CD7-targeting humanized antibody, chimeric antigen receptor, and use thereof

Abstract

The present application provides a CD7-targeting humanized antibody and a chimeric antigen receptor. The CD7-targeting humanized antibody comprises: an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO: 1 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 2; or an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO: 1 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 30; or an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO: 28 and an amino acid sequence of a light chain variable region as shown in SEQ ID NO: 30. Also provided is a use thereof in preparation of a reagent or a drug for diagnosing or treating diseases or conditions related to CD7 expression.

Description

A humanized antibody targeting CD7, a chimeric antigen receptor and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a humanized antibody targeting CD7, a chimeric antigen receptor, and their applications. Background Art

[0002] T-cell malignancies are a class of lymphoid malignancies that arise from T cells. Due to a lack of understanding of their biology, their classification remains elusive, with many categories remaining within the "provisional" classifications of the World Health Organization (WHO) classification of diseases. Currently, the WHO categorizes T-cell malignancies into two types: immature T-cell neoplasms, which include acute T-lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphoma (T-LBL), and mature T-cell neoplasms, which include various types of T-cell leukemia and T-cell lymphoma. T-cell malignancies are characterized by their aggressive nature, diverse symptoms, generally poor prognosis, high recurrence rates (30%-40%), unknown etiology, and a lack of biological understanding. Although current treatments for pediatric T-ALL are more effective than before, with event-free survival (EFS) rates reaching 85%, the EFS rate for relapsed T-ALL is less than 15%. The 5-year overall survival (OS) rate for adult T-ALL patients is 40% to 60%, indicating a poor prognosis. Therefore, new, targeted treatment options are still needed to improve the prognosis of T-cell malignancies, especially for patients with relapsed and refractory T-cell malignancies.

[0003] The chimeric antigen receptor (CAR) is the core component of CAR cell therapy, consisting of an antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. To date, the antigen recognition domain has been derived from the single-chain variable fragment (scFv) of an antibody, or from receptor-ligand interactions, TCR mimics, or variable lymphocyte receptors (VLRs); the most common source is the scFv antibody. CAR-T cell immunotherapy is considered one of the most promising approaches for combating cancer. CAR-T cells are genetically engineered to express a CAR protein, which has the ability to recognize intact proteins on the membrane independently of antigen presentation, leading to T cell activation and functional effects. Currently, CAR-T cell immunotherapy has achieved significant success in the treatment of various hematologic malignancies, with marketed drugs such as CD19 CAR-T for B-cell lymphoma and BCMA CAR-T for multiple myeloma.

[0004] CD7 is a transmembrane glycoprotein and a member of the immunoglobulin supergene family. CD7 is commonly overexpressed in immature T-cell tumors, while in mature T-cell tumors, the pattern and intensity of CD7 expression vary. In addition to T-cell malignancies, approximately 30% of acute myeloid leukemia (AML) patients also express CD7 on their leukemic blasts and malignant progenitor cells. Therefore, CAR-T cells targeting CD7 hold promise for treating these diseases. Summary of the Invention

[0005] The present application provides a humanized antibody targeting CD7, a chimeric antigen receptor, and its application. Based on the existing mouse antibody sequence, the inventors designed 5 humanized Th69 scFv antibodies to reduce their heterologousness. However, during the humanization process of the antibody, the binding affinity, specificity, and other functionalities of the antibody to the specific antigen may be lost. Therefore, it is of practical significance to obtain a humanized antibody with reduced immunogenicity and no loss of functionality or even enhanced functionality. The inventors used 5 humanized Th69 scFv antibodies as the extracellular antigen recognition domain of CD7 CAR, and evaluated the proliferation, specificity, and functionality of CAR-T cells using humanized Th69 scFv antibodies through in vitro experiments and in vivo animal experiments, and screened out the 3 humanized Th69 scFv antibodies with the best overall effect.

[0006] The present application provides a humanized antibody or antigen-binding fragment thereof targeting CD7, comprising: the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2;

[0007] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30;

[0008] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 28 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30.

[0009] In certain embodiments, the humanized antibody or antigen-binding fragment thereof is a scFv antibody, a sc(Fv)2 antibody, or a [sc(Fv)2]2 antibody.

[0010] In certain embodiments, the humanized antibody or antigen-binding fragment thereof is a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 3;

[0011] or, a scFv antibody having the amino acid sequence shown in SEQ ID NO: 35;

[0012] Or, a scFv antibody having the amino acid sequence shown in SEQ ID NO: 36.

[0013] In yet another aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned humanized antibody or antigen-binding fragment thereof.

[0014] In certain embodiments, the isolated nucleic acid molecule described above has a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, which is shown in SEQ ID NO: 4;

[0015] and / or, a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 2, which is shown in SEQ ID NO: 5;

[0016] and / or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 28, which is shown in SEQ ID NO: 37;

[0017] and / or, a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30, which is shown in SEQ ID NO:38.

[0018] In yet another aspect, the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

[0019] In yet another aspect, the present application further provides a cell comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof, isolated nucleic acid molecules or vectors.

[0020] In another aspect, the present application also provides a pharmaceutical composition comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof, an isolated nucleic acid molecule, a vector or a cell, and a pharmaceutically acceptable excipient.

[0021] In another aspect, the present application also provides the use of the above-mentioned humanized antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or cell in the preparation of a detection reagent for diagnosing a disease or condition related to the expression of CD7.

[0022] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0023] In certain embodiments of the above application, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T lymphoblastic leukemia (T-ALL), CD7 + T-cell lymphoma, CD7 + Acute myelocytic leukemia (AML).

[0024] In certain embodiments of the above application, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) and other acute T cell leukemias.

[0025] In certain embodiments of the above application, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T-lymphoblastic lymphoma (T-LBL), CD7+ extranodal NK / T-cell lymphoma, CD7 + Enteropathy-associated T-cell lymphoma, CD7 +Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma.

[0026] In another aspect, the present application also provides a use of the above-mentioned humanized antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or cell in the preparation of a medicament for treating a disease or condition associated with CD7 expression.

[0027] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0028] In certain embodiments of the above application, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0029] In certain embodiments of the above application, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0030] In certain embodiments of the above application, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0031] On the other hand, the present application also provides a method for treating a disease or condition, comprising the following steps: administering an effective amount of a drug comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof, isolated nucleic acid molecules, vectors or cells to a subject in need of treating a disease or condition associated with CD7 expression.

[0032] In certain embodiments of the above method, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0033] In certain embodiments of the above method, the CD7 +Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0034] In certain embodiments of the above method, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0035] In certain embodiments of the above method, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0036] On the other hand, the present application also provides a drug comprising the above-mentioned humanized antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or cell, for treating diseases or conditions related to CD7 expression.

[0037] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0038] In certain embodiments of the above-mentioned drug, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0039] In certain embodiments of the above-mentioned drug, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0040] In certain embodiments of the above-mentioned drug, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7+ Peripheral T-cell lymphoma.

[0041] In another aspect, the present application also provides an antibody drug comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof.

[0042] In another aspect, the present application also provides an antibody-drug conjugate comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof.

[0043] In another aspect, the present application further provides a chimeric antigen receptor targeting CD7, comprising an extracellular antigen recognition domain targeting CD7, a hinge region, a transmembrane region, and an intracellular domain, wherein the extracellular antigen recognition domain targeting CD7 comprises:

[0044] The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2;

[0045] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30

[0046] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 28 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30.

[0047] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the extracellular antigen recognition domain comprises any one selected from the following structures: an amino acid sequence as set forth in SEQ ID NO: 1-a linker-an amino acid sequence as set forth in SEQ ID NO: 2, an amino acid sequence as set forth in SEQ ID NO: 2-a linker-an amino acid sequence as set forth in SEQ ID NO: 1, an amino acid sequence as set forth in SEQ ID NO: 1-a linker-an amino acid sequence as set forth in SEQ ID NO: 30, an amino acid sequence as set forth in SEQ ID NO: 30-a linker-an amino acid sequence as set forth in SEQ ID NO: 1, an amino acid sequence as set forth in SEQ ID NO: 28-a linker-an amino acid sequence as set forth in SEQ ID NO: 30, an amino acid sequence as set forth in SEQ ID NO: 30-an linker-an amino acid sequence as set forth in SEQ ID NO: 28;

[0048] Optionally, the extracellular antigen recognition domain comprises any one selected from the following structures: an amino acid sequence as shown in SEQ ID NO: 2-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 30-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 30-an linker sequence-an amino acid sequence as shown in SEQ ID NO: 28;

[0049] Further optionally, the extracellular antigen recognition domain is any one selected from the following structures: the amino acid sequence shown in SEQ ID NO: 2-a connecting sequence-the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 30-a connecting sequence-the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 30-a connecting sequence-the amino acid sequence shown in SEQ ID NO: 28.

[0050] In certain embodiments of any of the above chimeric antigen receptors, the linker sequence is selected from one or more of the following sequences: SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0051] In certain embodiments of any of the above chimeric antigen receptors, the extracellular antigen recognition domain comprises: a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 3, or a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 35, or a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 36;

[0052] Optionally, the amino acid sequence of the extracellular antigen recognition domain is as shown in SEQ ID NO: 3, or as shown in SEQ ID NO: 35, or as shown in SEQ ID NO: 36.

[0053] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:9.

[0054] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence as shown in SEQ ID NO: 10; still further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 10.

[0055] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the intracellular domain comprises an intracellular signaling region; optionally, further comprises a costimulatory signaling region.

[0056] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:11; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:11.

[0057] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the costimulatory signaling region is derived from one, two or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; further optionally, the amino acid sequence of the costimulatory signaling region comprises the amino acid sequence shown in SEQ ID NO: 12; further optionally, the amino acid sequence of the costimulatory signaling region is as shown in SEQ ID NO: 12.

[0058] In certain embodiments, any of the above-mentioned chimeric antigen receptors further comprises a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO: 13; further optionally, the amino acid sequence of the guide peptide is as shown in SEQ ID NO: 13.

[0059] In certain embodiments of any of the above chimeric antigen receptors, the chimeric antigen receptor comprises any one of the following sequences: the amino acid sequence shown in SEQ ID NO: 14, or the amino acid sequence shown in SEQ ID NO: 18; or the amino acid sequence shown in SEQ ID NO: 19;

[0060] Optionally, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO: 14, or as shown in SEQ ID NO: 18, or as shown in SEQ ID NO: 19.

[0061] In yet another aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above-mentioned chimeric antigen receptors.

