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 signal transduction region and an intracellular signal transduction region; wherein the intracellular domain further comprises an enhanced structure connected to the C end of an intracellular signal transduction region, the enhanced structure comprises any one of the following structures: 1) an IL2RF amino acid sequence which simultaneously comprises an amino acid sequence derived from IL2 and an amino acid sequence which can be combined with the amino acid sequence derived from IL2 and is derived from an IL2 receptor; and 2) an amino acid sequence which simultaneously contains an amino acid sequence derived from IL2R gamma and an amino acid sequence derived from IL15RF, wherein the amino acid sequence derived from IL15 simultaneously contains an amino acid sequence derived from IL15 and an amino acid sequence which can be combined with the amino acid sequence derived from IL15 and is derived from an IL15 receptor.

Description

Technical Field

[0001] This application relates to the biomedical field, specifically to an enhanced chimeric antigen receptor targeting CLL1 and its applications. Background Technology

[0002] Chimeric antigen receptors (CARs) are the core components of CAR cell therapy drugs, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T cell immunotherapy is considered one of the most promising methods for conquering tumors. CAR-T cells utilize genetic modification to express CAR proteins on the surface of T cells. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without antigen presentation, thereby activating and functionally affecting T cells.

[0003] Currently approved CAR-T cell therapy drugs all use autologous T cells transduced and proliferated in vitro before being reinfused into the patient's own CAR-T cells. 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 do meet the requirements, personalized CAR-T cell preparation is time-consuming, complex, and very expensive. Using allogeneic CAR-T cells requires immune matching and also carries risks such as immune rejection, GvH (graft-versus-host disease) and HvG (host-versus-graft) reactions, thus its application has not yet been realized. If a universal CAR-T cell therapy drug could be developed that is suitable for a broad patient population, large-scale industrial production would be possible, resulting in standardized, universal products.

[0004] Most T cells in human peripheral blood express CD4 or CD8 molecules. Approximately 4% of them lack CD4 or CD8 molecules but express CD3 molecules (and therefore still belong to the T cell family). + CD4 - CD8 - Double-negative T cells (DNTs) have been reported to have a broad spectrum of tumor cell killing effects while exhibiting neither GvH nor HvG responses, making them an ideal cell type for universal allogeneic therapy. In CAR cell therapy, DNTs could be used to replace conventional T cells; however, DNTs constitute a relatively low proportion of T cells, and their proliferation and efficacy over a prolonged period depend on certain interleukins, such as IL-2. Currently, combination therapy with IL-2 is not recommended for treating hematological malignancies. Therefore, improving the duration of efficacy of CAR-DNT cells in the absence of IL-2 is of practical significance. Summary of the Invention

[0005] This application provides an enhanced chimeric antigen receptor targeting CLL1 and its application. The inventors introduced five candidate enhanced structures into a chimeric antigen receptor structure targeting CLL1, and obtained an enhanced chimeric antigen receptor structure targeting CLL1 that reduces dependence on exogenous interleukin through validation and screening on DNT cells. Compared to CLL1 CAR-DNT cells with conventional second-generation chimeric antigen receptor structures, CLL1 CAR-DNT cells with the enhanced chimeric antigen receptor structure of this application exhibit better persistence.

[0006] This application provides an enhanced chimeric antigen receptor targeting CLL1, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain targeting CLL1. The intracellular domain includes a co-stimulatory signal transduction region and an intracellular signal transduction region. The intracellular domain further includes an enhancement structure connected to the C-terminus of the intracellular signal transduction region, the enhancement structure comprising any one of the following structures:

[0007] 1) IL2RF amino acid sequence, wherein the IL2RF amino acid sequence is an amino acid sequence that simultaneously contains an amino acid sequence derived from IL2 and an amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2.

[0008] 2) An amino acid sequence that simultaneously contains an amino acid sequence derived from IL2Rγ and an amino acid sequence derived from IL15RF, wherein the IL15RF amino acid sequence is an amino acid sequence that simultaneously contains an amino acid sequence derived from IL15 and an amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15.

[0009] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, in the enhanced structure, a 2A peptide and a signal peptide are also attached to the N-terminus of the IL2RF amino acid sequence, and the 2A peptide, the signal peptide, and the IL2RF amino acid sequence are connected in sequence from the N-terminus to the C-terminus.

[0010] And / or, in the enhanced structure, the N-terminus of the IL15RF amino acid sequence is also connected to a 2A peptide and a signal peptide, which are linked to the IL15RF amino acid sequence in the order from the N-terminus to the C-terminus.

[0011] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the signal peptide linked to the IL2RF amino acid sequence is a first signal peptide as shown in SEQ ID NO:31, and / or the signal peptide linked to the IL15RF amino acid sequence is a second signal peptide as shown in SEQ ID NO:32.

[0012] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the 2A peptide is selected from one of T2A peptide, P2A peptide, E2A peptide, and F2A; optionally, the 2A peptide is a T2A peptide; further optionally, the amino acid sequence of the T2A peptide is as shown in SEQ ID NO:28.

[0013] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the 2A peptide linked to IL2RF is directly attached to the C-terminus of the intracellular signal transduction region.

[0014] And / or, the 2A peptide linked to IL15RF is directly attached to the C-terminus of an amino acid derived from IL2Rγ, and the N-terminus of an amino acid derived from IL2Rγ is directly attached to the C-terminus of the intracellular signal transduction region.

[0015] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, in the IL2RF amino acid sequence, the amino acid sequence derived from IL2 and the amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 are linked together in order from the N-terminus to the C-terminus by a linker sequence.

[0016] And / or, in the IL15RF amino acid sequence, amino acid sequences derived from IL15 and amino acid sequences derived from the IL15 receptor that can bind to amino acid sequences derived from IL15 are linked together by a linker sequence in the direction from the N-terminus to the C-terminus.

[0017] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 includes the amino acid sequence of the IL2 receptor α subunit; optionally, the amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 is the full-length amino acid sequence of the IL2 receptor α subunit; further optionally, the full-length amino acid sequence of the IL2 receptor α subunit is as shown in SEQ ID NO:24.

[0018] And / or, the amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15 includes the amino acid sequence of the IL15 receptor α subunit; optionally, the amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15 is the full-length amino acid sequence of the IL15 receptor α subunit; further optionally, the full-length amino acid sequence of the IL15 receptor α subunit is as shown in SEQ ID NO:26.

[0019] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the protein or protein fragment expressed by the amino acid sequence derived from IL2 can bind to the IL2 receptor on the surface of T cells and stimulate T cell proliferation.

[0020] In some embodiments, the amino acid sequence derived from IL2 in the aforementioned enhanced chimeric antigen receptor targeting CLL1 is the full-length amino acid sequence of IL2, as shown in SEQ ID NO:25; and / or, the amino acid sequence derived from IL15 is the full-length amino acid sequence of IL15, as shown in SEQ ID NO:27.

