WO2021208928A1 - Anticorps dirigé contre le facteur de croissance nerveuse humain - Google Patents

Anticorps dirigé contre le facteur de croissance nerveuse humain Download PDF

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WO2021208928A1
WO2021208928A1 PCT/CN2021/087015 CN2021087015W WO2021208928A1 WO 2021208928 A1 WO2021208928 A1 WO 2021208928A1 CN 2021087015 W CN2021087015 W CN 2021087015W WO 2021208928 A1 WO2021208928 A1 WO 2021208928A1
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antibody
ngf
antigen
binding fragment
seq
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廖化新
郑伟宏
王孝丽
王月明
龚惠珍
梁俊朗
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珠海泰诺麦博生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
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    • 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
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07—ORGANIC CHEMISTRY
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    • 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

Definitions

  • This application relates to the field of antibodies. Specifically, this application relates to antibodies that specifically bind to NGF, their preparation methods and uses.
  • Nerve growth factor is the earliest discovered neurotrophic protein (neurotrophic factor), widely distributed in brain, ganglion, iris, heart, spleen, placenta and other tissues and fibroblasts, smooth muscle, skeletal muscle, glial cells, Schwann cells and so on.
  • NGF is a member of neurotrophins (NT), which are a group of structurally related proteins that further include brain-derived neurotrophic factor (BDNF), NT-3, NT-4, and NT-5. (Wyman et al., Gene Therapy (1999), 6:1648-1660).
  • NGF tyrosine kinase A
  • TrkA tyrosine kinase A
  • p75 receptor tyrosine kinase A
  • NGF mediates a variety of diseases or disorders such as pain (RICHARDS, N.Br J Anaesth (2013), 111(1): 46-51), arthritis, collagen vascular disease, demyelinating disease, airway inflammatory disease ( Hoyle, Cytokine Growth Factor Rev (2003), 14: 551-8.; Lommatzsch, M. Ann NY Acad Sci (2003), 992: 241-9), neurological diseases, metabolic diseases (such as diabetes) (Yasuda, H.
  • NGF neurotrophic factor
  • Pain is a perception based on signals received from the surrounding environment and transmitted and interpreted by the nervous system (Millan, Prog. Neurobiol (1999). 57:1-164). Severe pain affects the quality of life of humans and causes social damage. A huge economic burden.
  • the treatment of pain is one of the most common clinical and social demands. However, the treatment of pain is only partially effective, and many of these treatments themselves have many adverse effects.
  • opioids have a good analgesic effect, but they are also kidney toxins. High doses often cause gastrointestinal irritation, ulcers, bleeding and mental confusion, and long-term use of opioids is accompanied by drug tolerance and dependence. sex.
  • anti-NGF antagonists Due to the huge clinical demand and market space in the pain field. It is very important and urgent to develop new drugs targeting pain pathway targets to treat pain. In recent years, due to the increasing understanding of the role of NGF in acute and chronic pain and hyperalgesia, anti-NGF antagonists have become a new hot spot in pain treatment research. Many murine and humanized anti-NGF antibodies have been reported, and clinical trial data of some anti-NGF antibody drugs have shown positive results. However, the murine anti-NGF antibodies and humanized anti-NGF antibodies have immunogenicity. And other issues.
  • compositions or methods that can be used to prevent or treat diseases related to NGF for example, compositions and methods that can be used to prevent or treat those diseases or conditions related to pain. Mentioned requirements.
  • the present invention provides a new antibody or antigen-binding fragment thereof that can specifically bind to NGF with high affinity, especially an antibody or antigen-binding fragment thereof that specifically binds to human NGF.
  • the anti-NGF antibody provided by the present invention is a human antibody, which can cross-react with non-human NGF (including non-human primate NGF and/or mouse NGF).
  • the human antibody or antigen-binding fragment thereof that specifically binds to human NGF according to the present invention is a neutralizing antibody that neutralizes NGF.
  • the present invention provides a human antibody or antigen-binding fragment thereof that specifically binds to human NGF, and is combined with neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), brain-derived neurotrophic factor (BDNF) cross-reaction occurs.
  • NT-3 neurotrophin-3
  • NT-4 neurotrophin-4
  • BDNF brain-derived neurotrophic factor
  • the anti-NGF antibody and antigen fragments thereof comprise one, two or three CDRs (preferably three CDRs) selected from the sequence of the VH region of the antibodies shown in Table 1.
  • the antibody of the present invention comprises one, two or three CDRs (preferably three CDRs) selected from the VL region sequences of the antibodies shown in Table I.
  • the antibody of the present invention comprises the 6 CDR region sequences of the antibodies shown in Table 1.
  • the CDR sequence of the antibody is the CDR sequence shown in Table II.
