Nerve growth factor specific antibodies and methods of making and using the same

Abstract

The present application relates to a nerve growth factor specific antibody and a preparation method and application thereof. The antibody provided by the present application can specifically recognize and detect a recombinant human nerve growth factor mutant, does not bind to a human nerve growth factor precursor protein and / or a wild-type human nerve growth factor and the precursor protein thereof, and has a good application prospect in the detection of the recombinant human nerve growth factor mutant.

Description

Technical Field

[0001] This invention belongs to the field of biodetection and analysis technology, and relates to an antibody that specifically binds to recombinant human nerve growth factor mutants, its preparation method and a kit containing the antibody, its use in detecting recombinant human nerve growth factor mutants, and a method for detecting the presence or level of recombinant human nerve growth factor mutants. Background Technology

[0002] Nerve growth factor (NGF) nourishes and protects nerve cells (neurons), inhibits neuronal apoptosis, promotes the formation of proper synaptic connections in retinal ganglion cells, and enhances the neuronal's adaptability to pathological environments. Given its nutritional and repairing effects in the nervous system, as well as other functions, NGF has long been extracted or prepared and extensively studied in non-clinical and clinical settings. It has been reported that NGF has shown significant research value in the treatment of ophthalmic diseases such as Alzheimer's disease, diabetic peripheral neuropathy, arthritis, pain, keratitis, dry eye syndrome, and glaucoma.

[0003] The complete NGF is a 7S NGF protein complex containing two α subunits, two β subunits, and one γ subunit. β-NGF alone can exert its biological activity, therefore, the NGF commonly referred to is β-NGF. In the human body, the complete β-NGF exon encodes a 241-amino acid protein, usually called preproNGF. Its first 18 amino acids form a signal peptide, which is cleaved in the endoplasmic reticulum to form the precursor protein proNGF (monomer of 223 amino acids). proNGF exists as a homodimer in the endoplasmic reticulum and then translocates to the Golgi apparatus, where it is cleaved by furin protease to form the mature NGF dimer (monomer of 120 amino acids), which is then transported extracellularly. Simultaneously, some uncleaved proNGF is secreted extracellularly. ProNGF plays a crucial role in the maturation and secretion of NGF and is also known as the molecular chaperone of NGF. Under normal circumstances, proNGF can also exert its biological activity independently and is indispensable in the body. Therefore, both forms of NGF and proNGF exist in the body, and their ratio and levels must be maintained in a certain balance. It has been confirmed that in the central nervous system of humans and rodents, NGF mainly exists in the form of proNGF, while the proportion of NGF is relatively low. The main function of NGF is to promote the growth and repair of neurons, playing a positive regulatory role. However, proNGF, acting alone on its receptor, can promote neuronal apoptosis, playing the opposite role to NGF, thereby maintaining homeostasis.

[0004] In the process of industrial-scale in vitro expression of NGF, it is necessary to first synthesize NGF in the form of proNGF within the host cell, then convert it into mature NGF through enzymatic cleavage before secretion outside the cell. ProNGF byproducts are unavoidable. A more unfavorable condition is that incomplete proNGF cleavage can produce various forms of incomplete splice variants. Immunoblotting assays of NGF supernatant expressed in CHO cells in vitro have also confirmed the presence of proNGF splice variants of different molecular weights, all of which can be recognized by NGF antibodies. These splice variants are similar in properties to mature NGF and often bind to NGF antibodies, thus interfering with ELISA quantitative analysis of NGF. Although SDS-PAGE can assess the expression level in the host cell supernatant, it only provides semi-quantitative results and has a significant margin of error. Quantification of some recombinant NGF protein expression levels uses SEC-HPLC or RP-HPLC methods, but due to the complex composition of the supernatant, various host proteins and multiple proNGF splice variants or product-related proteins with different modifications are difficult to separate from the target protein, leading to significant bias in the results. Meanwhile, the complexity of the supernatant components also leads to a significant reduction in the lifespan of the HPLC column, increasing production costs.

[0005] Furthermore, in non-clinical and clinical studies of NGF-related drugs, it is necessary to analyze the systemic distribution of NGF after administration, typically using ELISA. However, animal NGF shares a high similarity with human NGF, approximately 90%. Commercially available NGF antibodies often cannot distinguish between human and animal NGF, let alone differentiate between exogenously administered NGF and naturally occurring NGF in the human body. Therefore, in both non-clinical and clinical pharmacokinetic analyses, the distribution and metabolism of NGF drugs are often inaccurately quantified due to interference from innate NGF in animals or humans.

