WO2023234406A1 - カンナビノイド１型受容体に結合する抗体 - Google Patents

カンナビノイド１型受容体に結合する抗体 Download PDF

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WO2023234406A1
WO2023234406A1 PCT/JP2023/020578 JP2023020578W WO2023234406A1 WO 2023234406 A1 WO2023234406 A1 WO 2023234406A1 JP 2023020578 W JP2023020578 W JP 2023020578W WO 2023234406 A1 WO2023234406 A1 WO 2023234406A1
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amino acid
acid sequence
seq
antibody
variable region
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English (en)
French (fr)
Japanese (ja)
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舞 吉川
竜也 高橋
順二 小野田
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Shionogi and Co Ltd
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Shionogi and Co Ltd
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Priority to EP23816163.2A priority Critical patent/EP4534672A1/en
Priority to JP2024524954A priority patent/JPWO2023234406A1/ja
Priority to CN202380043799.XA priority patent/CN119384505A/zh
Priority to US18/870,866 priority patent/US20250326836A1/en
Publication of WO2023234406A1 publication Critical patent/WO2023234406A1/ja
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/50—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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

Definitions

  • the present invention relates to an antibody or an antibody fragment thereof that binds to a novel cannabinoid type 1 (CB1) receptor. More specifically, it relates to an antibody or an antibody fragment thereof that specifically binds to the CB1 receptor. More specifically, the present invention relates to an antibody or an antibody fragment thereof that selectively inhibits the CB1 receptor, a pharmaceutical composition containing the same, or a kit for detecting the CB1 receptor.
  • CB1 cannabinoid type 1
  • the CB1 receptor is a member of the G protein-coupled receptor (GPCR) family that is mainly expressed in the central nervous system, liver, kidneys, lungs, and adipose tissue, and it is a member of the G protein-coupled receptor (GPCR) family that is mainly expressed in the central nervous system, liver, kidney, lung, and adipose tissue. It mediates reactions to tetrahydrocannabinol.
  • GPCR G protein-coupled receptor
  • Gi/O proteins inhibits adenyl cyclase, activates mitogen-activated protein kinase (MAPK), and activates voltage-gated Ca ion channels. inhibition, and influence on NO signal transduction (Non-Patent Document 1).
  • CB1 receptors are involved in many diseases such as obesity, diabetes, pain, neurodegenerative diseases, fibrosis, liver diseases, and cardiovascular diseases.
  • CB1 receptor antagonist therapeutic drugs such as Rimonabant have been shown to have anti-obesity effects such as appetite reduction and lowering of triglyceride levels and metabolic improvement effects.
  • low-molecular-weight antagonists such as rimonabant cause central side effects such as psychoactive effects, depression, and suicidal thoughts (Non-Patent Document 2).
  • Non-Patent Document 3 it can be said that peripheral CB1 receptor inhibitors with low brain distribution can act equivalently to systemic antagonists. Therefore, CB1 receptor antagonists or CB1 receptor agonists without central side effects are useful drugs that broadly act on the periphery.
  • Patent Document 1 discloses an antibody against the CB1 receptor as an example of a compound capable of modulating the CB1 receptor signal.
  • Patent Document 2 discloses an antibody that binds to an epitope within the EC2 domain of the human CB1 receptor. Examples of evaluating the in vitro antagonist activity and neutralizing activity of these antibodies are described, but no in vivo evaluation is described at all.
  • Patent Documents 3 and 4 disclose antibodies that are inverse agonists of the CB1 receptor, and describe in vitro and in vivo evaluations of the CB1 antibody.
  • Patent Documents 3 [0361] to [0363] and Patent Documents 4 [0367] to [0369] disclose that administration of P1C4-h2-IgG4 to primates reduces triglyceride levels and other cardiovascular risk factors. Although it is stated that it has been shown to reduce insulin sensitivity and improve insulin sensitivity, no specific data are disclosed.
  • Patent Document 5 discloses an antibody that binds to the CB1 receptor. Examples of evaluating in vitro antagonist activity and neutralizing activity are described, but no in vivo evaluation is described at all. That is, none of the prior art documents discloses the CDR sequence, light chain variable region, heavy chain variable region, etc. of the antibody of the present invention.
  • An object of the present invention is to provide novel antibodies or antibody fragments thereof that bind to the CB1 receptor.
  • Another object of the present invention is to provide a novel CB1 receptor antibody or an antibody fragment thereof that can be used as a therapeutic agent for obesity, diabetes, and the like.
  • the present inventors have discovered an antibody or an antibody fragment thereof that specifically binds to one or more extracellular epitopes on the CB1 receptor and modulates CB1 receptor signaling.
  • the antibody of the present invention has an improvement in small intestinal transport ability or an anti-obesity effect, and is useful for treating obesity, diabetes, non-alcoholic steatohepatitis (NASH), etc.
