Anti-NGF antibody and antigen-binding fragment thereof, preparation method and application thereof

Abstract

The present invention provides an anti-NGF antibody or antigen-binding fragment thereof, a preparation method and use thereof; the present invention also provides an isolated polynucleotide encoding the anti-NGF antibody or antigen-binding fragment thereof, and a vector comprising the isolated polynucleotide. The present invention also provides the use of the antibody or antigen-binding fragment thereof in the preparation of a medicament for treating an NGF-mediated disease or condition.

Description

Technical Field

[0001] The present invention belongs to the field of biological immunology technology, and specifically relates to an anti-NGF antibody capable of specifically binding to human nerve growth factor and an antigen-binding fragment thereof. The present invention also relates to a preparation method and use of the antibody and the antigen-binding fragment thereof. Background Art

[0002] Nerve Growth Factor (NGF) is the first neurotrophic factor identified, and its role in the development and survival of peripheral and central neurons has been characterized. NGF has been shown to be a key 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 can upregulate the expression of neuropeptides in sensory neurons (Lindsay et al. (1989) Nature, 337: 362-364). NGF contains three subunits: α, β, and γ, and the β subunit is the active subunit of NGF. It is currently known that NGF regulates its activity through two different membrane surface receptors, TrkA tyrosine kinase receptor (Tropomyosin receptor kinase A, TrkA, also known as the "high affinity" NGF receptor) and p75 neurotrophin receptor (p75NTR, also known as the "low affinity" NGF receptor) (Chao et al. (1986) Science, 232: 518-521).

[0003] The NGF / TrkA signaling pathway is closely related to pain and can mediate the occurrence of pain. In injured and inflamed tissues, NGF is highly expressed, and the activation of nociceptive neurons by TrkA is triggered by multiple mechanisms and produces pain signals. After rats were injected with NGF, the latency of paw withdrawal caused by heat stimulation was significantly shortened (Lewin et al. (1994) Eur J Neurosci, 6: 1903-1912). In animal models, neutralizing 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. (1995) Net. Med. 1: 774-780; Koltzenburg M et al. (1999) Eur. J. Neurosci. 11: 1698-1704), suggesting that an increase in NGF levels is necessary for the generation of systemic hyperalgesia. NGF levels in the synovial fluid of patients with rheumatoid arthritis are significantly elevated, while NGF is not detected in the synovial fluid of non-inflammatory patients (Aloe et al. (1992) Arthritis and Rheumatism, 35:351-355). Injection of NGF in healthy individuals can cause hyperalgesia and localized pain (Petty et al. (1994) Ann Neurol, 36:244-246). Homozygous missense mutations in the NGFβ gene can cause HSAN5 symptoms in humans, and these 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 polymorphism is closely related to changes in pain perception. Autosomal recessive mutations in exon 17 of NTRK1 can cause congenital insensitivity to pain combined with anhidrosis (Indo et al. (1996) Nature genetics, 13:485-488).

[0004] Tens of millions of patients worldwide suffer from chronic pain, a number that continues to grow as the population grows. Current clinical treatments for chronic pain include nonsteroidal anti-inflammatory drugs (NSAIDs), anticonvulsants, and opioids. However, these drugs have numerous shortcomings. NSAIDs have limited efficacy and can have side effects such as gastrointestinal bleeding and renal toxicity, while opioids can cause addiction. The field urgently needs non-opioid analgesics that are pain-relieving, non-toxic, and abuse-resistant. Therefore, the value of inhibiting NGF in treating chronic pain is clear. Currently, numerous anti-human NGF antibodies are in research and development or clinical development, the most advanced of which include Pfizer / Lilly's anti-NGF monoclonal antibody Tanezumab and Regeneron / Sanofi's Fasinumab. Tanezumab is the first anti-NGF antibody drug developed. It has been reported to have a strong and broad analgesic effect on joint pain 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. Data from a Phase II / III clinical study of fasinumab for the treatment of osteoarthritis pain showed that patients in the four dose groups of fasinumab achieved statistically significant improvements in pain relief. On the other hand, clinical trials of multiple NGF inhibitors have also shown that NGF antibodies may face problems such as restrictions on use in seriously ill patients, long-term use, and dose restrictions, making the clinical application of NGF antibodies require further safety verification.

[0005] NGF is a crucial factor in neuronal development. When developing drugs to inhibit NGF function, the effects of NGF dosage on neurons must also be considered. On the one hand, the effective dose of an antibody drug depends on its neutralizing activity against the antigen and the amount of antigen present in the body. Increased neutralizing activity correlates with a reduced dosage. Research on anti-NGF antibodies requires obtaining CDR regions with varying affinities for different antigenic epitopes or the same epitope. Different CDRs have varying immunogenicity, resulting in varying rates of antibody tolerance and toxicity, which directly impacts drug efficacy. On the other hand, the immune response generated by subjects against the antibody itself can form immune complexes, altering its pharmacokinetics and causing allergic reactions, negating its therapeutic utility. Compared to murine and chimeric antibodies, the human immune system produces minimal antibody responses against humanized antibodies. Furthermore, humanized antibodies have similar half-lives to natural human antibodies, enabling less frequent dosing and lower dosages.

[0006] Therefore, developing anti-NGF antibodies with higher affinity and stronger specificity and performing humanized transformation are extremely important for the treatment or prevention of various diseases related to NGF. Summary of the Invention

[0007] Based on the deficiencies of the prior art, the main purpose of the present invention is to provide an anti-NGF antibody with high affinity, strong specificity and significant therapeutic effect. The present invention also provides a preparation method and use of the antibody.

[0008] In one aspect, the present invention provides an anti-NGF antibody or an antigen-binding fragment thereof that can specifically bind to NGF, wherein the antibody or antigen-binding fragment thereof comprises:

[0009] (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):

[0010] (i) a VH CDR1 consisting of SEQ ID NO: 13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0011] (ii) a VH CDR2 consisting of SEQ ID NO: 14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and

[0012] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0013] and / or

[0014] (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):

[0015] (iv) a VL CDR1 consisting of SEQ ID NO: 22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0016] (v) a VL CDR2 consisting of SEQ ID NO: 23, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and

[0017] (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0018] Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution;

[0019] Preferably, 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, and 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, and VL CDR3 as shown in SEQ ID NO: 24.

[0020] In one aspect, the present invention provides an anti-NGF antibody or an antigen-binding fragment thereof that can specifically bind to NGF, wherein the antibody or antigen-binding fragment thereof comprises:

[0021] (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):

[0022] (i) a VH CDR1 consisting of SEQ ID NO: 16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0023] (ii) a VH CDR2 consisting of SEQ ID NO: 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and

[0024] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 18, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0025] and / or

[0026] (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):

[0027] (iv) a VL CDR1 consisting of SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0028] (v) a VL CDR2 consisting of SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and

[0029] (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0030] Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution;

[0031] Preferably, 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, and 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, and VL CDR3 as shown in SEQ ID NO: 27.

[0032] In one aspect, the present invention provides an anti-NGF antibody or an antigen-binding fragment thereof that can specifically bind to NGF, wherein the antibody or antigen-binding fragment thereof comprises:

[0033] (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):

[0034] (i) a VH CDR1 consisting of SEQ ID NO: 19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0035] (ii) a VH CDR2 consisting of SEQ ID NO: 20, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and

[0036] (iii) a VH CDR3 consisting of the following sequence: SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0037] and / or

[0038] (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):

[0039] (iv) a VL CDR1 consisting of SEQ ID NO: 28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto,

[0040] (v) a VL CDR2 consisting of SEQ ID NO: 29, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, and

[0041] (vi) a VL CDR3 consisting of the following sequence: SEQ ID NO: 30, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0042] Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution;

[0043] Preferably, 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, and 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, and VL CDR3 as shown in SEQ ID NO: 30.

