WO2021246413A1 - 抗ｉｇｆ－１受容体ヒト化抗体 - Google Patents

抗ｉｇｆ－１受容体ヒト化抗体 Download PDF

Info

Publication number
WO2021246413A1
WO2021246413A1 PCT/JP2021/020890 JP2021020890W WO2021246413A1 WO 2021246413 A1 WO2021246413 A1 WO 2021246413A1 JP 2021020890 W JP2021020890 W JP 2021020890W WO 2021246413 A1 WO2021246413 A1 WO 2021246413A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
igf
seq
receptor
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2021/020890
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
博昭 松川
広志 江口
直子 並木
章 田野倉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Teijin Pharma Ltd
Original Assignee
Teijin Pharma Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP21817253.4A priority Critical patent/EP4159860A4/en
Priority to CA3176119A priority patent/CA3176119A1/en
Priority to JP2022528850A priority patent/JPWO2021246413A1/ja
Priority to IL296519A priority patent/IL296519A/en
Priority to US18/007,606 priority patent/US20250263489A1/en
Priority to MX2022015133A priority patent/MX2022015133A/es
Priority to KR1020227042511A priority patent/KR20230006006A/ko
Priority to BR112022022030A priority patent/BR112022022030A2/pt
Application filed by Teijin Pharma Ltd filed Critical Teijin Pharma Ltd
Priority to CN202180039577.1A priority patent/CN115605594A/zh
Priority to AU2021282629A priority patent/AU2021282629A1/en
Publication of WO2021246413A1 publication Critical patent/WO2021246413A1/ja
Priority to CONC2022/0017026A priority patent/CO2022017026A2/es
Anticipated expiration legal-status Critical
Priority to JP2024028737A priority patent/JP2024054410A/ja
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to an anti-IGF-1 receptor humanized antibody. Specifically, it relates to an anti-IGF-1 receptor humanized antibody that specifically binds to the IGF-1 receptor.
  • IGF-1 is an insulin-like growth factor that is secreted mainly from the liver and acts on the IGF-1 receptor through activation of GH receptors by growth hormone (GH) secreted from the pituitary gland. Therefore, various physiological functions are expressed in various organs. From this, IGF-1 is expected to be treated for various diseases. Since IGF-1 has a high homology of about 40% as compared with the amino acid sequence of proinsulin, it may also bind to the insulin receptor and exert an insulin-like action. In addition, since the IGF-1 receptor has a high homology of about 60% compared to the amino acid sequence of the insulin receptor, both receptors also exert a physiological effect by forming a heterodimer. Demonstrate. Insulin is used therapeutically as a hypoglycemic agent because it exerts a strong hypoglycemic effect by acting on the insulin receptor.
  • GH growth hormone
  • IGF-1 receptor The IGF-1 receptor is composed of ⁇ and ⁇ chains and contains six extracellular domains, transmembrane domains, and intracellular domains of L1, CR, L2, Fn1, Fn2, and Fn3. It is a penetrating protein.
  • the intracellular domain of the IGF-1 receptor has a tyrosine kinase.
  • the extracellular domain is involved in the activation of intracellular tyrosine kinases associated with changes in the conformation of IGF-1 when it binds to the IGF-1 receptor.
  • the IGF-1 receptor forms a homodimer complex (homotype), and when IGF-1 binds, it signals by activating the receptor kinase. It also forms a heterodimer complex (heterotype) with the insulin receptor, and when insulin or IGF-1 binds, it signals by activating the receptor kinase.
  • IGF-1 Physiological action of IGF-1 IGF-1 has been clarified to have a growth promoting action such as height and weight gain, and an insulin-like metabolic action such as glucose metabolism promoting action and blood glucose lowering action.
  • Mecasermin a human recombinant IGF-1, has been shown to improve symptoms of insulin receptor abnormalities such as hyperglycemia, hyperinsulinemia, acanthosis nigricans and hirsutism. It has also been found to improve growth failure in growth hormone-resistant short stature (Non-Patent Document 1).
  • IGF-1 is a major factor in promoting growth (Non-Patent Document 2 and Non-Patent Document 3).
  • mecasermin a human recombinant IGF-1
  • IGF-1 is known to enhance the DNA synthesis ability of human chondrocytes.
  • administration of IGF-1 increases the body weight of pituitary-excised rats and prolongs the length of the femoral bone.
  • IGF-1 Muscle mass-increasing action of IGF-1
  • the enhancement of cell proliferation activity mediated by IGF-1 requires continuous activation of the IGF-1 receptor. Muscle mass is increased in animals that overexpress the IGF-1 receptor.
  • continuous administration of IGF-1 / IGFBP3 enhances grip strength in patients with proximal femoral fractures and improves their ability to stand up from a sitting position without assistance. It is known that the IGF-1 concentration in the muscles of aged humans and mice is lower than that of young mice, but in aged mice in which IGF-1 is forcibly expressed in a muscle tissue-specific manner, it is wild. Compared with the type mouse, the muscle mass was improved (Non-Patent Document 4).
  • Anamorelin an agonist of the predecessor ghrelin receptor that increases muscle mass, increased lean body mass in a clinical trial of disuse muscular atrophy, Kakexia.
  • Myostatin is a negative regulator of skeletal muscle formation that acts on activin receptor II (ActRII) and inhibits Akt / mTOR.
  • ActRII activin receptor II
  • the anti-myostatin antibody LY2495655 increases muscle mass in patients and the elderly who have undergone total hip arthroplasty.
  • Vimaglumab an anti-ActRII antibody, also increases muscle mass in patients with neuromuscular disease.
  • IGF-1 As an insulin-like action of IGF-1, a hypoglycemic action is known. IGF-1 enhances glucose uptake in rat muscle-derived cells. In addition, administration of IGF-1 lowers blood glucose levels in rats. It has been reported that the hypoglycemic effect of IGF-1 causes hypoglycemic symptoms as a clinical side effect. Furthermore, since IGF-1 causes hypoglycemic symptoms when administered to humans, it is necessary to administer appropriate doses in order from the lowest dose at the start of treatment and observe various clinical findings including blood glucose level after administration. It becomes.
  • IGF-1 exerts a hypoglycemic effect through enhancement of Akt phosphorylation and the like. Active mutants of Akt enhance glucose uptake in 3T3-L1 cells. On the other hand, the blood glucose level of the mice lacking Akt2 increased. In addition, in rat muscle-derived cells, Akt inhibitors inhibit insulin-stimulated glucose uptake. Furthermore, IGF-1 is known to activate insulin receptors involved in hypoglycemic action. From these facts, it is considered that the hypoglycemic effect of IGF-1 involves excessive activation of Akt and activation of insulin receptor.
  • IGF-1 Short half-life of IGF-1
  • mecasermin a human recombinant IGF-1
  • IGFBP3 The free form of IGF-1 exhibits bioactivity. Binding to IGFBP3 maintains the blood half-life of IGF-1 from about 10 hours to 16 hours.
  • IPLEX which is a combination drug of IGF-1 and IGFBP3, has a half-life in blood of about 21 to 26 hours, which is longer than that of IGF-1, and is a drug that can be administered once a day.
  • IPLEX has withdrawn from the market.
  • PEGylated IGF-1 which has improved the dynamics of IGF-1, has also been attempted to be developed, but no drug has been used for treatment.
  • IGF-1 acts on various organs and its physiological functions are diverse. For example, IGF-1 has been reported to have a neuroprotective effect on mitochondrial protection and antioxidant activity in the central nervous system through activation of the IGF-1 receptor. IGF-1 promotes the formation of neurites after injury. IGF-1 is also believed to be useful in the treatment of liver cirrhosis. Cirrhosis is a disease that progresses from liver disorder or chronic liver disease and is accompanied by liver fibrosis. In liver cirrhosis model animals, administration of IGF-1 suppressed liver fibrosis. Furthermore, IGF-1 is known to be involved in the development and function of the kidney. In renal mesangial cells, IGF-1 has a protective effect against oxidative stress and apoptosis due to glycotoxicity. IGF-1 is expected as a therapeutic agent for nephropathy.
  • IGF-1 Diseases expected to be improved by administration of IGF-1 include sarcopenia, disused muscular atrophy, caquexia, short stature, laronosis, liver cirrhosis, liver fibrosis, aging, intrauterine fetal growth retardation (IUGR), Neurological disorders, stroke, spinal cord injury, cardiovascular protection, diabetes, insulin resistance, metabolic syndrome, nephropathy, osteoporosis, sarcopenia, wound healing, muscular tonic dystrophy, AIDS muscle atrophy, fat associated with HIV There are redistribution syndrome, burns, Crohn's disease, Werner's syndrome, X-chain complex immunodeficiency, hearing loss, neurological appetite and premature infant retinopathy. As described above, IGF-1 is expected as a therapeutic agent for various diseases due to its various physiological actions. However, the side effects of lowering blood glucose and multiple administrations with a short half-life are challenges for clinical use.
  • Anti-IGF-1 receptor agonist antibody antibody preparations generally have a long half-life and show efficacy when administered once or twice a month.
  • Anti-IGF-1 receptor agonist antibodies have been reported to have some receptor-activating effects in short-term treatment in vitro.
  • antibody 3B7 and antibody 2D1 promoted DNA synthesis in recombinant IGF-1 receptor-expressing cells cultured for 5 hours in vitro (Non-Patent Document 5).
  • anti-IGF-1 receptor antagonist antagonists 11A1, 11A4, 11A11, and 24-57 which have antiproliferative activity in cancer cell lines, are extremely weak in vitro but phosphorylate tyrosine of the IGF-1 receptor. Increased (Non-Patent Document 6).
  • Non-Patent Document 7 antibodies 16-13, 17-69, 24-57, 24-60, and 24-31 have been shown to have an effect of enhancing cellular DNA synthesis and glucose uptake in a short period of time in vitro. These antibodies may have a hypoglycemic effect (Non-Patent Document 7).
  • Non-Patent Documents 5 to 5 It cannot be an index of an agonist antibody having cell proliferation activity.
  • Non-Patent Documents 5 to 8 Furthermore, there were no reports of antibodies showing agonist activity against the IGF-1 receptor in vivo. In addition, since IGF-1 exerts both hypoglycemic action and cell proliferation action, it is necessary to avoid the hypoglycemic action in order to administer the anti-IGF-1 receptor agonist antibody as a therapeutic agent to humans. However, there have been no reports of such anti-IGF-1 receptor agonist antibodies so far.
  • the antibody has a large molecular weight and low tissue transferability, and the intracerebral transferability is about 0.1% and the muscle tissue transferability is about 2%. Therefore, in order to exert an action in a tissue having low transferability of the antibody, an antibody showing sufficient pharmacological activity in an extremely low concentration range (pM order) is required.
  • pM order pharmacological activity in an extremely low concentration range
  • Anti-IGF-1 receptor monoclonal mouse antibody We have succeeded in producing IGF11-16. Furthermore, it has been confirmed that the obtained monoclonal mouse antibody induces a muscle mass increasing action and a growth plate stretching action in vivo without causing hypoglycemic symptoms (Patent Document 1).
  • Anti-IGF-1 receptor antagonist antibody An antibody that binds to the IGF-1 receptor can be applied to the treatment of malignant tumors, etc. by utilizing the antagonistic action that inhibits the binding between IGF-1 and the IGF-1 receptor. Attempted.
  • the existing anti-IGF-1 receptor antagonist antibody not only has many side effects such as hyperglycemia in monotherapy (Non-Patent Document 9), but also increases the incidence of hyperglycemia when used in combination with other anticancer agents. (Non-Patent Document 10), its application to treatment is considered to be limited.
  • Teprotumumab has been approved for the indication of ophthalmopathy in hyperthyroidism (Non-Patent Document 11).
  • Musaro, A., et al. Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle, Nature Genetics, 2001, Vol.27, No.2, pp.195-200 Xiong, L., et al., Growth-stimulatory monoclonal antibody antibodies against human insulin-like growth factor I receptor. Proc Natl Acad Sci U S A, 1992. 89 (12): p. 5356-60. Runnels, H.A., et al., Human monoclonal antibody to the insulin-like growth factor 1 receptor inhibitor receptor activation and tumor growth in precursor clinical studies. Adv Ther, 2010. 27 (7): p.
  • Soos, MA, et al. A panel of monoclonal antibody for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity. J Biol Chem, 1992. 267 (18): p -63.
  • Kato, H., et al. Role of tyrosine kinase activity in signal transduction by the insulin-like growth factor-I (IGF-I) receptor.
  • the present invention is an anti-IGF-1 receptor humanized antibody or an anti-IGF-1 receptor humanized antibody thereof having a specificity equal to or higher than that of the previously reported anti-IGF-1 receptor mouse antibody IGF11-16 (Patent Document 1) and binding affinity or activity.
  • Patent Document 1 One of the purposes thereof is to provide a fragment, a derivative thereof, or a method for producing them.
  • the specific object of the present invention is not limited to these, but is, for example, specificity equal to or higher than that of the previously reported anti-IGF-1 receptor mouse antibody IGF11-16 (Patent Document 1), and binding.
  • Patent Document 1 In order to obtain humanized antibodies having affinity or activity, (1) provision of amino acids essential for the design of human framework, (2) CDR sequence which is an antigen binding site (identified by the Kabat method in the present invention). Examples thereof include provision of amino acid sites essential for maintaining activity, (3) provision of amino acid substitutions for reducing immunogenicity, and (4) provision of amino acid substitutions for avoiding the risk of deamidation.
  • an anti-IGF-1 receptor humanized antibody capable of increasing muscle mass via the human IGF-1 receptor without inducing hypoglycemic symptoms.
  • an anti-IGF-1 receptor that can be administered to a human for the purpose of ameliorating or treating a pathological condition or disease associated with an IGF-1 receptor signal, such as sarcopenia, disused muscular atrophy, or Kakexia.
  • Humanized antibodies can be obtained.
  • the present invention relates to the following.
  • CDRs heavy chain and light chain complementarity determining regions
  • FR heavy chain and light chain framework regions
  • CDR-1 CDR-H1
  • CDR-2 CDR-H2
  • amino acid sequence of the heavy chain variable region amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5
  • amino acid sequence of SEQ ID NO: 7 amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 7 are substituted.
  • CDR-1 CDR-L1 sequence of the light chain variable region
  • amino acid sequence of SEQ ID NO: 9 amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 9 are substituted
  • CDR-2 CDR-L2 sequence of the light chain variable region
  • amino acid sequence of SEQ ID NO: 11 or the amino acid sequence in which any one of the amino acid residues of SEQ ID NO: 11 is substituted.
  • a term comprising, as the CDR-3 (CDR-L3) sequence of the light chain variable region, the amino acid sequence of SEQ ID NO: 13 or the amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 13 are substituted.
  • the anti-IGF-1 receptor humanized antibody or fragment thereof according to [1] or [2], or a derivative thereof.
  • CDR-2 (CDR-H2) sequence of the heavy chain variable region an amino acid sequence having 82% or more homology with SEQ ID NO: 3 or SEQ ID NO: 5
  • CDR-3 (CDR-H3) sequence of the heavy chain variable region an amino acid sequence having 75% or more homology with SEQ ID NO: 7
  • As the CDR-1 (CDR-L1) sequence of the light chain variable region an amino acid sequence having 81% or more homology with SEQ ID NO: 9
  • CDR-2 (CDR-L2) sequence of the light chain variable region an amino acid sequence having 85% or more homology with SEQ ID NO: 11.
  • CDR-1 (CDR-H1) sequence of the heavy chain variable region the amino acid sequence in which the Trp at position 3 of SEQ ID NO: 1 is maintained or replaced with a similar amino acid residue is the 3
  • An amino acid sequence in which any one amino acid residue other than the amino acid residue at the position is substituted or has 80% or more homology with SEQ ID NO: 1.
  • CDR-2 CDR-H2 sequence of the heavy chain variable region
  • CDR-H2 CDR-2 sequence of the heavy chain variable region
  • the amino acid residues at any one, two, or three positions other than the amino acid residues at the 1st, 3rd, and 6th positions are substituted, or the amino acid residue is 82% or more homologous with SEQ ID NO: 3.
  • Amino acid sequence having sex or An amino acid sequence in which Glu at position 1 and Asn at position 3 of SEQ ID NO: 5 are maintained or replaced with similar amino acid residues, and Ser at position 6 is maintained or replaced with Asn or Gln, respectively.
  • the amino acid residues at any one, two, or three positions other than the amino acid residues at the 1, 3, and 6 positions are substituted, or the amino acid residue is 82% or more homologous with SEQ ID NO: 5.
  • Amino acid sequence with sex As the CDR-3 (CDR-H3) sequence of the heavy chain variable region, the amino acid sequence in which Arg at the 4-position of SEQ ID NO: 7 is maintained or substituted with a similar amino acid residue is the amino acid at the 4-position.
  • the amino acid sequence in which the Trp at position 9 of SEQ ID NO: 9 is maintained or substituted with a similar amino acid residue as the CDR-1 (CDR-L1) sequence of the light chain variable region is the amino acid at position 9.
  • CDR-3 (CDR-L3) sequence of the light chain variable region an amino acid residue at any one or two sites of SEQ ID NO: 13 is substituted, or an amino acid having 77% or more homology with SEQ ID NO: 13
  • the heavy chain variable region one or several places in the amino acid sequence of SEQ ID NO: 43, 47, 49, 53, 55, or 59, or the amino acid sequence of SEQ ID NO: 43, 47, 49, 53, 55, or 59.
  • the anti-IGF-1 receptor humanized antibody according to any one of Items [1] to [6], which comprises an amino acid sequence having 90% or more homology with SEQ ID NO: 65, 67, or 69. Or a fragment thereof, or a derivative thereof. [8] Any one of items [1] to [7], wherein the constant region of the heavy chain and / or the light chain includes the constant region of the heavy chain and / or the light chain of any class of human immunoglobulin.
  • the anti-IGF-1 receptor humanized antibody or fragment thereof according to the above, or a derivative thereof.
  • KD equilibrium dissociation constant
  • the anti-IGF-1 receptor according to any one of Items [1] to [15], which can induce growth plate cartilage elongation action without inducing hypoglycemic symptoms in a pituitary excision model animal. Humanized antibodies or fragments thereof, or derivatives thereof. [17] Any of the items [1] to [16], which does not lower the blood glucose level of the vertebrate when administered to the vertebrate at a dose that induces an increase in muscle mass and / or length of the vertebrate.
  • the anti-IGF-1 receptor humanized antibody or fragment thereof according to paragraph 1, or a derivative thereof.
  • the humanized antibody or fragment thereof according to any one of the above, or a derivative thereof.
  • a pharmaceutical composition comprising the recombinant cell according to item [27] as an active ingredient.
  • the pharmaceutical composition according to item [29] which is used for the treatment of amyotrophic disease or short stature.
  • the pharmaceutical composition according to item [30] wherein the amyotrophic disease is disuse muscular atrophy, sarcopenia, or cachexia.
  • the pharmaceutical composition according to item [28], wherein the short stature is Laron-type short stature or growth hormone-resistant short stature.
  • the pharmaceutical composition according to item [29] which is used for treating a disease associated with an IGF-1 receptor.
  • IGF-I receptor-related diseases include liver cancer, neuroblastoma, rhabdomyomyoma, osteosarcoma, pediatric cancer, apical giant disease, ovarian cancer, pancreatic cancer, and benign prostate enlargement.
  • Antibodies of the invention or fragments thereof, or derivatives thereof, can be used for the treatment or prevention of diseases acting via the human IGF-1 receptor by binding to the human IGF-1 receptor, anti-IGF-.
  • One receptor humanized antibody can be obtained.
  • 1A to 1F are graphs showing the human myoblast proliferation activity of various humanized antibodies of the present invention in comparison with the mouse parent antibody IGF11-16. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above.
  • FIG. 2 shows the reactivity of the humanized antibodies hIGF13_PS and hIGF25_PS of the present invention against IGF-1R of each animal species, measured by ELISA using HEK293T cells expressing IGF-1R of each animal species, in a human mouse chimera. It is a graph which shows the comparison with the antibody IGF11-16.
  • FIG. 3A is a graph showing changes in blood glucose level when the humanized antibody hIGF13_PS of the present invention is administered to a guinea pig.
  • FIG. 3B is a graph showing changes in blood glucose level when the humanized antibody hIGF25_PS of the present invention is administered to a guinea pig.