  • NASH non-alcoholic steatohepatitis
  • Sequence number 44 Xaa1-A-S-S-S-V-S-Xaa2-Xaa3-YL-H (where, Xaa1 is arginine or lysine, Xaa2 is threonine or serine, and Xaa3 is CDR1 consisting of an amino acid sequence in which one or two amino acids may be deleted, substituted, inserted and/or added to the amino acid sequence of arginine or serine), SEQ ID NO: 45: Deletion or substitution of 1 or 2 amino acids in the amino acid sequence of Xaa4-TSN-Xaa5-AS (where Xaa4 is glycine or serine, and Xaa5 is isoleucine or leucine); CDR2 consisting of an amino acid sequence that may be inserted and/or added and SEQ ID NO: 46: QQY-Xaa6-Xaa7-FPLT (where X
  • CB1 cannabinoid type 1 receptor
  • CB1 cannabinoid type 1 receptor
  • CB1 cannabinoid type 1 receptor
  • CB1 cannabinoid type 1 receptor
  • CB1 cannabinoid type 1 receptor
  • CB1 cannabinoid type 1 receptor
  • CB1 cannabinoid type 1 receptor
  • CB1 cannabinoid type 1 receptor
  • CDR3 consisting of an amino acid sequence in which two amino acids may be deleted, substituted, inserted, and/or added. .
  • CDR1 consisting of an amino acid sequence in which one or two amino acids may be deleted, substituted, inserted and/or added to the amino acid sequence of SEQ ID NO: 44
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 45, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 46
  • a light chain variable region including CDR3 consisting of an amino acid sequence that may have been substituted, inserted, and/or added
  • CDR1 consisting of an optional amino acid sequence
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 27, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 26
  • a heavy chain variable region including CDR3 consisting of an amino acid sequence that may be substituted, inserted and/or added
  • CDR1 consisting of an optional amino acid sequence
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 47, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 26,
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 20, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 46,
  • CDR1 consisting of
  • (1-3) a1) CDR1 consisting of an amino acid sequence in which one or two amino acids may be deleted, substituted, inserted and/or added to the amino acid sequence of SEQ ID NO: 44, CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 45, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 46, A light chain variable region including CDR3 consisting of an amino acid sequence that may have been substituted, inserted, and/or added; CDR1 consisting of an optional amino acid sequence, CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 36, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 26, A heavy chain variable region including CDR3 consisting of an amino acid sequence that may be substituted, inserted and/or added; b1) CDR1 consisting
  • CDR1 consisting of an optional amino acid sequence
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 47, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 26,
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 34, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 46,
  • CDR1 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added to the amino acid sequence of SEQ ID NO: 33
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 34, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 35,
  • a light chain variable region including CDR3 consisting of an amino acid sequence that may have been substituted, inserted, and/or added
  • CDR1 consisting of an optional amino acid sequence
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 36, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 26
  • a heavy chain variable region including CDR3 consisting of an amino acid sequence that may be substituted, inserted and/or added;
  • CDR1 consisting of an optional amino acid sequence
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 28, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 29,
  • Heavy chain variable region containing CDR3 consisting of an amino acid sequence that may be substituted, inserted and/or added (that is, heavy chain variable region and light chain variable region containing CDR of m2F8 or h2F8 of Examples or a variant thereof)
  • CDR1 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added to the amino acid sequence of SEQ ID NO: 19
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 20, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 21,
  • CDR1 consisting of an optionally added amino acid sequence
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 27, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 26,
  • CDR3 consisting of an amino acid sequence that may be substituted, inserted and/or added, or c2-2)
  • CDR1 consisting of a good amino acid sequence
  • CDR2 consisting of an amino acid sequence in which 1 or 2 amino acids may be deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 25, and 1 or 2 amino acids deleted in the amino acid sequence of SEQ ID NO: 26,
  • a heavy chain variable region including CDR3 consisting of an amino acid sequence that may be substituted, inserted and/or added; (That is, the heavy chain variable region and light chain variable region containing the heavy chain variable region and light chain variable region containing the heavy chain variable region containing the heavy chain variable region
  • (3-1) A light chain variable region consisting of the amino acid sequence of SEQ ID NOs: 13 to 15 or an amino acid sequence with 95% or more identity to the amino acid sequence of SEQ ID NOs: 13 to 15, and the amino acid sequence of SEQ ID NOs: 16 to 18, 52, or The antibody according to any one of (1-1) to (1-3) or (2), which comprises a heavy chain variable region consisting of an amino acid sequence with 95% or more identity to the amino acid sequence of SEQ ID NOs: 16 to 18, 52, or The antibody fragment.
  • a light chain variable region consisting of an amino acid sequence of SEQ ID NOs: 1 to 4 or an amino acid sequence with 95% or more identity to the amino acid sequence of SEQ ID NOs: 1 to 4, and an amino acid sequence of SEQ ID NOs: 7 to 12 or SEQ ID NO:
  • the antibody or antibody fragment thereof according to any one of (1-1) to (1-3) or (2), which comprises a heavy chain variable region consisting of an amino acid sequence with 95% or more identity to 7 to 12 amino acid sequences.
  • the pharmaceutical composition according to (6) which is a therapeutic and/or preventive agent for obesity, diabetes and/or non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • SEQ ID NO: 39 Xaa11-A-S-S-S-V-S-Xaa12-Xaa13-YL-H (where, Xaa11 is threonine, isoleucine, arginine or lysine, and Xaa12 is serine or CDR1 consisting of the amino acid sequence of threonine, Xaa13 is serine or arginine), SEQ ID NO: 45: CDR2 consisting of the amino acid sequence of Xaa14-TSN-Xaa15-AS (where Xaa14 is serine or glycine, Xaa15 is leucine or isoleucine) and SEQ ID NO: 41: Xaa16-Q- Y-Xaa17-Xaa18-Xaa19-P-Xaa20-T (where Xaa16 is histidine or glutamine, Xaa17 is histidine, threonine or se
  • the antibody or antibody fragment of the present invention specifically binds to the CB1 receptor, it can be used to detect the CB1 receptor in a biological sample. Furthermore, since the antibody or antibody fragment of the present invention has the activity of regulating CB1 receptor signaling, a pharmaceutical composition comprising the antibody or antibody fragment of the present invention can be used as a pharmaceutical, particularly for the treatment or treatment of CB1 receptor-related diseases. It is very useful as a preventive medicine.
