac. "Term" represents the terminating codon that is in the same reading frame as the preceeding initiating methionine codon.
  • ⁇ P R represents the rightward promoter, from bacteriophage lambda. Amp r represents the ampicillin resistance marker from pBR322. Ori represents the plasmid origin of DNA replication from pBR322. ⁇ -gal
  • FIG. 5 Complete nucleotide sequence (5'-3') of the synthetic recombinant IL-6 gene and its predicted amino acid sequence.
  • the coding sequence of the fused gene begins at nucleotide 3 (Met).
  • recombinant IL-6 protein extends to nucleotide 557 (Met) where it.is fused to the collagen linker of the fusion protein via a serine residue.
  • the asterisk designates the location of the TAG stop codon normally found in the native cDNA sequence. Modified amino acid residues are shown in script below the sequence.
  • FIG. 6 Assembly of the gene for the synthetic
  • Construct p334 was composed of three pairs of annealed oligonucleotides joined at residues 162-167 and 220-225.
  • Complementary overhangs for the assembly of p331 were located at nucleotides 312-315 and 358-361.
  • Construct p332 was ligated at positions 457-461 and 512-517.
  • the synthetic fragments were cloned into a modified pBS M13+ Stratogene vector by insertion into the multiple cloning site of the circular vector via synthetic 4-6 base overhangs (represented by the hatched boxes).
  • the coding sequence for the synthetic cysteine-free IL-6 like gene is shown in black. Open boxes represent the modified pBS M13+ sequences.
  • Fragments were assembled by digesting with appropriate enzymes, purifying the desired bands by electroelution from agarose gels and religating. Removal of the termination codon in p337 was accomplished by substituting a second synthetic fragment containing a serine residue and a
  • Nucleotide 564 identifies the position of the last base contained in the Bgl II site required to fuse the insert in p365 with the collagen linker of the expression vector. Restriction sites are identified as follows: B, Bam HI; Bg, Bgl II; RI, Eco RI; RV, Eco RV; H, HinD III; N, Nco I; S, Stu I; X, Xba I.
  • FIG. 7 Diagram showing the construction of plasmid p367, the successful expression vector for producing the synthetic cysteine-free IL-6 peptide. The steps in the construction of p367 are described in sections 5.2-5.5 in the text.
  • FIG. 8a Plasmid map of recombinant plasmid p367 showing the location of the IL-6 sequence (designated HSF).
  • the assembled synthetic cysteine-free recombinant IL-6 gene contained in an (Nco I - Bgl II) insert was introduced between the Nco I and Bam H I restriction sites of the vector.
  • the open box (C) indicates the location of the 60 amino acid collagen linker through which recombinant IL-6 is fused to ⁇ - galactosidase. Expression of the complete fusion gene produces a 130 kDa hybrid protein.
  • FIG. 8b Plasmid map of recombinant plasmid p367 showing the location of the sequence for the synthetic cysteirie-free IL-6 like peptide (designated HSF).
  • the vector shown is a significantly modified version of pJG200 as describing in the application in section 5.
  • the assembled synthetic IL-6 like gene contained in an (Nco I-BGl II) insert was introduced between the Nco I and Bam HI
  • the open box (C) indicates the location of the 60 amino acid collagen linker through which the synthetic IL-6 like peptide is fused to ⁇ - galactosidase. Expression of the complete fusion gene produces a hybrid protein of approximately 130 kDa (see
  • FIG. 9 NaDodSO 4 -PAGE analysis of synthetic cysteine- free IL-6 protein purification. Samples collected at various stages of purification were electrophoresed on 10%
  • polyacrylamide- NaDodSO 4 gels under reducing -conditions as shown. Protein molecular weight markers are shown in lanes M. E. coli cells grown in 10 L batch culture were lysed by addition of lysozyme followed by brief sonication (Lane 1). The unusually hydrophobic fusion protein was purified by repeated ammonium sulfate precipitation after solubilization in PBS- Sarkosyl (Lane 2). Partially purified fusion protein was then digested with collagenase to release the 23 kDa recombinant IL-6 moiety (Lane 3). After removing the
  • FIG. 10 Stimulation of fibrinogen sythesis by
  • Confluent FAZA cell monolayers were treated with varying concentrations of purified recombinant cysteine-free IL-6 in the presence of 10 -7 M dexamethasone. After five hours of stimulation, cell medium was recovered and filtered through a nitrocellulose membrane using a dot-blot apparatus. Secreted fibrinogen levels were determined by solid phase immunoassay using a polyclonal fibrinogen antiserum as primary antibody and alkaline phosphatase conjugated anti-IgG antiserum as secondary antibody. Bound antibody was visualized by adding chrom ⁇ genic substrates (BCIP and NBT) to the blots.
  • BCIP and NBT chrom ⁇ genic substrates
  • FIG. 11 B-cell differentiation assay. CH12.LX cells (2 x 10 5 cells/well) were co-cultured m the presence or absence of antigen (SRBC) prior to treatment with various concentrations of recombinant synthetic cysteine-free IL-6, as prepared according to the methods described in ⁇ 5.2 to
  • FIG. 12a and b Diagrammatic illustration of the construction of pTrpE/EK/cfIL-6. The details of the
  • FIG. 13 Graphic depiction of Time ⁇ colony assay for stimulation of progenitor cells by various growth factors and combinations thereof.
  • FIG. 14 Number of nonadherent cells after 7 days of liquid culture. This assay is described in Example 11.
  • FIG. 15. Imclone vs. Endogen IL-6 on 7td.1.
  • FIG. 16 Imclone vs. bm IL-6 on 7td.1.
  • FIG. 17 Imclone v. Genzyme IL-6 using 7td.1.
  • FIG. 18 The sequence in pTrpE/EK/cfIL-6 from the enterokinase site through the amino terminal sequence of cysteine-free IL-6 to the natural carboxy terminus of IL-6 followed by three stop codons.
  • interleukin-6 in bacterial cells resulted in the production of no detectable or commercially useful amounts of the protein.
  • One attempt to produce commercially useful amounts of protein resulted in the synthesis of plasmid p360.
  • the synthetic cysteine-free IL-6 sequences from p337-1 were inserted immediately downstream from the minicistron sequence produced by the fusion of oligo 737 [AGCTGATTAAATAAGGAGGAATAACCATGGCTGCA-3 ' ] and oligo 738 [GCCATGGTTATTCCTCCTTATTTAATC-3'].
  • the synthetic IL-6 peptide had- a termination codon and was not fused to the ⁇ -galactosidase protein.
  • Plasmid pJG200 was the starting material that was modified to produce a successful IL-6 expression vector.
  • the initial plasmid, pJG200 contained target cistrons that were fused in the correct reading frame to a marker peptide with a detectable activity via a piece of DNA that codes for a protease sensitive linker peptide (Germino and Bastia, 1984, Proc. Natl. Acad. Sci. USA 81:4692; Germino et al., 1983, Proc. Natl. Acad. Sci. USA 80 :6848).
