Anti-cd45 antibodies based on detection of leukocyte common antigen by flow cytometry and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular, to an anti-CD45 antibody for detecting leukocyte common antigen based on flow cytometry and application thereof. BACKGROUND

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

[0003] CD45 is composed of a heavily glycosylated extracellular domain, a single transmembrane domain and an intracellular domain. The N-terminal extracellular domain forms multiple isoforms (such as CD45RA, CD45RO, etc.) through selective splicing of exons 4, 5 and 6, and these variants differ in extracellular domain length and glycosylation pattern, affecting the localization, migration and signaling characteristics 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 controversy and potential immunogenicity risks. In addition, high-performance antibody varieties such as rabbit-derived monoclonal antibodies are scarce in the domestic market, and some multinational companies monopolize the supply of key raw materials. Therefore, it is urgent to break through the technical barriers by innovative development strategies (such as non-animal source recombinant technology, single B cell sequencing technology) to shorten the research and development time and screen CD45 antibodies with high affinity, high specificity and low batch-to-batch difference. SUMMARY

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

[0006] Therefore, in a first aspect of the present application, the present application provides an antibody or an antigen-binding fragment thereof. According to an embodiment of the present application, the antibody or the antigen-binding fragment thereof 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 version thereof; a light chain variable region CDR: an amino acid sequence of SEQ ID NO: 4~6 or a conservatively modified version thereof. The antibody or the antigen-binding fragment thereof according to the embodiment of the present application can specifically recognize CD45 with high efficiency, can effectively detect CD45, and can 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, can effectively detect CD45, and can 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, can effectively detect CD45, and can 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 a 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, and for those skilled in the art, other related drawings can also be obtained without creative labor.

[0017] Figure 1 Figure 1 is a protein purification and identification result diagram according to an embodiment of the present application, wherein:

[0018] Figure 1 A in figure 1 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 B in figure 1 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 Figure 2 is an animal immunization scheme and serum titer detection result diagram after three immunizations according to an embodiment of the present application, wherein:

[0021] Figure 2 A in figure 2 is an animal immunization scheme,

[0022] Figure 2 B is a serum titer detection result graph after three 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 adherent cells are removed by FSC and SSC area (A) and height (H), P4 indicates that lgG+ cells positive in the FITC channel are selected, and P6 indicates that Ag+lgG+ cells positive in the APC channel are selected;

[0024] Figure 4 is a result graph of CD45-P2-91 protein purity identification 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;

[0025] Figure 5 is a result graph 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);

[0026] Figure 6 is a result graph of flow test of rabbit recombinant monoclonal antibody CD45-P2-91 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-P2-91 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 document, the term “comprising” or “including” is an open expression, i.e. including the contents indicated by the present application, but not excluding other aspects.

[0031] In this document, the terms "optionally," "optional," or "may" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs, and instances where it does not.

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

[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 available that use these algorithms to perform the comparisons, 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 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 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 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 the DNA sequence of the present application, and can be used for the 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] In the present text, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. Except for any conventional excipient incompatible with the compound of the present application, for example, any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a deleterious manner, their use is also within the scope of the present application.

[0038] An antibody or antigen-binding fragment thereof

[0039] 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 some specific embodiments of the present application can recognize CD45 efficiently and specifically, can detect CD45 effectively, and can diagnose diseases related to abnormal expression of CD45.

[0040] 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.

[0041] 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 being able to specifically bind 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).

[0042] 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 ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been determined (Creighton, Proteins, Structure and Molecular Properties, W. H. Freeman & Co., 1983). 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, any 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.

[0043] 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:

[0044] According to some embodiments of the application, the antibody or antigen-binding fragment thereof comprises:

[0045] a heavy chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 1 or a conservatively modified form thereof;

[0046] a heavy chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 2 or a conservatively modified form thereof;

[0047] a heavy chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 3 or a conservatively modified form thereof;

[0048] a light chain variable region CDR1 having an amino acid sequence of SEQ ID NO: 4 or a conservatively modified form thereof;

[0049] a light chain variable region CDR2 having an amino acid sequence of SEQ ID NO: 5 or a conservatively modified form thereof; and

[0050] a light chain variable region CDR3 having an amino acid sequence of SEQ ID NO: 6 or a conservatively modified version thereof.