[0062] In certain embodiments of the isolated nucleic acid molecule described above, the nucleotide sequence encoding the chimeric antigen receptor comprises:

[0063] a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 1, which is set forth in SEQ ID NO: 4; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 2, which is set forth in SEQ ID NO: 5;

[0064] or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, which is shown in SEQ ID NO: 4; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30, which is shown in SEQ ID NO: 38;

[0065] Or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:28, which is shown in SEQ ID NO:37; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30, which is shown in SEQ ID NO:38.

[0066] In certain embodiments of the isolated nucleic acid molecule described above, the nucleotide sequence encoding the chimeric antigen receptor comprises:

[0067] a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 14, which is shown in SEQ ID NO: 15;

[0068] or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 18, which is shown in SEQ ID NO: 39;

[0069] Or, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 19 is shown in SEQ ID NO: 40.

[0070] In yet another aspect, the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

[0071] In some embodiments, the above-mentioned vector is an expression vector; in other embodiments, the vector is a viral vector; in other embodiments, the vector is a lentiviral vector.

[0072] In yet another aspect, the present application also provides an engineered immune effector cell comprising the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0073] In certain embodiments, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0074] In certain embodiments of the engineered immune effector cells, the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

[0075] In certain embodiments of the above-mentioned engineered immune effector cells, the allogeneic T lymphocytes comprise gene-edited CD7 / TRAC double-negative cells; further, the allogeneic T lymphocytes comprise 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more of gene-edited CD7 / TRAC double-negative cells.

[0076] In certain embodiments of the engineered immune effector cells, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0077] On the other hand, the present application also provides a pharmaceutical composition comprising the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.

[0078] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes a protective agent.

[0079] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes cell freezing solution.

[0080] In certain embodiments, the pharmaceutical composition is a cell suspension or frozen cells thereof.

[0081] In certain embodiments, the pharmaceutical composition is in the form of an intravenous injection.

[0082] In yet another aspect, the present application also provides a method for preparing engineered immune effector cells, comprising the following steps: introducing a nucleotide sequence encoding any one of the above-mentioned chimeric antigen receptors into the immune effector cells.

[0083] In certain embodiments of the above method, the immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), induced pluripotent stem cells (iPSC), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK) and embryonic stem cells.

[0084] In certain embodiments of the above method, the immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

[0085] In certain embodiments of the above method, the allogeneic T lymphocytes contain 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more of gene-edited CD7 / TRAC double negative cells.

[0086] In certain embodiments of the above method, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0087] In certain embodiments of the above method, the T cell Trac gene and CD7 gene are gene-edited by a gene editing tool to obtain CD7 / TRAC double-negative cells, and the gene editing tool is selected from one of the CRISPR / Cas system, zinc finger nuclease system, and transcription activator-like effector nuclease system; optionally, the gene editing tool is selected from the CRISPR / Cas system; further optionally, the CRISPR / Cas system includes Cas9 protein and sgRNA.

[0088] In certain embodiments of the above method, the sgRNA in the CRISPR / Cas system targeting the CD7 gene is shown as SEQ ID NO:33.

[0089] In certain embodiments of the above method, the sequence of chRDNA in the CRISPR / Cas system targeting the Trac gene is shown as SEQ ID NO:34.

[0090] In certain embodiments of the above-mentioned method, the method for introducing a nucleotide sequence encoding any of the above-mentioned chimeric antigen receptors into immune effector cells is selected from one or more of the following: a method using a virus or a non-viral method; optionally, the method using a virus includes using one or more of the following viral vectors: a γ-retroviral vector, a lentiviral vector, an adenovirus-associated viral vector; the non-viral method includes one or more of the following methods: gene transfer using a transposon, gene transduction mediated by mRNA, and electroporation.

[0091] In another aspect, 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 a drug for treating a disease or condition related to the expression of CD7.

[0092] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0093] In certain embodiments of the above application, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0094] In certain embodiments of the above application, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0095] In certain embodiments of the above application, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0096] On the other hand, the present application also provides a method for treating a disease or condition related to the expression of CD7, comprising the following steps: administering an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical composition to a subject in need of treating a disease or condition related to the expression of CD7.

[0097] In certain embodiments of the above methods, the administration can be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. For example, the administration can be administered to the subject by intravenous injection. In certain embodiments, an effective dose of the engineered immune effector cells or pharmaceutical composition can be administered to the subject in a single dose or in divided doses over a certain period of time, such as once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0098] In certain embodiments of the above method, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0099] In certain embodiments of the above method, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0100] In certain embodiments of the above method, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0101] In certain embodiments of the above method, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0102] In certain embodiments of the above method, the administration is by intravenous injection.

[0103] In certain embodiments of the above method, the effective amount of the engineered immune effector cells or pharmaceutical composition is 1×10 5 to 1×10 7 cells / kg dose.

[0104] On the other hand, the present application also provides a drug comprising the above-mentioned engineered immune effector cells or pharmaceutical composition, for treating diseases or conditions related to the expression of CD7.

[0105] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0106] In certain embodiments of the above-mentioned drug, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0107] In certain embodiments of the above-mentioned drug, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0108] In certain embodiments of the above-mentioned drug, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 shows the detection results of the CAR+ expression rate of CAR-T cells after T cells were transduced and expanded and cultured for different times in Example 2, wherein: UTD-NoEP represents the untransduced and non-knockout T cell group, UTD-KO TRAC+CD7 represents the untransduced but double-knockout T cell group, and the remaining 6 groups represent double-knockout T cell groups transduced with different CARs.

[0110] Figure 2 shows the detection results of the total T cell number after T cells were transduced and expanded and cultured for different times in Example 2, wherein: UTD-NoEP represents an untransduced and unknocked T cell group, UTD-KO TRAC+CD7 represents an untransduced but double-knocked T cell group, and the remaining 6 groups represent double-knocked T cell groups transduced with different CARs.

[0111] FIG3A shows the short-term killing of positive target cells MOLT4 by CAR-T cells using murine or five humanized Th69 scFvs at effector-target ratios of 1:3, 1:1, and 3:1 in Example 3;

[0112] Figure 3B shows the short-term killing of negative target cells KO CD7-MOLT4 by CAR-T cells using murine or five humanized Th69 scFvs at effector-target ratios of 1:3, 1:1, and 3:1 in Example 3;

[0113] Figure 3C shows the long-term killing of positive target cells MOLT4 by CAR-T cells using murine or five humanized Th69 scFvs at effector-target ratios of 1:32, 1:16, 1:4, and 1:1 in Example 3;

[0114] Figure 3D shows the long-term killing of positive target Jurkat cells by CAR-T cells using murine or five humanized Th69 scFvs at effector-target ratios of 1:32, 1:16, 1:4, and 1:1 in Example 3;

[0115] Figure 3E shows the long-term killing of negative target cells KO CD7-jurkat by CAR-T cells using mouse or five humanized Th69 scFvs at effector-target ratios of 1:32, 1:16, 1:4, and 1:1 in Example 3;

[0116] Among them: In Figure 3A-Figure 3E, NoEP represents untransduced and non-knockout T cells, UTD represents untransduced but double-knockout CD7 and TRAC T cells, and the remaining 6 groups represent double-knockout T cell groups transduced with different CARs.

[0117] Figure 4 shows the cumulative expansion multiples of each group of T cells in rounds 1-4 in the multiple rounds of stimulation experiment in Example 4, where: NoEP represents untransduced and non-knockout T cells, UTD represents untransduced but double-knockout CD7 and TRAC T cells, and the remaining 6 groups represent double-knockout T cell groups transduced with different CARs.

[0118] FIG5 shows the survival rates of mice at different times after the in vivo experiments in Example 6 were performed with the test articles in different groups as shown in Table 3.

[0119] FIG6 shows the in vivo tumor imaging signal values ​​of mice at different times after the test samples of different groups in Table 3 were reinfused in the in vivo experiment of Example 6. DETAILED DESCRIPTION

[0120] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0121] The present application is further described below. Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0122] In this application, the term "antibody" has the conventional meaning in the art and is used in the broadest sense in this application. Typically, antibodies have heavy (H) chains and / or light (L) chains. By analyzing the amino acid sequences of different antibody heavy and light chains, it was found that the amino acid sequences of the heavy and light chains near the N-terminus vary greatly, while the amino acid sequences of other parts are relatively constant. Therefore, the region near the N-terminus of the antibody light and heavy chains where the amino acid sequence varies greatly is called the variable region (V), and the region near the C-terminus where the amino acid sequence is relatively stable is called the constant region (C). The V regions of the heavy and light chains are referred to as VH and VL, respectively, and the C regions of the heavy and light chains are referred to as CH and CL, respectively. Within the variable region of an antibody, a small number of amino acid residues vary particularly strongly. These regions, where the composition and order of these amino acid residues are more susceptible to variation, are called hypervariable regions (HVRs). The V regions of the L and H chains each contain three hypervariable regions. Because these regions form precise spatial complementarity with antigenic determinants, they are also called complementarity determining regions (CDRs). Common CDR delineation rules for antibodies include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. Using websites that implement these rules, simply input the VH and VL sequences and select the corresponding rule to generate CDR sequences based on different rules.

[0123] In this application, the term "antigen-binding fragment" has the conventional meaning in the art, and refers to the key fragment of an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0124] In this application, the term "humanized antibody" also refers to an antibody that has undergone humanization. Methods for humanization are known (e.g., WO96 / 02576). The purpose of humanization is to substantially retain the affinity and specificity of the parent antibody while reducing its heterologous nature. For example, if the CDRs are obtained from a murine antibody, primers can be synthesized (referring to the method described in WO98 / 13388 for obtaining corresponding primers) and used to link the murine antibody CDRs to the framework regions (FRs) of a human antibody.

[0125] In this application, the terms "Th-69 antibody" and "Th69 antibody" refer to an existing mouse antibody, which is an IgG1 monoclonal antibody produced by Gramatzki M et al. in 1985 by immunizing mice with T-cell acute lymphoblastic leukemia (T-ALL) cell lines CEM and HSB-2. Th-69 can specifically bind to the extracellular domain of human CD7 with high affinity. If the scFv derived from mouse antibodies is directly used in CAR-T drugs, it may increase the immune risk. In order to reduce the immunogenicity of the scFv derived from mouse antibodies and improve the safety of treatment, it can be achieved by humanizing the scFv derived from mouse antibodies. However, in the process of humanization, the binding affinity, specificity and other functionalities of the antibody to the specific antigen may be lost. Therefore, it is of practical significance to obtain humanized antibodies with reduced immunogenicity and no loss of functionality or even enhanced functionality.

[0126] In this application, the term "scFv" has the conventional meaning in the art and refers to a single-chain variable fragment (scFv), which is an antibody composed of the heavy chain variable region and the light chain variable region of the antibody connected by a short peptide (linker).

[0127] In this application, the terms "Sc(Fv)2, [Sc(Fv)2]2" and other nouns not specifically explained have the conventional meanings in the art.

