An anti-NGF monoclonal antibody and its application

Abstract

This patent application belongs to the field of antibody technology, specifically relating to an anti-NGF monoclonal antibody and its applications. The anti-NGF monoclonal antibody of this patent application can specifically bind to TrkA and p75. The anti-NGF monoclonal antibody of this patent application can be used to treat or prevent NGF-mediated diseases, such as autoimmune arthritis, bone cancer pain, osteoarthritis, and fracture pain.

Description

Technical Field

[0001] This application belongs to the field of antibody technology, specifically relating to an anti-NGF monoclonal antibody and its application. Background Technology

[0002] Nerve growth factor (NGF) is the first active biomolecule discovered in the neurotrophic factor family, participating in the regulation of nerve cell growth, development, differentiation, and post-injury repair. NGF receptors mainly include tropomyosin receptor kinase A (TrkA) and the p75 receptor. TrkA is a transmembrane receptor produced by the fusion of tropomyosin and tyrosine kinase; its intracellular structure includes a transmembrane helical structure and a tyrosine kinase activation structure, exhibiting high affinity for mature NGF. p75 is a transmembrane glycoprotein receptor with similar affinities for different neurotrophic factors, but lower affinity for mature NGF. Currently, the main analgesics on the market are nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics, but these drugs generally have safety and tolerability issues, limiting their clinical use and causing side effects. NGF plays a crucial role in pain signal transmission, and the interaction between NGF and its receptor TrkA is a key step in initiating and maintaining pain. Numerous studies have shown that NGF levels are elevated in trauma, inflammation, and chronic pain. Blocking NGF pathways, including TrkA and p75, can significantly suppress pain, such as in autoimmune arthritis, bone cancer pain, OA, and fracture pain. Anti-NGF monoclonal antibodies hold promise as a safe and effective new approach for treating chronic pain. Summary of the Invention

[0003] The purpose of this application is to provide an anti-NGF monoclonal antibody, the technical solution of which is as follows:

[0004] 1. A monoclonal antibody, characterized in that the antibody comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0005] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 9;

[0006] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 12.

[0007] 2. The monoclonal antibody according to claim 1, characterized in that the antibody comprises an antibody heavy chain variable region HCVR, wherein:

[0008] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or 13, or is an amino acid sequence having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 or 13.

[0009] 3. The monoclonal antibody according to claim 1, characterized in that the antibody comprises an antibody light chain variable region (LCVR), wherein:

[0010] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6 or 14, or is an amino acid sequence having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6 or 14.

[0011] 4. The monoclonal antibody according to any one of claims 1-3, wherein the antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein:

[0012] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or 13, or is an amino acid sequence having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 or 13. The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6 or 14, or is an amino acid sequence having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6 or 14.

[0013] 5. The monoclonal antibody according to claim 1, characterized in that the antibody comprises a heavy chain and a light chain, wherein:

[0014] The heavy chain amino acid sequence is shown in SEQ ID NO: 3;

[0015] The light chain amino acid sequence is shown in SEQ ID NO: 4.

[0016] 6. The monoclonal antibody according to any one of items 1-5, wherein the antibody is a canine or feline-derived antibody or a whole-canine or whole-feline antibody.

[0017] 7. An isolated nucleic acid comprising a polynucleotide sequence encoding a monoclonal antibody as described in any one of items 1-6.

[0018] 8. A vector comprising the nucleic acid described in item 7.

[0019] 9. A host cell comprising the nucleic acid described in item 7 or the vector described in item 8.

[0020] The aforementioned monoclonal antibody is an anti-NGF monoclonal antibody, and therefore can be used to treat or prevent NGF-mediated diseases. The technical solution of this application also includes:

[0021] 10. A pharmaceutical composition comprising: a monoclonal antibody as described in any one of items 1-6, or an antibody expressed by a nucleic acid as described in item 7, or an antibody prepared from a vector as described in item 8, or an antibody produced by a host cell as described in item 9, and a pharmaceutically acceptable vector.

[0022] 11. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is used to treat or prevent NGF-mediated diseases.

[0023] 12. The pharmaceutical composition according to item 11, wherein the NGF-mediated related disease is selected from one or more of autoimmune arthritis, bone cancer pain, osteoarthritis, and fracture pain.

