Anti-CD45 antibody for detecting white blood cell common antigen based on flow cytometry and application of anti-CD45 antibody

Abstract

The invention relates to the technical field of antibodies, and provides an anti-CD45 antibody for detecting a white blood cell common antigen based on flow cytometry and application of the anti-CD45 antibody, 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, and can effectively detect the CD45.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an anti-CD45 antibody for detecting leukocyte common antigens based on flow cytometry 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] CD45 consists of a heavily glycosylated extracellular domain, a single transmembrane transmembrane domain, and an intracellular domain. The N-terminal extracellular domain is selectively spliced ​​at exons 4, 5, and 6 to form multiple isoforms (e.g., CD45RA and CD45RO). These variants differ in extracellular domain length and glycosylation patterns, affecting immune cell localization, migration, and signaling properties. The extracellular domain also consists of three membrane-proximal fibronectin type II domains and a cysteine-rich domain. The intracellular domain contains two tandem tyrosine phosphatase domains, only the first of which is catalytically active.

[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. Furthermore, high-performance antibodies, such as rabbit-derived monoclonal antibodies, are scarce in the domestic market, and some multinational companies have a monopoly on the supply of key raw materials. Therefore, innovative development strategies (such as non-animal-derived recombinant technologies 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 a first aspect, the present invention provides an antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises a CDR selected from at least one of the following: a heavy chain variable region CDR: an amino acid sequence of SEQ ID NOs: 1-3 or conservatively modified forms thereof; a light chain variable region CDR: an amino acid sequence of SEQ ID NOs: 4-6 or conservatively modified forms thereof. The antibody or antigen-binding fragment thereof according to an embodiment of the present invention can efficiently and specifically recognize CD45, can effectively detect CD45, and can 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, 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 its sixth aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the aforementioned antibody or antigen-binding fragment thereof; and a coupling moiety coupled thereto, wherein the coupling moiety is linked to the antibody or antigen-binding fragment thereof. The conjugate of the present invention can specifically recognize CD45, effectively detecting CD45 and diagnosing 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 an eighth aspect, 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 CD45-related diseases.

[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 protein purification and identification results according to an embodiment of the present invention, wherein: Figure 1 Figure A shows the results of cell supernatant purification by Strep column affinity chromatography. SDS-PAGE identification results show that the size of CD45 recombinant protein is about 140KD. Figure 1 Figure B shows the purification results of the protein after initial purification by gel filtration chromatography. The SDS-PAGE identification results show that the red box is the target protein and some aggregated proteins have been removed. Figure 2 This is a diagram showing the animal immunization scheme according to an embodiment of the present invention and the serum titer test results after three immunizations, wherein: Figure 2 A in the figure is the animal immunization program process. Figure 2 B in the figure is the result of serum titer test after three immunizations of experimental animals; Figure 3Figure 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 P6 indicates the selection of Ag+IgG+ cells positive in the APC channel. Figure 4 This is a graph showing the purity identification results of the CD45-P2-91 protein 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 5 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 6 Graph showing flow cytometry test results of the rabbit recombinant monoclonal antibody CD45-P2-91 in Daudi cells and Jurkat cells according to an embodiment of the present invention; Figure 7 This is a graph showing the results of staining of PBMC cells with the rabbit recombinant monoclonal antibody CD45-P2-91 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%, and 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] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0028] 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 antibodies or antigen-binding fragments thereof according to some specific embodiments of the present invention can efficiently and specifically recognize CD45, effectively detecting CD45 and diagnosing diseases associated with abnormal CD45 expression.

[0029] As used herein, the term "antibody" is used in the broadest sense to include 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 the 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.

[0030] 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 have biological activity 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 cleavage or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[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 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.

[0036] 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.

[0037] 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.

[0038] 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 conservatively modified form 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0043] 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.

[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, a primate antibody, a sheep antibody, a dog antibody, a cat antibody, an alpaca antibody, and mutants thereof.

[0045] 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.

[0046] 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.

[0047] According to some specific embodiments of the present invention, the heavy chain constant region and / or light chain constant region is derived from a rabbit antibody or a mutant thereof.

[0048] 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.

