Enhanced chimeric antigen receptor and application thereof

Abstract

The invention relates to an enhanced chimeric antigen receptor and application thereof, in particular to a nucleic acid molecule encoding a chimeric antigen receptor containing an NKG2D transmembrane region, a corresponding chimeric antigen receptor, a carrier, an immune effector cell, a preparation method and a product thereof, a pharmaceutical composition, pharmaceutical application and a tumor or cancer treatment method. The CAR has the advantages of high CAR expression efficiency, high NK cell proliferation speed, high killing power to tumor cells, high specificity, long killing effect duration and the like.

Description

Enhanced chimeric antigen receptor and its application

[0001] The present invention claims priority to a prior application with patent number 202210347351.4 filed with the State Intellectual Property Office of China on April 3, 2022, entitled “Enhanced Chimeric Antigen Receptor and Applications Thereof”; priority to a prior application with patent number 202210871138.3 filed with the State Intellectual Property Office of China on July 22, 2022, entitled “Chimeric Antigen Receptor Targeting GPRC5D and / or BCMA and Applications Thereof”; and priority to a prior application with patent number 202310296244.8 filed with the State Intellectual Property Office of China on March 24, 2023, entitled “Enhanced Chimeric Antigen Receptor and Applications Thereof”. The entire contents of the above-mentioned prior applications are incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of biotechnology, and particularly relates to an enhanced chimeric antigen receptor and applications thereof. Background Art

[0003] Natural killer cells (NK cells) are a type of lymphocyte that has a potent cytotoxic effect on tumor cells and is MHC-independent. Their recognition of tumor cells primarily relies on the cross-regulation of activating and inhibitory receptors on their surface. After recognizing tumor cells, NK cells kill tumor cells through various pathways, including releasing the cytotoxic mediators perforin and granzymes to induce target cell apoptosis, expressing membrane TNF family molecules to induce target cell apoptosis, and antibody-dependent cytotoxicity. Allogeneic NK cell transplantation rarely induces graft-versus-host disease (GVHD) or CRS, and can be used as a completely "off-the-shelf" product. However, due to the decline in the number and quality of NK cells in cancer patients and the existence of tumor escape mechanisms, their anti-tumor function in the body is not fully realized. Modifying NK cells with CARs is expected to enhance their ability to target and kill tumor cells and develop effector cells with powerful anti-tumor effects.

[0004] The structure of the functional chimeric antigen receptor (CAR) expressed by NK cells mainly includes: extracellular domain, transmembrane region and intracellular signaling domain. The selection of corresponding elements in the CAR structure, such as the transmembrane region and the intracellular activation signaling domain, affects the activity of CAR-NK cells. Currently, most CAR-NK products still use the CAR structure commonly used in traditional CAR-T cell therapy, and the intracellular co-activation signaling domain is CD28 or 4-1BB. Since the signaling pathways of NK cell activation and T cell activation are different, the CAR structure commonly used in traditional CAR-T cell therapy may not be able to play the optimal function in CAR-NK.

[0005] Summary of the Invention

[0006] Based on the unique signaling pathway of NK cell activation, the present invention designs a variety of CAR structure combinations containing different transmembrane regions and intracellular co-stimulatory domains. Through screening and verification of in vitro and in vivo experiments, the CAR structure most suitable for the activity of CAR-NK cells is obtained.

[0007] Furthermore, the present disclosure also optimizes the transmembrane region of CAR-NK. Commonly used TM regions for CAR-NK come from CD8 and CD28, and others such as NKG2D have also been used. For example, CN110684117B and CN112210016A disclose NK cells modified with chimeric antigen receptors, in which the transmembrane region of the chimeric antigen receptor is selected from NKG2D TM. NKG2D is an NK cell activator, and the NKG2D TM region is beneficial for enhancing the activation and killing effect of CAR-NK cells, but the current CAR-NK structure lacks a detailed design for the NKG2D TM region. The present disclosure found that optimizing the NKG2D TM region is of great significance for further improving the function of CAR-NK.

[0008] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily and is primarily expressed on the surface of terminally differentiated B cells. B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) are the primary ligands for BCMA. They interact with BCMA to transmit cell-stimulating signals, activating the TRAF-dependent NF-κB and JNK pathways, thereby increasing B-cell proliferation and survival. BCMA expression has been associated with various cancers, autoimmune disorders, and infectious diseases. Cancers with increased BCMA expression include hematologic cancers such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphomas, various leukemias, and glioblastoma. BCMA is an ideal therapeutic target.

[0009] ROR1 is a transmembrane receptor tyrosine kinase protein and a member of the receptor tyrosine kinase (RTK) family. ROR1 is lowly expressed or absent in normal human tissues but is highly expressed in a variety of malignant tumors or tissues, such as chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, and lung adenocarcinoma. Studies have shown that ROR1 plays an important role in promoting tumor growth and metastasis, inducing tumor cell drug resistance, and inhibiting cell apoptosis. Therefore, BCAM and ROR1 are ideal targets for tumor treatment. Developing corresponding chimeric antigen receptors for BCMA and ROR1, especially those suitable for NK cells, is particularly important.

[0010] In view of this, the present disclosure provides nucleic acid molecules encoding chimeric antigen receptors comprising an NKG2D transmembrane region, corresponding chimeric antigen receptors, vectors, immune effector cells, preparation methods and products thereof, pharmaceutical compositions, pharmaceutical uses, and methods for treating tumors or cancers.

[0011] In a first aspect, the present disclosure provides a nucleic acid molecule comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises: an extracellular region comprising an antigen binding region, an NKG2D transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the NKG2D transmembrane region has a sequence that is at least 80% identical or has no more than 9 mutations compared to SEQ ID NO: 48.

[0012] In some specific embodiments, the antigen binding region has the properties of at least one of groups (1)-(4):

[0013] (1) The antigen-binding region is selected from an antibody or a fragment thereof or a ligand that binds to the antigen, such as scFv, VHH, Fab, F(ab)'2 or ligand;

[0014] (2) the antigen binding region binds to or does not bind to NKG2DL;

[0015] (3) the antigen is selected from one or more of the following groups: BCMA, GPRC5D, CLDN18.2, ROR1, CD19, CD33, CD5, CD70, HER2, IL13Rα2, GCC or GPC3;

[0016] (4) The antigen-binding region binds to at least two different antigens, at least two different epitopes of the same antigen, or binds to more than two epitopes of the same antigen.

[0017] In some specific embodiments, the antigen binding region specifically binds BCMA, comprising:

[0018] (1) VHH, whose CDR1-3 respectively include:

[0019] The sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or

[0020] The sequences shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 11; or

[0021] The sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;

[0022] (2) VHH comprising a sequence having at least 80% identity or up to 25 mutations to SEQ ID NO: 1 or 3;

[0023] (3) VHH, whose CDR1-3 respectively include:

[0024] The sequences shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; or

[0025] The sequences shown in SEQ ID NO: 17, SEQ ID NO: 25 and SEQ ID NO: 19; or

[0026] The sequences shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:19; or

[0027] The sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24;

[0028] (4) a sequence having at least 80% identity or at most 25 mutations to SEQ ID NO: 2 or 4; or,

[0029] (5) VHH1-connecting peptide-VHH2, wherein the VHH1 has a sequence as shown in group (1) or group (2), and the VHH2 has a sequence as shown in group (3) or group (4); or

[0030] (6) VHH1-connecting peptide-VHH2, wherein the VHH1 has a sequence as shown in group (3) or group (4) and the VHH2 has a sequence as shown in group (1) or group (2); or

[0031] (7) VHH1-connector peptide-VHH2, having a sequence of at least 80% identity or at most 50 mutations compared to SEQ ID NO: 63.

[0032] In some specific embodiments, the antigen binding region specifically binds to ROR1, comprising:

[0033] (1) An antibody or a fragment thereof, comprising HCDR1-3 and LCDR1-3, wherein the HCDR1-3 respectively comprise:

[0034] The sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; or

[0035] The sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:32; or

[0036] The sequences shown in SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37;

[0037] And the LCDR1-3 respectively include:

[0038] The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or

[0039] The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or

[0040] The sequences shown in SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:40;

[0041] (2) an antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 26, and a light chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 27;

[0042] (3) scFv, said scFv having a sequence shown in said group (1) or group (2), optionally, said scFv having a light chain variable region having at least 80% identity or at most 50 mutations compared to SEQ ID NO: 28.

[0043] In some specific embodiments, the antigen binding region specifically binds to GPRC5D, comprising:

[0044] (1) An antibody or a fragment thereof, comprising HCDR1-3 and LCDR1-3, wherein the HCDR1-3 respectively comprise:

[0045] The sequences shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89; or

[0046] The sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92; or

[0047] The sequences shown in SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98;

[0048] And the LCDR1-3 respectively include:

[0049] The sequences shown in SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86; or

[0050] The sequences shown in SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95;

[0051] (2) An antibody or fragment thereof, comprising HCDR1-3, wherein the HCDR1-3 respectively comprise:

[0052] The sequences shown in SEQ ID NOs: 107-118;

[0053] (3) an antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NOs: 78, 80, 83, 131, and a light chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NOs: 77, 79, 81, 82;

[0054] (4) An antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NOs: 99-106.

[0055] In some specific embodiments, the extracellular region further comprises a hinge region, which is selected from one or more of the following groups: a CD8α hinge region, a 2B4 hinge region, a CD28 hinge region, an IgG1 hinge region, an IgD hinge region, an IgG4 hinge region, a GS hinge, a KIR2DS2 hinge, a KIR hinge, an NCR hinge, a SLAMF hinge, a CD16 hinge, a CD64 hinge or a LY49 hinge; optionally, the hinge region is selected from the CD8α hinge region, for example, the hinge region has a sequence that is at least 80% identical or has at most 10 mutations compared to SEQ ID NO: 45.

