Antibody specifically binding to Proinsulin and application thereof

Abstract

Amino acid sequences of LCDR1-3 of a light chain variable region VL of the antibody or the antigen binding fragment of the antibody are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and amino acid sequences of HCDR1-3 of a heavy chain variable region VH of the antibody or the antigen binding fragment of the antibody are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The antibody or the antigen fragment thereof disclosed by the invention can be used for accurately detecting the content of Proinsulin in a sample in a high-sensitivity and high-specificity manner and is used for diagnosing, preventing or treating diseases related to Proinsulin abnormality.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and specifically relates to an antibody that specifically binds to Proinsulin and its applications. Background Art

[0002] Proinsulin is a precursor protein of Insulin and C-peptide, and is a protein crucial in glucose metabolism. Proinsulin is synthesized in pancreatic islet β cells and processed into active mature insulin in the endoplasmic reticulum. The active mature insulin is then stored in secretory granules. An increase in blood glucose levels causes the release of active mature insulin from secretory granules into the blood. The level of proinsulin in the blood increases with age, type II diabetes, and β-cell regulatory disorders. In patients with type 1 diabetes or pancreatitis, the islets are damaged by immune attacks, and proinsulin is released from the broken islet β cells into the blood. Since insulin and C-peptide also continuously exist in large amounts in the blood of healthy people, the preparation of antibodies that specifically detect the level of free proinsulin protein in the blood and detection reagents based on such antibodies can help doctors clinically diagnose the health status of patients' islets.

[0003] Therefore, designing antibody molecules that can target and bind to Proinsulin with high affinity, good stability, and strong specificity helps to more effectively detect the expression level of Proinsulin in blood and tissues, and also helps to accurately image or administer drugs to Proinsulin-highly expressed lesions, which is of great significance for the diagnosis, treatment, and scientific research of various diseases such as diabetes and pancreatitis. Summary of the Invention

[0004] The objective of the present invention is to provide an antibody that specifically binds to Proinsulin and its applications, so as to achieve high-affinity, high-sensitivity, and high-specificity detection of Proinsulin.

[0005] To this end, the present invention provides the following technical solutions.

[0006] The first aspect of the present invention provides an antibody that specifically binds to Proinsulin or an antigen-binding fragment thereof, wherein the amino acid sequences of LCDR1-3 of the light chain variable region VL of the antibody or the antigen-binding fragment thereof are respectively as shown in SEQ ID NO: 1, 2, and 3, and the amino acid sequences of HCDR1-3 of the heavy chain variable region VH of the antibody or the antigen-binding fragment thereof are respectively as shown in SEQ ID NO: 4, 5, and 6.

[0007] In some embodiments, the VL comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7, and the VH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8.

[0008] In some embodiments, the light chain constant region CL of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9, and the heavy chain constant region CH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10.

[0009] In some embodiments, the antigen-binding fragment is selected from diabodies, and the antibody is a humanized antibody.

[0010] In some embodiments, the light chain of the antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, and the heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 12.

[0011] The second aspect of the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody or its antigen-binding fragment that specifically binds to Proinsulin as described above.

[0012] The third aspect of the present invention provides a vector comprising the nucleic acid as described above.

[0013] The fourth aspect of the present invention provides a host cell comprising the nucleic acid or vector as described above.

[0014] In some embodiments, the host cell is a eukaryotic cell.

[0015] In some embodiments, the host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

[0016] The fifth aspect of the present invention provides a pharmaceutical composition comprising: (i) an antibody or its antigen-binding fragment as described above; and (ii) a pharmaceutically acceptable carrier or adjuvant.

[0017] The sixth aspect of the present invention provides a method for preparing an antibody or its antigen-binding fragment as described above, comprising culturing the host cell as described above under conditions that allow the expression of the antibody and its antigen-binding fragment.

[0018] The seventh aspect of the present invention provides a detection reagent comprising an antibody or its antigen-binding fragment that specifically binds to Proinsulin as described above.

[0019] In some embodiments, the detection reagent further comprises one or more components of an enzyme-linked immunosorbent assay (ELISA) reagent.

[0020] In some embodiments, the components of the ELISA reagent are selected from a positive control solution, a sample diluent, a negative control solution, an enzyme-labeled reagent, an enzyme substrate solution, a chromogenic reagent, a washing solution, a blocking solution, and / or a termination solution.

[0021] In some embodiments, the detection reagent further comprises one or more components of a chemiluminescent immunoassay.

[0022] In some embodiments, the components of the chemiluminescent immunoassay include a magnetic microsphere solution, a biotinylated antibody solution, a luminescent label, and optionally a Proinsulin calibrator and a Proinsulin quality control product.

[0023] The eighth aspect of the present invention provides the use of an antibody or an antigen-binding fragment thereof that specifically binds to Proinsulin, or a detection reagent as described above, in the preparation of a reagent or product for detecting the content of Proinsulin in a sample, a reagent or product for diagnosing Proinsulin-related diseases, or a drug for preventing and / or treating Proinsulin-related diseases.

[0024] In some embodiments, the Proinsulin-related diseases are selected from: type I diabetes, type II diabetes, pancreatitis, pancreatic cancer.