[0021] In some embodiments, the enhanced chimeric antigen receptor targeting CLL1 described above has an amino acid sequence derived from IL2Rγ that is the intracellular amino acid sequence of IL2Rγ, as shown in SEQ ID NO:23.

[0022] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the IL2RF is as shown in SEQ ID NO:13; and / or, the amino acid sequence of the IL15RF is as shown in SEQ ID NO:14.

[0023] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the enhanced structure is selected from any one of the following two structures:

[0024] 1) SEQ ID NO:28-SEQ ID NO:31-SEQ ID NO:13 are directly connected in the direction from N end to C end;

[0025] 2) SEQ ID NO:23-SEQ ID NO:28-SEQ ID NO:32-SEQ ID NO:14 are directly connected in the direction from N end to C end.

[0026] In some embodiments of the aforementioned 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. 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:3, 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:4. 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:3, 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 antibody heavy chain variable region shown in SEQ ID NO:4. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the antibody light chain shown in NO:4.

[0027] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody heavy chain respectively include the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody light chain respectively include the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody heavy chain are as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody light chain are as shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0028] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the variable region of the CLL1 antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:3, and the variable region of the CLL1 antibody light chain contains the amino acid sequence shown in SEQ ID NO:4; optionally, the amino acid sequence of the variable region of the CLL1 antibody heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the variable region of the CLL1 antibody light chain is as shown in SEQ ID NO:4.

[0029] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:2.

[0030] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the co-stimulatory signal transduction region is derived from one, two, or more of the following co-stimulatory factors: 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 co-stimulatory signal transduction region is derived from CD28 or 4-1BB. Further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:21. Even more optionally, the amino acid sequence of the co-stimulatory signal transduction region is as shown in SEQ ID NO:21.

[0031] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the intracellular signal transduction 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 signal transduction region is derived from CD3ζ; further optionally, the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:22; even more optionally, the intracellular signal transduction region is as shown in SEQ ID NO:22.

[0032] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1 described above, the intracellular domain comprises an amino acid sequence as shown in SEQ ID NO:16 or SEQ ID NO:20; alternatively, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:16 or SEQ ID NO:20.

[0033] In some embodiments of the aforementioned 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:11; even more optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:11.

[0034] As shown in SEQ ID NO:11.

[0035] In some embodiments of the aforementioned 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:12; even more optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:12.

[0036] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1 described above, a guide peptide is further attached to the N-terminus of the extracellular antigen recognition domain; optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:1.

[0037] In some embodiments, the enhanced chimeric antigen receptor targeting CLL1 described above has an amino acid sequence as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:16, arranged in a direct chain of amino acids from the N-terminus to the C-terminus, or as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:20, arranged in a direct chain of amino acids from the N-terminus to the C-terminus.

[0038] This application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors targeting CLL1.

[0039] This application also provides a vector containing the isolated nucleic acid molecules described above.

[0040] This application also provides an engineered immune effector cell comprising the aforementioned enhanced chimeric antigen receptor, the aforementioned isolated nucleic acid molecule, or the aforementioned carrier, wherein the immune effector cell is derived from CD3. + CD4 - CD8 - DNT cells.

[0041] In some embodiments, the engineered immune effector cells described above are autologous DNT cells or allogeneic DNT cells.

[0042] This application also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients.

[0043] In some embodiments of the above-described pharmaceutical composition, pharmaceutically acceptable excipients include protective agents.

[0044] In some embodiments of the above-described pharmaceutical composition, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0045] In some embodiments, the pharmaceutical composition described above is an intravenous injection preparation.

[0046] This application also provides the use of the above-mentioned enhanced chimeric antigen receptor, isolated nucleic acid molecule, carrier or engineered immune effector cell in the preparation of a drug for treating diseases or conditions related to CLL1 expression.

[0047] In some implementations of the above application, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0048] In some implementations of the above application, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0049] This application also provides a method for treating a disease or condition associated with CLL1 expression, comprising the steps of: administering an effective amount of the above-described engineered immune effector cells or pharmaceutical composition to a subject who requires treatment for a disease or condition associated with CLL1 expression.

[0050] In some implementations of the above method, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0051] In some embodiments of the above method, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0052] In some embodiments of the above method, the administration is performed via intravenous injection.

[0053] In some embodiments of the above method, the administration is carried out by administering an effective amount of engineered immune effector cells or a pharmaceutical composition to the subject via a single injection.

[0054] In some embodiments of the above method, the effective amount of engineered immune effector cells or drug composition is 1 × 10⁻⁶. 5 Up to 1×10 7 A dose of cells / kg.

[0055] This application also provides a medicament comprising the above-described engineered immune effector cells or pharmaceutical composition for treating diseases or conditions associated with CLL1 expression.

[0056] In some embodiments of the above-mentioned drug, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0057] In some embodiments of the above-mentioned drug, the disease or condition associated with CLL1 expression is acute myeloid leukemia. Attached Figure Description

[0058] Figure 1 The locations of each component in the six candidate CAR structures in Example 1 are shown, where SP represents the guide peptide, Hinge represents the hinge region, and TM represents the transmembrane region.

[0059] Figure 2 The in vitro expansion of CAR-DNT cells with different structures in Example 1 is shown.

[0060] Figure 3 The results of short-term killing of CAR-DNT cells in Example 3 are shown at multiple effector-to-target ratios; from left to right are the target cell lysis rates at the following three effector-to-target ratios: 5:1, 10:1 and 20:1.

[0061] Figure 4 The expansion of effector cells in the multi-round stimulation experiment in Example 4 is shown.

[0062] Figure 5 The experiment in Example 4 demonstrates the killing effect of effector cells on target cells in a multi-round stimulation experiment. Each group consists of round-1, round-2, round-3, round-4, and round-5 from left to right. Detailed Implementation

[0063] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0064] The following further describes this application: In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0065] In this application, the term "Chimeric Antigen Receptor" (CAR) is a 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 one of the most promising methods for conquering tumors. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without relying on antigen presentation, thereby activating and functionally affecting T cells.

[0066] In this application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (ARD). CAR cell therapy products (such as CAR-T cells) rely on extracellular antigen recognition domains to specifically recognize and / or bind to target antigens expressed by tumor cells. To date, antigen recognition domains are derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). To date, the most common source is scFv antibodies.

[0067] In this application, the term "specific recognition and / or binding" refers to the recognition and / or binding between the CAR extracellular antigen recognition domain and a specific target, or the recognition and / or binding between the CAR extracellular antigen recognition domain and a specific detection antibody with greater affinity, stronger affinity, easier binding, and / or longer duration than the binding of the CAR to other targets or detection antibodies.

