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WO1998022577A1 - Proteines de fusion a demi-vie allongee - Google Patents

Proteines de fusion a demi-vie allongee Download PDF

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Publication number
WO1998022577A1
WO1998022577A1 PCT/IB1997/001508 IB9701508W WO9822577A1 WO 1998022577 A1 WO1998022577 A1 WO 1998022577A1 IB 9701508 W IB9701508 W IB 9701508W WO 9822577 A1 WO9822577 A1 WO 9822577A1
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protein
gly
stabilizing
prodrug
fusion
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PCT/IB1997/001508
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English (en)
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Maria Grazia Masucci
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Maria Grazia Masucci
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Priority to AU50651/98A priority Critical patent/AU5065198A/en
Publication of WO1998022577A1 publication Critical patent/WO1998022577A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to the production of fusion proteins with increased resistance to proteolytic degradation and their therapeutic and diagnostic uses, including the use of such fusion proteins in the therapeutic treatment of disease.
  • proteolytic enzymes play important roles in the regulation of the activities of other proteins in the cell.
  • proteolytic enzymes are well known to be critical in the formation of active enzymes from inactive proenzymes (zymogens) as found in the stomach or pancreas (for example, chymotrypsinogen, trypsinogen, pepsinogen, procarboxylase, proelastase) ; in the proteolytic conversions of zymogens to form fibrin to clot blood; in the complement cascade involved in lysing foreign cells and in the inflammatory response; in the synthesis of certain hormones
  • zymogens inactive proenzymes
  • pancreas for example, chymotrypsinogen, trypsinogen, pepsinogen, procarboxylase, proelastase
  • the eukaryotic cell contains at least two distinct systems of enzymes for the continuous degradation of cell proteins (see, for example, Zubay, Biochemistry, pp. 963 - 972 (Addison-Wesley Publishing Co. , Reading, Massachusetts, 1983)).
  • the ATP-dependent multisubunit protease also referred to as the proteosome
  • This ATP-dependent protease is also involved in the degradation of those proteins to which ubiquitin attaches (ubiquitination) as a signal for degradation (see, for example, Peters, Trends Biochem . Sci .
  • the other major proteolytic system of eukaryotic cells is the lysosome organelle which is primarily responsible for the degradation of relatively long-lived proteins, membrane proteins, and extracellular proteins.
  • the lysosomal degradative system may also be involved in protein degradation associated with certain pathological conditions, such as muscular dystrophy, denervated muscle conditions, and wounds from burns (Zubay, Biochemistry, 1983) .
  • the ATP-dependent protease and the lysosome comprise the primary proteolytic activities responsible for the rate of degradation of most proteins in the cell as reflected in and defined by the half-life of each protein which is the time during which 50 percent of the molecules of a particular protein is degraded.
  • the half-lives of particular proteins vary widely, for example, ranging from a couple of hours or less for key enzymes of metabolic pathways to days for other proteins. Two models have emerged for predicting whether a protein is likely to have a relatively short or long half-life.
  • certain amino acids at the amino-, or N- terminal, end of a particular protein serve as a signal to proteases of the cell and can be used to predict whether a protein will have a relatively short or long half-life (see, Bachmair et al., Science, 234 : 179 - 186 (1986)).
  • PEST domains proteins containing regions rich in one of these four amino acids are likely to have half-lives of 2 hours or less (see, Rogers et al., Science, 234 : 364 - 368 (1986)).
  • One object of the invention is to modify a protein so as to provide an increase in its half- life.
  • This invention provides the means and methods for producing fusion proteins that are modified to be more resistant to protease degradation.
  • fusion proteins comprise the amino acid sequence of a core protein that is normally susceptible to proteolytic degradation and a stabilizing polypeptide described herein.
  • Such fusion proteins are more resistant to degradation by proteases and, thus, have a longer half-life than the unfused core protein.
  • half-life refers to the time period over which 50% of the protein molecules are degraded by a protease.
  • the half-life of a fusion protein of this invention is at least approximately 10-20% and preferably 25% longer than the corresponding unfused core protein.
  • Fusion proteins of the invention will include a core protein and a stabilizing polypeptide sequence covalently linked thereto.
  • the stabilizing sequence may be joined to the core protein at the amino (n-) or carboxy (c-) terminus of the core protein or may be inserted within the core protein.