[0006] Therefore, there is an urgent need for an antibody that can specifically recognize rhNGF-V2 to avoid interference from proNGF, thereby accurately assessing the expression level of rh-NGFV2 in supernatants containing multiple precursor proteins. Summary of the Invention

[0007] The purpose of this invention is to provide an antibody that specifically binds to recombinant human nerve growth factor mutants. This antibody specifically recognizes rhNGF-V2, is unresponsive to NGF in humans or animals, and shows no cross-reactivity with proNGF. Therefore, an ELISA method can be established to accurately assess the expression level of rh-NGFV2 in supernatant samples containing various proteins. This method also provides an accurate method for evaluating drug metabolism in humans or animals after rhNGF-V2 administration in subsequent non-clinical and clinical studies, and is unaffected by naturally occurring NGF in vivo.

[0008] In a first aspect, the present invention provides an antibody that specifically binds to a recombinant human nerve growth factor mutant, which, according to the Kabat coding scheme, comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 is composed of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO: 2, HCDR3 is composed of the amino acid sequence shown in SEQ ID NO: 3, LCDR1 is composed of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO: 6;

[0009] The amino acid sequence of the recombinant human nerve growth factor mutant is shown in SEQ ID NO: 7.

[0010] The recombinant human nerve growth factor mutant (rhNGF-V2, SEQ ID NO: 7) described in this invention is NGF K34D as described in CN115942951A, the entire contents of which are incorporated herein by reference. This rhNGF-V2 differs from wild-type human NGF by one amino acid. In this context, unless otherwise specified, "recombinant human nerve growth factor mutant" refers to a mature recombinant human nerve growth factor mutant, excluding immature recombinant human nerve growth factor mutants (such as the precursor protein of human nerve growth factor mutants, i.e., proNGF).

[0011] The antibody of the present invention specifically binds to recombinant human nerve growth factor mutants, but does not bind to human nerve growth factor precursor protein and / or wild-type human nerve growth factor and its precursor protein.

[0012] In some embodiments, the antibody of the present invention that specifically binds to recombinant human nerve growth factor mutants comprises at least a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDR and spaced frame regions (FRs), with the domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, or comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, or comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 9. As described herein, the maximum 10% difference in amino acid sequence resulting from "at least 90% sequence identity" can exist in any frame region within the heavy chain variable region or light chain variable region, or in any domain or sequence outside the heavy chain variable region and light chain variable region of the antibody of the present invention. Such differences can be caused by amino acid substitutions, deletions, or insertions at any position.

[0013] In some embodiments, the antibody is any form, including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, bifunctional antibodies, single-domain antibodies, nanobodies, etc. In one specific embodiment, the antibody is a monoclonal antibody.

[0014] In some implementations, the antibody is a full-length antibody.

[0015] In some embodiments, the antibodies of the present invention are intact antibodies, such as IgG1, IgG2, IgG3 or IgG4 antibodies.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10, or comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; the light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, or comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11.

[0017] Secondly, the present invention provides a method for preparing an antibody that specifically binds to a human nerve growth factor mutant, comprising: Peripheral blood was obtained by immunizing animals with recombinant human nerve growth factor mutants; The peripheral blood is sorted to obtain single B cells with antigen-specific memory. The B cells can be fluorescently stained with the recombinant human nerve growth factor mutant but cannot be fluorescently stained with wild-type human nerve growth factor. The B cells are cultured to obtain a supernatant. Antibodies with relatively lower binding affinity to the precursor protein of the recombinant human nerve growth factor mutant and / or the wild-type human nerve growth factor are screened and isolated from the supernatant. These antibodies are the antibodies that specifically bind to the recombinant human nerve growth factor mutant. The amino acid sequence of the recombinant human nerve growth factor mutant is shown in SEQ ID NO: 7.

[0018] The term "relatively lower" refers to the fact that among the multiple antibodies obtained through screening, the binding affinity to the precursor protein of the recombinant human nerve growth factor mutant and / or the wild-type human nerve growth factor is relatively lower.

[0019] The recombinant human nerve growth factor mutant is as described above.

[0020] The antibody that specifically binds to human nerve growth factor mutants as described in the first aspect of the present invention can be prepared by the preparation method described in the second aspect, or by other methods known in the art.

[0021] The antibody prepared by the preparation method described in the second aspect of the present invention specifically binds to the recombinant human nerve growth factor mutant, but does not bind to human nerve growth factor precursor protein and / or wild-type human nerve growth factor and its precursor protein.

[0022] In some implementations, flow cytometry is used to sort the peripheral blood.

[0023] Thirdly, the present invention provides a biomaterial comprising: (i) A nucleic acid molecule encoding the antibody described in this invention; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) A host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell being transformed or transfected by the nucleic acid molecule described in (i) and / or the vector described in (ii).

[0024] In some embodiments, the nucleic acid molecule may be an isolated nucleic acid molecule. In some embodiments, the nucleic acid molecule is DNA, such as cDNA, genomic DNA, or recombinant DNA. In other embodiments, the nucleic acid molecule is RNA, such as mRNA or hnRNA.

[0025] The nucleic acid molecules of the present invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, the present invention also provides a vector comprising the nucleic acid molecules described in (i). In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector.

