Siglec-6-targeting CAR-T cell as well as preparation method and application thereof

Abstract

The invention discloses a CAR-T (Chimeric Antigen Receptor-T) cell targeting Siglec-6 as well as a preparation method and application of the CAR-T cell. The CAR provided by the invention contains an antigen binding fragment aiming at Siglec-6. The CAR-T cell provided by the invention can efficiently and specifically kill acute myelogenous leukemia cells. The invention provides a new scheme for the treatment of acute myelogenous leukemia, has great fundamental research and clinical application values, and has good industrial prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to CAR-T cells targeting Siglec-6 and a preparation method and application thereof. Background Art

[0002] Leukemia is a type of malignant clonal disease that originates from hematopoietic stem cells. According to the degree of differentiation of leukemia cells and the natural course of the disease, leukemia can be divided into two categories: acute and chronic. According to the main affected cell series, acute leukemia can be divided into acute myeloid leukemia and acute lymphocytic leukemia. Chronic leukemia is divided into chronic myeloid leukemia, chronic lymphocytic leukemia and rare types of leukemia. Acute myeloid leukemia is a tumor that invades the blood and bone marrow. It is one of the most common types of adults in the leukemia family. It progresses rapidly and is extremely invasive. Under normal circumstances, the bone marrow produces myeloid hematopoietic stem cells, which are then differentiated and eventually differentiated into various mature blood cells. However, if a gene mutation occurs in the body, it will cause the myeloid primitive cells to be unable to differentiate normally into normal blood cells (red blood cells, platelets, white blood cells), thereby causing acute myeloid leukemia. Most cases are acute and severe, with a dire prognosis. If not treated in time, it can often be life-threatening.

[0003] Chimeric antigen receptor T cell (CAR-T) therapy is a new type of immunotherapy that uses genetic engineering technology to artificially express antigen binding domains on the surface of T cells that can recognize specific tumor surface antigens, so that T cells acquire the specific tumor recognition ability of antibodies, and activate T cells through the intracellular signaling region of CAR after recognizing tumor cells, so that CAR-T cells can exert cytotoxic effects by releasing cytokines, granzymes, etc., thereby targeting and eliminating malignant tumor cells. Many clinical studies have confirmed that this engineered T cell can show efficient anti-tumor activity against a variety of refractory blood tumors. However, CAR-T cells can also cause some clinical side effects and toxicity due to off-target effects. Summary of the invention

[0004] In view of this, in order to make up for the shortcomings of the prior art, the purpose of the present invention is to provide a CAR-T cell targeting Siglec-6 and a preparation method and application thereof. The CAR-T cell targeting Siglec-6 can specifically recognize and kill malignant tumors such as myeloid tumors, and is expected to provide a basis for the design of other immune effector cell therapies targeting Siglec-6 in the future, and is expected to provide a new method for the treatment of acute myeloid leukemia.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention provides an antibody or antigen-binding fragment capable of binding to the Siglec-6 antigen, wherein the antibody or antigen-binding fragment comprises at least one sequence selected from the following: a light chain variable region CDR1 according to SEQ ID NO: 1-2, a light chain variable region CDR2 according to SEQ ID NO: 3-4, a light chain variable region CDR3 according to SEQ ID NO: 5-6, a heavy chain variable region CDR1 according to SEQ ID NO: 7-11, a heavy chain variable region CDR2 according to SEQ ID NO: 12-16, and a heavy chain variable region CDR3 according to SEQ ID NO: 17-22.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment comprises a combination of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, or a combination of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.

[0008] Further, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment includes an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with SEQ ID NO:23-25, SEQ ID NO:31-33. Further, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment includes a humanized amino acid sequence. Further, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment is as shown in SEQ ID NO:23-25, SEQ ID NO:31-33.

[0009] Further, the heavy chain variable region amino acid sequence of the antibody or antigen-binding fragment comprises a combination of SEQ ID NO:7, SEQ ID NO:12, and SEQ ID NO:17, or a combination of NO:7, SEQ ID NO:12, and SEQ ID NO:22, or a combination of NO:8, SEQ ID NO:13, and SEQ ID NO:18, or a combination of NO:9, SEQ ID NO:14, and SEQ ID NO:19, or a combination of NO:10, SEQ ID NO:15, and SEQ ID NO:20, or a combination of NO:11, SEQ ID NO:16, and SEQ ID NO:21.

[0010] Further, the heavy chain variable region amino acid sequence of the antibody or antigen-binding fragment includes an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with SEQ ID NO:26-30, SEQ ID NO:34-36. Further, the heavy chain variable region amino acid sequence of the antibody or antigen-binding fragment includes a humanized amino acid sequence. Further, the heavy chain variable region amino acid sequence of the antibody or antigen-binding fragment is as shown in SEQ ID NO:26-30, SEQ ID NO:34-36.

[0011] In some embodiments, the sequences of CDR1, CDR2, CDR3, CDR1, CDR2 and CDR3 of the light chain variable region and heavy chain variable region of the monoclonal antibody described in the present invention can be obtained based on the full-length sequences of the above-mentioned light chain variable region and heavy chain variable region according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system are also within the scope of protection of the present invention.

[0012] In some embodiments, the antibodies or antigen-binding fragments described herein also encompass amino acid sequence variants of the antibodies or antigen-binding fragments described above. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibodies or antigen-binding fragments, and the amino acid sequence variants of the antibodies or antigen-binding fragments may be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibodies or antigen-binding fragments, or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibodies or antigen-binding fragments, and any combination of deletion, insertion and substitution may be performed to obtain the final antibody or antigen-binding fragment variants, based on which the antibody or antigen-binding fragment variants obtained are also included within the scope of the present invention.

[0013] In the present invention, the term "antibody or antigen-binding fragment" generally refers to an immunoglobulin or antibody polypeptide fragment that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which they are derived) for binding to an antigen (i.e., specifically binding). The antibody or antigen-binding fragment may include, but is not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single-chain antibodies, diabodies, and multispecific antibodies formed by antibody fragments.

