WO2022028354A1 - 抗ngf抗体及其抗原结合片段、其制备方法和应用 - Google Patents

抗ngf抗体及其抗原结合片段、其制备方法和应用 Download PDF

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WO2022028354A1
WO2022028354A1 PCT/CN2021/109995 CN2021109995W WO2022028354A1 WO 2022028354 A1 WO2022028354 A1 WO 2022028354A1 CN 2021109995 W CN2021109995 W CN 2021109995W WO 2022028354 A1 WO2022028354 A1 WO 2022028354A1
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antibody
seq
antigen
amino acid
deletions
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PCT/CN2021/109995
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French (fr)
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原晓辉
王国永
刘玉姣
郑东红
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杭州熙源生物技术有限公司
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Priority to KR1020227030251A priority Critical patent/KR20220149909A/ko
Priority to EP21852225.8A priority patent/EP4083068A4/en
Priority to US17/906,190 priority patent/US20230109780A1/en
Priority to JP2022547093A priority patent/JP2023512313A/ja
Publication of WO2022028354A1 publication Critical patent/WO2022028354A1/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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/48—Nerve growth factor [NGF]

Definitions

  • the invention belongs to the technical field of biological immunity, in particular to an anti-NGF antibody that can specifically bind to human nerve growth factor and an antigen-binding fragment thereof, and also relates to a preparation method and use of the antibody and the antigen-binding fragment thereof.
  • Nerve Growth Factor was the first neurotrophic factor identified, and its role in peripheral and central neuron development and survival has been characterized.
  • NGF has been shown to be a critical survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and basal forebrain cholinergic neurons (Smeyne et al. (1994) Nature, 368:246-249; Crowley et al. (1994) Cell, 76:1001-1011), which upregulate neuropeptide expression in sensory neurons (Lindsay et al. (1989) Nature, 337:362-364).
  • NGF contains three subunits, ⁇ , ⁇ , and ⁇ , and the ⁇ subunit is the active subunit of NGF.
  • TrkA TrkA tyrosine kinase receptor
  • TrkA TrkA tyrosine kinase A
  • p75 neurotrophin receptor p75 neurotrophin receptor, p75 NTR, also known as “low affinity” NGF receptor
  • NGF/TrkA signaling pathway is closely related to pain and can mediate the occurrence of pain.
  • NGF is highly expressed, and activation of nociceptive neurons by TrkA is triggered by multiple mechanisms, resulting in pain signals.
  • the thermal stimulus-induced paw withdrawal latency was significantly shortened (Lewin et al. (1994) Eur J Neurosci, 6:1903-1912).
  • neutralization of NGF activity by administering NGF antibodies, TrkA-IgG, etc. can significantly reduce inflammation-related pain (Woolf CJ et al. (1994) Neuroscience, 62:327-331; McMahon SB et al.
  • NGF ⁇ Homozygous missense mutations in the NGF ⁇ gene can cause HSAN5 symptoms in humans, and such patients are insensitive to pain, cold, and heat (Larsson et al. (2009) Neurobiol Dis, 33: 221-228).
  • the NTRK1 gene encodes the TrkA protein, and its polymorphisms are closely related to changes in pain perception. Autosomal recessive mutations in exon 17 of NTRK1 cause congenital hypoalgesia with anhidrosis (Indo et al. (1996) Nature genetics, 13:485-488).
  • the drugs currently used clinically for the treatment of chronic pain include non-steroidal anti-inflammatory drugs, anticonvulsants, opioids, etc.
  • these drugs have many shortcomings. Among them, the efficacy of non-steroidal anti-inflammatory drugs is limited. And have side effects including gastrointestinal bleeding and kidney toxicity; while opioids have side effects such as addiction.
  • opioids have side effects such as addiction.
  • Tanezumab the first anti-NGF antibody drug to be developed, has been reported to exhibit potent and wide-ranging analgesic effects on arthralgia associated with degenerative joint disease, chronic low back pain, and bladder pain associated with interstitial cystitis. (Lane NE et al. (2010) N Engl J Med, 363: 1521-1531), the drug is currently undergoing Phase III clinical trials for indications such as osteoarthritis, back pain, and cancer pain.
  • NGF antibodies may face problems such as being restricted to severely ill people, not being used for a long time, and dose limitations, making the clinical application of NGF antibodies need further safety verification.
  • NGF is an extremely important factor for neuronal development, and the effect of NGF dose on neurons also needs to be considered when developing drugs that inhibit NGF function.
  • the effective dose of the antibody drug depends on the neutralizing activity on the antigen and the amount of the antigen present in the body, and an increase in the neutralizing activity correlates with a decrease in the dose administered.
  • Anti-NGF antibody research needs to obtain CDR regions with different affinities for different epitopes or the same epitope. Different CDRs have different immunogenicity, resulting in different antibody tolerance rates and toxicity, which directly affect drug efficacy.
  • the immune response of the subject to the antibody itself can form an immune complex, which alters the pharmacokinetics, produces an allergic response, etc., eliminating its therapeutic use.
  • the human immune system has minimal antibody response to humanized antibodies, and humanized antibodies have similar half-lives to natural human antibodies, which ensures less frequent dosing and lower the administered dose.
  • the main purpose of the present invention is to provide an anti-NGF antibody with high affinity, strong specificity and remarkable curative effect.
  • the present invention also provides the preparation method and use of the antibody.
  • the present invention provides an anti-NGF antibody or antigen-binding fragment thereof capable of specifically binding to NGF, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs 3 complementarity determining regions
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or having one or several amino acid substitutions, deletions or additions therefrom (e.g., 1, 2 or 3 amino acid substitutions, deletions) or added) sequence,
  • VH CDR2 consisting of the following sequence: SEQ ID NO: 14, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added), and
  • VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or having one or several amino acid substitutions, deletions or additions therefrom (e.g. 1, 2 or 3 amino acid substitutions, deletions) or added);
  • VL light chain variable region
  • CDRs 3 complementarity determining regions
  • VL CDR1 consisting of the following sequence: SEQ ID NO: 22, or having one or several amino acid substitutions, deletions or additions therefrom (e.g. 1, 2 or 3 amino acid substitutions, deletions) or added) sequence,
  • VL CDR2 consisting of the following sequence: SEQ ID NO: 23, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added), and
  • VL CDR3 consisting of the following sequence: SEQ ID NO: 24, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added);
  • substitutions described in any one of (i)-(vi) are conservative substitutions
  • the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 13, VH CDR2 as shown in SEQ ID NO: 14, VH CDR3 as shown in SEQ ID NO: 15 and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 22, VL CDR2 as shown in SEQ ID NO: 23, VL CDR3 as shown in SEQ ID NO: 24 .
  • the present invention provides an anti-NGF antibody or antigen-binding fragment thereof capable of specifically binding to NGF, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs 3 complementarity determining regions
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 16, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added) sequence,
  • VH CDR2 consisting of the following sequence: SEQ ID NO: 17, or having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions) therefrom or added), and
  • VH CDR3 consisting of the following sequence: SEQ ID NO: 18, or having one or several amino acid substitutions, deletions or additions therefrom (e.g., 1, 2 or 3 amino acid substitutions, deletions) or added);
  • VL light chain variable region
  • CDRs 3 complementarity determining regions
  • VL CDR1 consisting of the following sequence: SEQ ID NO: 25, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added) sequence,
  • VL CDR2 consisting of the following sequence: SEQ ID NO: 26, or having one or several amino acid substitutions, deletions or additions therefrom (e.g., 1, 2 or 3 amino acid substitutions, deletions) or added), and
  • VL CDR3 consisting of the following sequence: SEQ ID NO: 27, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added);
  • substitutions described in any one of (i)-(vi) are conservative substitutions
  • the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 16, VH CDR2 as shown in SEQ ID NO: 17, VH CDR3 as shown in SEQ ID NO: 18 and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:25, VL CDR2 as shown in SEQ ID NO:26, VL CDR3 as shown in SEQ ID NO:27 .
  • the present invention provides an anti-NGF antibody or antigen-binding fragment thereof capable of specifically binding to NGF, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs 3 complementarity determining regions
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 19, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added) sequence,
  • VH CDR2 consisting of the following sequence: SEQ ID NO: 20, or having one or several amino acid substitutions, deletions or additions therefrom (e.g. 1, 2 or 3 amino acid substitutions, deletions) or added), and
  • VH CDR3 consisting of the following sequence: SEQ ID NO: 21, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added);
  • VL light chain variable region
  • CDRs 3 complementarity determining regions
  • VL CDR1 consisting of the following sequence: SEQ ID NO: 28, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added) sequence,
  • VL CDR2 consisting of the following sequence: SEQ ID NO: 29, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added), and
  • VL CDR3 consisting of the following sequence: SEQ ID NO: 30, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions) therefrom or added);
  • substitutions described in any one of (i)-(vi) are conservative substitutions
  • the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:19, VH CDR2 as shown in SEQ ID NO:20, VH CDR3 as shown in SEQ ID NO:21 and, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:28, VL CDR2 as shown in SEQ ID NO:29, VL CDR3 as shown in SEQ ID NO:30 .
  • the antibody or its antigen-binding fragment capable of specifically binding to NGF according to the present invention
  • the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises the 3 CDRs contained in the heavy chain variable region shown in any one of SEQ ID NOs: 1, 5 and 9; and the light chain variable region comprises SEQ ID NO: 3 CDRs contained in the light chain variable region shown in any one of 3, 7 and 11;
  • the three CDRs contained in the variable region of the heavy chain, and/or the three CDRs contained in the variable region of the light chain are defined by the Kabat, Chothia or IMGT numbering systems.
  • the antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions
  • the antibody or antigen-binding fragment thereof comprises: a VH having the sequence shown in SEQ ID NO:1 and a VL having the sequence shown in SEQ ID NO:3.
  • the antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions
  • the antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO:5 and a VL having the sequence set forth in SEQ ID NO:7.
  • the antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • (iii) have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions
  • the antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO:9 and a VL having the sequence set forth in SEQ ID NO:11.
  • the antibody or antigen-binding fragment thereof according to the present invention wherein the antibody or antigen-binding fragment thereof further comprises:
  • amino acid substitutions, deletions or additions eg, up to 20 amino acids
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region;
  • the light chain constant region is a kappa light chain constant region.
  • the antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb); and/or, the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody;
  • the humanized antibody or antigen-binding fragment thereof comprises: SEQ ID NO: 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and The heavy chain of the sequence set forth in any one of 63 and/or the light chain of the sequence set forth in any one of SEQ ID NOs: 31 and 41.
