WO2021218574A1 - Antibody binding to human ngf, and preparation method therefor and use thereof - Google Patents

Abstract

The present invention provides an antibody binding to human NGF or an antigen-binding fragment thereof, capable of effectively binding to human NGF and blocking a signaling pathway of NGF-TrKA, used for preparing drugs for treating NGF overexpressed diseases (such as trauma, inflammation, and chronic pain), and having good clinical application prospects.

Description

Antibody binding to human NGF, its preparation method and application Technical field

The invention belongs to the field of pain treatment and biotechnology, and relates to an antibody that binds to human NGF, a preparation method and application thereof.

Background technique

Nerve growth factor (NGF) plays a very important role in the growth and development of the nervous system. It is necessary for neuron proliferation, differentiation and survival, and function maintenance, and can promote the repair and regeneration of nerves after injury. The main receptor for NGF is TrkA. NGF has the function of transmitting pain signals. Rare human gene mutations indicate that blocking NGF or its receptor TrkA can stop pain signals. A large number of animal and human experiments have shown that the expression level of NGF is elevated in trauma, inflammation and chronic pain. Blocking the NGF-TrkA signal can significantly inhibit pain and will not cause serious effects on the central nervous system.

Pain is a major disease that affects people's quality of life and disability, and is one of the major unmet medical needs. Although opioid analgesics are very effective, their addiction and abuse caused by large-scale use have become a serious social problem. The abuse of opioid analgesics in the United States has already affected the life expectancy of people. But pain is also an important mechanism for animals to perceive environmental risks, so it is extremely difficult to find highly selective analgesics. Clinical trial results show that NGF neutralizing antibody is a rare analgesic with curative effect comparable to morphine drugs but without the risk of addiction.

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) painkillers, Tanezumab, which theoretically has no risk of addiction, is highly anticipated by regulatory agencies and the industry. In June 2017, the FDA granted Tanezumab fast track qualification for the treatment of osteoarthritis and chronic low back pain. Currently, no product of this mechanism is 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 suffering from trauma, inflammation and chronic pain diseases, and provide patients with more and more effective Treatment programs.

Summary of the invention

In order to solve the above technical problems, the inventors of the present invention conducted a large number of experiments, ranging from antigen immunization, hybridoma screening, antibody expression and purification to biological activity identification, screening and obtaining murine antibodies that specifically bind to human NGF, and on this basis , Further construct and obtain its chimeric antibody and humanized antibody.

Therefore, the object of the present invention is to provide an antibody or antigen-binding fragment thereof that binds to human NGF; to provide a nucleotide molecule encoding the antibody or antigen-binding fragment thereof that binds to human NGF; Expression vector; providing host cells for the expression vector; providing a method for preparing the antibody or antigen-binding fragment thereof that binds human NGF; providing a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds human NGF; providing the binding Application of human NGF antibody or its antigen-binding fragment in the preparation of medicines.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

One aspect of the present invention provides an antibody or antigen-binding fragment thereof that binds to human NGF, comprising:

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

(b) Light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 10, and the amino acid sequence of L-CDR2 is shown in SEQ ID As shown in NO: 11, the amino acid sequence of the L-CDR3 is as shown in SEQ ID NO: 12.

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 and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region. Each light chain has a variable region (VL) at one end and a constant region at the other end; 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 (e.g., bispecific antibodies) formed from at least two antibodies, and the like.

The "monoclonal antibody" of the present invention refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually with different antibodies directed against 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 through hybridoma culture and will not be contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of the antibody, which is obtained from a substantially uniform antibody population, which should not be interpreted as requiring any special method to produce the antibody.

The "antigen-binding fragment" of the present invention refers to a fragment of an antibody capable of specifically binding to human NGF. Examples of the antigen-binding fragments of the present invention include Fab fragments, F(ab') ₂ fragments, Fv fragments, and the like. Fab fragments are fragments produced by digesting antibodies with papain. The F(ab') ₂ fragment is a fragment produced by digesting an antibody with pepsin. Fv fragments are composed of dimers in which the variable region of the heavy chain and the variable region of the light chain of an antibody are closely and non-covalently related.

As a preferred solution, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

The "murine antibody" of the present invention refers to an antibody derived from a rat or a mouse, preferably a mouse. The murine antibody of the present invention is obtained by immunizing mice with human NGF as the antigen and screening hybridoma cells.

