Anti-cd45 antibodies and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular, to an anti-CD45 antibody and application thereof. BACKGROUND

[0002] CD45 (Leukocyte Common Antigen) is a transmembrane protein tyrosine phosphatase that is essential for the immune system, and is widely expressed on the surface of all nucleated white blood cells, including T cells, B cells, natural killer cells (NK cells), macrophages and dendritic cells. As a core regulatory molecule of immune cell signaling, CD45 mediates the immune response of lymphocytes, and plays a key role in the development, activation, differentiation and tolerance of immune cells. Its structural diversity, functional complexity and association with diseases make it an important target for immunological research and clinical transformation.

[0003] Functionally, CD45 removes the inhibitory tyrosine residues (such as Tyr505 of Lck) at the C-terminus of Src family kinases (such as Lck and Fyn), thereby activating downstream signaling pathways. This process is crucial for T cell receptor (TCR) and B cell receptor (BCR) mediated immune response, and regulates the activation, proliferation and differentiation of T / B cells. In addition, CD45 is involved in the positive and negative selection of thymocytes, affects T cell maturation, and plays a role in cytokine production and immune response intensity regulation. Studies have shown that CD45 deficiency can cause severe immune dysfunction, and its abnormal expression in autoimmune diseases and leukemia also suggests its importance as a potential diagnostic marker or therapeutic target. The function of CD45 is environmentally dependent, and may exhibit a complex regulatory network through different isoforms or interacting molecules. Therefore, the development of CD45 antibodies is of great significance for immunological research and clinical diagnosis.

[0004] Currently, commercial CD45 antibodies have significant limitations. The mainstream products rely on traditional mouse hybridoma technology, and the production process is limited to animal-derived systems (such as ascites preparation), which has large batch-to-batch differences, animal ethics controversy and potential immunogenicity risks. Therefore, it is urgent to break through the technical barriers by innovative development strategies (such as non-animal source recombinant technology, single B cell sequencing technology) to shorten the research and development time and screen CD45 antibodies with high affinity, high specificity and low batch-to-batch difference. SUMMARY

[0005] The present application aims to at least partially solve at least one of the problems existing in the prior art.

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

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

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

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

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

[0011] In a sixth aspect, the present application provides an antibody conjugate. According to embodiments of the present application, the antibody conjugate comprises: the aforementioned antibody or antigen binding fragment thereof; and a conjugate moiety coupled to the antibody or antigen binding fragment thereof. The antibody conjugate of the present application can specifically recognize CD45, detect CD45, for example, IHC detection, and effectively detect CD45 and diagnose diseases related to abnormal expression of CD45.

[0012] In a seventh aspect, the present application provides a reagent or kit. According to embodiments of the present application, the reagent or kit comprises: the aforementioned antibody or antigen binding fragment thereof or the aforementioned antibody conjugate. The kit of the present application can specifically bind to CD45 and effectively detect CD45.

[0013] In an eighth aspect, the present application provides the use of the aforementioned antibody or antigen binding fragment thereof, the aforementioned antibody conjugate, or the reagent or kit in detecting CD45, preparing a product for detecting CD45, or diagnosing a CD45-related disease. As described above, the antibody or antigen binding fragment thereof according to embodiments of the present application is a high-affinity (pM level) rabbit recombinant antibody developed by single B cell sequencing and non-animal source recombinant technology, which can effectively detect CD45, for example, IHC detection.