  • the present invention provides a method for increasing the resistance of a core protein to proteolytic degradation, comprising linking or inserting a stabilizing polypeptide onto or into the core protein, wherein the stabilizing polypeptide has the general formula:
  • each Gly a , Gly b , Gly c may be one, two, three, four, five or six sequential glycine residues; each of X, Y and Z is, independently, selected from the group consisting of alanine, serine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and threonine; wherein X, Y and Z, respectively, need not be identical from n repeat to n repeat; wherein n is 1 to 66.
  • the present invention also provides a method for increasing the resistance of a core protein to proteolytic degradation, comprising linking or inserting a nucleotide sequence encoding a stabilizing polypeptide to a nucleotide sequence encoding a core protein to create a gene fusion which is expressible as a fusion protein, wherein the stabilizing polypeptide has the general formula: t(Gly a )X(Gly b )Y(Gly c )Z]n wherein each Gly a , Gly b , Gly c , independently, may be one, two, three, four, five or six sequential glycine residues; each of X, Y and Z is, independently, selected from the group consisting of alanine, serine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and threonine; wherein X, Y and Z, respectively, need not be identical from n repeat to n repeat;
  • the methods of the present invention can be used to increase the resistance of any core protein to proteolytic degradation.
  • the core protein retains its biological activity in the fusion protein.
  • the core protein used in the method of the present invention may be any enzyme or other protein such as the herpes virus type 1 Thymidine kinase, Clotting Factors, and insulin.
  • the invention thus relates to a recombinant protein containing a stabilizing amino acid sequence having the formula:
  • each Gly a , Gly b , Gly c may be one, two, three, four, five, or six sequential glycine residues; each of X, Y and Z is, independently, selected from the group consisting of alanine, serine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and threonine; more preferably, each of X, Y and Z is, independently, selected from the group consisting of alanine, serine, methionine and proline; most preferably, each of X, Y and Z is, independently, alanine and serine; more preferably, each of X and Y is, independently, serine and methionine; more preferably, each of X and Y is, independently, methionine and proline; more preferably, each of X and
  • Preferred stabilizing sequences of the invention include the 235 amino acid sequence provided in Figure 1 and a 17 amino acid glycine-alanine sequence having the amino acid sequence: Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Gly Ala Gly Gly Gly Gly Gly Gly (see Figure 2) .
  • the invention is particularly useful where administration of a protein is desired, for example, for a therapeutic or diagnostic purpose, where it is desired that the protein has a longer half-life in the recipient mammal, for example a human.
  • a stabilizing sequence is inserted into a foreign protein (also called a core protein) or at either the amino or carboxy terminal end of the foreign protein, thus rendering the resulting chimeric foreign protein (also called the fusion protein) relatively longer-lived upon administration to the recipient.
  • Proteins particularly useful according to the invention will be of therapeutic or diagnostic value to a mammal, such as a human.
  • the protein containing the stabilizing sequence not be a bacterial housekeeping protein such as an RNA poly erase, or beta- lactamase, a kanamycin resistance protein, or certain insect proteins such as silk fibroin proteins such as SLP1, SLP2 , SLP3, and SLP4 , the EBS1 protein (all as described in WO88/03533) .
  • a bacterial housekeeping protein such as an RNA poly erase, or beta- lactamase, a kanamycin resistance protein, or certain insect proteins such as silk fibroin proteins such as SLP1, SLP2 , SLP3, and SLP4 , the EBS1 protein (all as described in WO88/03533) .
  • the invention also features a reco binant fusion protein containing the above-described stabilizing amino acid sequence, wherein the recombinant fusion protein comprises a non-immunogenic core protein and the stabilizing sequence.
  • the stabilizing sequence may be joined to either the carboxy or amino terminal end of the core protein, or inserted anywhere within the protein to render that protein relatively longer-lived, it is preferred that the stabilizing sequence is inserted within the core protein at a distance wherein either terminal residue of the stabilizing sequence is at a distance of between about 1 and 300 residues from a proteolytic processing site of the protein.
  • the core protein contains one or more of such processing sites, the sites being ATP-dependent processing sites. In the case of a core protein containing multiple processing sites, it is believed that all such sites of the core protein will be rendered less susceptible to processing according to the invention.
  • the stabilizing sequence is present anywhere within or contiguous with the core protein, as the distance between the core protein and the stabilizing sequence is not believed to be critical to increase the half-life of the fusion protein.
  • the invention also encompasses a recombinant nucleic acid encoding the fusion protein described above, and bacterial or mammalian host cells containing such recombinant nucleic acids.
  • the invention also encompasses a vector containing a nucleotide sequence encoding the stabilizing sequence, wherein the 5 ' and 3 ' ends of this nucleotide sequence have been modified so as to permit insertion of this nucleotide sequence into the coding region of a target protein such that the insertion occurs in the same reading frame as the target protein.
