Humanized antibody targeting CD7, chimeric antigen receptor and application of humanized antibody and chimeric antigen receptor

Abstract

The invention 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 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, as well as application thereof in preparation of reagents or drugs for diagnosing or treating diseases or symptoms related to CD7 expression.

Description

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 type of lymphatic system malignancies that originate from T cells. Due to the lack of understanding of their biology, their classification has been difficult to complete, and many classifications still belong to the "provisional classification" in the World Health Organization (WHO) disease classification. Currently, WHO classifies T-cell malignancies into: immature T-cell tumors and mature T-cell tumors; among them: immature T-cell tumors include acute T-lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphoma (T-LBL), and mature T-cell tumors include various types of T-cell leukemia and T-cell lymphoma. T-cell malignancies are characterized by strong invasiveness, wide symptom variation, mostly poor prognosis, high recurrence rate (30%-40%), unknown pathogenic factors, and lack of biological research. Although the current treatments for pediatric T-ALL are more effective than before, with an event-free survival (EFS) rate of up to 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%, and the prognosis is poor. Therefore, new and 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] Chimeric Antigen Receptor (CAR) is the core component of CAR cell therapy drugs, which may include antigen recognition domain, hinge region, transmembrane region and intracellular domain. So far, the antigen recognition domain is derived from the single chain variable region (scFv) of the antibody, or from receptor ligand interaction, TCR mimics, variable lymphocyte receptors (VLR); the most common source is scFv antibody. CAR-T cell immunotherapy is considered to be one of the most promising means to conquer tumors. CAR-T cells use genetic modification methods to make T cells express CAR protein, which has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects. At present, CAR-T cell immunotherapy has achieved remarkable results in the treatment of various hematological tumors, such as: CD19CAR-T for the treatment of B cell lymphoma and BCMACAR-T for the treatment of multiple myeloma have been marketed.

[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 expression form and strength of CD7 vary. In addition to T-cell malignancies, approximately 30% of leukemic blasts and malignant progenitor cells in acute myeloid leukemia (AML) patients also express CD7. Therefore, CAR-T cells targeting CD7 are expected to treat the above-mentioned corresponding 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 heterology. 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 comprehensive effect.

[0006] The present application provides a humanized antibody or an antigen-binding fragment thereof targeting CD7, comprising: 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;

[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, sc(Fv) 2 Antibody or [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 an amino acid sequence as 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 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 also 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 yet another aspect, the present application also provides a pharmaceutical composition, which comprises 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 yet 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 associated with the expression of CD7.

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

[0023] In certain embodiments of the above application, the CD7 + Hematological neoplasms 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 yet 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 drug for treating a disease or condition associated with the expression of CD7.

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

[0028] In certain embodiments of the above application, 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.

[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 + Enteropathic 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 the expression of CD7.

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

[0033] In certain embodiments of the above method, 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.

[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 + Enteropathic 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 medicine 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 the expression of CD7.

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

[0038] In certain embodiments of the above-mentioned drug, 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.

[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+ Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

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

[0043] On the other hand, the present application also provides a chimeric antigen receptor targeting CD7, which comprises 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 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;

[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 shown in SEQ ID NO:1-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:2, 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:1-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:30, 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:28-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:30, 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;

[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 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;

[0049] 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.

[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 chimeric antigen receptors described above, 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

[0053] As shown in SEQ ID NO:35, or as shown in SEQ ID NO:36.

[0054] 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.

[0055] 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 shown in SEQ ID NO: 10; further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 10.

[0056] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the intracellular domain comprises an intracellular signaling region; optionally, it also comprises a co-stimulatory signaling region.

[0057] 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.

[0058] In certain embodiments of any of the above-mentioned chimeric antigen receptors, 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; further optionally, the amino acid sequence of the co-stimulatory signaling region is as shown in SEQ ID NO:12.

[0059] In certain embodiments, any of the above-mentioned chimeric antigen receptors further comprises a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide 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.

[0060] In certain embodiments of any of the above chimeric antigen receptors, the chimeric antigen receptor comprises any one of the following sequences: an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence as shown in SEQ ID NO: 18; or an amino acid sequence as shown in SEQ ID NO: 19;

[0061] 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.

[0062] 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.

[0063] In certain embodiments of the isolated nucleic acid molecule, the nucleotide sequence encoding the chimeric antigen receptor comprises:

[0064] 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;

[0065] 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;

[0066] 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.

[0067] In certain embodiments of the isolated nucleic acid molecule, the nucleotide sequence encoding the chimeric antigen receptor comprises:

[0068] A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 14, which is shown in SEQ ID NO: 15;

[0069] or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:18, which is shown in SEQ ID NO:39;

[0070] Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:19, which is shown in SEQ ID NO:40.

[0071] In yet another aspect, the present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

[0072] 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.

[0073] On the other hand, the present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] In certain embodiments of the engineered immune effector cells, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0078] On the other hand, the present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.

[0079] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes a protective agent.

[0080] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient comprises a cell freezing solution.

[0081] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutical composition is a cell suspension or frozen cells thereof.

[0082] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutical composition is an intravenous injection.

[0083] 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 of the above-mentioned chimeric antigen receptors into the immune effector cells.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In certain embodiments of the above method, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0088] In certain embodiments of the above method, the T cell Trac gene and CD7 gene are 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.

[0089] 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.

[0090] In certain embodiments of the above method, the sequence of chRDNA in the CRISPR / Cas system targeting the Trac gene is shown in SEQ ID NO:34.

[0091] In certain embodiments of the above method, the method for introducing a nucleotide sequence encoding any of the above 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 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.

[0092] On the other hand, 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.

[0093] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0094] In certain embodiments of the above application, 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.

[0095] 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.

[0096] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0097] On the other hand, the present application also provides a method for treating a disease or condition associated with 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 associated with the expression of CD7.

[0098] In certain embodiments of the above method, the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. For example, the administration method can be administered to the subject by intravenous injection. In certain embodiments, an effective dose of engineered immune effector cells or pharmaceutical compositions 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 3-6 months.

[0099] In certain embodiments of the above method, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0100] In certain embodiments of the above method, 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.

[0101] 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.

[0102] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0103] In certain embodiments of the above method, the administration is by intravenous injection.

[0104] In certain embodiments of the above method, the effective amount of the engineered immune effector cells or pharmaceutical composition is 1×10 5 Up to 1×10 7 The dose was cells / kg.

[0105] On the other hand, the present application also provides a medicine comprising the above-mentioned engineered immune effector cells or pharmaceutical composition, which is used to treat diseases or conditions related to the expression of CD7.