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

[0015] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0017] Figure 1 Fig. 1 shows the animal immunization scheme and serum titer detection results after four immunizations according to embodiments of the present application, wherein:

[0018] Figure 1 A in Fig. 1 is the animal immunization scheme,

[0019] Figure 1 B is a serum titer detection result chart after four immunizations on experimental animals in the above-mentioned figure;

[0020] Figure 2 is a result chart of flow sorting B cells according to an embodiment of the present application, wherein P1 indicates that the main cell population is selected by forward scattering light (FSC-A) and side scattering light (SSC-A), and cell fragments are removed, P2 and P3 indicate that the adherent cells are removed by FSC and SSC area (A) and height (H), P4 indicates that lgG+ cells positive in the FITC channel are selected, and P5 indicates that Ag+lgG+ cells positive in the APC channel are selected;

[0021] Figure 3 is an SDS-PAGE detection result chart of recombinant antibodies according to an embodiment of the present application, wherein M indicates protein marker, +DTT indicates that the protein loading buffer contains reducing agent DTT, and -DTT indicates that the protein loading buffer does not contain reducing agent DTT;

[0022] Figure 4 is a binding antibody affinity determination result chart according to an embodiment of the present application, wherein the abscissa represents time (s), and the ordinate represents relative displacement height (nm);

[0023] Figure 5 is an IHC detection result chart of CD45-58 rabbit recombinant monoclonal antibodies on various tissue sections according to an embodiment of the present application. DETAILED DESCRIPTION

[0024] Embodiments of the present application are described in detail below with reference to examples shown in the attached drawings. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present application, and cannot be understood as limiting the present application.

[0025] It should be noted that the terms “first”, “second” are only used for description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with “first”, “second” can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of “a plurality of” is two or more.

[0026] In this document, the term “comprising” or “including” is an open expression, i.e., including the indicated content of the present application, but not excluding other aspects.

[0027] In this document, the terms "optionally," "optional," or "may" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs, and instances where it does not.

[0028] In this document, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide having an N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and / or an internal deletion.

[0029] In the present context, the terms "identity", "homology" or "similarity" are used when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, in terms of the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional means, e.g., 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, D.C.). There are a number of algorithms that can be used to align sequences and determine 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 for 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 BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are also available that use these algorithms to perform the comparison, including, but 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 from Intelligenetics, Mountain View, California.

[0030] In the present text, the term "at least 80% identity" means at least 80%, which can 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 each reference sequence.

[0031] In the present text, the term "expression vector" generally refers to a nucleic acid molecule that is capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The expression vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for the transcription and / or translation of expression of DNA or RNA. The expression vector also includes a vector having a plurality of the above-mentioned functions. The expression vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0032] In the present text, the term "recombinant cell" generally refers to a cell having a unique trait stably inherited by modification or recombination of the genetic material of a host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein means the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with a DNA sequence of the present application, and can be used for expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application 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.

[0033] An antibody or antigen-binding fragment thereof

[0034] In some embodiments, the present application provides an antibody or an antigen binding fragment thereof, which comprises at least one CDR selected from the group consisting of: CDR of heavy chain variable region: amino acid sequence of SEQ ID NO: 1~3 or conservatively modified form thereof; CDR of light chain variable region: amino acid sequence of SEQ ID NO: 4~6 or conservatively modified form thereof. The antibody or an antigen binding fragment thereof according to the embodiments of the present application is a high-affinity (pM level) rabbit recombinant antibody developed by single B cell sequencing and non-animal source recombinant technology, which can specifically recognize and detect CD45, for example, for IHC detection, and can effectively detect and diagnose diseases related to abnormal expression of CD45.

[0035] In the present disclosure, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, without limitation as to structure, so long as they exhibit the desired biological activity. It generally includes a light chain of relatively low molecular weight and a heavy chain of relatively high molecular weight, and the antibody molecule is formed by the heavy chain (H chain) and the light chain (L chain) connected by a disulfide bond. Among them, the amino terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called variable region (V region); the carboxyl terminal (C-terminal) is relatively stable and varies little, which is called constant region (C region). The V region of L chain and H chain is called VL and VH, respectively. As used herein, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, which refers to the region containing one or more or even all of the major amino acid residues that affect the binding affinity of the antibody or its functional fragment to the antigen or epitope it recognizes. In the specific embodiments of the present disclosure, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0036] In the present disclosure, the term "antigen binding fragment" is a fragment containing part or all of the antibody, which lacks at least some of the amino acids present in the full-length chain but still has the performance activity of specifically binding to the antigen, for example, the fragment can contain part or all of the CDR of the antibody. Such fragments have biological activity because they bind to antigens 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 antibody. 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).

