CLL1-targeted double-epitope CAR, double-epitope CAR-T cell and application of CLL1-targeted double-epitope CAR-T cell

Abstract

The invention discloses a double-epitope CAR targeting CLL1, a double-epitope CAR-T cell and application of the double-epitope CAR and the double-epitope CAR-T cell, and belongs to the technical field of cellular immunotherapy. The technical problem to be solved by the invention is how to improve the killing activity of the CLL1-targeted chimeric antigen recipient cell on CLL1-positive tumor cells and / or the immunosuppression effect of resisting a tumor microenvironment. The invention specifically discloses a chimeric antigen receptor with an amino acid sequence as shown in SEQ ID No.1, a fusion protein with an amino acid sequence as shown in SEQ ID No.3, and a double-epitope CAR-T cell constructed by using the chimeric antigen receptor and the fusion protein. The double-epitope CAR-T cell disclosed by the invention can well secrete T cell specific effector molecules IFN-gamma, effectively and specifically kill CLL1 + target cells, and has good in-vivo tumor killing activity. The antibody can be used for immunotherapy of CLL1 target related diseases, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell immunotherapy, and specifically relates to a dual-epitope CAR targeting CLL1, a dual-epitope CAR-T cell and applications thereof. Background Art

[0002] 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 and in most AML cells, and is also expressed on the CD34 of most AML. + CD38 - On stem cells, normal CD34 + CD38 - Stem cells do not express it. CLL1 has become a potential target for the treatment and diagnosis of AML due to its special expression pattern. In addition, CLL1 is also expressed on cells of Myelodysplastic Syndromes (MDS) and Chronic Myeloid Leukemia (CML).

[0003] Chimeric antigen receptor T cell (CAR-T cell) therapy refers to the transfer of genetic material with specific antigen recognition domains and T cell activation signals into T cells through gene modification technology, so that T cells can directly bind to specific antigens on the surface of tumor cells and be activated and proliferate, thereby playing a role in targeted killing of tumor cells. With the development of tumor immunotherapy and clinical technology, CAR-T immunotherapy is one of the most promising tumor immunotherapy methods. Generally, chimeric antigen receptors (CARs) contain extracellular antigen binding domains, transmembrane domains, and intracellular signaling domains. The extracellular antigen binding domain can contain a single-chain variable fragment (scFv) or a single domain antibody (sdAb) targeting 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 (sdAbs) have only a single monomer 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 so far. Their antigen-binding ability and 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, arbitrary combination of building blocks, and low production costs.

[0004] At present, CAR-T cell therapy has achieved encouraging results in hematological tumors such as B cells, but the antigenic properties of the myeloid system are fundamentally different from those of the B system. The killing of normal myeloid cells by CAR-T will lead to bone marrow failure and lack of corresponding replacement therapy, which has made the progress of CAR-T treatment of AML very slow. The successful application of CAR-T technology in acute myeloid leukemia (AML) still faces many challenges. In addition, the development of multiple redirected CAR-T, by recognizing two non-overlapping epitopes of 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 of AML. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the killing activity of CLL1-targeted chimeric antigen receptor cells on CLL1-positive tumor cells and / or resist the immunosuppressive effect of the tumor microenvironment. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] To solve the above technical problems, the present invention first provides a chimeric antigen receptor (CAR), which may include an antigen binding domain, a transmembrane domain and an intracellular signaling domain. The antigen binding domain may include 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 may be as shown at positions 22-150 of SEQ ID No.1, and the amino acid sequence of the single domain antibody 2 may be as shown at positions 166-294 of SEQ ID No.1.

[0007] Furthermore, the chimeric antigen receptor includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain from the N-terminus to the C-terminus.

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

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

[0010] In the above chimeric antigen receptor, 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 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 above-mentioned chimeric antigen receptor, the intracellular signaling domain may include a co-stimulatory domain 4-1BB and an activation domain CD3ζ.

[0018] Furthermore, the amino acid sequence of the co-stimulatory domain 4-1BB may be as shown at 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 chimeric antigen receptor may further include a linker, and the linker 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 chimeric antigen receptor may also include a signal peptide (Signal) located at the N-terminus of the 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] In one embodiment of the present invention, the 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] Furthermore, the chimeric antigen receptor may be any of the following:

[0029] A1) the amino acid sequence is the protein shown in positions 22-522 of SEQ ID No. 1;

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

[0031] The amino acid sequence of the signal peptide in A2) may be positions 1 to 21 of SEQ ID No. 1.

[0032] Furthermore, the name of the chimeric antigen receptor may be CLL1 DE CAR, and the amino acid sequence of the CLL1 DE CAR may be SEQ ID No.1.

[0033] The chimeric antigen receptor described herein may be a bi-epitopic chimeric antigen receptor (bi-epitope CAR).

[0034] The present invention also provides a fusion protein, which may include any chimeric antigen receptor and chimeric switch receptor described herein, and the amino acid sequence of the chimeric switch receptor may be as shown in positions 545-788 of SEQ ID No.3.

