Anti-CD45 antibody and application thereof

Abstract

The invention relates to the technical field of antibodies, and provides an anti-CD45 antibody and application thereof, the antibody comprises at least one of the following CDRs: a heavy chain variable region CDR: an amino acid sequence of SEQ ID NO: 1-3 or a conservatively modified form thereof; and a light chain variable region CDR: SEQ ID NO: 4-6 or an amino acid sequence in a conservative modification form thereof. The antibody or the antigen binding fragment thereof can efficiently and specifically recognize CD45, can effectively detect CD45, and can be used for immunohistochemistry exemplarily.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an anti-CD45 antibody and applications thereof. Background Art

[0002] CD45 (Leukocyte Common Antigen) is a crucial transmembrane protein tyrosine phosphatase in the immune system, widely expressed on the surface of all nucleated leukocytes, including T cells, B cells, natural killer cells (NK cells), macrophages, and dendritic cells. As a core regulatory molecule in immune cell signaling, CD45 mediates lymphocyte immune responses and plays a key role in immune cell development, activation, differentiation, and tolerance. Its structural diversity, functional complexity, and relevance to disease make it a key target for immunology research and clinical translation.

[0003] Functionally, CD45 dephosphorylates inhibitory tyrosine residues (e.g., Tyr505 in Lck) at the C-terminus of Src family kinases (e.g., Lck and Fyn), releasing them from autoinhibition and activating downstream signaling pathways. This process is crucial for T cell receptor (TCR) and B cell receptor (BCR)-mediated immune responses, regulating T / B cell activation, proliferation, and differentiation. Furthermore, CD45 participates in positive and negative selection of thymocytes, influences T cell maturation, and plays a role in cytokine production and the intensity of immune responses. Studies have shown that CD45 deficiency can lead to severe immune dysfunction, and its abnormal expression in autoimmune diseases and leukemias suggests its importance as a potential diagnostic marker or therapeutic target. CD45 function is context-dependent, potentially exhibiting a complex regulatory network through different isoforms or interacting molecules. Therefore, the development of CD45 antibodies is of great significance for both immunological research and clinical diagnosis.

[0004] Currently, commercial CD45 antibodies face significant limitations. Mainstream products rely on traditional mouse hybridoma technology, and production processes are limited by animal-derived systems (such as ascites fluid preparation). This carries risks such as large batch-to-batch variability, animal ethics issues, and potential immunogenicity. Therefore, innovative development strategies (such as non-animal-derived recombinant technology and single B cell sequencing) are urgently needed to overcome technical barriers, shorten R&D time, and screen for CD45 antibodies with high affinity, high specificity, and low batch-to-batch variability. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] Therefore, in the first aspect of the present invention, an antibody or antigen-binding fragment thereof is provided. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 or a conservatively modified version thereof; a heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 or a conservatively modified version thereof; a heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 or a conservatively modified version thereof; a light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 or a conservatively modified version thereof; a light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 or a conservatively modified version thereof; and a light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 or a conservatively modified version thereof. The antibody or antigen-binding fragment thereof according to an embodiment of the present invention is a high-affinity (pM level) rabbit recombinant antibody developed through single B cell sequencing and non-animal recombinant technology. The antibody can efficiently and specifically recognize CD45, effectively detect CD45, and diagnose diseases associated with abnormal CD45 expression.

[0007] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof encoded by the nucleic acid molecule according to an embodiment of the present invention can efficiently and specifically recognize CD45, detect CD45, for example, by IHC testing, and can also effectively detect CD45 and diagnose diseases associated with abnormal CD45 expression.

[0008] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. Thus, the aforementioned antibody or antigen-binding fragment thereof is effectively expressed, thereby enabling the antibody or antigen-binding fragment thereof to be obtained in large quantities in vitro.

[0009] In a fourth aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or expression vector, or expressing the aforementioned antibody or antigen-binding fragment thereof. Under suitable conditions, the recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment thereof within the cell.

[0010] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect. According to an embodiment of the present invention, the method comprises culturing the recombinant cell according to the fourth aspect.