[0024] 13. A coupling, wherein the coupling comprises:

[0025] The monoclonal antibodies described in items 1-6, or the antibodies expressed by nucleic acids described in item 7, or the antibodies prepared by the vector described in item 8, or the antibodies produced by host cells described in item 9.

[0026] 14. The conjugate according to claim 13, wherein the conjugate further comprises a drug, cytokine, radionuclide, or enzyme linked to the antibody.

[0027] 15. A method of treating or preventing NGF-mediated diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition described in any one of items 10-12 to a user in need of treatment or prevention.

[0028] 16. The method according to claim 15, wherein the NGF-mediated related disease is selected from one or more of autoimmune arthritis, bone cancer pain, osteoarthritis, and fracture pain.

[0029] 17. Use of the monoclonal antibody according to any one of items 1-6 in the preparation of a medicament for the treatment or prevention of NGF-mediated diseases.

[0030] 18. The use according to item 17, wherein the NGF-mediated related diseases are selected from one or more of autoimmune arthritis, bone cancer pain, osteoarthritis, and fracture pain. Attached Figure Description

[0031] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein:

[0032] Figure 1 The results of ELISA for binding of anti-NGF monoclonal antibody with human NGF-His antigen to a substance that does not share the same epitope as *Gynostemma pentaphyllum*.

[0033] Figure 2 SDS-PAGE diagrams of the heavy and light chains of MB042.

[0034] Figure 3 The results of the claw extension force measurement for different groups are shown.

[0035] Figure 4 This is a schematic diagram of the Dog IgG1 in MB042.

[0036] Figure 5 The results show the binding activity of human MB042 antibody and canine MB042 antibody. Detailed Implementation

[0037] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0038] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0039] The technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art, and in case of any conflict, the definitions in this specification shall prevail.

[0040] Generally speaking, the terms used in this specification have the following meanings.

[0041] In this specification, "monoclonal antibody" means an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to this application can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0042] In this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using common methods known in the art.

[0043] In this specification, "Enzyme-Linked Immunosorbent Assay (EILSA)" refers to a detection method that utilizes the specific binding of antibody molecules to antigen molecules to bind free impurities and the target protein bound to a solid-phase carrier, and then uses special labels for qualitative or quantitative analysis. The principle is as follows: antigens or antibodies can be physically adsorbed onto a solid-phase surface while maintaining their immunological activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds, while maintaining their respective immunological or enzymatic activities; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of an immune reaction can be determined by the color reaction of the added substrate, and the intensity of the color reaction is directly proportional to the amount of the corresponding antigen or antibody in the sample. Various types of detection methods can be designed depending on the substance to be detected and the available detection conditions; the double-antibody sandwich method is the most commonly used method for antigen detection. The process involves adsorbing antiserum containing known antibodies into the wells of a microtiter plate, washing it once, adding the antigen to be tested, and if the two are specific, they will bind. Then, excess antibodies are washed away. An enzyme-linked antibody that specifically reacts with the antigen to be tested is added to form a "sandwich". The substrate of the enzyme is added. If a colored enzymatic digestion product is seen, it indicates the presence of the corresponding antigen.

[0044] In this specification, "avidin" is a glycoprotein, each molecule of which consists of four subunits and can bind tightly to four biotin molecules. Streptavidin, extracted from Streptomyces, is more commonly used.

[0045] In this instruction manual, "biotin" is also referred to as Vitamin H. Biotin-hydroxysuccinimide (BNHS), a chemically produced derivative, can form biotinylated products with various types of molecules, large and small, including proteins, carbohydrates, and enzymes. Although the binding of avidin and biotin is not an immune response, it is highly specific and has a strong affinity, making the combination extremely stable. Since one avidin molecule has four binding sites for biotin molecules, it can connect to more biotinylated molecules, forming a lattice-like complex. Therefore, combining avidin and biotin with ELISA can significantly improve the sensitivity of ELISA.

[0046] The biotin-avidin system has various applications in ELISA, including indirect coating and final reaction amplification. In conventional ELISA, enzyme-labeled antibodies can be replaced with biotinylated antibodies, which are then linked to avidin-enzyme conjugates to amplify the reaction signal.

[0047] In this manual, the term "dog" is used in a broad sense and may encompass various canines, such as domestic dogs, sheepdogs, or domestic dogs.

[0048] In this specification, "a chimeric antibody is an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody may comprise a canine variable region and a mouse constant region."

