WO2016116035A1 - Cd30靶向性的嵌合抗原受体和nkt细胞及其制法和应用 - Google Patents

Cd30靶向性的嵌合抗原受体和nkt细胞及其制法和应用 Download PDF

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WO2016116035A1
WO2016116035A1 PCT/CN2016/071371 CN2016071371W WO2016116035A1 WO 2016116035 A1 WO2016116035 A1 WO 2016116035A1 CN 2016071371 W CN2016071371 W CN 2016071371W WO 2016116035 A1 WO2016116035 A1 WO 2016116035A1
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cells
chimeric antigen
antigen receptor
seq
amino acid
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French (fr)
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伍志强
韩为东
王瑶
郭业磊
代汉仁
王晓慧
王春萌
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西比曼生物科技（上海）有限公司
西比曼生物科技（无锡）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present invention belongs to the field of biological products for tumor therapy, and in particular to CD30-targeted chimeric antigen receptors and NKT cells, and processes and applications thereof. More specifically, it relates to a chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ and its gene and recombinant expression vector, engineered CD30-targeted NKT cells (CAR30-NKT cells) in adoptive immunotherapy and application thereof .
  • the CD30 antigen is a 120 kDa glycoprotein that belongs to the TNF/nerve growth factor receptor family and is expressed almost exclusively on all Hodgkin's lymphoma, some non-Hodgkin's lymphoma, and the surface of virus-infected lymphocytes. Due to the above characteristics, CD30 antigen is an ideal target for antibody-based therapy.
  • CD30 monoclonal antibody combined with cytotoxic drugs or radioisotope schemes. This scheme also exists on the basis of certain clinical effects. Certain defects, such as the transient nature of the targeting effect of CD30 monoclonal antibodies and the lack of distribution of CD30 monoclonal antibodies in tumor sites.
  • the object of the present invention is to overcome the above-mentioned defects that the killing effect of NKT cells on tumors is weak, the specific killing activity needs to be improved, and the presence of CD30 monoclonal antibody in the existing clinical research, and a chimeric antigen receptor CD30ScFv is provided.
  • a chimeric antigen receptor (CAR) is provided, wherein the chimeric antigen receptor is formed by concatenating the hinge region and the transmembrane region of CD8 and the intracellular signal domains of CD137 and CD3 ⁇
  • the structure is a signaling domain.
  • the chimeric antigen receptor is CD30ScFv-CD8-CD137-CD3 ⁇ , from CD30ScFv, the hinge region and transmembrane region of CD8, the intracellular signal domain of CD137, and the intracellular signal domain of CD3 ⁇ Made up in series.
  • the extracellular domain of the chimeric antigen receptor is a single chain antibody against CD30.
  • amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO.
  • the hinge region and the transmembrane region of the CD8 are selected from the group consisting of:
  • the intracellular signal domain of CD137 is selected from the group consisting of:
  • (C) a derivative polypeptide formed by subjecting the amino acid sequence shown in any one of SEQ ID NO: 18 to substitution, deletion or addition of 1-5 amino acid residues.
  • the intracellular signal domain of the CD3 ⁇ is selected from the group consisting of:
  • (B) having ⁇ 80% homology to the amino acid sequence shown in SEQ ID NO: 19 (preferably, ⁇ 90% homology; etc. preferably ⁇ 95% homology; most preferably, ⁇ 97% Homology) and a polypeptide capable of transducing an effector function signal;
  • (C) a derivative polypeptide formed by subjecting the amino acid sequence shown in any one of SEQ ID NO: 19 to substitution, deletion or addition of 1-5 amino acid residues.
  • the CAR comprises the amino acid sequence set forth in SEQ ID NO:9.
  • the chimeric antigen receptor (CAR) is isolated.
  • the amino acid sequence of the chimeric antigen receptor (CAR) is set forth in SEQ ID NO.: 1.
  • a nucleic acid molecule encoding the chimeric antigen receptor (CAR) of the first aspect of the invention.
  • the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region and a transmembrane region of the CD8 selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence encoding an intracellular signal domain encoding the CD137 selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of an intracellular signal domain encoding the CD3 ⁇ :
  • the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:
  • the nucleic acid molecule is isolated.
  • the nucleic acid molecule further comprises a polynucleotide encoding a leader sequence (signal peptide), the amino acid sequence of the leader sequence being as shown in SEQ ID NO.: 21:
  • sequence of the nucleic acid molecule is set forth in SEQ ID NO.: 2.
  • a vector comprising the nucleic acid molecule of the second aspect of the invention is provided.
  • the vector is a lentiviral vector.
  • a cell comprising the vector of the third aspect of the invention or the nucleic acid molecule of the second aspect of the invention in which the exogenous source is integrated in the chromosome.
  • the cell is an isolated cell, and/or the cell is a genetically engineered cell.
  • the cell is a mammalian cell.
  • the cell is an NKT cell, or a T cell.
  • the T cell is an NKT cell.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor of the first aspect of the invention, the nucleic acid of the second aspect of the invention, is provided A molecule, the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention.
  • the chimeric antigen receptor of the first aspect of the invention the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, or the fourth aspect of the invention.
  • the tumor comprises a CD30 positive lymphoma.
  • the lymphoma is Hodgkin's lymphoma.
  • a seventh aspect of the invention provides a method for treating a disease comprising administering an appropriate amount of the chimeric antigen receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, to a subject in need of treatment,
  • the vector according to the third aspect of the invention, the cell according to the fourth aspect of the invention, or the pharmaceutical composition according to the fifth aspect of the invention are administered to a subject in need of treatment.
  • the disease is a tumor.
  • a chimeric antigen receptor comprising a structure in which a hinge region and a transmembrane region of CD8 and an intracellular signal domain of CD137 and CD3 ⁇ are connected in series is provided as a signal.
  • the conduction domain, the signal transduction domain amino acid sequence is set forth in SEQ ID NO:9.
  • the present invention also provides a chimeric antigen receptor which is CD30ScFv-CD8-CD137-CD3 ⁇ , which is composed of CD30 single-chain antibody CD30ScFv, hinge region (hinge region) and transmembrane region of CD8, CD137
  • CD30ScFv-CD8-CD137-CD3 ⁇ which is composed of CD30 single-chain antibody CD30ScFv, hinge region (hinge region) and transmembrane region of CD8, CD137
  • the intracellular signal domain and the intracellular signal domain of CD3 ⁇ are constructed in tandem.
  • the invention also provides a gene encoding the chimeric antigen receptor described above.
  • the present invention also provides a recombinant expression vector comprising the above gene.
  • the invention also provides an engineered CD30-targeted NKT cell which is a chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ modified NKT cell as described above.
  • the invention also provides the use of the above engineered CD30-targeted NKT cells for the preparation of a medicament for the treatment of tumors.
  • the chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ modified NKT cells of the present invention ie, engineered CD30-targeted NKT cells, are capable of Specific binding to CD30 antigen significantly prolongs the survival time of immune cells in patients, enhances the ability of immune cells to target Hodgkin's lymphoma cells and non-Hodgkin's lymphoma cells CD30 antigen, and strengthens Hodgkin's lymphoma cells.
  • the engineered CD30-targeted NKT cells of the invention provide a new choice for the treatment of CD30-positive Hodgkin's lymphoma and non-Hodgkin's lymphoma, and have good industrial application prospects.
  • Figure 1 shows the results of phenotypic analysis of isolated cultured NKT cells by flow cytometry.
  • Figure 2 is a diagram showing the electrophoresis identification of the restriction endonuclease MluI/SalI double-cut fragment of the lentiviral expression vector pWPT-CD8-CD137-CD3 ⁇ of the present invention.
  • Figure 3 is a diagram showing the electrophoresis identification of the restriction endonuclease BamHI/SalI double-cut fragment of the lentiviral expression vector pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ of the present invention.
  • Figure 4 is a schematic diagram showing the structure of the lentiviral expression vector pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ of the present invention, wherein the counterclockwise sequence is a forward gene fragment and the clockwise is a reverse gene fragment.
  • Figure 5 is a flow cytometry assay for the infection efficiency of NKT cells by a virus concentrate containing the chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ .
  • Figure 6 shows the results of phenotypic identification of chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ modified NKT cells (CAR30-NKT cells) by flow cytometry.
  • Figure 7 is a graph showing the cytotoxicity analysis of the killing effect of CAR30-NKT cells of the present invention on CD30-positive human Hodgkin lymphoma cells.