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, for example, having mouse heavy and light chain sequences linked to human constant regions. Variable region antibody. Preferably, the chimeric antibody of the present invention is obtained by splicing the murine antibody 183C6 heavy chain variable region and light chain variable region sequence with the human constant region. More preferably, the chimeric antibody of the present invention is selected from 183C6-Chimeric.

The "humanized antibody" of the present invention means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies. In addition, framework 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 some important residues. More preferably, the humanized antibody of the present invention is selected from 183C6-Humanized.

As a preferred solution, the antigen-binding fragments include Fab fragments, F(ab') ₂ fragments, and Fv fragments.

As a preferred solution, 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 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 Show.

As a preferred solution, 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.

Another aspect of the present invention provides a nucleotide molecule that encodes the antibody or antigen-binding fragment thereof that binds to human NGF.

As a preferred solution, 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.

As a preferred solution, the nucleotide sequence encoding the heavy chain of the nucleotide molecule is shown in SEQ ID NO: 16, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 18.

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

Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the above-mentioned human NGF-binding antibody or antigen-binding fragment thereof can be replaced, deleted, changed, inserted or added as appropriate to provide a polynucleotide homolog. The polynucleotide homologs of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the human NGF-binding antibody or its antigen-binding fragment within the scope of maintaining antibody activity.

Another aspect of the present invention provides an expression vector containing the above-mentioned nucleotide molecule.

Wherein, the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate regulatory sequences. Expression vector of the sequence. The expression vector may be a virus or a plasmid, such as a suitable phage or phagemid. For more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. For many known technologies and solutions for nucleic acid manipulation, please refer to Current Protocols in Molecular Biology, second edition, edited 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 pCHO 1.0.

The present invention additionally provides a host cell containing the above-mentioned expression vector.

The host cell of the present invention is a variety of conventional host cells in the field, as long as it can make the above-mentioned recombinant expression vector stably replicate itself and the nucleotides carried can be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21 (DE3) Or TG1, 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 the preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.

Another aspect of the present invention provides the above-mentioned method for binding human NGF antibodies or antigen-binding fragments thereof, characterized in that the method comprises the following steps:

a) Under expression conditions, culture the above-mentioned host cell to express the antibody or antigen-binding fragment thereof that binds to human NGF;

b) Isolation and purification of the antibody or antigen-binding fragment thereof that binds to human NGF as described in a).

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

The human NGF-binding antibody or antigen-binding fragment thereof 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, Changing the pH and other methods to elute the antibody or antigen-binding fragment thereof that binds to the human NGF bound on the affinity column. The inventors of the present invention conducted detection experiments on the antibodies or antigen-binding fragments thereof that bind to human NGF. The experimental results show that the antibodies or antigen-binding fragments thereof that bind to human NGF can well bind to the antigen. Has a high affinity.

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.

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 to achieve a more stable therapeutic effect. These preparations can ensure that the antibody or antigen-binding fragment disclosed in the present invention can bind to human NGF. The conformational integrity of its antigen-binding fragments, while also protecting the protein's multifunctional groups to prevent its degradation (including but not limited to aggregation, deamination or oxidation). Generally, for liquid preparations, it can be stored at 2°C-8°C for at least one year, and for freeze-dried preparations, it can be kept stable at 30°C for at least six months. The bispecific antibody preparation can be suspension, water injection, freeze-dried and other preparations commonly used in the pharmaceutical field.

For the water injection or lyophilized preparation of the antibody or antigen-binding fragment thereof that binds to human NGF disclosed in the present invention, pharmaceutically acceptable carriers preferably include, but are not limited to: surfactants, solution stabilizers, isotonic regulators, and buffers. One or a combination of liquids. The surfactant preferably includes but is not limited to: non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., the amount of which should be added to minimize the tendency of particles that bind to human NGF antibodies or their antigen-binding fragments . The solution stabilizer preferably includes, but is not limited to, one or a combination of the following: sugars, for example, reducing sugars and non-reducing sugars; amino acids, for example, monosodium glutamate or histidine; alcohols, For example: triols, higher sugar alcohols, propylene glycol, polyethylene glycol, etc. The amount of solution stabilizer added should enable the final formulation to maintain a stable state within a period of time considered by those skilled in the art to be stable. The isotonicity adjusting agent preferably includes, but is not limited to, one of sodium chloride, mannitol, or a combination thereof. The buffer preferably includes, but is not limited to, one of Tris, histidine buffer, phosphate buffer, or a combination thereof.

