WO2024051751A1 - 抗cd123的纳米抗体、嵌合抗原受体及其应用 - Google Patents

抗cd123的纳米抗体、嵌合抗原受体及其应用 Download PDF

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WO2024051751A1
WO2024051751A1 PCT/CN2023/117316 CN2023117316W WO2024051751A1 WO 2024051751 A1 WO2024051751 A1 WO 2024051751A1 CN 2023117316 W CN2023117316 W CN 2023117316W WO 2024051751 A1 WO2024051751 A1 WO 2024051751A1
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seq
cells
amino acid
chimeric antigen
vector
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French (fr)
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李建强
王琳
刘莹
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河北森朗生物科技有限公司
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Priority claimed from CN202211084440.0A external-priority patent/CN116041535A/zh
Priority claimed from CN202211084467.XA external-priority patent/CN115724972A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine technology. Specifically, the invention relates to anti-CD123 Nanobodies, chimeric antigen receptors and their applications.
  • CD123 also known as IL-3 receptor alpha chain (IL-3R ⁇ ), is a glycoprotein with a molecular weight of 75 kDa. It is only expressed on cells that bind IL-3, such as B lymphocytes, megakaryocytes, and hematopoietic stem/progenitor cells. , plasmacytoid dendritic cells and monocytes. CD123 can induce tyrosine phosphorylation after binding to its ligand IL-3, thereby promoting the proliferation and differentiation of hematopoietic cells and participating in innate and adaptive immune responses, inflammatory responses, etc. Relevant researchers have found that 45% of acute myelogenous leukemia (AML) patients have overexpression of CD123.
  • AML acute myelogenous leukemia
  • AML cells overexpressing CD123 have higher proliferative activity and resistance to apoptosis, and there is the presence of STAT5. With sustained phosphorylation, patients have a higher tumor burden at initial diagnosis and a worse clinical prognosis.
  • CD123 is highly expressed in leukemia stem cells and more differentiated leukemia blast cells, while it is low or not expressed in normal hematopoietic stem cells. Therefore, CD123 is a leukemia-related antigen and a specific antigen of AML, which makes CD123 a A promising target for the treatment of AML. Due to the high expression of CD123 in AML, immunotherapy targeting CD123 has theoretically safer and more effective therapeutic effects. Clinical trials of targeted drugs targeting CD123 are already underway at home and abroad, but their efficacy is limited and they are still safe. Sexual problems occur, therefore, it is of great significance to screen effective CD123-targeting Nanobodies.
  • CAR-T cell immunotherapy In recent years, more and more research has been conducted on translational medicine in the field of immunology, especially tumor immunity.
  • the most widely used cancer immunotherapy strategy is CAR-T cell immunotherapy. In the early 1990s, this immunotherapy strategy achieved good results in translational medicine.
  • CAR-T cells targeting CD123 molecules are widely used, especially in the treatment of hematological tumors such as AML.
  • Effective stimulation of T cells in normal bodies and their immunological functions require the regulation of multiple signals, which mainly include the recognition of T cell receptors and MHC-antigen peptide complexes as the first signal, and the recognition and activation of co-stimulatory molecules on the surface of T cells as The second signal even requires cytokines to participate in the formation of the third signal.
  • This chimeric antigen receptor mainly artificially integrates protein molecules required for the process of T cell stimulation and activation in series, thereby promoting the activation and specific killing of T cells.
  • the specific killing of CAR-T cells mainly relies on the recognition and binding of the antibody molecule part at the front end.
  • the most widely used method is the specific recognition of target proteins based on the single chain variable region (Single chain Fv, ScFv) of monoclonal antibodies from humans or other species.
  • ScFv has the same affinity as Nanobodies, ScFv has a larger molecular weight than Nanobodies, and has certain limitations in molecular expression and function; ScFv is derived from its parent monoclonal antibody and has There may be certain deficiencies in aspects such as activity and stability.
  • the CD7 molecule is a cell surface glycoprotein with a molecular weight of approximately 40kDa and is a member of the immunoglobulin superfamily. CD7 molecules are mainly expressed on the surface of most thymocytes, more than 85% of peripheral blood T lymphocytes, and natural killer cells. Although current research indicates that the specific function of the CD7 molecule is less clear, experimental studies have shown that CD7-deficient murine T lymphocytes respond normally to stimulation and that when antibodies bind to the CD7 molecule on human T lymphocytes, the cells grow and proliferate. and has no impact. At the same time, an important property of the CD7 molecule is that it undergoes rapid endocytosis after binding to antibodies.
  • CD7 CAR-T cells targeting CD7 have been verified in the treatment of malignant tumors derived from CD7+ T cells; at the same time, the tumor cells of approximately 1/3 of AML patients also express the CD7 antigen, and it has been Cases have confirmed that CD7 CAR-cells can be efficiently expanded in the body after being infused back into CD7+ AML patients, and can eliminate CD7+ AML cells very efficiently.
  • one object of the present invention is to provide anti-CD123 Nanobodies, chimeric antigen receptors and their applications.
  • the present invention obtained high affinity and high specificity anti-CD123 nanobodies VHH01 and VHH02 through screening, and used them as the antigen-binding domains of chimeric antigen receptor molecules to construct CAR-T cells. After experimental verification, it was found that the CAR -T cells can specifically recognize and kill CD123-positive cells and have important application prospects in the field of tumor treatment.
  • Another object of the present invention is to provide a chimeric antigen receptor targeting CD123 and CD7, and provide a new research strategy for the treatment of CD123 and/or CD7 related diseases in this field.
  • a first aspect of the invention provides anti-CD123 Nanobodies.
  • Nanobodies include VHH01 and VHH02;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the VHH01 are as shown in SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7 respectively or are the same as SEQ ID NO:3, SEQ ID NO:5 and SEQ respectively.
  • ID NO:7 Amino acid sequence with at least 75% identity
  • amino acid sequences of CDR1, CDR2 and CDR3 of the VHH02 are as shown in SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15 respectively or are the same as SEQ ID NO: 11, SEQ ID NO: 13 and SEQ respectively.
  • ID NO:15 Amino acid sequence with at least 75% identity.
  • nucleotide sequences of CDR1, CDR2, and CDR3 of VHH01 are as shown in SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 respectively or are the same as SEQ ID NO: 4 and SEQ ID NO respectively. :6.
  • SEQ ID NO:8 is a nucleotide sequence with at least 75% identity;
  • SEQ ID NO:12 The nucleotide sequences of CDR1, CDR2 and CDR3 of the VHH02 are as shown in SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16 respectively or are the same as SEQ ID NO:12 and SEQ ID NO:14 respectively.
  • SEQ ID NO:16 has a nucleotide sequence of at least 75% identity.
  • amino acid sequence of the heavy chain variable region of the VHH01 is as shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity with SEQ ID NO: 1;
  • amino acid sequence of the heavy chain variable region of the VHH02 is as shown in SEQ ID NO:9 or an amino acid sequence that has at least 75% identity with SEQ ID NO:9;
  • the nucleotide sequence of the heavy chain variable region of the VHH01 is as shown in SEQ ID NO:2 or a nucleotide sequence that is at least 75% identical to SEQ ID NO:2;
  • the nucleotide sequence of the heavy chain variable region of the VHH02 is as shown in SEQ ID NO: 10 or a nucleotide sequence having at least 75% identity with SEQ ID NO: 10.
  • the amino acid sequence or nucleotide sequence having at least 75% identity refers to having 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% identical amino acid sequences.
  • antibodies that include conservative sequence variants of the preferred antibody amino acid sequences are also included within the scope of the present invention.
  • Conservative amino acid sequence variants include modifications of the amino acid sequence that do not significantly change the binding properties and neutralizing properties of the monoclonal neutralizing antibodies of the invention, such as variants derived from similar amino acid substitutions well known in the art, resulting from the deletion or addition of amino acids. Variants are within the scope of the present invention.
  • the Nanobodies of the present invention also include human and non-human antibodies, as well as all antibodies with the same function as the monoclonal antibodies of the present invention or modified and optimized.
  • a second aspect of the invention provides anti-CD123 Nanobody-based chimeric antigen receptors.
  • the chimeric antigen receptor includes the Nanobody described in the first aspect of the present invention.
  • the chimeric antigen receptor further comprises a signal peptide
  • the chimeric antigen receptor further comprises a hinge region
  • the chimeric antigen receptor further comprises a transmembrane domain
  • the chimeric antigen receptor further comprises a costimulatory signaling domain
  • the chimeric antigen receptor further comprises an intracellular signaling domain
  • the chimeric antigen receptor also includes EF1 ⁇ , T2A, and tEGFR;
  • the chimeric antigen receptor further comprises a tEGFR signal peptide
  • the signal peptide includes the signal peptide of the following molecules: ⁇ chain and ⁇ chain of T cell receptor, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6;
  • the hinge region includes the hinge region of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3 ⁇ , IL-2 receptor, IL-7 receptor, IL -11 receptor;
  • the transmembrane domain includes the transmembrane domain of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3 ⁇ , CD8 ⁇ , IL-2 receptor, IL-7 Receptor, IL-11 receptor;
  • the costimulatory signal domain includes the costimulatory signal domain of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, CD4, CD8 ⁇ , CD8 ⁇ , HVEM, LIGHT, CD40, OX40, DR3, GITR , CD30, TIM1, CD2, CD226, CD278;
  • the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , CD4, CD5, CD8, CD21, CD22, CD79a, CD79b , CD278, Fc ⁇ RI, DAP10, DAP12, CD66d, FYN;
  • the hinge region is a CD8 hinge region
  • the transmembrane domain is a CD8 transmembrane domain
  • the costimulatory signal domain is a 4-1BB costimulatory signal domain
  • the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain
  • the chimeric antigen receptor is EF1 ⁇ , signal peptide, Nanobody according to the first aspect of the present invention, CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory signal domain, CD3 ⁇ intracellular
  • the signaling domain, T2A, tEGFR signal peptide, and tEGFR are sequentially connected in series;
  • the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO: 19 or has an amino acid sequence that is at least 75% identical to SEQ ID NO: 19;
  • the amino acid sequence of the CD8 transmembrane domain is as shown in SEQ ID NO:21 or has an amino acid sequence that is at least 75% identical to SEQ ID NO:21;
  • amino acid sequence of the 4-1BB costimulatory signal domain is as shown in SEQ ID NO: 23 or has an amino acid sequence that is at least 75% identical to SEQ ID NO: 23;
  • the amino acid sequence of the CD3 ⁇ intracellular signaling domain is as shown in SEQ ID NO:25 or has an amino acid sequence that is at least 75% identical to SEQ ID NO:25;
  • amino acid sequence of the signal peptide is as shown in SEQ ID NO: 17 or an amino acid sequence with at least 75% identity to SEQ ID NO: 17;
  • amino acid sequence of the T2A is as shown in SEQ ID NO: 27 or an amino acid sequence with at least 75% identity to SEQ ID NO: 27;
  • amino acid sequence of the tEGFR signal peptide is as shown in SEQ ID NO:29 or an amino acid sequence that has at least 75% identity with SEQ ID NO:29;
  • amino acid sequence of the tEGFR is as shown in SEQ ID NO:31 or has an amino acid sequence that is at least 75% identical to SEQ ID NO:31.
  • a third aspect of the invention provides nucleic acid molecules.
  • nucleic acid molecule encodes the Nanobody described in the first aspect of the present invention or encodes the chimeric antigen receptor described in the second aspect of the present invention.
  • nucleic acid molecule encoding the Nanobody according to the first aspect of the present invention or encoding the chimeric antigen receptor according to the second aspect of the present invention includes nucleic acid molecules having conservative nucleotide sequence variants of the above nucleotide sequence. So-called conservative nucleotide sequence variants are derived from degenerate and silent variants of the genetic code, and substitutions, deletions and additions of nucleotides are also included in the protection scope of the present invention.
  • the present invention also provides a DNA fragment of a nucleic acid molecule as described above.
  • the DNA fragment can encode any region of the heavy chain variable region of the Nanobody described in the first aspect of the present invention, including but not limited to the heavy chain. CDR1, CDR2, CDR3 of the variable region.
  • the nucleic acid molecules of the invention may be synthesized, for example, by standard chemical synthesis methods and/or recombinant methods, or produced semi-synthetically, for example, by combined chemical synthesis and recombinant methods.
  • Ligation of coding sequences to transcriptional regulatory elements and/or to other amino acid coding sequences can be performed using established methods, such as restriction digestion, ligation and molecular cloning.
  • a fourth aspect of the invention provides recombinant expression vectors.
  • the recombinant expression vector contains the nucleic acid molecule described in the third aspect of the present invention.
  • the expression vector includes a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, and a virus-derived vector;
  • the virus-derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated virus vector, a poxvirus vector, and a herpes virus vector.
  • the recombinant expression vector also contains an expression control sequence operatively linked to the nucleic acid molecule sequence.
  • An expression vector refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can transform, transduce or transfect the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • Types of vectors include bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors that are well known in the art. In short, any plasmid and vector can be used as long as it can replicate and be stable in the host body.
  • the expression vector may also contain marker genes and other translation control elements.
  • a fifth aspect of the invention provides engineered host cells.
  • the engineered host cell expresses the nucleic acid molecule described in the third aspect of the present invention and the recombinant expression vector described in the fourth aspect of the present invention;
  • the host cells include eukaryotic cells and prokaryotic cells;
  • the host cell is a eukaryotic cell
  • the eukaryotic cells include mammalian cells, plant cells, and yeast cells;
  • the eukaryotic cells are immune cells
  • the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof;
  • the immune cells are T cells.
  • a sixth aspect of the invention provides a Nanobody-drug conjugate or kit or composition.
  • Nanobody-drug conjugate is formed by covalently attaching the Nanobody described in the first aspect of the present invention to a small molecule drug;
  • the small molecule drugs include alkylating agents, antimetabolites, anti-tumor antibiotics, mitosis inhibitors, chromatin function inhibitors, anti-angiogenic agents, anti-estrogens, anti-androgens, and immunomodulators;
  • the alkylating agent includes bischloroethyl methylamine, chlorambucil, phenylalanine, bromopropazine, methapine, estramustine phosphate, cyclophosphamide, hexamethonium Melamine, cyclophosphamide chloride, isophosphamide, phosphotriamine, carboxamide Nitrogen mustard, streptozotomycin, fortimutine, cyclohexylnitrosourea, busulfan, sulfan, improsulfan, dacarbamide, cisplatin, oxaliplatin, carboplatin;
  • the antimetabolite includes methotrexate, 5-fluorouracil, fluoroside, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, cytarabine , 6-mercaptopurine (6-MP), 6-mercaptoguanine (6-TG), 2-chlorodeoxyadenosine, 5-azacytidine, 2,2-difluorodeoxycytosine nucleoside, cladrib Bin, deoxycoformycin, pentostatin;
  • the anti-tumor antibiotics include doxorubicin, daunorubicin, mitoxantrone, valrubicin, mitoxantrone hydrochloride, dactinomycin, mitoxantrone, mithramycin, Mitomycin C, bleomycin, procarbazine;
  • the mitosis inhibitor includes paclitaxel, docetaxel, vinblastine, vincristine, vindesamide, and vinorelbine;
  • the chromatin function inhibitor includes topotecan, irinotecan, etopoxa, etopoxa phosphate, and thiopodophyllin;
  • the anti-angiogenic agent includes proximine, mastat, bamamastat, prinostat, tannostat, ilomastat, CGS-27023A, bromocloperaquilone , COL-3, neovalastat, BMS-275291, thalidomide;
  • the anti-estrogens include anatozole, letrozole, tamoxifen, toremifene, raloxifene, droloxifene, oxydoxifene, and exemestane;
  • the anti-androgens include flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride, and cimetidine;
  • the immunomodulatory agent includes interferon, interleukin, tumor necrosis factor, mushroom polysaccharide, cizorose, roquinamac, pidotimod, methoxypolyethylene glycol succinamide adenosine deaminase, Thymosin preparations;
  • the kit or composition includes the Nanobody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the third aspect of the present invention.
  • the present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the Nanobody described in the first aspect of the present invention and the engineered host cell described in the fifth aspect of the present invention.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier, which includes but is not limited to any adjuvant, carrier, excipient, or excipient that has been approved by the U.S. Food and Drug Administration and can be used in humans or animals.
  • a pharmaceutically acceptable carrier which includes but is not limited to any adjuvant, carrier, excipient, or excipient that has been approved by the U.S. Food and Drug Administration and can be used in humans or animals.
  • the present invention also provides a detection product comprising the Nanobody or antigen-binding fragment thereof according to the first aspect of the present invention.
  • the detection products include but are not limited to detection reagents, test kits, chips or test strips. Any detection product that can detect CD123 including the Nanobody or antigen-binding fragment thereof described in the first aspect of the present invention is included in the protection scope of the present invention.
  • the seventh aspect of the present invention provides any of the following methods:
  • a method for producing the Nanobody according to the first aspect of the present invention comprising the following steps: culturing the engineered host cells according to the fifth aspect of the present invention, and isolating the nanobody from the culture. Nanobodies according to the first aspect;
  • a method for detecting CD123 in a sample to be tested for non-diagnostic and non-therapeutic purposes includes the following steps: contacting the sample to be tested with the Nanobody described in the first aspect of the present invention, and detecting the Nanobody Formation of complex with CD123;
  • the Nanobody is a Nanobody labeled with a label that can be used for detection;
  • the markers that can be used for detection include fluorescent dyes, avidin, paramagnetic atoms, and radioactive isotopes;
  • the fluorescent pigment is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, polydinophycoxanthin-chlorophyll protein;
  • the avidin is biotin, ovalvidin, streptavidin, vitellavidin, or avidin;
  • the radioactive isotope is radioactive iodine, radioactive cesium, radioactive iridium, and radioactive cobalt;
  • a method for preparing the engineered host cell according to the fifth aspect of the present invention comprising the following steps: introducing the recombinant expression vector according to the fourth aspect of the present invention into the host cell;
  • the introduced methods include physical methods, chemical methods, and biological methods;
  • the physical method includes calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation;
  • the chemical method includes colloidal dispersion systems, lipid-based systems;
  • the colloidal dispersion system includes macromolecular complexes, nanocapsules, microspheres, and beads;
  • the lipid-based system includes oil-in-water emulsions, micelles, mixed micelles, liposomes;
  • the biological method includes DNA vectors, RNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors;
  • a method for specifically inhibiting CD123 activity in vitro which method includes the following steps: introducing the nucleic acid molecule described in the third aspect of the present invention into biological cells, and expressing the nucleic acid molecule described in the first aspect of the present invention. Nanobodies inhibit the activity of CD123;
  • a method for detecting CD123 in a sample to be tested for diagnostic or therapeutic purposes includes the following steps: contacting the sample to be tested with the Nanobody according to the first aspect of the present invention, and detecting the interaction between the Nanobody and CD123. The formation of complex;
  • a method for diagnosing CD123-related diseases which uses the Nanobody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, The recombinant expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, the Nanobody-drug conjugate or kit or composition described in the sixth aspect of the present invention;
  • a method of treating CD123-expressing tumors comprising administering the Nanobody according to the first aspect of the present invention and the chimeric antigen receptor according to the second aspect of the present invention to a subject in need thereof , the nucleic acid molecule described in the third aspect of the present invention, the recombinant expression vector described in the fourth aspect of the present invention, the engineered host cell described in the fifth aspect of the present invention, the Nanobody described in the sixth aspect of the present invention - Drug conjugates or kits or compositions;.
  • the eighth aspect of the present invention provides applications in any of the following aspects:
  • the engineered host cells described in the fifth aspect of the present invention, the Nanobody-drug conjugate or kit or composition described in the sixth aspect of the present invention are used in the preparation of products for detecting CD123 protein or antigen fragments thereof Applications in;
  • Nanobodies according to the first aspect of the present invention chimeric antigen receptors according to the second aspect of the present invention, nucleic acid molecules according to the third aspect of the present invention, and recombinant expression vectors according to the fourth aspect of the present invention.
  • Nanobodies according to the first aspect of the present invention chimeric antigen receptors according to the second aspect of the present invention, nucleic acid molecules according to the third aspect of the present invention, and recombinant expression vectors according to the fourth aspect of the present invention , described in the fifth aspect of the present invention
  • the tumor is a hematological tumor
  • the tumor includes acute myeloid leukemia, acute B lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia, acute T lymphocytic leukemia, chronic B lymphocytic leukemia, chronic T lymphocyte leukemia, Richter syndrome, hairy cell leukemia, non-Hodgkin lymphoma, small lymphocytic lymphoma, Hodgkin lymphoma, Burkitt lymphoma.
  • CD123-related diseases refer to diseases with high expression of CD123, including hematological tumors with high expression of CD123, etc.
  • Nanobodies disclosed in the present invention may contain one or more glycosylation sites in the heavy chain variable region.
  • one or more glycosylation sites present in the weight variable region may Enhance antibody immunogenicity, or alter antibody pharmacokinetics due to altered antigen binding.
  • Nanobodies disclosed in the present invention can be conjugated to other factors chemically or through genetic engineering. These factors serve to target the antibody to a desired functional site or to enhance or provide other properties to the antibody.
  • Nanobodies according to the present invention can be labeled chemically or by genetic engineering to provide detectable Nanobodies, which include detectable moieties.
  • Detectable moieties include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions.
  • Labeling for detection and/or analysis and/or diagnostic purposes depends on the specific detection/analysis/diagnostic technology and/or method used such as immunohistochemical staining of (tissue) samples, flow cytometry, laser scanning cytometry detection, Fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), bioassays (e.g., phagocytosis assays), Western blot applications, etc.
  • Suitable labels for detection/analytical/diagnostic techniques and/or methods known in the art are well known to those skilled in the art.
  • a ninth aspect of the invention provides a chimeric antigen receptor targeting CD123 and CD7.
  • the chimeric antigen receptor includes Nanobody VHH01 that specifically binds to CD123 and Nanobody VHH10 that specifically binds to CD7;
  • amino acid sequences of CDR1, CDR2 and CDR3 of the Nanobody VHH01 are as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively or are the same as SEQ ID NO:3 and SEQ ID NO respectively. :4.
  • SEQ ID NO:5 is an amino acid sequence with at least 75% identity;
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the Nanobody VHH10 are as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44 respectively or are the same as SEQ ID NO: 42 and SEQ ID NO respectively.
  • SEQ ID NO:44 has an amino acid sequence with at least 75% identity;
  • the amino acid sequence of the Nanobody VHH01 is as shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity with SEQ ID NO: 1;
  • the amino acid sequence of the Nanobody VHH10 is as shown in SEQ ID NO: 45 or an amino acid sequence that has at least 75% identity with SEQ ID NO: 45;
  • the chimeric antigen receptor also includes a transmembrane domain and an intracellular signaling domain;
  • the chimeric antigen receptor further includes a hinge region
  • the chimeric antigen receptor further includes a costimulatory signaling domain
  • the chimeric antigen receptor further includes a signal peptide
  • the chimeric antigen receptor also includes EF1 ⁇ , T2A, and tEGFR;
  • the chimeric antigen receptor further includes a Linker connecting VHH01 and VHH10;
  • the chimeric antigen receptor further includes a tEGFR signal peptide
  • the transmembrane domain and hinge region include the transmembrane domain and hinge region of the following molecules: CD8, 4-1BB, PD-1, CD34, CD28, IgG1, IgG4, OX40, CD3 ⁇ ;
  • transmembrane domain and hinge region are CD8 transmembrane domain and CD8 hinge region;
  • amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO: 21;
  • amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 19;
  • the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278 , Fc ⁇ RI, DAP10, DAP12, CD66d, DAP10, DAP12, FYN;
  • the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain
  • amino acid sequence of the CD3 ⁇ intracellular signaling domain is shown in SEQ ID NO: 25;
  • the costimulatory signal domain includes the costimulatory signal domain of the following molecules: 4-1BB, ICOS, CD27, CD19, CD4, CD28, CD8 ⁇ , CD8 ⁇ , HVEM, LIGHT, CD40, 4-1BB, OX40 , DR3, GITR, CD30, TIM1, CD2, CD226, CD278;
  • the costimulatory signal domain is a 4-1BB costimulatory signal domain
  • amino acid sequence of the 4-1BB costimulatory signal domain is shown in SEQ ID NO: 23;
  • the signal peptide includes the signal peptide of the following molecules: ⁇ chain and ⁇ chain of T cell receptor, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6;
  • amino acid sequence of the signal peptide is as shown in SEQ ID NO: 17;
  • nucleotide sequence of EF1 ⁇ is shown in SEQ ID NO: 47;
  • amino acid sequence of T2A is shown in SEQ ID NO: 27;
  • amino acid sequence of tEGFR is shown in SEQ ID NO: 31;
  • amino acid sequence of the Linker is shown in SEQ ID NO: 48;
  • amino acid sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 29;
  • the chimeric antigen receptor is selected from any one of the following groups:
  • a chimeric antigen receptor formed by substituting, deleting or adding one or more amino acids on the basis of the amino acid sequence of the chimeric antigen receptor described in (1);
  • a chimeric antigen receptor formed by substituting, deleting or adding one or more amino acids on the basis of the amino acid sequence of the chimeric antigen receptor described in (2).