  • Figure 8 is a graph showing changes in the number of white blood cells, total lymphocytes, body temperature, and interleukin-6 levels in patients with CD30-positive Hodgkin's lymphoma treated with CAR30-NKT cells of the present invention.
  • Fig. 9 is a view showing changes in image of liver metastases of patients with CAR30-NKT cells of the present invention before and after treatment of patients with CD30-positive Hodgkin's lymphoma (liver metastases).
  • NKT cells modified with the chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ were used to treat CD30-positive Hodgkin's lymphoma and non-Hodgkin's lymphoma, and lymphoma cells.
  • CD30ScFv-CD8-CD137-CD3 ⁇ has a good specific killing activity, and has a certain number of CD30-positive Hodgkin's lymphoma patients who have undergone multiple treatments (such as CD30 monoclonal antibody combined with cytotoxic drugs or radioisotope treatment) but have no obvious effect. treatment effect.
  • the present invention has been completed on this basis.
  • the present invention provides a chimeric antigen receptor which is CD30ScFv-CD8-CD137-CD3 ⁇ , which is composed of a CD30 single-chain antibody CD30ScFv, a hinge region and a transmembrane region of CD8, and an intracellular signal structure of CD137.
  • the domain and the intracellular signal domain of CD3 ⁇ are constructed in series.
  • the amino acid sequence of the chimeric antigen receptor is as set forth in SEQ ID NO.
  • the invention provides a gene encoding the chimeric antigen receptor described above.
  • the nucleotide sequence of the gene encoding the above chimeric antigen receptor is as shown in SEQ ID NO.
  • the present invention provides a recombinant expression vector containing the above gene.
  • the recombinant expression vector is a lentiviral expression vector.
  • the lentiviral expression vector is not particularly limited as long as it can co-transfect packaging cells such as 293T packaging cells with the helper vector, and obtain the virus concentrate and the chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ modified NTK cells, preferably In this case, the lentiviral expression vector is pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ .
  • the preparation method of the lentiviral expression vector pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ is not particularly limited and may be various methods that can be thought of by those skilled in the art, and preferably, the lentiviral expression vector pWPT-CD30ScFv-CD8-CD137 -
  • the preparation method of CD3 ⁇ includes the following steps:
  • the method of amplifying the hinge region and the transmembrane region of CD8, the intracellular signal domain of CD137, and the intracellular signal domain of CD3 ⁇ from the NKT cell cDNA, respectively is not particularly limited, and may be in the field.
  • NKT cells can be obtained by isolating mononuclear cells in human venous blood and then culturing them.
  • the method for obtaining pWPT-CD8-CD137-CD3 ⁇ may include: extracting total RNA of NKT cells, obtaining NKT cell cDNA by reverse transcription, using the obtained NKT cell cDNA as a template, and using primers P1 (SEQ ID NO. 11) and P2. (SEQ ID NO. 12) PCR amplification was performed to obtain the hint region and transmembrane region of the CD8 gene (SEQ ID NO. 3); PCR amplification was performed using primers P3 (SEQ ID NO. 13) and P4 (SEQ ID NO. 14) to obtain CD137. Intracellular signal domain of the gene (SEQ ID NO. 4); PCR amplification using primers P5 (SEQ ID NO.
  • the method for synthesizing the nucleotide sequence encoding the rat growth hormone signal peptide and CD30ScFv is not particularly limited, and may be various methods commonly used in the art, for example, synthesis by a whole gene synthesis technique.
  • the method of obtaining the sequence correct pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ may comprise: synthesizing a nucleotide sequence encoding the rat growth hormone signal peptide and the CD30ScFv fusion gene (SEQ ID NO. 8) by a whole gene synthesis technique, Cloning into the vector pGSI to obtain pGSI-CD30ScFv; then pGSI-CD30ScFv was double-digested with BamHI/MluI, and ligated with the recombinant plasmid pWPT-CD8-CD137-CD3 ⁇ obtained in step (1) after BamHI/MluI double digestion.
  • the present invention also provides an engineered CD30-targeted NKT cell which is an NKT cell (i.e., CAR30-NKT cell) modified by the above chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ .
  • the preparation method of the engineered CD30-targeted NKT cells is not particularly limited and may be any method that can be thought of by those skilled in the art.
  • the method comprises: packaging carrying the pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ coding The lentivirus of the gene; the NKT cells are infected with the obtained lentivirus, and the NKT cells express the chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ .
  • the method for packaging the lentivirus carrying the pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ coding gene is not particularly limited and may be various methods commonly used by those skilled in the art, and preferably, the lentiviral expression vector pWPT-CD30ScFv-CD8 -CD137-CD3 ⁇ was co-transfected into 293T packaging cells with helper plasmids (such as psPAX2, pMD2.G). The virus supernatant was collected after transfection for 48-72 hours, centrifuged, filtered, and 5 ⁇ PEG6000-NaCl was added to the filtrate for mixing. After centrifugation, the supernatant was discarded, and the pellet was dissolved in sterile PBS precooled at 0-4 ° C to obtain a virus concentrate.
  • helper plasmids such as psPAX2, pMD2.G
  • the method for infecting NKT cells by lentivirus is not particularly limited and may be various methods commonly used in the art.
  • the method comprises: taking 1 ⁇ 10 7 -5 ⁇ 10 7 NKT cells, discarding the old culture solution.
  • the method for infecting NKT cells by lentivirus further comprises: inducing the lentivirus-infected NKT cells obtained by the above culture with GT-T551 culture medium having a final concentration of IL-2 of 800-1200 U/mL, in vitro, When the density of the cells is 80-90%, the cells are transferred into a cell culture bag, and the fresh GT-T551 culture medium with a final concentration of 800-2200 U/mL of IL-2 is added for 1.5-2.5 days to expand and culture the cells. The amplification was carried out to a total amount of 1 ⁇ 10 9 - 2 ⁇ 10 9 cells.
  • the mature protein amino acid sequence of the chimeric antigen receptor expressed by the chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ modified NKT cells is shown in SEQ ID NO.
  • the chimeric antigen receptor precursor protein includes a tandem signal peptide, a CD30 ScFv, a hinge region and a transmembrane region of CD8, an intracellular signal domain of CD137, and an intracellular signal structure of CD3 ⁇ . Domain, protein translation after the excision of the signal peptide in the intracellular rough endoplasmic reticulum into a mature chimeric antigen receptor protein, secreted output and localized on the cell membrane of NKT cells.
  • the gene coding sequence corresponding to the mature protein amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.
  • the chimeric antigen receptor has a structure in which a hinge region and a transmembrane region of the gene CD8 and an intracellular signal domain of CD137 and CD3 ⁇ are connected in series as a signal transduction domain, and the amino acid sequence thereof is represented by SEQ ID NO.
  • the gene coding sequence is shown in SEQ ID NO.
  • the invention also provides engineered CD30-targeted NKT cells prepared by the above method.
  • the invention also provides the use of engineered CD30-targeted NKT cells for the preparation of a formulation for treating a tumor.
  • the tumor is a CD30 positive lymphoma.
  • the lymphoma may be Hodgkin's lymphoma or non-Hodgkin's lymphoma, and further preferably, the lymphoma is Hodgkin's lymphoma.
  • the present invention provides compositions and methods for treating diseases such as cancer.
  • the cancer can be a solid tumor, a primary tumor or a metastatic tumor or a hematological malignancy.
  • Other diseases treatable using the compositions and methods of the invention include viral, bacterial and parasitic infections as well as autoimmune diseases.
  • NKT Natural killer cells
  • TCR Natural killer cells
  • NKR Natural killer cells
  • TCR and NKR can express the TCR of T cells and the NKR-P1 receptors of NK cells.
  • NKT cells Under the guidance of TCR and NKR, NKT cells can produce a large amount of IL-4 and INF ⁇ , and exert cell killing effect on tumor cells.
  • NKT cells bind to the Fc segment of a specific antibody via CD16 on their own surface, and exert an ADC- (antibody-dependent cell-mediated cytotoxicity) effect.
  • ADC- antibody-dependent cell-mediated cytotoxicity
  • NKT cells mainly include immunoregulation and cytotoxicity. After stimulation of NKT cells, they can secrete a large amount of IL-4, IFN- ⁇ , GM-CSF, IL-13 and other cytokines and chemokines to exert immunomodulation. Role, NKT cells are one of the bridges connecting innate immunity and acquired immunity. NKT cells have NK cell-like cytotoxic activity after activation, and can dissolve NK cell-sensitive target cells. The main effector molecules are perforin, Fas ligand and IFN- ⁇ .
