WO2024032247A1 - 一种cll1抗体及其应用 - Google Patents

一种cll1抗体及其应用 Download PDF

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Publication number
WO2024032247A1
WO2024032247A1 PCT/CN2023/104737 CN2023104737W WO2024032247A1 WO 2024032247 A1 WO2024032247 A1 WO 2024032247A1 CN 2023104737 W CN2023104737 W CN 2023104737W WO 2024032247 A1 WO2024032247 A1 WO 2024032247A1
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Prior art keywords
seq
amino acid
cll1
acid sequence
variable region
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PCT/CN2023/104737
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English (en)
French (fr)
Inventor
白大勇
张超
张其猛
周立
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合源康华医药科技(北京)有限公司
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Priority claimed from CN202210948681.9A external-priority patent/CN115850476B/zh
Priority claimed from CN202210949765.4A external-priority patent/CN115850505A/zh
Application filed by 合源康华医药科技(北京)有限公司 filed Critical 合源康华医药科技(北京)有限公司
Publication of WO2024032247A1 publication Critical patent/WO2024032247A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of biomedicine, specifically to a CLL1 antibody and its application.
  • AML Acute Myeloid Leukemia
  • myeloid hematopoietic stem/progenitor cells mainly characterized by abnormal proliferation of primitive and immature myeloid cells in bone marrow and peripheral blood. Its clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc. Most cases are in critical condition and the prognosis is dire. If not treated in time, Often life-threatening. Infants and young children are more likely to develop AML than adults, and this disease accounts for 30% of childhood leukemias. Current treatments include: chemotherapy, supportive care, and hematopoietic stem cell transplantation.
  • Chimeric Antigen Receptor is the core component of CAR cell therapy, which can include antigen recognition domain, hinge region, transmembrane region and intracellular domain. So far, the antigen recognition domain has been derived from the single chain variable region of an antibody (Single Chain Variable Fragment, abbreviated as scFv), or from receptor-ligand interactions, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR); the most common source is scFv antibodies.
  • CAR-T cell immunotherapy is considered to be one of the most promising methods to conquer tumors.
  • CAR-T cells use genetic modification methods to make T cells express CAR proteins.
  • This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.
  • CAR-T cell immunotherapy has achieved remarkable results in the treatment of various hematological tumors, such as CD19 CAR-T for the treatment of B-cell lymphoma and BCMA CAR-T for the treatment of multiple myeloma.
  • drugs on the market due to the heterogeneity of AML, it is difficult to find ideal CAR-T targets for the treatment of AML.
  • the existing targets for AML include CD33, CD123, LeY, NKG2D, etc., but there are no marketed CAR-T targets for these targets. T drugs.
  • CLL1 C-type Lectin-like Molecule 1, C-type lectin-like molecule-1
  • CLL1 C-type Lectin-like Molecule 1, C-type lectin-like molecule-1
  • This application provides a CLL1 antibody and its application.
  • the inventor has developed multiple antibodies targeting CLL1.
  • the corresponding scFv antibody was used as the extracellular antigen recognition domain of the CLL1CAR structure, thereby constructing a chimeric antigen receptor expression vector, preparing CAR-T cells targeting CLL1, and verifying CLL1CAR and CLL1CAR- at the cellular level.
  • a number of indicators of T cells were measured, and the three best-performing antibodies and three chimeric antigen receptors were selected.
  • 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 of the heavy chain variable region are respectively the same as those shown in SEQ ID NO: 54
  • the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are the same, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively the same as those in the light chain variable region of the antibody shown in SEQ ID NO:55.
  • the amino acid sequences of CDR1, CDR2, and CDR3 are the same;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO: 56, and the light chain can be
  • the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody light chain shown in SEQ ID NO:57;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO: 58
  • the light chain can be
  • the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody light chain shown in SEQ ID NO:59.
  • 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 of the heavy chain variable region respectively include SEQ ID NO: 36, SEQ ID NO:37, the amino acid sequence shown in SEQ ID NO:38, the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region respectively include SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO :The amino acid sequence shown in 41;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region respectively include the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the light chain variable region
  • amino acid sequences of CDR1, CDR2 and CDR3 of the region respectively include the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, and the light chain variable region
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the region include the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.
  • the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 54
  • the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:54.
  • amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 56
  • amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 57;
  • amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:58
  • amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:59.
  • the CLL1 antibody or its antigen-binding site is a CLL1 scFv antibody, a CLL1Sc(Fv) 2 antibody, or a CLL1[Sc(Fv) 2 ] 2 antibody.
  • the CLL1 scFv antibody includes any one selected from the following sequences: the amino acid sequence shown in SEQ ID NO: 54 - the connecting sequence - such as SEQ ID NO: The amino acid sequence shown in 55, the amino acid sequence shown in SEQ ID NO: 55 - the connecting sequence - the amino acid sequence shown in SEQ ID NO: 54, the amino acid sequence shown in SEQ ID NO: 56 - the connecting sequence - such as The amino acid sequence shown in SEQ ID NO: 57, the amino acid sequence shown in SEQ ID NO: 57 - the connecting sequence - the amino acid sequence shown in SEQ ID NO: 56, the amino acid sequence shown in SEQ ID NO: 58 - Connection sequence - an amino acid sequence as shown in SEQ ID NO: 59, amino acid sequence as shown in SEQ ID NO: 59 - connection sequence - an amino acid sequence as shown in SEQ ID NO: 58; optionally, the CLL1
  • the linking sequence is selected from one or more of the following sequences: SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned CLL1 antibody or its antigen-binding site.
  • the nucleotide sequence encoding the above-mentioned CLL1 antibody or its antigen-binding site includes:
  • the amino acids of the light chain variable region is set forth in SEQ ID NO: 61; or
  • 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 encoding as shown in SEQ ID NO:57 The nucleotide sequence of the amino acid sequence of the light chain variable region, optionally, is shown in SEQ ID NO: 63; or
  • the nucleotide sequence of the amino acid sequence of the light chain variable region optionally, is as shown in SEQ ID NO: 65.
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • the present application also provides a cell comprising any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof, isolated nucleic acid molecules or vectors.
  • This application also provides a pharmaceutical composition, which includes any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof, isolated nucleic acid molecules, vectors or cells, and pharmaceutically acceptable excipients.
  • the present application also provides an antibody drug, which contains any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof.
  • the antibody drug is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.
  • the present application also provides an antibody-drug conjugate, which contains any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof.
  • This application also provides the use of the above-mentioned CLL1 antibody or its antigen-binding site, isolated nucleic acid molecule, vector or cell in the preparation of a drug for treating diseases or conditions related to the expression of CLL1.
  • the disease or disorder associated with expression of CLL1 is a hematoma.
  • the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.
  • This application also provides the use of the above-mentioned CLL1 antibody or its antigen-binding site, isolated nucleic acid molecule, vector or cell in preparing detection reagents for diagnosing diseases or conditions related to the expression of CLL1.
  • the disease or disorder associated with expression of CLL1 is a hematoma.
  • the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.
  • the present application also provides a method for treating diseases or conditions related to the expression of CLL1, comprising the following steps: adding an effective amount of an antibody containing any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof, isolated nucleic acid molecules, vectors or cells.
  • the medicament is administered to a subject in need of treatment of a disease or disorder associated with expression of CLL1.
  • the disease or condition associated with expression of CLL1 is a hematoma.
  • the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.
  • the present application also provides a medicine, which contains the above-mentioned CLL1 antibody or its antigen-binding site, isolated nucleic acid molecule, vector or cell, for treating diseases or conditions related to the expression of CLL1.
  • the disease or disorder associated with the expression of CLL1 is a hematoma.
  • the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.
  • the present application provides a chimeric antigen receptor targeting CLL1, which includes a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain, and the CLL1 extracellular antigen recognition domain includes a CLL1 heavy chain variable region and CLL1 light chain variable region, where:
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO: 54.
  • the CLL1 light chain can The amino acid sequences of CDR1, CDR2 and CDR3 in the variable region are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody light chain shown in SEQ ID NO:55;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 56, and the CLL1 light
  • amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the chain are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the light chain of the antibody shown in SEQ ID NO:57;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 58, and the CLL1 light
  • amino acid sequences of CDR1, CDR2, and CDR3 in the chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO: 59.
  • the present application also provides a chimeric antigen receptor targeting CLL1, which includes a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain, and the CLL1 extracellular antigen recognition domain includes CLL1 Heavy chain variable region and CLL1 light chain variable region, where:
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region include the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38 respectively, and the CLL1 light chain variable region
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the region respectively include the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region respectively include the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the CLL1 light chain
  • amino acid sequences of CDR1, CDR2, and CDR3 of the variable region include the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 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 CLL1 light chain
  • amino acid sequences of CDR1, CDR2, and CDR3 of the variable region include the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53 respectively.
  • the amino acid sequence of the CLL1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 54, and the CLL light chain variable region sequence includes the amino acid sequence shown in SEQ ID NO.
  • amino acid sequence of the CLL1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:56, and the amino acid sequence of the CLL light chain variable region includes the amino acid sequence shown in SEQ ID NO:57;
  • amino acid sequence of the CLL1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:58
  • amino acid sequence of the CLL light chain variable region includes the amino acid sequence shown in SEQ ID NO:59.
  • the CLL1 extracellular antigen recognition domain includes any one selected from the following structures: the amino acid sequence shown in SEQ ID NO: 54 - the connecting sequence - such as SEQ ID NO : The amino acid sequence shown in SEQ ID NO: 55, the amino acid sequence shown in SEQ ID NO: 55 - the connecting sequence - the amino acid sequence shown in SEQ ID NO: 54, the amino acid sequence shown in SEQ ID NO: 56 - the connecting sequence - Amino acid sequence as shown in SEQ ID NO:57, Amino acid sequence as shown in SEQ ID NO:57 - connecting sequence - Amino acid sequence as shown in SEQ ID NO:56, Amino acid sequence as shown in SEQ ID NO:58 - connecting sequence - the amino acid sequence shown in SEQ ID NO: 59, the amino acid sequence shown in SEQ ID NO: 59 - connecting sequence - the amino acid sequence shown in SEQ ID NO: 58; optionally, the CLL1 The extracellular antigen recognition domain
  • the linking sequence is selected from one or more of the following sequences: SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.
  • the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ ; optionally, the amino acids in the hinge region Derived from CD8 ⁇ ; further optionally, the amino acid sequence of the hinge region includes the amino acid sequence shown in SEQ ID NO: 17.
  • the transmembrane region is derived from one of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70 or Multiple; optionally, the amino acid sequence of the transmembrane region is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 18.
  • the intracellular domain includes an intracellular signaling region; optionally, it also includes a costimulatory signaling region.
  • the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, Syk One or more of them; optionally, the intracellular signaling region is derived from CD3 ⁇ ; further optionally, the amino acid sequence of the intracellular signaling region includes the amino acid sequence shown in SEQ ID NO: 20 .
  • the costimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1 , one, two or more of ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3 and MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; further optionally Preferably, the amino acid sequence of the costimulatory signaling region includes the amino acid sequence shown in SEQ ID NO: 19.
  • the above chimeric antigen receptor also includes a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, wherein the guide peptide is derived from CD8 ⁇ ; further optionally , the amino acid sequence of the guide peptide includes the amino acid sequence shown in SEQ ID NO: 16.
  • the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 34.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned chimeric antigen receptor.
  • the nucleotide sequence encoding the chimeric antigen receptor includes:
  • the nucleotide sequence of the amino acid sequence of the region optionally, is shown in SEQ ID NO: 61; or
  • the nucleotide sequence of the amino acid sequence of the CLL1 light chain variable region shown, optionally, is shown in SEQ ID NO: 63; or
  • the nucleotide sequence of the amino acid sequence of the CLL1 light chain variable region is shown, optionally, as shown in SEQ ID NO: 65.
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • the above vector is an expression vector; in some embodiments, the vector is a viral vector; in some embodiments, the vector is a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which contains the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.
  • the engineered immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), multipotent One or more of stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells.
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes. Lymphocytes.
  • the T lymphocytes are ⁇ T lymphocytes or ⁇ T lymphocytes.
  • This application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include protective agents.
  • pharmaceutically acceptable excipients include cell cryopreservation solution.
  • the pharmaceutical composition is a cell suspension or cryopreserved cells thereof.
  • the pharmaceutical composition is an intravenous injection.
  • the present 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 medicines for treating diseases or conditions related to the expression of CLL1.
  • the disease or disorder associated with expression of CLL1 is a hematoma.
  • the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.
  • the present application also provides a method for treating diseases or conditions related to the expression of CLL1, comprising the following steps: applying an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical compositions to patients with a disease or disorder related to the expression of CLL1. Subjects in need of disease or condition.
  • the disease or condition associated with expression of CLL1 is a hematoma.
  • the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.
  • the administration is by intravenous injection.
  • the administration method is to administer an effective amount of engineered immune effector cells or pharmaceutical compositions to the subject in a single or multiple injections.
  • the effective amount of engineered immune effector cells or pharmaceutical composition is a dose of 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/kg.
  • the present application also provides a medicine, which contains the above-mentioned engineered immune effector cells or pharmaceutical composition, for treating diseases or conditions related to the expression of CLL1.
  • the disease or disorder associated with the expression of CLL1 is a hematoma.
  • the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.
  • Figure 1 shows a schematic structural diagram of various CLL1 CARs in Example 2 of the present application.
  • they are: CD8a signal peptide, anti-CLL1 scFv, CD8a hinge region and transmembrane region, 4-1BB, and CD3 ⁇ .
  • Figure 2 shows the continued proliferation of CD3 + cells in each group of CAR-T cells after antigen stimulation in Example 5 of the present application.
  • Figures 3A to 3C show the lysis and killing effects of various CLL1 CAR-T cells and positive control M26 CLL1 CAR-T cells on different target cells in Example 6 of the present application; wherein: the target cell in Figure 3A is HL60; The target cell in Figure 3B is K562-CLL1; the target cell in Figure 3C is K562.
  • Figure 4 shows the cytokine IFN- ⁇ release of various CLL1 CAR-T cells, positive control M26 CLL1 CAR-T cells, and UTD cells (T cells without CAR transduction) after being activated by positive target cells in Example 7 of the present application.
  • the term "antibody” has its conventional meaning in the art.
