WO2023046071A1 - 抗klb抗体及用途 - Google Patents

抗klb抗体及用途 Download PDF

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Publication number
WO2023046071A1
WO2023046071A1 PCT/CN2022/120887 CN2022120887W WO2023046071A1 WO 2023046071 A1 WO2023046071 A1 WO 2023046071A1 CN 2022120887 W CN2022120887 W CN 2022120887W WO 2023046071 A1 WO2023046071 A1 WO 2023046071A1
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Prior art keywords
amino acid
seq
acid sequence
variable region
antibody
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PCT/CN2022/120887
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English (en)
French (fr)
Inventor
张玲
应华
张明喜
金薪盛
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to EP22872115.5A priority Critical patent/EP4406968A1/en
Priority to CA3232472A priority patent/CA3232472A1/en
Priority to CN202280056275.XA priority patent/CN117813323A/zh
Priority to KR1020247012346A priority patent/KR20240067092A/ko
Priority to JP2024517530A priority patent/JP2024534531A/ja
Priority to US18/694,365 priority patent/US20240270874A1/en
Publication of WO2023046071A1 publication Critical patent/WO2023046071A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • 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
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/70—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

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to anti-KLB antibodies and applications thereof.
  • KLB (Klotho-beta, ⁇ -Klotho) is a member of the Klotho protein family, which consists of an extracellular domain, a transmembrane domain and an intracellular domain. KLB forms a complex with fibroblast growth factor receptors (FGFR, including FGFR1-4 and its alternative splicing bodies, such as FGFR1c, FGFR2c, FGFR3c), as a fibroblast growth factor (FGF for short, For example, co-receptors of FGF19 and FGF21).
  • FGFR fibroblast growth factor receptors
  • FGF fibroblast growth factor receptors
  • FGF fibroblast growth factor receptors
  • FGF fibroblast growth factor
  • Fibroblast growth factor receptors contain an intracellular tyrosine kinase domain, which is activated when it binds to a ligand, producing MAPK (Erk1/2), RAF1, AKT1, and STAT, etc., and activating downstream signaling pathways ( Kharitonenkov, A. et al. (2008) BioDrugs 22:37-44).
  • Fibroblast growth factor 21 (fibroblast growth factor 21, FGF21) is one of the members of the fibroblast growth factor family, which is highly expressed in the liver and pancreas (Itoh et al., (2004) Trend Genet.20:563-69 ). FGF21 binds to KLB through the C-terminus, and binds to FGFR at the N-terminus to form a receptor-ligand complex, thereby activating the signaling pathway downstream of FGFR.
  • FGF21 forms complexes with KLB&FGFR1c, KLB&FGFR2c and KLB&FGFR3c, thereby regulating glucose metabolism, lipid metabolism and energy consumption. And so on.
  • Transgenic mice overexpressing FGF21 exhibited low insulin, low cholesterol, and low triglycerides, improved insulin sensitivity, and prevented high-fat food-induced weight gain (Kharitonenkov et al., (2005) J Clin Invest 115:1627-35).
  • FGF21 analogs (such as AKR001, see SEQ ID NO: 47 in WO2010129503A1) have been fully confirmed in clinical trials, which can significantly reduce liver fat, improve NASH resolution, and improve fibrosis.
  • the disclosure constructs an anti-KLB antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, where,
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise the amino acid sequence of HCDR1, HCDR2 and HCDR3 of any sequence in SEQ ID NO: 40-48, respectively
  • the LCDR1 of the light chain variable region , LCDR2 and LCDR3 comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO:34 respectively
  • the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 3;
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 49
  • the LCDR1, LCDR2 and LCDR3 of the light chain variable region comprise respectively
  • the amino acid sequence of LCDR1, LCDR2 and LCDR3 of any sequence in SEQ ID NO: 56-68; or HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 4, respectively and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 5; or
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 18, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region comprise respectively Amino acid sequence of LCDR1, LCDR2 and LCDR3 in SEQ ID NO:19.
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 40, respectively, and LCDR1, HCDR1, and HCDR3 of the light chain variable region LCDR2 and LCDR3 respectively comprise the amino acid sequence of LCDR1, LCDR2 and LCDR3 among SEQ ID NO:34;
  • the amino acid sequence of HCDR3, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequence of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 57;
  • the HCDR1 of the heavy chain variable region , HCDR2 and HCDR3 respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 18, and LCDR1, LCDR2 and LCDR3 of the light chain variable region comprise respectively LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 19 amino acid
  • the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 40, respectively, and the LCDR1, LCDR2 and HCDR3 of the light chain variable region LCDR3 comprises the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 34, respectively.
  • the anti-KLB antibody according to any one of the above, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are selected from Kabat, IMGT, Chothia, AbM and Contact are defined by the same numbering sequence.
  • HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Kabat numbering convention; in some embodiments, the heavy chain can be HCDR1, HCDR2 and HCDR3 of the variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the IMGT numbering convention; in some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the light chain LCDR1, LCDR2, and LCDR3 of the variable region are defined according to the Chothia numbering convention; defined by the AbM numbering convention; in some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Contact numbering convention.
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 37, 38, 39 or 7
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 8
  • LCDR1 of said light chain variable region comprises the amino acid sequence of SEQ ID NO: 9
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 10
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 13
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 14
  • the light chain can LCDR1 of the variable region comprises the amino acid sequence of SEQ ID NO: 15, 69, 70, 71, 72, 73 or 74
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 16 or 75
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 17 ;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, HCDR2 comprises the amino acid sequence of SEQ ID NO: 25, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 26, and the light chain can LCDR1 of the variable region comprises the amino acid sequence of SEQ ID NO:27, LCDR2 comprises the amino acid sequence of SEQ ID NO:28, and LCDR3 comprises the amino acid sequence of SEQ ID NO:29.
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 37
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 8
  • the light chain can LCDR1 of the variable region comprises the amino acid sequence of SEQ ID NO: 9
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 10
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 13
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, and the light chain can
  • the LCDR1 of the variable region comprises the amino acid sequence of SEQ ID NO:69
  • the LCDR2 comprises the amino acid sequence of SEQ ID NO:75
  • the LCDR3 comprises the amino acid sequence of SEQ ID NO:17.
  • the anti-KLB antibody according to any one of the above, HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Kabat numbering convention.
  • the anti-KLB antibody according to any one of the above, said anti-KLB antibody is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a fully human antibody.
  • the anti-KLB antibody according to any one of the above, wherein (i) the heavy chain variable region comprises SEQ ID NO: 40, 30, 31, 32, 41, 42, 43, 44, 45, 46, 47 or 48, or an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto, and said light chain variable region comprising SEQ ID NO: 34, 33, 35 or 36, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 49, 50 or 51, or has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity therewith
  • a specific amino acid sequence and said light chain variable region comprises SEQ ID NO: 57, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68, or an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 18, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 19, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 2, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 3, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 4, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 5, or an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto.
  • the anti-KLB antibody according to any one of the above, the heavy chain variable region of the antibody comprises a frame region selected from positions 1, 24, 44, 71 and 91 (numbering according to the Kabat numbering system) ) in one or more amino acid back mutations, and the framework region of the light chain variable region contains one or More amino acid back mutations.
  • the anti-KLB antibody wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6, and HCDR2 comprises the amino acid sequence of SEQ ID NO: 37, 38, 39 or 7 , and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO : the amino acid sequence of 11; the framework region of the heavy chain variable region of the antibody comprises one or more amino acid backmutations selected from 1E, 24T, 44G, 71S and 91F (numbering according to the Kabat numbering system), and /or the frame region of the light chain variable region comprises one or more amino acid back mutations selected from 2V, 4I, 36F, 38E, 43T, 44N and 58I (numbered according to the Kabat numbering system).
  • the framework region of the heavy chain variable region of the antibody comprises positions selected from positions 1, 24, 48, 67, 69, 71 and 73 (according to One or more amino acid mutations in the Kabat numbering system numbering) containing one selected from positions 2, 45, 47, 49, 58 and 71 (numbering according to the Kabat numbering system) on the framework region of the light chain variable region or more amino acid back mutations.
  • the anti-KLB antibody wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 13
  • the amino acid sequence of NO: 14 and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, 69, 70, 71, 72, 73 or 74, and the LCDR2 comprises the amino acid sequence of SEQ ID NO: 16 or 75 sequence, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 17
  • the framework region of the heavy chain variable region of the antibody comprises a compound selected from the group consisting of 1E, 24G, 48I, 67A, 69V, 71V and 73K (numbering according to the Kabat numbering system)
  • One or more amino acid mutations in, and/or the frame region of the light chain variable region comprises one or more selected from 2N, 45K, 47W, 49Y, 58
  • the heavy chain variable region comprises any sequence of SEQ ID NO: 40, 30, 31, 32, 41, 42, 43, 44, 45, 46, 47 or 48, or at least 90% thereof (e.g. Amino acid sequences of at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity
  • said light chain variable region comprises any sequence of SEQ ID NO: 34, 33, 35 or 36, or an amino acid sequence having at least 90% (eg at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises any sequence of SEQ ID NO: 49, 50 or 51, or at least 90% thereof (e.g. at least 90%, 95%, 96%, 97%, 98% or 99%) ) sequence identity amino acid sequence
  • said light chain variable region comprises SEQ ID NO: 57, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66 , 67 or 68, or an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 18, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 19, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 2, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 3, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 4, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises an amino acid sequence with SEQ ID NO: 5, or with at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto .
  • the heavy chain variable region comprises SEQ ID NO: 40, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 34, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto;
  • said heavy chain variable region comprises SEQ ID NO: 49, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 57, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto; or
  • said heavy chain variable region comprises SEQ ID NO: 18, or an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto
  • said light chain variable region comprises SEQ ID NO: 19, or an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto.
  • the anti-KLB antibody of any one of the above wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 40, and the light chain variable region comprises SEQ ID NO: 34 amino acid sequence.
  • the anti-KLB antibody as described in any one of the above which includes a heavy chain constant region and a light chain constant region; in some embodiments, the heavy chain constant region is a human IgG heavy chain constant region; in some In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, IgG4 heavy chain constant region; in some embodiments, the light chain constant region is a human lambda, kappa light chain constant region; in some embodiments, The heavy chain constant region is a human IgG1 heavy chain constant region, and the light chain constant region is a human kappa light chain constant region; in some embodiments, the Fc region of the heavy chain constant region has one or more Fc regions capable of reducing Fc Amino acid substitutions in the region binding to Fc receptors.
  • the Fc region has L234A, L235A mutations, S228P mutations, and/or YTE mutations (M252Y, S254T, and T256E), and the numbering of the mutations is based on the EU index.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:22
  • the light chain constant region comprises the amino acid sequence of SEQ ID NO:23.
  • said heavy chain comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 78, and said light chain The chain comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 79; or
  • said heavy chain comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 80, and said light chain The chain comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 81; or
  • said heavy chain comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 20, and said light chain The chain comprises an amino acid sequence having at least 90% (eg, at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 21.
  • the anti-KLB antibody according to any one of the above, wherein the anti-KLB antibody comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 78, and the Said light chain comprises the amino acid sequence of SEQ ID NO: 79; (ii) said heavy chain comprises the amino acid sequence of SEQ ID NO: 80, and said light chain comprises the amino acid sequence of SEQ ID NO: 81; or (iii) said Said heavy chain comprises the amino acid sequence of SEQ ID NO: 20, and said light chain comprises the amino acid sequence of SEQ ID NO: 21.
  • the anti-KLB antibody as described in any one of the above, the anti-KLB antibody is an antigen-binding fragment, and in some embodiments, the antigen-binding fragment is selected from: Fab, F(ab')2, Fab', Fd, Fv, dsFv, scFv, Fab and diabodies.
  • the disclosure provides an isolated anti-KLB antibody that competes for binding to human and/or monkey KLB with any of the anti-KLB antibodies described above; in some embodiments, the disclosure provides an An isolated anti-KLB antibody that binds to the same epitope as any of the anti-KLB antibodies described above.
  • the anti-KLB antibody of any one of the above, said anti-KLB antibody has one or more of the following characteristics:
  • the anti-KLB antibody has activation activity to cells expressing hKLB &hFGFR1c; in some embodiments, the anti-KLB antibody has high activation activity to cells expressing hKLB &hFGFR1c; in some embodiments, the anti-KLB antibody has a high activation activity to cells expressing hKLB & hFGFR1c
  • the activation fold of CHOK1 cells expressing hKLB&hFGFR1c is greater than 10, and/or the activation fold of the anti-KLB antibody to HEK293T-hKLB&hFGFR1c cells is greater than 2; in some embodiments, the activation fold is the activation fold of the antibody relative to the blank control; In some embodiments, the activation factor is detected according to the methods of Test Examples 4 and 5 of the present disclosure;
  • the anti-KLB antibody has weak activation activity to recombinant cells expressing hFGFR1c (only hFGFR1c, not expressing hKLB); in some embodiments, the activation fold of the anti-KLB antibody to HEK293-hFGFR1c cells is less than 1; in some embodiments Wherein, the activation multiple is the activation multiple of the antibody relative to the blank control; in some embodiments, the activation multiple is detected according to the method of Test Example 6 of the present disclosure;
  • the anti-KLB antibody has weak activation activity to cells expressing hKLB&hFGFR2c, hKLB&hFGFR3c and hKLB&hFGFR4; in some embodiments, the anti-KLB antibody has weak activation activity to L6-hKLB&hFGFR2c and/or L6-hKLB&hFGFR4 cells; in some In some embodiments, the activation factor of the anti-KLB antibody to L6-hKLB&hFGFR2c and/or L6-hKLB&hFGFR4 cells is less than 2; The activation activity of the anti-KLB antibody is weak, in some embodiments, the activation fold of the anti-KLB antibody to L6-hKLB&hFGFR2c, hKLB&hFGFR3c and/or L6-hKLB&hFGFR4 cells is less than 2; in some embodiments, the anti-KLB antibody to L6-hKLB&hFGFR2c and the activation fold of L6-hKLB
  • the anti-KLB antibody can bind to human KLB, and can also bind to monkey KLB; in some embodiments, the anti-KLB antibody can bind to human and/or monkey KLB with an EC50 value of less than 2.0E-9M, The EC50 value is detected by a flow cytometry method;
  • the anti-KLB antibody can bind to human KLB with high affinity; in some embodiments, the antibody can bind to human KLB with a KD value of less than 3.0E-9M, and the KD value is detected by Biacore method.
  • the anti-KLB antibody has high activation activity on cells expressing hKLB&hFGFR1c, and has weak activation activity on cells expressing hKLB&hFGFR2c, hKLB&hFGFR3c, and hKLB&hFGFR4; in some embodiments, the anti-KLB antibody has high activation activity on cells expressing hKLB&hFGFR1c
  • the activation fold of CHOK1 cells is greater than 10
  • the activation fold of the anti-KLB antibody to L6-hKLB&hFGFR2c and/or L6-hKLB&hFGFR4 cells is less than 2
  • the activation fold is the activation fold of the antibody relative to the blank control.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a therapeutically effective amount of the anti-KLB antibody as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients .
  • the disclosure provides an isolated nucleic acid encoding an anti-KLB antibody as described above.
  • the present disclosure provides a vector comprising the aforementioned nucleic acid molecule.
  • the disclosure provides a host cell comprising a nucleic acid as described above.
  • the present disclosure provides a method for treating a disease, the method comprising the step of administering the above-mentioned anti-KLB antibody or the pharmaceutical composition to the subject.
  • the present disclosure also provides the use of the above-mentioned anti-KLB antibody or pharmaceutical composition in the preparation of medicines for treating diseases.
  • the present disclosure also provides an anti-KLB antibody or a pharmaceutical composition as described above for use as a medicament; in some embodiments, the medicament is used for treating a disease.
  • the present disclosure provides methods of treating a disease, disorder or condition associated with human FGF21 and/or human FGF19 comprising administering to a subject (eg, a patient) a therapeutically effective amount of an anti-KLB antibody as described above or pharmaceutical compositions.
  • the disease as described above is a FGF21 and/or FGF19 related disorder.
  • the disorder is selected from a metabolic disorder, an endocrine disorder, and a cardiovascular disorder.
  • the condition is selected from obesity, diabetes (type 1 diabetes, type 2 diabetes), pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin Resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, renal disease, complications of diabetes, Neurological disorders and gastroparesis.
  • the disease is selected from the group consisting of type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, and metabolic syndrome.
  • the disease is nonalcoholic steatohepatitis.
  • the present disclosure also provides a method of inducing FGF21 and/or FGF19 signal transduction, the method comprising contacting cells expressing KLB and FGFR with an anti-KLB antibody as described above.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Amino acid analogs are compounds that have the same basic chemical structure (i.e., the alpha carbon bonded to a hydrogen, carboxyl, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide , Methylsulfonium methionine.
  • Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.
  • amino acid mutation includes amino acid substitutions (also called amino acid substitutions), deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct so long as the final construct possesses the desired properties, such as reduced or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino and/or carboxyl termini of the polypeptide chain.
  • Specific amino acid mutations may be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, that is, replacing one amino acid with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) .
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various names may be used herein to refer to the same amino acid mutation.
  • amino acid residue at a specific position can be expressed in the form of position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at the 366th position is mutated from the original T to W.
  • antibody is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antibody Fragments (or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity.
  • Native antibody refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons composed of two identical light chains and two identical heavy chains joined by disulfide bonds.
  • each heavy chain has a variable region (VH), also known as variable heavy domain, heavy chain variable region, followed by a heavy chain constant region (CH), the native IgG heavy chain constant region is usually Contains three constant domains (CH1, CH2 and CH3).
  • VH variable region
  • CH heavy chain constant region
  • each light chain has a variable region (VL), also called variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL ).
  • VH variable heavy domain
  • CH2 and CH3 heavy chain constant region
  • CL constant light domain
  • full-length antibody “intact antibody” and “whole antibody” are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain with an Fc region as defined herein.
  • Natural complete antibody light chain includes light chain variable region VL and constant region CL, VL is at the amino terminal of light chain, light chain constant region includes ⁇ chain and ⁇ chain; heavy chain includes variable region VH and constant region (CH1, CH2 and CH3), VH is at the amino-terminus (also called N-terminus) of the heavy chain, and the constant region is at the carboxy-terminus (also called C-terminus), where CH3 is closest to the carboxy-terminus of the polypeptide, and the heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
  • IgG including IgG1, IgG2, IgG3 and IgG4 subtypes
  • IgA including IgA1 and IgA2 subtypes
  • IgM and IgE IgE.
  • variable region refers to the domain of an antibody that binds the antigen.
  • the heavy chain variable region VH and the light chain variable region VL each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3
  • VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also known as the N terminus) to the carboxyl terminus (also known as the C terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4.
  • Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen binding specificity.
  • amino acid sequence boundaries of CDRs can be determined by various known schemes, for example: “Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health , Bethesda, MD), “Chothia” numbering sequence, “ABM” numbering sequence, "contact” numbering sequence (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J].
  • antibody fragment refers to a molecule other than an intact antibody that comprises the portion of an intact antibody that binds to the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibody, single chain Fab (scFab), diabody, linear antibody, single chain antibody (scFv) , and multispecific antibodies formed from antibody fragments.
  • Fc region or “fragment crystallizable region” is used to define the C-terminal region of an antibody heavy chain, including native and engineered Fc regions.
  • the Fc region comprises the same or different two subunits.
  • the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • Suitable Fc regions for use in the antibodies described herein include the Fc regions of human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • the boundaries of the Fc region can also be varied, such as deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or deletion of the C-terminal glycine and lysine of the Fc region (residue 447 according to the EU numbering system). system residues 446 and 447).
  • the numbering convention for the Fc region is the EU numbering system, also known as the EU index.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chains is derived from a particular source or species, while the remaining portion of the heavy and/or light chains is derived from another, different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and the framework portion of the variable regions).
  • human antibody “human antibody”, “fully human antibody”, and “fully human antibody” are used interchangeably to refer to antibodies whose variable and constant regions are human sequences.
  • the term encompasses antibodies that are derived from human genes but have, for example, altered sequences that reduce potential immunogenicity, increase affinity, eliminate cysteines or glycosylation sites that might cause undesired folding.
  • the term encompasses such antibodies produced recombinantly in non-human cells which may confer glycosylation not characteristic of human cells.
  • the term also encompasses antibodies that have been raised in transgenic mice containing some or all of the immunoglobulin heavy and light chain loci.
  • the meaning of human antibody expressly excludes humanized antibodies comprising non-human antigen-binding residues.
  • affinity refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, binding “affinity” refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its ligand Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by routine methods known in the art, including those described herein.
  • the term “kassoc” or “ka” refers to the on-rate of a particular antibody-antigen interaction and the term “kdis” or “kd” refers to the dissociation rate of a particular antibody-antigen interaction.
  • KD refers to the dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and is expressed as molarity (M).
  • M molarity
  • the KD value of an antibody can be determined using methods well known in the art. For example, surface plasmon resonance is measured using biosensing systems such as the system, or affinity in solution is measured by solution equilibrium titration (SET). In some embodiments, the KD value is detected by Biacore.
  • effector function refers to those biological activities attributable to an antibody Fc region (either native sequence Fc region or amino acid sequence mutated Fc region) and which vary with the antibody isotype.
  • antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g., B cell receptors, body) downregulation; and B cell activation.
  • antibody-dependent cellular cytotoxicity refers to a mechanism of inducing cell death that relies on antibody coating of target cells with lytically active effector cells ( Cells such as natural killer (NK), monocytes, macrophages and neutrophils) interact via Fc ⁇ receptors (Fc ⁇ Rs) expressed on effector cells.
  • NK cells express FcyRIIIa
  • monocytes express FcyRI, FcyRII, and FcyRIIIa.
  • the ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on labels released from lysed cells, such as radioactive substrates, fluorescent dyes, or native intracellular proteins.