  • the 5' and/or 3' ends of the nucleotide sequence encoding the stabilizing sequence have been modified such that the sequence can be inserted into the coding region of a target core protein in any one of one, two or three upstream selected reading frames (i.e., upstream of the site of insertion in the protein of the stabilizing sequence) and any one of one, two or three corresponding downstream reading frames (i.e., downstream of the site of insertion).
  • the 5' and/or 3 1 ends of the nucleotide sequence encoding the stabilizing sequence have been modified such that the sequence can be inserted into the coding region of a target core protein in-frame.
  • the invention also encompasses the recombinant fusion protein or nucleic acid encoding the recombinant fusion protein for use in therapy.
  • the invention provides fusion proteins which comprise a non- immunogenic core protein covalently linked to a stabilizing polypeptide, the non-immunogenic core protein being any protein which retains its biological activity in the fusion protein form and which does not lead to an immunogenic reaction in a mammalian host in its unmodified form.
  • this invention provides fusion proteins which comprise an I ⁇ B regulator protein covalently linked to a stabilizing polypeptide, and genes encoding such proteins.
  • fusion proteins have a longer half-life, that is, at least approximately 10-20% and preferably 25% longer than the unfused I ⁇ B regulator, and retains the biological activity of the native protein, and are useful in treating inflammatory bowel disease (IBD) .
  • IBD inflammatory bowel disease
  • this invention provides fusion proteins which comprise a nitroreductase protein covalently linked to a stabilizing polypeptide which retains the biological activity of the native protein, and genes encoding such proteins.
  • fusion proteins are useful to activate nitro drugs such as CB1954, and thus are useful in enzyme/prodrug therapy to treat cancer or other pathological conditions.
  • DNA molecules encoding fusion proteins comprising an I «B regulator protein or nitroreductase linked to a stabilizing polypeptide are provided for use in gene therapy, the i B fusion protein to treat IBD and the nitroreductase fusion protein to treat cancer or a pathological disease.
  • fusion proteins of this invention also are useful as improved reagents in diagnostic methods, including in vivo imaging techniques such as MRI.
  • Figure 1 shows the amino acid sequence of the 235 residue glycine/alanine repeat region from Epstein Barr Virus EBNA1 protein.
  • Figure 3 shows the nucleotide sequence of the coding strand of DNA and deduced amino acid sequence for a 266 amino acid stabilizing polypeptide and is the FLGA insert described herein. Each amino acid of the amino acid sequence is depicted by its upper case, single letter abbreviation.
  • Figure 4A shows a schematic pBSFLGAl vector containing Gly- sequence coding region for protein engineering.
  • the plasmid is based on the pBS KS (+) cloning vector.
  • Figure 4B shows the nucleotide sequence of the stabilizing sequence-encoding insert with 5' and 3' flanking regions.
  • Figure 5 is a schematic map of the pBSFLGA2 vector containing Gly-sequence coding region for protein engineering.
  • the plasmid was created from pBSFLGAl by filling the protruding BamHI ends with Klenow enzyme and ligation. The BamHI site is substituted by an artificial
  • Figure 6 presents three upstream frames and 3 downstream frames which can be obtained using different combinations of restriction enzymes and/or Klenow enzyme and mung bean nuclease.
  • Figure 7 presents FLGA containing constructs with translation start (pBS-M-FLGA) or stop codons (p ⁇ hisMetFLGAstop) for expression of N-terminal and C- terminal FLGA fusion proteins.
  • Figure 11 is an autoradiogram of polyacrylamide gels showing in vitro ATP-dependent degradation of I ⁇ B and IcB fusion proteins.
  • Figure 12 is an autoradiogram of a polyacrylamide gel showing in vivo degradation of I «B and IcB fusion proteins.
  • Figure 13 shows the amino acid sequence for murine I ⁇ B- ⁇ .
  • Figure 14 shows a nucleotide sequence encoding murine I B- ⁇ .
  • the translational start codon (atg) and translation stop codon (taa) of the I/cB- ⁇ structural coding sequence are underlined.
  • Figure 17 is a schematic illustration of construction of a vector (pJG-N-NTR) containing a gene encoding a fusion protein according to the invention in which the stabilizing polypeptide is attached to the amino terminus of the fusion protein.