[0056] In some specific embodiments, the extracellular region further includes a signal peptide, and the signal peptide is selected from one or more of the following groups: CD8α signal peptide, IgG1 heavy chain signal peptide or GM-CSFR2 signal peptide; optionally, the signal peptide is a CD8α signal peptide, for example, the signal peptide has a sequence that is at least 80% identical or has at most 5 mutations compared to SEQ ID NO:43.

[0057] In some specific embodiments, the intracellular domain comprises an intracellular signaling domain and / or a costimulatory domain;

[0058] Optionally, the intracellular signaling domain is CD3ζ, for example, the intracellular signaling domain has a sequence that is at least 80% identical or has at most 35 mutations compared to SEQ ID NO: 55;

[0059] Optionally, the costimulatory domain is selected from one or more of the following groups: a CD27 costimulatory domain, a 4-1BB costimulatory domain, an OX40 costimulatory domain, a 2B4 costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, a DAP10 costimulatory domain or a DAP12 costimulatory domain; optionally, the costimulatory domain is a 2B4 costimulatory domain, for example, the costimulatory domain has a sequence that is at least 80% identical or has at most 25 mutations compared to SEQ ID NO: 53.

[0060] In some specific embodiments, the chimeric antigen receptor comprises: a CD8α hinge region, an NKG2D transmembrane region, a 2B4 co-stimulatory domain and a CD3ζ intracellular signaling domain, for example, a sequence having at least 80% identity or at most 50 mutations compared to SEQ ID NO: 58.

[0061] In some specific embodiments, the nucleic acid molecule further comprises a second nucleic acid sequence encoding IL15, optionally, the IL15 is selected from soluble IL15, membrane-bound IL15, or a complex of IL15 and its receptor or receptor fragment, optionally, the IL15 has a sequence having at least 80% identity or at most 35 mutations compared to SEQ ID NO: 57;

[0062] Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by an IRES or a sequence encoding a self-cleaving peptide, the self-cleaving peptide being selected from a 2A peptide, such as P2A, T2A, F2A or E2A, optionally, the self-cleaving peptide is P2A, such as a sequence having at least 80% identity or at most 5 mutations compared to SEQ ID NO: 56;

[0063] Optionally, the nucleic acid encodes a nucleic acid sequence having at least 85% identity or up to 100 mutations compared to SEQ ID NO:59.

[0064] In some specific embodiments, it includes a nucleic acid sequence encoding a sequence having at least 80% identity or up to 150 mutations compared to SEQ ID NO: 65 or SEQ ID NO: 70.

[0065] In some specific embodiments, the nucleic acid is DNA or RNA, and the RNA is preferably mRNA.

[0066] In a second aspect, the present disclosure provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a first nucleic acid sequence encoding a chimeric anti-receptor targeting BCMA, the chimeric antigen receptor comprising an extracellular region comprising an antigen binding region, a transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, the antigen binding region comprising:

[0067] (1) VHH, whose CDR1-3 respectively include:

[0068] The sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or

[0069] The sequences shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 11; or

[0070] The sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;

[0071] (2) VHH comprising a sequence having at least 80% identity or up to 25 mutations to SEQ ID NO: 1 or 3;

[0072] (3) VHH, whose CDR1-3 respectively include:

[0073] The sequences shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; or

[0074] The sequences shown in SEQ ID NO: 17, SEQ ID NO: 25 and SEQ ID NO: 19; or

[0075] The sequences shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:19; or

[0076] The sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24;

[0077] (4) a sequence having at least 80% identity or at most 25 mutations to SEQ ID NO: 2 or 4; or,

[0078] (5) VHH1-connecting peptide-VHH2, wherein the VHH1 has a sequence as shown in group (1) or group (2), and the VHH2 has a sequence as shown in group (3) or group (4); or

[0079] (6) VHH1-connecting peptide-VHH2, wherein the VHH1 has a sequence as shown in group (3) or group (4) and the VHH2 has a sequence as shown in group (1) or group (2); or

[0080] (7) VHH1-connector peptide-VHH2, having a sequence of at least 80% identity or at most 50 mutations compared to SEQ ID NO: 63.

[0081] In a third aspect, the present disclosure provides a nucleic acid molecule comprising a first nucleic acid sequence encoding a chimeric anti-receptor targeting ROR1, wherein the chimeric antigen receptor comprises an extracellular region comprising an antigen binding region, a transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the antigen binding region comprises:

[0082] (1) An antibody or a fragment thereof, comprising HCDR1-3 and LCDR1-3, wherein the HCDR1-3 respectively comprise:

[0083] The sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; or

[0084] The sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:32; or

[0085] The sequences shown in SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37;

[0086] And the LCDR1-3 respectively include:

[0087] The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or

[0088] The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or

[0089] The sequences shown in SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:40;

[0090] (2) an antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 26, and a light chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 27;

[0091] (3) scFv, said scFv having a sequence shown in said group (1) or group (2), optionally, said scFv having a light chain variable region having at least 80% identity or at most 50 mutations compared to SEQ ID NO: 28.

[0092] In some specific embodiments, the chimeric antigen receptor comprises: a CD8α hinge region, an NKG2D transmembrane region, a 2B4 co-stimulatory domain and a CD3ζ intracellular signaling domain, for example, the chimeric antigen receptor has a sequence that is at least 80% identical or has at most 50 mutations compared to SEQ ID NO: 58.

[0093] In some specific embodiments, the nucleic acid molecule further comprises a second nucleic acid sequence encoding IL15, optionally, the IL15 is selected from soluble IL15, membrane-bound IL15, or a complex of IL15 and its receptor or receptor fragment, optionally, the IL15 has a sequence having at least 80% identity or at most 35 mutations compared to SEQ ID NO: 57;

[0094] Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by an IRES or a sequence encoding a self-cleaving peptide, the self-cleaving peptide being selected from a 2A peptide, such as P2A, T2A, F2A or E2A, optionally, the self-cleaving peptide is P2A, such as a sequence having at least 80% identity or at most 5 mutations compared to SEQ ID NO: 56;

[0095] Optionally, the core encodes a nucleic acid sequence having at least 85% identity or up to 100 mutations compared to SEQ ID NO:59.

[0096] In some specific embodiments, it includes a nucleic acid sequence encoding a sequence having at least 80% identity or up to 150 mutations compared to SEQ ID NO: 65 or SEQ ID NO: 70.

[0097] In a fourth aspect, the present disclosure provides a chimeric antigen receptor encoded by the aforementioned nucleic acid.

[0098] In a fifth aspect, the present disclosure provides a vector, wherein the vector comprises the aforementioned nucleic acid molecule.

[0099] In a sixth aspect, the present disclosure provides immune effector cells, wherein the immune effector cells include the aforementioned nucleic acid molecule or chimeric antigen receptor or vector.

[0100] In some specific embodiments, the immune effector cells are NK cells, which are differentiated from iPSCs or derived from peripheral blood or umbilical cord blood.

[0101] In a seventh aspect, the present disclosure provides a method for preparing the aforementioned immune effector cells, which comprises the steps of providing immune effector cells and transferring the nucleic acid molecule into the immune effector cells.

[0102] In an eighth aspect, the present disclosure provides a product prepared according to the aforementioned method.

[0103] In a ninth aspect, the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the aforementioned nucleic acid molecule or chimeric antigen receptor or vector, immune effector cells or products, and a pharmaceutically acceptable carrier.

[0104] In a tenth aspect, the present disclosure provides the use of the aforementioned nucleic acid molecules or chimeric antigen receptors or vectors, immune effector cells, products or pharmaceutical compositions in the preparation of a medicament for treating cancer or tumors, wherein the cancer or tumor is selected from a blood tumor or a solid tumor; optionally, the blood tumor is selected from a myeloma, a lymphoma or a leukemia, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer or adrenal cancer.

[0105] In the eleventh aspect, the present disclosure provides the aforementioned nucleic acid molecules or chimeric antigen receptors or vectors, immune effector cells, products or pharmaceutical compositions for use in treating cancer or tumors, wherein the cancer or tumor is selected from a hematological tumor or a solid tumor; optionally, the hematological tumor is selected from a myeloma, a lymphoma or a leukemia, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer or adrenal cancer.

[0106] In a twelfth aspect, the present disclosure provides a method for treating cancer or tumors, comprising administering an effective amount of the aforementioned nucleic acid molecule or chimeric antigen receptor or vector, immune effector cell, product or pharmaceutical composition to a subject in need thereof, wherein the cancer or tumor is selected from a hematological tumor or a solid tumor; optionally, the hematological tumor is selected from a myeloma, a lymphoma or a leukemia, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer or adrenal cancer.

[0107] Beneficial effects: The present disclosure provides an optimized chimeric antigen receptor for the NKG2D transmembrane region, which has at least one of the following advantages: (1) high CAR expression efficiency after transfection; (2) rapid NK cell proliferation after transfection; (3) strong killing ability and specificity against tumor cells; (4) long-lasting killing effect.

[0108] Definitions and Explanations of Terms

[0109] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

[0110] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0111] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0112] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0113] The term "BCMA" in this article stands for B cell maturation antigen, a member of the tumor necrosis factor receptor family. BCMA is primarily expressed on the surface of late-stage B cells, short-lived proliferating plasmablasts, and long-lived plasma cells, but is not expressed on naive B cells, CD34-positive hematopoietic stem cells, and other normal tissue cells. However, it is highly expressed in MM cells and plays a key role in the survival, proliferation, metastasis, and drug resistance of MM cells by mediating downstream signaling pathways. Therefore, BCMA is an ideal antigen target for the treatment of MM. An exemplary human BCMA sequence can be found in GenBank Protein Accession No: NP_001183.2.

[0114] The term GPRC5D herein refers to the G protein-coupled receptor C5 family subtype D, an orphan receptor, a seven-transmembrane protein with no known ligand. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while expression in normal tissue is restricted to the hair follicle region. Studies have shown that 65% of multiple myeloma patients have GPRC5D expression exceeding the 50% threshold. This characteristic makes GPRC5D a potential target for the treatment of multiple myeloma. An exemplary human GPRC5D sequence can be found in GenBank Protein Accession No: NP_061124.1.