[0049] In this specification, "canine or feline-derived antibody" refers to an antibody in which at least one amino acid in a portion of a non-canine or feline variable region has been replaced by a corresponding amino acid from a canine or feline variable region. In some embodiments, the canine or feline-derived antibody comprises at least one canine or feline constant region (e.g., γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, the canine or feline-derived antibody is an antibody fragment such as Fab, scFv, F(ab')2, etc. The term "canine or feline-derived" also refers to a non-canine or feline (e.g., mouse) antibody form as a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of an antibody) containing the minimal sequence of a non-canine or feline immunoglobulin. Canine or feline-derived antibodies may include canine or feline immunoglobulins (receptor antibodies) in which residues of a receptor CDR are replaced by residues of a CDR from a non-canine or feline species such as mouse, rat, or rabbit (donor antibody) having the desired specificity, affinity, and capability. In some cases, the Fv framework region (FR) residues of canine or feline immunoglobulins are replaced with corresponding non-canine or feline residues. Furthermore, canine or feline-derived antibodies may contain residues that are not found in the receptor antibody or in the introduced CDR or framework sequence, but are included to further improve and optimize antibody performance.

[0050] In this specification, "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the above-described functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0051] In this specification, "nucleic acid," "polynucleotide," or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically defined, the term may include nucleic acids containing analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid (e.g., sequence information is shown) and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, the sequence of a nucleic acid may include variants modified in a conserved manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0052] In this specification, "identity" may be defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction. The methods and computer programs used for alignment are well known in the art. In this document, reference to an amino acid sequence having a percentage identity with an amino acid sequence means a sequence having said percentage identity across the entire length of the referenced amino acid sequence.

[0053] In this specification, "conjugate" refers to an antibody or its antigen-binding fragment linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, etc. The linking can be covalent or non-covalent, such as by electrostatic forces. Various connectors known in the art can be used to form conjugates. Alternatively, conjugates can be provided as fusion proteins encoding polynucleotide expressions of the conjugate.

[0054] In this specification, the light chain constant region and heavy chain constant region can be any light chain constant region or heavy chain constant region. The light chain constant region (CL) can be κ (kappa) or λ (lambda). The heavy chain constant region can be any heavy chain constant region of IgG, IgM, IgA, IgE, or IgD. It is known to those skilled in the art that each heavy chain variable region can consist of three complementarity-determining regions (CDRs) and four frame regions (FRs), and each light chain variable region can consist of three complementarity-determining regions (CDRs) and four frame regions (FRs). The complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. In some embodiments, from the N-terminus to the C-terminus, both the heavy chain variable region and the light chain variable region include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR sequences of heavy chain variable regions or light chain variable regions can be identified by referring to any numbering system known in the art, such as the Kabat system (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); the Chothia system (Chothia and Lesk, "Canonical Structures for the Hypervariable Regions of Immunoglobulins", J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc et al., "IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig Superfamily V-like Domains"). “Ig superfamily V-like domains”, Developmental Competitive Immunology (Dev. Comp. Immunol.) 27, 55-77 (2003)

[0055] In one specific embodiment of this application, the antibody is a full-length antibody, which typically refers to an antibody composed of two "heavy chains" and two "light chains". The "heavy chain" is usually a polypeptide composed of a heavy chain variable region (VH), a heavy chain constant region 1 (CH1), a hinge region (HR), an antibody heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3; in some embodiments, the "full-length antibody heavy chain" is a polypeptide composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. The "full-length antibody light chain" is usually a polypeptide composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.

[0056] Antibody

[0057] This application discloses an anti-NGF monoclonal antibody, characterized in that the antibody comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, CDR-L3).

[0058] In one specific embodiment of this application, CDR-H1 has the amino acid sequence SEQ ID NO: 7: SYEMN, CDR-H2 has the amino acid sequence SEQ ID NO: 8: YISSSGSTIYYADSVKG, and CDR-H3 has the amino acid sequence SEQ ID NO: 9: VGGINYYDSSGTTGAFDI; CDR-L1 has the amino acid sequence SEQ ID NO: 10: QASQDISDYLN, CDR-L2 has the amino acid sequence SEQ ID NO: 11: DASNLET, and CDR-L3 has the amino acid sequence SEQ ID NO: 12: QQYDNLPIT.