  • Figure 18 is a schematic illustration of construction of a vector containing a gene encoding a fusion protein in which the stabilizing polypeptide is attached at the carboxy terminal end of the protein
  • Figure 19 shows the predicted amino acid sequence of the junction region of two of the Gly-Ala minimal repeat constructs with 25 and 33 glycine/alanine residues, respectively, in the stabilizing polypeptide.
  • Figure 20 shows the predicted amino acid sequence of the junction region of two of the Pro-Ala minimal repeat constructs with 17 and 25 proline/alanine residues, respectively, in the stabilizing polypeptide.
  • Figure 21 (A) is a schematic representation of the chimeric IkB ⁇ constructs.
  • the coding sequence of the IkB ⁇ is shown as an open box with the five ankyrin repeats indicated by hatched boxes and the encoding region for three influenza hemagglutinin epitopes (3HA) indicated by a filled box.
  • Figure 21 (B) presents the amino acid sequences of the glycine/alanine inserts of Figure 21 A.
  • Figure 22 (A) is a western blot of total cell extracts probed with an IkB ⁇ -specific rabbit serum. Ectopically expressed IkB recombinant proteins are indicated by filled arrows. Open arrows indicate the endogenous IkB ⁇ which is recognized by the specific rabbit serum and serves as internal control for degradation.
  • Figure 22 (B) represents the ratio between the intensity of the IkB specific bands (as determined by scanning of the ECL developed blots) before treatment and after treatment with TNF ⁇ for the indicated time.
  • Figure 23 shows signal dependent degradation of IkB chimeras containing a 24 amino acid long Gly-Ala repeat.
  • Figure 24 shows results of Western blots of immunoprecipitation experiments in which IkB proteins containing the Gly-Ala repeat are bound to NFkB/RelA heterodimers .
  • Figure 25 (A) is a Western blot of HeLa cells transfected with GA24-X and 3HA-lkB and treated with 100 ng/ml of recombinant TNF ⁇ with or without addition of 10 nM of the peptide aldehide MG123 and LLnL to inhibit proteasome activity, and the ectopic IkB was then immunoprecipitated from ocadaic acid containing extracts using the anti-3HA mAbs, and the blots probed with the anti-IkB serum.
  • FIG. 26 is a Western blot showing that a ubiquitinated chimera containing Gly-Ala repeats is not targeted to proteasomes.
  • Figure 29 (B) shows results of densitometry analysis of Figure 29 (A) .
  • Figure 30 presents graphs showing sensitivity of EBNA4 chimeras to ubiquitin/proteasome dependent degradation.
  • This invention is based on the discovery that linking certain stabilizing polypeptides to a protein of interest (the core protein) , will produce a fusion protein which retains the biological activity of the core protein and which is more resistant to protease degradation (or processing) than the unmodified core protein.
  • the fusion proteins produced by the method of the invention are thus more stable, as evidenced by an increased half-life, and are able to exert their activity or function for a longer period of time than the corresponding unmodified core proteins.
  • a particular application of this invention is the use of the fusion proteins of the present invention to treat autoimmune disease such as inflammatory bowel disease (IBD) .
  • IBD inflammatory bowel disease
  • a stabilizing amino acid sequence for insertion or linkage to a core protein according to the invention will fall within the general formula: [(Gly a )X(Gly b )Y(Gly c )Z]n wherein each Gly a , Gly b , Gly c , independently, may be one, two, three, four, five, or six sequential glycine residues; each of X, Y and Z is, independently, selected from the group consisting of Ala, Ser, Val, lie, Leu, Met, Phe, Pro, and Thr; more preferably, each of X, Y and Z is, independently, selected from the group consisting of Ala, Ser, Met and Pro; most preferably, each of X, Y and Z is, independently, Ala and Ser; more preferably, each of X and Y is, independently, Ser and Met; more preferably, each of X and Y is, independently, Met and Pro; more preferably, each of X and Y is not Lys or Arg
  • Recombinant (i.e., made by recombinant DNA techniques) isolated peptides of the invention will not include synthetic (i.e., chemically synthesized) polypeptides which are used in the prior art for generation of antibodies to Epstein Barr Virus, for example, the synthetic peptides described in WO90/01495:
  • a preferred stabilizing sequence is the 235 amino acid glycine repeat sequence of Figure 1.
  • a fusion protein construct containing a putative stabilizing sequence is employed according to the methods described herein by which the GA24 constructs are tested for inhibition of degradation by proteases.
  • the half-life of polypeptides and proteins produced by the method of the present invention may also be determined in an in vivo system by measuring degradation of recombinant proteins expressed in cells which express or are induced to express a protease. For example, if an antibody to the expressed recombinant protein is available, the degradation of recombinant protein expressed in the cell can be followed using SDS-PAGE and standard Western blot analysis.