  • the antibody or antigen-binding fragment thereof wherein the antibody or antigen-binding fragment thereof bears a label; preferably, the antibody or antigen-binding fragment thereof bears a detectable label, such as an enzyme (such as root peroxidase), radionuclides, fluorescent dyes, luminescent substances (such as chemiluminescent substances) or biotin.
  • an enzyme such as root peroxidase
  • radionuclides such as radionuclides, fluorescent dyes, luminescent substances (such as chemiluminescent substances) or biotin.
  • the present invention also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof;
  • the polynucleotide comprises as SEQ ID NO: 2, 4, 6, 8, 10, 12, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
  • the present invention also provides a vector comprising the isolated nucleic acid molecule; preferably, the vector is a cloning vector or an expression vector.
  • the present invention also provides host cells comprising the isolated nucleic acid molecule or the vector.
  • the present invention also provides a method for preparing the antibody or antigen-binding fragment thereof, comprising, culturing the host cell under conditions that allow the expression of the antibody or antigen-binding fragment thereof, and extracting from the cultured host cell recovering the antibody or antigen-binding fragment thereof in culture;
  • the host cell is a mammalian cell, more preferably a human, murine, sheep, horse, dog or cat cell, further preferably a Chinese hamster ovary cell.
  • the present invention also provides bispecific or multispecific molecules comprising said antibodies or antigen-binding fragments thereof;
  • the bispecific or multispecific molecule specifically binds NGF, and additionally specifically binds one or more other targets;
  • the bispecific or multispecific molecule further comprises at least one molecule (eg, a second antibody) having a second binding specificity for a second target.
  • at least one molecule eg, a second antibody
  • the present invention also provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a therapeutic agent linked to the antibody or antigen-binding fragment thereof;
  • the therapeutic agent is selected from toxins, radioisotopes, drugs or cytotoxic agents;
  • the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof;
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule or immunoconjugate, and a pharmaceutically acceptable carrier and/or excipient;
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent;
  • the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.
  • the present invention also provides a kit, which contains the antibody or antigen-binding fragment thereof;
  • the antibody or antigen-binding fragment thereof bears a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin;
  • a detectable label such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin;
  • the kit further comprises a second antibody, which specifically recognizes the antibody or its antigen-binding fragment;
  • the second antibody further comprises a detectable label such as an enzyme (eg horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg a chemiluminescent substance) or biotin.
  • a detectable label such as an enzyme (eg horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg a chemiluminescent substance) or biotin.
  • the present invention also provides a chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof;
  • the antigen binding domain comprises the heavy chain variable region and the light chain variable region of the antibody or antigen binding fragment thereof;
  • the antigen binding domain is an scFv
  • the antigen-binding receptor comprises an antigen-binding fragment of the antibody
  • the antigen binding receptor is expressed by immune effector cells (eg T cells).
  • immune effector cells eg T cells.
  • the present invention also provides the use of the antibody or antigen-binding fragment thereof, or bispecific or multispecific molecule, or immunoconjugate, or pharmaceutical composition, or chimeric antigen receptor, in the preparation of medicine, wherein the The medicament is for the treatment of NGF-mediated diseases or conditions;
  • the disease or condition includes osteoarthritis pain, postoperative pain, rheumatoid arthritis pain, low back pain, cancer-related pain, neuropathic pain and visceral pain;
  • the subject is a mammal, such as a human.
  • the present invention also provides a method for preventing and/or treating a disease in a subject (eg, a human), the method comprising administering to a subject in need thereof an effective amount of said antibody or antigen thereof A binding fragment, or the bispecific or multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the host cell;
  • the disease is an NGF-mediated disease or disorder
  • the disease or condition includes osteoarthritis pain, postoperative pain, rheumatoid arthritis pain, low back pain, cancer-related pain, neuropathic pain and visceral pain;
  • the subject is a mammal, such as a human.
  • the present invention also provides a method for detecting the presence or amount of NGF (eg, human NGF) in a sample, comprising the steps of:
  • NGF eg, human NGF
  • the antibody or antigen-binding fragment thereof is detectably labeled
  • the NGF is human NGF.
  • the antibody provided by the present invention has high affinity, and the affinity constant K D value is less than or close to 10-12 M, which can effectively block the binding between NGF and its receptor, and block the response of pain;
  • the antibody provided by the present invention has extremely strong binding specificity to the antigen, and has no cross-binding with the same family of NGF proteins, so it can be expected that its clinical safety risk will also be reduced;
  • the antibody provided by the present invention has a significant animal analgesic effect, and is expected to have a significant clinical effect;
  • the antibody provided by the present invention is expressed in CHO cells, and has the advantages of high yield, high activity, simple purification process and low production cost.
  • Fig. 1 shows the results of an ELISA experiment in which the anti-NGF antibody of the present invention binds to human NGF protein.
  • Fig. 2 shows the effect of the anti-NGF antibody of the present invention on the binding of human NGF to receptors TrkA and p75.
  • Figure 3 shows that the anti-NGF antibodies of the present invention inhibit NGF-induced proliferation of TF-1 cells.
  • FIG. 4 shows that the anti-NGF antibodies of the present invention inhibit NGF-induced proliferation of TrkA/Ba/F3 cells.
  • FIG. 5 shows that the anti-NGF antibodies of the present invention inhibit reporter gene expression in TrkA/NFAT-bla/CHO cells.
  • Figure 6 shows that the anti-NGF antibodies of the present invention specifically bind to NGF and inhibit NGF-induced signaling pathways.
  • Figure 7 shows the competitive binding curve of the anti-NGF antibody 43E5 of the present invention and 138E12.
  • Figure 8 shows the effects of the anti-NGF antibodies 43E5 and 138E12 of the present invention on CFA-induced inflammatory pain in mice, wherein **P ⁇ 0.01vs saline; ***P ⁇ 0.001vs saline.
  • Figure 9 shows that the humanized anti-NGF antibody of the present invention binds to human NGF.
  • Figure 10 shows that the humanized anti-NGF antibodies of the present invention bind to NGF homofamily proteins.
  • Figure 11 shows that the humanized anti-NGF antibodies of the present invention inhibit NGF-induced proliferation of TF-1 cells.
  • the antibodies or antigen-binding fragments described herein are capable of specifically binding to NGF with a binding affinity (K D ) of less than or close to 10 ⁇ 12 M, as measured by biofilm layer optical interferometry.
  • the binding affinity value can be expressed as a K D value, which is calculated from the ratio of the off rate to the on rate (k off /k on ) when the antigen and antibody binding reaches equilibrium.
  • Said antigen-binding affinity (eg, KD ) may suitably be determined by suitable methods known in the art, including, for example, the use of instruments such as Fortebio's Biofilm Layer Optical Interferometry.
  • the antibodies or antigen-binding fragments described herein bind to NGF with an EC50 (ie, half-binding concentration) of 0.1 nM. Binding of an antibody or antigen-binding fragment to NGF can be determined by methods known in the art such as sandwich assays such as ELISA, Western blot, FACS or other binding assays.
  • the antibody is specific for NGF.
  • the antibody optionally does not interact with NGF homofamily proteins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), Neurotrophin-4 (neurotrophin-4, NT-4) binding.
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • Neurotrophin-4 neurotrophin-4, NT-4 binding.
  • Recombinant human NGF protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC) was mixed and emulsified with an equal amount of Freund's complete adjuvant (Sigma-Alderich, F5881) as an immunogen for initial immunization.
  • Freund's complete adjuvant (Sigma-Alderich, F5881)
  • Five 6-week-old BALB/c and C57 mice (Jiangsu Huafukang) were prepared, and each animal was subcutaneously injected with 50 ⁇ g of immunogen (excluding the adjuvant mass, the same below).
  • the immunogen was emulsified with incomplete Freund's adjuvant (Sigma-Alderich, F5506) for subsequent booster immunizations.
  • each animal was injected intraperitoneally with 25 ⁇ g immunogen for the first boost; 2 weeks later, each animal was injected subcutaneously with 25 ⁇ g immunogen for the second boost. 4-5 weeks after the last immune shock, 25 ⁇ g of immunogen was injected intraperitoneally.
  • the mouse B cells were isolated, mixed with SP2/0 cells (Cell Bank of the Chinese Academy of Sciences, TCM18), and fused according to the operation manual of the electroporator of BTX Company. After the fusion cells were cultured, an enzyme-linked immunosorbent assay (ELISA) was used to screen out hybridoma cells that bind to NGF and inhibit the binding of human NGF to the receptor TrkA. Subcloning was performed by limiting dilution method, and 3 positive hybridoma monoclonal cell lines were obtained by screening in the same ELISA method, which were named 43E5, 137H8 and 138E12 respectively.
  • ELISA enzyme-linked immunosorbent assay
  • the hybridoma monoclonal cell line was expanded and cultured with serum-free medium, and the medium was collected and purified by protein G column to obtain mouse-derived anti-human NGF monoclonal antibodies 43E5, 137H8, and 138E12.
  • the binding ability of anti-NGF antibodies was studied using human NGF (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC) as the antigen. Each well of a 96-well microtiter plate was coated with 50ng of human NGF. After washing and blocking, serially diluted antibodies were added and incubated at room temperature for 1 hour. After washing three times, add HRP-conjugated goat anti-mouse antibody (Biolegend, 405306), incubate at room temperature for 1 hour, add tetramethylbenzidine (TMB, Biolegend, 421101) after three washes to develop color, 1M HCl to stop color development, enzyme labeling The instrument reads the absorbance value at 450nm.
  • human NGF Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC
  • HRP-conjugated goat anti-mouse antibody Biolegend, 405306
  • TMB tetramethylbenzidine
  • the anti-NGF antibodies secreted by the three hybridomas all bound to human NGF in a dose-dependent manner (Figure 1), and the EC50 of the three antibodies for binding to human NGF were 0.082 nM, 0.112 nM, and 0.1 nM, respectively (Table 1).
  • Antibody affinity was determined using the biomolecular interaction detection platform ForteBio Octet Red96 (PALL). Biotin-labeled human NGF was immobilized using SA (Streptavidin) Biosensor (Fortebio, 18-5021), and then conjugated with gradient concentrations of anti-NGF antibodies. Add buffer (1X Kinetics Buffer:PBS+0.1%BSA+0.05%Tween20) for dissociation, and finally calculate the affinity kinetic constant of antigen-antibody binding by instrument algorithm (Table 2).
  • Example 4 Detection of blocking effect of murine anti-NGF antibody on binding of human NGF to receptor TrkA or p75
  • each well of 96-well microtiter plate is coated with 50ng of NGF receptor TrkA protein (fused to human Fc), washed three times and blocked with 3% BSA for 1 hour.
