WO2021096275A1 - Fusion protein including modified interleukin-7 and tgf beta receptor ii and use thereof - Google Patents

Fusion protein including modified interleukin-7 and tgf beta receptor ii and use thereof Download PDF

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WO2021096275A1
WO2021096275A1 PCT/KR2020/015948 KR2020015948W WO2021096275A1 WO 2021096275 A1 WO2021096275 A1 WO 2021096275A1 KR 2020015948 W KR2020015948 W KR 2020015948W WO 2021096275 A1 WO2021096275 A1 WO 2021096275A1
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methionine
glycine
fusion protein
cancer
stbrii
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French (fr)
Korean (ko)
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성영철
이승우
김지혜
박한욱
박수정
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주식회사 제넥신
포항공과대학교 산학협력단
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Priority to AU2020383176A priority Critical patent/AU2020383176B2/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/5418IL-7
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/20Interleukins [IL]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/20Interleukins [IL]
    • A61K38/2046IL-7
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a fusion protein comprising a modified interleukin-7 and TGF beta receptor II and uses thereof.
  • TGF beta receptor II (transforming growth factor beta receptor II, TBRII) is encoded by the TGFBR2 gene in humans. It is a membrane protein of 70 to 80 kDa. TBRII forms a heterodimer with TBRI (TGF beta receptor I), and by binding with TGF- ⁇ to transmit intracellular signals, it regulates the transcription of genes related to cell proliferation.
  • TGF beta receptor I transforming growth factor beta receptor II
  • TBRII consists of a C-terminal protein kinase domain and an N-terminal ecto domain.
  • the ecto domain is a domain of a membrane protein that extends into the extracellular space, and forms a folded structure comprising a single helix stabilized by 9 beta chains and 6 disulfide bonds in the strand.
  • TGF- ⁇ known as a ligand of TBRII
  • TGF- ⁇ is an immunosuppressive cytokine that is overexpressed in cancer cells, and is known as one of the mechanisms of immune evasion of cancer cells, such as inhibiting the proliferation of T cells due to TGF- ⁇ secreted from cancer cells.
  • interleukin-7 is a cytokine that promotes an immune response via B cells and T cells, and particularly plays an important role in the adaptive immune system. Specifically, IL-7 activates immune functions through survival and differentiation of T cells and B cells, survival of lymphoid cells, and promotion of natural killer cells (NK cells).
  • T It is important for the development of cells and B cells. It binds to HGF (hepatocyte growth factor) and is a cofactor of pre-pro-B cell growth-stimulating factor and V(D)J rearrangement of T cell receptor beta (TCR ⁇ ). ) (Muegge K, 1993, Science 261 (5117): 93-5).
  • An object of the present invention is to provide a fusion protein comprising a modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII).
  • IL-7 modified interleukin-7
  • TRII TGF beta receptor II
  • Another object of the present invention is to provide an isolated nucleic acid molecule encoding the fusion protein.
  • Another object of the present invention is to provide an expression vector comprising the nucleic acid molecule.
  • Another object of the present invention is to provide a host cell comprising the expression vector.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
  • Another object of the present invention is to provide the use of a fusion protein comprising a modified IL-7 and TBRII to produce a pharmaceutical preparation having a prophylactic or therapeutic effect of cancer or infectious disease.
  • Another object of the present invention is to provide a method for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
  • the present invention provides a fusion protein comprising a modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII).
  • IL-7 modified interleukin-7
  • TRII TGF beta receptor II
  • the present invention provides an isolated nucleic acid molecule encoding the fusion protein.
  • the present invention provides an expression vector containing the nucleic acid molecule.
  • the present invention provides a host cell comprising the expression vector.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
  • the present invention provides the use of a fusion protein comprising a modified IL-7 and TBRII to produce a pharmaceutical preparation having a prophylactic or therapeutic effect of cancer or infectious disease.
  • the present invention provides a method for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
  • the fusion protein containing the modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII) of the present invention has a high production yield and can effectively inhibit cancer, so it can be usefully used in the treatment of cancer or infectious diseases. I can.
  • FIG. 1 shows the gene constructs of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII.
  • FIG. 3 is a result of measuring the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the dose
  • (a) is the number of CD8+ T cells after administration of the sTBRII-hyFc-IL7 fusion protein to a mouse animal model. This is the result of measurement
  • (b) is the result of measuring the increase rate of CD8+ T cells on the 7th day of administration
  • (c) is the result of measuring the number of CD4+ T cells
  • (d) is the result of measuring CD4+CD25+Foxp3+ Treg cells.
  • NK cells ⁇ : PBS; ⁇ : sTBRII-hyFc-IL7, 10 mpk; ⁇ : sTBRII-hyFc-IL7, 30 mpk; ⁇ : sTBRII-hyFc-IL7, 100 mpk).
  • Figure 4 is a result of measuring the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the administration route, (a) is a mouse animal model after administration of the sTBRII-hyFc-IL7 fusion protein, immune cells (lymphocytes) It is the result of measuring the number, and (b) is the result of measuring the increase rate of immune cells different from intravenous administration and subcutaneous administration on the 7th day of administration.
  • FIG. 5 is a result of analyzing the in vivo activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins
  • (a) is a mouse tumor model after administration of the sTBRII-hyFc-IL7 fusion protein, CD8+ T It is the result of measuring the number of cells
  • (b) is the result of measuring the increase rate of CD8+ T cells on the 7th day of administration
  • (c) is the result of administration of IL-7-hyFc-sTBRII fusion protein to a mouse tumor model This is the result of measuring the number of CD8+ T cells
  • (d) is the result of measuring the increase rate of CD8+ T cells at the 7th day of administration.
  • FIG. 8 is a result showing the (a) cell proliferation diagram and (b) its standard curve after treatment with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in 2E8 cell line.
  • FIG. 9 is a result showing a standard curve of luminance of SBE reporter after treatment with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in the SMAD Signaling Pathway SBE Reporter-HEK293 cell line.
  • Figure 10 is a result of comparative analysis of the simultaneous binding power of TGF- ⁇ 1 and IL-7R ⁇ of each fusion protein, after binding to TGF- ⁇ 1 (1st ligand), according to the concentration of IL-7R ⁇ (2nd ligand) (a) This is the result of measuring the binding strength of sTBRII-hyFc-IL7 and (b) IL-7-hyFc-sTBRII fusion proteins.
  • Figure 11 is a result of comparative analysis of the simultaneous binding power of TGF- ⁇ 1 and IL-7R ⁇ of each fusion protein, after binding to IL-7R ⁇ (1st ligand), according to the concentration of TGF- ⁇ 1 (2nd ligand) (a) This is the result of measuring the binding strength of sTBRII-hyFc-IL7 and (b) IL-7-hyFc-sTBRII fusion proteins.
  • One aspect of the present invention provides a fusion protein comprising a modified interleukin-7 (interleukin 7, IL-7) and a TGF beta receptor II (transforming growth factor beta receptor II, TBRII).
  • a modified interleukin-7 interleukin 7, IL-7
  • TGF beta receptor II transforming growth factor beta receptor II, TBRII
  • the modified IL-7 may have the following structure:
  • A is an oligopeptide consisting of 1 to 10 amino acid residues
  • the modified IL-7 is IL-7 or a polypeptide having similar activity.
  • IL-7 or a polypeptide having similar activity refers to a polypeptide or protein having the same or similar sequence and activity as IL-7.
  • the IL-7 may include an IL-7 protein or a fragment thereof.
  • IL-7 may be derived from human, white mouse, mouse, monkey, cow, or sheep.
  • human IL-7 may have the amino acid sequence of SEQ ID NO: 1 (Genbank Accession No. P13232); Rat IL-7 is a Genbank Accession No. May have the amino acid sequence disclosed in P56478; Mouse IL-7 is a Genbank Accession No. It is disclosed in P10168 and may have an amino acid sequence; Monkey IL-7 is a Genbank Accession No. It may have an amino acid sequence disclosed in NP_001279008; Bovine IL-7 is Genbank Accession No. May have the amino acid sequence disclosed in P26895; Yang IL-7 is Genbank Accession No. It may have an amino acid sequence disclosed in Q28540.
  • SEQ ID NO: 1 Genbank Accession No. P13232
  • Rat IL-7 is a Genbank Accession No. May have the amino acid sequence disclosed in P56478
  • Mouse IL-7 is a Genbank Accession No. It is disclosed in P10168 and may have an amino acid sequence
  • Monkey IL-7 is a Genbank Accession No. It may have
  • the IL-7 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
  • the modified IL-7 is the sequence of SEQ ID NO: 1 and about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% or more homology.
  • the IL-7 protein or fragment thereof may contain variously modified proteins or peptides, ie, variants.
  • the modification may be performed through a method of substituting, deleting or adding one or more proteins to wild-type IL-7, which does not alter the function of IL-7.
  • These various proteins or peptides include wild-type proteins and 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % Or more of homology.
  • substitution of wild-type amino acid residues may be performed by alanine, or by conservative amino acid substitutions that do not affect or weaken the charge of the entire protein, that is, polarity or hydrophobicity.
  • IL-7 protein is also used as a concept including "IL-7 protein and fragments thereof".
  • A may be directly linked to the N-terminus of the IL-7 or linked through a linker.
  • the A may be linked to the N-terminus of IL-7.
  • the A is characterized in that it contains 1 to 10 amino acids, and the amino acid may be selected from the group consisting of methionine, glycine, and combinations thereof.
  • Methionine and glycine do not induce an immune response in the body. Protein therapeutics produced from E. coli necessarily contain methionine at the N-terminus, but no immune side effects have been reported. In addition, glycine is widely used in GS linkers, but it does not induce an immune response even in commercially available products such as Dulaglutide ( Cell Biophys. 1993 Jan-Jun:22(1-3): 189- 224).
  • A may be an oligopeptide containing 1 to 10 amino acids selected from the group consisting of methionine (Met, M), glycine (Gly, G), and combinations thereof.
  • it may be an oligopeptide including 2 to 10 amino acids, more preferably an oligopeptide including 3 to 10 amino acids, but is not limited thereto.
  • A is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine, Methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, glycine-glycine, methionine-methionine-methionine, methionine-glycine-methionine, methionine-glycine-methionine, methionine-glycine-methionine, methionine-glycine-methionine, methionine-glycine-methionine, methionine-glycine-methionine, methi
  • the modified IL-7 may consist of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
  • TGF beta receptor II refers to any vertebrae, including mammals such as primates (eg humans) and rodents (eg mice and rats). Refers to any wild-type TBRII obtained from animal sources.
  • the human TBRII may consist of the amino acid sequence of SEQ ID NO: 4.
  • the TBRII may be an extracellular domain of TBRII.
  • the extracellular domain of TBRII may have the amino acid sequence 24 to 159 of human TBRII (SEQ ID NO: 4), and the extracellular domain of TBRII may be composed of the amino acid sequence of SEQ ID NO: 5.
  • sTBRII in the present invention means soluble TBRII, and may be an extracellular domain of human TBRII.
  • the modified IL-7 and TBRII can be bound by an immunoglobulin Fc domain.
  • the Fc domain may be wild type or variant.
  • the Fc domain variant may be an Fc domain of a modified immunoglobulin.
  • the Fc domain of the modified immunoglobulin is modified to bind to the Fc receptor and/or complement, so that antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) This could be weakened.
  • the modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof.
  • the Fc domain of the modified immunoglobulin may include a hinge region, a CH2 domain, and a CH3 domain in the N-terminal to C-terminal direction.
  • the hinge region includes a human IgD hinge region
  • the CH2 domain includes a portion of an amino acid residue of a CH2 domain of human IgD and a portion of an amino acid residue of a CH2 domain of human IgG4, and the CH3 domain is of human IgG4. It may comprise a portion of the amino acid residues of the CH3 domain.
  • the hinge region may be an IgG1 hinge region, which may include the amino acid sequence of SEQ ID NO: 6.
  • Fc domain includes heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of immunoglobulin, but variable regions and light chains of heavy and light chains thereof
  • Constant region 1 refers to a protein that does not contain. It may further include a hinge region of the heavy chain constant region.
  • Hybrid Fc or hybrid Fc fragment is also referred to herein as “hFc” or “hyFc”.
  • Fc domain variant of the present invention means that some amino acids in the Fc domain are substituted or are prepared by combining different types of Fc domains.
  • the Fc domain variant can prevent cleavage at the hinge region.
  • the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 9 may be modified.
  • the 144th amino acid K of SEQ ID NO: 9 may be substituted with G or S (K144G, K144S), and the 145th amino acid E may be substituted with G or S (E145G, E145S).
  • Fc domain or Fc domain variant of the modified immunoglobulin can be represented by the following formula (I):
  • N' is the N-terminus of the polypeptide and C'is the C-terminus of the polypeptide;
  • p is an integer of 0 or 1;
  • Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from the 98 position among the amino acid residues at positions 90 to 98 of SEQ ID NO: 7,
  • Y is an amino acid sequence having 5 to 64 consecutive amino acid residues in the N-terminal direction from position 162 of the amino acid residues at positions 99 to 162 of SEQ ID NO: 7,
  • Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues in the C-terminal direction from position 163 of the amino acid residues at positions 163 to 199 of SEQ ID NO: 7,
  • Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues in the N-terminal direction from the 220 position among the amino acid residues at positions 115 to 220 of SEQ ID NO: 8,
  • Z4 is an amino acid sequence having an amino acid sequence of 80 to 107 in the C-terminal direction from position 221 among the amino acid residues at positions 221 to 327 of SEQ ID NO: 8.
  • the Fc fragment of the present invention may be a natural type sugar chain, an increased sugar chain compared to the natural type, a reduced sugar chain compared to the natural type, or a form in which the sugar chain has been removed.
  • the immunoglobulin Fc sugar chain can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment sharply decreases the binding affinity of the primary complement component C1 to C1q, leads to a decrease or loss of ADCC or CDC, and thereby does not induce an unnecessary immune response in vivo.
  • the immunoglobulin Fc fragment in the form of deglycosylated or aglycosylated sugar chain may be more suitable for the purposes of the present invention as a drug carrier.
  • deglycosylation as used herein means that sugars are enzymatically removed from an Fc fragment.
  • aglycosylation means that the Fc fragment is produced in an unglycosylated form by a prokaryote, preferably E. coli.
  • the Fc domain of the modified immunoglobulin contains the amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 10 (hyFcM1), SEQ ID NO: 11 (hyFcM2), SEQ ID NO: 12 (hyFcM3) or SEQ ID NO: 13 (hyFcM4). can do.
  • the Fc domain of the modified immunoglobulin may be described in U.S. Patent No. 7,867,491, and the production of the Fc domain of the modified immunoglobulin may be performed with reference to the bar described in U.S. Patent No. 7,867,491. .
  • the fusion protein may be a TBRII, Fc domain, and modified IL-7 are sequentially bound from the N-terminus to the C-terminus.
  • This fusion protein may be referred to as "sTBRII-hyFc-IL7".
  • a first linker may be further included between the TBRII and the Fc domain.
  • the first linker may consist of 20 to 60 contiguous amino acids, or 25 to 50 contiguous amino acids, or 30 to 40 amino acids. In one embodiment, the first linker may consist of 20 amino acids.
  • the first linker may include (G4S)n (here, n is an integer of 1 to 5). Preferably, the first linker may consist of the amino acid sequence of SEQ ID NO: 14.
  • a second linker may be further included between the Fc domain and the modified IL-7.
  • the second linker may consist of 1 to 30 contiguous amino acids, 3 to 20 contiguous amino acids, or 4 to 16 amino acids.
  • the second linker may include (SG3)n (here, n is an integer of 1 to 5).
  • the second linker may consist of the amino acid sequence of SEQ ID NO: 15.
  • the sTBRII-hyFc-IL7 fusion protein may have the following structure.
  • N' is the N-terminal
  • C' is the C-terminal
  • L1 is the first linker
  • L2 is the second linker
  • p and q are integers of 0 or 1.
  • the sTBRII-hyFc-IL7 fusion protein may consist of the amino acid sequence of SEQ ID NO: 17.
  • the fusion protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, in the amino acid sequence of SEQ ID NO: 17, Alternatively, it may have a sequence with 99% or more homology.
  • the fusion protein may be a modified IL-7, Fc domain, or TBRII is sequentially bound from the N-terminal to the C-terminal direction.
  • This fusion protein may be referred to as "IL7-hyFc-sTBRII”.
  • a first linker may be further included between the modified IL-7 and the Fc domain.
  • the first linker may consist of 20 to 60 contiguous amino acids, or 25 to 50 contiguous amino acids, or 30 to 40 amino acids. In one embodiment, the first linker may consist of 20 amino acids.
  • the first linker may include (G4S)n (here, n is an integer of 1 to 5). Preferably, the first linker may consist of the amino acid sequence of SEQ ID NO: 14.
  • a third linker may be further included between the Fc domain and TBRII.
  • the third linker may consist of 1 to 30 contiguous amino acids, or 3 to 20 contiguous amino acids, or 4 to 16 amino acids.
  • the third linker may be made of the amino acid sequence of SEQ ID NO: 16.
  • the IL7-hyFc-sTBRII fusion protein may have the following structure.
  • N' is the N-terminal
  • C' is the C-terminal
  • L1 is the first linker
  • L2 is the second linker
  • p and r are integers of 0 or 1.
  • the IL7-hyFc-sTBRII fusion protein may be composed of the amino acid sequence of SEQ ID NO: 18.
  • the fusion protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, in the amino acid sequence of SEQ ID NO: 18, Alternatively, it may have a sequence with 99% or more homology.
  • the present invention provides a nucleic acid molecule encoding the fusion protein.