[0014] The second aspect of the present invention provides a chimeric antigen receptor targeting Siglec-6, wherein the chimeric antigen receptor comprises an extracellular antigen binding domain composed of the antibody or antigen binding fragment described in the first aspect of the present invention.

[0015] Furthermore, the antibody or antigen-binding fragment that binds to Siglec-6 is composed of a heavy chain variable region and a light chain variable region. In one embodiment, the heavy chain variable region and the light chain variable region may be directly connected. In one embodiment, the heavy chain variable region and the light chain variable region may be connected by at least one linker, and the arrangement may be VH-linker-VL or VL-linker-VH.

[0016] Further, the linker includes but is not limited to glycine polymer (G)n, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, LRQKD(GGGS)2ERP, DGGGS, LRQRDGERP, TGEKP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKVD, GGRRGGGS, GGRR, LRQKDGGGSERP or (GGGGS)n. In one embodiment, the linker is (GGGGS)n, and in a specific embodiment of the present invention, the linker is (GGGGS)3.

[0017] Furthermore, the antigen binding fragment is a scFv that specifically binds to Siglec-6.

[0018] Furthermore, the amino acid sequence of the scFv is shown in SEQ ID NO: 37-39.

[0019] Furthermore, the chimeric antigen receptor also includes the following components: a transmembrane domain, a co-stimulatory signal transduction domain, and a primary signal transduction domain.

[0020] Further, the transmembrane domain includes human CD8 transmembrane region, human CD8α transmembrane region, human CD28 transmembrane region, human CD4 transmembrane region, human CD28 transmembrane region, human CD3ζ transmembrane region, human CD137 / 4-1BB transmembrane region, human CD134 / OX40 transmembrane region, human ICOS transmembrane region, human CD5 transmembrane region, human CD9 transmembrane region, human CD16 transmembrane region, human CD22 transmembrane region, human CD33 transmembrane region, human CD37 transmembrane region, human CD45 transmembrane region, human CD64 transmembrane region, human CD80 transmembrane region, human CD86 transmembrane region, human CD154 transmembrane region, human TCRα transmembrane region, and human TCRβ transmembrane region.

[0021] Furthermore, the transmembrane domain is selected from the transmembrane region of CD8.

[0022] Furthermore, the co-stimulatory signal transduction domain includes human CD28 intracellular co-stimulatory signal domain, human CD137 / 4-1BB intracellular co-stimulatory signal domain, human CD134 / OX40 intracellular co-stimulatory signal domain, human CD27 intracellular co-stimulatory signal domain, human ICOS intracellular co-stimulatory signal domain, human B7-H3 intracellular co-stimulatory signal domain, human 2B4 intracellular co-stimulatory signal domain, human DAP10 intracellular co-stimulatory signal domain, human DNAM1 intracellular co-stimulatory signal domain, and human DAP12 intracellular co-stimulatory signal domain.

[0023] Furthermore, the co-stimulatory signal transduction domain is selected from the human CD137 / 4-1BB intracellular co-stimulatory signal domain.

[0024] Furthermore, the primary signaling domain includes human CD3ζ and human FcεRIγ.

[0025] Further, the primary signaling domain is selected from human CD3ζ.

[0026] Furthermore, the chimeric antigen receptor also includes one or more of the following components: a detection tag, a cleavage protein, a signal peptide, and a hinge region.

[0027] Furthermore, the detection labels include protein labels, fluorescent dyes, colloidal gold, chemiluminescent markers, and chemiluminescent catalysts.

[0028] Furthermore, the detection tag is selected from a protein tag.

[0029] Furthermore, the protein tags include RQR8, EGFRt, CD20, c-MYC, 6×His, GST, Fc, Flag, and HA.

[0030] Furthermore, the protein tag is selected from RQR8.

[0031] Furthermore, the cleavage protein comprises cleavage protein 2A or furin.

[0032] Furthermore, the cleavage protein 2A includes cleavage protein T2A, cleavage protein P2A, cleavage protein E2A, and cleavage protein F2A.

[0033] Furthermore, the cleavage protein F2A is selected from FMD-2A.

[0034] Furthermore, the fluorescent dyes include total protein dyes, phosphorylated protein and glycosylated protein dyes propidium iodide (PI), DAPI, Hoechst 33342, thiazole orange, and acridine orange.

[0035] Furthermore, the chemiluminescent markers include luminol and its derivatives, isoluminol and its derivatives, acridinium esters and their derivatives, adamantane, rare earth elements, and bipyridine ruthenium complexes.

[0036] Furthermore, the chemiluminescent catalyst includes horseradish peroxidase and alkaline phosphatase.

[0037] Furthermore, the signal peptide also includes CD8α signal peptide, T2A, GMCSF signal peptide, EGFR DIII-DIV. In one embodiment, the signal peptide is selected from CD8α signal peptide. In a specific embodiment of the present invention, the signal peptide sequence is shown in SEQ ID NO:40.

[0038] Further, the hinge region includes the hinge region of CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor molecules. In one embodiment, the hinge region is the CD8 hinge region.

[0039] In the present invention, the term "chimeric antigen receptor" (Chimeric antigen receptor, CAR) generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is a core component of a chimeric antigen receptor T cell (CAR-T), which may include an antigen (e.g., a tumor-specific antigen and / or a tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. CAR is an engineered receptor that can implant any specific receptor into immune effector cells, especially T cells. In CAR, the scFv fragment of a monoclonal antibody that specifically recognizes a tumor antigen can be implanted into a T cell or a NK cell. For example, a retroviral vector can be used to introduce a nucleic acid encoding CAR into a T cell, a NK cell, or a NK T cell. In this way, a large number of cancer-specific T cells, NK cells, or NK T cells can be generated for adoptive cell transfer.

[0040] The third aspect of the present invention provides a nucleic acid molecule, which encodes the antibody or antigen-binding fragment as described in the first aspect of the present invention, or encodes the chimeric antigen receptor as described in the second aspect of the present invention.

[0041] Furthermore, the nucleic acid molecule further comprises a promoter, and / or a restriction enzyme cleavage site located after the promoter, and / or a kozak sequence located after the restriction enzyme cleavage site.