  • the promoter in the original vector pJG200 was the P R promoter of phage lambda.
  • Adjacent to the promoter is the C I 857 thermolabile repressor, followed by the ribosome-binding site and the AUG initiator triplet of the Cro gene of phage lambda.
  • Germino and Bastia inserted a fragment containing the triple helical region of the chicken pro-2 collagen gene into the Bam HI restriction site next to the ATG initiator, to produce a vector in which the collagen sequence was fused to the lacZ ⁇ -galactosidase gene sequence in the correct translational phase.
  • a single Bam HI restriction sites was regenerated and used to insert the plasmid R6K replication initiator protein coding
  • containing plasmids with inserts are not distinguishable from strains containing the parent vector with no insert.
  • the first alteration to pJG200 in this invention was the removal and replacement of the Eco RI-Bam HI fragment that contained the P R promoter, C I 857 repressor and amino terminus of the cro protein which provided the ATG start site for the fusion proteins.
  • An oligonucleotide linker was inserted to produce the p258 plasmid, which maintained the
  • This modification provided a new ATG start codon that was out of frame with the collagen/ ⁇ -galactosidase fusion. As a result, there is no ⁇ -galactosidase activity in cells transformed with the p258 plasmid. In addition this modification removed the cro protein amino terminus so that any resultant recombinant fusion products inserted adjacent to the ATG start codon will not have' cro encoded amino acids at their amino terminus. In contrast, recombinant proteins expressed from the original pJG200 vector all have cro encoded amino acids at their amino terminus.
  • the p277 plasmid contained the P TAC (also known as P TRC ) promoter of pKK233-2, the lacZ ribosome binding site and an ATG
  • the insertion of a target protein sequence allows its transcription from an IPTG inducible promoter in an appropriate strain background.
  • the appropriate strain background provides sufficient lac
  • the p277 plasmid provides a significant commercial advantage over promoters that require temperature shifts for induction such as the P R promoter of pJG200. Induction of commercial quantities of cell cultures containing temperature inducible promoters would otherwise require heating large volumes of cells and medium to produce the temperature shift necessary for induction. For example, induction by the P promoter requires a temperature shift to inactivate the C I 857
  • the p277 expression vector was further modified by insertion of twenty-nine base pairs, namely
  • modifications designed to introduce novel restriction sites for use in joining the gene sub-fragments, were incorporated within the coding sequence in such a manner as to avoid altering the amino acid composition of the synthetic gene with respect to the native IL-6 protein sequence.
  • oliqonucleotides were assembled in separate annealing and ligation reactions to produce four sub-fragments, each representing approximately one fourth of the recombinant synthetic cysteine-free IL-6 coding sequence.
  • a modified plasmid was constructed to allow for DNA amplification and ease in sequencing each oligomer.
  • a pBS M13+ cloning vector (Stratagene) was modified by insertion of a 28 base oligonucleotide adapter
  • the modified plasmid designated p287, no longer contains its original Sph I restriction site but encodes additional sites for Nco I, Nru I and Xho I.
  • the synthetic oligomers were separately cloned into the modified pBS M13+ vector p287 to allow DNA amplification and sequence verification by dideoxy-nucleotide sequencing.
  • Insertion of the assembled fragments into the modified vector produced recombinant plasmids p333, p334, p331, and p332, each containing a portion of the synthetic cysteine-free IL-6 protein coding region proceeding from amino to carboxy terminus respectively. Following ligation, each plasmid DNA was transformed separately into competent E. coli JM101.
  • inserts were subsequently combined to yield the entire coding sequence of the recombinant synthetic cysteine-free IL-6 gene.
  • the insert released from plasmid p335 was ligated into p336.
  • the resulting plasmid p365 contained the complete coding sequence of synthetic IL-6 inserted between the Nco I and Eco RI sites of the modified pBS M13+ vector p287.
  • the constructions are shown diagrammatically in Figure 6.
  • the assembled cysteine-free recombinant IL-6 gene was excised from p365, and inserted into plasmid p340 between the Nco I site, which encompasses the initiating methionine, and the BamH I site adjacent to the collagen linker as depicted in Figure 7.
  • the resulting vector p367 diagrammed in Figure 8 but not to scale, was used to transform E. coli JM101.
  • Recombinant colonies were selected on the basis of antibiotic resistance and by appearance of blue coloration in the presence of X-Gal.
  • the size of the insert DNA was confirmed by mini-lysate extraction followed by polyacrylamide gel electrophoresis.
  • a tripartite fusion protein composed of synthetic cysteine-free IL-6, a sixty amino acid collagen linker and ⁇ -galactosidase was produced in transformed bacteria (See Figure 3 and Figure 9).
  • ampicillin resistant transformants carrying the modified IL-6 expression plasmid produced blue colonies upon addition of the inducing agent IPTG and the chromogenic ⁇ -galactosidase substrate X-Gal.
  • Synthesis of the fusion protein by IPTG- induced transformants was independently confirmed by western blot analysis of a total E.coli lysate using a monoclonal anti- ⁇ -galactosidase antibody obtained from Promega Biotec.
  • the monoclonal anti- ⁇ -galactosidase antibody used in the Western blot recognized a band with an apparent molecular weight of 130,000 kDa.
  • E. coli cell pellets were processed in aliquots of 100 g (wet weight) by washing with TNS buffer (30 mM
  • TNS Tris.Cl, pH 7.4; 30 mM NaCl, 0.05% sodium lauroyl sarcosine). Washed cells were lysed in 450 ml TNS containing 1.5 mM EDTA and 0.5 mg/ml lysozyme. After incubating the suspension on ice for 30 minutes, complete lysis was ensured by subjecting cells to three cycles of freeze-thawing and brief sonication. Soluble proteins, devoid of ⁇ -galactosidase activity, were removed by three repeated washings in TNS followed by
  • the recombinant synthetic cysteine-free IL-6 protein was purified to homogeneity using reverse phase HPLC.
  • Thawed extract (4 mis) was sonicated briefly to disperse aggregates, added to pre- treated collagenase, and incubated for 45 minutes at room temperature.
  • the majority of the cleaved ⁇ - galactosidase was removed by adding 0.5 volumes of saturated ammonium sulfate, incubating on ice for 30 minutes and pelleting the insoluble material.
  • the cleaved recombinant cysteine-free IL-6 was concentrated by bringing the total volume of the supernatant to 13 mis with saturated ammonium sulfate, incubating on ice for 30 minutes, and centrifuging to compact the insoluble protein into a floating pellicle. Liquid was drained by puncturing the tube, and the remaining pellicle was resuspended in 0.5 mis of Tris buffered saline.