  • NKT cells can kill any target cells that have bound to the antibody, so the binding of the antibody to the antigen on the target cell is specific, but the killing effect of the NKT cells on the target cells is non-specific.
  • NKT cells Known NKT cells, methods for preparing T cells, and methods of activation and amplification have been known in the art, for example, by reference: Motohashi, S., Nagato, K., Kunii, N., Yamamoto, H., Yamasaki, K., Okita, K., Hanaoka, H., Shimizu, N., Suzuki, M., Yoshino, I., Taniguchi, M., Fujisawa, T.
  • the improved method of preparing NKT cells is as follows.
  • PBMCs Mononuclear cells
  • the final concentration of the cells was adjusted to 2 ⁇ 10 6 cells/mL using NKT cell culture medium GT-T551 containing 0.6% by volume of human autologous serum; the cells were seeded at a final concentration of 10 ⁇ g/ mL of retronectin coated in a 75 cm 2 cell culture flask. Then, recombinant human interleukin 2 at a final concentration of 500 U/mL, 50 ng/ml CD3 monoclonal antibody, 50 ng/mL recombinant human interleukin-15, and a CO 2 incubator at 37 ° C and a saturated humidity of 5% were added to the medium. Cultivate.
  • the ratio of CD3+CD8+NKT cells/CD3+CD4+NKT cells in the NKT cell population prepared by the method of the present invention is 10/1 to 4/1 (preferably 9/1 to 6/1); and CD3+
  • the ratio of CD56+ NKT cells/CD3+CD8+ NKT cells is 1/18 to 10/18 (preferably 5/18 to 10/18).
  • the CD3+ cell ratio is >95%; the CD3 + CD8 + cell ratio is >90.99%; the CD3 + CD56 + cell ratio is > 15% (preferably, >18%; more preferably >20%; most preferably ⁇ 22%, such as ⁇ 24%, ⁇ 26%, ⁇ 28%, ⁇ 30%); CD8 + CD56 + cell ratio ⁇ 15% (preferably, ⁇ 18%; more preferably ⁇ 20%; most preferably ⁇ 22%, such as ⁇ 24%, ⁇ 26%, ⁇ 28%, ⁇ 30%). .
  • NKT cells are CD3+ and CD56+, the variable region gene expressing T cell receptor (TCR) and the natural killer (NK) cell surface marker NK1 ⁇ 1 (NKR-P1C), and have strong T lymphocytes.
  • TCR T cell receptor
  • NK natural killer cell surface marker
  • Antitumor activity and non-MHC restrictive tumoricidal advantages of NK cells In the killing of the face, to make up for the limitations of T cell MHC restriction, tumor killing spectrum, killing ability are much better than T cells.
  • NKT cells are a group of specific immune cells that combine NK function with T cell characteristics.
  • NKT cells have a highly conserved TCR phenotype (TCRV ⁇ 24/V ⁇ 11-human) and co-express the NK cell-specific marker CD161, which does not recognize peptide antigens presented by classical MHC-I or class II molecules, but rather A non-classical MHC class I molecule presents a glycolipid molecular antigen.
  • NKT cells recognize specific glycolipid molecules presented by CD1d molecules and secrete a large number of cytokines, which are involved in the body's innate immunity and acquired immune response. At present, many studies have confirmed that NKT cells are anti-tumor immunity, anti-infective immunity, and self. Immune diseases and immune regulation play an important role, and many physiological and pathological processes involve NKT cells.
  • the survival time of the conventional T cells in vitro is relatively short, and the NKT cells according to the present invention survive for a long time in vitro, are capable of adherent infection, and have a high infection rate.
  • the T cell infection efficiency is low, and the T cell death rate after infection is also high.
  • the invention provides chimeric antigen receptors (CARs) comprising an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes a target-specific binding element (also known as an antigen binding domain).
  • the intracellular domain includes a costimulatory signaling region and a purine chain portion.
  • a costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise from 0 to 300 amino acids, preferably from 2 to 100 amino acids and most preferably from 3 to 50 amino acids.
  • the invention provides cells (e.g., T cells, NKT cells) genetically engineered to express CAR, which exhibit significant anti-tumor properties.
  • the CAR of the invention may also include an extracellular domain having an antigen binding domain fused to an intracellular signaling domain of a T cell antigen receptor complex ⁇ chain (eg, CD3 ⁇ ).
  • the CAR of the present invention when expressed in NKT cells, is capable of altering antigen recognition based on antigen binding specificity.
  • An exemplary antigen is CD30 because the antigen is expressed on lung cancer cells.
  • the CAR of the invention comprises a specific signaling domain comprising the invention (the hinge and transmembrane regions of CD8, the intracellular signal domains of CD137 and CD3 ⁇ are connected in series).
  • the signaling domain of the invention significantly increases anti-tumor activity and in vivo persistence of CART cells compared to other modes of CAR.
  • a CAR of the invention comprises a target-specific binding element referred to as an antigen binding domain.
  • the choice of antigen binding domain depends on the type and number of ligands that define the surface of the target cell.
  • an antigen binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state.
  • cell surface markers include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.
  • tumor antigen refers to a protein produced by a tumor cell that elicits an immune response, in particular a T-cell mediated immune response.
  • the antigen binding domain of the CAR of the invention targets CD30.
  • the antigen binding domain in the CAR of the invention is an anti-CD30 scFV (single-chain antibody fragment, scFv), wherein the nucleic acid sequence of anti-CD30 scFV comprises the sequence set forth in SEQ ID NO: 7.
  • the anti-CD30 scFV comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20.
  • the anti-CD30 scFV of the CAR of the invention is set forth in SEQ ID NO:20.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain that is naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing complexes with receptors. The interaction of other members.
  • Transmembrane domains can be derived from natural or synthetic sources. In natural sources, this domain can be derived from any membrane-bound protein or transmembrane protein.
  • the transmembrane region specifically used in the present invention may be derived from the ⁇ , ⁇ or ⁇ chain of the T-cell receptor, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80. , CD86, CD134, CD137, CD154 (ie, including at least the transmembrane region(s) in the above).
  • the hinge region and the transmembrane region in the CAR of the invention are the hinge region and the transmembrane region of CD8.
  • the nucleotide sequence encoding the hinge region and the transmembrane region of CD8 comprises the nucleic acid sequence of SEQ ID NO:3.
  • the hinge region and the transmembrane region of CD8 comprise the following amino acid sequences:
  • the intracellular domain of the CAR of the invention or the additional intracellular signaling domain is responsible for the activation of at least one normal effector function of the immune cells in which the CAR has been placed.
  • effector function refers to the proprietary function of a cell.
  • the effector function of a T cell can be a cytolytic activity or a helper activity including secretion of a cytokine.
  • intracellular signaling domain refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a proprietary function.
  • the entire intracellular signaling domain can generally be used, in many instances it is not necessary to use the entire chain.
  • intracellular signaling domain In the case of a truncated portion of an intracellular signaling domain, such a truncated portion can be used in place of the entire strand as long as it transduces an effector function signal.
  • the term intracellular signaling domain thus refers to any truncated portion comprising an intracellular signaling domain sufficient to transduce an effector function signal.
  • Preferred examples of the intracellular signaling domain of the CAR for use in the present invention include a cytoplasmic sequence of a T cell receptor (TCR) and a co-receptor that acts synergistically to initiate signal transduction after antigen receptor binding, and these sequences Any derivative or variant and any synthetic sequence that has the same functional ability.
  • TCR T cell receptor
  • co-receptor that acts synergistically to initiate signal transduction after antigen receptor binding
  • T cell activation can be thought of as mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by TCR (primary cytoplasmic signaling sequence) and in an antigen-independent manner Those that function to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).
  • Primary cytoplasmic signaling sequences regulate the primary activity of TCR complexes in a stimulatory or inhibitory manner Chemical.
  • the primary cytoplasmic signaling sequence that acts in a stimulatory manner can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • the cytosolic signaling molecule in the CAR of the invention comprises a cytosolic signaling sequence derived from CD3 ⁇ .
  • the cytoplasmic domain of the CAR can be designed to include the CD3- ⁇ signaling domain itself, or any other desired cytoplasmic domain that can be useful in the context of the CAR of the invention (a Or multiple) unions.
  • the cytoplasmic domain of a CAR can include a CD3 ⁇ chain portion and a costimulatory signaling region.