[0106] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0107] In certain embodiments of the above-mentioned drug, 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.

[0108] 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.

[0109] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 The results of the detection of CAR+ expression rate of CAR-T cells after T cells were transduced and expanded for different periods of time in Example 2 are shown, wherein: UTD-NoEP represents a non-transduced and non-knocked-out T cell group, UTD-KO TRAC+CD7 represents a non-transduced but double-knocked T cell group, and the remaining 6 groups represent double-knocked T cell groups transduced with different CARs.

[0111] Figure 2 The results of the total T cell number detection after T cells were transduced and expanded and cultured for different periods of time in Example 2 are shown, wherein: UTD-NoEP represents a non-transduced and non-knocked-out T cell group, UTD-KO TRAC+CD7 represents a non-transduced but double-knocked T cell group, and the remaining 6 groups represent double-knocked T cell groups transduced with different CARs.

[0112] Figure 3A The results show that in Example 3, under the conditions of effector-target ratios of 1:3, 1:1, and 3:1, CAR-T cells using mouse or 5 humanized Th69 scFvs can kill positive target cells MOLT4 in a short time.

[0113] Figure 3BThe results show that in Example 3, under the conditions of effector-target ratios of 1:3, 1:1, and 3:1, CAR-T cells using mouse or 5 humanized Th69 scFvs can kill negative target cells KO CD7-MOLT4 in a short time;

[0114] Figure 3C The long-term killing of positive target cells MOLT4 by CAR-T cells using mouse or 5 humanized Th69 scFvs in Example 3 at effector-target ratios of 1:32, 1:16, 1:4, and 1:1 was demonstrated;

[0115] Figure 3D The long-term killing of positive target cells Jurkat by CAR-T cells using mouse or 5 humanized Th69 scFvs in Example 3 at effector-target ratios of 1:32, 1:16, 1:4, and 1:1 was demonstrated;

[0116] Figure 3E The long-term killing of negative target cells KO CD7-jurkat by CAR-T cells using mouse or 5 humanized Th69 scFvs in Example 3 was demonstrated at effector-target ratios of 1:32, 1:16, 1:4, and 1:1;

[0117] in: Figure 3A-3E NoEP represents untransduced and non-knocked-out T cells, UTD represents untransduced but double-knocked-out CD7 and TRAC T cells, and the remaining 6 groups represent double-knocked-out T cell groups transduced with different CARs.

[0118] Figure 4 The cumulative expansion multiples of each group of T cells in rounds 1-4 in the multiple rounds of stimulation experiments in Example 4 are shown, wherein: NoEP represents non-transduced and non-knocked-out T cells, UTD represents non-transduced but double-knocked-out CD7 and TRAC T cells, and the remaining 6 groups represent double-knocked-out T cell groups transduced with different CARs.

[0119] Figure 5 The figure shows the survival rate of mice at different times after the in vivo experiment in Example 6 was reinfused with the test products in different groups in Table 2.

[0120] Figure 6 The in vivo tumor imaging signal values ​​of mice at different times after the different groups of test samples in Table 2 were reinfused in the in vivo experiment of Example 6 are shown. DETAILED DESCRIPTION

[0121] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0122] The present application is further described below: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0123] In this application, the term "antibody" has the conventional meaning in the art and is used in the broadest sense in this application. Antibodies generally have heavy (H) chains and / or light (L) chains. By analyzing the amino acid sequences of different antibody heavy chains and light chains, it is found that the amino acid sequences of the heavy 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 a variable region (variable region, V), and the region near the C-terminus where the amino acid sequence is relatively stable is called a constant region (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. There is a small part of amino acid residues in the variable region of antibodies that change particularly strongly. The residue composition and arrangement order of these amino acids are more prone to variation, which are called hypervariable regions (HVR); there are three hypervariable regions in the V region of the L chain and the H chain, which can form precise complementarity with the antigenic determinant in spatial structure, so the hypervariable region is also called complementarity determining region (CDR). In antibodies, the common rules for dividing CDRs are Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When using a website that executes these rules, you only need to input the VH and VL sequences and select the corresponding rules to obtain CDR sequences based on different rules.

[0124] In the present application, the term "antigen-binding fragment" has the conventional meaning in the art, and refers to a key fragment of an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0125] In the present application, the term "humanized antibody" also refers to an antibody that has undergone humanization modification. The methods of humanization modification are known (e.g., WO96 / 02576). The purpose of humanization modification is to reduce the heterologous nature of the parent antibody while substantially retaining the affinity and specificity of the parent antibody. For example, in the case where the CDR is obtained from a mouse antibody, a primer can be synthesized (the corresponding primer can be obtained by referring to the method described in WO98 / 13388) and used to connect the CDR of the mouse antibody to the framework region (FR) of the human antibody.

[0126] 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, the scFv derived from mouse antibodies can be humanized. However, in the process of humanization of antibodies, the binding affinity, specificity and other functionalities of antibodies to specific antigens may be lost. Therefore, it is of practical significance to obtain humanized antibodies with reduced immunogenicity and no loss or even enhanced functionality.

[0127] In the present application, the term "scFv" has the conventional meaning in the art, and refers to a single chain variable fragment (scFv), which is an antibody formed by connecting the heavy chain variable region and the light chain variable region of the antibody through a short peptide (linker).

[0128] In this application, the term "Sc(Fv) 2 , [Sc(Fv) 2 ] 2 " and other nouns not specifically explained also have the conventional meanings in this field.

[0129] In the present 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.

[0130] 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 connected to a monoclonal antibody through a chemical chain, and the monoclonal antibody acts as a carrier to transport the small molecule drug to the target cell.

[0131] 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 (e.g., 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, which have the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.

[0132] In this application, the term "extracellular antigen recognition domain" refers to the Antigen Recognition Domain (ARD). The ability of CAR cell therapy products (such as CAR-T cells) to specifically recognize and / or bind to target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. So far, the antigen recognition domain is derived from the single chain variable region (Single Chain Variable Fragment, abbreviated as scFv) of the antibody, or from receptor ligand interactions, TCR mimics, and variable lymphocyte receptors (VLR). So far, the most common source is the scFv segment of the antibody.

[0133] In the present application, the term "specific recognition and / or binding" refers to the recognition and / or binding between CAR and a specific target, which is binding to the target with greater affinity, avidity, more easily, and / or for a longer duration than CAR binds to other targets.