[0115] As used herein, "antigen chimeric receptor (CAR)" refers to an artificial immune effector cell surface receptor that is modified to express on immune effector cells and specifically binds to an antigen, comprising at least (1) an extracellular antigen binding domain, such as a variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune effector cells to selected targets, such as cancer cells, using the extracellular antigen binding domain in a non-MHC restricted manner.

[0116] The term "signal peptide" herein refers to a fragment of a protein or polypeptide that is used to guide the protein or polypeptide into the secretory pathway and transferred to the cell membrane and / or cell surface. In some embodiments, the signal peptide is a CD8α signal peptide, optionally having at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) to the following sequence: MALPVTALLLPLALLLHAARP.

[0117] As used herein, the "transmembrane (TM) region" of a chimeric antigen receptor refers to a polypeptide structure that enables the chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and guides the immune cell's cellular response to a target cell. The transmembrane domain may be natural or synthetic, and may also be derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor binds to the target antigen, the transmembrane domain is capable of signal transduction.

[0118] As used herein, the "hinge region" of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane region and the antigen binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antigen binding region. The hinge region can be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence.

[0119] As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs cells to perform designated functions. The intracellular signaling domain is responsible for primary signaling within the cell after the antigen binding domain binds to the antigen, leading to activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. Exemplary intracellular signaling domains include CD3ζ.

[0120] As used herein, the term "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes after binding to an antigen.

[0121] As used herein, the term "immune effector cell" or "effector cell" refers to a cell that participates in an immune response, such as a cell that promotes an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and phagocytes of myeloid origin.

[0122] The terms "T lymphocyte" and "T cell" are used interchangeably herein and refer to the main type of white blood cell that completes maturation in the thymus and has multiple roles in the immune system, including recognizing specific foreign antigens in the body and activating and inactivating other immune cells in an MHC class I restricted manner. T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, γδT cells (gamma delta T cell / γδT cell), etc. Other types of helper T cells include cells such as Th3 (Treg), Th17, Th9 or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express T cell receptors (TCR) or chimeric antigen receptors (CAR). T cells or T cell maintenance effector cells can also be differentiated by stem cells or progenitor cells. T cell-like derived effector cells can have T cell lineages in some aspects, but at the same time have one or more functional characteristics not present in primary T cells.

[0123] The term "NK cell" or "natural killer cell" herein refers to a subpopulation of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms "adaptive NK cell" and "memory NK cell" are interchangeable and refer to a subpopulation of NK cells whose phenotype is CD3 - and CD56 + , expresses at least one of NKG2C and CD57 and optionally CD16, but lacks expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some embodiments, the isolated CD56+ NK cell subsets include expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. + It can be weaker or stronger expression. NK cells or NK cell-like effector cells can be differentiated from stem cells or progenitor cells. NK cell-like derived effector cells can have NK cell lineage in some aspects, but at the same time have one or more functional characteristics not present in primary T cells.

[0124] As used herein, the term "NKT cell" or "natural killer T cell" refers to a T cell restricted to CD1d that expresses a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d (a non-classical MHC molecule). Two types of NKT cells are identified. Constant or type I NKT cells express a very limited TCR library: a combination of a typical α chain (Vα24-Jα18 in humans) and a limited spectrum of β chains (Vβ11 in humans). A second NKT cell population (called non-classical or non-constant type II NKT cells) shows a more uneven TCR α β utilization rate. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0125] As used herein, the term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual. "Allogeneic" refers to a transplant derived from a different individual of the same species.

[0126] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an antibody with a specific affinity of about 10 -6 M or lower, about 10 -7 M or lower about 10 -8 M or lower, about 1×10 -9 M or lower, about 1×10 -10 M or less, 1×10 -11 M or lower or 1×10 -12The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. For example, see the methods for obtaining KD values ​​described in the Examples herein.

[0127] The term "antibody" is used in the broadest sense herein to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0128] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0129] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe) and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0130] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0131] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, scFv, and VHH.

[0132] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, along with the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the light chain VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites (the two Fab fragments) and a portion of the Fc region.

[0133] The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0134] The term "nanoantibody" in this article refers to heavy chain antibodies that are naturally missing light chains and exist in camels and other organisms. Cloning their variable regions can produce single-domain antibodies consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0135] The terms "VHH domain" and "single domain antibody" (sdAb) are used interchangeably and have the same meaning. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy chain variable region by cloning the variable region of a heavy chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy chain variable region.

[0136] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0137] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0138] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0139] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0140] The term "IMGT numbering system" herein generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.

[0141] As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced into one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires achieving maximum alignment over the full length of the compared sequences. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0142] As used herein, "at least 80% identity" is preferably 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0143] As used herein, "mutation" includes insertion mutation, deletion mutation and substitution mutation, preferably, the substitution mutation is a conservative amino acid substitution. As used herein, "conservative amino acid" generally refers to amino acids belonging to the same class or having similar characteristics (such as charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation and rigidity). Exemplarily, the amino acids in each of the following groups belong to each other's conservative amino acid residues, and the replacement of amino acid residues in the group belongs to the replacement of conservative amino acids:

[0144] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0145] 1) Alanine (A), serine (S), threonine (T);

[0146] 2) Aspartic acid (D), glutamic acid (E);

[0147] 3) Asparagine (N), glutamine (Q);

[0148] 4) Arginine (R), Lysine (K), Histidine (H);

[0149] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0150] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0151] As used herein, "at most 5 mutations" is preferably at most 4, 3, 2, 1 or 0 mutations.

[0152] As used herein, "at most 9 mutations" is preferably at most 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

[0153] As used herein, "at most 10 mutations" is preferably at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

[0154] As used herein, "at most 25 mutations" is preferably at most 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

[0155] As used herein, "up to 30 mutations" is preferably up to 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

[0156] As used herein, "up to 50 mutations" is preferably at most 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 20, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

[0157] As used herein, “up to 100 mutations” is preferably up to 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 70, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

[0158] As used herein, “at most 150 mutations” is preferably at most 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 120, 129, 128, 127, 126, 125, 124, 123, 122, 121, 1 20, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84 , 83, 82, 81, 70, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 or 50 mutations, 49, 48, 47, 46, 45, 44, 43 or 0 mutations.

[0159] As used herein, a "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0160] As used herein, the terms "subject," "object," and "patient" refer to an organism that is being treated for a particular disease or condition, such as cancer or an infectious disease, as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as cattle, bison, buffalo, elk, and yaks, etc.), cattle, sheep, horses, and bison, etc., that are being treated for a disease or condition, such as a cell proliferative disorder, such as cancer or an infectious disease.

[0161] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a disease or disease, as well as subjects susceptible to a disease or disease, or subjects intending to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0162] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0163] FIG1 is a schematic diagram of the CAR-NK structure containing different NKG2D TM region elements.

[0164] Figure 2 shows the CAR expression rate in BCMA-CAR NK cells.

[0165] Figure 3 shows the CAR expression rate in ROR1-CAR NK cells.

[0166] Figure 4 shows the BCMA-CAR NK proliferation fold statistics.

[0167] Figure 5 shows the proliferation fold statistics of ROR1 CAR NK cells.

[0168] FIG6 shows the flow cytometry results of MOLP8 and NCI H929-hBCMA-KO cells.

[0169] Figure 7: Multiple rounds of killing effect of BCMA-CAR NK on MOLP8 cells.

[0170] Figures 8A to 8C show the multi-round killing effects of ROR1 CAR NK on A549, RPMI 8226, and 7860.

[0171] Figure 9 is a schematic diagram of the BCMA-CAR-NK-6 (BCMA-CAR6) structure.

[0172] Figure 10 shows the CAR expression rate in BCMA-CAR NK cells used in animal experiments.

[0173] Figures 11A-11E: Anti-tumor efficacy of different BCMA-CAR-NK cell lines in the NCI H929-luc multiple myeloma animal model. Figure 11A shows the bioluminescent signal detection results of the animal tumors; Figure 11B shows the animal body weight change (%); Figure 11C shows the photon count of the animal tumor bioluminescent signal; Figure 11D shows the animal tumor growth inhibition (%); and Figure 11E shows the animal survival (%).

[0174] Figure 12: Schematic diagram of the dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D.

[0175] Figure 13: Schematic diagram of the structure of the NKG2D_TM dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D.

[0176] Figure 14: Statistics of BI-CAR-NK cell proliferation fold.

[0177] FIG15A-FIG15B: The results of multiple rounds of killing experiments of MOLP8 cells and RPMI 8226 cells by BI-CAR-NK.

[0178] Figure 16: Schematic diagram of the dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D.

[0179] Figure 17: Statistics of BI-CAR-NK cell proliferation fold.

[0180] Figures 18A-18F: Results of multiple rounds of killing experiments of BI-CAR-NK cells against NCI H929 cells, RPMI 8226 cells, MOLP8 cells and tumor heterogeneous cells. DETAILED DESCRIPTION

[0181] The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0182] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0183] Unless otherwise specified, the target cells referred to in the following examples were all transfected with the luciferase gene to express luciferase. Fluorescence intensity was measured using a luciferase reporter gene assay to reflect cell viability and NK cell killing efficacy. The killing rate was calculated using the following formula:

[0184] Killing rate = (target cell well reading - test well reading) / target cell well reading × 100%.

[0185] Unless otherwise specified, the target cells used in the following examples are A549 cells, 786-O cells, RPMI 8226 cells, MOLP8 cells, and H929-hBCMA-KO cells. All cells contain luciferase reporter genes through conventional genetic manipulation methods, among which H929-hBCMA-KO is an H929 cell line in which BCMA is knocked out using conventional genetic manipulation methods.

[0186] Example 1 Screening and preparation of antibodies

[0187] 1. Screening and identification of BCMA antibodies

[0188] Alpacas were immunized with a protein containing the extracellular domain of human BCMA. Peripheral blood was collected after four and five immunizations (antibody titer and specificity verified by ELISA), and PBMCs were isolated. Total RNA was extracted, and VHH fragments were amplified by reverse transcription and nested PCR. Phage libraries were constructed, and clones cross-positive for human and monkey BCMA were selected and identified. The variable region sequences of the positive clones were sequenced and designated VHH1 and VHH2. Their CDR sequences were determined using the Kabat and Chothia numbering systems (http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) and the IMGT numbering system (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi).

