WO2013100580A1 - Novel fusion protein binding to wnt - Google Patents

Novel fusion protein binding to wnt Download PDF

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Publication number
WO2013100580A1
WO2013100580A1 PCT/KR2012/011488 KR2012011488W WO2013100580A1 WO 2013100580 A1 WO2013100580 A1 WO 2013100580A1 KR 2012011488 W KR2012011488 W KR 2012011488W WO 2013100580 A1 WO2013100580 A1 WO 2013100580A1
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fusion protein
seq
acid sequence
wnt
variant
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PCT/KR2012/011488
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French (fr)
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Yon-Rak Choi
Hak-Zoo Kim
Pan-Soo Kim
Mi-Gi LEE
Song-I IM
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Gyeonggi Institute Of Science & Technology Promotion
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    • 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
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention relates to a novel fusion protein capable of binding to WNT. More specifically, the invention relates to a novel fusion protein combining a frizzled motif of Type XVIII collagen (COL18) containing CRD and a Fc domain of human IgG.
  • the fusion protein according to the invention binds to Wnt and blocks its signaling pathway, thereby treating or preventing diseases associated with Wnt signaling pathway as well as diagnosing the diseases.
  • COL18 contains endostatin, which is an inhibitor of angiogenesis and tumor growth, at the carboxyl terminal (Felbor, U., et al., EMBO J 19: 11871194, 2000), and the endostatin can be used to treat cancers by inhibiting angiogenesis around the cancer cells (OReilly, M. S., et al., Cell 88: 277285, 1997).
  • the variant 3 of Type XVIII collagen can inhibit Wnt/ ⁇ -catenin signal transduction, thereby inhibiting the growth of cancer cells (Quelard, D., et al., PloS ONE e1878, 2008).
  • the variant 3 of COL18 and its frizzled motif are highly insoluble proteins, it raises a problem that the intracellular expression and extracellular secretion are decreased. Therefore, the variant 3 of COL18 and its frizzled motif known in the prior art per se are not efficient in using as therapeutic protein. There is a need to modify it for use in industry.
  • the purpose of the invention is to determine the most suitable sequence for binding to Wnt and blocking its signal transduction based on the frizzled motif of COL18, provide a fusion protein having such a sequence, and provide a pharmaceutical composition for preventing or treating diseases associated with Wnt signaling pathway comprising the fusion protein.
  • the further purpose of the invention is to elevate the intracellular expression and extracellular secretion by increasing the solubility of fusion protein, elevate the stability of protein by adjusting its secretion and introducing a linker and a thrombin cleavage site, and thus obtain effectively an effect of inhibiting Wnt/ ⁇ -catenin signaling pathway in vivo or intracellularly.
  • the invention provides a method of producing the fusion protein, comprising purifying the fusion protein more rapidly and easily. Thus, the resulting fusion protein can be used more effectively in view of industrial productivity.
  • the inventors designed various variants of COL18 as a form of fusion protein combining Fc domain, and then found fusion proteins capable of binding to Wnt and blocking Wnt/ ⁇ -catenin signal transduction.
  • the inventors found cysteine residues which are not involved in forming a disulfide bond in the frizzled motif of COL18, and then substituted the cysteine residues with other amino acid residues or inserted a thrombin cleavage site into the fusion protein in order to modify the sequence of fusion protein.
  • the inventors prepared new fusion proteins FZC18-Fc T0 and FZC18-Fc V0 by combining the frizzled motif of COL18 containing cysteine-rich domain (CRD) being a Wnt binding site (hereinafter, "FZC18") and the Fc domain of human IgG.
  • CCD cysteine-rich domain
  • FZC18 a Wnt binding site
  • the inventors prepared vectors comprising nucleic acid sequences encoding said fusion proteins.
  • the fusion proteins according to the invention were obtained from culture solutions of a Chinese Hamster Ovary cell transformed with the vector. Since the fusion protein according to the invention contains Fc domain specifically, it can be obtained in high yield even though only an affinity chromatography is used as the purification step.
  • the resulting fusion protein was not precipitated even when preserved for a long time, but maintained its natural form well. Thus, it represents a good stability. Particularly, in the FZC18-Fc T0 combining a thrombin domain to increase its structural stability, the cleavage of Fc occurred frequently was inhibited, and accordingly the stability and expression of protein were increased (see Fig. 8). Moreover, since the fusion protein according to the invention has a good solubility, it is expressed well in vivo or intracellularly, and also a solvent required for processing the fusion protein is not limited to the particular type.
  • the fusion protein according to the invention contains Fc domain specifically, the expression of fusion protein can be measured rapidly by using an anti-Fc antibody.
  • the pharmaceutical composition comprising the fusion protein of the invention can prevent, treat and diagnose cancer such as colon cancer, liver cancer or breast cancer, epithelioma such as squamous cell carcinoma, and fibrosis such as liver disorder which are known to be mainly caused by Wnt overexpression.
  • cancer such as colon cancer, liver cancer or breast cancer
  • epithelioma such as squamous cell carcinoma
  • fibrosis such as liver disorder which are known to be mainly caused by Wnt overexpression.
  • Fig. 1a shows a pCEZ expression vector for transient expression, which contains pUC replication origin and ampicillin resistant genes, and CMV promoter and SV40 polyadenylation signal for expression in mammalian cell.
  • the vector expresses EBNA-1 and contains OriP sequence, and thus the transient expression can be maintained for a long time and the amount of expression can be increased.
  • the vector contains a thymidine kinase promoter and neomycin resistant genes as a mammalian selection marker.
  • the upper part of Fig. 1a shows a restriction enzyme site located at a multi-cloning site in the pCEZ expression vector.