[0025] The ninth aspect of the present invention provides the application of an antibody or an antigen-binding fragment thereof that specifically binds to Proinsulin, a nucleic acid, a vector, a host cell, a pharmaceutical composition, or a detection reagent as described in the previous item in the preparation of any of the following products: (1) A product for detecting Proinsulin; (2) A product for stimulating or enhancing an immune response; (3) A product for preventing and / or treating Proinsulin-related diseases.

[0026] By virtue of the above technical solutions, the present invention has at least the following advantages: The present invention uses peripheral blood B cells of diabetic patients to screen for recombinant human proinsulin. Combining flow sorting and B cell receptor (BCR) sequencing techniques, after obtaining the BCR evolutionary tree, B cell clones with high occurrence frequencies are selected. After recombinantly expressing human antibodies, ELISA is used to detect their specificity and titer for proinsulin. At the same time, by using this antibody to detect plasma samples of several healthy people and known high levels of proinsulin, the content of proinsulin in plasma can be significantly detected. Thus, the present invention obtains an antibody with high affinity, high sensitivity, and high specificity for binding to Proinsulin. The product of the present invention solves the problems of poor specificity and low sensitivity of the antibody raw materials for Proinsulin detection items in the current market. The antibody disclosed in the present invention has high sensitivity and specificity and can be used as an auxiliary diagnosis for diseases related to abnormal proinsulin metabolism, such as diabetes, pancreatitis, pancreatic cancer, etc.

[0027] The above description is only an overview of the technical solution of the present invention. In order to be able to more clearly understand the technical means of the present invention and implement it in accordance with the content of the specification, the following details the preferred embodiments of the present invention as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a flow sorting diagram of B cells labeled with AF488-Proinsulin in Example 2; Figure 2 It is an SEC-HPLC detection diagram of the purity of the purified antibody GTH01 in Example 5; Figure 3 It is a comparison of detecting the level of Proinsulin in human plasma using the GTH01 antibody of the present invention and a commercial antibody respectively. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] In order to make the technical means, creative features, achieved purposes, and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0030] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0031] As used herein, the terms "first" and "second" are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present disclosure, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0032] As used herein, the term "comprising" or "including" is an open-ended expression, that is, it includes what is specified in the present invention, but does not exclude other aspects.

[0033] As used herein, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may but do not necessarily occur, and the description includes the cases where such events or conditions occur, as well as the cases where such events or conditions do not occur.

[0034] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to Proinsulin, wherein the amino acid sequences of LCDR1-3 of the light chain variable region VL of the antibody or the antigen-binding fragment thereof are shown as SEQ ID NO: 1, 2, and 3 respectively, and the amino acid sequences of HCDR1-3 of the heavy chain variable region VH of the antibody or the antigen-binding fragment thereof are shown as SEQ ID NO: 4, 5, and 6 respectively.

[0035] In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to Proinsulin disclosed herein, the amino acid sequence of VL is as shown in SEQ ID NO: 7 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto, and the amino acid sequence of VH is as shown in SEQ ID NO: 8 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

[0036] In some embodiments of the antibody or antigen-binding fragment thereof that specifically binds to Proinsulin disclosed herein, the amino acid sequence of VL comprises the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region having the sequence shown in SEQ ID NO: 7 or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof; the amino acid sequence of VH comprises the HCDR1, HCDR2, and HCDR3 sequences of the heavy chain variable region having the sequence shown in SEQ ID NO: 8 or having one or more conservative amino acid substitutions, deletions or insertions or any combination thereof.

[0037] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each heavy and light chain. The variable regions of the heavy and light chains of an antibody contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical pathway of complement activation). Thus, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

[0038] As used herein, the term "antibody analog" refers to a derivative produced by biological or chemical methods, which is based on the antibody structure and obtained by deleting, adding, or modifying chemical groups (such as amino acids). These derivatives still contain structures similar to the antibody variable region (or the CDR regions in the antibody variable region) and can undergo reactions similar to antigen-antibody binding through these structures.

[0039] As used herein, the term "bind" or "specifically bind" refers to a non-random binding reaction between two molecules, such as between an antibody and its target antigen. In certain embodiments, an antibody that specifically binds to a particular antigen refers to an antibody that binds to the antigen with an affinity corresponding to a KD of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less. As used herein, "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the KD, the higher the binding affinity between the antibody and the antigen.

[0040] As used herein, the term "antibody variable region" refers to a domain in an antibody heavy chain and light chain, including the variable light chain region (VL) and the variable heavy chain region (VH). In nature, the antibody variable region is encoded by the V, D (only applicable to heavy chains), and J segments in immunoglobulin (heavy chain and light chain) genes through gene recombination splicing. The amino acid sequences of the variable regions among different antibodies are highly diverse (the amino acid sequences of other regions of the antibody are relatively highly identical), and are responsible for the recognition and binding to specific antigenic determinants. In the antibody variable region, both the VL domain and the VH domain contain framework regions (FR) and CDR regions (complementarity determining region) from the amino terminus to the carboxyl terminus. A typical antibody variable region has 3 framework regions and 3 CDR regions, which are interspersed with each other: FR1, CDR1, FR2, CDR2, FR3, and CDR3. The framework region FR mainly plays a role in forming the framework of the protein domain, while the CDR region mainly plays a role in specific antigen-antibody recognition and binding. CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3 respectively herein; CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3 respectively herein.