Another aspect of the present invention provides the application of the above-mentioned antibody or antigen-binding fragment or pharmaceutical composition that binds to human NGF in the preparation of a medicament for treating trauma, inflammation, and chronic pain.

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.

When the antibody or antigen-binding fragment thereof that binds to human NGF of the present invention is administered to animals including humans, the dosage will vary depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. , Can refer to the results of animal experiments and various situations, the total dose cannot exceed a certain range. Specifically, the dose of intravenous injection is 1-1800 mg/day.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effects of the present invention are:

At present, there is an urgent need to develop new, specific, efficient, and addictive-free painkillers in clinical practice, which can 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 to human NGF, can effectively neutralize human NGF, and has a good clinical application prospect.

Description of the drawings

Figure 1: ELISA to detect the relative affinity of mouse anti-human NGF monoclonal antibodies to human NGF.

Figure 2: ELISA detects the relative affinity of 183C6-Chimeric and 183C6-Humanized to human NGF.

Figure 3A: Detection of the inhibition of 183C6-Chimeric and 183C6-Humanized on human NGF-induced TF-1 cell proliferation. Figure 3B: Detection of the inhibition of 183C6-Chimeric and 183C6-Humanized on human NGF-induced TF-1 cell proliferation. Figure 4: ELISA detects the ability of 183C6-Humanized to bind mouse and rat NGF.

Detailed ways

The following examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are useful in the art Persons with ordinary skills in this field are well known and described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 ^nd edition, Cold spring Harbor Laboratory Press.

The experimental materials and sources used in the following examples and the preparation method of the experimental reagents are specifically described as follows.

Experimental Materials:

HEK293F cells: purchased from Thermo Fisher Scientific.

Human NGF: purchased from Sino Biological, item number: 11050-HNAC.

Balb/c mice: purchased from Shanghai Lingchang Biological Technology Co., Ltd.

Hybridoma sp2/0 cells: purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.

Reverse transcription kit: purchased from Takara.

TF-1 cell line: purchased from ATCC, article number CRL-2003 ^TM .

Goat anti-mouse secondary antibody: purchased from Sigma, product number SAB3701283.

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

Rat NGF: purchased from R&D systems, item number 7815-NG-025.

Experimental reagents:

Sodium carbonate buffer: 1.59g Na ₂ CO ₃ and 2.93g NaHCO _{3 are} dissolved in 1L of pure water.

Phosphate buffer: 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.

Color development solution: Substrate color development solution A: sodium acetate·trihydrate 13.6g, citric acid·monohydrate 1.6g, 30% hydrogen peroxide 0.3ml, pure water 500ml; substrate color development solution B: ethylenediaminetetraacetic acid two Sodium 0.2g, citric acid·monohydrate 0.95g, glycerol 50ml, TMB0.15g dissolved in 3ml DMSO, 500ml pure water; mix A and B in equal volume before use.

Stop solution: 2M sulfuric acid solution.

RPMI-1640 medium: purchased from Thermo Fisher Scientific.

Free Style 293 Expression Medium: purchased from Thermo Fisher Scientific.

pcDNA4: purchased from Thermo Fisher Scientific.

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

Fetal bovine serum: purchased from Thermo Fisher Scientific.

HAT: purchased from Thermo Fisher Scientific.

Hybridoma-SFM: purchased from Thermo Fisher Scientific.

Trizol: purchased from Thermo Fisher Scientific.

GM-CSF: purchased from Xiamen Tebao Biological Engineering Co., Ltd.

CCK-8: purchased from Dojindo.

laboratory apparatus:

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

Biacore 8K: purchased from GE healthcare.

CO ₂ shaking incubator: purchased from INFORS.

Example 1 Preparation of positive control antibody

The heavy chain and light chain variable region amino acid sequences of the positive control antibody E3 described in the examples of the present invention are respectively derived from SEQ ID NOs 1 and 2 of US20170342143A1 in the US patent, that is, SEQ ID NOs 1 and 2 of the present invention.

