Cd34 antibody and use thereof

Abstract

An antibody or an antigen-binding fragment thereof, comprising a CDR selected from at least one of the following: a heavy chain variable region CDR having an amino acid sequence as shown in any one of SEQ ID NOs: 1-3 and 7-9, or a conservatively modified amino acid sequence thereof; or a light chain variable region CDR having an amino acid sequence as shown in any one of SEQ ID NOs: 4-6 and 10-12, or a conservatively modified amino acid sequence thereof.

Description

CD34 Antibody and Its Application Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology. Specifically, this invention relates to a CD34 antibody and its uses. More specifically, this invention relates to an antibody or antigen-binding fragment, recombinant protein, nucleic acid molecule, expression vector, recombinant cell, conjugate, reagent kit, and its uses. Background Technology

[0002] Thalassemia is generally classified into three clinical types: severe thalassemia (β-thalassemia), interstitial thalassemia, and mild thalassemia. Severe thalassemia is the most severe form, requiring continuous blood transfusions to sustain life, along with iron chelation therapy to address iron overload caused by prolonged transfusions. Currently, the only treatment option for severe β-thalassemia is hematopoietic stem cell transplantation (HSCCT). HSCCT can restore normal hematopoietic function and achieve the therapeutic goal. However, this treatment is expensive and faces challenges such as difficulty in bone marrow matching. Therefore, more rational approaches are needed to improve the efficiency of HSCCT, thereby reducing treatment costs and resolving bone marrow matching issues. The primary challenge in HSCCT is obtaining large quantities of purified hematopoietic stem cells to remove interference from impurity cells during transplantation. Since hematopoietic stem cells lack distinct morphological characteristics, identification relies solely on proteins on their cell surface.

[0003] CD34 is a transmembrane glycoprotein with a molecular weight of approximately 110 kDa. It is the earliest and most widely expressed antigen on human hematopoietic stem cells and is a good marker for measuring the quality and quantity of hematopoietic stem cells. CD34+ hematopoietic stem cells have shown hematopoietic reconstruction function in both experimental animals and humans. Therefore, with their unique biological characteristics and functions, they are becoming the most ideal target cells for hematopoietic stem cell transplantation.

[0004] Therefore, it is necessary to develop more monoclonal antibodies targeting CD34 to sort bone marrow hematopoietic stem cells for clinical hematopoietic stem cell transplantation, thereby treating thalassemia. Summary of the Invention

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

[0006] The inventors discovered that CD34 epitopes are distributed differently on normal hematopoietic stem cells. Furthermore, different CD34 epitopes may participate in regulating the differentiation, development, and function of hematopoietic stem cells, further influencing the formation and development of different cell lineages during hematopoiesis. Based on this, the inventors developed monoclonal antibodies targeting different CD34 epitopes. These monoclonal antibodies are conjugated to magnetic nanospheres, allowing for more specific sorting of bone marrow hematopoietic stem cells. These sorted bone marrow hematopoietic stem cells are then used in clinical hematopoietic stem cell transplantation to treat thalassemia.

[0007] In a first aspect, the present invention provides an antibody or antigen-binding fragment. According to embodiments of the invention, the antibody or antigen-binding fragment comprises a CDR selected from at least one of the following: heavy chain variable region CDR: any amino acid sequence shown in SEQ ID NO: 1-3, 7-9 or an amino acid sequence thereof with conserved modifications; light chain variable region CDR: any amino acid sequence shown in SEQ ID NO: 4-6, 10-12 or an amino acid sequence thereof with conserved modifications. The antibody or antigen-binding fragment according to embodiments of the invention can bind to CD34, enabling the sorting of bone marrow hematopoietic stem cells that highly express CD34, thereby facilitating clinical hematopoietic stem cell transplantation.

[0008] In a second aspect, the present invention provides a recombinant protein. According to embodiments of the present invention, the recombinant protein comprises: the antibody or antigen-binding fragment described in the first aspect of the present invention. The recombinant protein according to embodiments of the present invention can bind to CD34, enabling the sorting of bone marrow hematopoietic stem cells that highly express CD34, thereby facilitating its application in clinical hematopoietic stem cell transplantation.

[0009] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention. The nucleic acid molecule according to embodiments of the present invention can encode the aforementioned antibody or antigen-binding fragment and the aforementioned recombinant protein.

[0010] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule from a third aspect of the invention. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. Thus, the expression of the aforementioned antibody or antigen-binding fragment, or the aforementioned recombinant protein, is effectively achieved, thereby enabling the large-scale in vitro production of the antibody or antigen-binding fragment or recombinant protein.

[0011] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying a nucleic acid molecule as described in the third aspect of the present invention or an expression vector as described in the fourth aspect of the present invention; or, an antibody or antigen-binding fragment as described in the first aspect of the present invention or a recombinant protein as described in the second aspect of the present invention. Using this recombinant cell, under suitable conditions, the aforementioned antibody or antigen-binding fragment or recombinant protein can be effectively expressed intracellularly.