  • the anti-NGF antibody or antigen-binding fragment thereof of the present invention comprises A) heavy chain complementarity determining region (CDR): (i) CDR1, which comprises the amino acid sequence of SEQ ID NO:1, or is relative to the The sequence contains one or more and no more than 5 amino acid substitutions (such as conservative substitutions), deletions or insertions, (ii) CDR2, which includes the amino acid sequence of SEQ ID NO: 2, or is relative to the sequence Contains one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or inserted sequences, (iii) CDR3, which includes the amino acid sequence of SEQ ID NO: 3, or contains One or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 5 amino acids, and B) the light chain complementarity determining region (CDR): (i) CDR1, which includes SEQ ID NO : 4 amino acid sequence, or a sequence containing one or more and no more than 5 amino acid
  • the anti-NGF antibody or antigen-binding fragment thereof of the present invention comprises A) heavy chain complementarity determining region (CDR): (i) CDR1, which consists of the amino acid sequence of SEQ ID NO:1, (ii) CDR2 , Which consists of the amino acid sequence of SEQ ID NO: 2, (iii) CDR3, which consists of the amino acid sequence of SEQ ID NO: 3, and B) the light chain complementarity determining region (CDR): (i) CDR1, which consists of SEQ ID NO: 4 consists of the amino acid sequence, (ii) CDR2, which consists of the amino acid sequence of SEQ ID NO: 5, and (iii) CDR3, which consists of the amino acid sequence of SEQ ID NO: 6.
  • CDR heavy chain complementarity determining region
  • the anti-NGF antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH, which comprises the amino acid sequence of SEQ ID NO: 7 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% identity or higher identity amino acid sequence or composed of the sequence, the anti-NGF antibody comprising the VH has the ability to bind to NGF.
  • VH heavy chain variable region
  • the anti-NGF antibody or antigen-binding fragment thereof of the present invention includes the heavy chain variable region VH, which includes the three heavy chain variable region CDRs of the antibody shown in Table II, and is consistent with SEQ ID NO: 7
  • the amino acid sequence has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
  • the anti-NGF antibody containing the VH has the ability to bind to NGF.
  • the heavy chain variable region VH of the anti-NGF antibody comprises an amino acid sequence with one or more substitutions (such as conservative substitutions), insertions or deletions compared with the amino acid sequence of SEQ ID NO: 7, including all
  • the VH anti-NGF antibody has the ability to bind to NGF.
  • the anti-NGF antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region VL, which comprises the amino acid sequence of SEQ ID NO: 8 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% identity or higher identity amino acid sequence or composed of the sequence, the anti-NGF antibody comprising the VL has the ability to bind to NGF.
  • VL light chain variable region
  • the anti-NGF antibody or antigen-binding fragment thereof of the present invention includes the light chain variable region VL, which includes the 3 CDRs of the light chain variable region of the antibody shown in Table II, and is consistent with SEQ ID NO: 8
  • the amino acid sequence of has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, With 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
  • the anti-NGF antibody containing the VL has the ability to bind to NGF.
  • the light chain variable region VL of the anti-NGF antibody comprises an amino acid sequence with one or more substitutions (such as conservative substitutions), insertions or deletions compared with the amino acid sequence of SEQ ID NO: 8, including all
  • the VH anti-NGF antibody has the ability to bind to NGF.
  • the anti-NGF antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the VH of the heavy chain variable region contains at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and the amino acid sequence of SEQ ID NO: 7 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or higher identity
  • the light chain variable region VL contains at least 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence shown in SEQ ID NO: 8 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or higher identity amino acid sequence or consists of said sequence, or
  • the heavy chain variable region VH includes the heavy chain variable region CDRs shown in Table II, and has at least 75%, 76%, 77%, 78%, 79%, 80%, and the amino acid sequence of SEQ ID NO: 7 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% identity;
  • the light chain variable region VL includes the CDRs of the light chain variable region shown in Table II, and has at least 75%, 76%, 77%, 78% with the amino acid sequence of SEQ ID NO: 8 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, the anti-NGF antibody comprising said VH and VL has the ability to bind to NGF
  • the heavy chain variable region VH includes an amino acid sequence with one or more substitutions (such as conservative substitutions), insertions or deletions compared with the amino acid sequence of SEQ ID NO: 7;
  • the light chain variable region VL includes the amino acid sequence with SEQ ID NO: 7 Compared with the amino acid sequence of NO: 8 with one or more substitutions (eg conservative substitutions), insertions or deletions, the anti-NGF antibody comprising the VH and VL has the ability to bind to NGF.
  • the present invention provides an anti-NGF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region VL comprises SEQ The amino acid sequence shown in ID NO: 8 or consists of it.
  • the anti-NGF antibody or antigen-binding fragment thereof further comprises heavy and/or light chain constant region sequences derived from the germline consensus sequence of a human antibody.
  • the light chain constant region is preferably a human kappa (CK) or lambda (C ⁇ ) chain constant region.
  • the heavy chain constant region can be a gamma, mu, alpha, delta, or epsilon chain.
  • the heavy chain constant region is human-derived IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD Same type as IgE.
  • Each type of heavy chain and light chain is characterized by a specific constant region with a sequence well known in the art.