  • FIG. 4 is a graph showing changes in blood concentration when the humanized antibodies hIGF13_PS and hIGF25_PS of the present invention are administered to guinea pigs in comparison with those when the mouse parent antibody IGF11-16 is administered.
  • FIG. 5 shows changes in the mass of the extensor digitorum longus muscle after 2 weeks when the humanized antibody hIGF13_PS of the present invention was intravenously administered to a normal guinea pig by continuous subcutaneous administration of IGF-1 and the mouse parent antibody IGF11-16. It is a graph which shows the comparison with the single intravenous administration of.
  • FIG. 6 shows changes in the epiphyseal line thickness of the proximal tibia after 2 weeks when the humanized antibody hIGF13_PS of the present invention was intravenously administered to a pituitary gland excised guinea pig by continuous subcutaneous administration of IGF-1. It is a graph which shows the comparison with the subcutaneous continuous administration of the GH preparation.
  • FIG. 7 is a graph showing changes in blood glucose level when the humanized antibody hIGF13_PS of the present invention is administered to cynomolgus monkeys in comparison with the time of administration of IGF-1.
  • FIG. 8 is a graph showing changes in blood concentration when the humanized antibody hIGF13_PS of the present invention is administered to cynomolgus monkeys.
  • FIG. 9 is a graph showing the concentration-dependent effect of the mouse parent antibody IGF11-16 on HepG2 cell proliferation.
  • M indicating the concentration is a unit synonymous with "mol / L” indicating the molar concentration.
  • the present invention is an anti-IGF-1 receptor humanized antibody that specifically binds to the IGF-1 receptor.
  • the antibody of the present invention has an action of increasing muscle mass via the human IGF-1 receptor without inducing hypoglycemic symptoms. This makes it possible to ameliorate or treat conditions or diseases involving IGF-1 receptor signals such as sarcopenia, disuse muscular atrophy, and Kakexia. Further, the antibody of the present invention is a humanized antibody that ensures low immunogenicity and physical stability.
  • IGF insulin-like Growth Factor
  • IGF-1 and IGF-2 are present. IGF-1 and IGF-2 bind to the IGF-1 receptor (Insulin-like Growth Factor-I Receptor) described later, and signal intracellular cell division and metabolism. It is an in vivo ligand that has an agonistic activity to enter. IGF-1 and IGF-2 are also known to weakly and cross-link to the insulin receptor (INSR), which is structurally similar to the IGF-1 receptor. In the present specification, IGF-1 whose physiological function is better known is mainly dealt with, but when examining the action or disease through the binding between the IGF-1 receptor and the ligand, IGF is used. The effects of both -1 and IGF-2 may be included.
  • IGF-1 and IGF-2 may be included.
  • IGF-1 is also called somatomedin C and is a hormone of a single polypeptide consisting of 70 amino acids.
  • the sequence of human IGF-1 can be obtained by referring to NCBI Reference Sequence No .: NP_000609, or EMBL-EBI UniProt KB-Axon No. P05019 and the like.
  • the amino acid sequence of mature human IGF-1 is shown in SEQ ID NO: 83, and an example of the corresponding nucleic acid base sequence is shown in SEQ ID NO: 84.
  • This 70-amino acid sequence is conserved in many species. In the present invention, when only "IGF-1" is described, it means an IGF-1 protein having hormonal activity unless otherwise specified.
  • IGF-1 is produced in various cells in the living body including liver cells, and is also present in blood and other body fluids. Therefore, the natural form of IGF-1 can be obtained by purifying from the body fluid of an animal or a culture obtained by culturing primary cultured cells or cell lines separated from the animal. In addition, since IGF-1 is induced to be produced in cells by growth hormone, body fluids of animals to which growth hormone was administered, primary cultured cells separated from animals, established cells, etc. were cultured in the presence of growth hormone. It can also be obtained by purifying IGF-1 from the culture.
  • a nucleic acid molecule encoding the amino acid sequence of IGF-1 was incorporated into an expression vector and introduced into a host of prokaryotes such as Escherichia coli and eukaryotic cells such as yeast, insect cells or cultured cells derived from mammals. It is also possible to produce IGF-1 using recombinant cells, transgenic animals into which the IGF-1 gene has been introduced, transgenic plants, and the like.
  • human IGF-1 is a research reagent (Enzo Life Sciences, catalog: ADI-908-059-0100; Abnova, catalog: P3452, etc.), pharmaceuticals (Somazon (registered trademark) , mecasermin, INCRELEX (registered trademark), etc.).
  • IGF-1 used is 1 international unit / microgram of activity on the IGF-1 standard material of the NIBSC code: 91/554 of the World Health Organization's National Institute for Biological Standards and Control (NIBSC).
  • the specific activity can be evaluated by comparing with.
  • IGF-1 in the present invention shall be treated as having a specific activity equivalent to that of IGF-1 of the NIBSC code: 91/554.
  • IGF-1 receptor refers to an insulin-like Growth Factor-I Receptor.
  • IGF-1 receptor means an IGF-1 receptor protein unless otherwise specified.
  • the IGF-1 receptor is a protein with a structure in which two subunits consisting of an ⁇ chain and a ⁇ chain are associated.
  • SEQ ID NO: 71 the portion of the amino acid sequence consisting of the amino acid sequences at positions 31 to 735 corresponds to the ⁇ chain, and the ⁇ chain is the sequence after position 740. Corresponds to.
  • the ⁇ chain of the IGF-1 receptor has a binding portion of IGF-1, and the ⁇ chain has a transmembrane structure and functions to transmit a signal into the cell.
  • the ⁇ chain of the IGF-1 receptor is divided into L1, CR, L2, FnIII-1 and FnIII-2a / ID / FnIII-2b domains.
  • the portion from position 31 to 179 is the L1 domain
  • the portion from position 180 to position 328 is the CR domain
  • the portion from position 329 to position 491 is the L2 domain.
  • the portion from position 492 to position 607 corresponds to the FnIII-1 domain, and the portion from position 608 to position 735 corresponds to the FnIII-2a / ID / FnIII-2b domain.
  • the amino acid sequence of the human IGF-1 receptor can be referred to from UniProtKB-Axon No. P08069 of EMBL-EBI, etc., and is also shown in SEQ ID NO: 71 of the sequence listing.
  • the IGF-1 receptor is expressed in a wide range of tissues and cells in the living body, and is stimulated by IGF-1 such as induction of cell proliferation and activation of intracellular signals.
  • myoblasts can use the IGF-1 receptor-mediated action of IGF-1 for evaluation using the cell proliferation activity as an index. This makes myoblasts useful for analyzing the action of antibodies that bind to the IGF-1 receptor.
  • a recombinant in which a nucleic acid molecule encoding the amino acid sequence of the IGF-1 receptor of humans and other vertebrates is incorporated into an expression vector and introduced into a host of eukaryotic cells such as insect cells or cultured cells derived from mammals.
  • the IGF-1 receptor-expressing cells can be used for analysis of antibody binding and examination of intracellular signal transduction.
  • mouse parent antibody IGF11-16 The mouse parent antibody IGF11-16 is disclosed in Patent Document 1.
  • the amino acid sequence of CDR-H1 of IGF11-16 is SEQ ID NO: 85
  • the nucleic acid sequence of CDR-H2 is SEQ ID NO: 86
  • the amino acid sequence of CDR-H3 is SEQ ID NO: 87
  • the amino acid sequence of CDR-L1 is SEQ ID NO: 88
  • CDR- CDR-.
  • the amino acid sequence of L2 is SEQ ID NO: 89
  • the amino acid sequence of CDR-L3 is SEQ ID NO: 90
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 39 (an example of the corresponding nucleic acid base sequence is SEQ ID NO: 40), and the light chain variable region.
  • the amino acid sequence is shown in SEQ ID NO: 41 (an example of the corresponding nucleic acid base sequence is SEQ ID NO: 42).
  • the full-length amino acid sequence of the light chain of IGF11-16 is SEQ ID NO: 91 (an example of the corresponding nucleobase sequence, SEQ ID NO: 92), and the full-length amino acid sequence of the heavy chain is SEQ ID NO: 93 (an example of the corresponding nucleobase sequence).
  • All antibodies comprising the expression IGF11-16 mean this mouse parent antibody IGF11-16.
  • an “antibody” is a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region, and the heavy chain constant region contains three domains, CH1, CH2 and CH3.
  • Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region contains one domain, CL.
  • VHs and VLs are further referred to as framework regions (FRs), four more conserved regions (FR-1 (FR1), FR-2 (FR2), FR-3 (FR3), FR-4. (FR4)) and three hypervariable regions (CDR-1 (CDR1), CDR-2 (CDR2), CDR-3 (CDR3)) called complementarity determining regions (CDRs). ..
  • VH is from the amino terminus to the carboxy terminus, FR-1 (FR-H1), CDR-1 (CDR-H1), FR-2 (FR-H2), CDR-2 (CDR-H2), FR-3 ( It contains 3 CDRs and 4 FRs arranged in the order of FR-H3), CDR-3 (CDR-H3), FR-4 (FR-H4).
  • VL is from the amino terminus to the carboxy terminus, FR-1 (FR-L1), CDR-1 (CDR-L1), FR-2 (FR-L2), CDR-2 (CDR-L2), FR-3 ( It contains 3 CDRs and 4 FRs arranged in the order of FR-L3), CDR-3 (CDR-L3), FR-4 (FR-L4).
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the antibody of the present invention may be a fragment and / or a derivative of the antibody.
  • the antibody fragment include F (ab') 2 , Fab, Fv and the like.
  • the antibody derivative an antibody in which an amino acid mutation is artificially introduced into the constant region portion, an antibody in which the composition of the constant region domain is modified, an antibody having two or more Fc per molecule, a heavy chain only or light chain Chain-only antibodies, sugar chain-modified antibodies, bispecific antibodies, antibodies or antibody fragment compounds or antibody conjugates bound to proteins other than antibodies, antibody enzymes, nanobodies, tandem scFv, bispecific Examples include tandem scFv, Diabody, VHH and the like.
  • the term "antibody” also includes an antibody fragment and / or a derivative unless otherwise specified.
  • a monoclonal antibody is classically an antibody molecule obtained from a clone derived from a single antibody-producing cell, but is a single type of antibody molecule containing a combination of VH and VL having a specific amino acid sequence. It means that.
  • a monoclonal antibody it is possible to obtain a nucleic acid molecule having a gene sequence encoding an amino acid of the protein of the antibody, and it is also possible to prepare an antibody by genetic engineering using such a nucleic acid molecule.
  • genetic information such as H chain, L chain, their variable regions and CDR sequences can be used to modify the antibody to improve the binding property and specificity, and from animal antibodies such as mice to human type.
  • Producing an antibody having a structure suitable for use as a therapeutic agent by modifying it into an antibody is a technique well known to those skilled in the art in this field. It is also possible to obtain a human monoclonal antibody by using a non-human-transgenic animal into which a human antibody gene has been introduced as an animal that sensitizes the antigen. In addition, as a method that does not require sensitization to animals, a phage library (human antibody phage display) that expresses the antigen-binding region of a human antibody or a part thereof is used and specifically with the corresponding antigen.
  • a phage library human antibody phage display
  • a person skilled in the art can appropriately perform a technique for obtaining a phage clone consisting of an antibody to be bound or a specific amino acid sequence and producing a human antibody from the information (see, for example, Non-Patent Document 17 and the like). Further, when designing an antibody to be administered to an animal other than human, a person skilled in the art can design it by appropriately using the amino acid sequence information of CDR and the variable region, as in the technique of humanization. ..
  • the antibodies of the invention are the complementarity determining regions (CDRs) of the heavy and light chains derived from the mouse parent antibody IGF11-16 and the frames of the heavy and light chains derived from the human antibody.
  • CDRs complementarity determining regions
  • Anti-IGF-1 receptor humanization comprising working regions (FRs) and at least one CDR containing at least one amino acid residue substitution against the corresponding CDR of the mouse parent antibody IGF11-16. It is an antibody.
  • the heavy and light chain complementarity determining regions are derived from the corresponding CDRs of the mouse parent antibody IGF11-16, respectively.
  • the mouse parent antibody "IGF11-16” is an anti-IGF-1 receptor monoclonal mouse antibody previously produced by the present inventors as described above (Patent Document 1).
  • "derived" from the CDR of the mouse parent antibody means that the amino acid sequence of each CDR of the antibody of this embodiment is usually 75% or more of the amino acid sequence of the corresponding CDR of the mouse parent antibody IGF11-16.
  • the antibody of this embodiment requires that at least one of the CDRs comprises the substitution of at least one amino acid residue with respect to the corresponding CDR of the mouse parent antibody IGF11-16.
  • the amino acid sequence of the heavy chain CDR-2 (CDR-H2)
  • they are homologous (preferably the same) except for the difference of 3 amino acid residues or less, and further 2 amino acid residues or less.
  • each framework region (FR) of heavy and light chains is derived from the corresponding FR of each class of human immunoglobulin.
  • "derived" from the FR of a human immunoglobulin means that the amino acid sequence of each FR of the antibody of this embodiment is usually 80% or more, particularly 85, with the amino acid sequence of the corresponding FR of the human immunoglobulin. % Or more, and even 90% or more homology (preferably identity), and / or usually 4 amino acid residues or less, especially 3 amino acid residues or less, and further excluding the difference of 2 amino acid residues or less. Means that they are homologous (preferably the same).
  • Human immunoglobulin frameworks are available from public databases, and frameworks that are highly homologous to the mouse immunoglobulin framework can be selected.
  • IgBLAST can be used for identification of amino acid sequences having homology (Non-Patent Document 16).
  • the amino acid residue at position 25 of the heavy chain FR1 is preferably proline.
  • the amino acid residue at position 25 of the heavy chain FR1 of the mouse parent antibody IGF11-16 is substituted with proline in the same manner as the mouse parent antibody IGF11-16.
  • the activity is equal to or higher than that of the mouse parent antibody IGF11-16 ( It is preferable because the activity ratio can be exhibited within ⁇ 20% to the same extent.
  • an evolutionary engineering method of an antibody can be used using the sequence of the heavy chain and the light chain derived from the humanized antibody of the present invention as a template.
  • methods such as a site-specific mutagenesis method for CDR, a random mutagenesis method, chain shuffling, and CDR walking are used.
  • Random mutagenesis is a method of introducing a random mutation into a specific gene DNA to prepare a mutant.
  • a base mutation is introduced by selecting a condition with low replication strictness at the time of DNA amplification (error-prone PCR). Mutations are introduced at arbitrary sites for the entire area of DNA amplified by the PCR method.
  • the target gene can be first cut into pieces, and then the mutation can be introduced in the same manner by the PCR method.
  • Chain shuffling is to construct a library in which one of the VH or VL genes in the antibody variable region is immobilized and the other is bound to the V gene library, expressed on phage, and the original antigen is expressed. It is a method of screening a combination of antibody variable regions having high specificity for. It is the method of choice for in vitro affinity maturation of antibodies obtained from naive / non-immune libraries.
  • CDR walking introduces random mutations into each CDR of the VH and VL genes, selects antibodies with strong binding by adjusting the selection conditions from the population of the mutants, and combines the selected CDRs. It is a method to obtain a clone with a very high binding force. In general, random mutations are often introduced and examined only in CDR3.
  • the antibody of the present invention has a specific amino acid sequence as each CDR sequence. Specifically, it is as follows.
  • identity of an amino acid sequence means the ratio of matching amino acid residues
  • similarity means the ratio of matching or similar amino acid residues. do. Homology and identity can be determined, for example, by the BLAST method (the default condition of NCBI's BLAST).
  • similar amino acid residue as used herein means an amino acid residue having a side chain having similar chemical properties (eg, charge or hydrophobicity). Examples of similar amino acid residues include the following groups. For example, according to the following group, substituting alanine for a similar amino acid residue means substituting for a valine, leucine, isoleucine or methionine residue.
  • Amino acid residues having an aliphatic side chain alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I) and methionine (Met or M) residues.
  • Amino acid residues having an aliphatic hydroxyl side chain serine (Ser or S) and threonine (Thr or T) residues.
  • Amino acid residues having an amide-containing side chain asparagine (Asn or N) and glutamine (Gln or Q) residues.
  • Amino acid residues having an aromatic side chain phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W) and histidine (His or H) residues.
  • Amino acid residues having a basic side chain lysine (Lys or K), arginine (Arg or R) and histidine (His or H) residues.
  • Amino acid residues having an acidic side chain aspartic acid (Asp or D) and glutamic acid (Glu or E) residues.
  • Amino acid group having a sulfur-containing side chain cysteine (Cys or C), and methionine (Met or M) residue.
  • the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence in which any one of the amino acid residues of SEQ ID NO: 1 is substituted is preferable.
  • the CDR-H1 sequence preferably has 80% or more homology (preferably identity) with SEQ ID NO: 1.
  • the CDR-H1 sequence is an amino acid sequence in which the Trp at position 3 of SEQ ID NO: 1 is maintained or substituted with a similar amino acid residue, and any one other than the amino acid residue at position 3 is 1
  • the amino acid residue at the site is substituted or that the amino acid sequence has an amino acid sequence having 80% or more homology (preferably identity) with SEQ ID NO: 1.
  • An example of the nucleic acid base sequence corresponding to the amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • the CDR-2 (CDR-H2) sequence of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5, or any one, two, or three locations of SEQ ID NO: 3 or SEQ ID NO: 5. Amino acid sequences substituted with amino acid residues are preferred. Alternatively, the CDR-H2 sequence preferably has 82% or more homology (preferably identity) with SEQ ID NO: 3 or SEQ ID NO: 5.
  • Glu at position 1 and Asn at position 3 of SEQ ID NO: 3 are maintained or replaced with similar amino acid residues, and Asn at position 6 is maintained, or An amino acid sequence substituted with Ser or Gln, wherein any one, two, or three amino acid residues other than the amino acid residues at the 1, 3, and 6 positions are substituted or replaced. It is preferable to have an amino acid sequence having 82% or more homology with SEQ ID NO: 3.
  • Glu at position 1 and Asn at position 3 of SEQ ID NO: 5 are maintained, respectively, or are replaced with similar amino acid residues, and Ser at position 6 is maintained, or An amino acid sequence substituted with Asn or Gln, wherein any one, two, or three amino acid residues other than the amino acid residues at the 1, 3, and 6 positions are substituted or replaced. It is preferable to have an amino acid sequence having 82% or more homology with SEQ ID NO: 5. Examples of nucleic acid base sequences corresponding to the amino acid sequences of SEQ ID NOs: 3 and 5 are shown in SEQ ID NOs: 4 and 6, respectively.
  • the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 7 are substituted is preferable.
  • the CDR-H3 sequence preferably has 75% or more homology (preferably identity) with SEQ ID NO: 7.
  • the CDR-H3 sequence is an amino acid sequence in which Arg at position 4 of SEQ ID NO: 7 is maintained or substituted with a similar amino acid residue, and any one other than the amino acid residue at position 4 is 1 It is preferable that the amino acid residue at one or two sites is substituted, or that the amino acid sequence has 75% or more homology (preferably identity) with SEQ ID NO: 7.
  • An example of the nucleic acid base sequence corresponding to the amino acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 8.
  • the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 9 are substituted is preferable.
  • the CDR-L1 sequence preferably has 81% or more homology (preferably identity) with SEQ ID NO: 9.