[0035] The chimeric switch receptor may be located at the C-terminus of the chimeric antigen receptor.

[0036] The chimeric antigen receptor and the chimeric switch receptor may be linked by a self-cleaving peptide.

[0037] The self-cleaving peptide may be P2A, F2A, T2A or E2A.

[0038] The amino acid sequence of the P2A may be SEQ ID No. 5. The amino acid sequence of the F2A may be SEQ ID No. 6. The amino acid sequence of the T2A may be SEQ ID No. 7. The amino acid sequence of the E2A may be SEQ ID No. 8.

[0039] Specifically, the self-cleaving peptide is P2A.

[0040] Furthermore, the fusion protein may be any of the following:

[0041] B1) the amino acid sequence is the protein shown in positions 22-788 of SEQ ID No. 3;

[0042] B2) A fusion protein having the same function as B1) is obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of B1).

[0043] The amino acid sequence of the signal peptide in B2) may be positions 1 to 21 of SEQ ID No. 1.

[0044] Furthermore, the name of the fusion protein may be CLL1 DE CAR-CR, and the amino acid sequence of the CLL1 DE CAR-CR may be SEQ ID No.3.

[0045] The fusion protein described herein may also be referred to as a bi-epitope chimeric antigen receptor (bi-epitope CAR).

[0046] 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.

[0047] The present invention also provides a biomaterial, which may be any of the following:

[0048] C1) a nucleic acid molecule encoding any chimeric antigen receptor described herein;

[0049] C2) a nucleic acid molecule encoding the fusion protein;

[0050] C3) an expression cassette containing the nucleic acid molecule described in C1) or C2);

[0051] C4) a recombinant vector containing the nucleic acid molecule described in C1) or C2), or a recombinant vector containing the expression cassette described in C3);

[0052] C5) a recombinant microorganism containing the nucleic acid molecule described in C1) or C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4);

[0053] C6) A recombinant cell containing the nucleic acid molecule described in C1) or C2), or a recombinant cell containing the expression cassette described in C3), or a recombinant cell containing the recombinant vector described in C4).

[0054] In the above biological material, the nucleic acid molecule may be any of the following:

[0055] D1) the coding sequence is a DNA molecule shown in SEQ ID No. 2 or positions 64 to 1566 of SEQ ID No. 2, SEQ ID No. 4 or positions 64 to 2367 of SEQ ID No. 4;

[0056] D2) The nucleotide sequence is a DNA molecule shown in SEQ ID No. 2 or positions 64 to 1566 of SEQ ID No. 2, SEQ ID No. 4 or positions 64 to 2367 of SEQ ID No. 4.

[0057] The name of the DNA molecule shown in SEQ ID No. 2 may be CLL1 DE CAR gene, which encodes a chimeric antigen receptor CLL1 DE CAR having an amino acid sequence of SEQ ID No. 1. The DNA molecule shown at positions 64-1566 of SEQ ID No. 2 encodes a chimeric antigen receptor CLL1 DE CAR without a signal peptide.

[0058] The name of the DNA molecule shown in SEQ ID No. 4 can be CLL1 DE CAR-CR gene, which encodes the fusion protein CLL1 DE CAR-CR with the amino acid sequence of SEQ ID No. 3. The DNA molecule shown at positions 64-2367 of SEQ ID No. 4 encodes the fusion protein CLL1 DE CAR-CR without a signal peptide.

[0059] The present invention also provides a modified immune effector cell, which may comprise any chimeric antigen receptor, any fusion protein or any nucleic acid molecule described herein.

[0060] Furthermore, the modified immune effector cell may be an immune effector cell modified by any chimeric antigen receptor described herein, or an immune effector cell modified by any fusion protein described herein.

[0061] Furthermore, the modified immune effector cell may contain a nucleic acid molecule encoding any of the chimeric antigen receptors described herein.

[0062] Furthermore, the modified immune effector cell may contain a nucleic acid molecule encoding any of the fusion proteins described herein.

[0063] Furthermore, the immune effector cells may include T cells, NK cells, NKT cells, γδT cells, macrophages, peripheral blood mononuclear cells (PBMC) and pluripotent stem cells.

[0064] Specifically, the immune effector cells are T lymphocytes (T cells).

[0065] Further, the immune effector cells may include autologous or non-autologous immune effector cells. The non-autologous immune effector cells may be allogeneic immune effector cells. "Autologous" is intended to refer to cells derived from the same individual. "Allogeneic" is intended to refer to cells derived from individuals belonging to the same species but genetically different.

[0066] 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.

[0067] Furthermore, the modified immune effector cell may be a CAR-T cell, which may express any chimeric antigen receptor or fusion protein described herein, which targets C-type lectin-like molecule 1 (CLL1).