  • Fig. 1b shows a pIDZ expression vector for preparing stable cell lines, which contains pUC replication origin and ampicillin resistant genes, and CMV promoter and growth hormone polyadenylation signal for expression in mammalian cell.
  • the vector expresses DHFR by IRES, and its productivity can be improved using gene amplification by MTX.
  • the vector contains a SV40 promoter and neomycin resistant genes as a mammalian selection marker.
  • the upper part of Fig. 1b shows a restriction enzyme site located at a multi-cloning site in the pIDZ expression vector.
  • Fig. 1c shows simple schemes of the C18N-Fc, FZC18-Fc T0 and FZC18-Fc V0 designed according to the invention.
  • Fig. 2a shows a nucleic acid sequence of Exon 1 of variant 3 of human COL18.
  • Fig. 2b shows the amino acid sequence 1-450 of COL18, in which the frizzled motif is marked in red color at the site 216-450, the cysteine-rich domain (CRD) is marked in yellow color at the site 329-446, the cysteine is marked in green color, and the N-glycosylation site is marked in blue color.
  • CCD cysteine-rich domain
  • Fig. 3a shows a cloning scheme and sequence analysis performed on FZC18-Fc T0.
  • the restriction enzyme sites are HindIII, BamHI, KpnI, XhoI and XbaI.
  • the Kozak sequence is marked in green color
  • the signal sequence is marked in blue color
  • the thrombin cleavage site is marked in violet color.
  • the nucleic acid sequence 321-450 of COL18 is marked in yellow color
  • Fc is marked in gray color.
  • Fig. 3b shows structures of the proteins and the fusion proteins used in the invention.
  • Fig. 4 shows the probability of secretion signal obtained by SignalP-NN. More specifically, Fig. 4a relates to the signal sequence of COL18, and Fig. 4b relates to the signal sequence of Ig kappa chain.
  • Fig. 5 relates to the experiment measuring expressions of Fc fusion protein using ELISA. More specifically, Fig. 5a shows a schematic diagram of experimental method, and Fig. 5b shows the ELISA result measured on the Fc fusion protein, which is obtained after cells transfecting each of the vector construct are incubated in conditioned medium, i.e. serum containing medium (named as ‘SM-5’) or serum free medium (named as ‘SFM’).
  • serum containing medium named as ‘SM-5’
  • SFM serum free medium
  • Fig. 6a shows the results of immunoblotting performed using an anti-Fc antibody after SDS-PAGE electrophoresing the cell lysate sample, the medium sample and the sample immunoprecipitating the medium with Protein A bead (named as ‘Media IP’).
  • Fig. 6b shows the results measuring the expression in cell culture solutions by the immunoprecipitation using Protein A bead and the immunoblotting using an anti-Fc antibody (H represents 60kDa marker, and L represents 30kDa marker).
  • Fig. 7 relates to the experiment measuring the binding ability of Fc fusion protein and Wnt by using ELISA. More specifically, Fig. 7a shows a schematic diagram of experimental method (HRP represents horseradish peroxidase), and Fig. 7b shows the ELISA result measuring Wnt binding ability in each sample.
  • HRP horseradish peroxidase
  • Fig. 8 shows the result of that the proteins purified by an affinity chromatography using Protein A were electrophoresed by 4-12% gradient SDS-PAGE (with MOPS buffer), and then dyed by Coomassie Brilliant Blue. Frz8 was purchased from RnD company. Unlike the FZC18-Fc VO fusion protein, the FZC18-Fc T0 fusion protein did not show the cleavage of Fc domain.
  • Fig. 9 relates to the experiment measuring the binding ability of Wnt and each protein sample in vitro.
  • Fig. 10 relates to the experiment measuring with Wnt/ ⁇ -catenin reporter gene assay whether the Wnt induced activity is inhibited by each of the fusion protein.
  • each triangle indicates that the concentration of protein is higher in the order of 1, 3, 10 ⁇ g/ml toward the right direction.
  • the inventors used two methods to construct an expression system of animal cells. First, fusion proteins of COL18 were prepared, and then a transient expression system was used to determine their expressions rapidly. Next, stable cell lines were prepared for stable expressions of the selected fusion protein candidates.
  • HEK293E cell lines were transfected using Polyethylenimine (PEI) which is a cationic polymer. The culture solution was recovered 4 days after transfection, and then ELISA was used to determine the expression of fusion proteins and compare the relative expression levels.
  • PEI Polyethylenimine
  • the pCEZ vector shown in Fig. 1a is a mammalian expression vector containing pUC replication origin and ampicillin resistant genes and having a size 10.1 kb. It is expressed by CMV promoter and the BspEI-ClaI-BamHI-EcoRI-EcoRV-NotI-XhoI-XbaI-PvuII restriction enzyme site is incorporated in its multi-cloning site.
  • EBNA-1 Epstein-Barr nuclear antigen 1
  • neomycin resistant genes as a mammalian selection marker are incorporated.
  • the pIDZ vector shown in Fig. 1b is a mammalian expression vector containing pUC replication origin and ampicillin resistant genes and having a size 6.4 kb. It is regulated by CMV promoter and the BspEI-ClaI-BamHI-EcoRI-EcoRV-NotI-XhoI-XbaI-HpaI restriction enzyme site is incorporated in its multi-cloning site. It was designed such that dihydrofolate reductase (DHFR) is expressed via Internal Ribosome Entry Site (IRES), and the neomycin resistant genes as a mammalian selection marker are incorporated.
  • DHFR dihydrofolate reductase
  • IVS Internal Ribosome Entry Site
  • the Fc of human IgG was incorporated between XhoI and XbaI of each of the covector and the HindIII-Kozak sequence-secretion signal was incorporated between ClaI and BamHI, thereby producing Fc expression vectors.