  • fusion proteins which comprise a prodrug activating enzyme, nitroreductase, and a stabilizing polypeptide, and DNAs encoding such proteins.
  • fusion proteins which comprise an I/cB regulator protein and a stabilizing polypeptide and DNA molecules encoding such fusion proteins.
  • Such fusion proteins and the DNA molecules encoding such proteins are useful in the treatment of inflammatory bowel disease.
  • IBD Inflammatory bowel disease
  • IBD inflammatory bowel disease
  • Ultative colitis which is a predominantly superficial, ulcerative inflammation of the large intestine
  • Crohn's disease which is characterized as a transmural, granulomatous inflammation that may occur anywhere in the gastrointestinal tract.
  • the pathogenesis of IBD is correlated with an abnormal elevation in the level of expression in and secretion from lymphocytes and macrophages of cytokines that promote inflammation (the proinflammatory cytokines) .
  • lamina limbal macrophages isolated from patients with Crohn's disease produce and secrete into culture medium relatively high levels of the proinflammatory cytokines interleukin-1 (IL-1) , interleukin-6 (IL-6) , and tumor necrosis factor- ⁇ (TNF- ⁇ ) (see, for example, Neurath et al.. Nature Medicine, 2 : 998 - 1004 (1996)).
  • Animal models of IBD also have been shown to contain lymphocytes or macrophages in areas of inflammation which produce elevated levels of proinflammatory cytokines (Neurath et al., 1996).
  • the p65 and p50 subunits become free to associate and again form an active NF-/cB transcription factor.
  • the availability of adequate intracellular levels of intact (non-degraded) I «B- ⁇ regulator protein to bind the p65 and p50 subunits is critical to maintaining a control over the inflammation process in normal tissue. If an abnormality exists that results in the expression of unusually high levels of one or both of the p65 and p50 subunits, as in Crohn's disease, the formation of active NF- ⁇ B transcription factor may overwhelm the capacity of the normal levels of I/cB regulator protein to bind p65 and p50 subunits and prevent the onset of IBD.
  • I/cB- ⁇ regulator protein has a half-life of 1 - 2 hours when co plexed with NF- B and is less stable when free in the cytoplasm (see, for example, Rice et al., EMBO J. , 12 : 4685 - 4695 (1993); Scott et al.. Genes & Dev. , 7 : 1266 - 1276 (1993); Sun et al., Science, 259 : 1912 - 1915 (1993)). Beauparlant et al. (J " . Biol . Chem .
  • the nucleotide coding sequence and corresponding amino acid sequences for the I/cB- ⁇ protein from a number of species are known, including murine I ⁇ B- ⁇ (see, GenBank accession number MMU36277, Figures 13 and 14) and human IcB- ⁇ (see, GenBank accession number M69043, Figures 15 and 16).
  • Such sequence data permit the manipulation and use of I/cB- ⁇ protein in a variety of molecular studies. For example, Lin et al. (Mol . Cell . Biol .
  • the protein pl05 the precursor to the p50 subunit of NF- ⁇ B, contains a glycine-rich region (GRR) which includes the 23 amino acid sequence Gly Gly Gly Ser Gly Ala Gly Ala Gly Gly Gly Gly Gly Met Phe Gly Ser Gly Gly Gly Gly Gly Gly Ser Thr and which appears to act as a processing signal for the precursor pl05 by directing an endoproteolytic cleavage downstream of the GRR.
  • GRR glycine-rich region
  • fusion protein containing an amino terminal I/cB- ⁇ protein sequence linked to the GRR which in turn was linked to a p50 protein sequence as the carboxy terminal region of the fusion protein.
  • the fusion protein was processed in C0S1 cells to form both an I ⁇ B- ⁇ -like product (the I/cB- ⁇ protein sequence with the GRR and some sequences from p50) and a p50 peptide cleavage product (Lin et al., 1996).
  • a stabilizing polypeptide is covalently linked to the nitroreductase core protein to produce a fusion protein.
  • This fusion protein is especially useful in treating diseases involving depletion of a selected cell population, such as in activated prodrug treatment of cancer cells.
  • the EcoRI-Hindlll multiple cloning site can be used to encode a fusion protein having an amino terminal histidine tag polypeptide linked to the stabilizing polypeptide linked to a carboxy terminal core protein sequence.
  • the 6xHis tag can be used for the rapid checking of reading frames by expression in E. coli and for rapid specific purification on Ni beads. After checking for proper reading frame, the resulting gene fusion can be recloned in any eukaryotic vector.
