Fusion protein comprising biepitopic chimeric antigen receptor (CAR) and car-t cell modified by fusion protein

Abstract

Disclosed are a fusion protein comprising a biepitopic CAR and a CAR-T cell modified by the fusion protein. Specifically disclosed is a fusion protein having an amino acid sequence of SEQ ID NO: 5, and a CLL1 DE CAR-CD27-T cell constructed using the fusion protein. The CLL1 DE CAR-CD27-T cell can secrete T cell specific effector molecules IFN-γ, specifically kill CLL1+ target cells, has in-vivo antitumor activity, and has dual CLL1-binding sites.

Description

Fusion protein containing dual-epitope chimeric antigen receptor and CAR-T cells modified therewith Technical Field

[0001] The present application belongs to the field of cellular immunotherapy technology, and specifically relates to a fusion protein containing a dual-epitope chimeric antigen receptor and a modified CAR-T cell thereof. Background Art

[0002] Chimeric Antigen Receptor (CAR) contains an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen binding domain can contain a single-chain variable fragment (scFv) or a single-domain antibody (sdAb) that targets a tumor antigen. After binding to the target tumor antigen, CAR can activate T cells to produce a specific anti-tumor response in an antigen-dependent manner, without being restricted by the specific major histocompatibility complex (MHC) of the target tumor antigen. Single-domain antibodies (sdAb) only have a single monomeric antibody variable domain. Single-domain antibodies are also called nanobodies (Nb) because of their small molecular weight. They are the smallest antibody fragments with antigen-binding function to date. Their ability to bind to antigens and their stability are basically the same as those of complete antibodies or have higher specific antigen affinity. Compared with traditional antibodies, single-domain antibodies also have many unique properties, such as good stability, the ability to reach specific antigen epitopes, the ability to combine building blocks in any way, and low production costs. Chimeric antigen receptor T cell (CAR-T cell) therapy involves the transfer of genetic material containing specific antigen recognition domains and T cell activation signals into T cells through gene modification technology. This allows T cells to directly bind to specific antigens on the surface of tumor cells, activating and proliferating them, thereby achieving targeted killing of tumor cells. With the development of tumor immunotherapy and clinical technologies, CAR-T immunotherapy is currently one of the most promising tumor immunotherapy methods.

[0003] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem / progenitor cells, characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc. Most cases are acute and severe, with a poor prognosis. If not treated in time, it can often be life-threatening. In recent years, researchers have found that immune targeted therapy targeting CLL1 is effective in treating AML. C-type lectin-like molecule 1 (CLL1), also known as C-type lectin domain family 12 member A (CLEC12A), is a type II transmembrane glycoprotein that plays an important role in immune regulation as an inhibitory receptor. CLL1 is present in myeloid cells in peripheral blood and bone marrow, as well as in most AML cells, and is also expressed on CD34 in most AML cells. + CD38 - On stem cells, normal human CD34 + CD38 - Stem cells do not express CLL1, making it a potential target for the treatment and diagnosis of AML due to its unique expression pattern. Furthermore, CLL1 is also expressed on cells of myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML).

[0004] Currently, CAR-T cell therapy has achieved encouraging results in hematologic malignancies such as B-cells. However, the antigenic properties of myeloid cells differ fundamentally from those of B-cells. The elimination of normal myeloid cells by CAR-T cells can lead to bone marrow failure, and there is a lack of corresponding replacement therapies. This has led to very slow progress in CAR-T therapy for AML, and the successful application of CAR-T technology in acute myeloid leukemia (AML) still faces many challenges. Furthermore, the development of multiple redirecting CAR-Ts that recognize two non-overlapping epitopes on tumor antigens may be necessary to effectively eradicate tumor cells. Therefore, in-depth research and development of new and effective CAR-T cells and improving the efficacy of CAR-T cells have important theoretical significance and clinical application value for immune cell therapy for AML.

[0005] SUMMARY OF THE INVENTION

[0006] The technical problem to be solved by this application is to provide a fusion protein containing a dual-epitope chimeric antigen receptor, immune effector cells modified therewith, and their applications. The technical problem to be solved is not limited to the technical subject matter described herein, and those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0007] To solve the above technical problems, the present application first provides a fusion protein, which includes a dual-epitope chimeric antigen receptor and CD27. The dual-epitope chimeric antigen receptor includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain. The antigen binding domain includes a single-domain antibody 1 and a single-domain antibody 2 that specifically target CLL1. The amino acid sequence of the single-domain antibody 1 can be as shown in positions 22-150 of SEQ ID NO: 1, and the amino acid sequence of the single-domain antibody 2 can be as shown in positions 166-294 of SEQ ID NO: 1.

[0008] Furthermore, the single-domain antibody 1 may be located at the N-terminus or C-terminus of the single-domain antibody 2.

[0009] Furthermore, the single-domain antibody 1 may be located at the N-terminus of the single-domain antibody 2.

[0010] Furthermore, the transmembrane domain may be derived from CD8a, CD4, CD28, CD137, CD80, CD86 or CD152.

[0011] Furthermore, the transmembrane domain may be a CD8a transmembrane region.

[0012] Furthermore, the amino acid sequence of the CD8a transmembrane region may be as shown in positions 340-363 of SEQ ID NO:1.

[0013] Furthermore, the bi-epitope chimeric antigen receptor may further include a hinge domain, and the hinge domain may be located between the C-terminus of the antigen binding domain and the N-terminus of the transmembrane domain.

[0014] Furthermore, the hinge domain may be derived from CD8a.

[0015] Furthermore, the hinge domain may be a CD8a hinge region.

[0016] Furthermore, the amino acid sequence of the CD8a hinge region may be as shown in positions 295-339 of SEQ ID NO: 1.

[0017] In the dual-epitope chimeric antigen receptor described herein, the intracellular signaling domain may include the costimulatory domain 4-1BB and the activation domain CD3ζ.

[0018] Furthermore, the amino acid sequence of the costimulatory domain 4-1BB may be as shown in positions 364-410 of SEQ ID NO:1.