The DNA encoding the heavy chain variable region and the light chain variable region of the positive control was synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. Connect the synthetic positive control heavy chain variable region gene with the human IgG1 heavy chain constant region gene to obtain the full-length heavy chain gene, named E3-HC-IgG1; the above-mentioned light chain variable region gene is constant with the human Kappa chain Region genes are connected to obtain a full-length light chain gene, named E3-LC. The E3-HC-IgG1 and E3-LC genes were respectively constructed into pcDNA4 expression vector, and the resulting heavy and light chain expression vectors were transferred into HEK293F cells by PEI transfection method to express antibodies. HEK293F cells used Free Style 293Expression Medium nourish. The transfected HEK293F cells were cultured in a CO ₂ 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.

Example 2 Preparation and screening of mouse-derived anti-human NGF monoclonal antibodies

Step 1: Antigen immunize mice

Dilute the human NGF as the antigen with normal saline to an appropriate concentration, mix it with an equal volume of Freund’s complete adjuvant, and after complete phacoemulsification, perform subcutaneous multi-point injections into Balb/c mice aged 4-5 weeks , Each mouse was injected with 50μg antigen/100μl. Three weeks later, the same amount of protein was mixed with the same volume of Freund's incomplete adjuvant. After the phacoemulsification was completed, the mice were subcutaneously immunized at multiple points, and the immunization step was repeated again two weeks later. All mice took a drop of blood on the seventh day after the third immunization, separated the serum, and tested the serum titer by ELISA. For mice with serum antibody titer> 100,000, impulse immunization was performed one week after the titer determination: 10μg antigen protein/100μl normal saline/mouse was injected into the tail vein.

Among them, the method of using ELISA to detect serum titer is described as follows: dilute human NGF to 100ng/ml with sodium carbonate buffer, then add 100μl per well to the ELISA plate, incubate at room temperature for 2 hours; use 0.05% Tween-20 Wash the plate with PBST; 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 serially diluted mouse serum, and incubate for half an hour; wash the plate with PBST Two times, 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, stop the color reaction with stop solution; read OD450 with a microplate reader; use GraphPad Prism6 Perform data analysis, plot and calculate serum titer.

Step 2: Preparation and screening of hybridomas

Three days after the mice were immunized, spleen cells were taken for fusion. The well-growing hybridoma sp2/0 cells were cultured in a 37°C, 5% CO ₂ incubator, and the medium was changed one day before fusion. The process of fusion and selection is as follows: take mouse spleen, grind and wash, count; mix the two cells according to spleen cells: sp2/0 cells=2:1, discard the supernatant after centrifugation; then add 20ml of cell fusion buffer to wash the cells Three times; the cell pellet is ^{suspended in the cell fusion buffer at a density of 1×10 7} cells/ml; 2ml of the cell suspension is added to the fusion pool, placed on the electric fusion instrument ECM2001, within 30 seconds under certain conditions (AC 60V, 30S; DC 1700V, 40μS, 3X; POST AC 60V, 3S) for electrofusion; after electrofusion, gently transfer the fused cells to 37℃ preheated RPMI-1640 medium containing 10% fetal bovine serum, room temperature Continue to stand for 60 minutes; ^{inoculate the cells into a 96-well plate at 10 4} cells/well, 100 μl/well. On the next day, each well was supplemented with 100 μl of RPMI-1640 medium containing 2×HAT and 10% serum. On the fourth day after fusion, half of the old culture medium was replaced with fresh medium containing 1×HAT and RPMI-1640. On the seventh day after fusion, most of the old culture medium was replaced with fresh 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. The positive hybridoma clones were selected for expansion in 24-well plates and subcloned by limiting dilution method. The hybridoma strains stably expressing the target antibody are obtained by the aforementioned method, and these clones are expanded and the cells are cryopreserved. The aforementioned hybridoma strain was cultured with Hybridoma-SFM for 7 days, and then the mouse-derived anti-human NGF monoclonal antibody was purified from the culture supernatant using a Protein A/G affinity chromatography column. After purification, seven mouse monoclonal antibodies that can bind to human NGF were obtained. Their names were 183C6, 211D3, 27F4, 215F9, 147F10, 151G6, and 181H6. The antibody concentration was determined by ultraviolet spectrophotometry.

The relative affinity of the mouse-derived anti-human NGF monoclonal antibody to human NGF was detected by ELISA method. Refer to step 1 of Example 2 for the experimental method.

