Enhanced chimeric antigen receptor targeting CLL1 and application thereof

Abstract

The invention provides a CLL1-targeting enhanced chimeric antigen receptor and application thereof. The CLL1-targeting enhanced chimeric antigen receptor comprises a CLL1-targeting extracellular antigen recognition structural domain, a hinge region, a transmembrane region and an intracellular structural domain, and the intracellular structural domain comprises a costimulatory factor and an intracellular signal transduction structural domain; wherein the intracellular domain further comprises a cytokine receptor gamma chain amino acid sequence derived from IL2R gamma, or wherein the costimulatory domain comprises an amino acid sequence derived from 2B4. Compared with a CLL1CAR-DNT cell with a conventional second-generation chimeric antigen receptor structure, the CLL1CAR-DNT cell with the enhanced chimeric antigen receptor structure has the advantage that the proliferation level of the CLL1CAR-DNT cell with the enhanced chimeric antigen receptor structure is higher.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an enhanced chimeric antigen receptor targeting CLL1 and its application. Background Art

[0002] Chimeric Antigen Receptor (CAR) is the core component of CAR cell therapy drugs, which may include extracellular antigen recognition domains, hinge regions, transmembrane regions and intracellular domains. CAR-T cell immunotherapy is considered one of the most promising means to conquer tumors. CAR-T cells use genetic modification methods to express CAR proteins on the surface of T cells. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.

[0003] Currently, the CAR-T cell therapy drugs that have been approved for marketing all use autologous T cells that are transfected in vitro, proliferated, and then infused back into the patient's autologous CAR-T cell drugs. However, autologous CAR-T cell drugs have the following problems: some patients' autologous T cells do not meet the requirements. Even if the patient's autologous T cells meet the requirements, the preparation time of individualized CAR-T cells is long, the preparation process is complicated, and the cost is very high. However, the use of allogeneic CAR-T cells requires immune matching, and there are also risks such as immune rejection, GvH reaction (graft-versus-host reaction) and HvG reaction (host-versus-graft reaction), so it has not yet been used. If a universal CAR-T cell therapy drug can be prepared to make it universal for the patient population, it can be industrialized on a larger scale to obtain standardized universal products.

[0004] Most T cells in human peripheral blood express CD4 or CD8 molecules, and about 1-3% of them lack CD4 or CD8 molecules but express CD3 molecules (thus still belonging to T cells), that is, CD3 + CD4 - CD8 - Double Negative T cells (DNT). It is reported that DNT cells have a broad spectrum of tumor cell killing effects, and have no GvH reaction and HvG reaction. They are an ideal cell type for allogeneic universal treatment. DNT cells can be used to replace conventional T cells in CAR cell therapy drugs, but due to the low proportion of DNT cells in T cells, the proliferation rate of DNT cells after loading CAR may be further reduced. Therefore, how to improve the proliferation level of DNT cells loaded with CAR is of practical significance. Summary of the invention

[0005] The present application provides an enhanced chimeric antigen receptor targeting CLL1 and its application. The inventors designed 16 chimeric antigen receptor structures targeting CLL1, and obtained an enhanced chimeric antigen receptor structure targeting CLL1 by verification and screening on DNT cells. Compared with CLL1 CAR-DNT cells with conventional second-generation chimeric antigen receptor structures, CLL1 CAR-DNT cells with the enhanced chimeric antigen receptor structure have a higher proliferation level.

[0006] The present application provides an enhanced chimeric antigen receptor targeting CLL1, which comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain targeting CLL1, wherein the intracellular domain comprises a co-stimulatory domain and an intracellular signal transduction domain; wherein: the intracellular domain also comprises a cytokine receptor γ chain amino acid sequence derived from IL2Rγ.

[0007] In certain embodiments of the enhanced chimeric antigen receptor targeting CLL1, the N-terminus of the cytokine receptor γ chain amino acid sequence derived from IL2Rγ is connected to the C-terminus of the intracellular signaling domain.

[0008] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the cytokine receptor γ chain derived from IL2Rγ comprises the amino acid sequence shown in SEQ ID NO:32; alternatively, the amino acid sequence of the cytokine receptor γ chain derived from IL2Rγ is as shown in SEQ ID NO:32.

[0009] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 comprises a CLL1 antibody heavy chain variable region and a CLL1 antibody light chain variable region, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:8; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region are respectively The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in NO:8.

[0010] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0011] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the CLL1 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the CLL1 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; optionally, the amino acid sequence of the CLL1 antibody heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the CLL1 antibody light chain variable region is as shown in SEQ ID NO:8.

[0012] In certain embodiments of the enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:30.

[0013] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the costimulatory domain is derived from one, two or more costimulatory factors selected from the group consisting of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 2B4, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, DAP12, DNAM-1, HVEM-1, B7-H3, and MyD88; optionally, the costimulatory domain is derived from CD28 or 4-1BB; further optionally, the amino acid sequence of the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:36; further optionally, the amino acid sequence of the costimulatory domain is as shown in SEQ ID NO:35 or SEQ ID NO:36.

[0014] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:34; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:34.

[0015] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the intracellular domain comprises an amino acid sequence as shown in SEQ ID NO:28 or SEQ ID NO:29; optionally, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:28 or SEQ ID NO:29.

[0016] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:9.

[0017] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 11; further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 11.

[0018] In certain embodiments, the enhanced chimeric antigen receptor targeting CLL1 further comprises a signal peptide; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:31.

[0019] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the enhanced chimeric antigen receptor is shown as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:28, or as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:29.

[0020] The present application also provides an enhanced chimeric antigen receptor targeting CLL1, which comprises an extracellular antigen recognition domain targeting CLL1, a hinge region, a transmembrane region and an intracellular domain, wherein the intracellular domain comprises a co-stimulatory domain and an intracellular signaling domain; wherein: the co-stimulatory domain comprises an amino acid sequence derived from 2B4.

[0021] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence derived from 2B4 comprises the amino acid sequence shown in SEQ ID NO:33; optionally, the amino acid sequence derived from 2B4 is as shown in SEQ ID NO:33.

[0022] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 comprises a CLL1 antibody heavy chain variable region and a CLL1 antibody light chain variable region, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:8; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region are respectively The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in NO:8.

[0023] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0024] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the CLL1 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the CLL1 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; optionally, the amino acid sequence of the CLL1 antibody heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the CLL1 antibody light chain variable region is as shown in SEQ ID NO:8.

[0025] In certain embodiments of the enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:30.

[0026] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the co-stimulatory domain also includes one, two or more co-stimulatory factors derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, DAP12, DNAM-1, HVEM-1, B7-H3, and MyD88.

[0027] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:34; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:34.

[0028] In certain embodiments of the enhanced chimeric antigen receptor targeting CLL1, the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21; optionally, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:21.

[0029] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:9.

[0030] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α or 2B4; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence as shown in SEQ ID NO:11 or SEQ ID NO:13; further optionally, the amino acid sequence of the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:11 or SEQ ID NO:13.

[0031] In certain embodiments of the enhanced chimeric antigen receptor targeting CLL1, the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21.

[0032] In certain embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the enhanced chimeric antigen receptor is shown as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:21, or as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:13-SEQ ID NO:21.

[0033] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any one of the above-mentioned enhanced chimeric antigen receptors targeting CLL1.

[0034] The present application also provides a vector, which comprises the above-mentioned isolated nucleic acid molecule.

[0035] The present application also provides an engineered immune effector cell, which comprises the enhanced chimeric antigen receptor, the isolated nucleic acid molecule, or the vector, wherein the immune effector cell is derived from CD3 + CD4 - CD8 - of DNT cells.