  • the constant region is preferably a human IgG constant region, for example, a constant region of human IgG1, IgG2, IgG3 or IgG4 isotype.
  • the heavy chain and/or light chain constant region is described in, for example, Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, and any of them can be applied in the present invention.
  • the present invention provides an anti-NGF antibody or antigen-binding fragment thereof, wherein the heavy chain constant region sequence is a human IgG2 constant region, and the heavy chain constant region sequence includes the amino acid sequence shown in SEQ ID NO: 9 or is derived from it. Composition;
  • the heavy chain constant region sequence of the antibody can also be a human IgG4 constant region, and the heavy chain constant region sequence includes or consists of the amino acid sequence shown in SEQ ID NO: 10; in one implementation In the scheme, the light chain constant region of the antibody comprises or consists of the amino acid sequence shown in SEQ ID NO: 11.
  • sequence variants of these constant region domains can also be used, for example containing one or more amino acid modifications, where the amino acid positions are identified by the EU index system of Kabat et al. (1991).
  • this modification may be N297A or N297Q (Sazinsky, PNAS (2008), 105(51): 20167-20172) .
  • this modification in order to modify the Fc receptor interaction, such as modification in the constant region of human IgG, this modification may be L234A and/or L235E or L235A.
  • this modification in order to extend the half-life, such as modification in the constant region of human IgG, this modification may be R435H.
  • this modification may be S228P (Angal, S. Mol Immunol (1993), 30: 105-108)
  • such modification in order to enhance FcRn binding, such as modification in the constant region of human IgG, such modification may be M252Y, S254T, T256E (DallAcqua et al., J. Biol. Chem (2006), 281(33): 23514) -23524; Zalevsky et al., Nature Biotech (2010), 28(2): 157-159), M428L or N434S.
  • the human IgG constant region is modified, and such modifications are known (including but Not limited to: Natsume et al., Cancer Res (2008), 68(10): 3863-72; Idusogie et al., J. Immunol (2001), 166(4): 2571-5; Moore et al. mAbs (2010, 2 (2):181-189; Lazar et al., PNAS(2006), 103(11):4005-4010; Shields et al. J.Biol.Chem.
  • heterodimerization can also be induced by T366W modification, and optionally further by introducing disulfide bonds via S354C and Y349C modifications on the opposite CH3 domains (Carter, Journal of Immunological Methods). (2001), 248:7-15.).
  • the antibodies of the present invention also encompass antibodies that compete with any of the antibodies described above for binding to NGF, and antibodies that bind to the same epitope of NGF as any of the antibodies described above.
  • At least part of the framework sequence of the anti-NGF antibody is a human consensus framework sequence.
  • the anti-NGF antibodies of the invention are whole antibodies, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE antibodies.
  • the anti-NGF antibody of the present invention only covers its antigen binding portion, for example: Fab, Fab'-SH, Fv, scFv or (Fab') 2 fragments.
  • the anti-NGF antibody of the present invention can be modified to achieve a certain function, such as eliminating residual effector function (Glu can eliminate residual effector function (Reddy et al., 2000)).
  • the anti-NGF antibody of the present invention is a neutralizing antibody for neutralizing NGF.
  • the present invention provides a nucleic acid encoding any of the above anti-NGF antibodies or fragments thereof.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector.
  • the present invention provides a host cell containing the vector or the nucleic acid or antibody.
  • the host cell is a eukaryotic cell.
  • the host cell is selected from mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof, wherein the mammalian cells are, for example, CHO cells, HEK293 cells, or COS cells.
  • the host cell is a prokaryotic cell, such as an E. coli cell, a yeast cell.
  • the present invention provides a method for preparing an anti-NGF antibody or antigen-binding fragment thereof, wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody or antigen-binding fragment thereof, and any The antibody or antigen-binding fragment thereof is optionally isolated. In a certain embodiment, the method further comprises recovering the anti-NGF antibody or antigen-binding fragment thereof from the host cell.
  • the present invention provides an anti-NGF antibody or antigen-binding fragment thereof prepared by the method of the present invention.
  • the present invention provides a composition comprising any anti-NGF antibody or antigen-binding fragment thereof described herein, preferably the composition is a pharmaceutical composition.
  • the composition further comprises a pharmaceutical carrier.
  • the anti-NGF antibody and antigen-binding fragment thereof contained in the composition are coupled to the coupling portion.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
  • the present invention provides a method for treating diseases or disorders related to NGF, which comprises administering to a subject a therapeutically effective amount of the anti-NGF antibody of the present invention, or administering the pharmaceutical composition of the present invention.
  • the present invention also relates to a method for alleviating or ameliorating the symptoms associated with NGF-mediated diseases or conditions, including administering to a subject a therapeutically effective amount of the anti-NGF antibody of the present invention, or administering the pharmaceutical composition of the present invention.