  • a costimulatory signaling region refers to a portion of a CAR that includes an intracellular domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
  • cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other at random or in a defined order.
  • a short oligopeptide or polypeptide linker preferably 2 and 10 amino acids in length, can form the linkage.
  • the glycine-serine doublet provides a particularly suitable linker.
  • the cytoplasmic domain is designed to include a signaling domain of CD3 ⁇ and a signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to include a signaling domain of CD3[zeta] and a signaling domain of CD137.
  • the cytoplasmic domain of the CAR of the invention is designed to include a signaling domain of CD137 and a signaling domain of CD3 ⁇ , wherein the signaling domain of CD137 comprises the set forth in SEQ ID NO:
  • the nucleic acid sequence and the CD3- ⁇ signaling domain include the nucleic acid sequence set forth in SEQ ID NO: 5.
  • the cytoplasmic domain of the CAR of the invention is designed to include a signaling domain of CD137 and a signaling domain of CD3 ⁇ , wherein the signaling domain of CD137 comprises an amino acid encoding SEQ ID NO:18
  • the nucleic acid sequence of the sequence, and the signaling domain of CD3 ⁇ comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 19.
  • the cytoplasmic domain of the CAR of the invention is designed to include a signaling domain of CD137 and a signaling domain of CD3 ⁇ , wherein the signaling domain of CD137 comprises the set forth in SEQ ID NO:
  • the amino acid sequence, and the signaling domain of CD3 ⁇ include the amino acid sequence set forth in SEQ ID NO: 19.
  • the intracellular signal domain of CD137 comprises the following amino acid sequence:
  • the intracellular signal domain of CD3 ⁇ comprises the following amino acid sequence:
  • the invention includes a DNA construct comprising a CAR sequence, wherein the sequence comprises a nucleic acid sequence of an antigen binding domain of a nucleic acid sequence operably linked to a signaling domain.
  • Exemplary signaling domains for CARs useful in the present invention include anti-CD30 scFv, CD8 hinge and transmembrane regions, and CD137 and CD3 intracellular signaling domains.
  • the CAR of the invention comprises the nucleic acid sequence set forth in SEQ ID NO: 10.
  • the CAR of the invention comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:9.
  • the CAR of the invention comprises the amino acid sequence set forth in SEQ ID NO:9.
  • 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 a cell expressing the gene, by obtaining the gene from a vector known to include the gene, or by utilizing standard Techniques, isolated directly from cells and tissues containing the gene. Alternatively, the gene of interest can be produced synthetically.
  • the present invention also provides a vector into which the DNA of the present invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells.
  • Lentiviral vectors have the advantage over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • expression of a native or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector.
  • This vector is suitable for replication and integration of eukaryotic cells.
  • a typical cloning vector comprises a transcriptional and translational terminator, an initial sequence and a promoter that can be used to modulate expression of a desired nucleic acid sequence.
  • the expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5, 580, 859, 5, 589, 466, incorporated herein by reference.
  • the invention provides a gene therapy vector.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into such vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector comprises an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (eg, WO01/96584; WO01/29058; and the United States) Patent No. 6,326, 193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to a subject cell in vivo or ex vivo.
  • retroviral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • a lentiviral vector is used.
  • promoter elements can regulate the frequency of transcription initiation.
  • these are located in the 30-110 bp region 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 the promoter elements is often flexible to maintain the promoter function when the element is inverted or moved relative to the other.
  • tk thymidine kinase
  • the interval between promoter elements can be increased by 50 bp, and the activity begins to decrease.
  • a single element can function cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoter such as, but not limited to, actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention.
  • an inducible promoter provides a molecular switch capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or shutting down expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cell may also comprise any one or both of a selectable marker gene or reporter gene to facilitate seeking a population of cells that are transfected or infected by the viral vector. Identify and select expression cells.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • reporter genes were used to identify potentially transfected cells and to assess the functionality of regulatory sequences.
  • the reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and which encodes a polypeptide whose expression is clearly indicated by some readily detectable properties such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is determined at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (eg, Ui-Tei et al, 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and can be prepared using known techniques or commercially available.
  • a construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter.
  • Such a promoter region can be ligated to a reporter gene and used to assess the ability of the agent to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, for example, a mammalian, bacterial, yeast or insect cell by any method in the art.
  • a host cell for example, a mammalian, bacterial, yeast or insect cell by any method in the art.
  • an expression vector can be obtained by physical, chemical or biological hands. The segment is transferred into the host cell.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors particularly retroviral vectors, have become the most widely used method of inserting genes into mammals, such as human cells.
  • Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids. Platinum.
  • An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane sac).
  • an exemplary delivery tool is a liposome. It is contemplated to use a lipid formulation to introduce the nucleic acid into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid.
  • the nucleic acid associated with the lipid can be encapsulated into the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached via a linker molecule associated with both the liposome and the oligonucleotide
  • a linker molecule associated with both the liposome and the oligonucleotide
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets that occur naturally in the cytoplasm and in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • the vector is a lentiviral vector, more preferably a pWPT-GFP lentiviral vector.
  • the present inventors have confirmed that the use of the lentiviral vector to construct the CAR of the present invention has high transfection efficiency for NKT cells and is highly reproducible.
  • the vector further comprises a signal peptide sequence.
  • the signal peptide sequence is ligated upstream of the antigenic tuberculosis domain nucleic acid sequence.
  • the signal peptide is a rat growth hormone signal peptide; more preferably, the nucleic acid sequence of the rat growth hormone signal peptide is as shown in SEQ ID NO.: 6.
  • the present inventors have unexpectedly found that when the vector of the present invention is a lentiviral vector, the use of a rat growth hormone signal peptide can significantly increase the transfection efficiency of a lentiviral vector comprising the CAR of the present invention to NKT cells.
  • the invention encompasses cells transduced with a lentiviral vector (LV) (eg, NKT cells, T cells).
  • LV lentiviral vector
  • NKT cells T cells
  • LV coding links the antigen-binding domain of a specific antibody to the intracellular domain of CD3- ⁇ , CD137 Combined CAR.
  • transduced NKT cells can elicit a CAR-mediated NKT-cell response.
  • the invention also provides a method of stimulating an NKT cell-mediated immune response to a target cell population or tissue of a mammal comprising the step of administering to a mammal a CAR-expressing NKT cell, wherein the CAR comprises specifically A binding moiety that interacts with a predetermined target, including, for example, the ⁇ chain portion of the intracellular domain of human CD3 ,, and a costimulatory signaling region.
  • the invention encompasses a type of cell therapy wherein NKT cells are genetically modified to express CAR, and CAR-NKT cells are injected into a recipient in need thereof.
  • the injected cells are capable of killing the recipient's tumor cells.
  • CAR-NKT cells are able to replicate in vivo, producing long-lasting persistence that leads to sustained tumor control.
  • the CAR-NKT cells of the invention can undergo robust in vivo NKT cell expansion for an extended amount of time.
  • the CAR-mediated immune response can be part of a step of adoptive immunotherapy in which CAR-modified NKT cells induce an immune response specific for the antigen binding domain in the CAR.
  • anti-CD30 CAR-NKT cells elicit a specific immune response against cells expressing CD30.
  • Treatable cancers include tumors that have not been vascularized or have not been vascularized, as well as vascularized tumors.
  • Cancer can include non-solid tumors (such as hematological tumors such as leukemias and lymphomas) or can include solid tumors.
  • Types of cancer treated with the CAR of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Also included are adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancer is a cancer of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocyte-monocyte type).
  • monocyte and erythroleukemia monocyte and erythroleukemia
  • chronic leukemia such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia
  • polycythemia vera lymphoma
  • Hodgkin's disease non Hodgkin's lymphoma (painless and high-grade forms)
  • multiple myeloma Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • a solid tumor is an abnormal mass of tissue that usually does not contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, mucinous sarcoma, liposarcoma mesothelioma, lymphoid malignancy, pancreatic cancer, ovarian cancer.
  • the antigen binding domain of a CAR of the invention is designed to treat a particular cancer.
  • a CAR designed to target CD30 can be used to treat cancer and disorders including, but not limited to, lung cancer and the like.
  • treatment for cancer and disorders can be performed using a combination of CARs that target CD19, CD20, CD22, and ROR1.
  • the CAR-modified T cells of the invention can also be used as vaccine types for ex vivo immunity and/or in vivo therapy in mammals.
  • the mammal is a human.