  • the resuspended recombinant cysteine-free IL-6 was prepared for reverse phase HPLC by adding an equal volume of 60% acetonitrile, 0.1% trifluroacetic acid. Insoluble material was pelleted and the clarified supernatant was loaded onto a 250 mm, 4.6 mm ID reverse phase column (Vydac, 218Tp, C18, 10 ⁇ m- Alltech Associates) in an injection volume of 0.5 to 1 ml.
  • the mobile phase consisted of varying concentrations of solvent B (60% acetonitrile and 0.1% triflurooacetic acid) relative to solvent A (0.1
  • HPLC- purified material was subjected to direct N-terminal automated protein sequencing.
  • MVPPGED identified after seven cycles of sequential degradation coincided with the predicted N-terminal amino acid composition of the recombinant protein as deduced from the synthetic recombinant cysteine-free IL-6 gene.
  • the isolated active fraction consisted of a mixture of amino terminal methionine- containing and amino terminal methionine-free synthetic cysteine-free IL-6 proteins. Amino acid sequence analysis indicates that in one preparation of the mixture 90% of the mixture was methionine-free at the amino terminus and 10% contained an amino terminal methionine.
  • the active preparation of the cysteine-free protein varies from natural IL-6 in that 1) no intramolecular disulfide bonds occur; 2) a methionine amino acid at position one in the bioengineered protein replaces the signal sequence amino acids 1-28 of the natural unprocessed protein; 3) amino acid two in the bioengineered protein, glycine, replaces the proline amino acid present in the natural IL-6 protein; 4) the carboxy terminus contains additional amino acids.
  • Collagenase generally cleaves after Y in the sequence P-Y-G-P wherein Y represents a neutral amino acid. See Keil et al. FEBS Letters 56: 292-296 (1975).
  • the neutral amino represented by Y is valine.
  • the carboxy terminus of the protein produced by induction of the fusion protein coded by the p367 vector after digestion of the fusion protein with collagenase is expected to be mainly . . . P-G-P-V-G-P-V and/or . . . P-G-P-V. If sufficient
  • the carboxy terminus is exclusively . . . P-G-P-V. If the amino acid sequence starting with the third amino acid (i.e. V) in Figure 5 and ending with the
  • pep fourteenth amino acid from the end (i.e. M) is called pep, the following peptides are contemplated by the present invention:
  • a promoter is a region of DNA at which RNA polymerase attaches and initiates transcription.
  • promoter selected may be any one which has been isolated from the host cell organism.
  • E. coli a commonly used host system, has numerous promoters such as the lac or recA promoter associated with it, its bacteriophages or its plasmids.
  • synthetic or recombinantly produced promoters such as the ⁇ phage P L and P R promoters may be used to direct high level production of the segments of DNA adjacent to it. Similar promoters have also been identified for other bacteria, and eukaryotic cells.
  • a ribosome binding site includes the
  • SD-ATG sequences include, but are not limited to, the SD-ATG sequences of the cro gene or N gene of coliphage lambda, or the E. coli
  • tryptophane E, D, C, B or A genes tryptophane E, D, C, B or A genes.
  • DNA ligase is an enzyme which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain; this enzyme may therefore be used to covantly join the annealed cohesive ends produced by certain restriction enzymes.
  • DNA ligase can be used to catalyze the formation of phosphodiester bonds between blunt-ended fragments.
  • the enzyme terminal, deoxynucleotidyl transferase may be employed to form
  • oligo (dA) sequences to the 3' end of one population, and oligo (dT) blocks to 3 ' ends of a second population, the two types of molecules can anneal to form dimeric circles.
  • Any of these methods may be used to ligate the control elements into specific sites in the vector.
  • the sequence coding for the cysteine-free or cysteine-containing IL-6 fusion protein is ligated into the chosen vector in a specific relationship to the vector promoter and control elements, so that the sequence is in the correct reading frame with respect to the vector ATG
  • the vector employed will typically have a marker function, such as ampicillin resistance or tetracycline resistance, so that transformed cells can be identified.
  • the method employed may be any of the known expression vectors or their derivatives; among the most frequently used are plasmid vectors such as pBR 322, pAC 105, pVA 5, pACYC 177, pKH 47, pACYC 184, pUB 110, pmB9 , pBR325, col El, pSC101, pBR313, pML21, RSF2124, pCR1 or Rp4; bacteriophage vectors such as lambda gt11, lambda gt-WES-lambda B, chain 28, chain 4, lambda gt-I-lambda BC, lambda-gt-1 lambda B, Ml3mp7, M13mp8, M13mp9; SV40 and adenovirus vectors; and yeast vectors.
  • the vector is selected for its compatibility with the chosen host cell system.
  • bacteria particularly E. coli, have proven very useful in high yield production of the synthetic IL-6 peptide, and are the preferred host, the invention is not so limited.
  • the present method contemplates the use of any culturable unicellular organism as host; for example, eukaryotic hosts such as yeast, insect, and mammalian cells, are also .potential hosts for IL-6 production.
  • eukaryotic hosts such as yeast, insect, and mammalian cells
  • IL-6 production are also .potential hosts for IL-6 production.
  • the selection of an appropriate expression system, based on the choice of host cell, is well within the ability of the skilled artisan.
  • each of these segments may also be varied. For example, substantial variation is possible within and around the basic IL-6 peptide sequence.
  • any amino acid in the known sequence of IL-6 may be substituted with a chemically equivalent amino acid.
  • silent changes may be made in the amino acid sequence without affecting the activity of the molecule as a whole.
  • substitution of all cysteine residues with serine residues allows the modified IL-6 molecule to retain its biological activity.
  • Alternative choices as substitutes for cysteine are other neutral amino acids such as valine, proline, isoleucine and glycine, serine, threonine or tyrosine.
  • Negatively charged residues, such as aspartic acid and glutamic acid may be interchanged, as may be positively charged residues such as lysine or arginine.
  • phenylalanine, leucine, isoleucine, valine and alanine may also be exchanged. Alteration of the sequence by amino acid substitution, deletion, or addition and subsequent testing of the resultant molecule to determine if biological activity is retained is well within the ability of one skilled in the art, without necessity for undue experimentation.
  • the identity of the cleavable linker peptide sequence is also a matter of choice and may be accomplished using chemical or enzymatic means.
  • the sequence employed may be any one which can be chemically cleaved, so that the peptide with the biological activity of IL-6 can be released from the remainder of the fusion protein.
  • the cleavable sequence is one which is enzymatically
  • a collagenase-susceptible sequence is but one example.
  • Other useful sites include enterokinase- or Factor Xa-cleavable site.
  • enterokinase cleaves after the lysine in the sequence Asp-Asp-Asp-Lys.
  • Factor Xa is specific to a site having the sequence Ile-Glu-Gly-Arg, and cleaves after the arginine.
  • Another useful cleavage site is that of thrombin which recognizes the sequence Leu-Val-Pro- Arg-Gly-Ser-Pro. Thrombin cleaves between the Arg and Gly residues.