  • H chains antibody heavy chains
  • L chains light chains
  • V region in the antibody light chain and heavy chain near the N-terminal amino acid sequence that changes greatly
  • V region in the antibody light chain and heavy chain near the C-terminal amino acid sequence that is relatively stable
  • V constant region
  • C the V regions of heavy chain and light chain are abbreviated as VH and VL respectively
  • CH and CL the C regions of heavy chain and light chain
  • hypervariable regions HVR
  • CDRs complementarity determining regions
  • common CDR classification rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying the website that implements these rules, just enter the VH and VL sequences and select the corresponding rules. CDR sequences based on different rules can be obtained.
  • the protection scope of the present application covers combinations of CDR sequences obtained by analyzing using different rules.
  • the six CDR regions of the antibody jointly determine the antibody's recognition ability and specificity for the corresponding antigen.
  • the amino acid sequences of the six CDR regions are defined in this application, the recognition ability and specificity of the antibody to the corresponding antigen can be expected.
  • the term "antigen-binding site” has a conventional meaning in the art and refers to the key site on the antibody that can specifically recognize and bind to the antigen, including the VH and VL regions.
  • the term "monoclonal antibody” has a conventional meaning in the art and refers to an antibody that is highly uniform and only targets a specific antigenic epitope, which can be obtained through known methods such as hybridoma technology, antibody library technology, and transgene technology prepared using mouse technology or single-cell PCR technology.
  • scFv has a conventional meaning in the art and refers to a single chain variable region (Single Chain Variable Fragment, abbreviated as scFv), which is composed of an antibody heavy chain variable region and a light chain variable region.
  • CAR Chimeric Antigen Receptor
  • extracellular antigen recognition domains for example, binding to tumor-associated antigens (Tumor-Associated Antigen) , part of TAA), hinge region, transmembrane region and intracellular domain.
  • CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered to be one of the most promising methods to conquer tumors.
  • CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.
  • extracellular antigen recognition domain refers to the antigen recognition domain (Antigen Recognition Domain, ARD).
  • CAR cell therapy products such as CAR-T cells
  • the antigen recognition domain has been derived from the single antigen of an antibody.
  • Chain variable region Single Chain Variable Fragment, abbreviated as scFv
  • TCR single Chain Variable Fragment
  • VLR variable lymphocyte receptors
  • the most common source is the scFv segment of the antibody.
  • the scFv includes the antibody heavy chain variable region and the light chain variable region, which are connected by a peptide chain, such as: the connection sequence GSTGSSGKPGSGEGSTKG consisting of 18 amino acids.
  • the term "specific recognition and/or binding” refers to the recognition and/or binding between the CAR and the specific target with greater affinity, avidity, easier, and greater affinity than the CAR binding to other targets. and/or bind the target for a greater duration.
  • hinge region refers to the connecting segment between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by giving the antigen recognition domain a certain range of activity.
  • the hinge regions currently used are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ .
  • transmembrane region refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but they are not directly involved in signal transmission. They can improve downstream signal transmission through interactions.
  • the transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.
  • intracellular domain includes intracellular signaling regions and may also include costimulatory signaling regions. guidance area.
  • intracellular signaling region refers to the activation of at least one normal effector function of a CAR-expressing immune effector cell.
  • the intracellular signaling region can be derived from one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12, and Syk.
  • the term "costimulatory signaling region" exists because in addition to stimulation by antigen-specific signals, many immune effector cells require costimulation to promote cell proliferation, differentiation, and survival, as well as activation of cells. Effector functions.
  • the CAR may also include one or more costimulatory signaling domains, wherein the costimulatory signaling domains may be derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4- One, two or more of 1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3 and MyD88.
  • guide peptide refers to the short peptide before the extracellular antigen recognition domain (such as scFv sequence), which functions to guide the recombinant protein synthesized in the cell to be exported to the outside of the cell.
  • CLL1 cancer target antigen 1
  • KLR1 and CLEC12A KLR1 and CLEC12A.
  • CLL1 C-type Lectin-like Molecule 1
  • CLL1 C-type Lectin-like Molecule 1
  • 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 invention.
  • the linking sequence can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linker sequences can be used when it is desired to ensure that two adjacent domains do not interfere with each other spatially.
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolated does not exclude substances that have been artificially or synthetically obtained from their natural state by artificial means, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • isolated nucleic acid molecule generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, which may be isolated from its natural environment or a synthetic analog .
  • gene transduction/transfection methods mainly include viral and non-viral methods.
  • viral and non-viral methods For example: through gamma retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed.
  • the vector can transform, transduce or transfect the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • Viruses (such as SV40).
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site. Vectors may also contain components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but they are not the only ones.
  • transposon refers to a discontinuous segment of DNA that has the ability to migrate and carry genetic information between chromosomal sites, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects.
  • electroporation can also be used to transduce mRNA into T cells.
  • Immune effector cells generally refers to cells that participate in immune responses, such as promoting immune effector responses.
  • Immune effector cells may be selected from the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, pluripotent stem cells differentiated into One or more of NK cells and embryonic stem cells.
  • 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 : One or more of carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.
  • pharmaceutically acceptable excipients include protective agents, such as cell cryopreservation solutions.
  • the pharmaceutical composition of the present application is a cell suspension or cryopreserved cells thereof.
  • subject generally refers to a human or non-human animal, including but not limited to mouse, rat, cat, dog, rabbit, horse, pig, cow, sheep, or monkey.
  • the term "about” generally refers to a range of fluctuations above or below the specified value that is acceptable to those skilled in the art, such as: within the range of ⁇ 0.5%-10%, such as 0.5 above or below the specified value. %, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, Changes within the range of 9%, 9.5% or 10%.
  • CLL1 antibody or its antigen-binding site corresponding nucleic acid molecules, corresponding vectors, corresponding cells, and corresponding pharmaceutical compositions
  • the present application provides a CLL1 antibody or an antigen-binding site thereof, which includes a heavy chain variable region and a light chain variable region, and the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are respectively the same as SEQ.
  • the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in ID NO:54 are the same, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are respectively the same as those of the antibody shown in SEQ ID NO:55.
  • the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are identical;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO: 56, and the light chain can be
  • the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody light chain shown in SEQ ID NO:57;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO: 58
  • the light chain can be
  • the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody light chain shown in SEQ ID NO:59.
  • CDR classification rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying the website that implements these rules, just enter the VH and VL sequences and select the corresponding rules. , you can get CDR sequences based on different rules. Those skilled in the art will understand that even for the same heavy chain variable region and light chain variable region, different CDR sequences can be obtained according to different CDR classification rules. Those skilled in the art should understand that the protection scope of the present application covers combinations of CDR sequences obtained by analyzing using different rules.
  • the present application also provides a CLL1 antibody or an antigen-binding site thereof, which includes a heavy chain variable region and a light chain variable region, and the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are respectively Contains the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region respectively include SEQ ID NO:39, The amino acid sequence shown in SEQ ID NO:40 and SEQ ID NO:41;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the light chain variable region
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the region include SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47 respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 of 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 light chain variable region
  • amino acid sequences of CDR1, CDR2 and CDR3 of the region include the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53 respectively.
  • KABAT rules are used to classify CDRs.
  • the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region Contains the amino acid sequence shown in SEQ ID NO:55;
  • amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:56, and the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:57;
  • amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:58
  • amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:59.
  • 1-3 scFv antibodies include the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:54 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:55;
  • 1-28 scFv antibodies include the amino acid sequence A heavy chain variable region as shown in SEQ ID NO:56 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:57;
  • the 3-67scFv antibody includes a heavy chain with an amino acid sequence as shown in SEQ ID NO:58
  • the variable region and amino acid sequence are the light chain variable region shown in SEQ ID NO:59.
  • any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof are CLL1 scFv antibodies, CLL1Sc(Fv) 2 antibodies, and CLL1[Sc(Fv) 2 ] 2 antibodies.
  • the CLL1 scFv antibody comprises any one selected from the following sequences: the amino acid sequence shown in SEQ ID NO: 54 - the connecting sequence - such as SEQ ID
  • the linking sequence is selected from one or more of the following sequences: SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above-mentioned CLL1 antibodies or its antigen-binding site.
  • the nucleotide sequence encoding the above-mentioned CLL1 antibody or its antigen-binding site includes:
  • the nucleotide sequence of the amino acid sequence of the light chain variable region optionally, is shown in SEQ ID NO: 61; or
  • 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 encoding as shown in SEQ ID NO:57 The nucleotide sequence of the amino acid sequence of the light chain variable region, optionally, is shown in SEQ ID NO: 63; or
  • the nucleotide sequence of the amino acid sequence of the light chain variable region optionally, is as shown in SEQ ID NO: 65.
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • the vector can be arbitrarily selected from one or more of the following vectors: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses.
  • Viruses such as SV40
  • the present application also provides a cell comprising the above-mentioned CLL1 antibody or its antigen-binding site, isolated nucleic acid molecule or vector.
  • the present application also provides a pharmaceutical composition, which includes any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof, isolated nucleic acid molecules, vectors or cells, and pharmaceutically acceptable excipients.
  • Pharmacy Acceptable excipients include, but are not limited to: one or more of carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.
  • Antibody drugs and antibody drug conjugates containing CLL1 antibodies or their antigen-binding sites are included in the group consisting of CLL1 antibodies or their antigen-binding sites.
  • this application provides an antibody drug (Antibody-Drug Conjugate, ADC), which contains any of the above-mentioned CLL1 antibodies or its antigen-binding site.
  • ADC Antibody-Drug Conjugate
  • the antibody drug is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.
  • the present application also provides an antibody-drug conjugate, which contains any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof.
  • the present application provides a use of any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof, isolated nucleic acid molecules, vectors or cells in the preparation of medicaments for the treatment of diseases or conditions related to the expression of CLL1.
  • the disease or disorder associated with the expression of CLL1 is a hematoma.
  • the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.
  • 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 the expression of CLL1.
  • the disease or disorder associated with the expression of CLL1 is a hematoma.
  • the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.
  • the present application also provides a method for treating diseases or conditions related to the expression of CLL1, including the following steps: adding an effective amount of an effective amount of a nucleic acid molecule containing any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof, isolated nucleic acid molecules, The drug of the vector or cells is administered to a subject in need of treatment of a disease or disorder associated with expression of CLL1.
  • the disease or condition associated with expression of CLL1 is a hematoma.
  • the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.
  • the administration can be carried out in different ways, such as oral, intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the dosage may be different for different indications; the dosage may also be different for patients with different severity of illness.
  • the subjects may include humans and non-human animals.
  • the subject may include, but is not limited to, mouse, rat, cat, dog, horse, pig, cow, sheep, rabbit or monkey.
  • the present application also provides a medicine, which contains any of the above-mentioned CLL1 antibodies or antigen-binding sites thereof, isolated nucleic acid molecules, vectors or cells, for treating diseases or conditions related to the expression of CLL1.
  • the disease or condition associated with the expression of CLL1 is a hematoma.
  • the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.
  • CLL1 chimeric antigen receptor corresponding nucleic acid molecules, corresponding vectors, corresponding immune effector cells, and pharmaceutical compositions
  • the present application provides a chimeric antigen receptor targeting CLL1, which includes a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain, and the CLL1 extracellular antigen recognition domain Including CLL1 heavy chain variable region and CLL1 light chain variable region, where:
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO: 54.
  • the CLL1 light chain can The amino acid sequences of CDR1, CDR2 and CDR3 in the variable region are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody light chain shown in SEQ ID NO:55;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 56, and the CLL1 light
  • amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the chain are respectively the same as the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the light chain of the antibody shown in SEQ ID NO:57;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 58, and the CLL1 light
  • amino acid sequences of CDR1, CDR2, and CDR3 in the chain variable region are respectively the same as the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO: 59.
  • the present application also provides a chimeric antigen receptor targeting CLL1, which includes a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain, and the CLL1 extracellular antigen recognition domain
  • the structural domain includes the CLL1 heavy chain variable region and the CLL1 light chain variable region, where:
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 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, and the CLL1 light chain variable region
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the region include the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the CLL1 light chain
  • amino acid sequences of CDR1, CDR2, and CDR3 of the variable region include the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 of the CLL1 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 CLL1 light chain
  • amino acid sequences of CDR1, CDR2, and CDR3 of the variable region include the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53 respectively.
  • the amino acid sequence of the CLL1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 54
  • the CLL light chain variable region sequence includes the amino acid sequence shown in SEQ ID NO: 54.
  • amino acid sequence of the CLL1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:56, and the amino acid sequence of the CLL light chain variable region includes the amino acid sequence shown in SEQ ID NO:57;
  • amino acid sequence of the CLL1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:58
  • amino acid sequence of the CLL light chain variable region includes the amino acid sequence shown in SEQ ID NO:59.
  • the CLL1 extracellular antigen recognition domain includes any one selected from the following structures: the amino acid sequence shown in SEQ ID NO: 54 - the connecting sequence - such as SEQ The amino acid sequence shown in ID NO:55, the amino acid sequence shown in SEQ ID NO:55 - connecting sequence - the amino acid sequence shown in SEQ ID NO:54, the amino acid sequence shown in SEQ ID NO:56 - connecting Sequence - amino acid sequence as shown in SEQ ID NO: 57, amino acid sequence as shown in SEQ ID NO: 57 - linking sequence - amino acid sequence as shown in SEQ ID NO: 56, as shown in SEQ ID NO: 58 Amino acid sequence - connecting sequence - the amino acid sequence shown in SEQ ID NO: 59, amino acid sequence shown in SEQ ID NO: 59 - connecting sequence - the amino acid sequence shown in SEQ ID NO: 58; optionally, the The CLL1 scFv antibody includes any one selected from the following sequence
  • the linking sequence is selected from one or more of the following sequences: SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68.
  • the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8 ⁇ ; optionally, the hinge region amino acids come from CD8 ⁇ ; further optionally, the amino acid sequence of the hinge region includes the amino acid sequence shown in SEQ ID NO: 17.
  • the transmembrane region is derived from one of CD3, CD4, CD7, CD8 ⁇ , CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.
  • One or more species optionally, the amino acid sequence of the transmembrane region is derived from CD8 ⁇ ; further optionally, the amino acid sequence of the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 18.
  • the intracellular domain includes an intracellular signaling region; optionally, it also includes a costimulatory signaling region.
  • the intracellular signaling region is derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, FcR ⁇ , FcR ⁇ , CD66d, DAP10, DAP12 , one or more of Syk; optionally, the intracellular signaling region is derived from CD3 ⁇ ; further optionally, the amino acid sequence of the intracellular signaling region includes as shown in SEQ ID NO:20 Amino acid sequence.
  • the costimulatory signaling region is derived from CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA -1.
