Chimeric antigen receptors targeting cll1 and uses thereof

Abstract

This invention provides a chimeric antigen receptor targeting CLL1 and its application. The chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain of CLL1. The extracellular antigen recognition domain of CLL1 includes a CLL1 heavy chain variable region and a CLL1 light chain variable region. The amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable regions of the antibodies shown in SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable regions of the antibodies shown in SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:59, respectively.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a chimeric antigen receptor targeting CLL1 and its applications. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem / progenitor cells. It is characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, and metabolic abnormalities. Most cases are critically ill with a poor prognosis, and can be life-threatening if left untreated. Infants and young children are more susceptible to AML than adults, and it accounts for 30% of childhood leukemia cases. Current treatment methods include chemotherapy, supportive care, and hematopoietic stem cell transplantation.

[0003] Chimeric antigen receptors (CARs) are the core components of CAR cell therapy drugs, comprising antigen recognition domains, hinge regions, transmembrane regions, and intracellular domains. To date, antigen recognition domains have been derived from single-chain variable fragments (scFvs) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs); the most common source is scFv antibodies. CAR-T cell immunotherapy is considered one of the most promising approaches to combating cancer. CAR-T cells utilize genetic modification to express CAR proteins in 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. Currently, CAR-T cell immunotherapy has achieved significant success in treating various hematologic malignancies, such as CD19 CAR-T for B-cell lymphoma and BCMA CAR-T for multiple myeloma, for which there are already marketed drugs. However, due to the heterogeneity of AML, it is difficult to find ideal CAR-T targets for the treatment of AML. Existing targets for AML include CD33, CD123, LeY, NKG2D, etc., but there are no CAR-T drugs for these targets on the market.

[0004] Research has found that CLL1 (C-type Lectin-like Molecule 1) is an ideal target for the treatment of AML because it is not expressed in normal hematopoietic stem cells but is highly expressed in AML blast cells and leukemia stem cells. Therefore, it is of practical significance to find chimeric antigen receptors that target CLL1, especially chimeric antigen receptors that target CLL1 that are suitable for CAR-T cell therapy. Summary of the Invention

[0005] This application provides a chimeric antigen receptor targeting CLL1 and its application. The inventors developed multiple antibodies targeting CLL1 and used their corresponding scFv antibodies as the extracellular antigen recognition domain of the CLL1 CAR structure. In this way, a chimeric antigen receptor expression vector was constructed, and CAR-T cells targeting CLL1 were prepared. At the cellular level, multiple indicators of CLL1 CAR and CLL1 CAR-T cells were verified, and the three chimeric antigen receptors with the best performance were selected.

[0006] This application provides a chimeric antigen receptor targeting CLL1, comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. The CLL1 extracellular antigen recognition domain includes a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein:

[0007] The amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to those of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:54, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to those of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:55.

[0008] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:56, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:57.

[0009] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:58, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:59.

[0010] This application also provides a chimeric antigen receptor targeting CLL1, comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. The CLL1 extracellular antigen recognition domain includes a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein:

[0011] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of CLL1 respectively include the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of CLL1 respectively include the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively.

[0012] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47;

[0013] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of CLL1 may contain the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of CLL1 may contain the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively.

[0014] In some embodiments of the chimeric antigen receptor described above, the amino acid sequence of the CLL1 heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:54, and the CLL1 light chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO:55.

[0015] Alternatively, the amino acid sequence of the CLL1 heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:56, and the amino acid sequence of the CLL1 light chain variable region may contain the amino acid sequence shown in SEQ ID NO:57.

[0016] Alternatively, the amino acid sequence of the CLL1 heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:58, and the amino acid sequence of the CLL1 light chain variable region may contain the amino acid sequence shown in SEQ ID NO:59.

[0017] In some embodiments of the chimeric antigen receptor described above, the CLL1 extracellular antigen recognition domain includes any one of the following structures: amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:54-as shown in SEQ ID NO:55, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:54, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:56-as shown in SEQ ID NO:57, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:57-as shown in SEQ ID NO:56, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:58-as shown in SEQ ID NO:59, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:59-as shown in SEQ ID NO:58; optionally, the CLL1 scFv antibody includes any one of the following sequences: amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:55-as shown in SEQ ID NO:54, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:57-as shown in SEQ ID NO:58. The amino acid sequence shown in NO:56, the amino acid sequence shown in SEQ ID NO:59, the linker sequence, and the amino acid sequence shown in SEQ ID NO:58; further optionally, the CLL1 scFv antibody comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:14.

[0018] In some embodiments of the chimeric antigen receptor described above, the linker sequence is selected from one or more of the following sequences: SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68.

[0019] In some embodiments of the chimeric antigen receptor described above, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; 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:17.

[0020] In some embodiments of the chimeric antigen receptor described above, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; 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:18.

[0021] In some embodiments of the chimeric antigen receptor described above, the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a co-stimulatory signal transduction region.

[0022] In some embodiments of the chimeric antigen receptor described above, 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 amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:20.

[0023] In some embodiments of the chimeric antigen receptor described above, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, 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:19.

[0024] In some embodiments, the chimeric antigen receptor described above further includes a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide includes the amino acid sequence shown in SEQ ID NO:16.

[0025] In some embodiments, the chimeric antigen receptor described above comprises an amino acid sequence as shown in SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:34.

[0026] This application also provides an isolated nucleic acid molecule containing a nucleotide sequence encoding the chimeric antigen receptor described above.

[0027] In some embodiments, the isolated nucleic acid molecules described above, encoding the nucleotide sequence of the chimeric antigen receptor, include:

[0028] 1) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:54, optionally as shown in SEQ ID NO:60; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:55, optionally as shown in SEQ ID NO:61; or

[0029] 2) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:56, optionally as shown in SEQ ID NO:62; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:57, optionally as shown in SEQ ID NO:63; or

[0030] 3) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:58, optionally as shown in SEQ ID NO:64; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:59, optionally as shown in SEQ ID NO:65.

[0031] This application also provides a vector containing the isolated nucleic acid molecules described above.

[0032] In some embodiments, the aforementioned vector is an expression vector; in some embodiments, the vector is a viral vector; and in some embodiments, the vector is a lentiviral vector.

[0033] This application also provides an engineered immune effector cell comprising the chimeric antigen receptor, the isolated nucleic acid molecule, or the carrier described above.

[0034] In some embodiments, the engineered immune effector cells described above are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, and NK cells differentiated from pluripotent stem cells.

[0035] In some embodiments, the engineered immune effector cells described above are T lymphocytes; alternatively, the T lymphocytes may be derived from autologous T lymphocytes or allogeneic T lymphocytes.

[0036] In some embodiments, the engineered immune effector cells described above are αβT lymphocytes or γδT lymphocytes.

[0037] This application also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients.

[0038] In some embodiments of the above-described pharmaceutical composition, pharmaceutically acceptable excipients include protective agents.

[0039] In some embodiments of the above-described pharmaceutical composition, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0040] In some embodiments of the above-described pharmaceutical composition, the pharmaceutical composition is a cell suspension or frozen cells thereof.

[0041] In some embodiments, the above-described pharmaceutical composition is an intravenous injection.

[0042] This application also provides the use of the above-mentioned chimeric antigen receptor, isolated nucleic acid molecule, vector or engineered immune effector cell in the preparation of a drug for treating diseases or conditions associated with CLL1 expression.

[0043] In some implementations of the above application, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0044] In some implementations of the above application, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0045] 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.