[0134] 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 form 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. +Mixed phenotype AML, as well as most NK lymphomas and NKT lymphomas. However, while CD7 CAR-T cells kill tumor cells with high CD7 expression, they also kill CD7CAR-T "fellows" because CD7 genes are also expressed on the surface of T cells, resulting in a significant decrease in the number of CAR-T cells on the fifth day of in vitro culture.

[0135] 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) recognizing the antigen is mainly transmitted 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, avoiding the occurrence of graft-versus-host reaction (GVHD).

[0136] In the present 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 fact that after transplantation, the T lymphocytes in the allogeneic donor graft undergo a series of "cytokine storm" stimulations launched by the recipient, which greatly enhances their immune response to the recipient's antigens and launches cytotoxic attacks on the recipient's target cells, among which the skin, liver and intestines are the main targets.

[0137] In the present 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.

[0138] In the present 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.

[0139] In this application, the terms "gene editing" and "gene editing technology" have the usual meaning in the art, 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 this site. As a molecular biology technique, gene editing technology can achieve accurate 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, do not have any particularity 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, can be applied to more species and cells, is more efficient, has a shorter construction time, and has a lower cost.

[0140] In this application, the terms "CRISPR / Cas technology" and "CRISPR / Cas system" have the usual meaning in the art, and its full name is clustered regularly interspaced shortpalindromic repeats / CRISPR-associated proteins, which is an acquired immune system found in most bacteria and all archaea, which can direct the cutting of foreign gene fragments. Different types of CRISPR / Cas systems have been found, among which the second type has a relatively simple composition, with Cas9 protein and guide RNA (guide RNA, Grna, also known as sgRNA) as the core composition. Compared with the earlier ZFN technology and TALEN technology, CRISPR / Cas has the following advantages: low off-target rate, high efficiency, economical and affordable, and 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.

[0141] In this application, the terms "zinc finger nuclease technology" and "zinc finger nuclease system" have the usual meanings in the art. The core design concept of zinc finger nuclease technology is to cleverly chimerize two domains with specific functions, namely a specific recognition module and a functional module. The most classic zinc finger nuclease is a fusion of a non-specific nuclease Fok I with a domain containing zinc fingers, which can recognize specific DNA sequences.

[0142] 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 powerful tool 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 endonuclease function. The core principle of TALEN technology is to orderly realize the three different functions of guiding into the cell nucleus, specific recognition of target site DNA, and cutting of target site DNA on the same TALEN protein.

[0143] 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 CAR to recognize antigens by giving the antigen recognition domain a certain range of activity. The hinge regions currently used are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α.

[0144] 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 improve 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.

[0145] In the present application, the term "intracellular domain" includes the intracellular signaling region and may also include the co-stimulatory signaling region.

[0146] In the present application, the term "intracellular signaling region" refers to the activation of at least one normal effector function of the immune effector cell responsible for expressing CAR. The intracellular signaling region may be derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0147] In the present 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 functions 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.

[0148] In this application, the term "guide peptide" refers to a short peptide before the extracellular antigen recognition domain (such as scFv sequence), which guides the recombinant protein synthesized in the cell to be exported to the outside of the cell. Common guide peptides include human CD8α signal peptide or human GM-CSF receptor α signal peptide.

[0149] In this application, one of the key factors that determine 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, so 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. The scFv antibody against a single target includes an antibody heavy chain variable region (VH) and a light chain variable region (VL), which are connected by a connecting sequence; the scFv antibody against two or more targets includes VH regions and VL regions against different targets, and the different regions are also directly or indirectly connected by a connecting sequence, and the 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, the above “-” represents connection through a connecting sequence.

[0150] In the present application, the term "linker" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length, which links together any structure / region of the antibody or chimeric antigen receptor of the present invention. The linker can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. When it is desired to ensure that two adjacent domains do not interfere with each other in space, a longer linker can be used.

[0151] In this application, the term "isolated" generally refers to something obtained from a natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" does not exclude substances that have been artificially or synthetically obtained from a natural state, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0152] In this application, the term "isolated nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides of any length in isolated form, either separated from their natural environment or artificially synthesized analogs.

[0153] In the present application, when CAR gene transduction / transfection and target gene expression are used, the gene transduction / transfection method mainly includes viral and non-viral methods, such as: gene transduction via γ-retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

[0154] The term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce or transfect a host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not only these substances. However, the introduction of a polynucleotide encoding a protein into a cell does not necessarily have to be through a vector, such as a transposon, electroporation, etc. The term "transposon" refers to a discontinuous DNA fragment that has the ability to migrate between chromosome sites and carry genetic information, such as the Sleeping Beauty SB system and the PB system derived from Lepidoptera. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

[0155] In the present 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. The immune effector cell 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 from induced pluripotent stem cells, NK cells differentiated from induced pluripotent stem cells, and one or more of embryonic stem cells.

[0156] In the present 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 in the present application, and may also include one or more pharmaceutically acceptable excipients, such as: one or more of a carrier, a protective agent, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, and a preservative. In certain embodiments, the pharmaceutically acceptable excipient includes a protective agent, such as: a cell freezing solution. In certain embodiments, the pharmaceutical composition of the present application is a cell suspension or its frozen cells.

[0157] 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.

[0158] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0159] In this application, the term "about" generally refers to a fluctuation range acceptable to those skilled in the art above or below the specified value, such as: within the range of ±0.5%-10%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,

[0160] The range of fluctuation is 8.5%, 9%, 9.5% or 10%.

[0161] Humanized antibody or antigen-binding fragment thereof targeting CD7, corresponding nucleic acid molecule, corresponding vector, corresponding cell, corresponding pharmaceutical composition

[0162] In one aspect, the present application provides a humanized antibody or an antigen-binding fragment thereof targeting CD7, comprising:

[0163] 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;

[0164] 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;

[0165] 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.

[0166] In the present application, the biocomputational evaluation results showed that the humanized designs of the murine antibody Th69 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 the chimeric antigen receptor composed of 5 humanized scFv antibodies: the non-specific killing of negative target cells by 5 groups of humanized CAR-T cells was significantly weaker than that of mouse mTh69 CAR-T cells; humanized Th69 expressed higher CD107a than mouse mTh69 CAR-T after culturing with antigen-positive target cells, and there was no significant difference in CD107a expression after culturing with antigen-negative target cells, indicating that humanized Th69scFv improved the specific binding ability to CD7 antigen; the results of multiple rounds of stimulation and amplification ability detection experiments showed that the proliferation ability of humanized Th69 was significantly better than that of mouse CAR-T cells, among which hu11CAR-T cells, hu12 CAR-T cells and hu21CAR-T cells had more advantages in proliferation; the in vivo efficacy results showed that the humanized sequence hu21 had a stronger tumor killing effect and prolonged the survival time of mice. In summary, humanized hu21, hu11, and hu12 were selected as candidate sequences.