[0128] In this application, the term "antibody drug" has the conventional meaning in the art, that is, a drug with an antibody substance as an active ingredient, such as a monoclonal antibody drug and a bispecific antibody drug.

[0129] In this application, the term "antibody-drug conjugate" has the conventional meaning in the art, namely, antibody-drug conjugate, abbreviated as ADC. It is a small molecule drug with biological activity linked to a monoclonal antibody via a chemical linker. The monoclonal antibody acts as a carrier to deliver the small molecule drug to target cells.

[0130] In this application, the term "chimeric antigen receptor" (CAR) is the core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain (for example, a portion that binds to a tumor-associated antigen (TAA)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered to be one of the most promising means of conquering tumors. CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.

[0131] In this application, the term "extracellular antigen recognition domain" refers to the Antigen Recognition Domain (ARD). The ability of CAR cell therapy products (such as CAR-T cells) to specifically recognize and / or bind to target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. So far, the antigen recognition domain has been derived from the single chain variable region (scFv) of the antibody, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLR). So far, the most common source is the scFv segment of the antibody.

[0132] In this application, the term "specific recognition and / or binding" refers to the recognition and / or binding between CAR and a specific target, which is to bind to the target with greater affinity, avidity, more easily, and / or for a longer duration than CAR binds to other targets.

[0133] In this application, CD7 is a transmembrane glycoprotein containing 240 amino acid residues and is a member of the immunoglobulin supergene family. CD7 is commonly overexpressed in immature T cell tumors, while in mature T cell tumors, the expression pattern and strength of CD7 vary. In addition to T cell malignancies, approximately 30% of AML patients' leukemic blasts and malignant progenitor cells also express CD7, so drugs targeting CD7 can also be used to treat CD7. +However, while CD7 CAR-T cells kill tumor cells with high CD7 expression, they also kill their fellow CD7 CAR-T cells because the CD7 gene is also expressed on the surface of T cells. This results in a significant decrease in the number of CAR-T cells on the fifth day of in vitro culture.

[0134] In this application, the terms "Trac gene" and "TRAC gene" have the usual meaning in the art and refer to the constant region of the α chain of the T cell receptor. The signal of the T cell receptor (TCR) to recognize antigens is mainly through the TCR-CD3 complex (including the α chain, β chain, CD3γ, CD3δ, etc. of TCR). The TCR is composed of an α chain and a β chain. After the translated α chain and β chain are assembled into a heterodimer, they bind to multiple CD3 subtype molecules. In the cell, if a complete complex cannot be formed with the CD3 molecule, the excess TCR will be degraded. After the Trac gene is knocked out, the formation of the TCR-CD3 complex will be affected, which means that the T cell receptor on the surface of the T cell will be cleared, thereby avoiding the occurrence of graft-versus-host reaction (GVHD).

[0135] In this application, the term "GVHD (graft-versus-host disease)" has the usual meaning in the art, generally referring to graft-versus-host disease, which is caused by the T lymphocytes in the allogeneic donor graft undergoing a series of "cytokine storm" stimulation launched by the recipient after transplantation, greatly enhancing their immune response to the recipient's antigens and launching cytotoxic attacks against the recipient's target cells, among which the skin, liver and intestines are the main targets.

[0136] In this application, "CD7 / TRAC double-negative allogeneic T lymphocytes" refers to allogeneic T lymphocytes in which the CD7 gene and the TRAC gene cannot be normally expressed, or in which the CD7 protein and the T cell receptor α chain constant region are not correctly expressed.

[0137] In this application, the term "gene cannot be expressed normally" has the usual meaning in the art. Gene expression refers to the process of synthesizing genetic information from genes into functional gene expression products, and gene expression products usually refer to proteins.

[0138] In this application, the terms "gene editing" and "gene editing technology" have the usual meaning in the field and refer to a technology for site-specific modification of the genome. Using this technology, it is possible to accurately locate a certain site in the genome, cut the target DNA fragment, knock in or knock out the target gene fragment at that site. As a molecular biology technology, gene editing technology can achieve precise modification of chromosomes, thereby changing the existing functions of cells. Compared with the cell lines often used in basic research, T cells, as a primary cell, have no special characteristics except that they cannot proliferate for a long time, and can also be edited using gene editing technology. There are currently three main gene editing tools, namely zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology and RNA-guided CRISPR / Cas nuclease technology. Compared with traditional gene targeting technology, the new gene editing technology retains the characteristics of site-specific modification and can be applied to more species and cells, with higher efficiency, shorter construction time and lower cost.

[0139] In this application, the terms "CRISPR / Cas technology" and "CRISPR / Cas system" have the usual meanings in the art. Its full name is clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins, which is an acquired immune system found in most bacteria and all archaea that can direct the cutting of foreign gene fragments. Different types of CRISPR / Cas systems have been discovered, among which the second type is relatively simple, with Cas9 protein and guide RNA (guide RNA, Grna, also called sgRNA) as the core components. Compared with the earlier ZFN technology and TALEN technology, CRISPR / Cas has the following advantages: low off-target rate, high efficiency, cost-effectiveness, and a wide range of applications. In addition, there are also literature reports that CRISPR hybrid RNA-DNA (chRDNA) guide technology can significantly improve the specificity of Cas9 protein compared with all-RNA guide technology, thereby achieving a high level of expected genome editing in cells and minimizing off-target.

[0140] In this application, the terms "zinc finger nuclease technology" and "zinc finger nuclease system" have the common meanings in the art. The core design concept of zinc finger nuclease technology is the ingenious integration of two functional domains—a specific recognition module and a functional module. The most classic zinc finger nuclease is a fusion of the nonspecific endonuclease Fok I with a domain containing zinc fingers that can recognize specific DNA sequences.

[0141] In this application, the terms "transcription activator-like effector nuclease technology" and "transcription activator-like effector nuclease system" have the usual meaning in the art. TALE effectors were originally discovered as an invasion strategy for bacteria to infect plants. By connecting the Fok I nuclease to a man-made TALE with sequence-specific binding ability, researchers have formed a class of powerful tools with specific gene editing functions, namely TALEN proteins. A typical TALEN protein consists of an N-terminal domain containing a nuclear localization signal (NLS), a central domain containing a typical tandem TALE repeat sequence that can recognize a specific DNA sequence, and a C-terminal domain with Fok I nuclease function. The core principle of TALEN technology is to orderly realize the three different functions of guiding entry into the cell nucleus, specific recognition of target site DNA, and cutting of target site DNA on the same TALEN protein.

[0142] In this application, the term "hinge region" refers to the connecting segment between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize antigens by giving the antigen recognition domain a certain range of motion. The hinge regions currently used are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α.

[0143] In this application, "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to a certain extent, but do not directly participate in signal transduction, and can enhance downstream signal transduction through interaction. The transmembrane region can be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0144] In the present application, the term "intracellular domain" includes the intracellular signaling region and may also include the costimulatory signaling region.

[0145] In this application, the term "intracellular signaling region" refers to the activation of at least one normal effector function of the immune effector cells responsible for expressing CAR. The intracellular signaling region can be derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0146] In this application, the term "co-stimulatory signaling region" exists because, in addition to the stimulation of antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, as well as the effector function of activated cells. In certain embodiments, CAR may also include one or more co-stimulatory signaling regions, wherein the co-stimulatory signaling region may be derived from one, two or more than three of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0147] In this application, the term "guide peptide" refers to a short peptide preceding an extracellular antigen recognition domain (e.g., an scFv sequence) that guides the export of recombinant proteins synthesized within cells to the extracellular space. Commonly used guide peptides include the human CD8α signal peptide or the human GM-CSF receptor α signal peptide.

[0148] In this application, one of the key factors determining the therapeutic effect of CAR-immune effector cells is the selection of tumor target antigens, but the selection of antigen targets is not necessarily single. Therefore, the extracellular antigen recognition domain can also include an extracellular antigen recognition domain (such as scFv antibody) targeting any of the following targets: CD5, CD19, CD20, CD22, CD33, CD123, CLL1, BCMA, CD138, CS1. For example, in a dual-target CAR-T product, the extracellular antigen recognition domain includes scFv sequences targeting two targets. An scFv antibody targeting a single target includes an antibody heavy chain variable region (VH) and a light chain variable region (VL), which are connected by a linker sequence; an scFv antibody targeting two or more targets includes VH and VL regions targeting different targets, and the different regions are also directly or indirectly connected by a linker sequence, and their arrangement can be any of the following forms: target 1 VL-target 1 VH-target 2 VL-target 2 VH, target 2 VL-target 2 VH-target 1 VL-target 1 VH, target 1 VL-target 2 VL-target 2 VH-target 1 VH, target 2 VL-target 1 VL-target 1 VH-target 2 VH, and target 2 VL-target 1 VL-target 1 VH-target 2 VH. The above “-” represents connection through a linker sequence.

[0149] In this application, the term "linker" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure / region of an antibody or chimeric antigen receptor of the present invention. The linker can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains are free to move relative to each other. When it is desired to ensure that two adjacent domains do not interfere with each other spatially, a longer linker can be used.

[0150] In this application, the term "isolated" generally refers to a substance obtained from its natural state by artificial means. If a substance or component is "isolated" in nature, it may be that the natural environment in which it is located has changed, or 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 same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated. The term "isolated" does not exclude substances that have been artificially obtained from their natural state by artificial means or synthesized, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0151] As used herein, the term "isolated nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides, or ribonucleotides of any length in isolated form, either separated from their natural environment or artificially synthesized analogs.

[0152] In this application, when referring to CAR gene transduction / transfection and target gene expression, gene transduction / transfection methods mainly include viral and non-viral methods, such as gamma-retroviral vectors, lentiviral vectors, adenovirus-associated virus vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

[0153] The term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and protein expression can be achieved. A vector can transform, transduce, or transfect host cells, allowing the genetic material it carries to be expressed in host cells. Examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Examples of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A single vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication origin site. Vectors may also include components that facilitate cell entry, such as viral particles, liposomes, or protein coats, but are not limited to these. However, the introduction of a polynucleotide encoding a protein into cells does not necessarily require the use of a vector, such as a transposon or electroporation. The term "transposon" refers to a discrete DNA fragment with the ability to migrate between chromosomal loci and carry genetic information, such as the Sleeping Beauty SB system and the PB system derived from Lepidoptera. In certain embodiments, electroporation can also be used to transduce mRNA into T cells.

[0154] In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, such as a cell that promotes an immune effector response. Immune effector cells can be selected from the following group: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), induced pluripotent stem cells, T lymphocytes differentiated into by induced pluripotent stem cells, NK cells differentiated into by induced pluripotent stem cells, and one or more of embryonic stem cells.