[0011] In a sixth aspect, the present invention provides an antibody conjugate. According to an embodiment of the present invention, the antibody conjugate comprises: the aforementioned antibody or antigen-binding fragment thereof; and a coupling moiety coupled thereto, wherein the coupling moiety is connected to the antibody or antigen-binding fragment thereof. The antibody conjugate of the present invention can specifically recognize CD45 and detect CD45, for example, by IHC testing. It can also effectively detect CD45 and diagnose diseases associated with abnormal CD45 expression.

[0012] In a seventh aspect, the present invention provides a reagent or kit. According to an embodiment of the present invention, the reagent or kit comprises: the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned antibody conjugate. The kit of the present invention is capable of specifically binding to CD45 and effectively detecting CD45.

[0013] In its eighth aspect, the present invention provides the use of the aforementioned antibodies or antigen-binding fragments thereof, antibody conjugates, reagents, or kits for detecting CD45, preparing products for detecting CD45, or diagnosing CD45-related diseases. As previously described, the antibodies or antigen-binding fragments thereof according to embodiments of the present invention are high-affinity (pM-level) rabbit recombinant antibodies developed through single B cell sequencing and non-animal recombinant technology. These antibodies can effectively detect CD45, for example, by IHC testing.

[0014] In a ninth aspect, the present invention provides a method for detecting CD45 in a test sample. According to an embodiment of the present invention, the method comprises contacting the aforementioned antibody, antigen-binding fragment thereof, antibody conjugate, reagent, or kit with the CD45 antigen in the sample to be tested to form an immune complex.

[0015] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a diagram showing the animal immunization scheme according to an embodiment of the present invention and the serum titer test results after four immunizations, wherein: Figure 1 A in the figure is the animal immunization program process. Figure 1 B in the figure is the result of serum titer test after four immunizations of experimental animals; Figure 2 Figure 1 shows the results of flow cytometry sorting of B cells according to an embodiment of the present invention. P1 indicates that the main cell population was selected by forward scattered light (FSC-A) and side scattered light (SSC-A), and cell debris was removed. P2 and P3 indicate that adherent cells were removed by area (A) and height (H) of FSC and SSC, respectively. P4 indicates the selection of IgG+ cells positive in the FITC channel, and P5 indicates the selection of Ag+IgG+ cells positive in the APC channel. Figure 3 This is a diagram showing the SDS-PAGE detection results of the recombinant antibody according to an embodiment of the present invention, wherein M represents a protein marker, +DTT represents that the protein loading buffer contains the reducing agent DTT, and -DTT represents that the protein loading buffer does not contain the reducing agent DTT; Figure 4 This is a graph showing the affinity measurement results of the binding antibody according to an embodiment of the present invention, wherein the abscissa represents time (s) and the ordinate represents the relative displacement height (nm); Figure 5 Graphs showing the IHC detection results of CD45-58 rabbit recombinant monoclonal antibodies on various tissue sections according to an embodiment of the present invention. DETAILED DESCRIPTION

[0018] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0019] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0020] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0021] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0022] As used herein, the term "fragment" refers to a target protein or polypeptide, as well as a target protein or polypeptide with N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or internal deletion.

[0023] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see 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, DC). 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 search similarity method of Pearson et al. (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 that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410). 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.

[0024] As used herein, the term "at least 80% identity" refers to at least 80%, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to the respective reference sequence.

[0025] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0026] As used herein, the term "recombinant cell" generally refers to cells that have been modified or recombined using genetic engineering or cell fusion techniques to modify or reorganize the genetic material of a host cell, resulting in cells with unique, stably inherited traits. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected," as used herein, refer to the introduction of a nucleic acid (e.g., a vector) into a cell using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and used for the expression and / or secretion of the target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.