[0068] Furthermore, the CAR-T cells described herein may be CLL1 DE CAR-T cells or CLL1 DE CAR-CR-T cells, wherein the CLL1 DE CAR-T cells contain the CLL1 DE CAR gene shown in SEQ ID No.2; and the CLL1 DE CAR-CR-T cells contain the CLL1 DE CAR-CR gene shown in SEQ ID No.4.

[0069] The CAR-T cells described in this article can target dual epitopes of the CLL1 antigen and can also be called dual epitope CAR-T cells.

[0070] The CAR-T cells (bi-epitope CAR-T cells) described herein may have any of the following characteristics:

[0071] F1) secretes T cell-specific effector molecule IFN-γ;

[0072] F2) Specific killing of CLL1-positive tumor cells;

[0073] F3) Inhibits the growth of CLL1-positive tumors.

[0074] Furthermore, the CLL1 DE CAR-CR-T cells may have any of the following characteristics:

[0075] G1) secretes the T cell-specific effector molecule IFN-γ;

[0076] G2) specifically kills CLL1-positive tumor cells;

[0077] G3) inhibits the growth of CLL1-positive tumors;

[0078] G4) converts PD-1 inhibitory signals in CAR-T cells into CD27 stimulatory signals;

[0079] G5) Anti-immunosuppressive effects of the tumor microenvironment.

[0080] The present invention also provides the use of any of the chimeric antigen receptors, 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:

[0081] E1) Prevention or treatment of tumors;

[0082] E2) Prevent or treat CLL1 target-related diseases;

[0083] E3) Killing CLL1-positive tumor cells;

[0084] E4) inhibits the growth of CLL1-positive tumors;

[0085] E5) promotes the release of cytokine IFN-γ;

[0086] E6) Detection of cancer cells expressing CLL1.

[0087] In the above application, the tumor may be a CLL1-positive tumor.

[0088] In the above application, the CLL1 target-related disease may be CLL1-positive cancer (i.e., cancer expressing CLL1).

[0089] 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).

[0090] The products described herein may include reagents, kits, medicaments, or pharmaceutical compositions.

[0091] The present invention also provides a pharmaceutical composition for preventing or treating CLL1 target-related diseases, wherein the pharmaceutical composition comprises the modified immune effector cells and one or more pharmaceutically acceptable carriers.

[0092] The pharmaceutically acceptable carrier is selected from the group consisting of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants and lubricants.

[0093] In order to prepare the pharmaceutical composition into an injection 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, in order to prepare an isotonic injection, an appropriate amount of carriers such as sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional carriers such as cosolvents, buffers, pH adjusters, etc. can also be added.

[0094] The pharmaceutical composition may also include a cell freezing solution (containing dimethyl sulfoxide, sodium chloride, human serum albumin, etc.).

[0095] The present invention also provides a kit, which contains any of the chimeric antigen receptors, the fusion proteins or the modified immune effector cells described herein.

[0096] The present invention 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 the CLL1 target-related disease.

[0097] In the above method, the CLL1 target-related disease may be CLL1-positive cancer.

[0098] In the above method, the CLL1-positive cancer may be acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia.

[0099] The present invention 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.

[0100] 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.

[0101] In the above method, the immune effector cells may be T cells.

[0102] In the above method, the method may include the following steps:

[0103] (1) cloning the CLL1 DE CAR gene (SEQ ID No. 2) or the CLL1 DE CAR-CR gene (SEQ ID No. 4) into a retroviral vector to obtain a recombinant retroviral vector;

[0104] (2) introducing the recombinant retroviral vector into packaging cells for packaging to obtain a recombinant retrovirus;

[0105] (3) Infecting human T cells with the recombinant retrovirus to obtain recombinant cells.

[0106] The present invention also provides use of the modified immune effector cells in preparing a drug for preventing or treating CLL1 target-related diseases.

[0107] Furthermore, the retroviral vector may be the retroviral vector MP71.

[0108] Furthermore, the packaging cells may be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0109] The present invention 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 as follows Figure 1 On this basis, a chimeric conversion receptor (CR) PD-1 & CD27 was further connected by a self-cleaving peptide to design CLL1 DE CAR-CR (SEQ ID No. 3), the structure of which is shown in Figure 2 As shown. Furthermore, the T cells were modified using the CLL1 DE CAR encoding gene (SEQ ID No. 2) and CLL1 DE CAR-CR encoding gene (SEQ ID No. 4) designed above to obtain dual-epitope CAR-T cells targeting CLL1: CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells. The constructed CAR-T cells were functionally verified, and the experimental results showed that the dual-epitope CAR-T cells of the present invention can well secrete the T cell-specific effector molecule IFN-γ, effectively and specifically kill CLL1 + Target cells have good in vivo tumoricidal activity, which can not only significantly inhibit the proliferation of tumor cells in mice, but also significantly prolong the survival time of mice. Among them, CLL1 DE CAR-CR-T cells, because they contain chimeric conversion receptors, also have the function of reversing the PD-1 inhibitory signal in CAR-T cells to CD27 stimulation signals, and thus have the ability to resist the immunosuppressive tumor microenvironment, making it more persistent in vivo. Its effect of secreting IFN-γ, its specific cytotoxic effect and its in vivo tumoricidal activity are all better than CLL1 DE CAR-T cells. The dual-epitope CAR-T cells of the present invention 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 prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 Schematic diagram of the molecular structure of CLL1 DE CAR.