  • a tenth aspect of the invention provides a polynucleotide.
  • sequence of the polynucleotide is the coding sequence of the chimeric antigen receptor described in the ninth aspect of the present invention, or its complementary sequence.
  • nucleotide sequence of Nanobody VHH01 is shown in SEQ ID NO:2
  • nucleotide sequence of Nanobody VHH10 is shown in SEQ ID NO.
  • the nucleotide sequence of the CD8 transmembrane domain is shown in SEQ ID NO:22
  • the nucleotide sequence of the CD8 hinge region is shown in SEQ ID NO:20
  • the core of the CD3 ⁇ intracellular signaling domain The nucleotide sequence is shown in SEQ ID NO:26
  • the nucleotide sequence of the 4-1BB costimulatory signal domain is shown in SEQ ID NO:24
  • the nucleotide sequence of the signal peptide is shown in SEQ ID
  • the nucleotide sequence of EF1 ⁇ is shown in SEQ ID NO:47
  • the nucleotide sequence of T2A is shown in SEQ ID NO:28
  • the nucleotide sequence of tEGFR is shown in SEQ ID NO:32.
  • the nucleotide sequence of Linker is shown in SEQ ID NO:50
  • the nucleotide sequence of tEGFR signal peptide is shown in SEQ ID NO:30.
  • An eleventh aspect of the present invention provides a vector containing the polynucleotide of the tenth aspect of the present invention.
  • the vector includes a cloning vector and an expression vector
  • the vector includes a DNA vector, an RNA vector, a plasmid, and a virus-derived vector;
  • the virus-derived vectors include lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, poxvirus vectors, and herpes virus vectors.
  • a twelfth aspect of the present invention provides a genetically engineered host cell.
  • the genetically engineered host cell contains the polynucleotide described in the tenth aspect of the present invention or the vector described in the eleventh aspect of the present invention;
  • the host cell is selected from eukaryotic cells or prokaryotic cells;
  • the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells;
  • the prokaryotic cells include mycoplasma, chlamydia, rickettsia, bacteria, actinomycetes, and cyanobacteria;
  • the host cell is a eukaryotic cell
  • the eukaryotic cell is a mammalian cell
  • the mammalian cells are immune cells
  • the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof;
  • the immune cells are T cells.
  • a thirteenth aspect of the present invention provides a universal CAR-T cell targeting CD123 and CD7.
  • the universal CAR-T cell expresses the chimeric antigen receptor described in the ninth aspect of the present invention and does not express CD7 and TCR;
  • the T cells are derived from healthy volunteers or donors;
  • the CD7 encoding gene and the TCR encoding gene in the universal CAR-T cells are knocked out;
  • the ⁇ chain and/or ⁇ chain constant coding region (i.e., TRAC and/or TRBC) genes of the TCR are knocked out;
  • the alpha chain constant coding region (i.e., TRAC) gene of the TCR is knocked out;
  • the CD7 coding gene and the TCR coding gene are knocked out by a complex formed by the Cas9 protein, CD7-sgRNA and TRAC-sgRNA introduced into the T cell;
  • CD7-sgRNA is shown in SEQ ID NO: 51;
  • the sequence of the TRAC-sgRNA is shown in SEQ ID NO: 52.
  • the present invention reduces cell suicide by knocking out endogenous CD7 of T cells, improves the success rate of preparation, and eliminates the expression of TCR by knocking out the ⁇ chain constant coding region of TCR (ie, TRAC) , Reduce the risk of graft-versus-host disease (GvHD) caused by allogeneic T cells.
  • CAR targeting CD7 can specifically target the patient's own T cells and natural killer (NK) cells (alloreactive killer cells) to reduce host versus graft rejection (HvG), while CAR targeting CD123 can eliminate AML tumor cells.
  • the bispecific nano-universal CAR-T cell targeting CD123 and CD7 is UCAR0901T, which has significant ability to kill tumor cells, and the universal CAR-T cell product is used in patients It can be used directly when needed, without the need to extract T cells from patients who are already seriously ill and then customize the product.
  • a fourteenth aspect of the invention provides a derivative.
  • the derivatives include detectably labeled chimeric antigen receptors according to the ninth aspect of the present invention and/or polynucleotides according to the tenth aspect of the present invention, and antibiotic resistance-conferring chimeric antigen receptors according to the ninth aspect of the present invention.
  • the chimeric antigen receptor described above and/or the polynucleotide described in the tenth aspect of the present invention, the chimeric antigen receptor described in the ninth aspect of the present invention and/or the tenth aspect of the present invention combined or coupled with a therapeutic agent
  • the detectable label includes fluorescent dyes, chemiluminescent markers, colloidal gold, and chemiluminescent catalysts;
  • the chemiluminescent label includes luminol and its derivatives, acridinium ester or its derivatives, adamantane, isoluminol and its derivatives, rare earth elements, and bipyridyl ruthenium complexes;
  • the chemiluminescence catalyst includes horseradish peroxidase and alkaline phosphatase;
  • the antibiotic resistance genes include penicillin resistance genes, tetracycline resistance genes, chloramphenicol resistance genes, and kanamycin resistance genes;
  • the therapeutic agent includes radionuclides, cytokines, gold nanoparticles, virus particles, liposomes, nanomagnetic particles, prodrug activating enzymes, and chemotherapeutic agents;
  • the cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN - ⁇ , TNF- ⁇ , TNF- ⁇ , G-CSF, M-CSF;
  • the chemotherapeutic agent includes cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxalate platinum, and lexadine.
  • a fifteenth aspect of the present invention provides a pharmaceutical composition for treating CD123 and/or CD7 related diseases.
  • the pharmaceutical composition includes the polynucleotide according to the tenth aspect of the present invention, the vector according to the eleventh aspect of the present invention, the genetically engineered host cell according to the twelfth aspect of the present invention, and the polynucleotide according to the tenth aspect of the present invention.
  • the pharmaceutical composition further includes one or more pharmaceutically or physiologically acceptable carriers, diluents or excipient combinations;
  • the CD123 and/or CD7 related diseases include: acute myeloid leukemia, acute B lymphoblastic leukemia, lymphoblastic lymphoma, NKT cell leukemia, peripheral T cell lymphoma, NKT cell lymphoma, anaplastic large cell lymphoma neoplastic/blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia, acute T lymphoblastic leukemia, chronic B lymphocytic leukemia, chronic T lymphocytic leukemia, Richter syndrome, hairy cell leukemia, non-Hodgkin lymphoma tumour, small lymphocytic lymphoma, Hodgkin lymphoma, Burkitt lymphoma.
  • the CD123 and/or CD7 related diseases include but are not limited to the diseases listed above, as long as the diseases related to CD123 and/or CD7 are within the protection scope of the present invention.
  • the one or more pharmaceutically or physiologically acceptable carriers, diluents or excipient combinations may include: buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose Sugar, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants such as aluminum hydroxide; and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline, etc.
  • carbohydrates such as glucose, mannose Sugar, sucrose or dextran, mannitol
  • proteins such as polypeptides or amino acids, such as glycine
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants such as aluminum hydroxide
  • preservatives such as aluminum hydroxide
  • compositions disclosed in the present invention can be formulated for oral, intravenous, topical, enteral and/or parenteral administration of drugs according to actual needs.
  • a sixteenth aspect of the present invention provides a kit.
  • kit includes the polynucleotide described in the tenth aspect of the present invention or the vector described in the eleventh aspect of the present invention;
  • the kit further includes reagents for introducing the polynucleotide or vector into a host cell;
  • the kit further includes instructions for introducing the polynucleotide or vector into the host cell.
  • the seventeenth aspect of the present invention provides any of the following methods:
  • the method includes the following steps: introducing the polynucleotide according to the tenth aspect of the present invention or the vector according to the eleventh aspect of the present invention into the host cell;
  • the introduction method includes lipofection, microinjection, electroporation, DNA vector, RNA vector, retroviral vector, lentiviral vector, poxvirus vector, herpes simplex virus vector, adenovirus vector, adenovirus vector, Related viral vectors;
  • the method includes the following steps: administering to the mammal an effective amount of the genetically engineered host cell described in the twelfth aspect of the present invention or the universal CAR-T cell described in the thirteenth aspect of the present invention;
  • the method includes the following steps:
  • step 3 Transfect the universal T cells obtained in step 2 with a lentiviral vector encoding the expression of the chimeric antigen receptor targeting CD123 and CD7 as described in the ninth aspect of the present invention, to obtain a universal CAR targeting CD123 and CD7 -T cells;
  • the T cells described in step 1 are derived from healthy volunteers or donors;
  • the TCR described in step 2 is the ⁇ chain constant coding region of TCR (i.e., TRAC);
  • the genes encoding CD7 and TCR are knocked out by a complex formed by the Cas9 protein, CD7-sgRNA and TRAC-sgRNA introduced into the T cell;
  • CD7-sgRNA is shown in SEQ ID NO: 51;
  • the sequence of the TRAC-sgRNA is shown in SEQ ID NO: 52.
  • the present invention also provides a method for treating and/or preventing CD123 and/or CD7 related diseases.
  • the method includes administering to a subject in need an effective amount of the genetically engineered host cell described in the twelfth aspect of the present invention, the universal CAR-T cell described in the thirteenth aspect of the present invention, The derivative described in the fourteenth aspect or the pharmaceutical composition described in the fifteenth aspect of the present invention;
  • the CD123 and/or CD7 related diseases include: acute myeloid leukemia, acute B lymphoblastic leukemia, lymphoblastic lymphoma, NKT cell leukemia, peripheral T cell lymphoma, NKT cell lymphoma, anaplastic large cell lymphoma neoplastic/blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia, acute T lymphoblastic leukemia, chronic B lymphocytic leukemia, chronic T lymphocytic leukemia, Richter syndrome, hairy cell leukemia, non-Hodgkin lymphoma tumour, small lymphocytic lymphoma, Hodgkin lymphoma, Burkitt lymphoma.
  • the eighteenth aspect of the present invention provides applications in any of the following aspects:
  • the chimeric antigen receptor according to the ninth aspect of the present invention the polynucleotide according to the tenth aspect of the present invention, the vector according to the eleventh aspect of the present invention, and the gene according to the twelfth aspect of the present invention Engineered host cells, universal CAR-T cells according to the thirteenth aspect of the present invention, derivatives according to the fourteenth aspect of the present invention, pharmaceutical compositions according to the fifteenth aspect of the present invention, first Application of the kit described in the sixteenth aspect in preparing drugs for preventing and/or treating CD123 and/or CD7 related diseases;
  • the chimeric antigen receptor according to the ninth aspect of the present invention, the polynucleotide according to the tenth aspect of the present invention, the vector according to the eleventh aspect of the present invention, and the gene according to the twelfth aspect of the present invention The engineered host cells, the universal CAR-T cells described in the thirteenth aspect of the present invention, and the derivatives described in the fourteenth aspect of the present invention are used in the preparation of preparations for preventing and/or treating CD123 and/or CD7 related diseases. Application of immune cell kits;
  • kit described in the sixteenth aspect of the present invention in preparing immune cells for preventing and/or treating CD123 and/or CD7 related diseases;
  • the CD123 and/or CD7 related diseases include: acute myeloid leukemia, acute B lymphoblastic leukemia, lymphoblastic lymphoma, NKT cell leukemia, peripheral T cell lymphoma, NKT cell lymphoma, anaplastic large cell lymphoma neoplastic/blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia, acute T lymphoblastic leukemia, chronic B lymphocytic leukemia, chronic T lymphocytic leukemia, Richter syndrome, hairy cell leukemia, non-Hodgkin lymphoma tumour, small lymphocytic lymphoma, Hodgkin lymphoma, Burkitt lymphoma.
  • the two anti-CD123 nanobodies VHH01 and VHH02 provided by the present invention both have high affinity and high specificity for CD123, and have not yet been reported.
  • the present invention further utilizes genetic engineering technology to express the nanobody in immune cells.
  • the immune cells expressing anti-CD123 chimeric antigen receptor thus constructed have a significant killing effect on CD123-positive tumor cells and are useful in the treatment of CD123-positive acute It has important application prospects in myeloid leukemia.
  • the present invention has developed for the first time a bispecific chimeric antigen receptor that targets both CD7 and CD123, and provides a universal CAR-T cell product that targets both CD7 and CD123.
  • the bispecific CAR-T cells targeting both CD7 and CD123 are highly effective against CD7+ and CD123+ tumor cells.
  • the role of clearance has important application prospects in the treatment of CD7 and/or CD123-related diseases, especially in the treatment of acute myeloid leukemia.
  • the universal CAR-T cell product targeting both CD7 and CD123 provided by the present invention for the first time is not affected by the patient's own condition or treatment method during use. It can be prepared at any time and treated at the best time to ensure the effectiveness of the treatment. effectiveness.
  • Figure 1 is an alpaca immunization flow chart
  • Figure 2 is a statistical graph showing the ratio of the positive group to the negative group after phage panning
  • Figure 3 is a graph showing the statistical results of OD values of positive clones in single clone screening
  • FIG 4 is a graph showing the ELISA affinity detection results of anti-CD123 monoclonal Nanobody (VHH), in which Figure A: VHH01, Figure B: VHH02;
  • FIG. 5 is a graph showing the SPR method affinity detection results of anti-CD123 monoclonal Nanobody (VHH), in which Figure A: VHH01, Figure B: VHH02;
  • FIG. 6 shows the specificity detection results of anti-CD123 monoclonal Nanobody (VHH).
  • Picture A the binding of Nanobody to K562 cell line and CD123 positive cell line K562-CD123
  • Picture B Nanobody to MOLM-13 Cell line binding
  • Figure 7 is a graph showing the results obtained by flow cytometry to verify the CD123 expression of the constructed cell line K562-CD123;
  • Figure 8 is a schematic structural diagram of a single VHH CAR-T
  • Figure 9 is a flow chart of CAR-T cell culture
  • Figure 10 is a graph showing the statistical results of MFI binding to VHH on the surface of K562-NMC009 cells and human CD123-His antigen;
  • Figure 11 shows the representative results of flow cytometry detection of single VHH CAR-T cells.
  • Figure A the expression of tEGFR on the cell surface
  • Figure B the expression of nanobodies on the cell surface;
  • Figure 12 shows the statistical results of MFI binding between VHH and human CD123-His antigen on the surface of single VHH CAR-T cells
  • Figure 13 shows the statistical results of the CD4/CD8 ratio in the later stages of single VHH CAR-T cell culture
  • Figure 14 is a single VHH CAR-T cell expansion curve
  • Figure 15 shows the statistical results of the killing ratio of single VHH CAR-T cells
  • Figure 16 is a schematic structural diagram of dual VHH CAR-T
  • Figure 17 is a representative result of dual VHH CAR-T cell flow cytometry detection, in which Figure A: the expression of tEGFR on the cell surface, Figure B: the expression of nanobodies on the cell surface;
  • Figure 18 shows the statistical results of MFI binding between VHH and human CD123-His antigen on the surface of dual VHH CAR-T cells
  • Figure 19 shows the dual VHH CAR-T cell expansion results
  • Figure 20 shows the statistical results of the killing ratio of dual VHH CAR-T cells.
  • Figure 21 is a graph showing the results of detecting the affinity activity of antibody dNb0901-B by the SPR method, in which Figure A: human CD7 antigen, Figure B: human CD123;
  • Figure 22 is a schematic diagram of the specific structure of the 0901-CAR gene
  • Figure 23 is a schematic diagram of the specific structure of the 0109-CAR gene
  • Figure 24 is a specific preparation flow chart for preparing NS 0901-CAR-T cells according to the present invention.
  • Figure 25 is a specific preparation flow chart for preparing KO7-0901 CAR-T cells and KO7-0901 CAR-T cells according to the present invention.
  • Figure 26 is a representative flow chart of four CD7-targeting gRNA knockout T cells CD7;
  • Figure 27 shows the expansion fold curve of NS 0901-CAR-T, KO7-0901 CAR-T and KO7-0109 CAR-T cells cultured in vitro for 12 days;
  • Figure 28 shows the changes in CAR positivity rate (ERB) during the in vitro culture of NS 0901-CAR-T, KO7-0901 CAR-T and KO7-0109 CAR-T cells;
  • Figure 29 is a representative flow cytometry diagram of CAR expression in NS 0901-CAR-T, KO7-0901 CAR-T and KO7-0109 CAR-T cells;
  • Figure 30 shows the changes in cell viability during in vitro culture of NS 0901-CAR-T, KO7-0901 CAR-T and KO7-0109 CAR-T cells;
  • Figure 31 is a representative flow cytometry diagram of T cell CD7 knockout efficiency, wherein, Panel A: T cell control, Panel B: KO7T cells;
  • Figure 32 shows the results of the killing effect of NS 0901-CAR-T, KO7-0901 CAR-T and KO7-0109 CAR-T cells on MOLM-13 and CCRF-CEM.
  • Picture A MOLM-13 cells
  • Picture B CCRF-CEM cells;
  • Figure 33 shows the detection results of cytokines secreted by NS 0901-CAR-T, KO7-0901 CAR-T and KO7-0109 CAR-T cells after incubation with MOLM-13 and CCRF-CEM.
  • Figure A MOLM- 13 cells
  • Panel B CCRF-CEM cells
  • Figure 34 shows the fluorescence imaging of the anti-tumor effect in animals after administration of different doses of KO7-0901 CAR-T cells
  • Figure 35 shows the trend chart of fluorescence intensity in animals after administration of different doses of KO7-0901 CAR-T cells
  • Figure 36 is a graph showing the expression trend of CAR-T cells in animals after administration of different doses of KO7-0901 CAR-T cells;
  • Figure 37 is a graph of the expansion fold of UCAR0901T and mock-T cells cultured in vitro for 12 days;
  • Figure 38 is a graph showing changes in CAR positivity rate (ERB) during in vitro culture of UCAR0901T and mock-T cells;
  • Figure 39 is a representative flow cytometry diagram of CAR expression in UCAR0901T and mock-T cells;
  • Figure 40 is a representative flow cytometry diagram of T cell CD7 and TRAC double knockout efficiency, wherein, Panel A: T cell control, Panel B: T cells knocked out of CD7 and TRAC;
  • Figure 41 is a graph showing the results of the killing effect of UCAR0901T cells on MOLM-13 and CCRF-CEM.
  • Picture A MOLM-13 cells
  • Picture B CCRF-CEM cells;
  • Figure 42 shows the detection results of cytokines secreted by UCAR0901T cells after incubation with MOLM13 and CCRF-CEM.
  • Picture A MOLM-13 cells
  • Picture B CCRF-CEM cells.
  • the present invention successfully obtained Nanobodies VHH01 and VHH02. Specifically, the present invention uses CD123 antigen protein to immunize alpacas to obtain a high-quality immune Nanobody gene library. Then the antigen molecules are coupled to the enzyme plate, and the antigen in this form is screened using phage display technology to screen the immune Nanobody gene library (heavy chain antibody phage display gene library), thereby obtaining the CD123-specific Nanobody gene. Experimental results show that the Nanobody obtained in the present invention can effectively bind to the CD123 antigen, and the CAR-T cells prepared based on the Nanobody can effectively kill tumor cells. On this basis, the present invention was completed.
  • Nanobody In the present invention, the terms "Nanobody”, “VHH”, “VHH antibody fragment” are used without distinction and refer to the variable domain of a single heavy chain of those types of antibodies found in camelids.
  • Nanobodies In the absence of light chains, Nanobodies each have three CDRs, denoted CDR1, CDR2 and CDR3. It is the smallest fully functional antigen-binding fragment.
  • CDR1, CDR2 and CDR3 It is the smallest fully functional antigen-binding fragment.
  • CH1 light chain and heavy chain constant region 1
  • the variable region of the antibody heavy chain is cloned to construct a Nanobody (Nb) consisting of only one heavy chain variable region. Called VHH.
  • Nanobodies/single domain antibodies are a new type of small molecule antibody fragments, which are cloned from the heavy chain variable region (VHH) of camelid natural heavy chain antibodies. It has excellent biological properties, with a molecular weight of 12-15kDa, one-tenth that of a complete antibody, good tissue penetration, high specificity, and good water solubility.
  • VHH heavy chain variable region
  • the term “affinity” refers to the binding ability between a macromolecule and the antigen it binds to, in particular the binding ability between a Nanobody and the antigen it binds to, such as the Nanobody of the invention and the pathological form as defined above The binding ability between CD123 proteins.
  • the affinity of the Nanobodies of the present invention can be measured in vitro by several methods, including surface plasmon resonance or by ELISA.
  • variable refers to certain portions of the variable regions of antibodies that differ in sequence and contribute to the binding and specificity of various specific antibodies to their specific antigens.
  • the variability is concentrated in three segments in the heavy chain variable region called complementarity determining regions (CDRs) or hypervariable regions.
  • CDRs complementarity determining regions
  • the variable regions of the natural heavy chain each contain four FR regions (the more conserved parts of the variable regions). They are roughly in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop, which can form a partial ⁇ -sheet. structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody. Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • the invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the invention also encompasses fragments of the Nanobodies of the invention.
  • the fragment has at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
  • the "sample” herein means a portion of a larger component.
  • the sample is a substance of biological origin. It contains cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical and/or physiological characteristics. For example, it refers to any sample derived from a subject of interest, which is expected or known to be Contains cellular and/or molecular entities to be characterized.
  • Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid , semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extracts such as homogenized tissue, Tumor tissue, cell extracts, and combinations thereof.
  • the present invention constructs a chimeric antigen receptor that targets both CD123 and CD7, and applies it to the preparation of CAR-T cells. After experimental verification, it is found that the CAR-T cells are effective against tumor cells. With significant killing effect, the present invention provides the field with a brand new treatment plan for CD123 and/or CD7 related diseases.
  • the chimeric antigen receptor (CAR) provided by the present invention that simultaneously targets CD123 and CD7 dual targets includes not only the chimeric antigen receptor whose amino acid sequence is as described in the ninth aspect of the present invention, but also the chimeric antigen receptor as described in the ninth aspect of the present invention.
  • Mutants of the chimeric antigen receptor described above. These mutants include: having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% of the amino acid sequence of the chimeric antigen receptor according to the ninth aspect of the present invention. % sequence identity and retain the biological activity of the chimeric antigen receptor (such as activating T cells). acid sequence.
  • Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. That is to say, mutants obtained by mutation based on the amino acid sequence/nucleotide sequence corresponding to the chimeric antigen receptor described in the ninth aspect of the present invention are also included in the protection scope of the present invention.
  • Mutants also include: those with one or several mutations (insertions, deletions or substitutions) in the amino acid sequence corresponding to the chimeric antigen receptor as described in the ninth aspect of the present invention, while still retaining the chimeric antigen receptor.
  • Biologically active amino acid sequences The number of mutations usually refers to within 1-10, such as 1-8, 1-5 or 1-3.
  • Substitutions are preferably conservative substitutions. For example, in the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide. These conservative modifications refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain.
  • Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid ), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • Conservative modifications may be selected, for example, based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipath
  • the present invention synthesizes chimeric antigen receptor anti-0901 CAR (EF1 ⁇ -leader-anti CD123VHH (VHH01)-Linker-anti CD7VHH (VHH10)-CD8hinge-CD8TM-4-1BB-CD3 ⁇ -T2A-tEGFR) and 0109 CAR through full gene synthesis.
  • EF1 ⁇ -leader-anti CD7VHH(VHH10)-Linker-anti CD123VHH(VHH01)-CD8hinge-CD8TM-4-1BB-CD3 ⁇ -T2A-tEGFR gene fragment was inserted into the lentiviral vector NMC009-01.
  • the recombinant lentiviral vector NMC009-01 packages the virus in 293FT cells, infects T cells, and causes the T cells to express the chimeric antigen receptor.
  • the transduction method of T lymphocytes modified with chimeric antigen receptor genes of the present invention is based on the lentiviral transduction method. This method has high transduction efficiency, stable expression of foreign genes, high batch stability and can shorten in vitro culture. Advantages include the time it takes for T lymphocytes to reach clinical levels.
  • the transduced nucleic acid is expressed on the surface of CAR-T cells through transcription and translation.
  • the present invention uses lentivirus to transduce T lymphocytes to obtain a high proportion of CAR-positive T lymphocytes, which can be detected by enzyme-linked immunoreaction (ELISA) in vitro. It was found that the CAR-T cells prepared based on the chimeric antigen receptor could secrete large amounts of IFN- ⁇ , TNF- ⁇ and IL-8 into the culture supernatant, indicating that the lentivirus was successfully transduced into T cells and Expresses secreted IFN- ⁇ , TNF- ⁇ and IL-8.