  • any of the above chimeric antigen receptors also 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 CD8 ⁇ ; further optionally Preferably, the amino acid sequence of the guide peptide includes the amino acid sequence shown in SEQ ID NO:16.
  • the chimeric antigen receptor includes the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 34.
  • the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above chimeric antigen receptors.
  • the nucleotide sequence encoding the chimeric antigen receptor includes:
  • the nucleotide sequence of the amino acid sequence of the CLL1 light chain variable region shown, optionally, is shown in SEQ ID NO: 61; or
  • the nucleotide sequence of the amino acid sequence of the CLL1 light chain variable region shown, optionally, is shown in SEQ ID NO: 63;
  • the nucleotide sequence of the amino acid sequence of the CLL1 light chain variable region is shown, optionally, as shown in SEQ ID NO: 65.
  • the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.
  • the vector can be any one or more of the following: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome derived from P1 (PAC); phage Such as lambda phage or M13 phage and animal viruses.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses.
  • Viruses such as SV40
  • the above vector is an expression vector; in other embodiments, the vector is a viral vector; in other embodiments, the vector is a lentiviral vector.
  • the present application also provides an engineered immune effector cell, which contains the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.
  • the engineered immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), and pluripotent stem cells. , one or more of T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells.
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes. cell.
  • the above-mentioned engineered immune effector cells are ⁇ T lymphocytes or ⁇ T lymphocytes of T lymphocytes.
  • the surface of the engineered immune effector cells may express or express the chimeric antigen receptor described in the present application.
  • the present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients include, but are not limited to: one or more of carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.
  • pharmaceutically acceptable excipients include protective agents.
  • pharmaceutically acceptable excipients include cell cryopreservation solution.
  • the pharmaceutical composition is a cell suspension or cryopreserved cells thereof.
  • the pharmaceutical composition is an intravenous injection.
  • the present application also provides a method for preparing engineered immune effector cells, which includes the following steps: transducing the vector described in the application into the immune effector cells.
  • the engineered immune effector cells are selected from T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, and pluripotent stem cells differentiated into One or more of T cells, NK cells differentiated from pluripotent stem cells and embryonic stem cells.
  • NK cells natural killer cells
  • PBMC cells peripheral blood mononuclear cells
  • pluripotent stem cells pluripotent stem cells differentiated into One or more of T cells, NK cells differentiated from pluripotent stem cells and embryonic stem cells.
  • the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.
  • the T lymphocytes are ⁇ T lymphocytes or ⁇ T lymphocytes.
  • this application also provides the use of the above-mentioned chimeric antigen receptors, isolated nucleic acid molecules, vectors or engineered immune effector cells in the preparation of medicines for the treatment of diseases related to the expression of CLL1 or disease.
  • the disease or disorder associated with the expression of CLL1 is a hematoma.
  • the disease or disorder associated with the expression of CLL1 is acute myeloid leukemia.
  • the present application also provides a method for treating diseases or conditions related to the expression of CLL1, comprising the steps of: applying an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical compositions to patients with CLL1.
  • the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the method of administration may be by intravenous injection to the subject.
  • an effective dose of engineered immune effector cells or pharmaceutical compositions can be administered to the subject in a single time, or in divided doses within a certain period, such as once a week, Once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • the disease or condition associated with expression of CLL1 is a hematoma.
  • the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.
  • the administration method is intravenous injection.
  • the method of administration is to administer an effective amount of engineered immune effector cells or a pharmaceutical composition to the subject in a single injection.
  • the effective amount of engineered immune effector cells or pharmaceutical composition is a dose of 1 ⁇ 10 5 to 1 ⁇ 10 7 cells/kg.
  • the dosage may be different for different indications; the dosage may also be different for patients with different severity of illness.
  • the administered dose may range from 1 ⁇ 10 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,
  • the subjects may include humans and non-human animals.
  • the subject may include, but is not limited to, mouse, rat, cat, dog, horse, pig, cow, sheep, rabbit, or monkey.
  • the present application also provides a medicine, which contains the above-mentioned engineered immune effector cells or a pharmaceutical composition, for treating diseases or conditions related to the expression of CLL1.
  • the disease or condition associated with the expression of CLL1 is a hematoma.
  • the disease or condition associated with the expression of CLL1 is acute myeloid leukemia.
  • the following examples are only to illustrate the CLL1 antibodies, chimeric antigen receptors, engineered immune effector cells, preparation methods and uses of the present application, and are not intended to limit the invention of the present application. range.
  • the examples do not include a detailed description of traditional methods, such as those used to construct vectors and plasmids, insert genes encoding proteins into such vectors and plasmids, or introduce plasmids into host cells.
  • Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual ,2nd edition,Cold Spring Harbor Laboratory Press.
  • CLL1 antibody development process BALB/c and C57bl/6 mice were used as immunized animals, and human CLL1 was used as the antigen for immunization according to the procedure in Table 1.
  • the serum of each mouse was collected for FACS (flow cytometry) Fluorescence sorting technology) and ELISA detection, combining the results of ELISA detection and FACS detection, taking the mice with the best immune effect, enriching their plasma cells with CD138 antibodies and then seeding them on the Beacon screening chip to conduct antibody-positive B cells
  • FACS flow cytometry
  • ELISA detection Fluorescence sorting technology
  • the positive B cell clones were exported and V region sequenced. A total of 64 V region sequences of monoclonal antibodies were obtained. According to the sequence similarity classification, 14 representative sequences were selected for the synthesis of scFv in the CAR.
  • the CD8 ⁇ guide chain is 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 (shown) adopts the structure of CD8 ⁇ , with 4-1BB as the intracellular costimulatory signal (shown in SEQ ID NO:19), and CD3 ⁇ as the T cell activation signal (shown in SEQ ID NO:20).
  • the schematic diagram of the structure is shown in Figure 1 shown.
  • the above 15 CLL1 CAR structures were constructed into modified empty lentiviral vectors (manufacturer: SBI Company, product number: CD500-CD800, modified resistance as described in WO2021/121227 Example 1) to obtain the CAR expression vector, and then The CAR expression vector and three packaging plasmids were transfected into 293T cells, and functional lentiviral vectors were obtained after collection and purification.
  • the three packaging plasmids are pMD2.G (purchased from Biovector Company, product number Biovector012259), pMDLg/pRRE (purchased from Biovector Company, product number Biovector012251), and pRSV-Rev (purchased from Biovector Company, product number Biovector012253).
  • the transduction experiment was performed according to conventional methods known to those skilled in the art.
  • the transduction steps are briefly described as follows:
  • PBMC Peripheral blood mononuclear cells
  • T cell complete medium (X-VIVO15 medium + 5% FBS + 300 IU/ml IL-2 or X-VIVO15 medium + 5% FBS + 5ng/ml IL-15 + 10ng/ml IL -7) Resuspend to a final concentration of (1 ⁇ 3) ⁇ 10 6 cells/ml, add CD3/CD28 magnetic beads to stimulate 1ul beads/1 ⁇ 10 6 cells, mix well and place in an incubator for culture.
  • the culture conditions are 37°C + 5% CO 2 and the culture time is at least 24 hours.
  • the activated and cultured T cells resuspend them in simple X-VIVO15 medium containing polybrene at a final concentration of 8 ⁇ g/ml, mix well to obtain a cell suspension, and divide the cells into 800 ul cell suspension (containing 2 ⁇ 10 6 cells) slowly add 200ul lentiviral vector, mix well, put it into the well plate, and culture it in an incubator.
  • the culture conditions are 37°C + 5% CO 2 . at least 4-6 hours.
  • the transduced cells were taken out, expanded and cultured in T cell complete culture medium, and passaged every other day to maintain the cell density at (0.8-2) ⁇ 10 6 cells/ml for use in subsequent examples.
  • T cells obtained After infecting T cells with lentiviruses containing the CAR structures in Table 3, the T cells obtained are named according to their CAR serial numbers. For example, T cells obtained using 1-3CAR are named 1-3CAR-T cells, and T cells obtained using 2 The T cells obtained from -8CAR are named 2-8CAR-T cells.
  • T cells obtained using 1-3CAR are named 1-3CAR-T cells
  • the T cells obtained from -8CAR are named 2-8CAR-T cells.
  • Detection of CAR molecules To detect the CAR protein molecules expressed on the surface of 14 CLL1 CAR-T cells obtained in Example 2 and cultured for 7 days after transduction, we used PE fluorescently labeled CLL1 antigen (Manufacturer: ACRObiosystems, Cat. No. : CLA-PH2Q3) and FITC fluorescently labeled CLL1 antigen (manufacturer: ACRObiosystems, product number: CLA-HF247 25ug) on 14 types of CLL1 CAR-T cells, M26 CLL1 CAR-T cells and UTD cells (untransduced CAR) obtained in Example 2 T cells) were stained, and the positive proportion of CAR molecules was detected and analyzed by flow cytometry. The detection results are shown in Table 4.
  • Example 2 To test the antigen recognition ability of the 14 CAR protein molecules expressed on the surface of CLL1 CAR-T cells obtained in Example 2, we used FITC fluorescently labeled IgG (manufacturer: abcam, product number: ab98658 ) The 14 types of CLL1 CAR-T cells, M26 CLL1 CAR-T cells and UTD cells (T cells without CAR transduction) obtained in Example 2 were stained, and the antigen recognition ability was detected by flow cytometry. The detection results are as follows As shown in Table 4.
  • CAR expression rate is lower than 10%, it is considered as non-expression or extremely poor expression, which is lower than the screening standard; if the antigen binding recognition rate is lower than 10%, it is considered that the antigen recognition ability is poor, which is lower than the screening standard.
  • CLL1CAR Specific detection of CLL1CAR was performed at the protein level and cellular level.
  • CD19 protein (purchased from ACRObiosystems, Catalog No. CD9-HP2H3), CS1 protein (purchased from ACRObiosystems, Catalog No. SL7-HP2H3), BCMA protein (purchased from ACRObiosystems, Catalog No. BCA-HF254), GPC3 protein (purchased from ACRObiosystems, Catalog No. Catalog No. GP3-HF2H1), EGFRvIII protein (purchased from ACRObiosystems, Catalog No. EGI-HP2E3) and five antigenic proteins were screened according to the following method on the eight CLL1 CAR-T cells (1-3, 1-28, 1-16) obtained in Example 3.
  • the cell lines K562 (human chronic myelogenous leukemia cells), K562-CLL1 (K562 cells exogenously expressing 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 cutaneous melanoma cells), A549 ( Eight types of CLL1 CAR-T cells (1-3, 1-28, 1-16, 2) obtained by screening 11 cell lines from different tissues (human lung cancer cells) and HUH7 (human liver cancer cells) in Example 3 were screened according to the following method.
  • CD107a The expression of CD107a on the membrane is considered to be a sign of activated cytotoxic lymphocytes (CD8+ Markers of T cells and NK cells. When cytotoxic lymphocytes are stimulated by specific antigens, immune activation occurs. They express CD107a, so they can be used as a means to detect the specificity of CAR-T cells.
  • the specific steps are: count tumor cells and transfected and untransfected T cells, use X-VIVO medium to adjust tumor cells to 2 ⁇ 10 6 /ml, and adjust T cells to 1 ⁇ 10 6 /ml.
  • Add 20ul of CD107a antibody to each well of the killing plate then add 100ul of T cells and 100ul of tumor cells to each well, and centrifuge at 400r/min for 3 minutes after addition. Incubate in a 37°C 5% CO2 incubator for 60 minutes. After 60 minutes, add 20ul of golgi stop working solution (3ml Mix equal volumes of CD3 and CD8 antibodies, add 10ul to each well after mixing, and incubate in the incubator for 30 minutes.
  • 2-31CAR can be activated by negative target cells and exhibits CD107a signal. Therefore, it did not meet the screening criteria at the cellular level, so it was also excluded from subsequent experiments.
  • Antigen stimulation can activate CAR-T cells and cause CAR-T cell proliferation. However, continued activation of T cells will lead to cell exhaustion. The proliferation ability and effector function of exhausted T cells will be reduced. By detecting the presence of CLL1 CAR-T cells in multiple The proliferation of CD3+ cells (i.e. the proliferation of T cells) after the round antigen stimulation experiment was determined to determine their continued proliferation.
  • CLL1 CAR-T cells i.e., 1-3, 1-16, 1-28, 2-8, 3- 37, 3-64, 3-6
  • M26 CAR-T cells the CAR-positive proportions were adjusted by UTD to a level consistent with the group of CAR-T cells with the lowest CAR-positive proportion.
  • each group of CAR -T cells were co-cultured with the positive target cells HL60 in a 24-well plate at an effect-to-target ratio of 1:2, with 2 ml of X-VIVO15 medium per well, and each group of cells was repeated in 3 wells.
  • CD3 is a marker to distinguish whether they are T cells.
  • CD3 is a marker to distinguish whether they are T cells.
  • the volume multiple Convert and calculate the number of CD3-positive cells, and then take out a certain amount of CAR-T cells from each group according to the calculation results and add the corresponding positive target cells at an effect-to-target ratio of 1:2 for a new round of stimulation, once every 2 days, and repeat. Perform 3-4 rounds of stimulation. After repeated experiments many times, the 2-8 and 3-37CARs with the worst proliferation were eliminated.
  • CLL1 CAR-T cells Five types of CLL1 CAR-T cells (ie, 1-3, 1-16, 1-28, 3-64, 3-67) obtained through screening in Example 3, Example 4, and Example 5, M26CAR-T cells were co-cultured with target cells in X-VIVO15 medium for 16 to 24 hours according to different effector-target ratios (1:1, 3:1 or 9:1) of effector cells and target cells.
  • the luciferase activity stably expressed in the cells detects the killing ratio of CAR-T cells to target cells.
  • the target cells are HL60 cells, K562-CLL1 cells, and K562 cells, among which: HL60 cells are positive target cells endogenously expressing CLL1, K562-CLL1 is a positive target cell that expresses CLL1 exogenously, and K562 is a negative target cell that does not express CLL1.
  • the cell killing results are shown in Figure 3A- Figure 3C: in the HL60 tumor cell line, the killing effect of each CAR-T is very significant; in the K562-CLL1 tumor cell line, the killing effect of 1-16 CAR-T cells is poor, and the other CAR-T cells have a poor killing effect.
  • the T effect was very significant; in the K562 cell line, as expected, there was no killing effect in any group. Based on cell killing experiments, 1-16 can be further excluded.