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to a cell death-inducing mechanism in which the Fc effector domain of a target-binding antibody binds and activates the complement component C1q, which in turn activates the complement cascade, resulting in target cell death.
  • Activation of complement can also result in the deposition of complement components on the surface of target cells that promote CDC by binding to complement receptors (eg, CR3) on leukocytes.
  • complement receptors eg, CR3
  • the term “monoclonal antibody” refers to a population of substantially homogeneous antibodies, ie, the antibody molecules comprised in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts.
  • polyclonal antibody preparations typically comprise multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes.
  • “Monoclonal” denotes the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • the antibodies provided by the present disclosure are monoclonal antibodies.
  • antigen refers to a molecule or portion of a molecule capable of being bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody), and which can additionally be used in an animal to generate antibodies capable of binding the antigen.
  • a selective binding agent such as an antigen binding protein (including, for example, an antibody)
  • An antigen may have one or more epitopes capable of interacting with different antigen binding proteins (eg antibodies).
  • epitope refers to an area (area or region) on an antigen capable of specifically binding to an antibody or antigen-binding fragment thereof.
  • Epitopes may be formed from contiguous strings of amino acids (linear epitopes) or comprise non-contiguous amino acids (conformational epitopes), for example brought into spatial proximity by folding of the antigen, ie by tertiary folding of the proteinaceous antigen.
  • the difference between a conformational epitope and a linear epitope is that antibody binding to a conformational epitope is lost in the presence of denaturing solvents.
  • An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind a particular epitope can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, Chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.
  • an antibody such as an anti-KLB antibody
  • an antibody binds an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1 x 10 -7 M or less (eg, about 1 x 10 -8 M or less).
  • KD equilibrium dissociation constant
  • the antibody binds an antigen with a KD that is 10% or less (eg, 1%) of the antibody's KD for binding to a non-specific antigen (eg, BSA, casein).
  • KD can be measured using known methods, for example by measured by surface plasmon resonance.
  • antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, e.g. (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) are cross-reactive.
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring and non-naturally occurring, having similar binding properties to the reference nucleic acid, and defined in Metabolized in a manner similar to the reference nucleotide.
  • nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • An isolated nucleic acid encoding an anti-KLB antibody refers to one or more nucleic acid molecules encoding an anti-KLB antibody, including such one or more nucleic acid molecules in a single vector or in separate vectors, and one or more nucleic acid molecules present in a host cell Such one or more nucleic acid molecules at more positions.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is mixed with bases and/or deoxygenated Inosine residue substitution.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, and to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • sequence identity means that when two sequences are optimally aligned, gaps are introduced as necessary to obtain the maximum percent sequence identity and any conservative substitutions are not considered part of the sequence identity, two The degree (percentage) to which amino acids/nucleic acids of a sequence are identical at equivalent positions.
  • alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • fused or “linked” refer to the joining of components (eg, VH and VL in an Fv) by a covalent bond, either directly or via one or more linkers.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication).
  • vectors can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • expression vector or "expression construct” refers to a vector that can transform a host cell and contains a vector that directs and/or controls (along with the host cell) the expression of one or more heterologous coding regions operably linked thereto.
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when an intron is present, RNA splicing of the coding region to which it is operably linked.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster cells Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia willow salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomycescerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fus
  • Pichia any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Luke Mold (Chrysosporium lucknowense), any Fusarium species, Yarrowia lipolytica, and Neurospora crassa.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that not all progeny will have the exact same DNA content due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as the original transformed cell from which they were screened are included.
  • composition means a mixture comprising one or more anti-KLB antibodies described herein together with other chemical components such as physiological/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and that is nontoxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein unless otherwise indicated.
  • cyno or “cynomolgus” refers to Macaca fascicularis.
  • the individual or subject is a human.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the interaction of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human , subjects, cells, tissues, organs or biological fluids.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, faeces, sputum, mucous membrane secretions of secretory tissues and organs, vaginal secretions, ascites , pleura, pericardium, peritoneum, peritoneal and other body cavity fluids, fluid collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ condition culture medium), lavage fluid, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, faeces, sputum, mucous membrane
  • Treatment refers to clinical intervention that attempts to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation/reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or palliation of disease state, and regression or amelioration of prognosis.
  • the antibodies of the disclosure are used to delay the development of a disease or slow the progression of a disease.
  • the anti-KLB antibodies of the present disclosure have the effect of mimicking FGF21 and/or FGF19 in vivo and induce FGF21-like signal transduction and/or FGF19-like signaling
  • the unique properties of transduction in some embodiments, the antibodies exhibit activities consistent with the natural biological function of FGF21. This property makes anti-KLB antibodies useful for the treatment of FGF21 and/or FGF19-related metabolic diseases, such as type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, metabolic syndrome, and for the need to mimic or enhance FGF19 and Treatment of any disease, disorder or condition in which FGF21 acts in vivo.
  • an “effective amount” is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate the impairment caused by or associated with the disease state (e.g. lung disease).
  • the effective amount is a therapeutically or prophylactically effective amount.
  • a “therapeutically effective amount” is sufficient to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease state. The amount of progression of the desired symptoms.
  • a “prophylactically effective amount” is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms .
  • Complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose, but may occur after administration of a series of doses.
  • a therapeutically or prophylactically effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “prophylactically effective amount” can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.
  • Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of a patient.
  • the present disclosure obtains an antibody specifically binding to KLB through a large number of experiments.
  • the anti-KLB antibody of the present disclosure has many favorable properties, such as high affinity for antigen binding, human and monkey cross-reactivity, strong activation activity on hKLB&hFGFR1c cells but weak activation activity on hFGFR1c recombinant cells , selectivity to hKLB&hFGFR2c, hKLB&hFGFR3c and hKLB&hFGFR4 cells, pharmacokinetic properties and/or druggability, etc.
  • the present disclosure provides an antibody that specifically binds KLB, and in some embodiments, the antibody is a full-length antibody or an antigen-binding fragment thereof (e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibody, single chain Fab (scFab), diabody, linear antibody, single chain antibody (scFv)), in some embodiments, has one or more of the following functional activities:
  • the anti-KLB antibody has activation activity to cells expressing hKLB &hFGFR1c; in some embodiments, the anti-KLB antibody has high activation activity to cells expressing hKLB &hFGFR1c; in some embodiments, the anti-KLB antibody has a high activation activity to cells expressing hKLB & hFGFR1c
  • the activation fold of CHOK1 cells expressing hKLB&hFGFR1c is greater than 10, and/or the activation fold of the anti-KLB antibody to HEK293T-hKLB&hFGFR1c cells is greater than 2; in some embodiments, the activation fold is the activation fold of the antibody relative to the blank control; In some embodiments, the activation factor is detected according to the methods of Test Examples 4 and 5 of the present disclosure;
  • the anti-KLB antibody has weak activation activity to recombinant cells expressing hFGFR1c (only hFGFR1c, not expressing hKLB); in some embodiments, the activation fold of the anti-KLB antibody to HEK293-hFGFR1c cells is less than 1; in some embodiments Wherein, the activation multiple is the activation multiple of the antibody relative to the blank control; in some embodiments, the activation multiple is detected according to the method of Test Example 6 of the present disclosure;
  • the anti-KLB antibody has weak activation activity to cells expressing hKLB&hFGFR2c, hKLB&hFGFR3c and hKLB&hFGFR4; in some embodiments, the anti-KLB antibody has weak activation activity to L6-hKLB&hFGFR2c and/or L6-hKLB&hFGFR4 cells; in some In some embodiments, the activation factor of the anti-KLB antibody to L6-hKLB&hFGFR2c and/or L6-hKLB&hFGFR4 cells is less than 2; The activation activity of the anti-KLB antibody is weak, in some embodiments, the activation fold of the anti-KLB antibody to L6-hKLB&hFGFR2c, hKLB&hFGFR3c and/or L6-hKLB&hFGFR4 cells is less than 2; in some embodiments, the anti-KLB antibody to L6-hKLB&hFGFR2c and the activation fold of L6-hKLB
  • the anti-KLB antibody can bind to human KLB, and can also bind to monkey KLB; in some embodiments, the anti-KLB antibody can bind to human and/or monkey KLB with an EC50 value of less than 2.0E-9M, The EC50 value is detected by a flow cytometry method;
  • the anti-KLB antibody can bind to human KLB with high affinity; in some embodiments, the antibody can bind to human KLB with a KD value of less than 3.0E-9M, and the KD value is detected by Biacore method.
  • the anti-KLB antibody has high activation activity on cells expressing hKLB&hFGFR1c, and has weak activation activity on cells expressing hKLB&hFGFR2c, hKLB&hFGFR3c, and hKLB&hFGFR4; in some embodiments, the anti-KLB antibody has high activation activity on cells expressing hKLB&hFGFR1c
  • the activation fold of CHOK1 cells is greater than 10
  • the activation fold of the anti-KLB antibody to L6-hKLB&hFGFR2c and/or L6-hKLB&hFGFR4 cells is less than 2
  • the activation fold is the activation fold of the antibody relative to the blank control.
  • the anti-KLB antibody according to any one of the above, which comprises a heavy chain variable region and a light chain variable region, wherein i) the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively
  • the sequence of HCDR1, HCDR2 and HCDR3 of any sequence in SEQ ID NO: 40-48 is the same, and the sequence of LCDR1, LCDR2 and LCDR3 of the light chain variable region is the same as that of LCDR1, LCDR3 and LCDR1 in SEQ ID NO: 34, respectively.
  • amino acid sequences of LCDR2 and LCDR3 are the same; or the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively identical to the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 2, and the same as the amino acid sequences of the light chain variable region
  • sequences of LCDR1, LCDR2 and LCDR3 of the region are identical to the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 3, respectively; or
  • the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are identical to the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 49, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 of any sequence in SEQ ID NO: 56-68 are the same; or the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively identical to those of SEQ ID NO: 4
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 are identical, and the sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively identical to the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 5; or
  • sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are identical to the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 18, respectively, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region The sequences are identical to the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 19, respectively.
  • the anti-KLB antibody according to any one of the above, wherein the CDRs are defined according to a numbering convention selected from Kabat, IMGT, Chothia, AbM and Contact. In some embodiments,
  • HCDR1 of the heavy chain variable region is set forth in SEQ ID NO: 6
  • HCDR2 is set forth in SEQ ID NO: 37, 38, 39 or 7
  • HCDR3 is set forth in SEQ ID NO: 8
  • LCDR1 of the light chain variable region is shown in SEQ ID NO: 9
  • LCDR2 is shown in SEQ ID NO: 10
  • LCDR3 is shown in SEQ ID NO: 11; or
  • HCDR1 of the heavy chain variable region is shown in SEQ ID NO: 12
  • HCDR2 is shown in SEQ ID NO: 13
  • HCDR3 is shown in SEQ ID NO: 14 and
  • LCDR1 of the light chain variable region is shown in SEQ ID NO: 15, 69, 70, 71, 72, 73 or 74
  • LCDR2 is shown in SEQ ID NO: 16, or 75
  • LCDR3 is shown in SEQ ID NO: 17; or
  • HCDR1 of said heavy chain variable region is set forth in SEQ ID NO: 24
  • HCDR2 is set forth in SEQ ID NO: 25
  • HCDR3 is set forth in SEQ ID NO: 26 and
  • LCDR1 of the light chain variable region is shown in SEQ ID NO: 27
  • LCDR2 is shown in SEQ ID NO: 28
  • LCDR3 is shown in SEQ ID NO: 29.
  • HCDR1 of said heavy chain variable region is set forth in SEQ ID NO: 6
  • HCDR2 is set forth in SEQ ID NO: 37
  • HCDR3 is set forth in SEQ ID NO: 8
  • LCDR1 of the light chain variable region is shown in SEQ ID NO: 9
  • LCDR2 is shown in SEQ ID NO: 10
  • LCDR3 is shown in SEQ ID NO: 11; or
  • HCDR1 of said heavy chain variable region is set forth in SEQ ID NO: 12
  • HCDR2 is set forth in SEQ ID NO: 13
  • HCDR3 is set forth in SEQ ID NO: 14 and
  • LCDR1 of the light chain variable region is shown in SEQ ID NO:69
  • LCDR2 is shown in SEQ ID NO:75
  • LCDR3 is shown in SEQ ID NO:17.
  • the anti-KLB antibody according to any one of the above, is a heavy chain framework region of IGHV1-3*01/IGHJ6*01 comprising , 24, 44, 71 and 91 (according to Kabat numbering system numbering) in one or more amino acid back mutations, the framework region of the light chain variable region is in the IGKV1-39*01/IGKJ4*01 light chain framework region comprising one or more amino acid backmutations selected from positions 2, 4, 36, 38, 43, 44 and 58 (numbered according to the Kabat numbering system).
  • the anti-KLB antibody wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6, and HCDR2 comprises the amino acid sequence of SEQ ID NO: 37, 38, 39 or 7 , and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO : the amino acid sequence of 11;
  • the framework region of the heavy chain variable region of the antibody is that on the heavy chain framework region of IGHV1-3*01/IGHJ6*01, it also comprises the group selected from 1E, 24T, 44G, 71S and 91F (according to Kabat Numbering system numbering) in one or more amino acid back mutations, and/or the framework region of the light chain variable region is comprised of selected from 2V, 4I on the IGKV1-39*01/IG
  • the anti-KLB antibody according to any one of the above, is a heavy chain framework region of IGHV1-3*01/IGHJ6*01 comprising , 24, 48, 67, 69, 71 and 73 (numbering according to the Kabat numbering system) in one or more amino acid mutations, the framework region of the light chain variable region is in IGKV6-21*02/JGKJ2*01 or The IGKV3-20*02/JGKJ2*01 light chain framework region contains one or more amino acid back mutations selected from positions 2, 45, 47, 49, 58 and 71 (numbered according to the Kabat numbering system).
  • the anti-KLB antibody wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 13
  • the amino acid sequence of NO: 14 and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, 69, 70, 71, 72, 73 or 74, and the LCDR2 comprises the amino acid sequence of SEQ ID NO: 16 or 75 Sequence, and LCDR3 comprise the aminoacid sequence of SEQ ID NO: 17;
  • the framework region of the heavy chain variable region of described antibody is to comprise on IGHV1-3*01/IGHJ6*01 heavy chain framework region and be selected from 1E, 24G, 48I , 67A, 69V, 71V and 73K (numbering according to the Kabat numbering system), and/or the framework region of the light chain variable region is in IGKV6-21*02/J
  • amino acid sequence variants of the anti-KLB antibodies provided herein are contemplated.
  • Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the anti-KLB antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.
  • anti-KLB antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitution mutagenesis include CDRs and FRs.
  • Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions”. More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes.
  • Amino acid substitutions can be introduced into an antibody of interest, and the products screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions would entail replacing a member of one of these classes for a member of another class.
  • substitutional variant involves substituting one or more CDR residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg. a humanized or human antibody
  • the resulting variant selected for further study will have an altered (e.g. improved) certain biological property (e.g. increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Some of the biological properties of the parental antibody are retained.
  • An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (eg, binding affinity).
  • Alterations can be made to the CDRs, eg, to improve antibody affinity. Such changes can be made to CDR "hot spots", i.e. residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and/or residues that contact antigen, while making changes to the resulting variant VH or VL test for binding affinity.
  • affinity maturation diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity.
  • CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • HCDR3 and LCDR3 are frequently targeted.
  • substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes eg, conservative substitutions, as provided herein
  • Such changes may, for example, be outside antigen contacting residues in the CDRs.
  • alanine scanning mutagenesis One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis".
  • a residue or group of target residues e.g. charged residues such as Arg, Asp, His, Lys and Glu
  • neutral or negatively charged amino acids e.g. Ala or polyalanine
  • Further substitutions may be introduced at amino acid positions showing functional sensitivity to the initial substitution.
  • contact points between antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution.
  • Variants can be screened to determine whether they contain desired properties.
  • Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, and intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with an N-terminal methionyl residue.
  • Other insertional variants of antibody molecules include fusions of the N- or C-terminus of the antibody to enzymes or polypeptides that extend the serum half-life of the antibody.
  • the Fc region of an anti-KLB antibody of the disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, such as its binding to an Fc ⁇ receptor, and reduce or Eliminate effector functions.
  • a native IgG Fc region specifically an IgG 1 Fc region or an IgG 4 Fc region, may cause the anti-KLB antibodies of the present disclosure to target cells expressing Fc receptors, rather than cells expressing antigens.
  • the engineered Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and/or reduced effector function.
  • the engineered Fc region has a binding affinity for Fc receptors that is reduced by more than 50%, 80%, 90%, or 95% compared to a native Fc region.
  • the Fc receptor is an Fc gamma receptor.
  • the Fc receptor is a human Fc ⁇ receptor, eg, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the engineered Fc region also has reduced binding affinity for complement, such as C1q, compared to a native Fc region.
  • the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to a native Fc region.
  • the engineered Fc region has reduced effector function, which may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased interaction with NK cells decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling-induced apoptosis, decreased dendritic cell maturation, or decreased T cells primed.
  • CDC complement-dependent cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion decreased immune complex-mediated antigen uptake by antigen-presenting cells
  • decreased interaction with NK cells decreased binding to macrophages
  • monocytes decreased binding to monocytes
  • polymorphonuclear cells
  • amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function.
  • the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
  • amino acid residue substitutions at positions such as 228 may reduce effector function.
  • Anti-KLB antibodies may also contain disulfide bond modifications, eg, 354C of the first subunit and 349C of the second subunit.
  • Anti-KLB antibodies may contain different antigen-binding moieties fused to the two subunits of the Fc region, thus potentially leading to undesired homodimerization.
  • the Fc region of the present disclosure comprises modifications according to the knob-into-hole (KIH) technique, which involves the introduction of a knob at the interface of one subunit and A hole structure (hole) is introduced at the interface. This enables the protrusion structure to be positioned in the hole structure, promotes the formation of heterodimers and inhibits the generation of homodimers.
  • KIH knob-into-hole
  • the bulge structure is constructed by replacing small amino acid side chains from the interface of one subunit with larger side chains (such as tyrosine or tryptophan). Instead, the pore structure is created in the interface of another subunit by replacing large amino acid side chains with smaller ones, such as alanine or threonine.
  • the protrusion structure and hole structure are prepared by changing the nucleic acid encoding the polypeptide, and the optional amino acid substitutions are shown in Table 3 below:
  • knob-and-hole technique In addition to the knob-and-hole technique, other techniques for modifying the CH3 domain of the heavy chain of a multispecific antibody to achieve heterodimerization are known in the art, for example WO96/27011, WO98/050431, EP1870459, WO2007/ 110205, WO 007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO013/096291.
  • the C-terminus of the Fc region may be a complete C-terminus ending with the amino acid residue PGK; it may also be a shortened C-terminus in which, for example, one or two C-terminal amino acid residues have been removed.
  • the C-terminus of the heavy chain is a shortened C-terminus ending in PG.
  • a composition of intact antibodies can include a population of antibodies from which all K447 residues and/or G446+K447 residues have been removed.
  • a composition of intact antibodies can include a population of antibodies in which the K447 residue and/or the G446+K447 residues have not been removed.
  • the composition of whole antibodies has a population of antibodies with and without a K447 residue and/or a mixture of antibodies with G446+K447 residues.
  • Anti-KLB antibodies can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding anti-KLB antibodies are provided.
  • the present disclosure provides an isolated nucleic acid encoding an anti-KLB antibody as previously described. Such nucleic acid may be derived from an independent polypeptide chain encoding any of the foregoing.
  • the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.
  • the disclosure provides host cells comprising such nucleic acids.
  • a method of producing an anti-KLB antibody comprisin said method comprises, under conditions suitable for expression, culturing a host cell comprising a nucleic acid encoding said anti-KLB antibody, as provided above, and optionally The anti-KLB antibody is efficiently recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods or obtained by chemical synthesis.
  • Suitable host cells for cloning or expressing vectors encoding anti-KLB antibodies include prokaryotic or eukaryotic cells described herein. For example, it can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. After expression, it can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-KLB antibody-encoding vectors, including fungal and yeast strains.
  • Suitable host cells suitable for expressing anti-KLB antibodies may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells.
  • a number of baculovirus strains have been identified for use in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, e.g.
  • vertebrate cells can also be used as hosts, eg mammalian cell lines adapted for growth in suspension.
  • suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); Sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells ( BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • Suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as YO, NSO and Sp2/0.
  • CHO Chinese Hamster Ovary
  • myeloma cell lines such as YO, NSO and Sp2/0.
  • Anti-KLB antibodies provided herein can be identified, screened or characterized for their physical/chemical characteristics and/or biological activity by various assays known in the art.
  • the anti-KLB antibodies of the present disclosure are tested for activity, eg, by known methods such as ELISA, Western blot, and the like.
  • the anti-KLB antibodies provided by the present disclosure can be used to detect the presence of KLB in a biological sample.
  • the term “detection” encompasses quantitative or qualitative detection.
  • the biological sample comprises cells or tissue, such as tumor tissue.
  • an anti-KLB antibody for use in a method of diagnosis or detection.
  • methods of detecting the presence of KLB in a biological sample are provided.
  • the method comprises contacting the biological sample with an anti-KLB antibody under suitable conditions, and detecting whether a complex is formed between the detection reagent and the antigen. Such methods can be in vitro or in vivo methods.
  • an anti-KLB antibody is used to select subjects suitable for treatment, eg, KLB is a biomarker for patient selection.
  • Exemplary disorders that can be diagnosed using the anti-KLB antibodies of the present disclosure are eg diabetes, cardiovascular disease, insulin resistance, hypertension, thromboembolic disease or dyslipidemia; especially non-alcoholic steatohepatitis, diabetes.