[0111] Figure 2 Schematic diagram of the molecular structure of CLL1 DE CAR-CR.

[0112] Figure 3 These are the results of CAR expression detection in dual-epitope CAR-T cells in Example 4.

[0113] Figure 4This is the functional test result of the dual-epitope CAR-T cells secreting the specific effector molecule IFN-γ in Example 5.

[0114] Figure 5 This is the result of the cytotoxic (degranulated CD107a) function test of the dual-epitope CAR-T cells in Example 6.

[0115] Figure 6 These are the results of the cytotoxicity test of the dual-epitope CAR-T cells in Example 7.

[0116] Figure 7 These are the in vivo tumoricidal activity test results of the dual-epitope CAR-T cells in Example 8. DETAILED DESCRIPTION

[0117] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0118] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0119] The retroviral vector MP71 in the following examples is described in the following document: Engels B, Cam H, et al. Retroviral Vectors for High-Level Transgene Expression in T Lymphocytes [J]. Human Gene Therapy, 2003, 14 (12): 1155-1168. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0120] The human peripheral blood mononuclear cells (PBMC) in the following examples were derived from the venous blood of healthy volunteers.

[0121] Example 1. Design and structure of CLL1 DE CAR molecule

[0122] In this embodiment, a dual-epitope chimeric antigen receptor (dual-epitope CAR) that binds to two separate epitopes of a single tumor antigen (CLL1) is 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 (such as CD3ζ) of immune effector cells (such as T cells), and may also include a co-stimulatory signaling domain (such as 4-1BB or CD28). Furthermore, a hinge domain (such as 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 CD8a) located at the N-terminus.

[0123] An example of a dual-epitope CAR molecular structure designed in this embodiment is as follows Figure 1 As shown in the figure, the dual-epitope CAR is named CLL1DE 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 costimulatory domain 4-1BB, and an activation domain CD3ζ ( Figure 1 ).

[0124] The amino acid sequence of the dual-epitope chimeric antigen receptor CLL1 DE CAR is shown in SEQ ID No.1 (522aa). Among them: SEQ ID No.1 No. 1-21 is the amino acid sequence of the signal peptide (Signal); SEQ ID No.1 No. 22-150 is the amino acid sequence of the single-domain antibody CLL1-VHH-1; SEQ ID No.1 No. 151-165 is the amino acid sequence of the linker; SEQ ID No.1 No. 166-294 is the amino acid sequence of the single-domain antibody CLL1-VHH-16; SEQ ID No.1 No. 295-339 is the amino acid sequence of the CD8a hinge region (Hinge); SEQ ID No.1 No. 340-363 is the amino acid sequence of the CD8a transmembrane region; SEQ ID No.1 No. 364-410 is the amino acid sequence of the costimulatory domain 4-1BB; SEQ ID No.1 No. 411-522 is the amino acid sequence of the activation domain CD3ζ.

[0125] The coding gene of CLL1 DE CAR is CLL1 DE CAR gene, and its nucleotide sequence is shown in SEQ ID No. 2 (1566 bp). Among them: 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.

[0126] Example 2. Design and structure of CLL1 DE CAR-CR molecule

[0127] 1. Design of chimeric switch receptor PD-1 & CD27

[0128] When there is continuous antigen stimulation, in order to avoid excessive response, PD-1 is expressed on the surface of activated T cells, which binds to PD-L1 on the surface of APCs cells, and transmits negative regulatory signals to T cells, thereby inhibiting T cell activation, reducing T cell proliferation or apoptosis. This embodiment first designs a chimeric switch receptor (Chimeric Switch Receptor, CSR, referred to as CR in this article) to reverse the PD-1 signal of T cells and convert the above-mentioned inhibitory effect into an activation signal in T cells. The chimeric switch receptor (CR) is named PD-1&CD27, which includes the extracellular domain of PD-1 and the transmembrane region and cytoplasmic region of CD27, that is, the intracellular domain of PD-1 is replaced by the transmembrane region and cytoplasmic region of CD27, that is, the T cell inhibitory signal after the combination of PD-1 and PD-L1 is converted into a CD27-mediated activation signal, so that T cells can survive and exert cytotoxic effects under the conditions of the tumor microenvironment.

[0129] The amino acid sequence of the chimeric conversion receptor PD-1 & CD27 is shown in positions 545-788 of SEQ ID No. 3. Among them: positions 545-567 of SEQ ID No. 3 are the amino acid sequence of the human PD-1 signal peptide; positions 568-714 of SEQ ID No. 3 are the amino acid sequence of the PD-1 extracellular domain; positions 715-788 of SEQ ID No. 3 are the amino acid sequences of the CD27 transmembrane region and cytoplasmic region.