[0019] Furthermore, the amino acid sequence of the activation domain CD3ζ may be as shown in positions 411-522 of SEQ ID NO:1.

[0020] Furthermore, the bi-epitope chimeric antigen receptor may further include a linker, which can be used to connect the single-domain antibody 1 and the single-domain antibody 2.

[0021] Furthermore, the linker may be a flexible peptide linker.

[0022] Furthermore, the linker may be a peptide linker comprising glycine and / or serine residues.

[0023] Furthermore, the amino acid sequence of the linker may be as shown in positions 151-165 of SEQ ID NO: 1.

[0024] Furthermore, the dual-epitope chimeric antigen receptor may further include a signal peptide (Signal) located at the N-terminus of the dual-epitope chimeric antigen receptor.

[0025] Furthermore, the signal peptide may be derived from CD8.

[0026] Furthermore, the amino acid sequence of the signal peptide may be as shown in positions 1 to 21 of SEQ ID NO: 1.

[0027] Furthermore, the dual-epitope chimeric antigen receptor may be, from N-terminus to C-terminus, the signal peptide (Signal), the single-domain antibody 1 (also referred to as single-domain antibody CLL1-VHH-1), the linker, the single-domain antibody 2 (also referred to as single-domain antibody CLL1-VHH-16), the CD8a hinge region (Hinge), the CD8a transmembrane region, the co-stimulatory domain 4-1BB and the activation domain CD3ζ.

[0028] In the above fusion protein, the dual-epitope chimeric antigen receptor may be any of the following:

[0029] A1) the amino acid sequence is positions 22-522 of SEQ ID NO: 1 or the protein set forth in SEQ ID NO: 1;

[0030] A2) A fusion protein having the same function as A1) obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

[0031] Furthermore, the name of the dual-epitope chimeric antigen receptor (also known as dual-epitope CAR) may be CLL1 DE CAR, and the amino acid sequence of the CLL1 DE CAR may be SEQ ID NO: 1.

[0032] In the above fusion protein, the dual-epitope chimeric antigen receptor and the CD27 can be connected via a self-cleaving peptide.

[0033] Furthermore, the CD27 may be located at the C-terminus of the dual-epitope chimeric antigen receptor.

[0034] Furthermore, the CD27 may be full-length human CD27.

[0035] Specifically, the amino acid sequence of CD27 may be as shown in SEQ ID NO: 3.

[0036] In the above fusion protein, the self-cleavage peptide includes but is not limited to P2A, F2A, T2A and E2A.

[0037] The amino acid sequence of P2A may be SEQ ID NO: 7. The amino acid sequence of F2A may be SEQ ID NO: 8. The amino acid sequence of T2A may be SEQ ID NO: 9. The amino acid sequence of E2A may be SEQ ID NO: 10.

[0038] Furthermore, the self-cleaving peptide may be P2A.

[0039] Furthermore, the fusion protein may be any of the following:

[0040] B1) the amino acid sequence is positions 22-804 of SEQ ID NO:5 or the protein set forth in SEQ ID NO:5;

[0041] B2) A fusion protein having the same function as B1) obtained by connecting a tag to the N-terminus and / or C-terminus of B1).

[0042] Furthermore, the name of the fusion protein may be CLL1 DE CAR-CD27, and the amino acid sequence of the CLL1 DE CAR-CD27 may be SEQ ID NO:5.

[0043] The fusion protein contains a dual-epitope chimeric antigen receptor (CLL1 DE CAR).

[0044] The tags described herein include, but are not limited to, GST (glutathione sulfhydryltransferase) tag protein, His-tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0045] The present application also provides a biomaterial, which may be any of the following:

[0046] C1) a nucleic acid molecule encoding any one of the fusion proteins described herein;

[0047] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0048] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0049] C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

[0050] C5) A recombinant cell containing the nucleic acid molecule of C1), or a recombinant cell containing the expression cassette of C2), or a recombinant cell containing the recombinant vector of C3).

[0051] In the above-mentioned biological material, the nucleic acid molecule may be any of the following:

[0052] D1) the coding sequence is positions 64-2412 of SEQ ID NO:6 or the DNA molecule represented by SEQ ID NO:6;

[0053] D2) The nucleotide sequence is positions 64-2412 of SEQ ID NO: 6 or the DNA molecule shown in SEQ ID NO: 6.

[0054] The DNA molecule shown in SEQ ID NO: 6 can be named CLL1 DE CAR-CD27 gene, which encodes the fusion protein CLL1 DE CAR-CD27 with the amino acid sequence of SEQ ID NO: 5. The DNA molecule shown at positions 64-2412 of SEQ ID NO: 6 encodes the fusion protein CLL1 DE CAR-CD27 without a signal peptide.

[0055] The present application also provides modified immune effector cells, which may be immune effector cells modified with any of the fusion proteins described herein.

[0056] Furthermore, the modified immune effector cells comprise any one of the nucleic acid molecules described herein, and the immune effector cells include but are not limited to T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood mononuclear cells (PBMC) and pluripotent stem cells.

[0057] Furthermore, the immune effector cells may be T lymphocytes (T cells).

[0058] Furthermore, the immune effector cells may include autologous or non-autologous immune effector cells.

[0059] Furthermore, the modified immune effector cells may include CAR-T cells, CAR-NK cells, CAR-NKT cells, CAR-γδT cells, CAR-macrophages (CAR-M cells), CAR-iPSC cells and CAR-PSC cells.

[0060] Furthermore, the modified immune effector cell may be a CAR-T cell, which may express any of the fusion proteins described herein, targeting C-type lectin-like molecule 1 (CLL1).

[0061] Furthermore, the CAR-T cell may be a CLL1 DE CAR-CD27-T cell, and the CLL1 DE CAR-CD27-T cell contains the CLL1 DE CAR-CD27 gene shown in SEQ ID NO:6.

[0062] The CLL1 DE CAR-CD27-T cells can target dual epitopes of the CLL1 antigen.