[0012] In a sixth aspect, the present invention provides a conjugate. According to embodiments of the present invention, the conjugate comprises: an antibody or antigen-binding fragment as described in the first aspect of the present invention or a recombinant protein as described in the second aspect of the present invention; and a conjugation portion linked to the antibody or antigen-binding fragment or recombinant protein. The conjugate of the present invention can bind to CD34, enabling the sorting of bone marrow hematopoietic stem cells that highly express CD34, thereby facilitating clinical hematopoietic stem cell transplantation.

[0013] In a seventh aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises: an antibody or antigen-binding fragment as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a nucleic acid molecule as described in the third aspect of the present invention, an expression vector as described in the fourth aspect of the present invention, recombinant cells as described in the fifth aspect of the present invention, or a conjugate as described in the sixth aspect of the present invention. The kit of the present invention can effectively bind to CD34, thereby detecting CD34.

[0014] In an eighth aspect, the present invention provides for the use of the antibody or antigen-binding fragment of the first aspect of the invention, the recombinant protein of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention, the expression vector of the fourth aspect of the invention, the recombinant cell of the fifth aspect of the invention, or the conjugate of the sixth aspect of the invention in sorting stem cells, wherein the stem cells include at least one of hematopoietic stem cells and mesenchymal cells.

[0015] In a ninth aspect of the invention, the invention proposes the use of stem cells in the preparation of a medicament for the prevention and / or treatment of at least one of blood-related diseases and autoimmune diseases, wherein the stem cells are sorted using an antibody or antigen-binding fragment as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a nucleic acid molecule as described in the third aspect of the invention, an expression vector as described in the fourth aspect of the invention, a recombinant cell as described in the fifth aspect of the invention, or a conjugate as described in the sixth aspect of the invention, wherein the stem cells include at least one of hematopoietic stem cells and mesenchymal cells.

[0016] In a tenth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment described in the first aspect of the invention, the recombinant protein described in the second aspect of the invention, the conjugate described in the sixth aspect of the invention, or the kit described in the seventh aspect of the invention in the preparation of a product for the detection of CD34.

[0017] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0019] Figure 1 is an SDS-PAGE image of CD34 positive antibody expression in Example 1 (wherein, the left image in Figure 1 is a non-reducing gel, and the middle image is a reducing gel).

[0020] Figure 2 is an ELISA diagram of the candidate antibody binding to CD34 in Example 2.

[0021] Figure 3 shows the specificity of the candidate CD34 nanobody verified by Western blotting in Example 3 (where M in Figure 3 is the marker, lane 1 is a 293T cell, and lane 2 is a CD34 cell). + (cell, lane 3 contains recombinant CD34 protein).

[0022] Figure 4 is a flow cytometry plot of fluorescence activated cell sorting (FACS) in Example 6 to verify the specific recognition of the CD34 target on the cell surface by the candidate antibody. Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0024] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0025] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0026] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0027] In this paper, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide that has been truncated at an N-terminus (N-terminus) or C-terminus (C-terminus) and / or internally deleted.

[0028] In this article, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecule Lar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see AltschμL et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (AltschμL et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST AltschμL et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0029] In this paper, the term "at least 90% identity" means at least 90% identity with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0030] In this document, the term "at least 80% identity" refers to at least 90% identity with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 99.5%, 89.9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0031] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0032] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0033] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0034] This invention proposes an antibody or antigen-binding fragment, recombinant protein, nucleic acid molecule, expression vector, recombinant cell, conjugate, kit and its use, and the use of stem cells in drug preparation, which will be described in detail below.

[0035] Antibody or antigen-binding fragment

[0036] In a first aspect, the present invention provides an antibody or antigen-binding fragment. According to embodiments of the invention, the antibody or antigen-binding fragment comprises a CDR selected from at least one of the following: heavy chain variable region CDR: any amino acid sequence shown in SEQ ID NO: 1-3, 7-9 or an amino acid sequence thereof with conserved modifications; light chain variable region CDR: any amino acid sequence shown in SEQ ID NO: 4-6, 10-12 or an amino acid sequence thereof with conserved modifications. The antibody or antigen-binding fragment according to embodiments of the invention can bind to CD34, enabling the sorting of bone marrow hematopoietic stem cells that highly express CD34, thereby facilitating clinical hematopoietic stem cell transplantation.

[0037] In this document, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, with no specific structural limitations, as long as they exhibit the desired biological activity. Antibody molecules typically consist of a lighter light chain and a heavier heavy chain linked by disulfide bonds. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl-terminus (C-terminus) is relatively stable and varies little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are referred to as VL and VH, respectively. As used herein, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of the antibody or its functional fragments to the antigens or epitopes it recognizes. In specific embodiments of this disclosure, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0038] In this document, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody's CDR. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments can be generated using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0039] In this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0040] According to embodiments of the present invention, the above-described antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0041] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:1, 2, and 3, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:7, 8, and 9, respectively.

[0042] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively; or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively.