[0059] In one specific embodiment of this application, the antibody comprises a heavy chain variable region, wherein: the heavy chain variable region has a sequence

[0060] SEQ ID NO: 5:

[0061] The amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGL EWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCARVGGINYYDSSGTTGAFDIWGQGTMVTVSS, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% more identical to SEQ ID NO: 5.

[0062] In one specific embodiment of this application, the antibody comprises a light chain variable region, wherein: the light chain variable region has a sequence

[0063] SEQ ID NO: 6:

[0064] The amino acid sequence of DIVLTQSPASLAVSLGQRATISCKASQSVDSDGDSYMNWYQQKPGQ PPKFLLYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNE DPWTFGGGTKLEIK, or an amino acid sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with SEQ ID NO: 6.

[0065] In one specific embodiment of this application, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the same identity as SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the same identity as SEQ ID NO: 6.

[0066] In one specific embodiment of this application, the antibody is a canine or feline-derived antibody or a whole-canine or whole-cat antibody.

[0067] In one specific embodiment of this application, the heavy chain of the antibody further includes a heavy chain constant region, and the light chain further includes a light chain constant region.

[0068] In one specific embodiment of this application, the heavy chain of the antibody has a sequence.

[0069] SEQ ID NO: 3:

[0070] MHSSALLCCLVLLTGVRAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISSRDNAKNSLYLQMNSLRAEDTAVYYCARVGGINY YDSSGTTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE The amino acid sequence of PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% more identical to SEQ ID NO 3.

[0071] In one specific embodiment of this application, the heavy chain of the antibody has a sequence.

[0072] SEQ ID NO: 4:

[0073] The amino acid sequence of MHSSALLCCLVLLTGVRADIRVTQSPSSLSASVGDRVTITCQASQDISDYLNWYQHKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with SEQ ID NO: 4.

[0074] In one specific embodiment of this application, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:3, or is an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the same identity as SEQ ID NO:3, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:4, or is an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the same identity as SEQ ID NO:4.

[0075] Nucleic acid, vector, host cell

[0076] This application provides an isolated nucleic acid comprising a polynucleotide sequence encoding any of the aforementioned antibodies.

[0077] In one specific embodiment of this application, the nucleic acid molecule is an RNA or DNA molecule.

[0078] In one specific embodiment of this application, the nucleic acid is single-stranded or double-stranded.

[0079] In one specific embodiment of this application, the nucleic acid is linear or circular. The nucleic acid can be synthesized by cells or chemically.

[0080] In one specific embodiment of this application, the nucleic acid is a chemically modified nucleic acid molecule.

[0081] This application also provides a vector comprising the aforementioned nucleic acid.

[0082] In one specific embodiment of this application, the vector is a plasmid.

[0083] This application also provides a host cell containing the aforementioned nucleic acid or vector.

[0084] Pharmaceutical compositions, conjugates and treatment methods

[0085] This application provides a pharmaceutical composition comprising any one of the above-mentioned anti-NGF monoclonal antibodies, or antibodies expressed by the above-mentioned nucleic acids, or antibodies prepared by the above-mentioned vectors, or antibodies produced by the above-mentioned host cells, and a pharmaceutically acceptable vector.

[0086] Pharmaceutically acceptable carriers may include non-toxic buffers such as phosphoric acid, citric acid, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzyl chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoates). Parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight peptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants such as Tween or polyethylene glycol (PEG), etc.

[0087] The pharmaceutical composition may be used to treat or prevent NGF-mediated diseases. NGF-mediated diseases may be selected from autoimmune arthritis, bone cancer pain, osteoarthritis, and fracture pain.

[0088] This application also provides a conjugate comprising any one of the above-mentioned anti-NGF monoclonal antibodies, or antibodies expressed by the above-mentioned nucleic acids, or antibodies prepared by the above-mentioned vectors, or antibodies produced by the above-mentioned host cells.

[0089] Furthermore, the conjugate may also contain a drug, cytokine, radionuclide, or enzyme linked to the antibody or its antigen-binding fragment.

[0090] The pharmaceutical composition of this application can be administered alone or in combination with one or more therapeutic agents (or a second therapeutic agent). In the combination administration described herein, the pharmaceutical composition may be administered before, after, or simultaneously with one or more other agents.

[0091] This application also provides the use of the above-described monoclonal antibody in the preparation of medicaments for the treatment or prevention of NGF-mediated diseases.