[0044] According to the anti-NGF antibody or antigen-binding fragment thereof capable of specifically binding to NGF of the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein,

[0045] The heavy chain variable region comprises the three 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 the three CDRs contained in the light chain variable region shown in any one of SEQ ID NOs: 3, 7, and 11;

[0046] Preferably, the three CDRs contained in the heavy chain variable region, and / or the three CDRs contained in the light chain variable region, are defined by the Kabat, Chothia or IMGT numbering systems.

[0047] According to the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

[0048] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0049] (i) the sequence shown in SEQ ID NO: 1;

[0050] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 1; or

[0051] (iii) a sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1;

[0052] and / or,

[0053] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0054] (iv) the sequence shown in SEQ ID NO: 3;

[0055] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 3; or

[0056] (vi) a sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3;

[0057] Preferably, the substitutions described in (ii) or (v) are conservative substitutions;

[0058] Preferably, 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.

[0059] According to the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

[0060] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0061] (i) the sequence shown in SEQ ID NO: 5;

[0062] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 5; or

[0063] (iii) a sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:5;

[0064] and / or,

[0065] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0066] (iv) the sequence shown in SEQ ID NO:7;

[0067] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 7; or

[0068] (vi) a sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:7;

[0069] Preferably, the substitutions described in (ii) or (v) are conservative substitutions;

[0070] Preferably, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence shown in SEQ ID NO: 5 and a VL having the sequence shown in SEQ ID NO: 7.

[0071] According to the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

[0072] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0073] (i) the sequence shown in SEQ ID NO: 9;

[0074] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 9; or

[0075] (iii) a sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 9;

[0076] and / or,

[0077] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0078] (iv) the sequence shown in SEQ ID NO: 11;

[0079] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 11; or

[0080] (vi) a sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 11;

[0081] Preferably, the substitutions described in (ii) or (v) are conservative substitutions;

[0082] Preferably, the antibody or antigen-binding fragment thereof comprises: a VH having the sequence shown in SEQ ID NO: 9 and a VL having the sequence shown in SEQ ID NO: 11.

[0083] According to the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof further comprises:

[0084] (a) a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; and

[0085] (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions) compared to the sequence from which it is derived;

[0086] Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region;

[0087] Preferably, the light chain constant region is a kappa light chain constant region.

[0088] According to the antibody or antigen-binding fragment thereof of the present invention, 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, a chimeric antibody, a humanized antibody, a bispecific antibody or a multispecific antibody;

[0089] Preferably, the humanized antibody or antigen-binding fragment thereof comprises: a heavy chain having a sequence as shown in any one of SEQ ID NOs: 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63 and / or a light chain having a sequence as shown in any one of SEQ ID NOs: 31 and 41.

[0090] According to the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is labeled with a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance) or biotin.

[0091] The present invention also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof;

[0092] Preferably, the polynucleotide comprises a nucleotide coding sequence as shown in any one of 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 and 64.

[0093] The present invention also provides a vector comprising the isolated nucleic acid molecule; preferably, the vector is a cloning vector or an expression vector.

[0094] The present invention also provides a host cell comprising the isolated nucleic acid molecule or the vector.

[0095] The present invention also provides a method for preparing the antibody or antigen-binding fragment thereof, comprising culturing the host cell under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell;

[0096] Preferably, the host cell is a mammalian cell, more preferably a human, mouse, sheep, horse, dog or cat cell, further preferably a Chinese hamster ovary cell.

[0097] The present invention also provides bispecific or multispecific molecules comprising the antibody or antigen-binding fragment thereof;

[0098] Preferably, the bispecific or multispecific molecule specifically binds NGF and additionally specifically binds one or more other targets;

[0099] Preferably, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

[0100] 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;

[0101] Preferably, the therapeutic agent is selected from a toxin, a radioisotope, a drug or a cytotoxic agent;

[0102] Preferably, 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;

[0103] Preferably, the immunoconjugate is an antibody-drug conjugate (ADC).

[0104] The present invention also provides 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;

[0105] Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent;

[0106] Preferably, 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.

[0107] The present invention also provides a kit comprising the antibody or antigen-binding fragment thereof;

[0108] Preferably, the antibody or antigen-binding fragment thereof is detectably labeled, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin;

[0109] Preferably, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof;

[0110] Preferably, 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.

[0111] The present invention also provides a chimeric antigen receptor comprising the antigen-binding domain of the antibody or antigen-binding fragment thereof;

[0112] Preferably, the antigen-binding domain comprises the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment thereof;

[0113] Preferably, the antigen binding domain is a scFv;

[0114] Preferably, the antigen-binding receptor comprises an antigen-binding fragment of the antibody;

[0115] Preferably, the antigen binding receptor is expressed by immune effector cells (eg, T cells).

[0116] 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 a medicament for treating a disease or condition mediated by NGF;

[0117] Preferably, the disease or condition comprises osteoarthritis pain, postoperative pain, rheumatoid arthritis pain, low back pain, cancer-related pain, neuropathic pain and visceral pain;

[0118] Preferably, the subject is a mammal, such as a human.

[0119] The present invention also provides a method for preventing and / or treating a disease in a subject (e.g., a human), comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the host cell;

[0120] The disease is an NGF-mediated disease or condition;

[0121] Preferably, the disease or condition comprises osteoarthritis pain, postoperative pain, rheumatoid arthritis pain, low back pain, cancer-related pain, neuropathic pain and visceral pain;

[0122] Preferably, the subject is a mammal, such as a human.

[0123] The present invention also provides a method for detecting the presence or amount of NGF (such as human NGF) in a sample, comprising the following steps:

[0124] (1) contacting the sample with the antibody or antigen-binding fragment thereof;

[0125] (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and NGF or detecting the amount of the complex;

[0126] Preferably, the antibody or antigen-binding fragment thereof is detectably labeled;

[0127] Preferably, the NGF is human NGF.

[0128] The present inventors have discovered that the antibodies or antigen-binding fragments thereof provided by the present invention have the following advantages:

[0129] 1. The antibodies provided by the present invention have high affinity, affinity constant K D Value less than or close to 10 -12 M, which can effectively block the binding between NGF and its receptors, blocking the pain response;

[0130] 2. The antibodies provided by the present invention have extremely strong specificity for binding to antigens and have no cross-binding with NGF family proteins. It is expected that their clinical safety risks will also be reduced;

[0131] 3. The antibodies provided by the present invention have significant analgesic effects in animals and are expected to have significant clinical efficacy;

[0132] 4. The antibodies provided by the present invention are expressed in CHO cells, which have the advantages of high yield, high activity, simple purification process and low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 The ELISA test results show that the anti-NGF antibody of the present invention binds to the human NGF protein.

[0134] FIG2 shows the effect of the anti-NGF antibody of the present invention on the binding of human NGF to receptors TrkA and p75.

[0135] Figure 3 This indicates that the anti-NGF antibody of the present invention inhibits NGF-induced TF-1 cell proliferation.

[0136] Figure 4 This indicates that the anti-NGF antibody of the present invention inhibits NGF-induced proliferation of TrkA / Ba / F3 cells.

[0137] Figure 5 This indicates that the anti-NGF antibody of the present invention inhibits reporter gene expression in TrkA / NFAT-bla / CHO cells.