[0027] An antibody or antigen-binding fragment thereof In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof, comprising a CDR selected from at least one of the following: a heavy chain variable region CDR comprising an amino acid sequence of SEQ ID NOs: 1-3 or conservatively modified versions thereof; and a light chain variable region CDR comprising an amino acid sequence of SEQ ID NOs: 4-6 or conservatively modified versions thereof. The antibody or antigen-binding fragment thereof according to an embodiment of the present invention is a high-affinity (pM level) rabbit recombinant antibody developed through single B cell sequencing and non-animal recombinant technology. The antibody is capable of efficiently and specifically recognizing CD45, enabling detection of CD45, such as by IHC, and effectively detecting CD45 and diagnosing diseases associated with abnormal CD45 expression.

[0028] As used herein, the term "antibody" is used in the broadest sense and includes full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies. The specific structure is not limited, as long as they exhibit the desired biological activity. Antibodies typically consist of a light chain (lighter in molecular weight) and a heavy chain (heavy in molecular weight), with the heavy chain (H chain) and light chain (L chain) linked by disulfide bonds to form an antibody molecule. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is called the variable region (V region); the carboxyl terminus (C-terminus) is relatively stable and exhibits minimal variation and is called the constant region (C region). The V regions of the L chain and H chain are referred to as VL and VH, respectively. As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, encompassing one or more, or even all, of the amino acid residues that contribute primarily to the binding affinity of an antibody or its functional fragment to the antigen or epitope it recognizes. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0029] As used herein, the term "antigen-binding fragment" refers to a fragment comprising a portion or all of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. For example, the fragment may comprise a portion or all of an antibody CDR. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments can be produced by recombinant nucleic acid technology or can be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0030] As used herein, "conservatively modified forms of an amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of an antibody of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay method described herein. Preferably, conservative modifications do not exceed 1 or 2 in number.

[0031] According to some specific embodiments of the present invention, the above-mentioned antibody or antigen-binding fragment thereof may further include at least one of the following additional technical features: According to some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises: A heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence of a conservative modification thereof; A light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence of a conservative modification thereof; A light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence of a conservative modification thereof; and A light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence of a conservatively modified form thereof.

[0032] According to some specific embodiments of the present invention, the heavy chain variable region CDR1, CDR2, CDR3 and light chain variable region CDR1, CDR2, CDR3 are defined by any one of the Kabat, Chothia, IMGT, or AbM systems. Commonly used CDR numbering schemes in the art include: Kabat numbering, Chothia numbering, IMGT numbering, Martin numbering, and AHo numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, and AbM definition. As used herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described in Kabat et al., U.S. Patent No. 200,000, of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196:901-917 (1987). Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0033] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof includes a heavy chain framework region and / or a light chain framework region.

[0034] According to some specific embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of a rabbit antibody, a mouse antibody, a human antibody, a primate antibody, a sheep antibody, a dog antibody, a cat antibody, an alpaca antibody, and mutants thereof.

[0035] According to some specific embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from a rabbit antibody.

[0036] According to some specific embodiments of the present invention, the heavy chain framework region and / or the light chain framework region comprises a FR selected from at least one of the following: Heavy chain variable region FR: amino acid sequence of SEQ ID NOs: 7-10 or conservatively modified forms thereof; Light chain variable region FR: amino acid sequence of SEQ ID NOs: 11 to 14 or conservatively modified forms thereof.

[0037] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises: A heavy chain variable region FR1 having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region FR2 having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region FR3 having the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region FR4 having the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence of a conservative modification thereof; A light chain variable region FR1 having the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence of a conservative modification thereof; A light chain variable region FR2 having the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence of a conservative modification thereof; A light chain variable region FR3 having the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence of a conservative modification thereof; and The light chain variable region FR4 has the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence of a conservatively modified form thereof.

[0038] According to some specific embodiments of the present invention, the heavy chain variable region FR1, FR2, FR3, FR4 and the light chain variable region FR1, FR2, FR3, FR4 are defined by any one of the Kabat, Chothia, IMGT or AbM systems.

[0039] Likewise, those skilled in the art will appreciate that, given the variable region amino acid sequence of an antibody, those skilled in the art can routinely determine which residues comprise a particular FR.