  • CDR1 to CDR3 mean complementarity determining regions 1 to 3, respectively, and have the same meaning in FIGS.
  • the human cannabinoid (CB1) receptor is a protein (UniProtKB/Swiss-Prot: P21554: SEQ ID NO: 51) consisting of amino acids encoded by the gene CNR1.
  • the extramembrane domain of the human CB1 receptor consists of an N-terminal region consisting of amino acids 1 to 116, a loop 1 region (EC loop 1) consisting of amino acids 176 to 187, and a loop 2 region (EC loop 2) consisting of amino acids 256 to 273. , the loop3 region (EC loop 3) consisting of amino acids 366-377.
  • the terms "CB1 receptor” and "CB1" both mean cannabinoid type 1 receptor.
  • the antibody or antibody fragment of the present invention is an antibody or a fragment of the antibody having the CDR or heavy chain variable region/light chain variable region described herein.
  • the antibody or antibody fragment may be derived from any class (e.g., IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecules, such as mouse, rat, shark, rabbit, pig, hamster, camel, It may be obtained from any species including llama, goat or human.
  • the antibody or antibody fragment is preferably a humanized antibody or an antibody fragment of a humanized antibody.
  • antibody fragment of an antibody refers to a fragment that is a part of the antibody of the present invention and has the activity of specifically binding to the CB1 receptor and regulating CB1 receptor signal transduction like the antibody. means.
  • Fab fragment of antigen binding
  • Fab' fragment of antigen binding
  • F(ab')2 single chain antibody (single chain Fv; hereinafter referred to as scFv) that specifically binds to the human CB1 receptor.
  • dsFv disulfide stabilized Fv
  • Diabody dimerized V region fragments
  • CDR-containing peptides etc. ⁇ Therapeutic Patents, Vol. 6, No. 5, pp. 441-456, 1996).
  • Fab is obtained by decomposing the peptide part above the two disulfide bonds (SS bonds) that bridge the two H chains in the hinge region of IgG with the enzyme papain. It is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, consisting of one half and the entire L chain.
  • Fab used in the present invention can be obtained by treating the antibody of the present invention with papain.
  • Fab can also be produced by inserting DNA encoding the Fab of the antibody of the present invention into a cellular expression vector, and expressing the vector by introducing the vector into cells.
  • Fab' is an antibody fragment with a molecular weight of approximately 50,000 and having antigen-binding activity obtained by cleaving the SS bond between the hinges of F(ab')2.
  • Fab' used in the present invention can be obtained by treating F(ab')2 of the antibody of the present invention with a reducing agent dithiothreitol.
  • Fab' can also be produced by inserting the DNA encoding Fab' of the antibody of the present invention into a cellular expression vector, and expressing the vector by introducing it into E. coli, yeast, or animal cells.
  • F(ab')2 is obtained by decomposing the lower part of the two SS bonds in the hinge region of IgG with the enzyme pepsin, and is composed of two Fab' regions linked at the hinge region, and has a molecular weight of approximately It is an antibody fragment with an antigen binding activity of 100,000.
  • F(ab')2 used in the present invention can be obtained by treating the antibody of the present invention with pepsin.
  • F(ab')2 can also be expressed by inserting the DNA encoding F(ab')2 of the antibody of the present invention into a cellular expression vector and introducing the vector into E. coli, yeast, or animal cells. 2 can be manufactured.
  • scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P), and has antigenic activity. It is an antibody fragment with The VH and VL contained in the scFv used in the present invention may be those of the antibody of the present invention.
  • the scFv used in the present invention can be expressed and produced by constructing an scFv expression vector using cDNA encoding the VH and VL of the antibody of the present invention and introducing it into E. coli, yeast, or animal cells. .
  • dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue, which are linked via an SS bond.
  • Amino acid residues to be substituted for cysteine residues can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7, 697 (1994)).
  • the VH or VL contained in the dsFv used in the present invention may be those of the antibody of the present invention.
  • the dsFv used in the present invention is constructed by inserting the cDNA encoding the VH and VL of the antibody of the present invention into an appropriate expression vector, and then inserting the expression vector into Escherichia coli, yeast, or animal cells. It can be produced by introducing it into a cell and expressing it.
  • Diabody is an antibody fragment in which scFv with the same or different antigen-binding specificity forms a dimer, and has bivalent antigen-binding activity for the same antigen or two types of specific antigen-binding activity for different antigens. It is.
  • a bivalent diabody that specifically reacts with the antibody of the present invention can be prepared using cDNA encoding the VH and VL of the antibody of the present invention, and a DNA encoding an scFv having a peptide linker of 3 to 10 residues.
  • Diabodies can be expressed and produced by constructing the DNA, inserting the DNA into a cellular expression vector, and introducing the expression vector into E. coli, yeast, or animal cells.