  • Cytokine release experiment 5 types of CLL1 CAR-T cells (i.e. 1-3, 1-16, 1-28, 3-64, 3-67) obtained through screening in Example 3, Example 4, and Example 5 , M26CAR-T cells and UTD cells were co-cultured with target cells in X-VIVO15 medium for 24 hours at an effect-to-target ratio of 1:1, and then the concentration of IFN- ⁇ in the cell supernatant was detected by ELISA; the target cells were HL60 cells, K562-CLL1 cells, and K562 cells, among which: HL60 cells are positive target cells that endogenously express CLL1, K562-CLL1 are positive target cells that exogenously express CLL1, and K562 are negative target cells that do not express CLL1.
  • CARs with 1-3 scFv and 1-28 scFv as extracellular antigen recognition domains have a high expression rate on the surface of T cells, strong antigen recognition ability, and good expression at the protein level and cell level. It has good level specificity, good sustained proliferation, strong in vitro cell killing ability, and high cytokine release level; in particular, its sustained proliferation and cytokine release levels are significantly better than the positive control M26 scFv.
  • the CAR using 3-67 scFv as the extracellular antigen recognition domain has strong antigen recognition ability, good specificity at the protein level and cellular level, good sustained proliferation, strong in vitro cell killing ability, and high levels of cytokine release.
  • SEQ ID NO: 2 1-28 scFv amino acid sequence
  • SEQ ID NO:15 M26 scFv amino acid sequence
  • SEQ ID NO:16 Amino acid sequence of CD8 ⁇ guide chain as signal peptide
  • SEQ ID NO:17 Amino acid sequence of CD8 ⁇ hinge region
  • SEQ ID NO:18 Amino acid sequence of CD8 ⁇ transmembrane region
  • SEQ ID NO:19 Amino acid sequence of 4-1BB intracellular costimulatory signal
  • SEQ ID NO:20 Amino acid sequence of CD3 ⁇ T cell activation signal
  • SEQ ID NO:24 2-8CAR amino acid sequence
  • SEQ ID NO:25 2-25CAR amino acid sequence
  • SEQ ID NO:26 2-31CAR amino acid sequence
  • SEQ ID NO:28 3-11CAR amino acid sequence
  • SEQ ID NO:29 3-13CAR amino acid sequence
  • SEQ ID NO:30 3-16CAR amino acid sequence
  • SEQ ID NO:31 3-35CAR amino acid sequence
  • SEQ ID NO: 32 3-37CAR amino acid sequence
  • SEQ ID NO:33 3-64CAR amino acid sequence
  • SEQ ID NO:34 3-67CAR amino acid sequence
  • SEQ ID NO:35 M26CAR amino acid sequence
  • SEQ ID NO:60 Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:54;
  • SEQ ID NO:61 Nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO:55;
  • SEQ ID NO: 62 a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 56;
  • SEQ ID NO:63 Nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO:57;
  • SEQ ID NO:64 Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:58;
  • SEQ ID NO:65 Nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO:59;
  • SEQ ID NO:66 connection sequence
  • SEQ ID NO:67 connection sequence
  • SEQ ID NO:68 Linking sequence.

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Abstract

提供了一种CLL1抗体及其应用，该CLL1抗体或其抗原结合部位包含重链可变区和轻链可变区，其中，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:54、SEQ ID NO:56或SEQ ID NO:58所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:55、SEQ ID NO:57或SEQ ID NO:59所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同。

Description

一种CLL1抗体及其应用 技术领域
本申请涉及生物医药领域，具体涉及一种CLL1抗体及其应用。
背景技术
急性髓系白血病(Acute Myeloid Leukemia，AML)是髓系造血干/祖细胞恶性疾病。以骨髓与外周血中的原始和幼稚髓系细胞异常增生为主要特征，临床表现为贫血、出血、感染和发热、脏器浸润、代谢异常等，多数病例病情危重，预后凶险，如不及时治疗常可危及生命。婴幼儿比成人易发生AML，本病占小儿白血病的30％。目前的治疗方法有：化疗、支持治疗、造血干细胞移植。
嵌合抗原受体(Chimeric Antigen Receptor，CAR)是CAR细胞治疗药物的核心部件，其可包括抗原识别结构域、铰链区、跨膜区和细胞内结构域。到目前为止，抗原识别结构域从抗体的单链可变区(Single Chain Variable Fragment，缩写为scFv)、或者从受体配体相互作用、TCR模拟物、可变的淋巴细胞受体(Variable Lymphocyte Receptors，VLR)衍生而来；其中最为常见的来源就是scFv抗体。CAR-T细胞免疫疗法，被认为是最有希望攻克肿瘤的手段之一。CAR-T细胞就是利用基因改造的方法使T细胞表达CAR蛋白，这种CAR蛋白有能力在不依赖于抗原提呈的情况下识别膜表面的完整蛋白，进而引起T细胞的活化和功能效应。目前，CAR-T细胞免疫疗法在多种血液系统肿瘤的治疗中取得了显著的成效，如：用于治疗B细胞淋巴瘤的CD19CAR-T和用于治疗多发性骨髓瘤的BCMA CAR-T已有上市药物。然而，由于AML的异质性，较难发现治疗AML的理想CAR-T靶点，现有针对AML的靶点有CD33、CD123、LeY、NKG2D等，但这些靶点均未有上市的CAR-T药物。
研究发现，CLL1(C-type Lectin-like Molecule 1，C-型凝集素样分子-1)是有治疗AML前景的理想靶标，因为CLL1在正常造血干细胞中不表达，在AML原始细胞和白血病干细胞中高表达，因此寻找以CLL1为靶点的适合药物的抗体，尤其是以CLL1为靶点的适合CAR-T细胞治疗药物的抗体具有现实意义。
发明内容
本申请提供了一种CLL1抗体及其应用，发明人开发了多个靶向CLL1的抗体， 并将其相应scFv抗体作为CLL1CAR结构的胞外抗原识别结构域，以此构建了嵌合抗原受体表达载体、制备了靶向CLL1的CAR-T细胞，还在细胞水平验证了CLL1CAR以及CLL1CAR-T细胞的多项指标，并从中选择了表现最好的3条抗体和3种嵌合抗原受体。
一种CLL1抗体或其抗原结合部位，其包含重链可变区和轻链可变区，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:54所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:55所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:56所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:57所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:58所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:59所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同。
一种CLL1抗体或其抗原结合部位，其包含重链可变区和轻链可变区，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41所示的氨基酸序列；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47所示的氨基酸序列；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列。
上述CLL1抗体或其抗原结合部位在某些实施方式中，所述重链可变区的氨基酸序列包含如SEQ ID NO:54所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:55所示的氨基酸序列；
或，所述重链可变区的氨基酸序列包含如SEQ ID NO:56所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列；
或，所述重链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列。
上述CLL1抗体或其抗原结合部位在某些实施方式中，为CLL1 scFv抗体、CLL1Sc(Fv)2抗体、CLL1[Sc(Fv)2]2抗体。
上述CLL1抗体或其抗原结合部位在某些实施方式中，所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:54所示的氨基酸序列-连接序列-如SEQ ID NO:55所示的氨基酸序列、如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:56所示的氨基酸序列-连接序列-如SEQ ID NO:57所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:58所示的氨基酸序列-连接序列-如SEQ ID NO:59所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；可选地，所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；进一步可选地，所述CLL1 scFv抗体包含如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:14所示的氨基酸序列。
上述任一种CLL1抗体或其抗原结合部位在一些实施例中，连接序列选自以下序列的一种或多种：SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68。
本申请还提供了一种分离的核酸分子，其包含编码上述CLL1抗体或其抗原结合部位的核苷酸序列。
上述分离的核酸分子在某些实施方式中，编码上述CLL1抗体或其抗原结合部位的核苷酸序列包含：
1)编码如SEQ ID NO:54所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:60所示；和编码如SEQ ID NO:55所示的轻链可变区的氨基酸 序列的核苷酸序列，可选地，其如SEQ ID NO:61所示；或
2)编码如SEQ ID NO:56所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:62所示；和编码如SEQ ID NO:57所示的轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:63所示；或
3)编码如SEQ ID NO:58所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:64所示；和编码如SEQ ID NO:59所示的轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:65所示。
本申请还提供了一种载体，其包含上述分离的核酸分子。
本申请还提供了一种细胞，其包含上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子或载体。
本申请还提供了一种药物组合物，其包括上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞，以及药学上可接受的辅料。
本申请还提供了一种抗体药物，其包含上述任一种CLL1抗体或其抗原结合部位。
上述抗体药物在某些实施方式中，所述抗体药物为单特异性抗体药物、双特异性抗体药物、三特异性抗体药物或四特异性抗体药物。
本申请还提供了一种抗体药物偶联物，其包含上述任一种CLL1抗体或其抗原结合部位。
本申请还提供一种上述CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞在制备用于治疗与CLL1的表达相关的疾病或病症的药物中的应用。
上述应用在某些实施方式中，与CLL1的表达相关的疾病或病症为血液瘤。
上述应用在某些实施方式中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
本申请还提供了上述CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞在制备用于诊断与CLL1的表达相关的疾病或病症的检测试剂中的应用。
上述应用在某些实施方式中，与CLL1的表达相关的疾病或病症为血液瘤。
上述应用在某些实施方式中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
本申请还提供了一种治疗与CLL1的表达相关的疾病或病症的方法，包括以下步骤：将有效量的包含上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞的药物施用于具有治疗与CLL1的表达相关的疾病或病症的需求的受试者。
上述方法在某些实施方式中，与CLL1的表达相关的疾病或病症为血液瘤。
上述方法在某些实施方式中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
本申请还提供了一种药物，其包含上述CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞，用于治疗与CLL1的表达相关的疾病或病症。
上述药物在某些实施方式中，与CLL1的表达相关的疾病或病症为血液瘤。
上述药物在某些实施方式中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
本申请提供了一种靶向CLL1的嵌合抗原受体，其包含CLL1胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域，所述CLL1胞外抗原识别结构域包括CLL1重链可变区和CLL1轻链可变区，其中：
所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:54所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:55所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:56所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:57所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:58所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:59所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同。
本申请还提供了一种靶向CLL1的嵌合抗原受体，其包含CLL1胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域，所述CLL1胞外抗原识别结构域包括CLL1重链可变区和CLL1轻链可变区，其中：
所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38所示的氨基酸序列，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41所示的氨基酸序列；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示的氨基酸序列，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47所示的氨基酸序列；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:54所示的氨基酸序列，所述CLL轻链可变区序列包含如SEQ ID NO:55所示的氨基酸序列；
或，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:56所示的氨基酸序列，所述CLL轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列；