[0138] FIG6 shows that the anti-NGF antibody of the present invention specifically binds to NGF and inhibits the NGF-induced signaling pathway.

[0139] Figure 7 It shows the competition binding curve between the anti-NGF antibody 43E5 of the present invention and 138E12.

[0140] FIG8 shows the effects of the anti-NGF antibodies 43E5 and 138E12 of the present invention on CFA-induced inflammatory pain in mice, wherein **P<0.01 vs. normal saline; ***P<0.001 vs. normal saline.

[0141] FIG9 shows that the humanized anti-NGF antibody of the present invention binds to human NGF.

[0142] FIG10 shows that the humanized anti-NGF antibody of the present invention binds to NGF homologous family proteins.

[0143] FIG11 shows that the humanized anti-NGF antibody of the present invention inhibits NGF-induced proliferation of TF-1 cells. DETAILED DESCRIPTION

[0144] The following description of the present application is only for the purpose of illustrating various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive multiple equivalents, variations, and modifications without departing from the scope of the present application, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in this application, including publications, patents, and patent applications, are incorporated by reference in their entirety.

[0145] In some embodiments, the antibodies or antigen-binding fragments described herein can be expressed in a manner less than or close to 10 - 12 The binding affinity of M (K D ) binds specifically to NGF, which is measured by biofilm optical interferometry. The binding affinity value can be expressed as K D The value is expressed by the ratio of the dissociation rate to the association rate when the antigen and antibody binding reaches equilibrium (k off / k on ) is calculated. The antigen binding affinity (e.g., K D ) can be suitably determined by any suitable method known in the art, including, for example, biofilm optical interferometry using an instrument such as the Fortebio.

[0146] In certain embodiments, the antibodies or antigen-binding fragments described herein bind to NGF at an EC50 (i.e., half-binding concentration) of 0.1 nM. The binding of the antibodies or antigen-binding fragments to NGF can be determined by methods known in the art, such as sandwich assays, ELISA, Western blotting, FACS, or other binding assays.

[0147] The antibody is specific for NGF. In certain embodiments, the antibody optionally does not bind to NGF homologous family proteins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4).

[0148] Example 1: Preparation of mouse anti-NGF antibodies

[0149] Recombinant human NGF protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC) was used as an immunogen and mixed with an equal amount of Freund's complete adjuvant (Sigma-Alderich, F5881) for emulsification and used for initial immunization. Five 6-week-old BALB / c and C57 mice (Jiangsu Huafukang) were prepared, and each animal was subcutaneously injected with 50 μg of immunogen (excluding the adjuvant mass, the same below). The immunogen was mixed with Freund's incomplete adjuvant (Sigma-Alderich, F5506) for emulsification and used for subsequent booster immunization. Two weeks after the initial immunization, each animal was intraperitoneally injected with 25 μg of immunogen for the first booster immunization; two weeks later, each animal was subcutaneously injected with 25 μg of immunogen for the second booster immunization. The final immune shock was performed 4-5 weeks later, with an intraperitoneal injection of 25 μg of immunogen.

[0150] After the final immunization, mouse B cells were isolated and mixed with SP2 / 0 cells (TCM18, Cell Bank, Chinese Academy of Sciences). Fusion was performed according to the BTX electroporator operating manual. After culturing, the fused cells were screened using enzyme-linked immunosorbent assay (ELISA) to identify hybridoma cells that bind to NGF and inhibit the binding of human NGF to the receptor TrkA. Subcloning was then performed by limiting dilution, and three positive hybridoma monoclonal cell lines were obtained by the same ELISA method, designated 43E5, 137H8, and 138E12.

[0151] The hybridoma monoclonal cell lines were expanded and cultured in a serum-free medium, and the culture medium was collected and purified using a protein G column to obtain mouse anti-human NGF monoclonal antibodies 43E5, 137H8, and 138E12.

[0152] Example 2: ELISA detection of binding of mouse anti-NGF antibody to human NGF

[0153] Human NGF (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC) was used as an antigen to study the binding ability of anti-NGF antibodies. 50 ng of human NGF was coated per well of a 96-well microplate. After washing and blocking, gradient dilutions of the antibody were added and incubated at room temperature for 1 hour. After washing three times, HRP-conjugated goat anti-mouse antibody (Biolegend, 405306) was added and incubated at room temperature for 1 hour. After three washes, tetramethylbenzidine (TMB, Biolegend, 421101) was added for color development. 1M HCl was used to terminate color development, and the absorbance at 450 nm was read on a microplate reader.

[0154] The anti-NGF antibodies secreted by the three hybridomas all bound to human NGF ( Figure 1 ), the EC50 of the three antibodies binding to human NGF were 0.082nM, 0.112nM, and 0.1nM, respectively (Table 1).

[0155] Table 1 EC50 of anti-NGF antibodies binding to human NGF

[0156] Antibody 43E5 137H8 138E12 EC50 (nM) 0.082 0.112 0.100

[0157] Example 3: Detection of Binding Affinity between Mouse Anti-NGF Antibody and Human NGF

[0158] Antibody affinity was determined using the ForteBio Octet Red96 (PALL) biomolecular interaction detection platform. Biotinylated human NGF was immobilized using a SA (Streptavidin) Biosensor (Fortebio, 18-5021), followed by binding to a gradient of anti-NGF antibodies. Dissociation was then performed using a buffer (1X Kinetics Buffer: PBS + 0.1% BSA + 0.05% Tween 20). Finally, the affinity kinetic constants for antigen-antibody binding were calculated using the instrument algorithm (Table 2).

[0159] Table 2 Binding affinity of anti-NGF antibodies to human NGF

[0160]

[0161] Example 4: Mouse anti-NGF antibody against human NGF and receptor TrkA or p75 Binding blocking assay

[0162] A. Blocking effect of anti-NGF antibodies on the binding of human NGF to receptor TrkA

[0163] If the receptor TrkA coating method is used for detection, 50 ng of NGF receptor TrkA protein (fused to human Fc) is coated on each well of a 96-well ELISA plate, washed three times, and then blocked with 3% BSA for 1 hour. 10000 ng / mL (66.67 nM) anti-NGF antibody is diluted 3-fold into 10 concentrations to 0.17 ng / mL (0.0011 nM), 100 μl is taken and mixed with an equal volume of 1 μg / mL biotin-labeled human NGF, the two are incubated at room temperature for 0.5 hours, and then added to the blocked and washed ELISA plate. Incubate at room temperature for 1 hour and wash three times. Add streptavidin-HRP (Streptavidin-HRP, Biolegend, 405210), incubate for 0.5 hours, and then wash three times. Add TMB to the ELISA plate and read the results after color development is terminated.

[0164] If the ligand-coated NGF method is used for detection, 50 ng of human NGF is coated on each well of a 96-well enzyme-labeled plate and washed three times. Block with 3% BSA for 1 hour and then wash three times. Dilute 10,000 ng / mL (66.67 nM) anti-human NGF antibody by gradient 3 times to 0.17 ng / mL (0.0011 nM), add 100 μL to each well, incubate at room temperature for 1 hour, and then wash three times. Add 100 μL of 1 μg / mL TrkA fused to human Fc to each well, incubate at room temperature for 1 hour, and then wash three times. Add HRP-conjugated donkey anti-human IgG antibody (Biolegend, 410902) and react at room temperature for 1 hour. After washing, add TMB for color development and read the results after termination.

[0165] The results are as follows Figure 2A and Figure 2B As shown, anti-NGF antibodies can block the binding of human NGF to the receptor TrkA, and the blocking effect increases significantly with increasing antibody concentration. The IC50 values ​​of the three antibodies blocking the binding of human NGF to the receptor TrkA are shown in Tables 3 and 4.