[0040] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity thereto.

[0041] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0042] According to some specific embodiments of the present invention, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0043] According to some specific embodiments of the present invention, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of a rabbit antibody, a mouse antibody, a human antibody, a primate antibody, a sheep antibody, a dog antibody, a cat antibody, an alpaca antibody, and mutants thereof.

[0044] According to some specific embodiments of the present invention, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of a rabbit antibody, a mouse antibody, a human antibody, and a primate antibody.

[0045] According to some specific embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ type or λ type.

[0046] According to some specific embodiments of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region.

[0047] According to some specific embodiments of the present invention, the antibody includes at least one selected from a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, an Fv antibody, a single-chain antibody, a single-domain antibody and a minimum recognition unit; or, the antigen-binding fragment includes at least one selected from a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, an Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

[0048] As used herein, the terms "full-length antibody," "full-length monoclonal antibody," or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0049] Herein, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably and were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigen protein (e.g., CD45) through the heavy chain variable region.

[0050] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0051] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0052] In this article, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment composed only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds. It is the smallest functional fragment of an antibody that retains a complete antigen-binding site.

[0053] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0054] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, which has a very small molecular weight of only about 1% of the complete antibody.

[0055] Nucleic acid molecules, expression vectors and recombinant cells In some embodiments, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof encoded by the nucleic acid molecule according to embodiments of the present invention can efficiently and specifically recognize CD45, detect CD45, for example, by IHC, and can also effectively detect CD45 and diagnose diseases associated with abnormal CD45 expression.

[0056] According to some specific embodiments of the present invention, the nucleic acid molecule is DNA.

[0057] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is provided in most cases, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them implies disclosure of the other.

[0058] In some embodiments, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. When the aforementioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably connected. Herein, "operably connected" means that the exogenous gene is connected to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected function of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned antibody or its antigen-binding fragment can be effectively achieved under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the antibody or its antigen-binding fragment.

[0059] According to some specific embodiments of the present invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0060] According to some specific embodiments of the present invention, the expression vector is a plasmid expression vector.

[0061] In some embodiments, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or expression vector, or expressing the aforementioned antibody or antigen-binding fragment thereof. Under suitable conditions, the recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment thereof within the cell.

[0062] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the antibodies or antigen-binding fragments thereof of the present invention. It will be readily understood by those skilled in the art that conditions suitable for the expression of the antibodies or antigen-binding fragments thereof include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies or antigen-binding fragments thereof according to the specific environment of the laboratory.

[0063] According to some specific embodiments of the present invention, the recombinant cell is a eukaryotic cell According to some specific embodiments of the present invention, the recombinant cell is a mammalian cell.

[0064] Conjugates and kits In some embodiments, the present invention provides an antibody conjugate. According to an embodiment of the present invention, the antibody conjugate comprises: the aforementioned antibody or antigen-binding fragment thereof; and a coupling moiety conjugated thereto. The antibody conjugate of the present invention can specifically recognize CD45, detect CD45 protein, and effectively diagnose diseases associated with abnormal CD45 expression.

[0065] According to some specific embodiments of the present invention, the coupling moiety is selected from a purification tag or a label.

[0066] According to some specific embodiments of the present invention, the coupling moiety includes at least one selected from colloidal gold, radioactive labels, phosphorescent chemical agents, chemiluminescent agents, fluorescein, enzymes, natural toxins, nucleic acids and affinity labels.

[0067] According to some specific embodiments of the invention, the coupling moiety comprises a radioisotope.

[0068] According to some specific embodiments of the present invention, the coupling portion includes at least one selected from phycoerythrin, a fluorophore, rhodamine, luciferase, fluorescein isothiocyanate, green fluorescent protein, blue fluorescent protein, and red fluorescent protein.

[0069] According to some specific embodiments of the present invention, the coupling portion includes at least one selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase.

[0070] According to some specific embodiments of the present invention, the coupling moiety includes at least one selected from biotin and avidin.