[0068] In this application, the term "antibody" has its conventional meaning in the art and is used in the broadest sense. In the biosciences, analysis of the amino acid sequences of different antibody heavy and light chains reveals that the amino acid sequences near the N-terminus of both heavy and light chains vary considerably, while the amino acid sequences in other parts remain relatively constant. Therefore, the regions in the antibody light and heavy chains with significant amino acid sequence variation near the N-terminus are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. Within the antibody variable regions, a small subset of amino acid residues exhibits particularly strong variation; these regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions (HVRs). There are three hypervariable regions in the V regions of both the L and H chains. Because these regions can form precise complementarity with the antigenic determinants in their spatial structure, they are also called complementarity determining regions (CDRs). In antibodies, common CDR (Cellular Recognition Derivative) rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When using websites that apply these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of protection of this application covers combinations of CDR sequences obtained through analysis using different rules. The six CDR regions of an antibody collectively determine its recognition ability and specificity against the corresponding antigen. Those skilled in the art should understand that when this application defines the amino acid sequences of the six CDR regions, the antibody's recognition ability and specificity against the corresponding antigen are predictable.

[0069] In this application, the term "antigen binding site" has its conventional meaning in the art, referring to a key site on an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0070] In this application, the term "scFv" has the conventional meaning in the art and refers to a single-chain variable region, which is an antibody composed of antibody heavy chain variable regions and light chain variable regions linked by a short peptide (linker, connecting sequence).

[0071] In this application, the term "linking sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure / region of the antibody or chimeric antigen receptor of the present invention. Linking sequences may consist of different amino acid residues (such as glycine and serine) to allow adjacent protein domains to move freely relative to each other. Longer linking sequences may be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0072] In this application, the term "hinge region" refers to the connecting segment that acts 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 motion.

[0073] 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 some extent, but they do not directly participate in signal transduction; however, they can enhance downstream signal transduction through interactions.

[0074] In this application, the terms "intracellular domain," "intrinsic domain," and "intracellular region" have the same meaning, including a co-stimulatory signal transduction region, an intracellular signal transduction region, and an enhancement structure connected to the C-terminus of the intracellular signal transduction region. The term "intracellular signal transduction region" is responsible for activating at least one normal effector function of CAR-expressing immune effector cells. "Co-stimulatory signal transduction region" is used because, in addition to antigen-specific signal stimulation, many immune effector cells require co-stimulatory factors to promote cell proliferation, differentiation, survival, and activation of effector functions. In this application, the design of the enhancement structure prolongs the proliferation of DNT cells and CAR-DNT cells while avoiding the side effects caused by excessive interleukin release.

[0075] In this application, the terms "IL2" and "IL-2" have the same meaning as interleukin-2 (IL2); the terms "IL15" and "IL-15" have the same meaning as interleukin-15 (IL15).

[0076] In this application, the terms “IL2RF”, “IL-2RF”, or “IL2-RF” have the same meaning, all referring to an amino acid sequence that simultaneously contains (1) an amino acid sequence derived from IL2 and (2) an amino acid sequence derived from an IL2 receptor that can bind to the amino acid sequence derived from IL2.

[0077] In this application, the terms “IL15RF”, “IL-15RF” or “IL15-RF” have the same meaning, all referring to an amino acid sequence that simultaneously contains (1) an amino acid sequence derived from IL15 and (2) an amino acid sequence derived from an IL15 receptor that can bind to the amino acid sequence derived from IL15.

[0078] In this application, the terms “IL2Rγ”, “IL-2Rγ”, and “IL2-Rγ” have the same meaning as the γ subunit of the interleukin-2 receptor complex, which has also been identified as part of several cytokine receptor complexes including IL-4, IL-7, IL-9, IL-15, and IL-21. It participates 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.

[0079] In this application, the term "2A peptide" refers to a short peptide sequence that enables a transcript to be translated.

[0080] Multiple proteins are produced because no peptide bonds are formed between the C-terminus and N-terminus of the 2A peptide, allowing for protein self-cleavage. That is, by inserting the 2A peptide sequence between two genes, the protein can be automatically cleaved during translation, thus enabling the independent expression of multiple proteins. Currently, there are four commonly used 2A peptides: P2A, T2A, E2A, and F2A, which originate from four different viruses.

[0081] In this application, the term "signal peptide" refers to a recombinant protein synthesized intracellularly that is exported to the extracellular space. Because multiple protein molecules are involved in this application, there are multiple instances of signal peptides. For ease of distinction, the short peptide preceding the extracellular antigen recognition domain (such as the scFv sequence) is referred to as the "guide peptide," while other signal peptides are still referred to as signal peptides. Commonly used signal peptides include the human CD8α signal peptide or the human GM-CSF receptor α signal peptide.

[0082] In this application, the terms "sequentially linked," "sequentially connected," and "-" have similar meanings, all indicating the connection of different parts in a specific order, without limiting the connection to direct or indirect linking. In the description of amino acid sequences, unless specifically indicated, the direction of connection is from the N-terminus to the C-terminus, as is commonly used in the industry; in the description of nucleotide sequences, unless specifically indicated, the direction of connection is from the 5' end...

[0083] The direction from end 3' to end 4'. Similarly, the expressions "connected" and "linked" do not specify whether they are directly connected or indirectly connected.

[0084] In this application, the term "separated" generally refers to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" substance, it may be due to a change in its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated substance. The term "separated" does not exclude substances obtained artificially from their natural state and then synthesized, nor does it exclude the presence of other impurities that do not affect the substance's activity.

[0085] In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide of any length, which may be isolated from its natural environment or an analogue synthesized artificially.

[0086] The term "vector" generally refers to a nucleic acid delivery vehicle that inserts a polynucleotide encoding a protein into itself, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cell. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances. The term "transposon" refers to a discontinuous segment of DNA capable of migrating between chromosomal loci and carrying genetic information, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects. In some embodiments, electroporation can also be used to transfect mRNA into T cells.

[0087] In this application, the terms "comprising" or "including" generally mean including the explicitly specified features, but do not exclude other elements.

[0088] In this application, the term "about" generally refers to a range of fluctuation acceptable to a person skilled in the art above or below a specified value, such as a variation within ±0.5% to 10%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, or more above or below the specified value.

[0089] Variations within the range of 8.5%, 9%, 9.5%, or 10%.

[0090] Enhanced chimeric antigen receptor targeting CLL1

[0091] This application provides an enhanced chimeric antigen receptor targeting CLL1, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain targeting CLL1. The intracellular domain includes a co-stimulatory signal transduction region and an intracellular signal transduction region. The intracellular domain further includes an enhancement structure connected to the C-terminus of the intracellular signal transduction region, the enhancement structure comprising any one of the following structures:

[0092] 1) IL2RF amino acid sequence, wherein the IL2RF amino acid sequence is an amino acid sequence that simultaneously contains an amino acid sequence derived from IL2 and an amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2.

