Anti-cd45 antibodies and uses thereof

Abstract

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

Description

TECHNICAL FIELD

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

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

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

[0004] Currently, commercial CD45 antibodies have significant limitations. The mainstream products rely on traditional mouse hybridoma technology, and the production process is limited to animal-derived systems (such as ascites preparation), which has large batch-to-batch differences, animal ethics controversy and potential immunogenicity risks. Moreover, high-performance antibody varieties such as rabbit monoclonal antibodies are scarce in the domestic market. 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 problems existing in the prior art.

[0006] Therefore, in a first aspect of the present application, the present application provides an antibody or an antigen-binding fragment thereof. According to an embodiment of the present application, the antibody or the antigen-binding fragment thereof 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: 4~6 or a conservatively modified form thereof. The antibody or the antigen-binding fragment thereof according to the embodiment of the present application is a high-affinity (pM level) rabbit recombinant antibody developed by single B cell sequencing and non-animal source recombinant technology, which can efficiently and specifically recognize CD45, detect CD45, for example, perform IHC detection, and effectively detect CD45 and diagnose diseases related to abnormal expression of CD45.

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

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

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

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

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

[0012] In a seventh aspect, the present application provides a reagent or a kit. According to embodiments 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 or the kit of the present application can specifically bind to CD45 and can effectively detect CD45.

[0013] In an eighth aspect, the present application provides 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. As previously described, the antibody or antigen-binding fragment thereof according to embodiments of the present application is a rabbit recombinant antibody with high affinity (pM level) developed by single B cell sequencing and non-animal source recombinant technology, which can effectively detect CD45, for example, for IHC detection.

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

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

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as a limitation to 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 flow chart of an animal immunization scheme according to embodiments of the present application, wherein:

[0018] Figure 1 Fig. 1A is a flow chart of the animal immunization scheme of Fig. 1,

[0019] Figure 1 Fig. 1B is a graph showing the results of serum titer detection after three immunizations of the experimental animals of Fig. 1A;

[0020] Figure 2FIG. 4 is a result chart of flow sorting B cells according to an embodiment of the present application, wherein P1 indicates that a main cell population is selected by forward scattering light (FSC-A) and side scattering light (SSC-A), and cell debris is removed, P2 and P3 indicate that further adhesion cells are removed by area (A) and height (H) of FSC and SSC, P4 indicates that lgG+ cells positive in FITC channel are selected, and P5 indicates that Ag+lgG+ cells positive in APC channel are selected;

[0021] Figure 3 FIG. 6 is a result chart of SDS-PAGE detection of recombinant antibodies according to an embodiment of the present application, wherein M indicates protein marker, +DTT indicates that the protein loading buffer contains reducing agent DTT, and -DTT indicates that the protein loading buffer does not contain reducing agent DTT;

[0022] Figure 4 FIG. 7 is a result chart of binding antibody affinity determination according to an embodiment of the present application, wherein the abscissa represents time (s), and the ordinate represents relative displacement height (nm);

[0023] Figure 5 FIG. 8 is a result chart of IHC detection of CD45-61 rabbit recombinant monoclonal antibodies on various tissue sections according to an embodiment of the present application. DETAILED DESCRIPTION

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

[0025] It should be noted that the terms "first", "second" are used only for the purpose of description, and should not be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Thus, 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, the meaning of "a plurality of" is two or more, unless otherwise specified.

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

[0027] In this document, the term "optionally", "optional" or "optional" generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur.

[0028] In the present context, 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 internal deletions.

[0029] In the present context, the terms "identity", "homology" or "similarity" are used interchangeably when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and are determined by the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, determined by conventional methods, 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 are used in alignment and determination of 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 compare sequences, 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 provided by Intelligenetics, Mountain View, California.

[0030] In the present text, the term "at least 80% identity" means at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to each reference sequence.

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

[0032] In the present text, the term "recombinant cell" generally refers to a cell having a unique trait stably inherited by modification or recombination of the genetic material of a host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein means the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with a DNA sequence of the present application, and can be used for expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.

[0033] An antibody or antigen-binding fragment thereof

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

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

[0036] In the present disclosure, the term "antigen binding fragment" is a fragment comprising part 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 specifically binding to an antigen, for example, the fragment can comprise part or all of the CDR of the antibody. Such fragments have biological activity because they bind to antigens and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv or single domain antibody. Such fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic or chemical cleavage of antigen binding molecules (including intact antibodies).

[0037] In the present context, "conservatively modified amino acid sequence" refers to amino acid modifications that do 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, no more than one or two conservative modifications are made.