[0036] In certain embodiments of the engineered immune effector cells, the DNT cells are autologous DNT cells or allogeneic DNT cells.

[0037] The present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.

[0038] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes a protective agent.

[0039] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient comprises a cell freezing solution.

[0040] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutical composition is an intravenous injection.

[0041] The present application also provides a use of the above-mentioned enhanced chimeric antigen receptor, isolated nucleic acid molecule, vector or engineered immune effector cell in the preparation of a drug, wherein the drug is used to treat a disease or condition related to the expression of CLL1.

[0042] In certain embodiments of the above applications, the disease or disorder associated with the expression of CLL1 is a hematological tumor.

[0043] In certain embodiments of the above application, the disease or condition associated with the expression of CLL1 is acute myeloid leukemia.

[0044] The present application also provides a method for treating a disease or condition associated with the expression of CLL1, comprising the following steps: administering an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical composition to a subject in need of treating a disease or condition associated with the expression of CLL1.

[0045] In certain embodiments of the above method, the disease or disorder associated with the expression of CLL1 is a hematological tumor.

[0046] In certain embodiments of the above method, the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.

[0047] In certain embodiments of the above method, the administration is by intravenous injection.

[0048] In certain embodiments of the above method, the administration is to administer an effective amount of the engineered immune effector cells or the pharmaceutical composition to the subject in the form of a single injection.

[0049] In certain embodiments of the above method, the effective amount of the engineered immune effector cells or pharmaceutical composition is 1×10 5 Up to 1×10 7 The dose was cells / kg.

[0050] The present application also provides a drug comprising the engineered immune effector cells or pharmaceutical composition described above, for treating a disease or condition associated with the expression of CLL1.

[0051] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CLL1 is a hematological tumor.

[0052] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CLL1 is acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The positions of the components in the 16 candidate CAR structures in Example 1 are shown, SP represents the signal peptide, Hinge represents the hinge region, and TM represents the transmembrane region.

[0054] Figure 2 The in vitro expansion of CAR-DNT cells with different structures in Example 1 is demonstrated.

[0055] Figure 3A The short-term killing results of CAR-DNT cells with a CAR positive rate of 30%±5% in Example 3 at multiple effector-target ratios are shown; Figure 3BThe short-term killing results of CAR-DNT cells with a CAR positive rate of 15%±5% in Example 3 at multiple effector-target ratios are shown; Figure 3A and Figure 3B For each CAR-DNT group, from left to right are the target cell lysis rates at the following five effector-target ratios: 1.25:1, 2.5:1, 5:1, 10:1 and 20:1.

[0056] Figure 4 The expansion of effector cells in multiple rounds of stimulation experiments in Example 4 is shown. Baselines are provided based on the expansion fold of structure 1 CAR-DNT cells and CAR-T cells in the third round.

[0057] Figure 5 The changes in the CAR positivity rate in the effector cell gate during multiple rounds of stimulation experiments in Example 4 are shown.

[0058] Figure 6 The killing of target cells by effector cells in multiple rounds of stimulation experiments in Example 4 is shown, and in each group, from left to right, they are round-1, round-2, round-3, round-4, and round-5. DETAILED DESCRIPTION

[0059] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0060] The present application is further described below: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0061] In this application, the term "chimeric antigen receptor" (CAR) is the core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered to be one of the most promising means of conquering tumors. CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.

[0062] In this application, the term "extracellular antigen recognition domain" refers to the Antigen Recognition Domain (ARD). The ability of CAR cell therapy products (such as CAR-T cells) to specifically recognize and / or bind to target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. So far, the antigen recognition domain is derived from the single chain variable region (Single Chain Variable Fragment, abbreviated as scFv) of the antibody, or from receptor ligand interactions, TCR mimics, and variable lymphocyte receptors (VLR). So far, the most common source is the scFv antibody.

[0063] In the present application, the term "specific recognition and / or binding" refers to the recognition and / or binding between the extracellular antigen recognition domain of CAR and a specific target, or the recognition and / or binding between the extracellular antigen recognition domain of CAR and a specific detection antibody is with greater affinity, avidity, easier, and / or with greater duration than CAR binds to other targets or detection antibodies.

[0064] In this application, the term "antibody" has the conventional meaning in the art and is used in the broadest sense in this application. In the field of biological sciences, by analyzing the amino acid sequences of different antibody heavy chains and light chains, it is found that the amino acid sequences of the heavy chains and light chains near the N-terminus vary greatly, while the amino acid sequences of other parts are relatively constant. Therefore, the region in the antibody light chain and heavy chain where the amino acid sequence near the N-terminus varies greatly is called the variable region (variable region, V), and the region where the amino acid sequence near the C-terminus is relatively stable is called the constant region (constant region, C). The V region of the heavy chain and light chain is referred to as VH and VL, respectively, and the C region of the heavy chain and light chain is referred to as CH and CL, respectively. There is a small part of the amino acid residues in the variable region of the antibody that vary particularly strongly, and the residue composition and arrangement order of these amino acids are more prone to variation. The region is called hypervariable region (HVR); there are three hypervariable regions in the V region of the L chain and the H chain. Because this site can form precise complementarity with the antigenic determinant in terms of spatial structure, the hypervariable region is also called complementarity determining region (CDR). In antibodies, common CDR division rules include Kabat, AbM, Chothia, Contact, and IMGT, which are well known to those skilled in the art. When applying a website that executes these rules, as long as the VH and VL sequences are input and the corresponding rules are selected, CDR sequences according to different rules can be obtained. It should be understood by those skilled in the art that the scope of protection of the present application covers the combination of CDR sequences obtained by adopting different rule analyses. The 6 CDR regions of an antibody jointly determine the recognition ability and specificity of the antibody to the corresponding antigen. It should be understood by those skilled in the art that when the present application defines the amino acid sequences of the 6 CDR regions, the recognition ability and specificity of the antibody to the corresponding antigen are predictable.

[0065] In the present application, the term "scFv" has the conventional meaning in the art, and refers to a single-chain variable region, which is an antibody formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide (linker).

[0066] In the present application, the term "linker" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length, which links together any structure / region of the antibody or chimeric antigen receptor of the present invention. The linker can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. When it is desired to ensure that two adjacent domains do not interfere with each other in space, a longer linker can be used.

[0067] In the present application, the term "hinge region" refers to the connecting segment between the extracellular antigen recognition domain and the transmembrane region. This region allows CAR to recognize antigens by giving the antigen recognition domain a certain range of activity.

[0068] In this application, "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but do not directly participate in signal transduction, and can enhance downstream signal transduction through interaction.

[0069] In this application, the term "intracellular domain" includes an intracellular signaling domain, and also includes a co-stimulatory domain and / or a cytokine. The term "intracellular signaling domain" is responsible for activating at least one normal effector function of an immune effector cell expressing CAR. "Co-stimulatory domain" is because in addition to the stimulation of antigen-specific signals, many immune effector cells also require co-stimulatory factors to promote cell proliferation, differentiation, survival, and activate the effector function of cells; in some embodiments, the co-stimulatory domain of CAR may include one or more co-stimulatory factors. Cytokines, such as IL-7, IL-12, and IL-18, are connected to the intracellular signaling domain of ordinary CAR-T cells. These cytokines can locally stimulate the proliferation of CAR-T cells, prolong the survival time of T cells, recruit immune cells from the body, and improve the local immunosuppressive microenvironment, while avoiding the toxic side effects caused by cytokines when cytokines are used systemically in combination with adoptive immune cell therapy in order to enhance the efficacy of cell therapy. At present, according to the different intracellular domains, CAR includes four generations: the first generation of intracellular domain includes only one intracellular signal transduction domain (such as CD3ζ chain), the second generation includes one intracellular signal transduction domain (such as CD3ζ chain) and one co-stimulatory factor; the third generation includes one intracellular signal transduction domain (such as CD3ζ chain) and two or more different co-stimulatory factors; the fourth generation is the intracellular signal transduction domain of ordinary CAR-T cells (such as CD3ζ chain) connected to the cytokine IL-7, IL-12, IL-18 and other structures.