The results are shown in Figure 1. 183C6, 211D3, 27F4, 215F9, 147F10, 151G6 and 181H6 can effectively bind to human NGF, and their EC _{50 is 0.09205nM, 0.2433nM, 0.3459nM, 0.3355nM,}

0.8581nM, 0.409nM and 0.2988nM. The smaller the EC ₅₀ the higher the relative affinity of the highest affinity relative results show that mAb 183C6, 183C6 therefore chosen to develop the next step.

Example 3 Humanization of mouse-derived anti-human NGF monoclonal antibody

Step 1: Determination of the variable region sequence of the murine anti-human NGF monoclonal antibody

Trizol was used to extract total RNA from the 183C6 hybridoma monoclonal cell line, and the mRNA was reverse transcribed into cDNA with a reverse transcription kit. The combination of primers reported in the literature ("Antibody Engineering" Volume 1, Edited by Roland Kontermann and Stefan Dübel, combined The primer sequence is from page 323) The light chain variable region and heavy chain variable region genes of 183C6 were amplified by PCR, then the PCR product was cloned into the pMD18-T vector, and the variable region gene sequence was sequenced and analyzed. The murine 183C6 variable region sequence information is as follows: the heavy chain variable region gene sequence is 357bp 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 and encodes 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.

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

Through the analysis of the amino acid sequence of the variable region of the heavy chain and the variable region of the light chain of the murine 183C6 antibody, the antigen complementarity determining region (CDR) and the framework region (FR) of the heavy chain and light chain of the 183C6 monoclonal antibody were determined according to the Kabat rule. . The amino acid sequence of the heavy chain CDR of the 183C6 antibody is H-CDR1: SEQ ID NO: 7, H-CDR2: SEQ ID NO: 8 and H-CDR3: SEQ ID NO: 9, and the light chain CDR amino acid sequence is L-CDR1: SEQ ID NO: 10, L-CDR2: SEQ ID NO: 11 and L-CDR3: SEQ ID NO: 12.

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

The DNA encoding the humanized heavy chain and light chain variable regions was synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. Connect the synthetic humanized heavy chain variable region gene (SEQ ID NO: 15) with the human IgG1 heavy chain constant region to obtain the full-length humanized heavy chain gene (SEQ ID NO: 16), named 183C6- Hu-HC; link the humanized light chain variable region gene (SEQ ID NO: 17) with the human Kappa chain constant region to obtain the 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 examples. The heavy chain amino acid sequence is SEQ ID NO: 19, and the light chain amino acid sequence It is SEQ ID NO: 20, and the resulting antibody is named 183C6-Humanized.

In addition, the murine 183C6 heavy chain variable region was connected with the human IgG1 heavy chain constant region to obtain a chimeric heavy chain gene, named 183C6-Chi-HC; the murine 183C6 light chain variable region and the human Kappa chain constant region Connected 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 antibody was expressed and purified using the method described in the above examples, and the resulting antibody was named 183C6-Chimeric.

Example 4 Determination of the affinity of 183C6-Chimeric and 183C6-Humanized to antigen by enzyme-linked immunosorbent assay (ELISA)

ELISA method is used to detect the relative affinity of 183C6-Chimeric and 183C6-Humanized to human NGF. Refer to Step 1 of Example 2 for the experimental method. The difference is that the HRP-labeled goat anti-human secondary antibody is used here.

The results are shown in Figure 2. E3-IgG1, 183C6-Chimeric and 183C6-Humanized can effectively bind to human NGF, and their EC ₅₀ is 0.1118nM, 0.07049nM and 0.06848nM, respectively. The smaller the EC ₅₀ the higher the relative affinity of the results showed that the relative affinity of 183C6-Chimeric and 183C6-Humanized equivalent, and both were significantly higher than E3-IgG1.

Example 5 Determination of the affinity of 183C6-Chimeric and 183C6-Humanized to antigen by Biacore 8K

In this embodiment, Biacore 8K is used to detect the binding and dissociation kinetic parameters between the anti-NGF antibody and NGF, and the equilibrium dissociation constant. On Biacore 8K, use a chip coupled with ProteinA to capture anti-NGF antibodies. The captured antibodies are called immobilized ligands. Then, human NGF is injected as the analyte to obtain the binding-dissociation curve, and 6M guanidine hydrochloride is used to regenerate the buffer. After the solution is eluted, repeat the next cycle; use Biacore 8K Evaluation Software to analyze the data. The results are shown in Table 1.