[0049] 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.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 molecule with a complete antigen binding site.

[0055] 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.

[0056] 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.

[0057] 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, and can effectively detect CD45 and diagnose diseases associated with abnormal CD45 expression.

[0058] According to some specific embodiments of the present invention, the nucleic acid molecule is DNA.

[0059] 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.

[0060] 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.

[0061] According to some specific embodiments of the present invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0062] According to some specific embodiments of the present invention, the expression vector is a plasmid expression vector.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Conjugates and kits In some embodiments, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the aforementioned antibody or antigen-binding fragment thereof; and a conjugated moiety conjugated thereto. The conjugate of the present invention can specifically recognize CD45, effectively detecting CD45 and diagnosing diseases associated with abnormal CD45 expression.

[0067] According to some specific embodiments of the present invention, the coupling moiety is selected from a purification tag or a label.

[0068] 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.

[0069] According to some specific embodiments of the invention, the coupling moiety comprises a radioisotope.

[0070] 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.

[0071] 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.

[0072] According to some specific embodiments of the present invention, the coupling moiety includes at least one selected from biotin and avidin.

[0073] 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.

[0074] 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, luciferin, 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., phycoerythrin, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, blue 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.

[0075] 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.

[0076] 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.

[0077] 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 conjugate. As mentioned above, the aforementioned antibody or antigen-binding fragment thereof can specifically bind to CD45. In addition, under appropriate conditions, the nucleic acid molecule, expression vector or recombinant cell can express the antibody or antigen-binding fragment thereof. Furthermore, the reagent or 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.

[0078] 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.

[0079] 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 or the aforementioned kit with the sample to be tested to form an immune complex.

[0080] 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.

[0081] According to some specific embodiments of the present invention, the test sample includes cells, tissues, blood, etc.

[0082] According to some specific embodiments of the present invention, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0083] According to some specific embodiments of the present invention, the immune complex further comprises a second antibody, which binds to CD45.

[0084] The nucleic acid and amino acid sequences involved in this application are shown in Table 1.

[0085] Table 1

[0086] 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.

[0087] 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.

[0088] 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.).

[0089] 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 nucleic acid encoding the CD45 recombinant protein, a CD45_pcDNA3.4 expression vector was constructed (gene synthesis was performed by the synthesis platform of BGI Changzhou New Life 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. Plasmid CD45_pcDNA3.4 was transformed into Escherichia coli DH5α (purchased from TIANGEN) and the plasmid was fully extracted. The plasmid was then transfected into Expi293F™ cells (purchased from Thermo Fisher Scientific) using PEI (purchased from POLYSCIENCE) for protein expression. Five days after transfection, the cell supernatant containing the target CD45 recombinant protein was harvested. The results are shown in Figure 2. Figure 1 As shown, Figure 1 Figure A shows the results of cell supernatant purification by Strep column affinity chromatography (the horizontal axis is 0, 5, 10, 15, 20, 25, 30, 35, 40 ml, and the vertical axis is mAU). SDS-PAGE identification shows that the size of CD45 recombinant protein is about 140KD. Figure 1 Figure B is the purification result of the initially purified protein after gel filtration chromatography (the horizontal axis is 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 ml, and the vertical axis is mAU). The SDS-PAGE identification results show that the red box is the target protein, and some aggregated proteins are removed, that is, the cell supernatant is purified by Strep column affinity chromatography (Strep-Tactin XT column, IBA) and gel filtration chromatography (SuperoseTM 6 Increase10 / 300GL, GE) to obtain a relatively pure target protein.

[0090] 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 rabbits' 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 specific experimental results are shown in the figure. Figure 2 As shown, Figure 2 The first row of data in Figure B represents the reciprocals of the 11 dilutions of serum, and the second and third rows of data represent the absorbance values ​​of two parallel dilution wells of immune serum at OD450 wavelength. The results show that the titer of the three immune sera reaches above 78W. The OD of the serum samples at the same dilution is less than 0.1, which does not affect the experimental results.

[0091] 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.

[0092] 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 3 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 (P6).