[0155] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may include the immune effector cells described herein and may further include one or more pharmaceutically acceptable excipients, such as one or more of a carrier, a preservative, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, and a preservative. In certain embodiments, the pharmaceutically acceptable excipient includes a preservative, such as a cell freezing solution. In certain embodiments, the pharmaceutical composition of this application is a cell suspension or frozen cells thereof.

[0156] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cows, sheep, or monkeys.

[0157] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0158] In this application, the term "about" generally refers to a fluctuation range above or below the specified value that is acceptable to those skilled in the art, such as: varying within the range of ±0.5%-10%, for example, varying within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

[0159] Humanized antibodies or antigen-binding fragments thereof targeting CD7, corresponding nucleic acid molecules, corresponding vectors, corresponding cells, and corresponding pharmaceutical compositions

[0160] In one aspect, the present application provides a humanized antibody or antigen-binding fragment thereof targeting CD7, comprising:

[0161] The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2;

[0162] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30;

[0163] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 28 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30.

[0164] In this application, the biocomputational evaluation results showed that the humanization designs of the murine antibody Th69 all improved the degree of humanization of the antibody, and the immunogenicity prediction results showed that humanization reduced the immunogenicity of the antibody, and the immunogenicity risks of the five candidate antibodies were all low. The inventors also conducted experiments to verify the proliferation, specificity, and functionality of chimeric antigen receptors composed of five humanized scFv antibodies. The five groups of humanized CAR-T cells showed significantly weaker nonspecific killing of negative target cells than murine mTh69 CAR-T cells. Compared with murine mTh69 CAR-T cells, humanized Th69 cells expressed higher CD107a after culture with antigen-positive target cells, but no significant difference in CD107a expression after culture with antigen-negative target cells, suggesting that the humanized Th69 scFv enhanced its specific binding ability to the CD7 antigen. Multiple rounds of stimulation and expansion assays showed that humanized Th69 cells significantly outperformed murine CAR-T cells in proliferation, with hu11, hu12, and hu21 CAR-T cells showing greater proliferation advantages. In vivo efficacy results showed that the humanized hu21 sequence had a more potent anti-tumor effect and prolonged mouse survival. Therefore, humanized hu21, hu11, and hu12 were selected as candidate sequences.

[0165] In certain embodiments, the humanized antibody or antigen-binding fragment thereof is a scFv antibody, a sc(Fv)2 antibody, or a [sc(Fv)2]2 antibody.

[0166] In certain embodiments, the humanized antibody or antigen-binding fragment thereof is a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 3;

[0167] or, a scFv antibody having the amino acid sequence shown in SEQ ID NO: 35;

[0168] Or, a scFv antibody having the amino acid sequence shown in SEQ ID NO: 36.

[0169] In yet another aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned humanized antibody or antigen-binding fragment thereof.

[0170] In certain embodiments, the isolated nucleic acid molecule described above has a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, which is shown in SEQ ID NO: 4;

[0171] and / or, a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 2, which is shown in SEQ ID NO: 5;

[0172] and / or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 28, which is shown in SEQ ID NO: 37;

[0173] and / or, a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30, which is shown in SEQ ID NO:38.

[0174] In another aspect, the present application also provides a vector comprising the isolated nucleic acid molecule described above. 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); bacteriophage such as lambda phage or M13 phage and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).

[0175] In yet another aspect, the present application further provides a cell comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof, isolated nucleic acid molecules or vectors.

[0176] In another aspect, the present application further provides a pharmaceutical composition comprising any of the above-mentioned humanized antibodies or antigen-binding fragments thereof, an isolated nucleic acid molecule, a vector or cells, and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include, but are not limited to, one or more of a carrier, a protective agent, and a stabilizer.

[0177] Application of humanized antibodies or antigen-binding fragments thereof targeting CD7

[0178] In another aspect, the present application also provides the use of the above-mentioned humanized antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or cell in the preparation of a detection reagent for diagnosing a disease or condition related to the expression of CD7.

[0179] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0180] In certain embodiments of the above application, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0181] In certain embodiments of the above application, the CD7+ Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0182] In certain embodiments of the above application, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0183] In another aspect, the present application also provides a use of the above-mentioned humanized antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or cell in the preparation of a medicament for treating a disease or condition associated with CD7 expression.

[0184] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0185] In certain embodiments of the above application, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0186] In certain embodiments of the above application, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0187] In certain embodiments of the above application, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0188] On the other hand, the present application also provides a method for treating a disease or condition, comprising the following steps: administering an effective amount of a drug comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof, isolated nucleic acid molecules, vectors or cells to a subject in need of treating a disease or condition associated with CD7 expression.

[0189] In certain embodiments of the above method, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0190] In certain embodiments of the above method, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0191] In certain embodiments of the above method, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0192] In certain embodiments of the above method, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0193] In certain embodiments of the above methods, the administration can be performed by various routes, such as oral, intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0194] On the other hand, the present application also provides a drug comprising the above-mentioned humanized antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or cell, for treating diseases or conditions related to CD7 expression.

[0195] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0196] In certain embodiments of the above-mentioned drug, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 +T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0197] In certain embodiments of the above-mentioned drug, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0198] In certain embodiments of the above-mentioned drug, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0199] Antibody drugs and antibody-drug conjugates comprising humanized antibodies or antigen-binding fragments thereof targeting CD7

[0200] In another aspect, the present application provides an antibody drug comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof.

[0201] In certain embodiments of the above-mentioned antibody drug, the antibody drug is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.

[0202] In another aspect, the present application also provides an antibody-drug conjugate comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof.

[0203] Chimeric antigen receptor targeting CD7, corresponding nucleic acid molecule, corresponding vector, corresponding immune effector cell, and pharmaceutical composition

[0204] In another aspect, the present application further provides a chimeric antigen receptor targeting CD7, comprising an extracellular antigen recognition domain targeting CD7, a hinge region, a transmembrane region, and an intracellular domain, wherein the extracellular antigen recognition domain targeting CD7 comprises:

[0205] The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2;

[0206] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30

[0207] Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 28 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30.

[0208] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the extracellular antigen recognition domain comprises any one selected from the following structures: an amino acid sequence as set forth in SEQ ID NO: 1-a linker-an amino acid sequence as set forth in SEQ ID NO: 2, an amino acid sequence as set forth in SEQ ID NO: 2-a linker-an amino acid sequence as set forth in SEQ ID NO: 1, an amino acid sequence as set forth in SEQ ID NO: 1-a linker-an amino acid sequence as set forth in SEQ ID NO: 30, an amino acid sequence as set forth in SEQ ID NO: 30-a linker-an amino acid sequence as set forth in SEQ ID NO: 1, an amino acid sequence as set forth in SEQ ID NO: 28-a linker-an amino acid sequence as set forth in SEQ ID NO: 30, an amino acid sequence as set forth in SEQ ID NO: 30-an linker-an amino acid sequence as set forth in SEQ ID NO: 28;

[0209] Optionally, the extracellular antigen recognition domain comprises any one selected from the following structures: an amino acid sequence as shown in SEQ ID NO: 2-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 30-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 30-an linker sequence-an amino acid sequence as shown in SEQ ID NO: 28;

[0210] Further optionally, the extracellular antigen recognition domain is any one selected from the following structures: the amino acid sequence shown in SEQ ID NO: 2-a connecting sequence-the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 30-a connecting sequence-the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 30-a connecting sequence-the amino acid sequence shown in SEQ ID NO: 28.

[0211] In the above description, “-” indicates mutual connection, and in the above description, “-” has directionality, which indicates connection from the N-terminus to the C-terminus of the amino acid; alternatively, “-” indicates direct connection.

[0212] In certain embodiments of any of the above chimeric antigen receptors, the linker sequence is selected from one or more of the following sequences: SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0213] In certain embodiments of any of the above chimeric antigen receptors, the extracellular antigen recognition domain comprises: a scFv antibody with an amino acid sequence as shown in SEQ ID NO: 3, or a scFv antibody with an amino acid sequence as shown in SEQ ID NO: 35, or a scFv antibody with an amino acid sequence as shown in SEQ ID NO: 36;

[0214] Optionally, the amino acid sequence of the extracellular antigen recognition domain is as shown in SEQ ID NO: 3, or as shown in SEQ ID NO: 35, or as shown in SEQ ID NO: 36.

[0215] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence as shown in SEQ ID NO:9; further optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:9.

[0216] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence as shown in SEQ ID NO: 10; still further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 10.

[0217] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the intracellular domain comprises an intracellular signaling region; optionally, further comprises a costimulatory signaling region.

[0218] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO: 11; further optionally, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO: 11.

[0219] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the costimulatory signaling region is derived from one, two or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; further optionally, the amino acid sequence of the costimulatory signaling region comprises the amino acid sequence shown in SEQ ID NO: 12; further optionally, the amino acid sequence of the costimulatory signaling region is as shown in SEQ ID NO: 12.

[0220] In certain embodiments, any of the above-mentioned chimeric antigen receptors further comprises a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence as shown in SEQ ID NO: 13; further optionally, the amino acid sequence of the guide peptide is as shown in SEQ ID NO: 13.

[0221] In certain embodiments of any of the above chimeric antigen receptors, the chimeric antigen receptor comprises any one of the following sequences: the amino acid sequence shown in SEQ ID NO: 14, or the amino acid sequence shown in SEQ ID NO: 18; or the amino acid sequence shown in SEQ ID NO: 19;

[0222] Optionally, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO: 14, or as shown in SEQ ID NO: 18, or as shown in SEQ ID NO: 19.

[0223] In yet another aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above-mentioned chimeric antigen receptors.

[0224] In certain embodiments of the isolated nucleic acid molecule described above, the nucleotide sequence encoding the chimeric antigen receptor comprises: a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as set forth in SEQ ID NO: 1, which is set forth in SEQ ID NO: 4; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO: 2, which is set forth in SEQ ID NO: 5;

[0225] or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, which is shown in SEQ ID NO: 4; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30, which is shown in SEQ ID NO: 38;

[0226] Or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:28, which is shown in SEQ ID NO:37; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30, which is shown in SEQ ID NO:38.

[0227] In certain embodiments of the isolated nucleic acid molecule described above, the nucleotide sequence encoding the chimeric antigen receptor comprises:

[0228] a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 14, which is shown in SEQ ID NO: 15;

[0229] or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 18, which is shown in SEQ ID NO: 39;

[0230] Or, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 19 is shown in SEQ ID NO: 40.

[0231] In another aspect, the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule. The vector can be selected from any one or more of the following: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage and animal virus, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0232] In some embodiments, the above-mentioned vector is an expression vector; in other embodiments, the vector is a viral vector; in other embodiments, the vector is a lentiviral vector.