  • Each vector was named as "pCEZ-S-Fc” and “pIDZ-S-Fc” depending on its backbone, respectively.
  • a nucleic acid sequence corresponding to the amino acid sequence 1-450 of COL18 including Kozak sequence was incorporated between HindIII and XhoI of S-Fc expression vector, and named as "C18N-Fc" (see Fig. 1c).
  • the construct incorporating a nucleic acid sequence corresponding to the amino acid sequence 321-450 of COL18 between BamHI and XhoI of S-Fc expression vector was named as "FZC18-Fc V0”
  • the construct further incorporating a thrombin cleavage site between COL18 and Fc was named as "FZC18-Fc T0" (see Fig. 1c).
  • Frizzled receptor 8 - Fc fusion protein (Frz8-Fc) was also prepared.
  • a nucleic acid sequence corresponding to the amino acid sequence 1-156 of Frz8 and Kozak sequence was incorporated between HindIII and XhoI of S-Fc expression vector.
  • C18N gene was cloned by using the genomic DNA of HEK293E cell line. NCBI’s accession number NM_130444 was referred to as the mRNA sequence of variant 3 of human COL18, and UniProtKB P39060 sequence was referred to as its amino acid sequence.
  • the Exon 1 of genomic DNA sequence contains N-terminal sequences 1-450, and thus the human genomic DNA was used as a template to obtain C18N gene (see Fig. 2a).
  • the CRD of COL18 contains eleven cysteines (Cys), ten cysteines among them are believed to form disulfide bridges constituting of the frizzled motif’s topology, and an orphan cysteine is believed to influence the extracellular secretion and insolubility of fusion protein.
  • the inventors designed various constructs for finding the orphan Cys which is not involved in forming the disulfide bond, for example fusion proteins substituting cysteine (Cys) at the sites 408 and 409 with glycine (Gly) via mutagenesis of prototype, and fusion proteins whose length of linker shortens or lengthens.
  • Cys cysteine
  • Gly glycine
  • FZC18-Fc T0 (corresponding to the amino acid sequence 321-450 of COL18) (see Fig. 3a) was designed such that its amino terminal linker shortens by eight amino acids, and then the produced fusion protein (corresponding to the amino acid sequence 329-450 of COL18) was named as "FZC18-Fc T1".
  • FZC18-Fc T0 was designed such that its carboxyl terminal linker shortens by four amino acids, and then the produced fusion protein (corresponding to the amino acid sequence 321-446 of COL18) was named as "FZC18-Fc T2".
  • FZC18-Fc T0 was designed such that both its amino terminal linker and carboxyl terminal linker shorten, and then the produced fusion protein (corresponding to the amino acid sequence 329-446 of COL18) was named as "FZC18-Fc T3".
  • a fusion protein FZC18-Fc V0 not having a thrombin cleavage site was modified in the same manner, and then the produced fusion proteins were respectively named as “FZC18-Fc V1”, “FZC18-Fc V2” and “FZC18-Fc V3”.
  • C408G mutant, C409G mutant and C408,409G double mutant were designed for each of the fusion protein.
  • FIG. 3b shows the structures of FZC18-Fc M1, FZC18-Fc M3, FZC18-Fc M4, FZC18-Fc M6 and FZC18-Fc M7 naming said point mutations.
  • the expression and Wnt binding ability of each fusion protein were determined to compare the differences depending on the existence of thrombin cleavage site, the lengths of amino terminal linker and carboxyl terminal linker, and the mutant of orphan Cys.
  • UniProtKB Q7Z5W1 (the region 244-470) was referred to as the heavy chain of human IgG1, and NCBI’s accession number J00228 was referred to as the genes corresponding to 227 amino acids of P01857 (the region 104-330).
  • fusion protein was determined and quantified by using the property of Fc domain.
  • Cells were transfected with each of the vector construct, and then incubated for 4 days in serum containing medium (5%) or serum free medium, respectively. After that, ELISA was performed on the sample obtained from the conditioned medium.
  • the human IgG Fc-specific binding antibody was used in the Sandwich ELISA method (see Fig. 5a). More specifically, the captured antibody was immobilized and blocked on the plate, and then the sample was incubated and the signal was measured using Peroxidase conjugated anti-Fc antibody.
  • the signal in the level of background was detected in IgG of serum containing medium and bovine serum, whereas the specific signal was detected in the Fc fusion proteins (see Fig. 5b). All of the fusion proteins showed higher expression in serum containing medium than in serum free medium. Among the fusion proteins, S-Fc has the shortest length, and thus the level of expression is the highest. In the FZC18-Fc T0, FZC18-Fc T1 and FZC18-Fc T2, the level of expression decreased as the length of linker shortens. This pattern was also shown in the FZC18-Fc V0, FZC18-Fc V1 and FZC18-Fc V2.
  • the FZC18-Fc T0 prototype has a decreased expression when the length of either amino terminal linker or carboxyl terminal linker shortens, whereas it does not have a decreased expression when the length of both amino terminal linker and carboxyl terminal linker shorten. It is understood that both terminal linkers are structurally in connection each other, and thus involved in the skeletal structure.
  • Western blotting was performed using the fusion proteins selected through the analysis of the ELISA results. More specifically, each of the cell lysate sample, the medium sample and the sample immunoprecipitating the medium with Protein A bead was prepared in the conditioned serum-free medium after transfection, and then an anti-Fc antibody was used in the western blotting (see Fig. 6a). After immunoprecipitating using Protein A, the result of western blotting using an anti-Fc antibody is shown in Fig. 6b. Depending on the expression, various forms of proteins were identified.