[0130] The coding sequence of the chimeric switch receptor PD-1 & CD27 is shown in positions 1633-2367 of SEQ ID No. 4. Among them: positions 1633-1701 of SEQ ID No. 4 are the nucleotide sequence of the human PD-1 signal peptide; positions 1702-2142 of SEQ ID No. 4 are the nucleotide sequence of the PD-1 extracellular domain; positions 2143-2367 of SEQ ID No. 4 are the nucleotide sequences of the CD27 transmembrane region and cytoplasmic region.

[0131] 2. Design of CLL1 DE CAR-CR molecule

[0132] The CLL1 DE CAR designed in Example 1 is connected to the PD-1 & CD27 designed in step 1 of this example through a self-cleaving peptide (such as P2A) to obtain a CLL1 DE CAR-CR molecule ( Figure 2 ).

[0133] P2A (GSGATNFSLLKQAGDVEENPGP, SEQ ID No. 5) is a "self-cleaving" peptide. The "self-cleaving" 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 DECAR-CR molecule, for example, using F2A (VKQTLNFDLLKLAGCVESNPG, SEQ ID No. 6), T2A (EGRGSLLTCGDVEENPG, SEQ ID No. 7), E2A (QCTNYALLKLAGDVESNPG, SEQ ID No. 8), etc., which have the same "self-cleaving" function as P2A.

[0134] The amino acid sequence of CLL1 DE CAR-CR is shown in SEQ ID No.3 (788aa). Among them: positions 1-522 of SEQ ID No.3 are the amino acid sequence of the dual-epitope chimeric antigen receptor CLL1 DE CAR; positions 523-544 of SEQ ID No.3 are the amino acid sequence of the self-cleaving peptide P2A; positions 545-788 of SEQ ID No.3 are the amino acid sequence of the chimeric conversion receptor (CR) PD-1 & CD27.

[0135] The coding gene of CLL1 DE CAR-CR is CLL1 DE CAR-CR gene, and its nucleotide sequence is shown in SEQ ID No.4 (2367 bp). Among them: SEQ ID No.4 No. 1-1566 is the nucleotide sequence of the dual-epitope chimeric antigen receptor CLL1 DE CAR; SEQ ID No.4 No. 1567-1632 is the nucleotide sequence of the self-cleaving peptide P2A; SEQ ID No.4 No. 1633-2367 is the nucleotide sequence of the chimeric conversion receptor (CR) PD-1 & CD27.

[0136] Example 3. Construction of dual-epitope CAR-T cells

[0137] The bi-epitope CAR-T cells constructed using the CLL1 DE CAR designed in Example 1 are named CLL1 DE CAR-T cells; the bi-epitope CAR-T cells constructed using the CLL1 DE CAR-CR designed in Example 2 are named CLL1 DE CAR-CR-T cells. Specifically, the CAR gene is transferred into T cells for stable expression, and the steps are as follows:

[0138] 1. Construction of dual-epitope CAR vector

[0139] (1) The recombinant vectors pUC57-CLL1 DE CAR and pUC57-CL L1 DECAR-CR 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 CLL1DECAR gene (SEQ ID No. 2) into the pUC57 vector; the recombinant vector pUC57-CLL1 DE CAR-CR is a recombinant vector obtained by cloning the CLL1 DE CAR-CR gene (SEQ ID No. 4) into the pUC57 vector. The above recombinant vectors are synthesized and provided by Qingke Biotechnology Co., Ltd.

[0140] (2) The retroviral vector MP71 was double-digested with NotI and EcoRI, and the large vector fragment was recovered by gel excision.

[0141] (3) The target gene fragment and the large vector fragment were connected with T4 ligase to obtain recombinant retroviral vectors MP71-CLL1 DE CAR and MP71-CLL1 DECAR-CR carrying the CLL1 DECAR gene and CLL1 DE CAR-CR gene, respectively.

[0142] (4) The recombinant retroviral vectors MP71-CLL1 DE CAR and MP71-CLL1 DE CAR-CR were transformed into competent Escherichia coli DH5α, and the plasmids were extracted and purified using the plasmid purification kit of Qiagen to obtain MP71-CLL1 DE CAR plasmid and MP71-CLL1 DE CAR-CR plasmid.