[0070] In the present application, the term "IL2Rγ" refers to the γ subunit of the interleukin-2 (IL2) receptor complex, which has also been identified as part of the following cytokine receptor complexes: IL-4, IL-7, IL-9, IL-15 and IL-21. It is involved in the signal transduction and ligand binding of these cytokine receptors, and can transmit signals to the cell nucleus through the JAK-STAT pathway to regulate the proliferation and activation of immune cells.

[0071] In this application, the term "2B4" refers to the natural killer cell receptor 2B4, also known as CD244, which is a member of the signal lymphocyte activation molecule family and plays an important role in regulating the activity of NK cells, T cells, and eosinophils. 2B4 contains four receptor tyrosine conversion motifs in the cell, which transmit activation or inhibition signals by interacting with different signal receptors and regulating cytotoxicity.

[0072] In this application, the term "guide peptide" refers to a short peptide before the extracellular antigen recognition domain (such as scFv sequence), which guides the recombinant protein synthesized in the cell to be exported to the outside of the cell. Common guide peptides include human CD8α signal peptide or human GM-CSF receptor α signal peptide.

[0073] In this application, the term "isolated" generally refers to something obtained from a natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" does not exclude substances that have been artificially or synthetically obtained from a natural state, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0074] In this application, the term "isolated nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides of any length in isolated form, either separated from their natural environment or artificially synthesized analogs.

[0075] The term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce or transfect a host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not only these substances. The term "transposon" refers to a discontinuous DNA fragment that has the ability to migrate between chromosome sites and carry genetic information, such as the Sleeping Beauty SB system and the PB system from Lepidoptera. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

[0076] In this application, the terms "comprises", "comprising" and "including" generally mean including the features explicitly stated, but not excluding other elements.

[0077] In this application, the term "about" generally refers to a fluctuation range acceptable to those skilled in the art above or below the specified value, such as: varying within the range of ±0.5%-10%, for example, varying within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

[0078] Without intending to be bound by any theory, the following examples are only intended to illustrate the chimeric antigen receptors, immune effector cells, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, methods for inserting protein-encoding genes into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those with ordinary skills in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0079] The first enhanced chimeric antigen receptor targeting CLL1

[0080] The present application provides an enhanced chimeric antigen receptor targeting CLL1, which comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain targeting CLL1, wherein the intracellular domain comprises a co-stimulatory domain and an intracellular signal transduction domain; wherein: the intracellular domain also comprises a cytokine receptor γ chain amino acid sequence derived from IL2Rγ.

[0081] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1, the N-terminus of the cytokine receptor γ chain amino acid sequence derived from IL2Rγ is connected to the C-terminus of the intracellular signaling domain; the connection can be direct or indirect with other sequences in between.

[0082] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the cytokine receptor γ chain derived from IL2Rγ comprises the amino acid sequence shown in SEQ ID NO: 32; alternatively, the amino acid sequence derived from

[0083] The amino acid sequence of the cytokine receptor γ chain of IL2Rγ is shown in SEQ ID NO:32.

[0084] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 comprises a CLL1 antibody heavy chain variable region and a CLL1 antibody light chain variable region, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:8; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region are respectively The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in NO:8.

[0085] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0086] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the CLL1 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the CLL1 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; optionally, the amino acid sequence of the CLL1 antibody heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the CLL1 antibody light chain variable region is as shown in SEQ ID NO:8.

[0087] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 is an amino acid sequence shown in SEQ ID NO: 30. The extracellular antigen recognition domain targeting CLL1 is derived from a scFv antibody, which connects VH and VL through a connecting sequence.

[0088] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the costimulatory domain is derived from one, two or more costimulatory factors among CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 2B4, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, DAP12, DNAM-1, HVEM-1, B7-H3, and MyD88; optionally, the costimulatory domain is derived from CD28 or 4-1BB; further optionally, the amino acid sequence of the costimulatory domain comprises

[0089] The amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:36; further optionally, the amino acid sequence of the co-stimulatory domain is shown in SEQ ID NO:35 or SEQ ID NO:36.

[0090] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the intracellular signaling region comprises the amino acid sequence as shown in SEQ ID NO:34; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:34.

[0091] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1, the intracellular domain comprises

[0092] The amino acid sequence shown in SEQ ID NO:28 or SEQ ID NO:29; optionally, the amino acid sequence of the intracellular domain is shown in SEQ ID NO:28 or SEQ ID NO:29.

[0093] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:9.

[0094] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 11; further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 11.

[0095] In some embodiments, the enhanced chimeric antigen receptor targeting CLL1 further comprises a signal peptide; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:31.

[0096] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the enhanced chimeric antigen receptor is shown as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:28, or as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:29.

[0097] A second enhanced chimeric antigen receptor targeting CLL1

[0098] The present application provides an enhanced chimeric antigen receptor targeting CLL1, which comprises an extracellular antigen recognition domain targeting CLL1, a hinge region, a transmembrane region and an intracellular domain, wherein the intracellular domain comprises a co-stimulatory domain and an intracellular signaling domain; wherein: the co-stimulatory domain comprises an amino acid sequence derived from 2B4.

[0099] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence derived from 2B4 comprises the amino acid sequence shown in SEQ ID NO:33; optionally, the amino acid sequence derived from 2B4 is as shown in SEQ ID NO:33.

[0100] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 comprises a CLL1 antibody heavy chain variable region and a CLL1 antibody light chain variable region, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:8; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region are respectively The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in NO:8.

[0101] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0102] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the CLL1 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the CLL1 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; optionally, the amino acid sequence of the CLL1 antibody heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the CLL1 antibody light chain variable region is as shown in SEQ ID NO:8.

[0103] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:30.

[0104] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the co-stimulatory domain also includes one, two or more co-stimulatory factors derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, DAP12, DNAM-1, HVEM-1, B7-H3, and MyD88.

[0105] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the intracellular signaling region comprises the amino acid sequence as shown in SEQ ID NO:34; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:34.

[0106] In certain embodiments of the enhanced chimeric antigen receptor targeting CLL1, the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21; optionally, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:21.

[0107] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:9.

[0108] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α or 2B4; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:13; further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:11 or SEQ ID NO:13.

[0109] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1, the intracellular domain comprises

[0110] The amino acid sequence shown in SEQ ID NO:21.

[0111] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the enhanced chimeric antigen receptor is shown as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:21, or as the amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:13-SEQ ID NO:21.

[0112] Corresponding nucleic acid molecules, vectors, immune effector cells, and pharmaceutical compositions

[0113] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any one of the above-mentioned enhanced chimeric antigen receptors targeting CLL1.

[0114] The present application also provides a vector, which comprises the above-mentioned isolated nucleic acid molecule.

[0115] The present application also provides an engineered immune effector cell, which comprises the enhanced chimeric antigen receptor, the isolated nucleic acid molecule, or the vector, wherein the immune effector cell is derived from CD3 + CD4 - CD8 - of DNT cells.

[0116] In some embodiments of the engineered immune effector cells described above, the DNT cells are autologous DNT cells or allogeneic DNT cells.

