Antibodies binding to human NGF, preparation methods and uses thereof

Abstract

The present invention provides an antibody or an antigen-binding fragment thereof that binds to human NGF, which can effectively bind to human NGF and block the signaling pathway of NGF-TrKA, and is used in the preparation of drugs for treating diseases with overexpression of NGF (such as trauma, inflammation, and chronic pain), and has good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of pain treatment and biotechnology, and relates to an antibody binding to human NGF, a preparation method and use thereof. Background Art

[0002] Nerve growth factor (NGF) plays a very important role in the growth and development of the nervous system. It is necessary for neuronal proliferation, differentiation, survival and functional maintenance, and can promote repair and regeneration after nerve damage. The main receptor of NGF is TrkA. NGF has the function of transmitting pain signals, and rare human gene mutations indicate that blocking NGF or its receptor TrkA can terminate pain signals. A large number of animal and human experiments have shown that the expression level of NGF is increased in trauma, inflammation and chronic pain. Blocking the NGF-TrkA signal can significantly inhibit pain without causing serious effects on the central nervous system.

[0003] Pain is a major disease that affects people's quality of life and causes disability, and is one of the major unmet medical needs. Although opioid analgesics are very effective, their addictiveness and abuse caused by large-scale use have become a serious social problem. The abuse of opioid analgesics in the United States has reached the level of affecting life expectancy. However, pain is also an important mechanism for animals to perceive environmental risks, so the discovery of highly selective analgesics is extremely difficult. Clinical trial results show that NGF neutralizing antibodies are rare analgesics with efficacy comparable to morphine-like drugs but without the risk of addiction.

[0004] Tanezumab is the first NGF neutralizing antibody to enter the clinic, and was first developed by Rinat Neuroscience. Due to the abuse of opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) analgesics, Tanezumab, which theoretically has no risk of addiction, is highly anticipated by regulators and the industry. In June 2017, the FDA granted Tanezumab fast track status for the treatment of osteoarthritis and chronic low back pain. Currently, there are no products with this mechanism on the market. However, there is still an urgent need to develop new, specific, efficient, and risk-free NGF neutralizing antibodies to meet clinical treatment needs, so as to improve the quality of life of people with trauma, inflammation, and chronic pain diseases, and provide patients with more and more effective treatment options. Summary of the invention

[0005] In order to solve the above technical problems, the inventors of the present invention conducted a large number of experiments, from antigen immunization, hybridoma screening, antibody expression and purification to biological activity identification, and screened out mouse antibodies that specifically bind to human NGF. On this basis, they further constructed and obtained its chimeric antibodies and humanized antibodies.

[0006] Therefore, the purpose of the present invention is to provide an antibody or an antigen-binding fragment thereof that binds to human NGF; to provide a nucleotide molecule encoding the antibody or an antigen-binding fragment thereof that binds to human NGF; to provide an expression vector comprising the nucleotide molecule; to provide a host cell of the expression vector; to provide a method for preparing the antibody or an antigen-binding fragment thereof that binds to human NGF; to provide a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof that binds to human NGF; and to provide the use of the antibody or an antigen-binding fragment thereof that binds to human NGF in the preparation of drugs.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that binds to human NGF, comprising:

[0009] (a) heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of H-CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO: 9, and

[0010] (b) light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO: 12.

[0011] The "antibody (Ab)" of the present invention is a heterotetrameric glycoprotein of about 150,000 daltons, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. One end of each light chain has a variable region (VL) and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. The antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed by at least two antibodies, and the like.

[0012] The "monoclonal antibody" of the present invention refers to an antibody obtained from a class of substantially homogeneous populations, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies for different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring any special method to produce the antibody.

[0013] The "antigen-binding fragment" of the present invention refers to a fragment of an antibody that can specifically bind to human NGF. Examples of the antigen-binding fragment of the present invention include Fab fragments, F(ab') 2 Fragments, Fv fragments, etc. Fab fragments are fragments produced by digesting antibodies with papain. F(ab') 2 The Fv fragment is a fragment produced by digesting an antibody with pepsin. The Fv fragment is composed of a dimer of the heavy chain variable region and the light chain variable region of an antibody in tight non-covalent association.

[0014] As a preferred embodiment, the antibody is a mouse antibody, a chimeric antibody or a humanized antibody.

[0015] The "murine antibody" of the present invention refers to an antibody derived from rats or mice, preferably mice. The murine antibody of the present invention is obtained by immunizing mice with human NGF as an antigen and performing hybridoma cell screening.

[0016] The "chimeric antibody" of the present invention refers to an antibody comprising heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, such as an antibody having mouse heavy and light chain variable regions connected to human constant regions. Preferably, the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region and light chain variable region sequences of the mouse antibody 183C6 with the human constant region. More preferably, the chimeric antibody of the present invention is selected from 183C6-Chimeric.

[0017] The "humanized antibody" of the present invention refers to an antibody whose CDR is derived from a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework region and the constant region) is derived from a human antibody. In addition, the framework region residues can be changed to maintain binding affinity. Preferably, the humanized antibody of the present invention is obtained by recombining the CDR region of the murine antibody 183C6 and the non-CDR region derived from a human antibody, adding a fourth framework region and mutating the residues that have an important influence on the part. More preferably, the humanized antibody of the present invention is selected from 183C6-Humanized.