[0093] Example 4: Single B cell sequencing and antibody expression vector construction The sorted single B cells were subjected to single-cell sequencing using a platform based on BGI's independently developed C4 single-cell sequencing platform (DNBelab C Series High-Throughput Single-Cell RNA Library Preparation Kit). The sorted cells were first centrifuged at 300g for 10 minutes, resuspended, counted, and mixed with the relevant reagents for the reverse transcription reaction to form the cell phase. Large magnetic beads containing label information, Cell Beads, and small magnetic Index Carrier beads were then used as the magnetic bead phase. Using a negative pressure droplet microfluidics system, the cell and magnetic bead phases were encapsulated in oil droplets, generating oil-in-water droplets containing the single cells. Cell lysis, mRNA capture, and cDNA reverse transcription reactions were then performed within the droplets. After that, the emulsion was broken to recover the cDNA intermediate and oligo products, and the cDNA library, oligo library, and BCR library were constructed respectively. Finally, the prepared library products were subjected to DNB circularization (BGI) using the MGIEasy circularization kit and sequenced using the MGISEQ-2000RS high-throughput sequencing reagent (PE150). The sequencing data were then subjected to bioinformatics analysis.

[0094] 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.

[0095] Example 5: Expression and purification of recombinant antibodies Recombinant antibody expression: The antibody sequences (SEQ ID NOs: 15 and 16) were synthesized and incorporated into the pCDNA3.4(+) expression vector. The expression plasmid containing the light and heavy chain encoding genes for the specific antibody obtained in Example 3 was then extracted in bulk. The vectors containing the light and heavy chain variable regions 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 the mixture was allowed to stand at room temperature for half an hour, the cells were added dropwise. SMS293-SUPI feed solution (Sino Biological) was added 24 and 72 hours after transfection, and the cell supernatant was collected after 5 days.

[0096] Purification of recombinant antibodies: Collect cell supernatant and add 1 mL of Protein A filler (Sino Biologics). 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. Neutralize the eluate with 1M Tris (pH 9.0) solution to pH 7.0. Concentrate the neutralized eluate to 1 mL and dialyze into PBS. Determine protein concentration and run some representative antibodies on SDS-PAGE for purity identification. The results are as follows: Figure 4 shown.

[0097] 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 5μg / 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 5 As shown, the affinity of CD45-P2-91 (SEQ ID NO: 15, 16) involved in the present invention K D (M) is less than 1.00E-12, and the affinity reaches the picomolar (pM) level.

[0098] Example 7: Flow cytometry testing of cell lines using antibodies Cell preparation: In this example, the full-length CD45 sequence (M1-S1306) was constructed into the pEGFP-N1 vector by BGI Changzhou New Life Science Technology Co., Ltd. After plasmid extraction, the CD45_pEGFP-N1 plasmid was transfected into Expi293F™ cells using PEI. After two days of culture, 293F-CD45-overexpressing cells were obtained for flow cytometry analysis. Daudi cells (human Burkitt's lymphoma, a B lymphoblastoid cell line) and Jurkat cells (human acute T lymphoblastic leukemia, an immortalized human T lymphocyte line) were revived in a 37°C water bath, expanded, and passaged for flow cytometry analysis. 2. Cell washing: CD45-overexpressing cells with obvious green fluorescence or Daudi and Jurkat cells in the rapid proliferation phase were used for flow cytometry analysis. Cells were collected into 15 mL centrifuge tubes, centrifuged at 300 g for 5 min at room temperature, and resuspended in PBS. The cell density of each sample was 1E6.

[0099] 3. Rabbit antibody staining: Prepare rabbit antibody at a working concentration of 1 μg / mL for staining cells and incubate at 37°C in the dark for 30 minutes. 4. Cell washing: Centrifuge at 300 g for 5 min at room temperature, discard the supernatant, and wash the cells of each sample with PBS. Repeat the above steps twice. 5. Secondary antibody staining: Stain cells with anti-rabbit IgG / AF647 (Biolegend) at a ratio of 1:1000 and incubate at 37°C in the dark for 30 minutes. 6. Cell washing: Centrifuge at 300 g for 5 min at room temperature, discard the supernatant, and wash the cells with 200 μL of PBS for each sample. Repeat the above steps twice. 7. Analyze the washed cells using flow cytometry.