[0071] According to some specific embodiments of the present invention, the coupling part includes at least one selected from magnetic beads, magnetic microspheres, plastic microspheres, plastic particles, microporous plates, nylon and nitrocellulose membranes.

[0072] Herein, the coupling portion can be a substance that can be suspended or dispersed in a liquid phase (e.g., solid phase carriers such as particles and magnetic beads), or a solid phase that can accommodate or carry a liquid phase (e.g., supports such as plates, membranes, test tubes, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, and monolithic columns); it can also be a labeling carrier for labeling an antibody or antigen-binding fragment thereof, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of mutually complementary sense and antisense nucleic acids), a fluorescent substance (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), a luminescent substance (e.g., luciferin, aequorin, acridinium ester, tris(2,2 '-bipyridine) ruthenium, luminol), radioactive isotopes (e.g., 3H, 14C, 32P, 35S, 125I) and gold colloids, etc.

[0073] According to some specific embodiments of the present invention, the coupling moiety can be a protein tag, including but not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, a C-Myc tag, and the like.

[0074] It should be noted that methods known in the art can be used to bind the coupling moiety to the antibody or antigen-binding fragment thereof, for example, physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin), and ion binding.

[0075] In some embodiments, the present invention provides a reagent or kit. According to an embodiment of the present invention, the reagent or kit includes: the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell or antibody conjugate. As mentioned above, the aforementioned antibody or antigen-binding fragment thereof can bind to CD45, and the aforementioned antibody or antigen-binding fragment thereof can specifically bind to CD45. In addition, under appropriate conditions, the nucleic acid molecule, expression vector, recombinant cell or antibody conjugate can express the antibody or antigen-binding fragment thereof. Furthermore, the kit containing the above substances can effectively bind to CD45 and can be used to effectively detect CD45. The kit can be used for scientific research, such as qualitatively or quantitatively detecting CD45 in a biological sample, and can also be used to determine the status of a subject, such as determining whether the CD45 level of the subject is too high or too low after obtaining the CD45 level of the subject. The biological sample can be cells, tissues, blood, etc.

[0076] use In some embodiments, the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned antibody conjugate or reagent or kit in detecting CD45, preparing a product for detecting CD45, or a product for diagnosing a CD45-related disease.

[0077] method In some embodiments, the present invention provides a method for detecting CD45 in a test sample, comprising: contacting the aforementioned antibody or antigen-binding fragment thereof, antibody conjugate, or the aforementioned reagent or kit with the sample to be tested to form an immune complex.

[0078] According to some specific embodiments of the present invention, the test sample includes cells, tissues, blood, etc.

[0079] According to some specific embodiments of the present invention, whether the sample to be tested contains CD45 or the content of CD45 is determined based on the signal of the immune complex.

[0080] According to some specific embodiments of the present invention, the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment thereof.

[0081] According to some specific embodiments of the present invention, the immune complex further comprises a second antibody, which binds to CD45.

[0082] The nucleic acid and amino acid sequences involved in this application are shown in Table 1.

[0083] Table 1

[0084] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0085] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Colligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0086] In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder, GeneOptimizer online program, etc.).

[0087] Example 1: Gene synthesis and protein expression In this example, CD45 recombinant protein (sequence number: uniprot P08575) was prepared as an immunogen. Based on the encoding nucleic acid for this CD45 recombinant protein, the CD45_pcDNA3.4 expression vector was constructed (gene synthesis was performed by the synthesis platform of BGI Changzhou New Life Science Technology Co., Ltd.). The 3' end of the CD45 protein coding region (G25-S576) in the expression vector was labeled with a Twin-Strep-Tag. Codons were optimized according to the codon preference of Homo sapiens species and cloned into the pcDNA3.4 expression vector using the XbaI and HindIII restriction enzyme sites. The CD45_pcDNA3.4 plasmid was transformed into Escherichia coli DH5α (purchased from TIANGEN), and the plasmid was extracted in large quantities. The plasmid was then transfected into Expi293F™ cells (purchased from Thermo Fisher Scientific) using PEI (purchased from POLYSCIENCE) for protein expression. The cell supernatant containing the target protein CD45 recombinant protein was harvested on the 5th day after transfection. The cell supernatant was purified by Strep column affinity chromatography (Strep-Tactin XT column, IBA) and gel filtration chromatography (SuperoseTM 6 Increase 10 / 300GL, GE) to obtain a relatively pure target protein.