  • nucleic acid molecule may additionally include a signal sequence (or signal peptide) or a leader sequence.
  • signal sequence refers to a short peptide present at the N-terminus of a newly synthesized protein classified as a secretory pathway.
  • Signal sequences useful in the present invention include antibody light chain signal sequences, such as antibody 1418 (Gillies et al., J Immunol Meth 1989 125:191-202), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequences (Sakano et al, Nature 1980 286: 676-683), and other signal sequences known in the art (see, e.g., Watson et al, Nucleic Acid Research 1984 12:5145-5164).
  • the signal peptide is well known in the art, and is generally known to contain 16 to 30 amino acid residues, and may contain more or less amino acid residues.
  • a typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.
  • the central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide.
  • the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases.
  • the signal sequence may be a tissue plasma activation (tPa), HSV gDs, or a secretion signal sequence of growth hormone.
  • tPa tissue plasma activation
  • HSV gDs HSV gDs
  • secretion signal sequence of growth hormone e.gDs
  • the secretion signal sequence used in higher eukaryotic cells including mammals and the like can be used, more preferably, the tPa sequence (SEQ ID NO: 19) or the amino acid sequence of SEQ ID NO: 20 can be used.
  • the signal sequence of the present invention can be used by substituting codons with high expression frequency in host cells.
  • the present invention provides an expression vector containing the nucleic acid molecule.
  • vector of the present invention is understood as a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell, recombined and inserted into the host cell genome, or spontaneously replicated as an episome.
  • the vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors include, but are not limited to, retrovirus, adenovirus, and adeno-associated virus.
  • a useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof.
  • Useful expression vectors include a human CMV (cytomegalovirus) promoter to promote the continuous transcription of a gene of interest in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase the stable level of RNA after transcription. can do.
  • the expression vector is pAD15, a modified vector of RcCMV.
  • host cell refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.
  • an appropriate host cell may be transformed or transfected with the DNA sequence of the present invention, and may be used for expression and/or secretion of a protein of interest.
  • Currently preferred host cells that can be used in the present invention are immortal hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells. , CapT cells (human amniotic fluid derived cells) and COS cells.
  • transformation and “transfection” of the present invention mean the introduction of a nucleic acid (eg, a vector) into a cell by a number of techniques known in the art.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cancer or infectious diseases comprising a fusion protein containing modified IL-7 and TBRII as an active ingredient.
  • the cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. It may be selected from the group, but is not limited thereto.
  • the infectious disease may be selected from the group consisting of hepatitis B, hepatitis C, human papilloma virus infection, cytomegalovirus infection, viral respiratory disease, and influenza, but is not limited thereto.
  • the active ingredient may be in an arbitrary amount (effective amount) according to the use, formulation, purpose of combination, etc. It may be included, and a typical effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition.
  • effective amount refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such effective amounts can be determined empirically within the range of ordinary skill in the art.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohols, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmacological composition.
  • the pharmaceutical composition may be prepared in a parenteral formulation according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.
  • pharmaceutically acceptable means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.
  • the pharmaceutical composition When the pharmaceutical composition is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation, and a suppository according to a method known in the art together with a suitable carrier.
  • a suitable carrier When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterile water for injection. , An isotonic solution such as 5% dextrose may be used.
  • a pharmaceutical composition it is known in the art, and specifically, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered as part of this specification.
  • the preferred dosage of the pharmaceutical composition is in the range of 0.01 ug/kg to 10 g/kg per day, or 0.01 mg/kg to 1 g/kg per day depending on the patient's condition, weight, sex, age, patient severity, and route of administration. Can be Administration can be made once a day or divided into several times. Such dosages should not be construed as limiting the scope of the invention in any aspect.
  • compositions of the present application are mammals and humans, particularly preferably humans.
  • the pharmaceutical composition of the present application may further include any compound or natural extract, which has already been verified for safety and has a therapeutic effect on anticancer activity or infectious disease in order to increase and reinforce anticancer activity.
  • the present invention provides the use of a fusion protein comprising a modified IL-7 and TBRII to produce a pharmaceutical preparation having a prophylactic or therapeutic effect of cancer or infectious disease.
  • the present invention provides a method for preventing or treating cancer or infectious diseases comprising a fusion protein containing modified IL-7 and TBRII as an active ingredient.
  • the therapeutically effective amount is a specific composition including the type and degree of the reaction to be achieved, whether or not other agents are used in some cases, the individual's age, weight, general health status, sex and diet, administration time, administration route and composition. It is preferable to apply differently according to various factors including the secretion rate of the drug, the treatment period, drugs used with or concurrently with the specific composition, and similar factors well known in the field of medicine. Therefore, it is preferable to determine an effective amount of a composition suitable for the purposes of the present invention in consideration of the foregoing.
  • the individual is applicable to any mammal, and the mammal includes humans and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs and cats.
  • a fusion protein in which human-derived IL-7 and sTBRII soluble TGF beta receptor II
  • the present inventors fused IL-7 or sTBRII to the N-terminus of the Fc domain, and to the C-terminus of the Fc domain.
  • a gene construct in the form of fusion of IL-7 or sTBRII was prepared.
  • a fusion protein in which TBRII, Fc domain, and IL-7 are sequentially bound from the N-terminus to the C-terminus is expressed as "sTBRII-hyFc-IL7", and IL-7, Fc domain or TBRII is N-
  • the fusion proteins bound in order from the terminal to the C-terminus were denoted as "IL7-hyFc-sTBRII”.
  • sTBRII uses only the extracellular domain of 24-159 aa in the known amino acid sequence (Accession number: NP003233.4) of TGF beta receptor II. Thus, it was fused to the N-terminus of the Fc domain, and IL-7 was fused to the C-terminus of the Fc domain using a known amino acid sequence (Accession number: NP000871.1) to prepare a gene construct (Fig. 1 ).
  • IL-7 and sTBRII are fused to the N-terminus and C-terminus of the Fc domain, respectively, using the same sequence as above, and the gene construct is Was prepared (Fig. 1).
  • IL-7, sTBRII and Fc domains were prepared as sub-vectors by synthesizing each gene at Cosmo Genetech, and Golden Gateway assembly was performed to prepare the synthesized three gene segments into one gene segment. Then, the expression vector was obtained with the pGP30 vector.
  • the expression vector was transfected into CHO cells (suspension-adapted Chinese Hamster Ovary cells) adapted for suspension culture with the Neon Transfection system (Invitrogen, MPK1096), and a highly productive cell line was obtained through HT selection and amplification of Methotrexate (Sigma, M8407). Were selected. In addition, single cells were secured through limiting dilution cloning to obtain production cell lines producing sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII fusion proteins.
  • Each production cell line was cultured by 80 mL each in a 250 mL Erlenmeyer flask (Corning, 431144) using Hycell CHO medium (Hyclone, SH30949.02) to confirm the culture productivity. As a result, it was confirmed that the IL7-hyFc-sTBRII fusion protein was produced in an amount of 0.6 g/L, and the sTBRII-hyFc-IL7 fusion protein was produced in an amount of 0.95 g/L (FIG. 2).
  • the present inventors conducted an experiment to confirm the pharmacodynamics profile of the fusion protein according to intravenous administration. Briefly, after intravenous administration of sTBRII-hyFc-IL7 fusion protein at 10, 30 and 100 mg/kg (mpk) to a C57BL/6 (B6) mouse animal model, changes of immune cells in blood over time were analyzed. In addition, blood was collected through retro-orbital bleeding on days 3, 7, 10, 14, 17 and 21 after administration of the sTBRII-hyFc-IL7 fusion protein. Each value was calculated by checking the expression rate of the cell surface indicator and the composition ratio of each cell in the blood through flow cytometry, and multiplying this by the value measured using a complete blood count (CBC).
  • CBC complete blood count
  • mice administered with sTBRII-hyFc-IL7 fusion protein increased in a concentration-dependent manner, and the peak point was found at the 7th day of administration ( Fig. 3a).
  • the increase rate of CD8 + T cells at the 7th day of administration increased by 6.22, 12.45 and 35.04 times at concentrations of 10, 30 and 100 mpk, respectively, compared to the control group (PBS treatment) (Fig. 3b).
  • mice administered with the sTBRII-hyFc-IL7 fusion protein CD4 + T cells and CD4 + CD25 + Foxp3 + Treg cells, which are target cells of IL-7, also showed a tendency to increase (FIGS. 3C and 3D ).
  • neutrophils and NK cells other than the target cells no changes were observed according to the administration (FIGS. 3e and 3f).
  • sTBRII-hyFc-IL7 fusion protein (10 mpk) was administered intravenously (intravenous injection, iv) or subcutaneous (subcutaneous injection, sc) to a C57BL/6 (B6) mouse animal model, followed by immunity in the blood over time. Cellular changes were analyzed. Blood was collected through retro-orbital bleeding on days 0, 4, 7, 11, and 14 after administration of the sTBRII-hyFc-IL7 fusion protein. Each value was measured using a complete blood count (CBC).
  • CBC complete blood count
  • mice administered with the sTBRII-hyFc-IL7 fusion protein were increased in mice administered with the sTBRII-hyFc-IL7 fusion protein compared to the control group (PBS treatment) on the 7th day of administration (FIG. 4A).
  • the number of immune cells increased by about 1.51 times compared to the control group in the intravenous administration group on the 7th day of administration, and the number of immune cells increased by about 3.04 times compared to the control group in the subcutaneous administration group (Fig.
  • Example 4 In vivo of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model ( in vivo ) Activity assay
  • the present inventors performed an experiment to analyze the in vivo activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model.
  • a mouse tumor model was prepared by subcutaneously injecting 1 ⁇ 10 5 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Thereafter, on the 6th day of tumor formation, each fusion protein was administered subcutaneously (sc) at 10 mpk or 20 mpk, and then the changes in immune cells in the blood were analyzed using flow cytometry and CBC (complete blood count). I did. Blood was collected through retro-orbital bleeding on days 0, 3, 7, 11, 15 and 18 after administration of each fusion protein.
  • both sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins have an activity of increasing the proliferation of target cells such as CD8 + T cells in a concentration-dependent manner.
  • Example 5 Analysis of TGF beta inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model
  • the present inventors performed an experiment to analyze the inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins against TGF beta in a mouse tumor model.
  • a mouse tumor model was prepared by subcutaneously injecting 1 ⁇ 10 5 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Thereafter, on the 6th day of tumor formation, each fusion protein was administered subcutaneously (sc) at 20 mpk. Blood was collected through retro-orbital bleeding at 2, 6, 24, 48, 72 and 168 hours after administration of each fusion protein, and serum was isolated from the collected blood samples. The concentration of TGF beta present in the serum was measured using the Mouse TGF-beta 1 DuoSet ELISA Kit (R&D systems, catalog# DY1679-05).
  • TGF beta in serum was inhibited for about 48 hours after administration, and was detected again 72 hours after administration, and 168 hours after administration, control (PBS treatment) It was confirmed that the recovered to a level similar to (Fig. 6).
  • TGF beta in serum was inhibited for about 72 hours after administration, and it was confirmed that it recovered to a level similar to that of the control group (PBS treatment) 168 hours after administration (Fig. 6).
  • Example 6 Analysis of anticancer activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model
  • the present inventors performed an experiment to confirm the anticancer activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model.
  • a mouse tumor model was prepared by subcutaneously injecting 1 ⁇ 10 5 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Thereafter, on the 6th day of tumor formation, each fusion protein was administered subcutaneously (sc) at 20 mpk. Changes in tumor volume were measured at intervals of 2 to 3 days after administration. The tumor volume was calculated using the following formula.
  • Tumor volume ⁇ (long axis length) x (short axis length) 2 ⁇ / 2
  • tumor growth inhibition rate (% tumor growth inhibition) was calculated using the following formula (formula).
  • mice administered with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins the tumor volume was significantly reduced compared to the control group (PBS treatment) (FIG. 7).
  • the tumor growth inhibition rate was 61.79% compared to the control group (Fig. 7a), and IL7-hyFc-sTBRII fusion
  • the tumor growth inhibition rate was 83.14% compared to the control group (FIG. 7B).
  • Example 7 In vitro ( in vitro ), IL-7 bioactivity analysis of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins
  • IL-7 constituting the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in vitro
  • the present inventors conducted a 2E8 cell line (ATCC ® TIB) that proliferates dependently on IL-7 in vitro.
  • -239 TM Mouse B lymphocyte cell line
  • human-derived IL-7 has cross-reactivity between mouse and species, human-derived IL-7 can induce proliferation of the mouse cell line 2E8 cell line.
  • the 2E8 cell line was cultured using IMDM (ATCC ® 30-2005 TM ) medium containing mouse IL-7 (Cell Signaling, 5217SC) and FBS (Hyclone, SH30084.03). Thereafter, the cultured 2E8 cell line was suspended in IMDM medium containing no mouse IL-7 to make it starvation for IL-7, and then dispensed into a 96-well plate at 1 ⁇ 10 5 cells/well.
  • IMDM ATCC ® 30-2005 TM
  • mouse IL-7 Cell Signaling, 5217SC
  • FBS Hyclone, SH30084.03
  • sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins were treated on the cells in a concentration gradient sequentially diluted from 3 nM to 1/3, respectively, and in an incubator at 37° C., 5% CO 2 for 3 days. Cultured.
  • Cell proliferation was quantified using CellTiter 96® AQ ueous One Solution Assay (Promega, G3581). The cells were treated with the MTS reagent, and incubated for 4 hours in an incubator at 37° C. and 5% CO 2 , and absorbance at a wavelength of 490 nm was measured using an ELISA plate reader. Using the GraphPad Prism ® program (GraphPad Software), the standard curve of absorbance and the EC 50 (50% effective concentration) value of the two fusion proteins were calculated based on this.
  • sTBRII-hyFc-IL7 EC 50 of the fusion protein was measured in 52.52 pM
  • IL7-hyFc-sTBRII EC 50 of fusion protein was determined to be 56.21 pM ( Figure 8 and Table 2).
  • Example 8 In vitro ( in vitro ) Analysis of TGF beta inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins
  • SMAD Signaling which can measure the subtransmission signal induced by TGF beta as a luminescence signal.
  • the experiment was performed using the Pathway SBE Reporter-HEK293 cell line (BPS Bioscience, 60653).
  • the SMAD Signaling Pathway SBE Reporter-HEK293 cell line was cultured in a 37°C, 5% CO 2 incubator using Growth Medium 1B (BPS Bioscience, 79531) containing Geneticin (Invitrogen, 11811031). Thereafter, the cultured SMAD Signaling Pathway SBE Reporter-HEK293 cell line was suspended in Assay Medium 1B (BPS Bioscience, 79617-2) and dispensed into a white clear-bottom 96-well microplate at 3.5x10 4 cells/well.
  • the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins were treated on the cells in a concentration gradient sequentially diluted from 50 nM to 1/3, respectively, and at the same time 20 ng/mL of TGF beta (BPS Bioscience, After treating the cells with 90900-1), the cells were incubated for 18 hours in an incubator at 37°C and 5% CO 2. After culturing cells, ONE-Step TM Luciferase reagent was treated, and the plate was shaken for 30 minutes at room temperature and incubated. Thereafter, the degree of luminescence of the SBE reporter was measured with a Plate-reading Luminometer (TECAN SPARK 10M). Using the GraphPad Prism ® program (GraphPad Software), the luminance standard curve of the SBE reporter and the IC 50 values of the two fusion proteins were calculated.
  • SMAD Signaling Pathway SBE in Reporter-HEK293 cell line sTBRII-hyFc-IL-7 IC 50 values of the fusion proteins was measured in 1.874 nM
  • IL-7-hyFc -sTBRII IC 50 values of the fusion protein is measured by 0.4148 nM (Fig. 9 and Table 3).
  • the present inventors performed BLI (biolayer interferometry) to compare and analyze the simultaneous binding capacity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII to IL-7R ⁇ (CD127) and TGF- ⁇ 1.
  • BLI biological layer interferometry
  • sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII to IL-7R ⁇ CD127
  • TGF- ⁇ TGF- ⁇
  • Experiments were performed using an amine reactive second-generation biosensor (AR2G) and an AR2G reagent kit (AR2G Reagent Kit).
  • the sTBRII-hyFc-IL-7 and IL-7-hyFc-sTBRII fusion proteins are capable of simultaneous binding with the target proteins TGF- ⁇ 1 and IL-7R ⁇ .

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Abstract

The present invention relates to a fusion protein including modified interleukin-7 and TGF beta receptor II and use thereof. The fusion protein has a high production yield and can effectively inhibit cancer, and thus can be usefully utilized in the treatment of cancer or infectious diseases.

Description

변형된 인터루킨-7 및 TGF 베타 수용체 II를 포함하는 융합단백질 및 이의 용도Fusion protein containing modified interleukin-7 and TGF beta receptor II and uses thereof
본 발명은 변형된 인터루킨-7 및 TGF 베타 수용체 II를 포함하는 융합단백질 및 이의 용도에 관한 것이다.The present invention relates to a fusion protein comprising a modified interleukin-7 and TGF beta receptor II and uses thereof.