[0167] In certain embodiments, the humanized antibody or antigen-binding fragment thereof is a scFv antibody, sc(Fv) 2 Antibody or [sc(Fv) 2 ] 2 Antibody.

[0168] 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;

[0169] or, a scFv antibody having an amino acid sequence as shown in SEQ ID NO: 35;

[0170] Or, a scFv antibody having the amino acid sequence shown in SEQ ID NO:36.

[0171] 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.

[0172] 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;

[0173] 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;

[0174] 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;

[0175] 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.

[0176] On the other hand, the present application also provides a vector, which comprises the above-mentioned isolated nucleic acid molecule. The vector can be arbitrarily selected from one or more of the following vectors: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); 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, papillomaviruses (such as SV40).

[0177] In yet another aspect, the present application also provides a cell comprising any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof, isolated nucleic acid molecules or vectors.

[0178] In another aspect, the present application also provides a pharmaceutical composition, which includes any of the above humanized antibodies or antigen-binding fragments thereof, an isolated nucleic acid molecule, a vector or a cell, and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient includes, but is not limited to, one or more of a carrier, a protective agent, and a stabilizer.

[0179] Application of humanized antibodies or antigen-binding fragments thereof targeting CD7

[0180] In yet 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 associated with the expression of CD7.

[0181] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0182] In certain embodiments of the above application, 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.

[0183] 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.

[0184] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0185] On the other hand, 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 drug for treating a disease or condition associated with the expression of CD7.

[0186] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0187] In certain embodiments of the above application, 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.

[0188] 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.

[0189] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0190] 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 the expression of CD7.

[0191] In certain embodiments of the above method, the disease or disorder associated with the expression of CD7 is CD7 + Hematological tumors.

[0192] In certain embodiments of the above method, 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.

[0193] 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.

[0194] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0195] 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.

[0196] On the other hand, the present application also provides a medicine 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 the expression of CD7.

[0197] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0198] In certain embodiments of the above-mentioned drug, 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.

[0199] 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.

[0200] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0201] Antibody drugs and antibody-drug conjugates comprising humanized antibodies or antigen-binding fragments thereof targeting CD7

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

[0203] 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.

[0204] In yet another aspect, the present application also provides an antibody-drug conjugate, which comprises any one of the above-mentioned humanized antibodies or antigen-binding fragments thereof.

[0205] Chimeric antigen receptor targeting CD7, corresponding nucleic acid molecule, corresponding vector, corresponding immune effector cell, and pharmaceutical composition

[0206] On the other hand, the present application also provides a chimeric antigen receptor targeting CD7, which comprises 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:

[0207] 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;

[0208] 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

[0209] 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.

[0210] 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 shown in SEQ ID NO:1-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:2, 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:1-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:30, 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:28-a connecting sequence-an amino acid sequence as shown in SEQ ID NO:30, 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;

[0211] 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;

[0212] 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.

[0213] 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.

[0214] In certain embodiments of any of the chimeric antigen receptors described above, 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.

[0215] In certain embodiments of any of the chimeric antigen receptors described above, 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;

[0216] Optionally, the amino acid sequence of the extracellular antigen recognition domain is as shown in SEQ ID NO: 3, or as shown in

[0217] As shown in SEQ ID NO:35, or as shown in SEQ ID NO:36.

[0218] 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.

[0219] 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 shown in SEQ ID NO: 10; further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 10.

[0220] In certain embodiments of any of the above-mentioned chimeric antigen receptors, the intracellular domain comprises an intracellular signaling region; optionally, it also comprises a co-stimulatory signaling region.

[0221] 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.

[0222] In certain embodiments of any of the above-mentioned chimeric antigen receptors, 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; further optionally, the amino acid sequence of the co-stimulatory signaling region is as shown in SEQ ID NO:12.

[0223] In certain embodiments, any of the above-mentioned chimeric antigen receptors further comprises a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide 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.

[0224] In certain embodiments of any of the above chimeric antigen receptors, the chimeric antigen receptor comprises any one of the following sequences: an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence as shown in SEQ ID NO: 18; or an amino acid sequence as shown in SEQ ID NO: 19;

[0225] 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.

[0226] 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.

[0227] 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 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;

[0228] 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;

[0229] 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.

[0230] In certain embodiments of the isolated nucleic acid molecule described above, the nucleotide sequence encoding the chimeric antigen receptor comprises:

[0231] A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 14, which is shown in SEQ ID NO: 15;

[0232] or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:18, which is shown in SEQ ID NO:39;

[0233] Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:19, which is shown in SEQ ID NO:40.

[0234] On the other hand, the present application also provides a vector, which comprises 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).

[0235] 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.

[0236] On the other hand, the present application also provides an engineered immune effector cell, which comprises the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid molecule, or the above-mentioned vector.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] In certain embodiments, the engineered immune effector cells are αβT lymphocytes or γδT lymphocytes.

[0241] On the other hand, the present application also provides a pharmaceutical composition, which includes the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients.

[0242] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient includes a protective agent.

[0243] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutically acceptable excipient comprises a cell freezing solution.

[0244] In certain embodiments of the above-mentioned pharmaceutical composition, the pharmaceutical composition is a cell suspension or frozen cells thereof.

[0245] In certain embodiments of the above pharmaceutical composition, the pharmaceutical composition is an intravenous injection.

[0246] Method for preparing engineered immune effector cells

[0247] 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 of the above-mentioned chimeric antigen receptors into the immune effector cells.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] In certain embodiments of the above method, the T cell Trac gene and CD7 gene are 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.

[0252] 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.

[0253] 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.

[0254] In certain embodiments of the above method, the T lymphocytes are αβ T lymphocytes or γδ T lymphocytes.

[0255] 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 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.

[0256] Application of chimeric antigen receptors targeting CD7

[0257] On the other hand, 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.

[0258] In certain embodiments of the above application, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0259] In certain embodiments of the above application, 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.

[0260] 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.

[0261] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0262] On the other hand, the present application also provides a method for treating a disease or condition associated with 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 associated with the expression of CD7.

[0263] In certain embodiments of the above method, the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. For example, the administration method can be administered to the subject by intravenous injection. In certain embodiments, an effective dose of engineered immune effector cells or pharmaceutical compositions 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 3-6 months.

[0264] In certain embodiments of the above method, the disease or disorder associated with the expression of CD7 is CD7 + Hematological tumors.