  • the NGF-mediated disease or condition includes any medical disease or condition related to increased NGF levels or increased sensitivity to NGF, including but not limited to pain, arthritis, collagen vascular disease, Demyelinating diseases, airway inflammatory diseases, neurological diseases, metabolic diseases (such as diabetes), viral infection-related diseases, arrhythmia, psoriasis, and cancer.
  • the NGF-mediated disease or condition is a pain-related condition, inflammatory pain, postoperative incision pain, neuropathic pain, fracture pain, gout and joint pain, burn pain, cancer pain, compound local pain Syndrome, postherpetic neuralgia, or pain associated with sickle cell crisis.
  • the pain is pathophysiological pain.
  • the method improves, reduces, inhibits, or prevents any NGF-related diseases or disorders, or reduces symptoms related to the diseases, such as pain, by eliminating, inhibiting, or reducing the activity of NGF.
  • the present invention treats the NGF-related diseases or disorders by administering an anti-NGF antagonist alone, such as the antibody or antigen-binding fragment thereof of the present invention.
  • the treatment method disclosed in the present invention administers an anti-NGF antibody in combination with a second therapeutic agent to treat a disease or disorder related to NGF.
  • the second therapeutic agent is selected from other therapeutic agents, analgesics, anti-inflammatory agents, chemotherapeutic agents or immunotherapeutic agents.
  • the present invention provides a method for relieving or ameliorating NGF-mediated pain, comprising administering to a subject a therapeutically effective amount of the anti-NGF antibody of the present invention, or a therapeutically effective amount of a pharmaceutical composition.
  • the present invention provides a method for detecting NGF in an object or sample, the method comprising: (a) contacting the object or sample with any anti-NGF antibody or fragment thereof described herein; and (b) detecting anti-NGF The formation of complexes between antibodies or fragments thereof and NGF.
  • the anti-NGF antibody and antigen-binding fragment thereof of the present invention further include a detectable label.
  • the antibodies provided by the present invention can be used to detect, diagnose, and monitor diseases or disorders related to altered or abnormal NGF expression.
  • the present invention relates to the use of any anti-NGF antibody or fragment thereof described herein in the preparation of a medicament or kit for treating NGF-related diseases or disorders in a subject.
  • the present invention provides the use of a composition comprising the anti-NGF antibody in the preparation of a medicament or kit for treating a disease or disorder related to NGF in a subject.
  • the present invention also relates to the use of any anti-NGF antibody or fragment thereof described herein, or a composition comprising the anti-NGF antibody, in the preparation of a medicament for alleviating or ameliorating NGF-mediated pain.
  • any anti-NGF antibody or fragment thereof herein is for use as a medicine.
  • the present invention provides a kit containing the antibody or composition of the present invention, such as a diagnostic kit, a detection kit, a treatment kit, and the like.
  • the invention also encompasses any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof is applicable to any and all anti-NGF antibodies or fragments thereof, methods and uses of the invention described herein.
  • the present invention provides human antibodies and antigen-binding fragments thereof capable of specifically recognizing/binding NGF.
  • the antibodies and antigen-binding fragments thereof have a neutralizing effect against NGF and cross-react with NT-3, NT-4, and BDNF. Since the anti-NGF antibody of the present invention is a fully human antibody, it can treat NGF-related diseases or disorders, or relieve NGF-related pain, while having low immunogenicity to the subject, thereby effectively avoiding the test The patient produces a corresponding immune response against it, thus reducing the side effects associated with conventional anti-NGF antibodies.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” when used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence when referring to an antibody variable region that "comprises” a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.
  • NGF nerve growth factor
  • antibody is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they It suffices to show the desired antigen-binding activity.
  • Intact antibodies will generally contain at least two full-length heavy chains and two full-length light chains, but may include fewer chains in some cases, for example, antibodies naturally occurring in camels may contain only heavy chains.
  • monoclonal antibody refers to a single copy or cloned antibody derived, for example, from eukaryotes, prokaryotes or phage clones, ie, except for possible variant antibodies that are usually present in very small amounts (For example, a variant antibody that contains a natural mutation or is produced during the production of a monoclonal antibody product), each antibody constituting the population is the same and/or binds the same epitope.
  • the modifier “monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of the antibody by any specific method.
  • Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or a combination of such or other techniques known in the art.
  • “Native antibodies” refer to naturally occurring immunoglobulin molecules with different structures.
  • the "native sequence Fc domain” includes an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature.
  • the natural sequence human Fc domain includes, for example, the natural sequence human IgG1 Fc domain (non-A and A allotypes); the natural sequence human IgG2 Fc domain; the natural sequence human IgG3 Fc domain; and the natural sequence human IgG4 Fc domain; And its naturally occurring variants.
  • Human antibody refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source, which utilizes a human antibody library or other human antibodies Coding sequence. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • neutralizing antibody refers to an antibody or antibody fragment that reduces or inhibits the biological activity of NGF, such as reduces or inhibits the binding of NGF to one or more of its receptors (preferably TrkA).
  • the reduction in biological activity can be a partial reduction or a complete reduction.