[0042] Furthermore, the nucleic acid molecule sequence encoding the antibody or antigen-binding fragment corresponds to part or all of SEQ ID NOs: 45-47.

[0043] Furthermore, the nucleic acid molecule sequence encoding the chimeric antigen receptor corresponds to part or all of SEQ ID NOs: 48-50.

[0044] In the present invention, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0045] Those skilled in the art can use standard molecular biology techniques to obtain nucleic acid molecules of the present invention.Once the DNA fragments encoding the variable region of heavy chain and the variable region of light chain are obtained, these DNA fragments are further operated by standard recombinant DNA techniques, such as to convert the variable region gene into a full-length antibody chain gene, a Fab fragment gene or a scFv gene.In these operations, the DNA fragments encoding the variable region of heavy chain and the variable region of light chain are operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible joint.Wherein, the term "operably connected" is intended to represent the connection of two DNA fragments when used herein so that the amino acid sequence encoded by the two DNA fragments remains in the same reading frame (in-frame).

[0046] The fourth aspect of the present invention provides a vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0047] In some embodiments, the type of the carrier is not limited, for example, plasmid, phagemid, phage derivative, animal virus and clay, can be changed according to the host cell to be introduced. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals, and can be introduced by standard techniques, for example, infection, transfection, transduction or conversion. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE polydextrose, electroporation, protoplast fusion, liposome transfection, cell microinjection and viral vectors. Viruses that can be used as carriers include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. In addition, in order to evaluate the expression of target protein (for example, monoclonal antibody), the carrier introduced into the cell may also include any one or two of selectable marker genes or reporter genes, so as to identify and select expression cells from the cell group seeking to be transfected or infected by viral vectors.

[0048] In some embodiments, the vector can be an expression vector, a cloning vector or an integration vector. Usually, the expression of the nucleic acid molecule described in the third aspect of the present invention is achieved by operably connecting the nucleic acid molecule described in the third aspect of the present invention to the expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components that integrate the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequence usually includes one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multi-linker region and a selectable marker element for inserting a nucleic acid encoding a monoclonal antibody to be expressed.

[0049] The fifth aspect of the present invention provides a recombinant microorganism, which contains the nucleic acid molecule encoding the third aspect of the present invention, or the vector of the fourth aspect of the present invention.

[0050] A sixth aspect of the present invention provides any one of the following cells:

[0051] 1) A cell capable of expressing the antibody or antigen-binding fragment of the first aspect of the present invention;

[0052] 2) A modified cell, wherein the cell is modified to express the chimeric antigen receptor described in the second aspect of the present invention.

[0053] Furthermore, the cells include eukaryotic cells and prokaryotic cells.

[0054] Furthermore, the eukaryotic cells include lower eukaryotic cells and higher eukaryotic cells.

[0055] Furthermore, the lower eukaryotic cells include yeast cells.

[0056] Furthermore, the higher eukaryotic cells include mammalian cells.

[0057] Further, the mammalian cells include but are not limited to PER.C6 cells, monkey kidney cells 7 (COS7, in particular simian COS cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, Madin-Darby canine kidney (MDCK) cells, myeloma cell lines, HuT78 cells, 293T cells or 293F cells.

[0058] Furthermore, the prokaryotic cells include but are not limited to E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp.

[0059] Furthermore, the modified cells are immune cells.

[0060] Furthermore, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof. In a specific embodiment of the present invention, the immune cells are T cells.

[0061] Furthermore, the cell contains the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0062] The seventh aspect of the present invention provides a composition, which comprises the antibody or antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the recombinant microorganism described in the fifth aspect of the present invention, and the cell described in the sixth aspect of the present invention.

[0063] Furthermore, the composition also includes a pharmaceutically acceptable carrier.

[0064] A pharmaceutically acceptable carrier is one that has no deleterious side effects or toxicity under the conditions of use. Methods for preparing administrable pharmaceuticals are known or will be apparent to those skilled in the art.

[0065] The choice of pharmaceutically acceptable carrier will be determined in part by the specific antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, vector, immune cell and by the specific method for administering the antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, vector, immune cell. Therefore, there are a variety of suitable preparations of the pharmaceutical composition of the present invention. Suitable preparations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one approach can be used to administer the antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, vector, immune cell, and in some cases, a particular approach can provide a more direct and more effective response than another approach.

[0066] The compositions of the present invention may also include other pharmaceutically active agents or drugs administered in combination with the antibodies or antigen-binding fragments, chimeric antigen receptors, nucleic acid molecules, vectors, recombinant microorganisms, and cells of the present invention, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunomycin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, taxol, rituximab, vinblastine, vincristine, and the like.

[0067] When the antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, carrier, recombinant microorganism, cell is administered together with one or more other therapeutic agents, one or more other therapeutic agents can be co-administered to a mammal."Co-administered" means administering one or more other therapeutic agents and the antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, carrier, recombinant microorganism, cell close enough in time, so that the antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, carrier, recombinant microorganism, cell can enhance the effect of one or more other therapeutic agents, and vice versa. In this regard, the antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, carrier, recombinant microorganism, cell can be administered first, and then one or more other therapeutic agents can be administered, and vice versa. Alternatively, the antibody or antigen binding fragment of the present invention, chimeric antigen receptor, nucleic acid molecule, carrier, recombinant microorganism, cell and one or more other therapeutic agents can be administered simultaneously.

[0068] The eighth aspect of the present invention provides a kit, which comprises the antibody or antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the recombinant microorganism described in the fifth aspect of the present invention, the cell described in the sixth aspect of the present invention, and the composition described in the seventh aspect of the present invention.

[0069] In some embodiments, the kit may further include a sample diluent, a washing solution, a color developing solution, and a stop solution.