TGF 베타 수용체 II (transforming growth factor beta receptor II, TBRII)는 인간에서 TGFBR2 유전자에 의해 코딩되는 70 내지 80 kDa의 막단백질이다. TBRII는 TBRI(TGF beta receptor I)과 이종이량체를 이루며, TGF-α와 결합하여 세포 내 신호를 전달하여 세포의 증식과 관련된 유전자의 전사를 조절한다. TBRII는 C-말단의 단백질 인산화효소 도메인 및 N-말단의 엑토 도메인으로 구성된다. 엑토 도메인은 세포외 공간으로 연장되는 막 단백질의 도메인으로서, 9개의 베타 체인 및 가닥 내 6개의 이황화 결합에 의해 안정화된 단일 나선을 포함하는 접힘 구조를 이룬다. TGF beta receptor II (transforming growth factor beta receptor II, TBRII) is encoded by the TGFBR2 gene in humans. It is a membrane protein of 70 to 80 kDa. TBRII forms a heterodimer with TBRI (TGF beta receptor I), and by binding with TGF-α to transmit intracellular signals, it regulates the transcription of genes related to cell proliferation. TBRII consists of a C-terminal protein kinase domain and an N-terminal ecto domain. The ecto domain is a domain of a membrane protein that extends into the extracellular space, and forms a folded structure comprising a single helix stabilized by 9 beta chains and 6 disulfide bonds in the strand.
또한, TBRII의 리간드로 알려진 TGF-α는 암세포에서 과발현되는 면역억제성 사이토카인으로서, 암세포로부터 분비되는 TGF-α로 인해 T세포의 증식이 억제되는 등 암세포의 면역회피 기작 중 하나로 알려져 있다. In addition, TGF-α, known as a ligand of TBRII, is an immunosuppressive cytokine that is overexpressed in cancer cells, and is known as one of the mechanisms of immune evasion of cancer cells, such as inhibiting the proliferation of T cells due to TGF-α secreted from cancer cells.
최근, TBRII의 단편을 포함하는 TGF-α의 디코이 수용체(decoy receptor)에 대한 연구가 활발히 진행되고 있으나, TBRII의 단편의 세포 내 활성 및 안정성이 떨어진다는 문제점이 있다. Recently, studies on the decoy receptor of TGF-α including a fragment of TBRII have been actively conducted, but there is a problem that the intracellular activity and stability of the fragment of TBRII are poor.
한편, 인터루킨-7(interleukin 7, IL-7)은 B 세포와 T 세포를 매개로 면역 반응을 촉진시키는 사이토카인으로서, 특히 적응 면역체계에서 중요한 역할을 한다. 구체적으로, IL-7은 T세포와 B세포의 생존 및 분화, 임파상 세포(lymphoid cell)의 생존, 및 NK 세포(natural killer cell)의 활성 촉진 등을 통해 면역 기능을 활성화시키는데, 특히, T 세포와 B 세포의 발달에 중요하다. 이는 HGF(hepatocyte growth factor)와 결합하여 pre-pro-B 세포 성장-자극 인자(growth-stimulating factor), T 세포 수용체 베타(TCRβ)의 V(D)J 재배열(rearrangement)의 보조인자(cofactor)로서 작용한다(Muegge K, 1993, Science 261 (5117): 93-5).On the other hand, interleukin-7 (interleukin 7, IL-7) is a cytokine that promotes an immune response via B cells and T cells, and particularly plays an important role in the adaptive immune system. Specifically, IL-7 activates immune functions through survival and differentiation of T cells and B cells, survival of lymphoid cells, and promotion of natural killer cells (NK cells). In particular, T It is important for the development of cells and B cells. It binds to HGF (hepatocyte growth factor) and is a cofactor of pre-pro-B cell growth-stimulating factor and V(D)J rearrangement of T cell receptor beta (TCRβ). ) (Muegge K, 1993, Science 261 (5117): 93-5).
그러나, 의약적으로 활용하기 위하여 재조합 IL-7을 생산할 경우, 일반 재조합 단백질에 비해 불순물이 다량 생산되고, 변성이 쉬우며 대량생산이 용이하지 않은 문제가 있다.However, when the recombinant IL-7 is produced for medical use, there is a problem in that a large amount of impurities is produced compared to a general recombinant protein, it is easy to denature, and mass production is not easy.
본 발명의 목적은 변형된 인터루킨-7(IL-7) 및 TGF 베타 수용체 II(TBRII)를 포함하는 융합단백질을 제공하는 것이다.An object of the present invention is to provide a fusion protein comprising a modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII).
본 발명의 또 다른 목적은 상기 융합 단백질을 코딩하는 단리된 핵산 분자를 제공하는 것이다. Another object of the present invention is to provide an isolated nucleic acid molecule encoding the fusion protein.
본 발명의 또 다른 목적은 상기 핵산 분자를 포함하는 발현 벡터를 제공하는 것이다. Another object of the present invention is to provide an expression vector comprising the nucleic acid molecule.
본 발명의 또 다른 목적은 상기 발현 벡터를 포함하는 숙주 세포를 제공하는 것이다. Another object of the present invention is to provide a host cell comprising the expression vector.
본 발명의 다른 목적은 상기 융합단백질을 유효성분으로 포함하는 암 또는 감염성 질환의 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
본 발명의 또 다른 목적은 암 또는 감염성 질환의 예방 또는 치료 효과를 가진 의약제제를 생산하기 위한 변형된 IL-7 및 TBRII를 포함하는 융합단백질의 사용을 제공하는 것이다.Another object of the present invention is to provide the use of a fusion protein comprising a modified IL-7 and TBRII to produce a pharmaceutical preparation having a prophylactic or therapeutic effect of cancer or infectious disease.
본 발명의 또 다른 목적은 상기 융합단백질을 유효성분으로 포함하는 암 또는 감염성 질환의 예방 또는 치료 방법을 제공하는 것이다. Another object of the present invention is to provide a method for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 변형된 인터루킨-7(IL-7) 및 TGF 베타 수용체 II(TBRII)를 포함하는 융합단백질을 제공한다. In order to achieve the above object, the present invention provides a fusion protein comprising a modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII).
또한, 본 발명은 상기 융합 단백질을 코딩하는 단리된 핵산 분자를 제공한다. In addition, the present invention provides an isolated nucleic acid molecule encoding the fusion protein.
또한, 본 발명은 상기 핵산 분자를 포함하는 발현 벡터를 제공한다. In addition, the present invention provides an expression vector containing the nucleic acid molecule.
또한, 본 발명은 상기 발현 벡터를 포함하는 숙주 세포를 제공한다. In addition, the present invention provides a host cell comprising the expression vector.
또한, 본 발명은 상기 융합단백질을 유효성분으로 포함하는 암 또는 감염성 질환의 예방 또는 치료용 약학 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
또한, 본 발명은 암 또는 감염성 질환의 예방 또는 치료 효과를 가진 의약제제를 생산하기 위한 변형된 IL-7 및 TBRII를 포함하는 융합단백질의 사용을 제공한다. In addition, the present invention provides the use of a fusion protein comprising a modified IL-7 and TBRII to produce a pharmaceutical preparation having a prophylactic or therapeutic effect of cancer or infectious disease.
또한, 본 발명은 상기 융합단백질을 유효성분으로 포함하는 암 또는 감염성 질환의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.
본 발명의 변형된 인터루킨-7(IL-7) 및 TGF 베타 수용체 II(TBRII)를 포함하는 융합단백질은 생산 수율이 높고, 암을 효과적으로 억제시킬 수 있어, 암 또는 감염성 질환의 치료에 유용하게 사용될 수 있다.The fusion protein containing the modified interleukin-7 (IL-7) and TGF beta receptor II (TBRII) of the present invention has a high production yield and can effectively inhibit cancer, so it can be usefully used in the treatment of cancer or infectious diseases. I can.
도 1은 sTBRII-hyFc-IL7 및 IL7-hyFc-sTBRII의 유전자 컨스트럭트를 나타낸 것이다.1 shows the gene constructs of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII.
도 2는 회분 배양(batch culture)을 통한 sTBRII-hyFc-IL7 및 IL7-hyFc-sTBRII 융합단백질의 생산량을 측정한 결과이다. 2 is a result of measuring the production amount of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII fusion proteins through batch culture.
도 3은 투여량에 따른 sTBRII-hyFc-IL7 융합단백질의 생체 내 활성을 측정한 결과로서, (a)는 마우스 동물모델에 sTBRII-hyFc-IL7 융합단백질을 투여한 후, CD8+ T 세포의 수를 측정한 결과이고, (b)는 투여 7일 차의 CD8+ T 세포 증가율을 측정한 결과이며, (c)는 CD4+ T 세포의 수를 측정한 결과이고, (d)는 CD4+CD25+Foxp3+ Treg 세포의 수를 측정한 결과이며, (e)는 호중구(neutrophil)의 수를 측정한 결과이고, (f)는 NK 세포의 수를 측정한 결과이다 (○: PBS; ■: sTBRII-hyFc-IL7, 10 mpk; ▲: sTBRII-hyFc-IL7, 30 mpk; ▼: sTBRII-hyFc-IL7, 100 mpk). 3 is a result of measuring the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the dose, (a) is the number of CD8+ T cells after administration of the sTBRII-hyFc-IL7 fusion protein to a mouse animal model. This is the result of measurement, (b) is the result of measuring the increase rate of CD8+ T cells on the 7th day of administration, (c) is the result of measuring the number of CD4+ T cells, (d) is the result of measuring CD4+CD25+Foxp3+ Treg cells. Is the result of measuring the number of, (e) is the result of measuring the number of neutrophils, (f) is the result of measuring the number of NK cells (○: PBS; ■: sTBRII-hyFc-IL7, 10 mpk; ▲: sTBRII-hyFc-IL7, 30 mpk; ▼: sTBRII-hyFc-IL7, 100 mpk).
도 4는 투여 경로에 따른 sTBRII-hyFc-IL7 융합단백질의 생체 내 활성을 측정한 결과로서, (a)는 마우스 동물모델에 sTBRII-hyFc-IL7 융합단백질을 투여한 후, 면역세포(lymphocytes)의 수를 측정한 결과이고, (b)는 투여 7일 차에 정맥투여 및 피하투여에 다른 면역세포 증가율을 측정한 결과이다.Figure 4 is a result of measuring the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the administration route, (a) is a mouse animal model after administration of the sTBRII-hyFc-IL7 fusion protein, immune cells (lymphocytes) It is the result of measuring the number, and (b) is the result of measuring the increase rate of immune cells different from intravenous administration and subcutaneous administration on the 7th day of administration.
도 5는 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 생체 내 활성을 분석한 결과로서, (a)는 마우스 종양모델에 sTBRII-hyFc-IL7 융합단백질을 투여한 후, CD8+ T 세포의 수를 측정한 결과이고, (b)는 투여 7일 차의 CD8+ T 세포 증가율을 측정한 결과이며, (c)는 마우스 종양모델에 IL-7-hyFc-sTBRII 융합단백질을 투여한 후, CD8+ T 세포의 수를 측정한 결과이고, (d)는 투여 7일 차의 CD8+ T 세포 증가율을 측정한 결과이다. 5 is a result of analyzing the in vivo activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins, (a) is a mouse tumor model after administration of the sTBRII-hyFc-IL7 fusion protein, CD8+ T It is the result of measuring the number of cells, (b) is the result of measuring the increase rate of CD8+ T cells on the 7th day of administration, (c) is the result of administration of IL-7-hyFc-sTBRII fusion protein to a mouse tumor model This is the result of measuring the number of CD8+ T cells, and (d) is the result of measuring the increase rate of CD8+ T cells at the 7th day of administration.
도 6은 마우스 종양모델에 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 투여한 후, 혈청 내의 TGF beta의 농도를 측정한 결과이다. 6 is a result of measuring the concentration of TGF beta in serum after administration of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins to a mouse tumor model.
도 7은 마우스 종양모델에 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 투여한 후, 종양 부피(tumor volume)를 측정한 결과이다. 7 is a result of measuring tumor volume after administration of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins to a mouse tumor model.
도 8은 2E8 세포주에 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 처리한 후, (a) 세포증식도 및 (b) 이의 표준곡선을 나타낸 결과이다. 8 is a result showing the (a) cell proliferation diagram and (b) its standard curve after treatment with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in 2E8 cell line.
도 9는 SMAD Signaling Pathway SBE Reporter-HEK293 세포주에 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 처리한 후, SBE reporter의 발광도 표준 곡선을 나타낸 결과이다. 9 is a result showing a standard curve of luminance of SBE reporter after treatment with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in the SMAD Signaling Pathway SBE Reporter-HEK293 cell line.
도 10은 각 융합단백질의 TGF-β1 및 IL-7Rα의 동시 결합력을 비교 분석한 결과로서, TGF-β1 (1st 리간드)과 결합시킨 후, IL-7Rα (2nd 리간드)의 농도에 따른 (a) sTBRII-hyFc-IL7 및 (b) IL-7-hyFc-sTBRII 융합단백질의 결합력을 측정한 결과이다. Figure 10 is a result of comparative analysis of the simultaneous binding power of TGF-β1 and IL-7Rα of each fusion protein, after binding to TGF-β1 (1st ligand), according to the concentration of IL-7Rα (2nd ligand) (a) This is the result of measuring the binding strength of sTBRII-hyFc-IL7 and (b) IL-7-hyFc-sTBRII fusion proteins.
도 11은 각 융합단백질의 TGF-β1 및 IL-7Rα의 동시 결합력을 비교 분석한 결과로서, IL-7Rα (1st 리간드)와 결합시킨 후, TGF-β1 (2nd 리간드)의 농도에 따른 (a) sTBRII-hyFc-IL7 및 (b) IL-7-hyFc-sTBRII 융합단백질의 결합력을 측정한 결과이다. Figure 11 is a result of comparative analysis of the simultaneous binding power of TGF-β1 and IL-7Rα of each fusion protein, after binding to IL-7Rα (1st ligand), according to the concentration of TGF-β1 (2nd ligand) (a) This is the result of measuring the binding strength of sTBRII-hyFc-IL7 and (b) IL-7-hyFc-sTBRII fusion proteins.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명의 일 측면은, 변형된 인터루킨-7(interleukin 7, IL-7) 및 TGF 베타 수용체 II(transforming growth factor beta receptor II, TBRII)를 포함하는 융합단백질을 제공한다. One aspect of the present invention provides a fusion protein comprising a modified interleukin-7 (interleukin 7, IL-7) and a TGF beta receptor II (transforming growth factor beta receptor II, TBRII).
상기 변형된 IL-7은 하기 구조를 갖는 것일 수 있다:The modified IL-7 may have the following structure:
A - IL-7;A-IL-7;
이때, 상기 A는 1 내지 10 개의 아미노산 잔기로 구성된 올리고펩티드이고, 상기 변형된 IL-7은 IL-7 또는 이와 유사한 활성을 갖는 폴리펩티드이다.In this case, A is an oligopeptide consisting of 1 to 10 amino acid residues, and the modified IL-7 is IL-7 or a polypeptide having similar activity.
본 발명에서 사용된 용어 "IL-7 또는 이와 유사한 활성을 갖는 폴리펩티드"는 IL-7과 동일하거나 유사한 서열 및 활성을 갖는 폴리펩티드 또는 단백질을 의미한다. The term "IL-7 or a polypeptide having similar activity" as used herein refers to a polypeptide or protein having the same or similar sequence and activity as IL-7.
상기 IL-7은 IL-7 단백질 또는 이의 단편을 포함할 수 있다. 이때, IL-7은 인간, 흰쥐, 생쥐, 원숭이, 소 또는 양에서 유래된 것일 수 있다. The IL-7 may include an IL-7 protein or a fragment thereof. At this time, IL-7 may be derived from human, white mouse, mouse, monkey, cow, or sheep.
구체적으로, 인간 IL-7은 서열번호 1(Genbank Accession No. P13232)의 아미노산 서열을 가질 수 있고; 흰쥐(rat) IL-7은 Genbank Accession No. P56478에 개시되어 있는 아미노산 서열을 가질 수 있으며; 생쥐(mouse) IL-7은 Genbank Accession No. P10168에 개시되어 아미노산 서열을 가질 수 있고; 원숭이 IL-7은 Genbank Accession No. NP_001279008에 개시되어 있는 아미노산 서열을 가질 수 있으며; 소 IL-7은 Genbank Accession No. P26895에 개시되어 있는 아미노산 서열을 가질 수 있고; 양 IL-7은 Genbank Accession No. Q28540에 개시되어 있는 아미노산 서열을 가질 수 있다.Specifically, human IL-7 may have the amino acid sequence of SEQ ID NO: 1 (Genbank Accession No. P13232); Rat IL-7 is a Genbank Accession No. May have the amino acid sequence disclosed in P56478; Mouse IL-7 is a Genbank Accession No. It is disclosed in P10168 and may have an amino acid sequence; Monkey IL-7 is a Genbank Accession No. It may have an amino acid sequence disclosed in NP_001279008; Bovine IL-7 is Genbank Accession No. May have the amino acid sequence disclosed in P26895; Yang IL-7 is Genbank Accession No. It may have an amino acid sequence disclosed in Q28540.
상기 IL-7은 서열번호 1의 아미노산 서열로 이루어진 폴리펩티드일 수 있다. 또한, 상기 변형된 IL-7은 서열번호 1의 서열과 약 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 가진 것일 수 있다. The IL-7 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In addition, the modified IL-7 is the sequence of SEQ ID NO: 1 and about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% or more homology.
또한, IL-7 단백질 또는 이의 단편은 다양하게 변형된 단백질 또는 펩티드, 즉 변이체를 포함할 수 있다. 상기 변형은 IL-7의 기능을 변형시키지 않는, 야생형 IL-7에 하나 이상의 단백질을 치환, 결실 또는 추가하는 방법을 통하여 수행될 수 있다. 이러한 다양한 단백질 또는 펩티드는 야생형 단백질과 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 또는 99% 이상의 상동성을 가질 수 있다.In addition, the IL-7 protein or fragment thereof may contain variously modified proteins or peptides, ie, variants. The modification may be performed through a method of substituting, deleting or adding one or more proteins to wild-type IL-7, which does not alter the function of IL-7. These various proteins or peptides include wild-type proteins and 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % Or more of homology.