或，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列，所述CLL轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，CLL1胞外抗原识别结构域包括选自以下结构中的任意一种：如SEQ ID NO:54所示的氨基酸序列-连接序列-如SEQ ID NO:55所示的氨基酸序列、如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:56所示的氨基酸序列-连接序列-如SEQ ID NO:57所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:58所示的氨基酸序列-连接序列-如SEQ ID NO:59所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；可选地，所述CLL1胞外抗原识别结构域包含选自以下序列的任意一种：如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；进一步可选地，所述CLL1胞外抗原识别结构域包含如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:14所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，连接序列选自以下序列的一种或多种：SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68。
上述嵌合抗原受体在某些实施方式中，所述铰链区来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种；可选地，所述铰链区的氨基酸来源于CD8α；进一步可选地，所述铰链区的氨基酸序列包含如SEQ ID NO:17所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，所述跨膜区来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种；可选地，所述跨膜区的氨基酸序列来源于CD8α；进一步可选地，所述跨膜区的氨基酸序列包含如SEQ ID NO:18所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，其中所述细胞内结构域包含胞内信号传导区；可选地，还包括共刺激信号传导区。
上述嵌合抗原受体在某些实施方式中，其中所述胞内信号传导区来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种；可选地，所述胞内信号传导区来源于CD3δ；进一步可选地，所述胞内信号传导区的氨基酸序列包含如SEQ ID NO:20所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，其中所述共刺激信号传导区来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上；可选地，所述共刺激信号传导区来源于CD28或4-1BB；进一步可选地，所述共刺激信号传导区的氨基酸序列包含如SEQ ID NO:19所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，还包含位于所述嵌合抗原受体的氨基酸序列N-末端的引导肽；可选地，其中所述引导肽来源于CD8α；进一步可选地，所述引导肽的氨基酸序列包含如SEQ ID NO:16所示的氨基酸序列。
上述嵌合抗原受体在某些实施方式中，所述嵌合抗原受体包含如SEQ ID NO:21、SEQ ID NO:22或SEQ ID NO:34所示的氨基酸序列。
本申请还提供了一种分离的核酸分子，其包含编码上述嵌合抗原受体的核苷酸序列。
上述分离的核酸分子在某些实施方式中，编码所述嵌合抗原受体的核苷酸序列包含：
1)编码如SEQ ID NO:54所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:60所示；和编码如SEQ ID NO:55所示的CLL1轻链可变 区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:61所示；或
2)编码如SEQ ID NO:56所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:62所示；和编码如SEQ ID NO:57所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:63所示；或
3)编码如SEQ ID NO:58所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:64所示；和编码如SEQ ID NO:59所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:65所示。
本申请还提供了一种载体，其包含上述分离的核酸分子。
上述载体在某些实施方式中，为表达载体；在某些实施方式中，载体为病毒载体；在某些实施方式中，载体为慢病毒载体。
本申请还提供了一种经工程化的免疫效应细胞，其包含上述嵌合抗原受体、上述分离的核酸分子，或上述载体。
上述经工程化的免疫效应细胞在某些实施方式中，所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞和胚胎干细胞中的一种或多种。
上述经工程化的免疫效应细胞在某些实施方式中，所述经工程化的免疫效应细胞是T淋巴细胞；可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞。
上述经工程化的免疫效应细胞在某些实施方式中，T淋巴细胞为αβT淋巴细胞或γδT淋巴细胞。
本申请还提供了一种药物组合物，其包括上述经工程化的免疫效应细胞和药学上可接受的辅料。
上述药物组合物在某些实施方式中，药学上可接受的辅料包括保护剂。
上述药物组合物在某些实施方式中，药学上可接受的辅料包括细胞冻存液。
上述药物组合物在某些实施方式中，所述药物组合物为细胞悬液或其冻存细胞。
上述药物组合物在某些实施方式中，所述药物组合物为静脉注射剂。
本申请还提供上述嵌合抗原受体、分离的核酸分子、载体或经工程化的免疫效应细胞在制备药物中的应用，所述药物用于治疗与CLL1的表达相关的疾病或病症。
上述应用在某些实施方式中，与CLL1的表达相关的疾病或病症为血液瘤。
上述应用在某些实施方式中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
本申请还提供了一种治疗与CLL1的表达相关的疾病或病症的方法，包括以下步骤：将有效量的上述经工程化的免疫效应细胞或药物组合物施用于具有治疗与CLL1的表达相关的疾病或病症的需求的受试者。
上述方法在某些实施方式中，与CLL1的表达相关的疾病或病症为血液瘤。
上述方法在某些实施方式中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
上述方法在某些实施方式中，所述施用的方式为静脉注射。
上述方法在某些实施方式中，所述施用的方式为将有效量的经工程化的免疫效应细胞或药物组合物以单次或多次注射的方式施用于受试者。
上述方法在某些实施方式中，有效量的经工程化的免疫效应细胞或药物组合物为1×105至1×107个细胞/kg的剂量。
本申请还提供了一种药物，其包含上述经工程化的免疫效应细胞或药物组合物，用于治疗与CLL1的表达相关的疾病或病症。
上述药物在某些实施方式中，与CLL1的表达相关的疾病或病症为血液瘤。
上述药物在某些实施方式中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
附图说明
图1表示了本申请实施例2中各种CLL1CAR的结构示意图，在该示意图中从上到下分别为：CD8a信号肽、抗CLL1 scFv、CD8a铰链区和跨膜区、4-1BB、CD3δ。
图2表示了本申请实施例5中抗原刺激之后各组CAR-T细胞中CD3+细胞的持续增殖情况。
图3A-图3C表示了本申请实施例6中各种CLL1CAR-T细胞、阳性对照M26CLL1CAR-T细胞在不同效靶比对不同靶细胞的裂解杀伤效果；其中：图3A中靶细胞是HL60；图3B中靶细胞是K562-CLL1；图3C中靶细胞是K562。
图4表示了本申请实施例7中各种CLL1CAR-T细胞、阳性对照M26CLL1CAR-T细胞、UTD细胞(未转导CAR的T细胞)被阳性靶细胞激活后的细胞因子IFN-γ释放情况。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“抗体”具有本领域常规的含义。通过分析不同抗体重链(H链)和轻链(L链)的氨基酸序列发现，重链和轻链靠近N端的氨基酸序列变化很大，其他部分氨基酸序列相对恒定。因此，将抗体轻链和重链中靠近N端氨基酸序列变化较大的区域称为可变区(variable region，V)，将靠近C端的氨基酸序列相对稳定的区域，称为恒定区(constant region，C)，重链和轻链的V区分别简称为VH和VL，重链和轻链的C区分别简称为CH和CL。在抗体可变区内有一小部分氨基酸残基变化特别强烈，这些氨基酸的残基组成和排列顺序更易发生变异区域称高变区(hypervariable regions，HVR)；在L链、H链的V区中各有三个高变区，该部位因在空间结构上可与抗原决定簇形成精密的互补，故高变区又称互补性决定区(complementarity determining region，CDR)。抗体中，常见的划分CDR规则有Kabat、AbM、Chothia、Contact、IMGT，这些规则为本领域技术人员熟知的，当应用执行这些规则的网站时，只要将VH和VL序列输入并选择相应规则，即可得到依据不同规则的CDR序列。本领域技术人员应当理解，本申请的保护范围涵盖了通过采用不同规则分析获得的CDR序列的组合。抗体的6个CDR区共同决定了抗体对相应抗原的识别能力和特异性。本领域技术人员应当理解，当本申请限定了6个CDR区的氨基酸序列时，抗体对相应抗原的识别能力和特异性是可预期的。
在本申请中，术语“抗原结合部位”具有本领域常规的含义，是指抗体上可特异性识别并结合抗原的关键部位，包括VH和VL区。
在本申请中，术语“单克隆抗体”具有本领域常规的含义，是指高度均一、仅针对某一特定抗原表位的抗体，其可以通过已知的例如杂交瘤技术、抗体库技术、转基因小鼠技术或单细胞PCR技术来制备。
在本申请中，术语“scFv”具有本领域常规的含义，是指单链可变区(Single Chain Variable Fragment，缩写为scFv)，其是由抗体重链可变区和轻链可变区通过短肽(linker，连接序列)连接而成的抗体。
在本申请中，术语“Sc(Fv)2、[Sc(Fv)2]2”等未具体解释的名词也都具有本领域常规的含义。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)是CAR细胞治疗药物的核心部件，其可包括胞外抗原识别结构域(例如，结合肿瘤相关抗原(Tumor-Associated Antigen，TAA)的部分)、铰链区、跨膜区和细胞内结构域。CAR-T(Chimeric Antigen Receptor T)细胞免疫疗法，被认为是最有希望攻克肿瘤的手段之一。CAR-T细胞就是利用基因改造的方法使T细胞表达CAR蛋白，这种CAR蛋白有能力在不依赖于抗原提呈的情况下识别膜表面的完整蛋白，进而引起T细胞的活化和功能效应。
在本申请中，术语“胞外抗原识别结构域”是指抗原识别结构域(Antigen Recognition Domain，ARD)。CAR细胞治疗产品(如CAR-T细胞)之所以能特异性识别和/或结合到肿瘤细胞表达的靶抗原，依赖于胞外抗原识别结构域，到目前为止，抗原识别结构域从抗体的单链可变区(Single Chain Variable Fragment，缩写为scFv)、或者从受体配体相互作用、TCR模拟物、可变的淋巴细胞受体(Variable Lymphocyte Receptors，VLR)衍生而来。到目前为止，最为常见的来源就是抗体的scFv段，scFv包括抗体重链可变区和轻链可变区，它们之间由一段肽链连接，如：由18个氨基酸组成的连接序列GSTSGSGKPGSGEGSTKG。
本申请中，术语“特异性识别和/或结合”是指CAR和特异性靶标之间的识别和/或结合，是以比CAR结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标。
在本申请中，术语“铰链区”是指作用于胞外抗原识别结构域与跨膜结构域之间的连接段，这个区域通过给予抗原识别结构域一定的活动范围，允许CAR识别抗原。目前使用的铰链区主要来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种。
在本申请中，“跨膜区”是指连接着CAR结构的细胞内和细胞外成分的跨膜结构域。不同的跨膜结构域可以一定程度上影响CAR的表达和稳定性，但是并不直接参与信号传递，通过相互作用可以提高下游信号传递。所述跨膜区可以来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种。
在本申请内，术语“细胞内结构域”包括胞内信号传导区，还可以包括共刺激信号传 导区。
在本申请中，术语“胞内信号传导区”是指负责表达CAR的免疫效应细胞的至少一种正常效应子功能的活化。所述胞内信号传导区可以来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种。
在本申请中，术语“共刺激信号传导区”之所以存在，是因为除了抗原特异性信号的刺激之外，很多免疫效应细胞还需要共刺激来促进细胞增殖、分化和存活，以及活化细胞的效应子功能。在一些实施例中，CAR还可以包括一个或多个共刺激信号传导区，其中，共刺激信号传导区可以来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上。
在本申请中，术语“引导肽”，是指胞外抗原识别结构域(如scFv序列)前的短肽，其作用是引导细胞内合成的重组蛋白质输出到细胞外。
在本申请中，决定CAR-免疫细胞治疗效果的关键因素之一是对肿瘤靶抗原的选择。在本申请中，所选择的肿瘤靶抗原“CLL1”具有本领域常规的含义，其还被称为KLR1、CLEC12A，其是一种II型跨膜糖蛋白，是与免疫调节相关的C型凝集素样受体大家族的成员。研究发现，CLL1(C-type Lectin-like Molecule 1)是有治疗AML前景的理想靶标，因为CLL1在正常造血干细胞中不表达，在AML原始细胞和白血病干细胞中高表达。
在本申请中，术语“连接序列”通常是指长度为约1至100个氨基酸的寡肽或多肽区，其将本发明的嵌合抗原受体的任何结构/区域连接在一起。连接序列可由不同的氨基酸残基(如甘氨酸和丝氨酸)组成，以便相邻的蛋白质结构域相对于彼此自由移动。当期望确保两个相邻结构域在空间上不相互干扰时，可使用较长的连接序列。
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除从天然状态下经人工手段获得后，经过人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的核酸分子”通常指任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸，其可以是从其天然环境分离的或人工合成的类似物。
在本申请中，CAR基因转导/转染和靶基因表达时，基因转导/转染方法主要包括病毒和非病毒的方法。如：通过γ反转录病毒载体、慢病毒载体、腺病毒相关病毒载体、质粒DNA依赖的载体、转座子依赖的基因转移、mRNA介导的基因转导。
术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。术语“转座子”是指不连续的DNA片段，具有在染色体位点之间迁移和携带基因信息的能力，如：睡美人SB系统和来源于鳞翅目昆虫的PB系统。在一些实施例中，还可以使用电转的方法将mRNA转导进T细胞。
在本申请中，术语“免疫效应细胞”通常是指参与免疫应答，例如促进免疫效应应答的细胞。免疫效应细胞可以选自以下组：T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T淋巴细胞、多能干细胞分化成的NK细胞和胚胎干细胞中的一种或多种。
在本申请中，术语“药物组合物”通常指适合施用于患者的药物组合物，其可以包含本申请所述的免疫效应细胞，还可以包含一种或多种药学上可接受的辅料，如：载剂、保护剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂、防腐剂中的一种或多种。在一些实施例中，药学上可接受的辅料包括保护剂，如：细胞冻存液。在一些实施例中，本申请的药物组合物为细胞悬液或其冻存细胞。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于小鼠、大鼠、猫、狗、兔、马、猪、牛、羊或猴。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下本领域技术人员可接受的波动范围，如：在±0.5％-10％的范围内变动，例如在指定数值以上或以下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％或10％的范围内变动。
CLL1抗体或其抗原结合部位、相应核酸分子、相应载体、相应细胞、相应药物组合物
一方面，本申请提供了一种CLL1抗体或其抗原结合部位，其包含重链可变区和轻链可变区，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:54所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:55所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:56所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:57所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:58所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:59所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同。
在抗体中，常见的划分CDR规则有Kabat、AbM、Chothia、Contact、IMGT，这些规则为本领域技术人员熟知的，当应用执行这些规则的网站时，只要将VH和VL序列输入并选择相应规则，即可得到依据不同规则的CDR序列。本领域技术应当理解，即使针对相同的重链可变区和轻链可变区，根据不同的CDR划分规则可得到不同的CDR序列。本领域技术人员应当理解，本申请的保护范围涵盖了通过采用不同规则分析获得的CDR序列的组合。
另一方面，本申请还提供了一种CLL1抗体或其抗原结合部位，其包含重链可变区和轻链可变区，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41所示的氨基酸序列；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47 所示的氨基酸序列；
或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列。
在本申请中，采用了KABAT规则进行CDR的划分。
上述任一种CLL1抗体或其抗原结合部位在一些实施例中，所述重链可变区的氨基酸序列包含如SEQ ID NO:54所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:55所示的氨基酸序列；
或，所述重链可变区的氨基酸序列包含如SEQ ID NO:56所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列；
或，所述重链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列。
