WO2023193660A1 - 增强型嵌合抗原受体及其应用 - Google Patents

增强型嵌合抗原受体及其应用 Download PDF

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WO2023193660A1
WO2023193660A1 PCT/CN2023/085490 CN2023085490W WO2023193660A1 WO 2023193660 A1 WO2023193660 A1 WO 2023193660A1 CN 2023085490 W CN2023085490 W CN 2023085490W WO 2023193660 A1 WO2023193660 A1 WO 2023193660A1
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seq
nucleic acid
cells
cancer
sequence
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French (fr)
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杨翠青
王义芳
姜福伟
王超
王庆杨
曹卓晓
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上海先博生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present disclosure belongs to the field of biotechnology, and specifically relates to enhanced chimeric antigen receptors and their applications.
  • NK cells Natural killer cells
  • NK cells are a type of lymphocytes that have a powerful killing effect on tumor cells and are MHC-independent. Their recognition of tumor cells mainly relies on the cross-regulation of their surface activating receptors and inhibitory receptors. After recognizing tumor cells, NK cells kill tumor cells through multiple channels such as releasing the killing mediators perforin and granzymes to cause target cell apoptosis, expressing membrane TNF family molecules to induce target cell apoptosis, and antibody-dependent cytotoxicity. Allogeneic NK cell transplantation will hardly induce graft-versus-host disease (GVHD), will not lead to CRS, and can be used as a completely "off-the-shelf" product.
  • GVHD graft-versus-host disease
  • NK cells due to the decline in the number and quality of NK cells in tumor patients and the existence of tumor escape mechanisms, their anti-tumor functions in the body have not been fully exerted. Modifying NK cells with CAR is expected to enhance their ability to target and kill tumor cells and develop effector cells with powerful anti-tumor effects.
  • the structure of the functional chimeric antigen receptor (CAR) expressed by NK cells mainly includes: extracellular domain, transmembrane region and intracellular signaling domain.
  • Most CAR-NK products still use the CAR structure commonly used in traditional CAR-T cell therapy.
  • 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, traditional The CAR structure commonly used in CAR-T cell therapy may not necessarily function optimally in CAR-NK.
  • this disclosure has designed a variety of combinations of CAR structures containing different transmembrane regions and intracellular co-stimulatory domains. Through screening and verification in vitro and in vivo experiments, the most suitable CAR-NK cells have been obtained. Active CAR structure.
  • the present disclosure also optimizes the transmembrane region of CAR-NK.
  • the commonly used TM regions of CAR-NK are from CD8 and CD28, and others such as NKG2D are also used.
  • CN110684117B and CN112210016A disclose chimeric antigen receptor-modified NK cells, in which NKG2D TM is selected as the transmembrane region of the chimeric antigen receptor.
  • NKG2D is an NK cell activator.
  • the NKG2D TM region is beneficial to enhance the activation and killing effect of CAR-NK cells.
  • the current CAR-NK The structure lacks fine design for the NKG2D TM region.
  • the present disclosure finds that optimizing the NKG2D TM region is of great significance to further improve the function of CAR-NK.
  • BCMA B cell maturation antigen
  • BAFF B cell activating factor
  • APRIL proliferation-inducing ligand
  • BCMA expression has been associated with a variety of cancers, autoimmune disorders, and infectious diseases. Cancers with increased BCMA expression include blood cancers such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. BCMA is an ideal therapeutic target.
  • ROR1 is a transmembrane receptor tyrosine kinase protein and a member of the receptor tyrosine kinases (RTKs) family.
  • ROR1 has low or no expression 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, lung adenocarcinoma, etc.
  • CLL chronic lymphocytic leukemia
  • BCAM and ROR1 are ideal targets in the field of tumor treatment. It is particularly important to develop corresponding chimeric antigen receptors for BCMA and ROR1 targets, especially chimeric antigen receptors suitable for NK cells.
  • nucleic acid molecules encoding chimeric antigen receptors comprising the NKG2D transmembrane region, corresponding chimeric antigen receptors, vectors, immune effector cells, preparation methods and products thereof, pharmaceutical compositions, pharmaceutical uses and Tumor or cancer treatments.
  • the present disclosure provides a nucleic acid molecule comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: an extracellular region comprising an antigen-binding region, An NKG2D transmembrane region connecting the extracellular region and an intracellular domain connecting the transmembrane region, the NKG2D transmembrane region having at least 80% identity or at most 9 mutations compared to SEQ ID NO: 48 sequence.
  • CAR chimeric antigen receptor
  • the antigen-binding region has at least one of the properties of (1)-(4):
  • the antigen-binding region is selected from an antibody or a fragment thereof or a ligand that binds the antigen, such as scFv, VHH, Fab, F(ab)' 2 or ligand;
  • the antigen-binding region binds or does not bind to NKG2DL;
  • the antigen is selected from one or more of the following group: BCMA, GPRC5D, CLDN18.2, ROR1, CD19, CD33, CD5, CD70, HER2, IL13R ⁇ 2, GCC or GPC3;
  • the antigen-binding region binds at least two different antigens, at least two different epitopes of the same antigen, or the number of the same epitope is more than two.
  • the antigen-binding region specifically binds BCMA, including:
  • VHH its CDR1-3 include:
  • VHH including a sequence with at least 80% identity or up to 25 mutations to SEQ ID NO: 1 or 3;
  • (3)VHH, its CDR1-3 include:
  • VHH1-linking peptide-VHH2 the VHH1 has the sequence shown in the group (1) or the group (2), and the VHH2 has the sequence shown in the group (3) or the group (4); or,
  • VHH1-linking peptide-VHH2 said VHH1 has a sequence shown in the group (3) or group (4) and said VHH2 has a sequence shown in the group (1) or group (2); or,
  • VHH1-linker peptide-VHH2 a sequence having at least 80% identity or at most 50 mutations compared to SEQ ID NO: 63.
  • the antigen-binding region specifically binds ROR1, including:
  • Antibodies or fragments thereof including HCDR1-3 and LCDR1-3, where the HCDR1-3 respectively include:
  • LCDR1-3 respectively include:
  • An antibody or fragment thereof including a heavy chain variable region having at least 80% identity or up to 25 mutations compared to SEQ ID NO: 26, and at least 80% identity to SEQ ID NO: 27 or up to 25 mutated light chain variable regions;
  • the scFv has the sequence shown in the group (1) or group (2), optionally, the scFv has at least 80% identity or at most 50 mutations compared with SEQ ID NO: 28 The light chain variable region.
  • the antigen-binding region specifically binds GPRC5D, including:
  • Antibodies or fragments thereof including HCDR1-3 and LCDR1-3, where the HCDR1-3 respectively include:
  • LCDR1-3 respectively include:
  • Antibodies or fragments thereof, including HCDR1-3 which respectively include:
  • Antibodies or fragments thereof including heavy chain variable regions having at least 80% identity or up to 25 mutations compared to SEQ ID NO:78, 80, 83, 131, and SEQ ID NO:77, 79 , 81 , 82 Compared to a light chain variable region with at least 80% identity or at most 25 mutations;
  • the extracellular region further includes a hinge region selected from one or more of the following group: CD8 ⁇ hinge region, 2B4 hinge region, CD28 hinge region, IgG1 hinge region, IgD Hinge, 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 CD8 ⁇ hinge region, for example, the hinge region Sequences with at least 80% identity or up to 10 mutations compared to SEQ ID NO:45.
  • the extracellular region further includes a signal peptide selected from one or more of the following group: CD8 ⁇ signal peptide, IgG1 heavy chain signal peptide or GM-CSFR2 signal peptide;
  • the signal peptide is a CD8 ⁇ signal peptide, for example, the signal peptide has a sequence of at least 80% identity or at most 5 mutations compared to SEQ ID NO: 43.
  • the intracellular domain includes an intracellular signaling domain and/or a costimulatory domain
  • the intracellular signaling domain is CD3 ⁇ , for example, the intracellular signaling domain has a sequence of at least 80% identity or at most 35 mutations compared to SEQ ID NO: 55;
  • the costimulatory domain is selected from one or more of the following group: CD27 costimulatory domain, 4-1BB costimulatory domain, OX40 costimulatory domain, 2B4 costimulatory domain, CD28 costimulatory domain domain, ICOS co-stimulatory domain, DAP10 co-stimulatory domain or DAP12 co-stimulatory domain; optionally, the co-stimulatory domain is a 2B4 co-stimulatory domain, for example, the co-stimulatory domain has the same structure as SEQ ID NO:53 compared to sequences with at least 80% identity or at most 25 mutations.
  • the chimeric antigen receptor includes: CD8 ⁇ hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3 ⁇ intracellular signaling domain, for example, having the same structure as SEQ ID NO:58 Than sequences with at least 80% identity or at most 50 mutations.
  • the nucleic acid molecule further includes a second nucleic acid sequence encoding IL15.
  • the IL15 is selected from the group consisting of soluble IL15, membrane-bound IL15, or a complex of IL15 and its receptor or receptor fragment. , optionally, the IL15 has a sequence with at least 80% identity or at most 35 mutations compared with SEQ ID NO:57;
  • the first nucleic acid sequence and the second nucleic acid sequence are connected by an IRES or a sequence encoding a self-cleaving peptide selected from 2A peptides, such as P2A, T2A, F2A or E2A, optionally , the self-cleaving peptide is P2A, for example, having a sequence with at least 80% identity or at most 5 mutations compared to SEQ ID NO: 56;
  • the nucleic acid encodes a nucleic acid sequence having at least 85% identity or at most 100 mutations compared to SEQ ID NO: 59.
  • 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.
  • the nucleic acid is DNA or RNA
  • the RNA is preferably mRNA.
  • 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, and the antigen-binding region includes:
  • VHH its CDR1-3 include:
  • VHH including a sequence with at least 80% identity or up to 25 mutations to SEQ ID NO: 1 or 3;
  • (3)VHH, its CDR1-3 include:
  • VHH1-linking peptide-VHH2 the VHH1 has the sequence shown in the group (1) or the group (2), and the VHH2 has the sequence shown in the group (3) or the group (4); or,
  • VHH1-linking peptide-VHH2 said VHH1 has a sequence shown in the group (3) or group (4) and said VHH2 has a sequence shown in the group (1) or group (2); or,
  • VHH1-linker peptide-VHH2 a sequence having at least 80% identity or at most 50 mutations compared to SEQ ID NO: 63.
  • the present disclosure provides a nucleic acid molecule comprising a first nucleic acid sequence encoding a chimeric anti-receptor targeting ROR1, the chimeric antigen receptor comprising an extracellular region comprising an antigen-binding region, a linker
  • the transmembrane region of the extracellular region and the intracellular domain connecting the transmembrane region, the antigen-binding region includes:
  • Antibodies or fragments thereof including HCDR1-3 and LCDR1-3, where the HCDR1-3 respectively include:
  • LCDR1-3 respectively include:
  • An antibody or fragment thereof including a heavy chain variable region having at least 80% identity or up to 25 mutations compared to SEQ ID NO: 26, and at least 80% identity to SEQ ID NO: 27 or up to 25 mutated light chain variable regions;
  • the scFv has the sequence shown in the group (1) or the group (2), optionally, the scFv has the same A light chain variable region with at least 80% identity or up to 50 mutations compared to SEQ ID NO:28.
  • the chimeric antigen receptor includes: CD8 ⁇ hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3 ⁇ intracellular signaling domain.
  • 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.
  • the nucleic acid molecule further includes a second nucleic acid sequence encoding IL15.
  • the IL15 is selected from the group consisting of soluble IL15, membrane-bound IL15, or a complex of IL15 and its receptor or receptor fragment. , optionally, the IL15 has a sequence with at least 80% identity or at most 35 mutations compared with SEQ ID NO:57;
  • the first nucleic acid sequence and the second nucleic acid sequence are connected by an IRES or a sequence encoding a self-cleaving peptide selected from 2A peptides, such as P2A, T2A, F2A or E2A, optionally , the self-cleaving peptide is P2A, for example, having a sequence with at least 80% identity or at most 5 mutations compared to SEQ ID NO: 56;
  • the core encodes a nucleic acid sequence having at least 85% identity or at most 100 mutations to a sequence compared to SEQ ID NO: 59.
  • 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.
  • the present disclosure provides chimeric antigen receptors encoded according to the aforementioned nucleic acids.
  • the present disclosure provides a vector, wherein the vector includes the aforementioned nucleic acid molecule.
  • the present disclosure provides immune effector cells, wherein the immune effector cells include the aforementioned nucleic acid molecules or chimeric antigen receptors or vectors.
  • the immune effector cells are NK cells, which are differentiated from iPSCs or derived from peripheral blood or umbilical cord blood.
  • the present disclosure provides a method for preparing the aforementioned immune effector cells, which includes the steps of providing immune effector cells and transferring the nucleic acid molecules into the immune effector cells.
  • the present disclosure provides products prepared according to the aforementioned method.
  • the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition includes the aforementioned nucleic acid molecule or chimeric antigen receptor or carrier, immune effector cell or product, and a pharmaceutically acceptable carrier.
  • 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 medicaments for the treatment of cancer or tumors selected from Hematological tumor or solid tumor; optionally, the hematological tumor is selected from myeloma, lymphoma or leukemia, such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma , follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or hairy cell leukemia (HCL);
  • the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ova
  • 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 the treatment of cancer or tumors selected from the group consisting of hematomas or Solid tumor; optionally, the hematological tumor is selected from myeloma, lymphoma or leukemia, such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), Chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, lymph node Cancer of the rectum, stomach, pancreas, liver, skin, bladder, ovary, uterus
  • the present disclosure provides a method of treating cancer or tumors, the method comprising administering an effective amount of the aforementioned nucleic acid molecule or chimeric antigen receptor or vector, immune effector cell, product or drug to a subject in need thereof
  • the composition, the cancer or tumor is selected from a hematological tumor or a solid tumor; optionally, the hematological tumor is selected from myeloma, lymphoma or leukemia, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma Lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia Cellular leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is
  • the present disclosure provides an optimized chimeric antigen receptor targeting the NKG2D transmembrane region, which has at least one of the following advantages: (1) high CAR expression efficiency after transfection; (2) NK cell proliferation rate after transfection Fast; (3) Strong killing power and specificity against tumor cells; (4) The killing effect lasts for a long time.
  • compositions including A and B
  • compositions composed of A and B as well as a composition containing other components in addition to A and B, all fall into the category Within the scope of the aforementioned "a composition”.
  • BCMA B cell maturation antigen, which is a member of the tumor necrosis factor receptor family.
  • BCMA is mainly expressed on the surface of late B cells, short-lived proliferating plasmablasts and long-lived plasma cells. It is not expressed on initial B cells, CD34-positive hematopoietic stem cells and other normal tissue cells, but it is highly expressed on MM cells. , by mediating downstream signaling pathways, it plays a key role in the survival, proliferation, metastasis and drug resistance of MM cells, so BCMA is an ideal antigen target for the treatment of MM.
  • An exemplary human BCMA sequence can be found at GenBank Protein Accession No: NP_001183.2.
  • GPRC5D refers to G protein-coupled receptor C5 family subtype D, which is an orphan receptor and is a 7-transmembrane protein with no known ligand. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while its expression in normal tissues is limited to the hair follicle area. Studies have shown that 65% of multiple myeloma patients have more than 50% expression of GPRC5D. threshold. With this characteristic, GPRC5D has become a potential target for the treatment of MM. An exemplary human GPRC5D sequence can be found at GenBank Protein Accession No: NP_061124.1.
  • antigen chimeric receptor refers to an artificial immune effector cell surface receptor engineered to be expressed on immune effector cells and specifically bind an antigen, which contains at least (1) an extracellular antigen-binding structure Domains, such as the variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into immune effector cells, and (3) an intracellular signaling domain.
  • CARs are able to utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • signal peptide herein refers to the fragment of a protein or polypeptide used to guide the protein or polypeptide into the secretory pathway and transferred to the cell membrane and/or cell surface.
  • the signal peptide is a CD8 ⁇ signal peptide, optionally, the CD8 ⁇ signal peptide has 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%): MALPVTALLLPLALLLHAARP.