[0046] In some implementations of the above method, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0047] In some embodiments of the above method, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0048] In some embodiments of the above method, the administration is carried out via intravenous injection.

[0049] 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 or multiple injections.

[0050] 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.

[0051] 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.

[0052] In some embodiments of the above-mentioned drug, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0053] In some embodiments of the above-mentioned drug, the disease or condition associated with CLL1 expression is acute myeloid leukemia. Attached Figure Description

[0054] Figure 1 The diagram shows the structural schematics of various CLL1 CARs in Embodiment 2 of this application. From top to bottom, the schematics are: CD8a signal peptide, anti-CLL1 scFv, CD8a hinge region and transmembrane region, 4-1BB, and CD3ζ.

[0055] Figure 2 This indicates that CD3+ cells in each group of CAR-T cells after antigen stimulation in Example 5 of this application... + The continuous proliferation of cells.

[0056] Figures 3A-3C This demonstrates the lysis and killing effects of various CLL1 CAR-T cells and the positive control M26 CLL1 CAR-T cells on different target cells at different effector-to-target ratios in Example 6 of this application; wherein: Figure 3A The target cells are HL60 cells; Figure 3B The intermediate target cells are K562-CLL1 cells; Figure 3C The target cell is K562.

[0057] Figure 4 This illustrates the release of IFN-γ cytokine from various CLL1 CAR-T cells, positive control M26 CLL1 CAR-T cells, and UTD cells (T cells not transduced with CAR) after activation by positive target cells in Example 7 of this application. Detailed Implementation

[0058] 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.

[0059] 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.

[0060] In this application, the term "antibody" has its conventional meaning in the art, referring to an immunoglobulin molecule composed of four polypeptide chains, namely two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Analysis of the amino acid sequences of different antibody heavy and light chains revealed that the amino acid sequences near the N-terminus of both heavy and light chains exhibit significant variation, while the amino acid sequences in other parts remain relatively constant. Therefore, the region of significant variation in the amino acid sequence near the N-terminus in the antibody light and heavy chains is termed the variable region (V), and the region of relatively stable amino acid sequence near the C-terminus is termed the constant region (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 variable region of an antibody, there is a small subset of amino acid residues that exhibit particularly strong variations. 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 region 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 known as complementarity determining regions (CDRs).

[0061] 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 VL regions.

[0062] In this application, the term "monoclonal antibody" has its conventional meaning in the art, referring to a highly homogeneous antibody that targets only a specific antigenic epitope, which can be prepared by known techniques such as hybridoma technology, antibody library technology, transgenic mouse technology, or single-cell PCR technology.

[0063] In this application, the term "scFv" has the conventional meaning in the art and refers to a single chain variable fragment (scFv), which is an antibody composed of a heavy chain variable fragment and a light chain variable fragment linked by a short peptide (linker).

[0064] In this application, terms such as “Sc(Fv)2” and “[Sc(Fv)2]2” that are not specifically explained also have their conventional meanings in the art.

[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 (e.g., a portion that binds to tumor-associated antigens (TAAs)), 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 approaches to 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 have been derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). The most common source to date is the scFv segment of antibodies, which includes both heavy and light chain variable regions linked by a peptide chain, such as the 18-amino acid linker sequence GSTGSGSGKPGSGEGSTKG.

[0067] In this application, the term "specific recognition and / or binding" refers to the recognition and / or binding between a CAR and a specific target, which is to bind to the target with greater affinity, strength, ease, and / or duration than the CAR binds to other targets.

[0068] In this application, the term "hinge region" refers to the connecting segment that acts between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by providing a certain range of motion to the antigen recognition domain. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, typical hinge regions also contain residues that participate in CAR dimerization, which helps to enhance antigen sensitivity.

[0069] In this application, "transmembrane region" refers to a transmembrane domain connecting the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect CAR expression and stability to some extent, but do not directly participate in signal transduction; however, they can enhance downstream signal transduction through interactions. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0070] In this application, the term "intracellular domain" includes intracellular signal transduction regions and may also include co-stimulatory signal transduction regions.

[0071] In this application, the term "intracellular signal transduction region" refers to the activation of at least one normal effector function of an immune effector cell responsible for expressing CAR. The intracellular signal transduction region may originate from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0072] In this application, the term "co-stimulatory signal transduction region" is used because, in addition to antigen-specific signal stimulation, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. In some embodiments, the CAR may further include one or more co-stimulatory signal transduction regions, wherein the co-stimulatory signal transduction regions may be derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0073] In this application, the term "guide peptide" refers to a short peptide preceding an extracellular antigen recognition domain (such as the scFv sequence), which guides the export of intracellularly synthesized recombinant proteins to the extracellular space. Commonly used guide peptides include the human CD8α signal peptide or the human GM-CSF receptor α signal peptide.

[0074] In this application, one of the key factors determining the efficacy of CAR-immunotherapy is the selection of the tumor target antigen. In this application, the selected tumor target antigen "CLL1" has the conventional meaning in the art; it is also known as KLR1 or CLEC12A. It is a type II transmembrane glycoprotein and a member of the large family of C-type lectin-like receptors associated with immune regulation. Studies have found that CLL1 (C-type Lectin-like Molecule 1) is an ideal target for the treatment of AML because it is not expressed in normal hematopoietic stem cells but is highly expressed in AML blast cells and leukemia stem cells.

[0075] 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 chimeric antigen receptor of the present invention. The linking sequence may consist of different amino acid residues (such as glycine and serine) so that adjacent protein domains can 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.

[0076] 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.

[0077] 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.

[0078] In this application, the gene transduction / transfection methods for CAR gene transduction / transfection and target gene expression mainly include viral and non-viral methods. These include: gamma retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

[0079] The term "vector" generally refers to a nucleic acid delivery vehicle that inserts a polynucleotide encoding a protein into it, 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 transduce mRNA into T cells.

[0080] In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Immune effector cells may be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, and NK cells differentiated from pluripotent stem cells.

[0081] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives. In some embodiments, pharmaceutically acceptable excipients include protectants, such as cell cryopreservation solutions. In some embodiments, the pharmaceutical composition of this application is a cell suspension or its cryopreserved cells.

[0082] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cattle, sheep, or monkeys.

[0083] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0084] In this application, the term "about" generally refers to a range of fluctuations above or below a specified value that are acceptable to a person skilled in the art, such as a variation within ±0.5% to 10%, for example, a variation within a 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.

[0085] CLL1 antibody or its antigen-binding site, corresponding nucleic acid molecule, corresponding vector, corresponding cell, or corresponding pharmaceutical composition.

[0086] On one hand, this application provides a CLL1 antibody or its antigen-binding site, which includes a heavy chain variable region and a light chain variable region. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO:54, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO:55.

[0087] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO:56, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO:57.

[0088] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO:58, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO:59.

[0089] On the other hand, this application also provides a CLL1 antibody or its antigen-binding site, which includes a heavy chain variable region and a light chain variable region. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively include the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively include the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

[0090] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47;

[0091] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively include the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively include the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0092] In this application, the KABAT rule was used for CDR partitioning.

[0093] In some embodiments, the amino acid sequence of the heavy chain variable region of any of the above-mentioned CLL1 antibodies or their antigen-binding sites includes the amino acid sequence shown in SEQ ID NO:54, and the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:55.

[0094] Alternatively, the amino acid sequence of the heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:56, and the amino acid sequence of the light chain variable region may contain the amino acid sequence shown in SEQ ID NO:57.