[0063] The CLL1 DE CAR-CD27-T cells may have at least any one of the following characteristics:

[0064] F1) secretes T cell-specific effector molecule IFN-γ;

[0065] F2) specifically kills CLL1-positive tumor cells;

[0066] F3) Inhibits the growth of CLL1-positive tumors.

[0067] The present application also provides the use of any of the fusion proteins, the biomaterials, or the modified immune effector cells described herein in the preparation of a product having any of the following functions:

[0068] E1) Prevention or treatment of tumors;

[0069] E2) Prevent or treat CLL1 target-related diseases;

[0070] E3) Killing CLL1-positive tumor cells;

[0071] E4) inhibiting the growth of CLL1-positive tumors;

[0072] E5) promotes the release of cytokine IFN-γ;

[0073] E6) Detection of cancer cells expressing CLL1.

[0074] In the above application, the tumor may be a CLL1-positive tumor.

[0075] In the above application, the CLL1 target-related disease may be CLL1-positive cancer (i.e., cancer expressing CLL1).

[0076] Furthermore, the CLL1-positive tumor or the CLL1 target-related disease may be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) or chronic myeloid leukemia (CML).

[0077] The articles of manufacture described herein may include a reagent, a kit, a medicament, or a pharmaceutical composition.

[0078] The present application also provides a pharmaceutical composition for preventing or treating CLL1 target-related diseases, which comprises the modified immune effector cells and one or more pharmaceutically acceptable carriers.

[0079] The pharmaceutically acceptable carrier is selected from diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants and lubricants.

[0080] In order to prepare the pharmaceutical composition into an injectable preparation, such as a solution, emulsion, lyophilized powder injection, and suspension, all diluents (carriers) commonly used in the art can be used, for example, water, saline, phosphate-buffered saline, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, to prepare an isotonic injection, an appropriate amount of a carrier such as sodium chloride, glucose, or glycerol can be added to the injectable preparation. In addition, conventional carriers such as cosolvents, buffers, and pH adjusters can also be added.

[0081] The pharmaceutical composition may further include a cell freezing solution (containing dimethyl sulfoxide, sodium chloride, human serum albumin, etc.).

[0082] The present application also provides a kit containing any one of the fusion proteins or modified immune effector cells described herein.

[0083] The present application also provides a method for preventing or treating a CLL1 target-related disease, comprising administering the modified immune effector cell or the pharmaceutical composition to a subject suffering from a CLL1 target-related disease.

[0084] In the above method, the CLL1 target-related disease may be CLL1-positive cancer.

[0085] In the above method, the CLL1-positive cancer may be acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia.

[0086] The present application also provides the modified immune effector cells or the pharmaceutical composition described herein for use in preventing or treating CLL1 target-related diseases.

[0087] Depending on the modified immune effector cells or the pharmaceutical composition used, the CLL1 target-related disease may be CLL1-positive cancer.

[0088] Furthermore, the CLL1-positive cancer may be acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia.

[0089] The present application also provides a method for preparing the modified immune effector cells, which comprises: transferring the nucleic acid molecules described herein into immune effector cells for stable expression to obtain the modified immune effector cells.

[0090] In the above method, the immune effector cells are selected from T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood monocytes and pluripotent stem cells.

[0091] In the above method, the immune effector cells may be T cells.

[0092] In the above method, the method may include the following steps:

[0093] (1) cloning the CLL1 DE CAR-CD27 gene (SEQ ID NO: 6) into a retroviral vector to obtain a recombinant retroviral vector;

[0094] (2) introducing the recombinant retroviral vector into packaging cells for packaging to obtain a recombinant retrovirus;

[0095] (3) Infecting human T cells with the recombinant retrovirus to obtain recombinant cells.

[0096] Furthermore, the retroviral vector may be the retroviral vector MP71.

[0097] Furthermore, the packaging cells may be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0098] The present application also provides use of any of the fusion proteins described herein or the modified immune effector cells in the preparation of a drug for preventing or treating CLL1 target-related diseases.

[0099] This application first designs a dual-epitope chimeric antigen receptor (dual-epitope CAR) CLL1 DE CAR (SEQ ID NO: 1) that binds to two separate epitopes of a single tumor antigen (CLL1), and the structure is shown in Figure 1. On this basis, CLL1 DE CAR is fused (connected) with CD27 through a self-cleavage peptide (such as P2A) to obtain a fusion protein CLL1 DE CAR-CD27, the structure of which is shown in Figure 2. Furthermore, T cells are modified using the CLL1 DE CAR-CD27 gene (SEQ ID NO: 6) to obtain dual-epitope CAR-T cells targeting CLL1: CLL1 DE CAR-CD27-T cells. The constructed CAR-T cells were functionally verified, and the experimental results showed that the CLL1 DE CAR-CD27-T cells of this application can well secrete the T cell-specific effector molecule IFN-γ, effectively and specifically kill CLL1. + Target cells, with good in vivo tumoricidal activity, can not only significantly inhibit the proliferation of tumor cells in mice, but also significantly prolong the survival time of mice. Its IFN-γ secretion effect, its specific cytotoxic effect and its in vivo tumoricidal activity are better than CTR T cells and CLL1 DE CAR-T cells. The dual-epitope CAR-T cells of this application have dual CLL1 binding sites, bind more tightly to CLL1, have good anti-tumor ability, and can be used for immunotherapy of CLL1 target-related diseases (such as acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia), and have broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 is a schematic diagram of the molecular structure of CLL1 DE CAR.

[0101] Figure 2 is a schematic diagram of the molecular structure of CLL1 DE CAR-CD27.

[0102] Figure 3 shows the results of CAR expression detection in CLL1 DE CAR-CD27-T cells.

[0103] FIG4 shows the functional detection results of CLL1 DE CAR-CD27-T cells secreting specific effector molecule IFN-γ.

[0104] FIG5 shows the cytotoxicity (degranulation CD107a) function test results of CLL1 DE CAR-CD27-T cells.