[0088] Example 2: Animal Immunization In this example, the immunogen CD45 recombinant protein obtained in Example 1 was used to immunize animals. The specific procedures were as follows: The immunogen CD45 recombinant protein was mixed with an adjuvant and injected into New Zealand white rabbits for immunization. The first immunization was with Freund's complete adjuvant (Sigma, cat F5881), and the subsequent immunizations were with Freund's incomplete adjuvant (Sigma, cat F5506). The mixing ratio was 1:1. After multiple rounds of immunization, blood was collected from the rabbit's ears, and serum was separated. The antibody titer in the serum was tested by ELISA. After the serum titer met the requirements, B cells were sorted. The operation process is referenced. Figure 1 A. Specific experimental results are as follows Figure 1 As shown, Figure 1 B is the ELISA result of serum titer detection after four immunizations. The data in the first column represent the reciprocal of the 11 dilutions of serum dilution, and the data in the second and third columns represent the absorbance values ​​of two parallel wells at OD450 wavelength. The results show that the titer of the four-immune serum reaches above 78W, and B cell sorting can be started.

[0089] Example 3: B cell sorting of immunized rabbits 10 mL of whole blood was collected from the immune-qualified rabbit described in Example 2 for isolation of PBMCs. PBMCs were first labeled with biotin-T-lymphocyte antibody (purchased from Bio-Rad), biotin-IgM antibody (purchased from BD Pharmingen), and biotin-CD11b antibody (purchased from STEMCELL Technologies). Streptavidin magnetic beads (purchased from Miltenyi Biotec) were then added and incubated on ice for 15 minutes. Finally, B cells were negatively sorted using a magnetic column.

[0090] 1x10^6 B cells were taken, CD45 recombinant protein was added and incubated on ice for 30 minutes. After incubation, the cells were washed three times with PBS, and then AF488 Donkey Anti-Rabbit IgG H&L (purchased from Biolegend) and StrepMAB-ImmoDY-649 (purchased from IBA) antibodies were added. After incubation on ice for 30 minutes, the cells were washed three times with PBS. Finally, CD45+IgG+ B cells were sorted by FACSAriaTMII flow cytometry into 15mL centrifuge tubes for subsequent single-cell sequencing. The specific results are shown as follows: Figure 2 As shown, the main cell population was first selected by forward scattered light (FSC-A) and side scattered light (SSC-A), and cell debris was removed (P1). Then, the adherent cells (P2 and P3) were removed by area (A) and height (H) of FSC and SSC, respectively. Then, IgG+ cells positive in the FITC channel were selected (P4), and finally, Ag+IgG+ cells positive in the APC channel were further selected (P5).

[0091] Example 4: Single B cell sequencing and antibody expression vector construction The sorted single B cells are subjected to single-cell sequencing. First, the sorted cells are centrifuged at 300g for 10 minutes, resuspended and counted, and then passed through a microfluidic chip together with the oil phase to generate oil-in-water droplets. Each droplet encapsulates a single cell, lysis reagent, and magnetic beads carrying unique molecular labels. Cell lysis, mRNA capture, and cDNA reverse transcription reactions are then completed in the droplets. After that, the emulsion is broken to recover the cDNA product, and BCR enrichment is performed. Finally, the cDNA library and BCR library are established for sequencing on the machine, and the single-cell data information is obtained for bioinformatics analysis. After obtaining the transcriptome data, immune repertoire BCR analysis was performed to obtain the antibody sequence, and the antibody sequence with light and heavy chain pairing was selected and sent to BGI Changzhou New Life Technology Co., Ltd. to synthesize the antibody expression vector plasmid.