  • labeled anti-KLB antibodies include, but are not limited to, labels or moieties for direct detection (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties for indirect detection (e.g., indirect detection via enzymatic reactions or molecular interactions).
  • modules such as enzymes or ligands).
  • the present disclosure provides the use of an anti-KLB antibody in the manufacture or preparation of a medicament.
  • the disease is a disease or disorder associated with KLB.
  • the disease is selected from: diabetes, cardiovascular disease, insulin resistance, hypertension, thromboembolic disease or dyslipidemia; especially non-alcoholic steatohepatitis, diabetes.
  • the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents agent).
  • a "subject" according to any of the above embodiments may be a human.
  • the anti-KLB antibodies of the present disclosure (including antibodies that bind to human and/or cynomolgus monkey KLB) have the effect of mimicking FGF21 and/or FGF19 in vivo and induce FGF21-like signal transduction and/or FGF19-like signaling Unique properties of transduction; in some embodiments, the antibodies exhibit activities consistent with the native biological function of FGF21. This property makes the anti-KLB antibodies of the present disclosure useful for the treatment of FGF21 and/or FGF19 related metabolic diseases, such as type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, metabolic syndrome, and the need to mimic or enhance FGF19 and/or the in vivo effects of FGF21 for the treatment of any disease, disorder or condition.
  • FGF21 and/or FGF19 related metabolic diseases such as type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, metabolic syndrome, and the need to mimic or enhance FGF19 and/or the in vivo effects of FGF21 for the treatment of any disease, disorder or condition.
  • a pharmaceutical composition comprising the anti-KLB antibody, eg, for any of the above pharmaceutical uses or methods of treatment.
  • a pharmaceutical composition comprises any of the anti-KLB antibodies provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • anti-KLB antibodies of the present disclosure can be used alone or in combination with other agents for therapy.
  • an antibody of the present disclosure can be co-administered with at least one additional therapeutic agent.
  • Anti-KLB antibodies of the present disclosure may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if local treatment is desired, intralesional.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
  • a variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • anti-KLB antibodies of the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner. factor. Anti-KLB antibodies may or may not be formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of disorder or treatment, and other factors. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at other dosages, and any route empirically/clinically determined to be appropriate.
  • the appropriate dose of the anti-KLB antibody of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the therapeutic molecule Type, severity and course of disease, whether administered for prophylactic or therapeutic purposes, previous therapy, patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician.
  • the therapeutic molecule is suitably administered to the patient at one time or over a series of treatments.
  • a daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg, depending on the factors mentioned above.
  • an article of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of the disorders described above.
  • the article comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like.
  • Containers can be formed from various materials such as glass or plastic.
  • the container contains a composition effective, alone or in combination with another composition, for the treatment, prophylaxis and/or diagnosis of a condition, and may have a sterile access opening (e.g., the container may have a stopper pierceable by a hypodermic needle). IV solution bag or vial).
  • At least one active agent in the composition is an anti-KLB antibody of the present disclosure.
  • the label or package insert indicates that the composition is used to treat the condition of choice.
  • the article of manufacture may comprise: (a) a first container having a composition therein, wherein the composition comprises an anti-KLB antibody of the present disclosure; and (b) a second container having a composition therein, wherein the composition The drug contains an additional cytotoxic or other therapeutic agent.
  • the article of manufacture of this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. It may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • human KLB&FGFR1c complex (see Uniprot ID: Q86Z14 for human KLB sequence, see Uniprot ID: P11362 for human FGFR1c sequence), cynomolgus monkey KLB&FGFR1c complex (cynomolgus monkey KLB sequence, see Genebank ID: EHH53620.1, cynomolgus monkey FGFR1c For the sequence, see SEQ ID NO: 1), mouse KLB & FGFR1c complex (see Uniprot Q99N32 for the mouse KLB sequence, and Uniprot ID: P16092 for the mouse FGFR1c sequence), and a cell line expressing human FGFR1c alone.
  • CHOK1 cells ATCC, CCL-61) or HEK293 cells (ATCC, CRL-1573) lines were co-infected with packaged viruses containing KLB and FGFR1c genes at a certain ratio.
  • human FGFR1c overexpressing cell lines only CHOK1 or HEK293 cell lines were infected with viruses containing the FGFR1c gene. After 48 hours of infection, the virus-containing culture supernatant was removed, and the corresponding antibiotics were added for pressurized selection for 5 days, and CHOK1 or HEK293 monoclonal cells highly expressing KLB&FGFR1c complex or FGFR1c were obtained by flow sorting.
  • the hybridoma cell lines mAb-99 and mAb-18 with good activity were screened, sequenced and amplified, and the hybridoma cells in the logarithmic growth phase were collected, and Trizol (Invitrogen, Cat No. 15596-018) was used according to the kit instructions.
  • RNA was extracted and reverse transcribed using PrimeScript TM Reverse Transcription Kit (Takara, Cat No. 2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR with mouse Ig-Primer Set (Novagen, TB326Rev.B 0503), and then sent to a sequencing company for sequencing, and the variable regions of hybridoma cloned mouse antibodies mAb-99 and mAb-18 were measured The sequence looks like this:
  • B cells were isolated from human PBMC, spleen, and lymph node tissues, and RNA was extracted to construct a natural single-chain phage antibody library.
  • the constructed natural single-chain phage antibody library is packaged to form phage particles, the recombinant cell line overexpressing human KLB is used for panning, and CHOK1-hKLB (see Example 1) and HEK293T-hKLB (see Example 1) are used for recombination Cells were cross-panned. After 3 rounds of panning, single-clonal colonies were picked and packaged into phage scFv, and the binding activity of phage to human KLB protein was tested by ELISA.
  • variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 is formed, and then the variable region sequence is fused with a human constant region sequence to obtain a humanized antibody.
  • the humanization of mAb-99 and mAb-18 murine antibodies is exemplarily described as follows:
  • the template for the humanized heavy chain of the mAb-99 antibody is IGHV1-3*01/IGHJ6*01, that is, the FR1, FR2, FR3 of the human germline heavy chain IGHV1-3*01, and the JH6 region of IGHJ6*01 (as FR4) is used as the heavy chain framework region of the humanized antibody;
  • the light chain template is IGKV1-39*01/IGKJ4*01, that is, FR1, FR2, FR3, and IGKJ4*01 of the human germline light chain IGKV1-39*01 are selected
  • the JK4 region (as FR4) served as the humanized antibody light chain framework region.
  • the CDR region of the murine antibody mAb-99 was grafted onto the selected humanized template to replace the CDR region of the humanized template to obtain the humanized antibody variable region sequence of mAb-99.
  • the amino acid residues at positions 1, 24, 44, 71 and/or 91 (numbered according to the Kabat numbering system) on the heavy chain variable region of the humanized antibody were back mutated, and amino acid residues at positions 2, 44, 71 and/or 91 on the light chain variable region were Amino acid residues at positions 4, 36, 38, 43, 44 and/or 58 (numbered according to the Kabat numbering system) are back-mutated to obtain the variable region of the heavy/light chain of the humanized antibody, and the specific sequence is as follows:
  • variable region sequence 6th and 7th amino residues of HCDR2 (YIYIGNGDIEYNAKFKG) in the variable region of the heavy chain were also mutated from NG to NV, NL or QG to obtain a new humanized antibody variable region sequence, as follows:
  • HCDR2 mutant sequence of the heavy chain variable region of hAb99 is shown in Table 6 below:
  • the above heavy chain variable region is fused with the heavy chain constant region (as shown in SEQ ID NO: 22), and the light chain variable region is fused with the light chain constant region (as shown in SEQ ID NO: 23) to construct an anti-KLB antibody , and the binding activity of the obtained antibody to CHOK1-hKLB was detected (see Test Example 2 of this disclosure for the experimental method), and the experimental results are shown in Table 7.
  • VH Antibody Heavy Chain Variable Region
  • VL Light chain variable region
  • VH Light chain variable region
  • VL Light chain variable region
  • mAb99-hIgG1 is a chimeric antibody of mAb99, its heavy chain constant region is shown in SEQ ID NO: 22, and its light chain constant region is shown in SEQ ID NO: 23.
  • the light chain and heavy chain sequences of the hAb99-13 antibody are as follows:
  • the template for the humanized heavy chain of the mAb-18 antibody is IGHV1-3*01/IGHJ6*01, that is, the FR1, FR2, FR3 of the human germline heavy chain IGHV1-3*01, and the JH6 region of IGHJ6*01 (FR4 ) as the heavy chain framework region of the humanized antibody;
  • the light chain template is IGKV6-21*02/JGKJ2*01 or IGKV3-20*02/JGKJ2*01, that is, the human germline light chain IGKV6-21*02 or IGKV3- FR1, FR2, FR3 of 20*02, and JK2 region (as FR4) of JGKJ2*01 were used as the light chain framework region of the humanized antibody.
  • the CDR region of the murine antibody mAb-18 was grafted onto the selected humanized template to replace the CDR region of the humanized template to obtain the humanized antibody variable region sequence of mAb-18.
  • the amino acid residues at positions 24, 48, 67, 69, 71 and/or 73 (numbered according to the Kabat numbering system) of the heavy chain variable region of the humanized antibody are back mutated, and the first position ( Amino acid residues were mutated according to the Kabat numbering system, and amino acid residues at positions 2, 45, 47, 49, 58 and/or 71 of the light chain variable region (numbered according to the Kabat numbering system) were mutated back to obtain human Antibody variable region sequence, the specific sequence is as follows:
  • the above heavy chain variable region is fused with the heavy chain constant region (as shown in SEQ ID NO: 22), and the light chain variable region is fused with the light chain constant region (as shown in SEQ ID NO: 23) to construct an anti-KLB antibody , and detect the binding activity of the obtained antibody to CHOK1-hKLB (see test example 2 of this disclosure for the experimental method), and the experimental results are shown in Table 9:
  • VH Light chain variable region
  • VL Light chain variable region
  • SEQ ID NO: 52 1.34 hAb18-2 SEQ ID NO: 49 SEQ ID NO: 53 1.58 hAb18-3 SEQ ID NO: 50
  • SEQ ID NO: 52 1.9 hAb18-4 SEQ ID NO: 50
  • SEQ ID NO: 52 1.8 hAb18-6 SEQ ID NO: 51 SEQ ID NO: 53 2 hAb18-7
  • 49 SEQ ID NO: 56 2.4 hAb18-8 SEQ ID NO: 49 SEQ ID NO: 57 2.5 hAb18-9 SEQ ID NO: 49 SEQ ID NO: 58 2.4 hAb18-10 SEQ ID NO: 49 SEQ ID NO: 59 2.7 hAb18-11 SEQ ID NO: 49 SEQ ID NO: 60 2.7 hAb18-12 SEQ ID NO: 49 SEQ ID NO: 61 1.9
  • mAb18-hIgG1 is a chimeric antibody of mAb18, its heavy chain constant region is shown in SEQ ID NO:22, and its light chain constant region is shown in SEQ ID NO:23.
  • the light and heavy chain sequences of the hAb18-8 antibody are as follows:
  • the designed antibody sequence was codon-optimized to produce human codon-biased coding gene sequence, and primers were designed to PCR to build each antibody VH/VK gene fragment, and then combined with the expression vector pHr (with signal peptide and constant region gene (CH1-FC /CL) fragment) for homologous recombination to construct the antibody full-length expression plasmid VH-CH1-FC-pHr/VK-CL-pHr.
  • the plasmids expressing the heavy and light chains of the antibody were transfected into HEK293E cells at a ratio of 1:1.5. After 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The target protein was eluted with 100mM acetic acid, pH3.0, and neutralized with 1M Tris-HCl, pH8.0. After the eluted sample was properly concentrated, it was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, and monomer peaks were collected and separated for use.
  • GE gel chromatography Superdex200
  • Test example 1 Affinity experiment of BIAcore detecting anti-hKLB antibody
  • Test example 2 Flow cytometry detection of antibody binding to KLB on the cell membrane surface
  • the ability of the antibody to bind to CHOK1 cell lines CHOK1-hKLB and CHOK1-cynoKLB overexpressing human KLB and monkey KLB was detected by flow cytometry.
  • the details are as follows: the above cells were cultured in 10% FBS IMDM medium, placed in a 37°C, 5% CO 2 incubator, cultured for 2 days, and added to the cell plate according to the number of cells per well 1 ⁇ 10 5 Centrifuge at 300g for 5 minutes and wash once with 1% BSA.
  • Bio-FGF21/Bio-FGF19 (FGF21 is shown in SEQ ID NO: 76, FGF19 is shown in SEQ ID NO: 77, respectively, with Biotin Labeling Kit-NH2 (purchased from DOJINDO Molecular technologies Inc., LK03) for FGF21 and FGF19 (obtained by Biotin labeling) was immobilized onto the SA sensor at a concentration of 2ug/mL (incubated for 80 seconds), and then the sensor was transferred to a premix containing KLB (R&D, 5889-KB-1mg) or KLB+antibody premix (incubated for at least 15 minutes in advance). ) of the sample wells, combined for 180 seconds.
  • the ForteBio OCTET HTX instrument detects the reaction signal in real time and obtains the binding curve. The experimental results are shown in Table 12 below.
  • Test Example 4 Detection of agonistic activity of antibody against recombinant cell line CHOK1-hKLB&hFGFR1c
  • CHOK1-hKLB&hFGFR1c cells were seeded in a 96-well plate (Corning, #3903) at a density of 15,000 cells/well using DME/F12 medium containing 10% FBS, 10 ⁇ g/mL puromycin and 800 ⁇ g/mL G418. 100 ⁇ L of cell suspension was added to each well, and only 100 ⁇ L of PBS was added to the periphery of the 96-well plate. Place in a 37°C, 5% CO 2 cell incubator and culture overnight. The next day, the medium was discarded and 50 ⁇ L starvation medium (DME/F12 without FBS) was added to each well.
  • Cells were transfected with pFA2-Elk1 and pFR-Luc at a ratio of 1:6 with Lipofectamine 3000, and 10 ⁇ L of plasmid and Lipofectamine 3000 mixture was added to each well. After transfection, place the plate in a 37°C, 5% CO2 incubator for 24 hours. On the third day, add 60 ⁇ L of the antibody to be tested that was serially diluted with the starvation medium to each well. The final concentration of the antibody is 9 concentration points of 4-fold serial dilution starting from 200 nM. Set the starvation medium as the blank control well, and place the well plate Incubate in a 37°C, 5% CO 2 cell incubator for 24 hours.
  • Test Example 5 Antibody agonistic activity detection experiment on HEK293T expressing human KLB and human FGFR1c
  • HEK293T cells were seeded in 96-well plates (Corning BioCoat, #356692) at a density of 40,000 cells/well using DMEM/HIGH GLUCOSE medium containing 10% FBS, 100 ⁇ L cell suspension per well, 96-well plates Add only 100 ⁇ L of PBS to the periphery. The well plate was placed in a 37°C, 5% CO 2 cell incubator and cultured overnight. The next day, the original medium was discarded, and 50 ⁇ L starvation medium (DMEM/HIGH GLUCOSE without FBS) was added to each well.
  • HEK293-hFGFR1c cells were seeded in 96-well plates (Corning, #3903) at a density of 25,000 cells/well using DMEM/HIGH GLUCOSE medium containing 10% FBS, 100 ⁇ L cell suspension per well, 96 wells Add only 100 ⁇ L of PBS to the periphery of the plate. The well plate was placed in a 37°C, 5% CO 2 cell incubator and cultured overnight. The next day, cells were transfected with SRE-Luc2P with Lipofectamine 3000, and 10 ⁇ L of the mixture of plasmid and Lipofectamine 3000 was added to each well.
  • Test Example 7 Antibody Determination of ERK Phosphorylation Level in L6/hKLB Cells Expressing FGFR2c/FGFR3c/FGFR4
  • the selectivity of the antibody to FGFR2c, FGFR3c or FGFR4 is determined by measuring the effect of the tested antibody on the increase of ERK phosphorylation level of the L6/hKLB cell line expressing human FGFR2c, FGFR3c or FGFR4 through in vitro cell experiments.
  • the specific method is as follows:
  • L6-hKLB monoclonal cell line (L6 cells were derived from ATCC, CRL-1458) stably expressing human KLB (see Uniprot ID: Q86Z14) was constructed by lentivirus infection, and then respectively L6-hKLB&hFGFR2c, L6-hKLB&hFGFR3c and L6-hKLB&hFGFR4 cells stably expressing human FGFR2c (human FGF receptor 2c), human FGFR3c (human FGF receptor 3c) and human FGFR4 (human FGF receptor 4) were constructed.
  • L6-hKLB monoclonal cell line L6 cells were derived from ATCC, CRL-1458 stably expressing human KLB (see Uniprot ID: Q86Z14) was constructed by lentivirus infection, and then respectively L6-hKLB&hFGFR2c, L6-hKLB&hFGFR3c and L6-hKLB&hFGFR4 cells stably expressing human FGFR
  • Test Example 8 Antibody drug efficacy experiment in vivo
  • a rhesus monkey race spontaneous non-alcoholic steatohepatitis (NASH) model was selected, and MRI imaging technology and liver biopsy technology were used to evaluate the effect of antibodies on fat content in the liver, NAS (NAFLD Activity Score) score, body weight, BMI (Body Mass Index), abdominal circumference and glucose and lipid metabolism.
  • NASH non-alcoholic steatohepatitis
  • NASH rhesus monkeys were adapted to domestication and feeding for 35 days, during which the glucose and lipid metabolism indicators of the rhesus monkeys were detected, and the liver biopsy pathology was conducted under the guidance of ultrasound, and the fat content in the liver was quantitatively analyzed by MRI (GE, 3T) , to screen rhesus monkeys with stable hepatic fat content index, and divide them into three groups on average: hAb99-13 group (13mg/kg), hAb18-8 group (13mg/kg) and placebo group (vehicle control group, 10mM glacial acetic acid , 9% sucrose, pH 5.2). Each group was administered intravenously, once every 2 weeks, and administered 5 times in total.
  • liver biopsy was performed under the guidance of GE ultrasound, and the sitting height and abdominal circumference of rhesus monkeys were checked before and after administration; glucose and lipid metabolism and liver function of rhesus monkeys were regularly detected index and body weight, and calculate their average.
  • the experimental results are shown in Table 17-1 to Table 17-10, in which,
  • the experimental results of fat content in the liver are shown in Table 17-1.
  • the experimental results show that before administration and 60 days after administration (D60) in the placebo group, the percentage of ROI (Region of interest) was significantly changed; after administration of the hAb99-13 group At 60 days, the ROI% value decreased significantly, from 8.4% before administration (D0) to 6.6% (P ⁇ 0.05).
  • rate of change (current value-baseline value)/baseline value*100%, wherein, the baseline value is the ROI (%) before administration, and the current value is the ROI (%) at the time point to be detected (D60)
  • the pathological analysis results of the liver biopsy are shown in Table 17-2.
  • the experimental results showed that the pathological results of the liver tissue in the placebo group before and after administration had no obvious improvement;
  • the daily average NAS score decreased from 3.7 to 2.7, and the steatosis score also decreased significantly.
  • D0 refers to the detection value before administration
  • D65 refers to the detection value on the 65th day after the first administration.
  • rate of change (current value - baseline value) / baseline value * 100%, where the baseline value is AUC Ins0-30min (U*min/L) before administration, and the current value is the time point (D65) to be detected AUC Ins0-30min (U*min/L)
  • HOMA-IR change rate (current value - baseline value) / baseline value * 100%, where the baseline value is HOMA-IR before administration, and the current value is HOMA-IR at the time point to be detected after administration.
  • D0 is before administration
  • D7 is the 7th day after the first administration
  • FPG rate of change (current value-baseline value)/baseline value*100%, wherein, the baseline value is the FPG value before administration, and the current value is The FPG value at the time point to be detected after administration.
  • D0 is before administration
  • D7 is the 7th day after the first administration
  • rate of change (current value-baseline value)/baseline value*100%
  • the baseline value is the FPI value before administration
  • the current value is the given FPI value.
  • D0 is before administration
  • D7 is the 7th day after the first administration
  • rate of change (current value-baseline value)/baseline value*100%
  • the baseline value is the TG value before administration
  • the current value is the TG value given The TG value at the time point to be detected after the drug.
  • the experimental results of the effect of antibodies on NASH rhesus monkey adiponectin (Adiponectin) are shown in Table 17-8.
  • the experimental results show that the animal adiponectin in the placebo group has a downward trend; degree of increase.
  • D0 is before administration
  • D7 is the 7th day after the first administration
  • rate of change (current value-baseline value)/baseline value*100%
  • the baseline value is the value of Adiponectin before administration
  • the current value is given The Adiponectin value at the time point to be detected after the drug.
  • rate of change (current value - baseline value) / baseline value * 100%, where the baseline value is the detection value before administration, and the current value is the detection value at the time point (D60) to be detected after administration
  • BMI weight/sitting height
  • rate of change (current value-baseline value)/baseline value*100%, wherein the baseline value is the value detected before administration, and the current value is the time point (D60) to be detected after administration detection value.