  • 10000ng/mL (66.67nM) anti-NGF antibody was diluted 10 times successively to 0.17ng/mL (0.0011nM), and 100 ⁇ l was mixed with an equal volume of 1 ⁇ g/mL biotin-labeled human NGF, and the two were incubated at room temperature for 0.5 hours Then add it to the ELISA plate after blocking and washing. Incubate for 1 hour at room temperature and wash three times.
  • Streptavidin-HRP Streptavidin-HRP (Streptavidin-HRP, Biolegend, 405210) was added for 0.5 hour incubation and washed three times. Add TMB to the microtiter plate, and read after the color development is terminated.
  • each well of a 96-well microtiter plate is coated with 50ng of human NGF and washed three times. After blocking with 3% BSA for 1 hour and washing three times, 10000ng/mL (66.67nM) anti-human NGF antibody was serially diluted 3 times to 0.17ng/mL (0.0011nM), 100 ⁇ L was added to each well, incubated at room temperature for 1 hour and washed three times. 100 ⁇ L of 1 ⁇ g/mL TrkA fused to human Fc was added to each well, incubated for 1 hour at room temperature, and washed three times. HRP-conjugated donkey anti-human IgG antibody (Biolegend, 410902) was added and reacted at room temperature for 1 hour. After washing, add TMB to develop color, and read after termination.
  • 10000ng/mL (66.67nM) anti-human NGF antibody was serially diluted 3 times to 0.17ng/mL (0.0011nM)
  • 100 ⁇ L
  • the anti-NGF antibody can block the binding of human NGF to the receptor TrkA, and the blocking effect increases significantly with the increase of the antibody concentration.
  • the IC50s of the three antibodies blocking the binding of human NGF to the receptor TrkA are shown in Table 3 and Table 4;
  • Each well of the 96-well microtiter plate was coated with 50ng of human NGF, blocked with 3% BSA for 1 h, washed 3 times, and added with 10 ⁇ g/mL, 1 ⁇ g/mL, 0.1 ⁇ g/mL, 0.01 ⁇ g/mL, 0 ⁇ g/mL (66.67 nM, 6.67 ⁇ g/mL) nM, 0.67nM, 0.067nM, 0nM) anti-NGF antibody 40 ⁇ L, incubate for 15 minutes at room temperature, add 40 ⁇ L 2 ⁇ g/mL p75 fused to human Fc (Beijing Yiqiao Shenzhou Technology Co., Ltd., 13184-H02H) to each well and incubate for 1 hour After washing 3 times, 100uL of HRP-conjugated donkey anti-human Fc secondary antibody (Biolegend, 410902) was added, and the reaction was carried out at room temperature for 1 hour.
  • Tanezumab can simultaneously block the binding of NGF to TrkA and p75.
  • p75 receptor function is related to neuron development, osteoblast differentiation, proliferation, myoblast differentiation, muscle repair, etc.
  • GZ389988A a small molecule inhibitor of TrkA developed by Sanofi, showed no adverse reactions to RPOA during the clinical trial.
  • 43E5 cannot block the binding of human NGF to p75, and it is expected that the possibility of clinical occurrence of RPOA is reduced, and the safety is better than other antibodies with p75 blocking effect.
  • TF-1 cells human blood leukemia cells, ATCC, CRL-2003
  • GM-CSF granulocyte macrophage colony stimulating factor
  • TF-1 cells were cultured in RPMI1640 medium (HyClone, SH30027) containing 10% FBS (Gibco, 10091148), 2 ⁇ g/mL GM-CSF (R&D, 215-GM-010), and cells were collected in logarithmic growth phase, The GM-CSF in the original growth medium was washed thoroughly, resuspended in medium without GM-CSF, and 5000 cells per well were diluted in 80 ⁇ l medium and plated on a white transparent bottom 96-well cell culture plate (corning, 3610).
  • Anti-NGF antibody was prepared by 4-fold serial dilution of 400 ⁇ g/mL (2666.67nM) to prepare 10 concentrations, mixed with an equal volume of 50ng/mL human NGF, incubated for 0.5 hours, and 20 ⁇ L was added to cells in a 96-well plate, at 37°C , 100 ⁇ L was added after culturing the cells in a 5% CO 2 incubator for 72 hours Cell viability detection reagent (Promega, G7573) was used to disrupt cells by shaking, and the optical luminescence signal was read using a multifunctional microplate reader (SpectraMax).
  • the three anti-NGF antibodies were able to inhibit the proliferation of TF-1 cells induced by human NGF, and the IC50s were shown in Table 5.
  • the growth of Ba/F3 cells has two ways: IL3-dependent and IL3-independent.
  • the IL3-independent growth requires Ba/F3 cells to stably express active kinases.
  • a Ba/F3 (TrkA/Ba/F3) cell expressing full-length human TrkA was constructed, and the proliferation of the cells was induced by the addition of NGF.
  • TrkA/Ba/F3 cells were cultured in RPMI1640 medium containing 10% FBS, 100ng/mL NGF, cells were collected and washed thoroughly to remove NGF in the original growth medium, and 3000 cells per well were resuspended in 80 ⁇ L of NGF-free medium. culture medium and plated onto white transparent bottom 96-well cell culture plates (corning, 3610). Mix human NGF and serially diluted antibodies and add them to cells in a 96-well plate. The final concentration of NGF is 5ng/mL, and the final concentration of serially diluted anti-NGF antibodies is up to 40 ⁇ g/mL (266.67nM).
  • TrkA/Ba/F3 cells In the process of observing the growth of TrkA/Ba/F3 cells, the cells added with human NGF can proliferate normally; the three anti-NFG antibodies can inhibit the proliferation of TrkA/Ba/F3 cells induced by NGF, and the inhibitory effect increases with the increase of the concentration (Fig. 4), IC50 is shown in Table 6.
  • Anti-NGF antibody inhibits NGF-induced TrkA/Ba/F3 cell proliferation IC50
  • NGF-TrkA The binding of NGF to the transmembrane receptor TrkA can activate the downstream phospholipase C (PLC), which leads to the increase of intracellular calcium concentration and the activation of protein kinase C (PKC) through a series of signal transduction, and finally promotes the nuclear factor (nuclear factor) and activates T Nuclear factor (NFAT) is transported from the cytoplasm to the nucleus, turning on the expression of NFAT-dependent genes.
  • PLC phospholipase C
  • NFAT nuclear factor
  • the gene encoding human TrkA was integrated into the genome of TrkA/NFAT-bla/CHO cells (ThermoFisher, K1516), and an NFAT response element was inserted upstream of the reporter gene ⁇ -lactamase ( ⁇ -bla) gene.
  • TrkA-NFAT-bla/CHO cells were prepared according to the manufacturer's instructions, and 10,000 cells per well were plated into 384-well plates and cultured overnight. Mix human NGF and serially diluted antibodies, incubate at room temperature for 0.5 hours, and then add them to the cells, so that the final concentration of NGF is 80ng/mL, and the final concentration of serially diluted NGF antibodies is up to 40 ⁇ g/mL (266.67nM).
  • LiveBLAzer TM FRET-B/G ⁇ -lactamase substrate CCF4 (ThermoFisher, K1095) prepared according to the manual was added to the cells after culturing for 5 hours in a 37°C, 5% CO2 incubator, and allowed to stand at room temperature in the dark for 2 Hour. Signals were read by a multi-plate reader (SpectraMax).
  • Each well of the 96-well microtiter plate was coated with 50ng human NGF (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC), human BDNF (Beijing Yiqiao Shenzhou Technology Co., Ltd., 50240-MNAS), human NT-3 (Beijing Yiqiao Shenzhou Technology Co., Ltd., 50240-MNAS) Shenzhou Technology Co., Ltd., 10286-HNAE), or human NT-4 (Alomone, N-270), according to the method in Example 2 to detect the binding of mouse anti-NGF antibody to human NGF and three proteins of the same family.
  • human NGF Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC
  • human BDNF Beijing Yiqiao Shenzhou Technology Co., Ltd., 50240-MNAS
  • human NT-3 Beijing Yiqiao Shenzhou Technology Co.,
  • TrkA-NFAT-bla/CHO cells TrkB-NFAT-bla/CHO cells (ThermoFisher, K1491) and TrkC-NFAT-bla/CHO cells (ThermoFisher, K1515) were prepared as in Example 5C according to the manufacturer's instructions.
  • the three anti-NGF antibodies inhibited the subsequent signaling caused by the binding of NGF to TrkA, but did not affect the signaling of BDNF to TrkB and NT-3 to TrkC (Fig. 6E);
  • the three anti-NGF antibodies of this application do not bind to NGF's same family antibodies BDNF, NT-3, and NT-4, while Tanezumab It has obvious cross-binding effect with NT-3 and NT-4, that is to say, the specificity of 43E5 and 138E12 of the present application is better than that of Tanezumab, and the expected clinical adverse reaction performance is better than that of Tanezumab.
  • Example 7 Epitope grading of anti-NGF antibodies 43E5 and 138E12
  • the biomolecular interaction detection platform Fortebio Ocete RED96 was used to detect the competition between the two anti-NGF antibodies 43E5 and 138E12 for binding to NGF.
  • Biotin-labeled human NGF was immobilized with SA (Streptavidin) Biosensor.
  • SA Streptavidin
  • the first step was loading to monitor the binding of antibody 138E12.
  • the second step was loading to continue to monitor the binding of 138E12 or 43E5.
  • buffer (1X Kinetics Buffer:PBS) was added. +0.1%BSA+0.05%Tween20) for dissociation.
  • 43E5 and 138E12 can bind to human NGF simultaneously and in a non-competitive manner.
  • RNA of hybridoma cells was extracted using TRIzol kit (Ambion, 15596-026), and the first-strand cDNA (Takara) was synthesized using it as a template.
  • Antibody light chain and heavy chain fragments were obtained by rapid amplification of cDNA ends (RACE), and the amplified fragments were cloned into standard vectors respectively.
  • the heavy and light chain variable region sequences of anti-NGF antibodies 43E5, 137H8, 138E12 obtained by sequencing are:
  • Example 9 The effect of anti-NGF antibody on CFA-induced inflammatory pain in mice
  • C57BL/6 mice (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.), male, SPF grade, 6-8 weeks old, adaptively reared for 5 days.
  • the animals were divided into 3 groups, namely the saline group, the anti-NGF antibody 43E5 group, and the anti-NGF antibody 138E12 group, with 10 animals in each group.
  • VonFrey was used to measure the pain threshold 3 times (with an interval of not less than 10 minutes), and the average value of the 3 times was taken as the basal pain threshold (Fig. 8A).