[0189] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy chain variable region germline genes with high homology to the VHH1 antibody (IGHV3-64*04 and IGHJ3*01) or to the VHH2 antibody (IGHV3-7*01 and IGHJ6*01) as templates. The CDRs of VHH1 or VHH2 (determined by the IMGT numbering system) were transplanted into the corresponding humanized templates, resulting in a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, key amino acids in the framework sequence were backmutated to those corresponding to the VHH nanobody to maintain the original affinity. If the antibody contained sites susceptible to chemical modification, point mutations were performed to eliminate the risk of modification. This resulted in the humanized antibodies hu-VHH1 and hu-VHH2. The sequence information and CDR information of VHH antibodies and their humanized antibodies are shown in Tables 1 and 2.

[0190] The VHH1, VHH2, hu-VHH1, and hu-VHH2 sequences were recombined into the human IgG1 Fc expression vector, and chimeric and humanized antibodies VHH1-hFc, VHH2-hFc, hu-VHH1-hFc, and hu-VHH2-hFc were obtained through expression and purification. ELISA and FACS verification showed that the aforementioned antibodies all had good binding activity to human BCMA protein and monkey BCMA protein, and all had good binding to H929 and U266 cells that endogenously express BCMA. Affinity test results further verified the high affinity of VHH1-hFc, VHH2-hFc, hu-VHH1-hFc and hu-VHH2-hFc for human BCMA protein, with KD values ​​of 5.27E-10M (VHH1-hFc), 7.16E-11M (VHH2-hFc), 6.13E-10M (hu-VHH1-hFc) and 8.82E-11M (hu-VHH2-hFc), respectively.

[0191] Table 1 Antibody sequence information

[0192] Table 2 CDR information of VHH1 and VHH2

[0193] 2. Screening and Identification of ROR1 Antibodies

[0194] Mice were immunized with human ROR-1 fusion protein (ACRO, Cat.RO1-H5250), and positive clones ROR1-1 were screened by spleen cell fusion and hybridoma technology. The antibody variable region sequence was obtained by sequencing. The CDR region sequence was determined using the Kabat, Chothia, and IMGT numbering systems. The nucleic acid sequence encoding ROR1-1-scFv was cloned into an expression vector containing a signal peptide and human Fc, and the chimeric antibody ROR1-1-hFc was obtained by expression and purification. ELISA and FACS identification showed that the chimeric antibody specifically bound to human ROR1 protein and MDA-MB-231 cells that endogenously express ROR1. Affinity test results further verified that the chimeric antibody bound to human ROR1 protein, with a KD value of 3.23E-07M. Detailed sequence information is shown in Tables 3-4.

[0195] Table 3 ROR1 antibody variable region sequence information

[0196] Table 4 ROR1 antibody CDR region sequence information

[0197] Example 2 Design of different forms of chimeric antigen receptors

[0198] NKG2D TM1 was designed and loaded into a chimeric antigen receptor as a transmembrane region element to construct a new type of chimeric antigen receptor. At the same time, chimeric antigen receptors loaded with other NKG2D TM sequences were constructed, as well as chimeric antigen receptors in which the hinge region, transmembrane region, and co-stimulatory domain all used 2B4 or CD28, wherein NKG2D-TM4 was derived from WO2021071962A1, (see sequence 57 disclosed in WO2021071962A1 for details). In subsequent examples, the functional differences (CAR positivity, NK cell proliferation, and killing) of CAR NK cells loaded with NKG2D transmembrane regions and other CAR NK cells loaded with different transmembrane regions were evaluated, as well as the functional differences between CAR NK cells loaded with NKG2D TM1 elements and CAR NK cells loaded with other NKG2D TM sequences. The molecular structure of the chimeric antigen receptor is shown in Figures 1 and 9, the element sequence information is shown in Table 5, and the CAR sequence information is shown in Table 6. In Figures 1 and 9 , BCMA-CAR-NK-1 is hereinafter referred to as BCMA-CAR1, BCMA-CAR-NK-2 is hereinafter referred to as BCMA-CAR2, BCMA-CAR-NK-3 is hereinafter referred to as BCMA-CAR3, BCMA-CAR-NK-4 is hereinafter referred to as BCMA-CAR4, BCMA-CAR-NK-5 is hereinafter referred to as BCMA-CAR5, and BCMA-CAR-NK-6 is hereinafter referred to as BCMA-CAR6; ROR1-CAR-NK-1 is hereinafter referred to as ROR1-CAR1, ROR2-CAR-NK-2 is hereinafter referred to as ROR1-CAR2, ROR1-CAR-NK-3 is hereinafter referred to as ROR1-CAR3, ROR1-CAR-NK-4 is hereinafter referred to as ROR1-CAR4, and ROR1-CAR-NK-5 is hereinafter referred to as ROR1-CAR5.

[0199] Table 5 CAR element sequence information

[0200] Table 6 CAR sequence information

[0201] Example 3 Preparation of Chimeric Antigen Receptor Retrovirus

[0202] The conventional molecular biological methods in the art were used to load the nucleic acid sequence encoding the CAR described in Example 2 (Table 6) into a retroviral vector to construct the target plasmid. The day before virus packaging, 293T cells (purchased from ATCC) were trypsinized and inoculated into culture dishes at 1E7 cells / 10cm. When transfecting cells, the packaging plasmid and the target plasmid were mixed and added to α-MEM medium. In another centrifuge tube containing α-MEM medium, HD transfection reagent (Promega, E2311). Add the diluted transfection reagent dropwise to the diluted plasmid, mix well, and let it stand at room temperature for 15 minutes. Finally, add the mixture of plasmid and transfection reagent to a 10cm culture dish, shake gently 10 times, mix well, and place it in the incubator. 3 days after cell transfection, harvest the virus and transfer 10ml of the virus-containing culture supernatant to a 50ml centrifuge tube. Incubate at 4°C, 1250rpm, for 5 minutes to remove dead 293T cells. Filter the virus-containing supernatant and concentrate it using Retro-X Concentrator (Clontech, 631455). Aliquot and store at -80°C until use.

[0203] Example 4 Preparation of CAR-NK

[0204] 1. Isolation and Activation of NK Lymphocytes

[0205] Fresh PBMCs were centrifuged at 500 g for 7 minutes at room temperature. The supernatant was discarded, and NK cells were isolated using the Human NK Cell Isolation Kit (Stemcell, 17955). The isolated NK cells were activated with K562 cells using the following protocol: On Day 0, cells were counted using AO / PI, mixed at a 1:2 ratio of NK:K562, and added to a non-treated 6-well plate in NK cell culture medium (Miltenyi Biotec, 130-114-429) supplemented with 200 IU / ml human IL-2. The plate was then incubated in an incubator (37°C, 5% CO2). On Day 4, 3 mL of culture medium was added to each well. On Day 6, cell activation was complete and ready for transfection.

[0206] 2. Retroviral infection of NK cells

[0207] On Day 1, coat a 24-well plate with 500 μL of RetroNectin reagent (Takara, T202) at a concentration of 7 μg / mL per well at 4°C overnight. On Day 2, discard the upper RetroNectin layer and wash once with PBS. Infect NK cells at an MOI of 5. Calculate the amount of virus based on the viral titer and add the virus to the 24-well plate. Centrifuge the 24-well plate with virus at 2000g for 60 minutes at 4-8°C. Discard the upper viral layer. Count the NK cells and add 3E5 cells / well to the 24-well plate. Centrifuge at 400g for 5 minutes at room temperature. Place the 24-well plate in an incubator (37°C, 5% CO2). On Day 3, transfer the transfected NK cells to a non-treated 6-well plate. On Day 6, assay CAR expression and CAR NK cell proliferation.

[0208] Example 5 Detection of expression efficiency and proliferation efficiency of different forms of chimeric antigen receptors

[0209] On the 6th day after retroviral infection of NK cells, the expression rate of CAR on the NK cell membrane surface was detected by flow cytometry and the proliferation of CAR NK cells was detected by cell counter.

[0210] 1. Detection of CAR expression rate in NK cells

[0211] (1) BCMA: 2E5 cells were placed in a 96-well U-shaped plate, centrifuged, the supernatant was discarded, and after washing with buffer, 100 μl of FITC-hBCMA-his (ACROBiosystems, BCA-HF254) with a final concentration of 2 μg / ml was added and incubated at 4°C in the dark for 1 hour; after the incubation, centrifuged, the supernatant was discarded, and the cells were washed with buffer and resuspended; the BCMA-CAR expression rate was detected by BD FACS Canto II flow cytometer. The flow cytometric detection results of BCMA-CAR NK cell surface molecule expression are shown in Figure 2. The results showed that the expression efficiency of BCMA-CAR in NK cells was about 70-90%, and the expression efficiency from high to low was BCMA-CAR4 (89.7%) > BCMA-CAR5 (85.9%) > BCMA-CAR1 (73.8%) > BCMA-CAR2 (73%) > BCMA-CAR3 (65.1%).

[0212] (2) ROR1: 2E5 cells were transferred to a 96-well U-shaped plate, centrifuged, the supernatant was discarded, and after washing with buffer, 100 μl of human ROR1-his (ACROBiosystems, RO1-H522y) with a final concentration of 10 μg / ml was added, and incubated at 4°C in the dark for 1 hour; after the incubation, centrifuged, the supernatant was discarded, and after washing with buffer, 100 μl of the final concentration of 1 μg / ml of THE TM The cells were incubated with His Tag Antibody [iFluor 647] (GenScript, A01802) at 4°C in the dark for 1 hour. After incubation, the cells were centrifuged, the supernatant discarded, and the cells were washed with buffer and resuspended. ROR1-CAR expression was determined using a BD FACS Canto II flow cytometer. The flow cytometric analysis of ROR1CAR NK cell surface molecule expression is shown in Figure 3. The results showed that the expression efficiency of ROR1 CAR in NK cells was approximately 50-80%, with the expression efficiency ranking from highest to lowest as ROR1-CAR4 (83.5%) > ROR1-CAR5 (70.1%) > ROR1-CAR1 (69.8%) > ROR1-CAR3 (57.4%) > ROR1-CAR2 (52.2%).