本申请通过实验筛选出3条性能最好的抗体，分别为1-3、1-28和3-67抗体。其中，1-3 scFv抗体包含氨基酸序列如SEQ ID NO:54所示的重链可变区和氨基酸序列如SEQ ID NO:55所示的轻链可变区；1-28 scFv抗体包含氨基酸序列如SEQ ID NO:56所示的重链可变区和氨基酸序列如SEQ ID NO:57所示的轻链可变区；3-67scFv抗体包含氨基酸序列如SEQ ID NO:58所示的重链可变区和氨基酸序列如SEQ ID NO:59所示的轻链可变区。
上述任一种CLL1抗体或其抗原结合部位在一些实施例中，为CLL1 scFv抗体、CLL1Sc(Fv)2抗体、CLL1[Sc(Fv)2]2抗体。
上述任一种CLL1抗体或其抗原结合部位在一些实施例中，所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:54所示的氨基酸序列-连接序列-如SEQ ID NO:55所示的氨基酸序列、如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:56所示的氨基酸序列-连接序列-如SEQ ID NO:57所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:58所示的氨基酸序列-连接序列-如SEQ ID NO:59所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；可选地，所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID  NO:54所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；进一步可选地，所述CLL1 scFv抗体包含如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:14所示的氨基酸序列。在上述描述中，“-”表示相互连接，并且在上述描述中“-”具有方向性，其表示从氨基酸的N端向C端的连接。
上述任一种CLL1抗体或其抗原结合部位在一些实施例中，连接序列选自以下序列的一种或多种：SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68。
再一方面，本申请还提供了一种分离的核酸分子，其包含编码上述任一种CLL1抗体或其抗原结合部位的核苷酸序列。
上述分离的核酸分子在一些实施例中，编码上述CLL1抗体或其抗原结合部位的核苷酸序列包含：
1)编码如SEQ ID NO:54所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:60所示；和编码如SEQ ID NO:55所示的轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:61所示；或
2)编码如SEQ ID NO:56所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:62所示；和编码如SEQ ID NO:57所示的轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:63所示；或
3)编码如SEQ ID NO:58所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:64所示；和编码如SEQ ID NO:59所示的轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:65所示。
再一方面，本申请还提供了一种载体，其包含上述分离的核酸分子。载体可以任意地选自以下载体地一种或几种：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。
再一方面，本申请还提供了一种细胞，其包含上述CLL1抗体或其抗原结合部位、分离的核酸分子或载体。
再一方面，本申请还提供了一种药物组合物，其包括上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞，以及药学上可接受的辅料。药学上 可接受的辅料包括但不限于：载剂、保护剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂、防腐剂中的一种或多种。
包含CLL1抗体或其抗原结合部位的抗体药物、抗体药物偶联物
一方面，本申请提供了一种抗体药物(Antibody-Drug Conjugate,ADC)，其包含上述任一种CLL1抗体或其抗原结合部位。
上述抗体药物在一些实施例中，所述抗体药物为单特异性抗体药物、双特异性抗体药物、三特异性抗体药物或四特异性抗体药物。
另一方面，本申请还提供了一种抗体药物偶联物，其包含上述任一种CLL1抗体或其抗原结合部位。
CLL1抗体或其抗原结合部位的应用
一方面，本申请提供一种上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞在制备用于治疗与CLL1的表达相关的疾病或病症的药物中的应用。
上述应用在一些实施例中，与CLL1的表达相关的疾病或病症为血液瘤。
上述应用在一些实施例中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
另一方面，本申请还提供了上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞在制备用于诊断与CLL1的表达相关的疾病或病症的检测试剂中的应用。
上述应用在一些实施例中，与CLL1的表达相关的疾病或病症为血液瘤。
上述应用在一些实施例中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
再一方面，本申请还提供了一种治疗与CLL1的表达相关的疾病或病症的方法，包括以下步骤：将有效量的包含上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞的药物施用于具有治疗与CLL1的表达相关的疾病或病症的需求的受试者。
上述方法在一些实施例中，与CLL1的表达相关的疾病或病症为血液瘤。
上述方法在一些实施例中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
上述方法在一些实施例中，所述施用可以通过不同的方式进行，例如口服、静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
上述方法在一些实施例中，针对不同的适应症，给药剂量可以不同；针对病情严重程度不同的患者，给药剂量也可以不同。
上述方法在一些实施例中，所述受试者可以包括人类和非人类动物。例如，所 述受试者可以包括但不限于小鼠、大鼠、猫、狗、马、猪、牛、羊、兔或猴。
再一方面，本申请还提供了一种药物，其包含上述任一种CLL1抗体或其抗原结合部位、分离的核酸分子、载体或细胞，用于治疗与CLL1的表达相关的疾病或病症。
上述药物在一些实施例中，与CLL1的表达相关的疾病或病症为血液瘤。
上述药物在一些实施例中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
CLL1嵌合抗原受体、相应核酸分子、相应载体、相应免疫效应细胞、药物组合物
一方面，本申请提供了一种靶向CLL1的嵌合抗原受体，其包含CLL1胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域，所述CLL1胞外抗原识别结构域包括CLL1重链可变区和CLL1轻链可变区，其中：
所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:54所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:55所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:56所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:57所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:58所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:59所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同。
另一方面，本申请还提供了一种靶向CLL1的嵌合抗原受体，其包含CLL1胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域，所述CLL1胞外抗原识别结构域包括CLL1重链可变区和CLL1轻链可变区，其中：
所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38所示的氨基酸序列，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41所示的氨基酸序列；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示的氨基酸序列，所述CLL1轻链 可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47所示的氨基酸序列；
或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:54所示的氨基酸序列，所述CLL轻链可变区序列包含如SEQ ID NO:55所示的氨基酸序列；
或，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:56所示的氨基酸序列，所述CLL轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列；
或，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列，所述CLL轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，CLL1胞外抗原识别结构域包括选自以下结构中的任意一种：如SEQ ID NO:54所示的氨基酸序列-连接序列-如SEQ ID NO:55所示的氨基酸序列、如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:56所示的氨基酸序列-连接序列-如SEQ ID NO:57所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:58所示的氨基酸序列-连接序列-如SEQ ID NO:59所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；可选地，所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；进一步可选地，所述CLL1 scFv抗体包含如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:14所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，连接序列选自以下序列的一种或多种：SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68。
上述任一种嵌合抗原受体在一些实施例中，所述铰链区来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种；可选地，所述铰链区的氨基酸来源于 CD8α；进一步可选地，所述铰链区的氨基酸序列包含如SEQ ID NO:17所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，所述跨膜区来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种；可选地，所述跨膜区的氨基酸序列来源于CD8α；进一步可选地，所述跨膜区的氨基酸序列包含如SEQ ID NO:18所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，其中所述细胞内结构域包含胞内信号传导区；可选地，还包括共刺激信号传导区。
上述任一种嵌合抗原受体在一些实施例中，其中所述胞内信号传导区来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种；可选地，所述胞内信号传导区来源于CD3δ；进一步可选地，所述胞内信号传导区的氨基酸序列包含如SEQ ID NO:20所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，其中所述共刺激信号传导区来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上；可选地，所述共刺激信号传导区来源于CD28或4-1BB；进一步可选地，所述共刺激信号传导区的氨基酸序列包含如SEQ ID NO:19所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，还包含位于所述嵌合抗原受体氨基酸序列N-末端的引导肽；可选地，其中所述引导肽来源于CD8α；进一步可选地，所述引导肽的氨基酸序列包含如SEQ ID NO:16所示的氨基酸序列。
上述任一种嵌合抗原受体在一些实施例中，所述嵌合抗原受体包括如SEQ ID NO:21、SEQ ID NO:22或SEQ ID NO:34所示的氨基酸序列。
再一方面，本申请还提供了一种分离的核酸分子，其包含编码上述任一种嵌合抗原受体的核苷酸序列。
上述分离的核酸分子在一些实施例中，编码所述嵌合抗原受体的核苷酸序列包含：
1)编码如SEQ ID NO:54所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:60所示；和编码如SEQ ID NO:55所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:61所示；或
2)编码如SEQ ID NO:56所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:62所示；和编码如SEQ ID NO:57所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:63所示；
3)编码如SEQ ID NO:58所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:64所示；和编码如SEQ ID NO:59所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:65所示。
再一方面，本申请还提供了一种载体，其包含上述分离的核酸分子。载体可选子以下的任意一种或几种：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。
上述载体在一些实施例中，为表达载体；在另一些实施例中，载体为病毒载体；在另一些实施例中，载体为慢病毒载体。
再一方面，本申请还提供了一种经工程化的免疫效应细胞，其包含上述嵌合抗原受体、上述分离的核酸分子，或上述载体。
上述经工程化的免疫效应细胞在一些实施例中，所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞和胚胎干细胞中的一种或多种。
上述经工程化的免疫效应细胞在一些实施例中，所述经工程化的免疫效应细胞是T淋巴细胞；可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞。
上述经工程化的免疫效应细胞在一些实施例中，T淋巴细胞的αβT淋巴细胞或γδT淋巴细胞。
上述经工程化的免疫效应细胞在一些实施例中，所述经工程化的免疫效应细胞的表面可以表达或表达有本申请所述的嵌合抗原受体。
再一方面，本申请还提供了一种药物组合物，其包括上述经工程化的免疫效应细胞和药学上可接受的辅料。药学上可接受的辅料包括但不限于：载剂、保护剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂、防腐剂中的一种或多种。
上述药物组合物在一些实施例中，药学上可接受的辅料包括保护剂。
上述药物组合物在一些实施例中，药学上可接受的辅料包括细胞冻存液。
上述药物组合物在一些实施例中，药物组合物为细胞悬液或其冻存细胞。
上述药物组合物在一些实施例中，药物组合物为静脉注射剂。
制备方法和用途
一方面，本申请还提供了制备经工程化的免疫效应细胞的方法，其包括以下的步骤：向免疫效应细胞中转导本申请所述的载体。
上述方法在一些实施例中，所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞和胚胎干细胞中的一种或多种。
上述方法在一些实施例中，所述经工程化的免疫效应细胞是T淋巴细胞；可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞。
上述方法在一些实施例中，T淋巴细胞为αβT淋巴细胞或γδT淋巴细胞。
另一方面，本申请还提供上述嵌合抗原受体、分离的核酸分子、载体或经工程化的免疫效应细胞在制备药物中的应用，所述药物用于治疗与CLL1的表达相关的疾病或病症。
上述应用在一些实施例中，与CLL1的表达相关的疾病或病症为血液瘤。
上述应用在一些实施例中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
再一方面，本申请还提供了一种治疗与CLL1的表达相关的疾病或病症的方法，包括以下步骤：将有效量的上述经工程化的免疫效应细胞或药物组合物施用于具有治疗与CLL1的表达相关的疾病或病症的需求的受试者。
上述方法在一些实施例中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。例如，施用的方式可以通过静脉注射的方式施用于受试者。在一些实施例中，有效剂量的经工程化的免疫效应细胞或药物组合物可以单次施用于受试者，也可以在一定期间内分次施用于受试者，如：每周施用一次、两周一次、三周一次、四周一次、一个月一次、3个月一次、或3-6个月一次。
上述方法在一些实施例中，与CLL1的表达相关的疾病或病症为血液瘤。
上述方法在一些实施例中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
上述方法在一些实施例中，所述施用的方式为静脉注射。
上述方法在一些实施例中，所述施用的方式为将有效量的经工程化的免疫效应细胞或药物组合物以单次注射的方式施用于受试者。
上述方法在一些实施例中，有效量的经工程化的免疫效应细胞或药物组合物为1×105至1×107个细胞/kg的剂量。在一些实施例中，针对不同的适应症，给药剂量可以不同；针对病情严重程度不同的患者，给药剂量也可以不同。施用剂量范围可以是1×105个CAR阳性T细胞/kg至1×107个CAR阳性T细胞/kg，例如，1×105个CAR阳性T细胞/kg至1×106个CAR阳性T细胞/kg、1×106个CAR阳性T细胞/kg至1×107个CAR阳性T细胞/kg、0.5×106个CAR阳性T细胞/kg、0.6×106个CAR阳性T细胞/kg、0.7×106个CAR阳性T细胞/kg、0.8×106个CAR阳性T细胞/kg、0.9×106个CAR阳性T细胞/kg、1.0×106个CAR阳性T细胞/kg、1.1×106个CAR阳性T细胞/kg、1.2×106个CAR阳性T细胞/kg、1.3×106个CAR阳性T细胞/kg、1.4×106个CAR阳性T细胞/kg、1.5×106个CAR阳性T细胞/kg、1.6×106个CAR阳性T细胞/kg、1.7×106个CAR阳性T细胞/kg、1.8×106个CAR阳性T细胞/kg、1.9×106个CAR阳性T细胞/kg、2.0×106个CAR阳性T细胞/kg。
上述方法在一些实施例中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于小鼠、大鼠、猫、狗、马、猪、牛、羊、兔或猴。