[0117] The present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.

[0118] In some embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes a protective agent.

[0119] In some embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes cell freezing solution.

[0120] In some embodiments of the above-mentioned pharmaceutical composition, the pharmaceutical composition is an intravenous injection.

[0121] Preparation method and use

[0122] The present application also provides a use of the above-mentioned enhanced chimeric antigen receptor, isolated nucleic acid molecule, vector or engineered immune effector cell in the preparation of a drug, wherein the drug is used to treat a disease or condition related to the expression of CLL1.

[0123] In some embodiments of the above applications, the disease or disorder associated with the expression of CLL1 is a hematological tumor.

[0124] In some embodiments of the above application, the disease or condition associated with the expression of CLL1 is acute myeloid leukemia.

[0125] The present application also provides a method for treating a disease or condition associated with the expression of CLL1, comprising the following steps: administering an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical composition to a subject in need of treating a disease or condition associated with the expression of CLL1.

[0126] In some embodiments of the above method, the disease or disorder associated with the expression of CLL1 is a hematological tumor.

[0127] In some embodiments of the above method, the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.

[0128] In some embodiments of the above method, the administration is by intravenous injection.

[0129] In some embodiments of the above method, the administration is to administer an effective amount of the engineered immune effector cells or the pharmaceutical composition to the subject in the form of a single injection.

[0130] In some embodiments of the above method, the effective amount of the engineered immune effector cells or pharmaceutical composition is 1×10 5 Up to 1×10 7 The dose was cells / kg.

[0131] The present application also provides a drug comprising the engineered immune effector cells or pharmaceutical composition described above, for treating a disease or condition associated with the expression of CLL1.

[0132] In some embodiments of the above-mentioned drug, the disease or condition associated with the expression of CLL1 is a hematological tumor.

[0133] In some embodiments of the above-mentioned drug, the disease or condition associated with the expression of CLL1 is acute myeloid leukemia.

[0134] Example 1. Preparation of CAR-DNT cells

[0135] 1. Design of CAR structure

[0136] The inventors constructed a total of 16 candidate CAR structures and selected an enhanced CAR structure suitable for DNT cells through functional verification on DNT cells. In each candidate CAR structure, the position of each component is as follows Figure 1 The amino acid sequence of the signal peptide is shown in SEQ ID NO: 31; the scFv targeting CLL1 serves as the extracellular antigen recognition domain, and its amino acid sequence is shown in SEQ ID NO: 30 (wherein: in the scFv targeting CLL1, the amino acid sequence of CLL1 VH is shown in SEQ ID NO: 7, the amino acid sequence of CLL1VL is shown in SEQ ID NO: 8, and the CDR1, CDR2, and CDR3 of CLL1 VH are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, respectively.

[0137] NO:2, SEQ ID NO:3, CDR1, CDR2, and CDR3 of CLL1 VL are shown in SEQ ID

[0138] NO:4, SEQ ID NO:5, SEQ ID NO:6); in each candidate CAR structure, the amino acid sources and specific sequences of the hinge region, transmembrane region, and intracellular region are shown in Table 1.

[0139] In the CAR structure, there are two main families of co-stimulatory factors for T cells: the CD28 family and the tumor necrosis factor receptor superfamily (the latter includes 4-1BB and HVEM). In structure No. 1, a common second-generation CAR structure was used, using a co-stimulatory factor derived from 4-1BB; in structures No. 11 and No. 14, a co-stimulatory factor derived from HVEM and a co-stimulatory factor derived from CD28 were used to replace the co-stimulatory factor derived from 4-1BB in structure No. 1; in structure No. 13, CD28 and 4-1BB were used as co-stimulatory domains at the same time.

[0140] MyD88ΔTIR is a linker molecule for TLR and IL-1 receptor family signals, and CD40 is involved in the memory phenotype of T cells. In this application, MyD88ΔTIR and CD40 are used together as the costimulatory domain of the CAR molecule. In structure No. 2, MyD88ΔTIR and CD40 are used as costimulatory domains at the same time; in structure No. 3, CD28, MyD88ΔTIR and CD40 are used as costimulatory domains at the same time.

[0141] NKG2D is an activating receptor expressed on NK cells and certain types of T cells in humans and mice. In mice, NKG2D corresponds to two protein isoforms, called NKG2D-S and NKG2D-L. NKG2D-L mainly binds to the DAP 10 adaptor protein, while NKG2D-S binds to DAP10 or DAP12. DAP10 is a downstream activation signaling molecule of NKG2D that can promote cytokine release and T cell differentiation into memory precursor cells; DAP12 is involved in immune cell activation. DAP contains an intracellular signaling domain, and there is no need to add an additional intracellular signaling domain. In structure No. 4, DAP10 and DAP12 are used as co-stimulatory domains, and no additional intracellular signaling domains are used; in structure No. 5, relative to structure No. 4, the hinge region and transmembrane region derived from DAP10 are used to replace the hinge region and transmembrane region derived from CD8α; in structure No. 6, relative to structure No. 5, 4-1BB is used instead of DAP12 as a co-stimulatory factor, and an intracellular signaling domain derived from CD3ζ is also added.

[0142] The natural killer cell receptor 2B4 plays an important role in regulating the activity of NK cells, T cells, and eosinophils. In structure 7, 2B4 is used as a co-stimulatory factor; in structure 8, compared with structure 7, the transmembrane region derived from 2B4 is used to replace the transmembrane region derived from CD8α.

[0143] DNAM-1 is an activating receptor of the NK cell receptor family. DNAM-1 contains three tyrosine residues in the cell, which activate downstream signals through phosphorylation cascade reactions. In structure 9, DNAM-1-derived co-stimulatory factors and transmembrane regions were used; in structure 10, compared with structure 9, DNAM-1 and 2B4 were used as co-stimulatory domains.

[0144] In structure 12, ΔIL2Rβ is a truncated form of the IL-2 receptor β chain. CD3ζ(YXXQ) is the STAT3 binding motif tyrosine-XX-glutamine(YXXQ) used in the intracellular structure of CD3ζ.

[0145] In structures 15 and 16, IL2Rγ is included.

[0146] Table 1. Amino acid sequences of hinge region, transmembrane region and intracellular region in CAR structure

[0147]

[0148]

[0149] 2. Construction of Lentiviral Vector

[0150] According to the sequences in Table 1, the nucleotide sequences encoding the above 16 candidate CAR structures were artificially synthesized, and they were constructed into modified empty lentiviral vectors (manufacturer: SBI, product number: CD500-CD800, as described in Example 1 of WO2021 / 121227 for conventional resistance modification) to obtain 16 CAR vectors, and then the CAR vectors and 3 packaging plasmids were transfected into 293T cells together, and functional lentiviral vectors were obtained after collection and purification. The three packaging plasmids are pMD2.G (purchased from Biovector, product number Biovector012259), pMDLg / pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253).

[0151] 3. Preparation of CAR-DNT cells and CAR-T cells

[0152] Cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors were purchased and CD4 was isolated using CD4 / CD8 mixed magnetic beads (purchased from Miltenyi Biotec) after PBMC recovery. - CD8 - cells and CD4+ and / or CD8 + Mixed cells, including: CD4 - CD8 - Cells used for the preparation of CAR-DNT cells, CD4 + and / or CD8 + The mixed cells are used for the preparation of control CAR-T cells. In this step, no CD3 + Cells were sorted separately (CD3 + is a T cell sorting marker), because under the subsequent culture conditions, CD3 - The cells are almost unable to proliferate; they do not target CD4 + Cells and CD8 + Cells were sorted individually and used as mixed T cells.