[0213] 2. Detection of the proliferation rate of CAR NK cells

[0214] The proliferation of BCMA-CAR NK cells and ROR1 CAR NK cells was assessed by AO / PI counting, as shown in Figures 4 and 5. Six days after transfection, BCMA-CAR NK cells proliferated approximately 40-70 fold, with the proliferation rates ranking as follows: BCMA-CAR1 (73 fold) > BCMA-CAR2 (68 fold) > BCMA-CAR4 (66 fold) > BCMA-CAR3 (63 fold) > parental NK (53 fold) > BCMA-CAR5 (44 fold). Six days after transfection, ROR1-CAR NK cells proliferated approximately 60-120 fold, with the proliferation rates ranking as follows: ROR1-CAR1 (120 fold) > parental NK (110 fold) > ROR1-CAR2 (100 fold) > ROR1-CAR3 (86 fold) > ROR1-CAR4 (73 fold) > ROR1-CAR5 (67 fold).

[0215] Result description:

[0216] According to the above results, in terms of CAR expression rate, the expression rate of CAR loaded with NKG2D TM element in NK cells is lower than that of other forms of CAR; the expression rate of CAR loaded with NKG2D-TM1 element (CAR1) is higher than that of CAR loaded with NKG2D-TM2 and NKG2D-TM3 elements (CAR2 and CAR3). However, the proliferation rate of CAR NK cells loaded with NKG2D TM element is generally higher than that of CAR NK cells using other transmembrane elements. Among them, the proliferation rate of CAR NK cells loaded with NKG2D-TM1 element is the fastest, which is higher than that of CAR NK cells loaded with other transmembrane elements or other NKG2D TM sequences.

[0217] Example 6 4h in vitro killing functional evaluation of different forms of CAR NK

[0218] Flow cytometry was used to detect BCMA expression in multiple myeloma MOLP8 cells and H929-hBCMA-KO cells. The results are shown in Figure 6.

[0219] A 4-hour in vitro killing experiment was performed on the 6th day after retroviral infection of NK cells: target cells MOLP8 or H929-hBCMA-KO diluted in 1640 medium were added to a white opaque 96-well plate at 2E4 cells / 50 μl / well, and NK cells were added to the above target cells at effector-target ratios of 10:1, 5:1, and 2.5:1. The 96-well plate was placed in a 37°C, 5% CO2 incubator and cultured; after 4 hours, 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added, and the cells were incubated at room temperature in the dark for 10 minutes, and then the killing rate was measured using a microplate reader to calculate the killing rate.

[0220] Table 7 details the 4-hour in vitro cytotoxicity against MOLP8 cells. Compared with other CAR constructs, CAR constructs loaded with the NKG2D TM domain demonstrated stronger tumor cell cytotoxicity at effector-target ratios of 10:1, 5:1, and 2.5:1. The CAR loaded with NKG2D TM1 (BCMA-CAR1) exhibited the strongest cytotoxicity, outperforming other NKG2D TM constructs. Furthermore, NK cells transfected with BCMA-CARs 1 to 5 demonstrated comparable cytotoxicity against H929-hBCMA-KO cells as did partenal NK cells, indicating that BCMA-CARs exhibit no nonspecific cytotoxicity against H929-hBCMA-KO cells.

[0221] Table 7 4h in vitro killing rate of MOLP8 cells by BCMA-CAR NK (unit, %)

[0222] Note: ND stands for "Do Not Kill"

[0223] Example 7 Evaluation of multiple rounds of killing function of different forms of CAR NK

[0224] 1. BCMA-CAR NK cell multi-round killing test

[0225] On the 6th day after retroviral infection of NK cells, the in vitro multiple-round killing effect of BCMA-CAR NK cells on MOLP8 cells was detected: (1) First round of killing: Target cells MOLP8 diluted with 1640 culture medium were placed in a 12-well plate at a concentration of 2.5E5 cells / 500μl / well. NK cells were added to the 12-well plate at an effector-target ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator for 24 hours. After culturing for 24 hours, 100μl of the above mixed cells were added to a 96-well plate with an opaque white bottom, and 30μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the fluorescence intensity was measured using a microplate reader to calculate the NK cell killing efficiency. After that, the next round of killing test was directly entered, or the NK cell killing rate was measured again after culturing for 24 hours before entering the next round of killing test. (2) Next round of killing: Take the cells in the 12-well plate from the previous round, count the NK cells, add the NK cells from the previous round to the 12-well plate inoculated with new target cells at an effector-target ratio of 1:1, repeat step (1), determine the NK cell killing rate and proceed to the next round of killing test.

[0226] The results of multiple rounds of BCMA-CAR NK cell killing experiments are shown in Figure 7. As shown in Figure 7, the killing effect of BCMA-CAR loaded with NKG2DTM elements is better than that of other forms of BCMA-CAR. Among them, BCMA-CAR1 loaded with NKG2D-TM1 has the best killing effect, which is better than other forms of BCMA-CAR and CAR loaded with other NKG2D-TMs. It can still maintain a 98% cell killing rate in the third round of killing (R3-2d).

[0227] 2. ROR1-CAR NK cell multi-round killing test

[0228] Six days after retroviral infection of NK cells, the in vitro multi-round cytotoxicity of ROR1 CAR NK cells against A549 (ROR1 low expression), 786-O (ROR1 low expression), and RPMI 8226 cells (ROR1 medium expression) was tested: target A549, 786-O, or RPMI 8226 cells diluted in 1640 medium were added to a white-bottomed opaque 96-well plate at a rate of 2.0E4 cells / 50 μl / well. NK cells were then added to the 96-well plate at a target-to-effect ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours of culture, 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added, and the plate was incubated in the dark at room temperature for 10 minutes. The fluorescence value was measured using a microplate reader to calculate the NK cell cytotoxicity. After counting the NK cells in the previous round of 96-well plates, add the NK cells from the previous round to the 96-well plates seeded with new target cells at an effector-target ratio of 1:1, and repeat the above experimental steps.

[0229] The results of multiple rounds of ROR1-CAR NK cell killing experiments are shown in Figures 8A-8C. The results of the second and third rounds of killing showed that ROR-1 CAR1 NK cells were more effective than non-NKG2D-TM CARs and other NKG2D-TM CARs in killing 786-O cells, A549 cells, and RPMI 8226 cells.

[0230] Example 8 Evaluation of cytokine release function of different forms of CAR NK

[0231] After 14 days of culture, BCMA-CAR NK cells were transfected with MOLP8 cells at effector-target ratios of 10:1 and 2.5:1 for 24 hours. IFN-γ levels in the collected supernatants were measured using a human IFN-γ quantitative ELISA kit (BD, 555142) according to the manufacturer's instructions. IFN-γ levels in the supernatants of the test samples were calculated using a standard curve. The results are shown in Table 8.

[0232] ROR1-CAR NK cells were transfected and cultured until day 14. After incubation with RPMI 8226 cells at effector-target ratios of 1:1 and 1:3 for 24 hours, the supernatant was collected and the IFN-γ content in the supernatant was detected using a human IFN-γ quantitative ELISA kit (BD, 555142). The results are shown in Table 9.

[0233] The results in Tables 8 and 9 indicate that CAR1 can upregulate the expression of the tumor-killing cytokine IFN-γ at different effector-target ratios and against different target cells, and is generally superior to CARs using non-NKG2D TM elements, as well as CARs using other NKG2D-TM elements.

[0234] Table 8 Detection results of cytokine IFN-γ content

[0235] Note: ND stands for "Not Detected"

[0236] Table 9 Cytokine IFN-γ content (pg / mL) detection results

[0237] Note: ND stands for "Not Detected"

[0238] Example 9 In vivo efficacy evaluation of different forms of BCMA-BCMA CAR-NK

[0239] BCMA-CAR1 loaded with the NKG2D TM1 element, BCMA-CAR2 loaded with the NKG2D TM2 element, BCMA-CAR6 loaded with the NKG2D TM4 element, and BCMA-CAR4 and BCMA-CAR5 without the NKG2D TM element (see Figures 1 and 9 for CAR structures; see Table 6 for sequence information) were evaluated for efficacy in the NCI H929-luc mouse model.

[0240] 1. Preparation of BCMA CAR-NK Cells

[0241] Referring to the preparation method of Example 4, BCMA CAR-NK cells were prepared using NK cells from different sources than in Example 4. NK cells were infected at an MOI of 5, the amount of virus was calculated based on the viral titer, and the virus was added to a 24-well plate. The 24-well plate with the virus added was centrifuged at 2000g, 4-8°C for 60 minutes. The upper viral liquid was discarded. NK cells were counted and added to the 24-well plate at 3E5 cells / well. The cells were centrifuged at 400g for 5 minutes at room temperature. The 24-well plate was placed in an incubator and cultured (37°C, 5% CO2). On Day 3, the transfected NK cells were transferred to a non-treated 6-well plate. On Day 9, CAR expression was detected as shown in Figure 10.

[0242] According to the results in Figure 10, in this experiment, in terms of CAR expression rate, the expression rate of CAR loaded with NKG2D TM element in NK cells was lower than that of other forms of BCMA-CAR4 and BCMA-CAR5; the expression rate of BCMA-CAR1 and BCMA-CAR6 was higher than that of BCMA-CAR2.