[0233] In yet another aspect, the present application also provides an engineered immune effector cell comprising the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0234] In certain embodiments of the above-mentioned engineered immune effector cells, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, NK cells differentiated from induced pluripotent stem cells and embryonic stem cells.

[0235] In certain embodiments of the engineered immune effector cells, the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

[0236] In certain embodiments of the above-mentioned engineered immune effector cells, the allogeneic T lymphocytes comprise gene-edited CD7 / TRAC double-negative cells; further, the allogeneic T lymphocytes comprise 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more of gene-edited CD7 / TRAC double-negative cells.

[0237] In certain embodiments, the engineered immune effector cells are αβT lymphocytes or γδT lymphocytes.

[0238] On the other hand, the present application also provides a pharmaceutical composition comprising the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.

[0239] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes a protective agent.

[0240] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes cell freezing solution.

[0241] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutical composition is a cell suspension or frozen cells thereof.

[0242] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutical composition is in the form of an intravenous injection.

[0243] Method for preparing engineered immune effector cells

[0244] In yet another aspect, the present application also provides a method for preparing engineered immune effector cells, comprising the following steps: introducing a nucleotide sequence encoding any one of the above-mentioned chimeric antigen receptors into the immune effector cells.

[0245] In certain embodiments of the above method, the immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK) and embryonic stem cells.

[0246] In certain embodiments of the above method, the engineered immune effector cells are T lymphocytes; optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

[0247] In certain embodiments of the above method, the allogeneic T lymphocytes contain 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more of gene-edited CD7 / TRAC double-negative allogeneic T lymphocytes.

[0248] In certain embodiments of the above method, the T cell Trac gene and CD7 gene are gene-edited by a gene editing tool to obtain CD7 / TRAC double-negative cells, and the gene editing tool is selected from one of the CRISPR / Cas system, zinc finger nuclease system, and transcription activator-like effector nuclease system; optionally, the gene editing tool is selected from the CRISPR / Cas system; further optionally, the CRISPR / Cas system includes Cas9 protein and sgRNA.

[0249] In certain embodiments of the above method, the sgRNA in the CRISPR / Cas system targeting the CD7 gene is shown as SEQ ID NO: 33.

[0250] In certain embodiments of the above method, the sequence of chRDNA in the CRISPR / Cas system targeting the Trac gene is shown as SEQ ID NO:34.

[0251] In certain embodiments of the above method, the T lymphocytes are αβ T lymphocytes or γδ T lymphocytes.

[0252] In certain embodiments of the above-mentioned method, the method for introducing a nucleotide sequence encoding any of the above-mentioned chimeric antigen receptors into the immune effector cells is selected from one or more of the following: a method using a virus or a non-viral method; optionally, the method using a virus includes using one or more of the following viral vectors: a γ retroviral vector, a lentiviral vector, an adenovirus-associated viral vector; the non-viral method includes one or more of the following methods: gene transfer using a transposon, gene transduction mediated by mRNA, and electroporation.

[0253] Application of chimeric antigen receptors targeting CD7

[0254] In another aspect, 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 a drug for treating a disease or condition related to the expression of CD7.

[0255] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0256] In certain embodiments of the above application, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0257] In certain embodiments of the above application, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0258] In certain embodiments of the above application, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0259] On the other hand, the present application also provides a method for treating a disease or condition related to the expression of CD7, comprising the following steps: administering an effective amount of the above-mentioned engineered immune effector cells or pharmaceutical composition to a subject in need of treating a disease or condition related to the expression of CD7.

[0260] In certain embodiments of the above methods, the administration can be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. For example, the administration can be administered to the subject by intravenous injection. In certain embodiments, an effective dose of the engineered immune effector cells or pharmaceutical composition can be administered to the subject in a single dose or in divided doses over a certain period of time, such as once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0261] In certain embodiments of the above method, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0262] In certain embodiments of the above method, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0263] In certain embodiments of the above method, the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0264] In certain embodiments of the above method, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0265] In certain embodiments of the above methods, the administration is by intravenous injection.

[0266] In certain embodiments of the above method, the effective amount of the engineered immune effector cells or pharmaceutical composition is 1×10 5 to 1×10 7 In some embodiments, the dosage may be different for different indications; the dosage may also be different for patients with different disease severity. The dosage range may be 1×10 5 CAR-positive T cells / kg to 1×10 7 CAR-positive T cells / kg, for example, 1×105 CAR-positive T cells / kg to 1×10 6 CAR-positive T cells / kg, 1×10 6 CAR-positive T cells / kg to 1×10 7 CAR-positive T cells / kg, 0.5×10 6 CAR-positive T cells / kg, 0.6×10 6 CAR-positive T cells / kg, 0.7×10 6 CAR-positive T cells / kg, 0.8×10 6 CAR-positive T cells / kg, 0.9×10 6 CAR-positive T cells / kg, 1.0×10 6 CAR-positive T cells / kg, 1.1×10 6 CAR-positive T cells / kg, 1.2×10 6 CAR-positive T cells / kg, 1.3×10 6 CAR-positive T cells / kg, 1.4×10 6 CAR-positive T cells / kg, 1.5×10 6 CAR-positive T cells / kg, 1.6×10 6 CAR-positive T cells / kg, 1.7×10 6 CAR-positive T cells / kg, 1.8×10 6 CAR-positive T cells / kg, 1.9×10 6 CAR-positive T cells / kg, 2.0×10 6 CAR-positive T cells / kg.

[0267] On the other hand, the present application also provides a drug comprising the above-mentioned engineered immune effector cells or pharmaceutical composition, for treating diseases or conditions related to the expression of CD7.

[0268] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CD7 is CD7 + Hematologic malignancy.

[0269] In certain embodiments of the above-mentioned drug, the CD7 + Hematologic malignancies selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0270] In certain embodiments of the above-mentioned drug, the CD7 + Acute T-lymphoblastic leukemia including CD7+ Early pro-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0271] In certain embodiments of the above-mentioned drug, the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0272] Without intending to be bound by any theory, the following examples are merely illustrative of the chimeric antigen receptors, engineered immune effector cells, preparation methods, and uses of the present invention, and are not intended to limit the scope of the present invention. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into such vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled 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.

[0273] Example 1: Humanized transformation of mouse antibodies and preparation of CD7 CAR-T cells

[0274] In the present application, the VH amino acid sequence (as shown in SEQ ID NO: 31) and VL amino acid sequence (as shown in SEQ ID NO: 32) of the murine antibody sequence Th69 were divided into CDRs based on the KABAT rule, and the amino acid sequences of VH CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and the amino acid sequences of VL CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively, were obtained, and humanized transformation was performed based on this.

[0275] The template selected for the humanization of the Th69 VH region was IGHv3-21*07. During the humanization of VH, different combinations of multiple important amino acids were selected for back mutation, resulting in three humanized Th69 VH designs, namely Th69 VH-1, Th69 VH-2, and Th69 VH-3 (their amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 27, and SEQ ID NO: 28, respectively). Multiple important amino acids included amino acids at the PHI-ANGLE position, amino acids that are completely buried and have a low frequency of occurrence at this position in humans, and amino acids at the PHI-ANGLE position that are very close to the CDR region.

[0276] The template selected for humanization of the Th69 VL region was IGKv1-27*01. Different combinations of multiple important amino acids were selected for back mutation in the humanization of VL, resulting in three humanized Th69 VL designs: Th69 VL-1, Th69 VL-2, and Th69 VL-3 (their amino acid sequences are shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 2, respectively). Multiple important amino acids included those at the PHI-ANGLE position, the VH / VL interface, and the upper core amino acids.

[0277] The humanized heavy and light chains were connected through linker sequences to form hu01 scFv, hu10 scFv, hu11 scFv, hu12 scFv, and hu21 scFv, respectively. We chose to screen each candidate humanized scFv antibody based on the second-generation CAR structure, that is, the second-generation CAR structure obtained by directly connecting the signal peptide-extracellular antigen recognition domain-hinge region-transmembrane region-costimulatory signal transduction region-intracellular signal transduction region in the order from N-terminus to C-terminus. In each CAR structure, the CD8α guide chain was used as the signal peptide (as shown in SEQ ID NO: 13), hu01 scFv, hu10 scFv, hu11 scFv, hu12 scFv, hu21 scFv and murine control Th69 scFv were used as the extracellular antigen recognition domains, the hinge region (as shown in SEQ ID NO: 9) and the transmembrane region (as shown in SEQ ID NO: 10) adopted the structure of CD8α, 4-1BB was used as the costimulatory signal transduction region (as shown in SEQ ID NO: 12), and CD3ζ was used as the intracellular signal transduction region (as shown in SEQ ID NO: 11). The specific VH and VL amino acid sequences of each candidate humanized scFv and the amino acid sequence of the corresponding CAR are shown in Table 1.

[0278] Table 1. Various CD7 CAR sequences

[0279] The specific method for obtaining CD7 CAR-T cells is as follows:

[0280] 1. Construction of lentiviral vector

[0281] According to Table 1, the nucleotide sequences encoding the above 6 CD7 CAR structures were respectively constructed into empty lentiviral vectors (manufacturer: SBI, product number: CD500-CD800, as described in Example 1 of WO2021 / 121227 for conventional resistance modification) to obtain CAR expression vectors. Subsequently, the CAR expression vectors and three packaging plasmids were transfected into 293T cells together, and functional lentiviral vectors were obtained after collection and purification. The three packaging plasmids are pMD2.G (purchased from Biovector, product number Biovector012259), pMDLg / pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253).

[0282] 2. Preparation of T cells

[0283] 1) T cell sorting

[0284] Peripheral blood mononuclear cells (PBMCs) were isolated from human single-blood cells, and T cells were then sorted from the PBMCs.

[0285] 2) Activation of T cells

[0286] The isolated T cells were resuspended in complete lymphocyte culture medium (X-VIVO15 medium + 5% fetal bovine serum + 300 IU / ml IL-2 or X-VIVO15 medium + 5% fetal bovine serum + 5 ng / ml IL-15 + 10 ng / ml IL-7) to a final concentration of (1-3) × 10 6 cells / ml, 1ul beads / 1×10 6 The cells were stimulated by adding CD3 / CD28 magnetic beads, mixed and then cultured in an incubator at 37°C + 5% CO2 for at least 24 hours to obtain activated T cells.