[0143] 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 whose nucleotide sequence is SEQ ID No. 2 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0144] The recombinant retroviral vector MP71-CLL1 DE CAR-CR (i.e., MP71-CLL1 DE CAR-CR 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 whose nucleotide sequence is SEQ ID No. 4 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0145] 2. Retroviral packaging

[0146] The MP71-CLL1 DE CAR plasmid and the MP71-CLL1 DE CAR-CR 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:

[0147] a) Day 1: Phoenix Ecotropic (ECO) cells should be less than 20 generations and not overgrown. 6 / ml cell density, add 10mL DMEM medium (10% Fetal Bovine Serum, 2mM L-glutamine, 1% Penicillin / Streptomycin) to a 10cm cell culture dish, mix the cells thoroughly, and culture at 37°C overnight;

[0148] b) Day 2: When the confluency of ECO cells reaches about 90%, transfection is performed (usually about 14-18 hours after plating). Chloroquine is added half an hour before transfection. Prepare 15μg of plasmid, 250μL of 1.5M CaCl2, and 1ml of H2O to a total volume of 1.25mL. Add 2×HBS with an equal volume to the plasmid complex, mix well, and leave at room temperature for 15 minutes. Then add the mixture to the ECO cell dish. Culture at 37℃ for 6 hours, remove the culture medium, wash once with PBS, and add 10ml of preheated fresh culture medium again.

[0149] c) Day 4: 48 hours after transfection, collect the supernatant, filter with a 0.45 μm filter to obtain the retroviral solution, and store it in aliquots at -80°C;

[0150] d) Add 1.2 mL of 15 ug / mL Retronectin coating solution to each well of the NTC6-well plate and incubate at 4°C overnight;

[0151] e) Carefully remove the blocking solution, add 2 mL / well PBS for washing, add 5 mL of the above virus solution to each well, centrifuge at 32°C and 2000×g for 2 h, and discard the unbound virus supernatant;

[0152] f) Take logarithmic phase PG13 cells, wash them once with 10 mL PBS, add 1 mL 0.25% recombinant trypsin, and let stand at room temperature for 2-3 minutes;

[0153] g) Add 5 ml of complete medium containing 10% FBS to terminate digestion, and centrifuge at 1500 rpm for 5 min;

[0154] h) Discard the supernatant and adjust the cell density to 0.5×10 5 cell / mL, and add 3 mL / well to the NTC 6-well plate coated with the above virus to make the final cell number 1.5×10 5 cell / well;

[0155] i) culturing at 37°C, 5% CO2 for 48 h;

[0156] j) After 1-2 generations of subculture, the cells were transferred to T175 culture flasks and cultured in DEME medium containing 12% FBS for 2 days;

[0157] k) Replace with new DEME medium containing 12% FBS and continue culturing for 48 hours, then collect the supernatant, filter with a 0.45 μm filter to obtain a retrovirus solution, and store it in aliquots at -80°C.

[0158] 3. Retrovirus infection of human T cells

[0159] a) Thaw frozen healthy human peripheral blood PBMCs and adjust the cell density to 1×10 in RPMI-1640 medium containing 10% fetal bovine serum (FBS). 6 -2×10 6 cells / mL.

[0160] b) PBMCs were collected using Ficoll separation solution (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 a 24-well cell culture plate.

[0161] c) RetroNectin (TAKARA) diluted with PBS to a final concentration of 15 μg / mL was coated on non-tissue treated culture plates, 1.2 mL per well of a 6-well plate. Protect from light and store at 4°C overnight for later use.

[0162] d) After two days of T cell activation culture, the coated 6-well plate was taken out, the coating solution was discarded, and PBS was added to wash the plate once.

[0163] e) Add 5-6 mL of the retrovirus 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.

[0164] f) After cell infection, the cell density was observed every day, and T cell culture medium containing 500 U / mL IL-2 was added in time to maintain the T cell density at 5×10 5 / mL, which is convenient for cell expansion.

[0165] g) Thus, dual-epitope CAR-T cells infected with the retrovirus prepared in step 2 were obtained, and they were named CLL1 DE CAR-T cells (i.e., T cells expressing the CLL1 DE CAR gene with a nucleotide sequence of SEQ ID No. 2) and CLL1 DE CAR-CR-T cells (i.e., T cells expressing the CLL1 DE CAR-CR gene with a nucleotide sequence of SEQ ID No. 4).

[0166] The constructed CLL1 DE CAR-T cells, CLL1 DE CAR-CR-T cells and CTR T cells (i.e., T cells without virus transfection, as control) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and recorded as D0 day. Various functional tests were performed until D10 day.

[0167] Example 4. Detection of CAR expression in dual-epitope CAR-T cells

[0168] The constructed CLL1 DE CAR-T cells express CLL1 DE CAR with an amino acid sequence of SEQ ID No. 1. The constructed CLL1 DE CAR-CR-T cells express two artificial receptors under the action of the self-cleaving peptide P2A, one is the chimeric antigen receptor CLL1 DE CAR (SEQ ID No. 1), and the other is the chimeric conversion receptor PD-1 & CD27 (545-788 of SEQ ID No. 3). The detection steps of CAR expression in dual-epitope CAR-T cells are as follows:

[0169] 1. After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1×PBS containing 0.1% NaN3 and 2% FBS) to each well of a 96-well round bottom plate, resuspend, and centrifuge at 1500 rpm for 5 min;

[0170] 2. Add 60 μL of prepared fluorescent-labeled PD-L1(rp)-APC / CLL1(rp)-FITC to each well, resuspend and mix, and incubate at 4°C for 30 min;

[0171] 3. Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min.