[0243] 2. Anti-tumor efficacy test of BCMA-BCMA CAR-NK loaded with NKG2D-TM element in NCI H929-luc mouse tumor model

[0244] To evaluate the anti-tumor efficacy of BCMA-BCMA CAR-NK loaded with NKG2D-TM elements, an anti-tumor efficacy test was conducted using a mouse bone marrow cancer intravenous transplant tumor model. The details are as follows:

[0245] H929-Luc cells in the logarithmic growth phase and in good growth condition were collected and inoculated into the tail vein of NPG mice (combined immunodeficient mice) at a total of 2×10 6 cells. On the first day after tumor inoculation, the weight and inoculation status of mice were measured. Mice weighing about 18.85 to 23.52 g were randomly selected according to the random number principle, with an average weight of 21.92 g for random grouping. On the first day after tumor inoculation, freshly prepared CAR-NK cells (5×10 6The injection volume was 200 μl / mouse, and the CAR-NK cell injection date was Day 0. The mouse groups and CAR-NK cell injection status are shown in Table 10. Tumor growth fluorescence signal ROI values ​​and body weight changes were continuously monitored using an IVIS in vivo imager. Measurements were recorded twice a week and the tumor inhibition rate was calculated using the following formula: Tumor inhibition rate (TGI) (%) = (tumor photon signal value of mice in the PBS group - tumor photon signal value of mice in the experimental group) / tumor photon signal value of mice in the PBS group × 100%.

[0246] Table 10. Grouping of in vivo anti-tumor experiments

[0247] The results of mouse tumor growth fluorescence signal detection are shown in Figure 11A. The results show that: 76 days after the injection of CAR-NK cells, the BCMA-CAR 1 treatment group was able to significantly inhibit the tumor growth burden of mice, of which four animals were cured of tumors and only one animal had recurrence and growth of tumors; the BCMA-CAR 6 treatment group was able to significantly inhibit the tumor growth burden, but could not control the tumor progression of the animals in this group, of which two animals had recurrence and growth of tumors, two animals had complete tumor regression, and one animal died; the BCMA-CAR 2 treatment group was able to inhibit the growth rate of mouse tumors, of which one animal had a cured tumor, but four animals in the group had recurrence of tumors and two died; BCMA-CAR 4 and BCMA-CAR 5 were able to inhibit the growth rate of mouse tumors 39 days after administration. In the later stage of the experiment, as the tumor recurred, the tumor control effect was poor, and all BCMA-CAR 4 animals had died by 76 days.

[0248] At the same time, although the body weights of mice in each group treated with CAR-NK fluctuated after administration, they generally showed an upward trend (as shown in Figure 11B).

[0249] After drug administration, the photon counts of mouse tumors were continuously measured. Except for the BCMA-CAR 5 treatment group, the photon counts of the other CARNK treatment groups showed a decreasing trend compared with the Parental NK group (P < 0.05*, P < 0.001***). This was particularly evident in the BCMA-CAR1 and BCMA-CAR6 treatment groups, which demonstrated a highly significant therapeutic effect (as shown in Figure 11C).

[0250] On day 76, the tumor volume of the mice was measured and the tumor inhibition rate was calculated. As shown in Figure 11D, the tumor inhibition rates of the BCMA-CAR1, BCMA-CAR6, and BCMA-CAR4 treatment groups reached 93%, 94%, and 84%, respectively, demonstrating a good effect in inhibiting tumor growth. However, the tumor inhibition rate of the BCMA-CAR 5 treatment group was only 25%. Since all animals in the BCMA-CAR 4 group died before day 76, the tumor inhibition rate could not be calculated.

[0251] During the experiment, the survival rate was continuously monitored up to 130 days. As shown in Figure 11E, one animal in the Parental NK treatment group died starting on day 57, and the mortality rate was 100% by day 80; one animal in the BCMA-CAR 6 treatment group died starting on day 67, and the mortality rate was 60% by day 130; one animal in the BCMA-CAR 2 treatment group died starting on day 39 (the mouse died on the same day after the tumor growth fluorescence signal was detected on day 39), and the mortality rate was 60% by day 130; one animal in the BCMA-CAR4 treatment group died starting on day 48, and the mortality rate was 100% by day 76; one animal in the BCMA-CAR 5 treatment group died starting on day 63, and the mortality rate was 100% by day 108; only one animal in the BCMA-CAR 1 treatment group died on day 85, indicating that this group showed a lasting therapeutic effect and had a good safety profile.

[0252] Example 10 Preparation of GPRC5D Antibody

[0253] 10.1 Preparation of anti-GPRC5D scFv

[0254] Anti-human GPRC5D monoclonal antibodies were generated by immunizing mice using conventional hybridoma methods. Chimeric antibodies containing human IgG1 constant regions were constructed based on sequenced light and heavy chain variable region sequences. Using cell-based ELISA and FACS screening, familiar to those skilled in the art, monoclonal antibodies were identified that bound to human and monkey GPRC5D-overexpressing cells, as well as NCI-H929 cells, for subsequent humanization design.

[0255] Humanized monoclonal antibodies were generated by comparing the heavy and light chain variable region germline gene database of human antibodies maintained by IMGT (http: / / imgt.cines.fr) using conventional humanized antibody methods. The CDR amino acid residues were assigned and annotated using the Kabat numbering system. Subsequently, validation was performed using a cell-based ELISA. The resulting murine antibodies were designated GPRC5D-mab03 and GPRC5D-mab06, and their humanized counterparts were designated GPRC5D-Hab03 and GPRC5D-Hab06. The light and heavy chain variable region sequences are shown in Table 11, and the CDR sequences are shown in Table 12.

[0256] Table 11 VL and VH sequences of anti-GPRC5D mouse and humanized antibodies

[0257] Table 12 Kabat numbering of LCDRs and HCDRs sequences of anti-GPRC5D antibodies

[0258] 10.2 Preparation of Anti-GPRC5D Nanobodies

[0259] Alpaca-derived VHH antibodies were prepared using conventional VHH antibody screening methods in the art, using the human GPRC5D protein as the immunogen. The CDR amino acid residues of the antibodies were identified and annotated using the IMGT or Kabat numbering systems. This yielded two alpaca sequences, designated GPRC5D-Lab03 and GPRC5D-Lab04, and two humanized sequences, designated GPRC5D-Hab03-H10 and GPRC5D-Hab04-H5. The amino acid sequences are shown in Table 13, and the numbered CDR sequences are shown in Table 14.

[0260] Camel-derived VHH antibodies were prepared using conventional VHH antibody screening methods in the art, using the human GPRC5D protein as the immunogen. These antibodies were annotated according to IMGT or Kabat. After FACS verification, several camel antibodies with high affinity and their humanized counterparts were obtained. The camel antibodies were designated GPRC5D-Lab05 and GPRC5D-Lab06, and their humanized counterparts were designated GPRC5D-Hab05-H2 and GPRC5D-Hab06-H1. The Nanobody sequences are shown in Table 13, and the CDR sequences are shown in Table 14.

[0261] Table 13 Anti-GPRC5D Nanobody Amino Acid Sequences

[0262] Table 14 Anti-GPRC5D Nanobody CDRs Sequences

[0263] Example 11. Construction of 4-1BB chimeric antigen receptor and screening of extracellular antibody combinations

[0264] 11.1 Construction of 4-1BB CAR-NK

[0265] Using the BCMA-VHH antibodies obtained by screening in Example 1, i.e., hu-VHH2 and GPRC5D-scFv antibodies, a dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D as shown in Figure 12 was designed and constructed. The nucleic acid sequence of BI-CAR was loaded into a retroviral vector using the biological method of Example 3-4 to construct the target plasmid and viral vector, induce NK cells and prepare CAR-NK cells. The structures of BI-CAR01 to 04 are shown in Figures 12A to 12D, respectively, and the structure of BI-CAR05 is shown in Figure 12A. The amino acid sequences of each domain of BI-CAR and exemplary BI-CAR01 to 05 sequences are shown in Table 15.

[0266] Table 15 BI-CAR domains and exemplary BI-CAR amino acid sequence

[0267] 11.2 Detection of Chimeric Antigen Receptor Expression Rate

[0268] On day 6 after retroviral infection of NK cells, flow cytometry was used to detect the expression rate of CAR on the NK cell membrane surface. Detection of BCMA-CAR NK cells in BI-CAR and GPRC5D-CAR NK cells in BI-CAR was performed using the method for detecting the expression rate of CAR in NK cells according to Example 5.1.

[0269] The FACS expression detection results of BI-CAR are shown in Table 16. The results show that in the formats of Figures 12A and 12B, the expression of anti-BCMA and anti-GPRC5D is generally high and has no position dependence; while in the formats of Figures 12C and 12D, the expression of BCMA-CAR or GPRC5D-CAR is affected by its location.

[0270] Table 16 BI-CAR expression rate detection

[0271] 11.3 Detection of BI-CAR-NK Cell Killing Rate in Vitro within 4 Hours

[0272] Six days after retroviral infection of NK cells, a 4-hour in vitro killing experiment was performed: the target cells were NCI H929, RPMI 8226, MOLP8, and NCI H929-hBCMA-KO. All cells were genetically manipulated to contain a luciferase reporter gene. NCI H929-hBCMA-KO is an NCI H929 cell line in which BCMA was knocked out using conventional genetic manipulation methods. Target cells diluted in 1640 medium were plated at 2×10 4 50 μl / well were added to a white opaque 96-well plate, and NK cells were added to the target cells at effector-target ratios of 5:1, 2.5:1, and 1.25:1. The killing rate was measured and calculated according to the method of Example 6.

[0273] The 4-hour in vitro cell killing effect of BI-CAR on the above target cells is detailed in Table 17. At an effector-target ratio of 5:1, BI-CAR01 to BI CAR05 all showed specific killing effects on NCI H929-Lu and NCI H929-hBCMA-KO-Lu. BI-CAR01 to BI-CAR03 and BI-CAR05 all had good specific killing effects on the four tumor cell lines. Among them, BI-CAR01, BI-CAR-03 and BI-CAR05 showed stronger killing function against NCI H929-hBCMA-KO-Lu tumor cells, indicating that BI-CAR01, BI-CAR03 and BI-CAR05 have better killing effects on tumor cells with downregulated or missing BCMA expression.