[0018] As a preferred embodiment, the antigen-binding fragment includes Fab fragment, F(ab') 2 Fragment, Fv fragment.

[0019] As a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that binds to human NGF is as shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6; or the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that binds to human NGF is as shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 14.

[0020] As a preferred embodiment, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof that binds to human NGF is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0021] Another aspect of the present invention provides a nucleotide molecule, which encodes the above-mentioned antibody or antigen-binding fragment thereof that binds to human NGF.

[0022] As a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region of the nucleotide molecule is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 5; or the nucleotide sequence encoding the heavy chain variable region of the nucleotide molecule is shown in SEQ ID NO: 15, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 17.

[0023] As a preferred embodiment, the nucleotide sequence encoding the heavy chain of the nucleotide molecule is shown as SEQ ID NO: 16, and the nucleotide sequence encoding the light chain is shown as SEQ ID NO: 18.

[0024] The preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR method, or obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody by artificial full sequence synthesis method.

[0025] Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the above-mentioned antibody or antigen-binding fragment thereof that binds to human NGF can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a homologue of a polynucleotide. The homologue of the polynucleotide in the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the antibody or antigen-binding fragment thereof that binds to human NGF within the range of maintaining the activity of the antibody.

[0026] Another aspect of the present invention provides an expression vector, which contains the above-mentioned nucleotide molecule.

[0027] The expression vector is a conventional expression vector in the art, which refers to an expression vector containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. The expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, 1989. For many known techniques and protocols for nucleic acid manipulation, please refer to Current Protocols in Molecular Biology, Second Edition, compiled by Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pcDNA4, pDHFF, pGM-CSF or pCHO1.0.

[0028] The present invention further provides a host cell, wherein the host cell contains the above expression vector.

[0029] The host cell described in the present invention is any conventional host cell in the art, as long as the above-mentioned recombinant expression vector can be stably replicated by itself and the nucleotide carried can be effectively expressed. The host cell includes prokaryotic expression cells and eukaryotic expression cells, and the host cell preferably includes: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21 (DE3) or TG1, and more preferably E. coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies). The aforementioned expression vector is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0030] Another aspect of the present invention provides a method for producing the above-mentioned antibody or antigen-binding fragment thereof that binds to human NGF, characterized in that the method comprises the following steps:

[0031] a) culturing the above host cell under expression conditions to express the antibody or antigen-binding fragment thereof that binds to human NGF;

[0032] b) separating and purifying the antibody or antigen-binding fragment thereof that binds to human NGF as described in a).

[0033] The host cell culture method and the antibody separation and purification method described in the present invention are conventional methods in the art. For specific operating methods, please refer to the corresponding cell culture technical manual and antibody separation and purification technical manual. The method for preparing the antibody or its antigen-binding fragment that binds to human NGF disclosed in the present invention includes: culturing the above-mentioned host cells under expression conditions to express the antibody or its antigen-binding fragment that binds to human NGF; separating and purifying the antibody or its antigen-binding fragment that binds to human NGF. Using the above method, the recombinant protein can be purified into a substantially uniform substance, for example, as a single band on SDS-PAGE electrophoresis.

[0034] The antibody or antigen-binding fragment thereof that binds to human NGF disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high salt buffer, pH change, etc. can be used to elute the antibody or antigen-binding fragment thereof that binds to human NGF bound to the affinity column. The inventors of the present invention conducted a detection experiment on the obtained antibody or antigen-binding fragment thereof that binds to human NGF. The experimental results show that the antibody or antigen-binding fragment thereof that binds to human NGF can bind to the antigen well and has a high affinity.

[0035] Another aspect of the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment thereof that binds to human NGF and a pharmaceutically acceptable carrier.

[0036] The antibody or antigen-binding fragment thereof that binds to human NGF provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert the therapeutic effect more stably. These preparations can ensure the conformational integrity of the antibody or antigen-binding fragment thereof that binds to human NGF disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to coagulation, deamination or oxidation). Under normal circumstances, for liquid preparations, they can usually be stored stably for at least one year at 2°C-8°C, and for lyophilized preparations, they can remain stable at 30°C for at least six months. The bispecific antibody preparation can be a suspension, water injection, lyophilized preparation, etc. commonly used in the pharmaceutical field.

[0037] For the antibody or antigen-binding fragment water injection or lyophilized preparation binding to human NGF disclosed in the present invention, the pharmaceutically acceptable carrier preferably includes but is not limited to: one or a combination of surfactants, solution stabilizers, isotonic regulators and buffers. Among them, the surfactant preferably includes but is not limited to: non-ionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., and the amount added should minimize the particle tendency of the antibody or antigen-binding fragment binding to human NGF. The solution stabilizer preferably includes but is not limited to one or a combination of the following: sugars, such as reducing sugars and non-reducing sugars; amino acids, such as monosodium glutamate or histidine; alcohols, such as triols, higher sugar alcohols, propylene glycol, polyethylene glycol, etc., and the amount of solution stabilizer added should make the final preparation remain in a stable state within the time that the technicians in this field believe to reach stability. The isotonicity adjusting agent preferably includes, but is not limited to, sodium chloride, mannitol, or a combination thereof. The buffer preferably includes, but is not limited to, Tris, histidine buffer, phosphate buffer, or a combination thereof.