  • mice were injected with 25 ⁇ l of CFA into the left hind foot sole to induce inflammation (the ratio of CFA and liquid paraffin was 1:1). After the toes were significantly swollen (about 24 hours after modeling), the pain threshold of the left hind foot of the mice was detected three times by the same method, and the average value was taken as the pain threshold before administration (Fig. 8B).
  • each group was subcutaneously injected with normal saline, anti-NGF antibody 43E5, and anti-NGF antibody 138E12 at a dose of 10 mg/kg, and the pain thresholds at 24 hours, 48 hours, and 72 hours after administration were measured (Figure 8C-8E). The effect of the test substance on pain threshold was evaluated.
  • Example 10 Humanization and characterization of murine anti-NGF antibody
  • Humanization was carried out according to the light chain variable region (VL) and heavy chain variable region (VH) sequences of the antibody secreted by the hybridoma cells obtained above.
  • the amino acid sequences of VL and VH of the murine antibody were compared and searched in the human embryonic antibody amino acid sequence database, and the human IGHV and IGKV sequences with high homology were found as humanization templates.
  • the possible steric hindrance and interaction between the amino acids in the variable region and the framework region are analyzed, and the amino acids in the framework region that are critical to maintaining the activity of the humanized antibody are determined, and these amino acids are retained during the humanization process.
  • Humanization of the light and heavy chain variable regions was accomplished by CDR grafting technology.
  • the anti-NGF antibody 43E5 uses human IGHV1-2 as the heavy chain variable region and human IGKV2-30 as the light chain variable region template to obtain antibodies 43E5-01 and 43E5-02.
  • human IGHV1-69 as the heavy chain variable region and human IGKV2-30 as the light chain variable region template
  • 43E5-01, 43E5-02, 43E5-05 and 43E5-06 four The light chain variable region sequences of the two antibodies are identical.
  • Anti-NGF antibody 138E12 uses human IGHV4-59 as the heavy chain variable region and human IGKV1-39 as the light chain variable region to obtain antibodies 138E12-01 and 138E12-02.
  • 138E12-01 and 138E12-02 antibody light chain variable region amino acids The sequence is the same, but the amino acids of the heavy chain variable region are different.
  • 138E12-01 and 138E12-02 antibody heavy chain complementarity determining region sequences contain isomerization site DG and deamidation site NS, in order to remove the potential impact of the above sites on antibodies, 138E12-01 and 138E12- The heavy chain variable region of the 02 antibody was further engineered.
  • DG 54-55 in VH was mutated to EG 54-55
  • NS 60-61 was mutated to QS 60-61
  • DG 54-55 and NS 60-61 were mutated to EG 54-55 at the same time and QS 60-61 to obtain antibodies 138E12-08, 138E12-09 and 138E12-10, respectively.
  • DG 54-55 in VH was mutated to EG 54-55
  • DG 54-55 was mutated to DA 54-55
  • NS 60-61 was mutated to QS 60-61
  • NS 60-61 was mutated to Simultaneous mutation of NT 60-61
  • DG 54-55 and NS 60-61 to EG 54-55 and QS 60-61 yielded antibodies 138E12-03, 138E12-04, 138E12-05, 138E12-06 and 138E12-11.
  • 138E12- 07 Obtained by using human IGHV4-59 as the heavy chain variable region and human IGKV1-39 as the light chain variable region, the framework region sequence of the heavy chain variable region is fully human, and does not retain murine amino acids, but it is repeated.
  • DG 54-55 and NS 60-61 of the strand complementarity determining region were mutated to EG 54-55 and QS 60-61 at the same time.
  • the humanized antibody heavy chain and light chain were separately synthesized, and then ligated into the corresponding plasmid after restriction enzyme digestion.
  • the constructed plasmid was transiently transfected into CHO cells for expression. After 7-10 days of expression, the cell culture supernatant was equilibrated with a MabSelect column (GE Healthcare) equilibrated with a corresponding buffer (such as phosphate buffered saline (pH 7.4)). Purify and then elute with sodium citrate or other buffers.
  • the antibody obtained through this step can be identified by SDS-PAGE or SEC-HPLC for purity and other subsequent characterization studies.
  • variable region amino acid sequences of the above-mentioned antibodies are shown in Table 9:
  • Anti-NGF antibody 43E5 humanized antibody variable region sequence
  • Anti-NGF Antibody 43E5 Humanized antibodies 43E5-01, 43E5-02, 43E5-05, 43E5-06 contain the same light chain. Light chain variable region amino acid sequence:
  • Anti-NGF antibody 138E12 humanized antibody variable region sequence
  • Anti-NGF antibody 138E12 Humanized antibodies 138E12-01, 138E12-02, 138E12-03, 138E12-04, 138E12-05, 138E12-06, 138E12-07, 138E12-08, 138E12-09, 138E12-10, 138E12-11 contain the same light chain.
  • NGF Human NGF (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC) was used as the antigen to study the affinity and specificity of the antibody after humanization.
  • NGF was diluted with coating solution (PBS), and 50 ng per well of 96-well microtiter plate was coated. After washing and blocking, serially diluted antibodies were added, and incubated at room temperature for 1 hour. After washing three times, add HRP-conjugated donkey anti-human IgG antibody (Biolegend), and react at room temperature for 1 hour. After three washings, add tetramethylbenzidine (TMB, Biolegend) for color development, stop color development with 1M HCl, and read at 450 nm with a microplate reader absorbance value.
  • TMB tetramethylbenzidine
  • the neutralizing activity of the humanized anti-NGF antibodies was detected by proliferation of TF-1 cells as described in Example 5A.
  • the results are shown in FIG. 11 , all the humanized antibodies can inhibit the proliferation of TF-1 cells induced by NGF, and the inhibitory ability of most of the humanized antibodies is equivalent to that of the human-mouse chimeric antibody.
  • the IC50 is shown in Table 11.

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Abstract

本发明提供了一种抗NGF抗体或其抗原结合片段、其制备方法和应用；本发明还提供了编码所述抗NGF抗体或其抗原结合片段的分离的多核苷酸，以及包含所述分离的多核苷酸的载体。本发明还提供了本发明的抗体或其抗原结合片段在制备用于治疗NGF介导的疾病或病症的药物中的用途。

Description

抗NGF抗体及其抗原结合片段、其制备方法和应用 技术领域
本发明属于生物免疫技术领域，具体涉及能特异性结合人神经生长因子的抗NGF抗体及其抗原结合片段，本发明还涉及所述抗体及其抗原结合片段的制备方法和用途。
背景技术
神经生长因子(Nerve Growth Factor,NGF)是最先确定的神经营养因子，其在外周及中枢神经元的发育及存活中的作用已被表征。NGF已被证明在周围交感和胚胎感觉神经元和基底前脑胆碱能神经元的发育中是一种关键的存活和维持因子(Smeyne et al.(1994)Nature,368:246-249；Crowley et al.(1994)Cell,76:1001-1011)，其能上调感觉神经元中神经肽的表达(Lindsay et al.(1989)Nature,337:362-364)。NGF包含α、β、γ三个亚单位，β亚单位是NGF的活性亚单位。目前已知NGF通过两种不同的膜表面受体，TrkA酪氨酸激酶受体(Tropomyosin receptor kinase A,TrkA,也称为“高亲和性”NGF受体)和p75共同神经营养因子受体(p75 neurotrophin receptor,p75NTR,也称为“低亲和性”NGF受体)调节其活性(Chao et al.(1986)Science,232:518-521)。