[0093] 2) An amino acid sequence that simultaneously contains an amino acid sequence derived from IL2Rγ and an amino acid sequence derived from IL15RF, wherein the IL15RF amino acid sequence is an amino acid sequence that simultaneously contains an amino acid sequence derived from IL15 and an amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15.

[0094] In this application, the inventors explored five candidate enhancement structures, namely:

[0095] 1. Introducing the IL-2RF domain into the CAR structure: DNT cells themselves do not secrete IL-2, requiring additional IL-2 to maintain their proliferation. However, IL-2 is absent when DNT cells function in vivo, and the interleukin receptors on DNT cells, lacking ligand stimulation, induce cell death. To maintain the persistence of DNTs, we introduced the IL-2RF domain into the CAR structure. This structure, while overexpressing IL-2 or its fragments, allows the overexpressed IL-2 or its fragments to bind to IL-2 receptors (e.g., IL-2Rα). This structural design allows DNT cells to receive IL-2 signaling pathway stimulation, maintaining cell proliferation, while avoiding the side effects caused by excessive IL-2 release.

[0096] 2. Introducing IL-15RF into the CAR structure: IL-15 is added to the culture medium when culturing DNT cells; therefore, the inventors in this application also attempted to introduce the IL-15RF structure into the CAR structure. IL-15 has a very short half-life and exhibits transient toxicity at high plasma concentrations. This structure, while overexpressing IL-15 or its fragments, allows the overexpressed IL-15 or its fragments to bind to IL-15 receptors (such as IL-15Rα), reducing the possibility of adverse effects.

[0097] 3. Introducing IL2Rγ into the CAR structure: In the inventors’ previous research, it was found that introducing IL2Rγ into the CAR structure could improve the proliferation of CAR-DNT cells. In this application, the inventors also tested the sustained proliferation and sustained killing performance of this structure in a killing environment without the addition of interleukin.

[0098] 4. Simultaneous introduction of IL2Rγ and IL2RF into the CAR structure: Based on the above structures 1 and 3, we attempted to simultaneously introduce IL2Rγ and IL2RF into the CAR structure to determine the optimal enhancement structure.

[0099] 5. Simultaneously introduce IL2Rγ and IL15RF into the CAR structure; Based on the above structures 2 and 3, attempt to simultaneously introduce IL2Rγ and IL15RF into the CAR structure to determine the optimal enhancement structure.

[0100] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, in the enhanced structure, a 2A peptide and a signal peptide are also attached to the N-terminus of the IL2RF amino acid sequence, and the 2A peptide, the signal peptide, and the IL2RF amino acid sequence are connected in sequence from the N-terminus to the C-terminus.

[0101] And / or, in the enhanced structure, the N-terminus of the IL15RF amino acid sequence is also linked to a 2A peptide and a signal peptide, with the 2A peptide, signal peptide, and IL15RF amino acid sequence linked sequentially from the N-terminus to the C-terminus. Specifically, the 2A peptide enables the IL2RF and / or IL15R with the signal peptide to self-cleave during translation, while the signal peptide guides the export of intracellularly synthesized recombinant proteins to the extracellular space.

[0102] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1 described above, the signal peptide linked to the IL2RF amino acid sequence is a first signal peptide as shown in SEQ ID NO:31, and / or the signal peptide linked to the IL15RF amino acid sequence is a second signal peptide as shown in SEQ ID NO:32.

[0103] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1 described above, the 2A peptide is selected from one of T2A peptide, P2A peptide, E2A peptide, and F2A; optionally, the 2A peptide is a T2A peptide; further optionally, the amino acid sequence of the T2A peptide is as shown in SEQ ID NO:28.

[0104] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the 2A peptide linked to IL2RF is directly attached to the C-terminus of the intracellular signal transduction region.

[0105] And / or, the 2A peptide linked to IL15RF is directly attached to the C-terminus of an amino acid derived from IL2Rγ, and the N-terminus of an amino acid derived from IL2Rγ is directly attached to the C-terminus of the intracellular signal transduction region.

[0106] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1 described above, in the IL2RF amino acid sequence, the amino acid sequence derived from IL2 and the amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 are linked together in order from the N-terminus to the C-terminus by a linking sequence.

[0107] And / or, in the IL15RF amino acid sequence, amino acid sequences derived from IL15 and amino acid sequences derived from the IL15 receptor that can bind to amino acid sequences derived from IL15 are linked together by a linker sequence in the direction from the N-terminus to the C-terminus.

[0108] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 includes the amino acid sequence of the IL2 receptor α subunit; optionally, the amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 is the full-length amino acid sequence of the IL2 receptor α subunit; further optionally, the full-length amino acid sequence of the IL2 receptor α subunit is as shown in SEQ ID NO:24.

[0109] And / or, the amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15 includes the amino acid sequence of the IL15 receptor α subunit; optionally, the amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15 is the full-length amino acid sequence of the IL15 receptor α subunit; further optionally, the full-length amino acid sequence of the IL15 receptor α subunit is as shown in SEQ ID NO:26.

[0110] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the protein or protein fragment expressed by the amino acid sequence derived from IL2 can bind to the IL2 receptor on the surface of T cells and stimulate T cell proliferation.

[0111] In some embodiments, the amino acid sequence derived from IL2 in the aforementioned enhanced chimeric antigen receptor targeting CLL1 is the full-length amino acid sequence of IL2, as shown in SEQ ID NO:25; and / or, the amino acid sequence derived from IL15 is the full-length amino acid sequence of IL15, as shown in SEQ ID NO:27.

[0112] In some embodiments, the enhanced chimeric antigen receptor targeting CLL1 described above has an amino acid sequence derived from IL2Rγ that is the intracellular amino acid sequence of IL2Rγ, as shown in SEQ ID NO:23.

[0113] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequence of the IL2RF is as shown in SEQ ID NO:13; and / or, the amino acid sequence of the IL15RF is as shown in SEQ ID NO:14.

[0114] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the enhanced structure is selected from either of the following two structures:

[0115] 1) SEQ ID NO:28-SEQ ID NO:31-SEQ ID NO:13 are directly connected in the direction from N end to C end;

[0116] 2) SEQ ID NO:23-SEQ ID NO:28-SEQ ID NO:32-SEQ ID NO:14 are directly connected in the direction from N end to C end.

[0117] In some embodiments of the aforementioned 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. 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:3, 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:4. 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:3, 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 antibody heavy chain variable region shown in SEQ ID NO:4. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the antibody light chain shown in NO:4.