[0038] Another aspect of the present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof or pharmaceutical composition binding to human NGF in the preparation of drugs for treating trauma, inflammation and chronic pain.

[0039] The chronic pain described in the present invention preferably includes, but is not limited to, pain caused by arthritis or cancer, and chronic low back pain.

[0040] When the antibody or antigen-binding fragment thereof binding to human NGF and the composition thereof of the present invention is administered to animals including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and administration route. The results of animal experiments and various conditions can be referred to, and the total dosage cannot exceed a certain range. Specifically, the dosage for intravenous injection is 1-1800 mg / day.

[0041] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] The reagents and raw materials used in the present invention are commercially available.

[0043] The positive and progressive effects of the present invention are:

[0044] At present, there is an urgent need to develop new, specific, highly effective, and non-addictive analgesics in clinical practice, so as to improve the quality of life of people suffering from trauma, inflammation, and chronic pain diseases, and provide patients with more and more effective treatment options. The 183C6-Humanized of the present invention has a high affinity for human NGF, can effectively neutralize human NGF, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 :ELISA was used to detect the relative affinity of mouse anti-human NGF monoclonal antibody to human NGF.

[0046] Figure 2 : ELISA detected the relative affinity of 183C6-Chimeric and 183C6-Humanized to human NGF.

[0047] Figure 3A : Detect the inhibitory effects of 183C6-Chimeric and 183C6-Humanized on human NGF-induced TF-1 cell proliferation. Figure 3B : Detect the inhibitory effects of 183C6-Chimeric and 183C6-Humanized on human NGF-induced TF-1 cell proliferation. Figure 4 : ELISA detected the ability of 183C6-Humanized to bind to mouse and rat NGF. DETAILED DESCRIPTION

[0048] The following examples are provided to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert protein encoding genes into such vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.

[0049] The experimental materials and sources used in the following examples and the preparation methods of the experimental reagents are specifically described as follows.

[0050] Experimental Materials:

[0051] HEK293F cells: purchased from Thermo Fisher Scientific.

[0052] Human NGF: purchased from Sino Biological, catalog number: 11050-HNAC.

[0053] Balb / c mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0054] Hybridoma sp2 / 0 cells were purchased from the cell bank of Type Culture Collection Committee of the Chinese Academy of Sciences.

[0055] Reverse transcription kit: purchased from Takara.

[0056] TF-1 cell line: purchased from ATCC, catalog number CRL-2003 TM .

[0057] Goat anti-mouse secondary antibody: purchased from Sigma, catalog number SAB3701283.

[0058] Mouse NGF: purchased from Sino Biological, catalog number 50385-MNAC.

[0059] Rat NGF: purchased from R&D systems, catalog number 7815-NG-025.

[0060] Experimental reagents:

[0061] Sodium carbonate buffer: 1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 Dissolve in 1L of pure water.

[0062] Phosphate buffered saline: abbreviated as PBST, the formula is: KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5ml, add pure water to 1L.

[0063] Color developing solution: Substrate color developing solution A: 13.6g sodium acetate trihydrate, 1.6g citric acid monohydrate, 0.3ml 30% hydrogen peroxide, 500ml pure water; Substrate color developing solution B: 0.2g disodium ethylenediaminetetraacetic acid, 0.95g citric acid monohydrate, 50ml glycerol, 0.15g TMB dissolved in 3ml DMSO, 500ml pure water; mix equal volumes of solution A and solution B before use.

[0064] Stop solution: 2M sulfuric acid solution.

[0065] RPMI-1640 medium: purchased from Thermo Fisher Scientific.

[0066] Free Style 293Expression Medium: Purchased from Thermo Fisher Scientific.

[0067] pcDNA4: purchased from Thermo Fisher Scientific.

[0068] Electrofusion instrument ECM2001 and cell fusion buffer: purchased from BTX.

[0069] Fetal bovine serum: purchased from Thermo Fisher Scientific.

[0070] HAT: purchased from Thermo Fisher Scientific.

[0071] Hybridoma-SFM: purchased from Thermo Fisher Scientific.

[0072] Trizol: purchased from Thermo Fisher Scientific.

[0073] GM-CSF: purchased from Xiamen Tebao Biotechnology Co., Ltd.

[0074] CCK-8: purchased from Dojindo.

[0075] Experimental instruments:

[0076] Microplate reader: purchased from Molecular Devices, model SpectraMax 190.

[0077] Biacore 8K: purchased from GE healthcare.

[0078] CO 2 Shaking incubator: purchased from INFORS.

[0079] Example 1 Preparation of positive control antibody

[0080] The amino acid sequences of the heavy chain and light chain variable regions of the positive control antibody E3 described in the examples of the present invention are respectively from SEQ ID NOs 1 and 2 of US Pat. No. US20170342143A1, i.e., SEQ ID NOs 1 and 2 of the present invention.