[0041] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the composite of Chothia Kabat CDR, where CDR-H1 is the composite of Chothia CDR and Kabat CDR; the AbM definition used by the antibody modeling software of Oxford Molecular; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (Kabat numbering system), in which corresponding residues between different heavy chains or different light chains are given the same number. The present disclosure may use CDRs defined according to any one of these numbering systems, but the preferred embodiments use CDRs defined by Kabat or Chothia.

[0042] As used herein, the term "anti-Proinsulin antibody (or antibody analog)" exists in a single (or monoclonal) form, and the single antibody (or antibody analog) contains a single structure (such as a single amino acid sequence).

[0043] As used herein, the term "identity", when used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refers to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional methods. See, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.)). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0044] Without substantially affecting the antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the sequences of the antibody or its functional fragments. They are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention may have at least 80% identity (or homology) with the reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner.

[0045] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid with another residue that is biologically, chemically, or structurally similar. Biologically similar means that the substitution does not disrupt the biological activity of the Proinsulin antibody or the Proinsulin antigen. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine, or methionine are substituted with each other. Or polar amino acids are used, such as lysine is replaced with arginine, aspartic acid is replaced with glutamic acid, asparagine is replaced with glutamine, threonine is replaced with serine, and so on.

[0046] Therefore, the antibodies described in the present invention can also be directly synthesized according to the amino acid sequences of the antibody variable regions provided by the present invention, and can be further chemically modified to generate different antibody analogs.

[0047] In some embodiments of the antibodies or their antigen-binding fragments that specifically bind to Proinsulin disclosed herein, they further comprise a light chain constant region CL and / or a heavy chain constant region CH. The light chain constant region CL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10.

[0048] As used herein, the term "heavy chain constant region" includes amino acid sequences derived from immunoglobulin heavy chains. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure comprises a polypeptide chain containing a CH3 domain. Additionally, an antibody for use in the present disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, one of ordinary skill in the art will understand that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0049] Based on the amino acid sequence of the antibody heavy chain constant region, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and so on. Based on the amino acid sequence of the light chain, the light chain of an antibody can be divided into lambda (λ) chains and kappa (κ) chains. The antibodies disclosed herein can be of any of the above classes or subtypes.

[0050] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind Proinsulin disclosed herein, the antibody has an isotype selected from IgG. In some embodiments, the antibody has a subtype selected from IgG1.

[0051] As used herein, the term "antigen-binding fragment", also known as "antibody fragment", generally refers to an antigen-binding antibody fragment, which may include a part of a complete antibody, usually the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, Fab / c, complementarity-determining regions, single-chain antibodies, diabodies, or single-domain antibody molecules, etc. Among them, the "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab' fragment has one or more cysteine residues at the C-terminus of the CH1 domain of the Fab fragment; the F(ab')2 fragment is a bivalent fragment containing two Fab' fragments linked by a disulfide bond in the hinge region; the Fd fragment has VH and CH1 domains; the Fv fragment has VL and VH domains in a single arm of the antibody; the dAb fragment consists of a VH domain or a VL domain; the isolated CDR regions; and any modified forms of the above fragments that retain antigen-binding activity.

[0052] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to Proinsulin disclosed herein, the antigen-binding fragment is selected from diabodies.

[0053] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to Proinsulin disclosed herein, the antibody is a humanized antibody.

[0054] In some embodiments of the antibodies or antigen-binding fragments thereof that specifically bind to Proinsulin disclosed herein, the antibody is a humanized antibody, which further comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 11, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 12.

[0055] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, each antibody constituting the population is the same except for a small number of mutations that may occur naturally. Monoclonal antibodies are highly specific and directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by the hybridoma technique and should not be construed as requiring antibodies produced by any particular method.

[0056] As used herein, the term "humanized antibody" refers to an antibody that is re-expressed by genetically cloning and DNA recombination techniques from a murine monoclonal antibody, with most of its amino acid sequence replaced by human sequences, essentially retaining the affinity and specificity of the parental murine monoclonal antibody while reducing its heterogenicity, which is beneficial for application in the human body. The constant region portion of such an antibody (i.e., the CH and CL regions) or the entire antibody is encoded by human antibody genes. This can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Humanized antibodies include chimeric antibodies, reshaped antibodies, and fully humanized antibodies, etc. Chimeric antibodies are prepared using DNA recombination techniques by inserting the light and heavy chain variable region genes of a heterologous monoclonal antibody into an expression vector containing human antibody constant regions, and then transforming mammalian cells to express the chimeric antibody. In the antibody molecule expressed in this way, the V regions of the light and heavy chains are heterologous, while the C regions are human-derived. Therefore, nearly two-thirds of the entire antibody molecule is human-derived. The resulting antibody reduces the immunogenicity of the heterologous antibody while retaining the ability of the parental antibody to specifically bind to the antigen. Reshaped antibodies, also known as CDR-grafted antibodies, have CDRs in the variable region of the antibody, which are the regions for antibody recognition and binding to antigens and directly determine the specificity of the antibody. The CDRs of a murine monoclonal antibody are transplanted into the variable region of a human antibody to replace the human CDR, enabling the human antibody to acquire the antigen-binding specificity of the murine monoclonal antibody while reducing its heterogenicity. Fully humanized antibodies refer to antibodies obtained by transferring all human antibody-encoding genes to antibody-gene-deleted animals genetically engineered through transgenic or chromosome transfer techniques, enabling the animals to express human antibodies, thus achieving the goal of full humanization of the antibodies.