[0095] Alternatively, the amino acid sequence of the heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:58, and the amino acid sequence of the light chain variable region may contain the amino acid sequence shown in SEQ ID NO:59.

[0096] In some embodiments, any of the above-mentioned CLL1 antibodies or their antigen-binding sites are CLL1 scFv antibodies, CLL1Sc(Fv)2 antibodies, or CLL1 [Sc(Fv)2]2 antibodies.

[0097] In some embodiments, any of the CLL1 antibodies or their antigen-binding sites described above, the CLL1 scFv antibody comprises any one of the following sequences: amino acid sequence-linking sequence as shown in SEQ ID NO:54-amino acid sequence as shown in SEQ ID NO:55, amino acid sequence-linking sequence as shown in SEQ ID NO:54, amino acid sequence-linking sequence as shown in SEQ ID NO:56-amino acid sequence as shown in SEQ ID NO:57, amino acid sequence-linking sequence as shown in SEQ ID NO:57-amino acid sequence as shown in SEQ ID NO:56, amino acid sequence-linking sequence as shown in SEQ ID NO:58-amino acid sequence as shown in SEQ ID NO:59, amino acid sequence-linking sequence as shown in SEQ ID NO:59-amino acid sequence as shown in SEQ ID NO:58; optionally, the CLL1 scFv antibody comprises any one of the following sequences: amino acid sequence-linking sequence as shown in SEQ ID NO:55-amino acid sequence as shown in SEQ ID NO:54, amino acid sequence-linking sequence as shown in SEQ ID NO:57-amino acid sequence as shown in SEQ ID NO:58. The amino acid sequence shown in NO:56, the amino acid sequence shown in SEQ ID NO:59, the linker sequence, and the amino acid sequence shown in SEQ ID NO:58; further optionally, the CLL1 scFv antibody comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:14.

[0098] In some embodiments, the linker sequence of any of the CLL1 antibodies or their antigen-binding sites described above is selected from one or more of the following sequences: SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68.

[0099] Furthermore, this application also provides an isolated nucleic acid molecule containing a nucleotide sequence encoding any of the aforementioned CLL1 antibodies or their antigen-binding sites.

[0100] In some embodiments, the isolated nucleic acid molecules described above contain nucleotide sequences encoding the CLL1 antibody or its antigen-binding site that include:

[0101] 1) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:54, optionally as shown in SEQ ID NO:60; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:55, optionally as shown in SEQ ID NO:61; or

[0102] 2) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:56, optionally as shown in SEQ ID NO:62; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:57, optionally as shown in SEQ ID NO:63; or

[0103] 3) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:58, optionally as shown in SEQ ID NO:64; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:59, optionally as shown in SEQ ID NO:65.

[0104] Furthermore, this application also provides a vector containing the isolated nucleic acid molecules described above. The vector can be arbitrarily selected from one or more of the following vectors: 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, etc. 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).

[0105] In another aspect, this application also provides a cell that contains the above-mentioned CLL1 antibody or its antigen-binding site, nucleic acid molecule or vector.

[0106] Furthermore, this application also provides a pharmaceutical composition comprising any of the aforementioned CLL1 antibodies or their antigen-binding sites, isolated nucleic acid molecules, a carrier, cells, and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

[0107] Antibody drugs and antibody-drug conjugates containing CLL1 antibodies or their antigen-binding sites

[0108] On the one hand, this application provides an antibody drug comprising any of the above-mentioned CLL1 antibodies or their antigen-binding sites.

[0109] In some embodiments, the antibody drug described above is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.

[0110] On the other hand, this application also provides an antibody-drug conjugate comprising any of the above-mentioned CLL1 antibodies or their antigen-binding sites.

[0111] Application of CLL1 antibody or its antigen-binding site

[0112] On the one hand, this application provides the use of any of the above-mentioned CLL1 antibodies or their antigen-binding sites, isolated nucleic acid molecules, vectors or cells in the preparation of medicaments for treating diseases or conditions related to CLL1 expression.

[0113] In some embodiments of the above application, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0114] In some embodiments of the above application, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0115] On the other hand, this application also provides the use of any of the above-mentioned CLL1 antibodies or their antigen-binding sites, isolated nucleic acid molecules, vectors or cells in the preparation of detection reagents for diagnosing diseases or conditions related to CLL1 expression.

[0116] In some embodiments of the above application, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0117] In some embodiments of the above application, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0118] In another aspect, this application also provides a method for treating diseases or conditions related to CLL1 expression, comprising the following steps: administering an effective amount of a drug containing any of the above-mentioned CLL1 antibodies or their antigen-binding sites to a subject who requires treatment for diseases or conditions related to CLL1 expression.

[0119] In some embodiments of the above method, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0120] In some embodiments of the above method, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0121] In some embodiments of the above method, the application can be performed in different ways, such as oral, intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal application.

[0122] In some embodiments of the above method, the dosage may vary for different indications; the dosage may also vary for patients with different degrees of disease severity.

[0123] In some embodiments of the above method, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

[0124] In another aspect, this application also provides a drug comprising any of the above-mentioned CLL1 antibodies or their antigen-binding sites, isolated nucleic acid molecules, vectors or cells, for the treatment of diseases or conditions related to CLL1 expression.

[0125] In some embodiments of the above-mentioned drugs, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0126] In some embodiments of the above-mentioned drugs, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0127] CLL1 chimeric antigen receptor, corresponding nucleic acid molecule, corresponding carrier, corresponding immune effector cells, and pharmaceutical composition.

[0128] On one hand, this application provides a chimeric antigen receptor targeting CLL1, comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the CLL1 extracellular antigen recognition domain includes a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein:

[0129] The amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to those of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:54, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to those of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:55.

[0130] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:56, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:57.

[0131] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:58, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:59.

[0132] On the other hand, this application also provides a chimeric antigen receptor targeting CLL1, comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the CLL1 extracellular antigen recognition domain includes a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein:

[0133] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of CLL1 respectively include the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of CLL1 respectively include the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively.

[0134] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47;

[0135] Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of CLL1 may contain the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of CLL1 may contain the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively.

[0136] In this application, the KABAT rule was used for CDR partitioning.

[0137] In some embodiments of any of the chimeric antigen receptors described above, the amino acid sequence of the CLL1 heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:54, and the CLL1 light chain variable region sequence comprises the amino acid sequence shown in SEQ ID NO:55.

[0138] Alternatively, the amino acid sequence of the CLL1 heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:56, and the amino acid sequence of the CLL1 light chain variable region may contain the amino acid sequence shown in SEQ ID NO:57.

[0139] Alternatively, the amino acid sequence of the CLL1 heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:58, and the amino acid sequence of the CLL1 light chain variable region may contain the amino acid sequence shown in SEQ ID NO:59.

[0140] This application experimentally screened three chimeric antigen receptors, each containing three different CLL1 extracellular antigen recognition domains, corresponding to 1-3 scFv, 1-28 scFv, or 3-67 scFv antibodies, respectively. Specifically, the 1-3 scFv antibody contains a heavy chain variable region as shown in SEQ ID NO:54 and a light chain variable region as shown in SEQ ID NO:55; the 1-28 scFv antibody contains a heavy chain variable region as shown in SEQ ID NO:56 and a light chain variable region as shown in SEQ ID NO:57; and the 3-67 scFv antibody contains a heavy chain variable region as shown in SEQ ID NO:58 and a light chain variable region as shown in SEQ ID NO:59.