  • Other enzyme-cleavable sites will also be
  • sequence may be selected so as to contain a site cleavable by cyanogen bromide; cyanogen bromide attacks methionine
  • the IL-6 active portion is at the carboxy terminal end of the fusion protein, and the cleavage site is specific for a protease that is capable of leaving the natural pro-val-pro amino terminal peptide sequence. Examples of such cleavage sites are those that are cleaved by enterokinase or Factor Xa.
  • the identity of the third peptide sequence may also be varied.
  • This portion of the tripartite structure potentially serves two purposes: (1) the use of a correctly selected protein, capable of being expressed in the chosen host, can place the production of peptides having activity IL-6 under the control of a strong promoter, and thus facilitate the production of those peptides; and (2) it can provide a convenient means for identifying transformed clones producing the fusion protein.
  • the full sequence encoding ⁇ -galactosidase was used; this protein provides a visual means of detection by the addition of the proper substrate.
  • the third peptide portion can be a fraction of such a protein, provided that the portion
  • This portion can also be a peptide which is not necessarily visually detectable, but the presence of which may be
  • detectable by other means, such as by calculation of the expected molecular weight of the fusion protein or insertion into a vector with a detectable marker.
  • Another useful, alternative sequence for use in a prokaryotic host is the trpE gene product, or a portion thereof (Kleid et al.,
  • the quantity of production of IL-6 protein using this construct is also high, ranging from about 1-20% of total soluble cellular protein.
  • TrpE gene anthranilate synthase, followed by an enzymatic cleavage site, followed immediately by a synthetic IL-6 peptide sequence having C-terminus and N-terminus ends of the natural IL-6 peptide and all four cysteines replaced by serines, is expressed by a new recombinant plasmid
  • pTrpE/EK/cfIL6 To prepare pTrpE/EK/cfIL6, the plasmid p36 - which has been described in Section 5.5 and Figure 6 of this specification - is digested with the enzymes EcoRII (to cut the EcoRII site that is located 14 bases from the 5' NcoI site) and Bglll (to cut the Bglll site that is shown in
  • sequence B (approximately 10 ⁇ g) is digested with Hindlll and Bglll as described above at pH 7.5 in a buffer of 25 mM Tris HCl, 100 mM MgCl 2 , 10 mg/ml BSA and 2 mM BME.
  • the large (3.0 Kb) fragment that results from cutting the unique Hindlll site and the Bglll site referred to in Figure 13 as Bglll' is called sequence B.
  • sequence C is prepared and ligated to sequence A and sequence B.
  • the oligonucleotide starts with overlapping Hindlll and Bell sites, encodes a sequence of amino acids containing an enterokinase cleavage site followed immediately by the first three amino acids of natural IL-6, Pro-Val-Pro (PVP), and ends with an EcoRII site.
  • the sequence of the oligonucleotide is:
  • sequence A is coprecipitated with 200 ng of the synthetic oligonucleotide (sequence C) and ligated to the Hindlll/Bglll vector component (sequence B) of p365. Ligation is accomplished in a 20 ⁇ l reaction volume
  • pABC plasmid containing 20 mM Tris HCl, pH 7.6, 0.5 mM rATP, 10 mM MgCl 2 , 5 mM DTT at 16° overnight.
  • the new plasmid is pABC.
  • pABC is cloned by adding a 5 ⁇ l aliquot of the reaction mixture to competent HB101 bacteria. Ampicillin-resistant colonies are selected after overnight incubation at 37oC.
  • This oligonucleotide reading from left to right, starts with a Bglll site, encodes the natural amino acid sequence of IL-6 that follows the Bglll' site of p365, and concludes with a methionine residue that is followed immediately by three stop codons and a Kpnl site (sequence
  • step C is digested with Hindlll and Bglll (sequence E).
  • PATH 23 (available from A. Tzajaloff, Columbia University, New York City) is an ampicillin-resistance plasmid containing a gene that encodes the amino-terminal 337 amino acids of TrpE (anthranilate synthetase component I) adjacent, and in reading frame at its 3' end with, a
  • TrpE trpE
  • TrpE The Operon, Cold Spring Harbor Laboratory, pp. 263-302 (1978).
  • Other sources of DNA that encode all or part of trpE and lacZ are readily available. Such other sources may be found, for example, in Pouwels et al., Cloning Vectors, A Laboratory Manual, Elsevier, 1985. For example, trpE
  • sequences may be isolated from plasmids having the following identifying codes in the Pouwels et al. manual:
  • I-A-ii-3 (pDF41 and 42), I-A-iv-23 (pRK353), I-B-ii-4 (pMBL24), I-B-ii-1 (ptrpED5-1), I-D-i-3 (pEP70-pEP75), and I-D-i-4 (pEP165 and pEP168).
  • PATH 23 10 ⁇ g PATH 23 is digested with 5 units each of Hindlll and Kpnl at 37 °C for 2 hours. The large fragment is isolate by gel chromatography, followed by electro-elution and ethanol precipitation (sequence F).
  • sequence D Approximately 200 ng of sequence D are mixed with approximately 1 ⁇ g of sequence E. The resulting mixture is coprecipitated with ethanol in the presence of sequence F and ligated as described above. The resulting fragment is called pTrpE/EK/cfIL-6.
  • Competent E. coli host cells are transformed with pTrpE/EK/cfIL-6.
  • the E. coli HB101 strain is used as host cell for transformation in one embodiment.
  • Ampicillin-resistant colonies are gathered. These colonies express a fusion protein comprising a TrpE segment, an amino acid segment recognized and cleaved by enterokinase, and the synthetic cysteine-free IL-6 amino acid sequence that has the termini at both carboxy and amino ends of the natural IL-6 peptide, starting with PVP and ending with M.
  • TrpE enterokinase - cleavage site synthetic cysteine-free IL-6 fusion protein is followed, except the pEx-1 vector is substituted for PATH 23 in step f.
  • pEx-1 is digested with BamHI and Kpnl. BamHI and Bell have compatible restriction sites.
  • the resulting construct contains a thermoinducible ⁇ -galactosidase gene followed by a cloning polylinker.
  • the truncated gene produces a peptide that is approximately 48 Kd of the beta-galactosidase protein (Stanley, K.K. and Luzio, J.P., 1984, EMBO J., Vol. 3, pp. 1429-1434).
  • the resulting construct is called p ⁇ gal/EK/cfIL-6.
  • Another source of beta-galactosidase DNA includes pHg2000 described in ⁇ 5.2.1.
  • the fusion protein is produced after transformation of E.coli N4830 cells with pßgal/EK/cfIL-6 and thermoinduction. The plasmid is replicated in E.coli strain N99. N99 and N4830 are available from Pharmacia.
  • the fusion protein is cleaved by enterokinase using methods known and used in the art.
  • a factor Xa site is substituted for an enterokinase site by modifying step C of Section 5.10.1 and 2.