[0287] 3) Gene editing of activated T cells

[0288] 90% of T cells express CD7 on their surface. If CD7 CAR-T cells express CD7 on their surface, it will cause CD7 CAR-T cell suicide. Using gene editing to knock out the CD7 gene in T cells so that they do not express CD7 on their surface is an effective way to avoid suicide. At the same time, using gene editing to knock out the Trac gene in T cells allows them to be used as universal CAR-T products. The specific method is as follows:

[0289] Prepare RNP complexes. Each 11.5 μl of RNP complex consists of 10 μl Opti-MEM medium, 0.6 μl Cas9 (10 mg / ml), 0.45 μl CD7 (100 μM) gRNA, and 0.45 μl TRAC chRDNA (100 μM). Incubate at room temperature for 15 minutes. The sequence of the CD7 sgRNA is shown in SEQ ID NO: 33, and the sequence of the TRAC chRDNA is shown in SEQ ID NO: 34. Activated T cells were removed and electroporation buffer was added at a rate of 10 μl / mL cell. The cells were resuspended and mixed with the incubated RNP complexes. 11.5 μl of RNP complex was added per 1 mL of cells and transferred to an electroporation strip. Select EH100 as the electroporation program for the Lonza 4D electroporator and perform electroporation. After the electroporation is completed, quickly add pre-warmed T cell complete medium to the electroporation strip and immediately place the electroporation strip in the incubator for incubation for 15 minutes. Then transfer the cells to a 96-well plate and add 100ul of pre-warmed T cell complete medium. The culture conditions are 37°C + 5% CO2.

[0290] 3. Use lentiviral transduction to sorted, activated, and gene-edited (CD7 and Trac double gene knockout) T cells to prepare corresponding CD7 CAR-T cells.

[0291] The gene-edited T cells were removed and resuspended in a simple X-VIVO15 medium containing polybrene at a final concentration of 8 μg / ml. The cell suspension was obtained by mixing and the cells were plated at a rate of 2 × 10 6 200 μl of lentiviral vector was slowly added to the cells, mixed well, and placed in a well plate. The cells were cultured in an incubator at 37° C. + 5% CO 2 for at least 4-6 hours.

[0292] 4. Expansion and culture of transduced T cells

[0293] The transduced cells were removed and cultured with complete lymphocyte 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.

[0294] After T cells were infected with lentivirus containing the CAR structures in Table 1, the obtained T cells were named according to their CAR serial numbers. For example, T cells obtained using hu21 CAR were named hu21 CAR-T cells, and T cells obtained using hu12 CAR were named hu12 CAR-T cells.

[0295] On the 4th day after transduction, cells were stained with fluorescently labeled CD7 antibody (BioLegend, 395606) / CD3 antibody (BioLegend, 317318), and CD7 was detected by flow cytometry. - / CD3 - The cell ratio was calculated, and the CD7 knockout efficiency was 94.481±3.656% and the TRAC knockout efficiency was 96.201±1.437%.

[0296] Example 2. Detection of CAR-T cell CAR positive ratio and CAR-T cell expansion

[0297] 1. Detection of CAR-positive ratio in CAR-T cells

[0298] The 6 CAR-T cells obtained in Example 1, untransduced and unknocked-out T cells (UTD-NoEP group), and untransduced but double-knocked-out T cells (UTD-KO TRAC+CD7 group) were stained and labeled with FITC fluorescently labeled CD7 antigen (manufacturer: ACRO Biosystems, catalog number: CD7-HF258). The CAR molecule positive ratio of various cells was detected by flow cytometry at different days after transduction. The test results are shown in Figure 1: There was no significant difference in CAR expression on the surface of the 5 CAR-T cells using humanized scFv and the CAR-T cells using mouse scFv. The CAR positivity rates detected 4, 6, and 11 days after transduction were all at the same level; untransduced and unknocked-out T cells and untransduced but double-knocked-out T cells were used as two groups of negative controls, and no CAR expression was detected.

[0299] 2. Expansion detection of total T cells cultured for different days after transduction

[0300] The 6 CAR-T cells obtained in Example 1, the untransduced and unknocked-out T cells (UTD-NoEP group), and the untransduced but double-knocked-out T cells (UTD-KO TRAC+CD7 group) were sampled at different days after transduction, and stained with AO / PI double fluorescence (AO / PI double staining cell apoptosis detection kit can be purchased through commercial channels. It uses AO / PI probe double staining of cell nuclei to detect the state of apoptotic cells and can distinguish normal cells from apoptotic cells). The cells in each group were counted by a cell counter to calculate the total T cell expansion multiple during the culture process. The test results are shown in Figure 2: The expansion multiples of the 5 CAR-T cells using humanized scFv during the culture process were higher than those of the CAR-T cells using mouse scFv, but there was no significant difference in the expansion capacity of the 5 CAR-T cells using humanized scFv. In addition, the expansion capacity of the double-knocked T cells, regardless of whether they were transduced with a CAR structure, was significantly higher than that of the non-knocked-out T cells. It is speculated that the suicide injury to the T cells caused by the non-knockout of the CD7 gene affects the expansion capacity.

[0301] Example 3: In vitro killing ability of CAR-T cells

[0302] In vitro killing experiments are divided into short-term killing experiments and long-term killing experiments. Short-term killing refers to the use of mouse or humanized Th69 scFv CAR-T cells and target cells according to the effector cells (effector cells according to CAR + After co-culture with target cells at different effector-target ratios (1:3, 1:1, and 3:1) in X-VIVO15 medium for 4-6 hours, the killing ratio of CAR-T cells to target cells was detected by detecting the activity of luciferase stably expressed in the target cells. The method for establishing a target cell line stably expressing luciferase is as follows: target cells were infected with a virus carrying a luc-GFP sequence (conventional luciferase and fluorescent protein sequences in the prior art can be used), and monoclonal culture was performed by infinite dilution, and GFP-positive clones with good growth status were selected for culture; long-term killing refers to the use of CAR-T cells using mouse or humanized Th69 scFv to co-culture with target cells according to the effector cell (effector cells are expressed by CAR + After co-cultured in X-VIVO15 medium for 24 h under conditions of different effector-target ratios (cell count, CAR-T cell count, and target cell ratio, respectively) and target cells (1:32, 1:16, 1:4, and 1:1), the killing ratio of CAR-T cells to target cells was determined by detecting the activity of luciferase stably expressed in target cells.

[0303] In the above experiment, untransduced and non-knockout T cells (NoEP group) and untransduced but double-knockout T cells (UTD group) were used as negative effector cells; target cells included Molt-4 cells (human acute T lymphoblastic leukemia cells) and Jurkat cells (human T lymphoblastic leukemia cells) that endogenously express CD7, as well as negative controls KO CD7 Molt-4 cells (referring to the establishment of the KO CD7 Molt-4 cell line by selecting monoclonal CD7-Molt-4 cells by infinite dilution after CD7 was knocked out by gene editing using CRISPR / Cas9 technology, and the CD7 sgRNA used was the same as in Example 1) and KO CD7 Jurkat cells (referring to the establishment of the KO CD7 Jurkat cell line by selecting monoclonal CD7-Jurkat cells by infinite dilution after CD7 was knocked out by gene editing using CRISPR / Cas9 technology, and the CD7 sgRNA used was the same as in Example 1). The experimental results are as follows:

[0304] Short-term killing results: As shown in Figure 3A, at effector-target ratios of 1:3, 1:1, and 3:1, CAR-T cells using mouse or humanized Th69 scFv had the same level of killing ability against positive target cells MOLT4. However, as shown in Figure 3B, mouse Th69 CAR-T cells and humanized hu01 CAR-T cells had nonspecific killing effects on negative target cells KO CD7 MOLT4 at an effective target ratio of 3:1, while the four groups of humanized hu10 CAR-T cells, hu11 CAR-T cells, hu12 CAR-T cells, and hu21 CAR-T cells had no nonspecific killing effects on negative target cells, and the differences were significant (*** indicates p < 0.001, **** indicates p < 0.0001), indicating that these four groups of humanized CAR-T cells may have better safety.

[0305] Long-term killing results: As shown in Figures 3C and 3D, under the conditions of effector-target ratios of 1:32, 1:16, 1:4, and 1:1, the overall killing ability of CAR-T cells using mouse or five humanized Th69 scFvs against positive target cells MOLT4 and Jurkat was at the same level, but humanized hu21 CAR-T cells had a significant advantage over mouse Th69 CAR-T cells in killing positive target cells Jurkat at a low effector-target ratio of 1:32 (* indicates p < 0.05); In addition, as shown in Figure 3E, at higher effector-target ratios of 1:4 and 1:1, mouse Th69 CAR-T cells had nonspecific killing of negative target cells KO CD7 Jurkat, while the nonspecific killing of negative target cells by the five groups of humanized CAR-T cells was significantly weaker than that of mouse Th69 CAR-T cells (* indicates p < 0.05, * indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001), indicating better safety.

[0306] Example 4. Multiple rounds of stimulation experiments of CAR-T cells

[0307] Antigen stimulation can activate CAR-T cells and cause them to proliferate, but sustained T cell activation can lead to cell exhaustion, which reduces the proliferation and effector function of exhausted T cells. We determined the sustained proliferation capacity of CD3+ cells (i.e., T cell proliferation) in the CD7 CAR-T cell group after multiple rounds of antigen stimulation. Humanized scFvs with long-term proliferation capacity were selected as the preferred antibody screening criteria.

[0308] The CAR positive ratio of each humanized CAR-T cell was adjusted to the same level as the group of CAR-T cells with the lowest CAR positive ratio using UTD. In the antigen stimulation experiment, each group of CAR-T cells was respectively stimulated with CD7 +Molt4 target cells were co-cultured in 24-well plates at a 1:1 effector-target ratio, with 2 ml of X-VIVO15 medium per well, and triplicate wells were used for each group. T cells (CD3 is a marker for distinguishing T cells) were labeled with a fluorescently labeled CD3 antibody (Manufacturer: Biolegend, Catalog No.: 300312), and flow cytometry was used to analyze and measure T cell proliferation. The number of CD3-positive cells was calculated based on volume multiplication. Based on the calculated results, a certain amount of CAR-T cells was removed from each group and added to the corresponding positive target cells at a 1:1 effector-target ratio for a new round of stimulation. This stimulation cycle was repeated every 48 hours for four rounds. The proliferation results are shown in Figure 4: the proliferation ability of the humanized CAR-T cell group was significantly superior to that of the Th69 CAR-T cell group (** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001), among which the proliferation of hu21 CAR-T cells, hu11 CAR-T cells, and hu12 CAR-T cells was more advantageous.

[0309] Example 5: Specificity Analysis

[0310] To test whether humanized sequence modification would affect the specificity of CD7 CAR, we designed a cell-based experiment to evaluate the specificity of the five humanized Th69 CARs screened. Lysosomal associated membrane protein 1 (CD107a) expression on the membrane is considered to be a marker of cytotoxic lymphocytes (CD8 + CD107a is a marker of activation of T cells and NK cells. When cytotoxic lymphocytes are stimulated by specific antigens, immune activation occurs and they express CD107a. Therefore, the expression of CD107a can be used as a means to detect the specificity of CAR-T cells.