NGF/TrkA信号通路与疼痛密切相关，可介导疼痛的发生。在受伤和炎症组织中，NGF高表达，并且TrkA对伤害性感受神经元的激活通过多种机制引发，并产生疼痛信号。大鼠注射NGF后，热刺激引起的缩足潜伏期明显缩短(Lewin et al.(1994)Eur J Neurosci,6:1903-1912)。动物模型中，通过给予NGF抗体、TrkA-IgG等方式中和NGF活性，可显著减少炎症相关的疼痛(Woolf CJ et al.(1994)Neuroscience,62:327-331；McMahon SB et al.(1995)Net.Med.1:774-780；Koltzenburg M et al.(1999)Eur.J.Neurosci.11:1698-1704)，提示NGF水平的增加是产生全身痛觉过敏反应所必需的。患有类风湿关节炎患者滑液中NGF含量明显升高，非炎症患者滑液中未检测到NGF(Aloe et al.(1992)Arthritis and Rheumatism,35:351-355)。健康人注射NGF后，可产生痛觉敏感及局部性疼痛(Petty et al.(1994)Ann Neurol,36:244-246)。NGFβ基因发生纯合子错义突变会使人体出现HSAN5症状，这类患者对疼痛、冷、热不敏感(Larsson et al.(2009)Neurobiol Dis,33:221-228)。NTRK1基因编码TrkA蛋白，其多态性与痛觉改变密切相关。NTRK1的17号外显子常染色体隐性突变会引起先天性痛觉不敏感合并无汗症(Indo et al.(1996)Nature genetics,13:485-488)。
在全球范围内，有数以千万计的患者在遭受着慢性疼痛带来的痛苦，而这个数字随着人口增加还在不断增加。当前临床上使用的用于慢性疼痛治疗的药物包括非甾体类抗炎药、抗惊厥药、阿片类药物等，然而这些药物有很多的不足，其中，非甾体抗炎药的疗效有限，并且具有包括消化道出血和肾脏毒性等副作用；而阿片类药物具有成瘾等副作用。该领域亟需能缓解疼痛、无毒性、防滥用的非阿片类止痛药，因此通过抑制NGF治疗慢性疼痛的方法价值非常明确。到目前为止，有许多抗人NGF抗体处于研发或临床开发阶段，其中进展最快的包括Pfizer/Lilly公司的抗NGF单克隆抗体Tanezumab和Regeneron/Sanofi公司的Fasinumab。Tanezumab是最先开发的抗NGF抗体药物，据报道它对退行性关节病相伴的关节痛、慢性腰痛、与间质性膀胱炎相伴的膀胱痛等疼痛显示出了强大且范围广的镇痛效果(Lane NE et al.(2010)N Engl J Med,363:1521-1531)，目前该药物正在进行骨关节炎、背部疼痛、癌症疼痛等适应症的III期临床实验。Fasinumab治疗骨关节炎痛的II/III期临床研究数据显示，4种剂量Fasinumab治疗组的患者在疼痛缓解方面取得了具有统计学意义的显著改善。另一方面,多个NGF抑制剂的临床实验也表明，NGF抗体可能面临被限制用于重病人群、不能长期使用和剂量限制等问题，使得NGF抗体的临床应用需要进一步安全验证。
NGF是神经元发育极为重要的因子，在开发抑制NGF功能的药物时，也需要考虑NGF剂量对神经元的影响。一方面，抗体药物的有效剂量取决于对抗原的中和活性和存在于体内的抗原量，中和活性的提高与给药量降低相关。抗NGF抗体研究需要获得针对不同抗原表位或相同抗原表位而亲和力不同的CDR区。不同CDR的免疫原性不同，造成的抗体耐受速度和毒性也不同，直接影响药效。另一方面，受试者对抗体本身产生的免疫反应会形 成免疫复合物，使得药物动力学改变，产生变应性反应等，消除了它的治疗性用途。相对于鼠源抗体和嵌合抗体，人免疫系统针对人源化抗体的抗体反应最小，同时人源化抗体与天然人抗体具有相似的半衰期，这样就保证了更少的给药频率和更低的给药剂量。
因此，研发亲和力更高，特异性更强的抗NGF抗体，并进行人源化改造对于与NGF相关的各种疾病的治疗或预防而言是极其重要的。
发明内容
基于现有技术的不足，本发明的主要目的在于提供一种高亲和力、强特异性及疗效显著的抗NGF抗体。本发明还提供了所述抗体的制备方法和用途。
一方面，本发明提供了能够特异性结合NGF的抗NGF抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)包含下述3个互补决定区(CDR)的重链可变区(VH)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:13，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:14，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:15，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
和/或
(b)包含下述3个互补决定区(CDR)的轻链可变区(VL)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:22，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:23，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:24，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
优选地，(i)-(vi)任一项中所述的置换为保守置换；
优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:13所示的VH CDR1、如SEQ ID NO:14所示的VH CDR2、如SEQ ID NO:15所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:22所示的VL CDR1、如SEQ ID NO:23所示的VL CDR2、如SEQ ID NO:24所示的VL CDR3。
一方面，本发明提供了能够特异性结合NGF的抗NGF抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)包含下述3个互补决定区(CDR)的重链可变区(VH)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:16，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:17，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:18，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
和/或
(b)包含下述3个互补决定区(CDR)的轻链可变区(VL)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:25，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:26，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:27，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
优选地，(i)-(vi)任一项中所述的置换为保守置换；
优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:16所示的VH CDR1、如SEQ ID NO:17所示的VH CDR2、如SEQ ID NO:18所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:25所示的VL CDR1、如SEQ ID NO:26所示的VL CDR2、如SEQ ID NO:27所示的VL CDR3。
一方面，本发明提供了能够特异性结合NGF的抗NGF抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)包含下述3个互补决定区(CDR)的重链可变区(VH)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:19，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:20，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:21，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
和/或
(b)包含下述3个互补决定区(CDR)的轻链可变区(VL)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:28，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:29，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:30，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
优选地，(i)-(vi)任一项中所述的置换为保守置换；
优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:19所示的VH CDR1、如SEQ ID NO:20所示的VH CDR2、如SEQ ID NO:21所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:28所示的VL CDR1、如SEQ ID NO:29所示的VL CDR2、如SEQ ID NO:30所示的VL CDR3。
根据本发明所述的能够特异性结合NGF的抗NGF抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，
所述重链可变区包含SEQ ID NO:1、5和9中任一项所示的重链可变区中含有的3个CDR；并且，所述轻链可变区包含SEQ ID NO:3、7和11中任一项所示的轻链可变区中含有的3个CDR；
优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义。
根据本发明所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:1所示的序列；
(ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:3所示的序列；
(v)与SEQ ID NO:3所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换；
优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:1所示的序列的VH和具有如SEQ ID NO:3所示的序列的VL。
根据本发明所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:5所示的序列；
(ii)与SEQ ID NO:5所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:5所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:7所示的序列；
(v)与SEQ ID NO:7所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:7所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换；
优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:5所示的序列的VH和具有如SEQ ID NO:7所示的序列的VL。
根据本发明所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:9所示的序列；
(ii)与SEQ ID NO:9所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:11所示的序列；
(v)与SEQ ID NO:11所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:11所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换；
优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:9所示的序列的VH和具有如SEQ ID NO:11所示的序列的VL。
根据本发明所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含：
(a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具 有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和
(b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)；
优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；
优选地，所述轻链恒定区是κ轻链恒定区。
根据本发明所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体；
优选地，所述人源化抗体或其抗原结合片段包含：具有如SEQ ID NO:33、35、37、39、43、45、47、49、51、53、55、57、59、61和63的任一项所示的序列的重链和/或如SEQ ID NO:31和41的任一项所示的序列的轻链。
根据本发明所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段带有标记；优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
本发明还提供了分离的核酸分子，其编码所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区；
优选地，所述多核苷酸包含如SEQ ID NO:2、4、6、8、10、12、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62和64中任一项所示的核苷酸编码序列。
本发明还提供了载体，其包含所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
本发明还提供了宿主细胞，其包含所述的分离的核酸分子或所述的载体。
本发明还提拱了制备所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段；
优选地，所述宿主细胞是哺乳动物细胞，更优选为人、鼠、羊、马、狗或猫的细胞，进一步优选为中国仓鼠卵巢细胞。
本发明还提供了双特异性或多特异性分子，其包含所述的抗体或其抗原结合片段；
优选地，所述双特异性或多特异性分子特异性结合NGF，并且额外地特异性结合一个或多个其他靶标；
优选地，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
本发明还提供了免疫缀合物，其包含所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂；
优选地，所述治疗剂选自毒素、放射性同位素、药物或细胞毒剂；
优选地，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
本发明还提供了药物组合物，其含有所述的抗体或其抗原结合片段、双特异性或多特异性分子或者免疫缀合物，以及药学上可接受的载体和/或赋形剂；
优选地，药物组合物还包含另外的药学活性剂；
优选地，所述抗体或其抗原结合片段、双特异性或多特异性分子或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
本发明还提供了试剂盒，其含有所述的抗体或其抗原结合片段；
优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
优选地，所述试剂盒还包括第二抗体，其特异性识别所述的抗体或其抗原结合片段；
优选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