[0043] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively; or heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively.

[0044] According to embodiments of the present invention, the antibody or antigen-binding fragment includes a heavy chain framework region and / or a light chain framework region.

[0045] According to embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies.

[0046] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region of an amino acid sequence as shown in any one of SEQ ID NO:13 or SEQ ID NO:15, or an amino acid sequence having at least 90% homology therewith; and / or a light chain variable region of an amino acid sequence as shown in any one of SEQ ID NO:14 or SEQ ID NO:16, or an amino acid sequence having at least 90% homology therewith.

[0047] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having at least 90% homology therewith, and a light chain variable region of an amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 90% homology therewith; or a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 90% homology therewith, and a light chain variable region of an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 90% homology therewith.

[0048] According to an embodiment of the present invention, the antibody or antigen-binding fragment further includes a constant region; wherein the constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0049] According to embodiments of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0050] According to embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0051] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse antibodies or their mutants, or human antibodies or their mutants.

[0052] According to an embodiment of the present invention, the N-end of the heavy chain constant region is connected to the C-end of the heavy chain variable region; and / or

[0053] The N-terminus of the constant region of the light chain is connected to the C-terminus of the variable region of the light chain.

[0054] According to an embodiment of the present invention, the heavy chain constant region includes: a heavy chain constant region as shown in SEQ ID NO:17 or an amino acid sequence having at least 80% identity with it; and / or the light chain constant region includes or is a light chain constant region as shown in SEQ ID NO:18 or an amino acid sequence having at least 80% identity with it.

[0055] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain of an amino acid sequence as shown in any one of SEQ ID NO:19 or SEQ ID NO:21, or an amino acid sequence having at least 90% homology therewith; and / or a light chain of an amino acid sequence as shown in any one of SEQ ID NO:20 or SEQ ID NO:22, or an amino acid sequence having at least 90% homology therewith.

[0056] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence having at least 80% homology therewith, and a light chain having an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 80% homology therewith; a heavy chain having an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 80% homology therewith, and a light chain having an amino acid sequence as shown in SEQ ID NO:22 or an amino acid sequence having at least 80% homology therewith.

[0057] According to embodiments of the present invention, the antibody comprises at least one selected from polyclonal antibodies, full-length monoclonal antibodies, Fab antibodies, Fab' antibodies, F(ab')2 antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and minimal recognition units; or the antigen-binding fragment comprises 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, scFv-Fv fusion proteins, and minimal recognition units.

[0058] In this article, the terms “full-length antibody”, “full-length monoclonal antibody” or “full-length monoclonal antibody” refer to antibodies composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0059] In this document, the terms "polyclonal antibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes. For example, antibodies that recognize two antigenic epitopes (bispecific antibodies, or simply biantibodies), three antigenic epitopes, or four antigenic epitopes are used in a broad sense, and their specific structures are not limited, as long as they can recognize multiple antigenic epitopes. In this invention, at least one of the multiple antigenic epitopes is derived from CD34.

[0060] In this paper, the terms “single-domain antibody,” “nanobody,” and “VHH antibody” are used interchangeably. The antibody was originally described as an antigen-binding immunoglobulin (variable) domain of a “heavy chain antibody” (i.e., “antibody lacking light chains”) containing a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically binds to antigen proteins (e.g., CD34) through the heavy chain variable region.

[0061] In this article, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0062] In this paper, the term “F(ab')2 antibody” or “F(ab')2 fragment” has two antigen-binding F(ab') parts linked together by disulfide bonds.

[0063] In this article, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0064] In this paper, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by linking the variable regions of the antibody heavy chain and light chain through short peptides.

[0065] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, with a very small molecular weight, accounting for only about 1% of a complete antibody.

[0066] Recombinant protein

[0067] In a second aspect, the present invention provides a recombinant protein. According to embodiments of the present invention, the recombinant protein comprises: the antibody or antigen-binding fragment described in the first aspect of the present invention. The recombinant protein according to embodiments of the present invention can bind to CD34, enabling the sorting of bone marrow hematopoietic stem cells that highly express CD34, thereby facilitating its application in clinical hematopoietic stem cell transplantation.

[0068] According to embodiments of the present invention, the protein further comprises at least one selected from bioactive proteins or fragments thereof, bioactive polypeptides or fragments thereof.

[0069] According to embodiments of the present invention, the bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragment thereof, tumor necrosis factor or fragment thereof, interferon or fragment thereof, biological response regulators or fragment thereof, and Fc fragments.

[0070] Nucleic acid molecules

[0071] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect of the present invention or the recombinant protein described in the second aspect of the present invention. The nucleic acid molecule according to embodiments of the present invention can encode the aforementioned antibody or antigen-binding fragment and the aforementioned recombinant protein.