[0057] In yet another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to Proinsulin as disclosed herein.

[0058] In yet another aspect, the present disclosure provides a vector comprising the nucleic acid as disclosed herein.

[0059] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA fragments can be linked. Another type of vector is a viral vector, into which additional DNA fragments can be linked to the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of a gene operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some embodiments, vectors include, but are not limited to: (1) plasmids; (2) phagemids; (3) cosmids; (4) artificial chromosomes, such as yeast artificial chromosomes, bacterial artificial chromosomes, or artificial chromosomes derived from P1; (5) bacteriophages, such as λ phage or M13 phage; (6) animal viruses, such as retroviruses, adenoviruses, adeno-associated viruses, poxviruses, baculoviruses.

[0060] When the nucleic acids disclosed in the present invention are linked to a vector, the nucleic acids can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acids, etc. Of course, these control elements can be directly from the vector itself or can be exogenous, i.e., not from the vector itself. Of course, it is only necessary to operably link the polynucleotide to the control element. As used herein, "operably linked" means that an exogenous gene is linked to a vector such that the control elements in the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides encoding the heavy and light chains of an antibody can be inserted into different vectors separately and independently, and commonly are inserted into the same vector.

[0061] In another aspect, the present disclosure provides a host cell comprising the nucleic acids disclosed in the present invention or the vectors disclosed in the present invention.

[0062] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. The expression vector can be introduced into the host cell to construct a recombinant cell, and then these recombinant cells can be used to express the antibodies or antigen-binding fragments provided by the present invention. By culturing these recombinant cells, the corresponding antibodies can be obtained. In some embodiments, host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293F.

[0063] In some embodiments of the host cells disclosed herein, the host cells are eukaryotic cells.

[0064] In some embodiments of the host cells disclosed herein, the host cells are mammalian cells, including but not limited to 293F cells and CHO cells.

[0065] In another aspect, the present disclosure provides hybridoma cells that can produce antibodies or antigen-binding fragments that specifically bind to Proinsulin disclosed in the present invention.

[0066] As used herein, the term "hybridoma cell" refers to a cell formed by fusing myeloma cells and B lymphocytes during the preparation of monoclonal antibodies. In the classical method for preparing monoclonal antibodies, first, a suitable antigen is required and used to immunize an animal. To prepare an antibody (or antibody analog) against Proinsulin, the suitable antigen must contain Proinsulin. This antigen can be isolated and purified from natural human tissues or blood, or expressed and purified using prokaryotic or eukaryotic cells through recombinant protein expression. The antigen is used for animal immunization and antibody screening and detection.

[0067] In the classical method for preparing monoclonal antibodies, first, an animal is immunized with the Proinsulin antigen, and blood is collected at intervals to verify whether the animal has produced an antibody response to the Proinsulin antigen. Then, B cells isolated from the spleen of the animal with an antibody response are fused with immortalized myeloma cells in vitro to obtain hybridoma cells. Then, these hybridoma cells are serially diluted in a culture plate and regrown (monoclonal hybridoma cell lines), and the culture supernatant of these hybridoma cell lines is taken to detect whether it contains specific antibodies against the antigen. Based on antibody yield, quality, and cell line growth characteristics, the best monoclonal antibody-producing cell line can be selected for subsequent monoclonal antibody production.

[0068] Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells of the above animals can be isolated and then incubated with a labeled antigen (such as fluorescein-labeled Proinsulin). Since B cells that produce antibodies usually have antibody molecules presented on the cell membrane surface, these cells will be bound (stained) by the labeled antigen and thus can be sorted out by a fluorescence-activated cell sorter. The mRNA of these sorted B cells can be isolated, and a library of cDNA of the variable region of the antibody can be obtained through in vitro reverse transcription and specific PCR reactions. This cDNA library can be inserted into an expression plasmid (such as an antibody expression plasmid suitable for expression in mammalian cells or a phage expression plasmid suitable for expression in bacterial cells) and expressed in host cells suitable for this expression plasmid. These host cells (or phages) can be isolated and purified (cloned) by different methods (such as the cell limiting dilution culture method or the phage plaque plating method described above). These cell lines or phage clones can be used to produce antibodies and analyze and identify the antibodies.

[0069] In yet another aspect, the present disclosure provides a method for preparing the antibodies or antigen-binding fragments thereof disclosed in the present invention, comprising: culturing the host cells or hybridoma cells disclosed in the present invention under conditions that allow the expression of the antibodies, their antigen-binding fragments.

[0070] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that specifically binds to Proinsulin, a nucleic acid, a vector, a host cell or a hybridoma cell disclosed in the present invention; and (ii) a pharmaceutically acceptable carrier or adjuvant.