  • the CAR-T cells prepared by the present invention have strong killing function against CD123 or CD7-positive tumor cells, and when the effect-to-target ratio is 2:1, the killing value can reach more than 80%.
  • identity and “homology” are used without distinction and refer to the extent to which two (nucleotide or amino acid) sequences have the same residues at the same position in the alignment, and generally Expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with exactly the same sequence are 100% identical.
  • Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and ClustalW.
  • the term "administration" refers to the physical introduction of the product of the invention into a subject using any of various methods and delivery systems known to those skilled in the art, including intravenously, intramuscularly , subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.
  • the term "vector” means that a nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by selecting from cells known to contain the gene.
  • the gene is obtained in a vector or directly isolated from the cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.
  • the invention also provides vectors into which the expression cassette of the invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia virus, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of being low immunogenic;
  • the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector.
  • This vector is suitable for replication and integration into eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence;
  • the polynucleotides provided in the tenth aspect of the invention can be cloned into many types of vectors.
  • the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
  • Expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and others. Described in Handbook of Virology and Molecular Biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • a suitable vector will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193);
  • Additional promoter elements can modulate the frequency with which transcription is initiated.
  • these are located in a region of 30-110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased by 50 bp before activity begins to decrease.
  • an example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • EF-1 ⁇ elongation growth factor-1 ⁇
  • other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • inducible promoters are also considered part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the vector can be readily introduced into a host cell, eg, a mammalian, bacterial, yeast or insect cell, by any method known in the art.
  • expression vectors can be transferred into host cells by physical, chemical, or biological means;
  • a preferred method of introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors, especially retroviral vectors have become the most widely used method of inserting genes into mammalian, such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenovirus and adeno-associated virus, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362;
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Exemplary colloidal systems useful as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).
  • immune cell refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and/or NKT cells, or derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, Immune cells such as bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • T cells can be obtained from a subject's blood using a variety of techniques known to those skilled in the art, such as Ficoll isolation.
  • immune cells are engineered to express the chimeric antigen receptor targeting both CD123 and CD7 according to the ninth aspect of the present invention and to inhibit or silence endogenous CD7 and/or the alpha chain constant coding region of TCR (i.e. TRAC) gene expression.
  • TCR alpha chain constant coding region of TCR
  • the CAR-modified T cells provided by the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • a pharmaceutical composition of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as Glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins such as Glycine
  • antioxidants such as antioxidants
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives eg, aluminum hydroxide
  • the method for treating CD123 and/or CD7-related diseases includes administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.
  • the above therapeutic dose administered to the patient will vary with the treatment of the disease. The precise properties vary with the recipient of the treatment. Dosage proportions for human administration may be implemented in accordance with art-accepted practice. Typically, 1 ⁇ 10 6 to 1 ⁇ 10 10 CAR-modified T cells (such as KO7-0901 CAR T, KO7-0109 CAR-T and/or UCAR0901T), administered to the patient by, for example, intravenous infusion.
  • the means of administering the products described herein include any convenient means, including by spraying, injection, swallowing, infusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into the tumor, lymph node or site of infection.
  • the present invention can also be administered together with other therapeutic agents.
  • the other therapeutic agents include known anti-cancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium II (sorfimer sodium photofrin II), temozolomide, topotecan, trimesate glucuronate (trimetreate glucuronate), or auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and
  • the term "subject" refers to a mammal.
  • the mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans may advantageously be used as subjects representative of animal models of cancer. Preferably, the subject is human.
  • the diseases associated with CD123 and/or CD7 include non-solid tumors (such as hematological tumors, such as leukemias and lymphomas) and solid tumors.
  • Hematologic neoplasms are cancers of the blood or bone marrow, including, but not limited to, acute leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myelogenous leukemia, and myeloblastic, promyelocytic, myeloid -Monocytic, monocytic and erythroleukemia), Chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (painless and high graded form), multiple myeloma, Waldenström's macro
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid, and can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, pancreatic cancer, ovarian cancer, peritoneal, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, Small bowel cancer, melanoma, kidney cancer, laryngeal cancer, soft tissue cancer, stomach cancer, testicular cancer, colon cancer, esophageal cancer, cervical cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer , eye cancer, intrahepatic cholangiocarcinoma, joint cancer, neck cancer, gallblad
  • the universal CAR-T cell provided by the present invention is the first universal CAR-T cell obtained by simultaneously inhibiting the T cell antigen receptor TCR and the endogenous CD7 of T cells. It has the following characteristics: (1) By knocking Removing endogenous CD7 from T cells reduces cell suicide, improves the success rate of preparation, and further improves the killing effect of CAR-T cells on tumor cells; (2) Use CRISPR/Cas9 to targetedly knock out immune rejection-related genes (TCR), It avoids GVHD and potential TCR receptor signal interference that occurs when allogeneic T cells are injected, thereby achieving allogeneic treatment.
  • the present invention can be applied to the treatment of patients with recurrence after transplantation.
  • the present invention is a universal chimeric antigen receptor T cell (CAR-T) immunotherapy based on healthy donor T cells, and proposes CAR-T cell therapy for patients who relapse after transplantation. Since the patients received bone marrow hematopoietic stem cell transplantation from the donor, these patients can directly use T cells from healthy donors for modified cell therapy after relapse.
  • CAR-T universal chimeric antigen receptor T cell
  • RNA extraction kit Refer to the instruction manual of SuperScript TM II Reverse Transcriptase and use random primers to perform reverse transcription to obtain cDNA.
  • cDNA as a template, the extracellular region gene sequence of the antigen CD123 was obtained through PCR.
  • the CD123 extracellular region gene sequence was connected into a protein expression vector for expression, and Ni column purification was performed to obtain purified CD123-His protein.
  • the self-purified CD123-His protein of the present invention is used for alpaca immunization.
  • the specific alpaca immunization flow chart is shown in Figure 1. Immunization was performed once a week, with a total of 6 consecutive immunizations; 100 mL of peripheral blood was collected 7 days after the last immunization, and peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and RNA was extracted and reverse transcription kit was used.
  • Nanobodies were panned by ELISA method, and the recombinant CD123-His protein was coated on the enzyme plate and incubated at 4°C overnight; the enzyme plate was washed three times with 250 ⁇ L PBST, 200 ⁇ L blocking solution was added, and the enzyme plate was incubated at room temperature.
  • the construction results of the Nanobody library show that the present invention successfully constructed a Nanobody library for measuring CD123, with a library capacity of 4.8E7 and an insertion rate of nearly 95%.
  • the panning results are shown in Table 1 and Figure 2.
  • the results show that after three rounds of panning, the ratio of the positive group to the negative group reached 1656 times, which has reached the standard for screening monoclones. Therefore, after three rounds of panning, stop panning and proceed to the next step of screening and identification of single clones.
  • the screening and identification results of positive single clones are shown in Figure 3.
  • the results show that a total of 2 positive clones were screened in this example.
  • the OD450 values of the positive clones are shown in Figure 3.
  • the sequence-specific Nanobodies are numbered VHH01,
  • the variable region amino acid sequences of VHH02, VHH01 and VHH02 are shown in SEQ ID NO: 1 and SEQ ID NO: 9 respectively, and the variable region nucleotide sequences of VHH01 and VHH02 are shown in SEQ ID NO: 2 and SEQ ID NO: respectively.
  • the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH01 are respectively as shown in SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: As shown in 8, the nucleotide sequences of CDR1, CDR2, and CDR3 of VHH02 are shown in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16 respectively.
  • the Nanobody sequence was inserted into the pET-28a-Sumo-Nb-CH (retained by our laboratory, the sequence information is shown in SEQ ID NO: 33) plasmid through PstI and BstEII double enzyme digestion between the PstI and BstEII enzyme digestion sites. .
  • the N-terminus of the VHH sequence includes a SUMO tag (SUMO tag protein is a small molecule ubiquitin-like modified protein. Studies have found that SUMO can be used as a fusion tag and molecular chaperone for recombinant protein expression.
  • fusion proteins can not only further increase the expression of fusion proteins, but also It has the functions of resisting protease hydrolysis, promoting the correct folding of target proteins, and improving the solubility of recombinant proteins). It has a 6*His tag at the C-terminus of the VHH sequence for protein purification. After the sequencing is correct, the plasmid is extracted, then transformed into E. coli strain BL21, and protein expression is carried out under IPTG induction; after the expression is completed, the bacterial cells are collected and lysed by ultrasonic to obtain crude protein; purified by nickel column ion affinity chromatography Nanobodies with higher purity.
  • the ELISA detection method is used to determine the affinity of the antibody.
  • the prepared CD123-His protein is coated overnight.
  • Two antibodies VHH01 and VHH02
  • VHH01 and VHH02 with different dilutions after expression and purification are used as analytes to conduct experiments. Affinity between individual antibodies and CD123 antigen.
  • VHH01-His and VHH02-His antibodies were used for SPR assay.
  • the affinity of each VHH for the CD123 protein was determined by SPR using the Biacore 8K Assay System.
  • the anti-human IgG1Fc antibody was covalently coupled to the CM5 sensor chip at 25 ⁇ g/mL; then, the anti-human IgG1Fc antibody was used to capture the CD123-hFc protein as a stationary phase; then the diluted VHH01-His and VHH02-His
  • the mobile phase was injected at a flow rate of 20 ⁇ L/min, followed by dissociation; finally, association and dissociation rate constants were determined using Biacore Insight Evaluation Software 3.0.12.15655.
  • the results of detecting antibody affinity activity by ELISA method are shown in Figure 4A and 4B.
  • the results show that the EC50 value of VHH01 is 0.1318nM; the EC50 value of VHH02 is 0.7219nM.
  • the above results once again show that the two anti-CD123 Nanobodies (VHH01, VHH02) obtained through screening and identification of the present invention have good affinity with human CD123.
  • the results of detecting antibody affinity activity by SPR method are shown in Table 2 and Figures 5A and 5B.
  • the affinity constant between VHH01 and human CD123 protein is 1.16E-08; the affinity constant between VHH01 and human CD123 protein is 6.37E-08. .
  • the above results all show that the two anti-CD123 Nanobodies (VHH01, VHH02) obtained through screening and identification of the present invention have good affinity with human CD123.
  • Example 2 Using the cDNA obtained in Example 1 as a template, use PCR to obtain the full-length CD123 sequence, with Xho I and EcoR I enzyme cutting sites at both ends, and insert the sequence into the lentiviral vector through double enzyme digestion and ligation.
  • the recombinant plasmid In the pLVX-Puro vector, the recombinant plasmid is named pLVX-CD123-Puro. This recombinant plasmid uses the CMV promoter and contains a puromycin resistance gene.
  • the target plasmid pLVX-CD123-Puro and the lentiviral helper plasmid were used for lentiviral packaging.
  • lentivirus is first packaged: the target plasmid and three helper plasmids (pMD2.G, pRSV-REV, pMDLg) are co-transfected into 293FT cells under the action of PEI-Pro; packaging is performed for 6 hours. Liquid; packaged and shipped after 48 hours Harvest the lentivirus; the harvested lentivirus stock solution is concentrated by ultracentrifugation, and the lentivirus particles are resuspended in DMEM high-glucose medium and aliquoted for use.
  • antibiotic resistance testing was first performed. Add DMEM+10% FBS complete culture medium containing different concentrations of puromycin to the 24-well plate with the K562 cell line. When the puromycin concentration reaches 2 ⁇ g/mL, all K562 cells will die. This concentration was proved to be the maximum tolerated concentration of K562, and subsequent positive cell lines were screened using this concentration.
  • the packaged lentivirus is transduced into the library K562 cell line, and added to the transduced cell line according to the pre-tested antibiotic concentration, and a K562 control is performed at the same time. Stop screening when all cells in the control group die and cells in the experimental group are still alive. The cell lines in the experimental group continued to be cultured while adding the highest screening concentration of puromycin.
  • the cell line K562-CD123 which highly expresses CD123, was obtained.
  • flow cytometry was used to verify the expression of CD123 in the cell line K562-CD123, and the K562 cell line was used as a negative control.
  • Sequence-specific clones were used to construct single VHH CAR constructs. First, use PCR to amplify the VHH sequence of the positive clone; after the first round of PCR, use the first round of PCR products as a template to perform the second round of PCR; connect the second round of PCR products through homologous recombination. Into the vector Senl-S88BZ, the vector was digested with Not I single enzyme. At this point, the CAR structure containing a single VHH targeting CD123 has been successfully constructed. The structural diagram is shown in Figure 8, and the primer sequences are shown in Table 3.
  • NMC009-01 and NMC009-02 A total of two single VHH CAR structures were constructed, named NMC009-01 and NMC009-02 respectively.
  • the structures are shown in Figure 8.
  • EF1 ⁇ is the promoter of elongation factor 1 ⁇
  • Leader is the coding sequence of the signal peptide
  • VHH is the anti-CD123
  • the coding sequence of the Nanobody CD8H+TM is the CD8 hinge region and transmembrane region
  • the 4-1BB and CD3 ⁇ intracellular signal regions are the intracellular costimulatory domain
  • the tEGFR extracellular region is expressed through T2A peptide connection for lentiviral transduction. Then detect the expression of CAR.
  • the amino acid sequence and nucleotide sequence of the signal peptide are shown in SEQ ID NO:17 and SEQ ID NO:18 respectively, and the amino acid sequence and nucleotide sequence of the CD8 hinge region are shown in SEQ ID NO :19.
  • SEQ ID NO:20 the amino acid sequence and nucleotide sequence of the CD8 transmembrane region are shown in SEQ ID NO:21 and SEQ ID NO:22 respectively.
  • the amino acid sequence and nucleotide sequence of 4-1BB The amino acid sequence and nucleotide sequence of CD3 ⁇ are shown in SEQ ID NO:23 and SEQ ID NO:24 respectively.
  • the amino acid sequence and nucleotide sequence of T2A are shown in SEQ ID NO:25 and SEQ ID NO:26 respectively.
  • the amino acid sequence and nucleotide sequence of the tEGFR signal peptide are as shown in SEQ ID NO:29 and SEQ ID NO:30 respectively.
  • the amino acid sequence and nucleoside sequence of tEGFR The acid sequences are shown as SEQ ID NO:31 and SEQ ID NO:32 respectively.
  • lentivirus Before preparing CAR-T cells, lentivirus is first packaged: the target plasmid and three helper plasmids (pMD2.G, pRSV-REV, pMDLg) are co-transfected into 293FT cells under the action of PEI-Pro; packaging is performed for 6 hours. solution; lentivirus harvesting was performed 48 hours after packaging; the harvested lentivirus stock solution was concentrated by ultracentrifugation, and the lentiviral particles were resuspended in DMEM high-sugar medium and aliquoted for use.
  • the target plasmid and three helper plasmids pMD2.G, pRSV-REV, pMDLg
  • the lentivirus is transduced into the K562 cell line.
  • K562 transduced with NMC009 series lentivirus was cultured, and flow cytometry was performed after 3 days of culture.
  • CD123-His protein was used as the primary antibody
  • anti-His tag antibody was used as the secondary antibody to detect the expression of nanobodies on the cell surface. And make statistics on the MFI of VHH on the surface of K562.
  • CAR-T cells are prepared: peripheral blood mononuclear cells (PBMC) from patients or healthy donors are collected; ⁇ T cells are sorted using CD3 magnetic beads; the sorted ⁇ T cells are cultured in TexMACS GMP medium Culture in (MACS); perform lentiviral transduction 2 days later; continue culturing until 12-14 days to harvest CAR-T cells, and obtain VHH CAR-T cells targeting CD123 (named NMC009-01 and NMC009- respectively).
  • PBMC peripheral blood mononuclear cells
  • ⁇ T cells are sorted using CD3 magnetic beads
  • the sorted ⁇ T cells are cultured in TexMACS GMP medium Culture in (MACS)
  • MACS TexMACS GMP medium Culture in
  • VHH CAR-T cells targeting CD123 named NMC009-01 and NMC009- respectively.
  • the CAR-T cell culture process is shown in Figure 9; during the culture process, flow cytometry was performed to determine the proportion of CAR+ cells, anti-EGFR antibody was used to detect the expression of tEGFR, CD123-His protein was used as the primary antibody, and anti-His tag The antibody is a secondary antibody to detect the expression of nanobodies on the cell surface, and CD123-His antigen is used to detect VHH on the surface of CAR-T cells. MFI value for statistics. Cell counts were performed on the 5th, 8th, 11th, and 14th days of cell culture, and cell expansion was counted. And in the later stage of culture, the CD4/CD8 ratio of CAR-T cells was counted.
  • the MFI values of VHH on the surface of K562-NMC009-01 and K562-NMC009-02 cells are respectively: 5190 ⁇ 380.43 and 5160 ⁇ 468.11 .
  • CAR-T cells were prepared by transducing T cells with a single VHH structure.
  • the representative detection results of flow cytometry after 6 days of culture of CAR-T cells are shown in Figure 11.
  • the expression of tEGFR on the surface of CAR-T cells and the expression of nanobodies were respectively detected.
  • Figure 11A shows the expression of tEGFR on the cell surface
  • the positive rates of NMC009-01 and NMC009-02 are: 17.84%, 45.5% respectively
  • Figure 11B shows the expression of Nanobodies on the cell surface, NMC009- 01.
  • the positive rates of NMC009-02 are: 13.16% and 36.92% respectively
  • Figure 12 shows the statistics of nanobody MFI on the surface of CAR-T cells.
  • the VHH MFI values on the surface of NMC009-01 and NMC009-02 cells are: 683 ⁇ 8.48 and 671 ⁇ 36.06, both higher than the 131 ⁇ 23.33 of blank T cells.
  • the CD4/CD8 ratio at the late stage of cell culture is shown in Figure 13.
  • the CD4/CD8 ratios of NMC009-01 and NMC009-02 were 3.04 ⁇ 0.18 and 2.83 ⁇ 0.68 respectively.
  • the single VHH CAR-T amplification curve is shown in Figure 14. It can be seen from the figure that on the 14th day of amplification, the average amplification fold of NMC009-01 was 66.6 ⁇ 9.33 times, and the amplification fold of NMC009-02 was 63.25 ⁇ 4.03 times. The amplification factor of blank T cells was 77.3 ⁇ 4.38 times.
  • the Nanobodies VHH01 and VHH02 screened by the present invention are used to construct the target plasmid of the double VHH CAR structure.
  • the structural schematic diagram of the double VHH CAR is shown in Figure 16.
  • the primer sequences are shown in Table 4; then, the second round PCR product Through homologous recombination, it was connected into the vector NMC009-01.
  • the vector was digested with Not I single enzyme and the recombinant plasmid was named dNMC009-A.
  • CAR-T cells were cultured at the same time.
  • flow cytometry was performed to determine the proportion of CAR+ cells.
  • Anti-EGFR was used to Antibodies were used to detect the expression of tEGFR.
  • CD123-His protein was used as the primary antibody, and anti-His tag antibody was used as the secondary antibody to detect the expression of nanobodies on the cell surface.
  • the MFI value of VHH on the surface of CAR-T cells was measured using CD123-His antigen. .
  • Cell counts were performed on days 6, 9, 13, and 16 of CAR-T cell culture to observe the expansion of CAR-T cells.
  • CAR-T cells and blank T cells expressed positive expression of CD123 respectively.
  • Figure 17 The representative test results of flow cytometry after CAR-T cells were cultured for 6 days are shown in Figure 17.
  • Figure 17A shows the expression of tEGFR on the cell surface.
  • the positive rates of NMC009-01, NMC009-02, and dNMC009-A are: 84.1%, 76.1%, and 73.0% respectively.
  • Figure 17B shows the expression of nanobodies on the cell surface. NMC009-01, NMC009-02, and dNMC009-A The positive rates were: 22.42%, 23.5%, and 18.14% respectively.
  • Figure 18 shows the MFI statistics of nanobodies on the surface of CAR-T cells.
  • NMC009-01, NMC009-02, and dNMC009-AMFI values are: 481.5 ⁇ 55.86, 285.5 ⁇ 99.7, and 319 ⁇ 53.03 respectively, which are all higher than those of blank T cells. 88.8 ⁇ 8.06.
  • the amplification curve is shown in Figure 19. It can be seen from the figure that on the 16th day of amplification, the amplification factor of dNMC009-A is 76.4 ⁇ 1.40 times, the amplification factor of NMC009-01 is 71.77 ⁇ 3.62 times, and the amplification factor of NMC009-02 The multiple is 69.05 ⁇ 1.63 times.
  • the in vitro functional verification results are shown in Figure 20.
  • the results show that when the killing ratios are 0.3:1, 1:1, and 3:1, the average killing values of dNMC009-A against K562-CD123 are 16.60 ⁇ 1.70%, 34.50 ⁇ 1.84%, and 70.50 ⁇ 3.11%; the average killing values of NMC009-01 against K562-CD123 are 12.20 ⁇ 4.24%, 25.85 ⁇ 5.15%, and 67.75 ⁇ 2.19% respectively; the average killing values of NMC009-02 against K562-CD123 are 7.77 ⁇ 0.76% and 20.00 respectively.
  • the expressed and purified dNb0901-B antibody was used for SPR assay.
  • the affinity of dNb0901-B to human CD123 and CD7 proteins and mouse CD123 and CD7 was determined by SPR using the Biacore 8K analysis system.
  • the anti-human IgG1Fc antibody was covalently coupled to the CM5 sensor chip at 25 ⁇ g/mL; then, the anti-human IgG1Fc antibody was used to capture the dNb0901-B protein as a stationary phase; then the diluted human CD123, CD7 protein and Mouse CD123 and CD7 proteins were injected as the mobile phase at a flow rate of 30 ⁇ L/min, and then dissociated; finally, the association and dissociation rate constants were measured using Biacore Insight Evaluation Software 3.0.12.15655.
  • the results of detecting antibody affinity activity by SPR method are shown in Table 5 and Figure 21A and Figure 21B.
  • the affinity constant between dNb0901-B and human CD123 protein is 4.07E-09
  • the affinity constant between dNb0901-B and human CD7 protein is 4.07E-09. is 5.94E-09.
  • the results showed that dNb0901-B can specifically bind to human CD123 and CD7.
  • the above results show that the antibody used in the CAR structure constructed in the present invention has good affinity to both human CD123 and human CD7.
  • EF1 ⁇ is the promoter of elongation factor 1 ⁇
  • leader is the coding sequence of the signal peptide
  • anti-CD7VHH is the coding sequence of anti-CD7 Nanobody (VHH10)
  • anti-CD123VHH is the coding sequence of anti-CD123 Nanobody (VHH01)
  • CD8hinge is the hinge region
  • CD8TM is the transmembrane region
  • 4-1BB and CD3 ⁇ intracellular signaling regions are intracellular co-stimulatory domains
  • the tEGFR extracellular region is expressed through T2A peptide connection to detect CAR expression after viral transduction.
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the Nanobody VHH01 are shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively, and the amino acid sequence of the Nanobody VHH01 is shown in SEQ ID NO :1, and the nucleotide sequence is shown in SEQ ID NO:2; the amino acid sequences of CDR1, CDR2, and CDR3 of the Nanobody VHH10 are respectively SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO: 44, the The amino acid sequence of Nanobody VHH10 is shown in SEQ ID NO:45, and the nucleotide sequence is shown in SEQ ID NO:49; the amino acid sequence of CD8TM is shown in SEQ ID NO:21, and the amino acid sequence of CD8hinge is shown in SEQ ID NO: 19 is shown, the amino acid sequence of the CD3 ⁇ intracellular signal region is shown in SEQ ID NO: 25, and the amino acid sequence of the 4-1BB costimulatory signal domain
  • 0109-CAR gene expression vector lentiviral expression vector
  • VHH01 sequence obtained through PCR and connect it to the lentiviral vector NMC001-10 independently preserved in our laboratory through homologous recombination.
  • the sequence is SEQ ID NO:57.
  • the recombinant vector was named 0109-CAR.
  • the steps for lentivirus packaging are as follows: First, prepare the PEI transfection reagent and plasmid mixture, and combine the lentiviral expression vector, the packaging plasmid psPAX2 encoding the Rev protein, the envelope plasmid pMD2.G encoding the VSV-G protein, and the PEI transfection reagent. (Polyplus) mix evenly and let stand at room temperature for 20 minutes. Then the above mixture was added to 293FT cells, and after 72 hours of culture, the culture supernatant was collected as the virus stock solution. Centrifuge at 18300g high speed for 2 hours at 4°C. Discard the supernatant and add serum-free medium to resuspend the virus particles. This is the lentivirus solution.
  • T cells sorted from peripheral blood PBMC were activated by adding Dynabeads CD3/CD28 activation magnetic beads, and cultured in a 37°C 5% CO 2 cell incubator (marked as Day0). After 48 hours of culture, the cells prepared above were Add the lentiviral solution to the T cells, centrifuge for 2 hours (2000rpm, 35°C) for lentiviral transduction, and the transduction is completed. After completion, add MACS medium containing 200UI/mL IL-2 and place it in a 37°C 5% CO 2 cell incubator for culture. Samples were taken on Day 5 and the CAR+ ratio was detected by flow cytometry. Cells were harvested from Day12 to Day14 to obtain NS0901-CAR-T cells. The specific preparation process is shown in Figure 24.