[0072] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0073] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0074] expression carrier

[0075] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule from the third aspect of the invention. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. This effectively enables the expression of the aforementioned antibody or antigen-binding fragment, or the aforementioned recombinant protein, thereby achieving large-scale in vitro production of the antibody or antigen-binding fragment or recombinant protein. When the aforementioned nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked. In this context, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended function of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids, bacteriophages, etc. After the expression vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, or the aforementioned multispecific antibody can be effectively realized under the mediation of the regulatory system, thereby realizing the in vitro large-scale acquisition of antibody or antigen-binding fragment, recombinant protein, or multispecific antibody.

[0076] According to embodiments of the present invention, the expression vector includes those selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0077] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0078] Recombinant cells

[0079] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying a nucleic acid molecule as described in the third aspect of the present invention or an expression vector as described in the fourth aspect of the present invention; or, an antibody or antigen-binding fragment as described in the first aspect of the present invention or a recombinant protein as described in the second aspect of the present invention. Using this recombinant cell, under suitable conditions, the aforementioned antibody or antigen-binding fragment or recombinant protein can be effectively expressed intracellularly.

[0080] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the three antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies according to the specific environment of their laboratory.

[0081] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the expression vector described in the fourth aspect of the present invention into a host cell.

[0082] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells.

[0083] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0084] Coupled

[0085] In a sixth aspect, the present invention provides a conjugate. According to embodiments of the present invention, the conjugate comprises: an antibody or antigen-binding fragment as described in the first aspect of the present invention or a recombinant protein as described in the second aspect of the present invention; and a conjugation portion linked to the antibody or antigen-binding fragment or recombinant protein. The conjugate of the present invention can bind to CD34, enabling the sorting of bone marrow hematopoietic stem cells that highly express CD34, thereby facilitating clinical hematopoietic stem cell transplantation.

[0086] According to embodiments of the present invention, the coupling portion includes, but is not limited to, at least one of nanoparticles, carriers, drugs, cytokines, protein tags, and modifiers.

[0087] In this document, the carrier can be a substance capable of suspension or dispersion in a liquid phase (e.g., solid-phase carriers such as particles and magnetic beads), or a solid phase capable of containing or carrying a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic pathways, glass capillaries, nanopillars, and monolithic columns); it can also be a labeling carrier for labeling antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase). Glycosidases), affinity substances (e.g., one of streptavidin and biotin, one of complementary sense and antisense nucleic acids), fluorescent substances (e.g., luciferin, luciferin isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., insect luciferin, jellyfish luminescent protein (Aequorin), acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), radioactive isotopes (e.g., 3H, 14C, 32P, 35S, 125I), and gold colloids, etc.

[0088] According to embodiments of the present invention, the nanoparticles include, but are not limited to, at least one of magnetic nanospheres, metal nanoparticles, and polymer nanoparticles.

[0089] Reagent test kit

[0090] In a seventh aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises: an antibody or antigen-binding fragment as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a nucleic acid molecule as described in the third aspect of the present invention, an expression vector as described in the fourth aspect of the present invention, a recombinant cell as described in the fifth aspect of the present invention, or a conjugate as described in the sixth aspect of the present invention. As is known above, the aforementioned antibody or antigen-binding fragment can bind to CD34, and the recombinant protein containing the aforementioned antibody or antigen-binding fragment and the multispecific antibody can bind to CD34. Furthermore, under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can all express the antibody or antigen-binding fragment. Further, the kit containing the above substances can effectively bind to CD34 and can be used for the effective detection of CD34. The kit can be used for scientific research, such as qualitative or quantitative detection of CD34 in biological samples, and can also be used to assess individual status, such as determining whether an individual's CD34 level is too high or too low than normal after obtaining the individual's CD34 level. The biological sample can be cells, tissues, blood, etc.

[0091] use

[0092] In an eighth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment of the first aspect of the invention, the recombinant protein of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention, the expression vector of the fourth aspect of the invention, the recombinant cell of the fifth aspect of the invention, or the conjugate of the sixth aspect of the invention in sorting stem cells, said stem cells including at least one of hematopoietic stem cells and mesenchymal cells.

[0093] According to an embodiment of the present invention, the hematopoietic stem cells include at least one of bone marrow hematopoietic stem cells, umbilical cord blood hematopoietic stem cells, peripheral blood hematopoietic stem cells, and fetal liver hematopoietic stem cells.

[0094] According to an embodiment of the present invention, the hematopoietic stem cells include bone marrow hematopoietic stem cells.

[0095] Uses of stem cells in drug preparation

[0096] In a ninth aspect of the invention, the invention proposes the use of stem cells in the preparation of a medicament for the prevention and / or treatment of at least one of blood-related diseases and autoimmune diseases, wherein the stem cells are sorted using an antibody or antigen-binding fragment as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a nucleic acid molecule as described in the third aspect of the invention, an expression vector as described in the fourth aspect of the invention, a recombinant cell as described in the fifth aspect of the invention, or a conjugate as described in the sixth aspect of the invention, wherein the stem cells include at least one of hematopoietic stem cells and mesenchymal cells.