[0026] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells for purposes such as preservation or antibody expression. Therefore, the present invention also provides host cells comprising the vector described in (ii) or the nucleic acid molecule described in (i), or said host cells being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells. In some embodiments, the host cell is other cells suitable for preparing antibodies or their antigen-binding fragments.

[0027] The antibodies provided by this invention can be obtained using any method known in the art. For example, the heavy chain variable region and / or light chain variable region of the antibody can be obtained first from the nucleic acid molecule provided by this invention, or the heavy chain and / or light chain of the antibody can be obtained, and then assembled with optional other structural domains of the antibody to form an antibody. Alternatively, in a fourth aspect, this invention provides a method for preparing the antibodies described herein, the method comprising culturing the host cells described herein under conditions suitable for the expression of the antibody.

[0028] In some embodiments, the method further includes: recovering the antibody from the host cell or culture medium.

[0029] The antibodies, nucleic acid molecules, vectors, and / or host cells provided by this invention can be included in compositions, and more particularly in formulations, for use in various purposes as needed. Therefore, in a fifth aspect, this invention provides a composition comprising the antibodies of this invention or the biological materials of this invention (including nucleic acid molecules, vectors, and host cells).

[0030] In a sixth aspect, the present invention provides a kit comprising the antibody described herein.

[0031] In some embodiments, the kit further comprises a detection antibody for binding to the antigen (i.e., the recombinant human nerve growth factor mutant); preferably, the detection antibody is a nerve growth factor antibody. The nerve growth factor detection antibody of this invention can be any pairable nerve growth factor antibody known in the art, including human nerve growth factor antibodies, such as Sinocare's wild-type NGF antibody (catalog number 11050-R815), or mouse nerve growth factor antibodies, rabbit nerve growth factor antibodies, etc., and may also include other nerve growth factor mutant antibodies, as long as they can bind to the antibody of this invention.

[0032] In some embodiments, the kit further comprises at least one of a 96-well plate, a coating solution (e.g., PBS), a blocking solution, a chromogenic solution (e.g., TMB), a stop solution (e.g., 2M H2SO4), and a buffer (e.g., a washing buffer, such as PBST), wherein the coating solution, blocking solution, chromogenic solution, stop solution, and buffer are all common reagents in the art.

[0033] In some embodiments, the kit is an ELISA detection kit. ELISA detection methods known in the art can include double-antibody sandwich methods, indirect methods, and competitive methods. Therefore, the kit provided by this invention can adjust its components according to the specific ELISA detection type used.

[0034] In a seventh aspect, the present invention provides the use of the antibody, the kit, or the composition described herein in the detection of recombinant human nerve growth factor mutants, including but not limited to quantitative analysis of recombinant human nerve growth factor mutants, assessment of the expression level of recombinant human nerve growth factor mutants (e.g., expression level in in vitro cell supernatants), differentiation between natural NGF and recombinant human nerve growth factor mutants in vivo, and drug metabolism analysis of recombinant human nerve growth factor mutants.

[0035] Eighthly, the present invention provides a method for detecting the presence or level (e.g., expression level) of a recombinant human nerve growth factor mutant, comprising: Provide the sample to be tested; The antibody, the composition, and / or the antibody in the kit described in this invention are brought into contact with the sample to be tested, and it is detected whether the antibody forms a complex with the recombinant human nerve growth factor mutant in the sample to be tested; The amino acid sequence of the recombinant human nerve growth factor mutant is shown in SEQ ID NO: 7.

[0036] In some implementations, the sample to be tested includes cell culture supernatant, non-clinical / clinical plasma or serum, urine or tissue and tissue extracts, as well as other solutions or tissues containing rhNGF-V2.

[0037] In some implementations, the detection method is the ELISA method, the specific types of which are described above.

[0038] Compared with existing technologies, the antibody provided by this invention can specifically bind to mature recombinant human nerve growth factor mutants without binding to or recognizing wild-type human NGF and / or human NGF precursor protein (proNGF, including wild-type and mutant types). Therefore, when detecting the presence or level of recombinant human nerve growth factor mutants, interference from wild-type NGF and proNGF can be avoided, accurately assessing the presence or level of recombinant human nerve growth factor mutants. This solves the problems of low affinity between wild-type NGF antibodies and recombinant human nerve growth factor mutants and large deviations in in vitro recombinant human nerve growth factor mutant expression analysis. Simultaneously, the antibody provided by this invention can be used to develop non-clinical and clinical pharmacokinetic study methods, specifically analyzing exogenously administered recombinant human nerve growth factor mutants, thereby avoiding interference from in vivo natural NGF on the analytical results, improving the accuracy of detection and analysis, and possessing significant clinical analytical value. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the expression vector (heavy chain) structure of the specific antibody of this invention.