[0037] 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.

[0038] 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:

[0039] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0040] 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;

[0041] 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;

[0042] 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;

[0043] 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;

[0044] 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

[0045] a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 6 or a conservatively modified version thereof.

[0046] According to some embodiments of the present application, the heavy chain variable region CDR1, CDR2, CDR3 and the light chain variable region CDR1, CDR2, CDR3 are defined by any one of the Kabat, Chothia, IMGT or AbM system. 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 described herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). See Chothia et al., J Mol Biol 196:901-917 (1987) for "Chothia definition". One of skill in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of an antibody.

[0047] According to some embodiments of the present application, the antibody or antigen binding fragment thereof comprises a heavy chain framework region and / or a light chain framework region.

[0048] According to some embodiments of the present application, at least a portion of the heavy chain framework region and / or the light chain framework region is from at least one of a rabbit-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, a sheep-derived antibody, a dog-derived antibody, a cat-derived antibody, and a llama-derived antibody, and a mutant thereof.

[0049] According to some embodiments of the present application, at least a portion of the heavy chain framework region and / or the light chain framework region is from a rabbit-derived antibody.

[0050] According to some embodiments of the present application, the heavy chain framework region and / or the light chain framework region comprises a FR selected from at least one of:

[0051] a heavy chain variable region FR: an amino acid sequence of SEQ ID NO: 7~10 or a conservatively modified version thereof;

[0052] a light chain variable region FR: an amino acid sequence of SEQ ID NO: 11~14 or a conservatively modified version thereof.

[0053] According to some embodiments of the application, the antibody or antigen-binding fragment thereof comprises:

[0054] a heavy chain variable region FR1 having an amino acid sequence of SEQ ID NO: 7 or a conservatively modified version thereof;

[0055] a heavy chain variable region FR2 having an amino acid sequence of SEQ ID NO: 8 or a conservatively modified version thereof;

[0056] a heavy chain variable region FR3 having an amino acid sequence of SEQ ID NO: 9 or a conservatively modified version thereof;

[0057] a heavy chain variable region FR4 having an amino acid sequence of SEQ ID NO: 10 or a conservatively modified version thereof;

[0058] a light chain variable region FR1 having an amino acid sequence of SEQ ID NO: 11 or a conservatively modified version thereof;

[0059] a light chain variable region FR2 having an amino acid sequence of SEQ ID NO: 12 or a conservatively modified version thereof;

[0060] a light chain variable region FR3 having an amino acid sequence of SEQ ID NO: 13 or a conservatively modified version thereof; and

[0061] a light chain variable region FR4 having an amino acid sequence of SEQ ID NO: 14 or a conservatively modified version thereof.

[0062] According to some embodiments of the application, 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 systems of Kabat, Chothia, IMGT or AbM.

[0063] Likewise, it will be appreciated by a person skilled in the art that, given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a particular FR.

[0064] According to some embodiments of the application, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence at least 80% identical thereto; and / or a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence at least 80% identical thereto.

[0065] According to some embodiments of the present application, the antibody or antigen binding fragment thereof further comprises a constant region.

[0066] According to some embodiments of the present application, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0067] According to some embodiments of the present application, at least a portion of the heavy chain constant region and / or the light chain constant region is from at least one of a rabbit-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, a sheep-derived antibody, a dog-derived antibody, a cat-derived antibody, and a llama-derived antibody, and mutants thereof.

[0068] According to some embodiments of the present application, at least a portion of the heavy chain constant region and / or the light chain constant region is from at least one of a rabbit-derived antibody, a murine-derived antibody, a human-derived antibody, and a primate-derived antibody.