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Abstract

涉及抗KLB抗体及用途。

Description

抗KLB抗体及用途 技术领域
本披露属于生物技术领域，更具体地，本披露涉及抗KLB抗体及其应用。
背景技术
这里的陈述仅是提供与本披露有关的背景信息，而不必然地构成现有技术。
KLB(Klotho-beta，β-Klotho)是Klotho蛋白家族成员之一，其由胞外结构域、跨膜结构域和胞内结构域组成。KLB与成纤维细胞生长因子受体(FGFR，包括FGFR1-4及其可变剪切体，例如FGFR1c，FGFR2c，FGFR3c)一起形成复合物，作为成纤维细胞生长因子(fibroblast growth factor，简称FGF，例如FGF19和FGF21)的共同受体。KLB作为对FGF21/FGF19的细胞应答的决定簇，在肝脏、脂肪细胞和胰腺中高度表达(Kurosu，H.等人(2007)J Biol Chem[生物化学杂志]282，26687-95)。成纤维细胞生长因子受体含有细胞内酪氨酸激酶结构域，该结构域在与配体结合时被活化，产生MAPK(Erk1/2)、RAF1、AKT1和STAT等，激活下游信号传导通路(Kharitonenkov，A.等人(2008)BioDrugs[生物药物]22:37-44)。
成纤维细胞生长因子21(fibroblast growth factor 21，FGF21)是成纤维细胞生长因子家族成员之一，它在肝脏和胰中高度表达(Itoh et al.，(2004)Trend Genet.20:563-69)。FGF21通过C末端与KLB结合，N端与FGFR结合，形成受体-配体复合物，进而激活FGFR下游的信号通路。在多种代谢相关的靶器官上(例如肝脏、脂肪组织、胰腺、CNS中的肾上腺等)，研究显示FGF21通过与KLB&FGFR1c，KLB&FGFR2c和KLB&FGFR3c形成复合物，进而发挥调节糖代谢、脂代谢和能量消耗等作用。过表达FGF21的转基因小鼠展现低胰岛素、低胆固醇及低甘油三酯，胰岛素敏感性提高，阻止高脂食物引起的体重增加(Kharitonenkov等人，(2005)J Clin Invest115:1627-35)。FGF21类似物(例如AKR001，参见WO2010129503A1中SEQ ID NO：47)已在临床实验中被充分证实，其能显著降低肝脏脂肪，提高NASH resolution，改善纤维化等。
发明内容
本披露构建了一种抗KLB抗体，其包含重链可变区和轻链可变区，其中，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中，
(i)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40-48中的任一序列的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：34中的LCDR1、LCDR2 和LCDR3的氨基酸序列；或者所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：2中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：3中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
(ii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：49中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：56-68中的任一序列的LCDR1、LCDR2和LCDR3的氨基酸序列；或者所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：4中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：5中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
(iii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：18中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：19中的LCDR1、LCDR2和LCDR3的氨基酸序列。
在一些实施方案中，i)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：34中的LCDR1、LCDR2和LCDR3的氨基酸序列；或ii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：49中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：57中的的LCDR1、LCDR2和LCDR3的氨基酸序列；或iii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：18中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：19中的LCDR1、LCDR2和LCDR3的氨基酸序列。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：34中的LCDR1、LCDR2和LCDR3的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据选自Kabat、IMGT、Chothia、AbM和Contact的相同的编号规则定义的。在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Kabat编号规则定义的；在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据IMGT编号规则定义的；在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根 据Chothia编号规则定义的；在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据AbM编号规则定义的；在一些实施方案中，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Contact编号规则定义的。
在一些实施方案中，如上任一项所述的抗KLB抗体，其中：
i)所述重链可变区的HCDR1包含SEQ ID NO：6的氨基酸序列，HCDR2包含SEQ ID NO：37、38、39或7的氨基酸序列，和HCDR3包含SEQ ID NO：8的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：9的氨基酸序列，LCDR2包含SEQ ID NO：10的氨基酸序列，和LCDR3包含SEQ ID NO：11的氨基酸序列；或
ii)所述重链可变区的HCDR1包含SEQ ID NO：12的氨基酸序列，HCDR2包含SEQ ID NO：13的氨基酸序列，和HCDR3包含SEQ ID NO：14的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：15、69、70、71、72、73或74的氨基酸序列，LCDR2包含SEQ ID NO：16或75的氨基酸序列，和LCDR3包含SEQ ID NO：17的氨基酸序列；或
iii)所述重链可变区的HCDR1包含SEQ ID NO：24的氨基酸序列，HCDR2包含SEQ ID NO：25的氨基酸序列，和HCDR3包含SEQ ID NO：26的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：27的氨基酸序列，LCDR2包含SEQ ID NO：28的氨基酸序列，和LCDR3包含SEQ ID NO：29的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，其中，
i)所述重链可变区的HCDR1包含SEQ ID NO：6的氨基酸序列，HCDR2包含SEQ ID NO：37的氨基酸序列，和HCDR3包含SEQ ID NO：8的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：9的氨基酸序列，LCDR2包含SEQ ID NO：10的氨基酸序列，和LCDR3包含SEQ ID NO：11的氨基酸序列；或
ii)所述重链可变区的HCDR1包含SEQ ID NO：12的氨基酸序列，HCDR2包含SEQ ID NO：13的氨基酸序列，和HCDR3包含SEQ ID NO：14的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：69的氨基酸序列，LCDR2包含SEQ ID NO：75的氨基酸序列，和LCDR3包含SEQ ID NO：17的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述重链可变区的HCDR1、HCDR2和HCDR3以及轻链可变区的LCDR1、LCDR2和LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述的抗KLB抗体为鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。在一些实施方案中，所述抗体是人源抗体。在一些实施方案中，所述抗体是全人抗体。
在一些实施方案中，如上任一项所述的抗KLB抗体，其中，(i)所述重链可变区包含SEQ ID NO：40、30、31、32、41、42、43、44、45、46、47或48，或 与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：34、33、35或36，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(ii)所述重链可变区包含SEQ ID NO：49、50或51，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：57、52、53、54、55、56、58、59、60、61、62、63、64、65、66、67或68，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iii)所述重链可变区包含SEQ ID NO：18，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：19，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iv)所述重链可变区包含SEQ ID NO：2，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：3，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(v)所述重链可变区包含SEQ ID NO：4，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：5，或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗体的重链可变区的框架区上包含选自第1、24、44、71和91位(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变，轻链可变区的框架区上包含选自第2、4、36、38、43、44和58位(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。在一些实施方案中，所述的抗KLB抗体，其中，所述重链可变区的HCDR1包含SEQ ID NO：6的氨基酸序列，HCDR2包含SEQ ID NO：37、38、39或7的氨基酸序列，和HCDR3包含SEQ ID NO：8的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：9的氨基酸序列，LCDR2包含SEQ ID NO：10的氨基酸序列，和LCDR3包含SEQ ID NO：11的氨基酸序列；所述抗体的重链可变区的框架区上包含选自1E、24T、44G、71S和91F(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变，和/或所述轻链可变区的框架区上包含选自2V、4I、36F、38E、43T、44N和58I(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗体的重链可变区的框架区上包含选自第1、24、48、67、69、71和73位(根据Kabat编号系统编 号)中的一个或更多个氨基酸突变，轻链可变区的框架区上包含选自第2、45、47、49、58和71位(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。在一些实施方案中，所述的抗KLB抗体，其中，所述重链可变区的HCDR1包含SEQ ID NO：12的氨基酸序列，HCDR2包含SEQ ID NO：13的氨基酸序列，和HCDR3包含SEQ ID NO：14的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：15、69、70、71、72、73或74的氨基酸序列，LCDR2包含SEQ ID NO：16或75的氨基酸序列，和LCDR3包含SEQ ID NO：17的氨基酸序列；所述抗体的重链可变区的框架区上包含选自1E、24G、48I、67A、69V、71V和73K(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或所述轻链可变区的框架区上包含选自2N、45K、47W、49Y、58V和71Y(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。所述突变位点根据Kabat编号规则编号，例如，“2N”表示第2位(对应于Kabat编号规则编号)残基为“N”。
在一些实施方案中，如上任一项所述的抗KLB抗体，其中，
(i)所述重链可变区包含SEQ ID NO：40、30、31、32、41、42、43、44、45、46、47或48任一序列、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：34、33、35或36任一序列、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(ii)所述重链可变区包含SEQ ID NO：49、50或51任一序列、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：57、52、53、54、55、56、58、59、60、61、62、63、64、65、66、67或68任一序列、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iii)所述重链可变区包含SEQ ID NO：18、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：19、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iv)所述重链可变区包含SEQ ID NO：2、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：3、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(v)所述重链可变区包含SEQ ID NO：4、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含与SEQ ID NO：5、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，其中，
(i)所述重链可变区包含SEQ ID NO：40、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：34、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；
(ii)所述重链可变区包含SEQ ID NO：49、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：57、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iii)所述重链可变区包含SEQ ID NO：18、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：19、或与其具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，其中，所述重链可变区包含SEQ ID NO：40的氨基酸序列，和所述轻链可变区包含SEQ ID NO：34的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，其包括重链恒定区和轻链恒定区；在一些实施方案中，所述重链恒定区为人IgG重链恒定区；在一些实施方案中，所述重链恒定区为人IgG1、IgG2、IgG3、IgG4重链恒定区；在一些实施方案中，所述轻链恒定区为人λ、κ轻链恒定区；在一些实施方案中，所述重链恒定区为人IgG1重链恒定区，所述轻链恒定区为人κ轻链恒定区；在一些实施方案中，所述重链恒定区的Fc区具有一个或更多个能够减少Fc区与Fc受体结合的氨基酸取代。在一些实施方案中，所述Fc区具有L234A、L235A突变，S228P突变，和/或YTE突变(M252Y、S254T和T256E)，所述突变编号依据为EU索引。在一些实施方案中，所述重链恒定区包含SEQ ID NO：22的氨基酸序列，所述轻链恒定区包含SEQ ID NO：23的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗KLB抗体包含重链和轻链，其中，
(i)所述重链包含与SEQ ID NO：78具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：79具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(ii)所述重链包含与SEQ ID NO：80具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：81具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列；或
(iii)所述重链包含与SEQ ID NO：20具有至少90％(例如至少90％、95％、 96％、97％、98％或99％)序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：21具有至少90％(例如至少90％、95％、96％、97％、98％或99％)序列同一性的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗KLB抗体包含重链和轻链，其中，(i)所述重链包含SEQ ID NO：78的氨基酸序列，和所述轻链包含SEQ ID NO：79的氨基酸序列；(ii)所述重链包含SEQ ID NO：80的氨基酸序列，和所述轻链包含SEQ ID NO：81的氨基酸序列；或(iii)所述重链包含SEQ ID NO：20的氨基酸序列，和所述轻链包含SEQ ID NO：21的氨基酸序列。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗KLB抗体为抗原结合片段，在一些实施方案中，所述抗原结合片段选自：Fab、F(ab')2、Fab'、Fd、Fv、dsFv、scFv、Fab和双抗体。
在一些实施方案中，本披露提供一种分离的抗KLB抗体，其与前面任一项所述的抗KLB抗体竞争性结合人和/或猴KLB；在一些实施方案中，本披露提供一种分离的抗KLB抗体，其与前面任一项所述的抗KLB抗体结合相同在抗原表位。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗KLB抗体具有一种或更多种以下特征：
A.所述抗KLB抗体对表达hKLB&hFGFR1c的细胞具有激活活性；在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的细胞具有高的激活活性；在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的CHOK1细胞的激活倍数大于10，和/或所述抗KLB抗体对HEK293T-hKLB&hFGFR1c细胞的激活倍数大于2；在一些实施方案中，所述激活倍数为抗体相对于空白对照的激活倍数；在一些实施方案中，所述激活倍数根据本披露测试例4、5方法检测；
B.所述抗KLB抗体对表达hFGFR1c重组细胞(仅hFGFR1c，不表达hKLB)激活活性弱；在一些实施方案中，所述抗KLB抗体对HEK293-hFGFR1c细胞的激活倍数小于1；在一些实施方案中，所述激活倍数为抗体相对于空白对照的激活倍数；在一些实施方案中，所述激活倍数根据本披露测试例6方法检测；
C.所述抗KLB抗体对表达hKLB&hFGFR2c、hKLB&hFGFR3c和hKLB&hFGFR4的细胞的激活活性弱；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c和/或L6-hKLB&hFGFR4细胞的激活活性弱；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c和/或L6-hKLB&hFGFR4细胞的激活倍数小于2；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c、hKLB&hFGFR3c和/或L6-hKLB&hFGFR4细胞的激活活性弱，在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c、hKLB&hFGFR3c和/或L6-hKLB&hFGFR4细胞的激活倍数小于2；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c和L6-hKLB&hFGFR4细胞的激活倍数小于2，对 L6-hKLB&hFGFR3c细胞的激活倍数小于5；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c细胞的激活倍数小于1.4，对L6-hKLB&hFGFR4细胞的激活倍数小于1.6，对L6-hKLB&hFGFR3c细胞的激活倍数小于5；在一些实施方案中，所述激活倍数为抗体相对于空白对照的激活倍数；在一些实施方案中，所述激活倍数根据本披露测试例7方法检测；
D.所述抗KLB抗体能与人KLB结合，也能与猴KLB结合；在一些实施方案中，所述抗KLB抗体能以小于2.0E-9M的EC50值与人和/或猴KLB结合，所述EC50值通过流式细胞检测方法检测；
E.所述抗KLB抗体能高亲和力与人KLB结合；在一些实施方案中，所述抗体能以小于3.0E-9M的KD值与人KLB结合，所述KD值通过Biacore方法检测。
在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的细胞具有高激活活性，对表达hKLB&hFGFR2c、hKLB&hFGFR3c和hKLB&hFGFR4的细胞具有弱的激活活性；在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的CHOK1细胞的激活倍数大于10，所述抗KLB抗体对L6-hKLB&hFGFR2c和/或L6-hKLB&hFGFR4细胞的激活倍数小于2，所述激活倍数为抗体相对于空白对照的激活倍数。
另一方面，本披露提供一种药物组合物，其含有：治疗有效量的如上所述抗KLB抗体，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
另一方面，本披露提供分离的核酸，其编码如上所述抗KLB抗体。
另一方面，本披露提供一种载体，其包含前述的核酸分子。
另一方面，本披露提供宿主细胞，其包含如上所述的核酸。
另一方面，本披露提供一种治疗疾病的方法，所述方法包括向受试者施用如上所述抗KLB抗体或药物组合物的步骤。
另一方面，本披露还提供如上所述抗KLB抗体或药物组合物在制备治疗疾病的药物中的用途。
另一方面，本披露还提供用作药物的如上所述抗KLB抗体或药物组合物；在一些实施方案中，所述药物用于治疗疾病。
在一些实施方案中，本披露提供治疗与人FGF21和/或人FGF19有关的疾病、病症或病况的方法，所述方法包括给予受试者(例如患者)治疗有效量的如上所述抗KLB抗体或药物组合物。
在一些实施方案中，如上所述的疾病是FGF21和/或FGF19相关病症。在一些实施方案中，所述病症选自代谢病症、内分泌病症和心血管病症。在一些实施方案中，所述病症选自肥胖、糖尿病(1型糖尿病、2型糖尿病)，胰腺炎，血脂异常，非酒精性脂肪肝病(NAFLD)，非酒精性脂肪性肝炎(NASH)，胰岛素抵抗，高胰岛素血症，葡萄糖不耐受，高血糖，代谢综合征，急性心肌梗塞，高血压，动脉粥样硬化，外周动脉疾病，中风，心力衰竭，冠心病，肾脏疾病，糖尿 病并发症，神经系统疾病和胃轻瘫。在一些实施方案中，所述疾病选自由2型糖尿病、肥胖症、血脂异常、NASH、心血管疾病和代谢综合征组成的组。在一些实施方案中，所述疾病为非酒精性脂肪性肝炎。
另一方面，本披露还提供一种诱导FGF21和/或FGF19信号转导的方法，所述方法包括使表达KLB和FGFR的细胞与如上所述的抗KLB抗体接触。
具体实施方式
术语
为了更容易理解本披露，以下对某些技术和科学术语进行了描述。除非在本文中另有明确定义，本文使用的全部技术和科学术语具有与本领域的普通技术人员通常所理解的相同含义。
说明书和权利要求书中所用的单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文清楚表明并非如此。
