Antibodies and their use

Abstract

The application relates to the technical field of antibodies, and discloses an antibody and application thereof, wherein the antibody comprises CDRs selected from at least one of the following: 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: an amino acid sequence of SEQ ID NO: 4, 16 or 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 diagnose diseases related to CD45 mediation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular, to an antibody and application thereof. BACKGROUND

[0002] CD45 (leukocyte common antigen, LCA) is a type I transmembrane protein tyrosine phosphatase (PTP) that is highly expressed in a lineage-specific manner in the hematopoietic system, localized on the membrane surface of all nucleated white blood cells except mature red blood cells and platelets, covering T, B, NK cell subsets, and monocyte-macrophage and dendritic cell (DC) lineages. This molecule regulates the dephosphorylation of Src family kinases (SFK) through its intracellular PTP domain, constituting the "molecular switch" of the T cell receptor (TCR) and B cell receptor (BCR) signal transduction network, thus precisely controlling the lymphocyte development checkpoints (positive / negative selection), clonal expansion, effector differentiation, and peripheral tolerance establishment. CD45 generates multiple isoforms (such as CD45RA, RB, RO, etc.) through alternative splicing, conferring cell type-specific signal threshold regulation functions; its abnormal expression pattern is closely related to the occurrence, progression, and prognosis of severe combined immunodeficiency (SCID), autoimmune diseases, and various hematological malignancies, thus becoming a key molecular target for immunotherapy intervention and biomarker development.

[0003] CD45 is composed of a heavily glycosylated extracellular domain, a single transmembrane domain, and an intracellular domain. The N-terminal extracellular domain is formed by alternative splicing of exons 4, 5, and 6 to form multiple isoforms (such as CD45RA, CD45RO, etc.), which differ in extracellular domain length and glycosylation patterns, affecting the localization, migration, and signal transduction properties of immune cells. In addition, the extracellular domain is composed of three membrane-proximal fibronectin type II domains and a cysteine-rich domain, while the intracellular domain contains two tandem tyrosine phosphatase domains, of which only the first domain has catalytic activity.

[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 controversies, and potential immunogenicity risks. Moreover, high-performance antibody varieties such as rabbit-derived mAbs are scarce in the domestic market, and some multinational companies monopolize the supply of key raw materials. Therefore, it is urgent to break through technical barriers through 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 technical problems 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 comprises at least one CDR selected from the group consisting of: a heavy chain variable region CDR: an amino acid sequence of SEQ ID NO: 1~3 or a conservatively modified form thereof; a light chain variable region CDR: an amino acid sequence of SEQ ID NO: 4, 16, 6 or a conservatively modified form thereof. The antibody or the antigen-binding fragment thereof according to the embodiment of the present application can specifically recognize CD45 with high efficiency, and can effectively detect CD45 and diagnose a disease 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 specifically recognize CD45 with high efficiency, and can effectively detect CD45 and diagnose a disease 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, 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. Using the recombinant cell under suitable conditions, the aforementioned antibody or the antigen-binding fragment thereof can be effectively expressed in the cell.

[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 of the present application, the present application provides a conjugate. According to an embodiment of the present application, the conjugate comprises: the aforementioned antibody or the antigen-binding fragment thereof; and a conjugating moiety conjugated to the antibody or the antigen-binding fragment thereof. The conjugate of the present application can specifically recognize CD45, and can effectively detect CD45 and diagnose a disease related to abnormal expression of CD45.

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

[0013] In an eighth aspect of the present application, the present application provides the use of the aforementioned antibody or antigen binding fragment thereof, the aforementioned antibody conjugate, or the reagent or the kit in detecting CD45, preparing a product for detecting CD45, or diagnosing a CD45-related disease.

[0014] In a ninth aspect of the present application, the present application provides a method for detecting CD45 in a test sample. According to an embodiment 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 a sample to be detected to form an immune complex.