  • the synthetic oligonucleotide (sequence C) shown in step (C) of Section 5.10.1 comprises a DNA sequence that encodes
  • This DNA sequence is modified so as to encode the factor Xa cleavage recognition site
  • p ⁇ gal/Xa/cfIL-6 are expressed as described in ⁇ 5.10.1 and ⁇ 5.10.2.
  • the resulting fusion proteins are cleaved with factor Xa, which cleaves after the arg in its recognition site by methods known in the art. See, for example, Nagal & Thogerson, Nature 309: 810-812 (1984).
  • FAZA 967 rat hepatoma cells were grown in DMEM/F12 supplemented with 10% NuSerum (Collaborative Research), penicillin and streptomycin. Assays were performed on one day old confluent monolayers seeded in 48 well plates
  • Fibrinogen levels were determined by solid phase enzyme- linked immunoassay. Fibrinogen was detected using a 1:1000 dilution of rabbit anti-rat fibrinogen polyclonal antiserum (obtained from Dr. Gerald R. Crabtree, Stanford University). Secondary antibody was affinity purified alkaline
  • Positive controls consisted of cells treated with supernatant obtained from PMA stimulated MRC-5 fibroblasts. Quantitation of the assay was carried out by scanning laser densitometry.
  • Murine B-cell clone CH12.LX (N + , d + LY-1 + ) was grown and maintained in RPMI 1640 containing 5% heat-inactivated fetal bovine serum, 300 Ng/ml glutamine, 0.04 mM 2- mercaptoethanol and antibiotics CH12.LX cells bear surface IgM specific for the phosphatidyl choline moiety of sheep erythrocytes (SRBC).
  • SRBC sheep erythrocytes
  • the differentiation assay was performed by culturing 2 x 10 5 B-cells m the presence or absence of various
  • SRBC ASA Biological
  • the protocol for these assays is found in Figure 14. Particularly effective stimulation is observed when synthetic cysteine-free IL-6 is combined with IL-1, an also when these two cytokines are combined with either M-CSF or IL-3.
  • Tabular presentation of ⁇ values are found in Table 2 ; graphic depiction of Time ⁇ colony assays are shown in Figure 13.
  • Plasmid p478 is a plasmid construct identical to plasmid p367 except that in p478, the cysteines in the IL-6 sequence have not been replaced.
  • the IL-6 active protein fraction is the IL-6 peptide cut from the fusion protein ("p478 cut" in Table 2) with collagenase.
  • This peptide fraction for each deletion mutation tested is, therefore, a mixture of cysteine-containing IL-6 peptides having discrete amino acids added to the carboxy terminal end of the IL-6 peptide sequence.
  • the proteins tested are the fusion proteins themselves ("p478 uncut”).
  • the wild type, cysteine-containing, natural IL-6 sequence fusion protein produced by p478 (“478 uncut") retains the biological
  • 478 cut refers to the cleaned cysteine-containing IL-6 peptide that is produced from it.
  • the '478 cut eptide is the standard against which the nine deletion mutations are tested and as such, is 100% active in the test.
  • 478 cut refers to the wild type plasmid expressing cysteine-containing IL-6 fusion protem that has had the IL-6 cleaved from the fusion protein with collagenase and 478 uncut in Table 3 refers to the uncut fusion protein.
  • Mutant 1AB is a deletion mutation produced by deletion of amino acids 4 through 23 in the cysteine-containing IL-6 peptide where met is amino acid number one of the peptide sequence for the analogous IL-6 cysteine-free peptide found in Figure 8b.
  • Mutant 2AB, 3AB etc. all refer to the corresponding twenty amino acid
  • deletions at the appropriate position in the sequence as identified in Table 3 in the second column labeled amino acid residues deleted.
  • E. coli JM101 (P-L Pharmacia) was transformed as described in Hanahan, 1983, J. Mol. Biol. 166:557. Plasmid pKK233-2 was obtained from P-L Pharmacia; plasmid pBSt was from Stratogene. Other plasmid constructs are as described in this application.
  • Oligonucleotides were kinased with T4 polynucleotide kinase according to manufacturers suggestions (New England Biolabs). The kinase was inactivated by heating at 65°C.
  • Oligonucleotide mixtures were 'annealed by heating at* 85°C for 15 minutes and cooled slowly to room temperature.
  • the annealed oligonucleotides were ligated with 10 U T4 ligase, ligated products were separated on a 6% polyacrylamide gel, and the fragments were recovered by electroelution.
  • oligonucleotides were determined by the chain termination method of Sanger et al., 1977, Proc. Natl. Acad. Sci.
  • PROTEIN BLOT ANALYSIS Samples equivalent to 50 ⁇ L cell culture were run on 8% NaDodSO 4 -polyacrylamide gels under reducing conditions according to Laemlli, 1970, Nature 227:680. Gels were either stained with Coomasie blue or electroblotted onto two layers of nitrocellulose in order to have duplicate blots of the same gel. Prestained molecular weight markers (BRL) were used to monitor transfer. After being blocked with 0.25% gelatin, the blots were incubated with a commercial antibody to ⁇ -galactosidase (Promega Biotech).
  • cross reacting bands were visualized with a phosphatase-linked, affinitypurified, goat anti-mouse IgG antisera (1:7,500 dilution, Promega Biotech) using bromo-chloro-iodoyl phosphate and nitro-blue tetrazolium as recommended by Promega Biotech.
  • E. coli were harvested by centrifugation, and the cell pellets were suspended in one-fifth volume of 0.05 mol/L Tris-HCl pH8, 0.05 mol/L EDTA, 15% sucrose with freshly dissolved lysozyme at 1 mg/ml.
  • Trihybrid fusion protein was quantitated by colorimetric assay for ⁇ -galactosidase activity using 0- nitrophenyl- ⁇ -D-galactopyranoside as substrate.
  • Synthetic cysteine-free IL-6 as prepared according to the methods described in ⁇ 5.2 to 5.8, stimulates hepatocytes as shown in Figure 11.
  • the hepatocyte stimulating activity was detected according to the assay described in ⁇ 5.9.2
  • the hepatocyte stimulation exhibited by synthetic cysteine-free IL-6 occurs at concentrations of 10 -8 M
  • the peptide causes a decrease in the number of
  • the most sensitive method to evaluate the functionality of the synthetic cysteine-free IL-6 protein, as prepared according to the methods described in ⁇ 5.2 to 5.8 was the B-cell differentiation assay.
  • a hemolytic plaque assay assessed the ability of the synthetic IL-6 to induce Ig secretion in resting B-cells. The hemolytic assay is
  • EXAMPLE CYSTEINE-FREE IL-6 RETAINS BIOLOGICAL ACTIVITY
  • cysteine-free IL-6 retains biological activity.
  • Our invention has shown that it is the primary sequence of the peptide that is necessary to fold the peptide chain into an active conformation.