[0069] According to some embodiments of the present application, the heavy chain constant region comprises a heavy chain constant region selected from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; or the light chain constant region comprises a light chain constant region selected from kappa type or lambda type.

[0070] According to some embodiments of the present application, 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.

[0071] According to some embodiments of the present application, the antibody comprises at least one selected from a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a Fv antibody, a single-chain antibody, a single-domain antibody, and a minimal 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, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, and a minimal recognition unit.

[0072] In this context, the terms "full-length antibody", "full-length monoclonal antibody", or "full-length monoclonal antibody" each refer to an antibody formed by 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).

[0073] In the present context, the terms "single domain antibody", "nanobody" and "VHH antibody" are used interchangeably and were originally described as antigen binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e. "antibodies devoid of 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 antigenic protein (e.g. CD45) via the heavy chain variable region.

[0074] In the present context, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment comprising only a Fab molecule, which is composed of the VH and CH1 of a heavy chain and an entire light chain, which are connected by one disulfide bond between the light and heavy chain.

[0075] In the present context, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen binding F(ab') parts which are connected together by disulfide bonds.

[0076] In the present context, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment which is composed of only a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody which retains the entire antigen binding site.

[0077] In the present context, the term "single chain antibody" or "scFv fragment" is an antibody or fragment which is composed of an antibody heavy chain variable region and a light chain variable region connected by a short peptide.

[0078] In the present context, the terms "minimal recognition unit" and "MRU" both refer to an antibody or fragment which is composed of only one CDR, and has a molecular weight which is only about 1% of a complete antibody.

[0079] Nucleic acid molecules, expression vectors and recombinant cells

[0080] In some embodiments, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes an antibody or antigen binding fragment thereof as previously described. The antibody or antigen binding fragment thereof encoded by the nucleic acid molecule according to embodiments of the present application can efficiently and specifically recognize CD45, detect CD45, for example, for IHC detection, and can also effectively detect CD45 and diagnose diseases related to abnormal expression of CD45.

[0081] According to some embodiments of the present application, the nucleic acid molecule is DNA.

[0082] It should be noted that, for the nucleic acid molecule mentioned herein, it is understood by those skilled in the art that either one of the complementary double strands, or both, are actually included. For the convenience, in the present specification and claims, although only one strand is given in most cases, the other complementary strand is actually disclosed. In addition, the nucleic acid sequence in the present application includes either DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0083] In some embodiments, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the aforementioned nucleic acid molecule. In linking the aforementioned nucleic acid molecule to the vector, the nucleic acid molecule 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 acid molecule, 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, the nucleic acid molecule is operably linked to the control elements. "Operably linked" herein means that the exogenous gene is linked to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be plasmids, bacteriophages, etc. According to some embodiments of the present application, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned antibody or antigen-binding fragment thereof can be effectively realized under the mediation of the regulation system, and then the antibody or antigen-binding fragment thereof can be obtained in large quantities in vitro.

[0084] According to some embodiments of the present application, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0085] According to some embodiments of the present application, the expression vector is a plasmid expression vector.

[0086] In some embodiments, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell includes: carrying the aforementioned nucleic acid molecule or expression vector or expressing the aforementioned antibody or antigen-binding fragment thereof. Using the recombinant cell under suitable conditions, the aforementioned antibody or antigen-binding fragment thereof can be effectively expressed in the cell.

[0087] It is to be noted that the "suitable condition" described in the present application is a condition suitable for expression of the antibody or antigen-binding fragment thereof of the present application. It is readily understood by those skilled in the art that the condition suitable for expression of the antibody or antigen-binding fragment thereof includes, but is not limited to, a suitable transformation or transfection method, a suitable transformation or transfection condition, a healthy host cell state, a suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. The "suitable condition" is not particularly limited, and those skilled in the art can optimize the most suitable condition for expression of the antibody or antigen-binding fragment thereof according to the specific environment of the laboratory.