[0040] Figure 2 This is a schematic diagram of the expression vector (light chain) structure of the specific antibody of this invention.

[0041] Figure 3This is the result of Western blotting analysis of wild-type human NGF protein fermentation supernatant and purified samples (lanes 1 and 3), rhNGF-V2 protein fermentation supernatant and purified samples (lanes 2 and 4), and wild-type human proNGF (lane 5) and mouse NGF (lane 6) using commercially available NGF as the primary antibody. Solid boxes contain wild-type proNGF or mutant proNGF (the proNGF corresponding to rhNGF-V2) and / or NGF dimers, while dashed boxes contain wild-type NGF or mutant NGF (i.e., rhNGF-V2).

[0042] Figure 4 The primary antibody used in this invention, WM5-1-B0051, was analyzed by Western blotting. The sample and... Figure 3 Same. Only rhNGF-V2 dimer showed a positive signal within the solid box, while only rhNGF-V2 showed a positive signal within the dashed box.

[0043] Figure 5 This is the result of SDS-PAGE analysis of rhNGF-V2 expression in CHO cells. Lanes 1-4: fermentation supernatants of different rhNGF-V2 concentrations; Lanes 5-6: reference samples of 0.3 and 0.4 mg / mL rhNGF-V2, respectively.

[0044] Figure 6 This is a standard curve for ELISA analysis of CHO rhNGF-V2 expression levels using the specific antibody of this invention.

[0045] Figure 7 The results are SDS-PAGE analysis of rhNGF-V2 expression in CHO cells. Lanes 1-3: rhNGF-V2 reference values ​​are 0.3, 0.4, and 0.5 mg / mL, respectively; Lane 4: rhNGF-V2 fermentation supernatant. Detailed Implementation

[0046] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the invention will be practiced using conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of this art. For the purposes of this invention, the following terms are defined below.

[0047] Abbreviations and Definitions Unless otherwise stated, the following terms shall have the meanings described below. Other terms or abbreviations shall have meanings known in the art.

[0048] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0049] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps.

[0050] The term "antibody" refers to any form of antibody that exhibits desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting receptor signal transduction induced by the ligand). Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0051] The term "immunoglobulin" refers to a protein with a structure that contains naturally occurring antibodies, and is generally used interchangeably with the term "antibody" in this application. IgG immunoglobulins are heterotetrameric glycoproteins composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). In an IgG molecule, the VH-CH1 of the heavy chain typically pairs with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin essentially consists of two Fab molecules linked by an immunoglobulin hinge region and two dimerized Fc regions. Immunoglobulin heavy chains can be classified into one of five categories based on the type of their constant domains, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some of these categories can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ.

[0052] Those skilled in the art will understand that antibody heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000 Daltons. These four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region. Antibodies in the form of IgG1 are a subclass of IgG, with their heavy chain being the γ1 subtype. In some embodiments, the antibody disclosed in this invention is rabbit IgG.

[0053] The term "variable region" or "variable domain" of an antibody refers to the domain of the antibody's heavy or light chain involved in the binding of the antibody to the antigen. The variable region of an antibody can be further divided into hypervariable regions (i.e., complementarity-determining regions (CDRs)) and more conserved regions interspersed between the hypervariable regions (i.e., frame regions (FRs)). In the case of IgG immunoglobulins, the heavy or light chain variable regions, from the N-terminus to the C-terminus, consist of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively. In the case of heavy chain antibodies (also referred to herein as nanobodies), such as those derived from camelid heavy chains, the antigen-binding site consists of a single VH domain (i.e., the "VHH" domain). The VHH of natural heavy chain antibodies has a similar structure to the heavy chain variable region of natural IgG antibodies, containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs).

[0054] The "complementarity-determining region" (CDR), also known as the "hypervariant region," is a region within the antibody's variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. CDRs within the variable domain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.

[0055] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0056] "Affinity" or "binding affinity" refers to the intrinsic binding capacity that reflects the interaction between members of a binding pair. The affinity of molecule X for its binding partner Y can be expressed by the equilibrium dissociation constant (K). D This indicates that the equilibrium dissociation constant is the dissociation rate constant and the binding rate constant (k, k, k) respectively. dis and k on The ratio of binding affinity to the number of molecules. Binding affinity can be measured by common methods known in the art.

[0057] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of a single nucleotide: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. Nucleic acid molecules can be natural or synthetic nucleic acid molecules, or a combination of one or more natural nucleic acid molecules with one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0058] "Isolated nucleic acid molecules" are nucleic acid molecules that have been identified and separated from at least one contaminating nucleic acid molecule. Isolated nucleic acid molecules differ from their naturally occurring form or environment. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in their natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express antibodies, for example, where the chromosomal location of the nucleic acid molecule differs from its chromosomal location in natural cells.

[0059] "Monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, the individual antibodies constituting the group being identical. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically include multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen.