[0038] According to some embodiments of the application, the above antibody or antigen binding fragment thereof can further comprise at least one of the following additional technical features:

[0039] According to some embodiments of the application, the antibody or antigen binding fragment thereof comprises:

[0040] a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 1 or a conservatively modified form thereof;

[0041] a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 2 or a conservatively modified form thereof;

[0042] a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 3 or a conservatively modified form thereof;

[0043] a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 4 or a conservatively modified form thereof;

[0044] a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 5 or a conservatively modified form thereof; and

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0062] According to some embodiments of the application, the heavy chain variable region FR1, FR2, FR3, FR4 and the light chain variable region FR1, FR2, FR3, FR4 are defined by any one of the systems of Kabat, Chothia, IMGT, or AbM.

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

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

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

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

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

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

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

[0070] According to some embodiments of the present application, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region.

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

[0072] In this context, the terms "full-length antibody", "full-length monoclonal antibody", or "full-length monoclonal antibody" each refer to an antibody formed by at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0073] In the present context, the terms "single domain antibody", "nanobody" and "VHH antibody" are used interchangeably and were originally described as antigen binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e. "antibodies devoid of light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)) comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigenic protein (e.g. CD45) via the heavy chain variable region.

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

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

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

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

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

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

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

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

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

[0083] In some embodiments, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the aforementioned nucleic acid molecule. In linking the aforementioned nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the nucleic acid molecule is operably linked to the control elements. "Operably linked" herein means that the exogenous gene is linked to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be plasmids, bacteriophages, etc. According to some embodiments of the present application, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned antibody or antigen-binding fragment thereof can be effectively realized under the mediation of the regulation system, and then the antibody or antigen-binding fragment thereof can be obtained in large quantities in vitro.

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

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

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

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

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

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

[0090] Conjugate and kit

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

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

[0093] 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.

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

[0095] 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.

[0096] 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.

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

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

[0099] In the present specification, the coupling moiety can be a substance (e.g., a particle, a magnetic bead, or the like solid phase carrier) that can be suspended or dispersed in a liquid phase, or a solid phase (e.g., a plate, a membrane, a test tube, or the like support, and a container of a well plate, a microchannel, a glass capillary, a nanocolumn, a monolithic column, or the like) that can accommodate or carry a liquid phase; or a labeling carrier for labeling an antibody or an antigen-binding fragment thereof, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of a nucleic acid of a sense strand and an antisense strand complementary to each other), a fluorescent substance (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), a luminescent substance (e.g., luciferin, Aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), a radioisotope (e.g., 3H, 14C, 32P, 35S, 125I), and a gold colloid, or the like.

[0100] According to some embodiments of the present application, the coupling moiety can be a protein tag, including but not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag, or the like.

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

[0102] In some embodiments, the present application provides a reagent or a kit. According to embodiments of the present application, the reagent or the kit comprises: the aforementioned antibody or antigen binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned antibody conjugate. As known from the foregoing, the aforementioned antibody or antigen binding fragment thereof can specifically bind to CD45, and in addition, the aforementioned nucleic acid molecule, expression vector, recombinant cell, or antibody conjugate can express the aforementioned antibody or antigen binding fragment thereof under suitable conditions. Further, 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.

[0103] Use

[0104] In some embodiments, the present application provides use of the aforementioned antibody or antigen binding fragment thereof, the aforementioned antibody conjugate, or the aforementioned reagent or kit in detecting CD45, preparing a product for detecting CD45, or diagnosing a CD45-related disease.

[0105] Method

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

[0107] According to some specific embodiments of the present application, based on the signal of the immune complex, it is determined whether the sample to be detected contains CD45 or the content of the CD45.

[0108] According to some specific embodiments of the present application, the immune complex further comprises a second antibody, which binds to the antibody or antigen binding fragment thereof.

[0109] According to some specific embodiments of the present application, the immune complex further comprises a second antibody, which binds to CD45.

[0110] The nucleic acid and amino acid sequences involved in the present application are shown in Table 1.

[0111] Table 1

[0112]

[0113] The schemes of the present application will be explained below with reference to examples. Those skilled in the art will understand that the examples below are only for illustrating the present application and should not be considered as limiting the scope of the present application. In the examples, the specific techniques or conditions not mentioned are performed according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained commercially.

[0114] 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.

[0115] In the examples of the present application, the nucleotide sequence used for preparing the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder, etc.).

[0116] Example 1: Gene synthesis and protein expression

[0117] In this example, CD45 recombinant protein (its sequence number is uniprot P08575) was prepared as an immunogen, and thus, a 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 Yi Sheng Chuan Ke 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, codon optimization was performed according to the codon bias of the Homo sapiens species, and the expression vector was cloned into pcDNA3.4 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) through 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 after purification by Strep column affinity chromatography (Strep-Tactin XT column, IBA) and gel filtration chromatography (SuperoseTM 6 Increase 10 / 300 GL, GE), a relatively pure target protein was obtained.