  • CD7-targeting guide RNAs namely gRNA-T71, gRNA-T72, gRNA-T73, and gRNA-T74. Their sequence information is shown in SEQ ID NO: 53-56, and the activity is verified.
  • sgRNA was synthesized commercially, and 2′-O-methyl and 3′ phosphorothioate bases were incorporated at the three terminal bases of the 5′ and 3′ of the sgRNA to prevent nuclease activity.
  • sgRNA-T71 had the most effective CD7 knockout efficiency, reducing the percentage of CD7+ T cells from 92.9% to 11.1% (see Figure 26). Therefore, sgRNA-T71 was selected in the present invention. sgRNA-T71 was used in all subsequent CD7 knockdown experiments.
  • T cells Prepare T cells according to the above method of T cell isolation and activation. Add Dynabeads CD3/CD28 activation magnetic beads to the sorted T cells for activation, and place them in a 37°C 5% CO 2 cell incubator for culture. After 48 hours of culture, collect the cells, remove the magnetic beads, and resuspend them in EO buffer.
  • NS 0901-CAR-T, KO7-0901 CAR-T and KO7-0109 CAR-T cells were used as effector cells, CCRF-CEM-GFP-LUC cells (expressing CD7 antigen) and MOLM-13-GEP-LUC cells (expressing CD123 antigen) as target cells.
  • Cytokine test Mix effector cells and target cells at an effect-to-target ratio of 1:1, mix and incubate together for 18 hours, and take the supernatant to detect the secretion of cytokines.
  • the NPG mouse tumor-bearing model was used to conduct in vivo animal studies.
  • NPG mice were transplanted with human acute myeloid leukemia cells-luciferase (luciferase)-labeled cells (KG-1a-LUC) to construct a transplanted tumor model.
  • luciferase human acute myeloid leukemia cells-luciferase
  • KG-1a-LUC human acute myeloid leukemia cells-luciferase-labeled cells
  • a total of 24 male mice were used, and 1 ⁇ 10 6 KG-1a- LUC/only was injected into the tail vein of NPG mice. According to the fluorescence intensity, they were divided into 4 groups: model control group, CAR-T low, medium and high dose groups, with 6 mice in each group.
  • mice in each group were given vehicle, 0.1 ⁇ 10 7 , 0.33 ⁇ 10 7 , and 1.0 ⁇ 10 7 CAR+T cells/mouse via the tail vein respectively.
  • the fluorescence intensity of KG-1a-LUC cells in the body was detected on the day of group administration (i.e. D1) and once a week after administration, clinical symptoms were observed, and animal death was recorded; 2-4 hours after administration , D2, D5, D8, and D15, collect blood, detect the cell phenotype in the blood sample through flow cytometry, and evaluate the proliferation level of CAR-T cells in the body.
  • ERB staining is used as the CAR positive rate, and CD123 antigen and CD7 antigen are used to stain CAR-T cells.
  • CD123 antigen and CD7 antigen are used to stain CAR-T cells.
  • mice (4) The results of the study on the anti-tumor effect in mice are as follows:
  • the inventor used the characteristics of bispecific nanoCAR targeting CD123 and CD7 to develop a universal CAR-T cell and produce an off-the-shelf ready-to-use therapeutic product.
  • the detailed introduction of the CD123 and CD7 bispecific nanouniversal CAR-T cells (UCAR0901T) described in this example is as follows:
  • the inventor used CRISPR/Cas9 gene editing technology to destroy the T cell receptor ⁇ constant (TRAC) site , to eliminate the expression of TCR and reduce the risk of graft-versus-host disease (GvHD) caused by allogeneic T cells.
  • TCR T cell receptor ⁇ constant
  • CAR targeting CD7 can specifically target the patient's own T cells and natural killer (NK) cells (alloreactive killer cells) to reduce host versus graft rejection (HvG), while CAR targeting CD123 can eliminate AML tumor cells (acute myeloid leukemia cells).
  • NK natural killer
  • AML tumor cells acute myeloid leukemia cells
  • Cas9RNP-CD7&TRAC 9 ⁇ g Cas9 protein and 4.5 ⁇ g CD7-sgRNA (CAUCAUUUACUACGAGGACG) (SEQ ID NO: 51) and 9 ⁇ g TRAC-sgRNA (from Osborn et al. Obtained) (GAGAAUCAAAAUCGGUGAAU) (SEQ ID NO: 52) was added to 30 ⁇ L EO buffer and incubated at room temperature for 10 min to form the Cas9RNP-CD7&TRAC complex.
  • A, G, C and U are ribonucleotides
  • T is deoxyribonucleotide
  • the 2′-OH in the ribose sugar of nucleotides 1-3 are all replaced by methoxy groups, and the phosphates are all Replaced with phosphorothioate
  • nucleotides 1-3 from last The 2'-OH in the ribose sugar is replaced by methoxy group, and the phosphate is replaced by phosphorothioate
  • nucleotides 1-20 are the target nucleotide sequence of the gRNA of the TRAC gene.
  • UCAR0901T and mock-T cells were used as effector cells, and CCRF-CEM-GFP-LUC cells (expressing CD7 antigen) and MOLM-13-GEP-LUC cells (expressing CD123 antigen) were used as target cells.
  • Cytokine test Mix effector cells and target cells at an effect-to-target ratio of 1:1, mix and incubate together for 18 hours, and take the supernatant to detect the secretion of cytokines.

Abstract

本发明公开了抗CD123的纳米抗体、嵌合抗原受体及其应用，所述纳米抗体为VHH01、VHH02，所述纳米抗体VHH01、VHH02与CD123均具有较好的亲和力和专属性，基于所述纳米抗体VHH01、VHH02构建得到的嵌合抗原受体、嵌合抗原受体免疫细胞对CD123阳性细胞均具有显著的细胞毒性。本发明还公开了一种靶向CD123和CD7的嵌合抗原受体及其用途，所述嵌合抗原受体包含针对CD123的纳米抗体VHH01和针对CD7的纳米抗体VHH10，本发明还提供了一种同时靶向CD123和CD7的通用型CAR-T细胞，所述CAR-T细胞对肿瘤细胞具有显著的杀伤作用。

Description

抗CD123的纳米抗体、嵌合抗原受体及其应用 技术领域
本发明属于生物医药技术领域，具体地，本发明涉及抗CD123的纳米抗体、嵌合抗原受体及其应用。
背景技术
CD123又称IL-3受体α链(IL-3Rα)，是一种分子量为75kDa的糖蛋白，仅表达于和IL-3结合的细胞，例如B淋巴细胞、巨核细胞、造血干/祖细胞、浆细胞样树突状细胞及单核细胞。CD123与其配体IL-3结合后可诱发酪氨酸磷酸化，进而促进造血细胞的增殖和分化及参与固有和适应性免疫应答、炎症反应等。相关研究学者经研究发现，45％的急性髓系白血病(Acute myelogenous leukemia，AML)患者存在CD123的过表达，过表达CD123的AML细胞具有更高的增殖活性和耐凋亡能力，存在着STAT5的持续磷酸化，患者初诊时肿瘤负荷更高且临床预后更差。此外，CD123在白血病干细胞和更多分化的白血病母细胞中均存在高表达，而在正常造血干细胞中低表达或不表达，因此，CD123是白血病相关抗原也是AML的特异性抗原，这使得CD123成为有望治疗AML的靶标。由于CD123在AML高表达，理论上以CD123为靶标的免疫治疗具有更安全有效的治疗效果，国内外也已经在进行以CD123为靶标的靶向药物的临床试验，但是功效均有限并且仍有安全性问题的发生，因此，筛选有效的靶向CD123的纳米抗体具有重要意义。
近些年来，免疫学领域中对于转化医学的研究越来越多，尤其是肿瘤免疫。肿瘤免疫治疗中应用较为广泛的是CAR-T细胞免疫治疗策略。在90年代早期，这种免疫治疗策略在转化医学中就获得了较好的效果。其中，靶向CD123分子的CAR-T细胞应用较为广泛，尤其是治疗AML等血液系统肿瘤。正常机体内的T细胞被有效刺激并发挥免疫学功能需要多重信号的调控，主要包括T细胞受体与MHC-抗原肽复合物的识别作为第一信号、T细胞表面的共刺激分子识别活化作为第二信号，甚至还需要细胞因子参与形成第三信号。这种嵌合抗原受体主要是将T细胞刺激和活化的过程所需的蛋白分子进行人工串联整合，从而促进T细胞的活化和特异性杀伤。CAR-T细胞的特异性杀伤主要依赖于前端的抗体分子部分的识别和结合。目前使用较为广泛的是基于人或其它种属的单克隆抗体单链可变区(Single chain Fv，ScFv)对靶蛋白的特异性识别。但存在如下缺 陷：在ScFv与纳米抗体亲和力相当的基础上，ScFv相对于纳米抗体而言分子量较大，在分子表达和功能发挥上均存在一定的局限性；ScFv由其亲本单克隆抗体衍生而来，在活性和稳定性等方面都可能存在一定的不足。
鉴于此，有必要提供新型有效的抗CD123的纳米抗体，以及新的嵌合抗原受体和/或融合蛋白，以解决现有技术存在的不足。
CD7分子是一个分子量约40kDa细胞表面糖蛋白，属于免疫球蛋白超家族中的一员。CD7分子主要表达在大多数的胸腺细胞表面、85％以上的外周血T淋巴细胞表面以及自然杀伤细胞表面。尽管目前的研究表明CD7分子的具体功能还不太清楚，但实验研究显示CD7缺陷的鼠T淋巴细胞对刺激反应正常以及当抗体与人T淋巴细胞上的CD7分子结合后对细胞的生长和增殖并没有影响。同时，CD7分子的一个重要性质是当其和抗体结合后会快速发生内吞作用。已有研究表明靶向CD7的CAR-T细胞在CD7+的T细胞来源的恶性肿瘤治疗中临床疗效和安全性获得验证；同时，大约1/3的AML患者的肿瘤细胞也表达CD7抗原，已有病例证实CD7 CAR-细胞回输给CD7+的AML患者后，在体内可以获得高效扩增，并且能极高效的清除CD7+的AML细胞。
目前，尚未见同时靶向CD123和CD7的嵌合抗原受体的相关报道，更没有同时靶向CD123和CD7的CAR-T细胞在治疗AML中的应用。
发明内容
针对现有技术存在的不足和本领域的实际需求，本发明的一个目的在于提供抗CD123的纳米抗体、嵌合抗原受体及其应用。本发明经筛选得到了高亲和力和高专属性的抗CD123纳米抗体VHH01、VHH02，将其分别作为嵌合抗原受体分子的抗原结合结构域构建CAR-T细胞，经实验验证发现，所述CAR-T细胞能够特异性识别杀伤CD123阳性细胞，在肿瘤治疗领域中具有重要的应用前景。本发明的另一个目的是提供一种靶向CD123和CD7的嵌合抗原受体，为本领域提供一种全新的CD123和/或CD7相关疾病治疗的研究策略。
本发明的上述目的通过以下技术方案得以实现：
本发明的第一方面提供了抗CD123纳米抗体。
进一步，所述纳米抗体包括VHH01、VHH02；
所述VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7所示或分别为与SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7具有至少75％同一性的氨基酸序列；
所述VHH02的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15所示或分别为与SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15具有至少75％同一性的氨基酸序列。
进一步，所述VHH01的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8所示或分别为与SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8具有至少75％同一性的核苷酸序列；
所述VHH02的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16所示或分别为与SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16具有至少75％同一性的核苷酸序列。
进一步，所述VHH01的重链可变区的氨基酸序列如SEQ ID NO:1所示或与SEQ ID NO:1具有至少75％同一性的氨基酸序列；
所述VHH02的重链可变区的氨基酸序列如SEQ ID NO:9所示或与SEQ ID NO:9具有至少75％同一性的氨基酸序列；
优选地，所述VHH01的重链可变区的核苷酸序列如SEQ ID NO:2所示或与SEQ ID NO:2具有至少75％同一性的核苷酸序列；
优选地，所述VHH02的重链可变区的核苷酸序列如SEQ ID NO:10所示或与SEQ ID NO:10具有至少75％同一性的核苷酸序列。
在本发明的具体实施方案中，所述具有至少75％同一性的氨基酸序列或核苷酸序列是指与所述氨基酸序列或核苷酸序列具有75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
进一步，包括优选抗体氨基酸序列的保守序列变体的抗体也包括在本发明的范围之内。保守氨基酸序列变体包括不显著改变本发明的单克隆中和抗体结合性质和中和性质的氨基酸序列的修饰，如源于本领域熟知的相似氨基酸替代的变体，氨基酸的缺失、增加导致的变体均在本发明的保护范围内。此外，本发明的纳米抗体还包括人源与非人源抗体，以及具有与本发明的单克隆抗体相同功能或改造及优化的一切抗体。
本发明的第二方面提供了基于抗CD123纳米抗体的嵌合抗原受体。
进一步，所述嵌合抗原受体包含本发明第一方面所述的纳米抗体；
优选地，所述嵌合抗原受体还包含信号肽；
优选地，所述嵌合抗原受体还包含铰链区；
优选地，所述嵌合抗原受体还包含跨膜结构域；
优选地，所述嵌合抗原受体还包含共刺激信号结构域；
优选地，所述嵌合抗原受体还包含胞内信号传导结构域；
优选地，所述嵌合抗原受体还包含EF1α、T2A、tEGFR；
优选地，所述嵌合抗原受体还包含tEGFR信号肽；
更优选地，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD64、CD80、CD86、CD134、CD137、CD154、GITR、ICOS、IgG6；
更优选地，所述铰链区包括下列分子的铰链区：CD8、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体；
更优选地，所述跨膜结构域包括下列分子的跨膜结构域：CD8、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、CD3ε、CD8α、IL-2受体、IL-7受体、IL-11受体；
更优选地，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB、CD28、ICOS、CD27、CD19、CD4、CD8α、CD8β、HVEM、LIGHT、CD40、OX40、DR3、GITR、CD30、TIM1、CD2、CD226、CD278；
更优选地，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、TCRζ、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d、FYN；
最优选地，所述铰链区为CD8铰链区；
最优选地，所述跨膜结构域为CD8跨膜结构域；
最优选地，所述共刺激信号结构域为4-1BB共刺激信号结构域；
最优选地，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域；
最优选地，所述嵌合抗原受体为EF1α、信号肽、本发明第一方面所述的纳米抗体、CD8铰链区、CD8跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
最优选地，所述CD8铰链区的氨基酸序列如SEQ ID NO:19所示或与SEQ ID NO:19具有至少75％同一性的氨基酸序列；
最优选地，所述CD8跨膜结构域的氨基酸序列如SEQ ID NO:21所示或与SEQ ID NO:21具有至少75％同一性的氨基酸序列；
最优选地，所述4-1BB共刺激信号结构域的氨基酸序列如SEQ ID NO:23所示或与SEQ ID NO:23具有至少75％同一性的氨基酸序列；
最优选地，所述CD3ζ胞内信号传导结构域的氨基酸序列如SEQ ID NO:25所示或与SEQ ID NO:25具有至少75％同一性的氨基酸序列；
最优选地，所述信号肽的氨基酸序列如SEQ ID NO:17所示或与SEQ ID NO:17具有至少75％同一性的氨基酸序列；
最优选地，所述T2A的氨基酸序列如SEQ ID NO:27所示或与SEQ ID NO:27具有至少75％同一性的氨基酸序列；
最优选地，所述tEGFR信号肽的氨基酸序列如SEQ ID NO:29所示或与SEQ ID NO:29具有至少75％同一性的氨基酸序列；
最优选地，所述tEGFR的氨基酸序列如SEQ ID NO:31所示或与SEQ ID NO:31具有至少75％同一性的氨基酸序列。
本发明的第三方面提供了核酸分子。
进一步，所述核酸分子编码本发明第一方面所述的纳米抗体或编码本发明第二方面所述的嵌合抗原受体。
进一步，编码本发明第一方面所述的纳米抗体或编码本发明第二方面所述的嵌合抗原受体的核酸分子包括具有上述核苷酸序列的保守核苷酸序列变体的核酸分子。所谓的保守核苷酸序列变体源于遗传密码简并和沉默的变体，核苷酸的替代、缺失和增加也包含在本发明的保护范围内。
本发明还提供了一种如前所述的核酸分子的DNA片段，所述DNA片段可以编码本发明第一方面所述的纳米抗体的重链可变区的任何区域，包括但不限于重链可变区的CDR1、CDR2、CDR3。
在本发明的具体实施方案中，本发明所述的核酸分子可以通过例如标准化学合成方法和/或重组方法合成，或半合成地产生，例如通过组合化学合成和重组方法。编码序列与转录调控元件和/或与其他氨基酸编码序列的连接可以使用已确立的方法进行，例如限制酶切消化、连接和分子克隆。
本发明的第四方面提供了重组表达载体。
进一步，所述重组表达载体包含本发明第三方面所述的核酸分子；
优选地，所述表达载体包括DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体、病毒来源的载体；
更优选地，所述病毒来源的载体包慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体、疱疹病毒载体。
进一步，所述重组表达载体除了包含本发明第三方面所述的核酸分子外，还包含与所述核酸分子序列操作性相连的表达调控序列。
表达载体是指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。载体的种类包括本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以使用。在表达载体中，除了含有复制起点外，还可含有标记基因和其他翻译控制元件。
本发明的第五方面提供了经工程改造的宿主细胞。
进一步，所述经工程改造的宿主细胞表达含有本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体；
优选地，所述宿主细胞包括真核细胞、原核细胞；
更优选地，所述宿主细胞为真核细胞；
最优选地，所述真核细胞包括哺乳动物细胞、植物细胞、酵母细胞；
最优选地，所述真核细胞为免疫细胞；
最优选地，所述免疫细胞包括T细胞、B细胞、NK细胞、iNKT细胞、CTL细胞、树突状细胞、髓样细胞、单核细胞、巨噬细胞或其任意组合；
最优选地，所述免疫细胞为T细胞。
本发明的第六方面提供了一种纳米抗体-药物偶联物或试剂盒或组合物。
进一步，所述纳米抗体-药物偶联物是通过将本发明第一方面所述的纳米抗体共价附着至小分子药物上得以形成的；
优选地，所述小分子药物包括烷化剂、抗代谢物、抗肿瘤抗生素、有丝分裂抑制剂、染色质功能抑制剂、抗血管生成剂、抗雌激素、抗雄激素、免疫调节剂；
更优选地，所述烷化剂包括双氯乙基甲胺、苯丁酸氮芥、苯丙氨酸氮芥、溴丙哌嗪、松龙苯芥、磷雌氮芥、环磷酰胺、六甲密胺、氯乙环磷酰胺、异磷酰胺、三胺硫磷、卡 氮芥、链唑霉素、福替目丁、环己亚硝脲、白消安、苏消安、英丙舒凡、氮烯咪胺、顺铂、奥沙利铂、卡铂；
更优选地，所述抗代谢物包括甲氨喋呤、5-氟脲嘧啶、氟苷、5-氟脱氧尿嘧啶、卡培他滨、阿糖胞苷、氟达拉滨、阿糖胞苷、6-巯基嘌呤(6-MP)、6-巯基鸟嘌呤(6-TG)、2-氯脱氧腺苷、5-氮杂胞苷、2，2-二氟脱氧胞嘧啶核苷、克拉屈滨、脱氧柯福霉素、喷司他丁；
更优选地，所述抗肿瘤抗生素包括阿霉素、柔红霉素、去甲氧正定霉素、戊柔比星、盐酸米托蒽醌、更生霉素、光辉霉素、光神霉素、丝裂霉素C、博来霉素、甲基苄肼；
更优选地，所述有丝分裂抑制剂包括紫杉醇、紫杉萜、长春碱、长春新碱、长春酰胺、长春瑞滨；
更优选地，所述染色质功能抑制剂包括托泊替康、依立替康、依托扑沙、磷酸依托扑沙、鬼臼噻吩甙；
更优选地，所述抗血管生成剂包括丙亚胺、马马司他、巴马司他、普啉司他、坦诺司他、伊洛马司他、CGS-27023A、溴氯哌喹酮、COL-3、新伐司他、BMS-275291、沙立度胺；
更优选地，所述抗雌激素包括阿纳托唑、来曲唑、它莫西芬、托瑞米芬、雷洛昔芬、屈洛昔芬、奥多昔芬、依西美坦；
更优选地，所述抗雄激素包括氟他米特、尼鲁米特、比卡鲁胺、安体舒通、醋酸环丙氯地孕酮、非那司提、西咪替丁；
更优选地，所述免疫调节剂包括干扰素、白介素、肿瘤坏死因子、蘑菇多糖、西佐糖、罗喹美克、匹多莫特、甲氧聚乙二醇琥珀酰胺腺甙脱氨酶、胸腺肽制剂；
