WO2021096275A1 - Protéine de fusion comprenant l'interleukine -7 modifiée et le récepteur bêta ii du tgf et son utilisation - Google Patents

Protéine de fusion comprenant l'interleukine -7 modifiée et le récepteur bêta ii du tgf et son utilisation 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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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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5418—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2046—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-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

La présente invention concerne une protéine de fusion comprenant l'interleukine-7 et le récepteur bêta du TGF et une utilisation associée. La protéine de fusion présente un rendement de production élevé et peut efficacement inhiber le cancer, et peut donc être utilisée utilement dans le traitement du cancer ou de maladies infectieuses.
PCT/KR2020/015948 2019-11-15 2020-11-13 Protéine de fusion comprenant l'interleukine -7 modifiée et le récepteur bêta ii du tgf et son utilisation WO2021096275A1 (fr)

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WO2022086988A1 (fr) * 2020-10-20 2022-04-28 Cura Therapeutics, Inc. Polypeptides de fusion multifonctionnels et multivalents du récepteur de l'interleukine-tgf-bêta
US11407814B2 (en) 2020-10-20 2022-08-09 Cura Therapeutics, Inc. Multi-functional and multi-valent interleukin-TGF-beta receptor fusion polypeptides
US11672844B2 (en) 2021-02-17 2023-06-13 Cura Therapeutics, Inc. Method of treating or ameliorating a pathogenic infection by administering an interleukin-2/TGF-β receptor fusion polypeptide

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US11407814B2 (en) 2020-10-20 2022-08-09 Cura Therapeutics, Inc. Multi-functional and multi-valent interleukin-TGF-beta receptor fusion polypeptides
US11672844B2 (en) 2021-02-17 2023-06-13 Cura Therapeutics, Inc. Method of treating or ameliorating a pathogenic infection by administering an interleukin-2/TGF-β receptor fusion polypeptide

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