[0081] DNA encoding the positive control heavy chain variable region and light chain variable region was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The synthesized positive control heavy chain variable region gene was linked to the human IgG1 heavy chain constant region gene to obtain a full-length heavy chain gene, named E3-HC-IgG1; the above light chain variable region gene was linked to the human Kappa chain constant region gene to obtain a full-length light chain gene, named E3-LC. The E3-HC-IgG1 and E3-LC genes were constructed into pcDNA4 expression vectors, and the obtained heavy chain and light chain expression vectors were transferred into HEK293F cells by PEI transfection to express antibodies. HEK293F cells were cultured with Free Style 293 Expression Medium. The transfected HEK293F cells were placed in CO 2 The cells were cultured in a shaking incubator for 5 days, the cell supernatant was collected by centrifugation, and the antibody in the supernatant was purified by Protein A affinity chromatography. The obtained antibody was named E3-IgG1.

[0082] Example 2 Preparation and Screening of Mouse Anti-Human NGF Monoclonal Antibodies

[0083] Step 1: Immunize mice with antigen

[0084] Human NGF, which is used as an antigen, is diluted to an appropriate concentration with physiological saline, mixed with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple points into 4-5 week-old Balb / c mice after complete ultrasonic emulsification, with each mouse injected with 50μg antigen / 100μl. Three weeks later, an equal amount of protein is mixed with an equal volume of Freund's incomplete adjuvant, and mice are immunized subcutaneously at multiple points after complete ultrasonic emulsification. This immunization step is repeated again two weeks later. On the seventh day after the third immunization, a drop of blood is taken from all mice, and the serum is separated and the serum titer is tested using ELISA. For mice with serum antibody titers >100,000, shock immunization is performed one week after the titer is measured: 10μg antigen protein / 100μl physiological saline / mouse is injected into the tail vein.

[0085] Among them, the method of using ELISA for serum titer detection is described as follows: dilute human NGF to 100 ng / ml with sodium carbonate buffer, then add 100 μl to each well of the ELISA plate and incubate at room temperature for 2 hours; wash the plate with PBST containing 0.05% Tween-20; add PBST containing 1% bovine serum albumin (BSA) to each well for blocking, and incubate at room temperature for 1 hour; wash the plate twice with PBST, add gradient diluted mouse serum, and incubate for half an hour; wash the plate twice with PBST, add appropriately diluted HRP-labeled goat anti-mouse secondary antibody, and incubate for half an hour; after washing the plate, add color developing solution for color development, and use stop solution to stop the color development reaction; read OD450 with an enzyme reader; use GraphPad Prism6 for data analysis, graphing and calculation of serum titer.

[0086] Step 2: Preparation and screening of hybridomas

[0087] Three days after the mice were immunized, spleen cells were collected for fusion. Hybridoma sp2 / 0 cells with good growth status were kept at 37°C and 5% CO 2 The cells were cultured in an incubator and the medium was changed one day before fusion. The fusion and screening process was as follows: the mouse spleen was taken, ground and washed, and then counted; the two cells were mixed according to the ratio of spleen cells to sp2 / 0 cells = 2:1, and the supernatant was discarded after centrifugation; then 20 ml of cell fusion buffer was added to wash the cells three times; the cell pellet was centrifuged at 1×10 7 Cells were suspended in cell fusion buffer at a density of 100 μg / ml; 2 ml of cell suspension was added to the fusion pool, and placed on the electrofusion instrument ECM2001, and electrofusion was performed within 30 seconds under certain conditions (AC 60V, 30S; DC1700V, 40μS, 3X; POST AC 60V, 3S); after electrofusion, the fused cells were gently transferred to RPMI-1640 medium preheated at 37°C containing 10% fetal bovine serum, and kept at room temperature for 60 minutes; the cells were placed for 10 4 Each well was inoculated into a 96-well plate, 100 μl / well. The next day, 100 μl of RPMI-1640 medium containing 2×HAT and 10% serum was added to each well. On the fourth day after fusion, half of the old culture medium was replaced with fresh culture medium containing 1×HAT and RPMI-1640. On the seventh day after fusion, most of the old culture medium was replaced with fresh culture medium containing 1×HAT and RPMI-1640. On the ninth day after fusion, samples were taken for ELISA detection, and the method was as described in step 1 of Example 2 above. Positive hybridoma clones were selected for expansion culture in 24-well plates and subcloned by limiting dilution. Hybridoma strains that stably express the target antibody were obtained by the aforementioned method, and these clones were amplified and the cells were frozen. The aforementioned hybridoma strains were cultured with Hybridoma-SFM for 7 days, and then the mouse anti-human NGF monoclonal antibodies were purified from the culture supernatant using a ProteinA / G affinity chromatography column. After purification, 7 mouse monoclonal antibodies that can bind to human NGF were obtained. Their names are 183C6, 211D3, 27F4, 215F9, 147F10, 151G6 and 181H6. The antibody concentrations were determined by ultraviolet spectrophotometry.

[0088] The relative affinity of the above mouse anti-human NGF monoclonal antibody to human NGF was detected by ELISA method. The experimental method is shown in step 1 of Example 2.