[0118] Example 2: Animal immunization

[0119] In this example, the immunogen CD45 recombinant protein obtained in Example 1 was used for animal immunization, and the specific operation was as follows:

[0120] The immunogen CD45 recombinant protein was mixed with the adjuvant, and was injected into New Zealand white rabbits for immunization, wherein Freund's complete adjuvant (Freund's complete adjuvant: sigma, cat F5881) was used for the first immunization, and Freund's incomplete adjuvant (sigma, cat F5506) was used for subsequent immunization, and the mixing ratio was 1:1 for the immunogen and the adjuvant. After multiple rounds of immunization of the rabbits, blood was collected from the ears, the serum was separated, and the antibody titer in the serum was detected by ELISA, and after the serum titer was qualified, B cell sorting was performed, and the specific experimental results are shown in Table 1. Figure 1 Figure 1 In Table 1, B represents the ELISA results of serum titer detection after three immunizations, and the first column of data represents the reciprocal of 11 dilution degrees of serum dilution, and the second and third columns of data represent the absorbance values of two parallel wells at OD450 wavelength, and the results show that the serum titer after the third immunization reaches more than 15W, and the titer is qualified, and B cell sorting can be performed after the third immunization.

[0121] Example 3: B cell sorting of immunized rabbits​

[0122] 10 mL of whole blood from rabbits that had passed the immunization described in Example 2 was collected for the isolation of PBMCs. 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 cells were incubated on ice for 15 min. Finally, B cells were obtained by negative sorting using a magnetic column.

[0123] One x 10^6 B cells were collected, and recombinant CD45 protein was added. The cells were incubated on ice for 30 min. After incubation, the cells were washed three times with PBS. Then, AF488 Donkey Anti-Rabbit IgG H&L (purchased from Biolegend) and StrepMAB-ImmoDY-649 (purchased from IBA) antibodies were added, and the cells were incubated on ice for 30 min. After washing three times with PBS, CD45+IgG+ B cells were sorted into 15 mL centrifuge tubes using a FACSAria™ II flow cytometer for subsequent single-cell sequencing. Specific results are shown below. Figure 2 As shown, the principal cell population was first selected using forward scattered light (FSC-A) and side scattered light (SSC-A) to remove cell debris (P1). Then, adherent cells were removed using the area (A) and height (H) of FSC and SSC, respectively (P2 and P3). Next, FITC channel-positive IgG+ cells were selected (P4), and finally, APC channel-positive Ag+IgG+ cells were further selected (P5).

[0124] Example 4: Single B cell sequencing and antibody expression vector construction

[0125] The sorted single B cells were sequenced using the C4 single-cell sequencing platform developed by BGI (DNBelab C series high-throughput single-cell RNA library preparation kit, refer to the kit instructions for specific operation). First, the sorted cells were centrifuged at 300g for 10 min, resuspended, and counted. They were then passed through a microfluidic chip along with the oil phase to generate water-in-oil droplets. Each droplet encapsulated a single cell (or microorganism), lysis reagents, and magnetic beads carrying unique molecular tags. Cell lysis, mRNA capture, and cDNA reverse transcription were then performed within the droplets. Afterward, the cDNA product was recovered by demulsification, enriched with BCR, and finally, cDNA and BCR libraries were constructed for sequencing. Single-cell data were then obtained for bioinformatics analysis.

[0126] After obtaining the transcriptome data, immune repertoire BCR analysis was performed to obtain antibody sequences, and light and heavy chain paired antibody sequences were selected for synthesis of antibody expression vector plasmid by Huada Changzhou Xin Yi Production Technology Co., Ltd.

[0127] Example 5: Expression and purification of recombinant antibodies

[0128] Recombinant antibody expression: The antibody sequences (SEQ ID NO: 15, 16) were synthesized into pCDNA3.4(+) expression vectors, and expression plasmids containing the specific antibody light and heavy chain coding genes obtained in Example 3 were 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 feed solution (SinoBiological) was added for feeding, and 5 days later, the cell supernatant was collected.

[0129] Recombinant antibody purification: The cell supernatant was collected and added to Protein A filler (Yiqiao God State) 1 mL, incubated at room temperature for half an hour, then the filler was removed and loaded into a purification column empty column, 20 mL of PBS solution was added for flushing, then 10 mL of 100 mM glycine solution at pH 3.0 was added for elution, and the eluate was neutralized to pH 7.0 with 1M Tris (pH 9.0) solution. The neutralized eluate was concentrated to 1 mL, dialyzed into PBS, and the protein concentration was determined. Part of the representative antibody was run on SDS-PAGE for purity identification, and the results are shown in Figure 3 .