[0051] 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.

[0052] 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.

[0053] According to some embodiments of the present application, at least a portion of the heavy chain framework region and / or the light chain framework region is from at least one of a rabbit-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, a sheep-derived antibody, a dog-derived antibody, a cat-derived antibody, and a llama-derived antibody, and a mutant thereof.

[0054] According to some embodiments of the present application, at least a portion of the heavy chain framework region and / or the light chain framework region is from a rabbit-derived antibody.

[0055] 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:

[0056] a heavy chain variable region FR: an amino acid sequence of SEQ ID NO: 7~10 or a conservatively modified version thereof;

[0057] a light chain variable region FR: an amino acid sequence of SEQ ID NO: 11~14 or a conservatively modified version thereof.

[0058] According to some embodiments of the application, the antibody or antigen-binding fragment thereof comprises:

[0059] a heavy chain variable region FR1 having an amino acid sequence of SEQ ID NO: 7 or a conservatively modified version thereof;

[0060] a heavy chain variable region FR2 having an amino acid sequence of SEQ ID NO: 8 or a conservatively modified version thereof;

[0061] a heavy chain variable region FR3 having an amino acid sequence of SEQ ID NO: 9 or a conservatively modified version thereof;

[0062] a heavy chain variable region FR4 having an amino acid sequence of SEQ ID NO: 10 or a conservatively modified version thereof;

[0063] a light chain variable region FR1 having an amino acid sequence of SEQ ID NO: 11 or a conservatively modified version thereof;

[0064] a light chain variable region FR2 having an amino acid sequence of SEQ ID NO: 12 or a conservatively modified version thereof;

[0065] a light chain variable region FR3 having an amino acid sequence of SEQ ID NO: 13 or a conservatively modified version thereof; and

[0066] a light chain variable region FR4 having an amino acid sequence of SEQ ID NO: 14 or a conservatively modified version thereof.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] According to some embodiments of the present application, the antibody or antigen binding fragment thereof further comprises a constant region.

[0071] According to some embodiments of the present application, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0072] 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 mouse-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.

[0073] 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 mouse-derived antibody, a human-derived antibody, and a primate-derived antibody.

[0074] 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.

[0075] According to some embodiments of the present application, the heavy chain constant region and / or the light chain constant region is from a rabbit-derived antibody or mutants thereof.

[0076] 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.

[0077] 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.

[0078] In this context, the terms "full-length antibody", "full-length monoclonal antibody", or "full-length monoclonal antibody" all 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).

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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 that retains a complete antigen binding site.

[0083] In the present context, the term "single chain antibody", "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.

[0084] 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, accounting for only about 1% of a complete antibody.

[0085] Nucleic acid molecules, expression vectors and recombinant cells

[0086] 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.

[0087] According to some embodiments of the present application, the nucleic acid molecule is DNA.

[0088] 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.

[0089] 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, 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.

[0090] According to some embodiments of the present application, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0091] According to some embodiments of the present application, the expression vector is a plasmid expression vector.

[0092] 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.

[0093] 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.

[0094] According to some embodiments of the present application, the recombinant cell is a eukaryotic cell

[0095] According to some embodiments of the present application, the recombinant cell is a mammalian cell.

[0096] Conjugate and kit

[0097] 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, and is capable of effectively detecting CD45 and diagnosing a disease associated with abnormal expression of CD45.

[0098] According to some embodiments of the present application, the conjugating moiety is selected from a purification tag or label.

[0099] 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.

[0100] According to some embodiments of the present application, the conjugating moiety comprises a radioisotope.

[0101] 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.

[0102] 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, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.

[0103] According to some embodiments of the present application, the conjugating moiety comprises at least one selected from the group consisting of biotin and avidin.

[0104] 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.

[0105] 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 that are complementary to each other), a fluorescent substance (e.g., phycoerythrin, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, blue fluorescent protein, red fluorescent protein), a luminescent substance (e.g., luciferin, Aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), a radioisotope (e.g., 3H, 14C, 32P, 35S, 125I), and a gold colloid, or the like.

[0106] 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.

[0107] 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.

[0108] 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 specifically bind to CD45, and 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.

[0109] Use

[0110] 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.

[0111] Method

[0112] 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.