[0015] Additional aspects and advantages of the present application will be given in part in the following description, become apparent from the following description, or be understood through practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded 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 is a protein purification and identification result diagram according to an embodiment of the present application, wherein:

[0018] Figure 1 Fig. 1A is a result diagram of cell supernatant after Strep column affinity chromatography purification, and the SDS-PAGE identification result shows that the size of CD45 recombinant protein is about 140KD,

[0019] Figure 1 Fig. 1B is a result diagram of protein after gel filtration chromatography, and the SDS-PAGE identification result shows that the red box is the target protein, and part of the aggregated protein is removed;

[0020] Figure 2 Fig. 2 is an animal immunization scheme and serum titer detection result diagram after four immunizations according to an embodiment of the present application, wherein:

[0021] Figure 2 Fig. 2A is an animal immunization scheme flow,

[0022] Figure 2 B is the serum titer detection result after four immunizations on experimental animals;

[0023] Figure 3 is a result graph of flow sorting B cells according to an embodiment of the present application, wherein P1 indicates that main cell groups are selected by forward scattering light (FSC-A) and side scattering light (SSC-A), and cell fragments are removed, P2 and P3 indicate that the agglutinated cells are removed by FSC and SSC area (A) and height (H) again, P4 indicates that lgG+ cells positive in the FITC channel are selected, and P6 indicates that Ag+lgG+ cells positive in the APC channel are selected;

[0024] Figure 4 is a protein purity identification result graph of CD45-P2-67 and CD45-P2-91 according to an embodiment of the present application, wherein M indicates a protein marker, +DTT indicates that the protein loading buffer contains a reducing agent DTT, and -DTT indicates that the protein loading buffer does not contain a reducing agent DTT;

[0025] Figure 5 is a binding antibody affinity determination result graph according to an embodiment of the present application, wherein the abscissa represents time (s), and the ordinate represents relative displacement height (nm);

[0026] Figure 6 is a flow test result graph of rabbit recombinant monoclonal antibody CD45-P2-67 in Daudi cells and Jurkat cells according to an embodiment of the present application;

[0027] Figure 7 is a result graph of rabbit recombinant monoclonal antibody CD45-P2-67 binding PBMC cell staining according to an embodiment of the present application. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] In this text, the term "comprising" or "including" is an open expression, i.e. including the indicated aspects of the invention, but not excluding other aspects.

[0031] In this text, the term "optionally", "optional" or "optional" generally means that the event or circumstance subsequently described can or can not occur, and that the description includes situations where the event or circumstance occurs, as well as situations where it does not.

[0032] In this text, the term "fragment" refers to a target protein or polypeptide, as well as a target protein or polypeptide having an N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and / or an internal deletion.

[0033] 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.

[0034] 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%, 99.9% identity to each reference sequence.

[0035] In the present text, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule to a host cell 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 various functions described above. The expression vector can be a polynucleotide 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.

[0036] In the present text, the term "recombinant cell" generally refers to a cell having a unique trait stably inherited by modifying or recombining 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 introducing 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 the 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.

[0037] An antibody or antigen-binding fragment thereof

[0038] In some embodiments, the present application proposes an antibody or antigen-binding fragment thereof, which comprises at least one CDR selected from the following: 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, 16, 6 or conservatively modified form thereof. The antibody or antigen-binding fragment thereof according to some specific embodiments of the present application can efficiently and specifically recognize CD45, and can effectively detect CD45 and diagnose diseases related to abnormal expression of CD45.

[0039] In the present context, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, without structural limitation specific to the particular 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, the heavy chain (H chain) and the light chain (L chain) of the antibody molecule are connected by a disulfide bond. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called the variable region (V region); the carboxyl-terminal (C-terminal) is relatively stable, with little variation, called the constant region (C region). The V region of the L chain and the H chain are 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, referring to the region containing one or more or even all of the major amino acid residues that play a role in the binding affinity of the antibody or functional fragment thereof to the antigen or epitope it recognizes. In the specific embodiment of the present disclosure, CDRs refer to the highly variable region of the heavy and light chains of the antibody.

[0040] In the present context, the term "antigen-binding fragment" is a fragment comprising a portion or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but still has the performance activity of being able to specifically bind to an antigen, for example, the fragment can contain a portion or all of the CDRs of the antibody. Such fragments are biologically active 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 antibodies. Such fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0041] In the present context, "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of the antibody comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the application by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those substitutions that are conservative in the sense of the amino acid side chain properties following the substitution. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies of the application can be replaced with other amino acid residues from the same side chain family and the altered antibodies are tested for retained function using the functional assays described herein. Preferably, the conservative modifications are no more than 1 or 2 in number.