통상, 야생형 아미노산 잔기의 치환은 알라닌이나, 전체 단백질의 전하, 즉, 극성 또는 소수성에 영향을 주지 않거나 약하게 주는 보존적 아미노산 치환(conservative amino acid)에 의해 수행될 수 있다.In general, substitution of wild-type amino acid residues may be performed by alanine, or by conservative amino acid substitutions that do not affect or weaken the charge of the entire protein, that is, polarity or hydrophobicity.
보존적 아미노산 치환을 위해 하기 표 1이 참조될 수 있다.Refer to Table 1 below for conservative amino acid substitutions.
염기성Basic 아르기닌(Arg, R)라이신(Lys, K)히스티딘(His, H)Arginine (Arg, R) Lysine (Lys, K) Histidine (His, H)
산성acid 글루탐산(Glu, E)아스파르트산(Asp, D)Glutamic acid (Glu, E) Aspartic acid (Asp, D)
비전하 극성(uncharged polar)Uncharged polar 글루타민(Gln, O)아스파라진(Asn, N)세린(Ser, S)트레오닌(Thr, T)티로신(Tyr, Y)Glutamine (Gln, O) Asparagine (Asn, N) Serine (Ser, S) Threonine (Thr, T) Tyrosine (Tyr, Y)
무극성(non-polar)Non-polar 페닐알라닌(Phe, F)트립토판(Trp, W)시스테인(Cys, C)글리신(Gly, G)알라닌(Ala, A)발린(Val, V)프롤린(Pro, P)메티오닌(Met, M)루신(Leu, L)노르루신(norleucine)이소루신(isoleucine)Phenylalanine (Phe, F) Tryptophan (Trp, W) Cysteine (Cys, C) Glycine (Gly, G) Alanine (Ala, A) Valine (Val, V) Proline (Pro, P) Methionine (Met, M) Leucine ( Leu, L) norleucine isoleucine
각각의 아미노산에 대하여, 추가적인 보존적 치환은 아미노산의 "동족체(homolog)"를 포함한다. 이때, 상기 "동족체"는 아미노산의 곁사슬(side chain)의 베타 위치의 곁사슬에 메틸렌 그룹(CH2)이 삽입된 아미노산을 의미한다. 이러한 "동족체"의 예로는 호모페닐알라닌, 호모알지닌, 호모세린 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 용어, "IL-7 단백질"은 "IL-7 단백질 및 이의 단편"을 포함하는 개념으로 사용되기도 한다. 용어 "단백질", "폴리펩티드" 및 "펩티드"는, 별다른 설명이 없는 한, 상호 호환되는 개념으로 사용될 수 있다.For each amino acid, additional conservative substitutions include "homologs" of the amino acids. At this time, the "homolog" refers to an amino acid in which a methylene group (CH 2 ) is inserted into the side chain of the beta position of the side chain of the amino acid. Examples of such "homologs" include, but are not limited to, homophenylalanine, homoarginine, homoserine, and the like. In the present invention, the term "IL-7 protein" is also used as a concept including "IL-7 protein and fragments thereof". The terms “protein”, “polypeptide” and “peptide” may be used as interchangeable concepts unless otherwise specified.
상기 변형된 IL-7의 구조에서 A는 상기 IL-7의 N-말단에 직접 연결되거나, 또는 링커를 통해 연결될 수 있다. 상기 A는 IL-7의 N-말단에 연결될 수 있다. 상기 A는 1개 내지 10개의 아미노산을 포함하는 것을 특징으로 하며, 상기 아미노산은 메티오닌, 글리신 및 이들의 조합으로 이루어진 군에서 선택될 수 있다. In the structure of the modified IL-7, A may be directly linked to the N-terminus of the IL-7 or linked through a linker. The A may be linked to the N-terminus of IL-7. The A is characterized in that it contains 1 to 10 amino acids, and the amino acid may be selected from the group consisting of methionine, glycine, and combinations thereof.
메티오닌과 글리신은 인체에서 면역반응을 유도하지 않는다. E.coli로부터 생산되는 단백질 치료제들은 N-말단에 메티오닌이 반드시 포함되지만 이로 인한 면역 부작용은 보고된 바가 없다. 또한, 글리신은 GS 링커(linker)에 널리 사용되고 있는데, 둘라글루타이드(Dulaglutide)와 같이 상용화된 제품에서도 면역반응을 유도하지 않는다(Cell Biophys. 1993 Jan-Jun:22(1-3): 189-224).Methionine and glycine do not induce an immune response in the body. Protein therapeutics produced from E. coli necessarily contain methionine at the N-terminus, but no immune side effects have been reported. In addition, glycine is widely used in GS linkers, but it does not induce an immune response even in commercially available products such as Dulaglutide ( Cell Biophys. 1993 Jan-Jun:22(1-3): 189- 224).
일 실시양태에 따르면, 상기 A는 메티오닌(Met, M), 글리신(Gly, G) 및 이의 조합으로 이루어진 군에서 선택되는 1개 내지 10개의 아미노산을 포함한 올리고펩티드일 수 있다. 바람직하게는 2개 내지 10개의 아미노산을 포함한 올리고펩티드일 수 있고, 더욱 바람직하게는 3개 내지 10개의 아미노산을 포함한 올리고펩티드일 수 있으나, 이에 한정되는 것은 아니다. 구체적으로, 상기 A는 메티오닌, 글리신, 메티오닌-메티오닌, 글리신-글리신, 메티오닌-글리신, 글리신-메티오닌, 메티오닌-메티오닌-메티오닌, 메티오닌-메티오닌-글리신, 메티오닌-글리신-메티오닌, 글리신-메티오닌-메티오닌, 메티오닌-글리신-글리신, 글리신-메티오닌-글리신, 글리신-글리신-메티오닌, 글리신-글리신-글리신, 메티오닌-메티오닌-메티오닌-메티오닌, 메티오닌-글리신-메티오닌-메티오닌, 메티오닌-글리신-글리신-메티오닌, 메티오닌-글리신-글리신-글리신, 메티오닌-글리신-메티오닌-글리신, 글리신-메티오닌-메티오닌-메티오닌, 글리신-메티오닌-글리신-글리신, 글리신-글리신-글리신-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-메티오닌, 메티오닌-메티오닌-글리신-메티오닌-메티오닌, 메티오닌-메티오닌-글리신-글리신-메티오닌, 메티오닌-글리신-메티오닌-메티오닌-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-글리신, 글리신-글리신-글리신-글리신-글리신, 글리신-글리신-메티오닌-메티오닌-메티오닌, 글리신-글리신-글리신-메티오닌-글리신, 메티오닌-글리신-메티오닌-글리신-메티오닌-글리신, 메티오닌-메티오닌-메티오닌-글리신-글리신-글리신, 메티오닌-메티오닌-글리신-글리신-메티오닌-메티오닌, 글리신-글리신-메티오닌-메티오닌-글리신-글리신, 메티오닌-글리신-메티오닌-글리신-메티오닌-글리신-메티오닌-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-글리신-글리신-글리신-글리신, 메티오닌-메티오닌-글리신-글리신-메티오닌-메티오닌-글리신-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-글리신-글리신-글리신-글리신, 메티오닌-글리신-메티오닌-글리신-메티오닌-글리신-메티오닌-글리신-메티오닌-글리신 또는 메티오닌-메티오닌-메티오닌-메티오닌-메티오닌-글리신-글리신-글리신-글리신-글리신으로 이루어진 군에서 선택되는 하나의 N-말단 서열을 가질 수 있다.According to one embodiment, A may be an oligopeptide containing 1 to 10 amino acids selected from the group consisting of methionine (Met, M), glycine (Gly, G), and combinations thereof. Preferably, it may be an oligopeptide including 2 to 10 amino acids, more preferably an oligopeptide including 3 to 10 amino acids, but is not limited thereto. Specifically, A is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, Methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, glycine-glycine-glycine, methionine-methionine-methionine-methionine, methionine-glycine-methionine-methionine, methionine-glycine-glycine-methionine, methionine- Glycine-glycine-glycine, methionine-glycine-methionine-glycine, glycine-methionine-methionine-methionine, glycine-methionine-glycine-glycine, glycine-glycine-glycine-glycine, methionine-methionine-methionine-methionine-methionine, methionine- Methionine-glycine-methionine-methionine, methionine-methionine-glycine-glycine-methionine, methionine-glycine-methionine-methionine-glycine, methionine-methionine-methionine-methionine-glycine, glycine-glycine-glycine-glycine-glycine, glycine- Glycine-methionine-methionine-methionine, glycine-glycine-glycine-methionine-glycine, methionine-glycine-methionine-glycine-methionine-glycine, methionine-methionine-methionine-glycine-glycine-glycine, methionine-methionine-glycine-glycine- Methionine-methionine, glycine-glycine-methionine-methionine-glycine-glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine, methionine- Methionine-glycine-glycine-methionine-methionine-glycine-glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine or It may have one N-terminal sequence selected from the group consisting of methionine-methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine-glycine.
상기 변형된 IL-7은 서열번호 2 또는 서열번호 3의 아미노산 서열로 이루어진 것일 수 있다.The modified IL-7 may consist of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
본 발명의 용어, "TBRII" 또는 "TGF 베타 수용체 II"는 달리 언급되지 않는 한, 포유동물, 예를 들어, 영장류(예, 인간) 및 설치류(예, 마우스 및 래트)를 포함하여 임의의 척추동물 공급원으로부터 수득한 임의의 야생형 TBRII 를 말한다. 상기 인간 TBRII는 서열번호 4의 아미노산 서열로 이루어진 것일 수 있다. 구체적으로, 상기 TBRII는 TBRII의 세포외 도메인일 수 있다. 상기 TBRII의 세포외 도메인은 인간 TBRII(서열번호 4)의 24번째 내지 159번째 아미노산 서열을 갖는 것일 수 있으며, 상기 TBRII의 세포외 도메인은 서열번호 5의 아미노산 서열로 이루어진 것일 수 있다. The terms of the present invention, "TBRII" or "TGF beta receptor II", unless otherwise stated, include any vertebrae, including mammals such as primates (eg humans) and rodents (eg mice and rats). Refers to any wild-type TBRII obtained from animal sources. The human TBRII may consist of the amino acid sequence of SEQ ID NO: 4. Specifically, the TBRII may be an extracellular domain of TBRII. The extracellular domain of TBRII may have the amino acid sequence 24 to 159 of human TBRII (SEQ ID NO: 4), and the extracellular domain of TBRII may be composed of the amino acid sequence of SEQ ID NO: 5.
본 발의 용어, "sTBRII"는 용해성 TBRII를 의미하며, 인간 TBRII의 세포외 도메인일 수 있다. The term "sTBRII" in the present invention means soluble TBRII, and may be an extracellular domain of human TBRII.
상기 변형된 IL-7 및 TBRII는 면역글로불린 Fc 도메인에 의해 결합될 수 있다. The modified IL-7 and TBRII can be bound by an immunoglobulin Fc domain.
상기 Fc 도메인은 야생형 또는 변이체일 수 있다. 상기 Fc 도메인 변이체는 변형된 면역글로불린의 Fc 도메인일 수 있다. 이때, 변형된 면역글로불린의 Fc 도메인은 Fc 수용체 및/또는 보체(complement)와의 결합력이 변형되어 항체의존성 세포독성(antibody-dependent cellular cytotoxicity, ADCC) 또는 보체의존성 세포독성(complement-dependent cytotoxicity, CDC)이 약화된 것일 수 있다. 상기 변형된 면역글로불린은 IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE 및 이의 조합으로 이루어진 군에서 선택될 수 있다. 특히, 상기 변형된 면역글로불린의 Fc 도메인은 N-말단에서 C-말단 방향으로 힌지 영역, CH2 도메인 및 CH3 도메인을 포함할 수 있다. 이때, 상기 힌지 영역은 인간 IgD 힌지 영역을 포함하고, 상기 CH2 도메인은 인간 IgD의 CH2 도메인의 아미노산 잔기의 부분 및 인간 IgG4의 CH2 도메인의 아미노산 잔기의 부분을 포함하며, 상기 CH3 도메인은 인간 IgG4의 CH3 도메인의 아미노산 잔기의 부분을 포함할 수 있다. 상기 힌지 영역은 IgG1 힌지 영역일 수 있고, 이는 서열번호 6의 아미노산 서열을 포함하는 것일 수 있다. The Fc domain may be wild type or variant. The Fc domain variant may be an Fc domain of a modified immunoglobulin. At this time, the Fc domain of the modified immunoglobulin is modified to bind to the Fc receptor and/or complement, so that antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) This could be weakened. The modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. In particular, the Fc domain of the modified immunoglobulin may include a hinge region, a CH2 domain, and a CH3 domain in the N-terminal to C-terminal direction. In this case, the hinge region includes a human IgD hinge region, and the CH2 domain includes a portion of an amino acid residue of a CH2 domain of human IgD and a portion of an amino acid residue of a CH2 domain of human IgG4, and the CH3 domain is of human IgG4. It may comprise a portion of the amino acid residues of the CH3 domain. The hinge region may be an IgG1 hinge region, which may include the amino acid sequence of SEQ ID NO: 6.
본 발명의 용어, "Fc 도메인", "Fc 단편" 또는 "Fc"란 면역글로불린의 중쇄 불변 영역 2(CH2) 및 중쇄 불변 영역 3(CH3)을 포함하나, 이의 중쇄 및 경쇄의 가변 영역 및 경쇄 불변 영역 1(CL1)은 포함하지 않는 단백질을 말한다. 이는 중쇄 불변 영역의 힌지 영역을 더 포함할 수 있다. 하이브리드 Fc 또는 하이브리드 Fc 단편은 본원에서 "hFc" 또는 "hyFc"로 지칭되기도 한다.In the terms of the present invention, the term "Fc domain", "Fc fragment" or "Fc" includes heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of immunoglobulin, but variable regions and light chains of heavy and light chains thereof Constant region 1 (CL1) refers to a protein that does not contain. It may further include a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc fragment is also referred to herein as “hFc” or “hyFc”.
본 발명의 용어, "Fc 도메인 변이체"는 Fc 도메인 중 일부 아미노산이 치환되거나, 서로 다른 종류의 Fc 도메인을 조합하여 제조된 것을 의미한다. 상기 Fc 도메인 변이체는 힌지 부위에서 절단되는 것을 예방할 수 있다. 구체적으로, 서열번호 9의 144번째 아미노산 및/또는 145번째 아미노산이 변형될 수 있다. 바람직하게는 서열번호 9의 144번째 아미노산인 K를 G 또는 S로 치환(K144G, K144S)하고, 145번째 아미노산인 E를 G 또는 S로 치환(E145G, E145S)한 변이체일 수 있다.The term "Fc domain variant" of the present invention means that some amino acids in the Fc domain are substituted or are prepared by combining different types of Fc domains. The Fc domain variant can prevent cleavage at the hinge region. Specifically, the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 9 may be modified. Preferably, the 144th amino acid K of SEQ ID NO: 9 may be substituted with G or S (K144G, K144S), and the 145th amino acid E may be substituted with G or S (E145G, E145S).
또한, 상기 변형된 면역글로불린의 Fc 도메인 또는 Fc 도메인 변이체는 다음과 같은 식 (I)으로 나타낼 수 있다:In addition, the Fc domain or Fc domain variant of the modified immunoglobulin can be represented by the following formula (I):
[식 (I)][Equation (I)]
N'-(Z1)p-Y-Z2-Z3-Z4-C'N'-(Z1)p-Y-Z2-Z3-Z4-C'
상기 식에서, In the above formula,
N'은 폴리펩티드의 N-말단이고 C'는 폴리펩티드의 C-말단이고;N'is the N-terminus of the polypeptide and C'is the C-terminus of the polypeptide;
p는 0 또는 1인 정수이고; 및 p is an integer of 0 or 1; And
Z1은 서열번호 7의 90 내지 98 위치의 아미노산 잔기 중 98 위치로부터 N-말단 방향으로 5 내지 9개의 연속된 아미노산 잔기를 가지는 아미노산 서열이고,Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from the 98 position among the amino acid residues at positions 90 to 98 of SEQ ID NO: 7,
Y는 서열번호 7의 99 내지 162 위치의 아미노산 잔기 중 162 위치로부터 N-말단 방향으로 5 내지 64개의 연속된 아미노산 잔기를 가지는 아미노산 서열이고,Y is an amino acid sequence having 5 to 64 consecutive amino acid residues in the N-terminal direction from position 162 of the amino acid residues at positions 99 to 162 of SEQ ID NO: 7,
Z2는 서열번호 7의 163 내지 199 위치의 아미노산 잔기 중 163 위치로부터 C-말단 방향으로 4 내지 37개의 연속된 아미노산 잔기를 가지는 아미노산 서열이고,Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues in the C-terminal direction from position 163 of the amino acid residues at positions 163 to 199 of SEQ ID NO: 7,
Z3는 서열번호 8의 115 내지 220 위치의 아미노산 잔기 중 220 위치로부터 N-말단 방향으로 71 내지 106개의 연속된 아미노산 잔기를 가지는 아미노산 서열이고,Z3 is an amino acid sequence having 71 to 106 consecutive amino acid residues in the N-terminal direction from the 220 position among the amino acid residues at positions 115 to 220 of SEQ ID NO: 8,
Z4는 서열번호 8의 221 내지 327 위치의 아미노산 잔기 중 221 위치로부터 C-말단 방향으로 80 내지 107개의 아미노산 서열을 가지는 아미노산 서열이다.Z4 is an amino acid sequence having an amino acid sequence of 80 to 107 in the C-terminal direction from position 221 among the amino acid residues at positions 221 to 327 of SEQ ID NO: 8.