[0113] 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.

[0114] According to some specific embodiments of the present application, the test sample comprises cells, tissues, blood, etc.

[0115] 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.

[0116] According to some specific embodiments of the present application, the immune complex further comprises a second antibody, and the second antibody binds to CD45.

[0117] The nucleic acid and amino acid sequences involved in the present application are shown in Table 1.

[0118] Table 1

[0119]

[0120] The schemes of the present application will be explained below in connection with 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.

[0121] 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.

[0122] 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, the online program GeneOptimizer, etc.).

[0123] Example 1: Gene synthesis and protein expression

[0124] 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 Yizuo Genesyn 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 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 in the figure is the result of the cell supernatant after Strep column affinity chromatography (the horizontal coordinates are 0, 5, 10, 15, 20, 25, 30, 35, 40 ml in turn, and the vertical coordinates are in mAU units), and SDS-PAGE identification showed that the size of the CD45 recombinant protein was about 140 KD, Figure 1 B in the figure is the result of gel filtration chromatography of the protein after primary purification (the horizontal coordinates are 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 ml in turn, and the vertical coordinates are in mAU units), and the SDS-PAGE identification result showed that the red box was the target protein, and part of the aggregated protein was removed, that is, after the cell supernatant was 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 was obtained.

[0125] Example 2: Animal immunization

[0126] In this example, the immunogen CD45 recombinant protein obtained in Example 1 was used for animal immunization, and the specific operation was as follows:

[0127] ​The recombinant CD45 immunogen protein was mixed with an adjuvant and injected into New Zealand white rabbits for immunization. The initial immunization used Freund's complete adjuvant (Sigma, Cat F5881), and subsequent immunizations used Freund's incomplete adjuvant (Sigma, Cat F5506), with a 1:1 mixing ratio of immunogen and adjuvant. After multiple rounds of immunization, blood was collected from the ears, serum was separated, and antibody titers were detected by ELISA. Once the serum titers were satisfactory, B cells were sorted. Specific experimental results are as follows: Figure 2 As shown, where, Figure 2 In Figure B, the first row of data represents the reciprocal of the 11 dilutions of the serum. The second and third rows of data represent the absorbance values ​​of two parallel dilution replicates of the immune serum at an OD450 wavelength. The results show that the titer of the triple-immune serum reaches more than 78W. The OD of the serum sample at the same dilution is less than 0.1, which does not affect the experimental results.

[0128] Example 3: Sorting of B cells in immunized rabbits

[0129] 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.

[0130] 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 3 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 (P6).

[0131] Example 4: Single B cell sequencing and antibody expression vector construction

[0132] 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). First, the sorted cells were centrifuged at 300g for 10 min, then 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, the 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.

[0133] 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 antibody expression vector plasmid.

[0134] Example 5: Expression and purification of recombinant antibodies

[0135] Recombinant antibody expression: The antibody sequence (SEQ ID NO: 15, 16) 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 vector containing the light and heavy chain variable regions was 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 (Sino Biological) was added for feeding, and the cell supernatant was collected after 5 days.

[0136] Recombinant antibody purification: collect cell supernatant, add Protein A filler (Yiqiao God) 1 mL, incubate at room temperature for half an hour, take out the filler and load into a purification column empty column, add PBS solution to flush 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 .

[0137] Example 6: BLI screening of binding antibodies and identification of antibody affinity

[0138] The antibodies binding to CD45 recombinant protein were 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 strength (nm) of the interference spectrum. The experiment uses protein A probe to capture antibodies (capture amount greater than 0.2 nM), flowing antigen, PBST (0.2% Tween) as buffer. The antibody is diluted to 5 μg / 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 subtract the background change of the relative displacement of the interference spectrum during dissociation. The 1:1 binding model of the analysis software is used to calculate the kinetic parameters, to confirm whether the antibody binds to the antigen, and to determine the affinity KD of the antibody, and the experimental results are shown in Figure 5 . The affinity of CD45-P2-91 (SEQ ID NO: 15, 16) involved in the present application is K D (M) less than 1.00E-12, the affinity reaches the picomolar (pM) level.