[0042] According to some embodiments of the application, the above-mentioned antibody or antigen-binding fragment thereof can further comprise at least one of the following additional technical features:

[0043] According to some embodiments of the 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.

[0044] According to some embodiments of the application, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region and / or a light chain framework region.

[0045] According to some embodiments of the application, the heavy chain framework region and / or the light chain framework region comprises a FR selected from at least one of the following:

[0046] heavy chain variable region FR: SEQ ID NO: 7~10 or a conservatively modified amino acid sequence thereof;

[0047] light chain variable region FR: SEQ ID NO: 11~14 or a conservatively modified amino acid sequence thereof.

[0048] According to some embodiments of the application, the antibody or antigen-binding fragment thereof comprises:

[0049] a heavy chain variable region FR1 having an amino acid sequence of SEQ ID NO: 7 or a conservatively modified version thereof;

[0050] a heavy chain variable region FR2 having an amino acid sequence of SEQ ID NO: 8 or a conservatively modified version thereof;

[0051] a heavy chain variable region FR3 having an amino acid sequence of SEQ ID NO: 9 or a conservatively modified version thereof;

[0052] a heavy chain variable region FR4 having an amino acid sequence of SEQ ID NO: 10 or a conservatively modified version thereof;

[0053] a light chain variable region FR1 having an amino acid sequence of SEQ ID NO: 11 or a conservatively modified version thereof;

[0054] a light chain variable region FR2 having an amino acid sequence of SEQ ID NO: 12 or a conservatively modified version thereof;

[0055] a light chain variable region FR3 having an amino acid sequence of SEQ ID NO: 13 or a conservatively modified version thereof; and

[0056] a light chain variable region FR4 having an amino acid sequence of SEQ ID NO: 14 or a conservatively modified version thereof.

[0057] 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.

[0058] 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: 5 or an amino acid sequence at least 80% identical thereto.

[0059] According to some embodiments of the application, the antibody or antigen binding fragment thereof further comprises a constant region.

[0060] According to some embodiments of the application, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0061] According to some embodiments of the present application, at least one of the heavy chain constant region and / or the light chain constant region is derived from at least one of a rabbit-derived antibody, a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, a sheep-derived antibody, a dog-derived antibody, a cat-derived antibody, a rabbit-derived antibody, and a llama-derived antibody, and mutants thereof.

[0062] 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.

[0063] According to some embodiments of the present application, the heavy chain constant region and / or the light chain constant region is derived from a rabbit-derived antibody or mutants thereof.

[0064] 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.

[0065] 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.

[0066] 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 inter-chain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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 molecule which retains the complete antigen binding site.

[0071] 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.

[0072] 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 very small molecular weight of only about 1% of a complete antibody.

[0073] Nucleic acid molecules, expression vectors and recombinant cells

[0074] In some embodiments, the present application provides a nucleic acid molecule. According to embodiments of the present application, 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 application can efficiently and specifically recognize CD45, and can effectively detect CD45 and diagnose diseases related to abnormal expression of CD45.

[0075] According to some embodiments of the present application, the nucleic acid molecule is DNA.

[0076] It is 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 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.

[0077] 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 the control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, the 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, the expression vector introduced into a suitable recipient cell can effectively realize the expression of the aforementioned antibody or antigen-binding fragment thereof under the mediation of the regulation system, and further realize the in vitro mass acquisition of the antibody or antigen-binding fragment thereof.

[0078] According to some embodiments of the present application, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0079] According to some embodiments of the present application, the expression vector is a plasmid expression vector.

[0080] 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.

[0081] 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.

[0082] According to some embodiments of the present application, the recombinant cell is a eukaryotic cell

[0083] According to some embodiments of the present application, the recombinant cell is a mammalian cell.

[0084] Conjugate and kit

[0085] In some embodiments, the present application provides a conjugate. According to embodiments of the present application, the conjugate comprises: the aforementioned antibody or antigen-binding fragment thereof; and a conjugating moiety conjugated thereto. The conjugate of the present application can specifically recognize CD45, effectively detect CD45, and diagnose a disease related to abnormal expression of CD45.

[0086] According to some embodiments of the present application, the conjugating moiety is selected from a purification tag or label.

[0087] According to some embodiments of the present application, the conjugating 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.