[0141] In some embodiments, the CLL1 extracellular antigen recognition domain of any of the chimeric antigen receptors described above includes any one of the following structures: amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:54-as shown in SEQ ID NO:55, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:54, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:56-as shown in SEQ ID NO:57, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:57-as shown in SEQ ID NO:56, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:58-as shown in SEQ ID NO:59, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:59-as shown in SEQ ID NO:58; optionally, the CLL1scFv antibody includes any one of the following sequences: amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:55-as shown in SEQ ID NO:54, amino acid sequence-linking sequence-amino acid sequence as shown in SEQ ID NO:57-as shown in SEQ ID NO:58. The amino acid sequence shown in ID NO:56, the amino acid sequence shown in SEQ ID NO:59, the linking sequence, and the amino acid sequence shown in SEQ ID NO:58; further optionally, the CLL1 scFv antibody comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:14. In the above description, "-" indicates interconnection, and "-" is directional, indicating a connection from the N-terminus to the C-terminus of the amino acid.

[0142] In some embodiments, the linker sequence of any of the chimeric antigen receptors described above is selected from one or more of the following sequences: SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:68.

[0143] In some embodiments of any of the chimeric antigen receptors described above, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; 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:17.

[0144] In some embodiments of any of the chimeric antigen receptors described above, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; 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:18.

[0145] In some embodiments of any of the chimeric antigen receptors described above, the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a co-stimulatory signal transduction region.

[0146] In some embodiments of any of the chimeric antigen receptors described above, 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 amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:20.

[0147] In some embodiments of any of the chimeric antigen receptors described above, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, 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:19.

[0148] In some embodiments, any of the chimeric antigen receptors described above further includes a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide includes the amino acid sequence shown in SEQ ID NO:16.

[0149] In some embodiments, any of the chimeric antigen receptors described above includes an amino acid sequence as shown in SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:34.

[0150] Furthermore, this application also provides an isolated nucleic acid molecule containing a nucleotide sequence encoding any of the chimeric antigen receptors described above.

[0151] In some embodiments, the isolated nucleic acid molecules described above contain nucleotide sequences encoding the chimeric antigen receptor comprising:

[0152] 1) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:54, optionally as shown in SEQ ID NO:60; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:55, optionally as shown in SEQ ID NO:61; or

[0153] 2) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:56, optionally as shown in SEQ ID NO:62; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:57, optionally as shown in SEQ ID NO:63; or

[0154] 3) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:58, optionally as shown in SEQ ID NO:64; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:59, optionally as shown in SEQ ID NO:65.

[0155] Furthermore, this application also provides a vector containing the isolated nucleic acid molecules described above. The vector may be any one or more of the following: plasmids; phage particles; Cosmids; 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, etc. 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).

[0156] In some embodiments, the aforementioned vector is an expression vector; in other embodiments, the vector is a viral vector; and in still other embodiments, the vector is a lentiviral vector.

[0157] Furthermore, this application also provides an engineered immune effector cell comprising the aforementioned chimeric antigen receptor, the aforementioned isolated nucleic acid molecule, or the aforementioned carrier.

[0158] In some embodiments, the engineered immune effector cells described above are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, and NK cells differentiated from pluripotent stem cells.

[0159] In some embodiments, the engineered immune effector cells described above are T lymphocytes; optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes.

[0160] In some embodiments, the engineered immune effector cells described above are αβT lymphocytes or γδT lymphocytes of T lymphocytes.

[0161] In some embodiments, the engineered immune effector cells described above may express or have expressed the chimeric antigen receptor described in this application on their surface.

[0162] Furthermore, this application also provides a pharmaceutical composition comprising the aforementioned engineered immune effector cells and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

[0163] In some embodiments of the above-described pharmaceutical compositions, pharmaceutically acceptable excipients include protective agents.

[0164] In some embodiments of the above-described pharmaceutical compositions, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0165] In some embodiments, the pharmaceutical composition described above is a cell suspension or frozen cells thereof.

[0166] In some embodiments, the pharmaceutical composition described above is an intravenous injection.

[0167] Preparation method and uses

[0168] On the one hand, this application also provides a method for preparing engineered immune effector cells, which includes the following steps: transducing the vector described in this application into the immune effector cells.

[0169] In some embodiments of the above method, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, and NK cells differentiated from pluripotent stem cells.

[0170] In some embodiments of the above method, the engineered immune effector cells are T lymphocytes; optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes.

[0171] In some embodiments of the above method, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0172] On the other hand, this application also provides the use of the above-mentioned 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.

[0173] In some embodiments of the above application, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0174] In some embodiments of the above application, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0175] In another aspect, this application also provides a method for treating diseases or conditions related to 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 diseases or conditions related to CLL1 expression.

[0176] In some embodiments of the above method, the administration can be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration. For example, administration can be given to the subject via intravenous injection. In some embodiments, an effective dose of engineered immune effector cells or pharmaceutical composition can be administered to the subject once or in multiple doses over a period of time, such as once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0177] In some embodiments of the above method, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0178] In some embodiments of the above method, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0179] In some embodiments of the above method, the administration is performed via intravenous injection.

[0180] 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.

[0181] 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. In some embodiments, the dosage may vary for different indications; the dosage may also vary for patients with different disease severity. The dosage range may be 1×10⁻⁶. 5 CAR-positive T cells / kg to 1×10 7 CAR-positive T cells / kg, for example, 1×10 5 CAR-positive T cells / kg to 1×10 6 CAR-positive T cells / kg, 1×10 6 CAR-positive T cells / kg to 1×10 7 CAR-positive T cells / kg, 0.5×10 6 CAR-positive T cells / kg, 0.6×10 6 CAR-positive T cells / kg, 0.7×10 6 CAR-positive T cells / kg, 0.8×10 6 CAR-positive T cells / kg, 0.9 × 10 6 CAR-positive T cells / kg, 1.0×10 6 CAR-positive T cells / kg, 1.1×10 6 CAR-positive T cells / kg, 1.2 × 10 6 CAR-positive T cells / kg, 1.3×10 6 CAR-positive T cells / kg, 1.4 × 10 6 CAR-positive T cells / kg, 1.5 × 10 6 CAR-positive T cells / kg, 1.6 × 10 6 CAR-positive T cells / kg, 1.7 × 10 6 CAR-positive T cells / kg, 1.8 × 10 6 CAR-positive T cells / kg, 1.9 × 10 6 CAR-positive T cells / kg, 2.0 × 10 6 CAR-positive T cells / kg.

[0182] In some embodiments of the above method, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

[0183] In another aspect, 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.

[0184] In some embodiments of the above-mentioned drugs, the disease or condition associated with CLL1 expression is a hematologic malignancy.

[0185] In some embodiments of the above-mentioned drugs, the disease or condition associated with CLL1 expression is acute myeloid leukemia.

[0186] Not intended to be limited by any theory, the embodiments described below are merely illustrative of the chimeric antigen receptor, engineered 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, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0187] Example 1: CLL1 Antibody Development

[0188] CLL1 antibody development process: BALB / c and C57bl / 6 mice were used as immunization animals, and human CLL1 was used as the antigen, immunized according to the procedure in Table 1. Serum from each immunized mouse was collected for FACS (flow cytometry fluorescence sorting) and ELISA detection. Based on the combined results of ELISA and FACS, the mice with the best immunization effect were selected. Their plasma cells were enriched with CD138 antibody and seeded onto a Beacon selection chip for analysis and screening of antibody-positive B cell lines. A total of 8561 monoclonal cell lines and 1511 polyclonal cell lines were obtained, of which 690 lines had IgG antibodies. Based on the binding to the antigen, the positive B cell clones were exported and subjected to V region sequencing, yielding V region sequences of 64 monoclonal antibodies. Based on sequence similarity, 14 representative sequences were selected for the synthesis of scFv in CAR.