[0092] Among them, NGF-mediated diseases can be selected from autoimmune arthritis, bone cancer pain, osteoarthritis, and fracture pain.

[0093] Example 1: Screening and preparation of anti-NGF monoclonal antibodies

[0094] Using a fully human natural library as the source, and Bio-Human-NGF-His as the antigen, liquid-phase panning was performed. The output / input ratio indicates that specific enrichment of sequences occurred as panning progressed.

[0095] 1. Using the pre-constructed fully human natural phage library (Scfv) for panning and screening, with Bio-Human NGF-His as the antigen, 616 positive clones were initially obtained through three rounds of liquid phase panning and screening.

[0096] 2. Select the single clones after each panning, shake to the logarithmic phase, add IPTG to a final concentration of 0.5 mM, induce overnight at 30°C, centrifuge the next day to collect the cells, add 0.5 times the volume of TEST lysis buffer, and lyse on ice for 1 hour for later use. Coating antigen: The concentration of Bio-Human NGF-His antigen was 1 μg / mL, and the specific coating volume was 100 μL / well. Incubation was performed overnight at 4°C. Blocking: 5% BSA was used as the blocking buffer, with a volume of 200 μL / well. Blocking was performed at 25°C for 1 h. Sample: Monoclonal lysis buffer was diluted 1:1 with the blocking buffer, and the sample volume was 100 μL / well. Incubation was performed at 25°C for 1 h. Secondary antibody incubation: Anti-flag (HRP) was diluted 1:10000, and the secondary antibody volume was 100 μL / well. Incubation was performed at 25°C for 1 h. Color development: 100 μL of TMB was added to each well, and color development was performed in the dark for 5 min, followed by the addition of 1M HCl. The reaction was terminated with 50 μL / well; reading: the OD450 absorbance was read using a microplate reader, and 35 positive clones with OD450 > 0.5 were obtained; sequencing: the positive clones with OD450 > 0.5 were sent for sequencing, and a total of 28 unique sequences were obtained.

[0097] 3. The 28 unique sequences were amplified by PCR, recovered, and constructed into plasmid expression vectors. They were first transformed into E. coli. After colony PCR identification of positive clones, all positive clones were subjected to liquid amplification culture and low endotoxin plasmids were extracted. All plasmids were electroporated into mammalian cell expression systems, and 22 cell lines expressing monoclonal antibodies were successfully obtained. The cell culture supernatant was purified and subjected to purity and endotoxin detection to obtain 22 monoclonal antibodies.

[0098] Example 2: Affinity activity verification of anti-canine NGF monoclonal antibody

[0099] Affinity ELISA test

[0100] Binding ELISA testing solution

[0101] (1) Coating: Antigen 50385-MNAC, 2μg / mL, 100μL / well, 37℃ for 1h, wash 6 times, and pat dry.

[0102] (2) Blocking: Use self-made blocking solution, 200 μL / well, 25℃ for 1 hour, wash the plate 6 times, and pat dry.

[0103] (3) Antibody binding: 100 nM supernatant, 100 μL / well, three-fold serial dilution, eleven-fold serial dilution, if less than 100 nM, add 100 μL stock solution, 25℃ for 1 h, wash plate 6 times, pat dry.

[0104] (4) Binding of secondary antibody: Anti-human Fc specific HRP, 1:65000 dilution, 100 μL / well, 25℃ for 1 h, wash plate 6 times, pat dry.

[0105] (5) Color development: 100 μL / well color development solution, develop color at room temperature in the dark for 3-5 min.

[0106] (6) Termination: 50 μL / well of termination solution.

[0107] (7) Reading: Read OD450 using an enzyme-linked immunosorbent assay (ELISA) reader.

[0108] The prepared antibodies were subjected to affinity ELISA detection with the antigen proteins. Among the 22 purified antibodies, MNDDBD010 was the purified antibody that bound to Recombinant Mouseβ-NGF / Beat-NGF Protein.

[0109] Example 3: Determination of the equilibrium dissociation constant (KD)

[0110] The MNDDBD010 antibody was tested using the Yangshen Biacore system (1:1 binding mode). The results showed that the affinity constant of the MNDDBD010 antibody reached 10. -10 The order of magnitude, specifically 1.34 × 10 -10 M.

[0111] The simplified detection process of the Biacore system is as follows:

[0112] (1) Reagent preparation

[0113] Run buffer: Dilute 10×HBS-EP+Buffer with ultrapure water to 1×HBS-EP+Buffer, filter through a 0.22μm filter membrane and degas before use.