  • ELISA was performed to determine the Wnt binding ability.
  • the Wnt-3a protein was immobilized and blocked on the plate, and then the Fc fusion protein sample was incubated and the signal was measured using Peroxidase conjugated anti-Fc antibody (see Fig. 7a).
  • ELISA was performed on the conditioned serum containing medium after transfection, and the same samples as in the Fig. 5b were used. Even though the background signal was detected in high level, the signals in the Frz8, C18N-Fc, FZC18-Fc T0, FZC18-Fc V0 and FZC18-Fc V1 were detected in much higher level than that in the Fc sample (see Fig. 7b).
  • the inventors prepared CHO cell lines transfecting said fusion proteins via pIDZ vector.
  • stable CHO cell lines were prepared using the S-Fc and Frz8.
  • Each of the construct was transfected to DHFR-deficient CHO cell line using Effectene, and then parental clones were obtained by selection using G418 (500ng/ml) and methotrexate (MTX, 1nM) for three weeks. And then, the protein-producing cell lines adapted to suspension culture were established.
  • the purified fusion proteins were quantified by Bradford method, electrophoresed by 4-12% gradient SDS-PAGE (with MOPS buffer), and identified by Coomassie method (see Fig. 8).
  • the FZC18-Fc T0 fusion protein incorporating a thrombin cleavage site has higher expression than the FZC18-Fc V0 fusion protein, and Fc cleavage was not observed at about 30kDa, meaning that the FZC18-Fc T0 fusion protein has a high stability.
  • the fusion protein purified by an affinity chromatography using Protein A has about 95 % of purity. This result means that 90 % or more purity can be obtained by a purifying method consisting of only one single step.
  • the inventors performed a reporter assay in order to know whether said purified fusion proteins FZC18-Fc T0 and FZC18-Fc V0 influence a Wnt induced reporter.
  • T cell factor reporter (TCF-luc) and Renilla-luciferase were transfected to HEK293 cell by Effectene. 24 hours after transfection, it was treated with Wnt-3a (500ng/ml) in a new medium, or with Wnt-3a and the fusion protein (1, 3, and 10 ⁇ g/ml). After it was incubated for additional 24 hours, the cell culture solutions were recovered. The luminescence of each sample was measured using Dual luciferase reporter assay system.
  • the sample without adding Wnt-3a (negative control: NC) was normalized by Renilla-luciferase activity, and its fold induction was measured. Contrary to the negative control, the sample adding Wnt showed the induction of reporter activity. It was observed that Fc did not influence the reporter activity by Wnt, whereas Frz8, C18N, FZC18-Fc T0 and FZC18-Fc V0 inhibited the reporter activity by Wnt (see Fig. 10).

Abstract

The invention relates to a novel fusion protein capable of binding to WNT. More specifically, the invention relates to a novel fusion protein combining a frizzled motif of COL18 containing CRD and a Fc domain of human IgG. The fusion protein according to the invention binds to Wnt and blocks its signal transduction, thereby treating or preventing diseases associated with Wnt signaling pathway as well as diagnosing the diseases.

Description

NOVEL FUSION PROTEIN BINDING TO WNT
The invention relates to a novel fusion protein capable of binding to WNT. More specifically, the invention relates to a novel fusion protein combining a frizzled motif of Type XVIII collagen (COL18) containing CRD and a Fc domain of human IgG. The fusion protein according to the invention binds to Wnt and blocks its signaling pathway, thereby treating or preventing diseases associated with Wnt signaling pathway as well as diagnosing the diseases.
It has been reported that the secretory glycoprotein Wnt functions in establishing cell polarity during cytogenesis (Moon, R. T., et al., Science 296: 1644-1646, 2002) and plays an important role in the growth of stem cells and cancer cells (Reya, T., et al., Nature 434: 843-850, 2005). Therefore, methods for treating diseases associated with Wnt signaling pathway comprising blocking Wnt signal transduction have been researched (Moon, R. T., et al., Nat Rev Genet 5: 691-701, 2004). For example, frizzled proteins and secreted Frizzled Related Proteins (sFRPs), which are cell receptors known to bind to Wnt, have been developed (WO 2008/031009 and WO 98/54325).
It has been known that COL18 contains endostatin, which is an inhibitor of angiogenesis and tumor growth, at the carboxyl terminal (Felbor, U., et al., EMBO J 19: 11871194, 2000), and the endostatin can be used to treat cancers by inhibiting angiogenesis around the cancer cells (OReilly, M. S., et al., Cell 88: 277285, 1997). On the other hand, it has also been known that the variant 3 of Type XVIII collagen can inhibit Wnt/β-catenin signal transduction, thereby inhibiting the growth of cancer cells (Quelard, D., et al., PloS ONE e1878, 2008). This inhibition effect of Wnt/β-catenin signaling pathway results from the frizzled motif which is specifically present in the type XVIII collagen (hereinafter, "COL18"). The author of said literature compared expressions of collagen genes in fibrosis, particularly hepatocellular carcinoma, and then discovered that the variant 3 of COL18 can be used as an effective component for the diagnosis and treatment of hepatocellular carcinoma (PCT/EP2008/067779). In addition, it was discovered from the animal model transplanting cancer cells transducing collagen genes that the growth of cancer cell is slowed down when the frizzled motif of COL18 is expressed.
Since the variant 3 of COL18 and its frizzled motif are highly insoluble proteins, it raises a problem that the intracellular expression and extracellular secretion are decreased. Therefore, the variant 3 of COL18 and its frizzled motif known in the prior art per se are not efficient in using as therapeutic protein. There is a need to modify it for use in industry.