[0070] The ninth aspect of the present invention provides any of the following methods:

[0071] 1) A method for preparing the antibody or antigen-binding fragment of the first aspect of the present invention, the method comprising culturing the cell of the sixth aspect of the present invention to enable it to express the antibody or antigen-binding fragment of the first aspect of the present invention;

[0072] 2) A method for preparing the cell according to the sixth aspect of the present invention, the method comprising introducing the nucleic acid molecule according to the third aspect of the present invention or the vector according to the fourth aspect of the present invention into the cell;

[0073] 3) A method for in vitro targeted killing of cells expressing Siglec-6, the method comprising:

[0074] Contacting the Siglec-6-expressing cells with the antibody or antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the cells described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, or the kit described in the eighth aspect of the present invention, and observing the killing of the Siglec-6-expressing cells;

[0075] 4) A method for detecting Siglec-6, the method comprising:

[0076] The sample to be tested is contacted with the antibody or antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the cell described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, or the kit described in the eighth aspect of the present invention to form an antigen-antibody complex, and the complex is detected, wherein detection of the complex indicates the presence of Siglec-6 in the sample to be tested.

[0077] Furthermore, the Siglec-6 expressing cells include but are not limited to leukemia cells, lymphoma cells, mast cells, non-alcoholic steatohepatitis (NASH) cells, colitis cells, and neurofibroma cells.

[0078] Furthermore, the leukemia cells include acute lymphocytic leukemia (ALL) cells, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, and hairy cell leukemia cells.

[0079] Furthermore, the leukemia cells are selected from AML cells.

[0080] The tenth aspect of the present invention provides any one of the following applications:

[0081] 1) Use of the antibody or antigen-binding fragment of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the recombinant microorganism of the fifth aspect of the present invention, the cell of the sixth aspect of the present invention, or the composition of the seventh aspect of the present invention in the preparation of a drug for diagnosing and / or treating a disease or condition associated with Siglec-6 expression;

[0082] 2) Use of the antibody or antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the recombinant microorganism described in the fifth aspect of the present invention, the cell described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, and the kit described in the eighth aspect of the present invention in targeting and killing cells expressing Siglec-6 in vitro;

[0083] 3) Use of the antibody or antigen-binding fragment of the first aspect of the present invention, the chimeric antigen receptor of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the recombinant microorganism of the fifth aspect of the present invention, the cell of the sixth aspect of the present invention, the composition of the seventh aspect of the present invention, and the kit of the eighth aspect of the present invention in detecting Siglec-6;

[0084] 4) Use of the antibody or antigen-binding fragment described in the first aspect of the present invention in the preparation of the chimeric antigen receptor described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the recombinant microorganism described in the fifth aspect of the present invention, the cell described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, or the kit described in the eighth aspect of the present invention;

[0085] 5) Use of the chimeric antigen receptor described in the second aspect of the present invention in the preparation of the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the recombinant microorganism described in the fifth aspect of the present invention, the cell described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, and the kit described in the eighth aspect of the present invention;

[0086] 6) Use of the nucleic acid molecule described in the third aspect of the present invention in preparing the vector described in the fourth aspect of the present invention, the recombinant microorganism described in the fifth aspect of the present invention, the cell described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, and the kit described in the eighth aspect of the present invention;

[0087] 7) Use of the vector described in the fourth aspect of the present invention in preparing the recombinant microorganism described in the fifth aspect of the present invention, the cell described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, and the kit described in the eighth aspect of the present invention;

[0088] 8) Use of the recombinant microorganism described in the fifth aspect of the present invention in preparing the cell described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, and the kit described in the eighth aspect of the present invention;

[0089] 9) Use of the cell according to the sixth aspect of the present invention in preparing the composition according to the seventh aspect of the present invention or the kit according to the eighth aspect of the present invention;

[0090] 10) Use of the composition described in the seventh aspect of the present invention in the preparation of the kit described in the eighth aspect of the present invention.

[0091] In the present invention, the term "treatment" may refer to therapeutic treatment or preventive measures, wherein the goal is to prevent or slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. In the present invention, treatment may refer to both treatment and prevention. Beneficial or desired clinical results include, but are not limited to, relieving symptoms; alleviating the extent of a condition, disorder or disease; stabilizing (i.e., not worsening) the state of a condition, disorder or disease; delaying the onset of a condition, disorder or disease or slowing its progression; improving a condition, disorder or disease state; and alleviating (whether partial or complete) (whether detectable or undetectable) or improving or ameliorating a condition, disorder or disease. Treatment may include causing a clinically significant response without excessive side effects. Treatment also includes a prolonged survival period compared to the expected survival period when not receiving treatment.

[0092] Further, the diseases or conditions associated with Siglec-6 expression include leukemia, lymphoma, mastocytosis, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, fractures, Alzheimer's disease, chronic neuropathic pain, hyperalgesia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), graft-versus-host disease (GVH), colitis, hereditary α-trypsinemia, neurofibromatosis, Kunis syndrome, urticaria, atopic dermatitis, contact dermatitis, angioedema, nodular prurigo, cholangitis, psoriasis, irritable bowel disease, IBS, functional dyspepsia, asthma, allergic reactions, keloids, chronic sinusitis, aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitial cystitis, hidradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn's disease, rheumatoid arthritis, gastroesophageal reflux disease, viral infections, achalasia, orthostatic tachycardia syndrome, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), complex regional pain syndrome, Ehlers-Danlos syndrome.

[0093] Furthermore, the leukemia includes acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, myelodysplastic syndrome, and myeloproliferative diseases.

[0094] Furthermore, the leukemia is selected from AML.

[0095] Furthermore, the Siglec-6 expressing cells include leukemia cells, lymphoma cells, mast cells, non-alcoholic steatohepatitis (NASH) cells, colitis cells, and neurofibroma cells.

[0096] Furthermore, the leukemia cells include acute lymphocytic leukemia (ALL) cells, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, and hairy cell leukemia cells.

[0097] Furthermore, the leukemia cells are selected from AML cells.