[0097] According to embodiments of the present invention, the blood-related diseases include at least one of hemophilia, leukemia, and hemoglobinopathies.

[0098] According to embodiments of the present invention, the hemoglobinopathies include at least one of thalassemia syndrome and sickle cell disease.

[0099] According to an embodiment of the present invention, the autoimmune disease is at least one of severe rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

[0100] According to an embodiment of the present invention, the hematopoietic stem cells include at least one of bone marrow hematopoietic stem cells, umbilical cord blood hematopoietic stem cells, peripheral blood hematopoietic stem cells, and fetal liver hematopoietic stem cells.

[0101] According to an embodiment of the present invention, the hematopoietic stem cells include bone marrow hematopoietic stem cells.

[0102] use

[0103] In a tenth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment described in the first aspect of the invention, the recombinant protein described in the second aspect of the invention, the conjugate described in the sixth aspect of the invention, or the kit described in the seventh aspect of the invention in the preparation of a product for the detection of CD34.

[0104] The sequence details involved in this invention are shown in the table below:

[0105] sequence list

[0106] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0107] Example 1: Preparation of monoclonal antibodies by immunizing mice and cell fusion hybridomas

[0108] Commercial human CD34 recombinant antigen (SinoBiological, catalog number 10103-H08H) was combined with CD34-overexpressing cells to immunize BALA / c mice, inducing B lymphocytes in the mouse spleen to produce anti-CD34 antibodies. After the titer met the standard, B lymphocytes were isolated from the spleen and fused with mouse (SP2 / 0) myeloma cells. After selective culture, CD34-positive clones were obtained, and subclones were selected after cloning to ensure that single-clonal cells were produced for antibody experiments. The culture supernatant of single-clonal cells was then collected and purified by protein G column to obtain anti-human CD34 monoclonal antibodies, named M01 and M02. In addition, single-clonal cells were collected and sequenced using smart-seq to obtain sequence information. Using HindIII and EcoRI restriction endonucleases, the heavy and light chain variable regions of M01 and M02 were constructed into the backbones of human hIgG1CH1-CH3 (SEQ ID NO: 23) and human hIgG1CL (SEQ ID NO: 24), respectively, to form eukaryotic expression plasmids. Finally, chimeric antibodies were obtained through eukaryotic expression and named hM01 and hM02, respectively.

[0109] In this embodiment, two mouse monoclonal antibodies and two human monoclonal antibodies were obtained, namely, a mouse M01 antibody (hereinafter referred to as M01 antibody), a mouse M02 antibody (hereinafter referred to as M02 antibody), a human M01 antibody (hereinafter referred to as hM01 antibody), and a human M02 antibody (hereinafter referred to as hM02 antibody). The M01 antibody has HCDRs as shown in SEQ ID NO:1-3 and LCDRs as shown in SEQ ID NO:4-6, a heavy chain variable region as shown in SEQ ID NO:13 and a light chain variable region as shown in SEQ ID NO:14, a heavy chain as shown in SEQ ID NO:17, and a light chain as shown in SEQ ID NO:18. The M02 antibody has HCDRs as shown in SEQ ID NO:7-9 and LCDRs as shown in SEQ ID NO:10-12, a heavy chain variable region as shown in SEQ ID NO:15 and a light chain variable region as shown in SEQ ID NO:16, a heavy chain as shown in SEQ ID NO:19, and a light chain as shown in SEQ ID NO:20. The hM01 antibody has... The hM02 antibody has HCDRs as shown in SEQ ID NO:1-3 and LCDRs as shown in SEQ ID NO:4-6, a heavy chain variable region as shown in SEQ ID NO:13 and a light chain variable region as shown in SEQ ID NO:14, a heavy chain as shown in SEQ ID NO:25 and a light chain as shown in SEQ ID NO:26, and has HCDRs as shown in SEQ ID NO:7-9 and LCDRs as shown in SEQ ID NO:10-12, a heavy chain variable region as shown in SEQ ID NO:15 and a light chain variable region as shown in SEQ ID NO:16, a heavy chain as shown in SEQ ID NO:27 and a light chain as shown in SEQ ID NO:28.

[0110] Example 2: ELISA verification of the binding activity of candidate monoclonal antibody to CD34

[0111] Coat an ELISA plate with 50 ng of recombinant CD34 antigen and incubate overnight at 4°C. The next day, remove the ELISA plate, discard the supernatant, and wash three times with PBST (0.05% Tween-20). Add 100 μL of blocking buffer to each well, incubate at room temperature for 2 h, and wash three times with PBST. Dilute the prepared positive antibody protein 2-5 times with blocking buffer to 6-8 concentration gradients, repeating each gradient in 3 wells (50 μL per well), incubate at room temperature for 1 h, discard the supernatant, and wash three times with PBST. Add 50 μL of 3000-fold diluted Anti-mouse IgG secondary antibody, incubate at room temperature for 45 min, discard the supernatant, and wash three times with PBST. Add 100 μL of TMB chromogenic solution (abcam), develop for 10 min, add an equal volume of TMB stop buffer (abcam) to stop the development, read the OD450 value, and calculate the binding activity of the positive antibody and CD34.