  • the "transmembrane (TM) region" of a chimeric antigen receptor refers to the ability of the chimeric antigen receptor to be expressed on the surface of immune cells (such as lymphocytes, NK cells, or NKT cells) and to direct immune cells to target Polypeptide structure of cellular response of target cells.
  • the transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain enables signaling when the chimeric antigen receptor binds to the target antigen.
  • the "hinge region" of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane region and the antigen-binding region. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antigen-binding region.
  • the hinge region may be derived in whole or in part from a natural molecule, 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.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specified function.
  • the intracellular signaling domain is responsible for primary intracellular signal transmission after the antigen-binding domain binds the antigen, leading to the activation of immune cells and immune responses.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed.
  • Exemplary intracellular signaling domains include CD3 ⁇ .
  • 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 effective activation and function of T lymphocytes upon binding to an antigen.
  • immune effector cell refers to cells that participate in an immune response, such as promoting an immune effector response.
  • immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • T lymphocyte and "T cell” are used interchangeably herein and refer to the major type of white blood cell that matures in the thymus and has a variety of roles in the immune system, including recognition of specific foreign antigens in the body and Activating and inactivating other immune cells in an MHC class I-restricted manner.
  • the T cell can be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal.
  • T cells can be CD3+ cells.
  • T cells can be any type of T cell and can be in any culture 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 (PBMC), peripheral blood White blood cells (PBL), tumor-infiltrating lymphocytes (TIL), memory T cells, initial T cells, regulatory T cells, ⁇ T cells (gamma delta T cell/ ⁇ T cell), etc.
  • helper T cells include cells such as Th3 (Treg), Th17, Th9 or Tfh cells.
  • T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells).
  • T cells may also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • T cells or T-cell effector cells can also differentiate from stem cells or progenitor cells.
  • T-cell-like derived effector cells can have aspects of the T cell lineage but at the same time possess one or more functional characteristics not present in primary T cells.
  • NK cells or “natural killer cells” herein refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3).
  • adaptive NK cells and “memory NK cells” are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56 + -expressing NKG2C and CD57 and optionally CD16 at least one of, but lacks the expression of one or more of the following: PLZF, SYK, FceR ⁇ , and EAT-2.
  • the isolated CD56 + NK cell subset comprises expression of CD16, NKG2C, CD57, NKG2D, NCR ligand, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and/or DNAM-1.
  • CD56 + can be weakly or strongly expressed.
  • NK cells or NK cell-like effector cells can differentiate from stem cells or progenitor cells.
  • NK cell-like derived effector cells can be of the NK cell lineage in some aspects, but at the same time possess one or more functional characteristics not present in primary T cells.
  • NKT cells or “natural killer T cells” refers to CDld-restricted T cells that express the 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 repertoire of TCRs: a combination of canonical ⁇ chains (V ⁇ 24-J ⁇ 18 in humans) combined with a limited spectrum of ⁇ chains (V ⁇ 11 in humans).
  • TCR T cell receptor
  • a second NKT cell population termed non-classical or non-constant type II NKT cells, shows more uneven TCR ⁇ utilization.
  • Type I NKT cells are considered suitable for immunotherapy.
  • Adaptive or constant (type I) NKT cells can be identified using the expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.
  • autologous herein means any material derived from the same individual to which the material is subsequently reintroduced.
  • Allogeneic refers to a graft derived from a different individual of the same species.
  • an antigen-binding molecule eg, an antibody
  • Affinity is usually reflected in terms of equilibrium dissociation constant (KD), where a lower KD indicates higher affinity.
  • KD equilibrium dissociation constant
  • high affinity generally refers to having about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 1 ⁇ 10 -9 M or less, about 1 ⁇ KD of 10 -10 M or less, 1 ⁇ 10 -11 M or less, or 1 ⁇ 10 -12 M or less.
  • KD Kd/Ka, where Kd represents the dissociation rate and Ka represents the association rate.
  • the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (such as Biacore) or equilibrium dialysis measurement. For example, see the method for obtaining the KD value shown in the embodiments herein.
  • antibody is used herein in its broadest sense and refers to a polypeptide that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen. or peptide combinations.
  • This article “Antibody” encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • “Antibody” herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives.
  • Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, for example, biocompatible polymers.
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical “quadruple chain antibody”, which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to such a polypeptide chain, which It 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 in the direction from the N end to the C end; and, 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 composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain heavy chain constant region
  • CH3 domain heavy chain constant region in the direction from the N end to the C end
  • the full-length antibody is of the IgE isotype, it
  • Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.
  • Antibodies herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Alpaca ( Heavy-chain antibodies (HCAbs) produced by Vicugna pacos, etc., and immunoglobulin neoantigen receptors (Ig new antigen receptor, IgNAR) discovered in sharks and other cartilaginous fishes.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptor, IgNAR immunoglobulin neoantigen receptors
  • the "antibodies” herein can be derived from any animal, including but not limited to humans and non-human animals.
  • the non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camelids and llamas. , ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have the ability to bind Antigen capabilities.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , scFv, and VHH.
  • Papain digestion of intact antibodies generates two identical antigen-binding fragments, termed "Fab” fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy 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 domain carry free thiol groups. Pepsin treatment produces an F(ab') 2 fragment with two antigen binding sites (two Fab fragments) and part of the Fc region.
  • scFv single-chain variable fragment
  • 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 Page (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.
  • 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 provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).
  • anobody in this article refers to the natural heavy chain antibody lacking the light chain that exists in camels and other bodies. Cloning its variable region can obtain a single domain antibody 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.
  • VHH domain and “single domain antibody” (single domain antibody, sdAb) have the same meaning and can be used interchangeably. They refer to the variable region of a cloned heavy chain antibody, which is constructed from only one heavy chain variable region.
  • a single-domain antibody is the smallest fully functional antigen-binding fragment.
  • CH1 light chain and heavy chain constant region 1
  • variable region herein refers to the region of the heavy or light chain of an antibody involved in enabling the antibody to bind to the antigen.
  • Heavy chain variable region is used interchangeably with “VH” and “HCVR”
  • light chain variable region is used interchangeably.
  • VL can be used interchangeably with “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably in this article, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • HVR hypervariable region
  • VH heavy chain variable region
  • VL light chain variable region
  • This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • LCDR light chain variable region
  • frame region or "FR region” is used interchangeably and refers to those amino acid residues other than CDRs in the heavy or light chain variable region of an antibody.
  • FR region usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR CDR
  • 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 M.P.
  • CDR herein can be annotated and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but are not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of differently defined amino acid residues.
  • 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).
  • Chothia numbering system 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).
  • IMGT numbering system in this article generally refers to the 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.
  • IMGT ImMunoGeneTics information system
  • percent (%) sequence identity and “percent (%) sequence identity” are interchangeable and refer to the alignment of sequences and the introduction of gaps, if necessary, to achieve maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and nonhomologous sequences may be ignored for comparison purposes) followed by the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence.
  • alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% decrease in to 100% sequence identity.
  • the length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. .
  • 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.
  • At least 80% identity is preferably 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • substitution includes insertion mutations, deletion mutations and substitution mutations.
  • the substitution mutations are conservative amino acid substitutions.
  • conservative amino acids generally refer to amino acids that are in the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • conservative amino acids generally refer to amino acids that are in the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • conservative amino acids generally refer to amino acids that are in the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • the amino acids in each of the following groups belong to each other's conserved amino acid residues, and the substitution of amino acid residues within the group belongs to the substitution of conservative amino acids:
  • up to 5 mutations is preferably up to 4, 3, 2, 1 or 0 mutations.
  • up to 9 mutations is preferably up to 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.
  • up to 10 mutations is preferably at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.
  • up to 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.
  • up to 30 mutations is preferably at most 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.
  • 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.
  • 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, 52, 51 or 50 mutations, 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.
  • up to 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, 120 119, 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,
  • a "vector” is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell.
  • Many vectors are known in the art, including but not limited to linear polynucleotides, in combination with ionic or amphiphilic compounds. related polynucleotides, plasmids and viruses. Therefore, 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, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.
  • the terms "subject,” “subject,” and “patient” refer to an organism undergoing treatment for a particular disease or condition (eg, cancer or infectious disease) as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject, such as cell proliferative disorders (such as cancer) or infectious disease) progression.
  • Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented.
  • slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • the term "effective amount” refers to an amount of a therapeutic agent that is effective when administered alone or in combination with another therapeutic agent to a cell, tissue or subject to prevent or alleviate the symptoms of a disease or the progression of the disease. "Effective amount” also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • Figure 1 is a schematic diagram of the CAR-NK structure containing different NKG2D TM region elements.
  • Figure 2 shows the CAR expression rate in BCMA-CAR NK cells.
  • Figure 3 shows the CAR expression rate in ROR1-CAR NK cells.
  • Figure 4 shows -BCMA-CAR NK proliferation fold statistics.
  • Figure 5 shows the statistics of ROR1 CAR NK proliferation fold.
  • Figure 6 shows the cell flow cytometric detection results of MOLP8 and NCI H929-hBCMA-KO.
  • Figure 7 Multiple rounds of killing of MOLP8 cells by BCMA-CAR NK.
  • Figure 8A ⁇ Figure 8C shows the multiple rounds of killing effects of ROR1 CAR NK on A549, RPMI 8226 and 7860.
  • FIG. 9 is a schematic structural diagram of BCMA-CAR-NK-6 (BCMA-CAR6).
  • Figure 10 shows the CAR expression rate in BCMA-CAR NK cells used in animal experiments.
  • Figure 11A ?? Figure 11E Anti-tumor efficacy experiments of different forms of BCMA-CAR-NK on the NCI H929-luc multiple myeloma animal model.
  • Figure 11A shows the detection results of animal tumor bio-luminescence signal;
  • Figure 11B shows the animal body weight change rate %;
  • Figure 11C shows the animal tumor biosignal photon quantity;
  • Figure 11D shows the animal tumor growth inhibition rate %;
  • Figure 11E shows the animal survival rate %.
  • Figure 12 Schematic structural diagram of dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D.
  • BI-CAR dual-target chimeric antigen receptor
  • Figure 13 Schematic structural diagram of dual-target chimeric antigen receptor (BI-CAR) in the form of NKG2D_TM targeting BCMA and GPRC5D.
  • BI-CAR dual-target chimeric antigen receptor
  • Figure 15A- Figure 15B Results of multiple rounds of killing experiments by BI-CAR-NK on MOLP8 cells and RPMI 8226 cells.
  • Figure 16 Schematic structural diagram of dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D.
  • BI-CAR dual-target chimeric antigen receptor
  • Figure 18A- Figure 18F Results of multiple rounds of killing experiments by BI-CAR-NK on NCI H929 cells, RPMI 8226 cells, MOLP8 cells and tumor heterogeneous cells.
  • the target cells mentioned in the following examples were all transformed into luciferase genes and expressed luciferase.
  • the fluorescence intensity is detected by luciferase reporter gene detection reagent, which reflects the cell viability and the killing effect of NK cells.
  • the formula for calculating the kill rate is as follows:
  • Killing rate (target cell well reading value - test well reading value)/target cell well reading value ⁇ 100%.
  • the target cells used in the following examples are A549 cells, 786-O cells, RPMI 8226 cells, MOLP8 and H929-hBCMA-KO. All cells contain luciferase reporter genes through conventional gene manipulation methods. Among them, H929-hBCMA-KO is an H929 cell line that uses conventional gene manipulation methods to knock out BCMA.
  • the protein containing the extracellular domain of human BCMA was used as an antigen to immunize alpacas.
  • Peripheral blood was collected after the fourth and fifth immunizations (the antibody titer and specificity had been verified by ELISA), PBMCs were isolated, total RNA was extracted, and reverse transcription and Nested PCR amplified the VHH fragment, constructed a phage library, and panned and identified clones that were cross-positive with human and monkey BCMA.
  • variable region sequences of the positive clones were obtained by sequencing, named VHH1 and VHH2, and were numbered through the Kabat numbering system, Chothia numbering system (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT numbering system . (https://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) Determine its CDR region sequence.
  • IMGT http://imgt.cines.fr
  • human antibody heavy and light chain variable region germline gene database we selected the heavy chain variable region germline gene (IGHV3-64* with high homology to VHH1 antibody 04 and IGHJ3*01) or heavy chain variable region germline genes with high homology to VHH2 antibodies (IGHV3-7*01 and IGHJ6*01) as templates, using VHH1 or VHH2
  • the CDRs (determined by the IMGT numbering system) were transplanted into the corresponding human templates to form a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • VHH1, VHH2, hu-VHH1, and hu-VHH2 sequences into the expression vector of human IgG1 Fc, and obtain chimeric antibodies and humanized antibodies VHH1-hFc, VHH2-hFc, hu-VHH1-hFc and hu after expression and purification. -VHH2-hFc.
  • the aforementioned antibodies After verification by ELISA and FACS, the aforementioned antibodies all have good binding activity to human BCMA protein and monkey BCMA protein, and they all bind well to H929 and U266 cells that endogenously express BCMA.
  • VHH1-hFc , VHH2-hFc, hu-VHH1-hFc and hu-VHH2-hFc have high affinity to human BCMA protein, with KD values of 5.27E-10M (VHH1-hFc), 7.16E-11M (VHH2-hFc), and 6.13E respectively.
  • Human ROR-1 fusion protein (ACRO, Cat. RO1-H5250) was used to immunize mice, and the positive clone ROR1-1 was screened through spleen cell fusion and hybridoma technology, and the antibody variable region sequence was obtained through sequencing. And its CDR region sequence was determined through Kabat, Chothia and IMGT numbering systems.
  • the nucleic acid sequence encoding ROR1-1-scFv was cloned into an expression vector containing signal peptide and human Fc, and the chimeric antibody ROR1-1-hFc was obtained through expression and purification.
  • the chimeric antibody specifically binds to human ROR1 protein and MDA-MB-231, a cell endogenously expressing ROR1.