[0153] Preparation of CAR-DNT cells: CD3 monoclonal antibody OKT3 (purchased from Invitrogen) was used as an activation antibody to coat 24-well plates overnight, and then the residual OKT3 antibody was washed away; the CD4 - CD8 - Double negative T cells were treated with a medium containing recombinant human interleukin-2 (rhIL-2) at a final concentration of 250 IU / ml, recombinant human interleukin-15 (rhIL-15) at a final concentration of 10 ng / ml, and recombinant human interleukin-7 (rhIL-7) at a final concentration of 2 ng / ml. The cells were resuspended in the culture medium to a density of 2E6 / ml and inoculated into a coated 24-well plate for culture. After one day of culture, the 16 lentiviruses obtained in Example 1 were added to different wells of the 24-well plate for lentiviral transduction with an MOI of about 5. After culturing for another 2 days under the same conditions, the cells were transferred to a suitable culture container to maintain a culture density of 5E5 to 2E6 / ml in an incubator containing recombinant human interleukin-2 (rhIL-2) at a final concentration of 250 IU / ml, recombinant human interleukin-15 (rhIL-15) at a final concentration of 10 ng / ml, and recombinant human interleukin-7 at a final concentration of 2 ng / ml. Culture in the culture medium for another 3 days; when the CAR-DNT cells were harvested on the 7th day to the 10th day, No rhIL-7 was added to the culture medium, and other added components remained unchanged; during this period, cell counts were performed every other day, and fresh culture medium was added to maintain the cell density at 5E5-2E6 / ml; after harvesting on the 10th day, flow cytometry was used to detect the CAR positivity rate and expression of activation receptors on the surface of DNT cells. For the expansion of DNT cells used as blank controls, except for not infecting with lentivirus, other conditions were the same as the above-mentioned CAR-DNT cell expansion conditions. The expansion level of total DNT cells in each group is shown in Figure 2. Figure 2 As shown: Structures 9 and 10 were successfully transduced but the expression of the CAR gene was not detected, so these two groups were discarded on the fifth day of in vitro culture; CAR-DNT cells with structures 5 and 6 performed the worst in vitro expansion.

[0154] CAR-T cell preparation: CD4 + and / or CD8 + Anti-CD3 / CD28 magnetic beads (purchased from Thermo Fisher Scientific) were added to the mixed cells at a ratio of 1:1, and CD4 + and / or CD8 + Mix the cells for activation culture to obtain T cells: X-VIVO15 medium (Lonza) + 5% (v / v) heat-inactivated fetal bovine serum (GIBCO) + final concentration of 300IU / mL recombinant human IL-2 (Sino-biologic Inc.). After 24 hours of magnetic bead activation culture, lentiviral transduction was performed. During transduction, the T cell density was adjusted to 2E6 / ml, and the lentivirus obtained according to structure No. 1 in Example 1 was added at MOI=1 for infection. After mixing, the cells were placed in an incubator for culture at 37°C + 5% CO2 to obtain the T1 cell group. After 48 hours of infection, the positive rate of CAR-T cells can be observed early by flow cytometry, and the positive rate of CAR-T cells in the final product was detected again by flow cytometry at D10 harvest. The preparation of CAR-T cells is used to compare the relevant performance of CAR-T cells and CAR-DNT cells under the same CAR structure.

[0155] Example 2. Detection of CAR Positive Rate and Activation Receptor Expression

[0156] 1. Detection of CAR positivity rate

[0157] The CAR protein molecules expressed on the surface of the 16 CLL1 CAR-DNT cells obtained in Example 1 and cultured for 10 days after transduction were detected, and about (1-10)×10 5 The CAR-DNT cells were stained with APC fluorescently labeled mouse anti-human CD3 flow cytometry monoclonal antibody (Cat. No. 300312, purchased from BioLegend) and FITC fluorescently labeled human CLL1 protein (Cat. No. HF247, purchased from ACRO), and the CAR positivity rate was detected by flow cytometry; at the same time, the CAR positivity rate in the CAR-T cells obtained in Example 1 was detected by the same method as a control.

[0158] The results of the CAR positive rate detection are shown in Table 2. The CAR positive rates on the surface of CAR-DNT cells expressing different CAR structures are different. Except for structures 9 and 10, the CAR structures of the remaining structures can be normally expressed on the surface of DNT cells, among which: Since the expression of the CAR gene was not detected in the cell group where structures 9 and 10 were located in Example 1, structures 9 and 10 were not further verified in this experiment and subsequent experiments. In the following table, "---" means not detected.

[0159] Table 2. Positive rates of 16 enhanced CAR structures on the surface of DNT cells

[0160] Serial number CAR positive rate Serial number CAR positive rate 1 43.62％ 10 --- 2 26.82％ 11 26.38％ 3 32.61％ 12 20.36％ 4 70.22％ 13 25.60％ 5 15.81％ 14 23.79％ 6 35.56％ 15 32.48％ 7 25.84％ 16 17.84％ 8 13.14％ T1 (CAR-T cells) 32.02％ 9 ---

[0161] 2. Detection of activation receptor expression

[0162] The activation receptors NKG2D and DNAM-1 expressed on the surface of 16 CLL1 CAR-DNT cells cultured for 10 days after transduction obtained in Example 1 were detected. NKG2D and DNAM-1 are two major activation receptors on DNT cells, which play an important role in the anti-tumor process. DNT cells, as a type of natural immune cells, have more innate immune activation receptors NKG2D distributed on their surface compared to T cells, which plays an important role in anti-tumor immune response. CAR-DNT cells were stained with APC fluorescently labeled mouse anti-human NKG2D antibody (item number 320808, purchased from BioLegend) and FITC fluorescently labeled mouse anti-human DNAM-1 antibody (item number 338304, purchased from BioLegend) and then the expression of activation receptors NKG2D and DNAM-1 was detected by flow cytometry; at the same time, the NKG2D expression and DNAM-1 expression of CAR-T cells and UDT cells (uninfected T cells) obtained in Example 1 were detected by the same method as controls.

[0163] The expression of activation receptor DNAM-1 on DNT cells is shown in Table 3: The introduction of different CAR structures did not affect the high expression of DNAM-1 on the surface of DNT cells, and the expression rate of each group was above 90%. The expression of DNAM-1 in the CAR-T group and the UDT group was above 90%.

[0164] Table 3. Expression of activation receptor DNAM-1 on the surface of DNT cells

[0165] Serial number DNAM-1 expression rate Serial number DNAM-1 expression rate 1 96.16％ 10 --- 2 94.47％ 11 95.71％ 3 92.73％ 12 94.68％ 4 96.32％ 13 95.80％ 5 97.44％ 14 95.14％ 6 96.99％ 15 94.73％ 7 95.30％ 16 95.37％ 8 94.73％ T1 (CAR-T cells) 96.15％ 9 --- UDT 97.57％

[0166] The expression of the activating receptor NKG2D on DNT cells is shown in Table 4: The introduction of different CAR structures did not affect the high expression of NKG2D on the surface of DNT cells. Except for the NKG2D expression rate of the CAR-DNT cell group with structure No. 3 of 84.4%, the NKG2D expression rates of other CAR-DNT cell groups were all above 90%; the NKG2D expression levels in the CAR-T group and the UDT group were significantly lower.