[0118] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody heavy chain respectively contain the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody light chain respectively contain the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody heavy chain are as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CLL1 antibody light chain are as shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0119] In some embodiments of the above-mentioned enhanced chimeric antigen receptor targeting CLL1, the variable region of the CLL1 antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:3, and the variable region of the CLL1 antibody light chain contains the amino acid sequence shown in SEQ ID NO:4; optionally, the amino acid sequence of the variable region of the CLL1 antibody heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the variable region of the CLL1 antibody light chain is as shown in SEQ ID NO:4.

[0120] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:2.

[0121] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the co-stimulatory signal transduction region is derived from one, two, or more of the following co-stimulatory factors: 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 co-stimulatory signal transduction region is derived from CD28 or 4-1BB. Further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:21. Even more optionally, the amino acid sequence of the co-stimulatory signal transduction region is as shown in SEQ ID NO:21.

[0122] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CLL1, the intracellular signal transduction 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 signal transduction region is derived from CD3ζ; further optionally, the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:22; even more optionally, the intracellular signal transduction region is as shown in SEQ ID NO:22.

[0123] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1 described above, the intracellular domain comprises an amino acid sequence as shown in SEQ ID NO:16 or SEQ ID NO:20; alternatively, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:16 or SEQ ID NO:20.

[0124] In some embodiments of the aforementioned 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:11; even more optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:11.

[0125] In some embodiments of the aforementioned 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:12; even more optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:12.

[0126] In some embodiments of the enhanced chimeric antigen receptor targeting CLL1 described above, a guide peptide is also attached to the N-terminus of the extracellular antigen recognition domain; optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:1.

[0127] In some embodiments, the enhanced chimeric antigen receptor targeting CLL1 described above has an amino acid sequence as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:16, with amino acids directly linked from the N-terminus to the C-terminus, or as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:20, with amino acids directly linked from the N-terminus to the C-terminus.

[0128] Isolated nucleic acid molecules, vectors, immune effector cells, and drug compositions

[0129] This application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors targeting CLL1.

[0130] This application also provides a vector containing the isolated nucleic acid molecules described above.

[0131] This application also provides an engineered immune effector cell comprising the aforementioned enhanced chimeric antigen receptor, the aforementioned isolated nucleic acid molecule, or the aforementioned carrier, wherein the immune effector cell is derived from CD3. + CD4 - CD8 - DNT cells.

[0132] In some embodiments, the engineered immune effector cells described above are autologous DNT cells or allogeneic DNT cells.

[0133] This application also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients.

[0134] In some embodiments of the above pharmaceutical compositions, pharmaceutically acceptable excipients include protective agents.

[0135] In some embodiments of the above pharmaceutical compositions, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0136] In some embodiments, the pharmaceutical composition described above is an intravenous injection.

[0137] Applications, treatment methods and drugs

[0138] This application also provides the use of the above-mentioned enhanced chimeric antigen receptor, isolated nucleic acid molecule, carrier or engineered immune effector cell in the preparation of a drug for treating diseases or conditions related to CLL1 expression.

[0139] In some embodiments of the above application, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0140] In some embodiments of the above application, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0141] This application also provides a method for treating a disease or condition associated with CLL1 expression, comprising the steps of: administering an effective amount of the above-described engineered immune effector cells or pharmaceutical composition to a subject who requires treatment for a disease or condition associated with CLL1 expression.

[0142] In some embodiments of the above method, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0143] In some embodiments of the above method, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0144] In some embodiments of the above method, the administration is performed via intravenous injection.

[0145] In some embodiments of the above method, the administration is carried out by administering an effective amount of engineered immune effector cells or a pharmaceutical composition to the subject via a single injection.

[0146] In some embodiments of the above method, the effective amount of engineered immune effector cells or drug composition is 1 × 10⁻⁶. 5 Up to 1×10 7 A dose of cells / kg.

[0147] This application also provides a medicament comprising the above-described engineered immune effector cells or pharmaceutical composition for treating diseases or conditions associated with CLL1 expression.

[0148] In some embodiments of the above-mentioned drugs, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0149] In some embodiments of the above-mentioned drugs, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0150] Not intended to be limited by any theory, the embodiments described below are merely for illustrating the chimeric antigen receptor, immune effector cells, preparation methods, and uses of this application, and are not intended to limit the scope of the invention. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press.

[0151] Example 1: Preparation of CAR-DNT cells

[0152] 1. CAR structure design

[0153] The inventors constructed five enhanced CAR structures as candidates and one second-generation CAR structure as a control. Through functional validation on DNT cells, they selected an enhanced CAR structure suitable for targeting CLL1 on DNT cells. The positions of the components in each candidate CAR structure are shown below. Figure 1 As shown: The amino acid sequence of the guide peptide is as follows

[0154] As shown in SEQ ID NO:1; the scFv targeting CLL1 serves as an extracellular antigen recognition domain, and its amino acid sequence is as follows:

[0155] As shown in SEQ ID NO:2 (wherein: in the scFv targeting CLL1, the amino acid sequence of CLL1 VH is shown in SEQ ID NO:3, the amino acid sequence of CLL1 VL is shown in SEQ ID NO:4, CDR1, CDR2, and CDR3 of CLL1 VH are shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and CDR1, CDR2, and CDR3 of CLL1 VL are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively); the amino acid sources and specific sequences of the hinge region, transmembrane region, and intracellular region in each candidate CAR structure are shown in Table 1.

[0156] Table 1. Amino acid sequences of the hinge region, transmembrane region, and intracellular region in the CAR structure.

[0157]

[0158] Table 1 shows the intracellular regions, focusing only on the origins of the co-stimulatory signal transduction region and the intracellular signal transduction region, as well as the relative positions of the parts derived from IL2Rγ, IL2RF, and IL15RF in the enhancement region structure. The 2A peptide, signal peptide, and linker sequences between the parts are not shown, but their corresponding amino acid sequences (i.e., SEQ ID NO:16-20) all contain the complete intracellular sequences.

[0159] 2. Construction of lentiviral vectors

[0160] Based on the sequences in Table 1, nucleotide sequences encoding the six CAR structures were artificially synthesized and constructed into modified empty lentiviral vectors (manufacturer: SBI, catalog number: CD500-CD800, such as the conventional resistance modification described in Example 1 of WO2021 / 121227) to obtain six CAR vectors. Subsequently, the CAR vectors and three packaging plasmids were transfected into 293T cells. After collection and purification, functional lentiviral vectors were obtained. The three packaging plasmids were 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).

[0161] 3. Preparation of CAR-DNT cells

[0162] 1) Sorting CD4 - CD8 - cell

[0163] Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy donors were purchased. After PBMC thawing, CD4+ was isolated using CD4 / CD8 mixed magnetic beads (purchased from Miltenyi Biotechnology). - CD8 - Cells used for the preparation of CAR-DNT cells. This step does not target CD3. + Cells were sorted individually (CD3) + It is a sorting marker for T cells, because CD3 is important for subsequent culture conditions. - The cells are almost unable to proliferate; that is, only CD4 cells proliferate. - CD8 - CD3 + DNT cells.