优选地，所述试剂盒或组合物包含本发明第一方面所述的纳米抗体，本发明第二方面所述的嵌合抗原受体、本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体、本发明第五方面所述的经工程改造的宿主细胞。
本发明还提供了一种药物组合物，所述药物组合物包含治疗有效量的本发明第一方面所述的纳米抗体、本发明第五方面所述的经工程改造的宿主细胞。
进一步，所述药物组合物还包含药学上可接受的载体，所述载体包括但不限于已经被美国食品与药品管理局认可的而可用于人类或动物的任何佐剂、载体、赋形剂、助流剂、稀释剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗压剂、溶剂或乳化剂等对组成药物组合物无副作用的各种形式的载体。
本发明还提供了一种包含本发明第一方面所述纳米抗体或其抗原结合片段的检测产品。所述检测产品包括但不限于检测试剂、试剂盒、芯片或试纸。凡是包括本发明第一方面所述纳米抗体或其抗原结合片段能够检测出CD123的检测产品均包括在本发明的保护范围内。
本发明的第七方面提供了如下任一种方法：
(1)一种生产本发明第一方面所述的纳米抗体的方法，所述方法包括如下步骤：培养本发明第五方面所述的经工程改造的宿主细胞，从培养物中分离出本发明第一方面所述的纳米抗体；
(2)一种非诊断和非治疗目的地检测待测样品中CD123的方法，所述方法包括如下步骤：将待测样品与本发明第一方面所述的纳米抗体接触，检测所述纳米抗体与CD123的复合物的形成；
优选地，所述纳米抗体是被可用于检测的标记物标记的纳米抗体；
更优选地，所述可用于检测的标记物包括荧光色素、亲和素、顺磁原子、放射性同位素；
最优选地，所述荧光色素为荧光素、罗丹明、Texas红、藻红蛋白、藻蓝蛋白、别藻蓝蛋白、多甲藻黄素-叶绿素蛋白；
最优选地，所述亲和素为生物素、卵白亲和素、链亲和素、卵黄亲和素、类亲和素；
最优选地，所述放射性同位素为放射性碘、放射性铯、放射性铱、放射性钴；
(3)一种制备本发明第五方面所述的经工程改造的宿主细胞的方法，所述方法包括如下步骤：将本发明第四方面所述的重组表达载体引入到宿主细胞中；
优选地，所述引入的方法包括物理方法、化学方法、生物方法；
更优选地，所述物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔；
更优选地，所述化学方法包括胶体分散系统、基于脂质的系统；
最优选地，所述胶体分散系统包括大分子复合物、纳米胶囊、微球、珠；
最优选地，所述基于脂质的系统包括水包油乳剂、胶束、混合胶束、脂质体；
更优选地，所述生物方法包括DNA载体、RNA载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体；
(4)一种体外特异性地抑制CD123活性的方法，所述方法包括如下步骤：将本发明第三方面所述的核酸分子导入到生物体细胞中，通过表达本发明第一方面所述的纳米抗体抑制CD123的活性；
(5)一种诊断或治疗目的地检测待测样品中CD123的方法，所述方法包括如下步骤：将待测样品与本发明第一方面所述的纳米抗体接触，检测所述纳米抗体与CD123的复合物的形成；
(6)一种诊断CD123相关疾病的方法，其中使用本发明第一方面所述的纳米抗体、本发明第二方面所述的嵌合抗原受体、本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体、本发明第五方面所述的经工程改造的宿主细胞、本发明第六方面所述的纳米抗体-药物偶联物或试剂盒或组合物；
(7)一种治疗表达CD123的肿瘤的方法，所述方法包括向有此需要的受试者施用本发明第一方面所述的纳米抗体、本发明第二方面所述的嵌合抗原受体、本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体、本发明第五方面所述的经工程改造的宿主细胞、本发明第六方面所述的纳米抗体-药物偶联物或试剂盒或组合物；。
本发明的第八方面提供了如下任一方面的应用：
(1)本发明第一方面所述的纳米抗体、本发明第二方面所述的嵌合抗原受体、本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体、本发明第五方面所述的经工程改造的宿主细胞、本发明第六方面所述的纳米抗体-药物偶联物或试剂盒或组合物在检测CD123蛋白或其抗原片段中的应用；
(2)本发明第一方面所述的纳米抗体、本发明第二方面所述的嵌合抗原受体、本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体、本发明第五方面所述的经工程改造的宿主细胞、本发明第六方面所述的纳米抗体-药物偶联物或试剂盒或组合物在制备用于检测CD123蛋白或其抗原片段的产品中的应用；
(3)本发明第一方面所述的纳米抗体、本发明第二方面所述的嵌合抗原受体、本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体、本发明第五方面所述的经工程改造的宿主细胞、本发明第六方面所述的纳米抗体-药物偶联物或试剂盒或组合物在制备用于诊断CD123相关疾病的产品中的应用；
(4)本发明第一方面所述的纳米抗体、本发明第二方面所述的嵌合抗原受体、本发明第三方面所述的核酸分子、本发明第四方面所述的重组表达载体、本发明第五方面所述 的经工程改造的宿主细胞、本发明第六方面所述的纳米抗体-药物偶联物或试剂盒或组合物在制备用于治疗表达CD123的肿瘤的药物中的应用；
(5)本发明第三方面所述的核酸分子或本发明第四方面所述的重组表达载体在制备经工程改造的宿主细胞中的应用；
优选地，所述肿瘤为血液系统肿瘤；
更优选地，所述肿瘤包括急性髓系白血病、急性B淋巴细胞白血病、母细胞性浆细胞样树突细胞肿瘤、慢性髓系白血病、急性T淋巴细胞白血病、慢性B淋巴细胞白血病、慢性T淋巴细胞白血病、Richter综合征、毛细胞性白血病、非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤、霍奇金淋巴瘤、伯基特淋巴瘤。
进一步，所述CD123相关疾病是指高表达CD123的疾病，包括高表达CD123的血液系统肿瘤等。
本发明公开的纳米抗体可以在重链可变区包含一个或多个糖基化位点，如本领域技术人员熟知的，在重量可变区中存在的一个或多个糖基化位点可以增强抗体免疫原性，或者由于改变了抗原结合而改变抗体的药物动力学。
本发明公开的纳米抗体可以以化学方法或者通过基因工程与其他因子缀合。这些因子提供将抗体靶向所需功能位点的作用或者为抗体提高或提供其他性能。
根据本发明的纳米抗体可以以化学方法或者通过基因工程标记，以提供可检测的纳米抗体，可检测的抗体包括可检测部分。可检测的部分包括但不限于酶、辅基、荧光材料、发光材料，生物发光材料、放射性材料、正电子发射金属以及非放射性顺磁性金属离子。
为了检测和/或分析和/或诊断目的标记依赖于使用的特定检测/分析/诊断技术和/或方法例如免疫组织化学染色(组织)样品、流式细胞计量术、激光扫描细胞计量术检测、荧光免疫测定、酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)、生物测定(例如吞噬作用测定)、蛋白质印迹应用等。对于本领域已知的检测/分析/诊断技术和/或方法合适的标记为本领域技术人员所熟知。
本发明的第九方面提供了一种靶向CD123和CD7的嵌合抗原受体。
进一步，所述嵌合抗原受体包括与CD123特异性结合的纳米抗体VHH01和与CD7特异性结合的纳米抗体VHH10；
优选地，所述纳米抗体VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5所示或分别与SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5具有至少75％同一性的氨基酸序列；
优选地，所述纳米抗体VHH10的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示或分别与SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44具有至少75％同一性的氨基酸序列；
更优选地，所述纳米抗体VHH01的氨基酸序列如SEQ ID NO:1所示或与SEQ ID NO:1具有至少75％同一性的氨基酸序列；
更优选地，所述纳米抗体VHH10的氨基酸序列如SEQ ID NO:45所示或与SEQ ID NO:45具有至少75％同一性的氨基酸序列；
优选地，所述嵌合抗原受体还包括跨膜结构域、胞内信号传导结构域；
优选地，所述嵌合抗原受体还包括铰链区；
优选地，所述嵌合抗原受体还包括共刺激信号结构域；
优选地，所述嵌合抗原受体还包括信号肽；
优选地，所述嵌合抗原受体还包括EF1α、T2A、tEGFR；
优选地，所述嵌合抗原受体还包括连接VHH01和VHH10的Linker；
优选地，所述嵌合抗原受体还包括tEGFR信号肽；
更优选地，所述跨膜结构域和铰链区包括下列分子的跨膜结构域和铰链区：CD8、4-1BB、PD-1、CD34、CD28，IgG1、IgG4、OX40，CD3ε；
最优选地，所述跨膜结构域和铰链区为CD8跨膜结构域和CD8铰链区；
最优选地，所述CD8跨膜结构域的氨基酸序列如SEQ ID NO:21所示；
最优选地，所述CD8铰链区的氨基酸序列如SEQ ID NO:19所示；
更优选地，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d、DAP10、DAP12、FYN；
最优选地，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域；
最优选地，所述CD3ζ胞内信号传导结构域的氨基酸序列如SEQ ID NO:25所示；
更优选地，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB、ICOS、CD27、CD19、CD4、CD28、CD8α、CD8β、HVEM、LIGHT、CD40、4-1BB、OX40、DR3、GITR、CD30、TIM1、CD2、CD226、CD278；
最优选地，所述共刺激信号结构域为4-1BB共刺激信号结构域；
最优选地，所述4-1BB共刺激信号结构域的氨基酸序列如SEQ ID NO:23所示；
更优选地，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD64、CD80、CD86、CD134、CD137、CD154、GITR、ICOS、IgG6；
最优选地，所述信号肽的氨基酸序列如SEQ ID NO:17所示；
更优选地，所述EF1α的核苷酸序列如SEQ ID NO:47所示；
更优选地，所述T2A的氨基酸序列如SEQ ID NO:27所示；
更优选地，所述tEGFR的氨基酸序列如SEQ ID NO:31所示；
更优选地，所述Linker的氨基酸序列如SEQ ID NO:48所示；
更优选地，所述tEGFR信号肽的氨基酸序列如SEQ ID NO:29所示；
最优选地，所述嵌合抗原受体选自以下组中的任一种：
(1)由EF1α、信号肽、纳米抗体VHH01、Linker、纳米抗体VHH10、CD8铰链区、CD8跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次连接得到的嵌合抗原受体；
(2)由EF1α、信号肽、纳米抗体VHH10、Linker、纳米抗体VHH01、CD8铰链区、CD8跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次连接得到的嵌合抗原受体；
(3)在(1)中所述嵌合抗原受体的氨基酸序列的基础上经过取代、缺失或添加一个或多个氨基酸后形成的嵌合抗原受体；
(4)在(2)中所述嵌合抗原受体的氨基酸序列的基础上经过取代、缺失或添加一个或多个氨基酸后形成的嵌合抗原受体。
本发明的第十方面提供了一种多核苷酸。
进一步，所述多核苷酸的序列为本发明第九方面所述的嵌合抗原受体的编码序列、或其互补序列。
进一步，在如本发明第九方面所述的所述嵌合抗原受体中，纳米抗体VHH01的核苷酸序列如SEQ ID NO:2所示，纳米抗体VHH10的核苷酸序列如SEQ ID NO:49所示，CD8跨膜结构域的核苷酸序列如SEQ ID NO:22所示，CD8铰链区的核苷酸序列如SEQ ID NO:20所示，CD3ζ胞内信号传导结构域的核苷酸序列如SEQ ID NO:26所示，4-1BB共刺激信号结构域的核苷酸序列如SEQ ID NO:24所示，信号肽的核苷酸序列如SEQ ID  NO:18所示，EF1α的核苷酸序列如SEQ ID NO:47所示，T2A的核苷酸序列如SEQ ID NO:28所示，tEGFR的核苷酸序列如SEQ ID NO:32所示，Linker的核苷酸序列如SEQ ID NO:50所示，tEGFR信号肽的核苷酸序列如SEQ ID NO:30所示。
本发明的第十一方面提供了一种含有本发明第十方面所述多核苷酸的载体。
进一步，所述载体包括克隆载体、表达载体；
优选地，所述载体包括DNA载体、RNA载体、质粒、病毒来源的载体；
更优选地，所述病毒来源的载体包括慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体、疱疹病毒载体。
本发明的第十二方面提供了一种基因工程化的宿主细胞。
进一步，所述基因工程化的宿主细胞含有本发明第十方面所述的多核苷酸或本发明第十一方面所述的载体；
优选地，所述宿主细胞选自真核细胞或原核细胞；
更优选地，所述真核细胞包括哺乳动物细胞、昆虫细胞、植物细胞、酵母细胞；
更优选地，所述原核细胞包括支原体、衣原体、立克次氏体、细菌、放线菌、蓝细菌；
最优选地，所述宿主细胞为真核细胞；
最优选地，所述真核细胞为哺乳动物细胞；
最优选地，所述哺乳动物细胞为免疫细胞；
最优选地，所述免疫细胞包括T细胞、B细胞、NK细胞、iNKT细胞、CTL细胞、树突状细胞、髓样细胞、单核细胞、巨噬细胞或其任意组合；
最优选地，所述免疫细胞为T细胞。
本发明的第十三方面提供了一种靶向CD123和CD7的通用型CAR-T细胞。
进一步，所述通用型CAR-T细胞表达本发明第九方面所述的嵌合抗原受体并且不表达CD7和TCR；
优选地，所述T细胞来源于健康志愿者或供者；
更优选地，所述通用型CAR-T细胞中的CD7的编码基因和TCR的编码基因被敲除；
最优选地，所述TCR的α链和/或β链恒定编码区(即TRAC和/或TRBC)基因被敲除；
最优选地，所述TCR的α链恒定编码区(即TRAC)基因被敲除；
最优选地，所述CD7的编码基因和TCR的编码基因被引入到所述T细胞的Cas9蛋白、CD7-sgRNA和TRAC-sgRNA形成的复合物所敲除；
最优选地，所述CD7-sgRNA的序列如SEQ ID NO:51所示；
最优选地，所述TRAC-sgRNA的序列如SEQ ID NO:52所示。
在本发明的具体实施方案中，本发明通过敲除T细胞内源性的CD7降低细胞自杀，提高制备成功率，并通过敲除TCR的α链恒定编码区(即TRAC)，消除TCR的表达，降低同种异体T细胞引发的移植物抗宿主病(GvHD)风险。靶向CD7的CAR可以特异性靶向患者自身T细胞和自然杀伤(NK)细胞(同种异体反应性杀伤细胞)，减少宿主抗移植物排斥反应(HvG)，同时靶向CD123的CAR可以清除AML肿瘤细胞。在本发明的具体实施例中，所述靶向CD123和CD7双特异性纳米通用型CAR-T细胞为UCAR0901T，具有显著的杀伤肿瘤细胞的能力，且所述通用型CAR-T细胞产品在患者需要时即可直接取用，而不需要在已经罹患病重的患者体内提取T细胞后再定制产品。
本发明的第十四方面提供了一种衍生物。
进一步，所述衍生物包括可检测标记的本发明第九方面所述的嵌合抗原受体和/或本发明第十方面所述的多核苷酸、赋予抗生素抗性的本发明第九方面所述的嵌合抗原受体和/或本发明第十方面所述的多核苷酸、与治疗剂结合或偶联的本发明第九方面所述的嵌合抗原受体和/或本发明第十方面所述的多核苷酸；
优选地，所述可检测标记包括荧光染料、化学发光标记物、胶体金、化学发光催化剂；
更优选地，所述化学发光标记物包括鲁米诺及其衍生物、吖啶酯或其衍生物、金刚烷、异鲁米诺及其衍生物、稀土元素、联吡啶钌配合物；
更优选地，所述化学发光催化剂包括辣根过氧化物酶、碱性磷酸酶；
优选地，所述抗生素抗性的基因包括青霉素抗性基因、四环素抗性基因、氯霉素抗性基因、卡那霉素抗性基因；
优选地，所述治疗剂包括放射性核素、细胞因子、金纳米颗粒、病毒颗粒、脂质体、纳米磁粒、前药激活酶、化疗剂；
更优选地，所述细胞因子包括IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12、IL-13、IL-14、IFN-γ、TNF-β、TNF-α、G-CSF、M-CSF；
更优选地，所述化疗剂包括顺铂、紫杉醇、长春新碱、门冬酰胺酶、奥沙利铂、草酸铂、乐沙定。
本发明的第十五方面提供了一种用于治疗CD123和/或CD7相关疾病的药物组合物。
进一步，所述药物组合物包含本发明第十方面所述的多核苷酸、本发明第十一方面所述的载体、本发明第十二方面所述的基因工程化的宿主细胞、本发明第十三方面所述的通用型CAR-T细胞和/或本发明第十四方面所述的衍生物；
优选地，所述药物组合物还包括一种或多种药学或生理学上可接受的载体、稀释剂或赋形剂组合；
优选地，所述CD123和/或CD7相关疾病包括：急性髓系白血病、急性B淋巴细胞白血病、淋巴母细胞淋巴瘤、NKT细胞白血病、外周T细胞淋巴、NKT细胞淋巴瘤、间变性大细胞淋巴瘤/母细胞性浆细胞样树突细胞肿瘤、慢性髓系白血病、急性T淋巴细胞白血病、慢性B淋巴细胞白血病、慢性T淋巴细胞白血病、Richter综合征、毛细胞性白血病、非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤、霍奇金淋巴瘤、伯基特淋巴瘤。
在本发明的具体实施方案中，所述CD123和/或CD7相关疾病包括但不限于上述列举出的疾病，只要与CD123和/或CD7相关的疾病均在本发明的保护范围内。
进一步，所述一种或多种药学或生理学上可接受的载体、稀释剂或赋形剂组合可以包含：缓冲液，例如中性缓冲盐水、磷酸盐缓冲盐水等；碳水化合物，例如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸，例如甘氨酸；抗氧化剂；螯合剂，例如EDTA或谷胱甘肽；佐剂(例如氢氧化铝)；以及防腐剂。
本发明公开的药物组合物可根据实际需求被配制用于口服、静脉内、局部、肠内和/或肠胃外施用的药物。
本发明的第十六方面提供了一种试剂盒。
进一步，所述试剂盒包含本发明第十方面所述的多核苷酸或本发明第十一方面所述的载体；
优选地，所述试剂盒还包括将所述多核苷酸或载体引入到宿主细胞中的试剂；
优选地，所述试剂盒还包括将所述多核苷酸或载体引入到宿主细胞中的说明书。
本发明的第十七方面提供了如下任一种方法：
(1)一种制备本发明第十二方面所述基因工程化的宿主细胞的方法；
进一步，所述方法包括如下步骤：将本发明第十方面所述的多核苷酸或本发明第十一方面所述的载体引入到宿主细胞中；
优选地，所述引入的方法包括脂质转染法、微注射、电穿孔、DNA载体、RNA载体、逆转录病毒载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体；
(2)一种刺激哺乳动物靶细胞群或组织产生免疫应答的方法；
进一步，所述方法包括如下步骤：给哺乳动物施用有效量的本发明第十二方面所述的基因工程化的宿主细胞或本发明第十三方面所述的通用型CAR-T细胞；
(3)一种制备本发明第十三方面所述通用型CAR-T细胞的方法；
进一步，所述方法包括如下步骤：
①获得活化的T细胞；
②通过CRISPR/Cas9基因编辑技术敲除步骤①所述的T细胞中的CD7和TCR，得到CD7和TCR双敲除的通用型T细胞；
③用编码如本发明第九方面所述的靶向CD123和CD7的嵌合抗原受体表达的慢病毒载体来转染步骤②得到的通用型T细胞，获得靶向CD123和CD7的通用型CAR-T细胞；
优选地，步骤①中所述的T细胞来源于健康志愿者或供者；
优选地，步骤②中所述的TCR为TCR的α链恒定编码区(即TRAC)；
更优选地，所述CD7和TCR的编码基因被引入到所述T细胞的Cas9蛋白、CD7-sgRNA和TRAC-sgRNA形成的复合物所敲除；
最优选地，所述CD7-sgRNA的序列如SEQ ID NO:51所示；
最优选地，所述TRAC-sgRNA的序列如SEQ ID NO:52所示。
此外，本发明还提供了一种治疗和/或预防CD123和/或CD7相关疾病的方法。
进一步，所述方法包括给有需要的受试者施用有效量本发明第十二方面所述的基因工程化的宿主细胞、本发明第十三方面所述的通用型CAR-T细胞、本发明第十四方面所述的衍生物或本发明第十五方面所述的药物组合物；
优选地，所述CD123和/或CD7相关疾病包括：急性髓系白血病、急性B淋巴细胞白血病、淋巴母细胞淋巴瘤、NKT细胞白血病、外周T细胞淋巴、NKT细胞淋巴瘤、间变性大细胞淋巴瘤/母细胞性浆细胞样树突细胞肿瘤、慢性髓系白血病、急性T淋巴细胞白血病、慢性B淋巴细胞白血病、慢性T淋巴细胞白血病、Richter综合征、毛细胞性白血病、非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤、霍奇金淋巴瘤、伯基特淋巴瘤。
本发明的第十八方面提供了如下任一方面的应用：
(1)本发明第九方面所述的嵌合抗原受体、本发明第十方面所述的多核苷酸、本发明第十一方面所述的载体、本发明第十二方面所述的基因工程化的宿主细胞、本发明第十三方面所述的通用型CAR-T细胞、本发明第十四方面所述的衍生物、本发明第十五方面所述的药物组合物、本发明第十六方面所述的试剂盒在制备用于预防和/或治疗CD123和/或CD7相关疾病的药物中的应用；
(2)本发明第九方面所述的嵌合抗原受体、本发明第十方面所述的多核苷酸、本发明第十一方面所述的载体、本发明第十二方面所述的基因工程化的宿主细胞、本发明第十三方面所述的通用型CAR-T细胞、本发明第十四方面所述的衍生物在制备用于制备预防和/或治疗CD123和/或CD7相关疾病的免疫细胞的试剂盒中的应用；
(3)本发明第九方面所述的嵌合抗原受体、本发明第十方面所述的多核苷酸、本发明第十一方面所述的载体、本发明第十二方面所述的基因工程化的宿主细胞、本发明第十三方面所述的通用型CAR-T细胞、本发明第十四方面所述的衍生物、本发明第十五方面所述的药物组合物、本发明第十六方面所述的试剂盒在制备用于预防和/或治疗CD123和/或CD7相关疾病的生物制剂中的应用；
(4)本发明第十六方面所述的试剂盒在制备用于预防和/或治疗CD123和/或CD7相关疾病的免疫细胞中的应用；
优选地，所述CD123和/或CD7相关疾病包括：急性髓系白血病、急性B淋巴细胞白血病、淋巴母细胞淋巴瘤、NKT细胞白血病、外周T细胞淋巴、NKT细胞淋巴瘤、间变性大细胞淋巴瘤/母细胞性浆细胞样树突细胞肿瘤、慢性髓系白血病、急性T淋巴细胞白血病、慢性B淋巴细胞白血病、慢性T淋巴细胞白血病、Richter综合征、毛细胞性白血病、非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤、霍奇金淋巴瘤、伯基特淋巴瘤。
相对于现有技术，本发明具有的优点和有益效果如下：
本发明提供的两种抗CD123的纳米抗体VHH01和VHH02对CD123均具有高亲和力和高专属性，且目前尚未见报道。本发明进一步利用基因工程技术将所述纳米抗体表达于免疫细胞中，由此构建得到的表达抗CD123嵌合抗原受体的免疫细胞对CD123阳性肿瘤细胞具有显著的杀伤作用，在治疗CD123阳性急性髓系白血病方面具有重要的应用前景。
此外，本发明首次开发了一种同时靶向CD7和CD123的双特异性嵌合抗原受体，并提供了一种同时靶向CD7和CD123的通用型CAR-T细胞产品，经实验验证发现所述同时靶向CD7和CD123的双特异性CAR-T细胞对CD7+及CD123+的肿瘤细胞具有高效 清除的作用，在治疗CD7和/或CD123相关的疾病中具有重要的应用前景，尤其是在急性髓系白血病的治疗中。此外，本发明首次提供的同时靶向CD7和CD123的通用型CAR-T细胞产品在使用中不受病人自身病情或治疗方式的影响，可以随时制备，在最佳的时机给予治疗，确保治疗的有效性。
附图说明
图1为羊驼免疫流程图；
图2为噬菌体淘选后阳性组与阴性组比值的结果统计图；
图3为单克隆筛选阳性克隆OD值统计结果图；
图4为抗CD123单克隆纳米抗体(VHH)的ELISA亲和力检测结果图，其中，A图：VHH01，B图：VHH02；
图5为抗CD123单克隆纳米抗体(VHH)的SPR方法检测亲和力结果图，其中，A图：VHH01，B图：VHH02；
图6为抗CD123单克隆纳米抗体(VHH)的专属性检测结果图，其中，A图：纳米抗体与K562细胞系和CD123阳性细胞系K562-CD123的结合情况，B图纳米抗体与MOLM-13细胞系的结合情况；
图7为流式细胞术对构建得到的细胞系K562-CD123的CD123表达情况进行验证得到的结果图；
图8为单VHH CAR-T结构示意图；
图9为CAR-T细胞培养流程图；
图10为K562-NMC009细胞表面VHH与人CD123-His抗原结合MFI统计结果图；
图11为单VHH CAR-T细胞流式细胞术检测代表性结果图，其中，A图：细胞表面tEGFR的表达情况，B图：细胞表面纳米抗体的表达情况；
图12为单VHH CAR-T细胞表面VHH与人CD123-His抗原结合MFI统计结果图；
图13为单VHH CAR-T细胞培养后期CD4/CD8比例的统计结果图；
图14为单VHH CAR-T细胞扩增曲线图；
图15为单VHH CAR-T细胞杀伤比例统计结果图；
图16为双VHH CAR-T结构示意图；
图17为双VHH CAR-T细胞流式细胞术检测代表性结果图，其中，A图：细胞表面tEGFR的表达情况，B图：细胞表面纳米抗体的表达情况；
图18为双VHH CAR-T细胞表面VHH与人CD123-His抗原结合MFI统计结果；
图19为双VHH CAR-T细胞扩增结果图；
图20为双VHH CAR-T细胞杀伤比例统计结果。
图21为SPR方法检测抗体dNb0901-B亲和活性的结果图，其中，A图：人CD7抗原，B图：人CD123；
图22为0901-CAR基因具体结构示意图；
图23为0109-CAR基因具体结构示意图；
图24为本发明制备NS 0901-CAR-T细胞的具体制备流程图；
图25为本发明制备KO7-0901 CAR-T细胞和KO7-0901 CAR-T细胞的具体制备流程图；
图26为4个靶向CD7的gRNA敲除T细胞CD7的流式代表图；
图27为NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞体外培养12天的扩增倍数曲线图；
图28为NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞体外培养过程中CAR阳性率(ERB)的变化图；