[0167] Table 4. Expression of activating receptor NKG2D on the surface of DNT cells

[0168] Serial number NKG2D positive rate Serial number NKG2D positive rate 1 94.24％ 10 --- 2 91.17％ 11 93.58％ 3 84.40％ 12 94.60％ 4 95.57％ 13 94.50％ 5 97.50％ 14 94.02％ 6 98.45％ 15 91.87％ 7 94.19％ 16 94.38％ 8 94.24％ T1 (CAR-T cells) 50.65％ 9 --- UDT 31.24％

[0169] Example 3, in vitro killing experiment of CAR-DNT cells with different effector-target ratios (Luciferase detection method)

[0170] Among the 16 CAR-DNT cells obtained in Example 1 and cultured for 10 days after transduction, the positive rate of the CAR structure in each group of CAR-DNT cells is different (as shown in Table 2 in Example 2). Therefore, when performing functional experiments such as killing experiments, it is necessary to adjust the CAR-DNT cells of each structure to the same CAR positive rate so that the killing effects between different groups are comparable. According to the CAR positive rate of each structure CAR-DNT cell shown in Table 2, it was divided into two groups for subsequent functional verification: CAR positive rate of 30±5% was divided into 1 group, specifically including structures 1-4, 6-7, 11, 13, and 15; CAR positive rate of 15±5% was divided into another group, specifically including structures 5, 8, 12, 14, and 16; for groups with higher positive rates, DNT cells were added for adjustment. For both groups, DNT cells, T1 cells, and UDT cells were set as control groups.

[0171] Killing experiment: resuspend the target cells THP1-GFP (THP1-GFP refers to THP1 cells expressing luciferase, which can be purchased commercially. THP1 cells are human monocytic leukemia cells used as AML tumor target cell lines. The cell line expresses CLL1 protein on its surface. In the killing experiment, after the THP1-GFP cells are killed, the luciferase in the cells is released) with X-VIVO15 medium (Lonza) + 5% (v / v) heat-inactivated fetal bovine serum (Gibco) to make the target cell density 1E5 / ml, and inoculate the target cell suspension into a 96-well plate with 100ul per well. According to different effector-target ratios (total cells were counted as effector cells, not CAR+ cells), a total of 5 gradient effector-target ratios of 1.25:1, 2.5:1, 5:1, 10:1 and 20:1 were set, and different numbers of effector CAR-DNT cells, DNT cells, T1 cells or UDT cells were added respectively. Four replicates were made for each effector-target ratio, and the final reaction volume of each well was 200μl. Place in an incubator at 37°C + 5% CO2 for culture, take out after 20 hours, and perform short-term killing efficiency detection. During the efficiency test, 10μl Steady-glo luciferin substrate (purchased from Promega) was added to each well. After 3-5 minutes of reaction, the chemiluminescence fluorescence reading was detected using the microplate reader PerinElmer VICTOR X3. The tumor killing level was quantified by detecting the released luciferase content. The killing efficiency of effector cells on target cells was calculated based on the fluorescence value of each well:

[0172] Lysis rate (%) = (1-RUL 效应细胞+靶细胞 / RUL 靶细胞 )×100% (RUL: relative light unit)---Formula 1

[0173] Among them: RUL 效应细胞+靶细胞 Indicates the RUL of the effector cell + target cell sample well, RUL 靶细胞 It represents the RUL of the wells with only target cells but no effector cells; the effector cell background is almost 0 and can be ignored.

[0174] The killing results are as follows Figure 3A and Figure 3B As shown: DNT cells loaded with different CAR structures have a stronger killing advantage than unmodified DNT cells. Compared with other groups, the anti-tumor function of structure 3 is weaker. T1 cells did not kill THP1-GFP cells at 20 hours, while CAR-DNT cells loaded with the same CAR structure had a stronger killing function against THP1-GFP cells.

[0175] Example 4: Multiple rounds of antigen stimulation (Stress-Test) experiment

[0176] For each sample group, 1×10 6 Total CAR-DNT cells (total cells were used as effector cell counts, not CAR + Cell count) was co-cultured with THP1 tumor cells, the effector-target ratio was 2:1, and the reaction system was X-vivo medium containing 5% inactivated serum and a final concentration of 150IU / ml IL-2; one round of culture was performed every two days (the killing time was extended compared to Example 3), and then new tumor cells were added every other day to maintain the effector-target ratio of 2:1, and multiple rounds of antigen stimulation experiments were performed until the killing efficiency of CAR-T cells in different sample groups was significantly different. After each round of multiple rounds of stimulation experiments, cell counting was performed to calculate the number of effector cells and the proliferation multiples, and the effector cell ratio was analyzed by flow cytometry, and the killing effect of each round of experiments was investigated.

[0177] After each round, the proliferation results of effector cells in each sample group are as follows: Figure 4 As shown. The DNT cell group without CAR transfection proliferated very poorly, and there was almost no obvious expansion after 3 rounds of stimulation. The T1 cell group (ie, CAR-T group) expanded normally, and the expansion level could reach 13.21 times after the third round of stimulation. Among the CAR-DNT cell groups, the No. 2 structure CAR-DNT cell group expanded the best, exceeding the expansion level of the T1 cell group; followed by the No. 16 structure CAR-DNT cell group, which was equivalent to the expansion level of the T1 cell group; according to the amplification multiples, they were arranged from high to low, namely No. 2, No. 16, No. 15, No. 8, No. 7, No. 14, No. 3, and No. 1 structure CAR-DNT cell groups.

[0178] After each round, the CAR in the effector cell gate of each sample group + The cell ratio results are as follows Figure 5 As shown in the figure, the CAR positive rates in the CAR-DNT cells with structures 1, 4, 14, and 16 maintained an upward trend with the number of rounds of stimulation, while the CAR positive rates of the other CAR-DNT cell groups fluctuated around the initial value.

[0179] After each round, the killing efficiency was tested in each sample group. The specific steps are as follows: Use a pipette to mix the cell culture in each sample well, and then take out 150ul each. Distribute this 150ul evenly to three wells of an opaque 96-well culture plate, that is, add 50ul of cell culture to each well, and then add 150ul of cell culture medium to the 96-well plate, with a final volume of 200ul. Before using the microplate reader, use an electric gun to add 10ul of Steady-glo chemiluminescent substrate (Promega) to the 96-well plate for 3-5 minutes, then read the fluorescence signal value on the machine, and use formula 1 to calculate the killing efficiency.

[0180] The results of the killing level are as follows Figure 6 As shown in the figure, the killing ability of the CAR-DNT cell groups with structures 2 and 3 was significantly reduced in the 3rd to 5th rounds, and the killing rate was reduced to about 40% in the 5th round; the killing ability of the structure 14 was significantly reduced in the 4th round, and the other groups still maintained more than 70% of the anti-tumor function in the 4th and 5th rounds.

[0181] Comprehensive consideration: Although structures 2, 3, and 14 have good amplification performance, their killing function is weak in multiple rounds of stimulation, so they are excluded. The amplification ability of 1 is relatively poor, so it is excluded. Therefore, CAR structures 15, 16, 7, and 8 are finally selected as the preferred enhanced structures.

[0182] Example 5. GvH reaction and HvG reaction

[0183] To verify the universality of allogeneic cells, we used the in vitro mixed lymphocyte culture method. Different matching cells from different donors were used as effector cells and target cells respectively. Before in vitro mixed lymphocyte culture, CellTrace TM -CFSE cell proliferation kit (commercially available) was used to label cells from one of the donors to distinguish effector cells from target cells. Samples were taken at different time points for Annexin V staining, and apoptosis in the target cell gate was analyzed by flow cytometry.

[0184] 1. Verification of GvH response of DNT cells

[0185] DNT cells were selected as effector cells, PBMC and umbilical cord blood mononuclear cells (CB-MNC) from allogeneic healthy subjects were used as target cells, and AML cell lines HL60 and THP1 were used as positive target cell controls for immune response. The 24-hour killing of PBMC, CB-MNC and positive target cells by DNT cells was observed in vitro.