The purpose of the invention is to determine the most suitable sequence for binding to Wnt and blocking its signal transduction based on the frizzled motif of COL18, provide a fusion protein having such a sequence, and provide a pharmaceutical composition for preventing or treating diseases associated with Wnt signaling pathway comprising the fusion protein. In addition, the further purpose of the invention is to elevate the intracellular expression and extracellular secretion by increasing the solubility of fusion protein, elevate the stability of protein by adjusting its secretion and introducing a linker and a thrombin cleavage site, and thus obtain effectively an effect of inhibiting Wnt/β-catenin signaling pathway in vivo or intracellularly. Moreover, the invention provides a method of producing the fusion protein, comprising purifying the fusion protein more rapidly and easily. Thus, the resulting fusion protein can be used more effectively in view of industrial productivity.
To achieve the purposes of the invention, the inventors designed various variants of COL18 as a form of fusion protein combining Fc domain, and then found fusion proteins capable of binding to Wnt and blocking Wnt/β-catenin signal transduction. At the same time, to solve the problems of the prior art relating to the solubility, intracellular expression and extracellular secretion of fusion protein, the inventors found cysteine residues which are not involved in forming a disulfide bond in the frizzled motif of COL18, and then substituted the cysteine residues with other amino acid residues or inserted a thrombin cleavage site into the fusion protein in order to modify the sequence of fusion protein.
As a result, the inventors prepared new fusion proteins FZC18-Fc T0 and FZC18-Fc V0 by combining the frizzled motif of COL18 containing cysteine-rich domain (CRD) being a Wnt binding site (hereinafter, "FZC18") and the Fc domain of human IgG.
In addition, the inventors prepared vectors comprising nucleic acid sequences encoding said fusion proteins. The fusion proteins according to the invention were obtained from culture solutions of a Chinese Hamster Ovary cell transformed with the vector. Since the fusion protein according to the invention contains Fc domain specifically, it can be obtained in high yield even though only an affinity chromatography is used as the purification step.
The resulting fusion protein was not precipitated even when preserved for a long time, but maintained its natural form well. Thus, it represents a good stability. Particularly, in the FZC18-Fc T0 combining a thrombin domain to increase its structural stability, the cleavage of Fc occurred frequently was inhibited, and accordingly the stability and expression of protein were increased (see Fig. 8). Moreover, since the fusion protein according to the invention has a good solubility, it is expressed well in vivo or intracellularly, and also a solvent required for processing the fusion protein is not limited to the particular type.
Since the fusion protein according to the invention contains Fc domain specifically, the expression of fusion protein can be measured rapidly by using an anti-Fc antibody.
The pharmaceutical composition comprising the fusion protein of the invention can prevent, treat and diagnose cancer such as colon cancer, liver cancer or breast cancer, epithelioma such as squamous cell carcinoma, and fibrosis such as liver disorder which are known to be mainly caused by Wnt overexpression.
Fig. 1a shows a pCEZ expression vector for transient expression, which contains pUC replication origin and ampicillin resistant genes, and CMV promoter and SV40 polyadenylation signal for expression in mammalian cell. The vector expresses EBNA-1 and contains OriP sequence, and thus the transient expression can be maintained for a long time and the amount of expression can be increased. The vector contains a thymidine kinase promoter and neomycin resistant genes as a mammalian selection marker. The upper part of Fig. 1a shows a restriction enzyme site located at a multi-cloning site in the pCEZ expression vector.
Fig. 1b shows a pIDZ expression vector for preparing stable cell lines, which contains pUC replication origin and ampicillin resistant genes, and CMV promoter and growth hormone polyadenylation signal for expression in mammalian cell. The vector expresses DHFR by IRES, and its productivity can be improved using gene amplification by MTX. The vector contains a SV40 promoter and neomycin resistant genes as a mammalian selection marker. The upper part of Fig. 1b shows a restriction enzyme site located at a multi-cloning site in the pIDZ expression vector.
Fig. 1c shows simple schemes of the C18N-Fc, FZC18-Fc T0 and FZC18-Fc V0 designed according to the invention.
Fig. 2a shows a nucleic acid sequence of Exon 1 of variant 3 of human COL18.
Fig. 2b shows the amino acid sequence 1-450 of COL18, in which the frizzled motif is marked in red color at the site 216-450, the cysteine-rich domain (CRD) is marked in yellow color at the site 329-446, the cysteine is marked in green color, and the N-glycosylation site is marked in blue color.
Fig. 3a shows a cloning scheme and sequence analysis performed on FZC18-Fc T0. The restriction enzyme sites are HindIII, BamHI, KpnI, XhoI and XbaI. The Kozak sequence is marked in green color, the signal sequence is marked in blue color, and the thrombin cleavage site is marked in violet color. The nucleic acid sequence 321-450 of COL18 is marked in yellow color, and Fc is marked in gray color.
Fig. 3b shows structures of the proteins and the fusion proteins used in the invention.
Fig. 4 shows the probability of secretion signal obtained by SignalP-NN. More specifically, Fig. 4a relates to the signal sequence of COL18, and Fig. 4b relates to the signal sequence of Ig kappa chain.
Fig. 5 relates to the experiment measuring expressions of Fc fusion protein using ELISA. More specifically, Fig. 5a shows a schematic diagram of experimental method, and Fig. 5b shows the ELISA result measured on the Fc fusion protein, which is obtained after cells transfecting each of the vector construct are incubated in conditioned medium, i.e. serum containing medium (named as ‘SM-5’) or serum free medium (named as ‘SFM’).
Fig. 6a shows the results of immunoblotting performed using an anti-Fc antibody after SDS-PAGE electrophoresing the cell lysate sample, the medium sample and the sample immunoprecipitating the medium with Protein A bead (named as ‘Media IP’).