[0311] Cell co-culture was performed as follows, and the specificity of CD7 CAR was evaluated by flow cytometry detection of CD107a. The specific steps were as follows: tumor cells, CAR-T cells, and untransduced T cells were counted separately, and the tumor cells were adjusted to 2×10 6 / ml, and the volume of CAR-T cells and untransduced T cells was adjusted to 1×10 6 / ml. Add 20μl of fluorescently labeled CD107a antibody (purchased from Biolegend, model number: 328608) to each well of a 96-well plate. Then, add 100μl of T cells (referring to CAR-T cells or untransduced T cells) and 100μl of tumor cells to each well. Centrifuge at 400 rpm for 3 minutes. Incubate at 37°C in a 5% CO2 incubator for 60 minutes. After 60 minutes, add 20μl of Golgi stop working solution (i.e., 3ml X-VIVO15 medium plus 2μl of Golgi stop protein transport inhibitor, purchased from BD, model number 554724) to each well and incubate for 2.5 hours. Mix equal volumes of CD3 (purchased from Biolegend, model number: 300326) and CD8 antibody (purchased from Biolegend, model number: 344722). After thorough mixing, add 10μl to each well and incubate for 30 minutes. After 30 minutes, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 200 μl FACS buffer (PBS + 0.5% BSA), and mix thoroughly. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant. Add 200 μl FACS buffer (PBS + 0.5% BSA) to each well, mix thoroughly, transfer to a labeled flow cytometer, and analyze CD3. + CD8 + Fluorescence signal of CD107a in the cell population. In the above experiments, CAR-T cells refer to mouse Th69CAR-T cells, 5 humanized CAR-T cells, or h189-4 anti-CD7 scFv (VL and VH are derived from SEQ ID NO: 17 and SEQ ID NO: 18 in CN 112300282A, respectively, and the connection sequence is the same as the connection sequence of other groups), 3A1e-LH anti-CD7 scFv (VL and VH are derived from SEQ ID NO: 90 and SEQ ID NO: 91 in CN 113383071A, respectively, and the connection sequence is the same as the connection sequence of other groups) according to the same CAR component sequence as in Example 1. The empty lentiviral vector mentioned in the example is added and the CAR-T cells are prepared according to the method in Example 1; tumor cells refer to: cell lines that highly express CD7 (also referred to as CD7 in this application). + Target cells) CCRF-CEM (acute T lymphoblastic leukemia cells, commercial cell line), MOLT4 (acute T lymphoblastic leukemia cells, commercial cell line); cell lines with low expression of CD7 (also referred to as CD7 in this application) -Target cells) CD7 KO MOLT4 (after CD7 was knocked out using CRISPR / CAS9, CD7-monoclonal MOLT4 cells were selected and cultured to establish a cell line), Nalm6 (human acute lymphoblastic leukemia cells, commercial cell line), 786-O (human renal clear cell adenocarcinoma cells, commercial cell line), and U266 (human multiple myeloma cells, commercial cell line).

[0312] The detection results of CD107a are shown in Table 2: + Target cells, CD7 - After target cells were co-cultured with untransduced T cells (UTD group), there was no significant difference in the expression level of CD107a; the expression levels of CD7 CAR-T cells and CD7 + After CCRF / MOLT4 co-culture, a higher proportion of target cells expressed CD107a, while CAR-T cells and CD7 - The CD107a-positive ratio of target cells co-cultured with CD7 KO MOLT4 / Nalm6 / 786-O / U266 was very low, indicating that the five humanized Th69 CARs had excellent antigen recognition specificity. Furthermore, hu01 CAR-T, hu10CAR-T, hu11 CAR-T, hu12 CAR-T, and hu21 CAR-T cells expressed higher levels of CD107a than murine Th69 CAR-T cells after culture with antigen-positive target cells, suggesting that the humanized Th69 scFv enhanced its specific binding ability to the CD7 antigen.

[0313] Table 2. CD107a expression of UTD or different CD7 CAR-T cells after stimulation with various target cells

[0314] Example 6: In vivo functional experiment

[0315] To further screen and identify optimally functional humanized antibodies, we conducted in vivo pharmacodynamic studies of CD7 CAR-T cells. When selecting candidate sequences for in vivo functional studies, humanization scoring software (e.g., the classic humanization scoring system, T20 score) and computational immunogenicity prediction (using the AlphaMHC algorithm) revealed that the hu01 scFv and hu10 scFv sequences were highly humanized and had low immunogenicity, thus being selected as candidate sequences for in vivo functional studies. In vitro functional studies revealed that the hu21 CAR-T cell killing ability was strong, with weak nonspecific killing, and strong ability to expand after multiple rounds of stimulation, thus also being included as a candidate sequence for in vivo functional studies.

[0316] CCRF-CEM-GFP-Luc cells were cultured at a density of 2 × 105 The CCRF-CEM-GFP-Luc acute T lymphoblastic leukemia animal model was established by inoculating cells into the tail vein of female NSG mice at a volume of 0.2 ml / mouse. On the 4th day after cell inoculation, the mice were randomly divided into groups according to the tumor imaging signal intensity and the corresponding cells were reinfused through the tail vein. The experiment was divided into 8 groups (group information is shown in the table below), including: PBS group, untransduced and unknocked T cell group (T cell group), untransduced T cell group with double knockout of CD7 / TRAC (DKO-T cell group), positive control 3A1e group (as mentioned in Example 5), mouse Th69CAR-T cell group (mTh69 KO group), humanized hu01 CAR-T cell group (hu01 KO group), humanized hu10 CAR-T cell group (hu10 KO group) and humanized hu21CAR-T cell group (hu21 KO group). The T cells of the above groups 3-8 were all derived from CD7 / TRAC double knockout T cells. The CAR-T positive rates of each group were similar and were adjusted to the same positive rate before reinfusion. A single infusion of CAR-T cells was performed via the tail vein at a dose of 3×10 6 CAR+ cells were infused at a dose of 0.2 ml per mouse. From Day 0 to Day 14, in vivo imaging and blood collection for flow cytometry were performed twice weekly, and then weekly thereafter. Mouse body weights and tumor imaging signal values ​​were recorded to monitor and evaluate the tumor clearance efficacy of humanized hu01, hu10, and hu21 CAR-T cells in the animals.

[0317] Table 3. Grouping and dosage of each group in the mouse experiment

[0318] Note: Count the total number of viable cells after recovery and adjust the cell concentration to the dosing concentration with PBS.

[0319] The results of the mouse experiments are shown in Table 4, Figure 5, and Figure 6. The results in Table 4 and Figure 5 show the survival of the mice. The survival time of mice in the 3A1e group, mTh69 group, hu01 KO group, and hu10 KO group was longer than that in the PBS group, T cell group, and DKO-T cell group. The survival time of mice in the hu21 KO group was the longest (as shown in Figure 5), with a median survival time of 34.5 days (as shown in Table 4).

[0320] Table 4

[0321] The results of tumor imaging signal values ​​in mice are shown in Figure 6. Compared with the PBS group, T cell group, and DKO-T cell group, the CAR-T cells in the other groups were able to significantly inhibit tumor growth at the tested dose, among which the hu21 KO group had the best tumor inhibition effect.

[0322] A comprehensive comparison of the anti-tumor effects of CAR-T cells in each group and the survival time of each group of mice showed that the in vivo efficacy results showed that the humanized sequence hu21 had a stronger tumor killing effect than the mTh69, hu-10KO, hu-21KO and 3A1e groups. The survival time of mice was extended by 7.5 days compared with the PBS control and 3 days compared with the mouse-derived Th69 CAR-T.

[0323] Description of Sequence Listing

[0324] SEQ ID NO: 1----Th69 VH-1, amino acid sequence of the heavy chain variable region of the scFv in hu01 / 11 / 21CAR;

[0325] SEQ ID NO:2 --- Th69 VL-3, amino acid sequence of the light chain variable region of the scFv in hu21 CAR;

[0326] SEQ ID NO:3 --- Amino acid sequence of the scFv in hu21 CAR;

[0327] SEQ ID NO:4—A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:1;

[0328] SEQ ID NO:5—A nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO:2;

[0329] SEQ ID NO:6---connection sequence;

[0330] SEQ ID NO:7---connection sequence;

[0331] SEQ ID NO:8---connection sequence;

[0332] SEQ ID NO:9---Amino acid sequence of the hinge region;

[0333] SEQ ID NO:10---Amino acid sequence of the transmembrane region;

[0334] SEQ ID NO:11---Amino acid sequence of the intracellular signaling domain;

[0335] SEQ ID NO:12—Amino acid sequence of the co-stimulatory signaling domain;

[0336] SEQ ID NO:13 --- amino acid sequence of the guide peptide;

[0337] SEQ ID NO:14 --- amino acid sequence of hu21 CAR;

[0338] SEQ ID NO: 15—Nucleotide sequence encoding the amino acid sequence of hu21 CAR;

[0339] SEQ ID NO: 16 --- amino acid sequence of hu01 CAR;

[0340] SEQ ID NO:17 --- amino acid sequence of hu10 CAR;

[0341] SEQ ID NO:18 --- amino acid sequence of hu11 CAR;

[0342] SEQ ID NO: 19 --- amino acid sequence of hu12 CAR;

[0343] SEQ ID NO:20 --- amino acid sequence of Th69 CAR;

[0344] SEQ ID NO:21 --- Amino acid sequence of Th69 antibody VH CDR1;

[0345] SEQ ID NO:22—Amino acid sequence of Th69 antibody VH CDR2;

[0346] SEQ ID NO:23—Amino acid sequence of Th69 antibody VH CDR3;

[0347] SEQ ID NO:24—Amino acid sequence of Th69 antibody VL CDR1;

[0348] SEQ ID NO:25—Amino acid sequence of Th69 antibody VL CDR2;

[0349] SEQ ID NO:26—Amino acid sequence of Th69 antibody VL CDR3;

[0350] SEQ ID NO:27 --- Th69 VH-2, amino acid sequence of the heavy chain variable region of the scFv in the hu10 CAR;

[0351] SEQ ID NO:28----Th69 VH-3, amino acid sequence of the heavy chain variable region of the scFv in hu12 CAR;

[0352] SEQ ID NO:29 --- Th69 VL-1, amino acid sequence of the light chain variable region of the scFv in the hu01 CAR;

[0353] SEQ ID NO:30 --- Th69 VL-2, amino acid sequence of the light chain variable region of the scFv in hu10 / 11 / 12CAR;

[0354] SEQ ID NO:31—Amino acid sequence of the heavy chain variable region of the murine antibody Th69;

[0355] SEQ ID NO:32—Amino acid sequence of the light chain variable region of the murine antibody Th69;

[0356] SEQ ID NO:33---CD7 sgRNA;

[0357] SEQ ID NO:34 --- TRAC chRDNA;

[0358] SEQ ID NO: 36 --- Amino acid sequence of the scFv in hull CAR;

[0359] SEQ ID NO:36 --- Amino acid sequence of the scFv in hu12 CAR;

[0360] SEQ ID NO:37—Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:28

[0361] SEQ ID NO:38—Nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30

[0362] SEQ ID NO:39—Nucleotide sequence encoding the amino acid sequence of hu11 CAR

[0363] SEQ ID NO:40 --- Nucleotide sequence encoding the amino acid sequence of hu12 CAR

Claims

1. A humanized antibody or an antigen-binding fragment thereof targeting CD7, comprising: the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 2; or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30; Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:28 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:

30.