  • the above-described plasmid is useful for providing a DNA fragment encoding a stabilizing polypeptide sequence such that the DNA fragment can be inserted in-frame into a selected gene encoding a core protein of interest.
  • a DNA sequence encoding any stabilizing polypeptide as defined herein may be modified as described above such that the sequence is insertable in-frame into a selected protein- coding sequence.
  • the nucleotide sequence and corresponding amino acid sequence for a 17 amino acid stabilizing polypeptide may be used in place of the 235 amino acid EBNA-1 stabilizing polypeptide ( Figure 1) , or the 266 amino acid polypeptide ( Figure 3) described in the cassettes and plasmids.
  • the fragment may be used for PCR amplification, as follows.
  • the PCR products are cloned in the BamHI and EcoRI sites of the pGEX-T2 vector downstream of the glutathione S- transferase gene (GST) .
  • GST-GlyAla is expressed in bacteria using the tac promoter. Expression of fusion proteins containing the repeats inserted in-frame downstream of the GST gene is then screened by Western blotting of lysates from single transformed colonies using affinity purified human antibodies specific for the EBNA1 GlyAla repeat (Dillner et al., 1984, Proc. Nat. Aca. Sci. 81; 4652) .
  • Colonies expressing the GST-GlyAla polypeptide are further characterized to determine the size and coding capacity of the insert by restriction endonuclease analysis and sequencing.
  • a chimeric gene encoding the protein or polypeptide capable of activating a prodrug in the invention may encode any one of a variety of fusion proteins or polypeptides that retain the biological activity of the native protein in converting a prodrug to an active drug that is toxic to cells.
  • Genes encoding prodrug activating proteins or polypeptides include, but are not limited to: herpes simplex viral thymidine kinase (Genbank Accession No. J02224) , carboxypeptidase A (Genbank Accession No. M27717) , ⁇ - galactosidase (Genbank Accession No. M13571) , ⁇ - glucuronidase (Genbank Accession No.
  • alkaline phosphatase Genbank Accession No. J03252 J03512
  • cytochrome P-450 Genebank Accession No. D00003 N00003
  • Other proteins include plasmin, carboxypeptidase G2, cytosine deaminase, glucose oxidase, xanthine oxidase, ⁇ - glucosidase, azoreductase, or r-glutamyl transferase.
  • the polypeptide capable of activating a prodrug is DT diaphorase or a nitroreductase.
  • the gene encoding the prodrug-activating polypeptide may encode an enzyme used in the prodrug therapy described herein, and may specifically encode a nitroreductase.
  • the nitroreductase may be any naturally occurring, mutated or artificially produced nitroreductase which is capable of activating a prodrug by reducing a nitro group present on the prodrug to, for example, the hydroxylamino intermediate.
  • a particularly preferred nitroreductase is the E. coli nitroreductase (nucleotide sequence described WO 93/08288) .
  • nucleic acid condensing agents include the use of nucleic acid condensing agents, electroporation, complexation with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi, (1984), Crit. Rev. Biochem. ___, 349-379; Keown et al , (1990), Methods Enzymol. , 185. 527).
  • a self-assembling virus-like particle includes the DNA of interest and condensing peptides which are heteropeptides with respect to their amino acid composition (i.e., containing at least two different amino acids which are preferably basic and thus good DNA binding and DNA condensing peptides) and which have low polydispersion (i.e. , a given preparation of a heteropeptide which has low polydispersion contains peptides of very similar, if not identical lengths, such that the preparation is essentially monodispersed) .
  • a preferred prodrug and activating enzyme combination is the 2, 4-dinitro-5-ethyleneiminobenzamide (CB1954) and nitroreductase combination.
  • CB1954 has been shown to have an inhibiting effect on the growth of the Walker Rat Carcinoma this prodrug undergoes nitroreduction to a form a compound which is much more reactive with DNA than the prodrug, resulting in enhanced toxicity and mutagenicity (see Venitt et al . , (1987), Mutagenicity, 2 . (5), 375-381).
  • the use of CB1954 and analogs thereof as a prodrug in combination with a nitroreductase is described in PCT publication number WO 93/08288.
  • the E. coli NTR gene was excised as a Hindlll-EcoRI fragment from the pCR3-NTR-B/r plasmid (generated as a PCR fragment utilizing the Porton Down nitroreductase construct described by Drabek et al, 1997 supra, as a template, with primers as follows;
  • constructs were cloned into the transcription/translation vector pSP64Poly(A) .