[0098] The present invention has the following advantages and beneficial effects:

[0099] The present invention constructs a chimeric antigen receptor (CAR) targeting human Siglec-6 and T cells modified by the CAR based on the antigen binding fragment of anti-Siglec-6. After the CAR structure specifically recognizes Siglec-6 through the antigen binding fragment, it can activate T cells, start its cytotoxic killing effect, and specifically target and kill AML cells. In vitro experiments have shown that the CAR-T cells can accurately and effectively target and attack Siglec-6-positive AML tumor cells, and can be used to prepare biological preparations for tumor biological immunotherapy. The CAR-T cells targeting Siglec-6 provided by the present invention, and their preparation methods and applications provide a new solution for the treatment of malignant tumors, have great basic research and clinical application value, and have good industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 These are the results of Siglec-6 antibody screening and humanization transformation, among which 1A is the SDS-PAGE detection of Siglec-6 extracellular segment protein expression; 1B is the FACS identification of Siglec-6 expression level; 1C is the immune titer ELISA test result; 1D is the immune titer FACS test result; 1E is the antibody and cell binding ability test result; 1F is the antibody affinity test result; 1G is the antibody and MV411, U937 binding ability test result.

[0101] Figure 2 This is a diagram showing the expression and distribution of Siglec-6 in different tumor cells.

[0102] Figure 3The figure is a graph showing the killing function test results of Siglec-6 CAR-T cells, where 3A is a schematic diagram of the three Siglec-6CAR structures; 3B is a graph showing the killing effect of three types of Siglec-6 CAR-T cells on acute myeloid leukemia Siglec-6 positive cells U937 detected by flow cytometry.

[0103] Figure 4 It is a diagram of the construction structure of pHAGE-Siglec-6 CAR, wherein 4A is a schematic diagram of the molecular construction of pHAGE-Siglec-6 CAR cells; 4B is a diagram of PCR results; and 4C is a diagram of sequencing and plasmid construction results.

[0104] Figure 5 Figure 5 is a diagram of the construction and characterization of pHAGE-Siglec-6 CAR-T cells, where 5A is a diagram of the proliferation of pHAGE-Siglec-6 CAR-T cells after infection with CAR virus; 5B is a diagram of the cell viability of pHAGE-Siglec-6 CAR-T cells after infection with CAR virus; 5C is a diagram of the positive rate of pHAGE-Siglec-6 CAR-T cells detected by flow cytometry 11 days and 13 days after infection with CAR virus.

[0105] Figure 6 This is a diagram showing the killing effect of pHAGE-Siglec-6 CAR-T on acute myeloid leukemia positive cells.

[0106] Figure 7 This is a diagram showing the killing effect of pHAGE-Siglec-6 CAR-T cells on Siglec-6 target knockout cells. DETAILED DESCRIPTION

[0107] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods used in the following embodiments are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0108] Example 1 Siglec-6 antibody screening and humanization modification

[0109] 1. Experimental methods

[0110] (1) The Siglec-6 extracellular segment protein expression vector was constructed and transiently transfected into 293F cells to express the purified protein. The purity of the recombinant protein was identified by SDS-PAGE.

[0111] (2) Construct a Siglec-6 overexpression lentiviral expression vector, transfect the CHO-S cell line, screen the stable cell line overexpressing the target gene, and identify the expression level of the target gene by FACS for subsequent antibody screening.

[0112] (3) A total of 10 BALB / c mice were immunized, and the immunization strategy was as follows: first immunization: 100 μg Siglec-6 protein / mouse, subcutaneous multiple immunization, Freund's complete adjuvant; second immunization: 100 μg Siglec-6 protein / mouse, subcutaneous multiple immunization, Freund's incomplete adjuvant; third immunization: 100 μg Siglec-6 protein / mouse, subcutaneous multiple immunization, Freund's incomplete adjuvant. After immunization, the titer was measured by ELISA and FACS, and mice with high titers 213, 218 and 219 were selected for four immunizations, and the titer was measured by ELISA after four immunizations.

[0113] (4) After four immunizations, the cells were incubated with the hybridoma fusion supernatant and the secondary antibody was incubated with PE-anti mouse IgG. The fluorescence intensity was read by a multifunctional microplate reader to detect the titer of the fusion clone. The clones with high fluorescence intensity were selected for re-inspection. After four immunizations, the cells were incubated with the hybridoma fusion supernatant and the secondary antibody was incubated with PE-anti mouse IgG. The fluorescence intensity was read by a multifunctional microplate reader to detect the titer of the fusion clone. The clones with high fluorescence intensity were selected for subcloning. The clones with high fluorescence intensity were selected for re-inspection and cryopreservation.

[0114] (5) Sequencing the hybridoma.

[0115] (6) The heavy chain variable regions and light chain variable regions of the five antibodies were connected via (G4S)3, and the binding ability of the antibodies to cells was detected by flow cytometry.

[0116] (7) The heavy chain variable region and light chain variable region of the five antibodies were connected by (G4S)3, and after being fused with Fc, the scFv-Fc was immobilized using an AHC sensor for 80 seconds. The buffer was PBST (PBS+0.02% tween20), and the Siglec6-His antigen was diluted to 100, 50, 25, 12.5, 6.25, and 0 nM. Affinity detection: equilibrium 60 seconds, binding 180 seconds, dissociation 180 seconds, and detection temperature 25°C.

[0117] (8) Detect the binding of Siglec-6scFv-Fc protein to MV411 and U937.

[0118] (9) The amino acids in the heavy chain variable regions and light chain variable regions of antibodies No. 1, No. 2, and No. 4 with better binding activity were humanized.

[0119] 2. Experimental results

[0120] (1) SDS-PAGE results Figure 1 A) shows that the protein was successfully expressed and can be used for immunization.

[0121] (2) FACS results Figure 1 B) The positive rate of the screened cell lines is over 90%, and they can be used for antibody screening.

[0122] (3) ELISA test results Figure 1 C) showed that all mice were successfully immunized, and the immune titer basically reached 1:125K, among which 213, 218 and 219 had the highest immune titer. FACS results ( Figure 1 D) shows that all mice were successfully immunized, among which mice No. 211, 212, 213, and 215 had the highest immune titers. The ELISA results after four immunizations showed that the immune titers of 211 and 219 basically reached 1:625K after four immunizations, and fusion can be arranged.