  • the degree to which the antibody neutralizes NGF is called the neutralizing potency of the antibody.
  • the neutralizing potency of antibodies can be determined or measured using one or more tests known to those of ordinary skill and/or described or mentioned herein, including but not limited to competitive binding assays, direct and indirect sandwich assays, Immunoprecipitation assay and enzyme-linked immunosorbent assay (ELISA).
  • Anti-NGF antibody refers to an antibody that can bind to NGF and inhibit the biological activity of NGF and/or the downstream pathway mediated by NGF signal.
  • Anti-NGF antibodies include antibodies that block, antagonize, inhibit or reduce (including significantly reduce) the biological activity of NGF, including downstream pathways mediated by NGF signals, such as receptor binding and/or antibodies that trigger cell responses to NGF.
  • the invention also encompasses fragments of anti-NGF antibodies.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear antibodies, single-chain antibody molecules (such as scFv); and multispecific antibody fragments formed Sex antibody.
  • biological activity of NGF generally refers to the ability to bind to the NGF receptor and/or activate the NGF receptor signal pathway.
  • biological activity includes any one or more of the following: the ability to bind to NGF receptors (such as p75 and/or TrkA); the ability to promote TrkA receptor dimerization and/or TrkA autophosphorylation; activation of NGF receptors The ability of signal pathways; the ability to promote cell differentiation, proliferation, survival, growth and other changes in cell physiology; the ability to promote the survival of mouse E13.5 trigeminal neurons and the ability to mediate pain, including pain after surgery.
  • Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is composed of three domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (LCVR or VL) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL are composed of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDR region or “CDR” or “hypervariable region” is an amino acid region in the variable region of an antibody that is mainly responsible for binding to an epitope.
  • the CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • CDR complementarity determining region
  • AbM CDR is a compromise between Kabat CDR and Chothia structure loop, and is used by Oxford Molecular’s AbM antibody modeling software, "Contact "Contact” CDR is based on the analysis of available complex crystal structures. According to different CDR determination schemes, the residues of each of these CDRs are as follows.
  • variants in relation to antibodies refers herein to include at least one, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acid substitutions or deletions. And/or an antibody inserted into the target antibody region with amino acid changes (for example, a heavy chain variable region or a light chain variable region or a heavy chain CDR region or a light chain CDR region), wherein the variant basically retains the antibody molecule before the change Biological characteristics.
  • the invention encompasses variants of any of the antibodies described herein.
  • the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the antibody before the change.
  • variable region of the heavy chain or the variable region of the light chain, or each CDR region of an antibody can be changed individually or in combination.
  • amino acid changes are amino acid substitutions, preferably conservative substitutions.
  • substitution refers to the substitution of an amino acid by another amino acid in the same category, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another Neutral amino acid substitution. Exemplary substitutions are shown in the following table:
  • the antibody variant has at least 80%, 90% or 95% or 99% or higher amino acid identity with the parent antibody in the sequence region of the antibody of interest.
  • an “isolated” antibody is an antibody that has been separated from a component of its natural environment.
  • the antibody is purified to more than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) confirmed.
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component 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 the nucleic acid molecule exists outside the chromosome or at a chromosomal location different from its natural chromosomal location.
  • vector when used herein refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell refers to cells into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations.
  • the progeny of mutants having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.
  • Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required.
  • For the expression of antibody fragments and polypeptides in bacteria see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, and see also Charlton, Methods in Molecular Biology, Volume 248 (BKCLo, editor, Humana Press, Totowa, NJ, 2003) , Pages 245-254, which describe the expression of antibody fragments in E. coli).
  • the antibody can be separated from the bacterial cell paste in the soluble fraction and can be further purified.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains, whose glycosylation pathways have been "humanized", resulting in the production of partially or completely human Antibodies with glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • a mammalian cell line modified to be suitable for growth in suspension can be used.
  • useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, such as, for example, Graham et al., J. Gen Virol. 36:59 (1977) Described in) and so on.
  • Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad.
  • the "percent (%) amino acid sequence identity" relative to the reference polypeptide sequence is defined as when the sequence is aligned (and gaps are introduced when necessary) to obtain the maximum percent sequence identity, and any conservative substitutions are not considered After being part of sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence.
  • sequence alignment can be used for sequence alignment to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the measurement alignment, including any algorithm required to obtain the maximum alignment over the entire length of the sequence being compared.
  • Affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X to its partner Y can usually be expressed by the equilibrium dissociation constant (K D ).
  • the equilibrium dissociation constant is the ratio of the dissociation rate constant and the association rate constant (k dis and k on, respectively ). Affinity can be measured by common methods known in the art, including those known in the prior art and those described herein.
  • the equilibrium dissociation constant (KD) of the anti-NGF antibody of the present invention and NGF is less than or equal to 1 ⁇ 10 -7 M, less than or equal to 1 ⁇ 10 -8 M, and less than or equal to 1 ⁇ 10 -9 M, less than or equal to 1 ⁇ 10 -10 M, less than or equal to 1 ⁇ 10 -11 M, less than or equal to 1 ⁇ 10 -12 M, less than or equal to 1 ⁇ 10 -13 M, etc.