[0172] 4. Discard the supernatant, resuspend the cells with 400 μL FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BDCanto-II) to analyze the percentage of CLL1 antibody in CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells, and the percentage of PD-1 in CLL1 DE CAR-CR-T cells.

[0173] Test results such as Figure 3 As shown, CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells can well express the fusion of two single-domain antibodies, CLL1-VHH-1 and CLL1-VHH-16, and CLL1 DE CAR-CR-T cells can well express PD-1, indicating that the expression of CAR molecules in CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells meets the expected design.

[0174] Example 5. Functional detection of bi-epitope CAR-T cells secreting specific effector molecule IFN-γ

[0175] IFN-γ has anti-tumor, anti-viral and immunomodulatory properties. High levels of IFN-γ are an important indicator of a well-functioning CAR-T. Its expression is consistent with the cytotoxicity of T cells and can indicate the activation level of CAR-T cells. It is detected by flow cytometry after intracellular staining. The steps are as follows:

[0176] 1. Test cells CLL1 DE CAR-T cells, CLL1 DE CAR-CR-T cells and CTR T cells (i.e., T cells without virus transfection, as control) were adjusted to a cell density of 2×10 6 / mL, take 100 μL and add it to a 96-well U-bottom plate, add U937 cells at a ratio of test cells: target cells = 1:1, add Brefeldin A (Med ChemExpress, HY-16592) with a final concentration of 5 μg / mL to each well, and incubate in a 37°C incubator for 6 hours.

[0177] CTR+U937: 1 mL per well, 1×10 6 Add 1 mL of 1×10 T cells without virus transfection (CTR T cells) 6 U937 cells;

[0178] Test cells + U937: 1 mL per well, 1×10 6 Add 1 mL of 1 × 10 6 U937 cells.

[0179] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0180] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1×PBS containing 0.1% NaN3 and 2% FBS) to each well, resuspend, centrifuge at 1500 rpm for 5 min, and repeat this step twice;

[0181] (2) Add 60 μL of the prepared fluorescent-labeled CLL1(rp)-FITC to each well, resuspend and mix, and incubate at room temperature for 10 min in a dark environment;

[0182] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0183] (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 a dark place.

[0184] (5) Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Catalog No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0185] (6) Add 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, Catalog No. 506510) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark;

[0186] (7) Add 200 μL Perm buffer to each well and centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells with 400 μL 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-CR-T cells, and control cells.

[0187] Test results such as Figure 4 As shown, after co-culture with U937 target cells, both CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells can well secrete the T cell-specific effector molecule IFN-γ, and the effect of CLL1 DE CAR-CR-T in secreting IFN-γ is better than that of CLL1 DE CAR-T cells.

[0188] Example 6. Cytotoxicity (degranulation CD107a) function detection of dual-epitope CAR-T cells

[0189] The cell degranulation indicator CD107a is a marker of cytotoxic activity and is detected by flow cytometry as follows:

[0190] 1. Test cells CLL1 DE CAR-T cells, CLL1 DE CAR-CR-T cells and CTR T cells (i.e., T cells without virus transfection, as control) were adjusted to a cell density of 2×10 6 / mL, take 100μL and add it into a 96-well U-bottom plate;

[0191] 2. Add 100 μL of 2×10 cells / well at a ratio of 1:1. 5 U937 cells were used as CAR antigen-specific stimulation (+U937), and 100 μL Medium (10% FBS RPMI-1640 medium) was added to each well as a negative control (+Medium);

[0192] 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 h;

[0193] 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;

[0194] 5. After incubation, perform flow cytometry staining. The steps are as follows:

[0195] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1×PBS containing 0.1% NaN3 and 2% FBS) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and repeat this step twice;

[0196] (2) Add 60 μL of prepared fluorescent antibody (fluorescently labeled anti-human CD3 / CD4 / CD8 / CLL1(rp)) to each well, resuspend and mix, and incubate at room temperature for 10 min in the dark;

[0197] (3) Centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells in 400 μL FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) and analyze the percentage of cell degranulation molecule CD107a in T cells.

[0198] Test results such as Figure 5 As shown, after co-culture with target cells, both CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells can specifically upregulate CD107a degranulation. In addition, compared with CLL1 DE CAR-T cells, CLL1 DE CAR-CR-T cells have stronger specific cytotoxic effect (CD107a).

[0199] Example 7. Cytotoxicity detection of dual-epitope CAR-T cells

[0200] The cytotoxicity of CAR-T cells to tumor cells was detected by an in vitro killing experiment of dual-epitope CAR-T cells on tumor cells. The steps are as follows:

[0201] 1. Test cells CLL1 DE CAR-T cells, CLL1 DE CAR-CR-T cells and CTR T cells (i.e., T cells without virus transfection, as control) were adjusted to a cell density of 2×10 5 / mL, take 100 μL and add it to a 96-well U-bottom plate; add D-firefly luciferin sodium salt (Yeasen Biotechnology, product number 40901ES08, storage concentration is 100 mg / mL) at a final concentration of 100 μg / mL, and repeat for 3 wells;

[0202] 2. Add target cells U937-luc according to different effector-target ratios (1:1, 1:3, 1:9, 1:27) (the cell density of target cells under 1:1 condition is 2×10 4 cells) and cultured at 37°C for 16 h.