Table 1. Detect the binding and dissociation kinetic parameters between anti-NGF antibody and human NGF

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

Note: Kon means the binding constant; Koff means the dissociation constant; KD=Koff/Kon, means the equilibrium dissociation constant.

Experimental results show that E3-IgG1, 183C6-Chimeric, and 183C6-Humanized can effectively bind to human NGF, but 183C6-Chimeric and 183C6-Humanized bind to NGF at a faster rate (Kon) than E3-IgG1, and the dissociation rate is significantly slower Compared with E3-IgG1, the equilibrium dissociation constants of 183C6-Chimeric and 183C6-Humanized are significantly smaller than that of E3-IgG1, which indicates that the affinity of 183C6-Chimeric and 183C6-Humanized is significantly higher than that of E3-IgG1.

Example 6 The ability of 183C6-Chimeric and 183C6-Humanized to neutralize the biological activity of NGF

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

Wash TF-1 cells in the logarithmic growth phase twice with RPMI-1640 medium preheated at 37°C, and centrifuge at 300g for 5 minutes each time; to count TF-1 cells, use RPMI-1640 containing 10% fetal bovine serum Suspend the medium to an appropriate density and inoculate it in a 96-well cell culture plate, 10,000 cells/150μl/well; add human NGF to the RPMI-1640 complete medium to make the concentration reach 80ng/ml; Dilute the above antibodies to the appropriate concentration, and serially dilute 9 gradients at the appropriate ratio; 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; at 37℃ Incubate in a 5% CO ₂ incubator for 3 days; after 3 days, add 20μl of CCK-8 solution to each well of a 96-well plate, and continue to incubate in the incubator for 8 hours; shake and mix well and read the OD450 value with a microplate reader; GraphPad Prism6 data analysis, plotting and calculation of IC _50.

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

Example 7 183C6-Humanized species crossover ability

The ELISA method was used to detect the ability of the above-mentioned anti-human NGF monoclonal antibody to recognize NGF of other species. Refer to step 1 of Example 2 for the experimental method. The difference is that the HRP-labeled goat anti-human secondary antibody is used here.

The results are shown in Figure 4. Both 183C6-Humanized can effectively bind to NGF in mice and rats, and their EC ₅₀ is 0.2041 nM and 0.5310 nM, respectively. The isotype control antibody is a human IgG1 monoclonal antibody that does not bind to NGF.

Claims (13)

1. The antibody or antigen-binding fragment thereof that binds to human NGF is characterized in that it comprises:

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

   (b) Light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 10, and the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 11, 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, wherein the antibody is a murine 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, wherein 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, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to human NGF is as SEQ ID NO: 4 As shown, 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 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 according to 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 shown in SEQ ID NO: 19, the amino acid of the light chain The sequence is shown in SEQ ID NO: 20.
6. A nucleotide molecule, characterized in that the nucleotide molecule encodes the antibody or antigen-binding fragment thereof that binds to human NGF according to any one of claims 1-5.
7. The nucleotide molecule of claim 6, wherein the nucleotide sequence of the nucleotide molecule encoding the variable region of the heavy chain is shown in SEQ ID NO: 3, which encodes the nucleus of the variable region of the light chain. The nucleotide sequence is shown in SEQ ID NO: 5; or the nucleotide sequence of the nucleotide molecule encoding the heavy chain variable region is shown in SEQ ID NO: 15, the nucleotide sequence encoding the light chain variable region As shown in SEQ ID NO: 17.
8. The nucleotide molecule of claim 6, wherein the nucleotide sequence encoding the heavy chain of the nucleotide molecule is shown in SEQ ID NO: 16, and the nucleotide sequence encoding the light chain is shown in SEQ ID. NO: shown in 18.
9. An expression vector, characterized in that the expression vector contains the nucleotide molecule according to any one of claims 6-8.
10. A host cell, characterized in that the host cell contains the expression vector according to claim 9.
11. A method for preparing the antibody or antigen-binding fragment thereof that binds to human NGF according to any one of claims 1-5, wherein the method comprises the following steps:

    a) Under expression conditions, culturing the host cell according to claim 10 to express the antibody or antigen-binding fragment thereof that binds to human NGF;

    b) Isolation and purification of 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 according to any one of claims 1-5 and a pharmaceutically acceptable carrier.
13. The use of the antibody or antigen-binding fragment thereof that binds to human NGF according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 12 in the preparation of a medicine for treating trauma, inflammation, and chronic pain.

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