Fig. 6b shows the results measuring the expression in cell culture solutions by the immunoprecipitation using Protein A bead and the immunoblotting using an anti-Fc antibody (H represents 60kDa marker, and L represents 30kDa marker).
Fig. 7 relates to the experiment measuring the binding ability of Fc fusion protein and Wnt by using ELISA. More specifically, Fig. 7a shows a schematic diagram of experimental method (HRP represents horseradish peroxidase), and Fig. 7b shows the ELISA result measuring Wnt binding ability in each sample.
Fig. 8 shows the result of that the proteins purified by an affinity chromatography using Protein A were electrophoresed by 4-12% gradient SDS-PAGE (with MOPS buffer), and then dyed by Coomassie Brilliant Blue. Frz8 was purchased from RnD company. Unlike the FZC18-Fc VO fusion protein, the FZC18-Fc T0 fusion protein did not show the cleavage of Fc domain.
Fig. 9 relates to the experiment measuring the binding ability of Wnt and each protein sample in vitro.
Fig. 10 relates to the experiment measuring with Wnt/β-catenin reporter gene assay whether the Wnt induced activity is inhibited by each of the fusion protein. In the lower part of Fig. 10, each triangle indicates that the concentration of protein is higher in the order of 1, 3, 10 μg/ml toward the right direction.
The inventors used two methods to construct an expression system of animal cells. First, fusion proteins of COL18 were prepared, and then a transient expression system was used to determine their expressions rapidly. Next, stable cell lines were prepared for stable expressions of the selected fusion protein candidates.
In the transient expression system, HEK293E cell lines were transfected using Polyethylenimine (PEI) which is a cationic polymer. The culture solution was recovered 4 days after transfection, and then ELISA was used to determine the expression of fusion proteins and compare the relative expression levels.
The inventors prepared pCEZ (Fig. 1a) and pIDZ (Fig. 1b) as expression vectors for use in the invention. The pCEZ vector shown in Fig. 1a is a mammalian expression vector containing pUC replication origin and ampicillin resistant genes and having a size 10.1 kb. It is expressed by CMV promoter and the BspEI-ClaI-BamHI-EcoRI-EcoRV-NotI-XhoI-XbaI-PvuII restriction enzyme site is incorporated in its multi-cloning site. It was designed such that EBNA-1 (Epstein-Barr nuclear antigen 1) is prolonged-transiently expressed via OriP, and the neomycin resistant genes as a mammalian selection marker are incorporated. The pIDZ vector shown in Fig. 1b is a mammalian expression vector containing pUC replication origin and ampicillin resistant genes and having a size 6.4 kb. It is regulated by CMV promoter and the BspEI-ClaI-BamHI-EcoRI-EcoRV-NotI-XhoI-XbaI-HpaI restriction enzyme site is incorporated in its multi-cloning site. It was designed such that dihydrofolate reductase (DHFR) is expressed via Internal Ribosome Entry Site (IRES), and the neomycin resistant genes as a mammalian selection marker are incorporated.
The Fc of human IgG was incorporated between XhoI and XbaI of each of the covector and the HindIII-Kozak sequence-secretion signal was incorporated between ClaI and BamHI, thereby producing Fc expression vectors. Each vector was named as "pCEZ-S-Fc" and "pIDZ-S-Fc" depending on its backbone, respectively.
A nucleic acid sequence corresponding to the amino acid sequence 1-450 of COL18 including Kozak sequence was incorporated between HindIII and XhoI of S-Fc expression vector, and named as "C18N-Fc" (see Fig. 1c). In addition, the construct incorporating a nucleic acid sequence corresponding to the amino acid sequence 321-450 of COL18 between BamHI and XhoI of S-Fc expression vector was named as "FZC18-Fc V0", and the construct further incorporating a thrombin cleavage site between COL18 and Fc was named as "FZC18-Fc T0" (see Fig. 1c).
For comparative experiments, the expression vector of Frizzled receptor 8 - Fc fusion protein (Frz8-Fc) was also prepared. A nucleic acid sequence corresponding to the amino acid sequence 1-156 of Frz8 and Kozak sequence was incorporated between HindIII and XhoI of S-Fc expression vector.
In order to prepare an expression vector of Wnt, a nucleic acid sequence corresponding to the amino acid sequence of Kozak sequence, Wnt-3a full length (1-352) and thrombin cleavage site excluding stop codon was incorporated between expression vector HimdIII and XbaI, and flag-tagged between XbaI and HpaI.
C18N gene was cloned by using the genomic DNA of HEK293E cell line. NCBI’s accession number NM_130444 was referred to as the mRNA sequence of variant 3 of human COL18, and UniProtKB P39060 sequence was referred to as its amino acid sequence. The Exon 1 of genomic DNA sequence contains N-terminal sequences 1-450, and thus the human genomic DNA was used as a template to obtain C18N gene (see Fig. 2a). Using the frizzled motif (the amino acid sequence 329-446 of COL18) containing CRD, the FZC18-Fc T0 (containing a thrombin cleavage site) and the FZC18-Fc V0 (not containing a thrombin cleavage site) were respectively prepared and cloned (see Figs. 1c and 2b).
It was predicted from a sequence analysis that the C18N gene does not contain O-glycosylation site, but contain three N-glycosylation sites (at its amino acid sequences 68, 129 and 164, respectively). Said predicted N-glycosylation sites are not contained in the frizzled motif (see Fig. 2b). The CRD of COL18 contains eleven cysteines (Cys), ten cysteines among them are believed to form disulfide bridges constituting of the frizzled motif’s topology, and an orphan cysteine is believed to influence the extracellular secretion and insolubility of fusion protein. Accordingly, the inventors designed various constructs for finding the orphan Cys which is not involved in forming the disulfide bond, for example fusion proteins substituting cysteine (Cys) at the sites 408 and 409 with glycine (Gly) via mutagenesis of prototype, and fusion proteins whose length of linker shortens or lengthens.