[0071] As used herein, the term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other components of the composition and is not highly toxic to its recipient, and / or such carrier or adjuvant is approved or available for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0072] In some embodiments, the carriers or adjuvants used with the compositions disclosed herein include, but are not limited to, sterile liquids such as water and oils, including oils of petroleum, animal or vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, when the pharmaceutical composition is administered intravenously, the carrier can be water. Aqueous saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, especially for injection solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, which is hereby incorporated by reference into the present invention. Such compositions will contain a clinically effective dose of the antibody or antibody fragment, together with a suitable carrier, to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The formulation can be encapsulated in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0073] In yet another aspect, the present invention provides a detection reagent comprising an antibody or an antigen-binding fragment thereof that specifically binds to Proinsulin as disclosed herein.

[0074] In some embodiments of the detection reagent disclosed herein, the detection reagent further comprises one or more enzyme-linked immunosorbent assay (ELISA) reagent components.

[0075] In the disclosure of the present invention, the ELISA reagent components are selected from a positive control solution, a sample diluent, a negative control solution, an enzyme-labeled reagent, an enzyme substrate solution, a chromogenic reagent, a washing solution, a blocking solution, and / or a termination solution.

[0076] In the disclosure of the present invention, the detection reagent further comprises one or more chemiluminescent immunoassay components.

[0077] In the disclosure of the present invention, the chemiluminescent immunoassay components include a magnetic microsphere solution (such as streptavidin-conjugated magnetic beads), a biotinylated antibody solution (such as biotinylated polyclonal Proinsulin antibody), a luminescence label (such as acridinium ester, phycoerythrin, peridinin-chlorophyll-protein complex, fluorescein isothiocyanate or allophycocyanin-labeled monoclonal Proinsulin antibody), and / or a Proinsulin calibrator, and / or a Proinsulin quality control product.

[0078] In one aspect, the present invention provides a method for detecting the content of Proinsulin in a sample, comprising: mixing the sample with the antibody or its antigen-binding fragment as described above, or the detection reagent; incubating, and then detecting the luminescence value or absorbance.

[0079] In another aspect, the present disclosure provides the use of the antibody or antigen-binding fragment thereof that specifically binds to Proinsulin disclosed in the present invention, or a detection reagent, in the preparation of a reagent or product for detecting the content of Proinsulin in a sample, a reagent or product for diagnosing Proinsulin-related diseases, or a drug for preventing and / or treating Proinsulin-related diseases.

[0080] In some embodiments of the use disclosed herein, the Proinsulin-related diseases refer to diseases or disorders in which the patient exhibits abnormal Proinsulin expression levels during the onset and disease development process compared to healthy humans. In some embodiments, the Proinsulin-related diseases include: type I diabetes, type II diabetes, pancreatitis, pancreatic cancer.

[0081] In yet another aspect, the present disclosure provides the application of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, hybridoma cell, and / or pharmaceutical composition that specifically binds to Proinsulin disclosed in the present invention in the preparation of any of the following products: (1) A product for detecting Proinsulin; (2) A product for stimulating or enhancing the immune response; (3) A product for preventing and / or treating Proinsulin-related diseases.

[0082] The anti-Proinsulin antibody of the present invention can be used to detect Proinsulin on the surface of human peripheral blood lymphocytes, or substances containing Proinsulin, as well as substances that can specifically bind to Proinsulin (such as anti-Proinsulin antibodies). The detection principle is mainly based on the specific recognition and binding of the Proinsulin antibody of the present invention to Proinsulin, and chemical labeling techniques (such as labeled antibodies or labeled specific secondary antibodies) or physical detection techniques (such as light scattering techniques, surface plasmon resonance techniques, etc.) are used for detection.

[0083] The present invention provides a humanized antibody against Proinsulin, which has the characteristics of high affinity and specificity. At the same time, a flow cytometry detection method based on the above antibody is also provided. The present invention solves the problem of the relative shortage of antibody raw materials for Proinsulin detection items in the current market, can be used as an auxiliary diagnosis for related diseases, and fills the domestic market gap.

[0084] Example 1: Recombinant expression of human proinsulin (Proinsulin) First, the human Proinsulin gene was synthesized and inserted into the pcDNA4.1 plasmid (the above process was entrusted to Sangon Biotech (Shanghai) Co., Ltd.), and the expression vector for the target protein was obtained. The obtained expression vector was transfected into CHO cells (1×10 6 / well) using Lipofectamine 3000 reagent (Thermo catalog number L3000015). After culturing for 48 h, the transfected cells were transferred into a shake flask and cultured in a 500 ml culture medium (Thermo catalog number A3711001) system for 7 days, and then centrifuged and protein purified. After the cells were collected by centrifugation, the obtained cell pellet was lysed to obtain a cell lysate. The obtained cell lysate was loaded onto a pre-equilibrated nickel column and incubated at room temperature or 4 °C for 30 minutes to allow the His-tag protein to bind to nickel ions. Then, the nickel column was washed with 10 - 20 bed volumes of washing buffer to remove non-specifically bound proteins. Then, it was eluted with imidazole elution buffer. The eluate was collected and concentrated using an ultrafiltration tube (Sigma catalog number UFC5003). The concentration of the purified recombinant Proinsulin protein was 0.949 mg / ml as measured by Nanodrop One (ThermoFisher).