[0186] The results showed that DNT cells were co-cultured with PBMC and CB-MNC cells from 4 different healthy donors for 24 hours, and no significant killing reaction was detected from 6 hours to 24 hours. However, when DNT cells were co-cultured with two control AML tumor cells, more than 80% of THP1 and HL60 cell apoptosis was observed in 6 hours. This shows that the DNT cells used have normal immune function and have no immune response to allogeneic somatic cells, suggesting that the risk of GvH after allogeneic transfusion of DNT cells is low.

[0187] 2. Verification of whether DNT cells can resist HvG response

[0188] PBMCs from different donors were selected as effector cells, DNTs as target cells, and THP1 as a positive target cell control for immune response. T cells and gene-edited universal T cells (TCR and B2M double-knocked T cells, TCR single-knocked T cells) were also used as target cell controls. The killing ratio was observed in vitro for 24 hours.

[0189] From the results of allogeneic mixed lymphocyte in vitro culture, it can be seen that although T cells express MHC molecules, as the co-culture time increases, the T cells in the two allogeneic PBMCs are slowly activated, and both begin to kill after 144 hours of co-culture. However, no killing reaction of allogeneic PBMCs was detected in the DNT cell group. This shows that DNT cells can escape the immune rejection reaction of allogeneic PBMCs in vitro.

[0190] Sequence Description

[0191] SEQ ID NO: 1 --- CLL1 VH CDR1;

[0192] SYVMH

[0193] SEQ ID NO:2---CLL1 VH CDR2;

[0194] YINPYNDGTKYNEKFKG

[0195] SEQ ID NO:3---CLL1 VH CDR3;

[0196] GYYWYDEGHAMDS

[0197] SEQ ID NO:4---CLL1 VL CDR1;

[0198] KASQDVSTAVA

[0199] SEQ ID NO:5---CLL1 VL CDR2;

[0200] SASYRYT

[0201] SEQ ID NO:6---CLL1 VL CDR3;

[0202] QQHYSTPLT

[0203] SEQ ID NO:7 --- CLL1 VH amino acid sequence;

[0204] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMG

[0205] YINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGY

[0206] YWYDEGHAMDSWGQGTLVTVSS

[0207] SEQ ID NO:8 --- CLL1 VL amino acid sequence;

[0208] DIQMTQSLSSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSAS

[0209] YRYTGVPDRFSGSGSGTTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKL

[0210] EIK

[0211] SEQ ID NO:9---CD8α-derived hinge region amino acid sequence;

[0212] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0213] SEQ ID NO:10---Amino acid sequence of hinge region derived from DAP10;

[0214] QTTPGERSSLPAFYPGTSGSCSGCGSLSLP

[0215] SEQ ID NO:11---amino acid sequence of the transmembrane region derived from CD8α;

[0216] IYIWAPLAGTCGVLLLSLVITLYC

[0217] SEQ ID NO:12---Amino acid sequence of the transmembrane region derived from DAP10;

[0218] LLAGLVAADAVASLLIVGAVF

[0219] SEQ ID NO:13 --- amino acid sequence of the transmembrane region derived from 2B4;

[0220] FLVIIVILSALFLGTLACFCV

[0221] SEQ ID NO:14---amino acid sequence of the transmembrane region derived from DNAM-1;

[0222] GGTVLLLLFVISITTIIVIFL

[0223] SEQ ID NO:15---Amino acid sequence of the transmembrane region derived from HVEM;

[0224] WWFLSGSLVIVIVCSTVGLII

[0225] SEQ ID NO:16---Amino acid sequence of the intracellular region 4-1BB-CD3ζ;

[0226] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ

[0227] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0228] AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0229] SEQ ID NO: 17 --- Amino acid sequence of the intracellular region MyD88ΔTIR-CD40-CD3ζ;

[0230] MAAGGPGAGSAAPVSSTSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEM

[0231] DFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELLGPSIE

[0232] EDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDDPLGKKVAKKPTNK

[0233] APHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQRVKFS

[0234] RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY

[0235] NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0236] SEQ ID NO: 18 --- Amino acid sequence of the intracellular region CD28-MyD88ΔTIR-CD40-CD3ζ;

[0237] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSMAAGGPGAGSAAP

[0238] VSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQA

[0239] DPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEE

[0240] AEKPLQVAAVDSSVPRTAELAGITTLDDDPLGKKVAKKPTNKAPHPKQEPQEINFPD

[0241] DLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQRVKFSRSADAPAYQQGQN

[0242] QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

[0243] EIG

[0244] SEQ ID NO:19---Amino acid sequence of the intracellular region DAP10-DAP12;

[0245] QTTPGERSSLPAFYPGTSGSSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR

[0246] RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRS

[0247] DVYSDLNTQRPYYK

[0248] SEQ ID NO:20---Amino acid sequence of the intracellular region DAP10-4-1BB-CD3ζ;

[0249] LCARPRRSPAQEDGKVYINMPGRGKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF

[0250] PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM

[0251] GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD

[0252] TYDALHMQALPPR

[0253] SEQ ID NO:21 - Amino acid sequence of intracellular region 2B4 - CD3ζ;

[0254] WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAP

[0255] TSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELEN

[0256] FDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0257] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA

[0258] LHMQALPPR

[0259] SEQ ID NO:22 - Amino acid sequence of intracellular region DNAM - 1 - CD3ζ;

[0260] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRR

[0261] PKTRVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0262] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA

[0263] LHMQALPPR

[0264] SEQ ID NO:23---Amino acid sequence of the intracellular region DNAM1-2B4-CD3ζ;

[0265] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDDTREDIYVNYPTFSRR

[0266] PKTRVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQS

[0267] QSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSR

[0268] KELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE

[0269] MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT

[0270] KDTYDALHMQALPPR

[0271] SEQ ID NO:24---Amino acid sequence of the intracellular region of HVEM-CD3ζ;

[0272] CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGR

[0273] SPNHRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR

[0274] RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL

[0275] HMQALPPR

[0276] SEQ ID NO:25---Amino acid sequence of the intracellular region CD28-ΔIL2Rβ-3D3ζ(YXXQ);

[0277] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSNCRNTGPWLKKVL

[0278] KCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQL

[0279] LPLNTDAYLSLQELQGQDPTHLVRVKFSRSADAPAYQQGQNQLYNELNLGRREEY

[0280] DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG

[0281] HDGLYQGLSTATKDTYDAYRHQALPPR

[0282] SEQ ID NO:26---Amino acid sequence of intracellular region CD28-4-1BB-CD3ζ;

[0283] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPF

[0284] MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGR

[0285] REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR

[0286] GKGHDGLYQGLSTATKDTYDALHMQALPPR

[0287] SEQ ID NO:27---Amino acid sequence of intracellular region CD28-CD3ζ;

[0288] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA

[0289] PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN

[0290] ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL

[0291] PPR

[0292] SEQ ID NO:28---Amino acid sequence of intracellular region 4-1BB-CD3ζ-IL2Rγ;

[0293] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ

[0294] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0295] AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRERTMPRIPTL

[0296] KNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGAS

[0297] PCNQHSPYWAPPCYTLKPET

[0298] SEQ ID NO:29---Amino acid sequence of intracellular region CD28-CD3ζ-IL2Rγ;

[0299] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ

[0300] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0301] AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRERTMPRIPTL

[0302] KNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGAS

[0303] PCNQHSPYWAPPCYTLKPET

[0304] SEQ ID NO:30---Amino acid sequence of CLL1 scFv;

[0305] DIQMTQSLSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYT

[0306] GVPDRFSGSGSGTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKLEIKGGGGSG

[0307] GGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQG

[0308] LEWMGYINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARG

[0309] YYWYDEGHAMDSWGQGTLVTVSS

[0310] SEQ ID NO:31—Signal peptide amino acid sequence.