[0088] According to some embodiments of the present application, the recombinant cell is a eukaryotic cell

[0089] According to some embodiments of the present application, the recombinant cell is a mammalian cell.

[0090] Conjugate and kit

[0091] In some embodiments, the present application provides an antibody conjugate. According to embodiments of the present application, the antibody conjugate comprises: the aforementioned antibody or antigen-binding fragment thereof; and a conjugate moiety conjugated thereto. The antibody conjugate of the present application can specifically recognize CD45, can detect CD45 protein, and can effectively diagnose diseases related to abnormal expression of CD45.

[0092] According to some embodiments of the present application, the conjugate moiety is selected from a purification tag or label.

[0093] According to some embodiments of the present application, the conjugate moiety comprises at least one selected from the group consisting of colloidal gold, a radioactive label, a phosphorescent chemical agent, a chemiluminescent agent, a fluorescein, an enzyme, a natural toxin, a nucleic acid, and an affinity label.

[0094] According to some embodiments of the present application, the conjugate moiety comprises a radioisotope.

[0095] According to some embodiments of the present application, the conjugate moiety comprises at least one selected from the group consisting of phycoerythrin, a fluorophore, rhodamine, luciferase, fluorescein isothiocyanate, green fluorescent protein, blue fluorescent protein, and red fluorescent protein.

[0096] According to some embodiments of the present application, the conjugate moiety comprises at least one selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.

[0097] According to some embodiments of the present application, the coupling moiety comprises at least one selected from the group consisting of biotin and avidin.

[0098] According to some embodiments of the present application, the coupling moiety comprises at least one selected from the group consisting of magnetic beads, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, nylon, and nitrocellulose membranes.

[0099] In the present specification, the coupling moiety can be a substance (e.g., a particle, a magnetic bead, or the like solid phase carrier) that can be suspended or dispersed in a liquid phase, or a solid phase (e.g., a plate, a membrane, a test tube, or the like support, and a container of a well plate, a microchannel, a glass capillary, a nanocolumn, a monolithic column, or the like) that can accommodate or carry a liquid phase; or a labeling carrier for labeling an antibody or an antigen-binding fragment thereof, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of a nucleic acid of a sense strand and an antisense strand complementary to each other), 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'-bipyridyl)ruthenium, luminol), a radioisotope (e.g., 3H, 14C, 32P, 35S, 125I), and a gold colloid, or the like.

[0100] According to some embodiments of the present application, 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, and a C-Myc tag, or the like.

[0101] It should be noted that the method of binding the coupling moiety and the antibody or the antigen-binding fragment thereof can use a method known in the art. For example, a physical adsorption method, a covalent binding method, a method using an affinity substance (e.g., biotin, streptavidin), and an ionic binding method can be mentioned.

[0102] In some embodiments, the present application provides a reagent or a kit. According to embodiments of the present application, the reagent or the kit comprises: 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 known from the foregoing, the aforementioned antibody or antigen binding fragment thereof can bind to CD45, the aforementioned antibody or antigen binding fragment thereof can specifically bind to CD45, in addition, under suitable conditions, the nucleic acid molecule, the expression vector, the recombinant cell or the antibody conjugate can all express the antibody or antigen binding fragment thereof, further, the kit comprising the above-mentioned substances can effectively bind to CD45 and can be used for effectively detecting CD45. The kit can be used for scientific research, such as qualitative or quantitative detection of CD45 in a biological sample, and can also be used for judging the state of the subject, such as judging whether the CD45 level of the subject is higher or lower than the normal level after obtaining the CD45 level of the subject. The biological sample can be cells, tissues, blood, etc.

[0103] Use

[0104] In some embodiments, the present application provides 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 diagnosing a CD45-related disease.

[0105] Method

[0106] In some embodiments, the present application provides a method for detecting CD45 in a test sample, the method comprising: contacting the test sample to be detected with the aforementioned antibody or antigen binding fragment thereof, antibody conjugate or the aforementioned reagent or kit to form an immune complex.