本发明还提供了嵌合抗原受体，其包含所述的抗体或其抗原结合片段的抗原结合结构域；
优选地，所述抗原结合结构域包含所述的抗体或其抗原结合片段的重链可变区和轻链可变区；
优选地，所述抗原结合结构域是scFv；
优选地，所述抗原结合受体包含所述的抗体的抗原结合片段；
优选地，所述抗原结合受体由免疫效应细胞(例如T细胞)所表达。
本发明还提供了所述抗体或其抗原结合片段，或双特异性或多特异性分子，或免疫缀合物，或药物组合物，或嵌合抗原受体，在制备药物中的用途，所述药物用于治疗NGF介导的疾病或病症；
优选地，所述的疾病或病症包括骨关节炎疼痛、手术后疼痛、类风湿关节炎疼痛、低背痛、癌症相关性疼痛、神经病理性疼痛及内脏痛；
优选地，所述受试者为哺乳动物，例如人。
本发明还提供了一种用于在受试者(例如人)中预防和/或治疗疾病的方法，所述方法包括向有此需要的受试者施用有效量的所述的抗体或其抗原结合片段，或所述的双特异性或多特异性分子，或所述的免疫缀合物，或所述的药物组合物，或所述的嵌合抗原受体，或所述的宿主细胞；
所述疾病为NGF介导的疾病或病症；
优选地，所述的疾病或病症包括骨关节炎疼痛、手术后疼痛、类风湿关节炎疼痛、低背痛、癌症相关性疼痛、神经病理性疼痛及内脏痛；
优选地，所述受试者为哺乳动物，例如人。
本发明还提供了一种检测NGF(例如人NGF)在样品中的存在或其量的方法，其包括以下步骤：
(1)将所述样品与所述的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与NGF之间复合物的形成或检测所述复合物的量；
优选地，所述抗体或其抗原结合片段带有可检测的标记；
优选地，所述NGF是人NGF。
本发明人发现，本发明提供的抗体或其抗原结合片段具有以下优点：
1.本发明提供的抗体具有高的亲和力，亲和力常数K D值小于或者接近10 -12M，能够有效阻断NGF和其受体之间的结合，阻断疼痛的响应；
2.本发明提供的抗体与抗原结合的特异性极强，与NGF同家族蛋白无交叉结合，可以预期其临床安全性风险也将降低；
3.本发明提供的抗体具有显著的动物镇痛效果，预期临床疗效显著；
4.本发明提供的抗体采用CHO细胞表达，具有产量高、活性高、纯化工艺简单以及生产成本低的优势。
附图说明
图1表示本发明的抗NGF抗体与人NGF蛋白结合的ELISA实验结果。
图2表示本发明的抗NGF抗体对人NGF与受体TrkA、p75结合的影响。
图3表示本发明的抗NGF抗体抑制NGF诱导的TF-1细胞增殖。
图4表示本发明的抗NGF抗体抑制NGF诱导的TrkA/Ba/F3细胞增殖。
图5表示本发明的抗NGF抗体抑制TrkA/NFAT-bla/CHO细胞中的报告基因表达。
图6表示本发明的抗NGF抗体特异性结合NGF并抑制NGF诱导的信号通路。
图7表示本发明的抗NGF抗体43E5与138E12的竞争结合曲线。
图8表示本发明的抗NGF抗体43E5与138E12对CFA致小鼠炎症性疼痛的作用，其中**P<0.01vs生理盐水；***P<0.001vs生理盐水。
图9表示本发明的人源化抗NGF抗体与人NGF结合。
图10表示本发明的人源化抗NGF抗体与NGF同家族蛋白结合。
图11表示本发明的人源化抗NGF抗体抑制NGF诱导的TF-1细胞增殖。
具体实施方式
本申请的以下描述只为说明本申请的多种实施方式。因此，此处讨论的具体修改方式不应理解为对申请范围的限制。本领域的技术人员在不偏离本申请范围的情况下即可很容易地得出多种等同方式、变化和修改，应理解这样的等同实施方式包括在本发明范围内。在本申请中引用的所有文献，包括公开出版物、专利和专利申请都通过引用的方式全文并入。
在一些实施方式中，本申请所述的抗体或抗原结合片段能够以小于或者接近10 -12M的结合亲和性(K D)与NGF特异性结合，其通过生物膜层光学干涉技术测量。结合亲和性值可以用K D值表示，其通过当抗原和抗体结合达到平衡时的解离速率与结合速率的比值(k off/k on)计算得到。所述抗原结合亲和性(例如K D)可以通过本领域己知的适宜方法适宜地确定，例如包括使用仪器如Fortebio的生物膜层光学干涉技术。
在某些实施方式中，本申请所述抗体或抗原结合片段与NGF以0.1nM的EC50(即半数结合浓度)结合。抗体或抗原结合片段与NGF的结合可以通过本领域己知的方法如夹心法如ELISA，Western印迹，FACS或其他结合试验测定。
所述抗体是NGF特异性的。在某些实施方式中，所述抗体任选地不与NGF同家族蛋白脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、神经营养因子-3(neurotrophin-3,NT-3)、神经营养因子-4(neurotrophin-4,NT-4)结合。
实施例1:鼠源抗NGF抗体的制备
将重组人NGF蛋白(北京义翘神州科技有限公司，11050-HNAC)作为免疫原与等量弗氏完全佐剂(Sigma-Alderich，F5881)混合乳化，用于初始免疫。准备6周龄BALB/c和C57小鼠(江苏华阜康)各五只，每只动物皮下注射50μg免疫原(不包括佐剂质量，下同)。将免疫原与弗氏不完全佐剂(Sigma-Alderich，F5506)混合乳化，用于后续加强免疫。初始免疫2周后每只动物腹腔注射25μg免疫原进行第一次加免；2周后每只动物皮下注射25μg免疫原进行第二次加免。4-5周后最后一次免疫冲击，腹腔注射25μg免疫原。
最后一次免疫后分离小鼠B细胞，与SP2/0细胞(中国科学院细胞库，TCM18)混合，参照BTX公司电转仪操作手册进行融合。融合细胞经过培养后采用酶联免疫法(ELISA)筛选出与NGF结合并能抑制人NGF和受体TrkA结合的杂交瘤细胞。再通过有限稀释法进行亚克隆，以同样ELISA方式筛选获得3个阳性杂交瘤单克隆细胞株，分别命名为43E5、137H8、138E12。
将杂交瘤单克隆细胞株进行扩增并用无血清培养基培养，收集培养基并用蛋白G柱从中纯化，以获得鼠源抗人NGF单克隆抗体43E5、137H8、138E12。
实施例2：鼠源抗NGF抗体与人NGF结合的ELISA检测
使用人NGF(北京义翘神州科技有限公司，11050-HNAC)作为抗原研究抗NGF抗体的结合能力。96孔酶标板每孔包被50ng人NGF，洗涤及封闭后加入梯度稀释的抗体，室温孵育1小时。洗涤三次后加入HRP偶联山羊抗小鼠抗体(Biolegend,405306)，室温孵育1小时，三次洗涤后加入四甲基联苯胺(TMB，Biolegend，421101)显色，1M HCl终止显色，酶标仪读取450nm处吸光值。
三个杂交瘤分泌的抗NGF抗体均以剂量依赖的方式结合至人NGF(图1)，三个抗体与人NGF结合的EC50分别为0.082nM，0.112nM，0.1nM(表1)。
表1抗NGF抗体与人NGF结合的EC50
抗体 43E5 137H8 138E12
EC50(nM) 0.082 0.112 0.100
实施例3：鼠源抗NGF抗体与人NGF结合亲和力检测
应用生物分子相互作用检测平台ForteBio Octet Red96(PALL)测定抗体亲和力。利用SA(Streptavidin)Biosensor(Fortebio，18-5021)固定生物素标记的人NGF，然后结合梯度浓度的抗NGF抗体。再加缓冲液(1X Kinetics Buffer:PBS+0.1％BSA+0.05％Tween20)进行解离，最后通过仪器算法计算出抗原抗体结合的亲和动力学常数(表2)。
表2抗NGF抗体与人NGF结合亲和力
Figure PCTCN2021109995-appb-000001
实施例4:鼠源抗NGF抗体对人NGF与受体TrkA或p75结合的阻断作用检测
A.抗NGF抗体对人NGF与受体TrkA结合的阻断作用
如果采用包被受体TrkA方法检测，96孔酶标板每孔包被50ng NGF受体TrkA蛋白(融合人Fc)，洗涤三次后3％BSA封闭1小时。10000ng/mL(66.67nM)抗NGF抗体依次3倍稀释10个浓度至0.17ng/mL(0.0011nM)，取100μl与等体积1μg/mL的生物素标记人NGF混匀，二者室温孵育0.5小时后加入封闭洗涤完成的酶标板中。室温孵育1小时，洗涤三次。加入链霉亲和素标记HRP(Streptavidin-HRP，Biolegend，405210)孵育0.5小时后洗涤三次。酶标板中加TMB，显色终止后读数。
如果采用包被配体NGF方法检测，96孔酶标板每孔包被50ng人NGF，洗涤三次。3％BSA封闭1小时后洗涤三次，将10000ng/mL(66.67nM)抗人NGF抗体梯度稀释3倍至0.17ng/mL(0.0011nM)，每孔加入100μL，室温孵育1小时后洗涤三次。每孔加入100μL1μg/mL融合人Fc的TrkA，室温孵育1小时后洗涤三次。加入HRP偶联驴抗人IgG抗体(Biolegend，410902)，室温反应1小时。洗涤后加入TMB显色，终止后读数。
结果如图2A和图2B所示，抗NGF抗体可以阻断人NGF与受体TrkA的结合，阻断效果随抗体浓度的升高而明显升高。三个抗体阻断人NGF与受体TrkA结合的IC50如表3和表4；
表3阻断TrkA-NGF结合IC50
抗体 43E5 137H8 138E12
IC50(nM) 0.767 0.738 0.677
表4阻断NGF-TrkA结合IC50
抗体 43E5 137H8 138E12
IC50(nM) 0.967 0.791 0.865
B.抗NGF抗体对人NGF与受体p75结合的阻断作用
96孔酶标板每孔包被50ng人NGF，3％BSA封闭1h后洗涤3次，加入10μg/mL，1μg/mL，0.1μg/mL，0.01μg/mL，0μg/mL(66.67nM，6.67nM，0.67nM，0.067nM，0nM)的抗NGF抗体40μL，室温孵育15分钟，每孔加入40μL 2μg/mL的融合人Fc的p75(北京义翘神州科技有限公司，13184-H02H)孵育1小时后洗涤3次，加入100uL HRP偶联驴抗人Fc二抗，(Biolegend，410902)，室温反应1小时。洗涤后加入TMB显色，终止后读数。如图2C所示，所选三株抗NGF抗体中,43E5不能阻断人NGF与受体p75的结合,137H8及138E12可以阻断人NGF与受体p75的结合。
需要说明的是，Pfizer/Lilly的NGF抗体Tanezumab临床试验过程中出现骨关节炎进展加速(Rapidly Progressive Osteoarthritis,RPOA)的不良反应，Tanezumab可同时阻断NGF与TrkA及p75的结合。有研究显示，p75受体功能与神经元发育，成骨细胞分化、增殖，成肌细胞分化、肌肉修复等有关(Akiyama Y et al.(2014)Differentiation,87:111-118；Deponti et al.(2009)Molecular Biology of the Cell,20:3620-3627；Mikami et al.(2012)Differentiation,84:392-399)。此外，Sanofi公司开发的TrkA小分子抑制剂GZ389988A临床试验过程中未出现RPOA不良反应，研究人员认为与GZ389988A选择性作用于TrkA，保留了NGF-p75通路的完整功能相关(Krupka et al.(2019)Osteoarthritis and Cartilage,27:1599-1607)。本发明中，43E5不能阻断人NGF与p75的结合，预期临床出现RPOA的可能性降低，安全性优于其它具备p75阻断作用的抗体。
实施例5：鼠源抗NGF抗体的体外中和活性检测
A.NGF诱导的TF-1细胞增殖实验
TF-1细胞(人血液白血病细胞，ATCC,CRL-2003)的生长高度依赖GM-CSF(粒细胞巨噬细胞集落刺激因子)，但NGF与TF-1细胞表面的TrkA结合后也能诱导TF-1的生长，从而不需要依赖GM-CSF。
将TF-1细胞培养于含10％FBS(Gibco，10091148)，2μg/mL GM-CSF(R&D，215-GM-010)的RPMI1640培养基(HyClone，SH30027)，在对数生长期收集细胞，彻底洗涤清除原生长培养基中的GM-CSF,用不含GM-CSF的培养基重悬，每孔5000细胞稀释在80μl培养基中铺到白色透底96孔细胞培养板(corning，3610)。抗NGF抗体由400μg/mL(2666.67nM)进行4倍梯度稀释制备10个浓度，与等体积50ng/mL的人NGF混合，孵育0.5小时，取20μL加入96孔板内的细胞中，在37℃，5％CO 2培养箱中培养细胞72小时后加入100μL
Figure PCTCN2021109995-appb-000002
细胞活力检测试剂(Promega，G7573)，震荡破碎细胞，使用多功能酶标仪(SpectraMax)读取光学luminescence信号。
如图3所示，三个抗NGF抗体能够抑制人NGF诱导的TF-1细胞的增殖，IC50如表5所示。
表5抗NGF抗体抑制NGF诱导的TF-1细胞增殖IC50
抗体 43E5 137H8 138E12
IC50(nM) 0.083 0.123 0.077
B.NGF诱导的TrkA/Ba/F3细胞增殖实验
Ba/F3细胞的生长有依赖IL3及不依赖IL3两种途径，不依赖IL3的生长需要Ba/F3细胞稳定表达活性激酶。构建表达全长人TrkA的Ba/F3(TrkA/Ba/F3)细胞，该细胞增殖需要添加NGF诱导。
将TrkA/Ba/F3细胞培养于含10％FBS,100ng/mL NGF的RPMI1640培养基，收集细胞并彻底洗涤清除原生长培养基中的NGF，按照每孔3000细胞重悬于80μL不含NGF的培养基中，铺到白色透底96孔细胞培养板(corning，3610)。混合人NGF和梯度稀释的抗体，加入96孔板内的细胞中，NGF终浓度为5ng/mL，梯度稀释的抗NGF抗体终浓度最高为40μg/mL(266.67nM)。在37℃，5％CO2培养箱中培养细胞48小时后加入
Figure PCTCN2021109995-appb-000003
细胞活力检测试剂(Promega，G7573)，震荡破碎细胞，使用多功能酶标仪(SpectraMax)读取光学luminescence信号。
在观测TrkA/Ba/F3细胞生长的过程中，添加了人NGF的细胞可以正常增殖；三个抗NFG抗体能够抑制NGF诱导的TrkA/Ba/F3细胞增殖，抑制作用随浓度的升高而增强(图4)，IC50如表6所示。
表6抗NGF抗体抑制NGF诱导的TrkA/Ba/F3细胞增殖IC50
抗体 43E5 137H8 138E12
IC50(nM) 0.096 0.083 0.098
C.NGF诱导的TrkA/NFAT-bla/CHO细胞中报告基因的表达检测
NGF结合跨膜受体TrkA可激活下游磷脂酶C(PLC)，通过一系列信号转导引起胞内钙浓度升高和蛋白激酶C(PKC)活化，最终促进细胞核因子(nuclear factor)及活化T细胞核因子(NFAT)由胞质转运至细胞核，开启依赖NFAT的基因的表达。TrkA/NFAT-bla/CHO细胞(ThermoFisher，K1516)基因组中整合了编码人TrkA的基因，且报告基因β-内酰胺酶(β-bla)基因上游插入了NFAT响应元件，当NGF-TrkA通路激活，在NFAT控制下细胞将表达β-内酰胺酶，β-内酰胺酶的活性可以反应NGF-TrkA通路的激活程度。
按照产品说明准备TrkA-NFAT-bla/CHO细胞，每孔10,000细胞铺到384孔板，过夜培养。混合人NGF和梯度稀释的抗体，室温孵育0.5小时后加入到细胞中，使NGF最终作用浓度为80ng/mL，梯度稀释的NGF抗体终浓度最高为40μg/mL(266.67nM)。在37℃，5％CO 2培养箱中培养5小时后向细胞中加入按照操作手册配制的LiveBLAzer TMFRET-B/Gβ-内酰胺酶底物CCF4(ThermoFisher，K1095)，室温避光静置2小时。多功能酶标仪(SpectraMax)读取信号。
结果显示三个抗NGF抗体可以抑制人NGF引发的信号通路的激活(图5)，IC50如表7所示。
表7抗NGF抗体抑制TrkA/NFAT-bla/CHO细胞中报告基因表达IC50
抗体 43E5 137H8 138E12
EC50(nM) 0.0532 0.0859 0.0442
实施例6：抗NGF抗体特异性检测
A.鼠源抗NGF抗体与NGF同家族蛋白结合的ELISA检测