  • Immunoconjugate refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a carrier.
  • composition refers to a formulation that exists in a form that allows the biological activity of the active ingredients contained therein to be effective, and does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered .
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more monoclonal antibodies that bind to NGF or immunologically active fragments thereof.
  • the anti-NGF antibody or pharmaceutical composition provided by the present invention can be integrated into suitable carriers, excipients and other agents in the formulation for combined administration, thereby providing improved transfer, delivery, tolerance and the like.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant (for example, Freund's adjuvant (complete and incomplete)), excipient, or vehicle that is administered with the therapeutic agent.
  • Pharmaceutical carriers suitable for use in the present invention may be conventional, see “Handbook of Pharmaceutical Excipients”, seventh edition, RC Rowe, PJSeskey and SCOwen, Pharmaceutical Press, London, Chicago, described pharmaceutical preparation excipients; and “Remington's Pharmaceutical Sciences” , EW Martin, Mack Publishing Co, Easton, PA Publishing, 21st edition, 2012, describes compositions and formulations suitable for drug delivery of the disclosed antibodies.
  • an effective amount refers to an amount or dose sufficient to obtain or at least partially obtain the desired effect after administration in a single or multiple doses
  • therapeutically effective amount refers to an amount that produces the desired effect in the treated subject, including Improvement of the subject’s symptoms (for example, improvement of one or more symptoms) and/or delay of the progression of symptoms, etc.
  • An effective amount for preventing diseases refers to an amount sufficient to prevent, prevent, or delay the occurrence of diseases.
  • the therapeutically effective amount depends on the specific disease involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the organism to which the preparation is administered Utilization characteristics; selected dosing regimen; and use of any concomitant therapy, etc.
  • treatment refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • subject or “individual” is a primate (e.g., human and non-human primates such as monkeys). In certain embodiments, the individual or subject is a human.
  • primate e.g., human and non-human primates such as monkeys.
  • the individual or subject is a human.
  • “Relieving” pain or one or more symptoms of pain means reducing or ameliorating one or more symptoms of pain compared to not administering anti-NGF antibodies. "Relief” also includes shortening or reducing the duration of symptoms.
  • pain refers to any etiology, including acute and chronic pain and any pain with an inflammatory component.
  • Pain as used herein includes the nociception and sensation of pain, and pain can be assessed objectively and subjectively using pain scores and other methods known in the art.
  • hypoalgesia refers to the body's enhanced pain response to normal noxious or unpleasant stimuli.
  • Table I The sequence of the variable region of the heavy chain and the variable region of the light chain of the antibody TRN1028:
  • the specific operation is as follows: Take 10mL of venous blood, centrifuge at 400g, 22°C for 15min; aspirate the supernatant clear plasma layer after centrifugation, aliquot and store at -80°C for cryopreservation; mix the remaining part with the same amount of RPMI1640 medium and add slowly Into the sterile centrifuge tube containing the lymphocyte separation fluid Ficoll, and keep the liquid level intact; centrifuge horizontally at 2000 rpm for 20 minutes, use a capillary tube to suck the PBMC cells in the cloud layer, put it in another sterile centrifuge tube, and add more than 5 times The volume of RPMI1640 medium was centrifuged at 1500 rpm for 10 minutes, and the washing was repeated twice. After the cell count was 1 ⁇ 10 7 / branch, it was frozen and stored at -80°C for later use.
  • Dilute hNGF (Sino Biological) with pH 9.6 phosphate coating buffer to 2 ⁇ g/mL, add 0.1mL/well to a 96-well plate, coat at 4°C overnight, and block with blocking solution at 37°C for 2h.
  • a flow cytometer (BD, Facs Aria Sorp) was used to sort a single plasma cell from the PBMC in the corresponding labeled sample; set the gate CD3/CD14/CD16/cd235a-CD19+CD20+/- CD38hi CD27hi, sorting specific cell populations from PBMC. Then sort out the memory plasma cell population specific to hNGF protein (>3%), select a single plasma cell and place it in a 96-well PCR plate (20 ⁇ L single cell lysate per well), so that each well contains one plasma cell , Keep in refrigerator at -80°C for later use.