  • the affinity test results further verified that the chimeric antibody binds to human ROR1 protein, with a KD value of 3.23E-07M. Please see Table 3-Table 4 for specific sequence information.
  • NKG2D TM1 Design NKG2D TM1 and load it into the chimeric antigen receptor as a transmembrane domain element to construct a new chimeric antigen receptor.
  • chimeric antigen receptors loaded with other NKG2D TM sequences were constructed, as well as chimeric antigen receptors using 2B4 or CD28 in the hinge region-transmembrane region and costimulatory domain, in which NKG2D-TM4 was derived from WO2021071962A1, (see for details Sequence 57) disclosed in WO2021071962A1.
  • BCMA-CAR-NK-1 is referred to as BCMA-CAR1
  • BCMA-CAR-NK-2 is referred to as BCMA-CAR2
  • BCMA-CAR-NK-3 is referred to as BCMA-CAR3
  • BCMA-CAR- NK-4 is called BCMA-CAR4
  • BCMA-CAR-NK-5 is called BCMA-CAR5
  • BCMA-CAR-NK-6 is called BCMA-CAR6
  • ROR1-CAR-NK-1 is called ROR1-CAR1
  • ROR2- CAR-NK-2 will be referred to as ROR1-CAR2
  • ROR1-CAR-NK-3 will be referred to as ROR1-CAR3
  • ROR1-CAR-NK-4 will be referred to as ROR1-CAR4
  • ROR1-CAR-NK-5 will be referred to as ROR1-CAR5.
  • the nucleic acid sequence encoding the CAR described in Example 2 (Table 6) is loaded into a retroviral vector to construct a target plasmid.
  • the day before virus packaging 293T cells (purchased from ATCC) were trypsinized and inoculated into a culture dish at 1E7 cells/10cm.
  • When transfecting cells mix the packaging plasmid and the target plasmid, add them to ⁇ -MEM medium, and add them to another centrifuge tube containing ⁇ -MEM medium.
  • HD transfection reagent Promega, E2311).
  • NK cells were isolated according to the Human NK Cell isolation kit (Stemcell, 17955).
  • the isolated NK cells are activated with K562 cells.
  • the culture medium is NK cell culture medium (Miltenyi Biotec, 130-114-429) containing 200IU/ml human IL2, placed in an incubator (37°C, 5% CO 2 ); on Day 4, add 3mL to each well. Medium; Day 6, cell activation is complete and can be transfected.
  • RetroNectin reagent (Takara, T202) at a concentration of 7 ⁇ g/mL, 500 ⁇ L per well, and incubate at 4°C overnight.
  • RetroNectin reagent (Takara, T202) at a concentration of 7 ⁇ g/mL, 500 ⁇ L per well, and incubate at 4°C overnight.
  • Count NK cells add 3E5 cells/well to a 24-well plate, and centrifuge at 400g for 5 minutes at room temperature. Place the 24-well plate into an incubator (37°C, 5% CO 2 ).
  • Day 3 replace the transfected
  • BCMA Take 2E5 cells into a 96-well U-shaped plate, centrifuge, discard the supernatant, and after washing with buffer, add 100 ⁇ l of FITC-hBCMA-his (ACROBiosystems, BCA-HF254) with a final concentration of 2 ⁇ g/ml. Incubate in the dark at 4°C for 1 hour; after the incubation, centrifuge, discard the supernatant, wash with buffer and resuspend the cells; BD FACS Canto II flow cytometer detects BCMA-CAR expression rate. The flow cytometric detection results of BCMA-CAR NK cell surface molecule expression are shown in Figure 2.
  • ROR1 Take 2E5 cells into a 96-well U-shaped plate, centrifuge, discard the supernatant, and after washing with buffer, add 100 ⁇ l of human ROR1-his (ACROBiosystems, RO1-H522y) with a final concentration of 10 ⁇ g/ml, 4 Incubate at °C for 1 hour in the dark; after the incubation, centrifuge, discard the supernatant, and after washing with buffer, add 100 ⁇ l of THE TM His Tag Antibody [iFluor 647] (GenScript, A01802) with a final concentration of 1 ⁇ g/ml, and keep in the dark at 4°C.
  • human ROR1-his ACROBiosystems, RO1-H522y
  • BCMA-CAR NK cells proliferate about 40 to 70 times 6 days after transfection.
  • the proliferation rates from high to low are: BCMA-CAR1 (73 times) > BCMA-CAR2 (68 times) > BCMA-CAR4 (66 times) > BCMA -CAR3 (63 times)>parental NK (53 times)>BCMA-CAR5 (44 times).
  • the proliferation of ROR1-CAR NK is about 60-120 times after 6 days of transfection.
  • the proliferation speed from high to low is: ROR1-CAR1 (120 times)>parental NK (110 times)>ROR1-CAR2 (100 times)>ROR1- CAR3 (86 times)>ROR1-CAR4 (73 times)>ROR1-CAR5 (67 times).
  • the expression rate of CAR loaded with NKG2D TM elements in NK cells is lower than that of other forms of CAR; the expression rate of CAR (CAR1) loaded with NKG2D-TM1 elements is higher than that of CAR loaded with NKG2D-TM1 elements.
  • CARs of TM2 and NKG2D-TM3 elements CAR2 and CAR3.
  • the overall proliferation rate of CAR NK cells loaded with NKG2D TM elements is higher than that of CAR NK cells using other transmembrane elements.
  • the proliferation rate of CAR NK cells loaded with NKG2D-TM1 elements is the fastest, which is higher than that of other transmembrane elements. or other NKG2D TM sequence CAR NK cells.
  • Example 6 4h in vitro killing functional evaluation of different forms of CAR NK
  • target cells MOLP8 or H929-hBCMA-KO diluted in 1640 culture medium were added to a white opaque 96-well plate at 2E4 cells/50 ⁇ l/well, and the effect-to-target ratio was 10 :1, 5:1, 2.5:1
  • the 4h in vitro cell killing effect on MOLP8 cells is detailed in Table 7.
  • the CAR structure loaded with NKG2D TM region showed stronger tumor cell killing function at the effect-to-target ratio of 10:1, 5:1, and 2.5:1.
  • the CAR loaded with NKG2D TM1 BCMA-CAR1
  • BCMA-CAR1 has the strongest killing function and is superior to other NKG2D TM forms.
  • the killing of H929-hBCMA-KO cells by NK cells transfected with BCMA-CAR1 ⁇ 5 and partial NK was basically equivalent, indicating that BCMA-CAR has no non-specific killing of H929-hBCMA-KO cells.
  • ND stands for "no kill"
  • Next round of killing Take the cells from the previous round of 12-well plate, count the NK cells, and add the previous round of NK cells to the 12-well plate seeded with new target cells at an effective-to-target ratio of 1:1 , repeat step (1), measure the NK cell killing rate and continue the next round of killing test.
  • NK cells to the 96-well plate at an effective-to-target ratio of 1:1 and place at 37 °C, 5% CO2 incubator for 24 hours; after 24 hours of culture, add 30 ⁇ l FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1), incubate at room temperature for 10 minutes in the dark, and then measure with a microplate reader. Fluorescence value to calculate the killing efficiency of NK cells. After counting the NK cells in the previous round of 96-well plate, add the previous round of NK cells to the 96-well plate seeded with new target cells at an effective-to-target ratio of 1:1, and continue to repeat the above experimental steps.
  • FIREFLYGLO luciferase reporter gene detection reagent Meilun Bio, MA0519-1
  • BCMA-CAR NK was cultured for 14 days after transfection, and incubated with MOLP8 cells for 24 hours at an effective-to-target ratio of 10:1 and 2.5:1.
  • the supernatant was collected and detected according to the instructions of the human IFN- ⁇ quantitative ELISA kit (BD, 555142).
  • Medium IFN- ⁇ content Calculate the IFN- ⁇ content in the supernatant of the sample to be tested based on the standard curve of the standard product. The results are shown in Table 8.
  • ROR1-CAR NK was cultured until the 14th day after transfection. After incubation with RPMI 8226 cells for 24 hours at an effective-to-target ratio of 1:1 and 1:3, the supernatant was collected and used with human IFN- ⁇ quantitative ELISA kit (BD, 555142 ) to detect the IFN- ⁇ content in the supernatant. The results are shown in Table 9.
  • BCMA-CAR1 loaded with NKG2D TM1 elements
  • BCMA-CAR2 loaded with NKG2D TM2 elements
  • BCMA-CAR6 loaded with NKG2D TM4 elements
  • BCMA-CAR4 and BCMA-CAR5 not loaded with NKG2D TM elements (for the CAR structure, see Figure 1 and Figure 9; sequence information See Table 6), and the efficacy was evaluated on the NCI H929-luc mouse model.
  • BCMA CAR-NK cells were prepared using NK cells from different sources as in Example 4.
  • Count NK cells add 3E5 cells/well to a 24-well plate, and centrifuge at 400g for 5 minutes at room temperature. Place the 24-well plate into an incubator (37°C, 5% CO 2 ). On Day 3, replace the transfected NK into the Non-Treated 6-well plate. On Day9, CAR expression was detected as shown in Figure 10.
  • H929-Luc cells in the logarithmic growth phase and in good growth status were collected, and a total of 2 ⁇ 10 6 cells were inoculated into the tail veins of NPG mice (combined immunodeficient mice).
  • mice On the first day after tumor inoculation, the weight and inoculation status of the mice were measured, and mice with a weight of about 18.85-23.52g were selected based on the random number principle, with an average value of 21.92g for random grouping.
  • freshly prepared CAR-NK cells (5 ⁇ 10 6 cells/animal) were injected into the tail vein. The injection volume was 200 ⁇ l/animal.
  • the CAR-NK cell injection diary was Day 0.
  • mice and the injection of CAR-NK cells are shown in Table 10. Continuously monitor the tumor growth fluorescence signal ROI value and body weight changes with the IVIS intravital imager. Measure and record twice a week and calculate the tumor inhibition rate.
  • tumor inhibition rate TGI (%) (PBS group mouse tumor photon signal value - Photon signal value of mouse tumor in the experimental group)/photon signal value of mouse tumor in the PBS group ⁇ 100%.
  • the results of fluorescence signal detection of mouse tumor growth are shown in Figure 11A.
  • the results show that on the 76th day after injection of CAR-NK cells, the BCMA-CAR 1 treatment group was able to significantly inhibit the tumor growth burden of mice, and four of the animals had tumors. Cured, only 1 animal had tumor recurrence and growth; BCMA-CAR 6 treatment group could significantly inhibit tumor growth load, but could not control tumor progression in this group of animals. Among them, 2 animals had tumor recurrence and growth, 2 had tumors that completely disappeared, and 1 animal had tumor recurrence and growth. Death occurred; BCMA-CAR 2 treatment group was able to inhibit the growth rate of mouse tumors.
  • the mouse tumor volume was measured on day 76, and the tumor inhibition rate was calculated.
  • the tumor inhibition rates of the three treatment groups BCMA-CAR1, BCMA-CAR6, and BCMA-CAR4 reached 93%, 94%, and 84% respectively, showing good inhibitory effects on tumor growth, while BCMA-CAR 5 treatment
  • the tumor inhibition rate of the BCMA-CAR 4 group was only 25%.
  • the animals in this group of BCMA-CAR 4 were Since all died before 76 days, the tumor inhibition rate cannot be calculated;
  • the survival rate was continuously monitored until 130 days. As shown in Figure 11E, one animal died in the Parental NK treatment group starting from day 57, and the mortality rate was 100% by day 80; the BCMA-CAR 6 treatment group started from day 67 One animal died, and the mortality rate was 60% on day 130; one animal died in the BCMA-CAR 2 treatment group starting on day 39 (the mouse died on the same day after the tumor growth fluorescence signal was detected on day 39).
  • the mortality rate at 130 days was 60%; in the BCMA-CAR4 treatment group, one animal died from day 48, and the mortality rate was 100% at day 76; in the BCMA-CAR 5 treatment group, one animal died from day 63, and by day 108 The one-day mortality rate was 100%; only one animal died in the BCMA-CAR 1 treatment group on day 85. This group showed a durable therapeutic effect and good safety.
  • Anti-human GPRC5D monoclonal antibodies were generated by immunizing mice using conventional hybridoma methods.
  • a chimeric antibody containing the human IgG1 constant region was constructed based on the light chain and heavy chain variable region sequences obtained by sequencing, and human and monkey GPRC5D overexpression cells and NCI-H929 cells were screened using cell-based ELISA and FACS, which are well known to those skilled in the art. All have binding monoclonal antibodies for subsequent humanization design.
  • humanized monoclonal antibodies are obtained.
  • the CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system.
  • cell-based ELISA was used for verification, and the mouse-derived antibodies were finally named GPRC5D-mab03 and GPRC5D-mab06.
  • the humanized antibodies were named GPRC5D-Hab03 and GPRC5D-Hab06.
  • the light chain and heavy chain variable region sequences are as shown in the table. 11, and the CDRs sequences are shown in Table 12.
  • Alpaca-derived VHH antibodies were prepared using conventional VHH antibody screening methods in the field, in which the immunogen used was human GPRC5D protein.
  • the CDR amino acid residues of the antibody were determined and annotated by the IMGT or Kabat numbering system.
  • two alpaca sequences were obtained, named GPRC5D-Lab03 and GPRC5D-Lab04; and two humanized sequences were named GPRC5D-Hab03-H10. and GPRC5D-Hab04-H5.
  • the amino acid sequence is shown in Table 13, and the numbered CDRs sequence is shown in Table 14.
  • Camel-derived VHH antibodies were prepared using conventional VHH antibody screening methods in the field, in which the immunogen used was human GPRC5D protein.
  • the antibodies are annotated by IMGT or Kabat. After verification by FACS, several strains of camel antibodies and humanized antibodies with higher affinity were obtained.
  • the camel antibodies were named GPRC5D-Lab05 and GPRC5D-Lab06, and their humanized antibodies were named GPRC5D-Hab05-H2 and GPRC5D-Hab06-H1.
  • the Nanobody sequence is shown in Table 13, and the CDRs sequence is shown in Table 14.
  • Example 3-4 Using the BCMA-VHH antibodies screened in Example 1, namely hu-VHH2 and GPRC5D-scFv antibodies, a dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D was designed and constructed as shown in Figure 12.
  • the biological method of Example 3-4 was used to load the nucleic acid sequence of BI-CAR into the retroviral vector, 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.
  • the FACS expression detection results of BI-CAR are shown in Table 16.
  • the results show that in the forms of Figure 12A and Figure 12B, the expression of anti-BCMA and anti-GPRC5D is overall high and has no position dependence; in the forms of Figure 12C and Figure 12D, the expression of BCMA-CAR or GPRC5D-CAR Expression is affected by its location.
  • NCI H929, RPMI 8226, MOLP8 and NCI H929-hBCMA-KO were selected as target cells. All cells contained luciferase reporter genes through conventional gene manipulation methods. Among them, NCI H929-hBCMA-KO is an NCI H929 cell line that uses conventional gene manipulation methods to knock out BCMA. Add target cells diluted in 1640 culture medium to a white opaque 96-well plate at 2 ⁇ 10 4 cells/50 ⁇ l/well. Add NK cells to the above target cells at an effective-to-target ratio of 5:1, 2.5:1, and 1.25:1. Refer to The method of Example 6 measures and calculates the kill rate.
  • BI-CAR01 ⁇ BI CAR05 had specific killing effects on both NCI H929-Lu and NCI H929-hBCMA-KO-Lu.
  • BI-CAR01 ⁇ BI-CAR03 and BI-CAR05 have good specific killing effects on the four tumor cells as a whole.
  • BI-CAR01, BI-CAR-03 and BI-CAR05 are effective against NCI H929-hBCMA- KO-Lu tumor cells showed stronger killing function, indicating that BI-CAR01, BI-CAR03 and BI-CAR05 forms have better killing effects on tumor cells with down-regulated or deleted BCMA expression.
  • the dual-target chimeric antigen receptor targeting BCMA and GPRC5D includes CD8 ⁇ signal peptide (SP), anti-GPRC5D-scFv and anti-BCMA-VHH, CD8 hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3 ⁇ , and IL15 linked via the self-cleaving peptide P2A. See Table 18 for specific sequences. Refer to the molecular biology method in Example 3-4 to load the nucleic acid sequence into the retroviral vector to construct the target plasmid.
  • Example 3-4 use the same MOI to infect NK cells to prepare BI-CAR-NK cells with different transmembrane regions and intracellular stimulatory domain structures, and detect the expression of CAR with reference to the method in Example 5.1.
  • the results As shown in Table 19. The results show that the CAR expression shown in Figure 12A is better than the CAR expression shown in Figure 12C.
  • BI-CAR cells 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 from high to low were: BI-CAR06 (5566 times)>NK (3124 times)>BI-CAR03 (1705 times)>BI-CAR05 (1245.2 times)>BI -CAR01 (1012 times). Based on the above results, it can be seen that the proliferation rate of BI-CAR cells loaded with NKG2D TM elements after optimization is significantly higher than that of BI-CAR cells using other transmembrane elements.
  • target cells NCI H929, RPMI 8226, MOLP8 and NCI H929-hBCMA-KO diluted in 1640 medium were added at 2 ⁇ 10 4 cells/50 ⁇ l/well.
  • NK cells added to the above target cells at an effective-to-target ratio of 9:1 or 3:1, and measure and calculate the killing rate with reference to the method in Example 6.
  • BI-CAR01, BI-CAR-03, BI-CAR-05, and BI-CAR-06 on the 6th day after retrovirus infection of NK cells
  • Multiple rounds of killing effect on RPMI 8226 cells and MOLP8 cells in vitro Refer to the method of Example 7 to determine the in vitro multiple rounds of killing effect on RPMI 8226 cells and MOLP8 cells.
  • FIG. 15 The results of multiple rounds of killing tests of BI-CAR NK cells are shown in Figure 15. As shown in Figure 15, the killing effect of BI-CAR06 loaded with NKG2D TM elements is better than other forms of BI-CAR; BI-CAR06 can still maintain 50% cell killing in the third round of killing MOLP8 cells (R3-1d) rate, the third round of killing (R3-1d) of RPMI 8226 cells can still maintain a cell killing rate of 40%.
  • Example 5.1 Refer to the method of Example 5.1 to detect the expression of BI CAR18-21.
  • the results are shown in Table 22.
  • the positive rate of BCMA CAR can reach more than 60%; the positive rate of GPRC5D CAR is 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%).
  • BI-CAR21 has the best killing effect on NCI H929 cells, and can still maintain a cell killing rate of 30% in the fourth round of killing (R4-1d) experiments; BI-CAR19, BI-CAR20 and BI-CAR21 have the best killing effect on NCI H929+20 cells % (or 10% or 5%) H929-hBCMA-KO has the best killing effect on heterogeneous tumor cells, RPMI 8226 cells and MOLP8 cells, and NCI H929+20% (or 10% or 5%) H929-hBCMA- KO's heterogeneous tumor cells can still maintain a cell killing rate of more than 30% in the fourth round of killing (R4-1d) experiments, and can still maintain a cell killing rate of more than 30% in the fifth round of killing (R5-1d) experiments of RPMI 8226 cells and MOLP8 cells. Cell killing rate of about 10%.