[0107] According to some specific embodiments of the present application, the test sample comprises cells, tissues, blood, etc.

[0108] According to some specific embodiments of the present application, based on the signal of the immune complex, it is determined whether the test sample to be detected contains CD45 or the content of the CD45.

[0109] According to some specific embodiments of the present application, the immune complex further comprises a second antibody, and the second antibody binds to the antibody or antigen binding fragment thereof.

[0110] According to some specific embodiments of the present application, the immune complex further comprises a second antibody, and the second antibody binds to CD45.

[0111] The nucleic acid and amino acid sequences involved in the present application are shown in Table 1.

[0112] Table 1

[0113]

[0114] The schemes of the present application will be explained below with reference to examples. Those skilled in the art will appreciate that the examples below are for illustration only and should not be taken as limiting the scope of the present application. Unless otherwise indicated, the techniques or conditions in the examples are in accordance with those described in the literature or as per the product manual. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained commercially.

[0115] The 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 skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 2011), each of which is incorporated herein by reference in its entirety.

[0116] In the embodiments of the present application, the nucleotide sequence used for preparing the expression vector can be obtained according to its amino acid sequence by using a conventional method or a conventional software (such as the online program Vectorbuilder, the online program GeneOptimizer, etc.).

[0117] Example 1: Gene synthesis and protein expression

[0118] In this embodiment, a CD45 recombinant protein (its sequence number is uniprot P08575) is prepared as an immunogen, and thus a CD45_ pcDNA3.4 expression vector is constructed based on the coding nucleic acid of the CD45 recombinant protein (the gene synthesis is performed by the synthesis platform of Huada Changzhou Xin Yichuan Life Science and Technology Co., Ltd.), the 3' end of the CD45 protein (G25-S576) coding region in the expression vector is labeled with a Twin-Strep-Tag tag, the codon optimization is performed according to the codon bias of the Homo sapiens species, and the expression vector is cloned into a pcDNA3.4 expression vector through XbaI and HindIII restriction enzyme cutting sites. After the plasmid CD45_ pcDNA3.4 is transformed into Escherichia coli DH5α (purchased from TIANGEN), a large amount of plasmid extraction is performed, and then the plasmid is transfected into Expi293FTM cells (purchased from Thermo) by PEI (purchased from POLYSCIENCE) for protein expression. The cell supernatant containing the target protein CD45 recombinant protein is harvested after 5 days of transfection, and after purification by Strep column affinity chromatography (Strep-Tactin XT column, IBA) and gel filtration chromatography (SuperoseTM 6 Increase 10 / 300 GL, GE), a relatively pure target protein is obtained.

[0119] Example 2: Animal immunization

[0120] In this embodiment, the immunogen CD45 recombinant protein obtained in Example 1 is used for animal immunization, and the specific operation is as follows:

[0121] The immunogen CD45 recombinant protein is mixed with an adjuvant, and then injected into a New Zealand white rabbit for immunization, wherein a Freund's complete adjuvant (Freund's complete adjuvant: sigma, cat F5881) is used for the first immunization, and a Freund's incomplete adjuvant (sigma, cat F5506) is used for subsequent immunization, and the mixing ratio is 1:1 of the immunogen and the adjuvant. After multiple rounds of immunization of the rabbit, blood is collected from the ear, serum is separated, and antibody titer detection in the serum is performed by ELISA, and after the serum titer is qualified, B cell sorting is performed, and the operation process is referred to Figure 1 A, and the specific experimental results are shown in Figure 1 , wherein, Figure 1B 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.

[0122] Example 3: B cell sorting of immunized rabbits

[0123] 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.

[0124] 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).

[0125] Example 4: Single B cell sequencing and antibody expression vector construction

[0126] 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.

[0127] 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.

[0128] Example 5: Expression and purification of recombinant antibodies

[0129] 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.