  • cells are isolated from a mammal, preferably a human, and genetically modified (i.e., transduced or transfected in vitro) with a vector that expresses the CAR disclosed herein.
  • CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit.
  • Mammalian recipients can be human, and CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic relative to the recipient.
  • ex vivo culture and expansion of T cells includes: (1) collecting CD34+ hematopoietic stem and progenitor cells from mammals from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo.
  • other factors such as flt3-L, IL-1, IL-3 and c-kit ligands can also be used to culture and expand cells.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.
  • cells activated and expanded as described herein can be used to treat and prevent diseases produced in individuals without an immune response.
  • the CAR-modified T cells of the invention are used to treat CCL.
  • the cells of the invention are used to treat a patient at risk of developing CCL. Accordingly, the invention provides a method of treating or preventing CCL comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
  • the CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a population of target cells 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; Mixtures 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 polypeptides or amino acids
  • antioxidants such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives eg, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a form suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's condition - although appropriate dosages may be determined by clinical trials.
  • a pharmaceutical composition comprising a T cell as described herein may be at a dose of from 10 4 to 10 9 cells/kg body weight, preferably from 10 5 to 10 6 cells/kg body weight (including all integers in those ranges) Value) application. T cell compositions can also be administered multiple times in these doses.
  • Cells can be administered by using injection techniques well known in immunotherapy (see, eg, Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988).
  • Optimal dosages and treatment regimens for a particular patient can be readily determined by a person skilled in the medical arts by monitoring the patient's signs of disease and thus modulating the treatment.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.
  • the NKT cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection.
  • the NKT cell compositions of the invention are preferably administered by i.v. injection.
  • Compositions of NKT cells can be injected directly into tumors, lymph nodes or infected sites.
  • cells activated and expanded using the methods described herein or other methods known in the art to extend NKT cells to therapeutic levels are combined with any number of related therapeutic modalities (eg, prior Administering to the patient, concurrently or afterwards, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • the NKT cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies Or other immunotherapeutic agents.
  • the cell composition of the invention is administered to a bone marrow transplant, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • XRT external beam radiation therapy
  • cyclophosphamide eg, before, simultaneously or after
  • the subject may undergo standard treatment of high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an injection of the expanded immune cells of the invention after transplantation.
  • the expanded cells are administered prior to or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratios administered by a human can be carried out according to practices accepted in the art.
  • 1 x 10 6 to 1 x 10 10 modified NKT cells of the invention e.g., CARCD30-NKT cells
  • chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ modified NKT cells of the present invention to treat CD30-positive Hodgkin's lymphoma and non-Hodgkin's lymphoma, it has a good specific killing effect on lymphoma cells. Active, and has significant therapeutic effects on CD30-positive Hodgkin's lymphoma patients who have undergone multiple treatments (eg, CD30 monoclonal antibody combined with cytotoxic drugs or radioisotope therapy) but have no significant effect;
  • the present invention constructs for the first time a NKT cell expressing a chimeric antigen receptor having a significant killing effect on a CD30-positive tumor, and the NKT cell has a function of regulating an immune balance state within a Car-T cell population;
  • the T cells expressing the chimeric antigen receptor have a relatively short survival time in vitro, and the NKT cells expressing the chimeric antigen receptor of the present invention have a long survival time in vitro, and are capable of adherent infection, and the infection efficiency is high.
  • the T cell infection efficiency is low, and the T cell death rate after infection is also high.
  • the chimeric antigen receptor of the present invention which has a structure in which a hinge region and a transmembrane region of CD8 and an intracellular signal domain of CD137 and CD3 ⁇ are connected in series as a signal transduction domain, capable of maintaining efficient transduction of NKT cells. Dyeing and efficient expression in NKT cells;
  • the chimeric antigen receptor of the present invention is expressed in comparison to a conventional T cell expressing a chimeric antigen receptor. NKT cells have significantly higher in vitro oncolytic activity;
  • the NKT cells expressing the chimeric antigen receptor of the present invention are reinfused into a patient, can be largely expanded in peripheral blood and bone marrow, and can persist for a long time without depending on interleukin 2.
  • NKT cell culture medium GT-T551 was purchased from TaKaRa.
  • Lymphocyte separation fluid was purchased from TBD Corporation.
  • CD3 monoclonal antibodies recombinant decorin (retronectin) were purchased from TaKaRa.
  • retronectin recombinant decorin
  • Recombinant human interferon-gamma recombinant human interleukin 2 was purchased from protech.
  • RNA extraction kit RNAiso Reagent high fidelity DNA polymerase (HS DNA Polymerase), T4 DNA ligase was purchased from TaKaRa.
  • RevertAid TM First Strand cDNA Synthesis Kit was purchased from Fermentas,.
  • the agarose gel DNA recovery kit, the common DNA product purification kit, and the plasmid miniton kit were purchased from Tiangen Biochemical Technology Co., Ltd.
  • pWPT-GFP psPAX2, and pMD2.G were purchased from Addgene.
  • pGSI was purchased from Beijing Tianyi Huiyuan Biotechnology Co., Ltd.
  • Trans1-T1Phage Resistant chemically competent cells were purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • Lipofectamine TM 2000 Transfection Reagent transfection reagents were purchased from Invitrogen.
  • 293T packaging cells were purchased from ATCC, USA.
  • the final concentration of PEG6000 in PEG6000-NaCl was 25.5% by mass, and the final concentration of NaCl was 1.2M. Both PEG6000 and NaCl were purchased from Shanghai Suobao Biotechnology Co., Ltd.
  • Fetal bovine serum was purchased from PAA, Germany.
  • the CD30-positive lymphoma cell lines karpas 299 and L428 were provided by Southern Medical University.
  • 5-carboxyfluorescein succinimide ester was purchased from Shanghai Spectrum Biotechnology Co., Ltd.
  • the annexin V-RPE kit was purchased from BD Corporation of the United States.
  • PBMCs Mononuclear cells
  • the final concentration of the cells was adjusted to 2 ⁇ 10 6 cells/mL using NKT cell culture medium GT-T551 containing 0.6% by volume of fetal bovine serum; the cells were seeded at a final concentration of 5 ⁇ g/ mL CD3 monoclonal antibody and a 75 cm 2 cell culture flask in a retronectin-coated final concentration of 10 ⁇ g/mL. Then, recombinant human interferon- ⁇ at a final concentration of 1000 U/mL and recombinant human interleukin 2 at 1000 U/mL were added to the culture medium, and cultured in a CO 2 incubator at 37 ° C and a saturated humidity of 5%.
  • NKT cell culture medium GT-T551 containing 0.6% by volume of fetal bovine serum was added to the culture flask, and recombinant human interleukin 2 was added at a final concentration of 1000 U/mL.
  • the NKT cells were obtained by culturing for 4 days in a CO 2 incubator at 37 ° C and a saturated humidity of 5%, and the NKT cell phenotype was analyzed by flow cytometry. The results are shown in Figure 1, in which CD3 + : 49.64%; CD3 + CD4 + : 27.71%; CD3 + CD8 + : 21.93%; CD3 + CD56 + : 2.95%; CD8 + CD56 + : 2.86%.
  • the NKT cells cultured in Example 1 were pelleted by centrifugation, and the total RNA of the cells was extracted with a total RNA extraction kit RNAiso Reagent, and stored at -80 ° C until use.
  • Total RNA was extracted from reverse transcription kit RevertAid TM First Strand cDNA Synthesis Kit NKT cells obtained by reverse transcription cDNA, -20 °C stored for use.
  • primer sequences were designed and synthesized (wherein underlined to protect the base and the box is the cleavage site):
  • NKT cell cDNA in step (1) as a template and PCR amplification with primers P1 and P2, a hinge region and a transmembrane region of CD8 with a length of 287 bp were obtained, and the nucleotide sequence was as shown in SEQ ID NO. Containing MluI and BglII restriction sites and protecting bases; PCR amplification with primers P3 and P4 yields a 146 bp CD137 intracellular signal domain, the nucleotide sequence is shown in SEQ ID NO.
  • Double distilled water 32.5 ⁇ L
  • reaction buffer 10 ⁇ L
  • NKT cell cDNA (200ng/ul): 1 ⁇ L
  • the above PCR product was separated on a 1% agarose gel, and DNA fragment recovery was carried out using an agarose gel DNA recovery kit. After the fragment is obtained, the double digestion reaction is carried out separately, and the digested product is recovered by using a common DNA product purification kit.