[0274] Table 17 Results of BI-CAR-NK rapid killing rate of target cells in vitro within 4 hours

[0275] Note: “-” means no killing effect was detected.

[0276] Example 12. Design of NKG2D-F chimeric antigen receptor

[0277] 12.1 Construction of NKG2D-F chimeric antigen receptor.

[0278] To verify the effect of different transmembrane domains on the activity of chimeric antigen receptors, the antibodies BCMA-Hu-VHH2 and GPRC5D-Hab06 corresponding to BI-CAR01 and BI-CAR03 obtained by screening in Example 11 were used to design and construct a dual-target chimeric antigen receptor targeting BCMA and GPRC5D as shown in Figure 13, named BI-CAR06. The dual-target chimeric antigen receptor targeting BCMA and GPRC5D includes CD8α signal peptide (SP), anti-GPRC5D-scFv and anti-BCMA-VHH, CD8 hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3ζ, and IL15 connected by self-cleaving peptide P2A. The specific sequence is shown in Table 18. The nucleic acid sequence was loaded into a retroviral vector by the molecular biology method in Reference Example 3-4 to construct the target plasmid.

[0279] Table 18 BI-CAR domains and exemplary BI-CAR amino acid sequence

[0280] 12.2 Determination of CAR Expression Rate in CAR-NK Cells with Different Structures

[0281] With reference to the method shown in Examples 3-4, BI-CAR-NK cells with different transmembrane regions and intracellular stimulatory domain structures were prepared by infecting NK cells at the same MOI, and the expression of CAR was detected by the method in Reference Example 5.1. The results are shown in Table 19. The results show that the expression of the CAR shown in Figure 12A is better than the expression of the CAR described in Figure 12C.

[0282] Table 19 CAR expression rate results in different structures of CAR-NK

[0283] 12.3 Determination of the proliferation rate of CAR-NK cells with different structures

[0284] The proliferation of BI-CAR cells was detected by AO / PI counting, and the results are shown in Figure 14. After 14 days of NK activation, the proliferation rates of BI-CAR cells were as follows: BI-CAR06 (5566 times) > NK (3124 times) > BI-CAR03 (1705 times) > BI-CAR05 (1245.2 times) > BI-CAR01 (1012 times). According to the above results, the proliferation rate of BI-CAR cells loaded with NKG2D™ elements after optimization is significantly better than that of BI-CAR cells using other transmembrane elements.

[0285] 12.4 Determination of 4-hour Rapid Killing Rate of CAR-NK Cells with Different Structures

[0286] On the 6th day after retroviral infection of NK cells, a 4-hour in vitro rapid killing experiment was performed: target cells NCI H929, RPMI 8226, MOLP8 and NCI H929-hBCMA-KO diluted in 1640 medium were cultured at 2×10 4 50 μl / well were added to a white opaque 96-well plate, and NK cells were added to the target cells at effector-target ratios of 9:1 and 3:1. The killing rate was measured and calculated according to the method of Example 6.

[0287] The 4h in vitro cell killing effect of BI-CAR on the above target cells is detailed in Table 20. The results showed that BI-CAR05, BI-CAR01, BI-CAR-03, and BI-CAR-06 all showed strong tumor cell killing function.

[0288] Table 20 Results of 4h rapid killing rate of CAR-NK cells with different structures

[0289] Note: “-” means no killing effect was detected.

[0290] 12.5 Testing the multiple rounds of killing effects of CAR-NK cells with different structures

[0291] Six days after retroviral infection of NK cells, the in vitro multi-round killing effects of BI-CAR01, BI-CAR-03, BI-CAR-05, and BI-CAR-06 on RPMI 8226 cells and MOLP8 cells were measured. The in vitro multi-round killing effects on RPMI 8226 cells and MOLP8 cells were determined using the method of Example 7.

[0292] The results of the multi-round killing test of BI-CAR NK cells are shown in Figure 15. As shown in Figure 15, the killing effect of BI-CAR06 loaded with NKG2D™ element is better than that of other forms of BI-CAR; BI-CAR06 can still maintain a 50% cell killing rate in the third round of killing (R3-1d) of MOLP8 cells and a 40% cell killing rate in the third round of killing (R3-1d) of RPMI 8226 cells.

[0293] Example 13 NKG2D-F CAR constructed with humanized antibodies

[0294] 13.1 Construction of NKG2D-F CAR

[0295] The dual-target chimeric antigen receptor targeting BCMA and GPRC5D was designed and constructed, including the CD8α signal peptide (SP), anti-GPRC5D antibody and anti-BCMA, CD8α hinge region, NKG2D-F transmembrane region (NKG2D-TM1), 2B4 co-stimulatory domain and CD3ζ, as well as IL15 connected by the self-cleaving peptide P2A. The structures of BI-CAR18 and BI-CAR20 are shown in Figure 16A, and the structures of BI-CAR19 and BI-CAR21 are shown in Figure 16B. The sequences are shown in Table 21.

[0296] Table 21 NKG2D-F CAR amino acid sequence

[0297] 13.2 NKG2D-F CAR Expression Detection

[0298] Referring to the method in Example 5.1, the expression of BI CARs 18 to 21 was detected. The results are shown in Table 22. The results show that the expression of BCMA CAR and GPRC5D CAR can be detected after infecting NK cells at an MOI of 5. The BCMA CAR positive rate is relatively high, reaching more than 60%; the GPRC5D CAR positive rate is relatively low, and the expression rates from high to low are BI-CAR06 (69.60%) > BI-CAR21 (54.20%) > BI-CAR20 (54.00%) > BI-CAR18 (32.30%) > BI-CAR19 (13.80%).

[0299] Table 22 NKG2D-F CAR expression rate results

[0300] 13.3 NKG2D-F CAR-NK Cell Proliferation Assay

[0301] The proliferation of BI-CAR NK cells was detected by AO / PI counting, and the results are shown in Figure 17 and Table 23. On the 11th day after activation, BI-CAR NK cells proliferated by about 1473 to 2166 times, and the proliferation rates from high to low were: BI-CAR19 (2166 times) = BI-CAR20 (2166 times) > BI-CAR21 (1950 times) > Parental NK (1690 times) > BI-CAR18 (1538 times) > BI-CAR06 (1473 times). According to the above results, the proliferation rate of BI-CAR18, BI-CAR19, BI-CAR20 and BI-CAR21 cells constructed with humanized antibodies was better than that of BI-CAR06.

[0302] Table 23 NKG2D-F CAR-NK cell proliferation detection

[0303] 13.4 NKG2D-F CAR-NK Cell Cytotoxicity Test

[0304] On day 6 after retroviral infection of NK cells, the in vitro multi-round killing effects of BI-CAR18, BI-CAR19, BI-CAR20, BI-CAR21 and BI-CAR06 on heterogeneous tumor cells of NCI H929, RPMI 8226, MOLP8, NCI H929-hBCMA-KO, and NCI H929+20% (or 10% or 5%) H929-hBCMA-KO were detected. The in vitro multi-round killing effects on the above cells were determined by the method of Reference Example 7.

[0305] The results of the multi-round killing test of BI-CAR NK cells are shown in Figures 18A to 18F. The results show that the multi-round killing effects of BI-CAR19, BI-CAR20, and BI-CAR21 are better than those of BI-CAR18 and BI-CAR06. BI-CAR21 had the best killing effect on NCI H929 cells, maintaining a cell killing rate of 30% in the fourth round of killing (R4-1d) experiments; BI-CAR19, BI-CAR20 and BI-CAR21 had the best killing effect on NCI H929+20% (or 10% or 5%) H929-hBCMA-KO heterogeneous tumor cells, RPMI 8226 cells and MOLP8 cells, maintaining a cell killing rate of more than 30% in the fourth round of killing (R4-1d) experiments on NCI H929+20% (or 10% or 5%) H929-hBCMA-KO heterogeneous tumor cells, and maintaining a cell killing rate of about 10% in the fifth round of killing (R5-1d) experiments on RPMI 8226 cells and MOLP8 cells.

Claims

1. A nucleic acid molecule comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises: The invention relates to an extracellular region comprising an antigen binding region, an NKG2D transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the NKG2D transmembrane region has a sequence that is at least 80% identical to SEQ ID NO: 48 or has no more than 9 mutations.

2. The nucleic acid molecule according to claim 1, wherein The antigen binding region has the properties of at least one of groups (1) to (4): (1) The antigen-binding region is selected from an antibody or a fragment thereof or a ligand that binds to the antigen, such as scFv, VHH, Fab, F(ab)'2 or ligand; (2) the antigen binding region binds to or does not bind to NKG2DL; (3) the antigen is selected from one or more of the following groups: BCMA, GPRC5D, CLDN18.2, ROR1, CD19, CD33, CD5, CD70, HER2, IL13Rα2, GCC or GPC3; (4) The antigen-binding region binds to at least two different antigens, at least two different epitopes of the same antigen, or binds to more than two epitopes of the same antigen.

3. The nucleic acid molecule according to any one of claims 1 to 2, wherein The antigen binding region specifically binds to BCMA, comprising: (1) VHH, whose CDR1-3 respectively include: The sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; or The sequences shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 11; or The sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; (2) VHH comprising a sequence having at least 80% identity or up to 25 mutations to SEQ ID NO: 1 or 3; (3) VHH, whose CDR1-3 respectively include: The sequences shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; or The sequences shown in SEQ ID NO: 17, SEQ ID NO: 25 and SEQ ID NO: 19; or The sequences shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:19; or The sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; (4) a sequence having at least 80% identity or at most 25 mutations to SEQ ID NO: 2 or 4; or, (5) VHH1-connecting peptide-VHH2, wherein the VHH1 has a sequence as shown in group (1) or group (2), and the VHH2 has a sequence as shown in group (3) or group (4); or (6) VHH1-connecting peptide-VHH2, wherein the VHH1 has a sequence as shown in group (3) or group (4) and the VHH2 has a sequence as shown in group (1) or group (2); or (7) VHH1-connector peptide-VHH2, having a sequence of at least 80% identity or at most 50 mutations compared to SEQ ID NO:

63.