[0089] The results are as follows Figure 1 As shown, 183C6, 211D3, 27F4, 215F9, 147F10, 151G6 and 181H6 can effectively bind to human NGF.50 They are 0.09205nM, 0.2433nM, 0.3459nM, 0.3355nM,

[0090] 0.8581nM, 0.409nM and 0.2988nM. EC 50 The smaller the value, the higher the relative affinity. The above results show that the 183C6 monoclonal antibody has the highest relative affinity, so 183C6 was selected for the next step of development.

[0091] Example 3 Humanization of mouse anti-human NGF monoclonal antibody

[0092] Step 1: Determination of the variable region sequence of mouse anti-human NGF monoclonal antibody

[0093] Total RNA was extracted from the 183C6 hybridoma monoclonal cell line using Trizol, and the mRNA was reverse transcribed into cDNA using a reverse transcription kit. The light chain variable region and heavy chain variable region genes of 183C6 were amplified by PCR using a combination of primers reported in the literature (Antibody Engineering Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combination of primers is from page 323), and then the PCR product was cloned into the pMD18-T vector, and the variable region gene sequence was sequenced and analyzed. The variable region sequence information of mouse 183C6 is as follows: the heavy chain variable region gene sequence is 357 bp in length, encoding 119 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; the light chain variable region gene sequence is 321 bp in length, encoding 107 amino acid residues, the nucleotide sequence is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.

[0094] Step 2: Humanization of mouse anti-human NGF monoclonal antibody

[0095] The amino acid sequences of the heavy chain variable region and the light chain variable region of the mouse 183C6 antibody were analyzed, and the antigen complementarity determining regions (CDRs) and framework regions (FRs) of the heavy chain and light chain of the 183C6 monoclonal antibody were determined according to the Kabat rule. The amino acid sequences of the heavy chain CDRs of the 183C6 antibody are H-CDR1: SEQ ID NO: 7, H-CDR2: SEQ ID NO: 8, and H-CDR3: SEQ ID NO: 9, and the amino acid sequences of the light chain CDRs are L-CDR1: SEQ ID NO: 10, L-CDR2: SEQ ID NO: 11, and L-CDR3: SEQ ID NO: 12.

[0096] At https: / / www.ncbi.nlm.nih.gov / igblast / , the heavy chain variable region of the mouse 183C6 monoclonal antibody was compared for homology with the human IgG germline sequence, IGHV1-46*01 was selected as the heavy chain CDR transplantation template, the heavy chain CDR of the mouse 183C6 antibody was transplanted into the IGHV1-46*01 framework region, and WGQGTLVTVSS was added after H-CDR3 as the fourth framework region to obtain the CDR transplanted heavy chain variable region sequence. Similarly, the light chain variable region of the mouse 183C6 antibody was compared for homology with the human IgG germline sequence, IGKV1-39*01 was selected as the light chain CDR transplantation template, the light chain CDR of the mouse 183C6 antibody was transplanted into the IGKV1-39*01 framework region, and FGQGTKVEIK was added after L-CDR3 as the fourth framework region to obtain the CDR transplanted light chain variable region sequence. On the basis of CDR transplanted variable region, some amino acid sites of framework region are mutated. When mutating, the amino acid sequence is subjected to Kabat coding, and the position of the site is indicated by Kabat code. Preferably, for CDR transplanted heavy chain variable region, according to Kabat coding, the 30th T is mutated to S, the 37th V is mutated to I, the 48th M is mutated to I, the 67th V is mutated to A, the 69th M is mutated to L, the 71st R is mutated to A, and the 73rd T is mutated to Q. For CDR transplanted light chain variable region, the 43rd A is mutated to T, the 44th P is mutated to V, the 71st F is mutated to Y, and the 87th Y is mutated to F. The heavy chain variable region and light chain variable region with mutation sites were defined as humanized heavy chain variable region and light chain variable region, respectively, and named as 183C6-Hu-VH (SEQ ID NO: 13) and 183C6-Hu-VL (SEQ ID NO: 14), respectively.

[0097] The DNA encoding the humanized heavy chain and light chain variable regions was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The synthesized humanized heavy chain variable region gene (SEQ ID NO: 15) was linked to the human IgG1 heavy chain constant region to obtain a full-length humanized heavy chain gene (SEQ ID NO: 16), named 183C6-Hu-HC; the humanized light chain variable region gene (SEQ ID NO: 17) was linked to the human Kappa chain constant region to obtain a full-length humanized light chain gene (SEQ ID NO: 18), named 183C6-Hu-LC. The 183C6-Hu-HC and 183C6-Hu-LC genes were respectively constructed into the pcDNA4 expression vector, and the antibody was expressed and purified using the method described in the above example, the heavy chain amino acid sequence of which was SEQ ID NO: 19, and the light chain amino acid sequence of which was SEQ ID NO: 20, and the obtained antibody was named 183C6-Humanized.

[0098] In addition, the mouse 183C6 heavy chain variable region was linked to the human IgG1 heavy chain constant region to obtain a chimeric heavy chain gene, named 183C6-Chi-HC; the mouse 183C6 light chain variable region was linked to the human Kappa chain constant region to obtain a chimeric light chain gene, named 183C6-Chi-LC. The 183C6-Chi-HC and 183C6-Chi-LC genes were respectively constructed into the pcDNA4 expression vector, and the antibodies were expressed and purified using the method described in the above example, and the resulting antibodies were named 183C6-Chimeric.