FZC18-Fc T0 (corresponding to the amino acid sequence 321-450 of COL18) (see Fig. 3a) was designed such that its amino terminal linker shortens by eight amino acids, and then the produced fusion protein (corresponding to the amino acid sequence 329-450 of COL18) was named as "FZC18-Fc T1". FZC18-Fc T0 was designed such that its carboxyl terminal linker shortens by four amino acids, and then the produced fusion protein (corresponding to the amino acid sequence 321-446 of COL18) was named as "FZC18-Fc T2". FZC18-Fc T0 was designed such that both its amino terminal linker and carboxyl terminal linker shorten, and then the produced fusion protein (corresponding to the amino acid sequence 329-446 of COL18) was named as "FZC18-Fc T3". In addition, a fusion protein FZC18-Fc V0 not having a thrombin cleavage site was modified in the same manner, and then the produced fusion proteins were respectively named as “FZC18-Fc V1”, “FZC18-Fc V2” and “FZC18-Fc V3”. In addition, C408G mutant, C409G mutant and C408,409G double mutant were designed for each of the fusion protein. The table of Fig. 3b shows the structures of FZC18-Fc M1, FZC18-Fc M3, FZC18-Fc M4, FZC18-Fc M6 and FZC18-Fc M7 naming said point mutations. The expression and Wnt binding ability of each fusion protein were determined to compare the differences depending on the existence of thrombin cleavage site, the lengths of amino terminal linker and carboxyl terminal linker, and the mutant of orphan Cys.
SignalP-NN program was run in order to predict and design the extracellular secretion of fusion protein. It was observed that all constructs showed the extracellular secretion of seventy amino acids at the N-terminal and the cleavage after secretion. Even though the FZC18-Fc T0 and the FZC18-Fc V0 used Ig kappa signal sequence instead of signal sequence of COL18, they were observed to have higher probability score (0.998 vs 1.000, see Figs. 4a and 4b).
Frz8, Wnt-3a and Fc were cloned by a gene synthesization. NCBI’s accession number NP_114072 and UniProtKB Q9H461 were referred to as the nucleic acid sequence and the amino acid sequence of Frz8, and NCBI’s accession number NM_033131 and UniProtKB P56704 were referred to as the nucleic acid sequence and the amino acid sequence of Wnt-3a. Relating to Fc genes, UniProtKB Q7Z5W1 (the region 244-470) was referred to as the heavy chain of human IgG1, and NCBI’s accession number J00228 was referred to as the genes corresponding to 227 amino acids of P01857 (the region 104-330).
The expression of fusion protein was determined and quantified by using the property of Fc domain. Cells were transfected with each of the vector construct, and then incubated for 4 days in serum containing medium (5%) or serum free medium, respectively. After that, ELISA was performed on the sample obtained from the conditioned medium. The human IgG Fc-specific binding antibody was used in the Sandwich ELISA method (see Fig. 5a). More specifically, the captured antibody was immobilized and blocked on the plate, and then the sample was incubated and the signal was measured using Peroxidase conjugated anti-Fc antibody.
As a result, the signal in the level of background was detected in IgG of serum containing medium and bovine serum, whereas the specific signal was detected in the Fc fusion proteins (see Fig. 5b). All of the fusion proteins showed higher expression in serum containing medium than in serum free medium. Among the fusion proteins, S-Fc has the shortest length, and thus the level of expression is the highest. In the FZC18-Fc T0, FZC18-Fc T1 and FZC18-Fc T2, the level of expression decreased as the length of linker shortens. This pattern was also shown in the FZC18-Fc V0, FZC18-Fc V1 and FZC18-Fc V2. On the other hand, in the FZC18-Fc T3 and FZC18-Fc V3, the level of expression increased as the length of linker shortens. Thus, the FZC18-Fc T0 prototype has a decreased expression when the length of either amino terminal linker or carboxyl terminal linker shortens, whereas it does not have a decreased expression when the length of both amino terminal linker and carboxyl terminal linker shorten. It is understood that both terminal linkers are structurally in connection each other, and thus involved in the skeletal structure.
Western blotting was performed using the fusion proteins selected through the analysis of the ELISA results. More specifically, each of the cell lysate sample, the medium sample and the sample immunoprecipitating the medium with Protein A bead was prepared in the conditioned serum-free medium after transfection, and then an anti-Fc antibody was used in the western blotting (see Fig. 6a). After immunoprecipitating using Protein A, the result of western blotting using an anti-Fc antibody is shown in Fig. 6b. Depending on the expression, various forms of proteins were identified.
In addition, ELISA was performed to determine the Wnt binding ability. The Wnt-3a protein was immobilized and blocked on the plate, and then the Fc fusion protein sample was incubated and the signal was measured using Peroxidase conjugated anti-Fc antibody (see Fig. 7a). ELISA was performed on the conditioned serum containing medium after transfection, and the same samples as in the Fig. 5b were used. Even though the background signal was detected in high level, the signals in the Frz8, C18N-Fc, FZC18-Fc T0, FZC18-Fc V0 and FZC18-Fc V1 were detected in much higher level than that in the Fc sample (see Fig. 7b).
Based on the results, it was verified that the fusion proteins FZC18-Fc T0 and FZC18-Fc V0 have a high expression and a Wnt-3a binding ability. Therefore, the inventors prepared CHO cell lines transfecting said fusion proteins via pIDZ vector. For comparative experiments, stable CHO cell lines were prepared using the S-Fc and Frz8. Each of the construct was transfected to DHFR-deficient CHO cell line using Effectene, and then parental clones were obtained by selection using G418 (500ng/ml) and methotrexate (MTX, 1nM) for three weeks. And then, the protein-producing cell lines adapted to suspension culture were established.