[0130] 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.

[0131] Example 6: BLI screening of binding antibodies and identification of antibody affinity

[0132] The antibodies binding to CD45 recombinant protein were screened by bio-layer interferometry (BLI). The bio-layer interferometry (BLI) can monitor the interaction between molecules in real time, and the molecular change reaction is shown by the relative displacement intensity (nm) of the interference spectrum. The experiment used protein A probe to capture the antibody (the capture amount was greater than 0.2 nM), the flowing antigen, and PBST (0.2% Tween) as the buffer. The antibody was diluted to 5 ug / mL, the antigen was diluted to 200 nM, and the Gator Primer was sequentially flowed PBST, antibody, PBST, antigen, and PBST for binding and dissociation. PBST was added as a control for association in each group of experiments, which was used to deduct the background change of the relative displacement of the interference spectrum during dissociation. 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, and the experimental results are shown in Figure 4 The CD45-58 binding antibody (SEQ ID NO: 15, 16) of the present application has an affinity K D (M) less than 1.00E-12, the affinity reaches the picomolar (pM) level.

[0133] Example 7: IHC test of the binding antibody

[0134] First, the formalin-fixed paraffin-embedded tissue sample was made into a 3-micron-thick paraffin section, which was flattened at a water temperature of 40°C and baked in an oven at 60°C for 1 h. The section was dewaxed by xylene and treated by alcohol gradient, then the antigen was repaired by EDTA microwave heating, and the endogenous peroxidase was blocked by peroxidase blocking solution, then the tissue was blocked by adding goat serum, incubated at 37°C for 30 min, and the excess liquid was shaken off. Different concentrations of primary antibody (i.e., the above-mentioned antibody CD45-58 (SEQ ID NO: 15, 16) discovered by the present application) were added for incubation, and incubated at room temperature for 1-2 h. The NC group was replaced by PBS instead of the above-mentioned antibody. After the incubation of the primary antibody, the excess primary antibody was washed with PBST, and the secondary antibody Polymer HRP Goat anti-mouse / rabbit lgG (Ready-to-Use) (purchased from Chongqing Xin Yisheng Life Science and Technology Co., Ltd., cat LS-PA-03015T) was added and incubated at room temperature for 30 min, and the excess secondary antibody was washed with PBST. Then, DAB developing solution was configured for color development, and the color development time was observed and controlled under a microscope. After water washing to terminate the reaction, the cell nucleus was stained by hematoxylin, and the blueing treatment was performed. Finally, the dehydration was performed by alcohol gradient, the transparency was performed by xylene, and the section was dried after airing, and the staining results were observed and photographed by high-power lens after the section was sealed by neutral gum. The results are shown in Figure 5As shown, the CD45-58 rabbit recombinant monoclonal antibody of the present application exhibits high positive rate and strong specific membrane localization in paraffin sections of multiple tissues such as tonsil, spleen, etc., and its staining pattern (clear edge, continuous membrane signal) is consistent with the results of the widely verified commercial positive control antibody PC (Zhongshanjinqiao ZM-0183). Under the same experimental conditions, the rabbit recombinant monoclonal antibody CD45-58 has higher staining intensity at high concentration than the positive control antibody, and has no cytoplasmic or interstitial non-specific background, and still maintains the staining intensity comparable to the positive control at a low working concentration (0.1 μg / mL), indicating that the CD45-58 in the present application has the core advantages of high specificity and low background interference in IHC detection.

[0135] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "embodiment" or "specific embodiment" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment are included in at least one embodiment of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments. In addition, the person skilled in the art can combine and combine the different embodiments and the features of the different embodiments described in the present specification without contradiction.