除非上下文另外清楚要求，否则在专利说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为“包括但不仅限于”的意义，而不是排他性或穷举性意义。
术语“和/或”，意指包含“和”与“或”两种含义。例如短语“A、B和/或C”旨在涵盖以下方面中的每一个：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；和C(单独)。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“氨基酸”是指天然存在的和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构(即与氢、羧基、氨基和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构，但是以与天然存在的氨基酸类似的方式起作用的化学化合物。
术语“氨基酸突变”包括氨基酸取代(也称氨基酸替换)、缺失、插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来实现最终构建体，只要最终构建体拥有期望的特性，例如降低或对Fc受体的结合。氨基酸序列缺失和插入包括在多肽链的氨基端和/或羧基端的缺失和插入。具体的氨基酸突变可以是氨基酸取代。在一个实施方式中，氨基酸突变是非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸取代包括由非天然存在的 氨基酸或由20种天然氨基酸的衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变。遗传方法可以包括定点诱变、PCR，基因合成等。预计基因工程以外的改变氨基酸侧链基团的方法，如化学修饰也可能是可用的。本文中可使用各种名称来指示同一氨基酸突变。本文中，可采用位置+氨基酸残基的方式表示特定位点的氨基酸残基，例如366W，表示在366位点上的氨基酸残基为W。T366W则表示第366位点上的氨基酸残基由原来的T突变为了W。
术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)；全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。“天然抗体”指天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150，000道尔顿的异四聚糖蛋白，由二硫键结合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域、重链可变区，接着是重链恒定区(CH)，天然IgG重链恒定区通常含三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻域(轻链恒定区、CL)。术语“全长抗体”、“完整抗体”和“全抗体”在本文可互换使用，指具有与天然抗体结构基本类似的结构或具有如本文所限定的Fc区的重链的抗体。天然完整抗体轻链包括轻链可变区VL及恒定区CL，VL处于轻链的氨基末端，轻链恒定区包括κ链及λ链；重链包括可变区VH及恒定区(CH1、CH2及CH3)，VH处于重链的氨基末端(也称N端)，恒定区处于羧基末端(也称C端)，其中CH3最接近多肽的羧基末端，重链可属于任何同种型，包括IgG(包括IgG1、IgG2、IgG3及IgG4亚型)、IgA(包括IgA1及IgA2亚型)、IgM及IgE。
术语“可变区”或“可变域”指抗体中结合抗原的域。本文中，重链可变区VH和轻链可变区VL各包含四个保守的框架区(FR)和三个互补决定区(CDR)。术语“互补决定区”或“CDR”指可变结构域内主要促成与抗原结合的区域；“框架”或“FR”是指除CDR残基之外的可变结构域残基。VH包含3个CDR区：HCDR1、HCDR2和HCDR3；VL包含3个CDR区：LCDR1、LCDR2和LCDR3。每个VH和VL由从氨基末端(也称N末端)排到羧基末端(也称C末端)按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。每个VH和VL由从氨基末端排到羧基末端按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。对于某些抗体，单个VH或VL可能足以赋予抗原结合特异性。
可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia” 编号规则、“ABM”编号规则、“contact”编号规则(参见Martin，ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018 Oct 16；9:2278)等；各种编号系统之间的对应关系是本领域技术人员熟知的，示例性的，如下表1中所示。
表1.CDR编号系统之间的关系
CDR IMGT Kabat AbM Chothia Contact
HCDR1 27-38 31-35 26-35 26-32 30-35
HCDR2 56-65 50-65 50-58 52-56 47-58
HCDR3 105-117 95-102 95-102 95-102 93-101
LCDR1 27-38 24-34 24-34 24-34 30-36
LCDR2 56-65 50-56 50-56 50-56 46-55
LCDR3 105-117 89-97 89-97 89-97 89-96
术语“抗体片段”或“抗原结合片段”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分与完整抗体所结合的抗原相结合。抗体片段的实例包括但不限于Fv、Fab、Fab’、Fab’-SH、F(ab′) 2、单域抗体、单链Fab(scFab)、双抗体、线性抗体、单链抗体(scFv)，以及由抗体片段形成的多特异性抗体。
术语“Fc区”或“片段可结晶区”用于定义抗体重链的C末端区域，包括天然Fc区和改造的Fc区。在一些实施方式中，Fc区包含了相同或不同的两个亚基。在一些实施方式中，人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端。用于本文所述抗体的合适Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4的Fc区。在一些实施方式中，Fc区的边界还可以变化，例如缺失Fc区的C末端赖氨酸(根据EU编号系统的残基447)或缺失Fc区的C末端甘氨酸和赖氨酸(根据EU编号系统的残基446和447)。除非另有说明，Fc区的编号规则为EU编号系统，又称作EU索引。
术语“嵌合”抗体指抗体中的重和/或轻链的一部分自特定的来源或物种衍生，而重和/或轻链的剩余部分自另外的不同来源或物种衍生的抗体。
术语“人源化”抗体是保留非人抗体的反应性同时在人中具有较低免疫原性的抗体。例如，可以通过保留非人CDR区并用其人对应物(即，恒定区以及可变区的框架区部分)替换抗体的其余部分来实现。
术语“人抗体”、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，意指可变区及恒定区是人序列的抗体。该术语涵盖源自人基因但具有，例如，降低可能的免疫原性、增加亲和力、消除可能会引起不期望的折叠的半胱氨酸或糖基化位点等序列已发生改变的抗体。该术语涵盖这些在非人细胞(其可能会赋予不具人细胞特征的糖基化)中重组产生的抗体。该术语亦涵盖已在含有一些或所有人免疫球蛋白重链及轻链基因座的转基因小鼠中饲养的抗体。人抗体的含义明确排 除包含非人抗原结合残基的人源化抗体。
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，结合“亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些方法)测量。
如本文所使用的，术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合速率，术语“kdis”或“kd”指特定抗体-抗原相互作用的解离速率。术语“KD”指解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域公知的方法测定抗体的KD值。例如，使用生物传感系统例如系统测量表面等离子体共振，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。在一些实施方式中，KD值通过Biacore检测。
术语“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列突变的Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括但不限于：C1q结合和补体依赖性细胞毒性、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、吞噬作用、细胞表面受体(例如B细胞受体)下调；和B细胞活化。
术语“抗体依赖性细胞的细胞毒性”、“抗体依赖性细胞介导的细胞毒性”或“ADCC”是诱导细胞死亡的机制，该机制依赖于抗体包被靶细胞与具有裂解活性的效应细胞(诸如自然杀伤细胞(NK)、单核细胞、巨噬细胞和中性粒细胞)经由效应细胞上表达的Fcγ受体(FcγR)发生的相互作用。例如，NK细胞表达FcγRIIIa，而单核细胞表达FcγRI、FcγRII和FcγRIIIa。本文提供的抗体的ADCC活性可使用体外测定，使用表达抗原的细胞作为靶细胞和NK细胞作为效应细胞进行评定。根据从裂解的细胞中释放的标记物(例如放射性底物、荧光染料或天然胞内蛋白)来检测细胞裂解。
术语“抗体依赖性细胞吞噬作用”(“ADCP”)是指通过吞噬细胞(诸如巨噬细胞或树突状细胞)的内化作用消除抗体包被的靶细胞的机制。
术语“补体依赖性细胞毒性”或“CDC”是指诱导细胞死亡的机制，其中靶结合抗体的Fc效应域结合并激活补体成分C1q，C1q继而激活补体级联，从而导致靶细胞死亡。补体的激活也可导致补体成分沉积在靶细胞表面上，这些补体成分通过结合白细胞上的补体受体(例如，CR3)来促进CDC。
术语“单克隆抗体”指基本上均质的抗体的群，即在该群中包含的抗体分子的氨基酸序列是相同的，除了可能少量存在的天然突变以外。相比之下，多克隆抗体制剂通常包含在其可变结构域具有不同氨基酸序列的多种不同抗体，其通常特异性针对不同表位。“单克隆”表示从基本上均质的抗体群体获得的抗体的特征，并且不应解释为要求通过任何特定方法来生产抗体。在一些实施方式中，本披露提供 的抗体是单克隆抗体。
术语“抗原”是指能够由诸如抗原结合蛋白(包括例如抗体)的选择性结合剂结合，且另外能够用于动物中以产生能够结合该抗原的抗体的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合蛋白(例如抗体)相互作用的表位。
术语“表位”指能够与抗体或其抗原结合片段特异性结合的抗原上的区域(area或region)。表位可以由连续氨基酸串(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过蛋白质性质的抗原的三级折叠)而变成空间接近。构象表位和线性表位的差别在于：在变性溶剂的存在下，抗体对构象表位的结合丧失。表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如但不限于丙氨酸扫描，肽印迹，肽切割分析，表位切除，表位提取，抗原的化学修饰(见Prot.Sci.9(2000)487-496)，和交叉阻断。
术语“能够特异性结合”、“特异性结合”或“结合”是指相比其他抗原或表位，抗体(例如抗KLB抗体)能够以更高的亲和力结合至某个抗原或该抗原内的表位。通常地，抗体以约1×10 -7M或更小(例如约1×10 -8M或更小)的平衡解离常数(KD)结合抗原或抗原内的表位。在一些实施方式中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％或更低(例如1％)。可使用已知的方法来测量KD，例如通过
Figure PCTCN2022120887-appb-000001
表面等离子体共振测定法所测量的。然而，特异性结合至抗原或抗原内的表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp))或狨猴(Callithrix jacchus)(commonmarmoset，marmoset)的相应抗原具有交叉反应性。
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸，所述核酸是合成的、天然存在的和非天然存在的，具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。编码抗KLB抗体的分离的核酸指编码抗KLB抗体的一个或更多个核酸分子，包括在单一载体或分开的载体中的这样的一个或更多个核酸分子，和存在于宿主细胞中一个或更多个位置的这样的一个或更多个核酸分子。除非另有说明，否则特定的核酸序列还隐含地涵盖其保守修饰的变体 (例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代。
术语“多肽”和“蛋白质”在本文中可互换使用，指氨基酸残基的聚合物。该术语适用于氨基酸聚合物，其中一个或多个氨基酸残基是相应天然存在的氨基酸的人工化学模拟物，以及适用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。除非另外说明，否则特定的多肽序列还隐含地涵盖其保守修饰的变体。
术语序列“同一性”指，当对两条序列进行最佳比对时，必要时引入间隙，以获取最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，两条序列的氨基酸/核酸在等价位置相同的程度(百分比)。为测定序列同一性百分比，比对可以通过本领域技术已知的技术来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“融合”或“连接”是指部件(例如Fv中VH与VL)直接地或经由一个或多个连接子而通过共价键连接。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。术语“表达载体”或“表达构建体”是指可对宿主细胞进行转化，且含有指导和/或控制(连同宿主细胞一起)与其可操作地连接的一个或多个异源编码区的表达的核酸序列的载体。表达构建体可以包括但不限于影响或控制转录、翻译且在存在内含子时影响与其可操作地连接的编码区的RNA剪接的序列。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代，而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中包括具有与在初始转化细胞中筛选或选择的相同功能或生物学活性的突变体后代。宿主细胞包括原核和真核宿主细胞，其中真核宿主细胞包括但不限于哺乳动物细胞、昆虫细胞系植物细胞和真菌细胞。哺乳动物宿主细胞包括人、小鼠、大鼠、犬、猴、猪、山羊、牛、马和仓鼠细胞，包括但不限于中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝 细胞癌细胞(例如，Hep G2)、A549细胞、3T3细胞和HEK-293细胞。真菌细胞包括酵母和丝状真菌细胞，包括例如巴氏毕赤酵母(Pichiapastoris)、芬兰毕赤酵母(Pichia finlandica)、海藻毕赤酵母(Pichia trehalophila)、科克拉马毕赤酵母(Pichia koclamae)、膜状毕赤酵母(Pichia membranaefaciens)、小毕赤酵母(Pichia minuta)(Ogataea minuta、Pichia lindneri)、仙人掌毕赤酵母(Pichiaopuntiae)、耐热毕赤酵母(Pichia thermotolerans)、柳毕赤酵母(Pichia salictaria)、Pichia guercuum、皮杰普毕赤酵母(Pichia pijperi)、具柄毕赤酵母(Pichia stiptis)、甲醇毕赤酵母(Pichia methanolica)、毕赤酵母属、酿酒酵母(Saccharomycescerevisiae)、酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、克鲁维酵母属、乳酸克鲁维酵母(Kluyveromyces lactis)、白色念珠菌(Candida albicans)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)、勒克氏菌(Chrysosporium lucknowense)、镰刀菌属(Fusarium sp.)、禾谷镰刀菌(Fusarium gramineum)、菜镰刀菌(Fusarium venenatum)、小立碗藓(Physcomitrella patens)和粗糙脉孢菌(Neurospora crassa)。毕赤酵母属、任何酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、任何克鲁维酵母属、白色念珠菌(Candida albicans)、任何曲霉属、里氏木霉(Trichoderma reesei)、勒克霉菌(Chrysosporium lucknowense)、任何镰刀菌属、解脂耶氏酵母(Yarrowia lipolytica)和粗糙脉孢菌(Neurospora crassa)。
如在本申请中所使用的，表述“细胞”、“细胞系”和“细胞培养物”可以互换使用，并且所有这样的名称均包括子代。因而，词语“转化体”和“转化的细胞”包括原代受试者细胞和来源于其的培养物，而与传代的次数无关。还应理解的是，由于有意或无意的突变，使得并非所有子代均具有完全相同的DNA内容物。包括与筛选出其的原始转化细胞具有相同功能或生物活性的突变子代。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
术语“药物组合物”表示含有一种或多种本文所述的抗KLB抗体与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。
术语“药学上可接受的载体”指药学配制剂中与活性成分不同的，且对受试者无毒的成分。药学可接受载剂包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
术语“受试者”或“个体”包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。除非指出时，否则所述术语“患者”或“受试者”在本文中可互换地使用。如本文所使用的，术语“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些实施方案中，个体或受试者是人。
“施用”或“给予”，当其应用于动物、人、实验受试者、细胞、组织、器官或生 物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。
术语“样本”是指从受试者分离的类似流体、细胞、或组织的采集物，以及存在于受试者体内的流体、细胞或组织。示例性样本为生物流体，诸如血液、血清和浆膜液、血浆、淋巴液、尿液、唾液、囊液、泪液、排泄物、痰、分泌组织和器官的粘膜分泌物、阴道分泌物、腹水、胸膜、心包、腹膜、腹腔和其它体腔的流体、由支气管灌洗液收集的流体、滑液、与受试者或生物来源接触的液体溶液，例如细胞和器官培养基(包括细胞或器官条件培养基)、灌洗液等，组织活检样本、细针穿刺、手术切除的组织、器官培养物或细胞培养物。
“治疗(treatment或treat)”和“处理”(及其语法变型)指试图改变所治疗个体的天然过程的临床干预，并且可以为了预防或者在临床病理学的过程期间实施。治疗的期望效果包括但不限于预防疾病的发生或再发生，减轻症状，减轻/减少疾病的任何直接或间接病理后果，预防转移，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。在一些实施方案中，使用本披露的抗体来延迟疾病的形成或减缓疾病的进展。在一些实施方式中，本披露的抗KLB抗体(包括结合人和/或食蟹猴KLB的抗体)其具有模拟FGF21和/或FGF19的体内作用和诱导FGF21样信号转导和/或FGF19样信号转导的独特性质，在一些实施方案中，所述抗体显示与FGF21的天然生物功能一致的活性。这种性质使得抗KLB抗体可用于治疗FGF21和/或FGF19相关的代谢疾病，例如2型糖尿病、肥胖症、血脂异常、NASH、心血管疾病、代谢综合征，以及用于需要模拟或增强FGF19和/或FGF21的体内作用的任何疾病、病症或病况的治疗。
“有效量”一般是足以降低症状的严重程度及/或频率、消除这些症状及/或潜在病因、预防症状及/或其潜在病因出现及/或改良或改善由疾病状态引起或与其相关的损伤(例如肺病)的量。在一些实施例中，有效量是治疗有效量或预防有效量。“治疗有效量”是足以治疗疾病状态或症状、尤其与该疾病状态相关的状态或症状，或者以其他方式预防、阻碍、延迟或逆转该疾病状态或以任何方式与该疾病相关的任何其他不理想症状的进展的量。“预防有效量”是当给予受试者时将具有预定预防效应，例如预防或延迟该疾病状态的发作(或复发)，或者降低该疾病状态或相关症状的发作(或复发)可能性的量。完全治疗或预防效未必在给予一个剂量之后便发生，可能在给予一系列剂量之后发生。因而，治疗或预防有效量可以一次或多次给予的方式给予。“治疗有效量”和“预防有效量”可取决于多种因素变化：诸如个体的疾病状态、年龄、性别和体重，以及治疗剂或治疗剂组合在个体中引发期望的应答的能力。有效治疗剂或治疗剂组合的示例性指标包括例如患者改善的健康状况。
本披露的抗KLB抗体
在一个方面中，本披露通过大量的实验，获得了一种特异性结合KLB的抗体。在一些实施方案中，本披露的抗KLB抗体其具有诸多有利的特性，例如高亲和力与抗原结合、人和猴交叉反应活性、对hKLB&hFGFR1c细胞具有强激活活性而但对hFGFR1c重组细胞具有弱激活活性、对hKLB&hFGFR2c、hKLB&hFGFR3c和hKLB&hFGFR4细胞具有选择性、药物代谢动力学特性和/或成药性等。
在一些实施方案中，本披露提供一种特异性结合KLB的抗体，在一些实施方案中，所述抗体为全长抗体或其抗原结合片段(例如Fv、Fab、Fab’、Fab’-SH、F(ab′)2、单域抗体、单链Fab(scFab)、双抗体、线性抗体、单链抗体(scFv))，在一些实施方案中，其具有以下一个或更多个功能活性：
A.所述抗KLB抗体对表达hKLB&hFGFR1c的细胞具有激活活性；在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的细胞具有高的激活活性；在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的CHOK1细胞的激活倍数大于10，和/或所述抗KLB抗体对HEK293T-hKLB&hFGFR1c细胞的激活倍数大于2；在一些实施方案中，所述激活倍数为抗体相对于空白对照的激活倍数；在一些实施方案中，所述激活倍数根据本披露测试例4、5方法检测；
B.所述抗KLB抗体对表达hFGFR1c重组细胞(仅hFGFR1c，不表达hKLB)激活活性弱；在一些实施方案中，所述抗KLB抗体对HEK293-hFGFR1c细胞的激活倍数小于1；在一些实施方案中，所述激活倍数为抗体相对于空白对照的激活倍数；在一些实施方案中，所述激活倍数根据本披露测试例6方法检测；
C.所述抗KLB抗体对表达hKLB&hFGFR2c、hKLB&hFGFR3c和hKLB&hFGFR4的细胞的激活活性弱；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c和/或L6-hKLB&hFGFR4细胞的激活活性弱；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c和/或L6-hKLB&hFGFR4细胞的激活倍数小于2；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c、hKLB&hFGFR3c和/或L6-hKLB&hFGFR4细胞的激活活性弱，在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c、hKLB&hFGFR3c和/或L6-hKLB&hFGFR4细胞的激活倍数小于2；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c和L6-hKLB&hFGFR4细胞的激活倍数小于2，对L6-hKLB&hFGFR3c细胞的激活倍数小于5；在一些实施方案中，所述抗KLB抗体对L6-hKLB&hFGFR2c细胞的激活倍数小于1.4，对L6-hKLB&hFGFR4细胞的激活倍数小于1.6，对L6-hKLB&hFGFR3c细胞的激活倍数小于5；在一些实施方案中，所述激活倍数为抗体相对于空白对照的激活倍数；在一些实施方案中，所述激活倍数根据本披露测试例7方法检测；
D.所述抗KLB抗体能与人KLB结合，也能与猴KLB结合；在一些实施方案中，所述抗KLB抗体能以小于2.0E-9M的EC50值与人和/或猴KLB结合，所 述EC50值通过流式细胞检测方法检测；
E.所述抗KLB抗体能高亲和力与人KLB结合；在一些实施方案中，所述抗体能以小于3.0E-9M的KD值与人KLB结合，所述KD值通过Biacore方法检测。
在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的细胞具有高激活活性，对表达hKLB&hFGFR2c、hKLB&hFGFR3c和hKLB&hFGFR4的细胞具有弱的激活活性；在一些实施方案中，所述抗KLB抗体对表达hKLB&hFGFR1c的CHOK1细胞的激活倍数大于10，所述抗KLB抗体对L6-hKLB&hFGFR2c和/或L6-hKLB&hFGFR4细胞的激活倍数小于2，所述激活倍数为抗体相对于空白对照的激活倍数。