또한, 본 발명의 Fc 단편은 천연형 당쇄, 천연형에 비해 증가된 당쇄, 천연형에 비해 감소한 당쇄, 또는 당쇄가 제거된 형태일 수 있다. 화학적 방법, 효소적 방법 및 미생물을 사용한 유전공학적 엔지니어링 방법 등과 같이 통상적인 방법으로 면역글로불린 Fc 당쇄를 변형시킬 수 있다. Fc 단편으로부터 당쇄의 제거는 1차 보체 구성요소 C1의 C1q에의 결합 친화력을 급격하게 감소시키고, ADCC 또는 CDC의 감소 또는 소실을 가져오며, 그로 인하여 생체 내의 불필요한 면역반응을 유도하지 않는다. 이런 점에서, 당쇄가 제거되거나(deglycosylated) 비당쇄화된(aglycosylated) 형태에서의 면역글로불린 Fc 단편은, 약물의 담체로서 본 발명의 목적에 더 적합할 수 있다. 여기에서 사용된 용어 "당쇄의 제거(deglycosylation)"는 Fc 단편으로부터 효소적으로 당이 제거됨을 의미한다. 또한, 용어 "비당쇄화(aglycosylation)"는 Fc 단편이 원핵 생물, 바람직하게는 E.coli에 의하여 당쇄화 되지 않은(unglycosylated) 형태로 생성됨을 의미한다.In addition, the Fc fragment of the present invention may be a natural type sugar chain, an increased sugar chain compared to the natural type, a reduced sugar chain compared to the natural type, or a form in which the sugar chain has been removed. The immunoglobulin Fc sugar chain can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment sharply decreases the binding affinity of the primary complement component C1 to C1q, leads to a decrease or loss of ADCC or CDC, and thereby does not induce an unnecessary immune response in vivo. In this respect, the immunoglobulin Fc fragment in the form of deglycosylated or aglycosylated sugar chain may be more suitable for the purposes of the present invention as a drug carrier. The term "deglycosylation" as used herein means that sugars are enzymatically removed from an Fc fragment. In addition, the term "aglycosylation" means that the Fc fragment is produced in an unglycosylated form by a prokaryote, preferably E. coli.
또한, 상기 변형된 면역글로불린의 Fc 도메인은 서열번호 9 (hyFc), 서열번호 10 (hyFcM1), 서열번호 11 (hyFcM2), 서열번호 12 (hyFcM3) 또는 서열번호 13 (hyFcM4)의 아미노산 서열을 포함할 수 있다. In addition, the Fc domain of the modified immunoglobulin contains the amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 10 (hyFcM1), SEQ ID NO: 11 (hyFcM2), SEQ ID NO: 12 (hyFcM3) or SEQ ID NO: 13 (hyFcM4). can do.
본 발명에 따르면, 상기 변형된 면역글로불린의 Fc 도메인은 미국 특허 제7,867,491호에 기재된 것일 수 있으며, 상기 변형된 면역글로불린의 Fc 도메인의 생산은 미국 특허 제7,867,491호에 기재된 바를 참조하여 수행될 수 있다.According to the present invention, the Fc domain of the modified immunoglobulin may be described in U.S. Patent No. 7,867,491, and the production of the Fc domain of the modified immunoglobulin may be performed with reference to the bar described in U.S. Patent No. 7,867,491. .
또한, 상기 융합단백질은 TBRII, Fc 도메인 및 변형된 IL-7이 N-말단으로부터 C-말단 방향으로 순서대로 결합된 것일 수 있다. 이러한 융합단백질은 "sTBRII-hyFc-IL7"로 표기될 수 있다. In addition, the fusion protein may be a TBRII, Fc domain, and modified IL-7 are sequentially bound from the N-terminus to the C-terminus. This fusion protein may be referred to as "sTBRII-hyFc-IL7".
상기 sTBRII-hyFc-IL7 융합단백질의 경우, TBRII와 Fc 도메인의 사이에는 제1링커가 더 포함될 수 있다. 상기 제1링커는 20 내지 60개의 연속된 아미노산, 또는 25 내지 50개의 연속된 아미노산, 또는 30 내지 40개의 아미노산으로 이루어질 수 있다. 일 구체예로 제1링커는 20개의 아미노산으로 이루어질 수 있다. 또한, 제1링커는 (G4S)n(이때, n은 1 내지 5의 정수)를 포함할 수 있다. 바람직하게, 상기 제1링커는 서열번호 14의 아미노산 서열로 이루어진 것일 수 있다.In the case of the sTBRII-hyFc-IL7 fusion protein, a first linker may be further included between the TBRII and the Fc domain. The first linker may consist of 20 to 60 contiguous amino acids, or 25 to 50 contiguous amino acids, or 30 to 40 amino acids. In one embodiment, the first linker may consist of 20 amino acids. In addition, the first linker may include (G4S)n (here, n is an integer of 1 to 5). Preferably, the first linker may consist of the amino acid sequence of SEQ ID NO: 14.
또한, Fc 도메인과 변형된 IL-7의 사이에는 제2링커가 더 포함될 수 있다. 상기 제2링커는 1 내지 30개의 연속된 아미노산, 또는 3 내지 20개의 연속된 아미노산, 또는 4 내지 16개의 아미노산으로 이루어질 수 있다. 또한, 제2링커는 (SG3)n(이때, n은 1 내지 5의 정수)를 포함할 수 있다. 일 실시예로 상기 제2링커는 서열번호 15의 아미노산 서열로 이루어진 것일 수 있다.In addition, a second linker may be further included between the Fc domain and the modified IL-7. The second linker may consist of 1 to 30 contiguous amino acids, 3 to 20 contiguous amino acids, or 4 to 16 amino acids. In addition, the second linker may include (SG3)n (here, n is an integer of 1 to 5). In an embodiment, the second linker may consist of the amino acid sequence of SEQ ID NO: 15.
따라서, sTBRII-hyFc-IL7 융합단백질은 하기의 구조로 이루어진 것일 수 있다. Therefore, the sTBRII-hyFc-IL7 fusion protein may have the following structure.
N' - TBRII - (L1)p - Fc domain - (L2)q - A-IL-7 - C' N'-TBRII-(L1)p-Fc domain-(L2)q-A-IL-7-C'
상기에서, Above,
N'은 N-말단이고, C'는 C-말단이고, N'is the N-terminal, C'is the C-terminal,
L1은 제1링커이고, L2는 제2링커이며, L1 is the first linker, L2 is the second linker,
p 및 q는 0 또는 1인 정수이다. p and q are integers of 0 or 1.
상기 sTBRII-hyFc-IL7 융합단백질은 서열번호 17의 아미노산 서열로 이루어진 것일 수 있다. 상기 융합단백질은 서열번호 17의 아미노산 서열에 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 또는 99% 이상의 상동성을 갖는 서열을 가질 수 있다.The sTBRII-hyFc-IL7 fusion protein may consist of the amino acid sequence of SEQ ID NO: 17. The fusion protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, in the amino acid sequence of SEQ ID NO: 17, Alternatively, it may have a sequence with 99% or more homology.
또한, 상기 융합단백질은 변형된 IL-7, Fc 도메인 또는 TBRII가 N-말단으로부터 C-말단 방향으로 순서대로 결합된 것일 수 있다. 이러한 융합단백질은 "IL7-hyFc-sTBRII"로 표기될 수 있다. In addition, the fusion protein may be a modified IL-7, Fc domain, or TBRII is sequentially bound from the N-terminal to the C-terminal direction. This fusion protein may be referred to as "IL7-hyFc-sTBRII".
상기 IL7-hyFc-sTBRII 융합단백질의 경우, 변형된 IL-7과 Fc 도메인 사이에는 제1링커가 더 포함될 수 있다. 상기 제1링커는 20 내지 60개의 연속된 아미노산, 또는 25 내지 50개의 연속된 아미노산, 또는 30 내지 40개의 아미노산으로 이루어질 수 있다. 일 구체예로 제1링커는 20개의 아미노산으로 이루어질 수 있다. 또한, 제1링커는 (G4S)n(이때, n은 1 내지 5의 정수)를 포함할 수 있다. 바람직하게, 상기 제1링커는 서열번호 14의 아미노산 서열로 이루어진 것일 수 있다.In the case of the IL7-hyFc-sTBRII fusion protein, a first linker may be further included between the modified IL-7 and the Fc domain. The first linker may consist of 20 to 60 contiguous amino acids, or 25 to 50 contiguous amino acids, or 30 to 40 amino acids. In one embodiment, the first linker may consist of 20 amino acids. In addition, the first linker may include (G4S)n (here, n is an integer of 1 to 5). Preferably, the first linker may consist of the amino acid sequence of SEQ ID NO: 14.
또한, Fc 도메인과 TBRII 사이에는 제3링커가 더 포함될 수 있다. 상기 제3링커는 1 내지 30개의 연속된 아미노산, 또는 3 내지 20개의 연속된 아미노산, 또는 4 내지 16개의 아미노산으로 이루어질 수 있다. 일 실시예로 상기 제3링커는 서열번호 16의 아미노산 서열로 이루어진 것일 수 있다.In addition, a third linker may be further included between the Fc domain and TBRII. The third linker may consist of 1 to 30 contiguous amino acids, or 3 to 20 contiguous amino acids, or 4 to 16 amino acids. In an embodiment, the third linker may be made of the amino acid sequence of SEQ ID NO: 16.
따라서, IL7-hyFc-sTBRII 융합단백질은 하기의 구조로 이루어진 것일 수 있다. Therefore, the IL7-hyFc-sTBRII fusion protein may have the following structure.
N' - A-IL-7 - (L1)p - Fc domain - (L3)r - TBRII - C' N'-A-IL-7-(L1)p-Fc domain-(L3)r-TBRII-C'
상기에서, Above,
N'은 N-말단이고, C'는 C-말단이고, N'is the N-terminal, C'is the C-terminal,
L1은 제1링커이고, L2는 제2링커이며, L1 is the first linker, L2 is the second linker,
p 및 r는 0 또는 1인 정수이다.p and r are integers of 0 or 1.
상기 IL7-hyFc-sTBRII 융합단백질은 서열번호 18의 아미노산 서열로 이루어진 것일 수 있다. 상기 융합단백질은 서열번호 18의 아미노산 서열에 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 또는 99% 이상의 상동성을 갖는 서열을 가질 수 있다.The IL7-hyFc-sTBRII fusion protein may be composed of the amino acid sequence of SEQ ID NO: 18. The fusion protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, in the amino acid sequence of SEQ ID NO: 18, Alternatively, it may have a sequence with 99% or more homology.
또한, 본 발명은 상기 융합 단백질을 코딩하는 핵산 분자를 제공한다. In addition, the present invention provides a nucleic acid molecule encoding the fusion protein.
또한, 상기 핵산 분자는 신호 서열(또는 신호 펩티드) 또는 리더 서열을 추가적으로 포함할 수 있다.In addition, the nucleic acid molecule may additionally include a signal sequence (or signal peptide) or a leader sequence.
본 발명의 용어, "신호 서열(또는 신호 펩티드)"은 분비 경로로 분류된 새로 합성된 단백질의 N-말단에 존재하는 짧은 펩티드를 말한다. 본 발명에서 유용한 신호 서열은 항체 경쇄 신호 서열, 예를 들면 항체 1418(Gillies et al., J Immunol Meth 1989 125:191-202), 항체 중쇄 신호 서열, 예를 들면, MOPC141 항체 중쇄 신호 서열(Sakano et al, Nature 1980 286: 676-683), 및 당업계에 알려진 다른 신호 서열(예, Watson et al, Nucleic Acid Research 1984 12:5145-5164를 참조)을 포함한다. As used herein, the term "signal sequence (or signal peptide)" refers to a short peptide present at the N-terminus of a newly synthesized protein classified as a secretory pathway. Signal sequences useful in the present invention include antibody light chain signal sequences, such as antibody 1418 (Gillies et al., J Immunol Meth 1989 125:191-202), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequences (Sakano et al, Nature 1980 286: 676-683), and other signal sequences known in the art (see, e.g., Watson et al, Nucleic Acid Research 1984 12:5145-5164).
상기 신호 펩타이드는 당업계에 그 특징이 잘 알려져 있으며, 통상 16 내지 30개의 아미노산 잔기를 포함하는 것으로 알려져 있고, 그 보다 더 많은 또는 더 적은 아미노산 잔기를 포함할 수 있다. 통상적인 신호 펩타이드는 기본 N-말단 영역, 중심의 소수성 영역, 및 보다 극성인(polar) C-말단 영역의 세 영역으로 구성된다. The signal peptide is well known in the art, and is generally known to contain 16 to 30 amino acid residues, and may contain more or less amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.
중심 소수성 영역은 미성숙 폴리펩티드가 이동하는 동안 막지질 이중층을 통하여 신호 서열을 고정시키는 4 내지 12개의 소수성 잔기를 포함한다. 개시 이후에, 신호 서열은 흔히 신호 펩티다아제(signal peptidases)로 알려진 세포 효소에 의하여 ER의 루멘(lumen) 내에서 절단된다. 이때, 상기 신호 서열은 tPa(tissue Plasminogen Activation), HSV gDs, 또는 성장 호르몬의 분비신호 서열일 수 있다. 바람직하게, 포유동물 등을 포함하는 고등 진핵 세포에서 사용되는 분비 신호 서열을 사용할 수 있으며, 더욱 바람직하게는 tPa 서열(서열번호 19) 또는 서열번호 20의 아미노산 서열을 사용할 수 있다. 또한, 본 발명의 신호 서열은 숙주세포에서 발현 빈도가 높은 코돈으로 치환하여 사용할 수 있다.The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide. After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. In this case, the signal sequence may be a tissue plasma activation (tPa), HSV gDs, or a secretion signal sequence of growth hormone. Preferably, the secretion signal sequence used in higher eukaryotic cells including mammals and the like can be used, more preferably, the tPa sequence (SEQ ID NO: 19) or the amino acid sequence of SEQ ID NO: 20 can be used. In addition, the signal sequence of the present invention can be used by substituting codons with high expression frequency in host cells.
또한, 본 발명은 상기 핵산 분자를 포함하는 발현 벡터를 제공한다. In addition, the present invention provides an expression vector containing the nucleic acid molecule.
본 발명의 용어, "벡터"는 숙주 세포에 도입되어 숙주 세포 유전체 내로 재조합 및 삽입될 수 있거나, 또는 에피좀(episome)으로서 자발적으로 복제될 수 있는 뉴클레오타이드 서열을 포함하는 핵산 수단으로 이해된다. 상기 벡터는 선형(linear) 핵산, 플라스미드, 파지미드(phagemids), 코스미드(cosmids), RNA 벡터, 바이러스 벡터 및 이의 유사체들을 포함한다. 바이러스 벡터의 예로는 레트로바이러스(retrovirus), 아데노바이러스(adenovirus) 및 아데노-관련 바이러스(adeno-associated virus)를 포함하나, 이에 제한되지 않는다.The term "vector" of the present invention is understood as a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell, recombined and inserted into the host cell genome, or spontaneously replicated as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors include, but are not limited to, retrovirus, adenovirus, and adeno-associated virus.
본 발명에서, 유용한 발현 벡터는 RcCMV(Invitrogen, Carlsbad) 또는 이의 변이체일 수 있다. 유용한 발현 벡터는 포유류 세포에서 목적 유전자의 연속적인 전사를 촉진하기 위한 인간 CMV(cytomegalovirus) 프로모터, 및 전사 후 RNA의 안정상태 수준을 높이기 위한 우태 성장 인자(bovine growth hormone) 폴리아데닐레이션 신호 서열을 포함할 수 있다. 본 발명의 일 구체예로 발현 벡터는 RcCMV의 변형 벡터인 pAD15이다.In the present invention, a useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. Useful expression vectors include a human CMV (cytomegalovirus) promoter to promote the continuous transcription of a gene of interest in mammalian cells, and a bovine growth hormone polyadenylation signal sequence to increase the stable level of RNA after transcription. can do. In one embodiment of the present invention, the expression vector is pAD15, a modified vector of RcCMV.
본 발명의 용어, "숙주 세포"는 재조합 발현 벡터가 도입될 수 있는 원핵 및 진핵 세포를 나타낸다. The term "host cell" of the present invention refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.
본 발명에서, 적절한 숙주 세포는 본 발명의 DNA 서열로 형질 전환되거나 형질 감염시킬 수 있으며, 목적 단백질의 발현 및/또는 분비에 이용될 수 있다. 본 발명에 사용될 수 있는 현재 바람직한 숙주 세포는 불멸의 하이브리도마 세포(immortal hybridoma cells), NS/0 골수종 세포(NS/0 myeloma cells), 293 세포, 중국 햄스터 난소 세포(CHO cell), HeLa 세포, CapT 세포(인간 양수 유래 세포) 및 COS 세포를 포함한다.In the present invention, an appropriate host cell may be transformed or transfected with the DNA sequence of the present invention, and may be used for expression and/or secretion of a protein of interest. Currently preferred host cells that can be used in the present invention are immortal hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells. , CapT cells (human amniotic fluid derived cells) and COS cells.
본 발명의 용어, "형질전환" 및 "형질감염"은 당업계에 공지된 많은 기술에 의해 세포 내로 핵산(예를 들어, 벡터)을 도입하는 것을 의미한다.The terms "transformation" and "transfection" of the present invention mean the introduction of a nucleic acid (eg, a vector) into a cell by a number of techniques known in the art.