96孔酶标板每孔包被50ng人NGF(北京义翘神州科技有限公司，11050-HNAC)，人BDNF(北京义翘神州科技有限公司，50240-MNAS)，人NT-3(北京义翘神州科技有限公司，10286-HNAE)，或人NT-4(Alomone,N-270)，按照实施例2中方法检测鼠源抗NGF抗体与人NGF及三个同家族蛋白的结合情况。
如图6所示，三个鼠源抗NGF抗体与NGF(图6A)有高亲和力，抗体43E5、138E12与BDNF(图6B)，NT-3(图6C)，NT-4(图6D)基本无结合，抗体137H8与三个NGF同家族蛋白有微弱结合(图6B-6D)。
B.鼠源抗NGF抗体特异性抑制TrkA-NFAT-bla/CHO细胞中报告基因表达检测
如实施例5C中，按照产品说明准备TrkA-NFAT-bla/CHO细胞，TrkB-NFAT-bla/CHO细胞(ThermoFisher，K1491)和TrkC-NFAT-bla/CHO细胞(ThermoFisher，K1515)。
稀释本发明的抗体至40μg/mL(266.67nM)，与人NGF或者人BDNF(用于TrkB-NFAT-bla/CHO细胞)，或者人NT-3(用于TrkC-NFAT-bla/CHO细胞)混合,室温孵育0.5小时后加入到细胞中。培养5小时后向细胞中加入按照操作手册配制的LiveBLAzer TMFRET-B/Gβ-内酰胺酶底物CCF4，室温避光静置2小时。多功能酶标仪读取信号。
三个抗NGF抗体可抑制NGF与TrkA结合引起的后续信号传导，但并不影响BDNF与TrkB以及NT-3与TrkC的信号传导(图6E)；
需要说明的是，与Pfizer/Lilly的NGF抗体Tanezumab相比，本申请的三个抗NGF抗体，尤其是抗体43E5、138E12与NGF同家族抗体BDNF、NT-3、NT-4不结合，而Tanezumab与NT-3、NT-4交叉结合作用明显，也就是说，本申请的43E5、138E12特异性优于Tanezumab，预期临床不良反应表现优于Tanezumab。
实施例7：抗NGF抗体43E5与138E12的的抗原表位分级
应用生物分子相互作用检测平台Fortebio Ocete RED96检测两个抗NGF抗体43E5与138E12结合NGF是否有竞争。利用SA(Streptavidin)Biosensor固定生物素标记的人NGF，第一步上样监测抗体138E12的结合情况，第二步上样继续监测138E12或者43E5的结合情况，最后加缓冲液(1X Kinetics Buffer:PBS+0.1％BSA+0.05％Tween20)进行解离.
如图7所示，43E5与138E12可同时结合人NGF，且以非竞争方式结合。
实施例8：鼠源抗NGF抗体可变区测序及序列分析
使用TRIzol试剂盒(Ambion,15596-026)提取杂交瘤细胞总RNA，以之为模板合成第一链cDNA(Takara)。快速扩增cDNA末端(RACE)得到抗体轻链和重链片段，将扩增片段分别克隆至标准载体。测序得到抗NGF抗体43E5,137H8,138E12的重链和轻链可变区序列为：
抗NGF抗体43E5:
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000004
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000005
轻链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000006
轻链可变区核酸序列：
Figure PCTCN2021109995-appb-000007
抗NGF抗体137H8:
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000008
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000009
轻链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000010
轻链可变区核酸序列：
Figure PCTCN2021109995-appb-000011
Figure PCTCN2021109995-appb-000012
抗NGF抗体138E12:
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000013
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000014
轻链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000015
轻链可变区核酸序列：
Figure PCTCN2021109995-appb-000016
序列分析得到Kabat及IMGT系统界定的CDR，下表(表8)基于Kabat及IMGT系统的定义列出三个抗NGF抗体的CDR。
表8抗体CDR界定
Figure PCTCN2021109995-appb-000017
实施例9：抗NGF抗体对CFA致小鼠炎症性疼痛的作用
C57BL/6小鼠(浙江维通利华实验动物技术有限公司)，雄性，SPF级，6-8周龄，适应性饲养5天。适应期结束后将动物分成3组，分别为生理盐水组、抗NGF抗体43E5组、抗NGF抗体138E12组，每组10只动物。小鼠足底注射造模剂CFA之前，采用VonFrey测定3次痛阈(间隔不少于10分钟)，取3次平均值为其基础痛阈(图8A)。
次日于小鼠左后足足底注射25μl CFA致炎(CFA和液体石蜡比例为1：1)。待足趾肿胀明显后(造模后24小时左右)，同样方法3次检测小鼠左后足痛阈，取平均值为给药前的痛阈值(图8B)。
随后每组分别皮下注射生理盐水、抗NGF抗体43E5、抗NGF抗体138E12，剂量10mg/kg，并测定给药后24小时、48小时、72小时的痛阈(图8C-8E)，测定一次，评价受试物对痛阈的影响。
结果如图8所示，给药后24、48、72小时，抗NGF抗体43E5和138E12均能明显改善CFA引起的痛阈下降，与生理盐水组相比，具有统计学差异。
实施例10：鼠源抗NGF抗体人源化改造及表征
A.抗体的人源化改造
根据上述获得的杂交瘤细胞分泌的抗体的轻链可变区(VL)及重链可变区(VH)序列进行人源化改造。将鼠源抗体的VL、VH的氨基酸序列在人胚胎系抗体氨基酸序列数据库进行对比和检索，找出同源性高的人类IGHV和IGKV序列作为人源化模板。通过计算机模拟技术，分析可变区与框架区氨基酸存在的可能空间位阻及相互影响，确定对维持人源化抗体活性比较关键的框架区氨基酸，在人源化过程中对这些氨基酸予以保留。通过CDR移植技术完成对轻链和重链可变区的人源化改造。之后通过选定的抗体恒定区模板，得到以下多个人源化抗体。其中抗NGF抗体43E5以人IGHV1-2为重链可变区和人IGKV2-30为轻链可变区模板，获得抗体43E5-01和43E5-02。以人IGHV1-69为重链可变区和人IGKV2-30为轻链可变区模板，获得抗体43E5-05和43E5-06。43E5-01、43E5-02、43E5-05和43E5-06四个抗体的轻链可变区序列是相同的。抗NGF抗体138E12以人IGHV4-59为重链可变区和人IGKV1-39为轻链可变区获得抗体138E12-01和138E12-02。138E12-01和138E12-02抗体轻链可变区氨基酸序列相同，重链可变区氨基酸有区别。此外，由于138E12-01和138E12-02抗体重链互补决定区序列中含有异构化位点DG以及脱酰胺位点NS，为去除以上位点对抗体的潜在影响，对138E12-01和138E12-02抗体的重链可变区进行了进一步改造。以138E12-01为模板，将VH中的DG 54-55突变为EG 54-55、NS 60-61突变为QS 60- 61、同时将DG 54-55、NS 60-61突变为EG 54-55和QS 60-61，分别获得抗体138E12-08、138E12-09、138E12-10。以138E12-02为模板，将VH中的DG 54-55突变为EG 54-55、DG 54-55突变为DA 54- 55、NS 60-61突变为QS 60-61、NS 60-61突变为NT 60-61、DG 54-55和NS 60-61同时突变为EG 54-55和QS 60-61获得抗体138E12-03、138E12-04、138E12-05、138E12-06和138E12-11。138E12-07以人IGHV4-59为重链可变区和人IGKV1-39为轻链可变区获得，其重链可变区的框架区序列为全人源，未保留鼠源氨基酸，但将其重链互补决定区的DG 54-55和NS 60-61同时突变为EG 54-55和QS 60-61。
对人源化抗体重链和轻链分别进行基因合成，并经酶切后连入相应质粒中。将构建好的质粒瞬时转染至CHO细胞中进行表达，经过7-10天表达，细胞培养上清用相应缓冲液(如磷酸盐缓冲盐水(pH7.4))平衡的MabSelect柱(GE Healthcare)进行纯化，之后用柠檬酸钠或其它缓冲液进行洗脱，经此步骤获得的抗体，可通过SDS-PAGE或SEC-HPLC进行纯度等鉴定，并用于后续其它表征研究。
上述抗体的可变区氨基酸序列如表9所示：
表9抗NGF抗体43E5,138E12人源化抗体可变区序列
Figure PCTCN2021109995-appb-000018
抗NGF抗体43E5人源化抗体可变区序列:
抗NGF抗体43E5人源化抗体43E5-01、43E5-02、43E5-05、43E5-06含有相同的轻链。轻链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000019
轻链可变区核酸序列：
Figure PCTCN2021109995-appb-000020
43E5-01：
重链可变区氨基酸序列:
Figure PCTCN2021109995-appb-000021
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000022
Figure PCTCN2021109995-appb-000023
43E5-02:
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000024
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000025
43E5-05:
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000026
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000027
43E5-06：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000028
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000029
抗NGF抗体138E12人源化抗体可变区序列：
抗NGF抗体138E12人源化抗体138E12-01,138E12-02,138E12-03,138E12-04,138E12-05,138E12-06，138E12-07，138E12-08，138E12-09，138E12-10，138E12-11含有相同的轻链。
轻链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000030
轻链可变区核酸序列：
Figure PCTCN2021109995-appb-000031
138E12-01：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000032
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000033
138E12-02：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000034
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000035
138E12-03：
重链可变区氨基酸序列:
Figure PCTCN2021109995-appb-000036
重链可变区核酸序列:
Figure PCTCN2021109995-appb-000037
Figure PCTCN2021109995-appb-000038
138E12-04：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000039
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000040
138E12-05：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000041
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000042
138E12-06：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000043
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000044
138E12-07：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000045
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000046
Figure PCTCN2021109995-appb-000047
138E12-08：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000048
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000049
138E12-09：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000050
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000051
138E12-10：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000052
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000053
138E12-11：
重链可变区氨基酸序列：
Figure PCTCN2021109995-appb-000054
重链可变区核酸序列：
Figure PCTCN2021109995-appb-000055
B.人源化抗NGF抗体表征
1)人源化抗体与人NGF结合
使用人NGF(北京义翘神州科技有限公司，11050-HNAC)作为抗原研究人源化后抗体的亲和力及特异性。包被液(PBS)稀释NGF，96孔酶标板每孔包被50ng，洗涤及封闭后加入梯度稀释的抗体，室温孵育1小时。洗涤三次后加入HRP偶联驴抗人IgG抗体(Biolegend)，室温反应1小时，三次洗涤后加入四甲基联苯胺(TMB，Biolegend)显色，1M HCl终止显色，酶标仪读取450nm处吸光值。
结合情况如表10和图9所示，不同序列的人源化抗体均与人NGF有相似的结合，结合程度与人鼠嵌合抗体(含鼠源抗NGF抗体可变区和人抗体恒定区)相当。
表10人源化抗NGF抗体与人NGF结合EC50
抗体 43E5-01 43E5-02 43E5-05 43E5-06 43E5-chi -
EC50(nM) 0.229 0.222 0.214 0.192 0.104 -
抗体 138E12-01 138E12-02 138E12-03 138E12-04 138E12-05 138E12-06
EC50(nM) 0.0403 0.0420 0.0392 0.0570 0.0634 0.0500
抗体 138E12-07 138E12-08 138E12-09 138E12-10 138E12-11 138E12-Chi
EC50(nM) 0.0340 0.0253 0.0270 0.0184 0.0324 0.0454
2)人源化抗体与其他NGF家族蛋白结合