在一些实施方案中，如上任一项所述的抗KLB抗体，其包含重链可变区和轻链可变区，其中i)所述重链可变区的HCDR1、HCDR2和HCDR3的序列分别与SEQ ID NO：40-48中的任一条序列的HCDR1、HCDR2和HCDR3的序列相同，和所述轻链可变区的LCDR1、LCDR2和LCDR3的序列分别与SEQ ID NO：34中的LCDR1、LCDR2和LCDR3的氨基酸序列相同；或者所述重链可变区的HCDR1、HCDR2和HCDR3的序列分别与SEQ ID NO：2中的HCDR1、HCDR2和HCDR3的氨基酸序列相同，和所述轻链可变区的LCDR1、LCDR2和LCDR3的序列分别与SEQ ID NO：3中的LCDR1、LCDR2和LCDR3的氨基酸序列相同；或
ii)所述重链可变区的HCDR1、HCDR2和HCDR3的序列分别与SEQ ID NO：49中的HCDR1、HCDR2和HCDR3的氨基酸序列相同，和所述轻链可变区的LCDR1、LCDR2和LCDR3的序列分别与SEQ ID NO：56-68中的任一序列的LCDR1、LCDR2和LCDR3的氨基酸序列相同；或者所述重链可变区的HCDR1、HCDR2和HCDR3的序列分别与SEQ ID NO：4中的HCDR1、HCDR2和HCDR3的氨基酸序列相同，和所述轻链可变区的LCDR1、LCDR2和LCDR3的序列分别与SEQ ID NO：5中的LCDR1、LCDR2和LCDR3的氨基酸序列相同；或
iii)所述重链可变区的HCDR1、HCDR2和HCDR3的序列分别与SEQ ID NO：18中的HCDR1、HCDR2和HCDR3的氨基酸序列相同，和所述轻链可变区的LCDR1、LCDR2和LCDR3的序列分别与SEQ ID NO：19中的LCDR1、LCDR2和LCDR3的氨基酸序列相同。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述CDR是根据选自Kabat、IMGT、Chothia、AbM和Contact的编号规则定义的。在一些实施方案中，
i)所述重链可变区的HCDR1如SEQ ID NO：6所示，HCDR2如SEQ ID NO：37、38、39或7所示，和HCDR3如SEQ ID NO：8所示，和
所述轻链可变区的LCDR1如SEQ ID NO：9所示，LCDR2如SEQ ID NO：10所示，和LCDR3如SEQ ID NO：11所示；或
ii)所述重链可变区的HCDR1如SEQ ID NO：12所示，HCDR2如SEQ ID NO： 13所示，和HCDR3如SEQ ID NO：14所示，和
所述轻链可变区的LCDR1如SEQ ID NO：15、69、70、71、72、73或74所示，LCDR2如SEQ ID NO：16、或75所示，和LCDR3如SEQ ID NO：17所示；或
iii)所述重链可变区的HCDR1如SEQ ID NO：24所示，HCDR2如SEQ ID NO：25所示，和HCDR3如SEQ ID NO：26所示，和
所述轻链可变区的LCDR1如SEQ ID NO：27所示，LCDR2如SEQ ID NO：28所示，和LCDR3如SEQ ID NO：29所示。
优选地，
i)所述重链可变区的HCDR1如SEQ ID NO：6所示，HCDR2如SEQ ID NO：37所示，和HCDR3如SEQ ID NO：8所示，和
所述轻链可变区的LCDR1如SEQ ID NO：9所示，LCDR2如SEQ ID NO：10所示，和LCDR3如SEQ ID NO：11所示；或
ii)所述重链可变区的HCDR1如SEQ ID NO：12所示，HCDR2如SEQ ID NO：13所示，和HCDR3如SEQ ID NO：14所示，和
所述轻链可变区的LCDR1如SEQ ID NO：69所示，LCDR2如SEQ ID NO：75所示，和LCDR3如SEQ ID NO：17所示。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗体的重链可变区的框架区为在IGHV1-3*01/IGHJ6*01重链框架区上包含选自第1、24、44、71和91位(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变，轻链可变区的框架区为在IGKV1-39*01/IGKJ4*01轻链框架区上包含选自第2、4、36、38、43、44和58位(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。在一些实施方案中，所述的抗KLB抗体，其中，所述重链可变区的HCDR1包含SEQ ID NO：6的氨基酸序列，HCDR2包含SEQ ID NO：37、38、39或7的氨基酸序列，和HCDR3包含SEQ ID NO：8的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：9的氨基酸序列，LCDR2包含SEQ ID NO：10的氨基酸序列，和LCDR3包含SEQ ID NO：11的氨基酸序列；所述抗体的重链可变区的框架区为在IGHV1-3*01/IGHJ6*01重链框架区上还包含选自1E、24T、44G、71S和91F(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变，和/或所述轻链可变区的框架区为在IGKV1-39*01/IGKJ4*01轻链框架区上包含选自2V、4I、36F、38E、43T、44N和58I(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。
在一些实施方案中，如上任一项所述的抗KLB抗体，所述抗体的重链可变区的框架区为在IGHV1-3*01/IGHJ6*01重链框架区上包含选自第1、24、48、67、69、71和73位(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，轻链可变区的框架区为在IGKV6-21*02/JGKJ2*01或者IGKV3-20*02/JGKJ2*01轻链框 架区上包含选自第2、45、47、49、58和71位(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。在一些实施方案中，所述的抗KLB抗体，其中，所述重链可变区的HCDR1包含SEQ ID NO：12的氨基酸序列，HCDR2包含SEQ ID NO：13的氨基酸序列，和HCDR3包含SEQ ID NO：14的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：15、69、70、71、72、73或74的氨基酸序列，LCDR2包含SEQ ID NO：16或75的氨基酸序列，和LCDR3包含SEQ ID NO：17的氨基酸序列；所述抗体的重链可变区的框架区为在IGHV1-3*01/IGHJ6*01重链框架区上包含选自1E、24G、48I、67A、69V、71V和73K(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或所述轻链可变区的框架区为在IGKV6-21*02/JGKJ2*01或者IGKV3-20*02/JGKJ2*01轻链框架区上包含选自2N、45K、47W、49Y、58V和71Y(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变。所述突变位点根据Kabat编号规则编号，例如，“2N”表示第2位(对应于Kabat编号规则编号)残基为“N”。
抗KLB抗体的变体
在某些实施方案中，涵盖本文中提供的抗KLB抗体的氨基酸序列变体。例如，可以期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将合适的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包括例如对抗KLB抗体的氨基酸序列内的残基的删除、和/或插入、和/或取代。可以进行删除、插入、和取代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如抗原结合特性。
取代、插入、和删除变体
在某些实施方案中，提供了具有一处或多处氨基酸取代的抗KLB抗体变体。取代诱变感兴趣的位点包括CDR和FR。保守取代在表2中在“优选的取代”的标题下显示。更实质的变化在表2中在“例示性取代”的标题下提供，并且如下文参照氨基酸侧链类别进一步描述的。可以将氨基酸取代引入感兴趣的抗体中，并且对产物筛选期望的活性，例如保留/改善的抗原结合，降低的免疫原性，或改善的ADCC或CDC。
表2.氨基酸的取代
原始残基 示例性取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp，Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
依照常见的侧链特性，氨基酸可以如下分组:
(1)疏水性的：正亮氨酸，Met，Ala，Val，Leu，Ile；
(2)中性，亲水性的：Cys，Ser，Thr，Asn，Gln；
(3)酸性的：Asp，Glu；
(4)碱性的：His，Lys，Arg；
(5)影响链取向的残基：Gly，Pro；
(6)芳香族的：Trp，Tyr，Phe。
非保守取代会需要用这些类别之一的成员替换另一个类别的成员。
一类取代变体涉及取代亲本抗体(例如人源化或人抗体)的一个或多个CDR残基。一般地，经选择用于进一步研究的所得变体相对于亲本抗体会具有某些生物学特性(例如升高的亲和力，降低的免疫原性)的改变(例如改善)，和/或会基本上保留亲本抗体的某些生物学特性。一种例示性的取代变体是亲和力成熟的抗体，可以例如使用基于噬菌体展示的亲和力成熟技术(如本文所述的那些技术)，便利地产生所述抗体。简言之，将一个或多个CDR残基突变，并将变体抗体在噬菌体上展示，并对其筛选特定的生物学活性(例如结合亲和力)。可以对CDR做出改变(例如取代)，例如以改善抗体亲和力。可以对CDR“热点”，即在体细胞成熟过程期间以高频率经历突变的密码子所编码的残基，和/或接触抗原的残基做出此类改变，同时对所得的变体VH或VL测试结合亲和力。在亲和力成熟的一些实施方案中，通过多种方法(例如易错PCR、链改组、或寡核苷酸指导的诱变)的任一种，将多样性引入所选择用于成熟的可变基因中。然后，创建次级文库。然后，筛选文库以鉴定具有期望的亲和力的任何抗体变体。另一种引入多样性的方法涉及CDR定向的方法，其中将几个CDR残基(例如一次4-6个残基)随机化。可以例如使用丙氨酸扫描诱变或建模来特异性鉴定涉及抗原结合的CDR残基。特别地，经常靶向HCDR3和LCDR3。
在某些实施方案中，取代、插入或缺失可以在一个或多个CDR内发生，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对CDR做出保守变化(例如保守取代，如本文中提供的)，其不实质性降低结合亲和力。此类变化可以例 如在CDR中的抗原接触残基外部。
一种可用于鉴定抗体中可以作为诱变靶位的残基或区域的方法称作“丙氨酸扫描诱变”。在这种方法中，鉴定一个残基或靶残基组(例如带电荷的残基，诸如Arg、Asp、His、Lys和Glu)，并且用中性或带负电荷的氨基酸(例如，Ala或聚丙氨酸)替换以确定该抗体与抗原的相互作用是否受影响。可以在对初始取代显示功能敏感性的氨基酸位置引入进一步的取代。此外，可通过研究抗原-抗体复合物的晶体结构来鉴定抗体与抗原间的接触点。这些接触残基及邻近残基可以作为取代候选物被打靶或消除。可以筛选变体以确定它们是否含有期望的特性。
氨基酸序列插入包括长度范围为1个残基至含有100或更多个残基的多肽的氨基和/或羧基端融合，和单个或多个氨基酸残基的序列内插入。末端插入的例子包括具有N端甲硫氨酰基残基的抗体。抗体分子的其它插入变体包括抗体的N或C端与酶或延长抗体的血清半衰期的多肽的融合物。
Fc区的改造
在一个方面，本披露的抗KLB抗体的Fc区包含一个或多个氨基酸取代，所述一个或多个氨基酸取代减少其与Fc受体的结合，例如其与Fcγ受体的结合，并且降低或消除效应子功能。天然IgG Fc区，具体地是IgG 1Fc区或IgG 4Fc区，可能导致本披露的抗KLB抗体靶向表达Fc受体的细胞，而不是表达抗原的细胞。本披露改造的Fc区表现出降低的对Fc受体的结合亲和力和/或降低的效应子功能。在一些实施方案中，改造的Fc区与天然Fc区相比，对Fc受体的结合亲和力下降50％、80％、90％或95％以上。在一些实施方案中，所述的Fc受体是Fcγ受体。在一些实施方案中，所述Fc受体是人Fcγ受体，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些实施方案中，改造的Fc区与天然Fc区相比，对补体，如C1q的结合亲和力也降低。在一些实施方案中，改造的Fc区与天然Fc区相比，对新生儿Fc受体(FcRn)的结合亲和力不降低。在一些实施例中，改造的Fc区具有降低的效应子功能，所述降低的效应子功能可以包括但不限于以下中的一个或多个：降低的补体依赖性细胞毒性(CDC)、降低的抗体依赖性细胞介导的细胞毒性(ADCC)、降低的抗体依赖性细胞吞噬(ADCP)、减少的细胞因子分泌、减少的免疫复合物介导的抗原呈递细胞的抗原摄取、减少的与NK细胞的结合、减少的与巨噬细胞的结合、减少的与单核细胞的结合、减少的与多形核细胞的结合、减少的直接信号传导诱导性细胞凋亡、降低的树突细胞成熟或减少的T细胞引发。对于IgG 1Fc区，在238、265、269、270、297、327和329等位置的氨基酸残基取代可降低的效应子功能。在一些实施方案中，所述Fc区是人IgG 1Fc区，并且在234和235位置的氨基酸残基为A，编号依据为EU索引。对于IgG 4Fc区，在228等位置的氨基酸残基取代可降低的效应子功能。
抗KLB抗体还可包含二硫键改造，例如第一亚基的354C和第二亚基的349C。
抗KLB抗体可包含与Fc区的两个亚基融合的不同抗原结合模块，因此可能 导致不期望的同源二聚化。为了提高产率和纯度，因此在本披露的抗KLB抗体的Fc区中引入促进异源二聚化的修饰将是有利的。在一些实施方式中，本披露的Fc区包含根据杵臼(knob-into-hole，KIH)技术的改造，该方法涉及在一个亚基的界面处引入凸起结构(knob)以及在另一个亚基的界面处引入孔结构(hole)。使得所述凸起结构可以定位在孔结构中，促进异源二聚体的形成并抑制同源二聚体的产生。凸起结构是通过用较大侧链(例如酪氨酸或色氨酸)取代来自一个亚基的界面的小氨基酸侧链而构建的。而孔结构是通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大氨基酸侧链而在另一个亚基的界面中创建的。凸起结构和孔结构通过改变编码多肽的核酸来制备，可选的氨基酸取代如下表3所示：
表3.KIH突变组合
Figure PCTCN2022120887-appb-000002
除了杵臼技术外，用于修饰多特异性抗体的重链的CH3结构域以实现异源二聚化的其他技术也是本领域中已知的，例如WO96/27011、WO98/050431、EP1870459、WO2007/110205、WO 007/147901、WO2009/089004、WO2010/129304、WO2011/90754、WO2011/143545、WO2012/058768、WO2013/157954和WO013/096291。
Fc区的C末端可以是以氨基酸残基PGK结束的完整C末端；也可以是缩短的C末端，例如在所述缩短的C末端中已经去除了一个或两个C末端氨基酸残基。在一个优选的方面中，重链的C末端是以PG结束的缩短的C末端。因此，在一些实施方式中，完整抗体的组合物可以包括去除了所有K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物可以包括没有去除K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物具有带有和不带有K447残基和/或G446+K447残基的抗体混合物的抗体群体。
重组方法
抗KLB抗体(例如抗体)可以使用重组方法来产生。对于这些方法，提供编码抗KLB抗体的一个或更多个分离的核酸。
在一个实施方案中，本披露提供了编码如前所述的抗KLB抗体的分离的核酸。此类核酸可以给自独立的编码前述的任一多肽链。在另一方面中，本披露提供了包含此类核酸的一种或多种载体(例如表达载体)。在另一方面中，本披露提供了包含此类核酸的宿主细胞。在一个实施方案中，提供制备抗KLB抗体的方法，其中所述方法包括，在适合表达的条件下，培养包含编码所述抗KLB抗体的核酸的宿主细胞，如上文所提供的，和任选地从宿主细胞(或宿主细胞培养基)回收 所述抗KLB抗体。
为了重组产生抗KLB抗体，将编码蛋白的核酸分离并插入一个或更多个载体中，用于在宿主细胞中进一步克隆和/或表达。此类核酸可以使用常规程序容易地分离和测序，或者通过重组方法产生或通过化学合成获得。
用于克隆或表达编码抗KLB抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，可在细菌中产生，特别是当不需要糖基化和Fc效应子功能时。在表达后，可以在可溶级分中从细菌细胞糊状物分离，并且可进一步纯化。
除了原核生物以外，真核微生物诸如丝状真菌或酵母也是用于编码抗KLB抗体的载体的合适的克隆或表达宿主，包括真菌和酵母菌株。适于表达抗KLB抗体的合适的宿主细胞也可源自多细胞生物体(无脊椎动物和脊椎动物)；无脊椎动物细胞的例子包括植物和昆虫细胞。已经鉴定了许多杆状病毒株，其可与昆虫细胞联合使用，特别是用于草地贪夜蛾(Spodoptera frugiperda)细胞的转染；还可利用植物细胞培养物作为宿主，例如US5959177、US 6040498、US6420548、US7125978和US6417429；也可将脊椎动物细胞用作宿主，例如适应于在悬浮液中生长的哺乳动物细胞系。适宜的哺乳动物宿主细胞系的其它例子是经SV40转化的猴肾CVl系(COS-7)；人胚肾系(293或293T细胞)；幼仓鼠肾细胞(BHK)；小鼠塞托利(sertoli)细胞(TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人宫颈癌细胞(HELA)；犬肾细胞(MDCK)；水牛鼠(buffalo rat)肝细胞(BRL3A)；人肺细胞(W138)；人肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞；MRC 5细胞；和FS4细胞。其它适宜的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞；以及骨髓瘤细胞系，如Y0、NS0和Sp2/0。关于适合产生抗体的某些哺乳动物宿主细胞系的综述参见例如Yazaki，P.和Wu，A.M.，Methods in Molecular Biology，Vol.248，Lo，B.K.C.(编)，Humana Press，Totowa，NJ(2004)，第255-268页。
检测与诊断
本文提供的抗KLB抗体可以通过本领域已知的多种测定法对其物理/化学特征和/或生物学活性进行鉴定、筛选或表征。在一个方面中，例如通过已知方法如ELISA、蛋白印迹法等，测试本披露的抗KLB抗体活性。
在某些实施方案中，本披露提供的抗KLB抗体可用于检测生物学样品中KLB的存在。在用于本文时，术语“检测”涵盖定量或定性检测。在某些实施方案中，生物学样品包含细胞或组织，诸如肿瘤组织。
在一个实施方案中，提供了在诊断或检测方法中使用的抗KLB抗体。一方面，提供了检测生物学样品中KLB的存在的方法。在某些实施方案中，该方法包括在适宜条件下使生物学样品与抗KLB抗体接触，并检测是否在检测试剂与抗原之间形成复合物。此类方法可以是体外或体内方法。在一个实施方案中，使用抗KLB抗体来选择适合治疗的受试者，例如KLB是用于选择患者的生物标志物。
可使用本披露的抗KLB抗体来诊断的例示性病症，例如糖尿病，心血管疾病，胰岛素抗性，高血压，血栓栓塞性疾病或血脂异常；尤其是非酒精性脂肪性肝炎、糖尿病。
在某些实施方案中，提供了经标记的抗KLB抗体。标记物包括但不限于直接检测的标记物或模块(诸如荧光、发色、电子致密、化学发光、和放射性标记物)，和间接检测的模块(例如，经由酶反应或分子相互作用间接检测的模块，诸如酶或配体)。
治疗方法与施用途径
本文提供的任何抗KLB抗体(例如抗体)可用于治疗方法。在又一个方面，本披露提供抗KLB抗体在药物的制造或制备中的用途。在一些实施方案中，所述疾病是与KLB相关的疾病或病症。在一些实施方案中，所述疾病选自：糖尿病，心血管疾病，胰岛素抗性，高血压，血栓栓塞性疾病或血脂异常；尤其是非酒精性脂肪性肝炎、糖尿病。在一个此类实施方案中，所述用途进一步包括向受试者施用有效量的至少一种另外的治疗剂(例如一种、两种、三种、四种、五种或六种另外的治疗剂)。根据任意以上实施方案的“受试者”可以是人。在一些实施方式中，本披露的抗KLB抗体(包括结合人和/或食蟹猴KLB的抗体)其具有模拟FGF21和/或FGF19的体内作用和诱导FGF21样信号转导和/或FGF19样信号转导的独特性质；在一些实施方案中，所述抗体显示与FGF21的天然生物功能一致的活性。这种性质使得本披露的抗KLB抗体可用于治疗FGF21和/或FGF19相关的代谢疾病，例如2型糖尿病、肥胖症、血脂异常、NASH、心血管疾病、代谢综合征，以及需要模拟或增强FGF19和/或FGF21的体内作用的任何疾病、病症或病况的治疗。
在又一个的方面，提供包含所述抗KLB抗体的药物组合物，例如，其用于以上任何制药用途或治疗方法。在一个实施方案中，药物组合物包含本文提供的任何抗KLB抗体和药学上可接受的载体。在另一个实施方案中，药物组合物还包含至少一种另外的治疗剂。
本披露的抗KLB抗体可单独使用或与其他试剂联合用于治疗。例如，本披露的抗体可与至少一种另外的治疗剂共同施用。
本披露的抗KLB抗体(和任何另外的治疗剂)可通过任何合适的手段施用，包括肠胃外、肺内和鼻内，并且如果需要局部治疗，则病灶内施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。给药可以通过任何适当的途径，例如，通过注射，诸如静脉内或皮下注射，这部分取决于施用是短期的还是长期的。本文考虑多种给药时间方案，包括但不限于，单次或在多个时间点多次施用，推注施用和脉冲输注。
本披露的抗KLB抗体将以符合良好医疗实践的方式配制、给药和施用。在此背景下考虑的因素包括所治疗的具体病症、所治疗的具体哺乳动物、个体患者的 临床状况、病症的起因、试剂的递送部位、施用方法、施用时间安排以及医学从业者已知的其他因素。抗KLB抗体可以与或不与目前用于预防或治疗所述病症的一种或更多种试剂一起配制。此类其它试剂的有效量取决于药物组合物中存在的量、病症或治疗的类型以及其它因素。这些通常以与本文所述相同的剂量和施用路径使用，或以本文所述剂量的约1至99％使用，或以其它剂量使用，并通过经验/临床确定为合适的任何途径使用。
为了预防或治疗疾病，本披露的抗KLB抗体(当单独使用或与一种或更多种其他另外的治疗剂组合使用时)的适当的剂量将取决于待治疗的疾病的类型，治疗分子的类型，疾病的严重性和病程，是为预防还是治疗目的施用，之前的治疗，患者的临床病史和对治疗分子的响应，和主治医师的判断。治疗分子恰当地以一次或经过一系列治疗施用于患者。例如，每日剂量可能在约1μg/kg至100mg/kg，具体取决于上文提及的因素。
制品
在本披露的另一方面中，提供一种制品，所述制品包含可用于治疗、预防和/或诊断上述病症的材料。该制品包含容器和在容器上或与容器联合的标签或包装插页(package insert)。合适的容器包括，例如，瓶子、管形瓶、注射器、IV溶液袋等。容器可以自各种材料诸如玻璃或塑料形成。容器装有单独或与另一种组合物组合有效治疗，预防和/或诊断疾患的组合物，并且可具有无菌的存取口(例如，容器可以是具有由皮下注射针可刺穿的塞子的静脉内溶液袋或管形瓶)。组合物中的至少一种活性试剂是本披露的抗KLB抗体。标签或包装插页指示使用该组合物是来治疗选择的病况。此外，制品可以包含：(a)其中装有组合物的第一容器，其中所述组合物包含本披露的抗KLB抗体；和(b)其中装有组合物的第二容器，其中所述组合物包含另外的细胞毒性剂或其他方面的治疗剂。本披露的该实施方案中的制品可进一步包含包装插页，所述包装插页指示所述组合物可以用于治疗特定病况。备选地，或另外地，制品可进一步包含第二(或第三)容器，所述第二(或第三)容器包含药学上可接受的缓冲液。从商业和用户立场，它可进一步包括所需的其他材料，包括其他缓冲剂、稀释剂、滤器、针头和注射器。
实施例与测试例
以下结合实施例和测试例进一步描述本披露，但这些实施例和测试例并非限制着本披露的范围。本披露实施例和测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.重组细胞系的构建
构建表达人KLB&FGFR1c复合物(人KLB序列参见Uniprot ID:Q86Z14，人FGFR1c序列参见Uniprot ID:P11362)、食蟹猴KLB&FGFR1c复合物(食蟹猴KLB序列参见Genebank ID:EHH53620.1，食蟹猴FGFR1c序列参见SEQ ID NO：1)、鼠KLB&FGFR1c复合物(鼠KLB序列参见Uniprot Q99N32，鼠FGFR1c序列参见Uniprot ID:P16092)，以及单独表达人FGFR1c的细胞株。
方法：将人KLB、猴KLB、鼠KLB、人FGFR1c、猴FGFR1c和鼠FGFR1c的全长基因分别克隆到慢病毒表达载体pCDH(System bioscience，01.SBI.CD514B-1)上，其中KLB的病毒表达载体和FGFR1c的病毒表达载体带有不同的抗性基因，分别包装不同种属的KLB和FGFR1c病毒，用pVSV-G、pCMV-dR8.91、pCDH-KLB或pCDH-FGFR1c三种质粒共同转染HEK293T细胞(
Figure PCTCN2022120887-appb-000003
CRL-11268)包装病毒。对于构建复合物过表达细胞系，用包装好的含KLB和FGFR1c基因的病毒以一定比例共同侵染CHOK1细胞(ATCC，CCL-61)或HEK293细胞(ATCC，CRL-1573)系。对于构建人FGFR1c过表达细胞系，仅用含FGFR1c基因的病毒侵染CHOK1或HEK293细胞系。感染48小时后去除含病毒的培养上清，并加入对应抗生素加压筛选5天后，通过流式分选获取高表达KLB&FGFR1c复合物或FGFR1c的CHOK1或HEK293单克隆细胞。最后获得CHOK1-hKLB&hFGFR1c、CHOK1-mKLB&mFGFR1c、CHOK1-hFGFR1c、CHOK1-hKLB、HEK293-hKLB、HEK293T-hKLB&hFGFR1c等重组细胞。
食蟹猴FGFR1c的氨基酸序列：
Figure PCTCN2022120887-appb-000004