  • the CDR-L1 sequence is an amino acid sequence in which the Trp at position 9 of SEQ ID NO: 9 is maintained or is substituted with a similar amino acid residue, and any one other than the amino acid residue at position 9 is 1 It is preferable that the amino acid residue at one or two sites is substituted, or that the amino acid sequence has 81% or more homology (preferably identity) with SEQ ID NO: 9.
  • An example of the nucleic acid base sequence corresponding to the amino acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 10.
  • the amino acid sequence of SEQ ID NO: 11 or the amino acid sequence in which any one amino acid residue of SEQ ID NO: 11 is substituted is preferable.
  • the CDR-L2 sequence preferably has 85% or more homology (preferably identity) with SEQ ID NO: 11.
  • An example of the nucleic acid base sequence corresponding to the amino acid sequence of SEQ ID NO: 11 is shown in SEQ ID NO: 12.
  • the amino acid sequence of SEQ ID NO: 13 or the amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 13 are substituted is preferable.
  • the CDR-L3 sequence preferably has 77% or more homology (preferably identity) with SEQ ID NO: 13.
  • An example of the nucleic acid base sequence corresponding to the amino acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 14.
  • the antibody of the present invention preferably has the following combination of CDR sequences. That is, as the CDR-H1 sequence, the amino acid sequence of SEQ ID NO: 1, as the CDR-H2 sequence, the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5, as the CDR-H3 sequence, as the amino acid sequence of SEQ ID NO: 7, and as the CDR-L1 sequence.
  • the amino acid sequence of SEQ ID NO: 9, the CDR-L2 sequence is preferably a combination of the amino acid sequence of SEQ ID NO: 11 and the CDR-L3 sequence is preferably a combination of the amino acid sequence of SEQ ID NO: 13.
  • Non-Patent Document 13 Kabat method
  • Non-Patent Document 14 Chothia method
  • Non-Patent Document 15 a method obtained by further improving these methods
  • These methods are common technical knowledge for those skilled in the art in this area, but for example, you can get an overview from the Internet homepage (http://www.bioinf.org.uk/abs/) of Dr. Andrew CR Martin's Group. It is also possible.
  • Example 4 by performing an alanine scan, it is possible to identify a site important for binding activity in the amino acid sequence of CDR. From the results, it is clear that the amino acid residues shown in Tables 7 and 8 described later are extremely important. Substitution of at least the amino acid residue at this site with an amino acid other than an amino acid having similar properties is considered to lead to a decrease in binding property. Conversely, it may be possible to achieve improved affinity by substituting for amino acids with similar properties. On the other hand, of the 54 regions of the CDR regions substituted with alanine, 44 sites showed 80% or more of the binding activity even after the substitution with alanine. This suggests that amino acid substitutions at these sites do not significantly affect binding activity. In this way, by searching the amino acid sequences of the CDR regions and identifying the sites involved in binding, immunogenicity can be reduced, physical properties can be improved, and binding can be improved while maintaining binding. It is possible to do.
  • the antibody of the present invention preferably has a specific amino acid sequence as a heavy chain variable region and a light chain variable region. Specifically, it is as follows. In the present disclosure, “one place or several places” means one place, two places, three places, four places, five places, six places, seven places, eight places, nine places, or ten places, unless otherwise specified. It shall refer to any of the locations.
  • the antibody of the present invention has an amino acid sequence of SEQ ID NO: 47 or an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 47 as a heavy chain variable region. Is preferable. Alternatively, it is preferable to have an amino acid sequence having 90% or more homology (preferably identity) with SEQ ID NO: 47 as the heavy chain variable region.
  • VH13_PN SEQ ID NO: 43
  • VH13_PS SEQ ID NO: 47
  • VH23_PN SEQ ID NO: 49
  • VH23_PS SEQ ID NO: 53
  • VH25_PN SEQ ID NO: 55
  • VH25_PS SEQ ID NO: 59
  • nucleic acid base sequences corresponding to the amino acid sequences of SEQ ID NOs: 43, 47, 49, 53, 55, and 59 are shown in SEQ ID NOs: 44, 48, 50, 54, 56, and 60, respectively.
  • the antibody of the present invention contains, as a light chain variable region, an amino acid sequence of SEQ ID NO: 67 or an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 67. It preferably has an amino acid sequence. Alternatively, it is preferable to have an amino acid sequence having 90% or more homology (preferably identity) with SEQ ID NO: 67 as the light chain variable region.
  • the light chain variable region may have the amino acid sequence of VL13 (SEQ ID NO: 61), VL14 (SEQ ID NO: 63), VL22 (SEQ ID NO: 65), VL23 (SEQ ID NO: 67), or VL24 (SEQ ID NO: 69). preferable.
  • VL22 (SEQ ID NO: 65), VL23 (SEQ ID NO: 67), or VL24 (SEQ ID NO: 69).
  • Examples of nucleic acid base sequences corresponding to the amino acid sequences of SEQ ID NOs: 61, 63, 65, 67, and 69 are shown in SEQ ID NOs: 62, 64, 66, 68, and 70, respectively.
  • the antibody of the present invention has any of the above-mentioned amino acid sequences as the heavy chain variable region and the light chain variable region, respectively.
  • a particularly preferable antibody of the present invention is an antibody having an amino acid sequence of VH13_PS (SEQ ID NO: 47) as a heavy chain variable region and an amino acid sequence of VL23 (SEQ ID NO: 67) as a light chain variable region (hereinafter referred to as "hIGF13_PS").
  • hIGF25_PS an antibody having the amino acid sequence of VH25_PS (SEQ ID NO: 59) as the heavy chain variable region and the amino acid sequence of VL23 (SEQ ID NO: 67) as the light chain variable region (hereinafter referred to as "hIGF25_PS"). There is.).
  • the amino acid sequence of each constant region of the heavy chain and the light chain of the antibody of the present invention is selected from, for example, the amino acid sequences of each class of human IgG, IgA, IgM, IgE, and IgD, and the amino acid sequences of variants thereof. It is possible to do.
  • the amino acid sequence of the heavy chain constant region of the antibody of the present invention is an amino acid sequence of the heavy chain constant region of the human IgG4 class, or an amino acid sequence in which 1 to 10 amino acids thereof are substituted. Is preferable (Non-Patent Documents 19 and 20).
  • the amino acid sequence of the heavy chain constant region of the antibody of the present invention is an amino acid sequence of the heavy chain constant region of the human IgG1 class, or an amino acid sequence in which 1 to 10 amino acids thereof are substituted. Is preferable (Non-Patent Documents 19 and 20).
  • the antibody of the present invention is an antibody that causes an antigen-antibody reaction with a human IGF-1 receptor.
  • antigen-antibody reaction means that an antibody binds to an IGF-1 receptor with an affinity of equilibrium dissociation constant (KD) of 1 ⁇ 10 -7 M or less.
  • KD equilibrium dissociation constant
  • the antibody of the present invention preferably binds to the IGF-1 receptor with a KD of usually 1 ⁇ 10 -7 M or less, particularly 1 ⁇ 10 -8 M or less, and further preferably 1 ⁇ 10 -9 M or less. .. Most preferably, it is 1 ⁇ 10 -10 M or less.
  • the antibody of the present invention preferably specifically binds to the extracellular domain of the human IGF-1 receptor having the amino acid sequence of SEQ ID NO: 71.
  • an antibody having "specificity" to an antigen means that a high antigen-antibody reaction occurs between the antibody and the antigen.
  • IGF-1 receptor-specific antibody refers to a higher-order structure of the IGF-1 receptor at a concentration that exhibits a significant antigen-antibody reaction with cells expressing the IGF-1 receptor.
  • An antibody having an antigen-antibody reactivity to INSR having similarity of 1/100 or less.
  • the measurement of the antigen-antibody reaction can be carried out by a person skilled in the art by appropriately selecting the binding measurement in a solid phase or liquid phase system.
  • Such methods include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), surface plasmon resonance (SPR), and fluorescence resonance energy transfer method.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • SPR surface plasmon resonance
  • fluorescence resonance energy transfer method Fluorescence resonance energy transfer: FRET
  • emission resonance energy transfer method luminescence resonance energy transfer: LRET
  • the antibody and / or antigen is labeled with an enzyme, a fluorescent substance, a luminescent substance, a radioactive isotope, or the like, and the physical and / or chemical properties of the labeled substance. It is also possible to detect an antigen-antibody reaction using a measurement method suitable for the above.
  • the antibody of the present invention has an IGF-1 receptor signal activating ability equal to or higher than that of the mouse parent antibody IGF11-16.
  • “equivalent” in the ability to activate the IGF-1 receptor signal means that the EC 50 value is within 2 times and / or the E max value is within ⁇ 20%.
  • the antibody of the present invention preferably has a proliferation activity equal to or higher than that of the mouse parent antibody IGF11-16.
  • “equivalent” in proliferative activity means that the EC 50 value is within 10 times and the E max value is 90% or more in the myoblast proliferation test.
  • the antibody of the present invention preferably has a binding affinity for the recombinant soluble IGF-1 receptor equal to or higher than that of the mouse parent antibody IGF11-16.
  • "equivalent" binding affinity to recombinant soluble IGF-1 receptor is compared to mouse parent antibody IGF11-16 in the binding affinity analysis to recombinant soluble IGF-1 receptor by BIACORE. If this is the case, it means that the KD value is in the range of 1/3 or more to 3 times or less.
  • the antibody of the present invention has a long half-life in blood and exhibits a muscle mass increasing effect by a single administration to an animal.
  • the anti-IGF-1 receptor humanized antibody of the present invention was administered once to a guinea pig or a crab monkey as shown in Examples described later, and the IGF-1 was obtained. It showed the same level of muscle mass-increasing effect as when it was continuously administered.
  • the antibody of the present invention can induce a muscle mass increasing action without inducing hypoglycemic symptoms in normal mammals. According to one aspect, it is preferable that the antibody of the present invention can induce a growth plate cartilage elongation action without inducing hypoglycemic symptoms in a pituitary excision model animal.
  • the "blood glucose symptom” means, in the case of humans, symptoms such as cold sweat, palpitation, consciousness disorder, convulsions, and tremors of limbs caused by hypoglycemia. In vertebrates such as monkeys, a decrease in locomotor activity appears as an initial symptom, and when the symptom is strong, there is almost no movement, and when the blood glucose level drops, consciousness disorder occurs, leading to death.
  • the antibody of the present invention is administered to a vertebrate at a dose that induces an increase in vertebrate muscle mass and / or length, more preferably at a dose that is 10-fold or more higher than that dose. Even it has no effect on lowering the blood glucose level of the vertebrate. Further, according to one aspect, the antibody of the present invention was administered to the vertebrate so as to have a blood exposure amount of 10 times or more the effective dose for inducing an increase in muscle mass and / or body length of the vertebrate.
  • the antibody of the present invention does not induce hypoglycemic symptoms even when administered at a dose of 10 mg / kg in guinea pigs and cynomolgus monkeys, as shown in Examples described later. rice field.
  • the anti-IGF-1 receptor humanized antibody of the present invention can be used for various diseases associated with IGF-1 receptors such as disused muscular atrophy and short stature, which are expected for IGF-1. It has the potential to be a therapeutic or prophylactic agent, overcomes the problem of IGF-1 and has a hypoglycemic effect, and can prolong the half-life in blood.
  • the antibody of the present invention is a homozygous receptor in which the IGF-1 receptor forms a dimer by binding to the extracellular domain of the IGF-1 receptor, or the IGF-1 receptor and INSR are dimeric. It is thought to activate the heteromorphic receptors that formed the body.
  • Anti-Drug Antibody affects the efficacy and pharmacokinetics of therapeutic antibodies and can sometimes lead to serious side effects, so the usefulness and efficacy of therapeutic antibodies in clinical practice is ADA. Can be limited by the production of. Although many factors affect the immunogenicity of therapeutic antibodies, the importance of effector T cell epitopes present in therapeutic proteins has been widely reported. Epibase (Lonza), iTope / TCED (Antitope), EpiMatrix (EpiVax) and the like have been developed as in silico tools for predicting T cell epitopes.
  • Non-Patent Document 21 the presence of T cell epitopes in each amino acid sequence can be predicted (Non-Patent Document 21), and potential immunogenicity can be evaluated.
  • the potential immunogenicity was evaluated using Epibase (Lonza).
  • the NS sequence (heavy chain positions 55 and 56) was present in the heavy chain CDR-2 (CDR-H2) region of the mouse parent antibody IGF11-16, so that asparagine (N) at the heavy chain position 55 was present.
  • the asparagine (N) was replaced with serine (S) in consideration of the risk of deamidation and conversion to aspartic acid (D).
  • the anti-IGF-1 receptor humanized antibody of the present invention has a purity of 95% or more using a sample obtained by using a PBS solution of the present antibody, setting the temperature to 37 ° C. and incubating for 1 month. The stability of the physical properties was confirmed by confirming that no agglomerates were formed.
  • the antibody of the present invention uses the CR domain of the IGF-1 receptor as an epitope.
  • the antibody of the present invention binds to an epitope or a vicinity thereof containing a peptide having an amino acid sequence (ProSerGlyPheIleArgAsnGlySerGlnSerMet) corresponding to amino acid numbers 308 to 319 in the amino acid sequence (SEQ ID NO: 71) of the human IGF-1 receptor. do.
  • the antibody of the present invention is a homozygous receptor in which the IGF-1 receptor forms a dimer by binding to the CR domain of the IGF-1 receptor, or a dimer of the IGF-1 receptor and INSR. It is thought to activate the heterozygous receptor that formed the.
  • the agonist antibody of the present invention is preferably a human IgG class or a variant thereof, and is preferably a human IgG4 subclass or a variant thereof, or a human IgG1 subclass or a variant thereof.
  • the stabilized IgG4 constant region contains proline at position 241 of the hinge region by Kabat's system (Non-Patent Document 22). This position corresponds to the position 228 of the hinge region by the EU numbering method (Non-Patent Document 13). In human IgG4, this residue is generally serine and the substitution of serin with proline can induce stabilization.
  • the N297A mutation can be incorporated into the constant region of IgG1 to minimize the ability to bind to Fc receptors and / or immobilize complement.
  • the binding to FcRn can be regulated and the half-life in blood can be lengthened by introducing an amino acid substitution into the constant region.
  • amino acid substitutions in the constant region are not limited to these examples.
  • the agonist antibody of the present invention specifically and strongly binds to the IGF-1 receptor, and has an action of enhancing myoblast proliferation from an extremely low concentration in vitro.
  • the agonist antibody of the present invention exhibits a muscle mass-increasing effect similar to that of continuous administration of IGF-1 when administered once to an animal.
  • the agonist antibody of the present invention has a long half-life in blood and can exhibit a muscle mass-increasing effect by a single administration to an animal.
  • the muscle mass-increasing effect and the muscle mass-increasing effect when IGF-1 is continuously administered are shown.
  • the agonist antibody of the present invention is characterized in that it does not have a hypoglycemic effect at a dose showing a muscle mass increasing effect.
  • IGF-1 has a significant hypoglycemic effect when administered at a dose showing a muscle mass increasing effect.
  • the agonist antibody of the present invention has no effect of lowering the blood glucose level of the vertebrate at a dose that induces an increase in vertebrate muscle mass and / or body length.
  • the agonist antibodies of the invention are vertebrate blood glucose, even when administered to a blood exposure of at least 10 times the effective dose that induces an increase in vertebrate muscle mass and / or length. It has no effect of lowering the value.
  • the agonist antibody of the present invention in fact, in guinea pigs or crab monkeys, even when the agonist antibody of the present invention is administered so as to have a blood exposure of 10 times or more the effective dose that induces an increase in muscle mass, hypoglycemia and No hypoglycemic symptoms were observed.
  • the agonist antibody of the present invention has the potential to be a therapeutic or prophylactic agent for various diseases related to the IGF-1 receptor such as sarcopenia, disuse muscular atrophy, Kakexia and short stature.
  • various diseases related to the IGF-1 receptor such as sarcopenia, disuse muscular atrophy, Kakexia and short stature.
  • the anti-IGF-1 receptor humanized antibody of the present invention is an anti-IGF-1 receptor antagonist antibody having excellent activity and specificity by utilizing the properties of the variable region having extremely high binding and specificity. can do. In that case, in addition to IgG, it is conceivable to use it in the form of Fab, Fv, scFv, VHH, but the present invention is not limited to this.
  • the anti-IGF-1 receptor antagonist antibody thus produced can be evaluated using, for example, suppressing the IGF-1-dependent cell proliferation activity in a cancer cell line as an index.
  • the anti-IGF-1 receptor antagonist antibody thus selected is expected as an anti-cancer agent and an ameliorating and therapeutic agent for diseases and pathological conditions associated with abnormal cell proliferation.
  • bispecific antibodies and multispecific antibodies can be constructed by fusing with various antibodies recognizing other antigens or epitopes directly or via a linker or the like.
  • IgG immunoglobulin G
  • Bispecific antibodies and multispecific antibodies containing anti-IGF-1 receptor antagonist antibodies thus prepared can suppress IGF-1-dependent cell proliferation activity in cancer cell lines, for example. It can be evaluated as an index. Bispecific antibodies and multispecific antibodies containing the anti-IGF-1 receptor antagonist antibody thus selected are expected to be anti-cancer agents and ameliorating and therapeutic agents for diseases and pathological conditions associated with abnormal cell proliferation. Will be done.
  • the anti-IGF-1 receptor antagonist antibody of the present invention is expected as a therapeutic agent for a disease in which a pathological condition is induced by activation of an IGF-I receptor signal.
  • the ligands capable of activating the IGF-I receptor mainly include IGF-1, IGF-2, Insulin and the like, and RTK (receptor type tyrosine kinase) formed into a heterodimer with the IGF-1 receptor. It also includes ligands (such as EGF) and ligands for other receptors that crosstalk (such as TSH).
  • the antibody of the present invention has an action of suppressing the IGF-1 receptor signal activated by these ligands (has an allosteric antagonist action.
  • the antibody of the present invention binds to the IGF-1 receptor. It suppresses over-induced IGF-1 receptor signal activity and can be used to treat or prevent diseases induced by abnormally activated IGF-1 receptor.
  • Signal activation inhibitory ability is mouse. It is preferable to have the same or higher level as the parent antibody IGF11-16.
  • the signal activation inhibitory ability "equivalent to the mouse antibody IGF11-16" is defined as IGF-1, IGF-2, or IGF- such as Insulin in myoblast proliferation. It means an activity that suppresses the maximum cell proliferation activity that can be induced by a ligand capable of activating an I receptor by 10% or more, preferably 25% or more, particularly preferably 35% or more.
  • diseases induced by abnormal activation of IGF-1 receptors include liver cancer, neuroblastoma, rhabdomyomyoma, osteosarcoma, pediatric cancer, apical giant disease, and ovary. Cancer, pancreatic cancer, benign prostatic hypertrophy, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, bladder cancer, gastric cancer, Wilms tumor, metastatic cartinoids and Diarrhea associated with vasoactive intestinal peptide secretory tumors, bipoma, Werner-Morison syndrome, Beckwith-Wedemann syndrome, kidney cancer, renal cell cancer, transitional epithelial cancer, Ewing sarcoma, leukemia, acute lymphoblastic leukemia , Brain tumor, glioblastoma, non-glioblastic brain tumor, medullary tumor, pituitary adenoma, vestibular neurasemia, undifferentiated ectodermal tumor