[0139] Example 7: Flow test of binding antibodies on cell lines

[0140] Cell preparation: CD45 full-length sequence (M1-S1306) in this embodiment was constructed into pEGFP-N1 vector by Huada Changzhou Xin Yi Geng Technology Co., Ltd. After the plasmid was extracted, the CD45_pEGFP-N1 plasmid was transfected into Expi293F™ cells by PEI, and 293F-CD45 overexpression cells were obtained after 2 days of culture and were used for the next step of flow test; daudi cells (human Burkitt's lymphoma cells, belonging to B lymphoblastoid cell lines) and jurkat cells (human acute T lymphoblast leukemia cells, which are a kind of immortalized human T lymphocyte lines) were resuscitated in a 37°C water bath, and after amplification and passage, they were used for the next step of flow test;

[0141] 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.

[0142] 3. Rabbit anti-staining: configure the rabbit anti to a working concentration of 1 μg / mL for staining cells, and incubate at 37°C for 30 min in the dark;

[0143] 4. Cell washing: 300 g centrifugation at room temperature for 5 min, discard the supernatant, wash the cells with PBS for each sample, and repeat the above operation twice;

[0144] 5. Secondary antibody staining: stain the cells with anti-rabbit IgG / AF647 (Biolegend) at a ratio of 1:1000, and incubate at 37°C for 30 min in the dark;

[0145] 6. Cell washing: 300 g centrifugation at room temperature for 5 min, discard the supernatant, wash the cells with 200 μL of PBS for each sample, and repeat the above operation twice;

[0146] 7. Machine: analyze the washed cells by flow cytometry.

[0147] The experimental results are shown in Figure 6 , wherein the horizontal coordinate in the result graph of the 293F-CD45 overexpression cell line is FITC, the value is 0, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , and the vertical coordinate is APC-A, the value is 0, 10 3 , 10 4 , 10 5 , 10 6, Daudi cell result graph, the horizontal coordinate is APC-A, the value is 0, 10 3 , 10 4 , 10 5 , 10 6 , the vertical coordinate is FSC-A, the value is 50K, 100K, 150K, 200K, the horizontal coordinate of Jurkat cell result graph is APC-A, the value is 0, 10 3 , 10 4 , 10 5 , 10 6 , the vertical coordinate is FSC-A, the value is 50K, 100K, 150K, 200K, the flow result shows that CD45-P2-91 (SEQ ID NO: 15, 16) has high cell staining ratio and low background signal in 293F-CD45 overexpression cell line, B cell line Daudi and T cell line Jurkat, indicating that CD45-P2-91 can be used as a specific flow antibody in flow experiments.

[0148] Example 8: Flow cytometry test of binding antibody on human PBMC sample

[0149] Cell resuscitation: take PBMC cells (Aubio, PB005F-C) from the liquid nitrogen tank, quickly put into 37℃ water bath, and shake from time to time, and completely melt in 1-2 minutes. Use a pipette to suck the PBMC cells into a 15 mL centrifuge tube containing 5 mL 1640 culture medium (containing 10% FBS), and centrifuge at 400 g for 10 min. Discard the supernatant, add 5 mL PBS or antibody diluent to wash the cells, and centrifuge at 400 g for 5 min. Discard the supernatant, resuspend with 2 mL of antibody diluent, and add to a 96-well plate at 100 μL per well (i.e. 5E5 cells).

[0150] 2, the primary antibody is diluted to a working concentration of 1 μg / mL with PBS buffer (containing 3% BSA), and then added to 100 μL of diluted primary antibody resuspended in a 96-well plate with PBMCs, incubated at 4℃ for 1 h. Wash the cells with 500 μL of cold PBS (containing 2% FBS) for 3 times.

[0151] 3. Dilute the anti-rabbit IgG / PE (Biolegend) at a ratio of 1:3000 with DPBS buffer (1% BSA / 1 mM EDTA) and use it. Dilute the commercial antibody CD45-PC-APC at a ratio of 1:100 with DPBS buffer (1% BSA / 1 mM EDTA). Then, add 100 μL of the diluted secondary antibody to the resuspended and washed cells and incubate at 4°C in the dark for 30 min. Wash the cells three times with 500 μL of cold PBS (containing 2% FBS).

[0152] 4. Resuspend the cells in 200 μL PBS and perform analysis using a flow cytometer.