[0112] As shown in Figure 2, the two murine antibodies M01 and M02, as well as their corresponding chimeric antibodies hM01 and hM02, all showed good binding ability to CD34, with the maximum effective concentration (EC50) reaching the sub-nM level.

[0113] Example 3: Western blot verification of antigen specificity of candidate antibodies

[0114] Culture 293T cells and CD34 + Cells were brought to the logarithmic growth phase and washed once with physiological saline or serum-free culture medium. 100-200 μL of lysis buffer was added to each well to ensure complete cell lysis. The cells were centrifuged at 10000–14000 g for 3-5 minutes, and the supernatant was collected. 20 μL of the cell supernatant and 2 μg of recombinant CD34 protein were added to 5 μL of 5x native loading solution, and then subjected to SDS-PAGE at 100 V for 1.5 h. After gel running, the PVDF membrane was activated with methanol, then immersed in transfer equilibration buffer for about 1 min before transfer. The clamp was removed, and the following arrangement was placed at the positive electrode: dry sponge sheet + electrophoresis gel + PVDF membrane + dry sponge sheet. The membrane was then placed in the transfer apparatus for transfer, which took approximately 16 min. The membrane was washed 5 times with PBST for 5 min each time, and then blocked overnight at 4°C with 5% skim milk powder. Wash the membrane 5 times with PBST, 5 min each time. Prepare primary antibody with 5% skim milk at a concentration of 2.5 μg / mL, 10 mL per solution. Incubate at room temperature with shaking for 3.5 h. Wash the membrane 5 times with PBST, 5 min each time. Add 1:2000 diluted Anti-mouse IgG secondary antibody. Incubate at room temperature with shaking for 1.5 h. Wash the membrane 5 times with PBST, 5 min each time. Add ECL chromogenic solution for development.

[0115] Two murine antibodies, M01 and M02, and chimeric antibodies hM01 and hM02 can all specifically bind to CD34.+ The lysate and CD34 recombinant protein after cell treatment showed a single band, indicating good specificity. This example exemplifies the results of two murine antibodies, M01 and M02, and the chimeric antibody hM01, as shown in Figure 3.

[0116] Example 4: Determination of the affinity constant between monoclonal antibody and antigen using the SPR method

[0117] The CD34 recombinant antigen was conjugated to the CM5 sensor chip. Mouse antibodies M01 and M02, and chimeric antibodies hM01 and hM02 proteins were diluted with HBS-P buffer. The affinity of the candidate antibodies (mouse antibodies M01, M02, and chimeric antibodies hM01 and hM02) was detected using the SPR method. For each concentration of mouse antibody M01, M02, and chimeric antibodies hM01 and hM02, the injection time was 120 s, the dissociation time was 300 s, and the flow rate was 30 μL / min. Regeneration was performed with 10 mM pH 2.0 Glycine, and the cycle was repeated until all concentration gradients were injected. After the program ran, the built-in analysis program of the Biacore T200 (GE) instrument was used for fitting analysis to obtain the affinity constants of mouse antibodies M01, M02, and chimeric antibodies hM01 and hM02 to the antigen. The specific results are shown in Table 1. The results showed that the two murine antibodies M01 and M02, as well as the corresponding chimeric antibodies hM01 and hM02, all had high affinity for the CD34 antigen, reaching the sub-Namo level.

[0118] Table 1: Affinity constants of CD34 antibodies

[0119] Example 5: Predicting Monoclonal Antibody Binding Epitopes Using the SPR Method

[0120] In this embodiment, the CD34 antigen is coupled to the CM5 sensor chip. Candidate antibodies are prepared at 200 nM using HBS-P buffer. Antigen saturation tests are performed on M01 and M02, and the saturation time and response value are recorded. Then, competitive SPR detection is performed. By observing the response value and the trend of the real-time curve, the relationship between the epitopes recognized by the two antibodies M01 and M02 is determined. If the two antibodies M01 and M02 recognize different antigen epitopes, the response values ​​of both antibodies are consistent with the response values ​​of individual saturation. If the recognized epitopes are competing epitopes / the same epitope, then after antibody 1 saturates the epitope, antibody 2 cannot bind to the antigen, causing the response value of antibody 2 to decrease compared to individual loading. The degree of decrease is used to determine the competition.

[0121] The test results showed that M01 and M02 had virtually no competitive relationship, and that M01 and M02 recognized different epitopes, indicating that the two CD34 antibodies of this invention recognize two different types of epitopes.