[0164] 2) DNT cell activation treatment

[0165] The CD3 monoclonal antibody OKT3 (purchased from Invitrogen) was used as the activating antibody to coat 24-well plates overnight, after which residual OKT3 antibody was washed away; the CD4 obtained above were then... - CD8 - The cells were treated with 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 seeded into coated 24-well plates for culture for 1 day.

[0166] 3) Prepare CAR-DNT cells by transducing sorted and activated DNT cells with lentiviruses.

[0167] After activation, the six lentiviruses obtained in the above steps were added to different wells of a 24-well plate for lentiviral transduction, with an MOI of approximately 2. Subsequently, recombinant human interleukin-2 at a final concentration of 250 IU / ml, recombinant human interleukin-15 at a final concentration of 10 ng / ml, and recombinant human interleukin-7 at a final concentration of 2 ng / ml were added. The cells were cultured in the culture medium for 2 days; then transferred to a suitable culture vessel, maintaining a culture density of 5E5–2E6 / ml, and cultured for another 3 days under the same conditions; CAR-DNT cells were harvested from day 7 to day 10. Recombinant human interleukin-7 was no longer added to the culture medium, while other added components remained unchanged. Cell counting was performed every other day, and fresh culture medium was added to maintain the cell density at 5E5–2E6 / ml.

[0168] The total cell expansion fold was measured on days 0, 3, 5, 7, and 9. The expansion level of total DNT cells in each group was as follows: Figure 2 As shown, there was no significant difference in the fold increase of total DNT cells among the groups, which was almost equivalent to that of untransduced DNT cells. By day 9 of culture, the fold increase of total DNT cells in each group reached nearly 100-fold.

[0169] Example 2: In vitro cell killing assay of CAR-DNT cells with different effector-to-target ratios (Luciferase assay)

[0170] Six types of CAR-DNT cells obtained in Example 1 and cultured for 9 days after transduction were used for killing experiments, with untransduced DNT cells as a negative control. The killing experiment method is as follows: Target cells THP1-luc (THP1-luc refers to THP1 cells expressing luciferase, which can be purchased commercially; THP1 cells are human monocytic leukemia cells used as target cell lines for AML tumors; this cell line expresses CLL1 protein on its surface; in the killing experiment, after THP1-luc cells are killed, the luciferase in these cells is degraded and inactivated) were resuspended in X-VIVO15 medium (Lonza) + 5% (v / v) heat-inactivated fetal bovine serum (Gibco) to achieve a target cell density of 1E5 / ml. The target cell suspension was then seeded into 96-well plates at 100 μl per well. Three gradients of effector-target ratios (5:1, 10:1, and 20:1) were set up, with different numbers of effector CAR-DNT cells added for each ratio. Each ratio was performed in triplicate, with a final reaction volume of 200 μl per well. Cells were incubated at 37°C with 5% CO2 for 20 hours and then removed for short-term tumor killing efficiency assays. For efficiency assays, 10 μl of Steady-Glo luciferin substrate (Promega) was added to each well. After 3-5 minutes of reaction, the chemiluminescence fluorescence value was measured using a PerkinElmer VICTOR X3 microplate reader. The tumor killing level was quantified by detecting the amount of luciferase released. The killing efficiency of effector cells against target cells was calculated based on the fluorescence value of each well.

[0171] Pyrolysis rate (%) = (1-RUL) 效应细胞+靶细胞 / RUL 靶细胞 )×100% (RUL: relative light unit)---Formula 1

[0172] Among them: RUL 效应细胞+靶细胞 RUL represents the sample well of effector cells + target cells. 靶细胞This indicates that the RUL is only the target cell pore without effector cells; the effector cell background is almost 0 and therefore negligible.

[0173] The result of the killing is as follows Figure 3 As shown, DNT cells loaded with different CAR structures exhibited a greater killing advantage compared to untransduced DNT cells. However, no significant differences in short-term killing effects were observed among the groups loaded with different CAR structures.

[0174] Example 3: Multiple rounds of antigen stimulation (Stress-Test) experiment

[0175] For the six types of CAR-DNT cells obtained in Example 1 and cultured for 9 days after transduction, 1×10 6 Total CAR-DNT cells (using total DNT cells as effector cells, not CAR cells) + Cells were co-cultured with THP1-luc tumor cells at an effector-to-target ratio of 2:1 in X-VIVO15 medium containing 5% inactivated serum (without additional IL2). Tumor cell stimulation was performed every two days (extending the killing time compared to Example 3), meaning new tumor cells were added every two days to maintain a 2:1 effector-to-target ratio. Multiple rounds of antigen stimulation were conducted until significant differences in the killing efficiency or expansion of CAR-DNT cells were observed among different sample groups. After each round of stimulation, cell counting was performed to calculate the number and fold increase of effector cells, and flow cytometry was used to analyze the effector cell ratio and assess the killing effect of each round of experiments. The killing effect was detected as follows: Mix all cell cultures (the cell cultures here refer to the products after co-culturing effector cells and target cells), add 50 μl of each cell culture to an opaque 96-well plate, and then add 150 μl of cell culture medium to the 96-well plate, making a final volume of 200 μl. Add 10 μl of Steady-Glo fluorescein substrate (purchased from Promega) to each well. After 3-5 minutes, read the fluorescence signal value and calculate the killing efficiency using Formula 1 in Example 2.

[0176] The proliferation in each round of the experiment is as follows: After each round, the cumulative proliferation results of effector cells in each sample group are as follows: Figure 4 As shown, the DNT cell group without CAR transduction structure showed almost no proliferation after stimulation by tumor cells, while cells containing intracellular region 2 (amino acid sequence as shown in SEQ ID NO:16) and intracellular region 6 (amino acid sequence as shown in SEQ ID NO:20) had a significant expansion advantage compared to other groups.

[0177] The kill counts after each round of experiments are as follows: The kill levels are as follows: Figure 5As shown, except for unmodified DNT cells, all CLL1 CAR-DNT groups were able to completely eliminate THP1 tumor cells by the end of the 5th round of killing experiments.

[0178] Taking all factors into consideration, compared to other structures in this patent, CAR-DNT structures 2 and 6 can better maintain cell proliferation and persistence in IL-2-free tumor environments. Therefore, CAR structures 2 and 6 were ultimately selected as the preferred enhancement structures.