  • a peptide containing a CDR is composed of at least one region of a VH or VL CDR. Multiple CDRs can be linked directly or via a suitable peptide linker.
  • the CDR-containing peptide used in the present invention is obtained by constructing a CDR-encoding DNA using cDNA encoding the VH and VL of the antibody of the present invention, and inserting the DNA into an expression vector for animal cells. It can be produced by introducing a vector into E. coli, yeast, or animal cells and expressing it.
  • Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
  • the antibody or antibody fragment of the present invention is characterized by specifically binding to the CB1 receptor.
  • Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 ⁇ 10 ⁇ 6 M or less (eg, a lower KD indicates tighter binding).
  • the Kd value is preferably 1 ⁇ 10 ⁇ 7 M or less, more preferably 1 ⁇ 10 ⁇ 8 M or less, and still more preferably 1 ⁇ 10 ⁇ 9 M or less.
  • bispecific antibodies are considered antibodies that "specifically bind" to the CB1 receptor.
  • the antibody or antibody fragment of the present invention is characterized by having the activity of regulating CB1 receptor signal transduction.
  • the activity of regulating CB1 receptor signaling refers to the activity of inhibiting or antagonizing, inversely agonizing, or enhancing or agonizing CB1 receptor signaling.
  • the antibody or antibody fragment of the present invention is an antibody or antibody fragment having CB1 receptor inverse agonist activity or CB1 receptor antagonist activity.
  • CB1 receptor inverse agonist activity refers to an activity that reduces CB1 receptor activity below basal levels
  • CB1 receptor antagonist activity refers to an activity that inhibits, reduces, or blocks the signaling activity of a CB1 receptor ligand. Point.
  • CB1 receptor inverse agonist activity for example, CB1 receptor cells expressing human CB1 receptor are used, and Ca 2+ influx is measured by adding a CB1 receptor antagonist (CP55940, etc.).
  • the IC50 value can be determined by calculating the IC50 value, assuming that the inhibition rate without addition is 100% and the inhibition rate without addition of the CB1 receptor antagonist or antibody is 0% (see Example 4).
  • Other methods include determining whether signal transduction is suppressed using the cAMP assay described in Example 4, the aequorin assay, or the GTP-Eu assay.
  • the antibody or antibody fragment of the present invention has any or all of the following excellent characteristics. a) Shows strong anti-fibrotic effect. b) It has a strong medicinal effect on small intestinal transport inhibition. c) It has a strong anti-obesity medicinal effect. d) Low immunogenicity to humans. e) It has high stability. f) Good dynamics and high stability in blood.
  • the antibody of the present invention can be produced by a conventional method in the art using the CDRs or heavy chain variable region/light chain variable region described herein.
  • Antibodies of the invention include monoclonal, monospecific, single chain, chimeric, humanized, fully human, synthetic, recombinant, hybrid, mutated, grafted antibodies, antibody small molecule conjugates (ADCs) and bispecific antibodies.
  • Antibodies also includes humanized antibodies. Since humanized antibodies have reduced antigenicity in the human body, they are useful when administered to humans for therapeutic purposes.
  • a humanized antibody is one in which the complementarity determining regions (CDRs) of a non-human mammal, such as a mouse antibody, are grafted onto the framework regions (FRs) of a human antibody. Therefore, the FRs of a humanized antibody are of human origin.
  • a suitable FR is Kabat E. A.
  • FRs are selected that allow CDRs to form a good antigen-binding site.
  • amino acids in the FRs of the antibody variable region may be substituted so that the CDRs of the reconstituted humanized antibody form a suitable antigen-binding site (Sato, K. et al., Cancer Res. 1993). , Vol. 53, p. 851).
  • the proportion of FR amino acids to be replaced is 0-15%, preferably 0-5% of the total FR region.
  • a fully human antibody specific for the CB1 receptor can be obtained, for example, by immunizing a transgenic mouse containing a human immunoglobulin gene with the CB1 receptor antigen, obtaining lymphocytes from the antibody-expressing mouse, and then producing a myeloid cell line. It can be produced using hybridoma cells fused with lymphocytes.
  • constant region of a human antibody is used in the humanized antibody of the present invention.
  • Preferred constant regions of human antibodies include C ⁇ as a heavy chain, such as C ⁇ 1, C ⁇ 2, C ⁇ 3, and C ⁇ 4, and C ⁇ and C ⁇ as light chains. Additionally, the C region of human antibodies may be modified to improve the stability of the antibody or its production.
  • the human antibody used in humanization may be of any isotype such as IgG, IgM, IgA, IgE, IgD, etc., but in the present invention it is preferable to use IgG, and more preferably IgG1 or IgG4.
  • the humanized antibody of the present invention preferably has a light chain constant region having the amino acid sequence of SEQ ID NO: 38 and a heavy chain constant region having the amino acid sequence of SEQ ID NO: 37.
  • the C-terminal lysine or the C-terminal two amino acid residues (glycine-lysine) of SEQ ID NO: 37 may or may not be present.