[0123] (4) After hybridoma fusion, a total of 5 antibody sequences were obtained, which were labeled as antibodies 1 to 5. The heavy chain variable region amino acid sequence of antibody 1 is shown in SEQ ID NO:26, and the light chain variable region amino acid sequence is shown in SEQ ID NO:23; the heavy chain variable region amino acid sequence of antibody 2 is shown in SEQ ID NO:27, and the light chain variable region amino acid sequence is shown in SEQ ID NO:24; the heavy chain variable region amino acid sequence of antibody 3 is shown in SEQ ID NO:28, and the light chain variable region amino acid sequence is shown in SEQ ID NO:25; the heavy chain variable region amino acid sequence of antibody 4 is shown in SEQ ID NO:29, and the light chain variable region amino acid sequence is shown in SEQ ID NO:25; the heavy chain variable region amino acid sequence of antibody 5 is shown in SEQ ID NO:30, and the light chain variable region amino acid sequence is shown in SEQ ID NO:23.

[0124] (5) FACS results Figure 1 E) shows that antibodies No. 1, No. 2, and No. 4 bind well to the antigen, while antibodies No. 3 and No. 5 bind weakly to the antigen.

[0125] (6) Affinity test results are as follows Figure 1 As shown in F.

[0126] (7) The binding results of Siglec-6scFv-Fc protein with MV411 and U937 are as follows Figure 1 As shown in G.

[0127] (8) The sequences after humanization are shown in SEQ ID NO: 31-36.

[0128] Example 2 Flow cytometry detection of membrane expression of Siglec-6 in different tumor cell lines

[0129] 1. Experimental methods

[0130] Flow cytometry was used to detect the membrane expression of Siglec-6 on different tumor cells: U266, BxPC-3, 293T, H929, HGC-27, MV411, and U937 using CD327 (Siglec-6) flow cytometry antibody.

[0131] 2. Experimental results

[0132] The results showed that U937 cells were high-expressing Siglec-6 cells, and MV411 cells were low-expressing Siglec-6 cells ( Figure 2 ).

[0133] Example 3 Detection of the killing function of three CAR-T cells targeting Siglec-6

[0134] 1. Experimental methods

[0135] 1) The scFv sequences of humanized antibodies 1, 2, and 4 were sent to GeneCare to construct Siglec-6 CAR lentivirus containing three different scFv sequences ( Figure 3 A), the three scFv sequences are shown in SEQ ID NO:37-39. The amino acid sequences of the CARs constructed by the three scFv sequences are shown in SEQ ID NO:41-43, and the nucleotide sequences of the CARs constructed by the three scFv sequences are shown in SEQ ID NO:48-50. These three lentiviruses were used to infect primary human T cells to prepare three Siglec-6CAR-T cells, which were respectively recorded as CAR-T-1, CAR-T-2, and CAR-T-4.

[0136] 2) Flow cytometry was used to detect the killing function of Siglec-6 CAR-T. The acute myeloid leukemia Siglec-6-positive cells U937 were labeled with Cell Tracker. The three types of Siglec-6 CAR-T cells were co-incubated with acute myeloid leukemia Siglec-6-positive cells U937 at an effector-target ratio of 1:1. The killing effect was detected by flow cytometry after 24 hours.

[0137] 2. Experimental results

[0138] The results showed that the three Siglec-6 CAR-T constructed at 24 hours of killing had a killing effect on the acute myeloid leukemia Siglec-6 positive cells U937, while CAR-T-1 had the best killing effect, with the tumor cell viability decreasing from 42.34% to 2.11% after 24 hours of killing ( Figure 3 B).

[0139] Example 4 Construction of CAR structure containing Siglec-6 scFv sequence

[0140] 1. Experimental methods

[0141] The sequence of SEQ ID NO:45 was codon optimized using a T cell-specific codon optimization algorithm, and then the heavy chain and light chain were connected using a G4S linker to obtain a scFv sequence for Siglec-6 (SEQ ID NO:37). The above scFv sequence was constructed into a pHAGE vector, RQR8 was used as a detection tag, and 41BB was used as a co-stimulatory molecule to construct the following. Figure 4 The CAR structure shown in A, the constructed CAR nucleotide sequence and amino acid sequence are shown in SEQ ID NO: 51 and SEQ ID NO: 44, respectively. PCR and homologous recombination were performed on RQR8-FMD-2A, Siglec-6scFv and CD8-41BB-CD3ζ, and then sequencing was performed to verify whether the lentiviral shuttle plasmid of pHAGE-Siglec-6 CAR was successfully constructed.

[0142] 2. Experimental results

[0143] RQR8-FMD-2A, Siglec-6scFv and CD8-41BB-CD3ζ were successfully PCR- and homologous-recombined ( Figure 4 B).

[0144] The sequencing results showed that the pHAGE-Siglec-6 CAR lentiviral shuttle plasmid was successfully constructed ( Figure 4 C).

[0145] Example 5 Construction and Characterization of pHAGE-Siglec-6 CAR-T Cells

[0146] 1. Experimental methods

[0147] 1) The CAR vector was used to package lentivirus, and CAR-T cells were infected and constructed at MOI=5 and MOI=10. After infection, the proliferation and viability of Siglec-6 CAR-T cells were monitored.

[0148] 2) The CAR-T cell positivity rate was detected on the 11th and 13th days after CAR virus infection.

[0149] 2. Experimental results

[0150] 1) The results showed that pHAGE-Siglec-6 CAR-T cells proliferated well ( Figure 5 A), the cell viability was slightly lower than that of Mock T cells in the first 5 days after virus infection, but the two tended to be consistent in subsequent culture ( Figure 5 B).

[0151] 2) The results showed that pHAGE-Siglec-6 CAR-T cells were highly expressed on human primary T cells, and the positive rate reached more than 80% on the 13th day after virus infection ( Figure 5 C).