图29为NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞CAR表达的流式代表图；
图30为NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞体外培养过程中细胞活率的变化图；
图31为T细胞CD7敲除效率流式代表图，其中，A图：T细胞对照，B图：KO7T细胞；
图32为NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞对MOLM-13和CCRF-CEM杀伤作用的结果图，其中，A图：MOLM-13细胞，B图：CCRF-CEM细胞；
图33为NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞与MOLM-13和CCRF-CEM共同孵育后分泌的细胞因子检测结果图，其中，A图：MOLM-13细胞，B图：CCRF-CEM细胞；
图34为给药不同剂量的KO7-0901 CAR-T细胞后动物体内抗肿瘤作用的荧光成像图；
图35为给药不同剂量的KO7-0901 CAR-T细胞后动物体内荧光强度趋势图；
图36为给药不同剂量的KO7-0901 CAR-T细胞后动物体内CAR-T细胞表达趋势图；
图37为UCAR0901T和mock-T细胞体外培养12天的扩增倍数曲线图；
图38为UCAR0901T和mock-T细胞体外培养过程中CAR阳性率(ERB)的变化图；
图39为UCAR0901T和mock-T细胞CAR表达的流式代表图；
图40为T细胞CD7和TRAC双敲除效率流式代表图，其中，A图：T细胞对照，B图：敲除CD7和TRAC的T细胞；
图41为UCAR0901T细胞对MOLM-13和CCRF-CEM杀伤作用的结果图，其中，A图：MOLM-13细胞，B图：CCRF-CEM细胞；
图42为UCAR0901T细胞与MOLM13和CCRF-CEM共同孵育后分泌的细胞因子检测结果图，其中，A图：MOLM-13细胞，B图：CCRF-CEM细胞。
具体实施方式
本发明通过深入的研究，经过大量的筛选，成功获得纳米抗体VHH01和VHH02。具体地，本发明利用CD123抗原蛋白免疫羊驼，获得高质量的免疫纳米抗体基因文库。然后将抗原分子偶联在酶标板上，以此形式的抗原利用噬菌体展示技术筛选免疫纳米抗体基因库(重链抗体噬菌体展示基因库)，从而获得了CD123特异性的纳米抗体基因。实验结果表明，本发明获得纳米抗体能够有效地与CD123抗原结合，且基于所述纳米抗体制备得到的CAR-T细胞能够有效杀伤肿瘤细胞。在此基础上完成了本发明。
在本发明中，术语“纳米抗体”、“VHH”、“VHH抗体片段”无区别地使用，并且表示在骆驼科动物中发现的那些类型的抗体的单个重链的可变结构域。在没有轻链的情况下，纳米抗体各自具有三个CDR，分别表示为CDR1、CDR2和CDR3。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纳米抗体(Nanobody，Nb)也被称为VHH。纳米抗体/单域抗体作为一种新型的小分子抗体片段，由驼类天然的重链抗体重链可变区(VHH)克隆获得。其具有优良的生物学特性，分子量12-15kDa，是完整抗体的十分之一，具有很好的组织穿透性，特异性高，水溶性好。
在本发明中，术语“亲和力”是指大分子与其结合的抗原之间的结合能力，特别是纳米抗体与其结合的抗原之间的结合能力，例如本发明的纳米抗体和如上所定义的病理形式的CD123蛋白之间的结合能力。
本发明所述的纳米抗体的亲和力，可以通过几种方法在体外测量，包括表面等离子体共振或通过ELISA测定。
在本发明中，术语“可变”是指抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。可变性集中于重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。天然重链的可变区中各自包含四个FR区(可变区中较保守的部分)，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
本发明还提供了其他多肽，如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明纳米抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
本发明提供的非诊断和非治疗目的地检测待测样品中CD123的方法中，所述“样品”在本文中意指较大元件的一部分。优选地，样品是生物来源的物质。其包含有待根据例如物理，生化，化学和/或生理特点来表征和/或鉴定的细胞和/或其它分子实体例如，是指来源于感兴趣的受试者的任何样品，预计或已知其包含待表征的细胞和/或分子实体。样品包括但不限于组织样品(例如肿瘤组织样品)、原代或培养的细胞或细胞系、细胞上清、细胞裂解物、血小板、血清、血浆、玻璃体液、淋巴液、滑液、滤泡液、精液、羊水、乳、全血、血液衍生的细胞、尿液、脑脊髓液、唾液、痰、泪液、汗液，粘液、肿瘤裂解物、组织培养液、组织提取物如匀浆化的组织、肿瘤组织、细胞提取物、及其组合。
本发明首次构建了一种同时靶向CD123和CD7双靶点的嵌合抗原受体，并将其应用于CAR-T细胞的制备中，经实验验证发现，所述CAR-T细胞对肿瘤细胞具有显著的杀伤效果，本发明为本领域提供了一种全新的CD123和/或CD7相关疾病的治疗方案。
本发明提供的同时靶向CD123和CD7双靶点的嵌合抗原受体(CAR)不仅包括氨基酸序列如本发明第九方面中所述的嵌合抗原受体，也包括本发明第九方面所述的嵌合抗原受体的突变体。这些突变体包括：与本发明第九方面所述的嵌合抗原受体的氨基酸序列具有至少75％，优选至少80％，优选至少85％，优选至少90％，优选至少95％，优选至少97％的序列相同性并保留该嵌合抗原受体的生物学活性(如活化T细胞)的氨基 酸序列。可采用例如NCBI的BLASTp计算两条比对的序列之间的序列相同性。也即在本发明第九方面所述的嵌合抗原受体对应的氨基酸序列/核苷酸序列的基础上进行突变得到的突变体同样包含在本发明的保护范围内。
突变体还包括：在如本发明第九方面所述的嵌合抗原受体所对应的氨基酸序列中具有一个或数个突变(插入、缺失或取代)、同时仍保留该嵌合抗原受体的生物学活性的氨基酸序列。所述数个突变通常指1-10个以内，例如1-8个、1-5个或1-3个。取代优选是保守性取代。例如，在本领域中，用性能相近或相似的氨基酸进行保守性取代时，通常不会改变蛋白质或多肽的功能。这些保守性的修饰是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守修饰包括氨基酸取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。
本发明通过全基因合成嵌合抗原受体抗0901 CAR(EF1α-leader-anti CD123VHH(VHH01)-Linker-anti CD7VHH(VHH10)-CD8hinge-CD8TM-4-1BB-CD3ζ-T2A-tEGFR)和0109 CAR(EF1α-leader-anti CD7VHH(VHH10)-Linker-anti CD123VHH(VHH01)-CD8hinge-CD8TM-4-1BB-CD3ζ-T2A-tEGFR)的基因片段，插入到慢病毒载体NMC009-01中。重组慢病毒载体NMC009-01在293FT细胞中包装病毒，感染T细胞，使T细胞表达该嵌合抗原受体。本发明用嵌合抗原受体基因修饰T淋巴细胞的转导方法是基于慢病毒转导方法，该方法具有转导效率高，外源基因能够稳定表达，批次稳定性高且可以缩短体外培养T淋巴细胞到达临床级数量的时间等优点。转导的核酸通过转录、翻译表达在CAR-T细胞表面。用流式细胞术，通过检测与纳米抗体结合的蛋白的含量，可以计算出慢病毒感染的T淋巴细胞的比例和细胞表面CAR的表达情况。本发明通过慢病毒转导T淋巴细胞，得到CAR阳性T淋巴细胞的比例高，体外通过酶联免疫反应(ELISA)检测 发现，基于所述嵌合抗原受体制备得到的CAR-T细胞能分泌大量的IFN-γ、TNF-α和IL-8至培养基上清，表明了慢病毒成功转导至T细胞中并表达分泌型的IFN-γ、TNF-α和IL-8。本发明制备的CAR-T细胞对CD123或CD7阳性的肿瘤细胞具有强烈的杀伤功能，在效靶比为2:1的情况下，杀伤值可达80％以上。
在本发明中，术语“同一性”、“同源性”无区别地使用，是指两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25:3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215:403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147:195-197)和ClustalW。
在本发明中，术语“施用”，是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入到受试者体内，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
在本发明中，术语“载体”，是指编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了其中插入本发明的表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点；
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子；
本发明第十方面提供的多核苷酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他 病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)；
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以启动转录；合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
本发明中所述的将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞；
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)；
将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362；
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在本发明中，术语“免疫细胞”，是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞，或者是衍生自干细胞，例如成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞等的免疫细胞。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系,例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+/CD8+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)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离受试者的血液获得T细胞。在本发明中，免疫细胞被工程化以表达本发明第九方面所述的同时靶向CD123和CD7的嵌合抗原受体并抑制或沉默内源性CD7和/或TCR的α链恒定编码区(即TRAC)基因的表达。
本发明提供的CAR修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如 甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明提供的治疗CD123和/或CD7相关疾病的方法中，其包括施用给需要其的对象治疗有效量的本发明的CAR修饰的T细胞，施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×106个至1×1010个本发明提供的CAR修饰的T细胞(如，KO7-0901 CAR T、KO7-0109 CAR-T和/或UCAR0901T)，通过例如静脉回输的方式，施用于患者。
本发明中所述的施用产品的方式包括任何方便的方式，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。此外，本发明提供的药物组合物、CAR修饰的T细胞、衍生物等还可与其他治疗剂一起施用。所述其他治疗剂的优选实例包括已知的抗癌药物，例如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等。此外，本发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
在本发明中，术语“受试者”，是指哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。除人以外的哺乳动物可以有利地用作代表癌症动物模型的受试者。优选地，所述受试者是人。
在本发明的具体实施方案中，所述与CD123和/或CD7相关的疾病包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)和实体瘤。血液学肿瘤是血液或骨髓的癌症，包括但不限于急性白血病(诸如急性淋巴细胞白血病(ALL)、急性髓系白血病(AML)、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、 慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、骨髓增生异常综合征、多毛细胞白血病、伯基特淋巴瘤、弥漫性大细胞淋巴瘤、套细胞淋巴瘤、T淋巴母细胞性淋巴瘤(T-LBL)、早期前T淋巴母细胞白血病(ETP-ALL)、结外NK/T细胞淋巴瘤、小淋巴细胞淋巴瘤(SLL)和脊髓发育不良。实体瘤是通常不包含囊肿或液体区的组织的异常肿块，其可以是良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤的实例包括但不限于纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、胰腺癌、卵巢癌、腹膜、大网膜和肠系膜癌、咽癌、前列腺癌、直肠癌、肾癌、皮肤癌、小肠癌、黑素瘤、肾癌，喉癌、软组织癌、胃癌、睾丸癌、结肠癌，食道癌，宫颈癌、腺泡型横纹肌肉瘤、膀胱癌、骨癌、脑癌、乳腺癌、肛门癌、眼癌、肝内胆管癌、关节癌、颈癌、胆囊癌、胸膜癌、鼻癌、中耳癌、口腔癌、外阴癌、甲状腺癌和输尿管癌。也即与CD123和/或CD7相关的疾病均在本发明的保护范围内，在本发明的具体实施方案中，所述CD123和/或CD7相关的疾病优选为急性髓系白血病。
本发明提供的通用型CAR-T细胞，是一种首次同时抑制T细胞抗原受体TCR和T细胞内源性的CD7获得的通用型CAR-T细胞，其具有如下特点：(1)通过敲除T细胞内源性的CD7降低细胞自杀，提高制备成功率，进一步提高CAR-T细胞对肿瘤细胞的杀伤效果；(2)使用CRISPR/Cas9定向敲除免疫排斥反应的相关基因(TCR)，避免了输入同种异体的T细胞出现的GVHD以及潜在的TCR受体信号干扰，从而实现异体治疗。用同种异体供体来源的CAR-T细胞用来发展通用型产品，用来提供给那些因淋巴细胞数量较低或质量较差(以及体外扩增能力低)而没有机会进行自体细胞输注的患者；(3)本发明可适用于治疗移植后复发的患者。对于移植后复发的患者，国内外尚无有效的治疗手段。本发明基于健康供体T细胞的通用型嵌合抗原受体T细胞(CAR-T)免疫疗法，提出对移植后复发的患者进行CAR-T细胞治疗。由于患者接受了供体的骨髓造血干细胞移植，这部分患者复发后，可直接用健康供体的T细胞进行修饰后的细胞治疗。
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施 例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
实施例
实施例1抗CD123纳米抗体的筛选
1、CD123抗原制备
利用RNA提取试剂盒提取T细胞中的RNA。参考SuperScriptTMII Reverse Transcriptase使用说明书，利用random primers引物进行反转录，获得cDNA。以cDNA为模板，通过PCR获得抗原CD123的胞外区基因序列。将CD123胞外区基因序列连接入蛋白表达载体中进行表达，并进行Ni柱纯化，获得纯化的CD123-His蛋白。
2、纳米抗体文库的构建
利用本发明自主纯化的CD123-His蛋白进行羊驼免疫，具体羊驼免疫流程图见图1。每周进行1次免疫，共连续进行6次免疫；最后一次免疫7天后采集外周血100mL，通过Ficoll密度梯度离心法分离外周血单核细胞，并进行RNA的提取，并利用反转录试剂盒进行cDNA的制备；利用SOE-PCR获得VHH片段，并将其连接入pMES4噬菌体展示载体；将连接产物电转化至电转感受态细胞TG1中，所得菌库即为构建的CD123的单域重链抗体噬菌体展示文库；文库构建完成后，为检测文库的插入效率，随机选择25个克隆利用引物MP57以及GⅢ进行了菌落PCR，并将PCR产物进行Sanger测序。
3、纳米抗体的富集筛选
3.1、噬菌体纳米抗体文库的扩增
取TG1大肠杆菌纳米抗体文库转接到2-YT液体培养基中，37℃、200rpm培养至OD值为0.5，然后加入辅助噬菌体VCSM13对细胞进行侵染。轻轻混合后在37℃下孵育30分钟。离心菌液以去除微量葡萄糖，再将沉淀重悬于同时加有氨苄和卡那霉素抗性的2-YT培养基中，37℃、200rpm摇培过夜，以扩增展示纳米抗体的噬菌体；将过夜培养物转移到50mL离心管中，离心取上清液，并添加20％(wt/vol)PEG6000/2.5M NaCl溶液沉淀噬菌体。离心弃上清，PBS重悬沉淀，离心取上清至新的离心管中添加20％(wt/vol)PEG6000/2.5M NaCl溶液重新沉淀噬菌体。离心弃上清，并将沉淀重新悬浮于1mL PBS中。离心后将上清液转移到新的离心管中，添加甘油至终浓度为20％保存于-80℃中；噬菌体纳米抗体库滴度测定，将噬菌体按10倍梯度进行稀释，取不同稀释倍数的噬 菌体对对数生长期的TG1细菌进行侵染，37℃过夜培养，通过第二天的菌斑数目推算噬菌体纳米抗体库的滴度。
3.2、噬菌体富集筛选
通过ELISA方法对纳米抗体进行淘选，将重组CD123-His蛋白包被在酶标板上，在4℃下孵育过夜；用250μL PBST清洗酶标板三次，添加200μL封闭液，于室温孵育酶标板2h；在每个孔中加入相对应的噬菌体，室温孵育2h；250μL PBST洗板15次；每孔添加100μL浓度为0.25mg/mL的胰蛋白酶，室温700rpm孵育0.5h；用AEBSF洗脱噬菌体；将洗脱下的噬菌体进行滴度测定和噬菌体侵染扩增；洗脱下的噬菌体数量阳性：阴性≥100时，停止淘选。
4、阳性单克隆的筛选与鉴定
从经过3轮筛选后得到的TG1大肠杆菌文库中挑选单个克隆进行扩大培养，并利用辅助噬菌体VCSM13进行侵染，进行单克隆噬菌体的制备；将单克隆噬菌体加入到包被有CD123-His蛋白的培养板中，室温孵育2h；PBST清洗板子后，加入HA-HRP抗体，室温孵育1h；PBST清洗板子后，加入100μL TMB单组份显色液，室温孵育30min后加入100μL终止液；利用酶标仪检测450nm处的吸光度；当样品孔的OD450值与空白对照比例大于2时判定为阳性克隆；对阳性克隆进行菌液PCR，进行Sanger测序；经过Sanger测序的单克隆，利用软件DNAMAN进行序列比对。并筛选出序列特异性的克隆。
5、实验结果
纳米抗体文库的构建结果显示，本发明成功构建测定CD123的纳米抗体文库，库容量为4.8E7，插入率接近95％。
淘选结果见表1和图2，结果显示，淘选三轮后，阳性组与阴性组的比值达到了1656倍，已达到了筛选单克隆的标准。所以淘选三轮后，停止淘选，进行下一步单克隆的筛选和鉴定。
表1噬菌体淘选结果
阳性单克隆的筛选与鉴定结果见图3，结果显示，本实施例共筛选到阳性克隆2个，阳性克隆OD450值如图3所示，所述序列特异性的纳米抗体的编号分别为VHH01、 VHH02，VHH01、VHH02的可变区氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:9所示，VHH01、VHH02的可变区核苷酸序列分别如SEQ ID NO:2、SEQ ID NO:10所示，其中，VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7所示，VHH02的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15所示，VHH01的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8所示，VHH02的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16所示。
实施例2纳米抗体的表达及亲和力和专属性检测
1、重组抗体表达
通过PstI和BstEII双酶切将纳米抗体序列插入至pET-28a-Sumo-Nb-CH(本实验室留存，序列信息如SEQ ID NO:33所示)质粒的PstI和BstEII酶切位点之间。在VHH序列的N端包括SUMO标签(SUMO标签蛋白是一种小分子泛素样修饰蛋白，研究发现SUMO可以作为重组蛋白表达的融合标签和分子伴侣，不但可以进一步提高融合蛋白的表达量，还具有抗蛋白酶水解以及促进靶蛋白正确折叠，提高重组蛋白可溶性等功能)，在VHH序列的C端带有6*His标签，用于蛋白纯化。测序正确后提取质粒，然后转化至大肠杆菌菌株BL21中，并在IPTG诱导下进行蛋白表达；表达完成后收集菌体进行超声裂解菌体获得粗提蛋白；经过镍柱离子亲和层析纯化获得纯度较高的纳米抗体。
2、亲和力检测方法
2.1、ELISA检测抗体亲和力
本实施例中采用ELISA检测方法测定抗体的亲和力，将制备得到的CD123-His蛋白进行过夜包被，以经过表达纯化后不同稀释度的2个抗体(VHH01、VHH02)作为分析物进行实验，检测各个抗体与CD123抗原之间的亲和力。
2.2、SPR方法检测抗体亲和力
将表达纯化后的VHH01-His和VHH02-His抗体用于SPR测定。使用Biacore 8K分析系统通过SPR测定每种VHH对CD123蛋白的亲和力。首先，将抗人IgG1Fc抗体以25μg/mL共价偶联至CM5传感器芯片；然后，用抗人IgG1Fc抗体去捕获CD123-hFc蛋白作为固定相；再将倍比稀释的VHH01-His和VHH02-His作为流动相以20μL/min的流速注射，然后进行解离；最后，使用Biacore Insight Evaluation Software 3.0.12.15655软件测定缔合和解离速率常数。
2.3、专属性检测方法
以纯化后纳米抗体为一抗，以HIS-FITC抗体为二抗通过流式细胞术检测2个纳米抗体(VHH01、VHH02)与K562、K562-CD123、MOLM-13细胞系的结合情况，以此来确定纳米抗体VHH01、VHH02对人CD123抗原的专属性。
3、实验结果
ELISA方法检测抗体亲和活性结果见图4A、4B，结果显示，VHH01的EC50值为0.1318nM；VHH02的EC50值为0.7219nM。上述结果再次表明了本发明经筛选鉴定得到的2个抗CD123纳米抗体(VHH01、VHH02)与人CD123具有较好的亲和力。
SPR方法检测抗体亲和活性结果如表2和图5A、5B所示，VHH01与人CD123蛋白之间的亲和力常数为1.16E-08；VHH01与人CD123蛋白之间的亲和力常数为6.37E-08。以上结果均表明了本发明经筛选鉴定得到的2个抗CD123纳米抗体(VHH01、VHH02)与人CD123具有较好的亲和力。
表2 SPR方法测定抗体亲和力的结果统计
专属性检测结果见图6A和6B，流式结果显示，2个VHH(VHH01、VHH02)均未与K562发生特异性结合，但是均与CD123阳性细胞系K562-CD123、MOLM-13发生了特异性结合。表明了本发明筛选鉴定得到的2个单VHH均为人源CD123特异的。
实施例3 K562-CD123稳转细胞系的构建
1、实验方法
以实施例1中获得的cDNA为模板，用PCR的方式获得CD123全长序列，并且两端带有Xho I和EcoR I酶切位点，通过双酶切、连接方式将该序列插入慢病毒载体pLVX-Puro载体中，重组质粒命名为pLVX-CD123-Puro。该重组质粒采用CMV启动子，并带有嘌呤霉素抗性基因。
将目的质粒pLVX-CD123-Puro连同慢病毒辅助质粒一起进行慢病毒包装。CAR-T细胞制备前，首先进行慢病毒的包装：将目的质粒与三个辅助质粒(pMD2.G、pRSV-REV、pMDLg)在PEI-Pro作用下共转染293FT细胞；包装6小时进行换液；包装48小时后进 行慢病毒收获；收获的慢病毒原液进行超速离心浓缩，用DMEM高糖培养基重悬慢病毒颗粒，并进行分装备用。
构建阳性细胞系前，首先进行抗生素耐受性的测试。将铺有K562细胞系的24孔板中加入含有不同浓度的嘌呤霉素，DMEM+10％FBS完全培养基，当嘌呤霉素浓度到达2μg/mL时，K562细胞全部死亡。证明该浓度为K562最大耐受浓度，后续阳性细胞系用该浓度进行筛选。将包装完成的慢病毒进行库K562细胞系的转导，按照预先测试出的抗生素浓度加入到转导后的细胞系中，同时做K562的对照。当对照组细胞全部死亡，实验组细胞仍有存活时停止筛选。实验组细胞系继续进行培养，同时加入最高筛选浓度的嘌呤霉素。最终获得高表达CD123的细胞系K562-CD123。筛选完成后，利用流式细胞术对细胞系K562-CD123的CD123的表达情况进行验证，并用K562细胞系做阴性对照。
2、实验结果
结果见图7，结果显示，构建得到的K562-CD123稳转细胞系高表达CD123，即本发明成功构建得到了K562-CD123稳转细胞系。
实施例4单VHH CAR-T细胞的制备及体外功能验证
1、单VHH CAR结构构建
将序列特异性的克隆进行单VHH CAR结构构建。首先，利用PCR的方法扩增阳性克隆株的VHH序列；第一轮PCR结束后，以第一轮PCR产物为模板进行第二轮PCR；将第二轮PCR产物通过同源重组的方式，连接入载体Senl-S88BZ，载体用Not I单酶切。至此，包含有靶向CD123单VHH的CAR结构构建成功，结构示意图如图8，引物序列见表3。
表3单VHH CAR结构构建用引物列表
共构建2个单VHH CAR结构，分别命名为NMC009-01、NMC009-02，结构如图8所示，EF1α为延长因子1α的启动子，Leader是信号肽的编码序列，VHH是抗CD123 的纳米抗体的编码序列，CD8H+TM为CD8铰链区和跨膜区，4-1BB和CD3ζ胞内信号区为胞内共刺激域，通过T2A肽连接表达tEGFR胞外区域，以便慢病毒转导后检测CAR的表达。