[0105] FIG6 shows the cytotoxicity test results of CLL1 DE CAR-CD27-T cells.

[0106] FIG7 shows the results of in vivo tumoricidal activity detection of CLL1 DE CAR-CD27-T cells. Modes for Carrying Out the Invention

[0107] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0108] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0109] The retroviral vector MP71 in the following examples is described in the following literature: Engels B, Cam H, et al. Retroviral Vectors for High-Level Transgene Expression in TLymphocytes[J]. Human Gene Therapy, 2003, 14(12): 1155-1168. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this application and cannot be used for other purposes.

[0110] The human peripheral blood mononuclear cells (PBMCs) in the following examples were derived from the venous blood of healthy volunteers.

[0111] Example 1. Design of a fusion protein containing a dual-epitope chimeric antigen receptor

[0112] 1. Design and structure of the dual-epitope chimeric antigen receptor CLL1 DE CAR

[0113] In this example, a dual-epitope chimeric antigen receptor (dual-epitope CAR) that binds to two separate epitopes of a single tumor antigen (CLL1) is first designed. The dual-epitope CAR includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain contains two different anti-CLL1 single-domain antibodies (single-domain antibodies CLL1-VHH-1 and CLL1-VHH-16), which can target and recognize two different epitopes of the tumor antigen (CLL1); the transmembrane domain is used to connect the extracellular antigen-binding domain with the intracellular signaling domain, and can be derived from CD8a, CD4, CD28, CD137, CD80, CD86 or CD152, etc.; the intracellular signaling domain includes the main intracellular signaling domain of immune effector cells (such as T cells) (such as CD3ζ), and may also include a costimulatory signaling domain (such as 4-1BB or CD28). Furthermore, a hinge domain (such as a CD8a hinge region) may be included between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. Furthermore, the dual-epitope CAR may also include a signal peptide (such as a CD8a signal peptide) at the N-terminus.

[0114] An example of the dual-epitope CAR molecular structure designed above is shown in Figure 1. The dual-epitope CAR is named CLL1 DE CAR, which consists of a signal peptide (Signal), a single-domain antibody CLL1-VHH-1, a linker, a single-domain antibody CLL1-VHH-16, a CD8a hinge region (Hinge), a CD8a transmembrane region, a co-stimulatory domain 4-1BB, and an activation domain CD3ζ from the N-terminus to the C-terminus (Figure 1).

[0115] The amino acid sequence of the dual-epitope chimeric antigen receptor CLL1 DE CAR is shown in SEQ ID NO: 1 (522aa). Among them, positions 1-21 of SEQ ID NO: 1 are the amino acid sequence of the signal peptide (Signal); positions 22-150 of SEQ ID NO: 1 are the amino acid sequence of the single-domain antibody CLL1-VHH-1; positions 151-165 of SEQ ID NO: 1 are the amino acid sequence of the linker; positions 166-294 of SEQ ID NO: 1 are the amino acid sequence of the single-domain antibody CLL1-VHH-16; positions 295-339 of SEQ ID NO: 1 are the amino acid sequence of the CD8a hinge region (Hinge); positions 340-363 of SEQ ID NO: 1 are the amino acid sequence of the CD8a transmembrane region; positions 364-410 of SEQ ID NO: 1 are the amino acid sequence of the costimulatory domain 4-1BB; and positions 411-522 of SEQ ID NO: 1 are the amino acid sequence of the activation domain CD3ζ.

[0116] The gene encoding CLL1 DE CAR is the CLL1 DE CAR gene, and its nucleotide sequence is shown in SEQ ID NO: 2 (1566 bp). Wherein: positions 1-63 of SEQ ID NO: 2 are the nucleotide sequence of the signal peptide (Signal); positions 64-450 of SEQ ID NO: 2 are the nucleotide sequence of the single-domain antibody CLL1-VHH-1; positions 451-495 of SEQ ID NO: 2 are the nucleotide sequence of the linker; positions 496-882 of SEQ ID NO: 2 are the nucleotide sequence of the single-domain antibody CLL1-VHH-16; positions 883-1017 of SEQ ID NO: 2 are the nucleotide sequence of the CD8a hinge region (Hinge); positions 1018-1089 of SEQ ID NO: 2 are the nucleotide sequence of the CD8a transmembrane region; positions 1090-1230 of SEQ ID NO: 2 are the nucleotide sequence of the costimulatory domain 4-1BB; positions 1231-1566 of SEQ ID NO: 2 are the nucleotide sequence of the activation domain CD3ζ.

[0117] 2. Design and structure of fusion proteins containing dual-epitope chimeric antigen receptors

[0118] Based on the dual-epitope chimeric antigen receptor CLL1 DE CAR (Figure 1, SEQ ID NO: 1) designed in step 1, CLL1 DE CAR and CD27 were fused (connected) through a self-cleavage peptide (such as P2A) to obtain a fusion protein named CLL1 DE CAR-CD27 (Figure 2).

[0119] The CD27 is a full-length human CD27, the amino acid sequence of which is shown in SEQ ID NO: 3, and the nucleotide sequence of which is shown in SEQ ID NO: 4.

[0120] The amino acid sequence of the designed fusion protein CLL1 DE CAR-CD27 is shown in SEQ ID NO: 5 (804aa). Among them, positions 1-522 of SEQ ID NO: 5 are the amino acid sequence of the dual-epitope chimeric antigen receptor CLL1 DE CAR; positions 523-544 of SEQ ID NO: 5 are the amino acid sequence of the self-cleaving peptide P2A; and positions 545-804 of SEQ ID NO: 5 are the amino acid sequence of the full-length human CD27.

[0121] The gene encoding the fusion protein CLL1 DE CAR-CD27 is the CLL1 DE CAR-CD27 gene, and its nucleotide sequence is shown in SEQ ID NO: 6 (2412 bp). Among them, positions 1-1566 of SEQ ID NO: 6 are the nucleotide sequence of the dual-epitope chimeric antigen receptor CLL1 DE CAR; positions 1567-1632 of SEQ ID NO: 6 are the nucleotide sequence of the self-cleaving peptide P2A; and positions 1633-2412 of SEQ ID NO: 6 are the nucleotide sequence of the full-length human CD27.