[0179] Sequence Description

[0180] SEQ ID NO:1---Guide peptide amino acid sequence;

[0181] MALPVTALLLPLALLLHAARP

[0182] SEQ ID NO:2---Amino acid sequence of CLL1 scFv;

[0183] DIQMTQSLSSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYT

[0184] GVPDRFSGSGSGTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKLEIKGGGGSG

[0185] GGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQG

[0186] LEWMGYINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARG

[0187] YYWYDEGHAMDSWGQGTLVTVSS

[0188] SEQ ID NO:3---CLL1 VH amino acid sequence;

[0189] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMG

[0190] YINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGY

[0191] YWYDEGHAMDSWGQGTLVTVSS

[0192] SEQ ID NO:4---CLL1 VL amino acid sequence;

[0193] DIQMTQSLSSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSAS

[0194] YRYTGVPDRFSGSGSGTTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKL

[0195] EIK

[0196] SEQ ID NO:5---CLL1 VH CDR1;

[0197] SYVMH

[0198] SEQ ID NO:6---CLL1 VH CDR2;

[0199] YINPYNDGTKYNEKFKG

[0200] SEQ ID NO:7---CLL1 VH CDR3;

[0201] GYYWYDEGHAMDS

[0202] SEQ ID NO:8---CLL1 VL CDR1;

[0203] KASQDVSTAVA

[0204] SEQ ID NO:9---CLL1 VL CDR2;

[0205] SASYRYT

[0206] SEQ ID NO:10---CLL1 VL CDR3;

[0207] QQHYSTPLT

[0208] SEQ ID NO:11---Amino acid sequence of the hinge region derived from CD8α;

[0209] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0210] SEQ ID NO:12---Transmembrane amino acid sequence derived from CD8α;

[0211] IYIWAPLAGTCGVLLLSLVITLYC

[0212] SEQ ID NO:13---IL2RF amino acid sequence, the underlined part indicates the linker sequence, the N-terminus of the linker sequence is the full-length amino acid sequence of IL2, and the C-terminus of the linker sequence is the full-length amino acid sequence of IL2Rα;

[0213] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT GGGGSGGGGSGGG GS ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQ MVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI

[0214] SEQ ID NO:14---IL15RF amino acid sequence, the underlined part indicates the linker sequence, the N-terminus of the linker sequence is the full-length amino acid sequence of IL15, and the C-terminus of the linker sequence is the full-length amino acid sequence of IL15Rα;

[0215] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVP GSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

[0216] SEQ ID NO:15---Amino acid sequence of intracellular region 4-1BB-CD3ζ;

[0217] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0218] SEQ ID NO:16---Amino acid sequence of intracellular region 4-1BB-CD3ζ-IL2RF, gray shaded area represents T2A, underlined area represents signal peptide, C-terminus of signal peptide is IL2RF;

[0219] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGP MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSS HSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTH GKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI

[0220] SEQ ID NO:17---Amino acid sequence of intracellular region 4-1BB-CD3ζ-IL15RF, gray shaded area represents T2A; underlined area represents signal peptide, C-terminus of signal peptide is IL15RF;

[0221] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGP MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLN KATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

[0222] SEQ ID NO:18---Amino acid sequence of intracellular region 4-1BB-CD3ζ-IL2Rγ;

[0223] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0224] SEQ ID NO:19---Amino acid sequence of intracellular region 4-1BB-CD3ζ-IL2Rγ-IL2RF, double underlined part is IL2Rγ, gray shaded part is T2A, single underlined part is signal peptide, and C-terminus of signal peptide is IL2RF;

[0225] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCN QHSPYWAPPCYTLKPET EGRGSLLTCGDVEENPGP MYRMQLLSCIALSLALVTNS APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSS HSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTH GKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI

[0226] SEQ ID NO:20---The amino acid sequence of intracellular region 4-1BB-CD3ζ-IL2Rγ-IL15RF, the double underlined part is IL2Rγ, the gray shaded part is T2A, the single underlined part is the signal peptide, and the C-terminus of the signal peptide is IL2RF; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR EGRGSLLTCGDVEENPGP MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLN KATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

[0227] SEQ ID NO:21---Amino acid sequence derived from the co-stimulatory signal transduction region of 4-1BB;

[0228] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0229] SEQ ID NO:22---Amino acid sequence derived from the intracellular signal transduction region of CD3ζ;

[0230] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0231] SEQ ID NO:23---Amino acid sequence derived from IL2Rγ;

[0232] ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0233] SEQ ID NO:24---Full-length amino acid sequence of the α subunit of the IL2 receptor;

[0234] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI

[0235] SEQ ID NO:25---Amino acid sequence of IL2;

[0236] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0237] SEQ ID NO:26---Full-length amino acid sequence of the α subunit of IL15 receptor;

[0238] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

[0239] SEQ ID NO:27---Amino acid sequence of IL15;

[0240] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0241] SEQ ID NO:28---T2A;

[0242] EGRGSLLTCGDVEENPGP

[0243] SEQ ID NO:29---First Linking Sequence

[0244] GGGGSGGGGSGGGGS

[0245] SEQ ID NO:30---Second Linking Sequence

[0246] SGGGSGGGGSGGGGSGGGGSGGGSLQ

[0247] SEQ ID NO:31---First signal peptide

[0248] MYRMQLLSCIALSLALVTNS

[0249] SEQ ID NO:32---Second signal peptide

[0250] MDWTWILFLVAAATRVHS

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 signal transduction region and an intracellular signal transduction region; wherein the intracellular domain further comprises an enhanced structure connected to the C-terminus of the intracellular signal transduction region, the enhanced structure comprising any one of the following structures: 1) IL2RF amino acid sequence, wherein the IL2RF amino acid sequence is an amino acid sequence that simultaneously contains an amino acid sequence derived from IL2 and an amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2. 2) An amino acid sequence that simultaneously contains an amino acid sequence derived from IL2Rγ and an amino acid sequence derived from IL15RF, wherein the IL15RF amino acid sequence is an amino acid sequence that simultaneously contains an amino acid sequence derived from IL15 and an amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15.

2. The enhanced chimeric antigen receptor targeting CLL1 according to claim 1, In the enhanced structure, a 2A peptide and a signal peptide are also attached to the N-terminus of the IL2RF amino acid sequence, with the 2A peptide and signal peptide linked to the IL2RF amino acid sequence in a direction from the N-terminus to the C-terminus; and / or, In the enhanced structure, the N-terminus of the IL15RF amino acid sequence is also connected to a 2A peptide and a signal peptide, which are linked to the IL15RF amino acid sequence in the order from the N-terminus to the C-terminus.

3. The enhanced chimeric antigen receptor targeting CLL1 according to claim 2, wherein the signal peptide linked to the IL2RF amino acid sequence is a first signal peptide as shown in SEQ ID NO:31, and / or, the signal peptide linked to the IL15RF amino acid sequence is a second signal peptide as shown in SEQ ID NO:

32.