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Abstract

本发明涉及增强型嵌合抗原受体及其应用，包括编码包含NKG2D跨膜区的嵌合抗原受体的核酸分子、相应的嵌合抗原受体、载体、免疫效应细胞、制备方法及其产品、药物组合物、制药用途和肿瘤或癌症治疗方法，具有CAR表达效率高、NK细胞增殖速度快、对肿瘤细胞的杀伤力强、特异性强、杀伤效果持续时间长等优势。

Description

增强型嵌合抗原受体及其应用
本发明要求2022年4月3日向中国国家知识产权局提交的，专利申请号为202210347351.4，发明名称为“增强型嵌合抗原受体及其应用”的在先申请的优先权1；要求2022年7月22日向中国国家知识产权局提交的，专利申请号为202210871138.3，发明名称为“靶向GPRC5D和/或BCMA的嵌合抗原受体及其应用”的在先申请的优先权2；要求2023年3月24日向中国国家知识产权局提交的，专利申请号为202310296244.8，发明名称为“增强型嵌合抗原受体及其应用”的在先申请的优先权3。上述在先申请的全文通过引用的方式结合于本发明中。
技术领域
本公开属于生物技术领域，具体涉及增强型嵌合抗原受体及其应用。
背景技术
自然杀伤细胞(NK细胞)是一类对肿瘤细胞具有强力杀伤作用且MHC非依赖的淋巴细胞，其对肿瘤细胞的识别主要依赖于其表面活化性受体和抑制性受体的相互交叉调控。识别肿瘤细胞后，ΝK细胞通过释放杀伤介质穿孔素和颗粒酶使靶细胞凋亡、表达膜TNF家族分子诱导靶细胞凋亡和抗体依赖的细胞毒作用等多种途径杀伤肿瘤细胞。异体NK细胞移植几乎不会诱发移植物抗宿主病(GVHD)，不会导致CRS，可以作为完全“现货型”产品使用。但是由于肿瘤患者体内ΝK细胞数量、质量的下降和肿瘤逃逸机制的存在,其在体内的抗肿瘤功能未能得到充分发挥。通过CAR修饰ΝK细胞有望增强其靶向杀伤肿瘤细胞的能力并研制出具有强大抗肿瘤作用的效应细胞。
NK细胞表达的功能性嵌合抗原受体(Chimeric Antigen Receptor,CAR)的结构主要包括：胞外结构域、跨膜区以及胞内的信号结构域。CAR结构中相应元件的选择，例如跨膜区和细胞内激活信号结构域的选择影响CAR-NK细胞的活性。目前大多数CAR-NK产品仍然沿用传统的CAR-T细胞疗法常用的CAR结构，细胞内共激活信号结构域为CD28或者4-1BB，由于NK细胞激活和T细胞激活的信号通路不同，因此传统的CAR-T细胞疗法常用的CAR结构，在CAR-NK中不一定能够发挥最优的功能。
发明内容
本公开根据NK细胞激活的独特的信号通路，设计了多种包含不同跨膜区以及胞内共刺激域的CAR结构的组合，通过体外体内实验的筛选和验证，得到了最适合CAR-NK细胞发挥活性的CAR结构。
进一步地，本公开还对CAR-NK的跨膜区进行优化。CAR-NK常用的TM区来自CD8和CD28，其他如NKG2D也有被使用。例如，CN110684117B和CN112210016A公开了嵌合抗原受体修饰的NK细胞，其中嵌合抗原受体的跨膜区选取了NKG2D TM。NKG2D是NK细胞激活剂，NKG2D TM区有利于增强CAR-NK细胞的活化和杀伤作用，但目前的CAR-NK 结构缺少针对NKG2D TM区的精细设计。本公开发现优化NKG2D TM区对于进一步提高CAR-NK的功能具有重要意义。
B细胞成熟抗原(BCMA)是肿瘤坏死因子受体超家族的一员，主要表达于终末分化的B细胞表面。B细胞活化因子(BAFF)和增殖诱导配体(APRIL)为BCMA主要配体，通过与BCMA相互作用来传导细胞刺激信号，激活TRAF依赖的NF-κB，JNK途径，增加B细胞的增殖和存活率。BCMA的表达已与多种癌症、自身免疫性障碍和感染性疾病有关。BCMA表达增加的癌症包括血液癌，如多发性骨髓瘤、霍奇金氏(Hodgkin)淋巴瘤和非霍奇金氏(non-Hodgkin)淋巴瘤、各种白血病和胶质母细胞瘤。BCMA是一个理想的治疗靶点。
ROR1是一种跨膜受体酪氨酸激酶蛋白，属于受体酪氨酸激酶(RTKs)家族成员。ROR1在人正常组织中低表达或不表达，但在多种恶性肿瘤或组织中高度表达，如慢性淋巴细胞白血病(CLL)、乳腺癌、卵巢癌、黑色素瘤、肺腺癌等。研究表明，ROR1在促进肿瘤的生长和转移、诱导肿瘤细胞耐药和抑制细胞凋亡等方面发挥着重要作用。因此，BCAM和ROR1是肿瘤治疗领域的理想靶点，针对BCMA和ROR1靶点，开发相应的嵌合抗原受体，尤其是适合NK细胞的嵌合抗原受体尤为重要。
有鉴于此，本公开提供编码包含NKG2D跨膜区的嵌合抗原受体的核酸分子、相应的嵌合抗原受体、载体、免疫效应细胞、制备方法及其产品、药物组合物、制药用途和肿瘤或癌症治疗方法。
在第一方面，本公开提供核酸分子，其包括编码嵌合抗原受体(chimeric antigen receptor，CAR)的第一核酸序列，所述嵌合抗原受体包括：包含抗原结合区的胞外区、连接所述胞外区的NKG2D跨膜区和连接所述跨膜区的胞内结构域，所述NKG2D跨膜区具有与SEQ ID NO:48相比至少80％同一性或至多9个突变的序列。
在一些具体的实施方式中，所述抗原结合区具有(1)-(4)组中至少一组的性质：
(1)所述抗原结合区选自抗体或其片段或结合所述抗原的配体，例如scFv、VHH、Fab、F(ab)’2或ligand；
(2)所述抗原结合区结合或不结合NKG2DL；
(3)所述抗原选自下组中的一个或多个：BCMA、GPRC5D、CLDN18.2、ROR1、CD19、CD33、CD5、CD70、HER2、IL13Rα2、GCC或GPC3；
(4)所述抗原结合区至少结合两个不同的抗原、至少结合同一抗原的两个不同表位或结合同一表位的数量在两个以上。
在一些具体的实施方式中，所述抗原结合区特异性结合BCMA，包括：
(1)VHH，其CDR1-3分别包括：
如SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示的序列；或，
如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:11所示的序列；或，
如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的序列；
(2)VHH，包括与SEQ ID NO:1或3具有至少80％同一性或至多25个突变的序列；
(3)VHH，其CDR1-3分别包括：
如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示的序列；或，
如SEQ ID NO:17、SEQ ID NO:25和SEQ ID NO:19所示的序列；或，
如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:19所示的序列；或，
如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的序列；
(4)与SEQ ID NO:2或4具有至少80％同一性或至多25个突变的序列；或，
(5)VHH1-连接肽-VHH2，所述VHH1具有如所述组(1)或组(2)所示序列，所述VHH2具有如所述组(3)或组(4)所示序列；或，
(6)VHH1-连接肽-VHH2，所述VHH1具有如所述组(3)或组(4)所示序列和所述VHH2具有如所述组(1)或组(2)所示序列；或，
(7)VHH1-连接肽-VHH2，具有与SEQ ID NO:63相比至少80％同一性或至多50个突变的序列。
在一些具体的实施方式中，所述抗原结合区特异性结合ROR1，包括：
(1)抗体或其片段，包括HCDR1-3和LCDR1-3，所述HCDR1-3分别包括：
如SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:32所示的序列；或，
如SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:32所示的序列；或，
如SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37所示的序列；
和所述LCDR1-3分别包括：
如SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的序列；或，
如SEQ ID NO:38、SEQ ID NO39和SEQ ID NO:40所示的序列；或，
如SEQ ID NO:41、SEQ ID NO:42和SEQ ID NO:40所示的序列；
(2)抗体或其片段，包括与SEQ ID NO:26相比具有至少80％同一性或至多25个突变的重链可变区，和与SEQ ID NO:27相比至少80％同一性或至多25个突变的轻链可变区；
(3)scFv，所述scFv具有所述组(1)或组(2)所示序列，可选地，所述scFv具有与SEQ ID NO:28相比至少80％同一性或至多50个突变的轻链可变区。
在一些具体的实施方式中，所述抗原结合区特异性结合GPRC5D，包括：
(1)抗体或其片段，包括HCDR1-3和LCDR1-3，所述HCDR1-3分别包括：
如SEQ ID NO:87、SEQ ID NO:88和SEQ ID NO:89所示的序列；或，
如SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92所示的序列；或，
如SEQ ID NO:96、SEQ ID NO:97和SEQ ID NO:98所示的序列；
和所述LCDR1-3分别包括：
如SEQ ID NO:84、SEQ ID NO:85和SEQ ID NO:86所示的序列；或，
如SEQ ID NO:93、SEQ ID NO:94和SEQ ID NO:95所示的序列；
(2)抗体或其片段，包括HCDR1-3，所述HCDR1-3分别包括：
如SEQ ID NO:107-118所示的序列；
(3)抗体或其片段，包括与SEQ ID NO:78、80、83、131相比具有至少80％同一性或至多25个突变的重链可变区，和与SEQ ID NO:77、79、81、82相比至少80％同一性或至多25个突变的轻链可变区；
(4)抗体或其片段，包括与SEQ ID NO:99-106相比具有至少80％同一性或至多25个突变的重链可变区。
在一些具体的实施方式中，所述胞外区还包括铰链区，所述铰链区选自下组中的一个或多个：CD8α铰链区、2B4铰链区、CD28铰链区、IgG1铰链区、IgD铰链、IgG4铰链区、GS铰链、KIR2DS2铰链，KIR铰链，NCR铰链，SLAMF铰链，CD16铰链，CD64铰链或LY49铰链；可选地，所述铰链区选自CD8α铰链区，例如，所述铰链区具有与SEQ ID NO:45相比至少80％同一性或至多10个突变的序列。
在一些具体的实施方式中，所述胞外区还包括信号肽，所述信号肽选自下组中的一个或多个：CD8α信号肽、IgG1重链信号肽或GM-CSFR2信号肽；可选地，所述信号肽为CD8α信号肽，例如，所述信号肽具有与SEQ ID NO:43相比至少80％同一性或至多5个突变的序列。
在一些具体的实施方式中，所述胞内结构域包括胞内信号传导结构域和/或共刺激结构域；
可选地，所述胞内信号传导结构域为CD3ζ，例如，所述胞内信号传导结构域具有与SEQ ID NO:55相比至少80％同一性或至多35个突变的序列；
可选地，所述共刺激结构域选自下组中的一个或多个：CD27共刺激结构域、4-1BB共刺激结构域、OX40共刺激结构域、2B4共刺激结构域、CD28共刺激结构域、ICOS共刺激结构域、DAP10共刺激结构域或DAP12共刺激结构域；可选地，所述共刺激结构域为2B4共刺激结构域，例如，所述共刺激结构域具有与SEQ ID NO:53相比至少80％同一性或至多25个突变的序列。
在一些具体的实施方式中，所述嵌合抗原受体包括：CD8α铰链区、NKG2D跨膜区、2B4共刺激结构域和CD3ζ胞内信号传导结构域，例如，具有与SEQ ID NO:58相比至少80％同一性或至多50个突变的序列。
在一些具体的实施方式中，所述核酸分子还包括编码IL15的第二核酸序列，可选地，所述IL15选自可溶性IL15、膜结合型IL15或IL15与其受体或受体片段的复合物，可选地，所述IL15具有与SEQ ID NO:57相比至少具有80％同一性或至多35个突变的序列；
可选地，所述第一核酸序列和所述第二核酸序列通过IRES或编码自裂解肽的序列连接，所述自裂解肽选自2A肽，例如P2A、T2A、F2A或E2A，可选地，所述自裂解肽为P2A，例如具有与SEQ ID NO:56相比具有至少80％同一性或至多5个突变的序列；
可选地，所述核酸编码与SEQ ID NO:59相比具有至少85％同一性或至多100个突变的序列的核酸序列。
在一些具体的实施方式中，其包括编码与SEQ ID NO:65或SEQ ID NO:70相比具有至少80％同一性或至多150个突变的序列的核酸序列。
在一些具体的实施方式中，所述核酸为DNA或RNA，所述RNA优选为mRNA。
在第二方面，本公开提供核酸分子，其中，所述核酸分子包含编码靶向BCMA的嵌合抗受体的第一核酸序列，所述嵌合抗原受体包括包含抗原结合区的胞外区、连接所述胞外区的跨膜区和连接所述跨膜区的胞内结构域，所述抗原结合区包括：
(1)VHH，其CDR1-3分别包括：
如SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示的序列；或，
如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:11所示的序列；或，
如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的序列；
(2)VHH，包括与SEQ ID NO:1或3具有至少80％同一性或至多25个突变的序列；
(3)VHH，其CDR1-3分别包括：
如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示的序列；或，
如SEQ ID NO:17、SEQ ID NO:25和SEQ ID NO:19所示的序列；或，
如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:19所示的序列；或，
如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的序列；
(4)与SEQ ID NO:2或4具有至少80％同一性或至多25个突变的序列；或，
(5)VHH1-连接肽-VHH2，所述VHH1具有如所述组(1)或组(2)所示序列，所述VHH2具有如所述组(3)或组(4)所示序列；或，
(6)VHH1-连接肽-VHH2，所述VHH1具有如所述组(3)或组(4)所示序列和所述VHH2具有如所述组(1)或组(2)所示序列；或，
(7)VHH1-连接肽-VHH2，具有与SEQ ID NO:63相比至少80％同一性或至多50个突变的序列。
在第三方面，本公开提供核酸分子，所述核酸分子包含编码靶向ROR1的嵌合抗受体的第一核酸序列，所述嵌合抗原受体包括包含抗原结合区的胞外区、连接所述胞外区的跨膜区和连接所述跨膜区的胞内结构域，所述抗原结合区包括：
(1)抗体或其片段，包括HCDR1-3和LCDR1-3，所述HCDR1-3分别包括：
如SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:32所示的序列；或，
如SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:32所示的序列；或，
如SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37所示的序列；
和所述LCDR1-3分别包括：
如SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的序列；或，
如SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的序列；或，
如SEQ ID NO:41、SEQ ID NO:42和SEQ ID NO:40所示的序列；
(2)抗体或其片段，包括与SEQ ID NO:26相比具有至少80％同一性或至多25个突变的重链可变区，和与SEQ ID NO:27相比至少80％同一性或至多25个突变的轻链可变区；
(3)scFv，所述scFv具有所述组(1)或组(2)所示序列，可选地，所述scFv具有与 SEQ ID NO:28相比至少80％同一性或至多50个突变的轻链可变区。
在一些具体的实施方式中，所述嵌合抗原受体包括：CD8α铰链区、NKG2D跨膜区、2B4共刺激结构域和CD3ζ胞内信号传导结构域，例如，所述嵌合抗原受体具有与SEQ ID NO:58相比至少80％同一性或至多50个突变的序列。
在一些具体的实施方式中，所述核酸分子还包括编码IL15的第二核酸序列，可选地，所述IL15选自可溶性IL15、膜结合型IL15或IL15与其受体或受体片段的复合物，可选地，所述IL15具有与SEQ ID NO:57相比至少具有80％同一性或至多35个突变的序列；
可选地，所述第一核酸序列和所述第二核酸序列通过IRES或编码自裂解肽的序列连接，所述自裂解肽选自2A肽，例如P2A、T2A、F2A或E2A，可选地，所述自裂解肽为P2A，例如具有与SEQ ID NO:56相比具有至少80％同一性或至多5个突变的序列；
可选地，所述核编码与SEQ ID NO:59相比具有至少85％同一性或至多100个突变的序列的核酸序列。
在一些具体的实施方式中，其包括编码与SEQ ID NO:65或SEQ ID NO:70相比具有至少80％同一性或至多150个突变的序列的核酸序列。
在第四方面，本公开提供根据前述核酸编码的嵌合抗原受体。
在第五方面，本公开提供载体，其中，所述载体包括前述的核酸分子。
在第六方面，本公开提供免疫效应细胞，其中，所述免疫效应细胞包括前述核酸分子或嵌合抗原受体或载体。
在一些具体的实施方式中，所述免疫效应细胞为NK细胞，所述NK细胞由iPSC分化而来、或衍生自外周血或脐带血。
在第七方面，本公开提供前述免疫效应细胞的制备方法，其中，包括：提供免疫效应细胞以及将所述核酸分子转入所述免疫效应细胞的步骤。
在第八方面，本公开提供根据前述方法制备而成的产品。
在第九方面，本公开提供药物组合物，其中，所述药物组合物包括前述核酸分子或嵌合抗原受体或载体、免疫效应细胞或产品，以及药学上可接受的载体。
在第十方面，本公开提供前述核酸分子或嵌合抗原受体或载体、免疫效应细胞、产品或药物组合物在制备用于治疗癌症或肿瘤的药物中的用途，所述癌症或肿瘤选自血液瘤或实体瘤；可选地，所述血液瘤选自骨髓瘤、淋巴瘤或白血病，例如多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、急性髓细胞性白血病(AML)、慢性淋巴细胞性白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓细胞性白血病(CML)或毛细胞白血病(HCL)；可选的地，所述实体瘤选自肺癌、乳腺癌、结直肠癌、胃癌、胰腺癌、肝癌、皮肤癌、膀胱癌、卵巢癌、子宫癌、前列腺癌或肾上腺癌。
在第十一方面，本公开提供前述核酸分子或嵌合抗原受体或载体、免疫效应细胞、产品或药物组合物，用于治疗癌症或肿瘤的用途，所述癌症或肿瘤选自血液瘤或实体瘤；可选地，所述血液瘤选自骨髓瘤、淋巴瘤或白血病，例如多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、急性髓细胞性白血病(AML)、 慢性淋巴细胞性白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓细胞性白血病(CML)或毛细胞白血病(HCL)；可选的地，所述实体瘤选自肺癌、乳腺癌、结直肠癌、胃癌、胰腺癌、肝癌、皮肤癌、膀胱癌、卵巢癌、子宫癌、前列腺癌或肾上腺癌。