또한, 본 발명은 변형된 IL-7 및 TBRII를 포함하는 융합단백질을 유효성분으로 포함하는 암 또는 감염성 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer or infectious diseases comprising a fusion protein containing modified IL-7 and TBRII as an active ingredient.
상기 암은 위암, 간암, 폐암, 대장암, 유방암, 전립선암, 난소암, 췌장암, 자궁경부암, 갑상선암, 후두암, 급성 골수성 백혈병, 뇌종양, 신경모세포종, 망막 모세포종, 두경부암, 침샘암 및 림프종으로 이루어진 군에서 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다. The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. It may be selected from the group, but is not limited thereto.
상기 감염성 질환은 B형 간염, C형 간염, 인간 파필로마 바이러스 감염, 사이토메갈로바이러스 감염, 바이러스성 호흡기 질환 및 인플루엔자로 이루어진 군에서 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다. The infectious disease may be selected from the group consisting of hepatitis B, hepatitis C, human papilloma virus infection, cytomegalovirus infection, viral respiratory disease, and influenza, but is not limited thereto.
본 발명의 암 또는 감염성 질환 치료용 또는 예방용 조성물에서 그 유효성분은 항암 활성을 나타내거나, 감염성 질환에 치료 효과를 나타낼 수 있는 한, 용도, 제형, 배합 목적 등에 따라 임의의 양(유효량)으로 포함될 수 있는데, 통상적인 유효량은 조성물 전체 중량을 기준으로 할 때 0.001 중량 % 내지 20.0 중량% 범위 내에서 결정될 것이다. 여기서 "유효량"이란 항암 효과 또는 감염성 질환 치료 효과를 유도할 수 있는 유효성분의 양을 말한다. 이러한 유효량은 당업자의 통상의 능력 범위 내에서 실험적으로 결정될 수 있다.In the composition for treating or preventing cancer or infectious diseases of the present invention, the active ingredient may be in an arbitrary amount (effective amount) according to the use, formulation, purpose of combination, etc. It may be included, and a typical effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, "effective amount" refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such effective amounts can be determined empirically within the range of ordinary skill in the art.
이때, 상기 약학 조성물은 약학적으로 허용 가능한 담체를 더 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 환자에게 전달하기에 적절한 비-독성 물질이면 어떠한 담체라도 가능하다. 증류수, 알코올, 지방, 왁스 및 비활성 고체가 담체로 포함될 수 있다. 약물학적으로 허용되는 애쥬번트(완충제, 분산제) 또한 약물학적 조성물에 포함될 수 있다.In this case, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohols, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmacological composition.
구체적으로, 상기 약학 조성물은 유효성분 이외에 약제학적으로 허용되는 담체를 포함하여 당업계에 공지된 통상의 방법으로 투여 경로에 따라 비경구용 제형으로 제조될 수 있다. 여기서 "약제학적으로 허용되는" 의미는 유효성분의 활성을 억제하지 않으면서 적용(처방) 대상이 적응 가능한 이상의 독성을 지니지 않는다는 의미이다.Specifically, the pharmaceutical composition may be prepared in a parenteral formulation according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.
상기 약학 조성물이 비경구용 제형으로 제조될 경우, 적합한 담체와 함께 당업계에 공지된 방법에 따라 주사제, 경피 투여제, 비강 흡입제 및 좌제의 형태로 제제화될 수 있다. 주사제로 제제화할 경우 적합한 담체로서는 멸균수, 에탄올, 글리세롤이나 프로필렌 글리콜 등의 폴리올 또는 이들의 혼합물을 사용할 수 있으며, 바람직하게는 링거 용액, 트리에탄올 아민이 함유된 PBS(phosphate bufferedsaline)나 주사용 멸균수, 5% 덱스트로스 같은 등장 용액 등을 사용할 수 있다. 약제학적 조성물의 제제화와 관련하여서는 당업계에 공지되어 있으며, 구체적으로 문헌[Remington's Pharmaceutical Sciences(19th ed., 1995)] 등을 참조할 수 있다. 상기 문헌은 본 명세서의 일부로서 간주 된다.When the pharmaceutical composition is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation, and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterile water for injection. , An isotonic solution such as 5% dextrose may be used. Regarding the formulation of a pharmaceutical composition, it is known in the art, and specifically, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered as part of this specification.
상기 약학 조성물의 바람직한 투여량은 환자의 상태, 체중, 성별, 연령, 환자의 중증도, 투여 경로에 따라 1일 0.01 ug/kg 내지 10 g/kg 범위, 또는 0.01 mg/kg 내지 1 g/kg 범위일 수 있다. 투여는 1일 1회 또는 수회로 나누어 이루어질 수 있다. 이러한 투여량은 어떠한 측면으로든 본원 발명의 범위를 제한하는 것으로 해석되어서는 안 된다. The preferred dosage of the pharmaceutical composition is in the range of 0.01 ug/kg to 10 g/kg per day, or 0.01 mg/kg to 1 g/kg per day depending on the patient's condition, weight, sex, age, patient severity, and route of administration. Can be Administration can be made once a day or divided into several times. Such dosages should not be construed as limiting the scope of the invention in any aspect.
상기 약학 조성물이 적용(처방)될 수 있는 대상은 포유동물 및 사람이며, 특히 사람인 경우가 바람직하다. 본원의 약학적 조성물은 유효성분 이외에, 항암 활성의 상승·보강을 위하여 이미 안전성이 검증되고 항암 활성 또는 감염성 질환에 치료 효과를 갖는 것으로 공지된 임의의 화합물이나 천연 추출물을 추가로 포함할 수 있다.Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, particularly preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present application may further include any compound or natural extract, which has already been verified for safety and has a therapeutic effect on anticancer activity or infectious disease in order to increase and reinforce anticancer activity.
또한, 본 발명은 암 또는 감염성 질환의 예방 또는 치료 효과를 가진 의약제제를 생산하기 위한 변형된 IL-7 및 TBRII를 포함하는 융합단백질의 사용을 제공한다. In addition, the present invention provides the use of a fusion protein comprising a modified IL-7 and TBRII to produce a pharmaceutical preparation having a prophylactic or therapeutic effect of cancer or infectious disease.
또한, 본 발명은 변형된 IL-7 및 TBRII를 포함하는 융합단백질을 유효성분으로 포함하는 암 또는 감염성 질환의 예방 또는 치료 방법을 제공한다. In addition, the present invention provides a method for preventing or treating cancer or infectious diseases comprising a fusion protein containing modified IL-7 and TBRII as an active ingredient.
상기 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 조성물, 개체의 연령, 체중, 일반건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다. 따라서, 본 발명의 목적에 적합한 조성물의 유효량은 전술한 사항을 고려하여 결정하는 것이 바람직하다.The therapeutically effective amount is a specific composition including the type and degree of the reaction to be achieved, whether or not other agents are used in some cases, the individual's age, weight, general health status, sex and diet, administration time, administration route and composition. It is preferable to apply differently according to various factors including the secretion rate of the drug, the treatment period, drugs used with or concurrently with the specific composition, and similar factors well known in the field of medicine. Therefore, it is preferable to determine an effective amount of a composition suitable for the purposes of the present invention in consideration of the foregoing.
상기 개체는 임의의 포유동물에 적용가능하며, 상기 포유동물은 인간 및 영장류뿐만 아니라, 소, 돼지, 양, 말, 개 및 고양이 등의 가축을 포함한다.The individual is applicable to any mammal, and the mammal includes humans and primates, as well as livestock such as cattle, pigs, sheep, horses, dogs and cats.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.
실시예 1. sTBRII-hyFc-IL7 및 IL7-hyFc-sTBRII 융합단백질의 제조Example 1. Preparation of sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII fusion proteins
본 발명자들은 인간 유래 IL-7 및 sTBRII(soluble TGF beta receptor II)가 결합된 융합단백질을 제조하기 위하여, Fc 도메인의 N-말단에 IL-7 또는 sTBRII을 융합하고, Fc 도메인의 C-말단에 IL-7 또는 sTBRII을 융합한 형태의 유전자 컨스트럭트를 제조하였다. 본 발명에서는 TBRII, Fc 도메인 및 IL-7이 N-말단으로부터 C-말단 방향으로 순서대로 결합된 융합단백질은 "sTBRII-hyFc-IL7"로 표기하였고, IL-7, Fc 도메인 또는 TBRII가 N-말단으로부터 C-말단 방향으로 순서대로 결합된 융합단백질은 "IL7-hyFc-sTBRII"로 표기하였다. In order to prepare a fusion protein in which human-derived IL-7 and sTBRII (soluble TGF beta receptor II) are bound, the present inventors fused IL-7 or sTBRII to the N-terminus of the Fc domain, and to the C-terminus of the Fc domain. A gene construct in the form of fusion of IL-7 or sTBRII was prepared. In the present invention, a fusion protein in which TBRII, Fc domain, and IL-7 are sequentially bound from the N-terminus to the C-terminus is expressed as "sTBRII-hyFc-IL7", and IL-7, Fc domain or TBRII is N- The fusion proteins bound in order from the terminal to the C-terminus were denoted as "IL7-hyFc-sTBRII".
우선, sTBRII-hyFc-IL7 유전자 컨트스턱트의 제조에 있어, sTBRII은 TGF beta receptor II의 공지된 아미노산 서열(Accession number: NP003233.4)에서 24~159 aa의 세포 외 도메인(extracellular domain)만을 사용하여 Fc 도메인의 N-말단에 융합하였고, IL-7은 공지된 아미노산 서열(Accession number: NP000871.1)을 사용하여 Fc 도메인의 C-말단에 융합하여, 유전자 컨스트럭트를 제조하였다 (도 1). First of all, in the preparation of the sTBRII-hyFc-IL7 gene constraint, sTBRII uses only the extracellular domain of 24-159 aa in the known amino acid sequence (Accession number: NP003233.4) of TGF beta receptor II. Thus, it was fused to the N-terminus of the Fc domain, and IL-7 was fused to the C-terminus of the Fc domain using a known amino acid sequence (Accession number: NP000871.1) to prepare a gene construct (Fig. 1 ).
또한, IL7-hyFc-sTBRII 유전자 컨스트럭트에의 제조에 있어, IL-7 및 sTBRII은 상기와 동일한 서열을 사용하여 각각 Fc 도메인의 N-말단과 C-말단에 융합하여, 유전자 컨스트럭트를 제조하였다 (도 1). In addition, in the preparation of the IL7-hyFc-sTBRII gene construct, IL-7 and sTBRII are fused to the N-terminus and C-terminus of the Fc domain, respectively, using the same sequence as above, and the gene construct is Was prepared (Fig. 1).
IL-7, sTBRII 및 Fc 도메인은 코스모진텍에서 각각의 유전자를 합성하여 서브-벡터(sub-vector)로 제조하였고, 합성된 세 개의 유전자 절편을 하나의 유전자 절편으로 제조하기 위해서 Golden GATEway assembly를 진행하여 pGP30 vector로 발현 벡터를 확보하였다.IL-7, sTBRII and Fc domains were prepared as sub-vectors by synthesizing each gene at Cosmo Genetech, and Golden Gateway assembly was performed to prepare the synthesized three gene segments into one gene segment. Then, the expression vector was obtained with the pGP30 vector.
발현 벡터는 부유배양에 적응된 CHO 세포(suspension-adapted Chinese Hamster Ovary cell)에 Neon Transfection system(Invitrogen, MPK1096)으로 형질 주입하였고, HT selection 및 Methotrexate(Sigma, M8407) 증폭을 통해 생산성이 높은 세포주를 선별하였다. 또한, 한계희석 클로닝(limiting dilution cloning)을 통해 단일 세포를 확보하여 sTBRII-hyFc-IL7 및 IL7-hyFc-sTBRII 융합단백질을 생산하는 생산세포주를 확보하였다. The expression vector was transfected into CHO cells (suspension-adapted Chinese Hamster Ovary cells) adapted for suspension culture with the Neon Transfection system (Invitrogen, MPK1096), and a highly productive cell line was obtained through HT selection and amplification of Methotrexate (Sigma, M8407). Were selected. In addition, single cells were secured through limiting dilution cloning to obtain production cell lines producing sTBRII-hyFc-IL7 and IL7-hyFc-sTBRII fusion proteins.
각 생산세포주는 Hycell CHO medium(Hyclone, SH30949.02)을 사용하여 250 mL Erlenmeyer flask(Corning, 431144)를 사용하여 80 mL 씩 배양(batch culture)하여 배양 생산성을 확인하였다. 그 결과, IL7-hyFc-sTBRII 융합단백질은 0.6 g/L의 양으로 생산되었고, sTBRII-hyFc-IL7 융합단백질은 0.95 g/L의 양으로로 생산되었음을 확인하였다 (도 2). Each production cell line was cultured by 80 mL each in a 250 mL Erlenmeyer flask (Corning, 431144) using Hycell CHO medium (Hyclone, SH30949.02) to confirm the culture productivity. As a result, it was confirmed that the IL7-hyFc-sTBRII fusion protein was produced in an amount of 0.6 g/L, and the sTBRII-hyFc-IL7 fusion protein was produced in an amount of 0.95 g/L (FIG. 2).
실시예 2. 투여량에 따른 sTBRII-hyFc-IL7 융합단백질의 생체 내(Example 2. In vivo of sTBRII-hyFc-IL7 fusion protein according to dosage ( in vivoin vivo ) 활성 분석) Activity assay
본 발명자들은 정맥 투여에 따른 융합단백질의 약력학적 프로필(pharmacodynamics profile)을 확인하기 위한 실험을 수행하였다. 간단히, C57BL/6 (B6) 마우스 동물모델에 sTBRII-hyFc-IL7 융합단백질을 10, 30 및 100 mg/kg (mpk)로 정맥 투여한 후, 시간에 따른 혈액 내 면역세포의 변화를 분석하였다. 또한, 혈액은 sTBRII-hyFc-IL7 융합단백질의 투여 후 3, 7, 10, 14, 17 및 21일 차에 안와채혈(retro-orbital bleeding)을 통해 채취하였다. 각 수치는 유세포 분석(flow cytometry)을 통해 세포 표면 지표의 발현율 및 혈액 내 각 세포의 구성비를 확인하였고, 이를 CBC(complete blood count)를 이용해 측정한 값과 곱하여 산출하였다. The present inventors conducted an experiment to confirm the pharmacodynamics profile of the fusion protein according to intravenous administration. Briefly, after intravenous administration of sTBRII-hyFc-IL7 fusion protein at 10, 30 and 100 mg/kg (mpk) to a C57BL/6 (B6) mouse animal model, changes of immune cells in blood over time were analyzed. In addition, blood was collected through retro-orbital bleeding on days 3, 7, 10, 14, 17 and 21 after administration of the sTBRII-hyFc-IL7 fusion protein. Each value was calculated by checking the expression rate of the cell surface indicator and the composition ratio of each cell in the blood through flow cytometry, and multiplying this by the value measured using a complete blood count (CBC).
그 결과, sTBRII-hyFc-IL7 융합단백질이 투여된 마우스에서는 IL-7의 주요 타겟 세포인 CD8+ T 세포의 세포수가 농도-의존적으로 증가하였고, 투여 7일 차에 최고치(peak point)로 나타났다 (도 3a). 또한, 투여 7일 차의 CD8+ T 세포 증가율은 대조군(PBS 처리)에 비해 10, 30 및 100 mpk의 농도에서 각각 6.22, 12.45 및 35.04 배로 증가하였음을 확인하였다 (도 3b). As a result, in mice administered with sTBRII-hyFc-IL7 fusion protein, the number of cells of CD8 + T cells, the major target cells of IL-7, increased in a concentration-dependent manner, and the peak point was found at the 7th day of administration ( Fig. 3a). In addition, it was confirmed that the increase rate of CD8 + T cells at the 7th day of administration increased by 6.22, 12.45 and 35.04 times at concentrations of 10, 30 and 100 mpk, respectively, compared to the control group (PBS treatment) (Fig. 3b).
또한, sTBRII-hyFc-IL7 융합단백질이 투여된 마우스에서는 IL-7의 타겟 세포인 CD4+ T 세포 및 CD4+CD25+Foxp3+ Treg 세포 역시 증가하는 경향을 보였다 (도 3c 및 3d). 다만, 타겟 세포가 아닌 호중구(neutrophil) 및 NK 세포에서는 투여에 따른 어떠한 변화가 관찰되지 않았다 (도 3e 및 3f). In addition, in mice administered with the sTBRII-hyFc-IL7 fusion protein, CD4 + T cells and CD4 + CD25 + Foxp3 + Treg cells, which are target cells of IL-7, also showed a tendency to increase (FIGS. 3C and 3D ). However, in neutrophils and NK cells other than the target cells, no changes were observed according to the administration (FIGS. 3e and 3f).
실시예 3. 투여 경로에 따른 sTBRII-hyFc-IL7 융합단백질의 생체 내(Example 3. In vivo of sTBRII-hyFc-IL7 fusion protein according to the route of administration ( in vivoin vivo ) 활성 분석) Activity assay
본 발명자들은 투여 경로에 따른 sTBRII-hyFc-IL7 융합단백질의 생체 내 활성도를 측정하는 실험을 수행하였다. 간단히, C57BL/6 (B6) 마우스 동물모델에 sTBRII-hyFc-IL7 융합단백질(10 mpk)을 정맥(intravenous injection, i.v.) 또는 피하(subcutaneous injection, s.c.)로 투여한 후, 시간에 따른 혈액 내 면역세포의 변화를 분석하였다. 혈액은 sTBRII-hyFc-IL7 융합단백질의 투여 후 0, 4, 7, 11, 14일 차에 안와채혈(retro-orbital bleeding)을 통해 채취하였다. 각 수치는 CBC(complete blood count)를 이용해 측정하였다. The present inventors performed an experiment to measure the in vivo activity of the sTBRII-hyFc-IL7 fusion protein according to the administration route. Briefly, sTBRII-hyFc-IL7 fusion protein (10 mpk) was administered intravenously (intravenous injection, iv) or subcutaneous (subcutaneous injection, sc) to a C57BL/6 (B6) mouse animal model, followed by immunity in the blood over time. Cellular changes were analyzed. Blood was collected through retro-orbital bleeding on days 0, 4, 7, 11, and 14 after administration of the sTBRII-hyFc-IL7 fusion protein. Each value was measured using a complete blood count (CBC).