[0122] In the fusion protein CLL1 DE CAR-CD27, P2A (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 7) is a "self-cleavage" peptide. The "self-cleavage" function of the P2A peptide forms two proteins, an upstream product and a downstream product, by self-cleavage. Those skilled in the art can replace P2A in the CLL1 DE CAR-CD27 fusion protein molecule, for example, using F2A (VKQTLNFDLLKLAGCVESNPG, SEQ ID NO: 8), T2A (EGRGSLLTCGDVEENPG, SEQ ID NO: 9), E2A (QCTNYALLKLAGDVESNPG, SEQ ID NO: 10), etc., which have the same "self-cleavage" function as P2A.

[0123] Example 2. Construction of CAR-T cells modified with the fusion protein CLL1 DE CAR-CD27

[0124] The fusion protein CLL1 DE CAR-CD27 designed in Example 1 contains a dual-epitope chimeric antigen receptor CLL1 DE CAR, and the CAR-T cells modified by the fusion protein are also referred to as dual-epitope CAR-T cells. In this example, the dual-epitope CAR-T cells constructed using the CLL1 DE CAR gene (SEQ ID NO: 2) are named CLL1 DE CAR-T cells; the dual-epitope CAR-T cells constructed using the CLL1 DE CAR-CD27 gene (SEQ ID NO: 6) are named CLL1 DE CAR-CD27-T cells. The construction method is to transfer the CAR gene into T cells for stable expression, and the specific steps are as follows:

[0125] 1. Construction of dual-epitope CAR vector

[0126] (1) The recombinant vectors pUC57-CLL1 DE CAR and pUC57-CLL1 DE CAR-CD27 were double-digested with NotI and EcoRI, respectively, and the target gene fragments were recovered by gel cutting. The recombinant vector pUC57-CLL1 DE CAR is a recombinant vector obtained by cloning the CLL1 DE CAR gene (SEQ ID NO: 2) into the pUC57 vector; the recombinant vector pUC57-CLL1 DE CAR-CD27 is a recombinant vector obtained by cloning the CLL1 DE CAR-CD27 gene (SEQ ID NO: 6) into the pUC57 vector. The above recombinant vectors were synthesized and provided by Qingke Biotechnology Co., Ltd.

[0127] (2) The retroviral vector MP71 was double-digested with NotI and EcoRI, and the large vector fragment was recovered by gel cutting.

[0128] (3) The target gene fragment and the large vector fragment were connected using T4 ligase to obtain recombinant retroviral vectors MP71-CLL1 DE CAR and MP71-CLL1 DE CAR-CD27 carrying the CLL1 DE CAR gene and CLL1 DE CAR-CD27 gene, respectively.

[0129] (4) The recombinant retroviral vectors MP71-CLL1 DE CAR and MP71-CLL1 DE CAR-CD27 were transformed into competent Escherichia coli DH5α, and the plasmids were extracted and purified using the Qiagen plasmid purification kit to obtain MP71-CLL1 DE CAR plasmid and MP71-CLL1 DE CAR-CD27 plasmid.

[0130] The recombinant retroviral vector MP71-CLL1 DE CAR (i.e., MP71-CLL1 DE CAR plasmid) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment with a nucleotide sequence of SEQ ID NO: 2 in the sequence listing, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0131] The recombinant retroviral vector MP71-CLL1 DE CAR-CD27 (i.e., MP71-CLL1 DE CAR-CD27 plasmid) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment with the nucleotide sequence of SEQ ID NO: 6 in the sequence listing, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0132] 2. Retroviral packaging

[0133] The MP71-CLL1 DE CAR plasmid and MP71-CLL1 DE CAR-CD27 plasmid prepared in step 1 were respectively introduced into packaging cells for packaging to complete virus assembly and obtain retrovirus. The specific steps of virus packaging are as follows:

[0134] a) Day 1: Phoenix Ecotropic (ECO) cells should be less than 20 generations old and not overgrown. 6 Cells were plated at a density of 100 mL / ml and 10 mL of DMEM medium (10% Fetal Bovine Serum, 2 mM L-glutamine, 1% Penicillin / Streptomycin) was added to a 10 cm cell culture dish. The cells were thoroughly mixed and cultured at 37°C overnight.

[0135] b) Day 2: Transfect ECO cells when they reach approximately 90% confluency (usually 14-18 hours after plating). Add chloroquine half an hour before transfection. Prepare 15 μg of plasmid, 250 μL of 1.5 M CaCl2, and 1 mL of H2O to a total volume of 1.25 mL. Add an equal volume of 2× HBS to the plasmid complex, mix thoroughly, and let stand at room temperature for 15 minutes. Then, add the mixture to the ECO cell dish. Incubate at 37°C for 6 hours, remove the culture medium, wash once with PBS, and add 10 mL of prewarmed fresh culture medium.

[0136] c) Day 4: 48 hours after transfection, collect the supernatant, filter through a 0.45 μm filter to obtain the retroviral solution, and store in aliquots at -80°C.

[0137] d) Add 1.2 mL of 15 μg / mL Retronectin coating solution to each well of a 6-well NTC plate and incubate at 4°C overnight.