在第十二方面，本公开提供治疗癌症或肿瘤的方法，所述方法包括向有需要的受试者给予有效量的前述核酸分子或嵌合抗原受体或载体、免疫效应细胞、产品或药物组合物，所述癌症或肿瘤选自血液瘤或实体瘤；可选地，所述血液瘤选自骨髓瘤、淋巴瘤或白血病，例如多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、急性髓细胞性白血病(AML)、慢性淋巴细胞性白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓细胞性白血病(CML)或毛细胞白血病(HCL)；可选的地，所述实体瘤选自肺癌、乳腺癌、结直肠癌、胃癌、胰腺癌、肝癌、皮肤癌、膀胱癌、卵巢癌、子宫癌、前列腺癌或肾上腺癌。
有益效果：本公开提供针对NKG2D跨膜区的优化嵌合抗原受体，其至少具有以下优势之一：(1)转染后，CAR表达效率高；(2)转染后，NK细胞增殖速度快；(3)对肿瘤细胞的杀伤力强、特异性强；(4)杀伤效果持续时间长。
术语定义和说明
除非本公开另外定义，与本公开相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物，均落入前述“一种组合物”的范围内。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语“BCMA”全称B细胞成熟抗原(B cell maturation antigen)，属于肿瘤坏死因子受体家族成员。BCMA主要表达于晚期B细胞、短寿命增殖浆母细胞和长寿命浆细胞表面，而在初始B细胞、CD34阳性造血干细胞和其他正常组织细胞中不表达，但它在MM细胞中是高度表达的，通过介导下游信号通路，对MM细胞的存活、增殖、转移和耐药中起着关键性的作用，所以BCMA是治疗MM理想的抗原靶点。示例性的人BCMA序列可见于GenBank Protein Accession No:NP_001183.2。
本文术语GPRC5D是指G蛋白偶联受体C5家族亚型D，属于一种孤儿受体，为7次跨膜蛋白，目前没有已知的配体。GPRC5D在原代多发性骨髓瘤细胞表面高表达，而在正常组织的表达仅限于毛囊区域。有研究表明65％的多发性骨髓瘤患者GPRC5D有超过50％的表达 阈值，凭借这一特点，GPRC5D成为了治疗MM的潜在靶标。示例性的人GPRC5D序列可见于GenBank Protein Accession No:NP_061124.1。
如本文所用,“抗原嵌合受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工免疫效应细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“信号肽”是指蛋白或多肽中用于引导所述蛋白或多肽进入分泌路径，转移至细胞膜和/或细胞表面的片段。在一些实施方式中，所述信号肽为CD8α信号肽，任选地，所述CD8α信号肽具有与下述序列具有至少80％同一性(例如至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％)：MALPVTALLLPLALLLHAARP。
如本文所用，嵌合抗原受体的“跨膜(TM)区”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。
如本文所用，嵌合抗原受体的“铰链区”一般是指作用为连接跨膜区和抗原结合区的任何寡肽或多肽。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。胞内信号传导结构域负责在抗原结合结构域结合抗原以后的细胞内初级信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫细胞的正常的效应功能的至少一种。示例性的胞内信号传导结构域包括CD3ζ。
如本文所使用，术语“共刺激结构域”是指共刺激分子的细胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体之外的细胞表面分子，所述细胞表面分子提供在与抗原结合后使T淋巴细胞有效活化和起作用所需的第二信号。
如本文所用的术语“免疫效应细胞”或“效应细胞”是指参与免疫应答例如促进免疫效应子应答的细胞。免疫效应细胞的例子包括T细胞、例如α/βT细胞和γ/δT细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和髓系来源的吞噬细胞。
本文术语“T淋巴细胞”和“T细胞”可互换使用并且是指白血细胞的主要类型，其在胸腺中完成成熟并且在免疫系统中具有多种作用，包含识别体内的特异性外来抗原和使其它免疫细胞以MHC I类限制的方式激活和失活。T细胞可以是任何T细胞，例如培养的T细胞，例如原代T细胞，或来自培养的T细胞系的T细胞，例如Jurkat、SupT1等，或获自哺乳动物的T细胞。T细胞可以是CD3+细胞。T细胞可以是任何类型的T细胞并且可以处于任何发 育阶段，包含但不限于CD4+/CD8+双阳性T细胞、CD4+辅助T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如细胞毒性T细胞)、外周血单核细胞(PBMC)、外周血白细胞(PBL)、肿瘤浸润性淋巴细胞(TIL)、记忆T细胞、初始T细胞、调控T细胞、γδT细胞(gamma delta T cell/γδT cell)等等。其它类型的辅助T细胞包括例如Th3(Treg)、Th17、Th9或Tfh细胞的细胞。其它类型的记忆T细胞包括例如中枢记忆T细胞(Tcm细胞)、效应记忆T细胞(Tem细胞和TEMRA细胞)的细胞。T细胞还可以指经基因工程化的T细胞，例如经修饰以表达T细胞受体(TCR)或嵌合抗原受体(CAR)的T细胞。T细胞或T细保养效应细胞还可以由干细胞或祖细胞分化。T细胞样衍生效应细胞在一些方面可以具有T细胞谱系，但同时具有原代T细胞中不存在的一种或多种功能特征。
本文术语“NK细胞”或“自然杀伤细胞”是指由CD56或CD16的表达和T细胞受体(CD3)的不存在定义的外周血淋巴细胞的亚群。如本文中所使用，术语“适应性NK细胞”和“记忆NK细胞”可互换并且是指NK细胞的亚群，其表型为CD3-和CD56+，表达NKG2C和CD57和任选的CD16中的至少一种，但缺少以下中的一种或多种的表达：PLZF、SYK、FceRγ和EAT-2。在一些实施例中，分离的CD56+NK细胞亚群包含CD16、NKG2C、CD57、NKG2D、NCR配体、NKp30、NKp40、NKp46、活化和抑制性KIR、NKG2A和/或DNAM-1的表达。CD56+可以是较弱或较强的表达。NK细胞或NK细胞样效应细胞可以从干细胞或祖细胞分化。NK细胞样衍生效应细胞在一些方面可以具有NK细胞谱系，但同时具有原代T细胞中不存在的一种或多种功能特征。
如本文所使用，术语“NKT细胞”或“自然杀伤T细胞”是指受限于CD1d的T细胞，其表达T细胞受体(TCR)。不同于检测由常规主要组织相容性(MHC)分子呈递的肽抗原的常规T细胞，NKT细胞识别由CD1d(一种非经典MHC分子)呈递的脂质抗原。识别两种类型的NKT细胞。恒定或I型NKT细胞表达非常有限的TCR库：典型α链(在人类中为Vα24-Jα18)与有限光谱的β链(在人类中为Vβ11)的结合。第二NKT细胞群体(称为非经典或非恒定II型NKT细胞)显示更不均匀的TCRαβ利用率。I型NKT细胞被视为适合于免疫疗法。适应性或恒定(I型)NKT细胞可以利用以下标志物中的至少一种或多种的表达来鉴别：TCR Va24-Ja18、Vb11、CD1d、CD3、CD4、CD8、aGalCer、CD161和CD56。
本文术语“自体”意指来源于相同个体的任何材料，随后该材料被重新引入该个体。“同种异体”是指来源于相同物种的不同个体的移植物。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约10-6M或更低、约10-7M或更低约10-8M或更低、约1×10-9M或更低、约1×10-10M或更低、1×10-11M或更低或1×10-12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定，示例性地，可参见本文实施例所示KD值获得方法。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文 “抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2、scFv和VHH。
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此,本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段,和重链的VH域和第一恒定域(CH1)的抗体片段。Fab’片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同,包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’)2片段。
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315 页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。
本文术语“纳米抗体”是指骆驼等体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。
本文术语“VHH结构域”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health  Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
如本文所用，术语“百分比(％)序列一致性”和“百分比(％)序列同一性”可互换，是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
如本文所用，“至少80％同一性”优选为85％同一性，90％同一性，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性。
如本文所用，“突变”包括插入突变、缺失突变和替换突变，优选地，所述替换突变为保守氨基酸地替换。如本文所用，“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
如本文所用，“至多5个突变”优选为至多4个、3个、2个、1个或0个突变。
如本文所用，“至多9个突变”优选为至多8个、7个、6个、5个、4个、3个、2个、1个或0个突变。
如本文所用，“至多10个突变”优选为至多10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个突变。
如本文所用，“至多25个突变”优选为至多24个、23个、22个、21个、20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个突变。
如本文所用，“至多30个突变”优选为至多29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个突变。
如本文所用，“至多50个突变”优选为至多49个、48个、47个、46个、45个、44个、43个、42个、41个、40个、39个、38个、37个、36个、35个、34个、33个、32个、31个、20个、29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个突变。
如本文所用，“至多100个突变优选为至多99个、98个、97个、96个、95个、94个、93个、92个、91个、90个、89个、88个、87个、86个、85个、84个、83个、82个、81个、70个、79个、78个、77个、76个、75个、74个、73个、72个、71个、70个、69个、68个、67个、66个、65个、64个、63个、62个、61个、60个、59个、58个、57个、56个、55个、54个、53个、52个、51个或50个突变、49个、48个、47个、46个、45个、44个、43个、42个、41个、40个、39个、38个、37个、36个、35个、34个、33个、32个、31个、20个、29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个突变。
如本文所用，“至多150个突变优选为至多149个、148个、147个、146个、145个、144个、143个、142个、141个、140个、139个、138个、137个、136个、135个、134个、133个、132个、131个、120个、129个、128个、127个、126个、125个、124个、123个、122个、121个、120个、119个、118个、117个、116个、115个、114个、113个、112个、111个、110个、109个、108个、107个、106个、105个、104个、103个、102个、101个、100个、99个、98个、97个、96个、95个、94个、93个、92个、91个、90个、89个、88个、87个、86个、85个、84个、83个、82个、81个、70个、79个、78个、77个、76个、75个、74个、73个、72个、71个、70个、69个、68个、67个、66个、65个、64个、63个、62个、61个、60个、59个、58个、57个、56个、55个、54个、53个、52个、51个或50个突变、49个、48个、47个、46个、45个、44个、43个、42个、41个、40个、39个、38个、37个、36个、35个、34个、33个、32个、31个、20个、29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个突变。
如本文所用,“载体”是包含分离的核酸并且可以用于将分离的核酸递送至细胞内部的物质组成。许多载体是本领域已知的,包括但不限于线性多核苷酸,与离子或两亲性化合物相 关的多核苷酸,质粒和病毒。因此,术语“载体”包括自主复制质粒或病毒。该术语还应解释为包括有助于核酸转移到细胞中的非质粒和非病毒化合物,例如聚赖氨酸化合物,脂质体等。病毒载体的实例包括但不限于腺病毒载体,腺相关病毒载体,逆转录病毒载体等。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、牛、绵羊、马和野牛等。
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
如本文所用，术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
附图说明
图1为包含不同NKG2D TM区元件的CAR-NK结构示意图。
图2为BCMA-CAR NK细胞中CAR表达率。
图3为ROR1-CAR NK细胞中CAR表达率。
图4为-BCMA-CAR NK增殖倍数统计。
图5为ROR1 CAR NK增殖倍数统计。
图6为MOLP8及NCI H929-hBCMA-KO的细胞流式检测结果。
图7：BCMA-CAR NK对MOLP8细胞的多轮杀伤作用。
图8A～图8C为ROR1 CAR NK对A549、RPMI 8226和7860的多轮杀伤作用。
图9为BCMA-CAR-NK-6(BCMA-CAR6)结构示意图。
图10为动物实验用BCMA-CAR NK细胞中CAR表达率。
图11A～图11E：不同形式BCMA-CAR-NK在NCI H929-luc多发性骨髓瘤动物模型上抗肿瘤药效实验。图11A为动物肿瘤生物法发光信号检测结果；图11B为动物体重变化率％；图11C为动物肿瘤生物信号光子量；图11D为动物肿瘤生长抑制率％；图11E为动物生存率％。
图12：靶向BCMA和GPRC5D的双靶点嵌合抗原受体(BI-CAR)结构示意图。
图13：靶向BCMA和GPRC5D的NKG2D_TM形式双靶点嵌合抗原受体(BI-CAR)结构示意图。
图14：BI-CAR-NK细胞增殖倍数统计。
图15A-图15B：BI-CAR-NK对MOLP8细胞和RPMI 8226细胞的多轮杀伤实验结果。
图16：靶向BCMA和GPRC5D的双靶点嵌合抗原受体(BI-CAR)结构示意图。
图17：BI-CAR-NK细胞增殖倍数统计。
图18A-图18F：BI-CAR-NK对NCI H929细胞，RPMI 8226细胞，MOLP8细胞及肿瘤异质细胞的多轮杀伤实验结果。
具体实施方式
下面结合具体实施例来进一步描述本公开，本公开的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开实施例仅是范例性的，并不对本公开的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本公开的精神和范围下可以对本公开技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本公开的保护范围内。
如无特别指出，下述实施例中所指靶细胞均转入荧光素酶基因，表达荧光素酶。通过荧光素酶报告基因检测试剂检测荧光强度，反映细胞活率和NK细胞的杀伤效果。杀伤率计算公式如下所示：
杀伤率＝(靶细胞孔读值-试验孔读值)/靶细胞孔读值×100％。
如无特别指出，下述实施例采用的靶细胞为A549细胞、786-O细胞、RPMI 8226细胞、MOLP8和H929-hBCMA-KO，所有细胞均通过常规基因操作方法均包含荧光素酶报告基因，其中H929-hBCMA-KO为使用常规基因操作方法对BCMA敲除的H929细胞系
实施例1抗体的筛选和制备
1.筛选和鉴定BCMA抗体
以含人BCMA胞外结构域的蛋白为抗原免疫羊驼，取四免和五免后的外周血(已通过ELISA验证抗体效价和特异性)，分离PBMC，提取总RNA，经逆转录和巢式PCR扩增VHH片段，构建噬菌体文库，淘选和鉴定与人猴BCMA交叉阳性的克隆。测序获得阳性克隆的可变区序列，命名为VHH1和VHH2，并通过Kabat编号系统和Chothia编号系统 (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT编号 系统(https://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)确定其CDR区序列。
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，挑选与VHH1抗体同源性高的重链可变区种系基因(IGHV3-64*04和IGHJ3*01)或与VHH2抗体同源性高的重链可变区种系基因(IGHV3-7*01和IGHJ6*01)作为模板，将VHH1或VHH2的 CDR(由IMGT编号系统确定)分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列中关键氨基酸回复突变为VHH纳米抗体对应的氨基酸，以保证原有的亲和力；如抗体存在易发生化学修饰的位点，对这些位点进行点突变已消除修饰风险。获得人源化抗体hu-VHH1和hu-VHH2。VHH抗体及其人源化抗体的序列信息和CDR信息如表1-表2所示。
将VHH1、VHH2、hu-VHH1、hu-VHH2序列重组到人IgG1 Fc的表达载体中，经表达纯化获得嵌合抗体和人源化抗体VHH1-hFc、VHH2-hFc、hu-VHH1-hFc和hu-VHH2-hFc。经过ELISA和FACS验证，前述抗体均与人BCMA蛋白和猴BCMA蛋白有较好的结合活性，且均与内源性表达BCMA的H929和U266细胞有良好的结合，亲和力检测结果进一步验证VHH1-hFc、VHH2-hFc、hu-VHH1-hFc和hu-VHH2-hFc与人BCMA蛋白的高亲和力，KD值分别为5.27E-10M(VHH1-hFc)，7.16E-11M(VHH2-hFc)，6.13E-10M(hu-VHH1-hFc)和8.82E-11M(hu-VHH2-hFc)。
表1抗体序列信息