[0265] In certain embodiments of the above method, 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.

[0266] 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.

[0267] 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 + Enteropathic T-cell lymphoma, CD7 + Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0268] In certain embodiments of the above methods, the administration is by intravenous injection.

[0269] In certain embodiments of the above method, the effective amount of the engineered immune effector cells or the pharmaceutical composition is 1×10 5 Up 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, 1×10 5 CAR-positive T cells / kg to 1×10 6 CAR-positive T cells / kg, 1×10 6 CAR-positive T cells / kg to 1×10 7 CAR-positive T cells / kg, 0.5×10 6 CAR-positive T cells / kg, 0.6×10 6 CAR-positive T cells / kg, 0.7×10 6CAR-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.

[0270] On the other hand, the present application also provides a medicine comprising the above-mentioned engineered immune effector cells or pharmaceutical composition, which is used to treat diseases or conditions related to the expression of CD7.

[0271] In certain embodiments of the above-mentioned drug, the disease or condition associated with the expression of CD7 is CD7 + Hematological tumors.

[0272] In certain embodiments of the above-mentioned drug, 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.

[0273] 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.

[0274] 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 + Enteropathic T-cell lymphoma, CD7 +Primary Cutaneous T-Cell Lymphoma and CD7 + Peripheral T-cell lymphoma.

[0275] Without intending to be bound by any theory, the examples below are only intended to illustrate the chimeric antigen receptors, engineered immune effector cells, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, methods for inserting protein-encoding genes into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those with ordinary skills in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0276] Example 1. Humanized transformation design of mouse antibodies and preparation of CD7 CAR-T cells

[0277] In the present application, the VH amino acid sequence (as shown in SEQ ID NO:31) and the 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.

[0278] The template selected for the humanization of the Th69 VH region was IGHv3-21*07. In the humanization of VH, different combinations of multiple important amino acids were selected for back mutation, forming 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 SEQID NO:28, respectively). Multiple important amino acids included amino acids at the PHI-ANGLE position, amino acids that were completely buried inside and had a low frequency of occurrence at this position in humans, and amino acids at the PHI-ANGLE position that were very close to the CDR region.

[0279] The template selected for the humanization of the Th69 VL region was IGKv1-27*01. In the humanization of VL, different combinations of multiple important amino acids were selected for back mutation, forming three humanized Th69 VL designs, namely 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 SEQID NO:2, respectively). Multiple important amino acids included amino acids at the PHI-ANGLE position, amino acids at the VH / VL interface, and upper core amino acids.

[0280] The humanized heavy and light chains were connected through linker sequences to form hu01 scFv, hu10scFv, hu11 scFv, hu12 scFv, and hu21 scFv, respectively. We chose to screen each candidate humanized scFv antibody 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-co-stimulatory 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, hu11scFv, hu12 scFv, hu21 scFv and mouse 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 co-stimulatory 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.

[0281] Table 1. Various CD7 CAR sequences

[0282]

[0283] The specific method for obtaining CD7 CAR-T cells is as follows:

[0284] 1. Construction of lentiviral vector

[0285] 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, and then 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).

[0286] 2. Preparation of T cells

[0287] 1) T cell sorting

[0288] Peripheral blood mononuclear cells (PBMCs) were isolated from human single blood cells, and then T cells were sorted from PBMC cells.

[0289] 2) Activation of T cells

[0290] The separated 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 placed in an incubator at 37°C + 5% CO 2 , culture time at least 24 hours, to obtain activated T cells.

[0291] 3) Gene editing of activated T cells

[0292] 90% of T cells express CD7 on their surface. If CD7 CAR-T cells express CD7 on their surface, it will cause suicide injury of CD7 CAR-T cells. Using gene editing to knock out the CD7 gene of T cells so that they do not express CD7 on their surface is an effective way to avoid suicide injury. At the same time, using gene editing to knock out the Trac gene of T cells so that they can be used as universal CAR-T products. The specific methods are as follows:

[0293] Prepare RNP complex, each 11.5μl RNP complex consists of 10μl Opti-MEM medium + 0.6μl Cas9 (10mg / ml) + 0.45μl CD7 (100μM) gRNA + 0.45μl TRAC chRDNA (100μM)), incubate at room temperature for 15 minutes, wherein the sequence of CD7 sgRNA is shown in SEQ ID NO: 33, and the sequence of TRAC chRDNA is shown in SEQ ID NO: 34. Take the activated T cells, add electroporation solution at 10μl / M Cell, resuspend the cells, mix with the incubated RNP complex, add 11.5μl RNP complex per 1M cell, and transfer to the electroporation strip. Select EH100 as the electroporation program of the Lonza 4D electroporator and perform electroporation. After the electroporation is completed, quickly add pre-warmed T cell complete medium to the electroporation strip, immediately put the electroporation strip into the incubator and incubate 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℃+5% CO 2 .

[0294] 3. Use lentiviral transduction to select, activate and gene-edited (CD7 and Trac double gene knockout) T cells to prepare corresponding CD7 CAR-T cells.

[0295] The gene-edited T cells were taken out and resuspended in X-VIVO15 medium containing polybrene at a final concentration of 8 μg / ml. The cell suspension was obtained by mixing and 800 μl of cell suspension (containing 2×10 6 Slowly add 200ul of lentiviral vector to the cells, mix well and place in a well plate, and culture in an incubator at 37°C + 5% CO 2 , the incubation time is at least 4-6 hours.

[0296] 4. Expansion and culture of transduced T cells

[0297] The transduced cells were taken out and cultured with complete lymphocyte culture medium, and subcultured every other day to maintain the cell density at (0.8-2)×10 6 cells / ml for use in subsequent examples.

[0298] After T cells were infected with lentivirus containing the CAR structure in Table 1, the obtained T cells were named according to their CAR 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.

[0299] 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 to be 94.481±3.656% for CD7 knockout and 96.201±1.437% for TRAC knockout.

[0300] Example 2: Detection of CAR-T cell CAR positive ratio and CAR-T cell expansion

[0301] 1. Detection of CAR-positive ratio in CAR-T cells

[0302] The 6 CAR-T cells obtained in Example 1, the untransduced and non-knocked-out T cells (UTD-NoEP group), and the 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), and the CAR molecule positive ratio of various cells was detected by flow cytometry at different days after transduction. The test results are shown in the figure. Figure 1 As shown: There was no significant difference in CAR expression on the surface of the five CAR-T cells using humanized scFv and CAR-T cells using mouse scFv, and the CAR positivity rates detected 4, 6, and 11 days after transduction were all at the same level; untransduced and non-knockout T cells and untransduced but double-knockout T cells were used as two groups of negative controls, and no CAR expression was detected.