[0122] Table 2: Response values ​​when antibody is saturated alone

[0123] Table 3: Response values ​​during antibody competitive binding

[0124] Example 6: Flow Cytometry Validation of Candidate Antibody Binding Activity

[0125] This embodiment uses flow cytometry to verify whether two mouse antibodies M01 and M02, and two human antibodies hM01 and hM02, can bind to the CD34 target on the cell surface. Junkat cells cultured to the logarithmic growth phase and cells overexpressing CD34 were used. + Junkat cells were digested with trypsin, washed twice with 0.02% BSA / DPBS, and collected after 500g, 5min / cycle. Cells were then resuspended in 1% BSA / DPBS and blocked at 4℃ for 30min. The cells were then washed three times with 0.02% BSA / DPBS, 500g, 5min / cycle. Cells were then resuspended in 1% BSA / DPBS and the density was adjusted to 5x10⁻⁶ cells / mL. 6 Cells / mL; Take 100 μL of cells (i.e., 5 x 10⁶ cells / mL). 5 Add 1 μg of M01, M02, hM01 and hM02 antibody proteins to each cell, incubate at 4℃ for 1 h; wash 3 times with 0.02% BSA / DPBS, 500g, 5 min / time; add 6xhis fluorescently labeled secondary antibody (Invitrogen) according to the instructions, incubate at 4℃ for 20-30 min; wash 3 times with 0.02% BSA / DPBS, 500g, 5 min / time; resuspend cells in 200 μL DPBS, pass through a sieve, and analyze by flow cytometry.

[0126] The results showed that the M01 candidate antibody had good cell binding activity with a positive rate of 98%, followed by M02 with a positive rate of 79%; while the chimeric antibodies hM01 and hM02 both had a positive rate of over 98%, showing good cell binding activity.

[0127] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0128] 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 antigen-binding fragment, characterized in that, Includes CDRs selected from at least one of the following: Heavy chain variable region (CDR): any of the amino acid sequences shown in SEQ ID NO: 1-3, 7-9 or their conserved modified amino acid sequences; Light chain variable region CDR: any of the amino acid sequences shown in SEQ ID NO:4~6, 10~12 or their conserved modified amino acid sequences.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment includes: The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:1, 2, and 3, respectively; or The heavy chain variable regions CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:7, 8, and 9, respectively.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment includes: The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively; or The light chain variable regions CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively.

4. The antibody or antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment includes: The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively; or The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:7, 8, and 9, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The antibody or antigen-binding fragment includes a heavy chain framework region and / or a light chain framework region.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that, At least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The antibody or antigen-binding fragment includes: Heavy chain variable regions of amino acid sequences such as those shown in SEQ ID NO:13 or SEQ ID NO:15, or amino acid sequences having at least 90% homology with them; and / or The light chain variable region of any of the amino acid sequences shown in SEQ ID NO:14 or SEQ ID NO:16, or an amino acid sequence having at least 90% homology with them.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The antibody or antigen-binding fragment includes: The heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having at least 90% homology therewith, and the light chain variable region of an amino acid sequence as shown in any one of SEQ ID NO:14 or an amino acid sequence having at least 90% homology therewith; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 90% homology therewith, and the light chain variable region of the amino acid sequence shown in any one of SEQ ID NO:16 or an amino acid sequence having at least 90% homology therewith.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The antibody or antigen-binding fragment further includes a constant region; The constant region includes at least one of the heavy chain constant region and the light chain constant region.

10. The antibody or antigen-binding fragment according to claim 9, characterized in that, At least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

11. The antibody or antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; or The light chain constant region includes light chain constant regions selected from κ-type or λ-type.

12. The antibody or antigen-binding fragment according to claim 9, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse antibodies or their mutants, or human antibodies or their mutants.

13. The antibody or antigen-binding fragment according to claim 9, characterized in that, The N-end of the heavy chain constant region is connected to the C-end of the heavy chain variable region; and / or The N-terminus of the constant region of the light chain is connected to the C-terminus of the variable region of the light chain.

14. The antibody or antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region includes: Heavy chain constant regions as shown in SEQ ID NO:17 or amino acid sequences having at least 80% identity with it; and / or The light chain constant region includes or is the light chain constant region shown in SEQ ID NO:18 or an amino acid sequence having at least 80% identity with it.

15. The antibody or antigen-binding fragment according to claim 9, characterized in that, The antibody or antigen-binding fragment includes: The amino acid sequence shown in any one of SEQ ID NO:19 or SEQ ID NO:21, or a heavy chain having at least 90% homology with the amino acid sequence shown therein; and / or The amino acid sequence shown in either SEQ ID NO:20 or SEQ ID NO:22, or a light chain having at least 90% homology with the amino acid sequence shown therein.

16. The antibody or antigen-binding fragment according to claim 9, characterized in that, The antibody or antigen-binding fragment includes: Heavy chains of amino acid sequences as shown in SEQ ID NO:19 or having at least 80% homology with such amino acid sequences, and light chains of amino acid sequences as shown in SEQ ID NO:20 or having at least 80% homology with such amino acid sequences. Heavy chains of amino acid sequences as shown in SEQ ID NO:21 or amino acid sequences having at least 80% homology therewith, and light chains of amino acid sequences as shown in SEQ ID NO:22 or amino acid sequences having at least 80% homology therewith.