[0166] Table 3 Blocking TrkA-NGF binding IC50

[0167] Antibody 43E5 137H8 138E12 IC50(nM) 0.767 0.738 0.677

[0168] Table 4 Blocking NGF-TrkA binding IC50

[0169] Antibody 43E5 137H8 138E12 IC50(nM) 0.967 0.791 0.865

[0170] B. Blocking effect of anti-NGF antibody on the binding of human NGF to receptor p75

[0171] Each well of a 96-well ELISA plate was coated with 50 ng of human NGF, blocked with 3% BSA for 1 hour, and then washed three times. 40 μL of anti-NGF antibodies at 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0 μg / mL (66.67 nM, 6.67 nM, 0.67 nM, 0.067 nM, 0 nM) were added and incubated at room temperature for 15 minutes. 40 μL of 2 μg / mL human Fc-fused p75 (Beijing Yiqiao Shenzhou Technology Co., Ltd., 13184-H02H) was added to each well and incubated for 1 hour, followed by washing three times. 100 uL of HRP-conjugated donkey anti-human Fc secondary antibody (Biolegend, 410902) was added and reacted at room temperature for 1 hour. After washing, TMB was added for color development and the plate was read after termination. Figure 2C As shown, among the three selected anti-NGF antibodies, 43E5 could not block the binding of human NGF to receptor p75, while 137H8 and 138E12 could block the binding of human NGF to receptor p75.

[0172] It should be noted that during clinical trials of Pfizer / Lilly's NGF antibody, Tanezumab, there was an adverse reaction of accelerated osteoarthritis (RPOA). Tanezumab simultaneously blocks the binding of NGF to TrkA and p75. Studies have shown that p75 receptor function is associated with neuronal development, osteoblast differentiation and proliferation, myoblast differentiation, and muscle repair (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). Furthermore, no adverse reactions to RPOA were observed during clinical trials of GZ389988A, a small molecule TrkA inhibitor developed by Sanofi. Researchers believe this is due to GZ389988A's selective action on TrkA, which preserves the full functionality of the NGF-p75 pathway (Krupka et al. (2019) Osteoarthritis and Cartilage, 27:1599-1607). In the patented invention, 43E5 cannot block the binding of human NGF to p75, and the likelihood of clinical RPOA is expected to be reduced, with a safety profile superior to other antibodies with p75 blocking properties.

[0173] Example 5: In vitro neutralization activity detection of mouse anti-NGF antibodies

[0174] A. NGF-induced TF-1 cell proliferation experiment

[0175] The growth of TF-1 cells (human blood leukemia cells, ATCC, CRL-2003) is highly dependent on GM-CSF (granulocyte macrophage colony-stimulating factor), but NGF can also induce the growth of TF-1 after binding to TrkA on the surface of TF-1 cells, thus eliminating the need for GM-CSF.

[0176] TF-1 cells were cultured in RPMI1640 medium (HyClone, SH30027) containing 10% FBS (Gibco, 10091148) and 2 μg / mL GM-CSF (R&D, 215-GM-010). Cells were collected during the logarithmic growth phase, thoroughly washed to remove GM-CSF from the original growth medium, and resuspended in medium without GM-CSF. 5,000 cells per well were diluted in 80 μl of medium and plated onto white, clear-bottomed 96-well cell culture plates (corning, 3610). Anti-NGF antibody was diluted 4-fold from 400 μg / mL (2666.67 nM) to prepare 10 concentrations. The antibody was mixed with an equal volume of 50 ng / mL human NGF and incubated for 0.5 h. 20 μL of the antibody was then added to the cells in the 96-well plate. The cells were cultured in a 37°C, 5% CO2 incubator for 72 h, and then 100 μL of CellTiter- Cell viability detection reagent (Promega, G7573) was used to disrupt the cells, and the optical luminescence signal was read using a multifunctional microplate reader (SpectraMax).

[0177] like Figure 3 As shown, the three anti-NGF antibodies were able to inhibit the proliferation of TF-1 cells induced by human NGF, and the IC50 values ​​are shown in Table 5.

[0178] Table 5 IC50 of anti-NGF antibodies inhibiting NGF-induced TF-1 cell proliferation

[0179] Antibody 43E5 137H8 138E12 IC50(nM) 0.083 0.123 0.077

[0180] B. NGF-induced TrkA / Ba / F3 cell proliferation experiment

[0181] Ba / F3 cells can grow in two ways: IL3-dependent and IL3-independent. IL3-independent growth requires Ba / F3 cells to stably express active kinases. Ba / F3 cells expressing full-length human TrkA (TrkA / Ba / F3) were constructed, and their proliferation required the addition of NGF.

[0182] TrkA / Ba / F3 cells were cultured in RPMI1640 medium containing 10% FBS and 100 ng / mL NGF. The cells were collected and washed thoroughly to remove NGF from the original growth medium. 3000 cells per well were resuspended in 80 μL of medium without NGF and plated onto a white transparent 96-well cell culture plate (corning, 3610). Human NGF and gradient diluted antibodies were mixed and added to the cells in the 96-well plate. The final concentration of NGF was 5 ng / mL, and the final concentration of gradient diluted anti-NGF antibodies was up to 40 μg / mL (266.67 nM). Cells were cultured in a 37°C, 5% CO2 incubator for 48 hours and then CellTiter- Cell viability detection reagent (Promega, G7573) was used to disrupt the cells, and the optical luminescence signal was read using a multifunctional microplate reader (SpectraMax).

[0183] During the observation of TrkA / Ba / F3 cell growth, cells added with human NGF could proliferate normally; three anti-NFG antibodies could inhibit the proliferation of TrkA / Ba / F3 cells induced by NGF, and the inhibitory effect increased with the increase of concentration ( Figure 4 ), IC50 is shown in Table 6.

[0184] Table 6 IC50 of anti-NGF antibodies inhibiting NGF-induced TrkA / Ba / F3 cell proliferation

[0185] Antibody 43E5 137H8 138E12 IC50(nM) 0.096 0.083 0.098

[0186] C. Detection of reporter gene expression in NGF-induced TrkA / NFAT-bla / CHO cells

[0187] NGF binds to the transmembrane receptor TrkA, activating downstream phospholipase C (PLC). This, through a series of signal transduction pathways, leads to elevated intracellular calcium concentrations and activation of protein kinase C (PKC), ultimately promoting the translocation of nuclear factor (NF) and nuclear factor of activated T cells (NFAT) from the cytoplasm to the nucleus, thereby initiating the expression of NFAT-dependent genes. The gene encoding human TrkA has been integrated into the genome of TrkA / NFAT-bla / CHO cells (ThermoFisher, K1516), and an NFAT-responsive element has been inserted upstream of the reporter gene β-lactamase (β-bla). When the NGF-TrkA pathway is activated, cells express β-lactamase under the control of NFAT. β-lactamase activity reflects the degree of activation of the NGF-TrkA pathway.

[0188] TrkA-NFAT-bla / CHO cells were prepared according to the product instructions. 10,000 cells were plated per well in a 384-well plate and cultured overnight. Human NGF and serially diluted antibodies were mixed and incubated at room temperature for 0.5 hours before being added to the cells. The final concentration of NGF was 80 ng / mL and the final concentration of serially diluted NGF antibodies was up to 40 μg / mL (266.67 nM). After incubation at 37°C, 5% CO2 for 5 hours, LiveBLAzer prepared according to the operating instructions was added to the cells. TM The FRET-B / G β-lactamase substrate CCF4 (ThermoFisher, K1095) was incubated at room temperature in the dark for 2 hours, and the signal was read using a multi-function microplate reader (SpectraMax).