[0138] e) Carefully aspirate the blocking solution, add 2 mL / well PBS for washing, add 5 mL of the above virus solution to each well, centrifuge at 2000 × g at 32°C for 2 h, and discard the unbound virus supernatant;

[0139] f) Wash logarithmic phase PG13 cells once with 10 mL of PBS, add 1 mL of 0.25% recombinant trypsin, and incubate at room temperature for 2-3 minutes;

[0140] g) Add 5 ml of complete medium containing 10% FBS to terminate the digestion, and centrifuge at 1500 rpm for 5 minutes;

[0141] h) Discard the supernatant and adjust the cell density to 0.5×10 5cell / mL, and add 3 mL / well to the virus-coated NTC 6-well plate to make the final cell count 1.5×10 5 cell / hole;

[0142] i) incubation at 37°C, 5% CO2 for 48 h;

[0143] j) After 1-2 passages, the cells were transferred to T175 culture flasks and cultured in DEME medium containing 12% FBS for 2 days;

[0144] k) Replace with fresh DEME medium containing 12% FBS and continue culturing for 48 hours. Collect the supernatant, filter with a 0.45 μm filter to obtain a retroviral solution, and store in aliquots at -80°C.

[0145] 3. Retrovirus infection of human T cells

[0146] a) Thaw frozen healthy human peripheral blood PBMCs and adjust the cell density to 1×10 6 -2×10 6 cells / mL.

[0147] b) PBMCs were collected using Ficoll separation medium (Tianjin Haoyang) and CD3 + T cells, by magnetic beads: CD3 + Clinical-grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen) were added to activate T cells at a ratio of 3:1, and T cells were cultured in 24-well cell culture plates.

[0148] c) Coat non-tissue-treated culture plates with RetroNectin (TAKARA) diluted with PBS to a final concentration of 15 μg / mL, using 1.2 mL per well of a 6-well plate. Protect from light and incubate at 4°C overnight.

[0149] d) After two days of T cell activation culture, the coated 6-well plate was removed, the coating solution was discarded, and the plate was washed once with PBS.

[0150] e) Add 5-6 mL of the retroviral solution prepared in step 2 to each well. Centrifuge at 32°C, 2000 × g for 2 h, and discard the supernatant. Add 3 mL of activated T cell suspension containing hIL-2 (500 U / mL) to each well and continue culturing for 1 day.

[0151] f) After cell infection, the cell density was observed daily and T cell culture medium containing 500 U / mL IL-2 was added as appropriate to maintain the T cell density at 5×10 5 / mL, which is convenient for cell expansion.

[0152] g) Thus, dual-epitope CAR-T cells infected with the retrovirus prepared in step 2 were obtained, and were named CLL1 DE CAR-T cells (ie, T cells expressing the CLL1 DE CAR gene with a nucleotide sequence of SEQ ID NO: 2) and CLL1 DE CAR-CD27-T cells (ie, T cells expressing the CLL1 DE CAR-CD27 gene with a nucleotide sequence of SEQ ID NO: 6).

[0153] The constructed CLL1 DE CAR-T cells, CLL1 DE CAR-CD27-T cells and CTR T cells (i.e., T cells without virus transfection, as a control) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and recorded as day 0. Various functional tests were performed on day 10.

[0154] Example 3. Detection of CAR Expression in CLL1 DE CAR-CD27-T Cells

[0155] CLL1 DE CAR-CD27-T cells express the fusion protein CLL1 DE CAR-CD27 with the amino acid sequence of SEQ ID NO: 5, which expresses the dual-epitope chimeric antigen receptor CLL1 DE CAR (SEQ ID NO: 1) and CD27 (SEQ ID NO: 3) under the action of the self-cleavage peptide P2A. The steps for detecting CLL1 DE CAR expression are as follows:

[0156] 1. After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μl of the culture medium to each well of a 96-well round-bottom plate.

[0157] Resuspend in μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) and centrifuge at 1500 rpm for 5 min;

[0158] 2. Add 60 μL of prepared fluorescently labeled anti-human CD27-PE / CLL1(rp)-FITC to each well, resuspend and mix, and incubate at 4°C for 30 minutes;

[0159] 3. Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 minutes;

[0160] 4. Discard the supernatant, resuspend the cells in 400 μL FACS buffer, transfer them to a flow cytometer, read the cells using a flow cytometer (BD Canto-II), and analyze the percentage of CLL1 antibodies in CLL1 DE CAR-T cells and CLL1 DE CAR-CD27-T cells, and the percentage of CD27 in CLL1 DE CAR-CD27-T cells.

[0161] The test results are shown in Figure 3. CLL1 DE CAR-T cells and CLL1 DE CAR-CD27-T cells can well express CLL1-VHH-1+CLL1-VHH-16 bi-epitope antibody (a fusion of two single-domain antibodies, CLL1-VHH-1 and CLL1-VHH-16), and CLL1 DE CAR-CD27-T cells can well express CD27, indicating that the expression of CAR molecules in CLL1 DE CAR-T cells and CLL1 DE CAR-CD27-T cells meets the expected design.

[0162] Example 4. Functional detection of CLL1 DE CAR-CD27-T cells secreting specific effector molecule IFN-γ

[0163] IFN-γ has anti-tumor, antiviral, and immunomodulatory properties. High levels of IFN-γ are an important indicator of well-functioning CAR-T cells. Its expression is consistent with T cell cytotoxicity and can indicate the activation level of CAR-T cells. Detection is performed by flow cytometry after intracellular staining. The steps are as follows:

[0164] 1. The cell density of the test cells CLL1 DE CAR-T cells, CLL1 DE CAR-CD27-T cells and CTR T cells (i.e., T cells without virus transfection, as a control) was adjusted to 2×10 6 100 μL of the solution was added to a 96-well U-bottom plate, and U937 cells were added at a ratio of test cells: target cells = 1:1. Brefeldin A (Med Chem Express, HY-16592) was added to each well at a final concentration of 5 μg / mL, and the cells were incubated at 37°C in a culture medium for 6 hours.

[0165] CTR+U937: 1 mL per well, total 1×10 6 1×10 T cells without virus transfection (CTR T cells) were added with 1 mL of 6 U937 cells;

[0166] Test cells + U937: 1 mL per well, totaling 1×10 6 1 mL of 1 × 10 6U937 cells;

[0167] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0168] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) to each well for resuspending. Centrifuge at 1500 rpm for 5 min. Repeat this step twice.

[0169] (2) Add 60 μL of the prepared fluorescently labeled CLL1(rp)-FITC to each well, resuspend and mix thoroughly, and incubate at room temperature for 10 min in the dark.