  • Vaccines are often formulated and inoculated in
  • the adjuvants aid in attaining a more durable and higher level of immunity using smaller amounts of antigen or fewer doses than if the
  • immunogen were administered alone.
  • the mechanism of adjuvant action is complex. It may involve the stimulation of
  • cytokines such as the cytokine IL-6
  • phagocytosis and other activities of the reticuloendothelial system as well as a delayed release and degradation of the antigen.
  • adjuvants include Freund's adjuvant (complete or incomplete), Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), surface active substances such as lysolecithin, pluronic polyols,
  • polyanions such as polyanions, peptides, oil emulsions, and mineral gels such as aluminum hydroxide or aluminum phosphate.
  • Freund's adjuvant is no longer used in vaccine formulation for humans because it contains nonmetabolizable mineral oil and is a potential carcinogen.
  • Purified synthetic IL-6 like peptides of the present invention can be added to vaccine preparations to modulate an immune response, i.e., to act as an adjuvant; this includes vaccine preparations used to immunize animals such as mice, guinea pigs, rabbits, chickens, horses, goats, sheep, cows, chimpanzees as well as other primates, and humans.
  • Methods of introduction of the vaccine with peptides like IL-6 as adjuvant include oral, intradermal, intramuscular,
  • IL-6 active peptides can be used as an antigen for host immunization and ultimate production of IL-6 specific monoclonal antibodies.
  • Such antibodies in turn may be used as IL-6 inhibitors and as such may be useful in the treatment of certain conditions in which dysfunction in immunoglobulin production has been implicated. This includes treatment of multiple myeloma, and autoimmune disease. For example, intraarticular injection of these monoclonal antibodies could be employed in treating rheumatoid arthritis.
  • the purified synthetic IL-6 like peptide of either the cysteine-free form or the form with cysteines is adjusted to an appropriate concentration, formulated with any suitable additions such as other cytokine peptides or vaccines and packaged for use.
  • the peptide with IL-6 activity can also be incorporated into liposomes for use as an adjuvant in vaccine formulations or as a pharmaceutical product by itself.
  • the synthetic peptide with IL-6 activity can also be added to preformed antibodies that are provided for passive
  • the purified peptides with IL-6 activity can be used as an immunostimulant
  • IL-6 peptides are useful generally for stimulation of hemopoietic stem cells, and specifically for the stimulation of antibody production in disease-caused and drug or radiation-induced
  • the peptides are useful in the treatment of immunosuppressed AIDS patients. They are also useful for the stimulation of production of hepatic proteins in hepatic dysfunction.
  • the peptide can also be used as a reagent for inducing antibodies in vitro, or to modify the expression of other growth factors in culture.
  • the purified peptides with IL-6 activity preferably in combination with other antiviral compounds, can be used for the prevention and/or the treatment of viral diseases, including HIV and HBV.
  • viral diseases including HIV and HBV.
  • the purified peptides with IL-6 activity can be used, alone or in combination with other purified cytokines, to invoke the terminal differentiation of B-cells in
  • the synthetic IL-6 like peptide of either the cysteine-free form or the form containing cysteines can be used to induce antibodies that recognize any portion of the peptide.
  • Delta assay is designed to determine whether there is renewal of a highly proliferative population (HPP) in bone marrow (BM) stem cells that are stimulated with various growth factors. Mice are treated with 5-fluorouracil (5FU) to remove cells that are of low proliferative potential (LPP).
  • HPP highly proliferative population
  • BM bone marrow
  • 5FU 5-fluorouracil
  • the HPP cells are incubated in a first semi-solid agarose culture for 12 days in the presence of growth factors.
  • the growth factors used include IL-1, IL-6, and a
  • BM stem cells are grown in the presence of each of these growth factors in the absence of other growth factors, and in the presence of granulocyte colony stimulating factor (G-CSF), macrophage colony
  • G-CSF granulocyte colony stimulating factor
  • M-CSF M-CSF
  • GM-CSF granulocyte macrophage colony stimulating factors
  • IL-3 granulocyte macrophage colony stimulating factors
  • the next set of four bars represents the effect of G-CSF alone and in combination with IL-1, IL-6 (cysteine-free), an a 1:1 mixture of IL-1 and IL-6 (cysteine-free) on colony stimulating activity.
  • the next three sets of four bars each represent the colony stimulating activity of' M-CSF, GM-CSF, and IL-3 alone and with IL-1, IL-6 (cysteine-free), and a 1: mixture of IL-1 and IL-6 (cysteine-free).
  • the results show that IL-1, IL-6 (cysteine-free), and a combination of IL-1 and IL-6 (cysteine-free) stimulate colony formation when use in combination with M-CSF more than in combination with IL-3,
  • the HPP cells are incubated with the same growth factors and combination of growth factors in liquid culture.
  • the number of non-adherent cells are counte after 7 days, and the results are illustrated in Figure 15 labelled "Number of Non-Adherent Cells after 7 Days Liquid
  • the cells from the liquid culture are washed and again grown in the presence of the growth factors in semi-solid agarose, and subjected to a second double-layer agarose clonal assay.
  • This second agarose clonal assay is referred to as the "readout" assay.
  • a Delta value is calculated by dividing the number of bone marrow colonies in the readout assay by the number of bone marrow colonies in the Time O Clonal Assay. The results are shown in Table 2.
  • the data from the Delta assay describes which growth factors have synergizing activity in liquid culture when various factors are used in the readout assay.
  • the ability of the added cytokines to facilitate colony formation is IL-1 + IL-6 > IL-1 or IL-6.
  • This preference of IL-1 + IL-6 over the ability of IL-1 or IL-6 to synergize with other growth factors is evident when G-CSF, GM-CSF, M-CSF and IL-3 are used in the liquid culture.
  • the synergy is most evident when G-CSF is used in the readout assay.
  • IL-6 causes a greater Delta value than IL-1.
  • CSF-1 is used in conjunction with IL-1 or IL-6
  • IL-1 causes a greater synergistic value. The best results in the entire assay were seen when IL-1 + IL-6 were used in conjunction with IL-3 in the liquid assay and G-CSF was used in the readout.
  • cysteine-free IL-6 when used in conjunction with other growth factors, may be an important tool in bone marrow transplantation and cancer treatment.
  • a high Delta value indicates that a certain combination of growth factors yields both growth and renewal of stem cells. This particular characteristic would be required in a growth factor that would be used to stimulate bone marrow growth and differentiation in vivo.
  • BM cells are added to an overlayer at a concentration of 2.5 x 10 4 up to 2 x 10 5 BM cells m .5 ml per plate. This is called the Time O CConal Assay. Grow at 37°C under approximately 7% O 2 for 12 days.
  • BM cells are grown in 1 ml liquid culture (IMDM 20% FB
  • the cells are then diluted 20-100-fold and plated into the clonal agarose assay. These cultures are grown for 7-12 days at 37oC, 7% O 2 in a fully humidified atmosphere. This is called the Readout Assay.