[0189] The results showed that the three anti-NGF antibodies could inhibit the activation of the signaling pathway induced by human NGF ( Figure 5 ), IC50 is shown in Table 7.

[0190] Table 7 IC50 of anti-NGF antibodies inhibiting reporter gene expression in TrkA / NFAT-bla / CHO cells

[0191] Antibody 43E5 137H8 138E12 EC50 (nM) 0.0532 0.0859 0.0442

[0192] Example 6: Anti-NGF Antibody Specificity Detection

[0193] A. ELISA detection of binding of mouse anti-NGF antibody to NGF homologous proteins

[0194] Each well of a 96-well ELISA plate was coated with 50 ng of 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., 10286-HNAE), or human NT-4 (Alomone, N-270), and the binding of mouse anti-NGF antibody to human NGF and three homologous family proteins was detected according to the method in Example 2.

[0195] As shown in Figure 6, three mouse anti-NGF antibodies were detected by immunohistochemistry. Figure 6A ) have high affinity, antibodies 43E5 and 138E12 bind to BDNF ( Figure 6B ), NT-3( Figure 6C ), NT-4( Figure 6D ) had almost no binding, and antibody 137H8 had weak binding to three NGF family proteins ( Figures 6B-6D ).

[0196] B. Mouse anti-NGF antibody specifically inhibits reporter gene expression in TrkA-NFAT-bla / CHO cells

[0197] As in Example 5C, TrkA-NFAT-bla / CHO cells, TrkB-NFAT-bla / CHO cells (ThermoFisher, K1491) and TrkC-NFAT-bla / CHO cells (ThermoFisher, K1515) were prepared according to the manufacturer's instructions.

[0198] The antibody of the present invention was diluted to 40 μg / mL (266.67 nM), mixed with human NGF or human BDNF (for TrkB-NFAT-bla / CHO cells), or human NT-3 (for TrkC-NFAT-bla / CHO cells), incubated at room temperature for 0.5 hours, and then added to the cells. After 5 hours of incubation, the LiveBLAzer prepared according to the operating manual was added to the cells. TM The FRET-B / G β-lactamase substrate CCF4 was incubated at room temperature in the dark for 2 hours. The signal was read using a multifunctional microplate reader.

[0199] The three anti-NGF antibodies inhibited the subsequent signal transduction caused by the binding of NGF to TrkA, but did not affect the signal transduction between BDNF and TrkB and between NT-3 and TrkC ( Figure 6E );

[0200] It should be noted that compared with Pfizer / Lilly's NGF antibody Tanezumab, the three anti-NGF antibodies of the present application, especially antibodies 43E5 and 138E12, do not bind to NGF family antibodies BDNF, NT-3, and NT-4, while Tanezumab has obvious cross-binding effects with NT-3 and NT-4. In other words, the specificity of 43E5 and 138E12 of the present application is better than that of Tanezumab, and the expected clinical adverse reactions are better than those of Tanezumab.

[0201] Example 7: Epitope Fractionation of Anti-NGF Antibodies 43E5 and 138E12

[0202] The Fortebio Ocete RED96 biomolecular interaction assay platform was used to examine whether two anti-NGF antibodies, 43E5 and 138E12, compete for NGF binding. Biotinylated human NGF was immobilized using a SA (Streptavidin) Biosensor. In the first step, sample loading was used to monitor the binding of antibody 138E12. In the second step, sample loading was used to monitor the binding of either 138E12 or 43E5. Finally, the binding buffer (1X Kinetics Buffer: PBS + 0.1% BSA + 0.05% Tween 20) was added for dissociation.

[0203] like Figure 7As shown, 43E5 and 138E12 can bind to human NGF simultaneously and in a non-competitive manner.

[0204] Example 8: Sequencing and analysis of the variable regions of mouse anti-NGF antibodies

[0205] Total RNA from hybridoma cells was extracted using the TRIzol kit (Ambion, 15596-026) and used as a template for first-strand cDNA synthesis (Takara). Rapid amplification of cDNA ends (RACE) was performed to obtain antibody light and heavy chain fragments, which were then cloned into standard vectors. Sequencing revealed the following heavy and light chain variable region sequences for the anti-NGF antibodies 43E5, 137H8, and 138E12:

[0206] Anti-NGF antibody 43E5 :

[0207] Heavy chain variable region amino acid sequence:

[0208] QVQLQQSGAELVRPGASVTLSKASGYTFTDYEMHWVRQTPVHGLEWIGAIDPETGGTAYNQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCRRGANLNHYGNDEGSYWGQGTLVTVSA(SEQ ID NO:1)

[0209] Heavy chain variable region nucleic acid sequence:

[0210] CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAGGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACTGCCTACAATCAGA AGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTAGAAGAGGGGCAAATCTTAATCACTATGGTAACGACGAGGGTTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQID NO:2)

[0211] Light chain variable region amino acid sequence:

[0212] DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSVGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCWQGTHLPQTFGGGTKLEIK(SEQ ID NO:3)

[0213] Light chain variable region nucleic acid sequence:

[0214] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGTAAGTCAAGTCAGAGCCTCTTACATAGTGTTGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTTTGCTGGCAAGGTACACATCTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:4)

[0215] Anti-NGF antibody 137H8:

[0216] Heavy chain variable region amino acid sequence:

[0217] QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYAVNWVRQPPGKGLEWLGMIWFDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARDYYGSSWYFDVWGAGTTVTVSS(SEQ ID NO:5)

[0218] Heavy chain variable region nucleic acid sequence:

[0219] CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGCTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGTTTGATGGAAGCACAGACTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGACTACTACGGTAGTAGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:6)

[0220] Amino acid sequence of the light chain variable region:

[0221] DIQMTQTTSSLSASLGDRVTISCRASQDISYYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLDIK(SEQ ID NO:7)

[0222] Nucleic acid sequence of the light chain variable region:

[0223] GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCTATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGACATCAAA(SEQ ID NO:8)

[0224] Anti-NGF antibody 138E12:

[0225] Heavy chain variable region amino acid sequence:

[0226] QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWFDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCAREGYYYGTTYYFDYWGQGTTLTVSS(SEQ ID NO:9)

[0227] Heavy chain variable region nucleic acid sequence:

[0228] CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGTTTGATGGAAGCACAGACTATAATTCA GCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGAGGGTTATTACTACGGTACTACCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ IDNO:10)

[0229] Light chain variable region amino acid sequence:

[0230] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK(SEQ ID NO:11)

[0231] Light chain variable region nucleic acid sequence:

[0232] GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTA CACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:12)

[0233] Sequence analysis yielded CDRs defined by the Kabat and IMGT systems. The following table (Table 8) lists the CDRs of three anti-NGF antibodies based on the definitions of the Kabat and IMGT systems.

[0234] Table 8: Antibody CDR definitions

[0235]

[0236] Example 9: Effect of anti-NGF antibodies on CFA-induced inflammatory pain in mice

[0237] C57BL / 6 mice (Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.), male, SPF grade, 6-8 weeks old, were acclimated for 5 days. After the acclimation period, the animals were divided into three groups: saline group, anti-NGF antibody 43E5 group, and anti-NGF antibody 138E12 group, with 10 animals in each group. Before the intraplantar injection of the modeling agent CFA, the pain threshold of the mice was measured three times using Von Frey (with intervals of no less than 10 minutes), and the average of the three times was taken as the basal pain threshold ( Figure 8A ).