[0085] Example 2: Obtaining a monoclonal antibody against human proinsulin 1. Resuscitation of human PBMCs Mix PBS:FBS:EDTA in a ratio of 244:5:1 to prepare MACS, and store it at 4 °C for later use. Prepare a 15 mL centrifuge tube in a biosafety cabinet and add 3 mL of MACS to the centrifuge tube. Take out the cryopreserved human PBMC cells (provided by the Department of Endocrinology, Gulou Hospital) from liquid nitrogen, clamp the cryopreservation tube with hemostatic forceps, and quickly place it in a water bath until the cells thaw to obtain a resuscitated cell suspension. Transfer all the resuscitated cell suspension (nearly 5 mL) into the above 15 mL centrifuge tube and rinse the cryopreservation tube thoroughly. Then centrifuge at 400 g for 10 min, and then discard the supernatant to obtain a cell pellet. Pipette 10 mL of Gibco RPMI1640 medium (catalog number 61870036) and gently pipette and mix the cells to obtain a cell suspension. Take 10 μL of the cell suspension and add it to 90 μL of MACS buffer, then add 100 μL of trypan blue solution and count under a microscope. The counting method is: total number in four quadrants / 4 * dilution factor * volume * 10 4 . After counting, centrifuge the cell suspension at 400 g for 10 min, discard the supernatant to obtain a cell pellet. Then resuspend the cells at a density of 1E8 / mL to obtain a resuscitated PBMC cell suspension.

[0086] 3. Sorting antigen-specific B lymphocytes Take the above-mentioned resuscitated PBMC cell suspension, add Proinsulin labeled with AF488 and Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG Antibody for staining. After staining, wash the cell samples twice, then resuspend them with flow buffer and load them onto the machine. Before loading, add 7-AAD and incubate at room temperature for 10 min to distinguish dead / live cells. Use a BD FACSMelody TM flow cytometer, sort 4.4 E7 cells for loading. After removing cell debris, use the live / dead dye to count the final cell viability, which is 88.05%. Finally, about 20,000 positive cells are obtained. The results are as Figure 1 shown.

[0087] According to the flow cytometry analysis results, the antibody light and heavy chain sequences from the same cell will have the same barcode, and about 1000 Unique sequences are obtained. According to the evolutionary degree of the BCR phylogenetic tree, 30 Unique sequences are selected from the 1000 sequences for supernatant expression in mammalian cell system (Human IgG1-Kappa / Human IgG1-Lambda1), and the results show that all are successfully expressed. Then the successfully expressed supernatant is verified by ELISA, using the antibody anti-HEL-Human IgG1 Isotype-control in Binding ELISA as the negative reference. First, ELISA is used to detect the recognition of human proinsulin protein by the above 30 antibodies, and the results show that 4 clones of antibodies among the 30 antibodies can recognize proinsulin. Then, the commercial Insulin (MCE, catalog number HY-P73243) and C-peptide protein (MCE, catalog number HY-P1856) are used as the coating antigens respectively to detect the above 4 clones of antibodies. The specific operations are as follows: Coating: Antigen protein 1 µg / ml, 100 µL / well, coat at 37 °C for 1 h, then wash the plate 6 times and pat dry. Blocking: Blocking solution, 200 µL / well, block at 25 °C for 1 h, then wash the plate 6 times and pat dry. Binding: The cell supernatant in the first well is diluted 1:100; diluted 2-fold with casein blocking solution, 100 µL / well, incubate at 25 °C for 1 h, then wash the plate 6 times and pat dry. Secondary antibody: Anti-human IgG(Fc specific)HRP is diluted 1:5000 with casein blocking solution; 100 μL / well, incubate at 25 °C for 1 h, then wash the plate 6 times and pat dry. Color development: Add 100 µL / well chromogenic solution to each reaction well, and develop color in the dark at room temperature for 3 min. Termination: Add 50 µL / well termination solution to each reaction well to terminate the reaction. Reading: Measure the OD value to judge the result, use an enzyme-linked immunosorbent assay reader to measure the absorbance value (A450) at 450 nm, and the results are shown in Table 1.

[0088] Table 1 Absorbance values corresponding to different antibodies Table 1 shows the ELISA test results of four antibodies against three homologous antigen proteins under the condition of 100-fold dilution. As shown in Table 1, only 1 clone of the four antibodies does not recognize Insulin or C-peptide, which is a human antibody specifically binding to Proinsulin and is named GTH01.

[0089] Example 3: Sequencing of antibody GTH01 The antibody GTH01 obtained in Example 1 was sequenced by GenScript Biotech Corporation, and the results are shown in Table 2.

[0090] Table 2 Antibody sequence information Nucleic acids encoding the heavy chain and / or light chain of the antibody of the present invention are within the scope of the present invention. Based on the amino acid sequences of the heavy chain and / or light chain, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 2. It should be noted that the CDR sequences listed in Table 2 were obtained according to the IMGT database. Those skilled in the art should know that the CDR sequences analyzed by different databases may be different, but these variations should all be included within the protection scope of the present invention.

[0091] Example 4: Construction of the antibody GTH01 expression vector The hybridoma sequencing results were analyzed using NCBI blast, NCBI IGBlast, etc. to find the heavy chain variable region sequence and light chain variable region sequence of the antibody. The codons were optimized using a codon optimization software in a mammalian cell expression system. After synthesis by Sangon Biotech (Shanghai) Co., Ltd., the codon-optimized genes were respectively cloned into the pcDNA3.1 expression vector using the EcoRI / HinDIII restriction enzyme cleavage sites to construct monoclonal antibody expression plasmids, and the plasmids were sequenced to verify the correct construction.