[0189] Table 1. Animal Immunization Schedule

[0190]

[0191] Example 2: Obtaining CLL1 CAR-T cells

[0192] We obtained 14 candidate scFvs targeting CLL1, and their serial numbers and sequences are shown in Table 2. We also obtained one CLL1 positive control antibody, M26 (derived from Treatment of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type Lection-like Molecule 1). Molecular Therapy Vol. 25 No. 9 September 2017 )

[0193] Table 2. Candidate scFv numbers and sequences targeting CLL1 (14 in total)

[0194]

[0195] Because functional validation of antibodies at the protein level cannot reflect their function at the cellular level, we selected 14 candidate scFvs for screening based on second-generation CAR structures. In each CAR structure, the CD8α guide chain was used as the signal peptide (as shown in SEQ ID NO:16), the hinge region (as shown in SEQ ID NO:17) and the transmembrane region (as shown in SEQ ID NO:18) adopted the CD8α structure, 4-1BB was used as the intracellular co-stimulatory signal (as shown in SEQ ID NO:19), and CD3ζ was used as the T cell activation signal (as shown in SEQ ID NO:20). The structural schematic diagrams are shown below. Figure 1 As shown.

[0196] 1. Construction of lentiviral vectors

[0197] Based on the sequences of each candidate scFv in Table 1 and the sequences of CAR structural components, 14 CLL1CAR structures and 1 M26 CLL1CAR structure were artificially synthesized, and their amino acid sequences are shown in Table 3.

[0198] Table 3. 14 CLL1 CAR serial numbers and their sequences

[0199]

[0200] The 15 CLL1 CAR structures described above were constructed into modified empty lentiviral vectors (manufacturer: SBI, catalog numbers: CD500-CD800, such as the modified resistance described in Example 1 of WO2021 / 121227) to obtain CAR expression vectors. These CAR expression vectors, along with three packaging plasmids, were then 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).

[0201] 2. Fifteen CLL1-specific CAR-T cells were prepared via lentivirus transduction.

[0202] The transduction experiment was conducted according to conventional methods known to those skilled in the art. The transduction steps are briefly described below:

[0203] 1) Sorting T cells

[0204] Peripheral blood mononuclear cells (PBMCs) are isolated from human apheresis cells, and then T cells are sorted from the PBMCs.

[0205] 2) Activation treatment of T cells

[0206] The isolated T cells were resuspended in complete culture medium (X-VIVO15 medium + 5% FBS + 300 IU / ml IL-2 or X-VIVO15 medium + 5% FBS + 5 ng / ml IL-15 + 10 ng / ml IL-7) to a final concentration of (1~3) × 10⁻⁶. 6 Cells / ml, 1ul beads / 1×10 6 Cells were stimulated with CD3 / CD28 magnetic beads, mixed well, and then placed in an incubator at 37°C + 5% CO2 for at least 24 hours.

[0207] 3) Lentiviral transduction of T cells

[0208] Remove activated T cells from the culture and resuspend them in X-VIVO15 medium containing 8 µg / ml polybrene. Mix well to obtain a cell suspension, and then adjust the concentration of each cell suspension to 2 × 10⁻⁶ cells per 800 µL of cell suspension. 6Slowly add 200 μL of lentiviral vector to cells, mix well, place in a well plate, and incubate in an incubator at 37°C + 5% CO2 for at least 4-6 hours.

[0209] 4) Expansion and culture of transduced T cells

[0210] After transduction, the cells were cultured in complete T-cell culture medium and passaged every other day to maintain a cell density of (0.8~2)×10⁻⁶. 6 Cells / ml, for use in subsequent examples.

[0211] After infecting T cells with lentiviruses containing the CAR structures listed in Table 3, the resulting T cells were named according to their CAR sequence numbers. For example, T cells obtained using CAR 1-3 were named 1-3 CAR-T cells, and T cells obtained using CAR 2-8 were named 2-8 CAR-T cells. Next, we screened the 14 CLL1 CAR structures at the cellular level to determine the quality of each scFv.

[0212] Example 3: Detection of CAR molecules expressed on the surface of CLL1 CAR-T cells and their antigen recognition ability.

[0213] 1. Detection of CAR molecules: The CAR protein molecules expressed on the surface of 14 types of CLL1 CAR-T cells cultured for 7 days after transduction obtained in Example 2 were detected. We used PE fluorescently labeled CLL1 antigen (manufacturer: ACRObiosystems, catalog number: CLA-PH2Q3) and FITC fluorescently labeled CLL1 antigen (manufacturer: ACRObiosystems, catalog number: CLA-HF24725ug) to stain the 14 types of CLL1 CAR-T cells, M26 CLL1 CAR-T cells and UTD cells (untransduced T cells) obtained in Example 2, and analyzed the positive ratio of CAR molecules by flow cytometry. The detection results are shown in Table 4.

[0214] 2. Detection of CAR molecular antigen recognition ability: The antigen recognition ability of the 14 CAR protein molecules expressed on the surface of CLL1 CAR-T cells obtained in Example 2 was detected. We used FITC fluorescently labeled IgG (manufacturer: abcam, catalog number: ab98658) to stain the 14 CLL1 CAR-T cells, M26 CLL1 CAR-T cells and UTD cells (untransduced CAR T cells) obtained in Example 2, and detected the antigen recognition ability by flow cytometry. The detection results are shown in Table 4.

[0215] As shown in Table 4: Cells with gray backgrounds were discarded during screening due to poor CAR molecule expression and / or poor antigen recognition ability. Among them, cells 2-25, 2-40, and 3-35 did not express CAR; cells 3-11, 3-13, and 3-16 expressed CAR but had poor antigen recognition ability. These cells were all discarded in subsequent experiments.

[0216] Table 4. Detection results of CAR molecules expressed on cell surface and their antigen recognition ability.

[0217]

[0218] Note: A CAR expression rate of less than 10% is considered as no expression or extremely poor expression, below the screening criteria; an antigen binding recognition rate of less than 10% is considered as poor antigen recognition ability, below the screening criteria.

[0219] Example 4: Specific detection of CLL1 CAR

[0220] CLL1 CAR specificity was detected at both the protein and cellular levels.

[0221] Specific detection of protein levels

[0222] Five fluorescently labeled antigen proteins—CD19 (ACRO Biosystems, catalog number CD9-HP2H3), CS1 (ACRO Biosystems, catalog number SL7-HP2H3), BCMA (ACRO Biosystems, catalog number BCA-HF254), GPC3 (ACRO Biosystems, catalog number GP3-HF2H1), and EGFRvIII (ACRO Biosystems, catalog number EGI-HP2E3)—were used to specifically detect protein levels in eight CLL1 CAR-T cell lines (1-3, 1-28, 1-16, 2-8, 2-31, 3-37, 3-64, and 3-67 CAR-T cells) and M26 CAR-T cells screened in Example 3, as well as the CAR-T cells themselves. 0.5 × 10⁻⁶ cells of each CAR-T cell line were used. 6Cells were washed once with PBS, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. 0.1 μg of the fluorescently labeled antigen protein was added, and the cells were incubated at 4°C in the dark for 20 min. Cells were washed once with PBS, centrifuged at 200g for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. The binding ability was detected by flow cytometry. The results showed that none of the CAR-T cells could bind to CD19 protein, CS1 protein, BCMA protein, GPC3 protein, or EGFRvIII protein, showing good specificity. The detection results are shown in Table 5.