[0238] The next day, 25 μl of CFA was injected into the plantar of the left hind paw of the mouse to induce inflammation (the ratio of CFA to liquid paraffin was 1:1). After the toes became obviously swollen (about 24 hours after modeling), the pain threshold of the left hind paw of the mouse was measured three times using the same method, and the average value was taken as the pain threshold before administration ( Figure 8B ).

[0239] Subsequently, 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 threshold was measured 24 hours, 48 ​​hours, and 72 hours after administration ( Figures 8C-8E ), measure once, and evaluate the effect of the test substance on the pain threshold.

[0240] The results are shown in FIG8 . 24, 48, and 72 hours after administration, anti-NGF antibodies 43E5 and 138E12 could significantly improve the decrease in pain threshold induced by CFA, with statistically significant differences compared with the saline group.

[0241] Example 10: Humanized transformation and characterization of murine anti-NGF antibody

[0242] A. Humanization of Antibodies

[0243] Humanization was performed based on the light chain variable region (VL) and heavy chain variable region (VH) sequences of the antibodies secreted by the hybridoma cells obtained above. The amino acid sequences of the VL and VH of the mouse antibodies were compared and searched in the human embryonic antibody amino acid sequence database to identify highly homologous human IGHV and IGKV sequences as humanization templates. Computer simulation technology was used to analyze the possible steric hindrance and mutual influence between the amino acids in the variable and framework regions, and the framework region amino acids that were critical for maintaining the activity of the humanized antibody were identified. These amino acids were retained during the humanization process. The humanization of the light and heavy chain variable regions was completed using CDR transplantation technology. Subsequently, the following multiple humanized antibodies were obtained using the selected antibody constant region templates. Among them, the anti-NGF antibody 43E5 used 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. Antibodies 43E5-05 and 43E5-06 were generated using human IGHV1-69 as the heavy chain variable region and human IGKV2-30 as the light chain variable region template. The light chain variable region sequences of antibodies 43E5-01, 43E5-02, 43E5-05, and 43E5-06 are identical. Antibody 138E12, an anti-NGF antibody, was generated using human IGHV4-59 as the heavy chain variable region and human IGKV1-39 as the light chain variable region template. Antibodies 138E12-01 and 138E12-02 share the same light chain variable region amino acid sequence, but differ in their heavy chain variable region amino acids. In addition, since the heavy chain complementary determining region sequences of 138E12-01 and 138E12-02 antibodies contain isomerization sites DG and deamidation sites NS, in order to remove the potential effects of the above sites on the antibodies, the heavy chain variable regions of 138E12-01 and 138E12-02 antibodies were further modified. 54-55 Mutation to EG 54-55 NS 60-61 Mutation to QS 60-61 , while DG 54-55 NS 60-61 Mutation to EG 54-55 and QS 60-61, and obtained antibodies 138E12-08, 138E12-09, and 138E12-10, respectively. Using 138E12-02 as a template, the DG 54-55 Mutation to EG 54-55 , DG 54-55 Mutation to DA 54-55 NS 60-61 Mutation to QS 60-61 NS 60-61 Mutation to NT 60-61 , DG 54-55 and NS 60-61 Mutation to EG 54-55 and QS 60-61 Antibodies 138E12-03, 138E12-04, 138E12-05, 138E12-06, and 138E12-11 were obtained. 138E12-07 was obtained using human IGHV4-59 as the heavy chain variable region and human IGKV1-39 as the light chain variable region. The framework sequence of its heavy chain variable region was fully human, without retaining mouse amino acids, but the DG sequence of its heavy chain complementary determining region was 54-55 and NS 60-61 Mutation to EG 54-55 and QS 60-61 .

[0244] The humanized antibody heavy and light chains are synthesized separately and ligated into the corresponding plasmids after enzyme digestion. The constructed plasmids are transiently transfected into CHO cells for expression. After 7-10 days of expression, the cell culture supernatant is purified using a MabSelect column (GE Healthcare) equilibrated with a corresponding buffer (such as phosphate-buffered saline (pH 7.4)), followed by elution with sodium citrate or other buffers. The antibodies obtained in this step can be characterized for purity by SDS-PAGE or SEC-HPLC and used for subsequent characterization studies.

[0245] The variable region amino acid sequences of the above antibodies are shown in Table 9:

[0246] Table 9 Anti-NGF antibody 43E5, 138E12 humanized antibody variable region sequence:

[0247]

[0248] Anti-NGF antibody 43E5 humanized antibody variable region sequence:

[0249] The anti-NGF antibody 43E5 humanized antibodies 43E5-01, 43E5-02, 43E5-05, and 43E5-06 contain the same light chain.

[0250] Light chain variable region amino acid sequence:

[0251] DVVMTQSPLSLPVTLGQPASISCKSSQSLLHSVGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCWQGTHLPQTFGGGTKVEIK(SEQ ID NO:31)

[0252] Light chain variable region nucleic acid sequence:

[0253] GACGTGGTCATGACACAGAGCCCACTGTCTCTGCCTGTGACCCTGGGACAGCCAGCCTCTATCTCCTGCAAGTCCAGCCAGTCCCTGCTGCACAGCGTGGGCAAGACATACCTGAACTGGCTGCAGCAGAGGCCAGGACAGAGCCCAAGGCGGCTGATCTATCTGGTGTC TAAGCTGGACTCCGGCGTGCCTGATAGATTCAGCGGCTCTGGCTCCGGCACCGACTTTACACTGAAGATCTCTCGCGTGGAGGCTGAGGATGTGGGCGTGTACTTCTGTTGGCAGGGCACCCATCTGCCACAGACATTTGCCGGCGGCACCAAGGTGGAGATCAAG(SEQ ID NO:32)

[0254] 43E5-01:

[0255] Heavy chain variable region amino acid sequence:

[0256] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGAIDPETGGTAYNQKFKGRVTMTADKSISTAYMELSRLRSDDTAVYYCRRGANLNHYGNDEGSYWGQGTLVTVSS(SEQ ID NO:33)

[0257] Heavy chain variable region nucleic acid sequence:

[0258] CAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGAGCCTCCGTGAAGGTGTCTTGCAAGGCCTCCGGCTACACCTTCACAGACTATGAGATGCACTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAGTGGATGGGAGCTATCGATCCTGAGACCGGAGGAACAGCTTACAACCAGAAGTTTAAGGGCAGAGTGACCATGACAGCCGACAAGTCTATCTCCACCGCTTATATGGAGCTGAGCAGACTGCGCTCTGACGATACAGCCGTGTACTATTGTAGGCGGGGCGCTAACCTGAATCATTACGGCAATGATGAGGGCTCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCAGC(SEQID NO:34)

[0259] 43E5-02:

[0260] Heavy chain variable region amino acid sequence:

[0261] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWIGAIDPETGGTAYNQKFKGRATLTADKSISTAYMELSRLRSDDTAVYYCRRGANLNHYGNDEGSYWGQGTLVTVSS(SEQ ID NO:35)

[0262] Heavy chain variable region nucleic acid sequence:

[0263] CAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGAGCCTCCGTGAAGGTGTCTTGCAAGGCCTCCGGCTACACCTTCACAGACTATGAGATGCACTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAGTGGATCGGAGCTATCGATCCTGAGACCGGAGGAACAGCTTACAACCAGAAGTTTAAGGGCAGAGCCACCCTGACAGCTGACAAGTCTATCTCCACCGCCTATATGGAGCTGAGCAGACTGCGCTCTGACGATACAGCCGTGTACTATTGTAGGCGGGGCGCTAACCTGAATCATTACGGCAATGATGAGGGCTCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCAGC(SEQID NO:36)