  • Humanized antibodies can be produced by common manufacturing methods (for example, see Example 4 below, WO95/14041, WO96/02576, etc.). Specifically, first, a DNA sequence encoding a variable region designed to connect the CDRs of a mouse antibody and the FRs of a human antibody is prepared from several oligonucleotides prepared with overlapping portions at the ends. It is synthesized by PCR method (see WO98/13388). The obtained DNA is ligated with DNA encoding the constant region of a human antibody, and then incorporated into an expression vector. Alternatively, the DNA encoding the antibody variable region may be incorporated into an expression vector containing the antibody constant region DNA. To produce the antibody used in the present invention, the antibody gene is inserted into an expression vector so that it is expressed under the control of an expression control region, such as an enhancer/promoter. A host cell can then be transformed with this expression vector to express the antibody.
  • an expression control region such as an enhancer/promoter.
  • host cells for the above transformants include vertebrate cells such as COS cells and CHO cells, prokaryotic cells, and yeast.
  • the transformant can be cultured according to methods well known to those skilled in the art, and the antibody of the present invention is produced within or outside the transformant cells.
  • the culture medium used for the culture can be appropriately selected from various commonly used media depending on the host cell employed. For example, in the case of COS cells, RPMI-1640 medium, Dulbecco's modified Eagle's minimum essential medium (DMEM), etc.
  • DMEM Dulbecco's modified Eagle's minimum essential medium
  • a culture medium to which a serum component such as fetal bovine serum (FBS) is added, if necessary, can be used.
  • FBS fetal bovine serum
  • the culture temperature for culturing the transformant may be any temperature as long as it does not significantly reduce the intracellular protein synthesis ability, but it is preferably cultured at 32 to 42°C, most preferably at 37°C. It is preferable. Furthermore, if necessary, the culture can be carried out in air containing 1 to 10% (v/v) carbon dioxide gas.
  • the fraction containing the antibody of the present invention produced intracellularly or extracellularly of the transformant as described above is separated and purified by various known separation methods that utilize the physical and chemical properties of the protein. can do. Specifically, such methods include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC). ), various chromatography methods, dialysis methods, and combinations thereof can be employed. By this method, the antibody of the present invention can be easily produced in high yield and with high purity.
  • the antibodies of the present invention or active fragments thereof may be further modified with various molecules such as polyethylene glycol (PEG), radioactive substances, and toxins. Methods known in this field can be used to modify antibodies.
  • PEG polyethylene glycol
  • the antibody of the present invention may have another protein fused to its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). Proteins to be fused can be appropriately selected by those skilled in the art.
  • a pharmaceutical composition containing the antibody of the present invention or an antibody fragment thereof can be administered orally or parenterally, systemically or locally.
  • parenteral administration for example, intravenous injection such as drip, intramuscular injection, epidural injection, intraventricular injection, intraperitoneal injection, subcutaneous injection, intranasal administration, inhalation, etc. can be selected.
  • the pharmaceutical composition of the present invention is useful as a medicament for the treatment and/or prevention of CB1 receptor-related diseases.
  • CB1 receptor-related diseases include obesity, Alström syndrome, Praderwilli syndrome, Palde-Biedl syndrome, Albride hereditary osteodystrophy, symptomatic obesity including SIM1 deletion syndrome, diabetes and its related complications, and insulin.
  • Resistant type II diabetes, prediabetes, metabolic syndrome, dyslipidemia, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diabetic gastroparesis, gastroparesis, primary Liver diseases such as biliary cirrhosis, renal fibrosis, chronic kidney disease, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive kidney disease, nephrotic syndrome, steroid-resistant nephrotic syndrome, Minimal change nephrotic syndrome, membranous nephropathy, idiopathic membranous nephropathy, membranoproliferative glomerulonephritis (MPGN), immunocon
  • the antibody of the present invention or its antibody fragment or the pharmaceutical composition of the present invention is (1) Complementing and/or enhancing the therapeutic effect of the pharmaceutical composition of the present invention, (2) kinetics, absorption improvement, dosage reduction and/or of the pharmaceutical composition of the present invention; (3) Reduction of side effects of the pharmaceutical composition of the present invention, It may also be administered as a concomitant drug in combination with other active ingredients or drugs containing the active ingredients.
  • Examples of drugs containing other active ingredients include CB1 receptor antibodies other than the antibodies of the present invention or antibody fragments thereof, and CB1 receptor antagonists such as low-molecular-weight CB1 receptor inhibitors.
  • Examples of low-molecular CB1 receptor inhibitors include rimonabant, taranaban, AM251, AM1387, AM4113, cannabigerol, ibipinabant, otenabant, srinaban, tetrahydrocannabivarin, virodamine, AM6545, and the like.
  • a combination drug of the antibody of the present invention or its antibody fragment and other active ingredients may be administered in the form of a combination drug containing both components in one preparation, or may be administered in the form of a combination drug containing both components in one preparation, or in the form of a separate preparation (the pharmaceutical composition of the invention). It may also be administered as a drug containing other active ingredients.
  • Administration in separate formulations includes simultaneous administration and staggered administration.
  • administration at different times may be such that the pharmaceutical composition of the present invention is administered first and the other drug is administered later, or the other drug is administered first and the pharmaceutical composition of the present invention is administered later.
  • the respective administration methods may be the same or different.
  • the effective dosage of the pharmaceutical composition of the present invention is selected from the range of 0.01 mg to 100 mg per kg of body weight per dose. Alternatively, a dosage of 5 to 5000 mg, preferably 10 to 500 mg per patient can be chosen. However, the pharmaceutical composition of the present invention is not limited to these dosages. Furthermore, the administration period can be appropriately selected depending on the patient's age and symptoms, and the dosage can be scaled using methods well known in the art (for example, Mordenti et al. (1991)). Pharmaceut. Res. 8:1351-1359). The pharmaceutical composition of the present invention may also contain pharmaceutically acceptable carriers and additives depending on the route of administration.