[0099] Example 4 Determination of the affinity of 183C6-Chimeric and 183C6-Humanized for antigens by ELISA

[0100] The relative affinity of 183C6-Chimeric and 183C6-Humanized to human NGF was detected by ELISA. The experimental method is shown in step 1 of Example 2. The difference is that HRP-labeled goat anti-human secondary antibody is used here.

[0101] The results are as follows Figure 2 As shown in Figure 2, E3-IgG1, 183C6-Chimeric, and 183C6-Humanized can effectively bind to human NGF. 50 They are 0.1118nM, 0.07049nM and 0.06848nM respectively. 50 The smaller the value, the higher the relative affinity. The above results show that the relative affinities of 183C6-Chimeric and 183C6-Humanized are comparable, and both are significantly higher than E3-IgG1.

[0102] Example 5 Biacore 8K determination of the affinity of 183C6-Chimeric and 183C6-Humanized for antigens

[0103] In this example, Biacore 8K was used to detect the binding and dissociation kinetic parameters and equilibrium dissociation constant between anti-NGF antibody and NGF. On Biacore 8K, a chip coupled with ProteinA was used to capture anti-NGF antibody. The captured antibody was called immobilized ligand. Human NGF was then injected as the analyte to obtain a binding-dissociation curve. After elution with 6M guanidine hydrochloride regeneration buffer, the next cycle was repeated; the data was analyzed using Biacore 8K Evaluation Software. The results are shown in Table 1.

[0104] Table 1. Parameters of binding and dissociation kinetics between anti-NGF antibodies and human NGF

[0105] Antibody Name Kon(1 / Ms) Koff(1 / s) KD(M) E3-IgG1 8.76E+05 1.64E-04 1.87E-10 183C6-Chimeric 1.51E+07 3.57E-05 2.37E-12 183C6-Humanized 1.55E+07 3.71E-05 2.39E-12

[0106] Note: Kon represents binding constant; Koff represents dissociation constant; KD = Koff / Kon, represents equilibrium dissociation constant.

[0107] The experimental results showed that E3-IgG1, 183C6-Chimeric and 183C6-Humanized can all effectively bind to human NGF, but the binding rate (Kon) of 183C6-Chimeric and 183C6-Humanized to NGF is significantly faster than that of E3-IgG1, and the dissociation rate is significantly slower than that of E3-IgG1. Therefore, the equilibrium dissociation constants of 183C6-Chimeric and 183C6-Humanized are significantly smaller than those of E3-IgG1, indicating that the affinity of 183C6-Chimeric and 183C6-Humanized is significantly higher than that of E3-IgG1.

[0108] Example 6 Ability of 183C6-Chimeric and 183C6-Humanized to Neutralize the Bioactivity of NGF

[0109] TF-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO. 2Culture in an incubator, and 5ng / ml GM-CSF needs to be added to the culture medium; for specific culture and passage methods, refer to https: / / www.atcc.org / products / all / CRL-2003.aspx. Human NGF can induce TF-1 cell proliferation. In this example, TF-1 cells were used to detect the inhibitory effect of the above anti-NGF monoclonal antibody on NGF-induced TF-1 cell proliferation.

[0110] Wash TF-1 cells in the logarithmic growth phase twice with RPMI-1640 medium preheated at 37°C, centrifuge at 300g for 5 min each time; count TF-1 cells, suspend to an appropriate density with RPMI-1640 medium containing 10% fetal bovine serum, and inoculate into a 96-well cell culture plate, 10,000 cells / 150μl / well; add human NGF to the RPMI-1640 complete medium to make its concentration reach 80ng / ml; dilute the above antibodies to an appropriate concentration with medium supplemented with human NGF, and dilute them continuously for 9 steps at an appropriate multiple; add the diluted antigen and antibody mixture to a 96-well cell culture plate, 50μl / well; add distilled water around the 96-well plate, 200μl / well; incubate at 37°C, 5% CO 2 Incubate in an incubator for 3 days; after 3 days, add 20 μl of CCK-8 solution to each well of a 96-well plate and continue to culture in an incubator for 8 h; after shaking and mixing, read the OD450 value with an enzyme-labeled instrument; perform data analysis, plot, and calculate IC using GraphPad Prism6 50 .

[0111] Figure 3A and Figure 3B These are the results of two independent repeated experiments. Figure 3A and Figure 3B The results showed that E3-IgG1, 183C6-Chimeric and 183C6-Humanized could effectively inhibit the proliferation of TF-1 cells induced by NGF. Figure 3A Their IC 50 0.145nM, 0.1623nM and 0.1373nM respectively; Figure 3B Their IC 50 The results are as follows: 0.1162nM, 0.1273nM and 0.1104nM respectively, and their ability to neutralize the biological activity of NGF is basically the same. The isotype control antibody is a human IgG1 monoclonal antibody that does not bind to NGF.

[0112] Example 7: Cross-species capability of 183C6-Humanized

[0113] The recognition ability of the anti-human NGF monoclonal antibody to other species of NGF was detected by ELISA method. The experimental method is shown in step 1 of Example 2. The difference is that the HRP-labeled goat anti-human secondary antibody is used here.