再一方面，本申请还提供了一种药物，其包含上述经工程化的免疫效应细胞或药物组合物，用于治疗与CLL1的表达相关的疾病或病症。
上述药物在一些实施例中，与CLL1的表达相关的疾病或病症为血液瘤。
上述药物在一些实施例中，与CLL1的表达相关的疾病或病症为急性髓系白血病。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的CLL1抗体、嵌合抗原受体、经工程化的免疫效应细胞、制备方法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual,2nd edition,Cold Spring Harbor Laboratory Press。
实施例1、CLL1抗体开发
CLL1抗体开发过程：采用BALB/c和C57bl/6小鼠作为免疫动物，以人CLL1作为抗原按照表1中的程序进行免疫。取免疫后各只小鼠血清进行FACS(流式细胞 荧光分选技术)和ELISA检测，综合ELISA检测和FACS检测的结果，取免疫效果最好的小鼠，将其浆细胞经CD138抗体富集后铺种到Beacon筛选芯片上，进行抗体阳性B细胞株的分析和筛选，共获得单克隆细胞8561株、多克隆细胞1511株，其中抗体为IgG的690株。根据与抗原结合的情况，将阳性B细胞克隆导出、进行V区测序，共获得64条单克隆抗体的V区序列，根据序列相似性分类，选择14条代表序列用于合成CAR中的scFv。
表1、动物免疫程序
实施例2、CLL1CAR-T细胞的获得
我们获得14条靶向CLL1的候选scFv，候选scFv序号和其序列分别如表2所示。我们还获得1条CLL1阳性对照抗体M26(其来源于Treatmen 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)
表2、14条靶向CLL1的候选scFv序号及其序列
我们选择在二代CAR结构上对14条候选scFv进行筛选。在各个CAR结构中，均采用了CD8α引导链为信号肽(如SEQ ID NO:16所示)，铰链区(如SEQ ID NO:17所示)和跨膜区(如SEQ ID NO:18所示)采用CD8α的结构，以4-1BB为胞内共刺激信号(如SEQ ID NO:19所示)，CD3δ为T细胞激活信号(如SEQ ID NO:20所示)，结构示意图如图1所示。
1、慢病毒载体的构建
根据表1中各候选scFv的序列以及CAR结构组件的序列，分别人工合成14条CLL1CAR结构以及1条M26CLL1CAR结构，其氨基酸序列如表3所示。
表3、14条CLL1CAR序号及其序列
将上述15种CLL1CAR结构分别构建到经过改造的空慢病毒载体(厂家：SBI公司，货号：CD500-CD800，如WO2021/121227实施例1中记载的改造抗性)中获得CAR表达载体，随后将CAR表达载体和三种包装质粒一起转染293T细胞，经过收集纯化之后得到有功能性的慢病毒载体。三种包装质粒分别是pMD2.G(购自Biovector公司，产品号Biovector012259)，pMDLg/pRRE(购自Biovector公司，产品号Biovector012251)，pRSV-Rev(购自Biovector公司，产品号Biovector012253)。
2、通过慢病毒转导的方式制备相应15种CLL1特异性CAR-T细胞。
转导实验按照本领域技术人员已知的常规方法进行，简述转导步骤如下：
1)分选T细胞
从人单采血细胞中分离获得外周血单个核细胞(PBMC)，然后从PBMC细胞中分选获得T细胞。
2)对T细胞进行激活处理
将分离的T细胞用T细胞完全培养基(X-VIVO15培养基+5％FBS+300IU/ml IL-2或X-VIVO15培养基+5％FBS+5ng/ml IL-15+10ng/ml IL-7)进行重悬，使终浓度为(1～3)×106个细胞/ml，按1ul beads/1×106cells加入CD3/CD28磁珠刺激，混匀后置于培养箱培养，培养条件为37℃+5％CO2，培养时间至少24小时。
3)慢病毒转导T细胞
取出激活培养的T细胞，用含终浓度为8μg/ml的聚凝胺(polybrene)的单纯X-VIVO15培养基重悬，混匀获得细胞悬液，并按每800ul细胞悬液(含2×106细胞)缓慢加入200ul的慢病毒载体，混匀后放入孔板，并于培养箱培养，培养条件为37℃+5％CO2，培养时 间至少4-6小时。
4)转导后T细胞的扩增培养
取出转导后的细胞，用T细胞完全培养基扩增培养，隔天传代，使细胞密度维持在(0.8～2)×106个细胞/ml，以备后续实施例使用。
分别使用包含有表3中CAR结构的慢病毒感染T细胞后，获得的T细胞分别按其CAR序号进行命名，比如使用1-3CAR获得的T细胞即命名为1-3CAR-T细胞、使用2-8CAR获得的T细胞即命名为2-8CAR-T细胞。接下来，我们在细胞水平对14条CLL1CAR结构进行筛选，以确定各个scFv的优劣。
实施例3、CLL1CAR-T细胞表面表达的CAR分子的检测及其抗原识别能力检测
1、CAR分子的检测：对实施例2中获得的转导后培养7天的14种CLL1CAR-T细胞表面表达的CAR蛋白分子进行检测，我们用PE荧光标记的CLL1抗原(厂家：ACRObiosystems，货号：CLA-PH2Q3)和FITC荧光标记的CLL1抗原(厂家：ACRObiosystems，货号：CLA-HF247 25ug)对实施例2中获得的14种CLL1CAR-T细胞、M26CLL1CAR-T细胞和UTD细胞(未转导CAR的T细胞)进行染色，并通过流式细胞术进行CAR分子阳性比例检测分析，检测结果如表4所示。
2、CAR分子抗原识别能力的检测：对实施例2中获得的14种CLL1CAR-T细胞表面表达的CAR蛋白分子进行抗原识别能力检测，我们用FITC荧光标记的IgG(厂家：abcam，货号：ab98658)对实施例2中获得的14种CLL1CAR-T细胞、M26CLL1CAR-T细胞和UTD细胞(未转导CAR的T细胞)进行染色，并通过流式细胞术进行抗原识别能力的检测，检测结果如表4所示。
如表4所示：灰色底纹的细胞中，因CAR分子表达差和/或抗原识别能力差在筛选中被舍弃，其中：2-25、2-40、3-35的CAR不表达；3-11、3-13、3-16的CAR表达，但对抗原识别能力差，这些细胞在之后的实验中全部被舍弃。
表4、细胞表面表达的CAR分子的检测结果及其抗原识别能力检测结果

备注：CAR表达率低于10％认为不表达或者表达极差，低于筛选标准；抗原结合识别率低于10％认为抗原识别能力差，低于筛选标准。
实施例4、CLL1CAR的特异性检测
在蛋白水平和细胞水平分别进行了CLL1CAR的特异性检测。
1)蛋白水平的特异性检测
以荧光标记的CD19蛋白(购自ACRObiosystems，货号CD9-HP2H3)、CS1蛋白(购自ACRObiosystems，货号SL7-HP2H3)、BCMA蛋白(购自ACRObiosystems，货号BCA-HF254)、GPC3蛋白(购自ACRObiosystems，货号GP3-HF2H1)、EGFRvIII蛋白(购自ACRObiosystems，货号EGI-HP2E3)5种抗原蛋白按照如下方法对实施例3筛选得到的8种CLL1CAR-T细胞(1-3、1-28、1-16、2-8、2-31、3-37、3-64和3-67CAR-T细胞)以及M26CAR-T细胞进行蛋白水平的特异性检测：取每种CAR-T细胞0.5×106，用PBS洗涤细胞一次，200g离心5min，弃上清，细胞重悬到200ul PBS中，加入0.1ug上述荧光标记的抗原蛋白，4℃避光孵育20min，用PBS洗涤细胞一次，200g离心5min，弃上清，细胞重悬到200ul PBS中，以流式细胞术检测结合能力；结果显示：各个CAR-T细胞均不能与CD19蛋白、CS1蛋白、BCMA蛋白、GPC3蛋白、EGFRvIII蛋白结合，显示出好的特异性，检测结果如表5所示。
2)细胞水平的特异性
以细胞系K562(人慢性骨髓性白血病细胞)、K562-CLL1(外源表达CLL1蛋白的K562细胞)、HL60(人早幼粒急性白血病细胞)、THP1(人单核细胞白血病细胞)、OVCAR3(人卵巢腺癌细胞)、RAJI(人淋巴瘤细胞)、MM.1S(人多发性骨髓瘤细胞)、NALM6(人急性淋巴细胞白血病细胞)、SK-MEL1(人皮肤黑色素瘤细胞)、A549(人肺癌细胞)、HUH7(人肝癌细胞)这11种不同组织来源的细胞系按照如下方法对实施例3筛选得到的8种CLL1CAR-T细胞(1-3、1-28、1-16、2-8、2-31、3-37、3-64和3-67CAR-T细胞)以及M26CAR-T细胞进行细胞水平的特异性检测，其中：K562-CLL1、HL60、THP1、NALM6细胞系是CLL1阳性靶细胞，其余细胞系是阴性靶细胞。该研究中检测T细胞激活的方法为CD107a分析。CD107a在膜上的表达被认为是活化细胞毒性淋巴细胞(CD8+ T细胞和NK细胞)的标志物，当细胞毒性淋巴细胞给予特定抗原刺激即发生免疫激活，表达CD107a，因此可做为检测CAR-T细胞特异性的手段。具体步骤为：肿瘤细胞和转染及未转染的T细胞进行计数，用X-VIVO培养基将肿瘤细胞调整至2×106/ml，T细胞调整至1×106/ml。对于杀伤板每孔加20ul的CD107a抗体，然后每孔加100ul的T细胞和100ul的肿瘤细胞，加完后400r/min离心3分钟。37℃5％CO2培养箱培养60分钟。60分钟后每孔加入20ul的golgi stop工作液(3mlX-VIVO培养基加2ul golgi stop(BD GolgiStopTM Protein Transport Inhibitor，CAT：554724))培养箱培养2.5小时。取CD3和CD8抗体等体积混合，混匀后每孔加10ul，培养箱培养30分钟。30分钟后1500r/min离心5分钟，甩去上清，加250ul FACS buffer(PBS+0.5％BSA)，混匀。1500r/min离心5分钟，甩去上清。每孔加200ul FACS buffer(PBS+0.5％BSA)混匀，转移到已标记好的流式管中，再补200-300ul的含FACS buffer(PBS+0.5％BSA)上机检测。分析CD3+&CD8+阳性细胞群里CD107a的荧光信号。具体检测结果如表6所示。
表5、蛋白水平检测CAR-T细胞特异性结果

备注：“-”表示CAR-T细胞不与该蛋白结合，“+”表示CAR-T细胞与该蛋白结合。
表6、细胞水平检测CAR-T细胞特异性结果


备注：“-”表示未检测到CD107a信号，“+”表示检测到CD107a信号。
如表6所示：2-31CAR能被阴性靶细胞激活，表现出CD107a信号，因此在细胞水平没有达到筛选标准，因此在后续的实验中也将其排除。
实施例5、CLL1CAR-T细胞的持续增殖性
抗原刺激可以激活CAR-T细胞使CAR-T细胞增殖，而T细胞的持续激活会导致细胞耗竭，耗竭的T细胞的增殖能力和效应功能都会有所下降，我们通过检测CLL1CAR-T细胞在多轮抗原刺激实验后CD3+细胞的增殖情况(即T细胞的增殖情况)，确定其持续增殖性。
抗原刺激(以HL60细胞作为抗原)之前，将经过实施例3、实施例4中筛选获得的7种CLL1CAR-T细胞(即1-3、1-16、1-28、2-8、3-37、3-64、3-67)、M26CAR-T细胞的CAR阳性比例都利用UTD调整到了与CAR阳性比例最低的一组CAR-T细胞一致的水平，在抗原刺激实验中，将各组CAR-T细胞分别与阳性靶细胞HL60按照效靶比1:2在24孔板中进行共培养，每孔2ml X-VIVO15培养基，每组细胞重复3孔。用荧光标记的CD3抗体(厂家：BioLegend，货号：300312)进行CD3，通过流式细胞术进行检测分析，显示T细胞的增殖情况(CD3是区分是否为T细胞的标记物)，根据体积倍数的换算计算CD3阳性细胞的细胞数，然后根据计算结果各组再取出一定量CAR-T细胞按照效靶比1:2加入对应的阳性靶细胞进行新一轮刺激，每隔2天一次，如此重复进行3-4轮刺激。经过多次重复实验，排除掉增殖最差的2-8和3-37CAR。其余5种CLL1CAR-T细胞(即1-3、1-16、1-28、3-64、3-67)、M26CAR-T细胞再用上述方法进行多轮刺激和增殖统计后，发现1-3和1-28具有更强的持续增殖能力，显著高于阳性对照M26CAR-T细胞。1-16和3-67的持续增殖能力也不错，3-64的持续增殖性相对较差，结果如图2所示。在本申请中，经过多轮刺激后的细胞几乎全为CAR+细胞，因此CAR+表达率不再作 为重要指标。
实施例6、CLL1CAR-T细胞进行细胞杀伤实验
细胞杀伤实验：将经过实施例3、实施例4、实施例5中筛选获得的5种CLL1CAR-T细胞(即1-3、1-16、1-28、3-64、3-67)、M26CAR-T细胞分别与靶细胞按照效应细胞与靶细胞不同效靶比(1:1、3:1或9:1)的条件在X-VIVO15培养基中共培养16～24小时后，通过检测靶细胞中稳定表达的荧光素酶活性检测CAR-T细胞对靶细胞的杀伤比例，靶细胞分别采用HL60细胞、K562-CLL1细胞、K562细胞，其中：HL60细胞是内源表达CLL1的阳性靶细胞、K562-CLL1是外源表达CLL1的阳性靶细胞、K562是不表达CLL1的阴性靶细胞。细胞杀伤结果如图3A-图3C所示：在HL60肿瘤细胞系中各CAR-T杀伤效果都很显著；在K562-CLL1肿瘤细胞系中1-16CAR-T细胞的杀伤效果差，其余CAR-T效果非常显著；在K562细胞系中，如预期的，各组均没有杀伤效果。根据细胞杀伤实验，可以进一步排除1-16。
实施例7、CLL1CAR-T细胞进行细胞因子释放实验
细胞因子释放实验：将经过实施例3、实施例4、实施例5中筛选获得的5种CLL1CAR-T细胞(即1-3、1-16、1-28、3-64、3-67)、M26CAR-T细胞以及UTD细胞分别与靶细胞按照效靶比1:1的条件在X-VIVO15培养基中共培养24h后，通过ELISA方法检测细胞上清中IFN-γ的浓度；靶细胞分别采用HL60细胞、K562-CLL1细胞、K562细胞，其中：HL60细胞是内源表达CLL1的阳性靶细胞、K562-CLL1是外源表达CLL1的阳性靶细胞、K562是不表达CLL1的阴性靶细胞。细胞因子释放实验结果如图4所示：1-3和1-28CAR-T细胞因子释放量显著高于阳性对照M26CAR-T，3-67CAR-T细胞因子释放量也高于阳性对照M26和3-64。
综上，在体外药效学检测表明，以1-3 scFv、1-28 scFv作为胞外抗原识别结构域的CAR，在T细胞表面的表达率高、抗原识别能力强、在蛋白水平和细胞水平的特异性好、持续增殖性好、体外细胞杀伤能力强、细胞因子释放水平高；尤其是其持续增殖性和细胞因子释放水平都显著优于阳性对照M26 scFv。以3-67 scFv作为胞外抗原识别结构域的CAR，抗原识别能力强、在蛋白水平和细胞水平的特异性好、持续增殖性好、体外细胞杀伤能力强、细胞因子释放水平高。
序列描述
SEQ ID NO:1：1-3 scFv氨基酸序列；
SEQ ID NO:2：1-28 scFv氨基酸序列；
SEQ ID NO:3：1-16 scFv氨基酸序列；
SEQ ID NO:4：2-8 scFv氨基酸序列；
SEQ ID NO:5：2-25 scFv氨基酸序列；
SEQ ID NO:6：2-31 scFv氨基酸序列；
SEQ ID NO:7：2-40 scFv氨基酸序列；
SEQ ID NO:8：3-11 scFv氨基酸序列；
SEQ ID NO:9：3-13 scFv氨基酸序列；
SEQ ID NO:10：3-16 scFv氨基酸序列；
SEQ ID NO:11：3-35 scFv氨基酸序列；
SEQ ID NO:12：3-37 scFv氨基酸序列；
SEQ ID NO:13：3-64 scFv氨基酸序列；
SEQ ID NO:14：3-67 scFv氨基酸序列；
SEQ ID NO:15：M26 scFv氨基酸序列；
SEQ ID NO:16：CD8α引导链作为信号肽的氨基酸序列；
SEQ ID NO:17：CD8α铰链区的氨基酸序列；
SEQ ID NO:18：CD8α跨膜区的氨基酸序列；
SEQ ID NO:19：4-1BB胞内共刺激信号的氨基酸序列；
SEQ ID NO:20：CD3ζT细胞激活信号的氨基酸序列；
SEQ ID NO:21：1-3CAR氨基酸序列；
SEQ ID NO:22：1-28CAR氨基酸序列；
SEQ ID NO:23：1-16CAR氨基酸序列；
SEQ ID NO:24：2-8CAR氨基酸序列；
SEQ ID NO:25：2-25CAR氨基酸序列；
SEQ ID NO:26：2-31CAR氨基酸序列；
SEQ ID NO:27：2-40CAR氨基酸序列；
SEQ ID NO:28：3-11CAR氨基酸序列；
SEQ ID NO:29：3-13CAR氨基酸序列；
SEQ ID NO:30：3-16CAR氨基酸序列；
SEQ ID NO:31：3-35CAR氨基酸序列；
SEQ ID NO:32：3-37CAR氨基酸序列；
SEQ ID NO:33：3-64CAR氨基酸序列；
SEQ ID NO:34：3-67CAR氨基酸序列；
SEQ ID NO:35：M26CAR氨基酸序列；
SEQ ID NO:36:1-3 scFv VH CDR1氨基酸序列；
SEQ ID NO:37:1-3 scFv VH CDR2氨基酸序列；
SEQ ID NO:38:1-3 scFv VH CDR3氨基酸序列；
SEQ ID NO:39:1-3 scFv VL CDR1氨基酸序列；
SEQ ID NO:40:1-3 scFv VL CDR2氨基酸序列；
SEQ ID NO:41:1-3 scFv VL CDR3氨基酸序列；
SEQ ID NO:42:1-28 scFv VH CDR1氨基酸序列；
SEQ ID NO:43:1-28 scFv VH CDR2氨基酸序列；
SEQ ID NO:44:1-28 scFv VH CDR3氨基酸序列；
SEQ ID NO:45:1-28 scFv VL CDR1氨基酸序列；
SEQ ID NO:46:1-28 scFv VL CDR2氨基酸序列；
SEQ ID NO:47:1-28 scFv VL CDR3氨基酸序列；
SEQ ID NO:48:3-67 scFv VH CDR1氨基酸序列；
SEQ ID NO:49:3-67 scFv VH CDR2氨基酸序列；
SEQ ID NO:50:3-67 scFv VH CDR3氨基酸序列；
SEQ ID NO:51:3-67 scFv VL CDR1氨基酸序列；
SEQ ID NO:52:3-67 scFv VL CDR2氨基酸序列；
SEQ ID NO:53:3-67 scFv VL CDR3氨基酸序列；
SEQ ID NO:54：1-3 scFv VH氨基酸序列；
SEQ ID NO:55：1-3 scFv VL氨基酸序列；
SEQ ID NO:56：1-28 scFv VH氨基酸序列；
SEQ ID NO:57：1-28 scFv VL氨基酸序列；
SEQ ID NO:58：3-67 scFv VH氨基酸序列；
SEQ ID NO:59：3-67 scFv VL氨基酸序列；
SEQ ID NO:60：编码如SEQ ID NO:54所示的重链可变区的氨基酸序列的核苷酸序列；
SEQ ID NO:61：编码如SEQ ID NO:55所示的轻链可变区的氨基酸序列的核苷酸序列；
SEQ ID NO:62：编码如SEQ ID NO:56所示的重链可变区的氨基酸序列的核苷酸序列；
SEQ ID NO:63：编码如SEQ ID NO:57所示的轻链可变区的氨基酸序列的核苷酸序列；
SEQ ID NO:64：编码如SEQ ID NO:58所示的重链可变区的氨基酸序列的核苷酸序列；
SEQ ID NO:65：编码如SEQ ID NO:59所示的轻链可变区的氨基酸序列的核苷酸序列；
SEQ ID NO:66：连接序列；
SEQ ID NO:67：连接序列；
SEQ ID NO:68：连接序列。

Claims (37)

  1. 一种CLL1抗体或其抗原结合部位，其包含重链可变区和轻链可变区，其中，
    所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:54所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:55所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