其中，在VHH CAR结构中，信号肽的氨基酸序列和核苷酸序列分别如SEQ ID NO:17、SEQ ID NO:18所示，CD8铰链区的氨基酸序列和核苷酸序列分别如SEQ ID NO:19、SEQ ID NO:20所示，CD8跨膜区的氨基酸序列和核苷酸序列分别如SEQ ID NO:21、SEQ ID NO:22所示，4-1BB的氨基酸序列和核苷酸序列分别如SEQ ID NO:23、SEQ ID NO:24所示，CD3ζ的氨基酸序列和核苷酸序列分别如SEQ ID NO:25、SEQ ID NO:26所示，T2A的氨基酸序列和核苷酸序列分别如SEQ ID NO:27、SEQ ID NO:28所示，tEGFR信号肽的氨基酸序列和核苷酸序列分别如SEQ ID NO:29、SEQ ID NO:30所示，tEGFR的氨基酸序列和核苷酸序列分别如SEQ ID NO:31、SEQ ID NO:32所示。
2、慢病毒包装
CAR-T细胞制备前，首先进行慢病毒的包装：将目的质粒与三个辅助质粒(pMD2.G、pRSV-REV、pMDLg)在PEI-Pro作用下共转染293FT细胞；包装6小时进行换液；包装48小时后进行慢病毒收获；收获的慢病毒原液进行超速离心浓缩，用DMEM高糖培养基重悬慢病毒颗粒，分装备用。
3、K562-NMC009细胞制备
慢病毒包装完成后，将慢病毒进行K562细胞系的转导。并将转导NMC009系列慢病毒的K562进行培养，培养3天后进行流式细胞术检测，用CD123-His蛋白作一抗，抗His标签抗体为二抗检测细胞表面纳米抗体的表达情况。并对K562表面VHH的MFI进行统计。
4、CAR-T细胞制备
慢病毒包装完成后，进行CAR-T细胞的制备：采集患者或健康供者外周血单个核细胞(PBMC)；通过CD3磁珠进行αβT细胞分选；分选完成的αβT细胞在TexMACS GMP培养基(MACS)中进行培养；2天后进行慢病毒的转导；继续培养到12-14天进行CAR-T细胞收获，获得靶向CD123的VHH CAR-T细胞(分别命名为NMC009-01和NMC009-02)，CAR-T细胞培养流程如图9所示；培养过程中进行流式检测，测定CAR+细胞的比例，用抗EGFR抗体检测tEGFR的表达，用CD123-His蛋白作一抗，抗His标签抗体为二抗检测细胞表面纳米抗体的表达情况，并且对用CD123-His抗原检测CAR-T细胞表面VHH 的MFI值进行统计。在细胞培养的第5天、第8天、第11天、第14天分别进行细胞计数，统计细胞扩增情况。并在培养的后期进行CAR-T细胞CD4/CD8比例进行统计。
5、CAR-T细胞的体外功能验证
为了验证本实施例制备得到的抗CD123VHH CAR-T细胞的体外生物学活性，培养过程中进行了体外杀伤实验的验证：首先收集靶细胞K562-CD123(K562为人慢性髓原白血病细胞)，2000rpm离心5min，DPBS重悬计数，按1×105个/孔的数量加到96孔板中。然后根据不同效靶比(E：T＝0.3：1、1：1、3：1)向靶细胞中添加适量的效应细胞，混合后孵育4小时，流式细胞术检测细胞杀伤比例。
6、实验结果
将单VHH结构转导K562细胞后，K562细胞表面VHH的MFI统计结果如图10所示，K562-NMC009-01、K562-NMC009-02细胞表面VHH MFI值分别为：5190±380.43、5160±468.11。
将单VHH结构转导T细胞制备CAR-T细胞，CAR-T细胞培养6天后的流式细胞术代表性检测结果见图11，分别检测了CAR-T细胞表面tEGFR的表达，及纳米抗体的表达情况，其中图11A显示的是细胞表面tEGFR的表达情况，NMC009-01、NMC009-02的阳性率分别为：17.84％、45.5％，图11B显示的是细胞表面纳米抗体的表达情况，NMC009-01、NMC009-02的阳性率分别为：13.16％、36.92％；图12显示的是CAR-T细胞表面纳米抗体MFI统计情况，NMC009-01、NMC009-02细胞表面VHH MFI值分别为：683±8.48和671±36.06，均高于空白T细胞的131±23.33。细胞培养后期CD4/CD8比值如图13所示，NMC009-01、NMC009-02的CD4/CD8比值分别为3.04±0.18、2.83±0.68。
单VHH CAR-T扩增曲线见图14，由图可知，扩增到第14天，NMC009-01的平均扩增倍数为66.6±9.33倍，NMC009-02的扩增倍数为63.25±4.03倍，空白T细胞的扩增倍数为77.3±4.38倍。
CAR-T细胞的体外功能验证结果见图15，结果显示，在杀伤比例为0.3：1、1：1、3：1时，NMC009-01对K562-CD123的杀伤值分别为19.70±2.40％、45.15±3.04％、77.60±8.34％，NMC009-02对K562-CD123的杀伤比例分别为14.90±0.42％、36.05±8.56％、65.40±10.04％，空白T细胞对K562-CD123的杀伤比例分别为11.00±0.42％、11.25±1.91％、14.05±0.92％。
实施例5双VHH CAR-T细胞的制备
1、双VHH CAR结构构建
将本发明筛选到的纳米抗体VHH01和VHH02用于构建双VHH的CAR结构目的质粒，双VHH CAR的结构示意图如图16所示。首先，利用PCR的方法扩增阳性克隆株的VHH02序列；第一轮PCR结束后，以第一轮PCR产物为模板进行第二轮PCR，引物序列见表4；然后，将第二轮PCR产物通过同源重组的方式，连接入载体NMC009-01中，载体用Not I单酶切重组质粒命名为dNMC009-A。
表4单VHH CAR结构构建用引物列表
2、双VHH CAR-T细胞的制备
按照实施例4中CAR-T细胞的制备流程，同时进行dNMC009-A、NMC009-01、NMC009-02 CAR-T细胞的培养，培养过程中进行流式检测，测定CAR+细胞的比例，用抗EGFR抗体检测tEGFR的表达，用CD123-His蛋白作一抗，抗His标签抗体为二抗检测细胞表面纳米抗体的表达情况，并且对用CD123-His抗原检测CAR-T细胞表面VHH的MFI值进行统计。在CAR-T细胞培养的第6天、第9天、第13天、第16天进行细胞计数，用于观察CAR-T细胞的扩增情况。
3、dNMC009-A双VHH CAR-T细胞的体外功能实验验证
为比较dNMC009-A与单VHH CAR-T的体外功能，在dNMC009-A、NMC009-01、NMC009-02 CAR-T细胞培养的第11天，CAR-T细胞以及空白T细胞分别与CD123表达阳性细胞系K562-CD123按照不同比例进行细胞杀伤实验：首先收集靶细胞，2000rpm离心5min，DPBS重悬计数，进行CFSE染色，并按照1E5/孔的数量添加到96孔板中。然后根据不同效靶比(E：T＝0.3：1、1：1、3：1)向靶细胞中添加适量的效应细胞，混合后孵育4小时，流式细胞术检测细胞杀伤比例。
4、实验结果
CAR-T细胞培养6天后的流式细胞术代表性检测结果见图17，分别检测了CAR-T细胞表面tEGF及纳米抗体的表达情况，其中，图17A显示的是细胞表面tEGFR的表达情况，NMC009-01、NMC009-02、dNMC009-A的阳性率分别为：84.1％、76.1％、73.0％，图17B显示的是细胞表面纳米抗体的表达情况，NMC009-01、NMC009-02、dNMC009-A的阳性率分别为：22.42％、23.5％、18.14％。图18显示的是CAR-T细胞表面纳米抗体MFI统计情况，NMC009-01、NMC009-02、dNMC009-AMFI值分别为：481.5±55.86、285.5±99.7、319±53.03，均高于空白T细胞的88.8±8.06。
扩增曲线见图19，由图可知，扩增到第16天，dNMC009-A的扩增倍数为76.4±1.40倍，NMC009-01的扩增倍数为71.77±3.62倍，NMC009-02的扩增倍数为69.05±1.63倍。
体外功能验证结果见图20，结果显示，在杀伤比例为0.3：1、1：1、3：1时，dNMC009-A对K562-CD123的杀伤均值分别为16.60±1.70％、34.50±1.84％、70.50±3.11％；NMC009-01对K562-CD123的杀伤均值分别为12.20±4.24％、25.85±5.15％、67.75±2.19％；NMC009-02对K562-CD123的杀伤均值分别为7.77±0.76％、20.00±2.69％、55.15±2.90％，表明了本发明基于纳米抗体VHH01和/或VHH02构建得到的dNMC009-A与单VHH CAR-T对CD123阳性的肿瘤细胞均具有高效的杀伤效果。
实施例6纳米抗体亲和力检测
1、亲和力检测方法
将dNMC0901-B(实施例7中所述的0901-CAR)结构中包含的CD7(anti-CD7VHH10)和CD123(anti-CD123VHH01)纳米抗体用15个氨基酸的短肽(GGGGSGGGGSGGGGS)进行连接，然后再与人IgG1FC进行融合表达，重组抗体命名为dNb0901-B。将表达纯化后的dNb0901-B抗体用于SPR测定。使用Biacore 8K分析系统通过SPR测定dNb0901-B对人源CD123、CD7蛋白和鼠源CD123、CD7的亲和力。首先，将抗人IgG1Fc抗体以25μg/mL共价偶联至CM5传感器芯片；然后，用抗人IgG1Fc抗体去捕获dNb0901-B蛋白作为固定相；再将倍比稀释的人源CD123、CD7蛋白和鼠源CD123、CD7蛋白作为流动相以30μL/min的流速注射，然后进行解离；最后，使用Biacore Insight Evaluation Software 3.0.12.15655软件测定缔合和解离速率常数。
2、实验结果
SPR方法检测抗体亲和活性结果如表5和图21A和图21B所示dNb0901-B与人CD123蛋白之间的亲和力常数为4.07E-09，dNb0901-B与人CD7蛋白之间的亲和力常数 为5.94E-09。结果显示，dNb0901-B与人CD123和CD7能够发生特异性结合。以上结果表明了本发明构建得到的CAR结构所用抗体与人CD123和人CD7均具有较好的亲和力。
表5 SPR方法测定抗体亲和力的结果统计
实施例7制备NS0901、KO7-0901和KO7-0109 CAR-T细胞
1、构建慢病毒载体
(1)将靶向CD123的纳米抗体片段(VHH01)和靶向CD7的纳米抗体片段(VHH10)克隆至具有4-1BB和CD3ζ的第二代CAR结构骨架中，以T2A连接胞外tEGFR结构域，得到名称为0901的CAR。0901-CAR基因具体结构为EF1α-leader-anti CD123VHH(VHH01)-Linker-anti CD7VHH(VHH10)-CD8hinge-CD8TM-4-1BB-CD3ζ-T2A-tEGFR(如图22所示)。
(2)将靶向CD7的纳米抗体片段(VHH10)和靶向CD123的纳米抗体片段(VHH01)克隆至具有4-1BB和CD3ζ的第二代CAR结构骨架中，以T2A连接胞外tEGFR结构域，得到名称为0109的CAR。0109-CAR基因具体结构为EF1α-leader-anti CD7VHH(VHH10)-Linker-anti CD123VHH(VHH01)-CD8hinge-CD8TM-4-1BB-CD3ζ-T2A-tEGFR(如图23所示)。
其中，EF1α是延长因子1α的启动子，leader是信号肽的编码序列，anti-CD7VHH是抗CD7纳米抗体(VHH10)的编码序列，anti-CD123VHH是抗CD123纳米抗体(VHH01)的编码序列，CD8hinge是铰链区，CD8TM是跨膜区，4-1BB和CD3ζ胞内信号区是胞内共刺激域，通过T2A肽连接表达tEGFR胞外区域，以便病毒转导后检测CAR的表达。
其中，所述纳米抗体VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5所示，所述纳米抗体VHH01的氨基酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:2所示；所述纳米抗体VHH10的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示，所述 纳米抗体VHH10的氨基酸序列如SEQ ID NO:45所示，核苷酸序列如SEQ ID NO:49所示；所述CD8TM的氨基酸序列如SEQ ID NO:21所示，所述CD8hinge的氨基酸序列如SEQ ID NO:19所示，所述CD3ζ胞内信号区的氨基酸序列如SEQ ID NO:25所示，所述4-1BB共刺激信号结构域的氨基酸序列如SEQ ID NO:23所示，所述leader的氨基酸序列如SEQ ID NO:17所示，所述EF1α的核苷酸序列如SEQ ID NO:47所示，所述T2A的氨基酸序列如SEQ ID NO:27所示，所述tEGFR的氨基酸序列如SEQ ID NO:31所示，所述Linker的氨基酸序列如SEQ ID NO:48所示，所述tEGFR信号肽的氨基酸序列如SEQ ID NO:29所示；所述CD8TM的核苷酸序列如SEQ ID NO:22所示，所述CD8hinge的核苷酸序列如SEQ ID NO:20所示，所述CD3ζ胞内信号区的核苷酸序列如SEQ ID NO:26所示，所述4-1BB共刺激信号结构域的核苷酸序列如SEQ ID NO:24所示，所述leader的核苷酸序列如SEQ ID NO:18所示，所述T2A的核苷酸序列如SEQ ID NO:28所示，所述tEGFR的核苷酸序列如SEQ ID NO:32所示，所述Linker的核苷酸序列如SEQ ID NO:50所示，所述tEGFR信号肽的核苷酸序列如SEQ ID NO:30所示。
(3)0901-CAR基因表达载体(慢病毒表达载体)的构建，通过PCR获得VHH10序列，并通过同源重组的方式将其连接入本实验室自主保存的慢病毒载体NMC009-01，序列为SEQ ID NO:46。重组载体命名为0901-CAR。
(4)0109-CAR基因表达载体(慢病毒表达载体)的构建，通过PCR获得VHH01序列，并通过同源重组的方式将其连接入本实验室自主保存的慢病毒载体NMC001-10，序列为SEQ ID NO:57。重组载体命名为0109-CAR。
2、慢病毒包装
慢病毒包装操作步骤如下：首先进行PEI转染试剂和质粒混合物的制备，将慢病毒表达载体、编码Rev蛋白的包装质粒psPAX2、编码VSV-G蛋白的包膜质粒pMD2.G与PEI转染试剂(Polyplus)混合均匀，室温静置20min。然后将上述混合液加到293FT细胞中，培养72h后收取培养上清作为病毒原液。4℃18300g高速离心2h。将上清液弃净，加入无血清培养基重悬病毒颗粒，此即为慢病毒溶液。
3、NS 0901-CAR-T细胞的制备和KO7-0901 CAR-T、KO7-0109 CAR-T细胞的制备
(1)NS 0901-CAR-T细胞的制备
将外周血PBMC分选出来的T细胞，加入Dynabeads CD3/CD28活化磁珠进行激活，放入37℃5％CO2细胞培养箱进行培养(记为Day0)，培养48小时后，用上述制备的慢病毒溶液加入到T细胞中，离心2小时(2000rpm，35℃)进行慢病毒转导，转导完 成后加入含有200UI/mL IL-2的MACS培养基放入37℃5％CO2细胞培养箱进行培养，Day5取样，通过流式细胞术进行CAR+比例检测。Day12到Day14收获细胞，得到NS0901-CAR-T细胞。具体制备流程见图24。
(2)KO7-0901 CAR-T和KO7-0109 CAR-T细胞的制备
通过CRISPR/Cas9基因编辑敲除T细胞内源性的CD7降低细胞自杀，提高制备成功率。设计四个靶向CD7的向导RNA(gRNA)，分别为gRNA-T71、gRNA-T72、gRNA-T73、gRNA-T74，其序列信息分别如SEQ ID NO:53-56所示，并且验证活性。通过商业合成sgRNA，并在sgRNA的5’和3’的三个末端碱基处并入2′-O-甲基和3′硫代磷酸碱基以防止核酸酶活性。将9μg Cas9蛋白和4.5μg sgRNA加入到30μL EO buffer中，室温孵育10min，形成Cas9蛋白和CD7-sgRNA复合物(Cas9RNP-CD7)，然后电穿孔到T细胞中，在电转后的72小时通过流式细胞术进行CD7敲除率的测试，sgRNA-T71具有最有效的CD7敲除效率，将CD7+T细胞的百分比由92.9％降低到11.1％(见图26)，因此，在本发明中选择sgRNA-T71应用于后续所有敲除CD7的实验。
按照上述T细胞分离活化的方法制备T细胞。将分选出来的T细胞，加入Dynabeads CD3/CD28活化磁珠进行激活，放入37℃5％CO2细胞培养箱进行培养，培养48小时后，收集细胞，去除磁珠，用EO buffer重悬细胞，将Cas9RNP-CD7复合物加入到细胞中，对细胞进行电转，电转后的细胞继续培养24小时，用上述制备的慢病毒溶液(LV-0901或LV-0109)加入到T细胞中，进行慢病毒转导，继续培养至12-14天，获得KO7-0901CAR-T细胞或KO7-0109 CAR-T(具体制备流程见图25)，只进行敲除CD7未转导慢病毒的T细胞作为mock-T细胞对照。
4、NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞体外抗肿瘤能力的研究
采用NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞作为效应细胞，CCRF-CEM-GFP-LUC细胞(表达CD7抗原)和MOLM-13-GEP-LUC细胞(表达CD123抗原)作为靶细胞。
体外杀伤效果测试：按效靶比2：1将效应细胞和靶细胞混匀，混合后孵育18-24小时，孵育结束后，加入检测试剂，在荧光酶标仪上读取化学发光数值，计算杀伤率。杀伤率(％)＝(靶细胞RCL-实验细胞RCL)/靶细胞RCL。
细胞因子测试：按效靶比1：1将效应细胞和靶细胞混匀，混匀共同孵育18小时，取上清检测细胞因子的分泌。
5、KO7-0901 CAR-T细胞在动物体内抗肿瘤效果
为了评估KO7-0901 CAR-T在动物体内的抗肿瘤效果及安全性，采用NPG小鼠荷瘤模型进行动物体内研究。
采用NPG小鼠移植人急性髓系白血病细胞-荧光素酶(luciferase)标记细胞(KG-1a-LUC)构建移植瘤模型，共使用24只雄性小鼠，按照1×106个KG-1a-LUC/只注射到NPG小鼠尾静脉。根据荧光强度分为4组，模型对照组、CAR-T低、中、高剂量组，每组6只小鼠。造模后第5天，各组小鼠分别经尾静脉给予溶媒、0.1×107、0.33×107、1.0×107CAR+T细胞/只。通过活体成像法，于分组给药当天(即D1)、给药后每周1次检测体内KG-1a-LUC细胞荧光强度，并观察临床症状、记录动物死亡情况；于给药后2-4h、D2、D5、D8、D15采血，通过流式细胞术检测血液样本中细胞表型，评估CAR-T细胞在体内的增殖水平。
6、实验结果
(1)对于体外培养，结果显示，细胞从0天培养到第12天时，NS 0901-CAR-T细胞扩增7.3±2.6倍，KO7-0901 CAR-T细胞扩增22.4±7.4倍，KO7-0109 CAR-T细胞扩增18.6±2.2倍(见图27)；在收获时NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞的CAR阳性率分别为97.0±1.3％，57.9±19.3％和65.9±4.4％(见图28)；图29为CAR阳性率的流式代表图，用ERB染色作为CAR阳性率，用CD123抗原和CD7抗原染色，表示CAR-T细胞可以识别CD123和CD7抗原；细胞收获时，NS 0901-CAR T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞均具有较高的细胞活率(见图30)，其中敲除CD7的mock-T细胞的敲除效率为98.7％(见图31)。
(2)对于体外杀伤检测，结果显示，NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109CAR-T对CCRF-CEM和MOLM-13细胞都有强的杀伤力，在效靶比为2:1时，杀伤率可达80％以上(见图32A和32B)。
(3)对于细胞因子检测，结果显示，与靶细胞孵育18小时后，NS 0901-CAR-T、KO7-0901 CAR-T和KO7-0109 CAR-T细胞上清中均分泌较高的IFN-γ、TNF-α和IL-8(见图33A和33B)。
(4)对于小鼠体内抗肿瘤效果研究的结果如下：
①活体成像结果：给药后，D1、D8、D15进行活体成像。模型对照组动物体内荧光强度随着时间逐渐增强，表明了随试验进展动物体内肿瘤细胞数量逐渐增多(见图34)；CAR-T低、中、高剂量组动物总荧光值随着时间逐渐下降，在给药后D8时，CAR-T中、 高剂量组动物总荧光值与模型对照组相比均具有显著性差异(P<0.05)；在给药后D15时，CAR-T低、中、高剂量组动物总荧光值与模型对照组相比均具有显著性差异(P<0.05)(见图35)，表明了本发明构建的CAR-T细胞对于动物体内肿瘤具有一定的杀伤和/或抑制作用。
②CAR-T细胞在小鼠体内的表达结果：采用流式细胞术检测不同时间点(2-4h、D2、D5、D8、D15)外周血中CAR-T细胞的表达，反映血液中CAR-T细胞的动态变化情况。对于受试物CAR+CD3+/WBC，模型对照组在D2-D15无检出，CAR-T低、中、高剂量组在D2时表达量为最高值，随后呈下降趋势；D2时，CAR-T低、中、高剂量组CAR+占比相对模型对照组显著性升高(P<0.05)，D5、D8、D15时，CAR-T低、中、高剂量组CAR+占比相对模型对照组无显著变化(P>0.05)(见图36)；肿瘤造模NPG小鼠单次经尾静脉给予KO7-0109 CAR-T细胞，0.1×107、0.33×107、1.0×107 CAR+T细胞/只剂量范围内具有一定程度的抑制体内KG-1a-LUC肿瘤细胞增殖效果，对肿瘤相关的临床症状明显改善，且试验期间未出现与药物相关的不良反应。
实施例8通用型CAR0901T细胞的制备
在本实施例中，发明人利用靶向CD123和CD7双特异性纳米CAR的特点开发一种通用型CAR-T细胞，生产一种现成的(off-the-shelf)即用型治疗产品。本实施例中所述靶向CD123和CD7双特异性纳米通用型CAR-T细胞(UCAR0901T)的详细介绍如下：发明人利用CRISPR/Cas9基因编辑技术破坏T细胞受体α恒定(TRAC)位点，以消除TCR的表达，降低同种异体T细胞引发的移植物抗宿主病(GvHD)风险。靶向CD7的CAR可以特异性靶向患者自身T细胞和自然杀伤(NK)细胞(同种异体反应性杀伤细胞)，减少宿主抗移植物排斥反应(HvG)，同时靶向CD123的CAR可以清除AML肿瘤细胞(急性髓系白血病细胞)。
1、通用型CAR0901T(UCAR0901T)细胞的制备
Cas9蛋白、CD7-sgRNA和TRAC-sgRNA复合物(Cas9RNP-CD7&TRAC)的制备：将9μg Cas9蛋白和4.5μg CD7-sgRNA(CAUCAUUUACUACGAGGACG)(SEQ ID NO:51)和9μg TRAC-sgRNA(从Osborn等人获得)(GAGAAUCAAAAUCGGUGAAU)(SEQ ID NO:52)加入到30μL EO buffer中，室温孵育10min，形成Cas9RNP-CD7&TRAC复合物。上述序列中，A、G、C和U为核糖核苷酸，T为脱氧核糖核苷酸，第1-3位核苷酸的核糖中的2′-OH均替换为甲氧基，磷酸均被替换为硫代磷酸；倒数第1-3位核苷酸 的核糖中的2′-OH均替换为甲氧基，磷酸均被替换为硫代磷酸；第1-20位核苷酸为TRAC基因的gRNA的靶标核苷酸序列。
将PBMC分选出来的T细胞，加入Dynabeads CD3/CD28活化磁珠进行激活，放入37℃、5％CO2细胞培养箱进行培养，培养48小时后，收集细胞，去除磁珠，用EO buffer重悬细胞，将Cas9RNP-CD7&TRAC复合物加入到细胞中，混匀，室温孵育10min，对细胞进行电转，电转后继续培养24小时后，用上述制备的病毒浓缩(LV-0901)加入到T细胞中，离心2小时(2000rpm，35℃)进行慢病毒转染，继续培养至12-14天，获得UCAR0901T细胞，只进行敲除CD7和TCR未转导慢病毒的T细胞作为mock-T细胞对照。
2、UCAR0901T细胞体外抗肿瘤能力的研究
采用UCAR0901T和mock-T细胞作为效应细胞，用CCRF-CEM-GFP-LUC细胞(表达CD7抗原)和MOLM-13-GEP-LUC细胞(表达CD123抗原)作为靶细胞。
体外杀伤效果测试：按效靶比2：1将效应细胞和靶细胞混匀，混合后孵育18-24小时，孵育结束后，加入检测试剂，在荧光酶标仪上读取化学发光数值，计算杀伤率。杀伤率(％)＝(靶细胞RCL-实验细胞RCL)/靶细胞RCL。
细胞因子测试：按效靶比1：1将效应细胞和靶细胞混匀，混匀共同孵育18小时，取上清检测细胞因子的分泌。
3、实验结果
结果显示，细胞从0天培养到第12天时，UCAR0901T细胞扩增21.6±8.2倍，mock-T细胞扩增22.8±6.9倍(见图37)；在收获时UCAR0901T细胞的CAR阳性率为57.9±6.6％(见图38)；图19为CAR阳性率的流式代表图，用ERB染色作为CAR阳性率，用CD123抗原和CD7抗原染色，表示CAR-T细胞可以识别CD123和CD7抗原。CD7和TRAC敲除的mock-T细胞的双敲除效率为96.2％(见图40)。
对于体外杀伤效果检测，结果显示，与mock-T细胞相比，UCAR0901T对CCRF-CEM和MOLM-13细胞都有强的杀伤力，在效靶比为2:1时，杀伤率可达80％以上(见图41A和41B)。
对于细胞因子检测，结果显示，与靶细胞孵育18小时后，UCAR0901T细胞上清中分泌较高的IFN-γ、TNF-α和IL-8(见图42A和42B)。
上述实施例的说明只是用于理解本发明的方法及其核心思想，本领域技术人员还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (20)

  1. 抗CD123纳米抗体，其特征在于，所述纳米抗体包括VHH01、VHH02；
    所述VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7所示或分别为与SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7具有至少75％同一性的氨基酸序列；
    所述VHH02的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15所示或分别为与SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15具有至少75％同一性的氨基酸序列。