[0153] Experimental results are as follows Figure 7 As shown, in the blank finger cytometry experiment, the primary and secondary antibodies were both added to PBS as a control. In the NC control finger cytometry experiment, the primary antibody was added to PBS, and the secondary antibody staining was done with the same proportion of anti-rabbit IgG / PE. The flow cytometry results show that the CD45-P2-91 of this invention has good staining effect on PBMC cells, exhibiting a high staining ratio and low background signal. The commercial antibody CD45-PC-APC is Biolegend's APC anti-human CD45 (Cat 982304; CloneHI30), the most classic mouse CD45 flow cytometry antibody. This result indicates that the antibody of this invention (SEQ ID NO: 15, 16) can be applied to real samples at low concentrations, with a staining ratio of 99.9%, consistent with the classic mouse CD45 antibody.

[0154] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "embodiment," or "specific embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0155] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, include: The heavy chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO:1; The heavy chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO:2; The heavy chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO:3; The light chain variable region CDR1 of the amino acid sequence shown in SEQ ID NO:4; The light chain variable region CDR2 of the amino acid sequence shown in SEQ ID NO:5; and The light chain variable region CDR3 of the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, It includes a heavy chain framework region and a light chain framework region, wherein at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, sheep-derived antibodies, canine-derived antibodies, cat-derived antibodies, and alpaca-derived antibodies.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes: The heavy chain variable region FR1 of the amino acid sequence shown in SEQ ID NO:7; The heavy chain variable region FR2 of the amino acid sequence shown in SEQ ID NO:8; The heavy chain variable region FR3 of the amino acid sequence shown in SEQ ID NO:9; The heavy chain variable region FR4 of the amino acid sequence shown in SEQ ID NO:10; The light chain variable region FR1 of the amino acid sequence shown in SEQ ID NO:11; The light chain variable region FR2 of the amino acid sequence shown in SEQ ID NO:12; The light chain variable region FR3 of the amino acid sequence shown in SEQ ID NO:13; and The light chain variable region FR4 has the amino acid sequence shown in SEQ ID NO:

14.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, include: Heavy chain variable region of the amino acid sequence shown in SEQ ID NO:15; light chain variable region of the amino acid sequence shown in SEQ ID NO:

16.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, It includes a heavy chain constant region and a light chain constant region, and at least a portion of the heavy chain constant region and / or the light chain constant region is derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, sheep-derived antibodies, canine-derived antibodies, cat-derived antibodies, and alpaca-derived antibodies.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes at least one selected from full-length monoclonal antibodies, Fab antibodies, Fab' antibodies, F(ab')2 antibodies, Fv antibodies, and single-chain antibodies; or, the antigen-binding fragment includes at least one selected from F(ab')2 fragments, Fab' fragments, Fab fragments, F(ab)2 fragments, Fv fragments, scFv fragments, scFv-Fc fusion proteins, and scFv-Fv fusion proteins.

7. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6 and a conjugated portion thereof, wherein the conjugated portion is selected from a purification tag or label.

8. The antibody conjugate according to claim 7, characterized in that, The coupling portion includes at least one selected from nucleic acids, peptides, and magnetic microspheres.

9. The antibody conjugate according to claim 7, characterized in that, The coupling portion includes at least one selected from colloidal gold, radioactive labeling, phosphorescent agents, chemiluminescent agents, fluorescein, enzymes, and natural toxins.

10. The antibody conjugate according to claim 7, characterized in that, The coupling portion includes affinity markers.

11. The antibody conjugate according to claim 7, characterized in that, The coupling portion includes at least one selected from magnetic beads, plastic microspheres, plastic microparticles, microporous plates, nylon, and nitrocellulose membranes.

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

13. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 12.

14. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 12, the expression vector of claim 13, or the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, The method includes culturing the recombinant cells of claim 14.

16. A reagent or kit, characterized in that, The reagent or kit contains the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, or the antibody-drug conjugate as described in any one of claims 7 to 11.

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

18. A method for detecting CD45 in a test sample, characterized in that, The method is for non-diagnostic purposes, and the method includes contacting the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the antibody-drug conjugate of any one of claims 7 to 11, or the reagent or kit of claim 16 with the CD45 antigen in the sample to be tested to form an immune complex.

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