表2 VHH1和VHH2的CDR信息
2.筛选和鉴定ROR1抗体
使用人ROR-1融合蛋白(ACRO，Cat.RO1-H5250)免疫小鼠，通过脾细胞融合和杂交瘤技术筛选阳性克隆ROR1-1，经测序获得抗体可变区序列。并通过Kabat，Chothia和IMGT编号系统确定其CDR区序列。将编码ROR1-1-scFv的核酸序列克隆到包含信号肽和人Fc的表达载体上，通过表达纯化获得嵌合抗体ROR1-1-hFc。经ELISA和FACS鉴定，该嵌合抗体特异性结合人ROR1蛋白和内源性表达ROR1的细胞MDA-MB-231。亲和力检测结果进一步验证该嵌合抗体结合人ROR1蛋白，KD值为3.23E-07M。具体序列信息详见表3-表4。
表3 ROR1抗体可变区序列信息
表4 ROR1抗体CDR区序列信息
实施例2不同形式嵌合抗原受体的设计
设计NKG2D TM1，将其作为跨膜区元件装载至嵌合抗原受体，构建一种新型嵌合抗原 受体。同时构建装载有其他NKG2D TM序列的嵌合抗原受体，以及铰链区-跨膜区和共刺激结构域均使用2B4或CD28的嵌合抗原受体，其中NKG2D-TM4来源于WO2021071962A1,(详见WO2021071962A1公开的序列57)。在后续实施例中评价装载NKG2D跨膜区的CAR NK细胞与其他装载不同跨膜区的CARNK细胞在功能方面(CAR阳性率、NK细胞增殖和杀伤)的差异，以及评价装载NKG2D TM1元件的CAR NK细胞与装载其他NKG2D TM序列的CARNK细胞的功能差异。嵌合抗原受体的分子结构详见图1和图9，元件序列信息详见表5，CAR序列信息详见表6。图1和图9中，BCMA-CAR-NK-1下称BCMA-CAR1，BCMA-CAR-NK-2下称BCMA-CAR2，BCMA-CAR-NK-3下称BCMA-CAR3，BCMA-CAR-NK-4下称BCMA-CAR4，BCMA-CAR-NK-5下称BCMA-CAR5，BCMA-CAR-NK-6下称BCMA-CAR6；ROR1-CAR-NK-1下称ROR1-CAR1，ROR2-CAR-NK-2下称ROR1-CAR2，ROR1-CAR-NK-3下称ROR1-CAR3，ROR1-CAR-NK-4下称ROR1-CAR4，ROR1-CAR-NK-5下称ROR1-CAR5。
表5 CAR元件序列信息

表6 CAR序列信息



实施例3嵌合抗原受体逆转录病毒制备
采用本领域常规分子生物学方法，将编码实施例2(表6)所述CAR的核酸序列装载至逆转录病毒载体，构建目的质粒。病毒包装的前一天，胰酶消化293T细胞(购自ATCC)，按1E7个/10cm接种至培养皿。转染细胞时，将包装质粒和目的质粒混合后加入α-MEM培养基内，在另一个含α-MEM培养基离心管内加入HD转染试剂(Promega，E2311)。将稀释的转染试剂逐滴加入稀释的质粒上方，混匀，室温静置15分钟，最后将质粒和转染试剂形成的混合物加入10cm培养皿内，轻晃10次、混匀，放入孵箱。细胞转染3天后，收获 病毒，将10ml含病毒培养上清转入50ml离心管内，4℃，1250rpm，5分钟，去除死亡的293T细胞，过滤含病毒上清，采用Retro-X Concentrator浓缩试剂(Clontech，631455)进行浓缩，分装后于-80℃保存备用。
实施例4 CAR-NK的制备
1.NK淋巴细胞的分离与激活
新鲜PBMC在室温条件下500g离心7min，弃去上层培养液后按照Human NK Cell分离试剂盒(Stemcell,17955)分离NK细胞。分离后的NK细胞用K562细胞激活，激活方法为：Day0，用AO/PI计数，按照NK：K562＝1：2混合细胞，以2ml/孔将混合细胞添加至Non-Treated 6孔板中(培养基为含200IU/ml人IL2的NK细胞培养基(Miltenyi Biotec，130-114-429))，放入培养箱中培养(37℃，5％CO2)；Day4，在每孔中添加3mL培养基；Day6，细胞激活完成，可进行转染。
2.逆转录病毒感染NK细胞
Day1，用浓度为7μg/mL RetroNectin试剂(Takara，T202)包被24孔板，每孔500μL，4℃过夜。Day2，弃去上层RetroNectin，用PBS洗一次。采用MOI＝5时感染NK细胞，根据病毒滴度计算病毒量，添加病毒到24孔板中。将添加完病毒的24孔板，在2000g，4-8℃离心60min。弃去上层病毒液。NK细胞计数，按3E5个/孔添加至24孔板，400g室温离心5min。将24孔板放入培养箱中培养(37℃,5％CO2)。Day3，将转染的NK换入到Non-Treated 6孔板中。Day6，检测CAR表达及CAR NK细胞增殖。
实施例5不同形式嵌合抗原受体的表达效率和增殖效率的检测
逆转录病毒感染NK细胞后第6天，利用流式细胞术检测NK细胞膜表面CAR的表达率并通过细胞计数仪检测CAR NK细胞增殖。
1.检测NK细胞中CAR表达率
(1)BCMA：取2E5个细胞至96孔U型板中，离心，弃上清，缓冲液洗涤后，加入100μl终浓度为2μg/ml的FITC-hBCMA-his(ACROBiosystems，BCA-HF254)，4℃避光孵育1小时；孵育结束后，离心，弃上清，缓冲液洗涤后重悬细胞；BD FACS Canto II流式细胞仪检测BCMA-CAR表达率。BCMA-CAR NK细胞表面分子表达的流式检测结果如图2所示。结果显示，BCMA-CAR在NK细胞中的表达效率为70～90％左右，表达效率由高到低分别为BCMA-CAR4(89.7％)＞BCMA-CAR5(85.9％)＞BCMA-CAR1(73.8％)>BCMA-CAR2(73％)>BCMA-CAR3(65.1％)。
(2)ROR1：取2E5个细胞至96孔U型板中，离心，弃上清，缓冲液洗涤后，加入100μl终浓度为10μg/ml的人ROR1-his(ACROBiosystems，RO1-H522y)，4℃避光孵育1小时；孵育结束后，离心，弃上清，缓冲液洗涤后，加入100μl终浓度为1μg/ml的THETM His Tag Antibody[iFluor 647](GenScript，A01802)，4℃避光孵育1小时；孵育结束后，离心，弃上清，缓冲液洗涤后重悬细胞；BD FACS Canto II流式细胞仪检测ROR1-CAR表达率。ROR1CAR NK细胞表面分子表达的流式检测结果如图3所示。结果显示，ROR1 CAR在NK细胞 中的表达效率为50～80％左右，表达效率由高到低分别为ROR1-CAR4(83.5％)＞ROR1-CAR5(70.1％)＞ROR1-CAR1(69.8％)>ROR1-CAR3(57.4％)>ROR1-CAR2(52.2％)。
2.检测CAR NK细胞的增殖速度
通过AO/PI计数检测BCMA-CAR NK细胞和ROR1 CAR NK细胞的增殖，结果如图4-图5所示。BCMA-CAR NK细胞转染6天后增殖40～70倍左右，增殖速度由高到低分别为：BCMA-CAR1(73倍)＞BCMA-CAR2(68倍)＞BCMA-CAR4(66倍)＞BCMA-CAR3(63倍)＞parental NK(53倍)＞BCMA-CAR5(44倍)。ROR1-CAR NK转染6天后的增殖60～120倍左右，增殖速度由高到低分别为：ROR1-CAR1(120倍)＞parental NK(110倍)＞ROR1-CAR2(100倍)＞ROR1-CAR3(86倍)＞ROR1-CAR4(73倍)＞ROR1-CAR5(67倍)。
结果说明：
根据以上结果可知，就CAR表达率而言，装载NKG2D TM元件的CAR在NK细胞中的表达率低于其他形式的CAR；装载NKG2D-TM1元件的CAR(CAR1)的表达率高于装载NKG2D-TM2和NKG2D-TM3元件的CAR(CAR2和CAR3)。但装载NKG2D TM元件的CAR NK细胞在增殖速度整体上高于使用其他跨膜元件的CAR NK细胞，其中，装载NKG2D-TM1元件的CAR NK细胞增殖速度最快，均高于装载其他跨膜元件或其他NKG2D TM序列的CAR NK细胞。
实施例6不同形式CAR NK的4h体外杀伤功能性评价
流式细胞术检测多发性骨髓瘤MOLP8细胞、H929-hBCMA-KO细胞中BCMA的表达。结果参见图6。
逆转录病毒感染NK细胞后第6天进行4h体外杀伤实验：经1640培养基稀释的靶细胞MOLP8或H929-hBCMA-KO以2E4个/50μl/孔加入白色不透明96孔板，按效靶比10:1、5:1、2.5:1将NK细胞加入上述靶细胞中，并将96孔板放置于37℃，5％CO2培养箱中培养；4小时后，加入30μl FIREFLYGLO荧光素酶报告基因检测试剂(美仑生物，MA0519-1)，室温避光孵育10分钟后用酶标仪进行测量并计算杀伤率。
对MOLP8细胞的4h体外细胞杀伤效果详见表7。与其他CAR结构相比，装载NKG2D TM区的CAR结构整体上在10:1、5：1、2.5:1的效靶比下，显示更强的肿瘤细胞杀伤功能，同时装载NKG2D TM1的CAR(BCMA-CAR1)的杀伤功能最强，优于其他NKG2D TM形式。另外，转染BCMA-CAR1～5的NK细胞与partenal NK对H929-hBCMA-KO细胞的杀伤基本相当，表明BCMA-CAR对H929-hBCMA-KO细胞无非特异性杀伤。
表7 BCMA-CAR NK对MOLP8细胞的4h体外杀伤率(单位，％)