[0060] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from one source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0061] The antibodies of the present invention may contain amino acid mutations and / or conserved modifications. The antibodies of the present invention also include sequences having more than 90% sequence identity with the specifically provided amino acid sequence. Furthermore, the antibodies of the present invention also include variants thereof.

[0062] Amino acids are organic compounds that contain both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in their precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0063] Amino acid mutations can be amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, an amino acid mutation is the substitution of one or more amino acids, such as a single amino acid substitution or a combination of multiple amino acid substitutions. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. The amino acid substitutions of the present invention optionally include conservative substitutions of amino acids.

[0064] "Variations of conserved substitutions" or "conserved amino acid substitutions" refer to amino acid substitutions known to those skilled in the art that such substitutions generally do not alter the biological activity of the resulting molecule. Generally, it is generally accepted by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity. Conserved substitutions can be made by amino acid substitutions with chemically similar side chains, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0065] The term "identity" as used in this article can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When the positions in the compared sequences are occupied by the same bases or amino acids, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Sequence identity" of a polynucleotide or amino acid sequence with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0066] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein (e.g., a recombinant human nerve growth factor mutant) in a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0067] When applied to polynucleotides, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce mRNA containing a polypeptide and / or fragments thereof, is called "encoding" a polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0068] The term "composition" refers to a composition that exists in a form that allows for the effective biological activity of the active ingredient contained therein, and comprises one or more of the following: carrier, diluent, adjuvant, excipient, preservative, filler, disintegrant, wetting agent, emulsifier, suspending agent, sweetener, flavoring agent, fragrance, antimicrobial agent, antifungal agent, lubricant, dispersant, thermosensitive material, temperature regulator, adhesive, stabilizer, suspending agent, etc.

[0069] Biomaterials (nucleic acid molecules, carriers, and host cells) This invention provides nucleic acid molecules encoding any of the above-described antibody molecules. The polynucleotide sequences encoding the antibody molecules of this invention can be generated using methods well-known in the art, either through de novo solid-phase DNA synthesis or through genetic engineering methods. Furthermore, the polynucleotides and nucleic acids of this invention can include segments encoding secretion signal peptides and are operatively linked to segments encoding the antibody molecules of this invention, thereby guiding the secretory expression of the antibody molecules of this invention.

[0070] This invention also provides vectors comprising the nucleic acid molecules of this invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector and a prokaryotic expression vector. "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, including viscera, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0071] This invention also provides prokaryotic and eukaryotic host cells comprising the nucleic acid molecule or the vector. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells, and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues. Suitable host cells for replicating and supporting the expression of the antibody molecules of this invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of antibody molecules.

[0072] Composition The present invention provides compositions comprising the antibody molecules of the present invention.

[0073] In one embodiment, the composition of the present invention further comprises pharmaceutical excipients, such as carriers and excipients known in the art.

[0074] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used in conjunction with an active ingredient for therapeutic purposes. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.

[0075] Preparation of the antibody of the present invention In one embodiment, the present invention provides a method for preparing an antibody that specifically binds to a recombinant human nerve growth factor mutant, wherein the method comprises: Peripheral blood was obtained by immunizing animals with recombinant human nerve growth factor mutants; The peripheral blood is sorted to isolate individual B cells with antigen-specific memory. These B cells can be fluorescently stained with the recombinant human nerve growth factor mutant but cannot be fluorescently stained with wild-type human nerve growth factor. Therefore, individual B cells expressing specific antibodies are obtained. Theoretically, the antibodies secreted by these individual B cells only recognize rhNGF-V2 and cannot recognize human wild-type NGF. The B cells were cultured to obtain supernatant, and antibodies with relatively low affinity to wild-type human NGF or proNGF were screened again, which are the antibodies that specifically bind to the recombinant human nerve growth factor mutant. The amino acid sequence of the recombinant human nerve growth factor mutant is shown in SEQ ID NO: 7.

[0076] In addition, the antibodies of the present invention can be prepared by other methods, such as culturing host cells containing nucleic acids encoding the antibody or expression vectors containing the nucleic acids under conditions suitable for expressing nucleic acids encoding the antibody, and optionally isolating the antibody. In one embodiment, the method further includes recovering the antibody from the host cells (or host cell culture medium).

[0077] To recombinantly generate the antibodies of the present invention, the nucleic acid encoding the antibodies of the present invention is first isolated, and said nucleic acid is inserted into a vector for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using routine procedures, for example, by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the antibodies of the present invention.

[0078] The antibodies of the present invention prepared as described herein can be purified using known prior art techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0079] Example The following embodiments further illustrate the present invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art.