[0092] Example 5: Expression and purification of recombinant antibodies Recombinant antibody expression: The antibody sequence was synthesized into the pCDNA3.4(+) expression vector. The expression plasmid containing the light and heavy chain encoding genes of the specific antibody obtained in Example 3 was extracted in large quantities. The light and heavy chain vectors were then co-transfected into 293F cells at a molar ratio of 3:2. The plasmids were mixed with PEI (POLYSCIENCE) at a ratio of 1:3. After mixing, the cells were allowed to stand at room temperature for half an hour before being added dropwise to the cells. SMS293-SUPI feed solution (Sino Biological) was added 24 and 72 hours after transfection. The cell supernatant was collected after 5 days.

[0093] Purification of recombinant antibodies: Collect cell supernatant and add 1 mL of Protein A filler (Sino Biological), incubate at room temperature for half an hour, remove filler and load into empty purification column, rinse with 20 mL of PBS solution, then add 10 mL of 100 mM glycine solution (pH 3.0) for elution. The eluate is neutralized to pH 7.0 by adding 1M Tris (pH 9.0) solution. The neutralized eluate is concentrated to 1 mL and dialyzed into PBS. The protein concentration is determined and some representative antibodies are run on SDS-PAGE for purity identification. The results are as follows: Figure 3 As shown, the antibody is highly pure and has a single band.

[0094] Example 6: BLI screening of binding antibodies and identification of antibody affinity Antibodies that bind to CD45 recombinant protein were preliminarily screened using biomembrane interferometry (BLI). Biomembrane interferometry (BLI) can monitor intermolecular interactions in real time, and the molecular change response is displayed as the relative displacement intensity (nm) of the interference spectrum. The experiment used protein A probe to capture the antibody (capture amount greater than 0.2nM), flow antigen, and PBST (0.2% Tween) as a buffer. The antibody was diluted to 5ug / mL, the antigen was diluted to 200nM, and Gator Primer flowed PBST, antibody, PBST, antigen, and PBST in sequence for binding and dissociation. PBST was added to each group of experiments as an association control to deduct the background changes in the relative displacement of the interference spectrum during the dissociation process. The 1:1 Binding model of the analysis software was used to calculate the kinetic parameters to confirm whether the antibody binds to the antigen and to determine the affinity KD of the antibody. The experimental results are shown in the figure. Figure 4 As shown, the affinity of the CD45-58 binding antibody (SEQ ID NO: 15, 16) of the present invention is K D (M) is less than 1.00E-12, and the affinity reaches the picomolar (pM) level.

[0095] Example 7: IHC test with combined antibodies First, formalin-fixed paraffin-embedded tissue samples were prepared into 3-μm-thick paraffin sections. After flattening in 40°C water, the sections were baked in a 60°C oven for 1 h. The sections were dewaxed by xylene dewaxing and treated with an alcohol gradient. Then, the antigens were repaired by EDTA microwave heat repair, and the peroxidase in the tissue was blocked with an endogenous peroxidase blocking solution. Then, goat serum was added dropwise to block the tissue, and the sections were incubated at 37°C for 30 min. The excess liquid was discarded, and different concentrations of primary antibodies (i.e., the above-mentioned antibody CD45-58 (SEQ ID NOs: 15, 16) discovered by the present invention) were added and incubated at room temperature for 1 h-2 h. In the NC group, PBS was used instead of the above-mentioned antibody. After the primary antibody incubation is completed, the excess primary antibody is washed with PBST, and the secondary antibody Polymer HRP Goatanti-mouse / rabbit lgG (Ready-to-Use) (purchased from Chongqing Xinyisheng Life Technology Co., Ltd., cat LS-PA-03015T) is added and incubated at room temperature for 30 minutes. The excess secondary antibody is washed away with PBST. Then, DAB color development solution is prepared for color development, and the color development time is controlled under a microscope. After washing with water to terminate the reaction, the cell nucleus is stained with hematoxylin and then back to blue. Finally, it is dehydrated with alcohol gradient and transparentized with xylene. After drying, the slides are sealed with neutral gum, and the staining results are observed and photographed with a high-power microscope. The results are as follows Figure 5 As shown, the CD45-58 rabbit recombinant monoclonal antibody of the present invention exhibited high positive rates and strong specific membrane localization in paraffin sections of multiple tissues, including tonsils and spleen. Its staining pattern (clear edges and continuous membrane signal) was consistent with that of the extensively validated commercial positive control antibody PC (Zhongshan Jinqiao ZM-0183). Under the same experimental conditions, compared to the positive control antibody, the CD45-58 rabbit recombinant monoclonal antibody showed higher staining intensity at high concentrations and no cytoplasmic or interstitial nonspecific background. Even at a low working concentration (0.1 μg / mL), the staining intensity was comparable to that of the positive control, demonstrating the core advantages of CD45-58 of the present invention in IHC detection, namely high specificity and low background interference.