  • the junction regions were sequenced to confirm maintenance of the reading frame.
  • In vitro transcription/translation analyses are on-going as well as Western blotting of the expressed products.
  • the effect of protein stability is initially tested in vitro, by transient transfection of the GA-NTR constructs in C0S7 cells, utilizing the plasmid pTX0147 as a control (parental NTR driven by CMV and containing the jS-globin IVS and polyadenylation sequences) .
  • the cells are immunostained with a polyclonal antibody to NTR.
  • a comparison with parental NTR is made so as to estimate whether the GA repeat stabilizes the NTR protein in vitro.
  • the Gly-Ala tagged constructs should offer prolonged detection by immunostaining in the transient assay as compared to the parental NTR construct.
  • constructs are assessed by the in vitro proteolytic degradation assays as described herein.
  • the fusion protein will be incubated in the presence of a cell-free rabbit reticulocyte lysate containing the components of the ATP-dependent, proteasome and ubiquitin cascade system.
  • the percent protein degraded over time can be assessed by either separation of the proteins on SDS-PAGE followed by densitometry or analysis of trichloroacetic acid (TCA) precipitation of proteins.
  • TCA trichloroacetic acid
  • the full length Gly-Ala coding region (GA) was excised from the EBNA1 containing plasmid pBSEBNAl using Avail and Mspl restrictases. The ends were filled in by Klenow enzyme and the blunt-ended fragment was cloned into the pBS KS (+) vector previously linearized by Smal. The orientation of the insert was estimated by dideoxysequencing from two directions, using both, universal, and reverse sequencing primers. The resulting plasmid pBSFLGAl containing full length GA repeat is shown in Figure 4.
  • the derivative plasmid pBSFLGA2 ( Figure 6) contains an additional Clal site upstream of the 5 1 end of GA coding region and lacks the BamHI site in the same position. It was created by BamHI digest of pBSFLGAl, filling the cohesive termini by Klenow enzyme and ligating the blunt termini. The constructs allow cloning of the GA coding region in different sites of a target gene. Six possible frames could be obtained as is shown in Figure 7.
  • the efficiency of protein degradation was assessed by monitoring the radioactivity released into the TCA soluble fraction and by following the disappearance of labeled protein after separation in SDS-PAGE and visualization by PHOSPHOIMAGER® (Molecular Dynamics) .
  • Degradation was performed in standard 2 hrs assays. Background and non-specific levels of degradation were determined by incubation of the substrate on ice ( Figure 11, control) and in the presence of ATP- depletion system (ATP-) . The level of degradation was calculated as the difference between the degradation of the substrate in the presence of ATP-regenerating and ATP- depletion systems. In parallel assays, the level of degradation was assessed by TCA precipitation of the samples as the ratio between TCA soluble and total protein incorporated radioactivity. Between 15 to 25% specific ATP- dependent degradation was detected in repeated experiments performed with in vitro translated I/cB- ⁇ while no degradation was observed with the I ⁇ B ⁇ -NflGA chimera
  • tumor necrosis factor- ⁇ (TNF- ⁇ , Boehringer Mannhein Biochemical ⁇ , Indianapolis, Indiana) immediately before harvest in SDS-PAGE loading buffer.
  • the cell extracts were run in 8% polyacrylamide gels, transferred to nitrocellulose filers and probed with an anti-HA mouse monoclonal antibody (Boeringher Mannheim Biochemicals, Indianapolis, Indiana) .
  • the endogenous (not shown) and 3HA-tagged I ⁇ B- ⁇ are completely degraded in transfected Hela cells following treatment for 20-30 min with 10 ng/ml TNF- ⁇ (DiDonato et al., Mol . Cell .
  • TNF- a Human recombinant tumor necrosis factor-a (TNF- a) was obtained from Boehringer Mannheim Biochemica.
  • Antibodies used in this study are: anti-IkB rabbit immune serum (at dilution 1:2500 (kindly provided by J. DiDonato) , anti-hemagglutinin mouse antibody 12CA5 at 2mg/ml (Boehringer Mannheim Biochemica) anti-ubiquitin rabbit immune serum at dilution 1:800 ( )
  • IkB ⁇ constructs The coding sequence of the IkB ⁇ is shown as an open box with the five ankyrin repeats indicated by hatched boxes and the encoding region for three influenza hemagglutinin epitopes (3HA) indicated by a filled box.
  • the lysines, K21 and K22, and serines, S32 and S36, involved in signal dependent phosphorylation and ubiquitination are indicated.