  • Anti-IGF-1 receptor humanized antibody as a local delivery tool
  • Local delivery of drugs and antibodies to IGF-1 receptor-expressing cells, expression tissues, etc. is possible by utilizing the extremely high binding property and specificity of the anti-IGF-1 receptor antibody of the present invention.
  • the anti-IGF-1 receptor antibody of the present invention it is conceivable to use it in the form of Fab, Fv, scFv, VHH in addition to IgG, but the present invention is not limited thereto. ..
  • the drug can be delivered locally, so that the drug can exert its specific effect at a lower dose, which is a side effect. It also leads to the reduction of.
  • the drug or antibody to which the anti-IGF-1 receptor humanized antibody of the present invention thus prepared is ligated as a delivery tool is, for example, an IGF-1 receptor-expressing cancer cell when the drug is an apoptosis-inducing agent. It can be evaluated using the induction of apoptosis in the strain as an index.
  • the drug or antibody to which the anti-IGF-1 receptor antibody thus selected is ligated is expected as an anti-cancer drug or an ameliorating and therapeutic drug for diseases and pathological conditions associated with abnormal cell proliferation.
  • the anti-IGF-1 receptor antibody of the present invention and an antibody that competitively binds to the IGF-1 receptor are also included in the scope of the present invention.
  • “competitive binding” means a phenomenon in which when a plurality of types of monoclonal antibodies coexist with an antigen, the binding of one antibody to the antigen is inhibited by the binding of the other antibody to the antigen.
  • concentration concentration
  • concentration concentration of a monoclonal antibody
  • the monoclonal antibody that competitively binds to the anti-IGF-1 receptor antibody of the present invention has an IC 50 of usually 1000 nM or less when the antigen-antibody binding is detected using the anti-IGF-1 receptor antibody of the present invention at 10 nM. Above all, it refers to an antibody of 100 nM or less, and further 10 nM or less.
  • a part or all of the antibody to be used is labeled with an enzyme, a fluorescent substance, a luminescent substance, a radioactive isotope, etc., and the measurement is suitable for the physical and / or chemical properties of the labeled substance. It is also possible to make the measurement by detecting it using a method.
  • the antibodies of the invention have a cross-reactivity to the IGF-1 receptor of other vertebrates.
  • the cross-reaction means that the antibody exhibits binding property to an IGF-1 receptor of another animal species different from the animal species (for example, human) of the IGF-1 receptor that undergoes an antigen-antibody reaction.
  • the anti-IGF-1 receptor humanized antibody of the present invention exhibits cross-reactivity with IGF-1 receptors such as guinea pigs, monkeys and rabbits in addition to humans. On the other hand, it does not show cross-reactivity with mouse and rat IGF-1 receptors.
  • cells and animals expressing the IGF-1 receptor with which the antibody of the present invention cross-reacts by genetically engineering the cells and animals using a species of animal that does not cross-react with the antibody of the present invention. It is also possible to make.
  • the anti-IGF-1 receptor humanized antibody of the present invention has an extremely strong binding affinity at a level equal to or higher than that of the mouse parent antibody IGF11-16 (Patent Document 1).
  • the binding affinity can be evaluated, for example, by using the extracellular region of the recombinant IGF-1 receptor as an antigen and performing SPR (surface plasmon resonance) analysis.
  • SPR surface plasmon resonance
  • BIACORE is used to raise the reaction temperature to 40 ° C. to keep the amount of antigen fixed low to analyze the monovalent binding affinity, but the method is not limited to this method. Any analysis method that can quantitatively evaluate the strong binding affinity may be used.
  • the anti-IGF-1 receptor humanized antibody of the present invention has a level equal to or higher than that of the mouse parent antibody IGF11-16 (Patent Document 1) by using IGF-1 receptor signal activation as a primary evaluation system. It was obtained by selecting a humanized antibody.
  • IGF-1 receptor signaling activation was a commercially available PathHunter (TM) IGF1R Functional Assay (DiscoverX Co.).
  • TM PathHunter
  • phosphorylation directly under the IGF-1 receptor signal can be evaluated as enzyme activity, a chemiluminescent substance is used as a substrate, and the signal intensity is measured by the luminescence intensity.
  • an adapter protein SHC1-EnzymeAcceptor (EA) fusion protein having an SH2 domain that binds to the intracellular tyrosine kinase of the IGF-1 receptor and the IGF-1 receptor was forcibly expressed in HEK293 cells.
  • a cell line was used. In this cell line, ligand binding to the IGF-1 receptor causes receptor dimerization, followed by phosphorylation of the receptor to recruit adapter proteins with SH2 domains and accept receptor signals.
  • a transduction complex is formed, facilitating the binding of EA to spatially adjacent tyrosine kinases and reconstitution of active ⁇ -galactosidase.
  • the method for evaluating the IGF-1 receptor signal is not limited to this method, and any system may be used as long as it can directly or indirectly and quantitatively detect IGF-1 receptor tyrosine phosphorylation.
  • Human myoblast proliferation was evaluated as a secondary evaluation system for the humanized antibody of the present invention, and humanized antibodies having the same or higher agonist activity in the same concentration range as the mouse parent antibody IGF11-16 (Patent Document 1) were narrowed down. is. It was confirmed that the humanized antibody of the present invention thus selected did not show a hypoglycemic effect in vivo and had a muscle mass increasing effect. That is, the anti-IGF-1 receptor humanized antibody of the present invention in one embodiment has a growth-inducing activity of vertebrate-derived cells.
  • the "vertebrate-derived cell” is preferably a cell derived from a mammal, a bird, a reptile, an amphibian or a fish, more preferably a cell derived from a mammal or a bird, and even more preferably.
  • cells and animals modified to express certain IGF-1 receptors that are cross-reactive with the antibody of the present invention, or cells derived from the modified animals are also referred to as "cells derived from vertebrates" in the present disclosure. "include.
  • primary cultured cells As cells for investigating the growth-inducing activity of vertebrate-derived cells in vitro, primary cultured cells, cell lines, or transformed cells of those cells can be used.
  • primary cultured cell refers to a cell isolated from an organ or tissue of an organism, which is usually capable of subculture with a certain number of subcultures.
  • Primary cultured cells derived from vertebrates can be obtained from vertebrate organs and tissues by a method such as enzymatic treatment, dispersion by a physical method, or an expand method. It is also possible to use organs and tissues obtained from vertebrates, or fragments thereof.
  • the organs and tissues that prepare these primary cells are preferably endocrine tissues such as thyroid gland, parathyroid gland and adrenal gland, immune tissues such as pits, tonsils, lymph nodes and spleen, respiratory organs such as trachea and lung, and stomach.
  • the primary cultured cells used for investigating the growth-inducing activity of the antibody of the present invention include cells that express the IGF-1 receptor and whose growth is induced by IGF-1 that binds to the IGF-1 receptor. Used.
  • skeletal myoblasts which are primary cultured cells isolated from muscle tissue, are used.
  • primary cultured cells derived from humans or animals it is also possible to obtain and use commercially available cells.
  • Human primary culture cells for example, ATCC (R), available ECACC, Lonza, Gibco (R), Cell Applications, ScienCell research laboratories , the organizations and companies such PromoCell.
  • Examples of the method for investigating the cell proliferation-inducing activity of the antibody of the present invention in vertebrate-derived cells include measurement of cell number measurement, measurement of DNA synthesis amount, measurement of change amount of metabolic enzyme activity, and the like.
  • a method for measuring the number of cells there are a method using a blood cell count board and a method using a cell number measuring device such as a coulter counter, and as a method for measuring the amount of DNA synthesis, [3H] -thymidine or 5-bromo-
  • a method for measuring by taking in 2'-deoxyuridine (BrdU) and a method for observing the amount of change in metabolic enzyme activity there are colorimetric quantitative methods such as MTT method, XTT method and WST method. It can also be carried out by other methods.
  • the cell proliferation-inducing activity was determined by the fact that the cell proliferation was increased when the antibody of the present invention was reacted with the cultured cells used in the test as compared with the case where the antibody was not reacted. can. In this case, it is convenient to evaluate the activity by reacting IGF-1, which is a ligand of the original IGF-1 receptor, under the same conditions as a control of the inducing activity.
  • the EC 50 value is the concentration at which 50% of the maximum growth-inducing activity is exhibited when the antibody of the present invention and IGF-1 are reacted with the cultured cells to be tested at different concentrations. ..
  • cell proliferation-inducing activity of the antibodies of the present invention are The EC 50 values equal to or larger than that of the IGF-1, more preferably antibodies of the present invention
  • the EC 50 value is 1/10 or less of IGF-1, more preferably 1/20 or less, and even more preferably the EC 50 value of the antibody of the present invention is 1/50 or less of IGF-1.
  • the EC 50 value of the antibody of the present invention was preferably 0.5 nM or less, more preferably 0.3 nM or less, still more preferable. Is 0.1 nM or less.
  • the antibody of the present invention is administered to a vertebrate, and changes in the mass, size, cell number, etc. of an organ or tissue in the whole administered individual or in the administered individual are observed.
  • To measure, or to use an animal transplanted with the vertebrate cell measure changes in the mass, size, number of cells, etc. of the transplant containing the transplanted vertebrate cell in the transplanted individual. You can find out at. In the measurement of the whole individual, measurement of body weight, body length, circumference of limbs, etc., body composition measurement by impedance method, creatinine, height coefficient, etc. are used.
  • the target organs, tissues, or grafts in an individual in animals other than humans, the target organs, tissues, or grafts are directly collected, and the mass, size, and number of cells contained are calculated.
  • an X-ray image typified by the DXA (Dual-energy X-ray absorptiometry) method, image analysis by CT and MRI, and isotopes Or a contrast method using a tracer using a fluorescent substance or the like is used.
  • DXA Direct-energy X-ray absorptiometry
  • those skilled in the art can investigate the effect of the antibody of the present invention on the in vivo proliferation-inducing activity of vertebrate-derived cells by using other methods as appropriate.
  • an individual to which the antibody of the present invention was administered and an individual to which an antibody other than the antibody of the present invention or another control substance was administered were used. It can be evaluated by comparing the results of measurement and the like by the method shown above.
  • Example 14 guinea pig blood of the mouse antibody IGF11-16 antibody (Patent Document 1) which was the basis of the design of the antibody of the present invention and the antibodies hIGF13_PS and hIGF25_PS of the present invention. The kinetics were compared, and it was shown that the kinetics in the blood was improved compared with IGF11-16.
  • One of the in vivo effects of the antibody of the present invention is the effect of increasing muscle mass and / or body length.
  • IGF-1 In addition to acting on the proliferation and differentiation of the above-mentioned myoblasts in skeletal muscle, IGF-1 also has an action of thickening muscle fibers, and it is considered that IGF-1 has an effect of increasing muscle mass as such a comprehensive action. There is.
  • the antibody of the present invention also has the effect of increasing the muscle mass of the animal when administered to the animal in the same manner as IGF-1.
  • body composition measurement by the impedance method creatinine, height coefficient, etc. are used in the measurement of the whole individual, such as body weight, body length, and limb circumference.
  • methods such as X-ray imaging images represented by the DXA (Dual-energy X-ray absorptiometry) method, image analysis by CT and MRI, and imaging methods using tracers using isotopes and fluorescent substances are used.
  • Changes in muscle strength, etc. are also indicators.
  • the effect of increasing muscle mass is to compare the increase in muscle mass between an individual who received the antibody of the present invention and an individual who did not receive the antibody, or administer the antibody of the present invention in one individual. It can be evaluated by comparing the muscle mass before and after administration.
  • the effect of increasing muscle mass can be known if a difference in the increase in muscle mass is observed by administration of the antibody of the present invention.
  • IGF-1 is also involved in bone growth and also has the function of increasing body length (height in the case of humans). Therefore, the antibody of the present invention also has an effect of increasing the body length when administered to an animal.
  • the effect of increasing the body length by the antibody of the present invention can be measured by measuring the body weight, body length, perimeter of limbs, etc. of an individual.
  • the antibody of the present invention is characterized in that it does not affect the blood glucose level in vertebrates.
  • IGF-1 is known to cause a blood glucose lowering effect as part of the agonistic action on the IGF-1 receptor.
  • the agonist antibody of the present invention which functions as an anti-IGF-1 receptor agonist antibody, has a blood exposure of 10 times or more the effective dose that induces an increase in muscle mass when administered parenterally to an animal. , Shows the feature that the blood glucose level does not fluctuate.
  • the effect of the antibody of the present invention on vertebrates that does not induce a decrease in blood glucose can be evaluated, for example, in vitro.
  • cells for investigating the feature of vertebrate-derived cells that do not affect the uptake of glucose into cells in vitro primary cultured cells, cell lines, or transformed cells of those cells can be used. It is possible.
  • the effect on intracellular glucose uptake is such that the intracellular glucose uptake when the antibody of the present invention is reacted with the cultured cells used in the test is the same as when the antibody is not reacted. It can be judged by the existence. In this case, it is convenient to evaluate the activity by reacting IGF-1, which is a ligand of the original IGF-1 receptor, under the same conditions as a control of the inducing activity.
  • the antibody of the present invention is administered to the vertebrate and the change in glucose content in an organ or tissue in the administered individual is measured. Can be done. In the measurement of the whole individual, measurement of blood glucose level and the like, hemoglobin A1C and the like using a glycated protein as an index are used. In the measurement of the amount of glucose uptake in an organ or tissue in an individual, in an animal other than human, the target organ or tissue is directly collected, and the glucose content or tracer is calculated.
  • an X-ray image, image analysis by CT or MRI, a contrast method using a tracer using an isotope or a fluorescent substance, or the like is used. ..
  • glucose clamp or the like is also an index.
  • those skilled in the art can investigate the effect of the antibody of the present invention on glucose uptake in vivo by vertebrate-derived cells by using other methods as appropriate.
  • the vertebrate when the antibody of the present invention is administered to a vertebrate, the vertebrate has the same dose, preferably 10 times or more the effective dose for inducing an increase in muscle mass of the vertebrate. It is characterized in that it does not fluctuate the blood glucose level.
  • the animal for investigating the change in the blood glucose level of the antibody of the present invention in a vertebrate is preferably an animal belonging to a mammal, a bird, a reptile, an amphibian or a fish, and more preferably an animal belonging to a mammal or a bird. Even more preferred are humans, monkeys, rabbits, guinea pigs, cows, pigs, sheep, horses, dogs, rats, or mice.
  • animals modified to express certain IGF-1 receptors that are cross-reactive with the antibody of the present invention can also be used as animals for examining changes in blood glucose levels of the antibody of the present invention in vertebrates. included.
  • the colorimetric method, the electrode method, etc. are used as the invasive method, and the enzymes for detection include the glucose oxidase method (GOD method), the glucose dehydrogenase method (GDH method), etc.
  • GOD method glucose oxidase method
  • GDH method glucose dehydrogenase method
  • an invasive method there is an optical measurement method and the like, but other methods can be appropriately selected by those skilled in the art.
  • the blood glucose level is human, the normal range of the fasting blood glucose level is 100 mg / dL to 109 mg / dL.
  • Adverse events due to drug administration to blood glucose level are hypoglycemia when the blood glucose level is lower than the range of 77 mg / dL to 55 mg / dL, and 109 mg / dL to 160 mg / dL. If the value is higher than the range, it is defined as hyperglycemia.
  • No effect of drug administration on blood glucose level means that the blood glucose level after drug administration is in the range of above 55 mg / dL and below 160 mg / dL, more preferably above 77 mg / dL and below 109 mg / dL. Is to be within the range of.
  • the blood glucose level differs depending on the animal to which it is administered, and the range of fluctuation thereof differs, and even in humans, the blood glucose level at the time of administration is not always in the range of the normal value.
  • the blood glucose level of the vertebrate to which the antibody of the present invention was administered is preferably within 30%, more preferably within 20%, and even more preferably 10 as compared with the solvent administration control group. It means that the change is within%.
  • the antibody of the present invention can be obtained by humanizing the mouse monoclonal antibody IGF11-16 (Patent Document 1) against the IGF-1 receptor. Replacing the CDR regions with a human framework by CDR graphing using a monoclonal antibody obtained from a non-human animal species is called humanization (Non-Patent Document 12). Furthermore, by three-dimensional structural analysis, in order to avoid amino acid substitution to reduce immunogenicity (T cell antigenicity) to humans and post-translational modification risk such as deamidation and oxidation while maintaining the three-dimensional structure. By introducing the amino acid substitution of, we will produce a humanized antibody that ensures manufacturability and clinical safety while maintaining activity.
  • VH13_PN SEQ ID NO: 43
  • VH13_PS VH13_PS as heavy chain variable regions.
  • SEQ ID NO: 47 VH23_PN (SEQ ID NO: 49)
  • VH23_PS SEQ ID NO: 53
  • VH25_PN SEQ ID NO: 55
  • VH25_PS SEQ ID NO: 59
  • VL13 SEQ ID NO: 61
  • Examples thereof include humanized antibodies having VL14 (SEQ ID NO: 63), VL22 (SEQ ID NO: 65), VL23 (SEQ ID NO: 67), or VL24 (SEQ ID NO: 69).
  • the light chain variable region more preferably has VL22 (SEQ ID NO: 65), VL23 (SEQ ID NO: 67), or VL24 (SEQ ID NO: 69).
  • the antibodies of the invention are not limited to them.
  • the obtained anti-IGF-1 receptor humanized antibody it is also possible to obtain a nucleic acid molecule having a base sequence encoding the amino acid of the protein of the antibody, and the antibody is genetically engineered using such a nucleic acid molecule. It is also possible to produce.
  • the H chain, L chain, or variable regions thereof in the genetic information of the antibody can be modified to improve the binding property and specificity of the antibody by referring to the information such as the CDR sequences.
  • mammalian cells, insect cells, Escherichia coli, etc. into which a gene encoding the amino acid of the antibody of the antibody to be obtained is introduced are cultured, and the obtained culture supernatant is used by a conventional method.
  • the antibody can be purified and obtained.
  • the specific method is not limited to this, but the following methods are exemplified.