[0114] (2) Capture ligands

[0115] Antibody at a concentration of 2 μg / mL was injected into the experimental channel at a flow rate of 10 μL / min for 60 s, and the capture volume was approximately 128 RU.

[0116] (3) Multi-cycle analysis of analytes

[0117] The antigen was diluted to 50 nM with running buffer and injected into the experimental and reference channels at a flow rate of 30 μL / min for the corresponding binding and dissociation times. Both binding and dissociation steps were performed in the running buffer. After each concentration analysis, the chip was regenerated with 10 mM pH 1.5 Gly-HCl at a flow rate of 30 μL / min for 30 s to wash away any undissociated analyte. For the next concentration analysis, the experimental channels needed to recapture the same amount of ligand.

[0118] (4) Data Analysis

[0119] The ka, kd, and KD values ​​for each sample were calculated using Biacore 8K analysis software, Biacore Insight Evaluation Software. A reference channel was used for background subtraction.

[0120] Table 1. Affinity of MB042 antibody

[0121] Sample Name <![CDATA[k a (M -1 S -1 )]]> <![CDATA[k d (S -1 )]]> <![CDATA[K D (M)]]> MB042 antibody <![CDATA[1.48×10 6 ]]> <![CDATA[1.98×10 -4 ]]> <![CDATA[1.34×10 -10 ]]>

[0122] The antigen-specific monoclonal antibody numbered MNDDBD010 is named MB042. The affinity ELISA results for MB042 are as follows: Figure 1 As shown.

[0123] The heavy and light chain gene sequences of MB042 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 3; the amino acid sequence of the light chain is shown in SEQ ID NO: 4; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 5; the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 6; the amino acid sequences of the three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and the amino acid sequences of the three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3) are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0124] The purified MB042 was subjected to protein electrophoresis, and the results are as follows: Figure 2As shown in the figure. Lane M represents the molecular weight standard, lane R represents the reduction condition, and lane NR represents the non-reduction condition; the heavy chain size is 50 kDa, the light chain size is 24 kDa, and the total antibody size is 148 kDa.

[0125] SEQ ID NO: 1

[0126]

[0127] SEQ ID NO:2

[0128] GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGATATCAGGGTGACCCAGTCCCCCTCCTCCTTGTCTGCTTCCGTGGGTGACAGGGTGACCATTACCTGCCAGGCTTCCCAGGACATTTCCGACTACTTGAACTGGTACCAGCACAAGCCCGGCAAGGCTCCAAAGCTCCTCATCTACGACGCCTCTAACCTGGAGACAGGCGTGCCTTCCCGCTTTTCCGGTTCCGGTTCCGGCACAGACTTCACCTTCACTATCTCCTCCCTGCAGCCCGAAGATATCGCCACCTACTACTGCCAGCAGTACGACAACCTGCCCATCACCTTCGGCCAGGGTACCAGACTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATTCTAGA

[0129] SEQ ID NO:3

[0130] MHSSALLCCLVLLTGVRAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGGINYYDSSGTTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0131] SEQ ID NO:4

[0132] MHSSALLCCLVLLTGVRADIRVTQSPSSLSASVGDRVTITCQASQDISDYLNWYQHKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0133] SEQ ID NO:5

[0134] QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGGINYYDSSGTTGAFDIWGQGTMVTVSSSEQ

[0135] ID NO:6

[0136] DIRVTQSPSSSLSASVGDRVTITCQASQDISDYLNWYQHKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIK

[0137] SEQ ID NO: 7: SYEMN

[0138] SEQ ID NO: 8: YISSSGSTIYYADSVKG

[0139] SEQ ID NO: 9: VGGINYYDSSGTTGAFDI

[0140] SEQ ID NO: 10: QASQDISDYLN

[0141] SEQ ID NO: 11: DASNLET

[0142] SEQ ID NO: 12: QQYDNLPIT

[0143] Example 4: Pharmacodynamic test of a single subcutaneous administration of MB042 to an iodoacetic acid-induced knee osteoarthritis model in SD rats.

[0144] The purpose of this experiment was to observe the therapeutic effect of MB042 on a sodium iodoacetate-induced knee osteoarthritis model in SD rats by a single subcutaneous administration, and to compare it with the positive control drug PC, so as to provide a reference for subsequent experiments.