注释：上述序列中，依次为
Figure PCTCN2022120887-appb-000005
Figure PCTCN2022120887-appb-000006
人FGF21的氨基酸序列：
Figure PCTCN2022120887-appb-000007
Figure PCTCN2022120887-appb-000008
人FGF19的氨基酸序列：
Figure PCTCN2022120887-appb-000009
实施例2.抗人β-klotho(KLB)杂交瘤单克隆抗体的制备
1.小鼠免疫和杂交瘤融合
使用本披露实施例1构建的表达人KLB&FGFR1c复合物重组细胞系CHOK1-hKLB&hFGFR1c和过表达鼠KLB&FGFR1c复合物的重组细胞系CHOK1-mKLB&mFGFR1c混合或交叉免疫SJL或Balb/C小鼠，采用腹腔注射，持续免疫多次，直至效价达到理想值后，挑选血清中抗体滴度高小鼠进行脾细胞融合，筛选特异性结合CHOK1-hKLB&hFGFR1c重组细胞系，但不结合人FGFR1c重组细胞系CHOK1-hFGFR1c的克隆。筛选得到活性好的杂交瘤细胞株mAb-99和mAb-18，对其进行序列扩增，收集对数生长期杂交瘤细胞，用Trizol(Invitrogen，Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TM逆转录试剂盒(Takara，Cat No.2680A)反转录。将反转录得到的cDNA采用鼠Ig-Primer Set(Novagen，TB326Rev.B 0503)进行PCR扩增后送测序公司测序，测得杂交瘤克隆鼠源抗体mAb-99，mAb-18的可变区序列如下所示：
>mAb-99重链可变区的氨基酸序列
Figure PCTCN2022120887-appb-000010
>mAb-99轻链可变区的氨基酸序列
Figure PCTCN2022120887-appb-000011
>mAb-18重链可变区的氨基酸序列
Figure PCTCN2022120887-appb-000012
>mAb-18轻链可变区的氨基酸序列
Figure PCTCN2022120887-appb-000013
鼠源抗体mAb-99和mAb-18的CDR序列见表4：
表4.mAb-99和mAb-18抗体CDR序列表
Figure PCTCN2022120887-appb-000014
备注：表中CDR的氨基酸残基由Kabat编号系统确定并注释。
2.人天然噬菌体文库筛选
利用人PBMC、脾脏、淋巴结组织分离B细胞，并提取RNA，构建天然单链噬菌体抗体库。将构建的天然单链噬菌体抗体文库经过包装形成噬菌体颗粒后，用过表达人KLB的重组细胞系进行淘筛，采用CHOK1-hKLB(参见实施例1)和HEK293T-hKLB(参见实施例1)重组细胞交叉淘筛。经过3轮淘选，挑取单克隆菌落包装成噬菌体的单链抗体，用ELISA测试噬菌体与人KLB蛋白的结合活性。挑选出阳性克隆，将其轻重链可变区分别与抗体重/轻链恒定区(重链恒定区如SEQ ID NO：22，轻链恒定区如SEQ ID NO：23)融合，构建IgG形式抗体。检测抗体对CHOK1-hKLB&FGFR1c、CHOK1-cynoKLB&FGFR1c和CHOK1-hFGFR1c的结合活性，最终筛选获得具备良好生物活性的全人抗体hAb-23，具体序列如下：
>hAb-23重链可变区的氨基酸序列
Figure PCTCN2022120887-appb-000015
>hAb-23轻链可变区的氨基酸序列
Figure PCTCN2022120887-appb-000016
>hAb-23重链的氨基酸序列
Figure PCTCN2022120887-appb-000017
Figure PCTCN2022120887-appb-000018
>hAb-23轻链的氨基酸序列
Figure PCTCN2022120887-appb-000019
>人IgG1重链恒定区(含L234A，L235A突变)的氨基酸序列
Figure PCTCN2022120887-appb-000020
>人κ轻链恒定区的氨基酸序列
Figure PCTCN2022120887-appb-000021
人源抗体hAb-23的CDR见表5：
表5.hAb-23抗体的CDR序列
Figure PCTCN2022120887-appb-000022
备注：表中抗体CDR的氨基酸残基由Kabat编号系统确定并注释。
实施例3、抗人KLB鼠源抗体的人源化
通过比对人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别嫁接(graft)到 相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列，然后将可变区序列与人恒定区序列融合，获得人源化抗体。以下示范性地描述mAb-99和mAb-18鼠源抗体的人源化：
1.mAb-99的人源化
mAb-99抗体人源化重链的模版选IGHV1-3*01/IGHJ6*01，即选择人种系重链IGHV1-3*01的FR1，FR2，FR3，和IGHJ6*01的JH6区(作为FR4)作为人源化抗体重链框架区；轻链模版为IGKV1-39*01/IGKJ4*01，即选择人种系轻链IGKV1-39*01的FR1，FR2，FR3，和IGKJ4*01的JK4区(作为FR4)作为人源化抗体轻链框架区。首先，将鼠源抗体mAb-99的CDR区移植到选择的人源化模板上，替换人源模板的CDR区，获得mAb-99的人源化抗体可变区序列。然后，对人源化抗体的重链可变区上第1、24、44、71和/或91位(根据Kabat编号系统编号)氨基酸残基进行回复突变，轻链可变区上第2、4、36、38、43、44和/或58位(根据Kabat编号系统编号)氨基酸残基进行回复突变，得到人源化抗体重/轻链可变区，具体序列如下：
>hAb99VH1的氨基酸序列(Graft+Q1E、R71S)
Figure PCTCN2022120887-appb-000023
>hAb99VH2的氨基酸序列(Graft+Q1E、R44G、R71S)
Figure PCTCN2022120887-appb-000024
>hAb99VH3的氨基酸序列(Graft+Q1E、A24T、R44G、R71S、Y91F)
Figure PCTCN2022120887-appb-000025
>hAb99VL1的氨基酸序列(Graft+P44N)
Figure PCTCN2022120887-appb-000026
>hAb99VL2的氨基酸序列(Graft+Y36F、A43T、P44N)
Figure PCTCN2022120887-appb-000027
>hAb99VL3的氨基酸序列(Graft+I2V、M4I、Y36F、A43T、P44N)
Figure PCTCN2022120887-appb-000028
>hAb99VL4的氨基酸序列(Graft+I2V、M4I、Y36F、Q38E、A43T、P44N、V58I)
Figure PCTCN2022120887-appb-000029
此外，还对抗体重链可变区的HCDR2(YIYIGNGDIEYNAKFKG)的第6和第7位的氨基残基由NG突变为NV、NL或QG，获得新的人源化抗体可变区序列，具体如下：
>hAb99VH1-NV的氨基酸序列
Figure PCTCN2022120887-appb-000030
>hAb99VH2-NV的氨基酸序列
Figure PCTCN2022120887-appb-000031
>hAb99VH3-NV的氨基酸序列
Figure PCTCN2022120887-appb-000032
>hAb99VH1-NL的氨基酸序列
Figure PCTCN2022120887-appb-000033
>hAb99VH2-NL的氨基酸序列
Figure PCTCN2022120887-appb-000034
>hAb99VH3-NL的氨基酸序列
Figure PCTCN2022120887-appb-000035
Figure PCTCN2022120887-appb-000036
>hAb99VH1-QG的氨基酸序列
Figure PCTCN2022120887-appb-000037
>hAb99VH2-QG的氨基酸序列
Figure PCTCN2022120887-appb-000038
>hAb99VH3-QG的氨基酸序列
Figure PCTCN2022120887-appb-000039
hAb99重链可变区的HCDR2突变序列如下表6所示：
表6.hAb99重链可变区的HCDR2突变序列
重链可变区 可变区序列号 对应的HCDR2序列(Kabat编号系统)
hAb99VH1-NV SEQ ID NO：40 YIYIGNVDIEYNAKFKG(SEQ ID NO：37)
hAb99VH2-NV SEQ ID NO：41 YIYIGNVDIEYNAKFKG(SEQ ID NO：37)
hAb99VH3-NV SEQ ID NO：42 YIYIGNVDIEYNAKFKG(SEQ ID NO：37)
hAb99VH1-NL SEQ ID NO：43 YIYIGNLDIEYNAKFKG(SEQ ID NO：38)
hAb99VH2-NL SEQ ID NO：44 YIYIGNLDIEYNAKFKG(SEQ ID NO：38)
hAb99VH3-NL SEQ ID NO：45 YIYIGNLDIEYNAKFKG(SEQ ID NO：38)
hAb99VH1-QG SEQ ID NO：46 YIYIGQGDIEYNAKFKG(SEQ ID NO：39)
hAb99VH2-QG SEQ ID NO：47 YIYIGQGDIEYNAKFKG(SEQ ID NO：39)
hAb99VH3-QG SEQ ID NO：48 YIYIGQGDIEYNAKFKG(SEQ ID NO：39)
将上述重链可变区与重链恒定区(如SEQ ID NO：22所示)融合，轻链可变区与轻链恒定区(如SEQ ID NO：23所示)融合，构建抗KLB抗体，并检测所获得的抗体与CHOK1-hKLB结合活性(实验方法参见本披露测试例2)，实验结果见表7。
表7.mAb-99人源化抗体与CHOK1-hKLB结合实验结果
抗体 重链可变区(VH) 轻链可变区(VL) EC50(nM)
hAb99-1 SEQ ID NO：30 SEQ ID NO：33 0.61
hAb99-2 SEQ ID NO：30 SEQ ID NO：34 0.35
hAb99-3 SEQ ID NO：30 SEQ ID NO：35 0.35
hAb99-4 SEQ ID NO：30 SEQ ID NO：36 0.37
hAb99-5 SEQ ID NO：31 SEQ ID NO：33 0.50
hAb99-6 SEQ ID NO：31 SEQ ID NO：34 0.41
hAb99-7 SEQ ID NO：31 SEQ ID NO：35 0.35
hAb99-8 SEQ ID NO：31 SEQ ID NO：36 0.39
hAb99-9 SEQ ID NO：32 SEQ ID NO：33 0.47
hAb99-10 SEQ ID NO：32 SEQ ID NO：34 0.36
hAb99-11 SEQ ID NO：32 SEQ ID NO：35 0.33
hAb99-12 SEQ ID NO：32 SEQ ID NO：36 0.33
hAb99-13 SEQ ID NO：40 SEQ ID NO：34 0.32
hAb99-14 SEQ ID NO：43 SEQ ID NO：34 0.60
hAb99-15 SEQ ID NO：46 SEQ ID NO：34 0.41
hAb99-16 SEQ ID NO：41 SEQ ID NO：34 0.47
hAb99-17 SEQ ID NO：44 SEQ ID NO：34 0.43
hAb99-18 SEQ ID NO：47 SEQ ID NO：34 0.43
hAb99-19 SEQ ID NO：42 SEQ ID NO：34 0.38
hAb99-20 SEQ ID NO：45 SEQ ID NO：34 0.41
hAb99-21 SEQ ID NO：48 SEQ ID NO：34 0.45
mAb99-hIgG1 SEQ ID NO：2 SEQ ID NO：3 0.97
备注：mAb99-hIgG1为mAb99的嵌合抗体，其重链恒定区如SEQ ID NO：22所示，轻链恒定区如SEQ ID NO：23所示。
示例性地，hAb99-13抗体的轻链、重链序列如下：
>hAb99-13抗体的重链的氨基酸序列
Figure PCTCN2022120887-appb-000040
>hAb99-13抗体的轻链的氨基酸序列
Figure PCTCN2022120887-appb-000041
2、mAb-18的人源化
mAb-18抗体人源化重链的模版为IGHV1-3*01/IGHJ6*01，即选择人种系重链IGHV1-3*01的FR1，FR2，FR3，和IGHJ6*01的JH6区(FR4)作为人源化抗体重链框架区；轻链模版为IGKV6-21*02/JGKJ2*01或者IGKV3-20*02/JGKJ2*01，即选择人种系轻链IGKV6-21*02或者IGKV3-20*02的FR1，FR2，FR3，和JGKJ2*01 的JK2区(作为FR4)作为人源化抗体轻链框架区。首先，将鼠源抗体mAb-18的CDR区移植到选择的人源化模板上，替换人源模板的CDR区，获得mAb-18的人源化抗体可变区序列。另外，对人源化抗体的重链可变区上第24、48、67、69、71和/或73位(根据Kabat编号系统编号)氨基酸残基进行回复突变，还可对第1位(根据Kabat编号系统编号)氨基酸残基进行突变，轻链可变区的第2、45、47、49、58和/或71位(根据Kabat编号系统编号)氨基酸残基进行回复突变，得到人源化抗体可变区序列，具体序列如下：
>hAb18VH1的氨基酸序列(Graft+Q1E、R71V、T73K)
Figure PCTCN2022120887-appb-000042
>hAb18VH2的氨基酸序列(Graft+Q1E、A24G、I69V、R71V、T73K)
Figure PCTCN2022120887-appb-000043
>hAb18VH3的氨基酸序列(Graft+Q1E、A24G、M48I、V67A、I69V、R71V、T73K)
Figure PCTCN2022120887-appb-000044
>hAb18VL1的氨基酸序列(Graft(IGKV6-21*02/JGKJ2*01)+L47W、K49Y)
Figure PCTCN2022120887-appb-000045
>hAb18VL2的氨基酸序列(Graft(IGKV6-21*02/JGKJ2*01)+I2N、L47W、K49Y、F71Y)
Figure PCTCN2022120887-appb-000046
>hAb18VL3的氨基酸序列(Graft(IGKV3-20*02/JGKJ2*01)+R45K、L47W、I58V)
Figure PCTCN2022120887-appb-000047
>hAb18VL4的氨基酸序列(Graft(IGKV3-20*02/JGKJ2*01)+I2N、R45K、L47W、I58V、F71Y)
Figure PCTCN2022120887-appb-000048
Figure PCTCN2022120887-appb-000049
此外，还对hAb18VL2轻链可变区的LCDR1(RASSSVSSSYLH(SEQ ID NO：15))的第2、3、5、6、7、8位氨基酸残基以及LCDR2(STSNLAS(SEQ ID NO：16))的第4位氨基酸残基进行突变，获得新的人源化抗体轻链可变区序列，具体序列如下：
>hAb18VL2-1的氨基酸序列
Figure PCTCN2022120887-appb-000050
>hAb18VL2-2的氨基酸序列
Figure PCTCN2022120887-appb-000051
>hAb18VL2-3的氨基酸序列
Figure PCTCN2022120887-appb-000052
>hAb18VL2-4的氨基酸序列
Figure PCTCN2022120887-appb-000053
>hAb18VL2-5的氨基酸序列
Figure PCTCN2022120887-appb-000054
>hAb18VL2-6的氨基酸序列
Figure PCTCN2022120887-appb-000055
>hAb18VL2-7的氨基酸序列
Figure PCTCN2022120887-appb-000056
>hAb18VL2-8的氨基酸序列
Figure PCTCN2022120887-appb-000057
>hAb18VL2-9的氨基酸序列
Figure PCTCN2022120887-appb-000058
>hAb18VL2-10的氨基酸序列
Figure PCTCN2022120887-appb-000059
>hAb18VL2-11的氨基酸序列
Figure PCTCN2022120887-appb-000060
>hAb18VL2-12的氨基酸序列
Figure PCTCN2022120887-appb-000061
>hAb18VL2-13的氨基酸序列
Figure PCTCN2022120887-appb-000062
hAb18VL2轻链可变区突变序列的CDR如下表8所示：
表8.hAb18VL2轻链可变区突变序列的CDR序列表
Figure PCTCN2022120887-appb-000063
Figure PCTCN2022120887-appb-000064
将上述重链可变区与重链恒定区(如SEQ ID NO：22所示)融合，轻链可变区与轻链恒定区(如SEQ ID NO：23所示)融合，构建抗KLB抗体，并检测所获得的抗体与CHOK1-hKLB结合活性(实验方法参见本披露测试例2)，实验结果见表9：
表9.mAb-18人源化抗体与CHOK1-hKLB结合活性实验结果
抗体 重链可变区(VH) 轻链可变区(VL) EC50(nM)
hAb18-1 SEQ ID NO：49 SEQ ID NO：52 1.34
hAb18-2 SEQ ID NO：49 SEQ ID NO：53 1.58
hAb18-3 SEQ ID NO：50 SEQ ID NO：52 1.9
hAb18-4 SEQ ID NO：50 SEQ ID NO：53 2
hAb18-5 SEQ ID NO：51 SEQ ID NO：52 1.8
hAb18-6 SEQ ID NO：51 SEQ ID NO：53 2
hAb18-7 SEQ ID NO：49 SEQ ID NO：56 2.4
hAb18-8 SEQ ID NO：49 SEQ ID NO：57 2.5
hAb18-9 SEQ ID NO：49 SEQ ID NO：58 2.4
hAb18-10 SEQ ID NO：49 SEQ ID NO：59 2.7
hAb18-11 SEQ ID NO：49 SEQ ID NO：60 2.7
hAb18-12 SEQ ID NO：49 SEQ ID NO：61 1.9
hAb18-13 SEQ ID NO：49 SEQ ID NO：62 5.4
hAb18-14 SEQ ID NO：49 SEQ ID NO：63 1.6
hAb18-15 SEQ ID NO：49 SEQ ID NO：64 1.9
hAb18-16 SEQ ID NO：49 SEQ ID NO：65 2.4
hAb18-17 SEQ ID NO：49 SEQ ID NO：66 1.8
hAb18-18 SEQ ID NO：49 SEQ ID NO：67 1.7
hAb18-19 SEQ ID NO：49 SEQ ID NO：68 4.1
mAb18-hIgG1 SEQ ID NO：4 SEQ ID NO：5 2.16
备注：mAb18-hIgG1为mAb18的嵌合抗体，其重链恒定区如SEQ ID NO：22所示，轻链恒定区如SEQ ID NO：23所示。
示例性地，hAb18-8抗体的轻、重链序列如下：
>hAb18-8抗体的重链的氨基酸序列
Figure PCTCN2022120887-appb-000065
>hAb18-8抗体的轻链的氨基酸序列
Figure PCTCN2022120887-appb-000066
实施例4.抗体的制备
1.抗体的分子克隆
将设计的抗体序列，经过密码子优化后产生人密码子偏好的编码基因序列，设计引物PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达质粒VH-CH1-FC-pHr/VK-CL-pHr。
2.抗体的表达与纯化
分别将表达抗体重轻链的质粒以1：1.5的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH3.0洗脱目的蛋白，用1M Tris-HCl，pH8.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
以下用生化测试方法验证本披露抗体的技术效果
测试例1.BIAcore检测抗hKLB抗体的亲和力实验
用Protein A生物传感芯片(Cat.#29127556，GE)亲和捕获抗体，然后于芯片表面流经一定浓度的hKLB(R&D，货号：5889-KB)，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个试验循环解离完成后，用Glycine1.5(Cat.#BR-1003-54，GE)将生物传感芯片洗净再生。采用1:1模型对数据拟合，获得抗体亲和力数据，具体实验结果见下表10。
表10.待测抗体与hKLB亲和力测试结果
抗体 ka(1/Ms) kd(1/s) KD(M)
hAb99-13 2.98E+05 6.26E-04 2.10E-09
hAb18-8 1.06E+05 1.03E-04 9.77E-10
hAb-23 2.47E+05 4.91E-05 1.99E-10
实验结果表明，本披露抗体与人KLB具有良好的结合功能。
测试例2.流式细胞术检测抗体与细胞膜表面KLB的结合实验
用流式细胞术方法检测抗体与过表达人KLB和猴KLB的CHOK1细胞株CHOK1-hKLB、CHOK1-cynoKLB(具体制备参见实施例1)的结合能力。具体如下：上述细胞培养于10％FBS的IMDM的培养基中，放置于37℃，5％CO 2培养箱中，培养2天，按每孔细胞数1×10 5个将细胞加至细胞板中，300g离心5分钟，1％BSA洗一次。加入梯度稀释的抗体，按每孔100μL加入细胞板中，4℃孵育1小时，1％BSA洗一次，每孔加入100μL APC-羊抗人IgG Fc荧光二抗稀释液(1:400)，4℃孵育1个小时。1％BSA洗板三次，每孔加入100μL PBS读板。实验结果如下表11所示。
表11.抗体与细胞膜表面KLB的结合实验结果
抗体 人KLB抗原结合EC50(nM) 猴KLB抗原结合EC50(nM)
hAb99-13 0.44±0.01 0.27±0.06
hAb18-8 1.93±0.6 1.90±0.35
hAb-23 0.39±0.09 0.13±0.04
实验结果显示，本披露的抗体hAb99-13、hAb18-8和hAb-23均能以较强的结合能力与细胞膜表面的人KLB和猴KLB结合。
测试例3.抗体与FGF19/FGF21竞争结合KLB实验
将Bio-FGF21/Bio-FGF19(FGF21如SEQ ID NO：76所示，FGF19如SEQ ID NO：77所示，分别用Biotin Labeling Kit-NH2(购自DOJINDO Molecular technologies Inc.，LK03)对FGF21和FGF19进行Biotin标记获得)以2ug/mL的浓度固化至SA传感器(孵育80秒)，然后将传感器转移至含有KLB(R&D，5889-KB-1mg)或含有KLB+抗体预混合物(提前孵育至少15分钟)的样品孔，结合180秒。ForteBio OCTET HTX仪器实时检测反应信号，获得结合曲线。实验结果如下表12所示。
表12.抗体与FGF21或FGF19的竞争结合实验
组别 FGF21 FGF19
hAb99-13+KLB 108％ 99％
hAb18-8+KLB 108％ 101％
hAb-23+KLB 113％ 99％
KLB 100％ 100％
备注：将单独KLB蛋白与Bio-FGF21或Bio-FGF19的结合信号定义为100％
实验结果表明，本披露的hAb99-13、hAb18-8和hAb-23均不与FGF21或FGF19竞争结合KLB。
测试例4.抗体对重组细胞系CHOK1-hKLB&hFGFR1c的激动活性检测实验
以下通过体外细胞实验测定受试抗体对重组细胞系CHOK1-hKLB&hFGFR1c(见实施例1)的激活作用，重组细胞系中瞬时转入受GAL4结合元件(GAL4binding element)调控的Luciferase报告基因以及GAL4-Elk1融合蛋白基因，其激活活性用EC50值表示。实验方法如下：
实验第一天，使用含有10％FBS、10μg/mL puromycin和800μg/mL G418的DME/F12培养基将CHOK1-hKLB&hFGFR1c细胞以15000个/孔的密度种于96孔板(Corning，#3903)，每孔100μL细胞悬液，96孔板外围只加入100μL的PBS。放置37℃，5％CO 2细胞培养箱培养过夜。第二天，弃去培养基，每孔加入50μL饥饿培养基(无FBS的DME/F12)。用Lipofectamine 3000将pFA2-Elk1和pFR-Luc以1:6比例转染细胞，每孔加入10μL质粒和Lipofectamine 3000混合物。转染后，将孔板放置37℃，5％CO 2培养箱培养24小时。第三天，每孔加入60μL用饥饿培养基梯度稀释的待测抗体，抗体的终浓度是从200nM开始进行4倍梯度稀释的9个浓度点，设置饥饿培养基为空白对照孔，孔板放置37℃，5％CO 2细胞培养箱培养24小时。第四天，取出96孔细胞培养板，每孔加入55μL One-glo Luciferase Assay System(信号激活荧光检测试剂，Promega，E6120)，使用多功能微孔板酶标仪(EnVision2015，PerkinElmer)读取发光信号值。使用GraphPad Prism根据抗体的对数浓度和信号值进行曲线拟合并计算EC 50值。激活倍数＝样品最大信号值/空白对照信号值，在抗体最高浓度下，抗体对KLB&FGFR1c受体激活程度，激活倍数越大，说明激活程度越大。实验中加入MK3655(参见WO2015112886A2中的抗KLB抗体h5H23)作为对照，实验结果如下表13。