[0092] Example 5: Preparation of the antibody GTH01 The monoclonal antibody was obtained by transiently transfecting the 293F cells with the expression plasmid obtained in Example 3. One day before transfection, the 293F cells were inoculated into a cell culture flask at a density of 0.8×10 6 / mL and placed in a cell shaker at 37°C, 5% CO 2 , 120 rpm, and cultured in OPM-293 CD03 Medium until the cell confluence reached 80%-90% and the viability was greater than 95%. Then transfection was carried out. After mixing the pcDNA3.1 heavy chain expression plasmid and light chain expression plasmid (at a molar ratio of 1:1) with polyetherimide and incubating at room temperature for 15 min, it was slowly added to the HEK293 cells. An enhancer and excipients (purchased from Sangon Biotech (Shanghai) Co., Ltd.) were added, and it was cultured at 37°C, 5% CO 2, continue culturing in a cell shaker at 120 rpm. When the cell viability < 70%, collect the cell culture, centrifuge at 8000 g for 10 min, collect the supernatant, and then centrifuge at 12000 g, 4 °C for 30 min, collect the cell supernatant, perform antibody purification, and identify the purity of the purified antibody by SEC-HPLC to be 97.7% (the results are shown in Figure 2 ).

[0093] Example 6: Determination of the titer of antibody GTH01 Use the indirect ELISA method to detect the titer of the anti-human Proinsulin antibody GTH01. The specific method is as follows: Dilute the Proinsulin protein obtained in Example 1 with PBS and coat it on a 96-well ELISA plate at 0.2 μg / mL, 100 μL / well, and incubate overnight at 4 °C. Then wash the ELISA plate twice with PBST solution, 300 μL / well each time, and pat it dry after washing. Then block the ELISA plate with 1% BSA in PBS solution, 200 μL / well, and block it at room temperature for 2 hours, and pat it dry after blocking; Add the anti-Proinsulin antibody GTH01 diluted to 20 ng / mL and react in an incubator at 37 °C for 1 hour. After the reaction, wash the ELISA plate twice with PBST solution, 300 μL / well each time, and pat it dry after washing. Add the HRP-labeled Anti-human IgG (Fc specific) HRP antibody diluted 5000 times with PTB and react in an incubator at 37 °C for 1 hour. After the reaction, wash the ELISA plate twice with PBST solution, 300 μL / well each time, and pat it dry after washing. Add 100 µL / well chromogenic solution and develop color in the dark at room temperature for 3 min, then add 50 µL / well stop solution to terminate the reaction. Use an ELISA reader to detect the absorbance value at OD450nm. The results are shown in Table 3. It can be seen that the titer of the purified antibody is 1:33000.

[0094] Table 3 Determination of antibody titer Example 7: Determination of the sensitivity of antibody GTH01 The sensitivity of antibody GTH01 to antigen Proinsulin was detected by ELISA method, which specifically included the following steps: The Proinsulin protein obtained in Example 1 was diluted with PBS and coated on a 96-well enzyme-linked immunosorbent assay (ELISA) plate at each concentration in Table 4 overnight at 4 °C. Then, the ELISA plate was washed twice with PBST solution at 300 μL / well each time and patted dry after washing. Next, the ELISA plate was blocked with 200 μL / well of 1% BSA in PBS solution for 2 hours at room temperature and patted dry after blocking. Antibody GTH01 was added and reacted in an incubator at 37 °C for 1 hour. After the reaction, the ELISA plate was washed twice with PBST solution at 300 μL / well each time and patted dry after washing. HRP-labeled Anti-human IgG (Fc specific) HRP antibody diluted 5000 times with PTB was added and reacted in an incubator at 37 °C for 1 hour. After the reaction, the ELISA plate was washed twice with PBST solution at 300 μL / well each time and patted dry after washing. 100 μL / well of chromogenic solution was added, and the reaction was terminated by adding 50 μL / well of termination solution after 3 minutes of color development in the dark at room temperature. The absorbance value at OD450nm was detected using an enzyme-linked immunosorbent assay reader, and the results are shown in Table 4.

[0095] Table 4 Sensitivity data of antibody GTH01 to antigen Proinsulin detected by ELISA method The OD value in Table 4 represents the optical density value, which is the intensity of the detection signal. The higher the OD value, the more antibodies are bound. As shown in Table 4, according to the detection threshold (usually more than twice the average background OD value), the lowest antigen concentration that can be detected by this antibody was determined to be 0.313 ng / mL. Therefore, the sensitivity of this antibody is 0.313 ng / mL.