4. The enhanced chimeric antigen receptor targeting CLL1 according to claim 2, wherein the 2A peptide is selected from one of T2A peptide, P2A peptide, E2A peptide, and F2A; optionally, the 2A peptide is a T2A peptide; further optionally, the amino acid sequence of the T2A peptide is as shown in SEQ ID NO:

28.

5. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 2-4, wherein the 2A peptide linked to IL2RF is directly linked to the C-terminus of the intracellular signal transduction region; and / or, the 2A peptide linked to IL15RF is directly linked to the C-terminus of an amino acid derived from IL2Rγ, and the N-terminus of an amino acid derived from IL2Rγ is directly linked to the C-terminus of the intracellular signal transduction region.

6. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-5, In the IL2RF amino acid sequence, amino acid sequences derived from IL2 and amino acid sequences derived from the IL2 receptor that can bind to amino acid sequences derived from IL2 are linked together in a sequence from the N-terminus to the C-terminus by a linker sequence; and / or, In the IL15RF amino acid sequence, amino acid sequences derived from IL15 and amino acid sequences derived from the IL15 receptor that can bind to amino acid sequences derived from IL15 are linked together in sequence from the N-terminus to the C-terminus by a linker sequence.

7. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-6, The amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 includes the amino acid sequence of the IL2 receptor α subunit; optionally, the amino acid sequence derived from the IL2 receptor that can bind to the amino acid sequence derived from IL2 is the full-length amino acid sequence of the IL2 receptor α subunit; further optionally, the full-length amino acid sequence of the IL2 receptor α subunit is as shown in SEQ ID NO:24; and / or, The amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15 includes the amino acid sequence of the IL15 receptor α subunit; optionally, the amino acid sequence derived from the IL15 receptor that can bind to the amino acid sequence derived from IL15 is the full-length amino acid sequence of the IL15 receptor α subunit; further optionally, the full-length amino acid sequence of the IL15 receptor α subunit is as shown in SEQ ID NO:

26.

8. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-7, wherein the protein or protein fragment expressed by the amino acid sequence derived from IL2 can bind to the IL2 receptor on the surface of T cells and stimulate T cell proliferation.

9. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-8, wherein the amino acid sequence derived from IL2 is the full-length amino acid sequence of IL2 as shown in SEQ ID NO:25; and / or, the amino acid sequence derived from IL15 is the full-length amino acid sequence of IL15 as shown in SEQ ID NO:

27.

10. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-9, wherein the amino acid sequence derived from IL2Rγ is the intracellular amino acid sequence of IL2Rγ, as shown in SEQ ID NO:

23.

11. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-10, wherein the amino acid sequence of the IL2RF is as shown in SEQ ID NO:13; and / or, the amino acid sequence of the IL15RF is as shown in SEQ ID NO:

14.

12. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-11, The enhanced structure is selected from any one of the following two structures: 1) SEQ ID NO:28-SEQ ID NO:31-SEQ ID NO:13 are directly connected in the direction from N end to C end; 2) SEQ ID NO:23-SEQ ID NO:28-SEQ ID NO:32-SEQ ID NO:14 are directly connected in the direction from N end to C end.

13. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-12, wherein the extracellular antigen recognition domain targeting CLL1 comprises a CLL1 antibody heavy chain variable region and a CLL1 antibody light chain variable region, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody light chain variable region shown in SEQ ID NO:4; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO:4; The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the antibody light chain shown in NO:

4.

14. The enhanced chimeric antigen receptor targeting CLL1 according to claim 13, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody heavy chain variable region are shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 antibody light chain variable region are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

15. The enhanced chimeric antigen receptor targeting CLL1 according to claim 13 or 14, wherein the CLL1 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, and the CLL1 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; optionally, the amino acid sequence of the CLL1 antibody heavy chain variable region is as shown in SEQ ID NO:3, and the amino acid sequence of the CLL1 antibody light chain variable region is as shown in SEQ ID NO:

4.

16. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-15, wherein the extracellular antigen recognition domain targeting CLL1 is the amino acid sequence shown in SEQ ID NO:

2.

17. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-16, wherein the co-stimulatory signal transduction region is derived from one, two, or more co-stimulatory factors selected from 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 co-stimulatory signal transduction region is derived from CD28 or 4-1BB; further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:21; even further optionally, the amino acid sequence of the co-stimulatory signal transduction region is as shown in SEQ ID NO:

21.

18. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-17, wherein the intracellular signal transduction 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 signal transduction region is derived from CD3ζ; further optionally, the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:22; even more optionally, the intracellular signal transduction region is as shown in SEQ ID NO:

22.

19. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-18, wherein the intracellular domain comprises an amino acid sequence as shown in SEQ ID NO:16 or SEQ ID NO:20; optionally, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:16 or SEQ ID NO:

20.

20. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-19, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids of 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:11; even more optionally, the amino acid sequence of the hinge region is shown in SEQ ID NO:

11.

21. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-20, 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:12; even further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:

12.

22. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-21, wherein the N-terminus of the extracellular antigen recognition domain is further connected to a guide peptide; optionally, the amino acid sequence of the guide peptide is as shown in SEQ ID NO:

1.

23. The enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-22, wherein the amino acid sequence of the enhanced chimeric antigen receptor is as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:16 in a direct chain of amino acids from the N-terminus to the C-terminus, or as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:20 in a direct chain of amino acids from the N-terminus to the C-terminus.

24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an enhanced chimeric antigen receptor targeting CLL1 according to any one of claims 1-23.

25. A vector comprising the isolated nucleic acid molecule of claim 24.

26. An engineered immune effector cell comprising the enhanced chimeric antigen receptor of any one of claims 1-23, the isolated nucleic acid molecule of claim 24, or the vector of claim 25, wherein the immune effector cell is derived from CD3. + CD4 - CD8 - DNT cells.

27. The engineered immune effector cell of claim 26, wherein the DNT cell is an autologous DNT cell or an allogeneic DNT cell.

28. A pharmaceutical composition comprising the engineered immune effector cells of claim 26 or 27 and pharmaceutically acceptable excipients.

29. The pharmaceutical composition of claim 28, wherein pharmaceutically acceptable excipients include protective agents.

30. The pharmaceutical composition of claim 28, wherein pharmaceutically acceptable excipients include cell cryopreservation solutions.

31. The pharmaceutical composition according to any one of claims 28-30, wherein the pharmaceutical composition is an intravenous injection.

32. The use of the enhanced chimeric antigen receptor of any one of claims 1-23, the isolated nucleic acid molecule of claim 24, the vector of claim 25, or the engineered immune effector cell of claim 26 or 27 in the preparation of a medicament for treating a disease or condition associated with CLL1 expression.

33. The application according to claim 32, wherein the disease or condition associated with CLL1 expression is a hematologic malignancy.

34. The application according to claim 33, wherein the disease or condition associated with CLL1 expression is acute myeloid leukemia.

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