4. The nucleic acid molecule according to claim 1, wherein The antigen binding region specifically binds to ROR1, comprising: (1) An antibody or a fragment thereof, comprising HCDR1-3 and LCDR1-3, wherein the HCDR1-3 respectively comprise: The sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; or The sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:32; or The sequences shown in SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37; And the LCDR1-3 respectively include: The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or The sequences shown in SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:40; (2) an antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 26, and a light chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 27; (3) scFv, said scFv having a sequence shown in said group (1) or group (2), optionally, said scFv having a light chain variable region having at least 80% identity or at most 50 mutations compared to SEQ ID NO:

28.

5. The nucleic acid molecule according to any one of claims 1 to 3, wherein The antigen binding region specifically binds to GPRC5D, comprising: (1) An antibody or a fragment thereof, comprising HCDR1-3 and LCDR1-3, wherein the HCDR1-3 respectively comprise: The sequences shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89; or The sequences shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92; or The sequences shown in SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98; And the LCDR1-3 respectively include: The sequences shown in SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86; or The sequences shown in SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95; (2) An antibody or fragment thereof, comprising HCDR1-3, wherein the HCDR1-3 respectively comprise: The sequences shown in SEQ ID NOs: 107-118; (3) an antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NOs: 78, 80, 83, 131, and a light chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NOs: 77, 79, 81, 82; (4) An antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NOs: 99-106.

6. The nucleic acid molecule according to any one of claims 1 to 5, wherein The extracellular region also includes a hinge region, which is selected from one or more of the following groups: CD8α hinge region, 2B4 hinge region, CD28 hinge region, IgG1 hinge region, IgD hinge region, IgG4 hinge region, GS hinge, KIR2DS2 hinge, KIR hinge, NCR hinge, SLAMF hinge, CD16 hinge, CD64 hinge or LY49 hinge; optionally, the hinge region is selected from the CD8α hinge region, for example, the hinge region has a sequence that is at least 80% identical or has at most 10 mutations compared to SEQ ID NO:

45.

7. The nucleic acid molecule according to any one of claims 1 to 6, wherein The extracellular region also includes a signal peptide, which is selected from one or more of the following groups: CD8α signal peptide, IgG1 heavy chain signal peptide or GM-CSFR2 signal peptide; optionally, the signal peptide is a CD8α signal peptide, for example, the signal peptide has a sequence that is at least 80% identical or has at most 5 mutations compared to SEQ ID NO:

43.

8. The nucleic acid molecule according to any one of claims 1 to 7, wherein The intracellular domain includes an intracellular signaling domain and / or a costimulatory domain; Optionally, the intracellular signaling domain is CD3ζ, for example, the intracellular signaling domain has a sequence that is at least 80% identical or has at most 35 mutations compared to SEQ ID NO: 55; Optionally, the costimulatory domain is selected from one or more of the following groups: a CD27 costimulatory domain, a 4-1BB costimulatory domain, an OX40 costimulatory domain, a 2B4 costimulatory domain, a CD28 costimulatory domain, an ICOS costimulatory domain, a DAP10 costimulatory domain or a DAP12 costimulatory domain; optionally, the costimulatory domain is a 2B4 costimulatory domain, for example, the costimulatory domain has a sequence that is at least 80% identical or has at most 25 mutations compared to SEQ ID NO:

53.

9. The nucleic acid molecule according to any one of claims 1 to 8, wherein The chimeric antigen receptor comprises: a CD8α hinge region, an NKG2D transmembrane region, a 2B4 co-stimulatory domain and a CD3ζ intracellular signaling domain, for example, the chimeric antigen receptor has a sequence that is at least 80% identical to SEQ ID NO: 58 or has no more than 50 mutations.

10. The nucleic acid molecule according to any one of claims 1 to 9, wherein The nucleic acid molecule further comprises a second nucleic acid sequence encoding IL15, optionally, the IL15 is selected from soluble IL15, membrane-bound IL15, or a complex of IL15 and its receptor or receptor fragment, optionally, the IL15 has a sequence having at least 80% identity or at most 35 mutations compared to SEQ ID NO: 57; Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by an IRES or a sequence encoding a self-cleaving peptide, the self-cleaving peptide being selected from a 2A peptide, such as P2A, T2A, F2A or E2A, optionally, the self-cleaving peptide is P2A, such as a sequence having at least 80% identity or at most 5 mutations compared to SEQ ID NO: 56; Optionally, the nucleic acid encodes a nucleic acid sequence having at least 85% identity or up to 100 mutations compared to SEQ ID NO:

59.

11. The nucleic acid molecule according to any one of claims 1 to 10, comprising a nucleic acid sequence encoding a sequence having at least 80% identity or at most 150 mutations compared to SEQ ID NO: 65 or SEQ ID NO:

70.

12. The nucleic acid molecule according to any one of claims 1 to 11, wherein The nucleic acid is DNA or RNA, and the RNA is preferably mRNA.

13. A nucleic acid molecule comprising a first nucleic acid sequence encoding a chimeric antigen receptor targeting ROR1, wherein the chimeric antigen receptor comprises an extracellular region comprising an antigen binding region, a transmembrane region connected to the extracellular region, and an intracellular domain connected to the transmembrane region, wherein the antigen binding region comprises: (1) An antibody or a fragment thereof, comprising HCDR1-3 and LCDR1-3, wherein the HCDR1-3 respectively comprise: The sequences shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; or The sequences shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:32; or The sequences shown in SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37; And the LCDR1-3 respectively include: The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or The sequences shown in SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; or The sequences shown in SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:40; (2) an antibody or fragment thereof comprising a heavy chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 26, and a light chain variable region having at least 80% identity or at most 25 mutations compared to SEQ ID NO: 27; (3) scFv, said scFv having a sequence shown in said group (1) or group (2), optionally, said scFv having a light chain variable region having at least 80% identity or at most 50 mutations compared to SEQ ID NO:

28.

14. The nucleic acid molecule according to claim 12 or 13, wherein The chimeric antigen receptor comprises: a CD8α hinge region, an NKG2D transmembrane region, a 2B4 co-stimulatory domain and a CD3ζ intracellular signaling domain, for example, the chimeric antigen receptor has a sequence that is at least 80% identical to SEQ ID NO: 58 or has no more than 50 mutations.

15. The nucleic acid molecule according to any one of claims 12 to 14, wherein The nucleic acid molecule further comprises a second nucleic acid sequence encoding IL15, optionally, the IL15 is selected from soluble IL15, membrane-bound IL15, or a complex of IL15 and its receptor or receptor fragment, optionally, the IL15 has a sequence having at least 80% identity or at most 35 mutations compared to SEQ ID NO: 57; Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked by an IRES or a sequence encoding a self-cleaving peptide, the self-cleaving peptide being selected from a 2A peptide, such as P2A, T2A, F2A or E2A, optionally, the self-cleaving peptide is P2A, such as a sequence having at least 80% identity or at most 5 mutations compared to SEQ ID NO: 56; Optionally, the core encodes a nucleic acid sequence having at least 85% identity or up to 100 mutations compared to SEQ ID NO:

59.

16. The nucleic acid molecule according to any one of claims 12 to 15, comprising a nucleic acid sequence encoding a sequence having at least 80% identity or at most 150 mutations compared to SEQ ID NO: 65 or SEQ ID NO:

70.

17. A chimeric antigen receptor encoded by the nucleic acid molecule according to any one of claims 1 to 16.

18. A vector, wherein The vector comprises the nucleic acid molecule according to any one of claims 1 to 16.

19. Immune effector cells, wherein The immune effector cell comprises the nucleic acid molecule according to any one of claims 1 to 16, the chimeric antigen receptor according to claim 17, or the vector according to claim 18.

20. The immune effector cell according to claim 19, wherein The immune effector cells are NK cells, which are selected from NK cells differentiated from iPSCs, peripheral blood or umbilical cord blood-derived NK cells, or NK92 cells.

21. The method for preparing the immune effector cells according to any one of claims 19 to 20, wherein: include: The steps of providing immune effector cells and transferring the nucleic acid molecule into the immune effector cells.

22. The product prepared according to the method of claim 21.

23. A pharmaceutical composition, wherein The pharmaceutical composition comprises the nucleic acid molecule according to any one of claims 1 to 16, the chimeric antigen receptor according to claim 17, the vector according to claim 18, the immune effector cell according to any one of claims 19 to 20, or the product according to claim 22, and a pharmaceutically acceptable carrier.

24. Use of the nucleic acid molecule according to any one of claims 1 to 16, or the chimeric antigen receptor according to claim 17, or the vector according to claim 18, the immune effector cell according to any one of claims 19 to 20, the product according to claim 22, or the pharmaceutical composition according to claim 23 in the preparation of a medicament for treating a cancer or a tumor, wherein the cancer or tumor is selected from a hematological tumor or a solid tumor; optionally, the hematological tumor is selected from a myeloma, a lymphoma, or a leukemia, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer, or adrenal cancer.

25. The nucleic acid molecule of any one of claims 1 to 16, or the chimeric antigen receptor of claim 17, or the vector of claim 18, the immune effector cell of any one of claims 19 to 20, the product of claim 22, or the pharmaceutical composition of claim 23, for use in treating a cancer or a tumor, wherein the cancer or tumor is selected from a hematological tumor or a solid tumor; optionally, the hematological tumor is selected from a myeloma, a lymphoma, or a leukemia, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer, or adrenal cancer.

26. A method for treating cancer or tumor, comprising administering to a subject in need thereof an effective amount of the nucleic acid molecule of any one of claims 1 to 16, the chimeric antigen receptor of claim 17, the vector of claim 18, the immune effector cell of any one of claims 19 to 20, the product of claim 22, or the pharmaceutical composition of claim 23, wherein the cancer or tumor is selected from a hematological tumor or a solid tumor; optionally, the hematological tumor is selected from a myeloma, a lymphoma, or a leukemia, such as multiple myeloma. , Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer or adrenal cancer.

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