  • the Delta value is calculated by dividing the number of BM colonies in the Readout Assay by the number of BM colonies in the Time 0 Clonal Assay.
  • cysteine-free IL-6 peptide purified from the fusion product induced by cells harboring p367 was compared with cysteine-containing IL-6 from commercial sources using a 7td.l assay (Van Snick et al., Proc. Nat'l. Acad. Sci. USA 83:9679-9683, 1986).
  • Those commercial sources were Boehringer Mannheim (bm) which produced an E. coli derived IL-6; Endogen which was a non- recombinant product; and Genzyme which produced a yeast- derived IL-6.
  • Diluent medium is RPMI 1640 with 10% fetal calf serum.
  • Supplemented RPMI contains RPMI 1640 with 10% fetal calf serum, 0.1 mM 2-mercaptoethanol and 2x antibiotic solution.
  • the labeling reagent is 5 mg/mL MTT in PBS.
  • accession numbers The following plasmids have been deposited with the American Type Culture Collection (ATCC), Rockville, MD on November 29, 1988, and have been assigned the indicated accession numbers:

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Abstract

Gènes recombinants et leurs protéines codées qui sont des peptides présentant une activité IL-6. De telles protéines comprennent le peptide actif synthétique à activité IL-6 dépourvu de cystéine, ainsi que le peptide recombinant avec cystéines. L'invention se rapporte à la production commerciale peu coûteuse de grandes quantités de peptides à activité IL-6, sous une forme rendant superflue l'utilisation de dénaturants durs et ne devant pas être repliée après purification. Les protéines ci-décrites peuvent être utilisées pour stimuler la production de protéines par des cellules, y compris les cellules du système immunitaire et les hépatocytes. Les protéines décrites par la présente invention peuvent avoir une activité antivirale et peuvent empêcher l'infection virale des cellules. Dans une forme préférée d'exécution, le peptide synthétique à activité IL-6 dépourvu de cystéine est produit par des cellules microbiennes sous forme de fusion trihybride soluble comprenant un peptide synthétique à activité IL-6 dépourvu de cystéine, un peptide clivable chimiquement et une protéine assurant l'expression dans l'organisme hôte. Après purification de la grande protéine de fusion, le peptide synthétique à activité IL-6 est extrait par digestion de la partie collagène de la fusion avec une collagénase. On purifie par HPLC de la protéine synthétique à activité IL-6 dépourvue de cystéine, de manière à obtenir une protéine pure qui stimule la production d'immunoglobines par les cellules B, et qui stimule la production de protéines hépatiques.
PCT/US1989/005421 1988-12-01 1989-11-30 Interleucine-6 synthetique WO1990006370A1 (fr)

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WO1991011520A1 (fr) * 1990-02-03 1991-08-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. PROCEDE DE CLIVAGE ENZYMATIQUE DE PROTEINES RECOMBINANTES AU MOYEN DE PROTEASES D'IgA
WO1993001212A1 (fr) * 1991-07-02 1993-01-21 Imclone Systems Incorporated Muteines d'il-6 depourvues de cysteine_____________
US5210075A (en) * 1990-02-16 1993-05-11 Tanabe Seiyaku Co., Ltd. Interleukin 6 antagonist peptides
US5338833A (en) * 1992-07-23 1994-08-16 The University Of North Carolina At Chapel Hill Carboxy terminal IL-6 muteins
US5338834A (en) * 1993-01-26 1994-08-16 Allelix Biopharmaceuticals Inc. Ultrapure human interleukin-6
US5462731A (en) * 1991-10-20 1995-10-31 Yeda Research And Development Company Ltd. Use of IL-6 for the treatment of chronic lymphocyte leukemia (CLL) and B-cell lymphomas
EP0750040A2 (fr) * 1995-06-20 1996-12-27 Toyo Boseki Kabushiki Kaisha Variants de la protéine stimulatrice des macrophages et procédés de fabrication
EP0814154A2 (fr) 1993-09-15 1997-12-29 Chiron Corporation Vecteurs composés d'alphavirus recombinants
US6265204B1 (en) 1997-01-17 2001-07-24 Genencor International, Inc. DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi
EP1997900A2 (fr) 1996-04-05 2008-12-03 Novartis Vaccines and Diagnostics, Inc. Vecteurs à base d'alpha virus recombinants avec inhibition réduite de synthèse macromoléculaire cellulaire
EP2206785A1 (fr) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Expression améliorée de polypeptides HIV et production de particules de type virus
EP2266602A2 (fr) 2004-11-01 2010-12-29 Novartis Vaccines and Diagnostics, Inc. Approches combinatoires destinées à produire des réponses immunitaires
EP2281832A2 (fr) 2000-07-05 2011-02-09 Novartis Vaccines and Diagnostics, Inc. Polynucléotides codant pour des polypeptides antigéniques du type C du VIH, de tels polypeptides et leurs utilisations
EP2292772A1 (fr) 2001-07-05 2011-03-09 Novartis Vaccines and Diagnostics, Inc. Vaccination VIH avec un ADN codant un polypeptide VIH et un polypeptide VIH
EP2298900A1 (fr) 1996-09-17 2011-03-23 Novartis Vaccines and Diagnostics, Inc. Compositions et procédés de traitement de maladies intracellulaires
EP2339010A2 (fr) 2002-05-01 2011-06-29 Gbp Ip, Llc Particules de vecteur de lentivirus pour l'inactivation de complément
EP2412242A2 (fr) 2001-07-05 2012-02-01 Novartis Vaccines and Diagnostics, Inc. Polynucléotides codant pour des polypeptides antigènes de type C du VIH, polypeptides et leurs utilisations
US8486693B2 (en) 2006-05-23 2013-07-16 Bellicum Pharmaceuticals, Inc. Modified dendritic cells having enhanced survival and immunogenicity and related compositions and methods
WO2017040387A2 (fr) 2015-08-31 2017-03-09 Technovax, Inc. Vaccin à base de pseudoparticules virales (vlp) contre le virus syncytial respiratoire humain (hrsv)
WO2017180770A1 (fr) 2016-04-13 2017-10-19 Synthetic Genomics, Inc. Systèmes de réplicon d'artérivirus recombinant et utilisations correspondantes
EP3608401A1 (fr) 2012-07-05 2020-02-12 Ohio State Innovation Foundation Compositions et procédés liés aux vaccins viraux
US11020476B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Methods and compositions for inducing an immune response against Hepatitis B Virus (HBV)
US11021692B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11083786B2 (en) 2018-01-19 2021-08-10 Janssen Pharmaceuticals, Inc. Induce and enhance immune responses using recombinant replicon systems