  • Such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, water-soluble dextran, pectin, methylcellulose, ethylcellulose, casein, diglycerin, propylene glycol. , polyethylene glycol, petrolatum, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like.
  • the additives used are selected from the above as appropriate or in combination depending on the dosage form, but are not limited to these.
  • the present invention includes polynucleotides encoding the heavy chain variable region and/or light chain variable region of the antibody of the present invention.
  • the polynucleotide encoding the heavy chain variable region of the antibody of the present invention may further encode the heavy chain constant region.
  • the polynucleotide encoding the light chain variable region of the antibody of the present invention may further encode the light chain constant region.
  • the present invention further includes expression vectors containing at least one of the polynucleotides.
  • the polynucleotide is not particularly limited as long as it encodes the light chain variable region or heavy chain variable region of the antibody of the present invention, and may be a polymer consisting of multiple nucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). be. It may contain non-natural bases.
  • the polynucleotide of the present invention can be used to produce antibodies by genetic engineering techniques. It can also be used as a probe to screen for antibodies that have functions equivalent to the antibodies of the present invention.
  • a polynucleotide encoding the antibody of the present invention, or a portion thereof, is used as a probe and hybridized with the polynucleotide under stringent conditions by a technique such as hybridization or gene amplification technique (e.g. PCR), and DNA encoding an antibody having an activity equivalent to that of the antibody of the present invention can be obtained.
  • a technique such as hybridization or gene amplification technique (e.g. PCR)
  • DNA encoding an antibody having an activity equivalent to that of the antibody of the present invention can be obtained.
  • DNA is also included in the polynucleotide of the present invention.
  • Hybridization technology (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, (1989) is a technique well known to those skilled in the art.
  • Examples of hybridization conditions include low stringency conditions. Low stringency conditions include, for example, 42° C., 0.1 ⁇ SSC, 0.1% SDS in washing after hybridization, preferably 50° C., 0.1 ⁇ SSC, 0.1% SDS. This is a condition of SDS. More preferable hybridization conditions include highly stringent conditions. High stringency conditions are, for example, 65° C., 5 ⁇ SSC, and 0.1% SDS.
  • Antibodies that are functionally equivalent to the antibodies of the present invention and encoded by polynucleotides obtained by these hybridization techniques or gene amplification techniques usually have high homology with these antibodies in their amino acid sequences.
  • the antibodies of the present invention also include antibodies that are functionally equivalent to the antibodies of the present invention and have high homology to the amino acid sequence of the antibodies.
  • High homology generally refers to at least 75% identity, preferably 85% or more identity, and more preferably 95% or more identity at the amino acid level.
  • kits containing the antibody of the present invention can be used as a CB1 receptor detection kit.
  • the kit contains the antibody of the present invention or an antibody fragment thereof, and further includes a labeled secondary antibody, a substrate necessary for detecting the label, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, and a purified standard. It may also contain the CB1 receptor protein as a substance, instructions for use, and the like.
  • Example 1 Preparation of anti-human CB1 receptor mouse antibody by hybridoma method
  • Full length of human CB1 receptor (UniProtKB/Swiss-Prot: P21554) or extracellular N-terminal region (amino acids 1 to 116) of human CB1 receptor ICR female mice in which the CB1 receptor gene had been knocked out were immunized with DNA using the gene encoding ⁇ N-human CB1 receptor (T210A), which is a human CB1 receptor mutant in which the T210A mutation has been deleted and the T210A mutation has been introduced, as an antigen.
  • T210A ⁇ N-human CB1 receptor
  • DNA immunization was repeated two or three times at two-week intervals, and one week after the final immunization, Expi293 cells (Thermo Fisher SCIENTIFIC) that had transiently expressed human CB1 receptor were administered intraperitoneally to boost. Three days later, the spleen was removed, and the spleen cells and mouse myeloma cells (p3x6363-Ag8., Tokyo Tumor Research Institute) were fused using the PEG method, and selected using a medium containing hypoxanthine, aminopterin, and thymidine.
  • Expi293 cells which transiently expressed human CB1 receptor
  • Expi293 cells which transiently expressed human CB2 receptor
  • Clones exhibiting a signal specific to the human CB1 receptor were selected by reacting with cells and detecting with Alexa488-labeled anti-mouse IgG antibody (Thermo Fisher SCIENTIFIC) using Mirror Ball.
  • Anti-human CB1 receptor blocking antibodies were selected by cAMP assay using HitHunter (registered trademark) cAMP kit (DiscoverX).
  • CHO cells stably expressing human CB1 receptor were seeded at 1x10 4 cells/well on a 384 well plate and cultured O/N at 37°C under 5% CO 2 saturated water vapor pressure. Hybridoma culture supernatant was added and reacted at room temperature for 3 hours. After washing twice with 20mM Hepes-HBSS (pH 7.4), CP55940 (CB1 receptor agonist) was reacted at a final concentration of 10nM and Forskolin at a final concentration of 10 ⁇ M for 30 minutes at room temperature, and then cAMP antibody reagent, cAMP working detection solution was added, and the mixture was allowed to react at room temperature for 1 hour in the dark.