  • the lentiviral expression vector pWPT-GFP was digested with MluI/SalI, and the digested product was separated on a 1% agarose gel.
  • the large vector fragment was recovered by agarose gel DNA recovery kit, and then recovered with the previously recovered CD8.
  • the CD137 and CD3 ⁇ fragments were ligated by T4 DNA ligase, and the ligated products were transformed into Trans1-T1Phage Resistant chemically competent cells. After cultured at 37 °C for 16 hours, the monoclonals were picked, and cultured at 37 ° C, 250 rpm for 12 hours, and then the plasmid was extracted with a plasmid mini-kit.
  • the extracted plasmid was identified by restriction endonuclease MluI and SalI digestion, and the electrophoresis pattern was identified as shown in Fig. 2, wherein lane 1 was: DNA molecular weight marker D2000; lane 2: plasmid pWPT-GFP fragment (835 bp); Lane: Digested fragment (756 bp) of plasmid pWPT-CD8-CD137-CD3.
  • the correct plasmid was identified and sent to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing of the inserted fusion gene fragment.
  • the recombinant plasmid with the correct sequencing result was named pWPT-CD8-CD137-CD3 ⁇ .
  • the whole gene synthesizes the nucleotide sequence encoding the rat growth hormone signal peptide and CD30ScFv fusion gene, the sequence is shown in SEQ ID NO. 8, synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd., and its 5' end contains BamHI and kozak sequences.
  • the 3' end contains a MluI restriction site, and the aforementioned fusion gene was cloned into the plasmid pGSI and designated as pGSI-CD30ScFv.
  • the plasmid was digested with BamHI/MluI, and the digested product was separated on a 1% agarose gel, and the fragment of interest was recovered by using an agarose gel DNA recovery kit.
  • the pWPT-CD8-CD137-CD3 ⁇ plasmid was digested with restriction endonuclease BamHI/MluI, and the digested product was separated on a 1% agarose gel, and the vector fragment was recovered by agarose gel DNA recovery kit for use. Then, the recovered DNA fragment containing the rat growth hormone signal peptide and CD30ScFv is ligated by T4 DNA ligase, and the specific method is described in the specification.
  • the ligation product was transformed into Trans1-T1Phage Resistant chemically competent cells, and cultured at 37 ° C for 16 h, then picked up the monoclonal, and cultured at 37 ° C, 250 rpm for 12 h, and then the plasmid was extracted with a plasmid extraction kit.
  • the extracted plasmid was identified by restriction endonuclease BamHI/SalI double digestion, and the results were as shown in Fig.
  • M1 DNA molecular weight marker D2000
  • 1 lane plasmid pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ uncut Fragment (10232 bp);
  • Lane 2 restriction fragment of plasmid pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ ;
  • M2 DNA molecular weight marker D15000.
  • the correct plasmid was identified and sent to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing of the inserted fusion gene fragment.
  • the recombinant plasmid with the correct sequencing result was named pWPT-CD30ScFv-CD8-CD137-CD3 ⁇ , and its structural schematic diagram is shown in FIG.
  • SEQ ID NO. 4 which includes a rat growth hormone signal peptide (the nucleotide sequence is shown in SEQ ID NO. 6), An anti-CD30 single-chain antibody (nucleotide sequence as shown in SEQ ID NO. 7), a hinge region and a transmembrane region of CD8, an intracellular signal domain of CD137, and an intracellular signal domain of CD3 ⁇ , wherein the chimeric antigen
  • the receptor is a signal transduction domain in which the hinge region and the transmembrane region of the gene CD8 and the intracellular signal domain of CD137 and CD3 ⁇ are connected in series, and the amino acid sequence thereof is shown in SEQ ID NO. 9, and the corresponding gene coding sequence is as follows. SEQ ID NO. 10 is shown.
  • lentivirus expression plasmids were pWPT-CD30ScFv-CD8-CD137- CD3 ⁇ psPAX2 and a helper plasmid, the concentration pMD2.G, three plasmids of 4: 2: 1 mass ratio with Lipofectamine TM 2000 Transfection Reagent Transfection
  • the reagents were co-transfected into 293T packaging cells.
  • the virus supernatants were collected in 50 mL EP tubes at 48h and 72h, respectively, and centrifuged at 2000°C for 10min at 4°C, transferred to the new EP tube twice, and the virus supernatant was filtered with a 4.5 ⁇ m filter; the virus was filtered.
  • the supernatant was mixed with 5 ⁇ PEG6000-NaCl in a volume ratio of 4:1, allowed to stand at 4 ° C for 2 h, then centrifuged at 10000 g for 20 min at 4 ° C, the supernatant was discarded, and the precipitate was dissolved in 1 mL of 4° C precooled sterile PBS. That is, the virus concentrate of the chimeric antigen receptor was dispensed in 100 ⁇ L per tube, and stored at -80 ° C until use.
  • the 293T packaging cells were co-transfected with the lentiviral expression plasmid pWPT-GFP and the helper plasmids psPAX2 and pMD2.G according to the above method, and the virus supernatant was collected and concentrated to obtain a lentivirus concentrate expressing GFP green fluorescent protein.
  • NKT cells cultured in a 75 cm 2 flask of Example 1 were discarded, the old culture solution was discarded, 2 mL of fresh NKT cell culture medium GT-T551, 200 ⁇ L of the virus concentrate obtained in the step (1), 2 ⁇ L of 1 ⁇ 10 -6 mg/mL protamine, recombinant human interleukin 2 at a final concentration of 1000 U/mL, and placed in a CO 2 incubator at 37 ° C and a saturated humidity of 5% for 12 hours, the culture medium was discarded.
  • the resulting NKT cells are referred to as CAR30-NKT cells.
  • NKT cells were simultaneously infected with a lentiviral concentrate expressing GFP green fluorescent protein (the obtained NKT cells were referred to as CART-GFP cells) for calculating the infection efficiency of the virus.
  • the infected cells were transferred to a 75 cm 2 flask not coated with CD3 and retronectin, 20 mL of NKT cell culture medium GT-T551 was added, and recombinant human interleukin 2 was added at a final concentration of 1000 U/mL at 37 ° C. Incubate for 18 h in a CO 2 incubator with a saturated humidity of 5%.
  • the infection efficiency of the virus was examined by flow cytometry, and the results were as shown in Fig. 5, and the infection efficiency was 36.38%.
  • the cultured NKT cells were induced in vitro with NKT cell culture medium GT-T551 with a final concentration of recombinant human interleukin 2 of 1000 U/mL. When the density of the cells was 85%, the cells were transferred into a cell culture bag, 2 Fresh NKT cell culture medium GT-T551 with a final concentration of 1000 U/mL recombinant human interleukin 2 was added for expansion and culture. After the cells were expanded to a total of 1.5 ⁇ 10 9 cells, flow cytometry was used.
  • CD3 + 89.99%; CD3 + CD4 + : 58.18%; CD3 + CD8 + : 31.81%; CD3 + CD56 + : 6.38%; CD8 + CD56 + : 5.37%.
  • the CAR30-NKT cells prepared in Example 3, CART-GFP cells and the NKT cells cultured in Example 1 were inoculated into 96-well plates and stained with 5-carboxyfluorescein succinimidyl ester (CFSE).
  • control group was set to the lymphoma cell lines karpas299 and L428 which were not added to the immune cell killing treatment, and the cells were stained with the annexin V-RPE kit.
  • Flow cytometry was used to detect apoptosis.
  • the sample is the number of cells survived by the immune cell killing treatment of the corresponding effective target ratio (killer cells: target cells), see FIG.
  • the chimeric antigen receptor CD30ScFv-CD8-CD137-CD3 ⁇ modified NKT cells have specific killing activity against CD30-positive lymphoma cells, and the specific killing activity of CAR30-NKT cells is significantly better than that of NKT cells.
  • CD30ScFv-CD8-CD137-CD3 ⁇ -modified NKT cells were diluted with 100 ml of physiological saline and intravenously returned to the following CD30-positive Hodgkin's lymphoma patients for three consecutive days.
  • CAR30-NKT cells were diluted with 100 ml of physiological saline and intravenously returned to the following CD30-positive Hodgkin's lymphoma patients for three consecutive days.
  • Figure 8 is a trend analysis of the total number of white blood cells, total lymphocytes, body temperature changes and interleukin-6 levels in patients with CD30-positive Hodgkin's lymphoma treated with CAR30-NKT cells (FC is a clear marrow preconditioning regimen taken before treatment). ). The results showed that patients with Hodgkin's lymphoma showed hyperthermia, inflammation and bone pain before treatment (grade 7 bone pain according to VAS pain score). After treatment with CAR30-NKT cells, the number of white blood cells gradually decreased and became normal.