US11364310B2 (en) 2016-10-17 2022-06-21 Janssen Pharmaceuticals, Inc. Recombinant virus replicon systems and uses thereof
US11389531B2 (en) 2017-12-19 2022-07-19 Janssen Sciences Ireland Unlimited Company Methods and apparatus for the delivery of hepatitis B virus (HBV) vaccines
CN114805537A (zh) * 2022-04-26 2022-07-29 华南农业大学 一种表达犬白介素6的重组质粒、稳定表达犬白介素6蛋白的细胞株及其制备方法和应用
US11845939B2 (en) 2016-12-05 2023-12-19 Janssen Pharmaceuticals, Inc. Compositions and methods for enhancing gene expression

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US5427927A (en) * 1990-02-03 1995-06-27 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Process for the enzymatic cleavage of recombinant proteins using IgA proteases
WO1991011520A1 (fr) * 1990-02-03 1991-08-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. PROCEDE DE CLIVAGE ENZYMATIQUE DE PROTEINES RECOMBINANTES AU MOYEN DE PROTEASES D'IgA
US5210075A (en) * 1990-02-16 1993-05-11 Tanabe Seiyaku Co., Ltd. Interleukin 6 antagonist peptides
WO1993001212A1 (fr) * 1991-07-02 1993-01-21 Imclone Systems Incorporated Muteines d'il-6 depourvues de cysteine_____________
US5545537A (en) * 1991-07-02 1996-08-13 The Trustees Of Princeton University Method of making cysteine depleted IL-6 muteins
US5359034A (en) * 1991-07-02 1994-10-25 Imclone Systems Inc. Cysteine depleted IL-6 muteins
US5462731A (en) * 1991-10-20 1995-10-31 Yeda Research And Development Company Ltd. Use of IL-6 for the treatment of chronic lymphocyte leukemia (CLL) and B-cell lymphomas
US5338833A (en) * 1992-07-23 1994-08-16 The University Of North Carolina At Chapel Hill Carboxy terminal IL-6 muteins
EP0652901A1 (fr) * 1992-07-23 1995-05-17 The University Of North Carolina At Chapel Hill Muteines d'interleukine-6 a terminaison carboxy
US5565336A (en) * 1992-07-23 1996-10-15 University Of North Carolina At Chapel Hill Carboxy terminal IL-6 muteins
EP0652901A4 (fr) * 1992-07-23 1996-11-06 Univ North Carolina Muteines d'interleukine-6 a terminaison carboxy.
US5338834A (en) * 1993-01-26 1994-08-16 Allelix Biopharmaceuticals Inc. Ultrapure human interleukin-6
EP0982405A2 (fr) 1993-09-15 2000-03-01 Chiron Corporation Vecteurs composés d'alphavirus recombinants
EP0814154A2 (fr) 1993-09-15 1997-12-29 Chiron Corporation Vecteurs composés d'alphavirus recombinants
EP0750040A2 (fr) * 1995-06-20 1996-12-27 Toyo Boseki Kabushiki Kaisha Variants de la protéine stimulatrice des macrophages et procédés de fabrication
EP0750040A3 (fr) * 1995-06-20 1999-10-27 Toyo Boseki Kabushiki Kaisha Variants de la protéine stimulatrice des macrophages et procédés de fabrication
EP1997900A2 (fr) 1996-04-05 2008-12-03 Novartis Vaccines and Diagnostics, Inc. Vecteurs à base d'alpha virus recombinants avec inhibition réduite de synthèse macromoléculaire cellulaire
EP2298900A1 (fr) 1996-09-17 2011-03-23 Novartis Vaccines and Diagnostics, Inc. Compositions et procédés de traitement de maladies intracellulaires
US6265204B1 (en) 1997-01-17 2001-07-24 Genencor International, Inc. DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi
US6590078B2 (en) 1997-01-17 2003-07-08 Genencor International, Inc. DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi
EP2206785A1 (fr) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Expression améliorée de polypeptides HIV et production de particules de type virus
EP2281832A2 (fr) 2000-07-05 2011-02-09 Novartis Vaccines and Diagnostics, Inc. Polynucléotides codant pour des polypeptides antigéniques du type C du VIH, de tels polypeptides et leurs utilisations
EP2311958A2 (fr) 2000-07-05 2011-04-20 Novartis Vaccines and Diagnostics, Inc. Polynucléotides codant pour des polypeptides antigéniques du type C du VIH, de tels polypeptides et leurs utilisations
EP2412242A2 (fr) 2001-07-05 2012-02-01 Novartis Vaccines and Diagnostics, Inc. Polynucléotides codant pour des polypeptides antigènes de type C du VIH, polypeptides et leurs utilisations
EP2292772A1 (fr) 2001-07-05 2011-03-09 Novartis Vaccines and Diagnostics, Inc. Vaccination VIH avec un ADN codant un polypeptide VIH et un polypeptide VIH
EP2339010A2 (fr) 2002-05-01 2011-06-29 Gbp Ip, Llc Particules de vecteur de lentivirus pour l'inactivation de complément
EP2266602A2 (fr) 2004-11-01 2010-12-29 Novartis Vaccines and Diagnostics, Inc. Approches combinatoires destinées à produire des réponses immunitaires
US8486693B2 (en) 2006-05-23 2013-07-16 Bellicum Pharmaceuticals, Inc. Modified dendritic cells having enhanced survival and immunogenicity and related compositions and methods
EP3608401A1 (fr) 2012-07-05 2020-02-12 Ohio State Innovation Foundation Compositions et procédés liés aux vaccins viraux
WO2017040387A2 (fr) 2015-08-31 2017-03-09 Technovax, Inc. Vaccin à base de pseudoparticules virales (vlp) contre le virus syncytial respiratoire humain (hrsv)
WO2017180770A1 (fr) 2016-04-13 2017-10-19 Synthetic Genomics, Inc. Systèmes de réplicon d'artérivirus recombinant et utilisations correspondantes
US10538786B2 (en) 2016-04-13 2020-01-21 Janssen Pharmaceuticals, Inc. Recombinant arterivirus replicon systems and uses thereof
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US11845939B2 (en) 2016-12-05 2023-12-19 Janssen Pharmaceuticals, Inc. Compositions and methods for enhancing gene expression
US11021692B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11389531B2 (en) 2017-12-19 2022-07-19 Janssen Sciences Ireland Unlimited Company Methods and apparatus for the delivery of hepatitis B virus (HBV) vaccines
US11725194B2 (en) 2017-12-19 2023-08-15 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11020476B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Methods and compositions for inducing an immune response against Hepatitis B Virus (HBV)
US11083786B2 (en) 2018-01-19 2021-08-10 Janssen Pharmaceuticals, Inc. Induce and enhance immune responses using recombinant replicon systems
US11826416B2 (en) 2018-01-19 2023-11-28 Janssen Pharmaceuticals, Inc. Induce and enhance immune responses using recombinant replicon systems
CN114805537A (zh) * 2022-04-26 2022-07-29 华南农业大学 一种表达犬白介素6的重组质粒、稳定表达犬白介素6蛋白的细胞株及其制备方法和应用

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