그 결과, sTBRII-hyFc-IL7 융합단백질이 투여된 마우스에서는 투여 7일 차에 대조군(PBS 처리)에 비해 면역세포(lymphocytes)의 수가 증가하였음을 확인하였다 (도 4a). 구체적으로, 투여 7일 차의 정맥 투여군에서는 대조군에 비해 약 1.51 배로 면역세포의 수가 증가하였고, 피하 투여군에서는 대조군에 비해 약 3.04 배로 면역세포의 수가 증가하였음을 확인하였다 (도 4b). As a result, it was confirmed that the number of immune cells (lymphocytes) was increased in mice administered with the sTBRII-hyFc-IL7 fusion protein compared to the control group (PBS treatment) on the 7th day of administration (FIG. 4A). Specifically, it was confirmed that the number of immune cells increased by about 1.51 times compared to the control group in the intravenous administration group on the 7th day of administration, and the number of immune cells increased by about 3.04 times compared to the control group in the subcutaneous administration group (Fig.
실시예 4. 마우스 종양모델에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 생체 내(Example 4. In vivo of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model ( in vivoin vivo ) 활성 분석) Activity assay
본 발명자들은 마우스 종양모델에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 생체 내 활성을 분석하는 실험을 수행하였다. 간단히, C57BL/6 (B6) 마우스 동물모델에 1x105 개의 MC38 대장암 세포주를 피하 주입하여 마우스 종양모델을 제조하였다. 이후, 종양이 형성된 6일 차에 각 융합단백질을 10 mpk 또는 20 mpk로 피하 투여(s.c)한 후, 유세포 분석(flow cytometry) 및 CBC(complete blood count)를 이용해 혈액 내 면역세포의 변화를 분석하였다. 혈액은 각 융합단백질의 투여 후 0, 3, 7, 11, 15 및 18일 차에 안와채혈(retro-orbital bleeding)을 통해 채취하였다. The present inventors performed an experiment to analyze the in vivo activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1 ×10 5 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Thereafter, on the 6th day of tumor formation, each fusion protein was administered subcutaneously (sc) at 10 mpk or 20 mpk, and then the changes in immune cells in the blood were analyzed using flow cytometry and CBC (complete blood count). I did. Blood was collected through retro-orbital bleeding on days 0, 3, 7, 11, 15 and 18 after administration of each fusion protein.
그 결과, sTBRII-hyFc-IL7 융합단백질이 투여된 종양마우스에서는 CD8+ T 세포의 세포수가 농도-의존적으로 증가하였고, 투여 7일 차에서 최고치(peak point)로 나타났다 (도 5a). 또한, 투여 7일 차의 CD8+ T 세포 증가율은 대조군(PBS 처리)에 비해 10 및 20 mpk의 농도에서 각각 11.08 및 21.82 배로 증가하였음을 확인하였다 (도 5b). As a result, in tumor mice to which sTBRII-hyFc-IL7 fusion protein was administered, the number of CD8 + T cells increased in a concentration-dependent manner, and the peak point was found at the 7th day of administration (Fig. 5a). In addition, it was confirmed that the increase rate of CD8 + T cells at the 7th day of administration increased by 11.08 and 21.82 times at concentrations of 10 and 20 mpk, respectively, compared to the control (PBS treatment) (FIG. 5B).
또한, IL-7-hyFc-sTBRII 융합단백질이 투여된 종양마우스에서도 CD8+ T 세포의 세포수가 농도-의존적으로 증가하였고, 투여 7일 차에서 최고치(peak point)로 나타났다 (도 5a). 또한, 투여 7일 차의 CD8+ T 세포 증가율은 대조군(PBS 처리)에 비해 10 및 20 mpk의 농도에서 각각 18.74 및 29.56 배로 증가하였음을 확인하였다 (도 5b). In addition, in tumor mice administered with IL-7-hyFc-sTBRII fusion protein, the number of CD8 + T cells increased in a concentration-dependent manner, and the peak point was shown at the 7th day of administration (Fig. 5a). In addition, it was confirmed that the increase rate of CD8 + T cells at the 7th day of administration increased by 18.74 and 29.56 times at concentrations of 10 and 20 mpk, respectively, compared to the control group (PBS treatment) (FIG. 5B).
상기 결과로부터, sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질 모두에서 농도-의존적으로 CD8+ T 세포와 같은 타겟 세포의 증식을 증가시키는 활성이 있음을 확인하였다. From the above results, it was confirmed that both sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins have an activity of increasing the proliferation of target cells such as CD8 + T cells in a concentration-dependent manner.
실시예 5. 마우스 종양모델에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 TGF beta 억제 활성 분석Example 5. Analysis of TGF beta inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model
본 발명자들은 마우스 종양모델에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 TGF beta에 대한 억제 활성을 분석하는 실험을 수행하였다. 간단히, C57BL/6 (B6) 마우스 동물모델에 1x105 개의 MC38 대장암 세포주를 피하 주입하여 마우스 종양모델을 제조하였다. 이후, 종양이 형성된 6일 차에 각 융합단백질을 20 mpk로 피하 투여(s.c)하였다. 혈액은 각 융합단백질의 투여 후 2, 6, 24, 48, 72 및 168 시간에 안와채혈(retro-orbital bleeding)을 통해 채취하였고, 채취한 혈액 샘플에서 혈청(serum)을 분리하였다. 혈청 내에 존재하는 TGF beta의 농도는 Mouse TGF-beta 1 DuoSet ELISA Kit (R&D systems, catalog# DY1679-05)를 사용하여 측정하였다. The present inventors performed an experiment to analyze the inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins against TGF beta in a mouse tumor model. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1 ×10 5 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Thereafter, on the 6th day of tumor formation, each fusion protein was administered subcutaneously (sc) at 20 mpk. Blood was collected through retro-orbital bleeding at 2, 6, 24, 48, 72 and 168 hours after administration of each fusion protein, and serum was isolated from the collected blood samples. The concentration of TGF beta present in the serum was measured using the Mouse TGF-beta 1 DuoSet ELISA Kit (R&D systems, catalog# DY1679-05).
그 결과, sTBRII-hyFc-IL7 융합단백질이 투여된 종양마우스에서는 혈청 내의 TGF beta가 투여 후 약 48시간 동안 억제되었고, 투여 후 72시간에 다시 검출되었으며, 투여 후 168시간 후에는 대조군(PBS 처리)과 유사한 수준으로 회복되었음을 확인하였다 (도 6). 또한, IL7-hyFc-sTBRII 융합단백질이 투여된 종양마우스에서는 혈청 내의 TGF beta가 투여 후 약 72시간 동안 억제되었고, 투여 후 168시간 후에는 대조군(PBS 처리)과 유사한 수준으로 회복되었음을 확인하였다 (도 6). As a result, in tumor mice administered with sTBRII-hyFc-IL7 fusion protein, TGF beta in serum was inhibited for about 48 hours after administration, and was detected again 72 hours after administration, and 168 hours after administration, control (PBS treatment) It was confirmed that the recovered to a level similar to (Fig. 6). In addition, in tumor mice to which IL7-hyFc-sTBRII fusion protein was administered, TGF beta in serum was inhibited for about 72 hours after administration, and it was confirmed that it recovered to a level similar to that of the control group (PBS treatment) 168 hours after administration (Fig. 6).
상기 결과로부터, sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질은 피하 투여를 통해 48~72시간 동안 혈청 내 TGF beta를 억제하는 효과가 있음을 확인하였다. From the above results, it was confirmed that the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins have an effect of inhibiting TGF beta in serum for 48 to 72 hours through subcutaneous administration.
실시예 6. 마우스 종양모델에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 항암 활성 분석Example 6. Analysis of anticancer activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model
본 발명자들은 마우스 종양모델에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 항암 활성 여부를 확인하는 실험을 수행하였다. 간단히, C57BL/6 (B6) 마우스 동물모델에 1x105 개의 MC38 대장암 세포주를 피하 주입하여 마우스 종양모델을 제조하였다. 이후, 종양이 형성된 6일 차에 각 융합단백질을 20 mpk로 피하 투여(s.c)하였다. 투여 후 2~3일 간격으로 종양 부피(tumor volume)의 변화를 측정하였다. 종양 부피(tumor volume)는 하기 공식(formula)를 이용하여 산출하였다.The present inventors performed an experiment to confirm the anticancer activity of the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in a mouse tumor model. Briefly, a mouse tumor model was prepared by subcutaneously injecting 1 ×10 5 MC38 colon cancer cell lines into a C57BL/6 (B6) mouse animal model. Thereafter, on the 6th day of tumor formation, each fusion protein was administered subcutaneously (sc) at 20 mpk. Changes in tumor volume were measured at intervals of 2 to 3 days after administration. The tumor volume was calculated using the following formula.
Tumor volume = {(장축 길이) x (단축 길이)2} / 2Tumor volume = {(long axis length) x (short axis length) 2 } / 2
또한, 종양 생장 저해율(% tumor growth inhibition)은 하기 공식(formula)를 이용하여 산출하였다.In addition, the tumor growth inhibition rate (% tumor growth inhibition) was calculated using the following formula (formula).
% tumor growth inhibition (% TGI) = {(MTVcontrol - MTVtreated) / MTVcontrol} x 100 (* MTV = median tumor volume)% tumor growth inhibition (% TGI) = {(MTVcontrol-MTVtreated) / MTVcontrol} x 100 (* MTV = median tumor volume)
그 결과, sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 투여한 마우스에서는 대조군(PBS 처리)에 비해 종양 부피가 현저하게 감소된 효과가 있음을 확인하였다 (도 7). 특히, MC38 대장암 세포주를 이식 후 20일 차에 있어, sTBRII-hyFc-IL7 융합단백질을 투여한 마우스에서는 대조군에 비해 종양 생장 저해율이 61.79 %로 나타났고 (도 7a), IL7-hyFc-sTBRII 융합단백질을 투여한 마우스에서는 대조군에 비해 종양 생장 저해율이 83.14 %로 나타났다 (도 7b). As a result, it was confirmed that in mice administered with sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins, the tumor volume was significantly reduced compared to the control group (PBS treatment) (FIG. 7). In particular, in mice administered with the sTBRII-hyFc-IL7 fusion protein at the 20th day after transplantation of the MC38 colorectal cancer cell line, the tumor growth inhibition rate was 61.79% compared to the control group (Fig. 7a), and IL7-hyFc-sTBRII fusion In the mice to which the protein was administered, the tumor growth inhibition rate was 83.14% compared to the control group (FIG. 7B).
상기 결과로부터, sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질은 피하 투여를 통해 현저한 항암 효능이 있음을 확인하였다. From the above results, it was confirmed that the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins had remarkable anticancer efficacy through subcutaneous administration.
실시예 7. 시험관 내(Example 7. In vitro ( in vitroin vitro )에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 IL-7 생물학적 활성(bioactivity) 분석), IL-7 bioactivity analysis of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins
본 발명자들은 시험관 내에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 구성하고 있는 IL-7의 생물학적 활성도를 측정하기 위하여, IL-7에 의존적으로 증식하는 2E8 세포주(ATCC®TIB-239TM, Mouse B lymphocyte cell line)를 이용하여 실험을 수행하였다. 인간 유래 IL-7은 마우스와 종간 교차반응성이 존재하므로, 인간 유래 IL-7에 의해 마우스 세포주인 2E8 세포주의 증식을 유도할 수 있다.In order to measure the biological activity of IL-7 constituting the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins in vitro, the present inventors conducted a 2E8 cell line (ATCC ® TIB) that proliferates dependently on IL-7 in vitro. -239 TM , Mouse B lymphocyte cell line) was used to perform the experiment. Since human-derived IL-7 has cross-reactivity between mouse and species, human-derived IL-7 can induce proliferation of the mouse cell line 2E8 cell line.
간단히, 냉동 보관되었던 2E8 세포주를 녹인 후, T-75 플라스크에서 3회 이상의 계대 배양을 통해 안정화된 세포를 준비하였다. 2E8 세포주는 마우스 IL-7 (Cell Signaling, 5217SC) 및 FBS (Hyclone, SH30084.03)이 포함된 IMDM (ATCC® 30-2005TM) 배지를 이용하여 배양하였다. 이후, 배양된 2E8 세포주는 마우스 IL-7이 포함되지 않은 IMDM 배지에 현탁하여 IL-7에 대한 기아 상태로 만든 후, 96-well plate에 1x105 cells/well로 분주하였다. 이후, sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 각각 3 nM부터 1/3로 순차적으로 희석된 농도 구배로 세포에 처리하고, 37℃, 5% CO2 배양기에서 3일 동안 배양하였다. Briefly, after thawing the 2E8 cell line that had been stored frozen, stabilized cells were prepared by subculture at least three times in a T-75 flask. The 2E8 cell line was cultured using IMDM (ATCC ® 30-2005 TM ) medium containing mouse IL-7 (Cell Signaling, 5217SC) and FBS (Hyclone, SH30084.03). Thereafter, the cultured 2E8 cell line was suspended in IMDM medium containing no mouse IL-7 to make it starvation for IL-7, and then dispensed into a 96-well plate at 1×10 5 cells/well. Thereafter, the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins were treated on the cells in a concentration gradient sequentially diluted from 3 nM to 1/3, respectively, and in an incubator at 37° C., 5% CO 2 for 3 days. Cultured.
세포증식도는 CellTiter 96® AQueous One Solution Assay (Promega, G3581)를 이용하여 정량하였다. MTS 시약을 세포에 처리하고, 37℃, 5% CO2 배양기에서 4시간 동안 배양한 후, ELISA plate reader를 사용하여 490 nm 파장에서의 흡광도를 측정하였다. GraphPad Prism® 프로그램(GraphPad Software)을 이용하여 흡광도의 표준 곡선과 이를 기준으로 두 융합단백질의 EC50 (50 % effective concentration) 값을 산출하였다. Cell proliferation was quantified using CellTiter 96® AQ ueous One Solution Assay (Promega, G3581). The cells were treated with the MTS reagent, and incubated for 4 hours in an incubator at 37° C. and 5% CO 2 , and absorbance at a wavelength of 490 nm was measured using an ELISA plate reader. Using the GraphPad Prism ® program (GraphPad Software), the standard curve of absorbance and the EC 50 (50% effective concentration) value of the two fusion proteins were calculated based on this.
그 결과, sTBRII-hyFc-IL7 융합단백질의 EC50는 52.52 pM로 측정되었고, IL-7-hyFc-sTBRII 융합단백질의 EC50는 56.21 pM로 측정되었다 (도 8 및 표 2). As a result, sTBRII-hyFc-IL7 EC 50 of the fusion protein was measured in 52.52 pM, IL7-hyFc-sTBRII EC 50 of fusion protein was determined to be 56.21 pM (Figure 8 and Table 2).
EC50 (pM)EC 50 (pM) sTBRII-hyFc-IL-7sTBRII-hyFc-IL-7 IL-7-hyFc-sTBRIIIL-7-hyFc-sTBRII
52.5252.52 56.21 56.21
실시예 8.Example 8. 시험관 내(In vitro ( in vitroin vitro )에서 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 TGF beta 억제 활성 분석) Analysis of TGF beta inhibitory activity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins
본 발명자들은 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 구성하고 있는 sTBRII의 생물학적 활성도를 측정하기 위하여, TGF beta에 의해 유도된 하위 전달 신호를 발광 신호로 측정할 수 있는 SMAD Signaling Pathway SBE Reporter-HEK293 세포주(BPS Bioscience, 60653)를 이용하여 실험을 수행하였다. In order to measure the biological activity of sTBRII constituting the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins, the present inventors are SMAD Signaling, which can measure the subtransmission signal induced by TGF beta as a luminescence signal. The experiment was performed using the Pathway SBE Reporter-HEK293 cell line (BPS Bioscience, 60653).