[0311] MALPVTALLLPLALLLHAARP

[0312] SEQ ID NO:32—Amino acid sequence of a cytokine derived from IL2Rγ.

[0313] ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGG

[0314] ALGEGPGASPCNQHSPYWAPPCYTLKPET

[0315] SEQ ID NO:33—Amino acid sequence of a co-stimulatory factor derived from 2B4.

[0316] WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAP

[0317] TSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELEN

[0318] FDVY

[0319] SEQ ID NO:34—Amino acid sequence derived from the intracellular signal transduction region of CD3ζRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP

[0320] QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

[0321] ALPPR

[0322] SEQ ID NO:35 - Amino acid sequence derived from the costimulatory domain of CD28

[0323] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0324] SEQ ID NO:36 - Amino acid sequence derived from the costimulatory domain of 4-1BB

[0325] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Claims

1. An enhanced chimeric antigen receptor targeting CLL1, comprising an extracellular antigen recognition domain targeting CLL1, a hinge region, a transmembrane region and an intracellular domain, wherein the intracellular domain comprises a co-stimulatory domain and an intracellular signaling domain; wherein: The intracellular domain also comprises a cytokine receptor gamma chain amino acid sequence derived from IL2Rγ.

2. The enhanced chimeric antigen receptor targeting CLL1 according to claim 1, wherein the N-terminus of the cytokine receptor γ chain amino acid sequence derived from IL2Rγ is connected to the C-terminus of the intracellular signaling domain.

3. An enhanced chimeric antigen receptor targeting CLL1 according to claim 1 or 2, wherein the amino acid sequence of the cytokine receptor γ chain derived from IL2Rγ comprises the amino acid sequence shown in SEQ ID NO:32; optionally, the amino acid sequence of the cytokine receptor γ chain derived from IL2Rγ is as shown in SEQ ID NO:

32.

4. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1 to 3, wherein the extracellular antigen recognition domain targeting CLL1 comprises a CLL1 antibody heavy chain variable region and a CLL1 antibody light chain variable region, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region as shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region as shown in SEQ ID NO:8; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region as shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region are respectively The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in NO:

8.

5. An enhanced chimeric antigen receptor targeting CLL1 according to claim 4, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

6. An enhanced chimeric antigen receptor targeting CLL1 according to claim 4, wherein the CLL1 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the CLL1 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; optionally, the amino acid sequence of the CLL1 antibody heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the CLL1 antibody light chain variable region is as shown in SEQ ID NO:

8.

7. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1 to 6, wherein the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:

30.

8. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-7, wherein the costimulatory domain is derived from one, two or more costimulatory factors selected from the group consisting of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 2B4, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, DAP12, DNAM-1, HVEM-1, B7-H3, and MyD88; optionally, the costimulatory domain is derived from CD28 or 4-1BB; further optionally, the amino acid sequence of the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:36; further optionally, the amino acid sequence of the costimulatory domain is as shown in SEQ ID NO:35 or SEQ ID NO:

36.

9. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-8, wherein the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:34; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:

34.

10. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1 to 9, wherein the intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29; optionally, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO: 28 or SEQ ID NO:

29.

11. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-10, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:

9.

12. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-11, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 11; further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:

11.

13. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1 to 12, further comprising a signal peptide; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:

31.

14. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1 to 13, wherein the amino acid sequence of the enhanced chimeric antigen receptor is shown as amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:28, or as amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:

29.

15. An enhanced chimeric antigen receptor targeting CLL1, comprising an extracellular antigen recognition domain targeting CLL1, a hinge region, a transmembrane region and an intracellular domain, wherein the intracellular domain comprises a co-stimulatory domain and an intracellular signaling domain; wherein: The costimulatory domain comprises an amino acid sequence derived from 2B4.

16. An enhanced chimeric antigen receptor targeting CLL1 according to claim 15, wherein the amino acid sequence derived from 2B4 comprises the amino acid sequence shown in SEQ ID NO:33; optionally, the amino acid sequence derived from 2B4 is as shown in SEQ ID NO:

33.

17. An enhanced chimeric antigen receptor targeting CLL1 according to claim 15 or 16, wherein the extracellular antigen recognition domain targeting CLL1 comprises a CLL1 antibody heavy chain variable region and a CLL1 antibody light chain variable region, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region as shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region as shown in SEQ ID NO:8; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region as shown in SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 antibody light chain variable region are respectively The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in NO:

8.

18. An enhanced chimeric antigen receptor targeting CLL1 according to claim 17, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the CLL1 antibody are as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the CLL1 antibody are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

19. An enhanced chimeric antigen receptor targeting CLL1 according to claim 17, wherein the CLL1 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the CLL1 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; optionally, the amino acid sequence of the CLL1 antibody heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the CLL1 antibody light chain variable region is as shown in SEQ ID NO:

8.

20. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15 to 19, wherein the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:

30.

21. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15-20, wherein the co-stimulatory factors also include one, two or more co-stimulatory factors derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, DAP12, DNAM-1, HVEM-1, B7-H3, and MyD88.

22. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15-21, wherein the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:34; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:

34.

23. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15-22, wherein the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21; optionally, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:

21.

24. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15-23, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:

9.

25. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15-24, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α or 2B4; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:13; further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:11 or SEQ ID NO:

13.

26. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15 to 25, wherein the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:

21.

27. An enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 15-26, wherein the amino acid sequence of the enhanced chimeric antigen receptor is shown as amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:11-SEQ ID NO:21, or as amino acids connected in sequence by SEQ ID NO:31-SEQ ID NO:30-SEQ ID NO:9-SEQ ID NO:13-SEQ ID NO:

21.

28. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1 to 14 or any one of claims 15 to 27.

29. A vector comprising the isolated nucleic acid molecule of claim 28.

30. An engineered immune effector cell comprising the enhanced chimeric antigen receptor of any one of claims 1-14 or any one of claims 15-27, the isolated nucleic acid molecule of claim 28, or the vector of claim 29, wherein the immune effector cell is derived from a CD3 + CD4 - CD8 - of DNT cells.

31. The engineered immune effector cell according to claim 30, wherein the DNT cell is an autologous DNT cell or an allogeneic DNT cell.

32. A pharmaceutical composition comprising the engineered immune effector cells of claim 30 or 31 and a pharmaceutically acceptable excipient.

33. A pharmaceutical composition according to claim 32, wherein the pharmaceutically acceptable excipient comprises a protective agent.

34. The pharmaceutical composition according to claim 32, wherein the pharmaceutically acceptable excipient comprises a cell freezing solution.

35. The pharmaceutical composition according to any one of claims 32-34, wherein the pharmaceutical composition is an intravenous injection.

36. Use of the enhanced chimeric antigen receptor of any one of claims 1-14 or any one of claims 15-27, the isolated nucleic acid molecule described in claim 28, the vector described in claim 29, or the engineered immune effector cell described in claim 30 or 31 in the preparation of a medicament, wherein the medicament is used to treat a disease or condition associated with the expression of CLL1.

37. The use according to claim 36, wherein the disease or disorder associated with the expression of CLL1 is a hematological tumor.

38. The use according to claim 37, wherein the disease or condition associated with the expression of CLL1 is acute myeloid leukemia.

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