  • a nucleic acid molecule encoding an H chain variable region is bound to a nucleic acid molecule encoding an H chain signal peptide and a nucleic acid molecule encoding an H chain constant region. It is produced by binding a nucleic acid molecule encoding an L-chain signal peptide to a nucleic acid molecule encoding an L-chain variable region and a nucleic acid component encoding an L-chain constant region.
  • H-chain gene and L-chain gene into a vector suitable for expression in a selected host cell, for example, a cloning vector or an expression vector.
  • the H-chain gene and the L-chain gene may be integrated into one vector or may be integrated into different vectors as long as both genes are expressed.
  • the vector into which the H-chain gene and the L-chain gene are integrated is introduced into the host cell.
  • the host cell include eukaryotic cells such as mammalian cells, insect cells, yeast cells, or plant cells, or bacterial cells.
  • a method for introducing a gene into a host cell appropriately select from a chemical method such as calcium phosphate or lipofection method, a physical method such as electroporation method or particle gun method, or a method by infection with a virus or phage. Can be done.
  • Host cells into which the H-chain gene and L-chain gene have been introduced can be used for culture without selection, and recombinant cells into which the gene has been introduced using properties such as drug resistance and nutritional demand can be used. It is also possible to selectively concentrate or use a cloned recombinant cell established from a single host cell into which a gene has been introduced for culture.
  • Host cells into which the H-chain gene and L-chain gene have been introduced are cultured in an appropriate medium and culture conditions.
  • the products of the H chain gene and the L chain gene expressed in the host cell are usually secreted into the medium as an antibody protein, and the produced antibody protein can be obtained by recovering the medium.
  • the host cell may be destroyed and the antibody protein accumulated in the cell may be recovered if necessary, and the antibody protein may be recovered from the periplasmic fraction in the case of prokaryotic cells. You can choose.
  • concentration by salt precipitation method, dialysis, ultrafiltration, etc., exchange of solvent, protein A, protein G, antigen, etc. are immobilized.
  • Affinity chromatography and the like using the same carrier are generally used, but other methods such as ion exchange chromatography, hydrophobic chromatography, mixed mode chromatography and size exclusion chromatography can also be appropriately used for purification. All of the various techniques used in these procedures are well known to those of skill in the art.
  • the gene encoding the heavy chain and / or light chain of the immunoglobulin is genetically modified to introduce a desired trait, or the structure of the variable region or CDR region of the heavy chain and / or light chain of the immunoglobulin is performed. It is possible to produce an antibody chimeric protein, a small molecule antibody, a scaffold antibody, or the like by using information by using a technique known to those skilled in the art. Further, for the purpose of improving the performance of the antibody and avoiding side effects, it is also possible to modify the structure of the constant region of the antibody or to modify the sugar chain portion as appropriate by a technique well known to those skilled in the art. It can be carried out.
  • the antibody of the present invention can be used as a therapeutic agent, a prophylactic agent, or a diagnostic agent for a disease caused by an IGF-1 related condition or an action on an IGF-1 receptor.
  • the therapeutic / preventive drug or the diagnostic drug will be collectively referred to as “drug” or “drug”.
  • the IGF-1 related state or the disease to be treated or prevented with the anti-IGF-1 receptor agonist antibody includes muscular atrophy diseases (for example, disused muscular atrophy, sarcopenia, caquexia and the like). ), Short stature (eg Laron short stature, growth hormone resistant short stature, etc.), liver cirrhosis, liver fibrosis, diabetic nephropathy, chronic renal failure, aging, intrauterine fetal growth retardation (IUGR), nerves Diseases, strokes, spinal cord injuries, cardiovascular protection, diabetes, insulin resistance, metabolic syndrome, nephropathy, osteoporosis, cystic fibrosis, wound healing, muscular tonic dystrophy, AIDS muscle atrophy, fat relapse associated with HIV Distribution Syndrome, Burns, Crohn's Disease, Werner Syndrome, X-Chain Complex Immune Deficiency, Hearing, Neuropathic and Premature Infant Retinopathy, Turner Syndrome, Prader Willy Syndrome, Silver Russell Syndrome, Idiopathic Short Stature, Examples
  • the antibodies of the present invention treat muscular atrophy diseases (eg, disused muscular atrophy, sarcopenia, cahexia, etc.) and / or short stature (eg, laron-type short stature, growth hormone-resistant short stature, etc.). It is preferably used as a drug or a prophylactic agent.
  • the antibody of the present invention is excellent in that it does not cause fluctuations in blood glucose level by administration.
  • Neuroblastoma is a disease to be treated, prevented, or diagnosed with an antibody drug, an antibody drug complex, or a diagnostic drug fused or linked with a part or all of this anti-IGF-1 receptor antibody as a component.
  • the drug containing the antibody of the present invention may be formulated in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier and / or other additives in addition to the antibody of the present invention.
  • Formulations using pharmaceutically acceptable carriers and / or other additives include, for example, University of the Sciences in Philadelphia, “Remington: The Science and Practice of Pharmacy, 20th EDITION”, Lippincot Williams & Wilkins, 2000. It can be carried out by the method described.
  • a solution or lyophilizer prepared by dissolving, suspending, or emulsifying in a sterile aqueous or oily solution.
  • a solvent or solution include distilled water for injection, physiological saline and the like as an aqueous solution, and in addition, an osmotic pressure regulator (for example, D-glucose, D-sorbitol, D-mannitol, sodium chloride).
  • Etc. suitable solubilizing agents such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbitol 80, polyoxyethylene hydrogenated castor oil 50). ) Etc.
  • an oily liquid may be used as the solvent or the dissolution liquid, and examples of the oily liquid include sesame oil, soybean oil and the like, and benzyl benzoate, benzyl alcohol and the like may be used in combination as the dissolution aid.
  • buffers eg, phosphate buffers, acetate buffers
  • painkillers eg, benzalkonium chloride, prokine hydrochloride, etc.
  • stabilizers eg, human serum albumin
  • preservatives eg, ascorbic acid, erythorbic acid and salts thereof, etc.
  • colorants eg, copper chlorophyll, ⁇ -carotene, red No. 2, blue No. 1, etc.
  • preservatives eg, paraoxy.
  • thickeners eg, hydroxypropyl cellulose, carboxymethyl cellulose and their salts, etc.
  • stabilizers eg, human serum albumin, mannitol, sorbitol, etc.
  • odor Additives of agents eg, menthol, citrus fragrance, etc.
  • Another form is a drug for mucosal adaptation.
  • the main purpose is to impart adsorptivity, retention, etc. to the mucous membrane, and as additives, a pressure-sensitive adhesive, a pressure-sensitive adhesive, a thickening agent, a thickening agent, etc. (for example, mutin, canten, etc.)
  • a pressure-sensitive adhesive for example, mutin, canten, etc.
  • Gelatin pectin, carrageenan, sodium alginate, locust bin gum, xanthan gum, tragant gum, gum arabic, chitosan, purulan, waxy starch, scralfate, cellulose, and derivatives thereof (eg, hydroxypropylmethylcellulose, polyglycerin fatty acid ester, acrylic acid (meth).
  • Alkyl acrylate copolymer or a salt thereof, polyglycerin fatty acid ester, etc. may be contained.
  • the form, solvent and additive of the therapeutic agent or preventive agent provided to the living body are limited to these. It is not a product, and any person in the art can select it as appropriate.
  • the drug containing the antibody of the present invention may contain other existing drugs (active ingredients) in addition to the antibody of the present invention. Further, it may be fused or linked with other drugs such as antibody drug conjugates, bispecific antibodies and multispecific antibodies. Further, the drug containing the antibody of the present invention may be combined with other existing drugs to form a kit.
  • anti-IGF-1 receptor agonist antibodies growth hormone or its analog, insulin or its analog, IGF-2 or its analog, anti-myostatin antibody, myostatin antagonist, anti-activin type IIB receptor antibody, activin IIB receptor Included are body antagonists, soluble activin type IIB receptors or analogs thereof, grelin or analogs thereof, follistatin or analogs thereof, beta 2 agonists, and selective androgen receptor modulators.
  • the active ingredient combined with the anti-IGF-1 receptor antibody or the anti-IGF-1 receptor antibody is used together with the antibody drug or the antibody drug complex.
  • the active ingredients contained include corticosteroids, anti-antibodies, ondancetron hydrochloride, granisetron hydrochloride, metoclopramide, donperidone, haloperidol, cyclidine, lorazepam, prochlorperazine, dexamethasone, levomepromazine, tropicetron, and cancer.
  • Vaccines GM-CSF inhibitors, GM-CSF DNA vaccines, cell-based vaccines, dendritic cell vaccines, recombinant virus vaccines, heat shock protein (HSP) vaccines, allogeneic tumor vaccines, autologous tumor vaccines, analgesics, ibprofen, Naproxene, choline magnesium trisalicylate, oxycodon hydrochloride, anti-angiogenic drug, anti-thrombotic drug, anti-PD-1 antibody, nibolumab, pembrolizumab, anti-PD-L1 antibody, atezolizumab, anti-CTLA4 antibody, ipilimumab, anti-CD20 antibody, rituximab, anti HER2 antibody, trussumab, anti-CCR4 antibody, mogamurizumab, anti-VEGF antibody, bevasizumab, anti-VEGF receptor antibody, soluble VEGF receptor fragment, anti-TWEAK antibody, anti-TWEAK receptor antibody,
  • the agent of the present invention can be administered parenterally for the purpose of improving symptoms.
  • parenteral administration for example, it can be a nasal agent, and a liquid agent, a turbid agent, a solid preparation and the like can be selected.
  • Another form of parenteral administration may be an injection, and the injection may be a subcutaneous injection, an intravenous injection, a drip injection, an intramuscular injection, an intraventricular injection, an intraperitoneal injection, or the like. Can be selected.
  • other preparations used for parenteral administration include suppositories, sublingual agents, transdermal agents, transmucosal agents other than nasal agents, and the like. Further, it can be locally administered intravascularly by containing or applying it to a stent or an intravascular embolic agent.
  • the dose of the therapeutic or prophylactic agent in the present invention varies depending on the age, sex, body weight, symptom, therapeutic effect, administration method, treatment time, type of active ingredient contained in the pharmaceutical composition, and the like of the patient.
  • the main ingredient is in the range of 0.1 mg to 1 g, preferably 0.5 mg to 100 mg, once every 1 to 4 weeks, or 1 to 1 to 2 months.
  • a dose and the number of doses may be sufficient, and a dose and the number of doses exceeding the above range may be required. be.
  • Example 1 Design of humanized antibody of mouse antibody IGF11-16: -Selection of human framework IGF11-16, a mouse monoclonal antibody against the IGF-1 receptor, obtained by the hybridoma method of Kohler et al. (Nature, (1975), Vol.256, pp.495-497) (Patent Document 1). ) Complementarity determining region (CDR) amino acids in the heavy chain variable region (VH) and light chain variable region (VL) were transplanted into a template human antibody.
  • CDR complementarity determining region
  • an amino acid sequence having high homology to the VH and VL amino acid sequences (SEQ ID NO: 39 and SEQ ID NO: 41, respectively) of the mouse antibody IGF11-16 (mouse parent antibody) is used. From the germline of the human antibody having, VH-1-46 (SEQ ID NO: 95) and VH-1-e (SEQ ID NO: 96) as the heavy chain sequence, and JH4 (SEQ ID NO:) as the heavy chain J-segment.
  • VK1-L5 SEQ ID NO: 98
  • VK1-A20 SEQ ID NO: 99
  • JK2 SEQ ID NO: 100
  • Humanized antibodies were prepared by transplanting the necessary amino acid sequences from VH and VL of mouse antibodies IGF11-16 into the FRs of the above template human antibodies VH and VL.
  • VH the CDR amino acid sequence of the template human antibody VH described above and several FR amino acids were replaced with the corresponding amino acid sequences in the VH of the mouse antibody IGF11-16 to humanize the mouse antibody IGF11-16.
  • the amino acid sequence of the VH was designed, and the base sequence of the DNA encoding those amino acids was further designed.
  • VL the CDR amino acid sequence of the template human antibody VL and several FR amino acids were replaced with the amino acid sequences in the VL of the mouse antibody IGF11-16, and the amino acid sequence of the humanized mouse antibody IGF11-16 was designed. Then, the base sequence of the DNA encoding those amino acids was designed.
  • the composition of the heavy chain and light chain of the designed humanized antibody is shown in Table 2 below.
  • a humanized heavy chain composed by connecting a designed humanized antibody heavy chain variable region and a heavy chain constant region, and a designed humanized antibody light chain variable region.
  • VL22 is a light chain variable region and a human ⁇ chain constant region is linked thereto
  • VH13_PS is a heavy chain variable region
  • IgG4S228P heavy chain constant is used therein.
  • a humanized antibody constructed in combination with a heavy chain in which regions are linked.
  • Example 2 Preparation of humanized antibody: DNA encoding the heavy chain variable region of the designed humanized antibody and the heavy chain constant region of the human IgG4S228P mutant, which is a mutant stabilized by the human IgG4 subclass, were synthesized and integrated into the pcDNA3.4 system expression vector. Then, a plasmid expressing the humanized antibody heavy chain was used.
  • DNA encoding the humanized antibody light chain region linked to the ⁇ chain constant region was synthesized and incorporated into a pcDNA3.4 system expression vector to express the humanized antibody light chain. It was made into a plasmid to be used.
  • Various antibodies were expressed by mixing these humanized antibody heavy chain expression plasmids and humanized antibody light chain expression plasmids and introducing them into cells using ExpiCHO (registered trademark) Expression System (Thermo Fisher Scientific). ..
  • ExpiCHO registered trademark
  • a humanized antibody expressed by combining a heavy chain expression plasmid incorporating FW1_VH1 and a light chain expression plasmid incorporating FW1_VL1 was incorporated into a FW1_var1 antibody, and a heavy chain expression plasmid incorporating FW2_VH1 and FW2_VL2 were incorporated.
  • the humanized antibody expressed in combination with the light chain expression plasmid was named FW2_var2 antibody, respectively.
  • FW1_var9, FW1_var10, and FW1_var14 were similarly implemented and named.
  • the humanized antibody was obtained by affinity purification of the culture supernatant of the cells into which the humanized antibody heavy chain and the humanized antibody light chain expression plasmid were introduced using a protein A column. Subsequent preparation of humanized antibody was performed according to the above method.
  • PathHunter by (R) IGF-1 agonism To detect activation activity IGF-1 receptor designed humanized antibody, PathHunter (TM) IGF1R Functional using Assay (DiscoverX), activation of IGF-1 receptor signaling by the following steps was measured.
  • Cells expressing the IGF-1 receptor were placed in a poly-D-lysine-coated or collagen-I-coated 96-well plate (Black / clear or White / clear) at 90 ⁇ L / well (2 ⁇ 10 4 cells / well). Or seeded at 5 ⁇ 10 3 cells / well) and incubated at 37 ° C. and 5% CO 2 conditions. The next day, each concentration of drug was added at 10 ⁇ L / well and incubated at 37 ° C. and 5% CO 2 conditions.
  • the next day 30 ⁇ L of the culture supernatant was taken, 15 ⁇ L of the substrate solution was added, the reaction was carried out for 60 minutes, and the luminescence signal (RLU) was measured with a luminometer (Tristar, Berthold).
  • the relative value of the humanized antibody was calculated with the value obtained by subtracting the value at the time of adding the antibody 0.1 nM from the fluorescence intensity due to the addition of the 12.5 nM antibody as the activation intensity and the value of the mouse parent antibody IGF11-16 as 1. ..
  • A61, Q62, Q65, and G66 of the heavy chain CDR2 region which is a sequence portion different from that of the mouse, are replaced with N61, E62, K65, and S66, respectively, in mice.
  • Amino acid-substituted humanized antibodies (FW1_var10_NEKS, FW1_var14_NEKS) made the same as the parent antibody were prepared, and the mouse parent antibodies IGF11-16 and FW1_var1 were used as comparison targets to compare the IGF-1 receptor activation ability in the same manner as described above. did.
  • humanized antibodies in which FR1, FR2, and FR3 were replaced with mouse FRs were prepared.
  • the mouse FR substitutes of the prepared humanized antibody are shown in Table 5.
  • the IGF-1 receptor signaling activation of these substituents were evaluated by a system of previously described PathHunter (registered trademark).
  • PathHunter registered trademark
  • human chimeric IGF11-16 antibody (Chimera) which is a fusion of the variable region of mouse parent antibody IGF11-16 and the constant region of human IgG4 (S228P)
  • S228P human chimeric IGF11-16 antibody
  • S228P constant region of human IgG4
  • the signal intensities at an antibody concentration of 16.7 nM were compared in the same manner as described above.
  • the results are shown in Table 5.
  • examples of nucleic acid base sequences corresponding to the amino acid sequences of SEQ ID NOs: 29, 31, 33, 35, and 37 are shown in SEQ ID NOs: 30, 32, 34, 36, and 38, respectively
  • the substitute having the same signal intensity as human chimeric IGF11-16 (value within ⁇ 20%) is FW1_var9_mFR-H1, and the mouse heavy chain FR1 is important for maintaining the activity of the humanized antibody. I found out.
  • proline at position 25 in the heavy chain FR1 region is important for maintaining activity.
  • Subsequent heavy chains of humanized antibodies were all P (proline, Pro) substituted at position 25.
  • Example 4 Identification of CDR region amino acids important for maintaining activity by alanine substitution: For the purpose of identifying the amino acids required to retain activity in the CDR regions, each amino acid was replaced with alanine in the mouse parent antibody IGF11-16, and the signal activation ability of the substituted product was determined by EC 50 value and E max value. The binding activity was evaluated by the antigen ELISA. EC 50 value: within 2 times and E max value: within ⁇ 20% were defined as equivalent activity as compared with the mouse parent antibody IGF11-16.
  • IGF-1 receptor signaling activation ability was evaluated by a system of PathHunter (registered trademark) described in Example 3.
  • the EC 50 value and E max were calculated using the analysis software GraphPad Prism.
  • the binding activity was measured by the antigen ELISA in which the extracellular region of the recombinant IGF-1 receptor was immobilized as an antigen.
  • human recombinant IGF-1R manufactured by R & D
  • PBS phosphate buffered saline
  • the prepared human recombinant IGF-1R solution was added to the solidified plate at 50 ⁇ L / well. The reaction was carried out overnight at 4 ° C.
  • the reaction was carried out at room temperature for 45 minutes, washed three times with a washing solution, and then a substrate (pNPP (paranitrophenyl phosphate)) was added to initiate the reaction. After reacting at room temperature for 1 hour, the absorbances at 405 and 550 nm were measured, and the value of the difference between the absorbances of 405 nm and the absorbance of 550 nm was calculated. This value was used as the binding activity for analysis.
  • pNPP paranitrophenyl phosphate
  • Tables 7 and 8 show the prepared IGF11-16 antibody CDR substitutes and the measurement results of signal activation and binding activity. From the obtained results, in the CDR region, the amino acid moiety whose binding activity was reduced to about 10 to 20% by substitution with alanine, that is, tryptophan at position 32 of CDR-L1, tryptophan at position 33 of CDR-H1, and CDR- It has been identified that five amino acids, glutamic acid at position 50 of H2, asparagine at position 52 of CDR-H2 and arginine at position 102 of CDR-H3, are crucial for retention of activity.
  • histidine at position 35 of CDR-H1, serine at position 54 of CDR-H2, asparagine at position 55 of CDR-H2, serine at position 56 of CDR-H2, asparagine at position 59 of CDR-H2, and CDR Since the activity of phenylalanine at position 64 of -H2 decreased due to Ala substitution, it is considered that it contributes to the retention of activity. On the other hand, of the 54 amino acid residues in the alanine-substituted CDR regions, 44 showed 80% or more of the binding activity even after the alanine substitution.