注：ND表示“不杀伤”
实施例7不同形式CAR NK的多轮杀伤功能评价
1.BCMA-CAR NK细胞多轮杀伤试验
逆转录病毒感染NK细胞后第6天，检测BCMA-CAR NK细胞对MOLP8细胞的体外多轮杀伤效果：(1)第一轮杀伤：按2.5E5个/500μl/孔将经1640培养基稀释的靶细胞MOLP8置于12孔板中。按效靶比1:1在12孔板中加入NK细胞，37℃，5％CO2培养箱中培养24小时。培养24小时后，取100μl上述混合均匀细胞加入白色底不透的96孔板中，加入30μl FIREFLYGLO荧光素酶报告基因检测试剂(美仑生物，MA0519-1)，室温避光孵育10分钟后用酶标仪测量荧光强度，计算NK细胞杀伤效率。之后，直接进入下一轮杀伤试验，或培养24h后再次测定NK细胞杀伤率后进入下一轮杀伤试验。(2)下一轮杀伤：取上一轮12孔板中的细胞，对NK细胞进行计数，按效靶比1:1将上一轮NK细胞加入到接种有新靶细胞的12孔板中，重复步骤(1)，测定NK细胞杀伤率和继续下一轮杀伤试验。
BCMA-CAR NK细胞的多轮杀伤试验的结果参见图7。如图7所示，装载NKG2DTM元件的BCMA-CAR的杀伤效果优于其他形式的BCMA-CAR，其中，BCMA-CAR1装载NKG2D-TM1，其杀伤效果最佳，优于其他形式的BCMA-CAR和装载其他NKG2D-TM的CAR，在第三轮杀伤(R3-2d)仍能维持98％的细胞杀伤率。
2.ROR1-CAR NK细胞多轮杀伤试验
逆转录病毒感染NK细胞第6天，检测ROR1 CAR NK细胞对A549(ROR1低表达)、786-O(ROR1低表达)和RPMI 8226细胞(ROR1中表达)的体外多轮杀伤效果：按2.0E4个/50μl/孔将经1640培养基稀释的靶细胞A549、786-O或RPMI 8226加入白色底不透96孔板中，按效靶比1：1将NK细胞加入96孔板，置于37℃，5％CO2培养箱中培养24小时；培养24小时后，加入30μl FIREFLYGLO荧光素酶报告基因检测试剂(美仑生物，MA0519-1)，室温避光孵育10分钟后用酶标仪测量荧光值，计算NK细胞的杀伤效率。将上一轮96孔板中的NK细胞计数后，按效靶比1:1将上一轮的NK细胞加入到接种有新靶细胞的96孔板中，继续重复上述实验步骤。
ROR1-CAR NK细胞多轮杀伤试验的结果如图8A-图8C所示。第2轮和第3轮杀伤结果显示ROR-1 CAR1NK细胞对786-O细胞、A549细胞和RPMI 8226细胞的杀伤效果优于非NKG2D-TM型CAR，也优于其他NKG2D-TM型CAR。
实施例8不同形式CAR NK的细胞因子释放功能评价
BCMA-CAR NK转染后培养14天，与MOLP8细胞按照效靶比为10:1、2.5:1共孵育24小时，按照人IFN-γ定量ELISA试剂盒(BD,555142)说明书检测收集上清中IFN-γ含量。根据标准品的标准曲线计算待测样品的上清中IFN-γ的含量，结果如表8所示。
ROR1-CAR NK转染后培养至第14天，与RPMI 8226细胞按照效靶比为1:1、1:3共孵育24小时后，收集上清用人IFN-γ定量ELISA试剂盒(BD,555142)检测上清中IFN-γ含量，结果参见表9。
表8-表9结果表明，CAR1在不同的效靶比、针对不同靶细胞都能够上调具备肿瘤杀伤作用的细胞因子IFN-γ的表达量，且在整体上优于使用非NKG2D TM元件的CAR，同时优于使用其他NKG2D-TM元件的CAR。
表8细胞因子IFN-γ含量检测结果
注：ND表示“未检测到”
表9细胞因子IFN-γ含量(pg/mL)检测结果
注：ND表示“未检测到”
实施例9不同形式BCMA-BCMA CAR-NK体内药效评价
将装载NKG2D TM1元件的BCMA-CAR1，NKG2D TM2元件的BCMA-CAR2，NKG2D TM4元件BCMA-CAR6以及未装载NKG2D TM元件的BCMA-CAR4和BCMA-CAR5(CAR结构参见图1、图9；序列信息参见表6)，在NCI H929-luc小鼠模型上进行药效评价。
1.BCMA CAR-NK细胞制备
参照实施例4的制备方法，采用与实施例4中不同来源的NK细胞进行BCMA CAR-NK细胞制备，MOI＝5时感染NK细胞，根据病毒滴度计算病毒量，添加病毒到24孔板中。将添加完病毒的24孔板，在2000g，4-8℃离心60min。弃去上层病毒液。NK细胞计数，按3E5个/孔添加至24孔板，400g室温离心5min。将24孔板放入培养箱中培养(37℃,5％CO2)。Day3，将转染的NK换入到Non-Treated 6孔板中。Day9，检测CAR表达如图10所示。
根据图10结果可知，在本次实验中，就CAR表达率而言，装载NKG2D TM元件的CAR在NK细胞中的表达率低于其他形式的BCMA-CAR4和BCMA-CAR5；BCMA-CAR1，BCMA-CAR6的表达率高于装载BCMA-CAR2。
2.装载NKG2D-TM元件的BCMA-BCMA CAR-NK在NCI H929-luc小鼠肿瘤模型中的抗肿瘤药效试验
为了评估装载NKG2D-TM元件的BCMA-BCMA CAR-NK抗肿瘤药效，使用小鼠骨髓癌静脉移植瘤模型进行抗肿瘤药效试验。具体如下：
收集处于对数生长期且生长状态良好的H929-Luc细胞，于NPG小鼠(联合免疫缺陷小鼠)尾静脉共接种2×106个细胞。接种肿瘤后第1天测量小鼠体重和接种情况，根据随机数原则选择体重在18.85～23.52g左右的小鼠，均值为21.92g进行随机分组。接种肿瘤后第1天，即分组当天尾静脉注射新鲜制备CAR-NK细胞(5×106个/只)，注射体积为200μl/只，CAR-NK细胞注射日记为Day0。小鼠分组与CAR-NK细胞注射情况见表10。持续用IVIS活体成像仪监测肿瘤生长荧光信号ROI值及体重变化，每周测量记录2次并计算肿瘤抑制率，计算公式：抑瘤率TGI(％)＝(PBS组小鼠肿瘤光子信号值-实验组小鼠肿瘤光子信号值)/PBS组小鼠肿瘤光子信号值×100％。
表10.体内抗肿瘤实验分组情况
小鼠肿瘤生长荧光信号检测结果如图11A所示，结果表明：在注射CAR-NK细胞后的第76天，BCMA-CAR 1治疗组能够显著抑制小鼠肿瘤生长负荷，其中四只动物肿瘤出现治愈，仅1只动物肿瘤复发增长；BCMA-CAR 6治疗组能够显著抑制肿瘤生长负荷，但不能控制该组动物肿瘤进展，其中2只动物肿瘤复发增长，2只的肿瘤完全消退，1只动物出现死亡；BCMA-CAR 2治疗组能够抑制小鼠肿瘤生长速度，其中1只动物肿瘤治愈，但组内4只动物肿瘤复发及两只发生死亡；BCMA-CAR 4和BCMA-CAR 5在给药后39天能够抑制小鼠肿瘤生长速度，在实验后期随着肿瘤复发体现较差控制肿瘤效果，且BCMA-CAR 4在76天已经全部死亡。
同时，CAR-NK治疗各组小鼠体重在给药后虽有波动，但整体呈现上升增长趋势(如图11B所示)。
给药后连续测量小鼠肿瘤光子值，除了BCMA-CAR 5治疗组，其余CARNK治疗各组与Parental NK组相比，均呈现降低趋势，P<0.05*，P<0.001***，尤其体现在BCMA-CAR1和BCMA-CAR6治疗组，体现极显著治疗效果(如图11C所示)；
在第76天测量小鼠肿瘤体积，计算肿瘤抑制率。如图11D所示，BCMA-CAR1、BCMA-CAR6、BCMA-CAR4三个治疗组抑瘤率分别达到93％，94％，84％，表现良好的抑制肿瘤生长的效果，而BCMA-CAR 5治疗组组抑瘤率仅达25％，BCMA-CAR 4该组动物由 于在76天前全部死亡，无法进行计算抑瘤率；
实验过程中生存率连续监测至130天，如图11E所示，Parental NK治疗组从57天开始发生1只动物死亡，到80天死亡率为100％；BCMA-CAR 6治疗组从67天开始发生1只动物死亡，到130天死亡率为60％；BCMA-CAR 2治疗组从39天开始发生1只动物死亡(该小鼠在第39天检测肿瘤生长荧光信号后，当天死亡)，到130天死亡率为60％；BCMA-CAR4治疗组从48天开始发生1只动物死亡，到76天死亡率为100％；BCMA-CAR 5治疗组从63天开始发生1只动物死亡，到108天死亡率为100％；BCMA-CAR 1治疗组仅在85天发生一只动物死亡，该组表现持久治疗效果同时，有较好的安全性。
实施例10 GPRC5D抗体制备
10.1抗GPRC5D scFv的制备
抗人GPRC5D单克隆抗体通过免疫小鼠，使用常规的杂交瘤方法产生。根据测序得到的轻链和重链可变区序列构建包含人IgG1恒定区的嵌合抗体，使用本领域技术人员熟知的cell based ELISA和FACS筛选得到人和猴GPRC5D过表达细胞、NCI-H929细胞均有结合的单克隆抗体，进行后续人源化设计。
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，采用本领域常规的人源化抗体的方法，得到人源化单克隆抗体。其中抗体的CDR氨基酸残基由Kabat编号系统确定并注释。随后使用cell-based ELISA验证，最终得到鼠源抗体命名为GPRC5D-mab03、GPRC5D-mab06，其人源化抗体命名为GPRC5D-Hab03、GPRC5D-Hab06，其轻链和重链可变区序列如表11所示，CDRs序列如表12所示。
表11抗GPRC5D鼠源及其人源化抗体VL、VH序列

表12抗GPRC5D抗体Kabat编号LCDRs和HCDRs序列表
10.2抗GPRC5D纳米抗体的制备
羊驼来源VHH抗体的制备采用本领域常规的VHH抗体筛选方法，其中使用的免疫原为human GPRC5D蛋白。其中抗体的CDR氨基酸残基由IMGT或Kabat编号系统确定并注释，最终得到2条羊驼序列，命名为GPRC5D-Lab03和GPRC5D-Lab04；以及2条人源化序列，命名为GPRC5D-Hab03-H10和GPRC5D-Hab04-H5。氨基酸序列如表13所示，编号的CDRs序列如表14所示。
骆驼来源VHH抗体的制备采用本领域常规的VHH抗体筛选方法，其中使用的免疫原为人GPRC5D蛋白。所述抗体由IMGT或Kabat注释。经FACS验证后，得到若干株亲和力较高的骆驼抗体及其人源化抗体。骆驼抗体命名为GPRC5D-Lab05和GPRC5D-Lab06，其人源化抗体命名为GPRC5D-Hab05-H2和GPRC5D-Hab06-H1。纳米抗体序列如表13所示，CDRs序列如表14所示。
表13抗GPRC5D纳米抗体氨基酸序列

表14抗GPRC5D纳米抗体CDRs序列
实施例11.4-1BB结构嵌合抗原受体的构建与胞外抗体组合的筛选
11.1 4-1BB结构CAR-NK的构建
利用实施例1筛选得到的BCMA-VHH抗体，即hu-VHH2和GPRC5D-scFv抗体，设计构建如图12所示靶向BCMA和GPRC5D的双靶点嵌合抗原受体(BI-CAR)。采用实施例3-4的生物学方法将BI-CAR的核酸序列装载至逆转录病毒载体，构建目的质粒和病毒载体，诱导NK细胞并制备CAR-NK细胞。BI-CAR01～04的结构分别如图12A～图12D所示，BI-CAR05结构如图12A所示。BI-CAR各结构域的氨基酸序列和示例性的BI-CAR01～05序列如表15所示。
表15 BI-CAR的各结构域与示例性BI-CAR氨基酸序列表



11.2嵌合抗原受体表达率检测
逆转录病毒感染NK细胞后第6天，利用流式细胞术检测NK细胞膜表面CAR的表达率。BI-CAR中BCMA-CAR NK和BI-CAR中GPRC5D-CAR NK细胞表面分子表达检测参考实施例5.1检测NK细胞中CAR表达率的方法。
BI-CAR的FACS表达检测结果见表16。结果显示，在图12A和图12B形式中，anti-BCMA和anti-GPRC5D的表达在整体上均较高且没有位置依赖性；在而图12C和图12D形式中BCMA-CAR或者GPRC5D-CAR的表达受其所在位置的影响。
表16 BI-CAR表达率检测
11.3 BI-CAR-NK体外对靶细胞4h快速杀伤率检测
逆转录病毒感染NK细胞后第6天进行4h体外杀伤实验：靶细胞选择NCI H929、RPMI 8226、MOLP8和NCI H929-hBCMA-KO，所有细胞均通过常规基因操作方法均包含荧光素酶报告基因，其中NCI H929-hBCMA-KO为使用常规基因操作方法对BCMA敲除的NCI H929细胞系。经1640培养基稀释的靶细胞以2×104个/50μl/孔加入白色不透明96孔板，按效靶比5:1、2.5:1、1.25:1将NK细胞加入上述靶细胞中，参考实施例6的方法测量并计算杀伤率。
BI-CAR对上述靶细胞的4h体外细胞杀伤效果详见表17。在5:1的效靶比下，BI-CAR01～BI CAR05对NCI H929-Lu和NCI H929-hBCMA-KO-Lu均存在特异性杀伤效果。BI-CAR01～BI-CAR03和BI-CAR05在整体上对四种肿瘤细胞均有较好的特异性杀伤效果，其中，BI-CAR01、BI-CAR-03和BI-CAR05对NCI H929-hBCMA-KO-Lu肿瘤细胞显示更强的杀伤功能，表明BI-CAR01、BI-CAR03和BI-CAR05形式对于BCMA表达下调或缺失的肿瘤细胞具有较好的杀伤作用。
表17 BI-CAR-NK体外对靶细胞4h快速杀伤率结果

注：“-”代表没有检测到杀伤效果。
实施例12.NKG2D-F结构嵌合抗原受体设计
12.1 NKG2D-F结构嵌合抗原受体的构建。
为验证不同跨膜域对嵌合抗原受体活性的影响，利用实施例11筛选得到的BI-CAR01和BI-CAR03对应的抗体BCMA-Hu-VHH2和GPRC5D-Hab06，设计构建如图13所示结构的靶向BCMA和GPRC5D的双靶点嵌合抗原受体，命名为BI-CAR06。靶向BCMA和GPRC5D的双靶点嵌合抗原受体包括CD8α信号肽(SP)、anti-GPRC5D-scFv和anti-BCMA-VHH、CD8铰链区、NKG2D跨膜区、2B4共刺激结构域和CD3ζ，以及通过自切割肽P2A连接的IL15。具体序列详见表18。参考实施例3-4中的分子生物学方法将核酸序列装载至逆转录病毒载体，构建目的质粒。
表18 BI-CAR的各结构域与示例性BI-CAR氨基酸序列表

12.2不同结构CAR-NK中CAR表达率的测定
参考实施例3-4所示方法，采用相同MOI侵染NK细胞制备具有不同跨膜区和胞内刺激域结构的BI-CAR-NK细胞，参考实施例5.1中的方法检测CAR的表达，结果如表19所示。结果显示，图12A所示CAR表达优于图12C所述CAR表达。
表19不同结构CAR-NK中CAR表达率结果
12.3不同结构CAR-NK细胞增值速率的测定
通过AO/PI计数检测BI-CAR细胞的增殖情况，结果如图14所示。NK激活14天后，BI-CAR细胞染增殖速度由高到低分别为：BI-CAR06(5566倍)＞NK(3124倍)＞BI-CAR03(1705倍)＞BI-CAR05(1245.2倍)＞BI-CAR01(1012倍)。根据以上结果可知，优化后装载NKG2D TM元件的BI-CAR细胞增殖速度要显著优于高于使用其他跨膜元件的BI-CAR细胞。
12.4不同结构CAR-NK细胞4h快速杀伤率测定
逆转录病毒感染NK细胞后第6天进行4h体外快速杀伤实验：经1640培养基稀释的靶细胞NCI H929、RPMI 8226、MOLP8和NCI H929-hBCMA-KO以2×104个/50μl/孔加入白色不透明96孔板，按效靶比9:1、3:1将NK细胞加入上述靶细胞中，参考实施例6的方法测量并计算杀伤率。
BI-CAR对上述靶细胞的4h体外细胞杀伤效果详见表20。结果显示，BI-CAR05、BI-CAR01、BI-CAR-03、BI-CAR-06均显示较强的对肿瘤细胞杀伤功能。
表20不同结构CAR-NK细胞4h快速杀伤率结果
注：“-”代表没有检测到杀伤效果。
12.5不同结构CAR-NK多轮杀伤效果检测
逆转录病毒侵染NK细胞后第6天检测BI-CAR01、BI-CAR-03、BI-CAR-05、BI-CAR-06 对RPMI 8226细胞和MOLP8细胞的体外多轮杀伤效果。参考实施例7的方法测定对RPMI 8226细胞和MOLP8细胞的体外多轮杀伤效果
BI-CAR NK细胞的多轮杀伤试验的结果参见图15。如图15所示，装载NKG2D TM元件的BI-CAR06的杀伤效果优于其他形式的BI-CAR；BI-CAR06对MOLP8细胞的第三轮杀伤(R3-1d)仍能维持50％的细胞杀伤率，对RPMI 8226细胞的第三轮杀伤(R3-1d)仍能维持40％的细胞杀伤率。
实施例13人源化抗体构建的NKG2D-F CAR
13.1 NKG2D-F CAR的构建
设计构建靶向BCMA和GPRC5D的双靶点嵌合抗原受体包括CD8α信号肽(SP)、anti-GPRC5D抗体和anti-BCMA、CD8α铰链区、NKG2D-F跨膜区(NKG2D-TM1)、2B4共刺激结构域和CD3ζ，以及通过自切割肽P2A连接的IL15。BI-CAR18、BI-CAR20结构如图16A所示，BI-CAR19、BI-CAR21结构如图16B所示。序列如表21所示。
表21 NKG2D-F CAR氨基酸序列表