  • CP55940 CB1 receptor agonist
  • the inhibition rate was calculated by setting the signal value of CP55940+ and Forskolin+ as 0% inhibition rate and the signal value of CP55940- and Forskolin+ as 100% inhibition rate, and the inhibition rate was 50% or more. Clones exhibiting an inhibition rate of 1 were selected as human CB1 receptor-inhibiting antibodies.
  • Example 2 Preparation of anti-human CB1 receptor mouse antibody using immunophage antibody library
  • mRNA was extracted from spleen cells using Trizol and Dynabeads mRNA purification kit (Thermo Fisher SCIENTIFIC), and Superscript III first-strand cDNA synthesis was performed.
  • a cDNA library was synthesized using a kit (Thermo Fisher SCIENTIFIC).
  • Primer mixes encoding the N-terminal region and J chain of the mouse antibody germline registered in the IMGT (registered trademark) database were designed as a Forward primer set and a Reverse primer set, respectively, and these were used to perform PCR with KOD plus DNA polymerase (TOYOBO).
  • the K chain and H chain variable regions were each amplified.
  • These were linked by assembly PCR using a G4Sx3 linker to form an scFv antibody gene library, which was inserted into a phagemid vector by restriction enzyme treatment. This was transformed into E. coli TG1 strain by electroporation and cultured O/N on LB agar medium to obtain an scFv library (E. coli stock) with a diversity of about 109 .
  • this E. coli stock was amplified to the logarithmic growth phase in LB liquid medium, then infected with M13KO7 helper phage, induced into LB liquid medium culture supernatant at 26°C O/N, and concentrated with PEG-NaCl. It was prepared by The phage antibody library was first reacted with Expi293 cells, and an operation (subtraction) in which unbound phages were recovered as "phage that do not non-specifically bind to cells" was repeated 5 times.
  • the enriched phage antibody library was infected with TG1 and monocloned in a 96-well plate, and the anti-human CB1 receptor-specific antibody was isolated using the same method as described in Example 1 using the scFv protein induced in the culture supernatant. Antibodies and anti-human CB1 receptor blocking antibodies were selected.
  • Example 3 Determination of antibody sequence Antibodies were prepared using conventional methods from phagemid vectors derived from hybridoma cells identified as human CB1 receptor-inhibiting antibodies in Example 1 and phage antibodies identified as human CB1 receptor-inhibiting antibodies in Example 2. The amino acid sequences of the light chain variable region and heavy chain variable region of The identified sequences are shown in Table 1.
  • Example 4 Evaluation of neutralizing activity
  • the established hybridoma was cultured in a serum-free medium, and the culture supernatant was subjected to affinity purification using a Protein G column and gel filtration purification to obtain a purified antibody.
  • the neutralizing activity of this purified antibody was measured by the method shown below using cAMP activity and Ca 2+ influx as indicators.
  • cAMP activity was evaluated using the HitHunter cAMP kit.
  • the hCB1 receptor/CHO described in Example 1 was seeded on a 384-well plate at 7.5x10 3 cells/well, cultured O/N at 37°C under 5% CO 2 saturated water vapor pressure, and incubated with 0.1% BSA/CHO.
  • Antibodies diluted with 20mM HEPES-HBSS (pH 7.4) were added and allowed to react at room temperature for 1.5 hours. After washing twice with 20mM HEPES-HBSS (pH 7.4), react at room temperature for 30 minutes with CP55940 at a final concentration of 3nM and Forskolin at a final concentration of 10 ⁇ M, followed by cAMP antibody reagent, cAMP working detection sol. Add ution Then, the mixture was allowed to react at room temperature for 1 hour in the dark. After adding cAMP solution A and reacting at room temperature for 3 hours while shielding from light, luminescence was detected.
  • the inhibition rate was determined by setting the signal value of CP55940+ and Forskolin+ as 0% inhibition rate and the signal value of CP55940- and Forskolin+ as 100% inhibition rate, and the antibody concentration showing 50% inhibition was set as IC50 (nM). It was calculated and expressed as Average ⁇ SD (Table 2). Ca 2+ influx was assessed using FLIPR Penta. A diluted antibody solution diluted with a medium was added to human CB1 receptor-expressing G ⁇ 16CHO cells that had been preloaded with a Ca 2+ indicator, and allowed to react for 60 minutes at 37° C. in a 5% CO 2 incubator.
  • Ca 2+ influx was measured by addition of CP55940 at a final concentration of 10 ⁇ M after washing twice with 20 mM HEPES-HBSS (pH 7.4).
  • the inhibition rate was calculated by setting the signal when CP55940 was not added as an inhibition rate of 100%, and the signal when CP55940 and antibody were not added as an inhibition rate of 0%, and the antibody concentration showing a 50% inhibition rate was defined as IC50. Evaluations were performed at least three times and IC50s were expressed as Average ⁇ SD (Table 2). As a result, in both evaluation systems, it had much stronger human CB1 receptor inhibitory activity than Rimonabant, a small molecule inhibitor used as a positive control, and exhibited an inverse agonist-like effect. .
  • Example 5 Humanization of antibodies Humanization of mIML1-2 and m2F8 was carried out using the method described below. Numbering according to Kabat definition using antibody sequence analysis software abYsis (Kabat numbering (Wu, T.T. and Kabat, E.A., J Exp. Med. Aug1; 132 (2): 211-50. (1970)) Next, we used the sequence analysis software AbYsis to search for human germline acceptor sequences that are highly homologous to the heavy chain and light chain variable region sequences of the mouse antibody amino acid sequences.