  • Example 5 Design of humanized heavy chain variable region: Since it was found from the results of Example 3 that proline at the 25th position of the heavy chain is important for maintaining the activity, the design was performed with the 25th position of the heavy chain as P and the humanized heavy chain variable regions FW1_VH1 and FW2_VH1 as the basic skeleton. .. Since the amino acid substitution product was examined based on FR1 of FW1_VH1, the amino acid substitution of S16A was performed in order to make the FR1 portion of FW2-VH1 the same as FW1_FR1. The amino acid substitutions introduced for immunogenicity reduction was performed on the basis of Epibase (R) (Lonza, Inc.) immunogenic score analysis results. The designed heavy chain variable region list is shown in Table 9 below.
  • Example 6 Design of humanized light chain variable region: The humanized light chain variable region was designed with FW1_VL1 and FW2_VL2 as the basic skeletons. Amino acid substitutions introduced for immunogenicity score reduction was carried out based on the result of the analysis of Epibase (R) (Lonza, Inc.). A list of designed light chain variable regions is shown in the table.
  • Example 7 Humanized antibody design with deamidation risk amino acid substitution product: If deamidation occurs during the production of humanized antibodies, quality control becomes difficult. Therefore, it is necessary to replace the amino acid at risk of deamidation with another amino acid that does not affect the activity in advance. Sequences generally at risk of deamidation include NG, NT, NS, and NN. The NS sequence is present in the CDR-H2 region of the heavy chain of this humanized antibody. Therefore, amino acid substitution was performed in consideration of the risk that asparagine (N) at position 55 is deamidated and converted to aspartic acid (D). A list of replaced heavy chains is shown in Table 11. Further, examples of the nucleic acid base sequences corresponding to the amino acid sequences of SEQ ID NOs: 45, 51, and 57 are shown in SEQ ID NOs: 46, 52, and 58, respectively.
  • Example 8 Selection of humanized antibody by IGF-1 receptor signal activating action: Humanized antibodies were evaluated based on the activating effect of the IGF-1 receptor, and humanized antibodies having the same activity as the mouse parent antibody IGF11-16 were selected.
  • IGF-1 receptor of the anti IGF-1 receptor agonist antibodies was determined the activation of IGF-1 receptor signaling using PathHunter (TM) IGF1R Functional Assay (DiscoverX) ..
  • Cells expressing the IGF-1 receptor were placed in a poly-D-lysine-coated or collagen-I-coated 96-well plate (Black / clear or White / clear) at 90 ⁇ L / well (2 ⁇ 10 4 cells / well). Or seeded at 5 ⁇ 10 3 cells / well) and incubated at 37 ° C. and 5% CO 2 conditions. The next day, each concentration of drug was added at 10 ⁇ L / well and incubated at 37 ° C. and 5% CO 2 conditions. The next day, 30 ⁇ L of the culture supernatant was taken, 15 ⁇ L of the substrate solution was added, the reaction was carried out for 60 minutes, and the luminescence signal (RLU) was measured with a luminometer (Tristar, Berthold).
  • RLU luminescence signal
  • the humanized antibody confirmed to have the same activity as the mouse parent antibody IGF11-16 (EC 50 value: within 2 times, E max value: within ⁇ 20% compared with the mouse parent antibody IGF11-16) is shown in the table. Shown in 12.
  • Example 9 Selection of humanized antibody by human myoblast proliferation activity: Humanized antibodies were evaluated based on human myoblast proliferation activity, and humanized antibodies having activity equivalent to that of mouse parent antibody IGF11-16 were selected.
  • the 96-well plate incubated for 4 days was allowed to stand at room temperature for 30 minutes or more after removing the supernatant so that the culture solution became 50 ⁇ L / well.
  • the emission signal was measured with a luminometer (Tristar, Berthold).
  • Table 13 shows the humanized antibodies confirmed to have the same activity as the mouse parent antibody IGF11-16 (EC 50 value: 10 times or less, E max : 90% or more as compared with the mouse parent antibody IGF11-16). show.
  • the graphs of the measurement results are shown in FIGS. 1A to 1F.
  • Example 10 Evaluation of immunogenicity: In order to analyze the immunogenicity of the humanized antibody, using Epibase (TM) in Silico of Lonza, it was calculated immunogenicity score. Lonza's Epibase (TM) in Silico platform, using structural characteristics of the HLA class II receptors previously with binding affinity of the peptide and HLA class II receptors 10-mer which is determined experimentally, the antibody It is an immunogenicity prediction method that predicts potential peptide / HLA binding, which is a condition necessary for activation of T cells present in the contained amino acid sequence, and calculates it as an immunogenicity score.
  • TM Epibase
  • siico platform using structural characteristics of the HLA class II receptors previously with binding affinity of the peptide and HLA class II receptors 10-mer which is determined experimentally, the antibody It is an immunogenicity prediction method that predicts potential peptide / HLA binding, which is a condition necessary for activation of T cells present in the contained amino acid sequence, and calculates it as an immunogenicity score.
  • HLA class II allotypes 43 types of DRB1, 8 types of DRB3 / 4/5, 22 types of DQ, 12 types of DP
  • the immunogenicity score was scored by taking into account the frequency of appearance in addition to the binding affinity of allotypes.
  • Example 11 Evaluation of binding activity to mammalian IGF-1 receptor: Anti-IGF against IGF-1 receptors in humans (SEQ ID NO: 71), crab monkeys (SEQ ID NO: 73), rabbits (SEQ ID NO: 75), guinea pigs (SEQ ID NO: 77), rats (SEQ ID NO: 79) and mice (SEQ ID NO: 81). In order to examine the binding activity of the -1 receptor agonist antibody, Cell-based ELISA was performed using cells expressing various IGF-1 receptors.
  • HEK293T cells After lipofection was added to HEK293T cells were incubated over night in 96 well plates at 4 ⁇ 10 4 cells / well (poly -D- lysine-coated), 10% buffered formalin (Mildform (registered trademark) 10 nm, Wako) The one that was fixed using and blocked with a phosphate buffer solution containing 3% BSA was used for ELISA.
  • ELISA was prepared by adding 100 ⁇ L of each humanized antibody solution prepared to 5 nM with 1% BSA / 1% FBS / PBS to each well and reacting at 37 ° C. for about 1 hour. It was washed 3 times with a washing solution. 100 ⁇ L of anti-human IgG antibody HRP conjugated solution prepared at each concentration with 1% BSA / 1% FBS / PBS was added to each well, reacted at 37 ° C. for about 1 hour, and washed 3 times with a washing solution. .. 100 ⁇ L of substrate (TMB) was added to each well to initiate the reaction.
  • TMB substrate
  • FIG. 2 shows the results of reactivity of humans, guinea pigs, cynomolgus monkeys, and rabbits with each IGF-R.
  • the humanized antibodies hIGF13_PS and hIGF25_PS increased the binding activity of human, guinea pig, cynomolgus monkey, and rabbit IGF-1 receptors by about twice as much as those of Mock cells, and the human mouse chimera. It was as reactive as antibody IGF11-16.
  • the binding activity to cells expressing the IGF-1 receptor in rats and mice was similar to that of Mock cells.
  • Binding affinity for IGF-1 receptor by surface plasmon resonance method To examine the binding properties (binding rate and dissociation rate) of the drug to the IGF-1 receptor, it was measured by the surface plasmon resonance (SPR) method.
  • the measurement system used was the BIACORE T200 system.
  • Anti-histidine-tagged monoclonal antibody was applied to all flow cells of the sensor chip CM3 (BR-1005-36, GE) with AmineCouplingKit (BR-1000-50, GE) and HisCaptureKit (28-9950-56, GE). , Approximately 3000 RU fixed and used.
  • HBS-EP + (BR-1006-69, GE) was used as the running buffer.
  • IGF-1R-His a recombinant human IGF-1 receptor histidine tag (305-GR-050, R & D SYSTEMS, hereinafter IGF-1R-His) was captured and used in the measurement system.
  • Analite used each concentration of drug.
  • a flow cell that did not capture IGF-1R-His was used as a ligand negative control.
  • PBS PBS pH 7.4 (1x), # 10010049, Gibco
  • the measurement temperature of the measurement system was set to 40 ° C.
  • the anti-histidine-tagged monoclonal antibody of Frocel (2 and 4) was reacted with IGF-1R-His ( ⁇ 2 ⁇ 10 -8 M) so as to be 100 RU or less.
  • IGF-1R-His ⁇ 2 ⁇ 10 -8 M
  • 10 nM purified mice IgG2a, ⁇ , isotype Ctrl, Clone: MG2a-53 (401502, BioLegend, hereinafter ctrl IgG2a) were reacted for 1 minute, and HBS-EP + was allowed to flow for 10 minutes or longer.
  • Analite was serially diluted with HBS-EP + (0.5-8 ⁇ 10 -10 M) and reacted with all flow cells.
  • Example 13 In vivo hypoglycemic action (hypoglycemic action in guinea pigs): In order to confirm the presence or absence of the in vivo hypoglycemic effect of the anti-IGF-1 receptor agonist antibody, hIGF13_PS and hIGF25_PS were administered once to guinea pigs, and the blood glucose level was measured over time to determine the hypoglycemic effect. The presence or absence was examined. The hypoglycemic action is an action of lowering the blood glucose level to 50 mg / dL or less or causing hypoglycemic symptoms.
  • the guinea pig was fasted for 12 hours, and each humanized antibody: hIGF13_PS and hIGF25_PS was intravenously administered at 10 mg / kg in a single dose.
  • Guinea pigs were fasted until 24 hours after dosing.
  • Wakeful guinea pigs were collected before administration (0 hours), 1, 2, 4, 8, 24, 48, 72, and 144 hours after administration, and used with a glutest sensor (Sanwa Chemical Laboratory). The blood glucose level was measured. The results are shown in FIGS. 3A and 3B.
  • each humanized antibody does not have a remarkable hypoglycemic effect like IGF-1, and does not affect the blood glucose level. Therefore, as a drug for overcoming hypoglycemia, which is a side effect of IGF-1. The possibility of was shown.
  • Example 14 Blood kinetics of humanized antibody in guinea pigs: The guinea pigs were fasted for 12 hours and a single intravenous dose of 1 and 10 mg / kg of humanized antibodies hIGF13_PS and hIGF25_PS or IGF11-16 (mouse parent antibody) was administered. Guinea pigs were fasted until 24 hours after dosing and re-fed 24 hours later. Awake guinea pigs were collected before administration (0 hours), 2, 4, 8, 24, 48, 72, 96, 120, and 144 hours after administration, and the plasma humanized antibody concentration was measured by ELISA. ..
  • the antibody having a known concentration administered to the guinea pig was serially diluted with guinea pig plasma to obtain a standard substance.
  • the standard substance and plasma were diluted 10 to 1000 times and measured.
  • a 0.5 ⁇ g / mL PBS solution of recombinant IGF-1R was added to a 96-well plate (MaxiSorp (NUNC)) and fixed at 4 ° C. overnight. Further blocking was performed with 3% BSA / PBS to prepare a recombinant IGF-1R fixation plate.
  • guinea pig plasma to which no antibody was administered was used, and the administered antibody was serially diluted to obtain a standard substance. Plasma and standard material were diluted 10-fold and added to recombinant IGF-1R fixation plates at 50 ⁇ L / well.
  • the reaction was carried out at room temperature for 1 hour and 30 minutes, and then a washing operation was carried out with PBS-T (PBS, 0.025% Tween 20). Then, a solution of alkaline phosphatase-labeled anti-human IgG (H + L) polyclonal antibody (Southern Biotechnology Associates, Cat # 2087-04) diluted 2000 times with 3% BSA / PBS was added at 50 ⁇ L / well, and the mixture was added at room temperature for 1 hour. It was reacted.
  • PBS-T PBS, 0.025% Tween 20
  • H + L alkaline phosphatase-labeled anti-human IgG polyclonal antibody
  • the results are shown in Fig. 4.
  • the plasma humanized antibody concentration increased depending on the dose, and even in the low dose group, the plasma humanized antibody concentration up to 144 hours after administration was maintained at about 50% or more as compared with 24 hours after administration.
  • the blood kinetics of the humanized antibody was shown to be superior in sustainability as compared with the mouse parent antibody IGF11-16.
  • Example 15 Muscle mass increasing action of humanized antibody in normal guinea pigs: A single dose of hIGF13_PS was intravenously administered to normal guinea pigs, and the muscle mass was measured 2 weeks later. did.
  • hIGF13_PS and mouse parent antibody IGF11-16 was intravenously administered to normal guinea pigs at 0.1 mg / kg.
  • human IGF-I (mecasermin) was subcutaneously implanted using an osmotic pump (Arzet) and continuously administered at 1 mg / kg / day.
  • osmotic pump As a control, only the solvent was administered intravenously.
  • the guinea pig was exsanguinated under anesthesia, the extensor digitorum longus muscle was removed, and the muscle mass was measured.
  • the results are shown in Fig. 5.
  • the drug efficacy was similar to that of the group in which human IGF-I was continuously administered at 1 mg / kg / day and the drug efficacy intensity in which the mouse parent antibody IGF11-16 was intravenously administered.
  • hIGF13_PS can be expected to have the same efficacy as the continuous administration of human IGF-1 for 2 weeks by a single dose.
  • Example 16 Growth plate cartilage elongation action in pituitary excised guinea pigs of humanized antibody: The epiphyseal line thickness of the proximal tibia was evaluated using a guinea pig pituitary excision (HPX) model as an evaluation of the growth plate cartilage proliferation effect of hIGF13_PS.
  • HPX guinea pig pituitary gland removal
  • HIGF13_PS was subcutaneously administered once to pituitary excised guinea pigs at 0.3 mg / kg and 1.0 mg / kg, and the right lower limb was collected 2 weeks later.
  • a tissue specimen of the growth plate cartilage in the proximal part of the tibia was prepared, and the thickness of the growth plate cartilage (epiphyseal plate thickness) was measured by Truisin Blue.
  • the IGF-1 (mecasermin) preparation was continuously subcutaneously administered at 1 mg / kg / day with an osmotic pump, and the GH (somatropin) preparation was subcutaneously administered at 1 mg / kg / day once daily.
  • the hIGF13_PS antibody has an effect of recovering the obstruction of the epiphyseal line due to the decrease in IGF-1 concentration due to the pituitary excision (HPX) treatment by activating the signal mediated by IGF-1R.
  • HPX pituitary excision
  • Example 17 Hypoglycemic effect of humanized antibody in cynomolgus monkey: In order to confirm the presence or absence of the hypoglycemic effect of the anti-IGF-1 receptor agonist antibody in cynomolgus monkeys, hIGF13_PS was administered once to cynomolgus monkeys, and the blood glucose level was measured sequentially to measure IGF-1 (1 mg / kg). ) was compared with the hypoglycemic effect of a single dose.
  • the hypoglycemic action is an action that lowers the blood glucose to less than 50% or causes hypoglycemic symptoms as compared with the solvent-administered group.
  • Humanized antibody was administered to cynomolgus monkeys at 10 mg / kg once intravenously or subcutaneously. Blood was collected before (0 hours), 5 minutes, 30 minutes, 1, 2, 4, 8, and 24 hours after administration, and the blood glucose level was measured using Medisafe Fit (Terumo Corporation).
  • Example 18 Blood kinetics of humanized antibody in cynomolgus monkeys: The humanized antibody hIGF13_PS was administered once intravenously or subcutaneously to cynomolgus monkeys at 1 and 10 mg / kg. Blood was collected before administration (0 hours), 2, 4, 8, 24, 48, 72, and 144 hours after administration, and the concentration of humanized antibody in plasma was measured by ELISA.
  • the calibration curve for quantification was a standard substance obtained by serially diluting an antibody of known concentration administered to monkeys with monkey plasma. The standard substance and plasma were diluted 10 to 1000 times and measured.
  • a 0.5 ⁇ g / mL PBS solution of recombinant IGF-1R was added to a 96-well plate (MaxiSorp (NUNC)) and fixed at 4 ° C. overnight. Further blocking was performed with 3% BSA / PBS to prepare a recombinant IGF-1R fixation plate.
  • monkey plasma to which no antibody was administered was used, and the administered antibody was serially diluted to obtain a standard substance. Plasma and standard material were diluted 10-fold and added to recombinant IGF-1R fixation plates at 50 ⁇ L / well. The reaction was carried out at room temperature for 1 hour and 30 minutes, and then a washing operation was carried out with PBS-T (PBS, 0.025% Tween 20).
  • the absorbance at 405 nm and 550 nm was measured with a plate reader, and the value of the difference between the absorbances of 405 nm and 550 nm was calculated.
  • a calibration curve was drawn with the antibody concentration as the standard substance, and the antibody concentration in plasma was calculated.
  • Muscle mass increasing action of humanized antibody in cynomolgus monkey Two cynomolgus monkeys were intravenously administered with hIGF13_PS at 1 mg / kg. Muscle mass was measured by the DXA (Dual Energy X-ray Absorptiomyomy) method before administration and 3 to 4 weeks after administration.
  • DXA Direct Energy X-ray Absorptiomyomy
  • ketamine hydrochloride (Arevipharma GmbH, 50 mg / mL, 0.2 mL / kg) and an aqueous solution of medetomidine hydrochloride (domitol, Orion Corporation, 1 mg / mL, 0.08 mL / kg) were used.
  • General anesthesia is performed by intramuscular administration (buttock), and using a dual energy X-ray absorption measuring device (Discovery-A, HOLOGIC), fat mass (Fat Mass, g) and non-fat mass (Lean Body Mass, g) are used.
  • Bone mass Bone Mineral Content (BMC), g) was measured, and non-fat mass was analyzed as muscle mass.
  • BMC bone mineral content
  • Lean + BMC g
  • muscle mass g was calculated, and compared with the muscle mass before administration.
  • hIGF13_PS was subcutaneously administered to 2 cynomolgus monkeys at 10 mg / kg. Muscle mass was measured by the DXA (Dual Energy X-ray Absorptiomyomy) method before administration and 3 to 4 weeks after administration.
  • DXA Direct Energy X-ray Absorptiomyomy
  • ketamine hydrochloride (Arevipharma GmbH, 50 mg / mL, 0.2 mL / kg) and an aqueous solution of medetomidine hydrochloride (domitol, Orion Corporation, 1 mg / mL, 0.08 mL / kg) were used.
  • General anesthesia is performed by intramuscular administration (buttock), and using a dual energy X-ray absorption measuring device (Discovery-A, HOLOGIC), fat mass (Fat Mass, g) and non-fat mass (Lean Body Mass, g) are used.
  • Bone mass Bone Mineral Content (BMC), g) was measured, and non-fat mass was analyzed as muscle mass.
  • BMC bone mineral content
  • Lean + BMC g
  • Example 20 Effect of IGF11-16 on HepG2 cell proliferation The concentration-dependent effect of the mouse parent antibody IGF11-16 on HepG2 cell proliferation was evaluated by a cell survival assay.
  • HepG2 cell lines were suspended in DMEM (Gibco, 11995) with 1% FBS and seeded in collagen-I coated 96-well plates (Corning, 356650) at 0.25 ⁇ 10 4 cells / well. The next day, BSA / PBS, IGF-1 (mecasermin), control mouse IgG1 antibody (mIgG1), IGF11-16 antibody, and Sixtumumab (IGF-1 receptor antagonist antibody) were diluted from 50 nM at a ratio of 1/10. And added.
  • the mouse parent antibody IGF11-16 showed an inhibitory effect on HepG2 cell proliferation. From this result, it was considered that IGF11-16 could exhibit an antagonistic effect on at least certain cancer cells.
  • Example 21 Effect of IGF11-16 on the proliferative activity of human breast cancer cell line (MCF7) by IGF-1 to evaluate the effect of IGF11-16 on the proliferative activity of human breast cancer cell line (MCF7) by IGF-1, 50 nM The concentration-dependent proliferative activity of hIGF-1 (mechaselmin) under the condition of coexistence of IGF11-16 was measured by the amount of intracellular ATP 2 days after the addition.
  • Human breast cancer cell lines were cultured in DMEM / F12 medium containing 10% FBS. The next day, using DMEM / F12 medium containing 10% FBS, seeded in 96-well plates (Collagen-type I coated) at 0.1 mL / well (2.5 ⁇ 10 3 cells / well) at 37 ° C. Incubated under 5% CO 2 conditions. The day after cell seeding, the medium was replaced with DMEM / F12 medium containing 1% BSA, and the cells were incubated at 37 ° C. and 5% CO 2 for about 8 hours.
  • the mouse parent antibody IGF11-16 has an inhibitory effect on reducing the maximum activity of the human breast cancer cell line (MCF7) by IGF-1. From this result, it was considered that IGF11-16 has the action of an allosteric antagonist.
  • the present invention provides an anti-IGF-1 receptor humanized antibody that specifically binds to the IGF-1 receptor in vertebrates and does not lower blood glucose levels while increasing muscle mass via the IGF-1 receptor. It can be used for the treatment, prevention, or diagnosis of diseases associated with the IGF-1 receptor. The present invention can also be used for the treatment, prevention, or diagnosis of diseases associated with cell overgrowth or activation by suppressing the excessive signal of the IGF-1 receptor, and is industrially available. The value is extremely high.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Neurology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2021/020890 2020-06-02 2021-06-01 抗ｉｇｆ－１受容体ヒト化抗体 Ceased WO2021246413A1 (ja)