[0136] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. An anti-CD45 antibody or antigen-binding fragment thereof, characterized in that, a heavy chain variable region CDR1 of the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain variable region CDR2 of the amino acid sequence set forth in SEQ ID NO: 2; a heavy chain variable region CDR3 of the amino acid sequence set forth in SEQ ID NO: 3; a light chain variable region CDR1 of the amino acid sequence set forth in SEQ ID NO: 4; a light chain variable region CDR2 of the amino acid sequence STS; and a light chain variable region CDR3 of the amino acid sequence set forth in SEQ ID NO:

6. comprises a heavy chain framework region and a light chain framework region, at least a portion of the heavy chain framework region and / or the light chain framework region being from at least one of a rabbit-derived antibody, a murine-derived antibody, a primate-derived antibody, a goat-derived antibody, a dog-derived antibody, a cat-derived antibody, and a llama-derived antibody.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, comprises a heavy chain framework region and a light chain framework region, at least a portion of the heavy chain framework region and / or the light chain framework region being from a human-derived antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof comprises:

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, a heavy chain variable region FR1 of the amino acid sequence set forth in SEQ ID NO: 7; a heavy chain variable region FR2 of the amino acid sequence set forth in SEQ ID NO: 8; a heavy chain variable region FR3 of the amino acid sequence set forth in SEQ ID NO: 9; a heavy chain variable region FR4 of the amino acid sequence set forth in SEQ ID NO: 10; a light chain variable region FR1 of the amino acid sequence set forth in SEQ ID NO: 11; a light chain variable region FR2 of the amino acid sequence set forth in SEQ ID NO: 12; a light chain variable region FR3 of the amino acid sequence set forth in SEQ ID NO: 13; and a light chain variable region FR4 of the amino acid sequence set forth in SEQ ID NO:

14. comprises:

5. The antibody or antigen-binding fragment thereof of claim 1, wherein, a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 15; a light chain variable region of the amino acid sequence set forth in SEQ ID NO:

16. The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, at least a portion of the heavy chain constant region and / or the light chain constant region being from at least one of a rabbit-derived antibody, a murine-derived antibody, a primate-derived antibody, a goat-derived antibody, a dog-derived antibody, a cat-derived antibody, and a llama-derived antibody.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, at least a portion of the heavy chain constant region and / or the light chain constant region being from a human-derived antibody.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody comprises at least one selected from the group consisting of a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a Fv antibody, and a single-chain antibody; or the antigen-binding fragment comprises at least one selected from the group consisting of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, and a scFv-Fv fusion protein.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof of any one of claims 1-8.

9. A nucleic acid molecule, characterized in that, comprises the nucleic acid molecule of claim 9.

10. An expression vector, characterized in that, ​ 11. A recombinant cell, characterized in that The recombinant cell comprises the nucleic acid molecule of claim 9, the expression vector of claim 10, or expresses the antibody or antigen-binding fragment thereof of any one of claims 1-8.

12. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that: The method comprises culturing the recombinant cell of claim 11.

13. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody or antigen-binding fragment thereof of any one of claims 1-8 and a conjugating moiety coupled thereto, the conjugating moiety being selected from a purification tag or label.

14. The antibody conjugate of claim 13, wherein, The conjugating moiety comprises at least one selected from a nucleic acid, a polypeptide, and a magnetic microsphere.

15. The antibody conjugate of claim 13, wherein, The conjugating moiety comprises at least one selected from colloidal gold, a radioactive label, a phosphorescent chemical agent, a chemiluminescent agent, a fluorescein, an enzyme, and a natural toxin.

16. The antibody conjugate of claim 13, wherein, The conjugating moiety comprises an affinity label.

17. The antibody conjugate of claim 13, wherein, The conjugating moiety comprises at least one selected from a magnetic bead, a plastic microsphere, a plastic microparticle, a microwell plate, nylon, and a nitrocellulose membrane.

18. A reagent or kit characterized in that, The reagent or kit comprises the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the antibody conjugate of any one of claims 13-17.

19. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8, the antibody conjugate of any one of claims 13-17, or the reagent or kit of claim 18 in the manufacture of a product for detecting CD45.

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