[0152] Example 6 Validation of pHAGE-Siglec-6 CAR-T killing of acute myeloid leukemia positive cells

[0153] 1. Experimental methods

[0154] Using Cell Tracker to mark myeloid leukemia Siglec-6-positive cells U937 and MV411, the positive cells were co-incubated with Siglec-6 CAR-T at an effector-target ratio of 1:1 for 48 hours, and the killing effect was detected by flow cytometry.

[0155] 2. Experimental results

[0156] The results showed that after 48 hours of co-culture, pHAGE-Siglec-6 CAR-T cells could effectively kill both cells, but the killing effect on U937 was more obvious, which may be related to its higher target antigen ( Figure 6 ).

[0157] Example 7 pHAGE-Siglec-6 CAR-T killing specificity detection

[0158] 1. Experimental methods

[0159] 1) CRISPR-Cas9 gene editing technology was used to knock out the Siglec-6 target in U937 cells to obtain U937Siglec-6KO cells.

[0160] 2) Cell Tracker was used to label U937 and U937 Siglec-6KO cells, and pHAGE-Siglec-6 CAR-T cells were co-cultured with tumor cells at effector-target ratios of 1:1 and 1:3 for 24 hours, and the killing was detected by flow cytometry.

[0161] 2. Experimental results

[0162] The results show:

[0163] 1) Successfully obtained U937 Siglec-6KO cells ( Figure 7 A)

[0164] 2) When the effector-target ratio was 1:1, after 24 hours of CAR-T killing, the proportion of U937 cells in living cells dropped from 48.20% to 8.10%, while the proportion of U937 cells with Siglec-6 gene knockout remained at around 40%. A similar phenomenon was observed when the effector-target ratio was 1:3 ( Figure 7 B).

[0165] The above data indicate that pHAGE-Siglec-6 CAR-T has intrinsic target-dependent cytotoxic activity.

[0166] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment capable of binding to a Siglec-6 antigen, wherein the antibody or antigen-binding fragment comprises at least one sequence selected from the following: a light chain variable region CDR1 according to SEQ ID NO: 1-2, a light chain variable region CDR2 according to SEQ ID NO: 3-4, a light chain variable region CDR3 according to SEQ ID NO: 5-6, a heavy chain variable region CDR1 according to SEQ ID NO: 7-11, a heavy chain variable region CDR2 according to SEQ ID NO: 12-16, and a heavy chain variable region CDR3 according to SEQ ID NO: 17-22; Preferably, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment comprises a combination of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, or a combination of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6; Preferably, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment is as shown in SEQ ID NO:23-25, SEQ ID NO:31-33; Preferably, the heavy chain variable region amino acid sequence of the antibody or antigen-binding fragment comprises a combination of SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:17, or a combination of NO:7, SEQ ID NO:12, SEQ ID NO:22, or a combination of NO:8, SEQ ID NO:13, SEQ ID NO:18, or a combination of NO:9, SEQ ID NO:14, SEQ ID NO:19, or a combination of NO:10, SEQ ID NO:15, SEQ ID NO:20, or a combination of NO:11, SEQ ID NO:16, SEQ ID NO:21; Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is as shown in SEQ ID NO: 26-30, SEQ ID NO: 34-36.

2. A chimeric antigen receptor targeting Siglec-6, characterized in that: The chimeric antigen receptor comprises an extracellular antigen binding domain consisting of the antibody or antigen binding fragment of claim 1; Preferably, the antigen-binding fragment is a scFv that specifically binds to Siglec-6; Preferably, the amino acid sequence of the scFv is as shown in SEQ ID NO:37-39; Preferably, the chimeric antigen receptor further comprises the following components: a transmembrane domain, a co-stimulatory signal transduction domain, and a primary signaling domain; Preferably, the transmembrane domain includes a human CD8 transmembrane region, a human CD8α transmembrane region, a human CD28 transmembrane region, a human CD4 transmembrane region, a human CD28 transmembrane region, a human CD3ζ transmembrane region, a human CD137 / 4-1BB transmembrane region, a human CD134 / OX40 transmembrane region, a human ICOS transmembrane region, a human CD5 transmembrane region, a human CD9 transmembrane region, a human CD16 transmembrane region, a human CD22 transmembrane region, a human CD33 transmembrane region, a human CD37 transmembrane region, a human CD45 transmembrane region, a human CD64 transmembrane region, a human CD80 transmembrane region, a human CD86 transmembrane region, a human CD154 transmembrane region, a human TCRα transmembrane region, and a human TCRβ transmembrane region; Preferably, the transmembrane domain is selected from the transmembrane region of CD8; Preferably, the costimulatory signal transduction domain includes a human CD28 intracellular costimulatory signal domain, a human CD137 / 4-1BB intracellular costimulatory signal domain, a human CD134 / OX40 intracellular costimulatory signal domain, a human CD27 intracellular costimulatory signal domain, a human ICOS intracellular costimulatory signal domain, a human B7-H3 intracellular costimulatory signal domain, a human 2B4 intracellular costimulatory signal domain, a human DAP10 intracellular costimulatory signal domain, a human DNAM1 intracellular costimulatory signal domain, and a human DAP12 intracellular costimulatory signal domain; Preferably, the costimulatory signal transduction domain is selected from the human CD137 / 4-1BB intracellular costimulatory signal domain; Preferably, the primary signaling domain includes human CD3ζ and human FcεRIγ; Preferably, the primary signaling domain is selected from human CD3ζ; Preferably, the chimeric antigen receptor further comprises one or more of the following components: a detection tag, a cleavage protein, a signal peptide, a hinge region; Preferably, the detection tags include RQR8, EGFRt, CD20, c-MYC, 6×His, GST, Fc, Flag, and HA; Preferably, the detection tag is selected from RQR8; Preferably, the cleavage protein comprises cleavage protein 2A or furin; Preferably, the cleavage protein 2A includes cleavage protein T2A, cleavage protein P2A, cleavage protein E2A, and cleavage protein F2A; Preferably, the cleavage protein F2A is selected from FMD-2A.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment of claim 1, or encodes the chimeric antigen receptor of claim 2; Preferably, the nucleic acid molecule further comprises a promoter, and / or a restriction enzyme cleavage site located after the promoter, and / or a kozak sequence located after the restriction enzyme cleavage site; Preferably, the nucleic acid molecule sequence encoding the antibody or antigen-binding fragment corresponds to part or all of SEQ ID NO:45-47; Preferably, the nucleic acid molecule sequence encoding the chimeric antigen receptor corresponds to part or all of SEQ ID NO: 48-50.