[0145] 1. Test Plan

[0146] Twenty-four SD rats were randomly divided into four groups of six each based on their claw retraction pressure: sham-operated group, model control group (PBS), positive control group (PC, 1 mg / kg), and test sample group (MB042, 1 mg / kg). The sham-operated group received no drug, while the other groups received subcutaneous injections. Clinical observation, weighing, and Von Frey tests were performed on the animals in this experiment.

[0147] 2. Test Results

[0148] Regarding clinical observation and body weight, no significant abnormalities were observed in the animals during the experiment.

[0149] Paw retraction pressure: Significant changes in paw retraction pressure were observed in the sham-operated group before and after modeling (P>0.05); the paw retraction pressure in the modeled group was significantly lower after modeling than before (11.10±3.19g vs 28.89±4.70g, P<0.001), indicating a decrease in paw retraction pressure in MIA-induced knee osteoarthritis SD rats, i.e., hyperalgesia. Compared with the model control group, a single subcutaneous injection of 1 mg / kg of the positive control product and the test product MB042 increased the paw retraction pressure in MIA-induced knee osteoarthritis SD rats within 3–7 days. At the same dose, the analgesic duration of the test product MB042 was superior to that of the positive control product. Figure 3 ).

[0150] Example 5: Canine Derivative Design and Plasmid Construction of MB042

[0151] The heavy and light chain sequences of the MB042 antibody were input into an antibody structure simulation and optimization analysis platform to search for homologous proteins in the V region. Results from several databases (such as Kabat, Igblast, and IMGT) were compared to identify two canine germline framework sequences as templates for the MB042 heavy chain (SEQ ID NO: 13) and one canine germline framework sequence as a template for the MB042 light chain (SEQ ID NO: 14). The canine-derived heavy and light chain antibody genes were cloned into plasmids and expressed as follows: Figure 4 The form of Dog IgG1 shown below has its variable region amino acid sequence as follows.

[0152] SEQ ID NO: 13:

[0153] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYEMNWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARVGGINYYDSSGTTGAFDIWGQGTLVTVSS

[0154] SEQ ID NO: 14:

[0155] EIVMTQSPASLSLSQEEKVTITCQASQDISDYLNWYQHKPGQAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDVAVYYCQQYDNLPITFGQGTKLEIK

[0156] Example 6: Affinity Detection of Canine MB042 Antibody

[0157] 1. Surface Plasmon Resonance (SPR) Detection

[0158] The kinetics and affinity properties of antibodies were tested using the Biacore 8K platform. The dissociation equilibrium constant (KD) reflects the affinity of the antibody for the target; a smaller value indicates stronger affinity. KD is determined by the ratio of the dissociation rate constant (Koff) to the association rate constant (Kon), i.e., KD = Koff / Kon. The KD(M) of the human MB042 maternal antibody is 4.99 * 10⁻⁶. -09 The KD(M) of canine-derived MB042 is 2.44 * 10⁻⁶. -09 This indicates that the affinity of the canine antibody is higher than that of the human maternal antibody.

[0159] 2. ELISA test

[0160] Coating antibody, 2 μg / ml, 100 μL / well, incubated at 37℃ for 1 h, then washed 5 times with 0.05% PBST; blocked at 25℃ for 1 h, then washed 5 times with 0.05% PBST; added Bio-Recombinant Mouse Beta-NGF antigen, 2 μg / ml, serially diluted 3-fold for 11 spots, with blank at the end of each well, incubated at 37℃ for 1 h, then washed 5 times with 0.05% PBST; SA-HRP diluted 1:2000, 100 μL / well, incubated at 25℃ for 1 h, then washed 5 times with 0.05% PBST; TMB chromogenic solution, 100 μL / well, incubated at 25℃ in the dark for 3 min, added 50 μL of stop solution to each well, and read OD450 using a microplate reader. Figure 5 As shown, the canine-derived MB042 antibody exhibits comparable binding activity to the maternal MB042 antibody.

Claims

1. An anti-NGF monoclonal antibody, characterized in that, The antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of CDR-H1 is shown as SEQ ID NO: 7, the amino acid sequence of CDR-H2 is shown as SEQ ID NO: 8, and the amino acid sequence of CDR-H3 is shown as SEQ ID NO: 9; the amino acid sequence of CDR-L1 is shown as SEQ ID NO: 10, the amino acid sequence of CDR-L2 is shown as SEQ ID NO: 11, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO:

12.