[0170] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0171] (4) Add 150 μL of Cytofix / Cytoperm (BD Biosciences, Cat. No. 55472) to each well, resuspend and mix, and incubate at room temperature for 15 min in the dark.

[0172] (5) Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Cat. No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0173] (6) Add 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, Cat. No. 506510) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark.

[0174] (7) Add 200 μL of Perm buffer to each well and centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells in 400 μL of FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) to analyze the percentage of the functional effector molecule IFN-γ in CLL1 DE CAR-T cells, CLL1 DE CAR-CD27-T cells, and control cells.

[0175] The test results are shown in Figure 4. After co-culture with U937 target cells, both CLL1 DE CAR-T cells and CLL1 DE CAR-CD27-T cells can well secrete the T cell-specific effector molecule IFN-γ, and the effect of CLL1 DE CAR-CD27-T cells in secreting IFN-γ is better than that of CLL1 DE CAR-T cells.

[0176] Example 5. Cytotoxicity (degranulation CD107a) function detection of CLL1 DE CAR-CD27-T cells

[0177] CD107a, a cell degranulation indicator, is a marker of cytotoxic activity and is detected by flow cytometry after intracellular staining. The steps are as follows:

[0178] 1. The cell density of the test cells CLL1 DE CAR-T, CLL1 DE CAR-CD27-T and CTR T cells (i.e., T cells without virus transfection, as a control) was adjusted to 2×10 6 / mL, take 100 μL and add it into 96-well U-bottom plate;

[0179] 2. Add 100 μL of 2×10 cells to each well at a ratio of 1:1. 5 U937 cells were used as CAR antigen-specific stimulation (+U937), and 100 μL of Medium (10% FBS RPMI-1640 medium) was added to each well as a negative control (+Medium);

[0180] 3. Add 1 μL of APC-labeled anti-human CD107a antibody (Biolegend, Cat. No. 328620) to each well and incubate at 37°C for 1 hour.

[0181] 4. Add 10 μL of 1:50 diluted Monensin Solution (Invitrogen, Cat. No. 00-4505-51) to each well and continue incubation for 3 h.

[0182] 5. After incubation, perform flow cytometry staining. The steps are as follows:

[0183] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) to each well, resuspend and mix, and centrifuge at 1500 rpm for 5 min. Repeat this step twice;

[0184] (2) Add 60 μL of prepared fluorescent antibody (fluorescently labeled anti-human CD3 / CD4 / CD8 / CLL1(rp)-FITC) to each well, resuspend and mix, and incubate at room temperature for 10 min in the dark.

[0185] (3) Centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend the cells in 400 μL FACS buffer, transfer the cells to a flow cytometer, and analyze the percentage of CD107a in T cells using a flow cytometer (BD Canto-II).

[0186] The test results are shown in Figure 5 . After co-culture with target cells, both CLL1 DE CAR-T and CLL1 DE CAR-CD27-T cells can specifically upregulate CD107a degranulation.

[0187] Example 6. Cytotoxicity Detection of CLL1 DE CAR-CD27-T Cells

[0188] In vitro killing assay was used to detect the cytotoxicity of CLL1 DE CAR-CD27-T cells against tumor cells. The steps are as follows:

[0189] 1. The cell density of the test cells CLL1 DE CAR-T, CLL1 DE CAR-CD27-T and CTR T cells (i.e., T cells without virus transfection, as a control) was adjusted to 2×10 5 100 μL was added to a 96-well U-bottom plate; D-firefly luciferin sodium salt (Yeasen Biotechnology, Catalog No. 40901ES08, stock concentration 100 mg / mL) was added to a final concentration of 100 μg / mL, and the mixture was repeated for three wells.

[0190] 2. Target cells U937-luc were added according to different effector-target ratios (1:1, 1:3, 1:9, 1:27) (the cell density of target cells under 1:1 condition was 2×10 4 cells) and cultured at 37°C for 16 h.

[0191] 3. Measure the fluorescence value using a TECAN spark microplate reader and take the average value of three replicate wells to calculate the specific cytotoxicity of the test cells:

[0192] Specific lysis%=100-100×(Eexp-Emin) / (Tmax-Tmin)

[0193] Eexp: RLU value when effector cells and target cells are co-cultured;

[0194] Emin: RLU value of spontaneous death of effector cells in the absence of cells;

[0195] Tmax: RLU value of spontaneous death of target cells in the absence of effector cells;

[0196] Tmin: RLU value under conditions of maximum killing rate.

[0197] The test results are shown in Figure 6. Both CLL1 DE CAR-T cells and CLL1 DE CAR-CD27-T cells can effectively and specifically kill CLL1+ target cells (U937 cells), while control CTR T cells were + No killing effect on target cells.

[0198] Example 7. Detection of in vivo tumoricidal activity of CLL1 DE CAR-CD27-T cells

[0199] 1. Fifteen female NSG mice (Shanghai Model Organisms Science Co., Ltd., catalog number NM-NSG-001, 8-10 weeks old) were inoculated with 2.0 × 10 6 U937-Luc-GFP cells;

[0200] 2. Six days after inoculation, the rats were randomly divided into three groups and each group was injected with 3.0×10 6 CLL1 DE CAR-T cells, CLL1 DE CAR-CD27-T cells or CTR T cells (i.e., T cells without virus transfection, as control) cells were injected into the 400 cells and recorded as D0 day;

[0201] 3. Use Photo Acquisition small animal imager to capture tumor luminescence signals on D-1, the day before administration, and D4, D8, D14, D21, D26, and D33 after administration.

[0202] The test results, shown in Figure 7, show that compared to the CTR T cell control group, both CLL1 DE CAR-T cells and CLL1 DE CAR-CD27-T cells significantly inhibited tumors and prolonged mouse lifespan. All control mice died by D21, while CAR-T-treated mice survived to D33 or even longer. Compared to CLL1 DE CAR-T cells, mice injected with CLL1 DE CAR-CD27-T cells had significantly fewer residual human myeloid leukemia tumor cells, demonstrating that CLL1 DE CAR-CD27-T cells have a more potent tumor-suppressing effect.