表13.抗体对CHOK1-hKLB&hFGFR1c细胞的激活活性实验结果
抗体 EC50(nM) 激活倍数
hAb99-13 0.19 14.28
MK3655 0.64 5.62
实验结果显示，本披露抗体对CHOK1-hKLB&hFGFR1c细胞具有明显的激活活性，且激活程度强于对照分子。
测试例5.抗体对表达人KLB和人FGFR1c的HEK293T的激动活性检测实验
以下通过体外细胞实验测定受试抗体对瞬时表达人KLB和人FGFR1c的HEK293T细胞HEK293T-hKLB&hFGFR1c(参见实施例1)的激活作用，细胞系中瞬时转入人KLB、人FGFR1c和受磷酸化Elk调控的Luciferase报告基因，其激 活活性用EC50值来表示。具体方法如下：
实验第一天，使用含有10％FBS的DMEM/HIGH GLUCOSE培养基将HEK293T细胞以40000个/孔的密度种于96孔板(Corning BioCoat，#356692)，每孔100μL细胞悬液，96孔板外围只加入100μL的PBS。孔板放置37℃，5％CO 2细胞培养箱培养过夜。第二天，弃去原有培养基，每孔加入50μL饥饿培养基(无FBS的DMEM/HIGH GLUCOSE)。用Lipofectamine 3000将SRE-Luc2P(Promega，E1340)、hKLB(序列参见Uniprot ID:Q86Z14)和hFGFR1c(序列参见Uniprot ID:P11362)以30：9：1的比例转染细胞。每孔加入10μL质粒和Lipofectamine 3000的混合物。转染后，将孔板放置37℃，5％CO 2培养箱培养24小时。第三天，每孔加入60μL用饥饿培养基梯度稀释的待测抗体，抗体的终浓度是从200nM开始进行4倍梯度稀释的9个浓度点，设置饥饿培养基为空白对照孔，孔板放置37℃，5％CO 2细胞培养箱培养24小时。第四天，取出96孔细胞培养板，每孔加入60μL One-glo Luciferase Assay System(Promega，E6120)，使用多功能微孔板酶标仪(EnVision2015，PerkinElmer)读取发光信号值。使用GraphPad Prism根据抗体的对数浓度和信号值进行曲线拟合并计算EC50值。激活倍数＝样品最大信号值/空白对照信号值，在抗体最高浓度下，抗体对KLB&FGFR1c受体激活程度，激活倍数越大，说明激活程度越大。实验结果见表14。
表14.抗体对重组细胞系HEK293T-hKLB&hFGFR1c的激动活性检测结果
抗体 EC50(nM) 激活倍数
hAb99-13 2.62 3.57
hAb18-8 5.35 2.29
MK3655 5.06 1.65
实验结果显示，本披露抗体对HEK293T-hKLB&hFGFR1c具有明显的激活活性，且强于对照分子。
测试例6.抗体对重组细胞系HEK293-hFGFR1c的激活作用检测实验
为了验证抗体对人KLB和人FGFR1c稳定细胞系的激活是人KLB依赖的，本测试例测定受试抗体对稳定表达人FGFR1c(序列参见Uniprot ID:P11362)的HEK293(ATCC，CRL-1573)细胞HEK293-hFGFR1c的激活作用。具体方法如下：
实验第一天，使用含有10％FBS的DMEM/HIGH GLUCOSE培养基将HEK293-hFGFR1c细胞以25000个/孔的密度种于96孔板(Corning，#3903)，每孔100μL细胞悬液，96孔板外围只加入100μL的PBS。孔板放置37℃，5％CO 2细胞培养箱培养过夜。第二天，用Lipofectamine 3000将SRE-Luc2P转染细胞，每孔加入10μL质粒和Lipofectamine 3000的混合物。转染后，将孔板放置37℃，5％CO 2培养箱培养24小时。第三天，每孔加入10μL用铺板培养基梯度稀释的待测抗体，抗体的终浓度是从200nM开始进行4倍梯度稀释的第9个浓度点，设置铺 板培养基为空白对照孔，孔板放置37℃，5％CO 2细胞培养箱培养16小时。第四天，取出96孔细胞培养板，每孔加入60μL One-glo Luciferase Assay System(信号激活荧光检测试剂，Promega，E6120)，使用多功能微孔板酶标仪(EnVision2015，PerkinElmer)读取发光信号值。待测抗体信号值与空白对照读值的比值表示抗体对FGFR1c的激活活性，激活倍数＝样品最大信号值/空白对照信号值，在样品最高浓度下，抗体对HEK293-hFGFR1c激活程度，激活倍数越大，说明激活程度越大。实验中加入FGF2(Sino Biological，10014-HNAE)作为对照。实验结果见表15。
表15.抗体对HEK293-hFGFR1c细胞的激活活性检测结果
单抗 激活倍数
hAb99-13 0.77
hAb18-8 0.81
hAb-23 0.86
FGF2 11.59
实验结果表明，本披露抗体对HEK293-hFGFR1c激活活性弱，说明本披露抗体对人KLB和人FGFR1c稳定细胞系的激活是人KLB依赖的。
测试例7.抗体对表达FGFR2c/FGFR3c/FGFR4的L6/hKLB细胞ERK磷酸化水平测定
以下通过体外细胞实验测定受试抗体对表达人FGFR2c、FGFR3c或FGFR4的L6/hKLB细胞株ERK磷酸化水平的升高的作用，来测定抗体对FGFR2c、FGFR3c或FGFR4的选择性。具体方法如下：
首先，通过慢病毒感染的方法构建稳定表达人KLB(序列参见Uniprot ID:Q86Z14)的L6-hKLB单克隆细胞株(L6细胞来源于ATCC，CRL-1458)，然后再通过慢病毒感染的方法分别构建稳定表达人FGFR2c(human FGF receptor 2c)、人FGFR3c(human FGF receptor 3c)和人FGFR4(human FGF receptor 4)的L6-hKLB&hFGFR2c、L6-hKLB&hFGFR3c和L6-hKLB&hFGFR4细胞。实验第一天，使用含有10％FBS(Gibco，10099-141C)的DMEM/HIGH GLUCOSE培养基(GE，SH30243.01)分别将L6-hKLB&hFGFR2c、L6-hKLB&hFGFR3c和L6-hKLB&hFGFR4细胞以每孔25000个的密度种于96孔板，每孔加入100μL细胞悬液，放置37℃，5％CO 2细胞培养箱培养过夜。第二天，将孔板中的完全培养基甩掉，每孔加入90μL含有0.1％Fatty-free BSA(Sangon Biotech，A602448)的DMEM/HIGH GLUCOSE培养基，置于37℃，5％CO 2的培养箱孵育1小时。每孔加入10μL用PBS梯度稀释的待测抗体，抗体的终浓度是从50或500nM开始4倍梯度稀释的9个浓度点，设置不含抗体的空白对照细胞孔，孔板放置37℃，5％CO 2的培养箱孵育15分钟。使用Advanced ERK phospho-T202/Y204kit(Cisbio，64AERPEH)和ERK total Kit(Cisbio，64NRKPEH)按照说明书操作步骤检测细 胞裂解液中pERK和total ERK的水平。用PHERAstar多功能酶标仪(BMG Labtech)读取337nm波长激发，665nm和620nm波长发射的荧光值。根据比值＝信号值 665nm/信号值 620nm的公式分别计算化合物各浓度对应的pERK和total ERK比值。用抗体最高浓度的pERK/total ERK的比值除以空白对照孔的比值计算S/N值，也即激活倍数，激活倍数越大，说明激活程度越大。本披露抗体对ERK磷酸化水平的激活活性由上述分析所得，实验中加入AKR001(参见WO2010129503A1中SEQ ID NO：47的FGF21类似物)作为对照。实验结果如下表16所示：
表16.抗体对细胞ERK磷酸化水平的激活检测实验结果
Figure PCTCN2022120887-appb-000067
实验结果显示，本披露抗体对L6-hKLB&hFGFR2c、L6-hKLB&hFGFR3c和L6-hKLB&hFGFR4细胞的激活作用弱于对照分子AKR001。
测试例8.抗体的体内药效实验
本实验选用恒河猴种族自发的非酒精性脂肪性肝炎(NASH)模型，运用MRI影像技术和肝活检技术评价抗体对肝脏中脂肪含量、NAS(NAFLD Activity Score)分数、体重、BMI(Body Mass Index)、腹围以及糖脂代谢的影响。
实验方法：将NASH恒河猴进行35天适应驯化饲养，期间对恒河猴糖脂代谢指标进行检测，并进行超声指引下的肝脏活检病理，利用MRI(GE，3T)定量分析肝内脂肪含量，筛选肝内脂肪含量指标稳定的恒河猴，平均分为三组：hAb99-13组(13mg/kg)，hAb18-8组(13mg/kg)和安慰剂组(溶媒对照组，10mM冰乙酸，9％蔗糖，pH5.2)。各组采用静脉注射给药，每2周给药1次，共给药5次。给药后，通过MRI(GE，3T)定量分析肝内脂肪含量，GE超声引导下进行肝脏活检，检查给药前后恒河猴坐高和腹围；定期检测恒河猴糖脂代谢、肝功指标和体重，并计算其平均值。实验结果见表17-1至表17-10，其中，
肝内脂肪含量实验结果见表17-1，实验结果显示，安慰剂组给药前和给药后60天(D60)，ROI(Region of interest)百分数见明显改变；hAb99-13组给药后60天时，ROI％值明显下降，从给药前(D0)的8.4％降低到6.6％(P<0.05)。
表17-1.抗体对NASH恒河猴肝脏脂肪含量的影响检测结果
组别 给药前ROI(％) D60 ROI(％) 变化率(％)
hAb99-13组 8.4 6.6 -20.84
安慰剂组 7.3 7.4 0.62
注：表中，变化率＝(当前值-基线值)/基线值*100％，其中，基线值为给药前ROI (％)，当前值为待检测时间点(D60)的ROI(％)
肝组织活检病理分析结果见表17-2，实验结果显示，安慰剂组给药前后的肝脏组织病理结果未见明显改善；hAb99-13给药组，与给药前比较，给药后第65天平均NAS评分由3.7降至2.7，脂肪变评分也明显下降。
表17-2.抗体对NASH恒河猴肝脏病理的影响
Figure PCTCN2022120887-appb-000068
注：D0指给药前检测值，D65为首次给药后第65天检测值。
抗体对胰岛功能影响实验结果见表17-3和表17-4。实验结果显示，各实验组AUC Ins0-30min均有下降，hAb99-13和hAb18-8下降幅度超过50％；hAb99-13组和hAb18-8组给药后，HOMA-IR(稳态模型评估胰岛素抵抗指数，用于评价胰岛素抵抗水平，其随着胰岛素抵抗水平升高而升高)有较大的降幅。
表17-3.抗体对NASH恒河猴IVGTT胰岛素指标的影响
Figure PCTCN2022120887-appb-000069
备注：变化率＝(当前值-基线值)/基线值*100％，其中基线值为给药前的AUC Ins0-30min(U*min/L)，当前值为待检测时间点(D65)的AUC Ins0-30min(U*min/L)
表17-4.抗体对NASH恒河猴HOMA-IR变化率(％)影响实验结果
组别 给药前 D14 D28 D35 D49
hAb99-13组 0.00 -25.15 -15.72 -31.16 -52.41
hAb18-8组 0.00 -40.75 -35.15 -48.31 -49.26
安慰剂组 0.00 25.11 11.34 103.23 57.79
备注：HOMA-IR变化率＝(当前值-基线值)/基线值*100％，其中基线值为给药前HOMA-IR，当前值为给药后待检测时间点的HOMA-IR。
抗体对NASH恒河猴的空腹血糖(FPG)和空腹胰岛素(FPI)影响实验结果见表17-5和表17-6。实验结果显示，安慰剂组给药前后动物FPG和FPI未见明显改变；抗体给药组的FPG和FPI均有明显的降低。
表17-5.抗体对NASH恒河猴FPG的影响实验结果
Figure PCTCN2022120887-appb-000070
备注：D0为给药前，D7为首次给药后第7天；FPG变化率＝(当前值-基线值)/基线值*100％，其中，基线值为给药前FPG值，当前值为给药后待检测时间点的FPG值。
表17-6.抗体对NASH恒河猴FPI的影响实验结果
Figure PCTCN2022120887-appb-000071
备注：D0为给药前，D7为首次给药后第7天；变化率＝(当前值-基线值)/基线值*100％，其中，基线值为给药前FPI值，当前值为给药后待检测时间点的FPI值。
抗体对NASH恒河猴TG影响实验结果见表17-7。实验结果显示，安慰剂组TG未见明显改变；hAb99-13给药组的TG有一定的下降。
表17-7.抗体对NASH恒河猴TG的影响
Figure PCTCN2022120887-appb-000072
备注：D0为给药前，D7为首次给药后第7天；变化率＝(当前值-基线值)/基线值*100％，其中，基线值为给药前TG值，当前值为给药后待检测时间点的TG值。
抗体对NASH恒河猴脂联素(Adiponectin)的影响实验结果见表17-8，实验结果显示，安慰剂组动物脂联素有下降趋势；本披露抗体给药组的脂联素却有一定程度的升高。
表17-8.抗体对NASH恒河猴Adiponectin的影响
Figure PCTCN2022120887-appb-000073
备注：D0为给药前，D7为首次给药后第7天；变化率＝(当前值-基线值)/基线值*100％，其中，基线值为给药前Adiponectin值，当前值为给药后待检测时间点的Adiponectin值。
抗体对恒河猴腹围和BMI的影响实验结果见表17-9和表17-10。实验结果表明，安慰剂组动物腹围和BMI未见明显改变；本披露的抗体给药组的腹围和BMI均有明显的降低。
表17-9.抗体对NASH恒河猴腹围的影响
Figure PCTCN2022120887-appb-000074
备注：变化率＝(当前值-基线值)/基线值*100％，其中基线值为给药前检测值，当前值为给药后待检测时间点(D60)的检测值
表17-10.抗体对NASH恒河猴BMI的影响
Figure PCTCN2022120887-appb-000075
备注：BMI＝体重/坐高；变化率＝(当前值-基线值)/基线值*100％，其中基线值为给药前检测值，当前值为给药后待检测时间点(D60)的检测值。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本披露的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (15)

  1. 一种抗KLB抗体，其包含重链可变区和轻链可变区，其中，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中，
    i)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：40-48中的任一序列的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：34中的LCDR1、LCDR2和LCDR3的氨基酸序列；或所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：2中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：3中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
    ii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：49中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：56-68中的任一序列的LCDR1、LCDR2和LCDR3的氨基酸序列；或所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：4中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：5中的LCDR1、LCDR2和LCDR3的氨基酸序列；或
    iii)所述重链可变区的HCDR1、HCDR2和HCDR3分别包含SEQ ID NO：18中的HCDR1、HCDR2和HCDR3的氨基酸序列，和所述轻链可变区的LCDR1、LCDR2和LCDR3分别包含SEQ ID NO：19中的LCDR1、LCDR2和LCDR3的氨基酸序列。
  2. 根据权利要求1所述的抗KLB抗体，其中：
    i)所述重链可变区的HCDR1包含SEQ ID NO：6的氨基酸序列，HCDR2包含SEQ ID NO：37、38、39或7的氨基酸序列，和HCDR3包含SEQ ID NO：8的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：9的氨基酸序列，LCDR2包含SEQ ID NO：10的氨基酸序列，和LCDR3包含SEQ ID NO：11的氨基酸序列；或
    ii)所述重链可变区的HCDR1包含SEQ ID NO：12的氨基酸序列，HCDR2包含SEQ ID NO：13的氨基酸序列，和HCDR3包含SEQ ID NO：14的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：69、15、70、71、72、73或74的氨基酸序列，LCDR2包含SEQ ID NO：75或16的氨基酸序列，和LCDR3包含SEQ ID NO：17的氨基酸序列；或
    iii)所述重链可变区的HCDR1包含SEQ ID NO：24的氨基酸序列，HCDR2包含SEQ ID NO：25的氨基酸序列，和HCDR3包含SEQ ID NO：26的氨基酸序 列，和所述轻链可变区的LCDR1包含SEQ ID NO：27的氨基酸序列，LCDR2包含SEQ ID NO：28的氨基酸序列，和LCDR3包含SEQ ID NO：29的氨基酸序列；
    优选地，
    i)所述重链可变区的HCDR1包含SEQ ID NO：6的氨基酸序列，HCDR2包含SEQ ID NO：37的氨基酸序列，和HCDR3包含SEQ ID NO：8的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：9的氨基酸序列，LCDR2包含SEQ ID NO：10的氨基酸序列，和LCDR3包含SEQ ID NO：11的氨基酸序列；或
    ii)所述重链可变区的HCDR1包含SEQ ID NO：12的氨基酸序列，HCDR2包含SEQ ID NO：13的氨基酸序列，和HCDR3包含SEQ ID NO：14的氨基酸序列，和所述轻链可变区的LCDR1包含SEQ ID NO：69的氨基酸序列，LCDR2包含SEQ ID NO：75的氨基酸序列，和LCDR3包含SEQ ID NO：17的氨基酸序列。
  3. 根据权利要求1或2所述的抗KLB抗体为鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。
  4. 根据权利要求1至3中任一项所述的抗KLB抗体，其中，
    i)所述重链可变区包含SEQ ID NO：40、30、31、32、41、42、43、44、45、46、47或48，或与其具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：34、33、35或36，或与其具有至少90％序列同一性的氨基酸序列；或
    ii)所述重链可变区包含SEQ ID NO：49、50或51，或与其具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：57、52、53、54、55、56、58、59、60、61、62、63、64、65、66、67或68，或与其具有至少90％序列同一性的氨基酸序列；或
    iii)所述重链可变区包含SEQ ID NO：18，或与其具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：19，或与其具有至少90％序列同一性的氨基酸序列；或
    iv)所述重链可变区包含SEQ ID NO：2，或与其具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：3，或与其具有至少90％序列同一性的氨基酸序列；或
    v)所述重链可变区包含SEQ ID NO：4，或与其具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：5，或与其具有至少90％序列同一性的氨基酸序列。
  5. 根据权利要求1至4中任一项所述的抗KLB抗体，其中，
    i)所述重链可变区包含SEQ ID NO：40或与其具有至少90％序列同一性的氨 基酸序列，和所述轻链可变区包含SEQ ID NO：34或与其具有至少90％序列同一性的氨基酸序列；优选地，所述重链可变区在其框架区上包含选自1E、24T、44G、71S和91F(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变，和所述轻链可变区在其框架区上包含选自2V、4I、36F、38E、43T、44N和58I(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变；
    ii)所述重链可变区包含SEQ ID NO：49或与其具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：57或与其具有至少90％序列同一性的氨基酸序列；优选地，所述重链可变区在其框架区上包含选自1E、24G、48I、67A、69V、71V和73K(根据Kabat编号系统编号)中的一个或更多个氨基酸突变，和/或所述轻链可变区在其框架区上包含选自2N、45K、47W、49Y、58V和71Y(根据Kabat编号系统编号)中的一个或更多个氨基酸回复突变；或
    iii)所述重链可变区包含SEQ ID NO：18或与其具有至少90％序列同一性的氨基酸序列，和所述轻链可变区包含SEQ ID NO：19或与其具有至少90％序列同一性的氨基酸序列。
  6. 根据权利要求1至5中任一项所述的抗KLB抗体，其包括重链恒定区和轻链恒定区；
    优选地，所述重链恒定区为人IgG1重链恒定区，所述轻链恒定区为人κ轻链恒定区；
    更优选地，所述重链恒定区包含SEQ ID NO：22的氨基酸序列，所述轻链恒定区包含SEQ ID NO：23的氨基酸序列。
  7. 根据权利要求6所述的抗KLB抗体，其中所述的抗KLB抗体包含重链和轻链，其中：
    i)所述重链包含与SEQ ID NO：78具有至少90％序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：79具有至少90％序列同一性的氨基酸序列；或
    ii)所述重链包含与SEQ ID NO：80具有至少90％序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：81具有至少90％序列同一性的氨基酸序列；或
    iii)所述重链包含与SEQ ID NO：20具有至少90％序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：21具有至少90％序列同一性的氨基酸序列。
  8. 根据权利要求6所述的抗KLB抗体，其中所述的抗KLB抗体包含重链和轻链，其中：
    i)所述重链包含SEQ ID NO：78的氨基酸序列，和所述轻链包含SEQ ID NO：79的氨基酸序列；
    ii)所述重链包含SEQ ID NO：80的氨基酸序列，和所述轻链包含SEQ ID NO： 81的氨基酸序列；或
    iii)所述重链包含SEQ ID NO：20的氨基酸序列，和所述轻链包含SEQ ID NO：21的氨基酸序列。
  9. 分离的抗KLB抗体，其与权利要求1至8中任一项所述的抗KLB抗体竞争性结合人KLB或猴KLB。
  10. 根据权利要求1至9中任一项所述的抗KLB抗体，其中所述的抗KLB抗体具有一种或更多种以下特征：
    A.所述抗KLB抗体对表达hKLB&hFGFR1c的细胞具有激活活性；优选地，所述抗KLB抗体对CHOK1-hKLB&hFGFR1c的激活倍数大于10，和/或所述抗KLB抗体对HEK293T-hKLB&hFGFR1c细胞的激活倍数大于2；
    B.所述抗KLB抗体对表达hFGFR1c的重组细胞激活活性弱；优选地，所述抗KLB抗体对HEK293-hFGFR1c细胞的激活倍数小于1；
    C.所述抗KLB抗体对表达hKLB&hFGFR2c、hKLB&hFGFR3c和/或hKLB&hFGFR4的细胞的激活活性弱；优选地，所述抗KLB抗体对L6-hKLB&hFGFR2c和/或L6-hKLB&hFGFR4的激活倍数小于2；
    D.所述抗KLB抗体能与人KLB结合，也能与猴KLB结合；优选地，所述抗KLB抗体能以小于2.0E-9M的EC50值与人KLB和猴KLB结合，所述EC50值通过流式细胞检测方法检测；和
    E.所述抗KLB抗体能与人KLB结合；优选地，所述抗体能以小于3.0E-9M的KD值与人KLB结合，所述KD值通过Biacore方法检测。
  11. 一种药物组合物，其含有：
    治疗有效量的如权利要求1至10中任一项所述的抗KLB抗体，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂，
    优选地，所述的药物组合物中还包含至少一种第二治疗剂。
  12. 分离的核酸，其编码如权利要求1至10中任一项所述的抗KLB抗体。
  13. 宿主细胞，其包含如权利要求12所述的核酸。
  14. 一种治疗疾病的方法，所述方法包括向有需要的受试者施用治疗有效量的如权利要求1至10中任一项所述的抗KLB抗体或权利要求11所述的药物组合物的步骤；
    优选地，所述疾病是与FGF21和/或FGF19相关的疾病；
    更优选地，所述疾病选自NASH、2型糖尿病、肥胖症、血脂异常、心血管疾 病和代谢综合征。
  15. 一种诱导FGF21和/或FGF19信号转导的方法，所述方法包括使表达KLB和FGFR的细胞与如权利要求1至10中任一项所述的抗KLB抗体接触。
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