  • the number of lymphocytes gradually increased indicates that CAR30-NKT cells can gradually expand in patients with CD30-positive Hodgkin's lymphoma
  • patients with bone pain Symptoms were alleviated (grade 1 bone pain according to the VAS pain score).
  • CAR30-NKT cells are safe and tolerable in the treatment of CD30-positive lymphoma patients, and that CAR30-NKT cells are not effective in treating CD30-positive Hodgkin's lymphoma patients after various treatments. After treatment, the patient's disease has a certain therapeutic effect.
  • Figure 9 is a CT analysis of CD30-positive Hodgkin's lymphoma liver metastases. After treatment with CAR30-NKT cells (one month), the patient's liver site changes. The results showed that patients with Hodgkin's lymphoma treated with liver lesions (see arrows) were significantly reduced, further indicating that CAR30-NKT cells were positive for CD30-positive Hodgkin's lymphoma (no significant treatment after treatment with various treatments). Efficacy) After treatment, there is a significant therapeutic effect on the patient's disease.
  • the CAR30-NKT cells prepared in Example 3 were seeded in a 25 cm cell culture flask for culture, and the cell supernatant was continuously collected, and the level of interleukin 2 secreted by the cells was analyzed using an ELISA kit.
  • the results show that the CAR30-NKT cells of the present invention can continuously secrete interleukin 2 cytokines in vitro, and have unique non-IL-2-like amplification ability, thereby avoiding the in vitro supplementation of IL-2 in clinical use.

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Abstract

一种嵌合抗原受体及其基因和重组表达载体、工程化CD30靶向性的NKT细胞及其应用，所述嵌合抗原受体为CD30-ScFv-CD8-CD137-CD3ζ，由CD30ScFv、CD8的铰链区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域串联构成。嵌合抗原受体CD30-ScFv-CD8-CD137-CD3ζ修饰的NKT细胞治疗CD30阳性的霍奇金淋巴瘤及非霍奇金淋巴瘤时，对淋巴瘤细胞具有很好的特异杀伤活性，且对已经过多次治疗但均无明显疗效的CD30阳性的霍奇金淋巴瘤患者有一定的治疗效果。

Description

CD30靶向性的嵌合抗原受体和NKT细胞及其制法和应用 技术领域
本发明属于用于肿瘤治疗的生物制品领域，具体地，涉及CD30靶向性的嵌合抗原受体和NKT细胞及其制法和应用。更具体地，涉及过继免疫治疗中的一种嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ及其基因和重组表达载体、工程化CD30靶向性的NKT细胞(CAR30-NKT细胞)及其应用。
背景技术
CD30抗原是一个120kDa糖蛋白，属于TNF/nerve生长因子受体家族成员，几乎表达于所有的霍奇金淋巴瘤、部分非霍奇金淋巴瘤及被病毒感染的淋巴细胞表面。由于以上特性，CD30抗原成为抗体为基础治疗的理想靶点。目前，在治疗复发难治性霍奇金淋巴瘤患者过程中，临床研究通常采用CD30单克隆抗体结合细胞毒素药物或者放射性同位素的方案，这种方案在产生一定临床效应的基础上，同时也存在一定的缺陷，如：CD30单克隆抗体靶向效应的短暂性及CD30单克隆抗体在肿瘤部位分布的不足等。
因此，本领域技术人员致力于开发新的更有效的针对CD30阳性肿瘤的治疗方案。
发明内容
本发明的目的是为了克服现有技术中NKT细胞对肿瘤的杀伤作用比较弱、特异杀伤活性有待提高以及现有临床研究中CD30单克隆抗体存在的上述缺陷，提供一种嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ及其基因和重组表达载体、工程化CD30靶向性的NKT细胞(CAR30-NKT细胞)及其应用。
本发明的第一方面，提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体以CD8的绞链区和跨膜区及CD137和CD3ζ的胞内信号结构域串联而成的结构为信号传导结构域。
在另一优选例中，所述嵌合抗原受体为CD30ScFv-CD8-CD137-CD3ζ，由CD30ScFv、CD8的铰链区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域串联构成。
在另一优选例中，所述嵌合抗原受体的胞外结构域为抗CD30的单链抗体。
在另一优选例中，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.1所示。
在另一优选例中，所述CD8的铰链区和跨膜区选自下组：
(A)具有SEQ ID NO:17所示氨基酸序列的多肽；
(B)具有与SEQ ID NO:17所示氨基酸序列≥80％同源性(优选地，≥90％ 的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性)的多肽；(C)将SEQ ID NO:17中任一所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的衍生多肽。
在另一优选例中，所述CD137的胞内信号结构域选自下组：
(A)具有SEQ ID NO:18所示氨基酸序列的多肽；
(B)具有与SEQ ID NO:18所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；优选地≥95％的同源性；最优选地，≥97％的同源性)，并且能够转导效应子功能信号的多肽；
(C)将SEQ ID NO:18中任一所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的衍生多肽。
在另一优选例中，所述CD3ζ的胞内信号结构域选自下组：
(A)具有SEQ ID NO:19所示氨基酸序列的多肽；
(B)具有与SEQ ID NO:19所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性)，并且能够转导效应子功能信号的多肽；
(C)将SEQ ID NO:19中任一所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的衍生多肽。
在另一优选例中，所述CAR包括SEQ ID NO:9所示的氨基酸序列。
在另一优选例中，所述嵌合抗原受体(CAR)为分离的。
在另一优选例中，所述嵌合抗原受体(CAR)的氨基酸序列如SEQ ID NO.:1所示。
本发明的第二方面，提供了一种核酸分子，所述核酸分子编码本发明第一方面所述的嵌合抗原受体(CAR)。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD8的铰链区和跨膜区的核酸序列：
(a)编码如SEQ ID NO.:17所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:3所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:3所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.:17所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD137的胞内信号结构域的核酸序列：
(a)编码如SEQ ID NO.:18所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:4所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:4所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.:18所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD3ζ的胞内信号结构域的核酸序列：
(a)编码如SEQ ID NO.:19所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:5所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:5所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.:19所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的核酸序列：
(a)编码如SEQ ID NO.:9所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:10所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:10所示序列的同源性≥95％(较佳地≥98％)，并且编码SEQ ID NO.:9所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子为分离的。