[0130] Example 6: BLI primary screening of binding antibodies and identification of antibody affinity

[0131] The antibodies binding to CD45 recombinant protein were primary screened by bio-layer interferometry (BLI). Bio-layer interferometry (BLI) can monitor molecular interactions in real time, and molecular changes are displayed as relative displacement intensity (nm) of interference spectrum. In the experiment, protein A probe was used to capture antibodies (capture amount greater than 0.2 nM), flowing antigen, and PBST (0.2% Tween) was used as buffer. The antibody was diluted to 5 ug / mL, the antigen was diluted to 200 nM, and the Gator Primer was used to flow PBST, antibody, PBST, antigen and PBST in turn for binding and dissociation. PBST was added as a control for association in each experiment to subtract the background change of interference spectrum relative displacement during dissociation. The 1:1 Binding model of the analysis software was used to calculate the kinetic parameters, to confirm whether the antibody binds to the antigen, and to determine the affinity KD of the antibody. The experimental results are shown inFigure 4 As shown, the CD45-61 binding antibody (SEQ ID NO: 15, 16) involved in the present application has a KD(M) of less than 1.00E-12, and the affinity reaches the picomolar (pM) level.

[0132] Example 7: IHC test of the binding antibody

[0133] First, the formalin-fixed paraffin-embedded tissue sample was made into a 3-micron-thick paraffin section, which was flattened at a water temperature of 40°C and baked in an oven at 60°C for 1 h, dewaxed by xylene and treated by alcohol gradient, then the antigen was repaired by EDTA microwave heating, and the endogenous peroxidase was blocked by peroxidase blocking solution, then the tissue was blocked by adding goat serum, incubated at 37°C for 30 min, and the excess liquid was shaken off, then different concentrations of primary antibody were added for incubation, and incubated at room temperature for 1-2 h. After the incubation of the primary antibody, the excess primary antibody was washed with PBST, and the secondary antibody Polymer HRP Goat anti-mouse / rabbit IgG (Ready-to-Use) (purchased from Chongqing Xin Yisheng Technology Co., Ltd., cat LS-PA-03015T) was added and incubated at room temperature for 30 min, and the excess secondary antibody was washed with PBST. Then DAB developing solution was prepared for color development, and the color development time was observed and controlled under a microscope, and the reaction was stopped by water washing, then the cell nuclei were stained by hematoxylin, and the blueing process was performed. Finally, dehydration was performed by alcohol gradient, xylene was used for transparency treatment, and the section was dried with neutral resin, and the staining results were observed and photographed under a high-power microscope. The results are shown in Figure 5 As shown, in the order from left to right and from top to bottom, the tissues in the figure are in turn: stomach, breast, esophagus, endometrium, placenta, liver, kidney, spleen, testis, and the CD45-61 rabbit recombinant monoclonal antibody (SEQ ID NO: 15, 16) of the present application exhibits high positive rate and strong specific membrane localization in parotid gland, spleen and other paraffin sections, and its staining mode (clear edge, continuous membrane signal) is consistent with the results of the widely verified commercial positive control antibody PC (Zhongshang Jinqiao ZM-0183). Under the same experimental conditions, compared with the positive control antibody, the rabbit recombinant monoclonal antibody of CD45-61 has higher staining intensity at high concentration, and has no cytoplasm or interstitial non-specific background, and still maintains the staining intensity comparable to the positive control at a low working concentration (0.1 μg / mL), indicating that the CD45-61 in the present application has the core advantages of high specificity and low background interference in IHC detection.

[0134] 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.

[0135] Although the embodiments of the application have been shown and described above, it should be understood that the above-described embodiments are exemplary and are not to be construed as limiting the application, and those ordinarily skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the application.

Claims

1. A CD45 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: 5; 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:

16. The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, at least a portion of 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.

5. 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.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody conjugate comprises the antibody or antigen-binding fragment thereof of any one of claims 1-6 conjugated to a conjugation moiety selected from a purification tag or label.

7. An antibody conjugate, characterized in that, The purification tag or label 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 nucleic acid, and an affinity label.

8. The antibody conjugate of claim 7, wherein, The purification tag or label comprises a polypeptide.

9. The antibody conjugate of claim 7, wherein, ​ 10. An antibody conjugate, characterized in that, The conjugate moiety comprises at least one member selected from the group consisting of magnetic microspheres and plastic microparticles.

11. The antibody conjugate of claim 10, wherein, The conjugate moiety comprises at least one member selected from the group consisting of magnetic beads, plastic microspheres, microwell plates, nylon and nitrocellulose membranes.

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

13. An expression vector, characterized by, The nucleic acid molecule of claim 12.

14. A recombinant cell, wherein, The recombinant cell comprises the nucleic acid molecule of claim 12, the expression vector of claim 13 or expresses the antibody or antigen-binding fragment thereof of any one of claims 1-6.

15. A method of producing an 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 14.

16. 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 7-11.

17. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the antibody conjugate of any one of claims 7-11 or the reagent or kit of claim 16 in the manufacture of a product for detecting CD45.

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