Priority Applications (12)

Application Number Priority Date Filing Date Title
KR1020227042511A KR20230006006A (ko) 2020-06-02 2021-06-01 항 igf-1 수용체 인간화 항체
JP2022528850A JPWO2021246413A1 (https=) 2020-06-02 2021-06-01
IL296519A IL296519A (en) 2020-06-02 2021-06-01 Anti-igf-1 receptor humanized antibody
US18/007,606 US20250263489A1 (en) 2020-06-02 2021-06-01 Anti-igf-1 receptor humanized antibody
MX2022015133A MX2022015133A (es) 2020-06-02 2021-06-01 Anticuerpo humanizado anti-receptor de igf-1.
BR112022022030A BR112022022030A2 (pt) 2020-06-02 2021-06-01 Anticorpo humanizado do receptor anti-igf-1, molécula de ácido nucleico, vetor de clonagem ou vetor de expressão, célula recombinante, processo para produzir um anticorpo humanizado do receptor anti-igf-1, e, composição farmacêutica
CN202180039577.1A CN115605594A (zh) 2020-06-02 2021-06-01 抗igf-1受体人源化抗体
EP21817253.4A EP4159860A4 (en) 2020-06-02 2021-06-01 HUMANIZED ANTI-IGF-1 RECEPTOR ANTIBODY
CA3176119A CA3176119A1 (en) 2020-06-02 2021-06-01 Anti-igf-1 receptor humanized antibody
AU2021282629A AU2021282629A1 (en) 2020-06-02 2021-06-01 Anti-IGF-1 receptor humanized antibody
CONC2022/0017026A CO2022017026A2 (es) 2020-06-02 2022-11-28 Anticuerpo humanizado anti– receptor de igf–1
JP2024028737A JP2024054410A (ja) 2020-06-02 2024-02-28 抗ｉｇｆ－１受容体ヒト化抗体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020-096344 2020-06-02
JP2020096344 2020-06-02