간단히, SMAD Signaling Pathway SBE Reporter-HEK293 세포주는 Geneticin (Invitrogen, 11811031)이 포함된 Growth Medium 1B (BPS Bioscience, 79531)를 이용하여 37℃, 5% CO2 배양기에서 배양하였다. 이후, 배양된 SMAD Signaling Pathway SBE Reporter-HEK293 세포주는 Assay Medium 1B (BPS Bioscience, 79617-2)에 현탁하여 white clear-bottom 96-well microplate 에 3.5x104 cells/well로 분주하였다. 이후, sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질을 각각 50 nM부터 1/3로 순차적으로 희석된 농도 구배로 세포에 처리하고, 동시에 20 ng/mL의 TGF beta (BPS Bioscience, 90900-1)를 함께 세포에 처리한 후, 37℃, 5% CO2 배양기에서 18시간 동안 배양하였다. 배양이 끝난 세포에는 ONE-StepTM Luciferase reagent를 처리하고, 상온에서 30분 동안 플레이트를 흔들며 배양하였다. 이후, SBE reporter의 발광 정도를 Plate-reading Luminometer(TECAN SPARK 10M)로 측정하였다. GraphPad Prism® 프로그램(GraphPad Software)을 이용하여 측정된 SBE reporter의 발광도 표준 곡선 및 두 융합단백질의 IC50 값을 산출하였다. Briefly, the SMAD Signaling Pathway SBE Reporter-HEK293 cell line was cultured in a 37°C, 5% CO 2 incubator using Growth Medium 1B (BPS Bioscience, 79531) containing Geneticin (Invitrogen, 11811031). Thereafter, the cultured SMAD Signaling Pathway SBE Reporter-HEK293 cell line was suspended in Assay Medium 1B (BPS Bioscience, 79617-2) and dispensed into a white clear-bottom 96-well microplate at 3.5x10 4 cells/well. Thereafter, the sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins were treated on the cells in a concentration gradient sequentially diluted from 50 nM to 1/3, respectively, and at the same time 20 ng/mL of TGF beta (BPS Bioscience, After treating the cells with 90900-1), the cells were incubated for 18 hours in an incubator at 37°C and 5% CO 2. After culturing cells, ONE-Step TM Luciferase reagent was treated, and the plate was shaken for 30 minutes at room temperature and incubated. Thereafter, the degree of luminescence of the SBE reporter was measured with a Plate-reading Luminometer (TECAN SPARK 10M). Using the GraphPad Prism ® program (GraphPad Software), the luminance standard curve of the SBE reporter and the IC 50 values of the two fusion proteins were calculated.
그 결과, SMAD Signaling Pathway SBE Reporter-HEK293 세포주에서 sTBRII-hyFc-IL-7 융합단백질의 IC50 값은 1.874 nM로 측정되었고, IL-7-hyFc-sTBRII 융합단백질의 IC50 값은 0.4148 nM로 측정되었다 (도 9 및 표 3). As a result, SMAD Signaling Pathway SBE in Reporter-HEK293 cell line sTBRII-hyFc-IL-7 IC 50 values of the fusion proteins was measured in 1.874 nM, IL-7-hyFc -sTBRII IC 50 values of the fusion protein is measured by 0.4148 nM (Fig. 9 and Table 3).
IC50 (nM)IC 50 (nM) sTBRII-hyFc-IL-7sTBRII-hyFc-IL-7 IL-7-hyFc-sTBRIIIL-7-hyFc-sTBRII
1.8741.874 0.41480.4148
실시예 9.Example 9. sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII 융합단백질의 동시 결합력 분석Simultaneous avidity analysis of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII fusion proteins
본 발명자들은 sTBRII-hyFc-IL7 및 IL-7-hyFc-sTBRII의 IL-7Rα (CD127)와 TGF-β1에 대한 동시 결합력을 비교 분석하기 위하여 BLI (biolayer interferometry)를 수행하였다. 아민 반응성 2세대 바이오 센서 (Amine Reactive Second-Generation Biosensor, AR2G)와 AR2G 시약 키트 (AR2G Reagent Kit)를 사용하여 실험을 수행하였다. The present inventors performed BLI (biolayer interferometry) to compare and analyze the simultaneous binding capacity of sTBRII-hyFc-IL7 and IL-7-hyFc-sTBRII to IL-7Rα (CD127) and TGF-β1. Experiments were performed using an amine reactive second-generation biosensor (AR2G) and an AR2G reagent kit (AR2G Reagent Kit).
각각의 융합 단백질을 재조합 인간 TGF-β1 단백질 (2 μg/mL) (고정 리간드 (1st 리간드)) - 융합 단백질 (1000 nM) - 재조합 인간 IL-7Rα 단백질 (1, 2, 4, 8 μg/mL) (2nd 리간드) 순으로; 또는 재조합 인간 IL-7Rα 단백질 (2 μg/mL) (고정 리간드 (1st 리간드)) - 융합 단백질 (1000 nM) - 재조합 인간 TGF-β1 단백질 (0.25, 0.5, 1, 2 μg/mL) (2nd 리간드) 순으로 샘플을 넣은 후, 동시 결합 및 1st 리간드에 결합된 융합 단백질의 2nd 리간드의 농도에 따른 결합을 비교 분석하였다. Recombinant human TGF-β1 protein (2 μg/mL) (fixed ligand (1 st ligand))-fusion protein (1000 nM)-recombinant human IL-7R α protein (1, 2, 4, 8 μg) in / mL) (2 nd ligand) in order; Or recombinant human IL-7R α protein (2 μg/mL) (fixed ligand (1 st ligand))-fusion protein (1000 nM)-recombinant human TGF-β1 protein (0.25, 0.5, 1, 2 μg/mL) ( After putting the samples in the order of 2 nd ligand), the simultaneous binding and binding of the fusion protein bound to the 1 st ligand according to the concentration of the 2 nd ligand were compared and analyzed.
각 융합 단백질과 고정된 1st 리간드 (TGF-β1, IL-7Rα)의 결합을 확인한 결과, 시간에 따라 회합(association)이 잘 되었으며, 두 융합단백질 모두 결합속도가 비슷하였다. 또한, 상대적으로 TGF-β1과의 결합이 IL-7Rα와의 결합보다 더 좋은 것을 확인하였다. As a result of confirming the binding of each fusion protein to the immobilized 1 st ligand (TGF-β1, IL-7R α ), the association was well performed over time, and the binding rates of both fusion proteins were similar. In addition, it was confirmed that the binding with TGF-β1 is relatively better than that with IL-7R α.
이후, TGF-β1 (1st 리간드)과 결합한 뒤, IL-7Rα (2nd 리간드)의 농도에 따른 융합단백질에서의 결합을 확인한 결과, 두 융합단백질 모두에서 농도 의존적으로 결합이 증가하는 것을 확인하였다 (도 10). 또한, IL-7Rα (1st 리간드)와 결합한 뒤, TGF-β1 (2nd 리간드)의 농도에 따른 융합단백질에서의 결합을 확인한 결과, 두 융합단백질 모두에서 농도 의존적으로 결합이 증가하였다 (도 11).Thereafter, after binding with TGF-β1 (1 st ligand), binding in the fusion protein according to the concentration of IL-7R α (2 nd ligand) was confirmed, and as a result, binding increased in a concentration-dependent manner in both fusion proteins. (Fig. 10). In addition, after binding to IL-7R α (1 st ligand), as a result of confirming the binding in the fusion protein according to the concentration of TGF-β1 (2 nd ligand), the binding increased in a concentration-dependent manner in both fusion proteins (Fig. 11).
상기 결과로부터, sTBRII-hyFc-IL-7 및 IL-7-hyFc-sTBRII 융합단백질은 타겟 단백질인 TGF-β1과 IL-7Rα과의 동시 결합이 가능함을 확인하였다.From the above results, it was confirmed that the sTBRII-hyFc-IL-7 and IL-7-hyFc-sTBRII fusion proteins are capable of simultaneous binding with the target proteins TGF-β1 and IL-7R α.

Claims (27)

  1. 변형된 인터루킨-7(interleukin 7, IL-7) 및 TGF 베타 수용체 II(transforming growth factor beta receptor II, TBRII)를 포함하는 융합단백질.Modified interleukin-7 (interleukin 7, IL-7) and TGF beta receptor II (transforming growth factor beta receptor II, TBRII) containing a fusion protein.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 변형된 IL-7은 하기의 구조를 갖는 것인, 융합단백질:The modified IL-7 is a fusion protein having the following structure:
    A - IL-7A-IL-7
    상기 A가 1 내지 10 개의 아미노산 잔기로 이루어진 올리고펩티드이고, Wherein A is an oligopeptide consisting of 1 to 10 amino acid residues,
    상기 변형된 IL-7이 IL-7 또는 이와 유사한 활성을 갖는 폴리펩티드이다.The modified IL-7 is IL-7 or a polypeptide having similar activity.
  3. 제 2 항에 있어서,The method of claim 2,
    상기 IL-7은 서열번호 1의 아미노산 서열로 이루어진 것인, 융합단백질.The IL-7 is a fusion protein consisting of the amino acid sequence of SEQ ID NO: 1.
  4. 제 2 항에 있어서,The method of claim 2,
    상기 A는 IL-7의 N-말단에 연결되는 것인, 융합단백질.The A is to be linked to the N-terminus of IL-7, a fusion protein.
  5. 제 2 항에 있어서,The method of claim 2,
    상기 A는 메티오닌, 글리신, 메티오닌-메티오닌, 글리신-글리신, 메티오닌-글리신, 글리신-메티오닌, 메티오닌-메티오닌-메티오닌, 메티오닌-메티오닌-글리신, 메티오닌-글리신-메티오닌, 글리신-메티오닌-메티오닌, 메티오닌-글리신-글리신, 글리신-메티오닌-글리신, 글리신-글리신-메티오닌, 글리신-글리신-글리신, 메티오닌-메티오닌-메티오닌-메티오닌, 메티오닌-글리신-메티오닌-메티오닌, 메티오닌-글리신-글리신-메티오닌, 메티오닌-글리신-글리신-글리신, 메티오닌-글리신-메티오닌-글리신, 글리신-메티오닌-메티오닌-메티오닌, 글리신-메티오닌-글리신-글리신, 글리신-글리신-글리신-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-메티오닌, 메티오닌-메티오닌-글리신-메티오닌-메티오닌, 메티오닌-메티오닌-글리신-글리신-메티오닌, 메티오닌-글리신-메티오닌-메티오닌-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-글리신, 글리신-글리신-글리신-글리신-글리신, 글리신-글리신-메티오닌-메티오닌-메티오닌, 글리신-글리신-글리신-메티오닌-글리신, 메티오닌-글리신-메티오닌-글리신-메티오닌-글리신, 메티오닌-메티오닌-메티오닌-글리신-글리신-글리신, 메티오닌-메티오닌-글리신-글리신-메티오닌-메티오닌, 글리신-글리신-메티오닌-메티오닌-글리신-글리신, 메티오닌-글리신-메티오닌-글리신-메티오닌-글리신-메티오닌-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-글리신-글리신-글리신-글리신, 메티오닌-메티오닌-글리신-글리신-메티오닌-메티오닌-글리신-글리신, 메티오닌-메티오닌-메티오닌-메티오닌-글리신-글리신-글리신-글리신, 메티오닌-글리신-메티오닌-글리신-메티오닌-글리신-메티오닌-글리신-메티오닌-글리신 또는 메티오닌-메티오닌-메티오닌-메티오닌-메티오닌-글리신-글리신-글리신-글리신-글리신으로 이루어진 군에서 선택되는 것인, 융합단백질.A is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine -Glycine, glycine-methionine-glycine, glycine-glycine-methionine, glycine-glycine-glycine, methionine-methionine-methionine-methionine, methionine-glycine-methionine-methionine, methionine-glycine-glycine-methionine, methionine-glycine-glycine -Glycine, methionine-glycine-methionine-glycine, glycine-methionine-methionine-methionine, glycine-methionine-glycine-glycine, glycine-glycine-glycine-glycine, methionine-methionine-methionine-methionine-methionine, methionine-methionine-glycine -Methionine-methionine, methionine-methionine-glycine-glycine-methionine, methionine-glycine-methionine-methionine-glycine, methionine-methionine-methionine-methionine-glycine, glycine-glycine-glycine-glycine-glycine, glycine-glycine-methionine -Methionine-methionine, glycine-glycine-glycine-methionine-glycine, methionine-glycine-methionine-glycine-methionine-glycine, methionine-methionine-methionine-glycine-glycine-glycine, methionine-methionine-glycine-glycine-methionine-methionine , Glycine-glycine-methionine-methionine-glycine-glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine, methionine-methionine-glycine -Glycine-methionine-methionine-glycine-glycine, methionine-methionine-methionine-methionine-glycine-glycine-glycine-glycine, methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine-methionine-glycine or methionine-methionine -Methionine-methionine-methionine-glycine-glycine-glycine-glycine-glycine that is selected from the group consisting of, a fusion protein.
  6. 제 1 항에 있어서,The method of claim 1,
    상기 TBRII는 서열번호 4의 아미노산 서열로 이루어진 것인, 융합단백질.The TBRII is a fusion protein consisting of the amino acid sequence of SEQ ID NO: 4.
  7. 제 1 항에 있어서,The method of claim 1,
    상기 TBRII는 TBRII의 세포외 도메인인 것인, 융합단백질.The TBRII is the extracellular domain of TBRII, a fusion protein.
  8. 제 7 항에 있어서, The method of claim 7,
    상기 TBRII의 세포외 도메인은 서열번호 5의 아미노산 서열로 이루어진 것인, 융합단백질.The extracellular domain of the TBRII is a fusion protein consisting of the amino acid sequence of SEQ ID NO: 5.
  9. 제 1 항에 있어서,The method of claim 1,
    상기 변형된 IL-7 및 TBRII는 면역글로불린 Fc 도메인에 의해 결합된 것인, 융합단백질.The modified IL-7 and TBRII are bound by an immunoglobulin Fc domain, a fusion protein.
  10. 제 9 항에 있어서,The method of claim 9,
    상기 Fc 도메인은 야생형 또는 변이체인 것인, 융합단백질.The Fc domain is a wild type or a mutant, a fusion protein.
  11. 제 9 항에 있어서, The method of claim 9,
    TBRII, Fc 도메인 및 변형된 IL-7가 N-말단으로부터 C-말단 방향으로 순서대로 결합된 것인, 융합단백질.TBRII, Fc domain and modified IL-7 are bound in order from the N-terminus to the C-terminus, a fusion protein.
  12. 제 11 항에 있어서, The method of claim 11,
    TBRII와 Fc 도메인의 사이에 제1링커가 더 포함되는 것인, 융합단백질.A fusion protein that further comprises a first linker between the TBRII and the Fc domain.
  13. 제 12 항에 있어서, The method of claim 12,
    상기 제1링커는 서열번호 14의 아미노산 서열로 이루어진 것인, 융합단백질.The first linker is consisting of the amino acid sequence of SEQ ID NO: 14, fusion protein.
  14. 제 11 항에 있어서, The method of claim 11,
    Fc 도메인과 변형된 IL-7의 사이에 제2링커가 더 포함되는 것인, 융합단백질.A fusion protein that further comprises a second linker between the Fc domain and the modified IL-7.
  15. 제 14 항에 있어서, The method of claim 14,
    상기 제2링커는 서열번호 15의 아미노산 서열로 이루어진 것인, 융합단백질.The second linker is consisting of the amino acid sequence of SEQ ID NO: 15, fusion protein.
  16. 제 9 항에 있어서, The method of claim 9,
    변형된 IL-7, Fc 도메인 및 TBRII가 N-말단으로부터 C-말단 방향으로 순서대로 결합된 것인, 융합단백질.Modified IL-7, Fc domain and TBRII are bound in order from the N-terminus to the C-terminus, a fusion protein.
  17. 제 16 항에 있어서, The method of claim 16,
    변형된 IL-7과 Fc 도메인의 사이에 제1링커가 더 포함되는 것인, 융합단백질.A fusion protein that further comprises a first linker between the modified IL-7 and the Fc domain.
  18. 제 17 항에 있어서, The method of claim 17,
    상기 제1링커는 서열번호 14의 아미노산 서열로 이루어진 것인, 융합단백질.The first linker is consisting of the amino acid sequence of SEQ ID NO: 14, fusion protein.
  19. 제 16 항에 있어서, The method of claim 16,
    Fc 도메인과 TBRII의 사이에 제3링커가 더 포함되는 것인, 융합단백질.A fusion protein that further comprises a third linker between the Fc domain and TBRII.
  20. 제 19 항에 있어서, The method of claim 19,
    상기 제3링커는 서열번호 16의 아미노산 서열로 이루어진 것인, 융합단백질.The third linker is consisting of the amino acid sequence of SEQ ID NO: 16, fusion protein.
  21. 제 1 항 내지 제 20 항 중 어느 한 항에 따른 융합 단백질을 코딩하는 단리된 핵산 분자.An isolated nucleic acid molecule encoding a fusion protein according to claim 1.
  22. 제 21 항에 있어서, The method of claim 21,
    상기 핵산 분자는 신호 서열 또는 리더 서열을 더 포함하는 것인, 핵산 분자.The nucleic acid molecule further comprises a signal sequence or a leader sequence, a nucleic acid molecule.
  23. 제 21 항 또는 제 22 항에 따른 핵산 분자를 포함하는 발현 벡터.An expression vector comprising the nucleic acid molecule according to claim 21 or 22.
  24. 제 23 항의 발현 벡터를 포함하는 숙주 세포.A host cell comprising the expression vector of claim 23.
  25. 제 1 항 내지 제 21 항 중 어느 한 항에 따른 융합단백질을 유효성분으로 포함하는 암 또는 감염성 질환의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for the prevention or treatment of cancer or infectious diseases comprising the fusion protein according to any one of claims 1 to 21 as an active ingredient.
  26. 제 25 항에 있어서,The method of claim 25,
    상기 암은 위암, 간암, 폐암, 대장암, 유방암, 전립선암, 난소암, 췌장암, 자궁경부암, 갑상선암, 후두암, 급성 골수성 백혈병, 뇌종양, 신경모세포종, 망막 모세포종, 두경부암, 침샘암 및 림프종으로 이루어진 군에서 선택되는 것인, 약학 조성물.The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. Which is selected from the group, a pharmaceutical composition.
  27. 제 25 항에 있어서,The method of claim 25,
    상기 감염성 질환은 B형 간염, C형 간염, 인간 파필로마 바이러스 감염, 사이토메갈로바이러스 감염, 바이러스성 호흡기 질환 및 인플루엔자로 이루어진 군에서 선택되는 것인, 약학 조성물.The infectious disease is selected from the group consisting of hepatitis B, hepatitis C, human papilloma virus infection, cytomegalovirus infection, viral respiratory disease and influenza.
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