[0088] According to some embodiments of the present application, the conjugating moiety comprises a radioisotope.

[0089] According to some embodiments of the present application, the conjugating 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.

[0090] According to some embodiments of the present application, the conjugating 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.

[0091] According to some embodiments of the present application, the conjugating moiety comprises at least one selected from the group consisting of biotin and avidin.

[0092] According to some embodiments of the present application, the coupling moiety includes at least one selected from the group consisting of magnetic beads, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, nylon, and nitrocellulose membranes.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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 the aforementioned 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, the aforementioned nucleic acid molecule, expression vector or recombinant cell can express the aforementioned antibody or antigen binding fragment thereof under suitable conditions, further, the reagent or the kit comprising the aforementioned substances can effectively bind to CD45 and can be used for effectively detecting 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 for judging the state of a 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.

[0097] Use

[0098] In some embodiments, the present application provides use of the aforementioned antibody or antigen binding fragment thereof, the aforementioned antibody conjugate or the aforementioned reagent or kit in detecting CD45, preparing a product for detecting CD45 or diagnosing a CD45 related disease.

[0099] Method

[0100] 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 or the aforementioned kit to form an immune complex.

[0101] According to some specific embodiments of the present application, the test sample comprises cells, tissues, blood, etc.

[0102] 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.

[0103] 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 functional fragment thereof.

[0104] According to some specific embodiments of the present application, the immune complex further comprises a second antibody, and the second antibody binds to CD45.

[0105] The nucleic acid and amino acid sequences involved in the present application are shown in Table 1.

[0106] Table 1

[0107]

[0108] The schemes of the present application will be explained below in connection with 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, technical or conditions not specified in the examples were performed according to techniques or conditions described in the literature or according to the product manual. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained commercially.

[0109] Unless otherwise indicated, 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.

[0110] In the embodiments of the present application, 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.).

[0111] Example 1: Gene synthesis and protein expression

[0112] In this embodiment, the CD45 recombinant protein (its sequence number is uniprot P08575) was prepared as an immunogen, and thus, the CD45_ pcDNA3.4 expression vector was constructed based on the coding nucleic acid of the CD45 recombinant protein (gene synthesis was 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 was labeled with a Twin-Strep-Tag tag, the codon was optimized according to the codon bias of the Homo sapiens species, and was cloned into the pcDNA3.4 expression vector through XbaI and HindIII restriction enzyme sites. After the plasmid CD45_ pcDNA3.4 was transformed into Escherichia coli DH5α (purchased from TIANGEN), a large amount of plasmid extraction was performed, and then the plasmid was 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 was harvested after 5 days of transfection, and the results are shown in Figure 1 Figure 1 A is the result chart of the cell supernatant after Strep column affinity chromatography purification, and SDS-PAGE identification shows that the size of the CD45 recombinant protein is about 140KD, Figure 1 B is the result chart of the gel filtration chromatography of the protein after the initial purification, and the SDS-PAGE identification result shows that the red box is the target protein, and part of the aggregated protein is removed, that is, after the cell supernatant is purified by Strep column affinity chromatography (Strep-Tactin XT column, IBA) and gel filtration chromatography (SuperoseTM 6 Increase 10 / 300 GL, GE), the target protein is obtained.

[0113] Example 2: Animal immunization

[0114] In this embodiment, the immunogen CD45 recombinant protein obtained in Example 1 was used for animal immunization, and the specific operation was as follows:

[0115] ​The immunogen CD45 recombinant protein was mixed with adjuvant and injected into New Zealand white rabbits for immunization, wherein the first immunization was performed with Freund's complete adjuvant (Freund's complete adjuvant: sigma, cat F5881), and the subsequent immunization was performed with Freund's incomplete adjuvant (sigma, cat F5506) at a mixing ratio of 1:1 of the immunogen and the adjuvant. After four rounds of immunization, the rabbit was bled from the ear, and the serum was separated. The antibody titer in the serum was detected by ELISA, and the serum titer was qualified for B cell sorting. The specific experimental results are shown in Table 2, wherein, Figure 2 Figure 2 B is the serum titer detection result after four immunizations, the first row of data represents the reciprocal of 11 dilution degrees of serum dilution, and the second and third rows of data represent the absorbance values of two parallel dilution wells of the serum at the OD450 wavelength, and the results show that the serum titer after four immunizations reaches more than 390W.