[0303] 2. Expansion detection of total T cells cultured for different days after transduction

[0304] The 6 types of CAR-T cells obtained in Example 1, the untransduced and unknocked T cells (UTD-NoEP group), and the untransduced but double knocked T cells (UTD-KO TRAC+CD7 group) were sampled at different days after transduction, and AO / PI double fluorescence staining (AO / PI double staining cell apoptosis detection kit can be purchased through commercial channels, which uses AO / PI probe double staining of cell nuclei to detect the state of apoptotic cells, and can distinguish normal cells from apoptotic cells) was used. 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 as follows Figure 2As shown in the figure: The expansion times of the five CAR-T cells using humanized scFv during culture were higher than those of the CAR-T cells using mouse scFv, but there was no significant difference in the expansion capacity of the five CAR-T cells using humanized scFv. In addition, the expansion capacity of double-knocked T cells, regardless of whether they were transduced with CAR structure, was significantly higher than that of non-knocked T cells. It is speculated that the suicide injury to T cells caused by non-knockout of CD7 gene affects the expansion capacity.

[0305] Example 3. In vitro killing ability of CAR-T cells

[0306] 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-derived or humanized Th69 scFv CAR-T cells to target cells according to the effector cells (effect 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 target cells. The method for establishing a target cell line stably expressing luciferase is as follows: target cells are infected with a virus carrying a luc-GFP sequence (the conventional luciferase and fluorescent protein sequence in the prior art can be used), and monoclonal culture is performed by infinite dilution, and clones with GFP positive and good growth status are selected for culture; long-term killing refers to the CAR-T cells using mouse or humanized Th69 scFv are co-cultured with target cells according to effector cells (effector cells are CAR + After co-cultured in X-VIVO15 medium for 24 h under conditions of different effector-target ratios (cell count, 1:32, 1:16, 1:4, and 1:1) with target cells, the killing ratio of CAR-T cells to target cells was detected by detecting the activity of luciferase stably expressed in target cells.

[0307] In the above experiment, untransduced and unknocked-out T cells (NoEP group) and untransduced but double-knocked-out T cells (UTD group) were used as negative effector cells; target cells included positive target cells Molt-4 cells (human acute T lymphocytic leukemia cells) and Jurkat cells (human T lymphocytic leukemia cells) that endogenously express CD7, as well as negative controls KO CD7 Molt-4 cells (referring to the establishment of KO CD7 Molt-4 cell line by selecting monoclonal CD7-Molt-4 cells by infinite dilution after knocking out CD7 using CRISPR / Cas9 gene editing method, and the CD7 sgRNA used was the same as in Example 1), and KO CD7 Jurkat cells (referring to the establishment of KO CD7 Jurkat cell line by selecting monoclonal CD7-Jurkat cells by infinite dilution after knocking out CD7 using CRISPR / Cas9 gene editing method, and the CD7 sgRNA used was the same as in Example 1). The experimental results are as follows:

[0308] Short-term killing results: Figure 3A As shown in the figure, under the conditions of effector-target ratio of 1:3, 1:1, and 3:1, the killing ability of CAR-T cells using mouse or humanized Th69 scFv on positive target cells MOLT4 was at the same level; Figure 3B As shown in the figure, mouse Th69 CAR-T cells and humanized hu01 CAR-T cells had nonspecific killing effects on negative target cells KO CD7 MOLT4 at an efficient target ratio of 3:1, while the four groups of humanized hu10 CAR-T cells, hu11CAR-T cells, hu12 CAR-T cells, and hu21 CAR-T had no nonspecific killing effects on negative target cells, and the difference was significant (*** indicates p<0.001, **** indicates p<0.0001), indicating that these four groups of humanized CAR-T cells may have better safety.

[0309] Long-term killing results: Figure 3C and Figure 3D As shown in the figure, under the conditions of target-efficiency ratio of 1:32, 1:16, 1:4, and 1:1, the overall killing ability of CAR-T cells using mouse or 5 humanized Th69 scFvs on 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 target-efficiency ratio of 1:32 (* indicates p<0.05); in addition, Figure 3EAs shown in the figure, at the higher efficient 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 by mouse Th69 CAR-T cells (* indicates p<0.05, * indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001), indicating better safety.

[0310] Example 4. Multiple rounds of stimulation experiments of CAR-T cells

[0311] Antigen stimulation can activate CAR-T cells and make them proliferate, but continuous activation of T cells will lead to cell exhaustion. The proliferation ability and effector function of exhausted T cells will decrease. We determined the continuous proliferation ability of CD7 CAR-T cells by detecting the proliferation of CD3+ cells (i.e., the proliferation of T cells) after multiple rounds of antigen stimulation experiments. Humanized scFv with long-term proliferation ability was used as the screening criterion for preferred antibodies.

[0312] 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 by UTD. In the antigen stimulation experiment, each group of CAR-T cells was stimulated with CD7 + The target cells Molt4 were co-cultured in a 24-well plate at an effector-target ratio of 1:1, with 2 ml of X-VIVO15 medium in each well, and each group of cells was repeated 3 wells. T cells (CD3 is a marker to distinguish whether they are T cells) were labeled with a fluorescently labeled CD3 antibody (manufacturer: Biolegend, catalog number: 300312), and detected and analyzed by flow cytometry to count the proliferation of T cells. The number of CD3-positive cells was calculated based on the conversion of volume multiples, and then a certain amount of CAR-T cells was taken out from each group according to the calculation results, and the corresponding positive target cells were added at an effector-target ratio of 1:1 for a new round of stimulation. This was repeated every 48 hours for 4 rounds of stimulation. The proliferation results are shown in the figure. Figure 4 As shown: 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 hu12CAR-T cells was more advantageous.

[0313] Example 5: Specificity analysis

[0314] In order 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 signaling pathway for cytotoxic lymphocytes (CD8 + CD107a is a marker of activation of CAR-T cells and NK cells. When cytotoxic lymphocytes are stimulated by specific antigens, immune activation occurs and CD107a is expressed. Therefore, the expression of CD107a can be used as a means to detect the specificity of CAR-T cells.