[0203] 3. Measure the fluorescence value with TECAN spark microplate reader, and take the average value of three replicate wells to calculate the specific killing activity (Cytotoxicity) of the tested cells:

[0204] Specific lysis%=100-100×(Eexp-Emin) / (Tmax-Tmin)

[0205] Eexp: RLU value when effector cells and target cells are co-cultured;

[0206] Emin: RLU value of spontaneous death of effector cells in the absence of cells;

[0207] Tmax: RLU value of spontaneous death of target cells in the absence of effector cells;

[0208] Tmin: RLU value under conditions of maximum killing rate.

[0209] Test results such as Figure 6 As shown in Figure 2, both CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells can effectively and specifically kill CLL1 + target cells (U937 cells), while control CTR T cells were + No killing effect on target cells.

[0210] Example 8. Detection of in vivo tumoricidal activity of dual-epitope CAR-T cells

[0211] 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;

[0212] 2. Six days after inoculation, the mice were randomly divided into three groups and each group was injected with 3.0×10 6CLL1DE CAR-T cells, CLL1DE CAR-CR-T cells, or CTR T cells were expressed, and recorded as D0 day;

[0213] 3. On D4, D8, D14, D21, D26, and D33 days, use the Photo Acquisition small animal imager to capture tumor luminescence signals.

[0214] Test results Figure 7 As shown, both CLL1 DE CAR-T cells and CLL1 DE CAR-CR-T cells can significantly inhibit tumors and prolong the lifespan of mice; compared with CLL1 DE CAR-T cells, the residual human myeloid leukemia tumor cells in mice injected with CLL1 DE CAR-CR-T cells were significantly reduced, indicating that CLL1 DE CAR-CR-T cells have a better tumor inhibition effect.

[0215] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A chimeric antigen receptor, characterized in that The 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 chimeric antigen receptor according to claim 1, characterized in that The transmembrane domain is derived from CD8a, CD4, CD28, CD137, CD80, CD86 or CD152.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that The intracellular signaling domain includes the co-stimulatory domain 4-1BB and the activation domain CD3ζ.

4. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that The chimeric antigen receptor is any of the following: A1) the amino acid sequence is the protein shown in positions 22-522 of SEQ ID No. 1; A2) A fusion protein having the same function as A1) is obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of A1).

5. A fusion protein, characterized in that The fusion protein comprises the chimeric antigen receptor and the chimeric switch receptor according to any one of claims 1 to 4, and the amino acid sequence of the chimeric switch receptor is shown at positions 545 to 788 of SEQ ID No.

3.

6. The fusion protein according to claim 5, characterized in that The fusion protein is any of the following: B1) the amino acid sequence is the protein shown in positions 22-788 of SEQ ID No. 3; B2) A fusion protein having the same function as B1) is obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of B1).

7. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 4; C2) a nucleic acid molecule encoding the fusion protein of claim 5 or 6; C3) an expression cassette containing the nucleic acid molecule described in C1) or C2); C4) a recombinant vector containing the nucleic acid molecule described in C1) or C2), or a recombinant vector containing the expression cassette described in C3); C5) a recombinant microorganism containing the nucleic acid molecule described in C1) or C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4); C6) A recombinant cell containing the nucleic acid molecule described in C1) or C2), or a recombinant cell containing the expression cassette described in C3), or a recombinant cell containing the recombinant vector described in C4).

8. The biomaterial according to claim 7, characterized in that The nucleic acid molecule is any of the following: D1) the coding sequence is the DNA molecule shown in SEQ ID No. 2, positions 64 to 1566 of SEQ ID No. 2, SEQ ID No. 4, or positions 64 to 2367 of SEQ ID No. 4; D2) The nucleotide sequence is a DNA molecule shown in SEQ ID No. 2, positions 64 to 1566 of SEQ ID No. 2, SEQ ID No. 4 or positions 64 to 2367 of SEQ ID No.

4.

9. A modified immune effector cell, characterized in that The modified immune effector cell comprises the chimeric antigen receptor according to any one of claims 1 to 4, the fusion protein according to claim 5 or 6, or the nucleic acid molecule according to any one of claims 7 or 8.

10. Use of the chimeric antigen receptor according to any one of claims 1 to 4, the fusion protein according to claim 5 or 6, the biomaterial according to claim 7 or 8, or the modified immune effector cell according to claim 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) inhibits the growth of CLL1-positive tumors; E5) promotes the release of cytokine IFN-γ; E6) Detection of cancer cells expressing CLL1.

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