4. A carrier, characterized in that It contains the nucleic acid molecule according to claim 3.

5. A recombinant microorganism, characterized in that It contains a nucleic acid molecule encoding the nucleic acid of claim 3, Or the vector described in claim 4.

6. Any of the following cells: 1) A cell, characterized in that: The cell is capable of expressing the antibody or antigen-binding fragment of claim 1; 2) A modified cell, characterized in that the cell is modified to express the chimeric antigen receptor according to claim 2; Preferably, the cell contains the nucleic acid molecule of claim 3 or the vector of claim 4.

7. A composition comprising the antibody or antigen-binding fragment of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the recombinant microorganism of claim 5, and the cell of claim 6; Preferably, the composition further comprises a pharmaceutically acceptable carrier.

8. A kit comprising the antibody or antigen-binding fragment of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the recombinant microorganism of claim 5, the cell of claim 6, and the composition of claim 7.

9. Any of the following methods: 1) A method for preparing the antibody or antigen-binding fragment according to claim 1, characterized in that: The preparation method comprises culturing the cell described in claim 6 so that it can express the antibody or antigen-binding fragment described in claim 1; 2) A method for preparing the cell according to claim 6, characterized in that the preparation method comprises introducing the nucleic acid molecule according to claim 3 or the vector according to claim 4 into the cell; 3) A method for in vitro targeted killing of cells expressing Siglec-6, the method comprising: contacting the Siglec-6-expressing cells with the antibody or antigen-binding fragment of claim 1, the chimeric antigen receptor of claim 2, the cells of claim 6, the composition of claim 7, or the kit of claim 8, and observing the killing of the Siglec-6-expressing cells; 4) A method for detecting Siglec-6, the method comprising: contacting the test sample with the antibody or antigen-binding fragment according to claim 1, the chimeric antigen receptor according to claim 2, the cell according to claim 6, the composition according to claim 7, or the kit according to claim 8, thereby forming an antigen-antibody complex, and detecting the complex, wherein detection of the complex indicates the presence of Siglec-6 in the test sample; Preferably, the Siglec-6 expressing cells include leukemia cells, lymphoma cells, mast cells, non-alcoholic fatty liver inflammation cells, colitis cells, and neurofibroma cells; Preferably, the leukemia cells include acute lymphocytic leukemia (ALL) cells, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, and hairy cell leukemia cells; Preferably, the leukemia cells are selected from AML cells.

10. Any of the following applications: 1) Use of the antibody or antigen-binding fragment of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the recombinant microorganism of claim 5, the cell of claim 6, or the composition of claim 7 in the preparation of a drug for diagnosing and / or treating a disease or condition associated with Siglec-6 expression; 2) Use of the antibody or antigen-binding fragment of claim 1, the chimeric antigen receptor of claim 2, the cell of claim 6, the composition of claim 7, or the kit of claim 8 in vitro for targeted killing of cells expressing Siglec-6; 3) Use of the antibody or antigen-binding fragment of claim 1, the chimeric antigen receptor of claim 2, the cell of claim 6, the composition of claim 7, or the kit of claim 8 in detecting Siglec-6; 4) Use of the antibody or antigen-binding fragment of claim 1 in the preparation of the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the vector of claim 4, the recombinant microorganism of claim 5, the cell of claim 6, the composition of claim 7, or the kit of claim 8; 5) Use of the chimeric antigen receptor according to claim 2 in the preparation of the nucleic acid molecule according to claim 3, the vector according to claim 4, the recombinant microorganism according to claim 5, the cell according to claim 6, the composition according to claim 7, or the kit according to claim 8; 6) Use of the nucleic acid molecule according to claim 3 in preparing the vector according to claim 4, the recombinant microorganism according to claim 5, the cell according to claim 6, the composition according to claim 7, or the kit according to claim 8; 7) Use of the vector according to claim 4 in the preparation of the recombinant microorganism according to claim 5, the cell according to claim 6, the composition according to claim 7, or the kit according to claim 8; 8) Use of the recombinant microorganism according to claim 5 in preparing the cell according to claim 6, the composition according to claim 7, or the kit according to claim 8; 9) Use of the cell according to claim 6 in preparing the composition according to claim 7 or the kit according to claim 8; 10) Use of the composition according to claim 7 in the preparation of the kit according to claim 8; Preferably, the diseases or disorders associated with Siglec-6 expression include leukemia, lymphoma, mastocytosis, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, fractures, Alzheimer's disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease, nonalcoholic fatty hepatitis, graft-versus-host disease, colitis, hereditary α-trypsinemia, neurofibromatosis, Kunis syndrome, urticaria, atopic dermatitis, contact dermatitis, angioedema, nodular prurigo, cholangitis, psoriasis. disease, irritable bowel syndrome, functional dyspepsia, asthma, allergic reactions, keloids, chronic sinusitis, aspirin-exacerbated respiratory disease, chronic obstructive pulmonary disease, bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitial cystitis, hidradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn's disease, rheumatoid arthritis, gastroesophageal reflux disease, viral infections, achalasia, orthostatic tachycardia syndrome, amyotrophic lateral sclerosis, multiple sclerosis, complex regional pain syndrome, Ehlers-Danlos syndrome; Preferably, the leukemia includes acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, myelodysplastic syndrome, myeloproliferative disease; Preferably, the leukemia is selected from AML; Preferably, the Siglec-6 expressing cells include leukemia cells, lymphoma cells, mast cells, non-alcoholic steatohepatitis (NASH) cells, colitis cells, neurofibroma cells; Preferably, the leukemia cells include acute lymphocytic leukemia (ALL) cells, acute myeloid leukemia (AML) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, and hairy cell leukemia cells; Preferably, the leukemia cells are selected from AML cells.

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