[0315] 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 with X-VIVO15 medium. 6 / ml, and the CAR-T cells and untransduced T cells were adjusted to 1×10 6 / ml. Add 20μl of fluorescently labeled CD107a antibody (purchased from: Biolegend, model: 328608) to each well of a 96-well plate, then add 100μl of T cells (T cells refer to CAR-T cells or untransduced T cells) and 100μl of tumor cells to each well, and centrifuge at 400r / min for 3 minutes after adding. Incubate in a 37℃5% CO2 incubator for 60 minutes. After 60 minutes, add 20μl of Golgi stop working solution (ie: 3ml X-VIVO15 culture medium plus 2μl Golgi stop protein transport inhibitor, Golgi stop purchased from BD, model 554724) to each well and incubate for 2.5 hours. Take CD3 (purchased from: Biolegend, model: 300326) and CD8 antibody (purchased from: Biolegend, model: 344722) and mix them in equal volumes, add 10μl to each well after mixing, and incubate for 30 minutes. After 30 minutes, centrifuge at 1500r / min for 5 minutes, discard the supernatant, add 200μl FACS buffer (PBS + 0.5% BSA), and mix. Centrifuge at 1500r / min for 5 minutes, discard the supernatant. Add 200μl FACS buffer (PBS + 0.5% BSA) to each well, mix, transfer to the labeled flow tube, and test on the machine. Analysis of CD3 + CD8 +Fluorescence signal of CD107a in the cell population. In the above experiment, 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-CD7scFv (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. CAR-T cells 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 by CRISPR / CAS9, CD7-monoclonal MOLT4 cells were selected for culture to establish cell lines), Nalm6 (human acute lymphoblastic leukemia cells, commercial cell lines), 786-O (human renal clear cell adenocarcinoma cells, commercial cell lines), U266 (human multiple myeloma cells, commercial cell lines).

[0316] The detection results of CD107a are shown in Table 1: + Target cells, CD7 - After co-culture of target cells with untransduced T cells (UTD group), there was no significant difference in the expression level of CD107a; + After CCRF / MOLT4 co-culture, a high proportion of target cells expressed CD107a, while CAR-T cells and CD7 - The CD107a positive ratio of target cells CD7 KO MOLT4 / Nalm6 / 786-O / U266 after co-culture was very low, indicating that the five humanized Th69 CARs have good antigen recognition specificity. In addition, hu01 CAR-T, hu10CAR-T, hu11 CAR-T, hu12 CAR-T, and hu21 CAR-T cells expressed higher CD107a than mouse Th69 CAR-T cells after culture with antigen-positive target cells, indicating that humanized Th69scFv has improved the specific binding ability to CD7 antigen.

[0317] Table 1. CD107a expression of UTD or different CD7 CAR-T cells after stimulation by various target cells

[0318]

[0319]

[0320] Example 6: In vivo functional experiment

[0321] In order to further screen and determine the humanized antibodies with optimal functions, we conducted an in vivo pharmacodynamics test of CD7 CAR-T. When selecting candidate sequences for in vivo functional experiments, through humanization scoring software (such as: using the classic humanization scoring system T20score) and computer immunogenicity prediction (using the AlphaMHC algorithm), hu01 scFv and hu10 scFv sequences have a high degree of humanization and low immunogenicity, so they were selected as candidate sequences for in vivo functional experiments; the results of in vitro functional experiments showed that hu21 CAR-T cells have strong in vitro killing ability and weak nonspecific killing, and strong multi-round stimulation amplification ability, so they were also included in the candidate sequences for in vivo functional experiments.

[0322] CCRF-CEM-GFP-Luc cells were cultured at 2×10 5 The cells were inoculated into the tail vein of female NSG mice at a volume of 0.2 ml / mouse to establish an animal model of CCRF-CEM-GFP-Luc acute T lymphoblastic leukemia. On the 4th day after cell inoculation, the mice were randomly grouped according to the tumor imaging signal intensity, and the corresponding cells were reinfused through the tail vein. The experiment was divided into 8 groups (grouping information is shown in the table below), including: PBS group, non-transduced non-knockout T cell group (T cell group), non-transduced T cell group with double knockout of CD7 / TRAC (DKO-Tcell 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 3-8 groups 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. CAR-T cells were reinfused once through the tail vein at a dose of 3×10 6 / 0.2ml / CAR+ cell. From Day 0 to Day 14, in vivo imaging was performed twice a week, and blood was collected for flow cytometry twice, and then changed to once a week. The weight of mice and tumor imaging signal values ​​were recorded to monitor and evaluate the tumor clearance effect of humanized hu01, hu10, and hu21 CAR-T in animals.

[0323] Table 2. Grouping and dosage of each group in the mouse experiment

[0324]

[0325]

[0326] Note: The total number of viable cells after recovery was used to adjust the cell concentration to the dosing concentration using PBS.

[0327] The results of the mouse experiments are shown in Table 3. Figure 5 and Figure 6 As shown in Table 3 and Figure 5 The results showed the survival of 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 (e.g. Figure 5 The median survival time was 34.5 days (as shown in Table 3).

[0328] Table 3

[0329]

[0330] The results of tumor imaging signal values ​​in mice are as follows Figure 6 As shown, compared with the PBS group, T cell group and DKO-T cell group, the other groups of CAR-T cells were able to significantly inhibit tumor growth at the tested dose, among which the hu21 KO group had the best tumor inhibition effect.

[0331] 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 tumor killing effect of the humanized sequence hu21 was stronger than that of 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 Th69 CAR-T.

[0332] Description of Sequence Listing

[0333] SEQ ID NO: 1----Th69 VH-1, amino acid sequence of the heavy chain variable region of the scFv in hu01 / 11 / 21CAR;

[0334] EVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSS

[0335] SEQ ID NO:2---Th69 VL-3, amino acid sequence of the light chain variable region of the scFv in hu21 CAR;

[0336] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPDKTVKLLIYYTSSLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIK

[0337] SEQ ID NO:3---Amino acid sequence of scFv in hu21 CAR;

[0338] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPDKTVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVSSI SSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSS

[0339] SEQ ID NO:4---a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:1;

[0340] GAGGTCCAACTGGTGGAGTCCGGCGGGGGCCTCGTGAAGCCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCTCCGGCCTGACCTTCAGCAGCTACGCCATGAGCTGGGTGAGACAAGCCCCCGAGAAGAGACTGGAGTGGGTGAGCAGCATCAGCAGCGGCGGCTTCACCTACTACCCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGACGAGGTGAGAGGCTACCTGGACGTGTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC

[0341] SEQ ID NO:5 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:2;

[0342] GACATTCAGATGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTGCAGCGCCTCCCAAGGCATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGACAAGACCGTGAAGCTGCTGATCTACTACACAAGCAGCCTGCACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCAGCAGTACAGCAAGCTGCCCTACACATTTGGGGGCGGGACCAAACTCGAGATTAAG