按照实施例6A中所述的ELISA方法检测人源化抗NGF抗体与人NGF及三个同家族蛋白的结合情况。结果如图10A-H所示，人源化后抗体与NGF(图10A和10B)有高亲和力，与BDNF(图10C和图10D)，NT-3(图10E和图10F)，NT-4(图10G和图10H)基本无结合。
3)NGF诱导的TF-1细胞增殖实验
如实施例5A中所述，通过TF-1细胞的增殖来检测人源化抗NGF抗体的中和活性。结果如图11所示，人源化抗体均可抑制NGF诱导的TF-1细胞的增殖，且多数人源化抗体的抑制能力与人鼠嵌合抗体相当，IC50如表11所示。
表11人源化抗NGF抗体抑制NGF诱导的TF-1细胞增殖IC50
抗体 43E5-01 43E5-02 43E5-05 43E5-06 43E5-chi -
IC50(nM) 0.582 0.534 0.701 0.568 0.255 -
抗体 138E12-01 138E12-02 138E12-03 138E12-04 138E12-05 138E12-06
IC50(nM) 0.030 0.064 0.130 0.112 0.136 0.125
抗体 138E12-07 138E12-08 138E12-09 138E12-10 138E12-11 138E12-Chi
IC50(nM) 0.214 0.067 0.035 0.031 0.062 0.074
尽管已经对本发明的具体实施方式进行了详细说明和描述，但应当认识到，本发明不受所述具体实施方式的限制。在不脱离本发明主旨和范围的情况下，可以对本发明做出各种改进、修饰和变化，而这些改进、修饰和变化均在本发明的范围之内。
Figure PCTCN2021109995-appb-000056
Figure PCTCN2021109995-appb-000057
Figure PCTCN2021109995-appb-000058
Figure PCTCN2021109995-appb-000059
Figure PCTCN2021109995-appb-000060
Figure PCTCN2021109995-appb-000061
Figure PCTCN2021109995-appb-000062
Figure PCTCN2021109995-appb-000063
Figure PCTCN2021109995-appb-000064
Figure PCTCN2021109995-appb-000065
Figure PCTCN2021109995-appb-000066
Figure PCTCN2021109995-appb-000067
Figure PCTCN2021109995-appb-000068
Figure PCTCN2021109995-appb-000069
Figure PCTCN2021109995-appb-000070
Figure PCTCN2021109995-appb-000071
Figure PCTCN2021109995-appb-000072
Figure PCTCN2021109995-appb-000073
Figure PCTCN2021109995-appb-000074
Figure PCTCN2021109995-appb-000075

Claims (20)

  1. 能够特异性结合NGF的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)包含下述3个互补决定区(CDR)的重链可变区(VH)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:13，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:14，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:15，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)包含下述3个互补决定区(CDR)的轻链可变区(VL)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:22，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:23，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:24，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:13所示的VH CDR1、如SEQ ID NO:14所示的VH CDR2、如SEQ ID NO:15所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:22所示的VL CDR1、如SEQ ID NO:23所示的VL CDR2、如SEQ ID NO:24所示的VL CDR3。
  2. 能够特异性结合NGF的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)包含下述3个互补决定区(CDR)的重链可变区(VH)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:16，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:17，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:18，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)包含下述3个互补决定区(CDR)的轻链可变区(VL)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:25，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:26，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:27，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:16所示的VH CDR1、如SEQ ID NO:17所示的VH CDR2、如SEQ ID NO:18所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:25所示的VL CDR1、如SEQ ID NO:26所示的VL CDR2、如SEQ ID NO:27所示的VL CDR3。
  3. 能够特异性结合NGF的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)包含下述3个互补决定区(CDR)的重链可变区(VH)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:19，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:20，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:21，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)包含下述3个互补决定区(CDR)的轻链可变区(VL)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:28，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:29，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:30，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:19所示的VH CDR1、如SEQ ID NO:20所示的VH CDR2、如SEQ ID NO:21所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:28所示的VL CDR1、如SEQ ID NO:29所示的VL CDR2、如SEQ ID NO:30所示的VL CDR3。
  4. 能够特异性结合NGF的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，
    所述重链可变区包含SEQ ID NO:1、5和9中任一项所示的重链可变区中含有的3个CDR；并且，所述轻链可变区包含SEQ ID NO:3、7和11中任一项所示的轻链可变区中含有的3个CDR；
    优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义。
  5. 权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:1所示的序列；
    (ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:3所示的序列；
    (v)与SEQ ID NO:3所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％ 的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:1所示的序列的VH和具有如SEQ ID NO：3所示的序列的VL。
  6. 权利要求2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:5所示的序列；
    (ii)与SEQ ID NO:5所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:5所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:7所示的序列；
    (v)与SEQ ID NO:7所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:7所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:5所示的序列的VH和具有如SEQ ID NO:7所示的序列的VL。
  7. 权利要求3所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:9所示的序列；
    (ii)与SEQ ID NO:9所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:11所示的序列；
    (v)与SEQ ID NO:11所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:11所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:9所示的序列的VH和具有如SEQ ID NO：11所示的序列的VL。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段 进一步包含：
    (a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和
    (b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；
    优选地，所述轻链恒定区是κ轻链恒定区。
  9. 权利要求1-8任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体；
    优选地，所述人源化抗体或其抗原结合片段包含：具有如SEQ ID NO:33、35、37、39、43、45、47、49、51、53、55、57、59、61和63的任一项所示的序列的重链和/或如SEQ ID NO:31和41的任一项所示的序列的轻链。
  10. 权利要求1-9任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段带有标记；优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  11. 分离的核酸分子，其编码权利要求1-9任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区；
    优选地，所述多核苷酸包含如SEQ ID NO:2、4、6、8、10、12、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62和64中任一项所示的核苷酸编码序列。
  12. 载体，其包含权利要求11所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  13. 宿主细胞，其包含权利要求11所述的分离的核酸分子或权利要求12所述的载体。
  14. 制备权利要求1-10任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求13所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段；
    优选地，所述宿主细胞是哺乳动物细胞，更优选为人、鼠、羊、马、狗或猫的细胞，进一步优选为中国仓鼠卵巢细胞。
  15. 双特异性或多特异性分子，其包含权利要求1-10中任一项所述的抗体或其抗原结合片段；
    优选地，所述双特异性或多特异性分子特异性结合NGF，并且额外地特异性结合一个或多个其他靶标；
    优选地，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
  16. 免疫缀合物，其包含权利要求1-20任一项所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂；
    优选地，所述治疗剂选自毒素、放射性同位素、药物或细胞毒剂；
    优选地，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
    优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
  17. 药物组合物，其含有权利要求1-10任一项所述的抗体或其抗原结合片段、权利要求15所述的双特异性或多特异性分子或者权利要求16所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述抗体或其抗原结合片段、双特异性或多特异性分子或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
  18. 试剂盒，其含有权利要求1-10中任一项所述的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述试剂盒还包括第二抗体，其特异性识别权利要求1-10任一项所述的抗体或其抗原结合片段；
    优选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  19. 嵌合抗原受体，其包含权利要求1-10任一项所述的抗体或其抗原结合片段的抗原结合结构域；
    优选地，所述抗原结合结构域包含权利要求1-10任一项所述的抗体或其抗原结合片段的重链可变区和轻链可变区；
    优选地，所述抗原结合结构域是scFv；
    优选地，所述抗原结合受体包含权利要求1-10任一项所述的抗体的抗原结合片段；
    优选地，所述抗原结合受体由免疫效应细胞(例如T细胞)所表达。
  20. 权利要求1-10任一项所述的抗体或其抗原结合片段，或权利要求15所述的双特异性或多特异性分子，或权利要求16所述的免疫缀合物，或权利要求17所述的药物组合物，或权利要求19所述的嵌合抗原受体，在制备药物中的用途，所述药物用于治疗NGF介导的疾病或病症；
    优选地，所述的疾病或病症包括骨关节炎疼痛、手术后疼痛、类风湿关节炎疼痛、低背痛、癌症相关性疼痛、神经病理性疼痛及内脏痛；
    优选地，所述受试者为哺乳动物，例如人。
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