  • BD Facs Aria Sorp
  • the first round of PCR 50 ⁇ L system, add 5 ⁇ L reverse transcription reaction product, 25 ⁇ L Taq Enzyme Mix, 25 ⁇ M heavy chain and light chain antibody constant region primers of each subtype. Reaction conditions:
  • Cycle conditions denaturation at 94°C for 30s, annealing at 55°C for 60s, extension at 72°C for 90s; repeat the cycle 35 times;
  • Second round of PCR 50 ⁇ L system, add 3 ⁇ L of the first round of PCR reaction product, 25 ⁇ L Taq Enzyme Mix, 20 ⁇ M of each subtype heavy chain and light chain antibody variable region primers. Reaction conditions:
  • Cycle conditions denaturation at 94°C for 30s, annealing at 58°C for 60s (or Lambda chain annealing at 64°C for 60s), extension at 72°C for 90s; repeat the cycle 35 times;
  • variable region gene was identified by 1.2% agarose gel electrophoresis to obtain the variable region gene. Its heavy chain variable region amino acid sequence VH is shown in SEQ ID NO: 7 and its light chain variable region amino acid sequence VL is shown in SEQ ID NO: 8 is shown.
  • the PCR product of the antibody variable region gene identified above is cloned into the pcDNA3.1+/C-(K)-DYK plasmid containing the human IgG constant region by conventional methods to construct human anti-NGF
  • the antibody expression vector is then transformed into Escherichia coli DH5 ⁇ competent bacteria for vector amplification, and the vector (recombinant plasmid) is extracted.
  • the obtained recombinant plasmid was co-transfected into HEK293 cells, and then HEK293 cells were cultured according to conventional methods to express the antibody.
  • the culture supernatant was collected, centrifuged at 4000 rpm for 1 h, and the supernatant was purified by Protein A affinity chromatography.
  • the expression and purification of the purified antibody were tested by SDS-PAGE.
  • the SDS-PAGE detection result showed that the antibody was successfully expressed, and the antibody was named TRN1028, with a relative molecular weight of about 180KD, a heavy chain of about 55KD, and a light chain of about 30KD.
  • the binding activity of antibody TRN1028 and hNGF was detected by ELISA test. Specifically, 2 ⁇ g/mL of hNGF was used as an antigen to be diluted and coated with a carbonate buffer of pH 9.6 and coated in a 96-well plate at 4° C. overnight; the blocking solution was blocked at 37° C. for 2 hours. Dilute the antibody TRN1028 with blocking solution times, starting at 10 ⁇ g/mL, and dilute 12 gradients of 3 times; then add each gradient of antibody TRN1028 to a 96-well plate, and then incubate at 37°C for 1h.
  • the binding force between an antibody and an antigenic determinant is called antibody affinity, which reflects the ability of an antibody molecule to bind to an antigenic determinant.
  • surface plasmon resonance analysis (BIACORE3000) was used to detect the antigen binding affinity of the antibody TRN1028.
  • the anti-human IgG (Fc) antibody was immobilized on the surface of the CM5 sensor chip (GE) by the method of amino coupling, and the anti-NGF antibody was captured as a ligand, so that the capture level reached about 400 RU; hNGF protein of different concentrations was used as the analytical flow.
  • Detection channel real-time monitoring of the binding of antigen and antibody and the dissociation of the bound complex.
  • the antigen dissociation constant of TRN1028 is as low as 2.52*10 -11 M, and the dissociation rate is 2.63*10 -04 /s, indicating that the antibody TRN1028 has a very strong binding affinity to the antigen hNGF.
  • TF-1 cells human erythroid leukemia cells
  • NGF can bind to TrkA.
  • TrkA binds to NGF to form a dimer, activates the intracellular domain tyrosine kinase region, and causes the corresponding tyrosine phosphorylation, thereby activating the downstream signal transduction system. Perform the function of regulating target cells, thereby inducing the proliferation of TF-1 cells.
  • the TF-1 cells (ATCC, CRL-2003 TM ) in the logarithmic growth phase were hung and cultured at 37° C. and 5% CO 2 for 24 h.
  • a 32ng/mL hNGF solution was prepared with minimal medium, and 25uL per well was added to a 96-well cell culture plate.
  • a solution of the antibody of the present invention with a concentration of 6 ug/mL was prepared with a minimal medium, and this was used as the starting concentration, and then a 3-fold dilution was performed to obtain 8 concentration gradients.
  • Add 25uL of human anti-NGF antibody serial dilution solution to each 96-well plate, and incubate at 37°C for 30min.
  • the dose-response curve of antibody TRN1028 and the control group is a typical S-type dose-response curve.
  • the EC50 of the antibody TRN1028 and the control group to block NGF signal is 12.41ng/mL and 14.35ng/mL, respectively. It shows that the antibody of the present invention can inhibit the proliferation of TF-1 cells induced by NGF and has better neutralizing activity.
  • this example also used PC-12 cells to detect the neutralizing activity of the antibody TRN1028.
  • the PC-12 cell line is derived from a transplantable murine pheochromocytoma and has a reversible neuronal phenotype response to NGF. This cell line highly expresses the NGF receptor TrkA. After being induced by physiological levels of NGF, PC-12 cells differentiate into cells with the characteristics of sympathetic neurons, which are often used to analyze growth factor-related mechanisms and research on neuronal differentiation.
  • NGF promotes the differentiation and synapse formation of PC-12 cells by combining with TrkA, and enhances the metabolic activity of PC-12 cells.