[0100] The experimental results are as follows Figure 6 As shown, the horizontal axis of the 293F-CD45 overexpression cell line result is FITC, and the values ​​are 0, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , the vertical axis is APC-A, and the values ​​are 0, 10 3 , 10 4 , 10 5 , 10 6 In the Daudi cell results, the horizontal axis is APC-A, and the values ​​are 0, 10 3 , 10 4 , 10 5 , 10 6 The vertical axis is FSC-A, and the values ​​are 50K, 100K, 150K, and 200K. The horizontal axis in the Jurkat cell result graph is APC-A, and the values ​​are 0, 10 3 , 10 4 , 10 5 , 10 6The vertical axis is FSC-A, and the values ​​are 50K, 100K, 150K, and 200K. The flow cytometry results showed that CD45-P2-91 (SEQ ID NO: 15, 16) exhibited a higher cell staining ratio and lower background signal in the 293F-CD45 overexpressing cell line, the B cell line Daudi, and the T cell line Jurkat, indicating that CD45-P2-91 can be used as a specific flow cytometry antibody in flow cytometry experiments.

[0101] Example 8: Flow cytometry testing of binding antibodies in human PBMC samples Cell Thawing: Remove PBMCs (Aoneng Biotech, PB005F-C) from the liquid nitrogen tank and quickly place in a 37°C water bath with occasional shaking to completely thaw within 1–2 minutes. Pipette the PBMCs into a 15 mL centrifuge tube containing 5 mL of 1640 medium (containing 10% FBS) and centrifuge at 400 g for 10 minutes. Discard the supernatant, add 5 mL of PBS or antibody diluent to wash the cells, and centrifuge at 400 g for 5 minutes. Discard the supernatant, reconstitute the cells with 2 mL of antibody diluent, and add 100 μL per well (i.e., 5E5 cells) to a 96-well plate.

[0102] 2. Dilute the primary antibody to a working concentration of 1 μg / mL in PBS buffer (containing 3% BSA). Add 100 μL of the diluted primary antibody to resuspend the PBMCs plated in a 96-well plate and incubate at 4°C for 1 hour. Wash the cells three times with 500 μL of cold PBS (containing 2% FBS).

[0103] 3. Dilute anti-rabbit IgG / PE (Biolegend) at a ratio of 1:3000 in DPBS buffer (1% BSA / 1 mM EDTA). Dilute the commercial antibody CD45-PC-APC at a ratio of 1:100 in DPBS buffer (1% BSA / 1 mM EDTA). Add 100 μL of the diluted secondary antibody to the resuspended and washed cells and incubate at 4°C in the dark for 30 minutes. Wash the cells three times with 500 μL of cold PBS (containing 2% FBS).

[0104] 4. Resuspend the cells in 200 μL PBS and analyze on a flow cytometer.

[0105] The experimental results are as follows Figure 7As shown, "blank" refers to a control in which both the primary and secondary antibodies were added with PBS during the flow cytometry experiment. "NC" refers to a control in which the primary antibody was added with PBS and the secondary antibody was stained with the same ratio of anti-rabbit IgG / PE. The flow cytometry results show that the CD45-P2-91 of the present invention stains PBMC cells well, exhibiting a high staining ratio and low background signal. The commercial antibody CD45-PC-APC is Biolegend's APC anti-human CD45 (Cat 982304; CloneHI30), the most classic mouse CD45 flow cytometry antibody. These results demonstrate that the antibodies of the present invention (SEQ ID NOs: 15, 16) can be applied to real samples at low concentrations, achieving a staining ratio of 99.9%, consistent with classic mouse CD45 antibodies.

[0106] 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.

[0107] 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 conservatively modified form 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 conservative modification 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 It 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. 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.

8. The antibody conjugate according to claim 7, characterized in that The coupling moiety is selected from a purification tag or a label.

9. The antibody conjugate according to claim 7, 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.

10. 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.

11. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 10.

12. A recombinant cell, characterized in that The recombinant cell comprises the nucleic acid molecule of claim 10, the expression vector of claim 11, or expresses the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

13. 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 12.

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 7 to 9.

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 7 to 9, 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 7 to 9, 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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