[0080] Unless otherwise expressly stated, the present invention will be practiced using conventional chemical, biochemical, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology methods in the art. Unless otherwise specified, the experimental methods described herein are conventional methods using default parameters and procedures; the experimental materials used are commercially available products. Where specific techniques or conditions are not specified in the examples, they shall be performed according to the techniques or conditions described in the literature in the art, or according to the corresponding product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0081] Example 1: Preparation of specific antibodies Antibody preparation was performed using animal immunization. B cells expressing specific antibodies were sorted by flow cytometry, further identified and screened by ELISA, sequenced, and then recombinantly expressed to obtain antibody samples for pairing. The specific preparation method is as follows: (1) Animal immunization: Rabbits were immunized with rhNGF-V2 protein (SEQ ID NO: 7) or rhNGF-V2 polypeptide (SEQ ID NO: 12) conjugated with human papillomavirus VLP from Yiqiao Shenzhou after Freund's complete adjuvant emulsification. Booster immunization was performed every two weeks for three to five rounds. Blood was collected to obtain serum, and the titer was measured by ELISA. The method for detecting serum titer by ELISA was as follows: 96-well plates were coated with different antigens (including wild-type NGF protein (SEQ ID NO: 13), rhNGF-V2 protein or commercially available proNGF protein (Yiqiao wild-type human proNGF)), and then the rabbit serum samples collected after immunization were added and incubated. Then, the samples were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit detection antibody (Yiqiao HRP-goat anti-rabbit IgG antibody, catalog number 111-035-003). Finally, the expression level and affinity of the antibody in the culture supernatant were indirectly analyzed by the strength of the HRP signal. Animal serum with high initial titers will be used for further testing.

[0082] (2) Flow cytometry sorting: Peripheral blood from animals with high titers was sorted by flow cytometry. APC fluorescently labeled rhNGF-V2 peptide (SEQ ID NO: 12, in which an amidated cysteine ​​C was added to the C-terminus for coupling, and the N-terminal amino acid was acetylated) and PE labeled wild-type NGF peptide (SEQ ID NO: 14, in which an amidated cysteine ​​C was added to the C-terminus and the N-terminal amino acid was acetylated) were mixed with peripheral blood cells, and single B cells that could be stained by APC but not by PE were sorted to obtain antigen-specific memory B cells.

[0083] (3) Supernatant identification and screening: The antigen-specific B cells selected by flow cytometry were cultured, and the culture supernatant was used for ELISA expression identification. Antibodies with poor ELISA reaction signals with wild-type NGF, poor or weak reaction signals with wild-type human proNGF, and relatively strong reaction signals with rhNGF-V2 were selected for sequence identification.

[0084] (4) Sequencing, expression and purification: The B cells identified and screened above were lysed and reverse transcribed to obtain the light chain and heavy chain variable regions of the antibody, and an expression vector was constructed. Figure 1 and Figure 2 The antibodies were then transfected into 293 cells for recombinant expression to obtain purified antibody samples. The titer was then detected again using the same ELISA method. The results are shown in Table 1. Several antibodies with good specificity were screened: WM5-1-B001, WM5-1-B0023, WM5-1-B0035, WM5-1-B0051, and WM5-1-B0052, which can basically not distinguish between wild-type human proNGF and wild-type NGF.

[0085] Table 1. Titer and specificity of purified antibody samples screened by ELISA analysis

[0086] Note: All OD values ​​in the table are after deducting Blank.

[0087] (5) Antibody pairing and preparation: Antibodies with relatively good specificity to wild-type NGF were selected as coating antibodies and paired with other prepared antibodies and commercial NGF antibodies. Pairable antibodies were screened out. Among them, the specific antibody WM5-1-B0051 can be paired with the Sinocare NGF antibody (catalog number 11050-R815), and has good specificity to both wild-type human proNGF and wild-type NGF.

[0088] The specific antibody WM5-1-B0051 has the following amino acid sequences: HCDR1 is composed of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO: 2, HCDR3 is composed of the amino acid sequence shown in SEQ ID NO: 3, LCDR1 is composed of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO: 5, LCDR3 is composed of the amino acid sequence shown in SEQ ID NO: 6, the heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO: 8, the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO: 9, the heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 10, and the light chain is composed of the amino acid sequence shown in SEQ ID NO: 11.

[0089] Example 2: Antibody Specificity Analysis The screened antibody WM5-1-B0051 was subjected to Western blotting for specificity analysis. Non-reducing SDS-PAGE analysis was performed using the supernatant of rhNGF-V2 protein expressed in CHO cells, the supernatant of wild-type NGF protein expressed in 293 cells, purified rhNGF-V2 protein and wild-type NGF protein, as well as wild-type human proNGF (catalog number 11050-HNAH) and mouse NGF (catalog number 50385-MNAC) from Yiqiao. Western blotting was then performed on both antibodies. The primary antibodies used in the Western blotting analysis were a commercially available NGF antibody (Yiqiao, catalog number 11050-T16) and the WM5-1-B0051 antibody of this invention. The secondary antibodies were both HRP-labeled goat anti-rabbit antibodies (Beyotime, catalog number A0208).