Each of the cell line was incubated to recover cell culture solutions, and then the solutions were purified by an affinity chromatography using Protein A. The purified fusion proteins were quantified by Bradford method, electrophoresed by 4-12% gradient SDS-PAGE (with MOPS buffer), and identified by Coomassie method (see Fig. 8). In particular, the FZC18-Fc T0 fusion protein incorporating a thrombin cleavage site has higher expression than the FZC18-Fc V0 fusion protein, and Fc cleavage was not observed at about 30kDa, meaning that the FZC18-Fc T0 fusion protein has a high stability. In addition, it was observed that the fusion protein purified by an affinity chromatography using Protein A has about 95 % of purity. This result means that 90 % or more purity can be obtained by a purifying method consisting of only one single step.
After the purified Fc fusion protein was incubated with flag-tagged Wnt-3a and immunoprecipitated by an anti-Flag bead, the immunoblotting using an anti-Fc antibody was performed to determine Wnt binding ability. The Wnt-3a bound to Frz8, FZC18-Fc T0 and FZC18-Fc V0, but did not bind to Fc (seeFig. 9).
The inventors performed a reporter assay in order to know whether said purified fusion proteins FZC18-Fc T0 and FZC18-Fc V0 influence a Wnt induced reporter. T cell factor reporter (TCF-luc) and Renilla-luciferase were transfected to HEK293 cell by Effectene. 24 hours after transfection, it was treated with Wnt-3a (500ng/ml) in a new medium, or with Wnt-3a and the fusion protein (1, 3, and 10 μg/ml). After it was incubated for additional 24 hours, the cell culture solutions were recovered. The luminescence of each sample was measured using Dual luciferase reporter assay system. The sample without adding Wnt-3a (negative control: NC) was normalized by Renilla-luciferase activity, and its fold induction was measured. Contrary to the negative control, the sample adding Wnt showed the induction of reporter activity. It was observed that Fc did not influence the reporter activity by Wnt, whereas Frz8, C18N, FZC18-Fc T0 and FZC18-Fc V0 inhibited the reporter activity by Wnt (see Fig. 10).

Claims (14)

  1. A fusion protein FZC18-Fc T0 having an amino acid sequence of SEQ ID NO: 2 or its variant having at least 95 % identity with the amino acid sequence of SEQ ID NO: 2 and having a Wnt binding activity.
  2. A fusion protein FZC18-Fc V0 having an amino acid sequence of SEQ ID NO: 3 or its variant having at least 95 % identity with the amino acid sequence of SEQ ID NO: 3 and having a Wnt binding activity.
  3. The fusion protein according to claim 1 or 2, characterized in that the variant is a variant in which the length of carboxyl terminal linker of Type XVIII collagen in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 shortens by 1 to 8 amino acids.
  4. The fusion protein according to claim 1 or 2, characterized in that the variant is a variant in which the length of amino terminal linker of Type XVIII collagen in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 shortens by 1 to 8 amino acids.
  5. The fusion protein according to claim 1 or 2, characterized in that the variant is a variant in which the length of amino terminal linker of Type XVIII collagen in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 lengthens by 105 amino acids at the site 216-320 of C18N.
  6. The fusion protein according to claim 1 or 2, characterized in that the variant is a variant in which at least one of cysteines in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 is substituted with glycine.
  7. The fusion protein according to claim 6, characterized in that the substitution is performed at the site 408, 409, or 408 and 409 in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
  8. A gene encoding the fusion protein according to claim 1 or 2.
  9. A recombinant vector comprising a nucleic acid sequence of the gene according to claim 8.
  10. A Chinese Hamster Ovary cell transformed with the recombinant vector according to claim 9.
  11. A pharmaceutical composition for preventing or treating cancer, epithelioma and fibrosis, comprising the fusion protein according to claim 1 or 2.
  12. A kit for diagnosis of cancer, epithelioma and fibrosis, comprising the fusion protein according to claim 1 or 2 and a human IgG Fc-specific antibody.
  13. A method of producing the fusion protein according to claim 1 or 2, comprising purifying the fusion protein by an affinity chromatography using Protein A.
  14. A method of detecting a Wnt binding ability of the fusion protein according to claim 1 or 2, comprising combining the fusion protein with Wnt.
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Citations (4)

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US20100092477A1 (en) * 2007-03-27 2010-04-15 Christopher Hovens Methods and compositions for treating prostate cancer
US20100098716A1 (en) * 2006-01-27 2010-04-22 Novagen Holding Corporation Recombinant human epo-fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
US20100196370A1 (en) * 2006-12-21 2010-08-05 Mogam Biotechnology Research Institute Fusion protein of immunoglobulin fc and human apolipoprotein(a) kringle fragment
US20110091416A1 (en) * 2007-05-30 2011-04-21 Genexine Co., Ltd Immunoglobulin fusion proteins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100098716A1 (en) * 2006-01-27 2010-04-22 Novagen Holding Corporation Recombinant human epo-fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
US20100196370A1 (en) * 2006-12-21 2010-08-05 Mogam Biotechnology Research Institute Fusion protein of immunoglobulin fc and human apolipoprotein(a) kringle fragment
US20100092477A1 (en) * 2007-03-27 2010-04-15 Christopher Hovens Methods and compositions for treating prostate cancer
US20110091416A1 (en) * 2007-05-30 2011-04-21 Genexine Co., Ltd Immunoglobulin fusion proteins

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