Publications (1)

Publication Number Publication Date
WO2021246413A1 true WO2021246413A1 (ja) 2021-12-09

Family

ID=78831168

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/020890 Ceased WO2021246413A1 (ja) 2020-06-02 2021-06-01 抗ｉｇｆ－１受容体ヒト化抗体

Country Status (15)

Country Link
US (1) US20250263489A1 (https=)
EP (1) EP4159860A4 (https=)
JP (2) JPWO2021246413A1 (https=)
KR (1) KR20230006006A (https=)
CN (1) CN115605594A (https=)
AR (1) AR122499A1 (https=)
AU (1) AU2021282629A1 (https=)
BR (1) BR112022022030A2 (https=)
CA (1) CA3176119A1 (https=)
CL (1) CL2022003399A1 (https=)
CO (1) CO2022017026A2 (https=)
IL (1) IL296519A (https=)
MX (1) MX2022015133A (https=)
TW (1) TW202210513A (https=)
WO (1) WO2021246413A1 (https=)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230357384A1 (en) * 2020-06-18 2023-11-09 Nextcure, Inc. Compositions and methods for modulating flrt3 mediated signal transduction
WO2024206858A1 (en) 2023-03-30 2024-10-03 Revolution Medicines, Inc. Compositions for inducing ras gtp hydrolysis and uses thereof
WO2024229406A1 (en) 2023-05-04 2024-11-07 Revolution Medicines, Inc. Combination therapy for a ras related disease or disorder
WO2025034702A1 (en) 2023-08-07 2025-02-13 Revolution Medicines, Inc. Rmc-6291 for use in the treatment of ras protein-related disease or disorder
WO2025080946A2 (en) 2023-10-12 2025-04-17 Revolution Medicines, Inc. Ras inhibitors
WO2025171296A1 (en) 2024-02-09 2025-08-14 Revolution Medicines, Inc. Ras inhibitors
WO2025240847A1 (en) 2024-05-17 2025-11-20 Revolution Medicines, Inc. Ras inhibitors
WO2025255438A1 (en) 2024-06-07 2025-12-11 Revolution Medicines, Inc. Methods of treating a ras protein-related disease or disorder
WO2025265060A1 (en) 2024-06-21 2025-12-26 Revolution Medicines, Inc. Therapeutic compositions and methods for managing treatment-related effects
WO2026006747A1 (en) 2024-06-28 2026-01-02 Revolution Medicines, Inc. Ras inhibitors
WO2026015796A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Methods of treating a ras related disease or disorder
WO2026015801A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Methods of treating a ras related disease or disorder
WO2026015790A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Methods of treating a ras related disease or disorder
WO2026015825A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Use of ras inhibitor for treating pancreatic cancer
WO2026050446A1 (en) 2024-08-29 2026-03-05 Revolution Medicines, Inc. Ras inhibitors
WO2026072904A2 (en) 2024-09-26 2026-04-02 Revolution Medicines, Inc. Compositions and methods for treating lung cancer

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114561337B (zh) * 2022-03-09 2023-10-03 广州源井生物科技有限公司 一种提高HepG2细胞克隆形成率的单克隆增强培养基和方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015516985A (ja) * 2012-04-27 2015-06-18 ミレニアム ファーマシューティカルズ， インコーポレイテッドＭｉｌｌｅｎｎｉｕｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 抗ｇｃｃ抗体分子、およびｇｃｃ標的化療法への感受性を試験するためのその使用
WO2018221521A1 (ja) 2017-05-30 2018-12-06 帝人ファーマ株式会社 抗ｉｇｆ－ｉ受容体抗体
WO2019152571A1 (en) * 2018-02-01 2019-08-08 Merck Sharp & Dohme Corp. Anti-pd-1 antibodies
WO2019156565A1 (en) * 2018-02-12 2019-08-15 Fast Forward Pharmaceuticals B.V. Improved antagonistic anti-human cd40 monoclonal antibodies
WO2019175186A1 (en) * 2018-03-12 2019-09-19 UltraHuman Six Limited Anti c-met antibodies
JP2020508666A (ja) * 2017-02-27 2020-03-26 帝人ファーマ株式会社 認知障害を治療または予防するためのヒト化抗体、その製造プロセス、及びそれを用いた認知障害を治療または予防するための薬剤
WO2020116398A1 (ja) * 2018-12-03 2020-06-11 帝人ファーマ株式会社 抗ｉｇｆ－ｉ受容体ヒト化抗体

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7538195B2 (en) * 2002-06-14 2009-05-26 Immunogen Inc. Anti-IGF-I receptor antibody
WO2007064911A1 (en) * 2005-12-02 2007-06-07 Biogen Idec Inc. Anti-mouse cd20 antibodies and uses thereof
US20150353643A1 (en) * 2013-09-24 2015-12-10 Universite De La Mediterranee - Aix-Marseille Ii Anti-cd277 antibodies and uses thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015516985A (ja) * 2012-04-27 2015-06-18 ミレニアム ファーマシューティカルズ， インコーポレイテッドＭｉｌｌｅｎｎｉｕｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 抗ｇｃｃ抗体分子、およびｇｃｃ標的化療法への感受性を試験するためのその使用
JP2020508666A (ja) * 2017-02-27 2020-03-26 帝人ファーマ株式会社 認知障害を治療または予防するためのヒト化抗体、その製造プロセス、及びそれを用いた認知障害を治療または予防するための薬剤
WO2018221521A1 (ja) 2017-05-30 2018-12-06 帝人ファーマ株式会社 抗ｉｇｆ－ｉ受容体抗体
WO2019152571A1 (en) * 2018-02-01 2019-08-08 Merck Sharp & Dohme Corp. Anti-pd-1 antibodies
WO2019156565A1 (en) * 2018-02-12 2019-08-15 Fast Forward Pharmaceuticals B.V. Improved antagonistic anti-human cd40 monoclonal antibodies
WO2019175186A1 (en) * 2018-03-12 2019-09-19 UltraHuman Six Limited Anti c-met antibodies
WO2020116398A1 (ja) * 2018-12-03 2020-06-11 帝人ファーマ株式会社 抗ｉｇｆ－ｉ受容体ヒト化抗体

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. NP 000609
"Remington: The Science and Practice of Pharmacy, 20th EDITION", 2000, LIPPINCOTT WILLIAMS & WILKINS
"UniProtKB", Database accession no. P05019
ABHINANDAN, K.R. ET AL., MOLECULAR IMMUNOLOGY, vol. 45, 2008, pages 3832 - 3839
ABUZZAHAB, M.J. ET AL.: "IGF-1 receptor mutations resulting in intrauterine and postnatal growth retardation", N ENGL J MED, vol. 349, no. 23, 2003, pages 2211 - 22
AL-LAZIKANI ET AL., JOURNAL OF MOLECULAR BIOLOGY, vol. 273, no. 4, 1997, pages 927 - 948
ATZORI, F. ET AL.: "A Phase I Pharmacokinetic and Pharmacodynamic Study of Dalotuzumab (MK-0646), an Anti-Insulin-like Growth Factor-1 Receptor Monoclonal Antibody, in Patients with Advanced Solid Tumors", CLIN CANCER RES., vol. 17, no. 19, 2011, pages 6304 - 12
BURKS, E. A. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, no. 2, 1997, pages 412 - 417
DE BONO J. S. ET AL.: "Phase II randomized study of figitumumab plus docetaxel and docetaxel alone with crossover for metastaticcastration- resistant prostate cancer", CLIN CANCER RES., vol. 20, no. 7, 2014, pages 1925 - 34, XP055687495, DOI: 10.1158/1078-0432.CCR-13-1869
DUMET, C. ET AL., MABS, vol. 11, no. 8, 2019, pages 1341 - 1350
HUMAN SOMATOMEDIN C: "Somazon Formulation for Injection 10mg", May 2015, article "Drug interview form"
JIAN, Y. ET AL., NUCLEIC ACIDS RESEARCH, vol. 41, 2013, pages W34 - W40
KABAT ET AL., THE JOURNAL OF IMMUNOLOGY, vol. 147, no. 5, 1991, pages 1709 - 1719
KATO, H. ET AL.: "Role of tyrosine kinase activity in signal transduction by the insulin-like growth factor-I (IGF-1) receptor. Characterization of kinase-deficient IGF-1 receptors and the action of an IGF-1-mimetic antibody (alpha IR-3).", J BIOL CHEM, vol. 268, no. 4, 1993, pages 2655 - 61, XP008114108
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495 - 497
MARKHAM. A: "Teprotumumab: First Approval", DRUGS, vol. 80, no. 5, 2020, pages 509 - 512
MUSARO, A. ET AL.: "Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle", NATURE GENETICS, vol. 27, no. 2, 2001, pages 195 - 200, XP001199471, DOI: 10.1038/84839
RIECHMAN, L.CLARK, M.WALDMANN, H.WINTER, G.: "Reshaping human antibodies for therapy.", NATURE, vol. 332, 1988, pages 323 - 327
RUNNELS, H.A. ET AL.: "Human monoclonal antibodies to the insulin-like growth factor 1 receptor inhibit receptor activation and tumor growth in preclinical studies", ADV THER, vol. 27, no. 7, 2010, pages 458 - 75, XP002751208, DOI: 10.1007/s12325-010-0026-5
SAUNDERS, K. O., FRONTIERS IN IMMUNOLOGY, vol. 10, 2019, pages 1296
SILVA, J-P. ET AL., THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 290, no. 9, 2015, pages 5462 - 5469
SOOS, M.A. ET AL.: "A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity", J BIOL CHEM, vol. 267, no. 18, 1992, pages 12955 - 63, XP002477383
WALLE ET AL., EXPERT OPIN. BIOL. THER., vol. 7, no. 3, 2007, pages 405 - 418
WOODS, K.A. ET AL.: "Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene", N ENGL J MED, vol. 335, no. 18, 1996, pages 1363 - 7
XIONG, L. ET AL.: "Growth-stimulatory monoclonal antibodies against human insulin-like growth factor I receptor", PROC NATL ACAD SCI USA, vol. 89, no. 12, 1992, pages 5356 - 60, XP002423617, DOI: 10.1073/pnas.89.12.5356
YAMADA, T. ET AL.: "Therapeutic monoclonal antibodies", KEIO JOURNAL OF MEDICINE, vol. 60, no. 2, 2011, pages 37 - 46

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230357384A1 (en) * 2020-06-18 2023-11-09 Nextcure, Inc. Compositions and methods for modulating flrt3 mediated signal transduction
WO2024206858A1 (en) 2023-03-30 2024-10-03 Revolution Medicines, Inc. Compositions for inducing ras gtp hydrolysis and uses thereof
WO2024229406A1 (en) 2023-05-04 2024-11-07 Revolution Medicines, Inc. Combination therapy for a ras related disease or disorder
WO2025034702A1 (en) 2023-08-07 2025-02-13 Revolution Medicines, Inc. Rmc-6291 for use in the treatment of ras protein-related disease or disorder
WO2025080946A2 (en) 2023-10-12 2025-04-17 Revolution Medicines, Inc. Ras inhibitors
WO2025171296A1 (en) 2024-02-09 2025-08-14 Revolution Medicines, Inc. Ras inhibitors
WO2025240847A1 (en) 2024-05-17 2025-11-20 Revolution Medicines, Inc. Ras inhibitors
WO2025255438A1 (en) 2024-06-07 2025-12-11 Revolution Medicines, Inc. Methods of treating a ras protein-related disease or disorder
WO2025265060A1 (en) 2024-06-21 2025-12-26 Revolution Medicines, Inc. Therapeutic compositions and methods for managing treatment-related effects
WO2026006747A1 (en) 2024-06-28 2026-01-02 Revolution Medicines, Inc. Ras inhibitors
WO2026015796A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Methods of treating a ras related disease or disorder
WO2026015801A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Methods of treating a ras related disease or disorder
WO2026015790A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Methods of treating a ras related disease or disorder
WO2026015825A1 (en) 2024-07-12 2026-01-15 Revolution Medicines, Inc. Use of ras inhibitor for treating pancreatic cancer
WO2026050446A1 (en) 2024-08-29 2026-03-05 Revolution Medicines, Inc. Ras inhibitors
WO2026072904A2 (en) 2024-09-26 2026-04-02 Revolution Medicines, Inc. Compositions and methods for treating lung cancer

Also Published As

Publication number Publication date
EP4159860A1 (en) 2023-04-05
JPWO2021246413A1 (https=) 2021-12-09
CL2022003399A1 (es) 2023-08-11
IL296519A (en) 2022-11-01
JP2024054410A (ja) 2024-04-16
CA3176119A1 (en) 2021-12-09
CO2022017026A2 (es) 2022-12-09
CN115605594A (zh) 2023-01-13
KR20230006006A (ko) 2023-01-10
AU2021282629A1 (en) 2022-12-01
MX2022015133A (es) 2023-01-11
AR122499A1 (es) 2022-09-14
TW202210513A (zh) 2022-03-16
US20250263489A1 (en) 2025-08-21
BR112022022030A2 (pt) 2023-01-17
EP4159860A4 (en) 2024-09-25

Similar Documents

Publication Publication Date Title
WO2021246413A1 (ja) 抗ｉｇｆ－１受容体ヒト化抗体
JP7798489B2 (ja) 抗ｉｇｆ－ｉ受容体抗体
JP7246409B2 (ja) 抗ｉｇｆ－ｉ受容体ヒト化抗体
RU2836869C1 (ru) Гуманизированное антитело против рецептора igf-1
CA3120740C (en) Anti-igf-i receptor humanized antibody
HK40087036A (zh) 抗igf-1受体人源化抗体
RU2785305C2 (ru) Антитело против рецептора igf-i
JP2024121816A (ja) 抗成長ホルモン抗体を含有する医薬
WO2023032955A1 (ja) 抗成長ホルモン抗体
HK40104653A (zh) 抗生长激素抗体
HK40021781A (en) Anti-igf-i receptor antibody

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21817253

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022528850

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 3176119

Country of ref document: CA

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112022022030

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 202217065681

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: NC2022/0017026

Country of ref document: CO

ENP Entry into the national phase

Ref document number: 2021282629

Country of ref document: AU

Date of ref document: 20210601

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20227042511

Country of ref document: KR

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: NC2022/0017026

Country of ref document: CO

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01E

Ref document number: 112022022030

Country of ref document: BR

Free format text: COM BASE NA PORTARIA INPI 48/2022, APRESENTAR NOVO CONTEUDO ELETRONICO DE LISTAGEM DE SEQUENCIAS, POIS O CONTEUDO APRESENTADO NA PETICAO INICIAL NAO POSSUI TODOS OS CAMPOS OBRIGATORIOS (CAMPO 130). INCLUIR CAMPOS 140-141 EM RESPOSTA A EXIGENCIA, UMA VEZ QUE O DEPOSITANTE JA POSSUI O NUMERO DO PEDIDO NA FASE NACIONAL.

ENP Entry into the national phase

Ref document number: 2021817253

Country of ref document: EP

Effective date: 20230102

ENP Entry into the national phase

Ref document number: 112022022030

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20221028

WWE Wipo information: entry into national phase

Ref document number: 522440892

Country of ref document: SA

WWE Wipo information: entry into national phase

Ref document number: 522440892

Country of ref document: SA

WWG Wipo information: grant in national office

Ref document number: 2022131340

Country of ref document: RU

WWP Wipo information: published in national office

Ref document number: 18007606

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 522440892

Country of ref document: SA