[0203] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art. Industrial Applicability

[0204] The CLL1 DE CAR-CD27-T cells of the present application can effectively secrete the T cell-specific effector molecule IFN-γ and effectively kill CLL1 + Target cells, with good in vivo tumoricidal activity, can not only significantly inhibit the proliferation of tumor cells in mice, but also significantly prolong the survival time of mice. Its IFN-γ secretion effect, its specific cytotoxic effect and its in vivo tumoricidal activity are better than CTR T cells and CLL1DE CAR-T cells. The dual-epitope CAR-T cells of this application have dual CLL1 binding sites, bind more tightly to CLL1, have good anti-tumor ability, and can be used for immunotherapy of CLL1 target-related diseases (such as acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia), and have broad clinical application prospects.

[0205] CROSS-REFERENCE TO RELATED APPLICATIONS

[0206] This application claims priority to Chinese patent application No. 202410291882.5 filed with the Patent Office of China on March 14, 2024, entitled “Fusion protein containing a dual-epitope chimeric antigen receptor and its modified CAR-T cells”, the entire contents of which are incorporated by reference into this application.

Claims

1. A fusion protein, characterized in that The fusion protein includes a dual-epitope chimeric antigen receptor and CD27, wherein the dual-epitope chimeric antigen receptor includes an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen binding domain includes a single-domain antibody 1 and a single-domain antibody 2 that specifically target CLL1, wherein the amino acid sequence of the single-domain antibody 1 is shown at positions 22-150 of SEQ ID NO:1, and the amino acid sequence of the single-domain antibody 2 is shown at positions 166-294 of SEQ ID NO:

1.

2. The fusion protein according to claim 1, characterized in that The dual-epitope chimeric antigen receptor is any one of the following: A1) the amino acid sequence is positions 22-522 of SEQ ID NO: 1 or the protein set forth in SEQ ID NO: 1; A2) A fusion protein having the same function as A1) obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

3. The fusion protein according to claim 1 or 2, characterized in that The bi-epitope chimeric antigen receptor and the CD27 are connected via a self-cleaving peptide.

4. The fusion protein according to claim 3, characterized in that The self-cleaving peptides include P2A, F2A, T2A and E2A.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The fusion protein is any one of the following: B1) the amino acid sequence is positions 22-804 of SEQ ID NO:5 or the protein set forth in SEQ ID NO:5; B2) A fusion protein having the same function as B1) obtained by connecting a tag to the N-terminus and / or C-terminus of B1).

6. Biomaterial, characterized in that The biological material is any one of the following: C1) a nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 5; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A recombinant cell containing the nucleic acid molecule of C1), or a recombinant cell containing the expression cassette of C2), or a recombinant cell containing the recombinant vector of C3).

7. The biomaterial according to claim 6, characterized in that The nucleic acid molecule is any of the following: D1) the coding sequence is positions 64-2412 of SEQ ID NO:6 or the DNA molecule represented by SEQ ID NO:6; D2) The nucleotide sequence is positions 64-2412 of SEQ ID NO: 6 or the DNA molecule shown in SEQ ID NO:

6.

8. A modified immune effector cell, characterized in that The modified immune effector cell is an immune effector cell modified by the fusion protein according to any one of claims 1 to 5.

9. The modified immune effector cell according to claim 8, characterized in that The modified immune effector cells comprise the nucleic acid molecule according to any one of claims 6 or 7, and the immune effector cells include T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood monocytes and pluripotent stem cells.

10. Use of the fusion protein according to any one of claims 1 to 5, the biomaterial according to claim 6 or 7, or the modified immune effector cell according to claim 8 or 9 in the preparation of a product having any of the following functions: E1) Prevention or treatment of tumors; E2) Prevent or treat CLL1 target-related diseases; E3) Killing CLL1-positive tumor cells; E4) inhibiting the growth of CLL1-positive tumors; E5) promotes the release of cytokine IFN-γ; E6) Detection of cancer cells expressing CLL1.

11. A pharmaceutical composition for preventing or treating CLL1 target-related diseases, characterized in that: The pharmaceutical composition comprises the modified immune effector cell according to claim 8 or 9 and one or more pharmaceutically acceptable carriers.

12. A kit, characterized in that The kit contains the fusion protein according to any one of claims 1 to 5 or the modified immune effector cell according to claim 8 or 9.

13. A method for preventing or treating CLL1 target-related diseases, characterized in that: The method comprises administering the modified immune effector cell of claim 8 or 9 or the pharmaceutical composition of claim 11 to a subject suffering from a CLL1 target-related disease.

14. The method according to claim 13, characterized in that The CLL1 target-related disease is CLL1-positive cancer.

15. The method according to claim 14, characterized in that The CLL1-positive cancer is acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

16. A method for preparing the modified immune effector cells according to claim 8 or 9, characterized in that: The method comprises: transferring the nucleic acid molecule according to claim 6 or 7 into immune effector cells for stable expression, thereby obtaining the modified immune effector cells.

17. The method according to claim 16, characterized in that The immune effector cells are selected from T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood monocytes and pluripotent stem cells.

18. The method according to claim 17, characterized in that The immune effector cells are T cells.

19. The method according to claim 18, characterized in that The method comprises the following steps: (1) cloning the CLL1 DE CAR-CD27 gene (SEQ ID NO: 6) into a retroviral vector to obtain a recombinant retroviral vector; (2) introducing the recombinant retroviral vector into packaging cells for packaging to obtain a recombinant retrovirus; (3) Infecting human T cells with the recombinant retrovirus to obtain recombinant cells.

20. Use of the fusion protein according to any one of claims 1 to 5 or the modified immune effector cell according to claim 8 or 9 in the preparation of a medicament for preventing or treating a CLL1 target-related disease.

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