[0116] Example 3: B cell sorting of immunized rabbits

[0117] 10 mL of whole blood of the qualified rabbit in Example 2 was collected for PBMCs separation. First, PBMCs were 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), then streptavidin magnetic beads (purchased from Miltenyi Biotec) were added, and the mixture was incubated on ice for 15 min. Finally, B cells were obtained by negative sorting through a magnetic column.

[0118] 1x10^6 B cells were taken, and after adding CD45 recombinant protein, they were incubated on ice for 30 min. After incubation, the cells were washed with PBS for 3 times, then AF488 Donkey Anti-Rabbit IgG H&L (purchased from Biolegend) and StrepMAB-ImmoDY-649 (purchased from IBA) antibodies were added, and the mixture was incubated on ice for 30 min. After washing with PBS for 3 times, CD45+lgG+B cells were sorted into 15 mL centrifuge tubes by flow cytometry FACSAriaTMII for subsequent single-cell sequencing. The specific results are shown in FIG. 3, wherein the main cell population was first selected by forward scatter light (FSC-A) and side scatter light (SSC-A), and cell fragments were removed (P1). Then, adherent cells were removed by FSC and SSC area (A) and height (H) (P2 and P3), respectively. After that, lgG+cells positive in the FITC channel (P4) were selected, and finally Ag+lgG+cells positive in the APC channel (P6) were further selected. Figure 3 ​​

[0119] Example 4: Single B cell sequencing and antibody expression vector construction

[0120] The sorted single B cells were subjected to single cell sequencing, which was based on the C4 single cell sequencing platform independently developed by Huada (DNBelab C series high-throughput single cell RNA library preparation kit set). The sorted cells were centrifuged at 300g for 10 min, resuspended, counted, mixed with the relevant reagents for reverse transcription reaction as the cell phase, and then the large magnetic beads Cell Beads and small magnetic beads Index Carrier containing label information were used as the magnetic bead phase. Through the droplet microfluidic system based on negative pressure, the cell phase and the magnetic bead phase were wrapped with oil droplets, and then the oil-in-water droplets containing single cells were generated. Then, cell lysis, mRNA capture and cDNA reverse transcription reaction were completed in the droplets. After that, the cDNA intermediate product and Oligo product were recovered, and the cDNA library, Oligo library and BCR library were constructed, respectively. Finally, the above prepared library products were subjected to DNB circularization (BGI) through the MGIEasy circularization kit, sequencing was performed through the MGISEQ-2000RS high-throughput sequencing reagent (PE150), and bioinformatics analysis was performed on the sequencing data.

[0121] After obtaining the transcriptome data, the BCR analysis was performed to obtain the antibody sequence, and the light and heavy chain paired antibody sequence was selected to be sent to Huada Changzhou Xin Yi Production Technology Co., Ltd. for synthesis of the antibody expression vector plasmid.

[0122] Example 5: Expression and purification of recombinant antibodies

[0123] Recombinant antibody expression: The antibody sequence (SEQ ID NO: 15, 5) was synthesized into the pCDNA3.4(+) expression vector, and the expression plasmid containing the specific antibody light and heavy chain coding genes obtained in Example 3 was extracted in large quantities. Then, the light and heavy chain vectors were co-transfected into 293F at a molar ratio of 3:2. The mixing ratio of plasmid and PEI (POLYSCIENCE) was 1:3, and after mixing, it was incubated at room temperature for half an hour, then added dropwise into the cells. 24h and 72h after transfection, SMS293-SUPI feeding liquid (SinoBiological) was added for feeding, and the cell supernatant was collected after 5 days.

[0124] Recombinant antibody purification: collect cell supernatant and add Protein A filler (Yiqiao God State) 1 mL, incubate at room temperature for half an hour, take out the filler and load it into a purification column empty column, add PBS solution to rinse 20 mL, then add 10 mL of 100 mM glycine solution of pH 3.0 for elution, and the eluate is neutralized to pH 7.0 with 1M Tris (pH 9.0) solution. The neutralized eluate is concentrated to 1 mL, dialyzed into PBS, the protein concentration is determined, and part of the representative antibody is run SDS-PAGE for purity identification, and the results are shown in Figure 4 .