2. The humanized antibody or antigen-binding fragment thereof according to claim 1, which is a scFv antibody, a sc(Fv)2 antibody or a [sc(Fv)2]2 antibody.

3. The humanized antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 3; or, a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 35; Or, a scFv antibody having the amino acid sequence shown in SEQ ID NO:

36.

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, wherein: a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, which is shown in SEQ ID NO: 4; and / or, a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:2, which is shown in SEQ ID NO:5; and / or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:28, which is shown in SEQ ID NO:37; And / or, a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30, which is shown in SEQ ID NO:

38. A vector comprising the nucleic acid molecule according to claim 4 or 5.

7. A cell comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the isolated nucleic acid molecule according to claim 4 or 5, or the vector according to claim 6.

8. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the isolated nucleic acid molecule according to claim 4 or 5, the vector according to claim 6 or the cell according to claim 7, and a pharmaceutically acceptable excipient.

9. Use of the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the isolated nucleic acid molecule according to claim 4 or 5, the vector according to claim 6 or the cell according to claim 7 in the preparation of a detection reagent for diagnosing a disease or condition associated with the expression of CD7.

10. The use according to claim 9, wherein the disease or condition associated with the expression of CD7 is CD7 + Hematological tumor; Optionally, the CD7 + Hematological neoplasms selected from one or more of the following: CD7 + Acute T-lymphocytic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

11. The use according to claim 10, wherein the CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T lymphoblastic leukemias; and / or wherein the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

12. Use of the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the isolated nucleic acid molecule according to claim 4 or 5, the vector according to claim 6 or the cell according to claim 7 in the preparation of a medicament for treating a disease or condition associated with the expression of CD7.

13. The use according to claim 12, wherein the disease or condition associated with the expression of CD7 is CD7 + Hematological tumor; Optionally, the CD7 + Hematological neoplasms selected from one or more of the following: CD7 + Acute T-lymphocytic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

14. The use according to claim 13, wherein CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T lymphoblastic leukemias; and / or wherein the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

15. An antibody drug comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

16. An antibody-drug conjugate comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

17. A chimeric antigen receptor targeting CD7, comprising an extracellular antigen recognition domain targeting CD7, a hinge region, a transmembrane region and an intracellular domain, wherein the extracellular antigen recognition domain targeting CD7 comprises: The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 2; Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30 The amino acid sequence of the variable region Or, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:28 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:

30.

18. The chimeric antigen receptor according to claim 17, wherein the extracellular antigen recognition domain comprises any one selected from the following structures: an amino acid sequence as shown in SEQ ID NO: 1-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 2, an amino acid sequence as shown in SEQ ID NO: 2-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 1-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 30, an amino acid sequence as shown in SEQ ID NO: 30-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 28-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 30, an amino acid sequence as shown in SEQ ID NO: 30-a linker sequence-an amino acid sequence as shown in SEQ ID NO: 28; Optionally, the extracellular antigen recognition domain comprises any one selected from the following structures: an amino acid sequence as shown in SEQ ID NO: 2-a connecting sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 30-a connecting sequence-an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence as shown in SEQ ID NO: 30-a connecting sequence-an amino acid sequence as shown in SEQ ID NO: 28; Further optionally, the extracellular antigen recognition domain is any one selected from the following structures: an amino acid sequence as shown in SEQ ID NO:2-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:1, an amino acid sequence as shown in SEQ ID NO:30-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:1, an amino acid sequence as shown in SEQ ID NO:30-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:

28.

19. The chimeric antigen receptor according to claim 18, wherein the linker sequence is selected from one or more of the following sequences: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:

8.

20. The chimeric antigen receptor according to claim 19, wherein the extracellular antigen recognition domain comprises: a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 3, or a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 35, or a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 36; Optionally, the amino acid sequence of the extracellular antigen recognition domain is as shown in SEQ ID NO:3, or as shown in SEQ ID NO:35, or as shown in SEQ ID NO:

36.

21. The chimeric antigen receptor according to any one of claims 17-20, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:

9.

22. The chimeric antigen receptor according to any one of claims 17-20, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

10.

23. The chimeric antigen receptor according to any one of claims 17-20, wherein the intracellular domain comprises an intracellular signaling region; optionally, further comprises a co-stimulatory signaling region.

24. The chimeric antigen receptor of claim 23, wherein the intracellular signaling region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signaling region is derived from CD3ζ; further optionally, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO:

11.

25. The chimeric antigen receptor of claim 23, wherein the co-stimulatory signaling region is derived from one, two or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the co-stimulatory signaling region is derived from CD28 or 4-1BB; further optionally, the amino acid sequence of the co-stimulatory signaling region comprises the amino acid sequence shown in SEQ ID NO:

12.

26. The chimeric antigen receptor according to any one of claims 17-20, further comprising a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:

13.

27. The chimeric antigen receptor according to any one of claims 17 to 20, wherein the chimeric antigen receptor comprises any one of the following sequences: the amino acid sequence shown in SEQ ID NO: 14, or the amino acid sequence shown in SEQ ID NO: 18; or the amino acid sequence shown in SEQ ID NO: 19; Optionally, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:14, or as shown in SEQ ID NO:18, or as shown in SEQ ID NO:

19.

28. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 17-20.

29. The nucleic acid molecule of claim 28, wherein the nucleotide sequence comprises: a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, which is shown in SEQ ID NO: 4; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 2, which is shown in SEQ ID NO: 5; or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1, which is shown in SEQ ID NO: 4; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30, which is shown in SEQ ID NO: 38; Or, a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:28, which is shown in SEQ ID NO:37; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30, which is shown in SEQ ID NO:

38.

30. The nucleic acid molecule of claim 28, wherein the nucleotide sequence comprises: A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 14, which is shown in SEQ ID NO: 15; or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:18, which is shown in SEQ ID NO:39; Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:19, which is shown in SEQ ID NO:

40.

31. A vector comprising the isolated nucleic acid molecule of any one of claims 28-30; optionally, the vector is an expression vector.

32. The vector according to claim 31, wherein the vector is a viral vector; optionally, the vector is a lentiviral vector.

33. An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 17-27, the isolated nucleic acid molecule of any one of claims 28-30, or the vector of any one of claims 31-32.

34. The engineered immune effector cells according to claim 33, which are selected from one or more of T lymphocytes, natural killer cells, peripheral blood mononuclear cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, NK cells differentiated from induced pluripotent stem cells and embryonic stem cells; optionally, they are selected from T lymphocytes; further optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes; further optionally, the allogeneic T lymphocytes contain gene-edited CD7 / TRAC double-negative cells; further optionally, the allogeneic T lymphocytes contain 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more of gene-edited CD7 / TRAC double-negative cells.

35. The engineered immune effector cell of claim 34, wherein the T lymphocyte is an αβ T lymphocyte or a γδ T lymphocyte.

36. A pharmaceutical composition comprising the engineered immune effector cell of any one of claims 33-35 and a pharmaceutically acceptable excipient.

37. The pharmaceutical composition of claim 36, wherein the pharmaceutically acceptable excipient comprises a protective agent.

38. The pharmaceutical composition of claim 37, wherein the pharmaceutically acceptable excipient comprises a cell freezing solution.

39. A method for preparing an engineered immune effector cell, comprising the step of introducing into the immune effector cell a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 26-36.

40. The method according to claim 39, wherein the immune effector cells are selected from one or more of T lymphocytes, natural killer cells, peripheral blood mononuclear cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, NK cells differentiated from induced pluripotent stem cells and embryonic stem cells; optionally, the immune effector cells are T lymphocytes; further optionally, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes; further optionally, the allogeneic T lymphocytes contain gene-edited CD7 / TRAC double-negative cells; further optionally, the allogeneic T lymphocytes contain 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more of gene-edited CD7 / TRAC double-negative cells. The method according to claim 39 , wherein the T lymphocytes are αβ T lymphocytes or γδ T lymphocytes.

42. According to the method of claim 40, the T cell Trac gene and CD7 gene are gene-edited by a gene editing tool to obtain CD7 / TRAC double-negative cells, and the gene editing tool is selected from one of the CRISPR / Cas system, zinc finger nuclease system, and transcription activator-like effector nuclease system; optionally, the gene editing tool is selected from the CRISPR / Cas system; further optionally, the CRISPR / Cas system includes Cas9 protein and sgRNA.

43. According to the method of claim 39, the sgRNA in the CRISPR / Cas system targeting the CD7 gene is shown as SEQ ID NO:33; and / or the sequence of chRDNA in the CRISPR / Cas system targeting the Trac gene is shown as SEQ ID NO:

34.

44. The method according to any one of claims 39-43, wherein the method for introducing the nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 26-36 into the immune effector cells is selected from one or more of the following: a method using a virus or a non-viral method; optionally, the method using a virus includes using one or more of the following viral vectors: a gamma retrovirus vector, a lentivirus vector, an adenovirus-associated virus vector; the non-viral method includes one or more of the following methods: gene transfer using a transposon, gene transduction mediated by mRNA, and electroporation.

45. Use of the chimeric antigen receptor of any one of claims 17-27, the isolated nucleic acid molecule of any one of claims 28-30, the vector of any one of claims 31-32, or the engineered immune effector cell of any one of claims 33-35 in the preparation of a medicament for treating a disease or condition associated with the expression of CD7.

46. ​​The use according to claim 45, wherein the disease or condition associated with the expression of CD7 is CD7 + Hematological tumor; Optionally, the CD7 + Hematological neoplasms selected from one or more of the following: CD7 + Acute T-lymphocytic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

47. The use according to claim 46, wherein CD7 + Acute T-lymphoblastic leukemia including CD7 + Early pro-T lymphoblastic leukemia and other acute T lymphoblastic leukemias; and / or wherein the CD7 + T-cell lymphoma selected from one or more of the following: CD7 + T lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

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