  • humanization was performed by transplanting mouse antibody heavy chain CDR1, CDR2, CDR3 and mouse antibody light chain CDR1, CDR2, CDR3, respectively.
  • the J chain region has homology with the J chain region sequence of mouse antibody. Sequences with a high level of 100% were selected by searching IMGT (http://www.imgt.org/).As a result, humanized monoclonal antibodies (hIML1-2, hIML1-2, hIML1-2-2, h2F8).Also, for hIML1-2-2, we comprehensively created mutants in which single mutations were introduced in the framework and CDR, and the affinity for the human CB1 receptor was improved. Mutations were identified. Next, by superimposing these mutations, a humanized monoclonal antibody (hIML1-2-OP1) with improved human CB1 receptor inhibitory activity was obtained. These sequences are shown in Table 3.
  • human CB1 receptor inhibitory activity was evaluated using cAMP activity and Ca 2+ influx as indicators in the same manner as in Example 4.
  • Nimacimab BIRD ROCK BIO
  • M5 and M7 described in Patent Document 5 WO2019211665
  • cAMP activity was determined using CP55940 at a final concentration of 10 nM and Forskolin at a final concentration of 10 ⁇ M.
  • Ca 2+ influx evaluation was performed using CP55940 at a final concentration of 10 ⁇ M.
  • Human CB1 receptor inhibitory activity was comparatively evaluated. As shown in Table 4, the antibody exhibited a very strong human CB1 receptor inhibitory activity several to several tens of times higher, indicating that it had an inhibitory activity superior to that of the preceding antibody.
  • Example 6 Single-dose POM test (small intestinal transport ability evaluation) in human CB1 receptor knock-in mice (hereinafter referred to as hCB1 receptor-KI (KI/KI) mice)
  • hCB1 receptor-KI KI/KI mice
  • the mouse CB1 receptor gene (Gene ID: 12801) is composed of multiple exons, and the final exon contains the entire length of the ORF (open reading frame).
  • ORF of the human CB1 receptor gene CCDS ID: 5015.1
  • ORF CCDS ID: 18025.1
  • DYKDDDDK SEQ ID NO: 53
  • hCB1 receptor-KI KI/KI
  • the DNA fragments used as homologous recombination arms were obtained by PCR amplifying mouse genome sequences of approximately 2 kb (kilobases) upstream and downstream of the ORF of the mouse CB1 receptor gene. Designed to be removed.
  • a targeting vector is created by seamlessly connecting homologous recombination arms before and after the full-length ORF sequence of the human CB1 receptor gene with a DYKDDDDK (SEQ ID NO: 53) tag added to the C-terminus, and subjected to homologous recombination.
  • hCB1 receptor-KI (KI/+) C57/BL6 mice were generated.
  • hCB1 receptor-KI (KI/+) C57/BL6 mouse In the created hCB1 receptor-KI (KI/+) C57/BL6 mouse, the full length ORF of the mouse CB1 receptor gene and the full length ORF of the human CB1 receptor gene are correctly replaced, and there is no insertion of unnecessary sequences. It was confirmed by PCR and DNA sequence analysis that there was no deletion.
  • hCB1 receptor-KI (KI/KI) C57/BL6 mice were generated by crossing hCB1 receptor-KI (KI/+) mice through normal mating.
  • (2) Single dose hCB1 receptor POM test in hCB1 receptor-KI (KI/KI) mice The anti-human CB1 receptor human shown in Example 5 was administered to 13-week-old hCB1 receptor KI (KI/KI) mice.
  • hIML1-2-OP1 0.3 mg/kg, 1 mg/kg
  • Saline which is a modified antibody
  • CB1 receptor agonist CP55940 0.05 mg/kg
  • a 10% charcoal suspension was orally administered at 0.1 mL/mouse.
  • the small intestine was removed 30 minutes after administration of the charcoal suspension, and the small intestine transport ability was evaluated using the distance traveled by the charcoal suspension as an index.
  • Example 7 Anti-obesity effect in hCB1 receptor-KI (KI/KI) mouse DIO model Seven-week-old hCB1 receptor KI (KI/KI) mice were loaded with a 60% high-fat diet (58Y1), and loading was started. At 5 weeks, the mice were assigned to a PBS administration group and an antibody administration group based on body weight. Thereafter, hIML1-2-OP1, an anti-human CB1 receptor humanized antibody prepared in Example 5, and Isotype Control antibody were repeatedly administered subcutaneously once a week for a total of 6 times at 10 mg/kg or 30 mg/kg. . Body weight and food intake were monitored once a week to evaluate anti-obesity effects.
  • h2F8 was subcutaneously administered 5 times in total in the same manner, and the end point was set at about 5 weeks later (Day 36), and its anti-obesity effect and fatty liver improving effect were evaluated.
  • hIML1-2-OP1 h2F8 administration group, a suppressive effect on weight gain due to high-fat diet loading was observed, and a dose-dependent anti-obesity effect was confirmed.
  • the antibodies or antibody fragments of the present invention can be used to detect CB1 receptors in biological samples. Furthermore, a pharmaceutical composition containing the antibody or antibody fragment of the present invention is very useful as a medicament for treating or preventing CB1 receptor-related diseases.

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