[0223] Specificity at the cellular level

[0224] Eleven cell lines from different tissue sources—K562 (human chronic myeloid leukemia cells), K562-CLL1 (K562 cells expressing exogenous CLL1 protein), HL60 (human promyelocytic acute leukemia cells), THP1 (human monocytic leukemia cells), OVCAR3 (human ovarian adenocarcinoma cells), RAJI (human lymphoma cells), MM.1S (human multiple myeloma cells), NALM6 (human acute lymphoblastic leukemia cells), SK-MEL1 (human skin melanoma cells), A549 (human lung cancer cells), and HUH7 (human liver cancer cells)—were used to screen eight CLL1 CAR-T cells (1-3, 1-28, 1-16, 2-8, 2-31, 3-37, 3-64, and 3-67 CAR-T cells) obtained in Example 3, as well as M26 CAR-T cells, according to the following method. CAR-T cells underwent cell-level specific assays. K562-CLL1, HL60, THP1, and NALM6 cell lines were CLL1-positive target cells, while the remaining cell lines were CLL1-negative target cells. The method used to detect T cell activation in this study was CD107a analysis. CD107a expression on the membrane is considered a marker of activated cytotoxic lymphocytes (CD8+ T cells and NK cells). When cytotoxic lymphocytes are stimulated by specific antigens, they undergo immune activation and express CD107a, thus serving as a method for detecting CAR-T cell specificity. The specific steps were as follows: tumor cells and transfected and untransfected T cells were counted, and tumor cells were adjusted to 2 × 10⁶ cells / year using X-VIVO medium. 6 / ml, T cells adjusted to 1×10 6 / ml. Add 20ul of CD107a antibody to each well of the killing plate, followed by 100ul of T cells and 100ul of tumor cells per well. Centrifuge at 400 rpm for 3 minutes. Incubate at 37°C with 5% CO2 for 60 minutes. After 60 minutes, add 20ul of Golgi Stop working solution (3ml X-VIVO medium plus 2ul of Golgi Stop (BD GolgiStop™ Protein Transport Inhibitor, CAT: 554724)) to each well and incubate for 2.5 hours. Mix equal volumes of CD3 and CD8 antibodies, add 10ul to each well, and incubate for 30 minutes. After 30 minutes, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 250ul of FACS buffer (PBS + 0.5% BSA), and mix well. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant. Add 200 μL of FACS buffer (PBS + 0.5% BSA) to each well, mix well, transfer to a labeled flow cytometry tube, and then add another 200-300 μL of FACS buffer (PBS + 0.5% BSA) for flow cytometry analysis. Analyze the fluorescence signal of CD107a in the CD3+ & CD8+ positive cell population. Specific detection results are shown in Table 6.

[0225] Table 5. CAR-T cell-specific results of protein level detection

[0226]

[0227] Note: "-" indicates that CAR-T cells do not bind to this protein, and "+" indicates that CAR-T cells bind to this protein.

[0228] Table 6. Results of CAR-T cell-specific detection at the cellular level

[0229]

[0230] Note: "-" indicates that the CD107a signal was not detected, and "+" indicates that the CD107a signal was detected.

[0231] As shown in Table 6: 2-31 CAR can be activated by negative target cells and exhibit CD107a signaling. Therefore, it did not meet the screening criteria at the cellular level and was excluded in subsequent experiments.

[0232] Example 5: Sustained proliferation of CLL1 CAR-T cells

[0233] Antigen stimulation can activate CAR-T cells and cause them to proliferate. However, continuous activation of T cells can lead to cell exhaustion. Exhausted T cells will have reduced proliferative capacity and effector function. We determined the sustained proliferative capacity of CLL1 CAR-T cells by detecting the proliferation of CD3+ cells (i.e., T cell proliferation) after multiple rounds of antigen stimulation experiments.

[0234] Before antigen stimulation (using HL60 cells as the antigen), the CAR-positive ratios of the seven CLL1 CAR-T cells (i.e., 1-3, 1-16, 1-28, 2-8, 3-37, 3-64, 3-67) and M26 CAR-T cells obtained in Examples 3 and 4 were adjusted using UTD to be consistent with the group of CAR-T cells with the lowest CAR-positive ratio. In the antigen stimulation experiment, each group of CAR-T cells was co-cultured with positive target cells HL60 at an effector-to-target ratio of 1:2 in 24-well plates with 2 ml of X-VIVO15 medium per well, and each group of cells was replicated in 3 wells. CD3 was detected using a fluorescently labeled CD3 antibody (manufacturer: BioLegend, catalog number: 300312), and the proliferation of T cells was analyzed by flow cytometry (CD3 is a marker for distinguishing T cells). The number of CD3-positive cells was calculated based on the volume fraction. Then, a certain amount of CAR-T cells from each group was added to the corresponding positive target cells at an effector-to-target ratio of 1:2 for a new round of stimulation, once every 2 days, and this was repeated for 3-4 rounds of stimulation. After repeated experiments, the 2-8 and 3-37 CAR cells with the worst proliferation were excluded. The remaining 5 types of CLL1 CAR-T cells (i.e., 1-3, 1-16, 1-28, 3-64, 3-67) and M26 CAR-T cells were stimulated and their proliferation was statistically analyzed using the above method. It was found that 1-3 and 1-28 had stronger sustained proliferation ability, significantly higher than the positive control M26 CAR-T cells. The sustained proliferative capacity of 1-16 and 3-67 was also good, while the sustained proliferative capacity of 3-64 was relatively poor, as shown in the results. Figure 2 As shown. In this application, almost all cells after multiple rounds of stimulation were CAR+ cells, therefore CAR+ expression rate is no longer used as an important indicator.

[0235] Example 6: Cell killing experiment using CLL1 CAR-T cells

[0236] Cell killing assay: Five types of CLL1 CAR-T cells (i.e., 1-3, 1-16, 1-28, 3-64, and 3-67) and M26 CAR-T cells obtained through screening in Examples 3, 4, and 5, were co-cultured with target cells in X-VIVO15 medium for 16-24 hours at different effector-to-target cell ratios (1:1, 3:1, or 9:1). The killing ratio of CAR-T cells to target cells was detected by measuring the activity of luciferase stably expressed in the target cells. Target cells used were HL60 cells, K562-CLL1 cells, and K562 cells, where HL60 cells were endogenously CLL1-expressing positive target cells, K562-CLL1 cells were exogenously CLL1-expressing positive target cells, and K562 cells were CLL1-negative target cells. The cell killing results are as follows: Figures 3A-3C As shown: In the HL60 tumor cell line, all CAR-T cells exhibited significant killing effects; in the K562-CLL1 tumor cell line, CAR-T cells 1-16 showed poor killing effects, while the remaining CAR-T cells showed very significant effects; in the K562 cell line, as expected, none of the groups showed any killing effect. Based on the cell killing experiments, CAR-T cells 1-16 can be further excluded.

[0237] Example 7: Cytokine Release Experiment Using CLL1 CAR-T Cells

[0238] Cytokine release assay: Five types of CLL1 CAR-T cells (i.e., 1-3, 1-16, 1-28, 3-64, and 3-67), M26 CAR-T cells, and UTD cells obtained through screening in Examples 3, 4, and 5, were co-cultured with target cells at an effector-to-target ratio of 1:1 in X-VIVO15 medium for 24 h. The concentration of IFN-γ in the cell supernatant was detected by ELISA. Target cells included HL60 cells, K562-CLL1 cells, and K562 cells, where HL60 cells were endogenously CLL1-expressing positive target cells, K562-CLL1 cells were exogenously CLL1-expressing positive target cells, and K562 cells were CLL1-negative target cells. The results of the cytokine release assay are as follows: Figure 4 As shown, the release levels of CAR-T cytokines in 1-3 and 1-28 were significantly higher than those in the positive control M26 CAR-T, and the release levels of CAR-T cytokines in 3-67 were also higher than those in the positive controls M26 and 3-64.