  • the light signal is directly proportional to the amount of ATP in the system, and ATP is positively related to the number of living cells; therefore, the ATP content can be detected to detect cell activity.
  • the calculation results show that the dose-response curves of antibodies TRN1028 and E3 are typical S-type dose-response curves, with EC50 of 65.19ng/mL and 32.21ng/mL, respectively; indicating that the antibody of the present invention can neutralize NGF and block its binding effect. Somatic TrkA can, thereby inhibiting the active differentiation of PC-12 cells induced by NGF, and has a neutralizing activity.
  • the present invention determines the cross-reactivity of the antibody TRN1028 to other neurotrophic factors by ELISA.
  • hBDNF, hNT-3, and hNT-4 (Absin) as antigens, respectively, coat a 96-well plate at 200ug/well at 4°C overnight; then, block with a blocking solution at room temperature for 2 hours.
  • the antibody TRN1028 (range 10ug/ml-0ug/ml) with decreasing concentration was added at 100ul/well and incubated at 37°C for 1h.
  • the human anti-NGF antibody of the present invention can cross-react with neurotrophic factors such as BDNF, NT-3 and NT-4, and has a strong broad spectrum.
  • NGF belongs to the neurotrophic family with BDNF, NT-3, and NT-4, and has certain homology in amino acid sequence.
  • BDNF has important protective and therapeutic effects in diseases such as AD, cerebral ischemia injury, PD, depression and anxiety.
  • BDNF may be more closely related to pain related to endometriosis than NGF, and BDNF is also It is found that it has a role in the occurrence and development of tumors; NT-3 gene can cause obvious loss of skin touch-pressure receptors, and NT-3 also has a role in congenital neurological diseases; NTs can induce or induce neurogenic inflammation. Cytokine imbalance and autoimmune response are involved in the pathogenesis of inflammatory skin diseases characterized by cell death (stress-induced alopecia) or hyperproliferation (atopic dermatitis, psoriasis, etc.). The combination of TRN1028 with BDNF, NT-3 and NT-4 may provide unexpected effects in the treatment of multiple, recurrent diseases or cancer pain.
  • mice C57BL/6N, male
  • VonFrey manufactured by VonFrey
  • IITC Withdrawal Threshold
  • mice in each group were injected intracutaneously with 30 ⁇ l CFA (3 mg/ml) into the left hind plantar to cause inflammation.
  • CFA 3 mg/ml
  • the 3rd, 4th, and 5th groups were given 10mg/kg of the test substance TRN1028, E3, and negative control antibody; and measured 24h (day 3), 48h (day 4) after administration ), 72h (the 5th day) pain threshold, calculate the pain threshold increase rate after administration.
  • Pain threshold increase rate% (MWT administration group-MWT model group)/MWT model group ⁇ 100%.

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Abstract

L'invention concerne un anticorps neutralisant ou un fragment de liaison à l'antigène de celui-ci qui se lie de manière spécifique à un NGF, et un procédé de préparation pour produire cet anticorps. L'invention concerne également une utilisation de cet anticorps dans le traitement de maladies associées au NGF, et en particulier pour soulager ou atténuer la douleur individuelle.
PCT/CN2021/087015 2020-04-17 2021-04-13 Anticorps dirigé contre le facteur de croissance nerveuse humain WO2021208928A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2364728A1 (fr) * 2003-07-15 2011-09-14 Amgen, Inc Anticorps humains à neutralisation anti-ngf en tant que qu'inhibiteurs sélectifs de chemin ngf
CN102762228A (zh) * 2009-10-09 2012-10-31 安姆根有限公司 作为选择性ngf途径抑制剂的人抗ngf中和抗体
CN104910274A (zh) * 2015-02-12 2015-09-16 北京华安科创生物技术有限公司 抗人神经生长因子的中和性单克隆抗体12c11及其杂交瘤细胞株

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1846451B1 (fr) * 2005-01-24 2013-05-15 Medimmune Limited Anticorps humains contre le ngf humain
PT2187964E (pt) * 2007-08-10 2015-01-14 Regeneron Pharma Anticorpos humanos de elevada afinidade para o factor de crescimento neural humano
TWI701258B (zh) * 2014-12-19 2020-08-11 美商再生元醫藥公司 流行性感冒病毒血球凝集素之人類抗體
CN108623687A (zh) * 2018-04-17 2018-10-09 中山康方生物医药有限公司 神经生长因子的单克隆抗体及其编码基因和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2364728A1 (fr) * 2003-07-15 2011-09-14 Amgen, Inc Anticorps humains à neutralisation anti-ngf en tant que qu'inhibiteurs sélectifs de chemin ngf
CN102762228A (zh) * 2009-10-09 2012-10-31 安姆根有限公司 作为选择性ngf途径抑制剂的人抗ngf中和抗体
CN104910274A (zh) * 2015-02-12 2015-09-16 北京华安科创生物技术有限公司 抗人神经生长因子的中和性单克隆抗体12c11及其杂交瘤细胞株

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