  • a cassette encoding for the 244 amino acid long Gly-Ala repeat for the B95.8 virus EBNA1 gene (flGA) was inserted in frame at the carboxy terminal end of the IkB sequence (-C) or at the amino terminal end immediately after the 3HA tag (-N) .
  • Phosphorylated IkB species with a slower migration in SDS-PAGE are indicated by arrows.
  • B Ubiquitination of 3HA-IkB and GA24- X in TNFa treated HeLa cells, TNF ⁇ treatment was performed in the presence of the peptide aldehide MG123 and LLnL. IkB was imrnunoprecipitated from cell extracts containing 20 mM of the isopeptidase inhibitor NEM using the anti IkB rabbit serum and Western blots were probed with the anti-HA antibody. Presence of unbiquitinated IkB is visualized by the appearance of a smear of high moleculartive weight species. One representative experiment out of three.
  • C The high molecular weight IkB contains ubiquitin conjugates.
  • TNF ⁇ Treatment with TNF ⁇ induces apoptosis of HeLa cells expressing Gly-Ala containing IkB chimeras.
  • HeLa cells were cotransfected with the indicated IkB construct and with a J-gal plasmid. After 48 hrs the transfected cells were treated with 10 ng/ml TNP ⁇ for 18 hrs and apoptotic cells were then detected by Hoechst staining. The % transfected cells was estimated by staining of /?-gal positive cells (A) % apoptosis in ⁇ -gal positive cells. (B) Representative Hoechst staining illustrating the induction of apoptosis in cells expressing the Gly-Ala containing IkB chimeras.
  • Suitable viruses for practice of the invention include but are not limited to, for example, adenoviruses, adeno- associated virus, retroviruses, and vaccinia viruses, representative examples of which follow.
  • T cells and lamina intestinal macrophages may be isolated from the mice to examine the levels of expression of proinflammatory cytokines, such as IL-1, IL-6, and TNF- ⁇ , for example, by standard ELISA or Western blot analysis coupled with densitometry (see, for example, Neurath et al., Nature Medicine, 2 : 998 - 1004 (1996)).
  • proinflammatory cytokines such as IL-1, IL-6, and TNF- ⁇
  • Antibodies to proinflammatory cytokines are commercially available (for example, PharMingen, San Diego, California) .
  • Fusion proteins according to the invention may be tested in any of the above-described animal models by administering the fusion protein directly to the animal and observing the animal for amelioration of any of the above-described indications of inflammatory bowel disease, or by administering a recombinant DNA encoding the fusion protein and observing the animal for signs of disease amelioration, or by creating a transgenic animal using known transgenic techniques, inducing the disease as described above, and observing the animal for amelioration of indications of inflammatory bowel disease.
  • transgenic mice (Gordon et al., 1980, PNAS 77:7380; Harbers et al., 1981, Nature 293:540; Wagner et al., 1981, PNAS 78:5016; and Wagner et al., 1981, PNAS 78:6376) , sheep, pigs, chickens (see Hammer et al., 1985, Nature 315:680), etc., are well- known in the art and are contemplated for use according to the invention.
  • Nuclear proteins are extracted from macrophages, for example, by the method of Neurath et al. (Proc . Natl . Acad . Sci . USA, 92 : 5336 - 5340 (1995)) and blotted on nitrocellulose filters (0.45 ⁇ m) and p65 and p50 NF- ⁇ B subunits detected using rabbit anti-p65 and anti-p50 antibodies (Santa Cruz Biotechnology, Santa Cruz, California) and a detecting system such as antibody such as alkaline phosphatase-labeled goat anti-rabbit antibody and alkaline phosphatase color substrate (Promega, Madison, Wisconsin) .
  • Western blots are analyzed with a densitometer and an appropriate instrument program (for example, IMAGEQUANT, Molecular Dynamics) .
  • Table 4 Recovery of stable geneticin resistant clones of HeLa cells transfected with GAr containing IKB chimeras.

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Abstract

L'invention concerne des protéines de fusion et des molécules d'ADN codant pour lesdites protéines. La protéine de fusion est constituée d'une protéine noyau liée par une liaison covalente à un polypeptide stabilisant. Elle est plus résistante à la dégradation protéolytique que la protéine noyau. L'invention concerne également des méthodes permettant d'utiliser à des fins thérapeutiques et diagnostiques les protéines de fusion et les molécules d'ADN codant pour lesdites protéines.
PCT/IB1997/001508 1996-11-15 1997-11-17 Proteines de fusion a demi-vie allongee WO1998022577A1 (fr)

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