[0239] In summary, in vitro pharmacodynamic assays showed that CARs with 1-3 scFv and 1-28 scFv as the extracellular antigen recognition domain exhibited high expression rates on T cell surfaces, strong antigen recognition ability, good specificity at both protein and cellular levels, good sustained proliferation, strong in vitro cell-killing ability, and high cytokine release levels; in particular, their sustained proliferation and cytokine release levels were significantly superior to the positive control M26 scFv. CARs with 3-67 scFv as the extracellular antigen recognition domain also showed strong antigen recognition ability, good specificity at both protein and cellular levels, good sustained proliferation, strong in vitro cell-killing ability, and high cytokine release levels.

[0240] Sequence Description

[0241] SEQ ID NO:1: 1-3 scFv amino acid sequence;

[0242] SEQ ID NO:2: 1-28 scFv amino acid sequence;

[0243] SEQ ID NO:3: 1-16 scFv amino acid sequence;

[0244] SEQ ID NO:4: 2-8 scFv amino acid sequence;

[0245] SEQ ID NO:5: 2-25 scFv amino acid sequence;

[0246] SEQ ID NO:6: 2-31 scFv amino acid sequence;

[0247] SEQ ID NO:7: 2-40 scFv amino acid sequence;

[0248] SEQ ID NO:8: 3-11 scFv amino acid sequence;

[0249] SEQ ID NO:9:3-13 scFv amino acid sequence;

[0250] SEQ ID NO:10: 3-16 scFv amino acid sequence;

[0251] SEQ ID NO:11: 3-35 scFv amino acid sequence;

[0252] SEQ ID NO:12: 3-37 scFv amino acid sequence;

[0253] SEQ ID NO:13: 3-64 scFv amino acid sequence;

[0254] SEQ ID NO:14: 3-67 scFv amino acid sequence;

[0255] SEQ ID NO:15: M26 scFv amino acid sequence;

[0256] SEQ ID NO:16: Amino acid sequence of the CD8α guide chain as a signal peptide;

[0257] SEQ ID NO:17: Amino acid sequence of the CD8α hinge region;

[0258] SEQ ID NO:18: Amino acid sequence of the CD8α transmembrane region;

[0259] SEQ ID NO:19: Amino acid sequence of 4-1BB intracellular co-stimulatory signal;

[0260] SEQ ID NO:20: Amino acid sequence of CD3ζ T cell activation signal;

[0261] SEQ ID NO:21: 1-3 CAR amino acid sequence;

[0262] SEQ ID NO:22: 1-28 CAR amino acid sequence;

[0263] SEQ ID NO:23: 1-16 CAR amino acid sequence;

[0264] SEQ ID NO:24: 2-8 CAR amino acid sequence;

[0265] SEQ ID NO:25: 2-25 CAR amino acid sequence;

[0266] SEQ ID NO:26: 2-31 CAR amino acid sequence;

[0267] SEQ ID NO:27:2-40 CAR amino acid sequence;

[0268] SEQ ID NO:28:3-11 CAR amino acid sequence;

[0269] SEQ ID NO:29:3-13 CAR amino acid sequence;

[0270] SEQ ID NO:30: 3-16 CAR amino acid sequence;

[0271] SEQ ID NO:31: 3-35 CAR amino acid sequence;

[0272] SEQ ID NO:32: 3-37 CAR amino acid sequence;

[0273] SEQ ID NO:33: 3-64 CAR amino acid sequence;

[0274] SEQ ID NO:34: 3-67 CAR amino acid sequence;

[0275] SEQ ID NO:35: M26 CAR amino acid sequence;

[0276] SEQ ID NO:36: 1-3 scFv VH CDR1 amino acid sequence;

[0277] SEQ ID NO:37: 1-3 scFv VH CDR2 amino acid sequence;

[0278] SEQ ID NO:38: 1-3 scFv VH CDR3 amino acid sequence;

[0279] SEQ ID NO:39: 1-3 scFv VL CDR1 amino acid sequence;

[0280] SEQ ID NO:40: 1-3 scFv VL CDR2 amino acid sequence;

[0281] SEQ ID NO:41: 1-3 scFv VL CDR3 amino acid sequence;

[0282] SEQ ID NO:42: 1-28 scFv VH CDR1 amino acid sequence;

[0283] SEQ ID NO:43: 1-28 scFv VH CDR2 amino acid sequence;

[0284] SEQ ID NO:44: 1-28 scFv VH CDR3 amino acid sequence;

[0285] SEQ ID NO:45: 1-28 scFv VL CDR1 amino acid sequence;

[0286] SEQ ID NO:46: 1-28 scFv VL CDR2 amino acid sequence;

[0287] SEQ ID NO:47: 1-28 scFv VL CDR3 amino acid sequence;

[0288] SEQ ID NO:48: 3-67 scFv VH CDR1 amino acid sequence;

[0289] SEQ ID NO:49: 3-67 scFv VH CDR2 amino acid sequence;

[0290] SEQ ID NO:50: 3-67 scFv VH CDR3 amino acid sequence;

[0291] SEQ ID NO:51: 3-67 scFv VL CDR1 amino acid sequence;

[0292] SEQ ID NO:52: 3-67 scFv VL CDR2 amino acid sequence;

[0293] SEQ ID NO:53: 3-67 scFv VL CDR3 amino acid sequence;

[0294] SEQ ID NO:54: 1-3 scFv VH amino acid sequence;

[0295] SEQ ID NO:55: 1-3 scFv VL amino acid sequences;

[0296] SEQ ID NO:56: 1-28 scFv VH amino acid sequence;

[0297] SEQ ID NO:57: 1-28 scFv VL amino acid sequence;

[0298] SEQ ID NO:58:3-67 scFv VH amino acid sequence;

[0299] SEQ ID NO:59:3-67 scFv VL amino acid sequence;

[0300] SEQ ID NO:60: A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:54;

[0301] SEQ ID NO:61: A nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:55;

[0302] SEQ ID NO:62: A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:56;

[0303] SEQ ID NO:63: A nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:57;

[0304] SEQ ID NO:64: A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:58;

[0305] SEQ ID NO:65: A nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:59;

[0306] SEQ ID NO:66: Linkage sequence;

[0307] SEQ ID NO:67: Linkage sequence;

[0308] SEQ ID NO:68: Linkage sequence.

Claims

1. A chimeric antigen receptor targeting CLL1, comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the CLL1 extracellular antigen recognition domain includes a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to those of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:54, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to those of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:

55. Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:56, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:

57. Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 heavy chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:58, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CLL1 light chain variable region are identical to the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:

59. The KABAT rule was used to divide the variable region (CDR) of the CLL1 heavy chain and the CLL1 light chain.

2. A chimeric antigen receptor targeting CLL1, comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the CLL1 extracellular antigen recognition domain includes a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of CLL1 are as shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of CLL1 are as shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively. Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of CLL1 are as shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of CLL1 are as shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively. Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of CLL1 are as shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of CLL1 are as shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively. The KABAT rule was used to divide the variable region (CDR) of the CLL1 heavy chain and the CLL1 light chain.