[0090] Depend on Figure 3It can be seen that, except for proNGF which only showed a positive signal at 20-25 kDa (samples without NGF), all other samples showed positive signals at both 10-15 kDa and 20-25 kDa. Therefore, commercially available NGF antibodies can indiscriminately recognize wild-type human NGF and rhNGF-V2 mutants, as well as wild-type proNGF, mutant proNGF (the proNGF corresponding to rhNGF-V2), and / or NGF dimers, and can also recognize murine NGF.

[0091] Depend on Figure 4 It can be seen that the antibody WM5-1-B0051 prepared in this invention only showed positive signals in lanes 2 and 4 containing rhNGF-V2 protein, mainly at 10-15 kDa. In addition, there were also positive signals at 20-25 kDa. N-terminal sequencing of the 20-25 kDa band in the purified rhNGF-V2 sample confirmed that its N-terminal sequence was consistent with NGF and did not contain proNGF (proNGF forms NGF after cleaving some amino acids at the N-terminus), suggesting it is an NGF dimer. Therefore, the antibody of this invention only recognizes rhNGF-V2, including the purified rhNGF-V2 protein and the rhNGF-V2 protein in the culture supernatant containing complex proteins, and cannot recognize wild-type NGF; nor can it recognize wild-type or the mutant proNGF, and it also does not respond to mouse NGF.

[0092] In summary, the screened WM5-1-B0051 antibody exhibits good specificity, recognizing only rhNGF-V2 and unaffected by proNGF, wild-type NGF, or other species-specific NGFs. This antibody can be used for the analysis of rhNGF-V2 expression levels in rhNGF-V2 supernatant without being affected by human proNGF; it can also be used for the development of analytical methods for non-clinical and clinical assessment of rhNGF-V2 distribution and metabolism after administration, without cross-reactivity with naturally occurring wild-type NGF in humans or animals.

[0093] Comparative Example: Analysis of CHO rhNGF-V2 Expression Levels Using Commercially Available ELISA Kits Initial screening of imported (e.g., Invitrogen, Abcam) and domestic (e.g., Sinocare, Dayou) commercially available ELISA kits revealed significantly low detection results, suggesting that the antibody pairs in these kits were prepared using wild-type NGF, resulting in weak binding affinity to rhNGF-V2. Subsequently, commercially available antibody pairs with better affinity for rhNGF-V2 were screened, and an ELISA method was established to relatively accurately assess rhNGF-V2 levels. However, this ELISA method often yielded high results when assessing rhNGF-V2 levels in fermentation supernatants. This may be because proNGF in the supernatant can also bind to the antibody pairs, leading to some false positives.

[0094] The ELISA method was as follows: the primary antibody was a mouse monoclonal antibody against stigma (catalog number 11050-MM06), and the secondary antibody was a rabbit polyclonal antibody labeled with HRP against stigma (catalog number 11050-RP02-H). The standard curves for rhNGF-V2, after content calibration, were set at 0, 37.5, 75, 150, 300, 600, and 1200 ng / mL. The results showed that the expression level in the supernatant was approximately 0.6–0.7 mg / mL (Table 2), significantly higher than the 0.3–0.4 mg / mL assessed by SDS-PAGE. The SDS-PAGE results are shown below. Figure 5 The SDS-PAGE method involves mixing standards of different concentrations with supernatants and stock solutions, then separating them by vertical electrophoresis and staining with Coomassie brilliant blue.

[0095] Table 2. Analysis of rhNGF-V2 expression levels in fermentation supernatant

[0096] Example 3: Specific antibody ELISA analysis of CHO rhNGF-V2 expression level The antibody WM5-1-B0051 of this invention was used as the coating antibody, and the rhNGF antibody (catalog number 11050-R815, HRP-labeled) was used as the detection antibody. The rhNGF-V2 standard curves were set at 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ng / ml. ELISA analysis was performed, and the results are shown in Table 3. The standard curves are shown in […]. Figure 6 .

[0097] Table 3. Expression level analysis of fermentation supernatant ELISA

[0098] ELISA showed that the expression level of rhNGF-V2 in the expression supernatant of CHO cells was 0.300 mg / mL, which was consistent with the result of 0.311 mg / mL obtained by non-reducing SDS-PAGE. Figure 7 Therefore, the antibody that specifically binds to human nerve growth factor mutants provided by this invention can accurately detect rhNGF-V2 and is not affected by human nerve growth factor precursor proteins and wild-type human nerve growth factor and its precursor proteins.