[0264] 43E5-05:

[0265] Heavy chain variable region amino acid sequence:

[0266] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGAIDPETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCRRGANLNHYGNDEGSYWGQGTLVTVSS(SEQ ID NO:37)

[0267] Heavy chain variable region nucleic acid sequence:

[0268] CAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGCTCCAGCGTGAAGGTGTCTTGCAAGGCTTCCGGCTACACCTTCACAGACTATGAGATGCACTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAGTGGATGGGAGCTATCGATCCTGAGACCGGAGGAACAGCTTACAACCAGAAGTTTAAGGGCAGAGTGACCATCACAGCCGACAAGTCCACCAGCACAGCTTATATGGAGCTGTCTTCCCTGCGCAGCGAGGATACCGCCGTGTACTATTGTAGGCGGGGCGCTAACCTGAATCATTACGGCAATGACGAGGGCTCTTATTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCT(SEQID NO:38)

[0269] 43E5-06:

[0270] Heavy chain variable region amino acid sequence:

[0271] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEMHWVRQAPGQGLEWIGAIDPETGGTAYNQKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCRRGANLNHYGNDEGSYWGQGTLVTVSS(SEQ ID NO:39)

[0272] Heavy chain variable region nucleic acid sequence:

[0273] CAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGCTCCAGCGTGAAGGTGTCTTGCAAGGCTTCCGGCTACACCTTCACAGACTATGAGATGCACTGGGTGAGGCAGGCTTCCAGGACAGGGACTGGAGTGGATCGGAGCTATCGATCCTGAGACCGGAGGAACAGCTTACAACCAGA AGTTTAAGGGCAGAGCCACCCTGACAGCTGACAAGTCCACCAGCACAGCTTATATGGAGCTGTCTTCCCTGCGCAGCGAGGATACCGCCGTGTACTATTGTAGGCGGGGCGCTAACCTGAATCATTACGGCAATGACGAGGGCTCTTATTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCT(SEQID NO:40)

[0274] Anti-NGF antibody 138E12 humanized antibody variable region sequence:

[0275] The 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, and 138E12-11 contain the same light chain.

[0276] Light chain variable region amino acid sequence:

[0277] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPRTFGGGTKVEIK(SEQ ID NO:41)

[0278] Light chain variable region nucleic acid sequence:

[0279] GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCAGAGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCAGAACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG(SEQ ID NO:42)

[0280] 138E12-01:

[0281] Heavy chain variable region amino acid sequence:

[0282] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGMIWFDGSTDYNSALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:43)

[0283] Heavy chain variable region nucleic acid sequence:

[0284] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCATGATCTGGTTCGACGGCAGCACCGACTACAACAGCGCCCTGAAGAGCAGAGTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:44)

[0285] 138E12-02:

[0286] Heavy chain variable region amino acid sequence:

[0287] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWLGMIWFDGSTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:45)

[0288] [[ID=,12]]Heavy chain variable region nucleic acid sequence:

[0289] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCATGATCTGGTTCGACGGCAGCACCGACTACAACAGCGCCCTGAAGAGCAGACTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:46)

[0290] 138E12-03:

[0291] Amino acid sequence of the heavy chain variable region:

[0292] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWLGMIWFEGSTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:47)

[0293] Nucleic acid sequence of the heavy chain variable region:

[0294] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCATGATCTGGTTCGAGGGCAGCACCGACTACAACAGCGCCCTGAAGAGCAGACTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:48)

[0295] 138E12-04:

[0296] Heavy chain variable region amino acid sequence:

[0297] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWLGMIWFDASTDYNSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:49)

[0298] Heavy chain variable region nucleic acid sequence:

[0299] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCATGATCTGGTTCGACGCCAGCACCGACTACAACAGCGCCCTGAAGAGCAGACTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:50)

[0300] 138E12-05:

[0301] Heavy chain variable region amino acid sequence:

[0302] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWLGMIWFDGSTDYQSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:51)

[0303] Heavy chain variable region nucleic acid sequence:

[0304] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCATGATCTGGTTCGACGGCAGCACCGACTACCAGAGCGCCCTGAAGAGCAGACTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:52)

[0305] 138E12-06:

[0306] Heavy chain variable region amino acid sequence:

[0307] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWLGMIWFDGSTDYNTALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:53)

[0308] Heavy chain variable region nucleic acid sequence:

[0309] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCATGATCTGGTTCGACGGCAGCACCGACTACAACACCGCCCTGAAGAGCAGACTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:54)

[0310] 138E12-07:

[0311] Heavy chain variable region amino acid sequence:

[0312] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGMIWFEGSTDYQSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:55)

[0313] Heavy chain variable region nucleic acid sequence:

[0314] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCATGATCTGGTTCGAGGGCAGCACCGACTACCAGAGCGCCCTGAAGAGCAGAGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:56)

[0315] 138E12-08:

[0316] Amino acid sequence of the heavy chain variable region:

[0317] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGMIWFEGSTDYNSALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:57)

[0318] Nucleic acid sequence of the heavy chain variable region:

[0319] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCATGATCTGGTTCGAGGGCAGCACCGACTACAACAGCGCCCTGAAGAGCAGAGTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:58)

[0320] 138E12-09:

[0321] Heavy chain variable region amino acid sequence:

[0322] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGMIWFDGSTDYQSALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:59)

[0323] Heavy chain variable region nucleic acid sequence:

[0324] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCATGATCTGGTTCGACGGCAGCACCGACTACCAGAGCGCCCTGAAGAGCAGAGTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:60)

[0325] 138E12-10:

[0326] Heavy chain variable region amino acid sequence:

[0327] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWIGMIWFEGSTDYQSALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:61)

[0328] Heavy chain variable region nucleic acid sequence:

[0329] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCATGATCTGGTTCGAGGGCAGCACCGACTACCAGAGCGCCCTGAAGAGCAGAGTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:62)

[0330] 1--138E12-11:

[0331] Heavy chain variable region amino acid sequence:

[0332] QVQLQESGPGLVKPSETLSLTCTVSGFSLTGYGVNWIRQPPGKGLEWLGMIWFEGSTDYQSALKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAREGYYYGTTYYFDYWGQGTTVTVSS(SEQ ID NO:63)

[0333] Heavy chain variable region nucleic acid sequence:

[0334] Note: In the translation of line 3, "138E12 - 11" is translated as "1--138E12-11" to make the format more in line with the overall style. You can adjust it according to specific needs.CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCTTCAGCCTGACCGGCTACGGCGTGAACTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCATGATCTGGTTCGAGGGCAGCACCGACTACCAGAGC GCCCTGAAGAGCAGACTGACCATCAGCAAGGACAACAGCAAGAGCCAGGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAGAGGGCTACTACTACGGCACCACCTACTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:64)

[0335] B. Characterization of Humanized Anti-NGF Antibodies

[0336] 1) Humanized antibody binds to human NGF

[0337] Human NGF (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11050-HNAC) was used as an antigen to study the affinity and specificity of the humanized antibody. NGF was diluted in PBS and coated with 50 ng per well of a 96-well microplate. After washing and blocking, a gradient dilution of the antibody was added and incubated at room temperature for 1 hour. After washing three times, HRP-conjugated donkey anti-human IgG antibody (Biolegend) was added and reacted at room temperature for 1 hour. After three washes, tetramethylbenzidine (TMB, Biolegend) was added for color development. 1M HCl was used to terminate the color development, and the absorbance at 450 nm was read on a microplate reader.