[0096] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "an embodiment," or "a specific embodiment" means that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments. Furthermore, those skilled in the art may combine and integrate different embodiments and features of different embodiments described in this specification, unless otherwise inconsistent.

[0097] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that: include A heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence of a conservative modification thereof; A light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence of a conservative modification thereof; A light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence of a conservative modification thereof; and A light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence of a conservatively modified form thereof.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein It includes a heavy chain framework region and / or a light chain framework region, at least a portion of which is derived from at least one of a rabbit antibody, a mouse antibody, a human antibody, a primate antibody, a sheep antibody, a dog antibody, a cat antibody, and an alpaca antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region FR1 having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region FR2 having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region FR3 having the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence of a conservative modification thereof; A heavy chain variable region FR4 having the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence of a conservative modification thereof; A light chain variable region FR1 having the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence of a conservative modification thereof; A light chain variable region FR2 having the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence of a conservative modification thereof; A light chain variable region FR3 having the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence of a conservative modification thereof; and The light chain variable region FR4 has the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence of a conservatively modified form thereof.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein include: The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 80% identity thereto; and / or A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, or an amino acid sequence at least 80% identical thereto.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof further includes a heavy chain constant region and / or a light chain constant region, at least a portion of which is derived from at least one of a rabbit antibody, a mouse antibody, a human antibody, a primate antibody, a sheep antibody, a dog antibody, a cat antibody, and an alpaca antibody.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody comprises at least one selected from a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, an Fv antibody, a single-chain antibody, a single-domain antibody, and a minimum recognition unit; or the antigen-binding fragment comprises at least one selected from a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, an Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, and a minimum recognition unit.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 7.

9. A recombinant cell, characterized in that The recombinant cell comprises the nucleic acid molecule of claim 7, the expression vector of claim 8, or expresses the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

10. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The method comprises culturing the recombinant cell of claim 9.

11. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a coupling portion coupled thereto.

12. The antibody conjugate according to claim 11, characterized in that The coupling moiety is selected from a purification tag or a label.

13. The antibody conjugate according to claim 12, characterized in that The coupling moiety comprises at least one selected from colloidal gold, radioactive labels, phosphorescent chemicals, chemiluminescent agents, fluorescein, enzymes, natural toxins, nucleic acids, polypeptides and affinity labels; and / or The coupling part includes at least one selected from magnetic beads, magnetic microspheres, plastic microspheres, plastic particles, microporous plates, nylon and nitrocellulose membranes.

14. A reagent or kit, characterized in that The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or the antibody conjugate according to any one of claims 11 to 13.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the antibody conjugate according to any one of claims 11 to 13, or the reagent or kit according to claim 14 in detecting CD45, preparing a product for detecting CD45, or a product for diagnosing a CD45-related disease.

16. A method for detecting CD45 in a test sample, characterized in that: The method comprises contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the antibody conjugate according to any one of claims 11 to 13, or the reagent or kit according to claim 14 with the CD45 antigen in the sample to be detected to form an immune complex.

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