3. The chimeric antigen receptor according to claim 1 or 2, wherein, The amino acid sequence of the CLL1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:54, and the amino acid sequence of the CLL1 light chain variable region includes the amino acid sequence shown in SEQ ID NO:

55. Alternatively, the amino acid sequence of the CLL1 heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:56, and the amino acid sequence of the CLL1 light chain variable region may contain the amino acid sequence shown in SEQ ID NO:

57. Alternatively, the amino acid sequence of the CLL1 heavy chain variable region may contain the amino acid sequence shown in SEQ ID NO:58, and the amino acid sequence of the CLL1 light chain variable region may contain the amino acid sequence shown in SEQ ID NO:

59.

4. The chimeric antigen receptor according to claim 1 or 2, wherein, The CLL1 extracellular antigen recognition domain includes any one of the following structures: amino acid sequence-linked sequence as shown in SEQ ID NO:54-amino acid sequence as shown in SEQ ID NO:55; amino acid sequence-linked sequence as shown in SEQ ID NO:55-amino acid sequence as shown in SEQ ID NO:54; amino acid sequence-linked sequence as shown in SEQ ID NO:56-amino acid sequence as shown in SEQ ID NO:57; amino acid sequence-linked sequence as shown in SEQ ID NO:57-amino acid sequence as shown in SEQ ID NO:56; amino acid sequence-linked sequence as shown in SEQ ID NO:58-amino acid sequence as shown in SEQ ID NO:59; amino acid sequence-linked sequence as shown in SEQ ID NO:59-amino acid sequence as shown in SEQ ID NO:

58.

5. The chimeric antigen receptor according to claim 4, wherein, The CLL1 extracellular antigen recognition domain comprises any one of the following sequences: amino acid sequence-linking sequence as shown in SEQ ID NO:55-amino acid sequence as shown in SEQ ID NO:54, amino acid sequence-linking sequence as shown in SEQ ID NO:57-amino acid sequence as shown in SEQ ID NO:56, amino acid sequence-linking sequence as shown in SEQ ID NO:59-amino acid sequence as shown in SEQ ID NO:

58.

6. The chimeric antigen receptor according to claim 5, wherein, The CLL1 extracellular antigen recognition domain contains an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

14.

7. The chimeric antigen receptor according to claim 6, wherein, The connection sequence is selected from one or more of the following sequences: SEQ ID NO:66, SEQ ID NO:67 and SEQ ID NO:

68.

8. The chimeric antigen receptor according to claim 1 or 2, wherein, The hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α.

9. The chimeric antigen receptor according to claim 8, wherein, The amino acid sequence of the hinge region is derived from CD8α.

10. The chimeric antigen receptor according to claim 9, wherein, The amino acid sequence of the hinge region includes the amino acid sequence shown in SEQ ID NO:

17.

11. The chimeric antigen receptor according to claim 1 or 2, wherein, The transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

12. The chimeric antigen receptor according to claim 11, wherein, The amino acid sequence of the transmembrane region is derived from CD8α.

13. The chimeric antigen receptor according to claim 12, wherein, The amino acid sequence of the transmembrane region includes the amino acid sequence shown in SEQ ID NO:

18.

14. The chimeric antigen receptor according to claim 1 or 2, wherein, The intracellular domain contains intracellular signal transduction regions.

15. The chimeric antigen receptor according to claim 14, 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.

16. The chimeric antigen receptor according to claim 15, wherein, The intracellular signal transduction region originates from CD3ζ.

17. The chimeric antigen receptor according to claim 16, wherein, The amino acid sequence of the intracellular signal transduction region includes the amino acid sequence shown in SEQ ID NO:

20.

18. The chimeric antigen receptor according to claim 1 or 2, wherein, The intracellular domain also includes a co-stimulatory signal transduction region.

19. The chimeric antigen receptor according to claim 18, wherein, The co-stimulatory signal transduction region is derived from one, two, or more of the following: CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

20. The chimeric antigen receptor according to claim 19, wherein, The co-stimulatory signal transduction region originates from CD28 or 4-1BB.

21. The chimeric antigen receptor according to claim 20, wherein, The amino acid sequence of the co-stimulatory signal transduction region includes the amino acid sequence shown in SEQ ID NO:

19.

22. The chimeric antigen receptor according to claim 1 or 2, wherein, It also contains a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence.

23. The chimeric antigen receptor according to claim 22, wherein, The guide peptide mentioned above is derived from CD8α.

24. The chimeric antigen receptor according to claim 23, wherein, The amino acid sequence of the guide peptide includes the amino acid sequence shown in SEQ ID NO:

16.

25. The chimeric antigen receptor according to claim 1 or 2, wherein, The chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:

34.

26. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor as described in any one of claims 1-25.

27. The isolated nucleic acid molecule according to claim 26, wherein, The nucleotide sequence encoding the chimeric antigen receptor includes: 1) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:54; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:55; or 2) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:56; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:57; or 3) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:58; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:

59.

28. The isolated nucleic acid molecule according to claim 27, wherein, The nucleotide sequence encoding the chimeric antigen receptor includes: 1) The nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:54 is shown in SEQ ID NO:60; and the nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:55 is shown in SEQ ID NO:61; or 2) The nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:56 is shown in SEQ ID NO:62; and the nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:57 is shown in SEQ ID NO:63; or 3) The nucleotide sequences encoding the amino acid sequence of the CLL1 heavy chain variable region as shown in SEQ ID NO:58 are shown in SEQ ID NO:64; and the nucleotide sequences encoding the amino acid sequence of the CLL1 light chain variable region as shown in SEQ ID NO:59 are shown in SEQ ID NO:

65.

29. A vector comprising the isolated nucleic acid molecule of any one of claims 26-28.

30. The carrier according to claim 29, wherein, The carrier is an expression carrier.

31. The carrier according to claim 30, wherein, The vector is a viral vector.

32. The carrier according to claim 31, wherein, The vector is a lentiviral vector.

33. An engineered immune effector cell comprising a chimeric antigen receptor as described in any one of claims 1-25, an isolated nucleic acid molecule as described in any one of claims 26-28, or a vector as described in any one of claims 29-32; in, The engineered immune effector cells are selected from one or more of T lymphocytes and natural killer cells.

34. The engineered immune effector cells according to claim 33, wherein, The engineered immune effector cells are T lymphocytes.

35. The engineered immune effector cells according to claim 34, wherein, The T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes.

36. The engineered immune effector cells according to claim 35, wherein, The T lymphocytes are either αβT lymphocytes or γδT lymphocytes.

37. A pharmaceutical composition comprising engineered immune effector cells as described in any one of claims 33-36 and pharmaceutically acceptable excipients.

38. The pharmaceutical composition according to claim 37, wherein, Pharmaceutically acceptable excipients include protective agents.

39. The pharmaceutical composition according to claim 38, wherein, Pharmaceutically acceptable excipients include cell cryopreservation solutions.

40. The pharmaceutical composition according to claim 37, wherein, The pharmaceutical composition is a cell suspension or frozen cells thereof.

41. The pharmaceutical composition according to claim 40, wherein, The pharmaceutical composition is an intravenous injection.

42. The use of the chimeric antigen receptor of any one of claims 1-25, the isolated nucleic acid molecule of any one of claims 26-28, the vector of any one of claims 29-32, or the engineered immune effector cell of any one of claims 33-36 in the preparation of a medicament for the treatment of acute myeloid leukemia.

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