[0096] Example 8: Specificity determination of antibody GTH01 The cell lysate of human HEK293 cells transfected with plasmid and overexpressing proinsulin was used as the positive control, and the cell lysate transfected with the blank control plasmid and not expressing proinsulin was used as the negative control. First, use Nanodrop to detect the protein content in the sample and quantify it to 20 µg / ml protein, and then perform the following operations: Coating: 100 µL / well of the cell lysate with 20 µg / ml protein, coat at 37 °C for 1 h, wash the plate 6 times, and pat dry. Blocking: 200 µL / well of the blocking solution, block at 25 °C for 1 h, wash the plate 6 times, and pat dry. Binding: Dilute the first well of cell supernatant 1:100; dilute 2-fold with casein blocking solution, 100 µL / well, incubate at 25 °C for 1 h, wash the plate 6 times, and pat dry. Secondary antibody: Dilute Anti-human IgG(Fc specific)HRP 1:5000 with casein blocking solution; 100 μL / well, incubate at 25 °C for 1 h, wash the plate 6 times, and pat dry. Chromogenic reaction: Add 100 µL / well of the chromogenic solution and develop color in the dark at room temperature for 3 min. Termination: Add 50 µL / well of the termination solution to terminate the reaction. Reading: Measure the absorbance value (A450) at 450 nm using a microplate reader, and the results are shown in Table 5.

[0097] Table 5 Identification of antibody specificity As can be seen from the results in Table 5, the antibody GTH01 only recognizes Proinsulin and does not recognize the proteins of human cells HEK293. Therefore, this antibody has high specificity.

[0098] Example 9: Application of antibody GTH01 in detecting the level of Proinsulin in human plasma.

[0099] Using the ELISA method, compare and detect the levels of Proinsulin in the plasma donated by 5 healthy people and 5 T1DM diabetic patients using the GTH01 antibody of the present invention and a commercial antibody (Novus Biologicals catalog number NB100-73013). The detection results are as Figure 3 . As Figure 3 shown, both antibodies can detect the content of Proinsulin in plasma well, and the detection sensitivity of the antibody of the present invention is not inferior to that of the commercial antibody.

[0100] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical content disclosed above within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to proinsulin, wherein the amino acid sequences of LCDR1-3 of the light chain variable region VL of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 1, 2 and 3, respectively, and the amino acid sequences of HCDR1-3 of the heavy chain variable region VH of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 4, 5 and 6, respectively.

2. An antibody or antigen-binding fragment thereof that specifically binds Proinsulin according to claim 1, wherein the amino acid sequence of the VL is as shown in SEQ ID NO: 7 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto, and the amino acid sequence of the VH is as shown in SEQ ID NO: 8 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity thereto.

3. An antibody or antigen-binding fragment thereof that specifically binds to Proinsulin according to claim 2, wherein the light chain constant region CL of the antibody or antigen-binding fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 9, and the heavy chain constant region CH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:

10.

4. The antibody or antigen-binding fragment thereof that specifically binds to Proinsulin according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from a double-chain antibody, and the antibody is a humanized antibody.

5. An antibody or antigen-binding fragment thereof that specifically binds Proinsulin according to claim 4, wherein the light chain of the antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 11, and the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

12.

6. A nucleic acid comprising a protein encoding an antibody or an antigen-binding fragment thereof that specifically binds to proinsulin according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid according to claim 6.

8. A host cell comprising the nucleic acid according to claim 6 or the vector according to claim 7.

9. The host cell according to claim 8, wherein the host cell is a eukaryotic cell.

10. The host cell according to claim 9, wherein the host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

11. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; and (ii) a pharmaceutically acceptable carrier or adjuvant.

12. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising: The host cell according to any one of claims 8 to 10 is cultured under conditions such that the antibody or antigen-binding fragment thereof is expressed.

13. A detection reagent comprising the antibody or antigen-binding fragment thereof that specifically binds to proinsulin according to any one of claims 1 to 5. The detection reagent according to claim 13 , wherein the detection reagent further comprises one or more enzyme-linked immunosorbent assay components.

15. The detection reagent according to claim 14, wherein the enzyme-linked immunosorbent assay reagent components are selected from a positive control solution, a sample diluent, a negative control solution, an enzyme-labeled reagent, an enzyme substrate solution, a colorimetric reagent, a washing solution, a blocking solution, and / or a stop solution.

16. The detection reagent according to claim 13, wherein the detection reagent further comprises one or more chemiluminescent immunoassay components.

17. The detection reagent according to claim 16, wherein the chemiluminescent immunoassay component comprises a magnetic microsphere solution, a biotinylated antibody solution, a luminescent marker, and optionally a Proinsulin calibrator and a Proinsulin quality control product.

18. Use of the antibody or antigen-binding fragment thereof that specifically binds to proinsulin according to any one of claims 1 to 5, or the detection reagent according to any one of claims 13 to 17, in the preparation of a reagent or product for detecting the content of proinsulin in a sample, a reagent or product for diagnosing a proinsulin-related disease, or a drug for preventing and / or treating a proinsulin-related disease.

19. The use according to claim 18, wherein the proinsulin-related disease is selected from the group consisting of type I diabetes, type II diabetes, pancreatitis, and pancreatic cancer.

20. Use of the antibody or antigen-binding fragment thereof that specifically binds to proinsulin according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, the host cell according to any one of claims 8 to 10, the pharmaceutical composition according to claim 11, or the detection reagent according to any one of claims 13 to 17 in the preparation of any of the following products: (1) Testing of Proinsulin products; (2) Products that stimulate or enhance immune response; (3) Products for preventing and / or treating proinsulin-related diseases.

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