[0114] The results are as follows Figure 4 As shown in Figure 2, 183C6-Humanized can effectively bind to mouse and rat NGF, and their EC 50 The isotype control antibody is a human IgG1 monoclonal antibody that does not bind to NGF. SEQUENCE LISTING <110> Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd. <120> Antibodies binding to human NGF, preparation methods and uses thereof <130> SH363-21P450334 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Composite <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr 20 25 30 Asp Leu Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Tyr Trp Tyr Ala Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Composite <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Phe His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Glu His Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 357 <212> DNA <213> Mus musculus <400> 3 caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagttg 60 tcctgcaagg cttctggcta cacctttagt aggtactgga tgcagtggat aaaacagagg 120 cctggacagg gtctggaatg gattggggct attatcctg gagatggtga tacaaggtac 180 agtcagaggg tcaagggcaa ggccacattg actgcagatc aatcctccag tacagtctcc 240 atgcaactca gcagcttggc atctgaggac tctgcggtct attactgtgc aagagagggga 300 aactttgcta actctttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 4 <211> 119 <212> PRT <213> mus musculus <400> 4 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 <h2 style=";text-align:left;direction:ltr">Trp Met Gln Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gln Arg Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50 55 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Lys Gly Lys Ala Thr Leu Thr Ala Asp Gln Ser Ser Ser Thr Val Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 65 70 75 80<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 85 90 95<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Arg Glu Gly Asn Phe Ala Asn Ser Phe Asp Tyr Trp Gly Gln Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 100 105 110<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Thr Thr Leu Thr Val Ser Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 115<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 321<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> muscular muscle<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatattcaga tgacacagac tacatcctcc ctgtctgcct ctctggggga cagagtcacc 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atccgttgca ggccaagtca ggacattggt aatcatttaa actggtatca gcagaagcca 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatggaactg ttaaactcct gatctacttc acatcaagat tatactcagg agtcccatca 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa tctggagcaa 240<h2 style=";text-align:left;direction:ltr"> gaagatattg ccacttactt ttgccaccag ggttatacgc ttccgtacac gttcggaggg 300 gggaccaagc tggaaataaa a 321 <210> 6 <211> 107 <212> PRT <213> mus musculus <400> 6 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Arg Cys Arg Pro Ser Gln Asp Ile Gly Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys His Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 5 <212> PRT <213> mus musculus <400> 7 Arg Tyr Trp Met Gln 1 5 <210> 8 <211> 17 <212> PRT <213> mus musculus <400> 8 Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gln Arg Val Lys 1 5 10 15 Gly <210> 9 <211> 10 <212> PRT <213> mus musculus <400> 9 Glu Gly Asn Phe Ala Asn Ser Phe Asp Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> mus musculus <400> 10 Arg Pro Ser Gln Asp Ile Gly Asn His Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> mus musculus <400> 11 Phe Thr Ser Arg Leu Tyr Ser 1 5 <210> 12 <211> 9 <212> PRT <213> mus musculus <400> 12 His Gln Gly Tyr Thr Leu Pro Tyr Thr 1 5 <210> 13 <211> 119 <212> PRT <213> Composite <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gln Arg Val 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Gln Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asn Phe Ala Asn Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 14 <211> 107 <212> PRT <213> Composite <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Pro Ser Gln Asp Ile Gly Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys His Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 357 <212> DNA <213> Composite <400> 15 caggtgcagc tcgtgcagtc tggcgctgaa gtgaaaaaac ctggagcttc tgtgaaggtg 60 tcttgtaagg ccagtggcta cacattttct agatactgga tgcagtggat tagacaggct 120 cctggacagg gtctggagtg gattggcgct atctatccag gcgatggtga cacaagatat 180 tctcagagag tgaagggcag ggccacactg actgctgatc agtctacatc tactgtgtat 240 atggaactgt cttctctgag atccgaggac accgccgtgt actactgcgc cagggagggc 300 aacttcgcca actccttcga ctactggggc cagggcaccc tggtgaccgt gtcctcc 357 <210> 16 <211> 1347 <212> DNA <213> Composite <400> 16 caggtgcagc tcgtgcagtc tggcgctgaa gtgaaaaaac ctggagcttc tgtgaaggtg 60 tcttgtaagg ccagtggcta cacattttct agatactgga tgcagtggat tagacaggct 120 cctggacagg gtctggagtg gattggcgct atctatccag gcgatggtga cacaagatat 180 tctcagagag tgaagggcag ggccacactg actgctgatc agtctacatc tactgtgtat 240 atggaactgt cttctctgag atccgaggac accgccgtgt actactgcgc cagggagggc 300 aacttcgcca actccttcga ctactggggc cagggcaccc tggtgaccgt gtcctccgcg 360 agcaccaagg gaccttccgt gtttcccctc gcccccagct ccaaaagcac cagcggcgga 420 acagctgctc tcggctgtct cgtcaggat tactccccg agccgtgac cgtgagctgg 480 aacagcggag ccctgacaag cggcgtccac accttccctg ctgtcctaca gtcctccgga 540 ctgtacagcc tgagcagcgt ggtgacagtc cctagcagct ccctgggcac ccagacatat 600 atttgcaacg tgaatcacaa gcccagcaac accaaggtcg atagaaggt ggagcctaag 660 tcctgcgaca agacccacac atgtccccc tgtcccgctc ctgaactgct gggaggccct 720 tccgtgttcc tgttcccccc taagcccaag gaccctga tgatttccag gaacccgag 780 gtgacctgtg tggtggtgga cgtcagccac gaggaccccg aggtgaatt caacggtac 840 gtcgatggcg tggaggtgca caacgctaag accaagccca gggaggagca gtacaattcc 900 acctacaggg tggtgtccgt gctgaccgtc ctccatcagg actggctgaa cggcaagag 960 tataagtgca aggtgagcaa caggccctc cctgctccca tcgagagac catcagcaa 1020 gccaagggcc agcccaggga acccaagctc tataccctgc ctcccagcag ggaggagatg 1080 accaagaacc aagtgagcct cacatgccctc gtcaagggct tctatccttc cgatattgcc 1140 gtcgagtggg agtccaacgg acagcccgag aacaactaca agacaacacc ccccgtgctc 1200 gattccgatg gcagcttctt cctgtactcc aagctgaccg tggacaagtc cagatggcaa 1260 caaggcaacg tcttcagttg cagcgtcatg catgaggccc tccacaacca ctacacccag 1320 aagagcctct ccctgagccc tggaaag 1347 <210> 17 <211> 321 <212> DNA <213> Composite <400> 17 gatatccaga tgacccagtc tccttcttct ctgtctgctt ccgtgggaga cagagtgact 60 atcacctgta gaccttctca ggacattggc aaccacctga attggtacca gcagaagcca 120 ggcaagaccg tgaagctgct gatctacttt acctctagac tgtattctgg cgtgccatct 180 agattttctg gatccggctc tggaacagat tataccctga ccatcagctc tctgcagcct 240 gaggattttg ctacatactt ctgtcatcag ggctatacac tgccttatac atttggccag 300 ggcacaaagg tggagatcaa g 321 <210> 18 <211> 642 <212> DNA <213> Composite <400> 18 gatatccaga tgacccagtc tccttcttct ctgtctgctt ccgtggggaga cagagtgact 60 atcacctgta gaccttctca ggacattggc aaccacctga attggtacca gcagaagcca 120 ggcaagaccg tgaagctgct gatctacttt acctctagac tgtattctgg cgtgccatct 180 agattttctg gatccggctc tggaacagat tataccctga ccatcagctc tctgcagcct 240 gaggattttg ctacatactt ctgtcatcag ggctatacac tgccttatac atttggccag 300 ggcacaaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360 agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420 ccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480 gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600 ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 19 <211> 449 <212> PRT <213> Composite <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Ser Gln Arg Val 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Gln Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asn Phe Ala Asn Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 20 <211> 214 <212> PRT <213> Composite <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Pro Ser Gln Asp Ile Gly Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys His Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An antibody or antigen-binding fragment thereof that binds to human NGF, characterized in that: include: (a) heavy chain complementary determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of H-CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO: 9, and (b) light chain complementary determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof that binds to human NGF according to claim 1, characterized in that: The antibody is a mouse antibody, a chimeric antibody or a humanized antibody.