[0125] Example 6: BLI primary screening of binding antibodies and identification of antibody affinity

[0126] The antibodies binding to CD45 recombinant protein were primary screened by biofilm interference technology (BLI). Biofilm interference technology (BLI) can monitor the interaction between molecules in real time, and the molecular change reaction is displayed as the relative displacement intensity (nm) of interference spectrum. The experiment uses protein A probe to capture antibodies (capture amount greater than 0.2 nM), flowing antigen, and PBST (0.2% Tween) as buffer. The antibody is diluted to 5 ug / mL, the antigen is diluted to 200 nM, and the Gator Primer is sequentially flowed PBST, antibody, PBST, antigen and PBST for binding and dissociation. PBST is added as a control for association in each experiment, which is used to deduct the background change of the relative displacement of the interference spectrum in the dissociation process. The 1:1 Binding model of the analysis software is used for kinetic parameter calculation to confirm whether the antibody binds to the antigen and determine the affinity KD of the antibody. The experimental results are shown in Figure 5 , the affinity KD(M) of CD45-P2-67 (SEQ ID NO: 15, 5) involved in the present application is less than 1.00E-12, and the affinity reaches the picomolar (pM) level.

[0127] Example 7: Flow test of cell lines for binding antibodies

[0128] Cell preparation; in this embodiment, the CD45 full-length sequence (M1-S1306) is constructed into pEGFP-N1 vector by Huada Changzhou Xin Yi Production Technology Co., Ltd. After large-scale plasmid, the CD45_pEGFP-N1 plasmid is transfected into Expi293FTM cells by PEI, and 293F-CD45 overexpression cells are obtained after 2 days of culture, which are used for the next step of flow test; daudi cells (human Burkitt's lymphoma cells, belonging to B lymphoblast cell lines) and jurkat cells (human acute T lymphoblastic leukemia cells, which are an immortalized human T lymphocyte cell line) are resuscitated in a 37°C water bath, and after amplification and passage, they are used for the next step of flow test;

[0129] 2. Cell washing: CD45 overexpression cells have obvious green fluorescence or daudi cells and jurkat cells are in the rapid proliferation period for flow test; collect the cells into a 15 mL centrifuge tube, centrifuge at 300 g at room temperature for 5 min, resuspend the cells with PBS, and the cell density of each sample is 1E6.

[0130] 3. Rabbit anti-staining: configure the rabbit anti to a working concentration of 1 μg / mL for staining cells, incubate at 37°C in the dark for 30 min;

[0131] 4. Cell washing: centrifuge at 300 g at room temperature for 5 min, discard the supernatant, wash the cells of each sample with PBS, and repeat the above operation twice;

[0132] 5. Secondary antibody staining: stain the cells with anti-rabbit IgG / AF647 (Biolegend) at a ratio of 1:1000, incubate at 37°C in the dark for 30 min;

[0133] 6. Cell washing: centrifuge at 300 g at room temperature for 5 min, discard the supernatant, wash the cells of each sample with 200 μL of PBS, and repeat the above operation twice;

[0134] 7. Machine: analyze the washed cells by flow cytometry.

[0135] The experimental results are shown in Figure 6 , wherein blank refers to the control in which PBS is added during the flow experiment, and NC refers to the addition of PBS during the flow experiment. The flow results show that in B cell line daudi cells and T cell line jurkat cells, CD45-P2-67 (SEQ ID NO: 15, 5) can stain more than 99% of cells, and show a high proportion of cell staining and a low background signal, indicating that CD45-P2-67 can be used as a specific flow antibody for flow experiments.

[0136] Example 8: Flow cytometry test of binding antibody on human PBMC sample

[0137] Cell resuscitation: PBMC cells (Aubio, PB005F-C) were taken out from the liquid nitrogen tank, quickly placed in a 37℃ water bath, and shaken from time to time, so that they were completely melted within 1-2 minutes. The PBMC cells were sucked into a 15 mL centrifuge tube containing 5 mL of 1640 medium (containing 10% FBS) using a pipette, and centrifuged at 400 g for 10 min. The supernatant was discarded, and the cells were washed with 5 mL of PBS or antibody diluent, and centrifuged at 400 g for 5 min. The supernatant was discarded, and 2 mL of antibody diluent was used for resuspension, and 100 μL (i.e. 5E5 cells) was added to a 96-well plate.