  2. 根据权利要求1所述的纳米抗体，其特征在于，所述VHH01的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8所示或分别为与SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8具有至少75％同一性的核苷酸序列；
    所述VHH02的CDR1、CDR2、CDR3的核苷酸序列分别如SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16所示或分别为与SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16具有至少75％同一性的核苷酸序列。
  3. 根据权利要求1所述的纳米抗体，其特征在于，所述VHH01的重链可变区的氨基酸序列如SEQ ID NO:1所示或与SEQ ID NO:1具有至少75％同一性的氨基酸序列；
    所述VHH02的重链可变区的氨基酸序列如SEQ ID NO:9所示或与SEQ ID NO:9具有至少75％同一性的氨基酸序列；
    优选地，所述VHH01的重链可变区的核苷酸序列如SEQ ID NO:2所示或与SEQ ID NO:2具有至少75％同一性的核苷酸序列；
    优选地，所述VHH02的重链可变区的核苷酸序列如SEQ ID NO:10所示或与SEQ ID NO:10具有至少75％同一性的核苷酸序列。
  4. 基于抗CD123纳米抗体的嵌合抗原受体，其特征在于，所述嵌合抗原受体包含权利要求1-3中任一项所述的纳米抗体；
    优选地，所述嵌合抗原受体还包含信号肽；
    优选地，所述嵌合抗原受体还包含铰链区；
    优选地，所述嵌合抗原受体还包含跨膜结构域；
    优选地，所述嵌合抗原受体还包含共刺激信号结构域；
    优选地，所述嵌合抗原受体还包含胞内信号传导结构域；
    优选地，所述嵌合抗原受体还包含EF1α、T2A、tEGFR；
    优选地，所述嵌合抗原受体还包含tEGFR信号肽；
    更优选地，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD64、CD80、CD86、CD134、CD137、CD154、GITR、ICOS、IgG6；
    更优选地，所述铰链区包括下列分子的铰链区：CD8、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、OX40、CD3ε、IL-2受体、IL-7受体、IL-11受体；
    更优选地，所述跨膜结构域包括下列分子的跨膜结构域：CD8、CD28、IgG1、IgG4、4-1BB、PD-1、CD34、CD3ε、CD8α、IL-2受体、IL-7受体、IL-11受体；
    更优选地，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB、CD28、ICOS、CD27、CD19、CD4、CD8α、CD8β、HVEM、LIGHT、CD40、OX40、DR3、GITR、CD30、TIM1、CD2、CD226、CD278；
    更优选地，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、TCRζ、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d、FYN；
    最优选地，所述铰链区为CD8铰链区；
    最优选地，所述跨膜结构域为CD8跨膜结构域；
    最优选地，所述共刺激信号结构域为4-1BB共刺激信号结构域；
    最优选地，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域；
    最优选地，所述嵌合抗原受体为EF1α、信号肽、权利要求1-3中任一项所述的纳米抗体、CD8铰链区、CD8跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次串联得到；
    最优选地，所述CD8铰链区的氨基酸序列如SEQ ID NO:19所示或与SEQ ID NO:19具有至少75％同一性的氨基酸序列；
    最优选地，所述CD8跨膜结构域的氨基酸序列如SEQ ID NO:21所示或与SEQ ID NO:21具有至少75％同一性的氨基酸序列；
    最优选地，所述4-1BB共刺激信号结构域的氨基酸序列如SEQ ID NO:23所示或与SEQ ID NO:23具有至少75％同一性的氨基酸序列；
    最优选地，所述CD3ζ胞内信号传导结构域的氨基酸序列如SEQ ID NO:25所示或与SEQ ID NO:25具有至少75％同一性的氨基酸序列；
    最优选地，所述信号肽的氨基酸序列如SEQ ID NO:17所示或与SEQ ID NO:17具有至少75％同一性的氨基酸序列；
    最优选地，所述T2A的氨基酸序列如SEQ ID NO:27所示或与SEQ ID NO:27具有至少75％同一性的氨基酸序列；
    最优选地，所述tEGFR信号肽的氨基酸序列如SEQ ID NO:29所示或与SEQ ID NO:29具有至少75％同一性的氨基酸序列；
    最优选地，所述tEGFR的氨基酸序列如SEQ ID NO:31所示或与SEQ ID NO:31具有至少75％同一性的氨基酸序列。
  5. 核酸分子，其特征在于，所述核酸分子编码权利要求1-3中任一项所述的纳米抗体或编码权利要求4所述的嵌合抗原受体。
  6. 重组表达载体，其特征在于，所述重组表达载体包含权利要求5所述的核酸分子；
    优选地，所述表达载体包括DNA载体、RNA载体、质粒、转座子载体、CRISPR/Cas9载体、病毒来源的载体；
    更优选地，所述病毒来源的载体包慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体、疱疹病毒载体。
  7. 经工程改造的宿主细胞，其特征在于，所述经工程改造的宿主细胞表达含有权利要求5所述的核酸分子或权利要求6所述的重组表达载体；
    优选地，所述宿主细胞包括真核细胞、原核细胞；
    更优选地，所述宿主细胞为真核细胞；
    最优选地，所述真核细胞包括哺乳动物细胞、植物细胞、酵母细胞；
    最优选地，所述真核细胞为免疫细胞；
    最优选地，所述免疫细胞包括T细胞、B细胞、NK细胞、iNKT细胞、CTL细胞、树突状细胞、髓样细胞、单核细胞、巨噬细胞或其任意组合；
    最优选地，所述免疫细胞为T细胞。
  8. 一种纳米抗体-药物偶联物或试剂盒或组合物，其特征在于，所述纳米抗体-药物偶联物是通过将权利要求1-3中任一项所述的纳米抗体共价附着至小分子药物上得以形成的；
    优选地，所述小分子药物包括烷化剂、抗代谢物、抗肿瘤抗生素、有丝分裂抑制剂、染色质功能抑制剂、抗血管生成剂、抗雌激素、抗雄激素、免疫调节剂；
    更优选地，所述烷化剂包括双氯乙基甲胺、苯丁酸氮芥、苯丙氨酸氮芥、溴丙哌嗪、松龙苯芥、磷雌氮芥、环磷酰胺、六甲密胺、氯乙环磷酰胺、异磷酰胺、三胺硫磷、卡氮芥、链唑霉素、福替目丁、环己亚硝脲、白消安、苏消安、英丙舒凡、氮烯咪胺、顺铂、奥沙利铂、卡铂；
    更优选地，所述抗代谢物包括甲氨喋呤、5-氟脲嘧啶、氟苷、5-氟脱氧尿嘧啶、卡培他滨、阿糖胞苷、氟达拉滨、阿糖胞苷、6-巯基嘌呤(6-MP)、6-巯基鸟嘌呤(6-TG)、2-氯脱氧腺苷、5-氮杂胞苷、2，2-二氟脱氧胞嘧啶核苷、克拉屈滨、脱氧柯福霉素、喷司他丁；
    更优选地，所述抗肿瘤抗生素包括阿霉素、柔红霉素、去甲氧正定霉素、戊柔比星、盐酸米托蒽醌、更生霉素、光辉霉素、光神霉素、丝裂霉素C、博来霉素、甲基苄肼；
    更优选地，所述有丝分裂抑制剂包括紫杉醇、紫杉萜、长春碱、长春新碱、长春酰胺、长春瑞滨；
    更优选地，所述染色质功能抑制剂包括托泊替康、依立替康、依托扑沙、磷酸依托扑沙、鬼臼噻吩甙；
    更优选地，所述抗血管生成剂包括丙亚胺、马马司他、巴马司他、普啉司他、坦诺司他、伊洛马司他、CGS-27023A、溴氯哌喹酮、COL-3、新伐司他、BMS-275291、沙立度胺；
    更优选地，所述抗雌激素包括它莫西芬、托瑞米芬、雷洛昔芬、屈洛昔芬、奥多昔芬、阿纳托唑、来曲唑、依西美坦；
    更优选地，所述抗雄激素包括氟他米特、尼鲁米特、比卡鲁胺、安体舒通、醋酸环丙氯地孕酮、非那司提、西咪替丁；
    更优选地，所述免疫调节剂包括干扰素、白介素、肿瘤坏死因子、蘑菇多糖、西佐糖、罗喹美克、匹多莫特、甲氧聚乙二醇琥珀酰胺腺甙脱氨酶、胸腺肽制剂；
    优选地，所述试剂盒或组合物包含权利要求1-3中任一项所述的纳米抗体、权利要求4所述的嵌合抗原受体、权利要求5所述的核酸分子、权利要求6所述的重组表达载体、权利要求7所述的经工程改造的宿主细胞。
  9. 如下任一种方法，其特征在于，所述方法包括：
    (1)一种生产权利要求1-3中任一项所述的纳米抗体的方法，其特征在于，所述方法包括如下步骤：培养权利要求7所述的经工程改造的宿主细胞，从培养物中分离出权利要求1-3中任一项所述的纳米抗体；
    (2)一种非诊断和非治疗目的地检测待测样品中CD123的方法，其特征在于，所述方法包括如下步骤：将待测样品与权利要求1-3中任一项所述的纳米抗体接触，检测所述纳米抗体与CD123的复合物的形成；
    优选地，所述纳米抗体是被可用于检测的标记物标记的纳米抗体；
    更优选地，所述可用于检测的标记物包括荧光色素、亲和素、顺磁原子、放射性同位素；
    最优选地，所述荧光色素为荧光素、罗丹明、Texas红、藻红蛋白、藻蓝蛋白、别藻蓝蛋白、多甲藻黄素-叶绿素蛋白；
    最优选地，所述亲和素为生物素、卵白亲和素、链亲和素、卵黄亲和素、类亲和素；
    最优选地，所述放射性同位素为放射性碘、放射性铯、放射性铱、放射性钴；
    (3)一种制备权利要求7所述的经工程改造的宿主细胞的方法，其特征在于，所述方法包括如下步骤：将权利要求6所述的重组表达载体引入到宿主细胞中；
    优选地，所述引入的方法包括物理方法、化学方法、生物方法；
    更优选地，所述物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔；
    更优选地，所述化学方法包括胶体分散系统、基于脂质的系统；
    最优选地，所述胶体分散系统包括大分子复合物、纳米胶囊、微球、珠；
    最优选地，所述基于脂质的系统包括水包油乳剂、胶束、混合胶束、脂质体；
    更优选地，所述生物方法包括DNA载体、RNA载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体；
    (4)一种体外特异性地抑制CD123活性的方法，其特征在于，所述方法包括如下步骤：将权利要求5所述的核酸分子导入到生物体细胞中，通过表达权利要求1-3中任一项所述的纳米抗体抑制CD123的活性。
  10. 如下任一方面的应用，其特征在于，所述应用包括：
    (1)权利要求1-3中任一项所述的纳米抗体、权利要求4所述的嵌合抗原受体、权利要求5所述的核酸分子、权利要求6所述的重组表达载体、权利要求7所述的经工程改造的宿主细胞、权利要求8所述的纳米抗体-药物偶联物或试剂盒或组合物在检测CD123蛋白或其抗原片段中的应用；
    (2)权利要求1-3中任一项所述的纳米抗体、权利要求4所述的嵌合抗原受体、权利要求5所述的核酸分子、权利要求6所述的重组表达载体、权利要求7所述的经工程改 造的宿主细胞、权利要求8所述的纳米抗体-药物偶联物或试剂盒或组合物在制备用于检测CD123蛋白或其抗原片段的产品中的应用；
    (3)权利要求1-3中任一项所述的纳米抗体、权利要求4所述的嵌合抗原受体、权利要求5所述的核酸分子、权利要求6所述的重组表达载体、权利要求7所述的经工程改造的宿主细胞、权利要求8所述的纳米抗体-药物偶联物或试剂盒或组合物在制备用于诊断CD123相关疾病的产品中的应用；
    (4)权利要求1-3中任一项所述的纳米抗体、权利要求4所述的嵌合抗原受体、权利要求5所述的核酸分子、权利要求6所述的重组表达载体、权利要求7所述的经工程改造的宿主细胞、权利要求8所述的纳米抗体-药物偶联物或试剂盒或组合物在制备用于治疗表达CD123的肿瘤的药物中的应用；
    (5)权利要求5所述的核酸分子或权利要求6所述的重组表达载体在制备经工程改造的宿主细胞中的应用；
    优选地，所述肿瘤为血液系统肿瘤；
    更优选地，所述肿瘤包括急性髓系白血病、急性B淋巴细胞白血病、母细胞性浆细胞样树突细胞肿瘤、慢性髓系白血病、急性T淋巴细胞白血病、慢性B淋巴细胞白血病、慢性T淋巴细胞白血病、Richter综合征、毛细胞性白血病、非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤、霍奇金淋巴瘤、伯基特淋巴瘤。
  11. 一种靶向CD123和CD7的嵌合抗原受体，其特征在于，所述嵌合抗原受体包括与CD123特异性结合的纳米抗体VHH01和与CD7特异性结合的纳米抗体VHH10；
    优选地，所述纳米抗体VHH01的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5所示或分别与SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5具有至少75％同一性的氨基酸序列；
    优选地，所述纳米抗体VHH10的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44所示或分别与SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44具有至少75％同一性的氨基酸序列；
    更优选地，所述纳米抗体VHH01的氨基酸序列如SEQ ID NO:1所示或与SEQ ID NO:1具有至少75％同一性的氨基酸序列；
    更优选地，所述纳米抗体VHH10的氨基酸序列如SEQ ID NO:45所示或与SEQ ID NO:45具有至少75％同一性的氨基酸序列；
    优选地，所述嵌合抗原受体还包括跨膜结构域、胞内信号传导结构域；
    优选地，所述嵌合抗原受体还包括铰链区；
    优选地，所述嵌合抗原受体还包括共刺激信号结构域；
    优选地，所述嵌合抗原受体还包括信号肽；
    优选地，所述嵌合抗原受体还包括EF1α、T2A、tEGFR；
    优选地，所述嵌合抗原受体还包括连接VHH01和VHH10的Linker；
    优选地，所述嵌合抗原受体还包括tEGFR信号肽；
    更优选地，所述跨膜结构域和铰链区包括下列分子的跨膜结构域和铰链区：CD8、4-1BB、PD-1、CD34、CD28、IgG1、IgG4、OX40、CD3ε；
    最优选地，所述跨膜结构域和铰链区为CD8跨膜结构域和CD8铰链区；
    最优选地，所述CD8跨膜结构域的氨基酸序列如SEQ ID NO:21所示；
    最优选地，所述CD8铰链区的氨基酸序列如SEQ ID NO:19所示；
    更优选地，所述胞内信号传导结构域包括下列分子的胞内信号传导结构域：CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD4、CD5、CD8、CD21、CD22、CD79a、CD79b、CD278、FcεRI、DAP10、DAP12、CD66d、DAP10、DAP12、FYN；
    最优选地，所述胞内信号传导结构域为CD3ζ胞内信号传导结构域；
    最优选地，所述CD3ζ胞内信号传导结构域的氨基酸序列如SEQ ID NO:25所示；
    更优选地，所述共刺激信号结构域包括下列分子的共刺激信号结构域：4-1BB、ICOS、CD27、CD19、CD4、CD28、CD8α、CD8β、HVEM、LIGHT、CD40、4-1BB、OX40、DR3、GITR、CD30、TIM1、CD2、CD226、CD278；
    最优选地，所述共刺激信号结构域为4-1BB共刺激信号结构域；
    最优选地，所述4-1BB共刺激信号结构域的氨基酸序列如SEQ ID NO:23所示；
    更优选地，所述信号肽包括下列分子的信号肽：T细胞受体的α链及β链、CD3ζ、CD3ε、CD4、CD5、CD8、CD9、CD28、CD16、CD22、CD64、CD80、CD86、CD134、CD137、CD154、GITR、ICOS、IgG6；
    最优选地，所述信号肽的氨基酸序列如SEQ ID NO:17所示；
    更优选地，所述EF1α的核苷酸序列如SEQ ID NO:47所示；
    更优选地，所述T2A的氨基酸序列如SEQ ID NO:27所示；
    更优选地，所述tEGFR的氨基酸序列如SEQ ID NO:31所示；
    更优选地，所述Linker的氨基酸序列如SEQ ID NO:48所示；
    更优选地，所述tEGFR信号肽的氨基酸序列如SEQ ID NO:29所示；
    最优选地，所述嵌合抗原受体选自以下组中的任一种：
    (1)由EF1α、信号肽、纳米抗体VHH01、Linker、纳米抗体VHH10、CD8铰链区、CD8跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次连接得到的嵌合抗原受体；
    (2)由EF1α、信号肽、纳米抗体VHH10、Linker、纳米抗体VHH01、CD8铰链区、CD8跨膜结构域、4-1BB共刺激信号结构域、CD3ζ胞内信号传导结构域、T2A、tEGFR信号肽、tEGFR依次连接得到的嵌合抗原受体；
    (3)在(1)中所述嵌合抗原受体的氨基酸序列的基础上经过取代、缺失或添加一个或多个氨基酸后形成的嵌合抗原受体；
    (4)在(2)中所述嵌合抗原受体的氨基酸序列的基础上经过取代、缺失或添加一个或多个氨基酸后形成的嵌合抗原受体。
  12. 一种多核苷酸，其特征在于，所述多核苷酸的序列为权利要求11所述的嵌合抗原受体的编码序列、或其互补序列；
    优选地，在所述嵌合抗原受体中，纳米抗体VHH01的核苷酸序列如SEQ ID NO:2所示，纳米抗体VHH10的核苷酸序列如SEQ ID NO:49所示，CD8跨膜结构域的核苷酸序列如SEQ ID NO:22所示，CD8铰链区的核苷酸序列如SEQ ID NO:20所示，CD3ζ胞内信号传导结构域的核苷酸序列如SEQ ID NO:26所示，4-1BB共刺激信号结构域的核苷酸序列如SEQ ID NO:24所示，信号肽的核苷酸序列如SEQ ID NO:18所示，EF1α的核苷酸序列如SEQ ID NO:47所示，T2A的核苷酸序列如SEQ ID NO:28所示，tEGFR的核苷酸序列如SEQ ID NO:32所示，Linker的核苷酸序列如SEQ ID NO:50所示，tEGFR信号肽的核苷酸序列如SEQ ID NO:30所示。
  13. 一种含有权利要求12所述多核苷酸的载体，其特征在于，所述载体包括克隆载体、表达载体；
    优选地，所述载体包括DNA载体、RNA载体、质粒、病毒来源的载体；
    更优选地，所述病毒来源的载体包括慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体、疱疹病毒载体。
  14. 一种基因工程化的宿主细胞，其特征在于，所述基因工程化的宿主细胞含有权利要求12所述的多核苷酸或权利要求13所述的载体；
    优选地，所述宿主细胞选自真核细胞或原核细胞；
    更优选地，所述真核细胞包括哺乳动物细胞、昆虫细胞、植物细胞、酵母细胞；
    更优选地，所述原核细胞包括支原体、衣原体、立克次氏体、细菌、放线菌、蓝细菌；
    最优选地，所述宿主细胞为真核细胞；
    最优选地，所述真核细胞为哺乳动物细胞；
    最优选地，所述哺乳动物细胞为免疫细胞；
    最优选地，所述免疫细胞包括T细胞、B细胞、NK细胞、iNKT细胞、CTL细胞、树突状细胞、髓样细胞、单核细胞、巨噬细胞或其任意组合；
    最优选地，所述免疫细胞为T细胞。
  15. 一种靶向CD123和CD7的通用型CAR-T细胞，其特征在于，所述通用型CAR-T细胞表达权利要求11所述的嵌合抗原受体并且不表达CD7和TCR；
    优选地，所述T细胞来源于健康志愿者或供者；
    更优选地，所述通用型CAR-T细胞中的CD7的编码基因和TCR的编码基因被敲除；
    最优选地，所述TCR的α链和/或β链恒定编码区(即TRAC和/或TRBC)基因被敲除；
    最优选地，所述TCR的α链恒定编码区(即TRAC)基因被敲除；
    最优选地，所述CD7的编码基因和TCR的编码基因被引入到所述T细胞的Cas9蛋白、CD7-sgRNA和TRAC-sgRNA形成的复合物所敲除；
    最优选地，所述CD7-sgRNA的序列如SEQ ID NO:51所示；
    最优选地，所述TRAC-sgRNA的序列如SEQ ID NO:52所示。
  16. 一种衍生物，其特征在于，所述衍生物包括可检测标记的权利要求11所述的嵌合抗原受体和/或权利要求12所述的多核苷酸、赋予抗生素抗性的权利要求11所述的嵌合抗原受体和/或权利要求12所述的多核苷酸、与治疗剂结合或偶联的权利要求11所述的嵌合抗原受体和/或权利要求12所述的多核苷酸；
    优选地，所述可检测标记包括荧光染料、化学发光标记物、胶体金、化学发光催化剂；
    更优选地，所述化学发光标记物包括鲁米诺及其衍生物、吖啶酯或其衍生物、金刚烷、异鲁米诺及其衍生物、稀土元素、联吡啶钌配合物；
    更优选地，所述化学发光催化剂包括辣根过氧化物酶、碱性磷酸酶；
    优选地，所述抗生素抗性的基因包括青霉素抗性基因、四环素抗性基因、氯霉素抗性基因、卡那霉素抗性基因；
    优选地，所述治疗剂包括放射性核素、细胞因子、金纳米颗粒、病毒颗粒、脂质体、纳米磁粒、前药激活酶、化疗剂；
    更优选地，所述细胞因子包括IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12、IL-13、IL-14、IFN-γ、TNF-β、TNF-α、G-CSF、M-CSF；
    更优选地，所述化疗剂包括顺铂、紫杉醇、长春新碱、门冬酰胺酶、奥沙利铂、草酸铂、乐沙定。
  17. 一种用于治疗CD123和/或CD7相关疾病的药物组合物，其特征在于，所述药物组合物包含权利要求12所述的多核苷酸、权利要求13所述的载体、权利要求14所述的基因工程化的宿主细胞、权利要求15所述的通用型CAR-T细胞和/或权利要求16所述的衍生物；
    优选地，所述药物组合物还包括一种或多种药学或生理学上可接受的载体、稀释剂或赋形剂组合；
    优选地，所述CD123和/或CD7相关疾病包括：急性髓系白血病、急性B淋巴细胞白血病、淋巴母细胞淋巴瘤、NKT细胞白血病、外周T细胞淋巴、NKT细胞淋巴瘤、间变性大细胞淋巴瘤/母细胞性浆细胞样树突细胞肿瘤、慢性髓系白血病、急性T淋巴细胞白血病、慢性B淋巴细胞白血病、慢性T淋巴细胞白血病、Richter综合征、毛细胞性白血病、非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤、霍奇金淋巴瘤、伯基特淋巴瘤。
  18. 一种试剂盒，其特征在于，所述试剂盒包含权利要求12所述的多核苷酸或权利要求13所述的载体；
    优选地，所述试剂盒还包括将所述多核苷酸或载体引入到宿主细胞中的试剂；
    优选地，所述试剂盒还包括将所述多核苷酸或载体引入到宿主细胞中的说明书。
  19. 如下任一种方法，其特征在于，所述方法包括：
    (1)一种制备权利要求14所述基因工程化的宿主细胞的方法，其特征在于，所述方法包括如下步骤：将权利要求12所述的多核苷酸或权利要求13所述的载体引入到宿主细胞中；
    优选地，所述引入的方法包括脂质转染法、微注射、电穿孔、DNA载体、RNA载体、逆转录病毒载体、慢病毒载体、痘病毒载体、单纯疱疹病毒载体、腺病毒载体、腺相关病毒载体；
    (2)一种刺激哺乳动物靶细胞群或组织产生免疫应答的方法，其特征在于，所述方法包括如下步骤：给哺乳动物施用有效量的权利要求14所述的基因工程化的宿主细胞或权利要求15所述的通用型CAR-T细胞；
    (3)一种制备权利要求15所述通用型CAR-T细胞的方法，其特征在于，所述方法包括如下步骤：
    ①获得活化的T细胞；
    ②通过CRISPR/Cas9基因编辑技术敲除步骤①所述的T细胞中的CD7和TCR，得到CD7和TCR双敲除的通用型T细胞；
    ③用编码如权利要求11所述的靶向CD123和CD7的嵌合抗原受体表达的慢病毒载体来转染步骤②得到的通用型T细胞，获得靶向CD123和CD7的通用型CAR-T细胞；
    优选地，步骤①中所述的T细胞来源于健康志愿者或供者；
    优选地，步骤②中所述的TCR为TCR的α链恒定编码区(即TRAC)；
    更优选地，所述CD7和TCR的编码基因被引入到所述T细胞的Cas9蛋白、CD7-sgRNA和TRAC-sgRNA形成的复合物所敲除；
    最优选地，所述CD7-sgRNA的序列如SEQ ID NO:51所示；
    最优选地，所述TRAC-sgRNA的序列如SEQ ID NO:52所示。
  20. 如下任一方面的应用，其特征在于，所述应用包括：
    (1)权利要求11所述的嵌合抗原受体、权利要求12所述的多核苷酸、权利要求13所述的载体、权利要求14所述的基因工程化的宿主细胞、权利要求15所述的通用型CAR-T细胞、权利要求16所述的衍生物、权利要求17所述的药物组合物、权利要求18所述的试剂盒在制备用于预防和/或治疗CD123和/或CD7相关疾病的药物中的应用；
    (2)权利要求11所述的嵌合抗原受体、权利要求12所述的多核苷酸、权利要求13所述的载体、权利要求14所述的基因工程化的宿主细胞、权利要求15所述的通用型CAR-T细胞、权利要求16所述的衍生物在制备用于制备预防和/或治疗CD123和/或CD7相关疾病的免疫细胞的试剂盒中的应用；
    (3)权利要求11所述的嵌合抗原受体、权利要求12所述的多核苷酸、权利要求13所述的载体、权利要求14所述的基因工程化的宿主细胞、权利要求15所述的通用型CAR-T细胞、权利要求16所述的衍生物、权利要求17所述的药物组合物、权利要求18所述的试剂盒在制备用于预防和/或治疗CD123和/或CD7相关疾病的生物制剂中的应用；
    (4)权利要求18所述的试剂盒在制备用于预防和/或治疗CD123和/或CD7相关疾病的免疫细胞中的应用；
    优选地，所述CD123和/或CD7相关疾病包括：急性髓系白血病、急性B淋巴细胞白血病、淋巴母细胞淋巴瘤、NKT细胞白血病、外周T细胞淋巴、NKT细胞淋巴瘤、间变性大细胞淋巴瘤/母细胞性浆细胞样树突细胞肿瘤、慢性髓系白血病、急性T淋巴细胞白血病、慢性B淋巴细胞白血病、慢性T淋巴细胞白血病、Richter综合征、毛细胞性白血病、非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤、霍奇金淋巴瘤、伯基特淋巴瘤。
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