2. The monoclonal antibody according to claim 1, characterized in that, The antibody comprises an antibody heavy chain variable region HCVR, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 5 or 13.

3. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO: 5 or 13.

4. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 91% or more identity with SEQ ID NO: 5 or 13.

5. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 92% or more identity with SEQ ID NO: 5 or 13.

6. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 93% or more identity with SEQ ID NO: 5 or 13.

7. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 94% or more identity with SEQ ID NO: 5 or 13.

8. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 95% or more identity with SEQ ID NO: 5 or 13.

9. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 96% or more identity with SEQ ID NO: 5 or 13.

10. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 97% or more identity with SEQ ID NO: 5 or 13.

11. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 98% or more identity with SEQ ID NO: 5 or 13.

12. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody heavy chain variable region HCVR, wherein the amino acid sequence of the heavy chain variable region is an amino acid sequence having 99% or more identity with SEQ ID NO: 5 or 13.

13. The monoclonal antibody of claim 1, wherein, The antibody comprises an antibody light chain variable region LCVR, wherein: the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 6 or 14. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6 or 14.

14. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO 6 or 14.

15. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 91% or more identity with SEQ ID NO 6 or 14.

16. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 92% or more identity with SEQ ID NO 6 or 14.

17. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 93% or more identity with SEQ ID NO 6 or 14.

18. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 94% or more identity with SEQ ID NO 6 or 14.

19. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 95% or more identity with SEQ ID NO 6 or 14.

20. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 96% or more identity with SEQ ID NO 6 or 14.

21. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 97% or more identity with SEQ ID NO 6 or 14.

22. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 98% or more identity with SEQ ID NO 6 or 14.

23. The monoclonal antibody of claim 1, wherein, The antibody comprises a light chain variable region LCVR, wherein the amino acid sequence of the light chain variable region is an amino acid sequence having 99% or more identity with SEQ ID NO 6 or 14.

24. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5 or 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6 or 14.

25. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO: 5 or 13; The amino acid sequence of the light chain variable region is an amino acid sequence having 90% or more identity with SEQ ID NO: 6 or 14.

26. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence having 91% or more identity with SEQ ID NO: 5 or 13; The amino acid sequence of the light chain variable region is an amino acid sequence having 91% or more identity with SEQ ID NO: 6 or 14. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

27. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

28. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

29. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

30. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

31. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

32. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

33. The monoclonal antibody of any one of claims 1-23, wherein, The antibody comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein: The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13. The amino acid sequence of the heavy chain variable region is an amino acid sequence that is 99% or more identical to SEQ ID NO: 5 or 13.

34. The monoclonal antibody of any one of claims 1-23, wherein, ​ ​ The amino acid sequence of the light chain variable region is an amino acid sequence having 99% or more identity with SEQ ID NO: 6 or 14.

35. The monoclonal antibody of claim 1, wherein, The antibody comprises a heavy chain and a light chain, wherein: The heavy chain amino acid sequence is shown in SEQ ID NO: 3; The light chain amino acid sequence is shown in SEQ ID NO:

4.

36. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is a caninized or felineized monoclonal antibody.

37. An isolated nucleic acid which is a polynucleotide sequence encoding the monoclonal antibody of any one of claims 1-36.

38. A vector comprising the nucleic acid of claim 37.

39. A host cell comprising the nucleic acid of claim 37 or the vector of claim 38.

40. A pharmaceutical composition, wherein, The pharmaceutical composition comprises: The monoclonal antibody of any one of claims 1-36, or an antibody expressed from the nucleic acid of claim 37, or an antibody produced from the vector of claim 38, or an antibody produced from the host cell of claim 39, and a pharmaceutically acceptable carrier.

41. The pharmaceutical composition of claim 40, wherein, The pharmaceutical composition is used for treating or preventing an NGF-mediated related disease.

42. The pharmaceutical composition of claim 41, wherein, The NGF-mediated related disease is selected from one or more than two of autoimmune arthritis, bone cancer pain, osteoarthritis, and fracture pain.

43. Use of a monoclonal antibody according to any one of claims 1 to 36 for the manufacture of a medicament for the treatment or prevention of a disease associated with NGF, wherein, The NGF-mediated related disease is selected from one or two of bone cancer pain and osteoarthritis.

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