13.2 NKG2D-F CAR表达检测
参考实施例5.1方法，检测BI CAR18～21的表达。结果如表22所示，结果显示，采用MOI＝5时侵染NK细胞后均可检测到BCMA CAR和GPRC5D CAR的表达。BCMA CAR阳性率较高均可达60％以上；GPRC5D CAR阳性率偏低，表达率从高到低分别为BI-CAR06(69.60％)>BI-CAR21(54.20％)>BI-CAR20(54.00％)>BI-CAR18(32.30％)>BI-CAR19(13.80％)。
表22 NKG2D-F CAR表达率结果
13.3 NKG2D-F CAR-NK细胞增殖检测
通过AO/PI计数检测BI-CAR NK细胞的增殖，结果如图17和表23所示。BI-CAR NK 细胞激活后第11天增殖1473～2166倍左右，增殖速度由高到低分别为：BI-CAR19(2166倍)＝BI-CAR20(2166倍)＞BI-CAR21(1950倍)＞Parental NK(1690倍)＞BI-CAR18(1538倍)>BI-CAR06(1473倍)。根据以上结果可知，人源化抗体构建的BI-CAR18、BI-CAR19、BI-CAR20和BI-CAR21细胞增殖速度优于BI-CAR06。
表23 NKG2D-F CAR-NK细胞增殖检测
13.4 NKG2D-F CAR-NK多轮杀伤效果检测
逆转录病毒侵染NK细胞后第6天检测BI-CAR18、BI-CAR19、BI-CAR20、BI-CAR21和BI-CAR06对NCI H929、RPMI 8226、MOLP8、NCI H929-hBCMA-KO、NCI H929+20％(或者10％或者5％)H929-hBCMA-KO的异质肿瘤细胞的体外多轮杀伤效果。参考实施例7的方法测定对上述细胞的体外多轮杀伤效果
BI-CAR NK细胞的多轮杀伤试验的结果参见图18A-图18F结果显示，BI-CAR19、BI-CAR20、BI-CAR21的多轮杀伤效果优于BI-CAR18和BI-CAR06。BI-CAR21对NCI H929细胞的杀伤效果最优，第四轮杀伤(R4-1d)实验中仍能维持30％的细胞杀伤率；BI-CAR19，BI-CAR20和BI-CAR21对NCI H929+20％(或者10％或者5％)H929-hBCMA-KO的异质肿瘤细胞、RPMI 8226细胞和MOLP8细胞的杀伤效果最优，对NCI H929+20％(或者10％或者5％)H929-hBCMA-KO的异质肿瘤细胞的第四轮杀伤(R4-1d)实验中仍能维持30％以上的细胞杀伤率，对RPMI 8226细胞和MOLP8细胞第五轮杀伤(R5-1d)实验中仍能维持10％左右的细胞杀伤率。

Claims (26)

  1. 核酸分子，其包括编码嵌合抗原受体(chimeric antigen receptor，CAR)的第一核酸序列，所述嵌合抗原受体包括：包含抗原结合区的胞外区、连接所述胞外区的NKG2D跨膜区和连接所述跨膜区的胞内结构域，所述NKG2D跨膜区具有与SEQ ID NO:48相比至少80％同一性或至多9个突变的序列。
  2. 根据权利要求1所述的核酸分子，其中，所述抗原结合区具有(1)-(4)组中至少一组的性质：
    (1)所述抗原结合区选自抗体或其片段或结合所述抗原的配体，例如scFv、VHH、Fab、F(ab)’2或ligand；
    (2)所述抗原结合区结合或不结合NKG2DL；
    (3)所述抗原选自下组中的一个或多个：BCMA、GPRC5D、CLDN18.2、ROR1、CD19、CD33、CD5、CD70、HER2、IL13Rα2、GCC或GPC3；
    (4)所述抗原结合区至少结合两个不同的抗原、至少结合同一抗原的两个不同表位或结合同一表位的数量在两个以上。
  3. 根据权利要求1-2任一项所述的核酸分子，其中，所述抗原结合区特异性结合BCMA，包括：
    (1)VHH，其CDR1-3分别包括：
    如SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示的序列；或，
    如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:11所示的序列；或，
    如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的序列；
    (2)VHH，包括与SEQ ID NO:1或3具有至少80％同一性或至多25个突变的序列；
    (3)VHH，其CDR1-3分别包括：
    如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示的序列；或，
    如SEQ ID NO:17、SEQ ID NO:25和SEQ ID NO:19所示的序列；或，
    如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:19所示的序列；或，
    如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的序列；
    (4)与SEQ ID NO:2或4具有至少80％同一性或至多25个突变的序列；或，
    (5)VHH1-连接肽-VHH2，所述VHH1具有如所述组(1)或组(2)所示序列，所述VHH2具有如所述组(3)或组(4)所示序列；或，
    (6)VHH1-连接肽-VHH2，所述VHH1具有如所述组(3)或组(4)所示序列和所述VHH2具有如所述组(1)或组(2)所示序列；或，
    (7)VHH1-连接肽-VHH2，具有与SEQ ID NO:63相比至少80％同一性或至多50个突变的序列。
  4. 根据权利要求1-2所述的核酸分子，其中，所述抗原结合区特异性结合ROR1，包括：
    (1)抗体或其片段，包括HCDR1-3和LCDR1-3，所述HCDR1-3分别包括：
    如SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:32所示的序列；或，
    如SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:32所示的序列；或，
    如SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37所示的序列；
    和所述LCDR1-3分别包括：
    如SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的序列；或，
    如SEQ ID NO:38、SEQ ID NO39和SEQ ID NO:40所示的序列；或，
    如SEQ ID NO:41、SEQ ID NO:42和SEQ ID NO:40所示的序列；
    (2)抗体或其片段，包括与SEQ ID NO:26相比具有至少80％同一性或至多25个突变的重链可变区，和与SEQ ID NO:27相比至少80％同一性或至多25个突变的轻链可变区；
    (3)scFv，所述scFv具有所述组(1)或组(2)所示序列，可选地，所述scFv具有与SEQ ID NO:28相比至少80％同一性或至多50个突变的轻链可变区。
  5. 根据权利要求1-3任一项所述的核酸分子，其中，所述抗原结合区特异性结合GPRC5D，包括：
    (1)抗体或其片段，包括HCDR1-3和LCDR1-3，所述HCDR1-3分别包括：
    如SEQ ID NO:87、SEQ ID NO:88和SEQ ID NO:89所示的序列；或，
    如SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92所示的序列；或，
    如SEQ ID NO:96、SEQ ID NO:97和SEQ ID NO:98所示的序列；
    和所述LCDR1-3分别包括：
    如SEQ ID NO:84、SEQ ID NO:85和SEQ ID NO:86所示的序列；或，
    如SEQ ID NO:93、SEQ ID NO:94和SEQ ID NO:95所示的序列；
    (2)抗体或其片段，包括HCDR1-3，所述HCDR1-3分别包括：
    如SEQ ID NO:107-118所示的序列；
    (3)抗体或其片段，包括与SEQ ID NO:78、80、83、131相比具有至少80％同一性或至多25个突变的重链可变区，和与SEQ ID NO:77、79、81、82相比至少80％同一性或至多25个突变的轻链可变区；
    (4)抗体或其片段，包括与SEQ ID NO:99-106相比具有至少80％同一性或至多25个突变的重链可变区。
  6. 根据权利要求1-5任一项所述的核酸分子，其中，所述胞外区还包括铰链区，所述铰链区选自下组中的一个或多个：CD8α铰链区、2B4铰链区、CD28铰链区、IgG1铰链区、IgD铰链、IgG4铰链区、GS铰链、KIR2DS2铰链，KIR铰链，NCR铰链，SLAMF铰链，CD16铰链，CD64铰链或LY49铰链；可选地，所述铰链区选自CD8α铰链区，例如，所述铰链区具有与SEQ ID NO:45相比至少80％同一性或至多10个突变的序列。
  7. 根据权利要求1-6任一项所述的核酸分子，其中，所述胞外区还包括信号肽，所述信号肽选自下组中的一个或多个：CD8α信号肽、IgG1重链信号肽或GM-CSFR2信号肽；可选地，所述信号肽为CD8α信号肽，例如，所述信号肽具有与SEQ ID NO:43相比至少80％同一性或至多5个突变的序列。
  8. 根据权利要求1-7任一项所述的核酸分子，其中，所述胞内结构域包括胞内信号传导结构域和/或共刺激结构域；
    可选地，所述胞内信号传导结构域为CD3ζ，例如，所述胞内信号传导结构域具有与SEQ ID NO:55相比至少80％同一性或至多35个突变的序列；
    可选地，所述共刺激结构域选自下组中的一个或多个：CD27共刺激结构域、4-1BB共刺激结构域、OX40共刺激结构域、2B4共刺激结构域、CD28共刺激结构域、ICOS共刺激结构域、DAP10共刺激结构域或DAP12共刺激结构域；可选地，所述共刺激结构域为2B4共刺激结构域，例如，所述共刺激结构域具有与SEQ ID NO:53相比至少80％同一性或至多25个突变的序列。
  9. 根据权利要求1-8任一项所述的核酸分子，其中，所述嵌合抗原受体包括：CD8α铰链区、NKG2D跨膜区、2B4共刺激结构域和CD3ζ胞内信号传导结构域，例如，所述嵌合抗原受体具有与SEQ ID NO:58相比至少80％同一性或至多50个突变的序列。
  10. 根据权利要求1-9任一项所述的核酸分子，其中，所述核酸分子还包括编码IL15的第二核酸序列，可选地，所述IL15选自可溶性IL15、膜结合型IL15或IL15与其受体或受体片段的复合物，可选地，所述IL15具有与SEQ ID NO:57相比至少具有80％同一性或至多35个突变的序列；
    可选地，所述第一核酸序列和所述第二核酸序列通过IRES或编码自裂解肽的序列连接，所述自裂解肽选自2A肽，例如P2A、T2A、F2A或E2A，可选地，所述自裂解肽为P2A，例如具有与SEQ ID NO:56相比具有至少80％同一性或至多5个突变的序列；
    可选地，所述核酸编码与SEQ ID NO:59相比具有至少85％同一性或至多100个突变的序列的核酸序列。
  11. 根据权利要求1-10任一项所述的核酸分子，其包括编码与SEQ ID NO:65或SEQ ID NO:70相比具有至少80％同一性或至多150个突变的序列的核酸序列。
  12. 根据权利要求1-11任一项所述的核酸分子，其中，所述核酸为DNA或RNA，所述RNA优选为mRNA。
  13. 核酸分子，所述核酸分子包含编码靶向ROR1的嵌合抗受体的第一核酸序列，所述嵌合抗原受体包括包含抗原结合区的胞外区、连接所述胞外区的跨膜区和连接所述跨膜区的胞内结构域，所述抗原结合区包括：
    (1)抗体或其片段，包括HCDR1-3和LCDR1-3，所述HCDR1-3分别包括：
    如SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:32所示的序列；或，
    如SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:32所示的序列；或，
    如SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37所示的序列；
    和所述LCDR1-3分别包括：
    如SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的序列；或，
    如SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示的序列；或，
    如SEQ ID NO:41、SEQ ID NO:42和SEQ ID NO:40所示的序列；
    (2)抗体或其片段，包括与SEQ ID NO:26相比具有至少80％同一性或至多25个突变的重链可变区，和与SEQ ID NO:27相比至少80％同一性或至多25个突变的轻链可变区；
    (3)scFv，所述scFv具有所述组(1)或组(2)所示序列，可选地，所述scFv具有与SEQ ID NO:28相比至少80％同一性或至多50个突变的轻链可变区。
  14. 根据权利要求12或13所述的核酸分子，其中，所述嵌合抗原受体包括：CD8α铰链区、NKG2D跨膜区、2B4共刺激结构域和CD3ζ胞内信号传导结构域，例如，所述嵌合抗原受体具有与SEQ ID NO:58相比至少80％同一性或至多50个突变的序列。
  15. 根据权利要求12-14任一项所述的核酸分子，其中，所述核酸分子还包括编码IL15的第二核酸序列，可选地，所述IL15选自可溶性IL15、膜结合型IL15或IL15与其受体或受体片段的复合物，可选地，所述IL15具有与SEQ ID NO:57相比至少具有80％同一性或至多35个突变的序列；
    可选地，所述第一核酸序列和所述第二核酸序列通过IRES或编码自裂解肽的序列连接，所述自裂解肽选自2A肽，例如P2A、T2A、F2A或E2A，可选地，所述自裂解肽为P2A，例如具有与SEQ ID NO:56相比具有至少80％同一性或至多5个突变的序列；
    可选地，所述核编码与SEQ ID NO:59相比具有至少85％同一性或至多100个突变的序列的核酸序列。
  16. 根据权利要求12-15任一项所述的核酸分子，其包括编码与SEQ ID NO:65或SEQ ID NO:70相比具有至少80％同一性或至多150个突变的序列的核酸序列。
  17. 根据权利要求1-16任一项所述核酸分子编码的嵌合抗原受体。
  18. 载体，其中，所述载体包括权利要求1-16任一项所述的核酸分子。
  19. 免疫效应细胞，其中，所述免疫效应细胞包括权利要求1-16任一项所述核酸分子或权利要求17所述嵌合抗原受体或权利要求18所述载体。
  20. 根据权利要求19所述的免疫效应细胞，其中，所述免疫效应细胞为NK细胞，所述NK细胞选自iPSC分化而来的NK细胞、外周血或脐带血衍生NK细胞或NK92细胞。
  21. 权利要求19-20任一项所述免疫效应细胞的制备方法，其中，包括：提供免疫效应细胞以及将所述核酸分子转入所述免疫效应细胞的步骤。
  22. 根据权利要求21所述方法制备而成的产品。
  23. 药物组合物，其中，所述药物组合物包括权利要求1-16任一项所述核酸分子或权利要求17所述嵌合抗原受体或权利要求18所述载体、权利要求19-20任一项所述免疫效应细胞或权利要求22所述的产品，以及药学上可接受的载体。
  24. 权利要求1-16任一项所述核酸分子或权利要求17所述嵌合抗原受体或权利要求18所述载体、权利要求19-20任一项所述免疫效应细胞、权利要求22所述的产品或权利要求23所述药物组合物在制备用于治疗癌症或肿瘤的药物中的用途，所述癌症或肿瘤选自血液瘤或实体瘤；可选地，所述血液瘤选自骨髓瘤、淋巴瘤或白血病，例如多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、急性髓细胞性白血病(AML)、慢性淋巴细胞性白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓细胞 性白血病(CML)或毛细胞白血病(HCL)；可选的地，所述实体瘤选自肺癌、乳腺癌、结直肠癌、胃癌、胰腺癌、肝癌、皮肤癌、膀胱癌、卵巢癌、子宫癌、前列腺癌或肾上腺癌。
  25. 权利要求1-16任一项所述核酸分子或权利要求17所述嵌合抗原受体或权利要求18所述载体、权利要求19-20任一项所述免疫效应细胞、权利要求22所述的产品或权利要求23所述药物组合物，用于治疗癌症或肿瘤的用途，所述癌症或肿瘤选自血液瘤或实体瘤；可选地，所述血液瘤选自骨髓瘤、淋巴瘤或白血病，例如多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、急性髓细胞性白血病(AML)、慢性淋巴细胞性白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓细胞性白血病(CML)或毛细胞白血病(HCL)；可选的地，所述实体瘤选自肺癌、乳腺癌、结直肠癌、胃癌、胰腺癌、肝癌、皮肤癌、膀胱癌、卵巢癌、子宫癌、前列腺癌或肾上腺癌。
  26. 治疗癌症或肿瘤的方法，所述方法包括向有需要的受试者给予有效量的权利要求1-16任一项所述核酸分子或权利要求17所述嵌合抗原受体或权利要求18所述载体、权利要求19-20任一项所述免疫效应细胞、权利要求22所述的产品或权利要求23所述药物组合物，所述癌症或肿瘤选自血液瘤或实体瘤；可选地，所述血液瘤选自骨髓瘤、淋巴瘤或白血病，例如多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、急性髓细胞性白血病(AML)、慢性淋巴细胞性白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓细胞性白血病(CML)或毛细胞白血病(HCL)；可选的地，所述实体瘤选自肺癌、乳腺癌、结直肠癌、胃癌、胰腺癌、肝癌、皮肤癌、膀胱癌、卵巢癌、子宫癌、前列腺癌或肾上腺癌。
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