    或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:56所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:57所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
    或，所述所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:58所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:59所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同。
  2. 一种CLL1抗体或其抗原结合部位，其包含重链可变区和轻链可变区，其中，
    所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41所示的氨基酸序列；
    或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47所示的氨基酸序列；
    或，所述重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列，所述轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列。
  3. 根据权利要求1或2所述的CLL1抗体或其抗原结合部位，其中，
    所述重链可变区的氨基酸序列包含如SEQ ID NO:54所示的氨基酸序列，所述轻 链可变区的氨基酸序列包含如SEQ ID NO:55所示的氨基酸序列；
    或，所述重链可变区的氨基酸序列包含如SEQ ID NO:56所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列；
    或，所述重链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列，所述轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列。
  4. 根据权利要求3所述的CLL1抗体或其抗原结合部位，其中，
    所述CLL1抗体或其抗原结合部位为CLL1 scFv抗体、CLL1Sc(Fv)2抗体、CLL1[Sc(Fv)2]2抗体。
  5. 根据权利要求4所述的CLL1抗体或其抗原结合部位，其中，
    所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:54所示的氨基酸序列-连接序列-如SEQ ID NO:55所示的氨基酸序列、如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:56所示的氨基酸序列-连接序列-如SEQ ID NO:57所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:58所示的氨基酸序列-连接序列-如SEQ ID NO:59所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；
    可选地，所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；
    进一步可选地，所述CLL1 scFv抗体包含如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:14所示的氨基酸序列；
    更进一步可选地，所述连接序列选自以下序列的一种或多种：SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68。
  6. 一种分离的核酸分子，其包含编码权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位的核苷酸序列。
  7. 根据权利要求6所述的核酸分子，其中，编码上述CLL1抗体或其抗原结合部位的核苷酸序列包含：
    1)编码如SEQ ID NO:54所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:60所示；和编码如SEQ ID NO:55所示的轻链可变区的氨基酸 序列的核苷酸序列，可选地，其如SEQ ID NO:61所示；或
    2)编码如SEQ ID NO:56所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:62所示；和编码如SEQ ID NO:57所示的轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:63所示；或
    3)编码如SEQ ID NO:58所示的重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:64所示；和编码如SEQ ID NO:59所示的轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:65所示。
  8. 一种载体，其包含权利要求6或7所述的分离的核酸分子。
  9. 一种细胞，其包含权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位、权利要求6或7所述的分离的核酸分子或权利要求8所述的载体。
  10. 一种药物组合物，其包括权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位、权利要求6或7所述的分离的核酸分子、权利要求8所述的载体或权利要求9所述的细胞，以及药学上可接受的辅料。
  11. 一种抗体药物，其包含权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位；
    可选地，所述抗体药物为单特异性抗体药物、双特异性抗体药物、三特异性抗体药物或四特异性抗体药物。
  12. 一种抗体药物偶联物，其包含权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位。
  13. 权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位、权利要求6或7所述的分离的核酸分子、权利要求8所述的载体或权利要求9所述的细胞在制备用于治疗与CLL1的表达相关的疾病或病症的药物中的应用；
    可选地，与CLL1的表达相关的疾病或病症为血液瘤；
    进一步可选地，与CLL1的表达相关的疾病或病症为急性髓系白血病。
  14. 权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位、权利要求6或7所述的分离的核酸分子、权利要求8所述的载体或权利要求9所述的细胞在制备用于诊断与CLL1的表达相关的疾病或病症的检测试剂中的应用；
    可选地，与CLL1的表达相关的疾病或病症为血液瘤；
    进一步可选地，与CLL1的表达相关的疾病或病症为急性髓系白血病。
  15. 一种治疗与CLL1的表达相关的疾病或病症的方法，其包括以下步骤：将有 效量的包含权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位、权利要求6或7所述的分离的核酸分子、权利要求8所述的载体或权利要求9所述的细胞的药物施用于具有治疗与CLL1的表达相关的疾病或病症的需求的受试者；
    可选地，与CLL1的表达相关的疾病或病症为血液瘤；
    进一步可选地，与CLL1的表达相关的疾病或病症为急性髓系白血病。
  16. 一种药物，其包含权利要求1-5中任一项所述的CLL1抗体或其抗原结合部位、权利要求6或7所述的分离的核酸分子、权利要求8所述的载体或权利要求9所述的细胞，用于治疗与CLL1的表达相关的疾病或病症；
    可选地，与CLL1的表达相关的疾病或病症为血液瘤；
    进一步可选地，与CLL1的表达相关的疾病或病症为急性髓系白血病。
  17. 一种靶向CLL1的嵌合抗原受体，其包含CLL1胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域，所述CLL1胞外抗原识别结构域包括CLL1重链可变区和CLL1轻链可变区，其中：
    所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:54所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:55所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
    或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:56所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:57所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同；
    或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:58所示抗体重链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别与SEQ ID NO:59所示抗体轻链可变区中的CDR1、CDR2、CDR3的氨基酸序列相同。
  18. 一种靶向CLL1的嵌合抗原受体，其包含CLL1胞外抗原识别结构域、铰链区、跨膜区和细胞内结构域，所述CLL1胞外抗原识别结构域包括CLL1重链可变区和CLL1轻链可变区，其中：
    所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38所示的氨基酸序列，所述CLL1轻链可变区 的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41所示的氨基酸序列；
    或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示的氨基酸序列，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47所示的氨基酸序列；
    或，所述CLL1重链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列，所述CLL1轻链可变区的CDR1、CDR2、CDR3的氨基酸序列分别包含如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列。
  19. 根据权利要求17或18所述的嵌合抗原受体，其中，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:54所示的氨基酸序列，所述CLL轻链可变区序列包含如SEQ ID NO:55所示的氨基酸序列；
    或，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:56所示的氨基酸序列，所述CLL轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列；
    或，所述CLL1重链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列，所述CLL轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列。
  20. 根据权利要求17-19中任一项所述的嵌合抗原受体，其中，CLL1胞外抗原识别结构域包括选自以下结构中的任意一种：如SEQ ID NO:54所示的氨基酸序列-连接序列-如SEQ ID NO:55所示的氨基酸序列、如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:56所示的氨基酸序列-连接序列-如SEQ ID NO:57所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:58所示的氨基酸序列-连接序列-如SEQ ID NO:59所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；
    可选地，所述CLL1 scFv抗体包含选自以下序列的任意一种：如SEQ ID NO:55所示的氨基酸序列-连接序列-如SEQ ID NO:54所示的氨基酸序列、如SEQ ID NO:57所示的氨基酸序列-连接序列-如SEQ ID NO:56所示的氨基酸序列、如SEQ ID NO:59所示的氨基酸序列-连接序列-如SEQ ID NO:58所示的氨基酸序列；
    进一步可选地，所述CLL1 scFv抗体包含如SEQ ID NO:1或SEQ ID NO:2或SEQ  ID NO:14所示的氨基酸序列；
    更进一步可选地，所述连接序列选自以下序列的一种或多种：SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68。
  21. 根据权利要求17-20中任一项所述的嵌合抗原受体，其中，所述铰链区来源于IgG1、IgG4、CD4、CD7、CD28、CD84、CD8α中的一种或多种；
    可选地，所述铰链区的氨基酸序列来源于CD8α；
    进一步可选地，所述铰链区的氨基酸序列包含如SEQ ID NO:17所示的氨基酸序列。
  22. 根据权利要求17-21中任一项所述的嵌合抗原受体，其中，所述跨膜区来源于CD3、CD4、CD7、CD8α、CD28、CD80、CD86、CD88、4-1BB、CD152、OX40、Fc70中的一种或多种；
    可选地，所述跨膜区的氨基酸序列来源于CD8α；
    进一步可选地，所述跨膜区的氨基酸序列包含如SEQ ID NO:18所示的氨基酸序列。
  23. 根据权利要求17-22中任一项所述的嵌合抗原受体，其中，所述细胞内结构域包含胞内信号传导区；
    可选地，所述胞内信号传导区来源于CD3δ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、FcRγ、FcRβ、CD66d、DAP10、DAP12、Syk中的一种或多种；
    进一步可选地，所述胞内信号传导区来源于CD3δ；
    更进一步可选地，所述胞内信号传导区的氨基酸序列包含如SEQ ID NO:20所示的氨基酸序列。
  24. 根据权利要求17-23中任一项所述的嵌合抗原受体，其中，所述细胞内结构域还包括共刺激信号传导区；
    可选地，所述共刺激信号传导区来源于CD2、CD3、CD7、CD27、CD28、CD30、CD40、CD83、CD244、4-1BB、OX40、LFA-1、ICOS、LIGHT、NKG2C、NKG2D、DAP10、B7-H3、MyD88中的一种、两种或三种以上；
    进一步可选地，所述共刺激信号传导区来源于CD28或4-1BB；
    更进一步可选地，所述共刺激信号传导区的氨基酸序列包含如SEQ ID NO:19所示的氨基酸序列。
  25. 根据权利要求17-24中任一项所述的嵌合抗原受体，其中，还包含位于所述 嵌合抗原受体氨基酸序列N-末端的引导肽；
    可选地，其中所述引导肽来源于CD8α；
    进一步可选地，所述引导肽的氨基酸序列包含如SEQ ID NO:16所示的氨基酸序列。
  26. 根据权利要求17-25中任一项所述的嵌合抗原受体，其中，所述嵌合抗原受体包含如SEQ ID NO:21、SEQ ID NO:22或SEQ ID NO:34所示的氨基酸序列。
  27. 一种分离的核酸分子，其包含编码权利要求1-10中任一项所述的嵌合抗原受体的核苷酸序列。
  28. 根据权利要求27所述的分离的核酸分子，其中，编码所述嵌合抗原受体的核苷酸序列包含：
    1)编码如SEQ ID NO:54所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:60所示；和编码如SEQ ID NO:55所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:61所示；或
    2)编码如SEQ ID NO:56所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:62所示；和编码如SEQ ID NO:57所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:63所示；或
    3)编码如SEQ ID NO:58所示的CLL1重链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:64所示；和编码如SEQ ID NO:59所示的CLL1轻链可变区的氨基酸序列的核苷酸序列，可选地，其如SEQ ID NO:65所示。
  29. 一种载体，其包含权利要求27或28所述的分离的核酸分子；
    可选地，所述载体为表达载体；
    进一步可选地，所述载体为病毒载体；
    更进一步可选地，载体为慢病毒载体。
  30. 一种经工程化的免疫效应细胞，其包含权利要求17-26中任一项所述的嵌合抗原受体、权利要求27或28所述的分离的核酸分子，或权利要求29所述的载体。
  31. 根据权利要求30所述的经工程化的免疫效应细胞，其中，所述经工程化的免疫效应细胞选自T淋巴细胞、自然杀伤细胞(NK细胞)、外周血单个核细胞(PBMC细胞)、多能干细胞、多能干细胞分化成的T细胞、多能干细胞分化成的NK细胞和胚胎干细胞中的一种或多种；
    可选地，所述经工程化的免疫效应细胞是T淋巴细胞；
    进一步可选地，所述T淋巴细胞的来源为自体T淋巴细胞或同种异体T淋巴细胞；
    更进一步可选地，所述T淋巴细胞为αβT淋巴细胞或γδT淋巴细胞。
  32. 一种药物组合物，其包括权利要求30或31所述的经工程化的免疫效应细胞和药学上可接受的辅料；
    可选地，所述药学上可接受的辅料包括保护剂。
    进一步可选地，药学上可接受的辅料包括细胞冻存液。
  33. 根据权利要求32所述的药物组合物，所述药物组合物为细胞悬液或其冻存细胞；
    可选地，所述药物组合物为静脉注射剂。
  34. 权利要求17-26中任一项所述的嵌合抗原受体、权利要求27或28所述的分离的核酸分子、权利要求29所述的载体或权利要求30或31所述的经工程化的免疫效应细胞在制备药物中的应用，所述药物用于治疗与CLL1的表达相关的疾病或病症；
    可选地，与CLL1的表达相关的疾病或病症为血液瘤；
    进一步可选地，与CLL1的表达相关的疾病或病症为急性髓系白血病。
  35. 一种治疗与CLL1的表达相关的疾病或病症的方法，其包括以下步骤：将有效量的权利要求30或31所述的经工程化的免疫效应细胞或权利要求32或33所述的药物组合物施用于具有治疗与CLL1的表达相关的疾病或病症的需求的受试者。
    可选地，与CLL1的表达相关的疾病或病症为血液瘤；
    进一步可选地，与CLL1的表达相关的疾病或病症为急性髓系白血病。
  36. 根据权利要求35所述的方法，其中，所述施用的方式为静脉注射；
    可选地，所述施用的方式为将有效量的权利要求30或31所述的经工程化的免疫效应细胞或权利要求32或33所述的药物组合物以单次或多次注射的方式施用于受试者；
    进一步可选地，有效量的经工程化的权利要求30或31所述的经工程化的免疫效应细胞或权利要求32或33所述的药物组合物为1×105至1×107个细胞/kg的剂量。
  37. 一种药物，其包含权利要求30或31所述的经工程化的免疫效应细胞或权利要求32或33所述的药物组合物，用于治疗与CLL1的表达相关的疾病或病症；
    可选地，与CLL1的表达相关的疾病或病症为血液瘤；
    进一步可选地，与CLL1的表达相关的疾病或病症为急性髓系白血病。
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