3. The antibody or antigen-binding fragment thereof that binds to human NGF according to claim 1, characterized in that: The antigen binding fragments include Fab fragments, F(ab')2 fragments, and Fv fragments.

4. The antibody or antigen-binding fragment thereof that binds to human NGF according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to human NGF is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; or The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to human NGF is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

14.

5. The antibody or antigen-binding fragment thereof that binds to human NGF as described in claim 1, wherein the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof that binds to human NGF is as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

20.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes an antibody or antigen-binding fragment thereof that binds to human NGF as described in any one of claims 1-5.

7. The nucleic acid molecule according to claim 6, wherein The nucleotide sequence encoding the heavy chain variable region of the nucleic acid molecule is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 5; or The nucleotide sequence encoding the heavy chain variable region of the nucleic acid molecule is shown in SEQ ID NO: 15, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

17.

8. The nucleic acid molecule according to claim 6, wherein The nucleotide sequence encoding the heavy chain of the nucleic acid molecule is shown in SEQ ID NO: 16, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO:

18.

9. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to any one of claims 6 to 8.

10. A host cell, characterized in that The host cell contains the expression vector according to claim 9.

11. A method for preparing an antibody or antigen-binding fragment thereof that binds to human NGF as claimed in any one of claims 1 to 5, characterized in that: The method comprises the following steps: a) culturing the host cell according to claim 10 under expression conditions, thereby expressing the antibody or antigen-binding fragment thereof that binds to human NGF; b) separating and purifying the antibody or antigen-binding fragment thereof that binds to human NGF as described in a).

12. A composition, characterized in that The composition contains the antibody or antigen-binding fragment thereof that binds to human NGF as described in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

13. Use of the antibody or antigen-binding fragment thereof that binds to human NGF as described in any one of claims 1 to 5 or the composition as described in claim 12 in the preparation of a drug for treating chronic pain.

Images