17. The antibody or antigen-binding fragment according to claim 9, characterized in that, The antibody includes at least one selected from polyclonal antibodies, full-length monoclonal antibodies, Fab antibodies, Fab' antibodies, F(ab')2 antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and the smallest recognition unit; or The antigen-binding fragment includes at least one selected from the following: F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, and minimum recognition unit.

18. A recombinant protein, characterized in that, include: The antibody or antigen-binding fragment according to any one of claims 1 to 17.

19. The recombinant protein according to claim 18, characterized in that, It further includes at least one selected from bioactive proteins or fragments thereof, bioactive peptides or fragments thereof.

20. The recombinant protein according to claim 19, characterized in that, The bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

21. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment as described in any one of claims 1 to 17 or the recombinant protein as described in any one of claims 18 to 20.

22. The nucleic acid molecule according to claim 21, characterized in that, The nucleic acid molecule in question is DNA.

23. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 21 or 22.

24. The expression vector according to claim 23, characterized in that, The expression vectors include those selected from eukaryotic expression vectors or prokaryotic expression vectors.

25. The expression vector according to claim 23, characterized in that, The expression vector is a plasmid expression vector.

26. A recombinant cell, characterized in that, include: Carrying the nucleic acid molecule as described in claim 21 or 22 or the expression vector as described in any one of claims 23 to 25; or, Expresses the antibody or antigen-binding fragment according to any one of claims 1 to 17 or the recombinant protein according to any one of claims 18 to 20.

27. The recombinant cell according to claim 26, characterized in that, The recombinant cells are obtained by introducing the expression vector according to any one of claims 23 to 25 into a host cell.

28. The recombinant cell according to claim 26, characterized in that, The recombinant cells are eukaryotic cells.

29. The recombinant cell according to claim 26, characterized in that, The recombinant cells are mammalian cells.

30. A coupling agent, characterized in that, Include: The antibody or antigen-binding fragment according to any one of claims 1 to 17, or the recombinant protein according to any one of claims 18 to 20; and The coupling portion is linked to the antibody or antigen-binding fragment or recombinant protein.

31. The coupling according to claim 30, characterized in that, The coupling portion includes at least one selected from nanoparticles, carriers, drugs, cytokines, protein tags, and modifiers.

32. The coupling according to claim 31, characterized in that, The nanoparticles include at least one of magnetic nanospheres, metal nanoparticles, and polymer nanoparticles.

33. A reagent kit, characterized in that, include: The antibody or antigen-binding fragment according to any one of claims 1 to 17, the recombinant protein according to any one of claims 18 to 20, the nucleic acid molecule according to any one of claims 21 to 22, the expression vector according to any one of claims 23 to 25, the recombinant cell according to any one of claims 26 to 29, or the conjugate according to any one of claims 30 to 32.

34. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 17, the recombinant protein according to any one of claims 18 to 20, the nucleic acid molecule according to any one of claims 21 to 22, the expression vector according to any one of claims 23 to 25, the recombinant cell according to any one of claims 26 to 29, or the conjugate according to any one of claims 30 to 32 in the sorting of stem cells, wherein the stem cells include at least one of hematopoietic stem cells and mesenchymal cells.

35. The use according to claim 34, characterized in that, The hematopoietic stem cells include at least one of bone marrow hematopoietic stem cells, umbilical cord blood hematopoietic stem cells, peripheral blood hematopoietic stem cells, and fetal liver hematopoietic stem cells.

36. The use according to claim 34, characterized in that, The hematopoietic stem cells include bone marrow hematopoietic stem cells.

37. Use of stem cells in the preparation of a medicament for the prevention and / or treatment of at least one of blood-related diseases and autoimmune diseases, wherein the stem cells are sorted using an antibody or antigen-binding fragment according to any one of claims 1-17, a recombinant protein according to any one of claims 18-20, a nucleic acid molecule according to any one of claims 21-22, an expression vector according to any one of claims 23-25, a recombinant cell according to any one of claims 26-29, or a conjugate according to any one of claims 30-32, wherein the stem cells include at least one of hematopoietic stem cells and mesenchymal cells.

38. The use according to claim 37, wherein the blood-related disease includes at least one of hemophilia, leukemia, and hemoglobinopathies.

39. The use according to claim 38, wherein the hemoglobinopathies include at least one of thalassemia syndrome and sickle cell disease.

40. The use according to claim 37, characterized in that, The autoimmune disease is at least one of severe rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

41. The use according to claim 37, wherein the hematopoietic stem cells include at least one of bone marrow hematopoietic stem cells, umbilical cord blood hematopoietic stem cells, peripheral blood hematopoietic stem cells, and fetal liver hematopoietic stem cells.

42. The use according to claim 37, characterized in that, The hematopoietic stem cells include bone marrow hematopoietic stem cells.

43. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 17, the recombinant protein according to any one of claims 18 to 20, the conjugate according to any one of claims 30 to 32, or the kit according to claim 33 in the preparation of a product for the detection of CD34.

Images