[0099] Sequence information: HCDR1 (SEQ ID NO: 1): GFSLSSYA HCDR2 (SEQ ID NO: 2): ISPGGRT HCDR3 (SEQ ID NO: 3): ARDGGNPDYNYFNL LCDR1 (SEQ ID NO: 4): QSISNA LCDR2 (SEQ ID NO: 5): AAS LCDR3 (SEQ ID NO: 6): QCTTHISDGNA Recombinant human nerve growth factor mutant protein (SEQ ID NO: 7): SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKG D EVMVLGEVNINSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA Heavy chain variable region (SEQ ID NO: 8): QSVKESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGEGLEYIGWISPPGGRTYYATWAKGRFTISKTSTTVGLKITSPTTEDTATYFCARDGGNPDYNYFNLWGPGTLVTVSS Light chain variable region (SEQ ID NO: 9): AQVTTQTPASVEAAVGGTVTIACQASQSISNALAWYQQKPGQPPKLLIYAASNLASGVSSRFKGSRSGTEHTLTISDLDCADAATYYCQCTTHISDGNAFGGGTEVLVK Heavy chain (SEQ ID NO: 10): QSVKESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGEGLEYIGWISPGGRTYYATWAKGRFTISKTSTTVGLKITSPTTEDTATYFCARDGGNPDYNYFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK Light chain (SEQ ID NO: 11): AQVTTQTPASVEAAVGGTVTIACQASQSISNALAWYQQKPGQPPKLLIYAASNLASGVSSRFKGSRSGTEHTLTISDLDCADAATYYCQCTTHISDGNAFGGGTEVLVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC NGF mutant (rhNGF-V2) polypeptide (SEQ ID NO: 12): KTTATDIKGDEVMVLC (with C added at the end for conjugation) Wild-type NGF protein (SEQ ID NO: 13): SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA Wild-type NGF polypeptide (SEQ ID NO: 14): KTTATDIKGKEVMVLC (Add C at the end for coupling)

Claims

1. An antibody that specifically binds to a recombinant human nerve growth factor mutant, comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:1, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:2, HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:3, LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:4, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:6; The amino acid sequence of the recombinant human nerve growth factor mutant is shown in SEQ ID NO:

7.

2. The antibody according to claim 1, wherein, The antibody does not bind to the precursor protein of human nerve growth factor mutants and / or wild-type human nerve growth factor and its precursor protein.

3. The antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, or comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, or comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

9.

4. The antibody according to any one of claims 1-3, wherein, The antibody is a monoclonal antibody, polyclonal antibody, multispecific antibody, single-chain antibody, bifunctional antibody, single-domain antibody, or nanobody.

5. The antibody according to claim 4, comprising a heavy chain and a light chain, wherein, The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, or comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, or comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

11.

6. A method for preparing an antibody that specifically binds to a human nerve growth factor mutant, comprising: Peripheral blood was obtained by immunizing animals with recombinant human nerve growth factor mutants; The peripheral blood is sorted to obtain single B cells with antigen-specific memory. The B cells can be fluorescently stained with the recombinant human nerve growth factor mutant but cannot be fluorescently stained with wild-type human nerve growth factor. The B cells are cultured to obtain a supernatant. Antibodies with relatively lower binding affinity to the precursor protein of the recombinant human nerve growth factor mutant and / or the wild-type human nerve growth factor are screened and isolated from the supernatant. These antibodies are the antibodies that specifically bind to the recombinant human nerve growth factor mutant. The amino acid sequence of the recombinant human nerve growth factor mutant is shown in SEQ ID NO:

7.

7. A biomaterial comprising: (i) A nucleic acid molecule encoding the antibody as described in any one of claims 1-5 or the antibody prepared by the method described in claim 6; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) A host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell being transformed or transfected by the nucleic acid molecule described in (i) and / or the vector described in (ii).

8. A kit comprising the antibody according to any one of claims 1-5 or the antibody prepared by the preparation method of claim 6; preferably, the kit further comprises a detection antibody; more preferably, the detection antibody is a nerve growth factor antibody; preferably, the kit further comprises at least one of a 96-well plate, a coating solution, a blocking solution, a chromogenic solution, a stop solution, and a buffer; more preferably, the kit is an ELISA detection kit.

9. Use of the antibody according to any one of claims 1-5, the antibody prepared by the preparation method of claim 6, and / or the kit according to claim 8 in the detection of recombinant human nerve growth factor mutants.

10. A method for detecting the presence or level of a recombinant human nerve growth factor mutant, comprising: Provide the sample to be tested; The antibody according to any one of claims 1-5, the antibody prepared by the preparation method of claim 6, and / or the antibody in the kit of claim 8 are brought into contact with the sample to be tested, and it is detected whether the antibody forms a complex with the recombinant human nerve growth factor mutant in the sample to be tested; The amino acid sequence of the recombinant human nerve growth factor mutant is shown in SEQ ID NO: 7; Preferably, the detection method is the ELISA method; Preferably, the sample to be tested includes cell culture supernatant, non-clinical / clinical plasma / serum, urine or tissue and tissue extract, as well as other solutions or tissues containing rhNGF-V2.

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