[0138] 2, the primary antibody was diluted to a working concentration of 1 μg / mL using PBS buffer (containing 3% BSA), and then 100 μL of the diluted primary antibody was added to the PBMC cells resuspended in the 96-well plate, and incubated at 4℃ for 1 h. The cells were washed 3 times with 500 μL of cold PBS (containing 2% FBS).

[0139] 3, first dilute the secondary antibody anti-rabbit IgG / PE (Biolegend) at a ratio of 1:3000 using DPBS buffer (1% BSA / 1 mM EDTA), and dilute the commercial antibody CD45-PC-APC at a ratio of 1:100 using DPBS buffer (1% BSA / 1 mM EDTA). Then add 100 μL of the diluted secondary antibody to the washed cells, and incubate at 4℃ in the dark for 30 min. Wash the cells 3 times with 500 μL of cold PBS (containing 2% FBS).

[0140] 4, resuspend the cells with 200 μL of PBS, and analyze and test them using a flow cytometer.

[0141] The experimental results are shown in Figure 7 The CD45-P2-67 (SEQ ID NO: 15, 5) of the present application has good staining effect on PBMC cells, and shows high staining ratio and low background signal. The commercial antibody CD45-PC-APC is an APC anti-human CD45 (Cat982304; Clone HI30) from Biolegend, which is the most classic CD45 mouse-derived flow cytometry antibody. The results show that the antibody of the present application can be applied to real samples, and has performance and effect comparable to the classic mouse CD45 antibody.

[0142] In the description of the specification, the description using terms such as "one embodiment", "some embodiments", "an embodiment" or "the embodiments" etc. means that the particular feature, structure, material or characteristic described in connection with the embodiment is included in at least one embodiment of the application. The illustrative representations of the above terms in the specification are not necessarily directed to the same embodiment. Moreover, the particular features, structures, materials or characteristics can be combined in any suitable manner in one or more embodiments. In addition, different embodiments described in the specification and the features of different embodiments can be combined and combined by those skilled in the art without contradiction, within the scope of the application.

[0143] Although the embodiments of the application have been shown and described above, it is to be understood that the above-described embodiments are exemplary, and are not to be construed as limiting the application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the application.

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that, comprises: 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 set forth in SEQ ID NO: 16; and a light chain variable region CDR3 of the amino acid sequence set forth in SEQ ID NO:

6.

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

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

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

5.

5. 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 / or a light chain constant region, at least a portion of which is from at least one of a rabbit-derived antibody, a murine-derived antibody, a primate-derived antibody, a sheep-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 / or a light chain constant region, at least a portion of which is 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, a scFv-Fv fusion protein, and a minimal recognition unit.

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

9. An expression vector, characterized by, comprises the nucleic acid molecule of claim 8.

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

11. A method of producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized by, The method comprises culturing the recombinant cell of claim 10.

12. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody or antigen-binding fragment thereof of any one of claims 1-7 and a conjugating moiety coupled thereto, the conjugating moiety selected from a purification tag or label.

13. The antibody conjugate of claim 12, wherein, The conjugating 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, and a nucleic acid.

14. The antibody conjugate of claim 12, wherein, The conjugating moiety comprises at least one of a polypeptide and a magnetic microsphere.

15. The antibody conjugate of claim 12, wherein, The conjugating moiety comprises an affinity label.

16. The antibody conjugate of claim 12, wherein, The conjugating moiety comprises at least one selected from the group consisting of a magnetic bead, a plastic microparticle, a microwell plate, nylon, and a nitrocellulose membrane.

17. The antibody conjugate of claim 12, wherein, The conjugating moiety comprises a plastic microsphere.

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 1-6 or the antibody conjugate of any one of claims 11-17.

19. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the antibody conjugate of any one of claims 11-17, or the reagent or kit of claim 18 in the manufacture of a product for detecting CD45.

20. A method of detecting CD45 in a test sample, the method comprising: The method is for non-diagnostic purposes, the method comprising contacting the antibody of any one of claims 1-6, the antibody conjugate of any one of claims 11-17, or the reagent or kit of claim 18 with a CD45 antigen in a sample to be tested, to form an immunocomplex.

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