[0343] SEQ ID NO:6 - Linker sequence;

[0344] GGGGSGGGGSGGGGS

[0345] SEQ ID NO:7 - Linker sequence;

[0346] GSTSGSGKPG SGEGSTKG

[0347] SEQ ID NO:8 - Linker sequence;

[0348] GGGGS

[0349] SEQ ID NO:9---Amino acid sequence of hinge region;

[0350] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0351] SEQ ID NO:10---Amino acid sequence of the transmembrane region;

[0352] IYIWAPLAGTCGVLLLSLVITLYC

[0353] SEQ ID NO:11---Amino acid sequence of the intracellular signaling region;

[0354] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0355] SEQ ID NO:12---Amino acid sequence of the co-stimulatory signaling region;

[0356] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0357] SEQ ID NO:13 --- amino acid sequence of the guide peptide;

[0358] MALPVTALLLPLALLLHAARP

[0359] SEQ ID NO:14 --- Amino acid sequence of hu21 CAR;

[0360] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPDKTVKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0361] SEQ ID NO:15 - Nucleotide sequence encoding the amino acid sequence of hu21 CAR;

[0362]

[0363] SEQ ID NO:16---Amino acid sequence of hu01 CAR;

[0364] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0365] SEQ ID NO:17---Amino acid sequence of hu10 CAR;

[0366] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNW YQQKPGKTVKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPGKGLEWVSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0367] SEQ ID NO:18 --- Amino acid sequence of hu11 CAR;

[0368] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKTVKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0369] SEQ ID NO:19---Amino acid sequence of hu12 CAR;

[0370] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKTVKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGAGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0371] SEQ ID NO:20---Amino acid sequence of Th69 CAR;

[0372] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNW YQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLK LSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0373] SEQ ID NO:21---Amino acid sequence of Th69 antibody VH CDR1;

[0374] SYAMS

[0375] SEQ ID NO:22---Amino acid sequence of Th69 antibody VH CDR2;

[0376] SISSGGFTYYPDSVKG

[0377] SEQ ID NO:23---Amino acid sequence of Th69 antibody VH CDR3;

[0378] DEVRGYLDV

[0379] SEQ ID NO:24---Amino acid sequence of Th69 antibody VL CDR1;

[0380] SASQGISNYLN

[0381] SEQ ID NO:25---Amino acid sequence of Th69 antibody VL CDR2;

[0382] YTSSLHS

[0383] SEQ ID NO:26---Amino acid sequence of Th69 antibody VL CDR3;

[0384] QQYSKLPYT

[0385] SEQ ID NO:27---Th69 VH-2, amino acid sequence of the heavy chain variable region of the scFv in hu10 CAR;

[0386] EVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPGKGLEWVSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSS

[0387] SEQ ID NO:28----Th69 VH-3, amino acid sequence of the heavy chain variable region of the scFv in hu12 CAR;

[0388] EVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGAGTTVTVSS

[0389] SEQ ID NO:29---Th69 VL-1, amino acid sequence of the light chain variable region of the scFv in hu01 CAR;

[0390] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAPKLLIYYTSSLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIK

[0391] SEQ ID NO:30---Th69 VL-2, amino acid sequence of the light chain variable region of the scFv in hu10 / 11 / 12CAR;

[0392] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKTVKLLIYYTSSLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIK

[0393] SEQ ID NO:31---Amino acid sequence of the heavy chain variable region of murine antibody Th69;

[0394] EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSS

[0395] SEQ ID NO:32---Amino acid sequence of the light chain variable region of murine antibody Th69;

[0396] DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHS GVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKR

[0397] SEQ ID NO:33---CD7 sgRNA;

[0398] CGGAACCGUCUGUCCGUAGU

[0399] SEQ ID NO:34---TRAC chRDNA;

[0400] AGAGTCUCTCAGCUGGUACA

[0401] SEQ ID NO:35---Amino acid sequence of scFv in hu11 CAR;

[0402] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKTVKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGG GGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQGTTVTVSS

[0403] SEQ ID NO:36 --- Amino acid sequence of scFv in hu12 CAR;

[0404] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKTVKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGGGGSGG GGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGAGTTVTVSS

[0405] SEQ ID NO:37---Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:28

[0406] GAGGTCCAACTGGTGGAGTCCGGCGGGGGCCTCGTGAAGCCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCTCCGGCCTGACCTTCAGCAGCTACGCCATGAGCTGGGTGAGACAAGCCCCGAGAAGAGACTGGAGTGGGTGGCCTCCATCAGCAGCGGCGGCTTCACCTACT ACCCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACAGCCTGTACCTGCGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCTAGAGACGAGGTGAGAGGCTACCTGGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGC

[0407] SEQ ID NO:38---Nucleotide sequence encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO:30

[0408] GACATTCAGATGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTGCAGCGCCTCCCAAGGCATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGACCGTGAAGCTGCTGATCTACTACACAAGCAGCCTGCACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCAGCAGTACAGCAAGCTGCCCTACACATTTGGGGGCGGGACCAAACTCGAGATTAAG

[0409] SEQ ID NO:39 - Nucleotide sequence encoding the amino acid sequence of hu11 CAR

[0410]

[0411] SEQ ID NO:40---Nucleotide sequence encoding the amino acid sequence of hu12 CAR

[0412]

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, sc(Fv) 2 Antibody or [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, in: 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, which comprises the humanized antibody or its antigen-binding fragment as described in any one of claims 1-3.

16. An antibody-drug conjugate, which comprises the humanized antibody or its antigen-binding fragment as described in any one of claims 1-3.

17. A chimeric antigen receptor targeting CD7, which comprises an extracellular antigen recognition domain targeting CD7, a hinge region, a transmembrane region and an intracellular domain, and 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 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 of the following structures: the amino acid sequence as shown in SEQ ID NO:1 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:2, the amino acid sequence as shown in SEQ ID NO:2 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:1, the amino acid sequence as shown in SEQ ID NO:1 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:30, the amino acid sequence as shown in SEQ ID NO:30 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:1, the amino acid sequence as shown in SEQ ID NO:28 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:30, the amino acid sequence as shown in SEQ ID NO:30 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:28; Optionally, the extracellular antigen recognition domain comprises any one of the following structures: the amino acid sequence as shown in SEQ ID NO:2 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:1, the amino acid sequence as shown in SEQ ID NO:30 - a linker sequence - the amino acid sequence as shown in SEQ ID NO:1, the amino acid sequence as shown in SEQ ID NO:30 - a linker sequence - the 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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