[0338] The binding conditions are shown in Table 10 and Figure 9. Humanized antibodies with different sequences all have similar binding to human NGF, and the degree of binding is comparable to that of human-mouse chimeric antibodies (containing the variable region of mouse anti-NGF antibody and the constant region of human antibody).

[0339] Table 10 EC50 of humanized anti-NGF antibodies binding to human NGF

[0340] Antibody 43E5-01 43E5-02 43E5-05 43E5-06 43E5-chi - EC50 (nM) 0.229 0.222 0.214 0.192 0.104 - Antibody 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 Antibody 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

[0341] 2) Humanized antibodies bind to other NGF family proteins

[0342] The binding of humanized anti-NGF antibody to human NGF and three homologous proteins was detected by ELISA method described in Example 6A. Figure 10A -H, the humanized antibody and NGF ( Figure 10A and 10B ) has high affinity with BDNF ( Figure 10C and D), NT-3( Figure 10E and F), NT-4 ( Figure 10G There is essentially no binding between H).

[0343] 3) NGF-induced TF-1 cell proliferation experiment

[0344] As described in Example 5A, the neutralizing activity of humanized anti-NGF antibodies was tested by TF-1 cell proliferation. As shown in Figure 11, all humanized antibodies inhibited NGF-induced TF-1 cell proliferation, and most showed inhibitory abilities comparable to those of human-mouse chimeric antibodies. IC50 values ​​are shown in Table 11.

[0345] Table 11 IC50 of humanized anti-NGF antibodies inhibiting NGF-induced TF-1 cell proliferation

[0346] Antibody 43E5-01 43E5-02 43E5-05 43E5-06 43E5-chi - IC50(nM) 0.582 0.534 0.701 0.568 0.255 - Antibody 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 Antibody 138E12-07 138E12-08 138E12-09 138E12-10 138E12-11 138E 12-Chi IC50(nM) 0.214 0.067 0.035 0.031 0.062 0.074

[0347] Although specific embodiments of the present invention have been described in detail, it should be understood that the present invention is not limited to the specific embodiments described. Various improvements, modifications, and variations may be made to the present invention without departing from the spirit and scope of the present invention, and these improvements, modifications, and variations are within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to NGF, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): (i) VH CDR1 consisting of the following sequence: SEQ ID NO: 13, (ii) VH CDR2 consisting of the following sequence: SEQ ID NO: 14, and (iii) VH CDR3 consisting of the following sequence: SEQ ID NO: 15; and (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): (iv) VL CDR1 consisting of SEQ ID NO: 22, (v) VL CDR2 consisting of SEQ ID NO: 23, and (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:

24.

2. An antibody or antigen-binding fragment thereof capable of specifically binding to NGF, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises the three CDRs contained in the heavy chain variable region shown in SEQ ID NO: 1; and the light chain variable region comprises the three CDRs contained in the light chain variable region shown in SEQ ID NO: 3, Wherein, the three CDRs in the heavy chain variable region are as follows: (i) VH CDR1 consisting of the following sequence: SEQ ID NO: 13, (ii) VH CDR2 consisting of SEQ ID NO: 14, and (iii) VH CDR3 consisting of the following sequence: SEQ ID NO: 15; The three CDRs in the light chain variable region are as follows: (iv) VL CDR1 consisting of SEQ ID NO: 22, (v) VL CDR2 consisting of SEQ ID NO: 23, and (vi) VL CDR3 consisting of the following sequence: SEQ ID NO: 24; The three CDRs contained in the heavy chain variable region and the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering systems.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 1; (ii) a sequence having one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 1; or (iii) a sequence having at least 80% sequence identity to the sequence shown in SEQ ID NO: 1; and / or, (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) the sequence shown in SEQ ID NO: 3; (v) a sequence having one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 3; or (vi) a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO:

3.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein The substitutions described in (ii) or (v) are conservative substitutions.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein The antibody or antigen-binding fragment thereof comprises: a VH having a sequence as shown in SEQ ID NO: 1 and a VL having a sequence as shown in SEQ ID NO:

3.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; and (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids compared to the sequence from which it is derived.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein The heavy chain constant region is an IgG heavy chain constant region, which is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; The light chain constant region is a kappa light chain constant region.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, and scFv; and / or the antibody is a murine antibody or a chimeric antibody.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein The Fv is a disulfide-bonded Fv; and the chimeric antibody is a humanized antibody.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein The humanized antibody or antigen-binding fragment thereof comprises: a heavy chain having a sequence as shown in any one of SEQ ID NOs: 33, 35, 37 and 39 and / or a light chain having a sequence as shown in SEQ ID NO:

31.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antibody or antigen-binding fragment thereof is labeled.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein The antibody or antigen-binding fragment thereof carries a detectable label selected from enzymes, radionuclides, luminescent substances and biotin.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein The enzyme is horseradish peroxidase.

14. The antibody or antigen-binding fragment thereof according to claim 12, wherein The luminescent substance is selected from fluorescent dyes and chemiluminescent substances.

15. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or the heavy chain variable region and light chain variable region thereof, according to any one of claims 1 to 14.

16. The nucleic acid molecule according to claim 15, wherein The nucleic acid molecule comprises a nucleotide coding sequence as shown in any one of SEQ ID NOs: 2, 4, 32, 34, 36, 38 and 40.

17. A vector comprising the isolated nucleic acid molecule of claim 15 or 16.

18. The vector according to claim 17, wherein The vector is a cloning vector or an expression vector.

19. A host cell comprising the isolated nucleic acid molecule of claim 15 or 16 or the vector of claim 17 or 18.

20. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, comprising culturing the host cell according to claim 19 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

21. The method of claim 20, wherein The host cell is a mammalian cell.

22. The method of claim 21, wherein The mammalian cells are cells of humans, mice, sheep, horses, dogs or cats.

23. The method of claim 21, wherein the mammalian cell is a Chinese hamster ovary cell.

24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, and a pharmaceutically acceptable carrier and / or excipient.

25. The pharmaceutical composition according to claim 24, wherein The pharmaceutical composition may also contain an additional pharmaceutically active agent.

26. The pharmaceutical composition according to claim 25, wherein The antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.

27. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

28. The kit according to claim 27, wherein The antibody or antigen-binding fragment thereof is detectably labeled.

29. The kit according to claim 28, wherein The antibody or antigen-binding fragment thereof carries a detectable label selected from enzymes, radionuclides, luminescent substances and biotin.

30. The kit according to claim 29, wherein The enzyme is horseradish peroxidase.

31. The kit according to claim 29, wherein The luminescent substance is selected from fluorescent dyes and chemiluminescent substances.

32. The kit according to claim 29, wherein The kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

33. The kit according to claim 32, wherein The secondary antibody further comprises a detectable label.

34. The kit according to claim 33, wherein The second antibody carries a detectable label selected from the group consisting of an enzyme, a radionuclide, a luminescent substance and biotin.

35. The kit according to claim 34, wherein The enzyme is horseradish peroxidase.

36. The kit according to claim 34, wherein The luminescent substance is selected from fluorescent dyes and chemiluminescent substances.

37. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-14, or the pharmaceutical composition according to any one of claims 24 to 26, in the preparation of a medicament for treating an NGF-mediated disease or condition; the disease or condition is selected from osteoarthritis pain, postoperative pain, rheumatoid arthritis pain, low back pain, cancer-related pain, neuropathic pain and visceral pain.

38. The use according to claim 37, wherein The subject is a mammal.

39. The use according to claim 38, wherein The mammal is a human.

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