在另一优选例中，所述核酸分子还包括编码前导序列(信号肽)的多核苷酸，所述前导序列的氨基酸序列如SEQ ID NO.:21所示：
在另一优选例中，所述核酸分子的序列如SEQ ID NO.:2所示。
本发明的第三方面，提供了一种载体，所述的载体含有本发明第二方面所述的核酸分子。
在另一优选例中，所述载体为慢病毒载体。
本发明的第四方面，提供了一种细胞，所述的细胞中含有本发明第三方面所述的载体或染色体中整合有外源的本发明第二方面所述的核酸分子。
在另一优选例中，所述细胞为分离的细胞，和/或所述细胞为基因工程化的细胞。
在另一优选例中，所述细胞为哺乳动物细胞。
在另一优选例中，所述细胞为NKT细胞、或T细胞。
在另一优选例中，所述T细胞为NKT细胞。
本发明的第五方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞。
本发明的第六方面，提供了本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞的用途，用于制备治疗肿瘤的药物或制剂。
在另一优选例中，所述的肿瘤包括CD30阳性的淋巴瘤。
在另一优选例中，所述淋巴瘤为霍奇金淋巴瘤。
本发明的第七方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、 本发明第三方面所述的载体、本发明第四方面所述的细胞、或本发明第五方面所述的药物组合物。
在另一优选例中，所述疾病为肿瘤。
本发明的第八方面，提供了一种嵌合抗原受体，所述嵌合抗原受体以CD8的hinge区和跨膜区及CD137和CD3ζ的胞内信号结构域串联而成的结构为信号传导结构域，所述信号传导结构域氨基酸序列如SEQ ID NO：9所示。
本发明还提供了一种嵌合抗原受体，所述嵌合抗原受体为CD30ScFv-CD8-CD137-CD3ζ，由CD30单链抗体CD30ScFv、CD8的铰链区(hinge区)和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域串联构成。
本发明还提供了编码上述嵌合抗原受体的基因。
本发明还提供了含有上述基因的重组表达载体。
本发明还提供了一种工程化CD30靶向性的NKT细胞，所述NKT细胞是上述嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞。
本发明还提供了上述工程化CD30靶向性的NKT细胞在制备用于治疗肿瘤的制剂中的应用。
在治疗CD30阳性的霍奇金淋巴瘤及非霍奇金淋巴瘤时，本发明的嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞，即工程化CD30靶向性的NKT细胞能够特异性结合CD30抗原，明显延长免疫细胞在患者体内的存活时间，增强免疫细胞靶向识别霍奇金淋巴瘤细胞及非霍奇金淋巴瘤细胞CD30抗原的能力，加强对霍奇金淋巴瘤细胞及非霍奇金淋巴瘤细胞的特异杀伤活性，而且对已经过多次治疗(如采用CD30单克隆抗体结合细胞毒素药物或者放射性同位素治疗等)但均无明显疗效的CD30阳性的霍奇金淋巴瘤患者有一定的治疗效果。本发明的工程化CD30靶向性的NKT细胞为治疗CD30阳性的霍奇金淋巴瘤及非霍奇金淋巴瘤提供了一种新的选择，具有良好的产业应用前景。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说明。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为流式细胞术对分离培养的NKT细胞表型分析的结果。
图2为本发明的慢病毒表达载体pWPT-CD8-CD137-CD3ζ的限制性内切酶MluI/SalI双酶切片段的电泳鉴定图。
图3为本发明的慢病毒表达载体pWPT-CD30ScFv-CD8-CD137-CD3ζ的限制性内切酶BamHI/SalI双酶切片段的电泳鉴定图。
图4为本发明的慢病毒表达载体pWPT-CD30ScFv-CD8-CD137-CD3ζ的结构示意图，其中，逆时针序列为正向基因片度，顺时针为反向基因片段。
图5为流式细胞术检测含有嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ的病毒浓缩液对NKT细胞的感染效率。
图6为流式细胞术检测嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞(CAR30-NKT细胞)表型鉴定的结果。
图7为本发明的CAR30-NKT细胞对CD30阳性的人霍奇金淋巴瘤细胞的杀伤作用的细胞毒性分析图。
图8为本发明的CAR30-NKT细胞对CD30阳性的霍奇金淋巴瘤患者治疗过程中，患者白细胞总数、淋巴细胞总数、患者体温水平及白介素6水平的变化趋势图。
图9为本发明的CAR30-NKT细胞对CD30阳性的霍奇金淋巴瘤患者(肝转移)治疗前后，患者肝转移病灶的影像图变化。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
本发明的发明人在研究中意外发现，采用嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞治疗CD30阳性的霍奇金淋巴瘤及非霍奇金淋巴瘤时，对淋巴瘤细胞具有很好的特异杀伤活性，且对已经过多次治疗(如采用CD30单克隆抗体结合细胞毒素药物或者放射性同位素治疗等)但均无明显疗效的CD30阳性的霍奇金淋巴瘤患者有一定的治疗效果。在此基础上完成了本发明。
本发明提供了一种嵌合抗原受体，所述嵌合抗原受体为CD30ScFv-CD8-CD137-CD3ζ，由CD30单链抗体CD30ScFv、CD8的铰链区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域串联构成。优选情况下，嵌合抗原受体的氨基酸序列如SEQ ID NO.1所示。
本发明提供了编码上述嵌合抗原受体的基因。优选情况下，编码上述嵌合抗原受体的基因的核苷酸序列如SEQID NO.2所示。
本发明提供了含有上述基因的重组表达载体。优选情况下，重组表达载体为慢病毒表达载体。对于慢病毒表达载体没有特别的限定，只要能够与辅助载体共转染包装细胞如293T包装细胞，获得病毒浓缩液及嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NTK细胞即可，优选情况下，慢病毒表达载体为pWPT-CD30ScFv-CD8-CD137-CD3ζ。
对于慢病毒表达载体pWPT-CD30ScFv-CD8-CD137-CD3ζ的制备方法没有特别的限定，可以为本领域技术人员能够想到的各种方法，优选情况下，慢病毒表达载体pWPT-CD30ScFv-CD8-CD137-CD3ζ的制备方法包括以下步骤：
(1)从NKT细胞cDNA中分别扩增CD8的hinge区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域，并克隆至载体pWPT-GFP中，构建得到pWPT-CD8-CD137-CD3ζ；
(2)合成编码大鼠生长激素信号肽和CD30ScFv的核苷酸序列，并克隆至 pWPT-CD8-CD137-CD3ζ中，经测序验证后得到序列正确的pWPT-CD30ScFv-CD8-CD137-CD3ζ。
步骤(1)中，对于从NKT细胞cDNA中分别扩增CD8的hinge区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域的方法没有特别的限定，可以为本领域常用的各种方法，例如可以为RT-PCR法。其中，NKT细胞可以通过分离人静脉血中的单个核细胞，然后进行培养获得。
具体地，得到pWPT-CD8-CD137-CD3ζ的方法可以包括：提取NKT细胞的总RNA，逆转录获得NKT细胞cDNA，以得到的NKT细胞cDNA为模板，利用引物P1(SEQID NO.11)和P2(SEQID NO.12)进行PCR扩增获得CD8基因的hinge区和跨膜区(SEQID NO.3)；利用引物P3(SEQID NO.13)和P4(SEQID NO.14)进行PCR扩增获得CD137基因的胞内信号结构域(SEQID NO.4)；利用引物P5(SEQID NO.15)和P6(SEQID NO.16)进行PCR扩增获得CD3ζ基因的胞内信号结构域(SEQID NO.5)，将获得的PCR产物分别进行双酶切，然后与MluI/SalI双酶切后的慢病毒表达载体pWPT-GFP连接。
步骤(2)中，对于合成编码大鼠生长激素信号肽和CD30ScFv的核苷酸序列的方法没有特别的限定，可以为本领域常用的各种方法，例如可以通过全基因合成技术合成。
具体地，得到序列正确的pWPT-CD30ScFv-CD8-CD137-CD3ζ的方法可以包括：通过全基因合成技术合成编码大鼠生长激素信号肽和CD30ScFv融合基因的核苷酸序列(SEQID NO.8)，克隆至载体pGSI中，得到pGSI-CD30ScFv；然后将pGSI-CD30ScFv进行BamHI/MluI双酶切，与BamHI/MluI双酶切后的步骤(1)得到的重组质粒pWPT-CD8-CD137-CD3ζ连接，经测序鉴定，得到序列正确的pWPT-CD30ScFv-CD8-CD137-CD3ζ。其中，大鼠生长激素信号肽的核苷酸序列如SEQID NO.6所示，CD30ScFv核苷酸序列如SEQID NO.7所示。
本发明还提供了一种工程化CD30靶向性的NKT细胞，所述NKT细胞是由上述嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞(即CAR30-NKT细胞)。
对于工程化CD30靶向性的NKT细胞的制备方法没有特别的限定，可以为本领域技术人员能够想到的任何方法，优选情况下，该方法包括：包装携带pWPT-CD30ScFv-CD8-CD137-CD3ζ编码基因的慢病毒；利用得到的慢病毒感染NKT细胞，使NKT细胞表达嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ。
对于包装携带pWPT-CD30ScFv-CD8-CD137-CD3ζ编码基因的慢病毒的方法没有特别的限定，可以为本领域技术人员常用的各种方法，优选情况下，将慢病毒表达载体pWPT-CD30ScFv-CD8-CD137-CD3ζ与辅助质粒(如psPAX2、pMD2.G)共转染293T包装细胞，转染48-72h时收集病毒上清，离心、过滤，在滤液中添加5×PEG6000-NaCl进行混匀，离心后弃上清，沉淀用0-4℃预冷的无菌PBS溶解，获得病毒浓缩液。
对于慢病毒感染NKT细胞的方法没有特别限定，可以为本领域常用的各种方法，优选情况下，该方法包括：取1×107-5×107个NKT细胞，弃掉旧的培养液，加入2-4mL新鲜GT-T551培养液，再加入200-400μL病毒浓缩液、2-4μL 1×10-6mg/mL鱼精蛋白和终浓度为800-1200U/mL的IL-2，置于30-37℃、饱和湿度为3-6％的CO2培养箱中感染12-16h后，弃培养液，将细胞转至未包被的培养瓶中，加入20-50mL的GT-T551培养基，再加入终浓度为800-1200U/mL的IL-2，于30-37℃、饱和湿度为3-6％的CO2培养箱中培养12-18h，获得嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞。
进一步优选地，慢病毒感染NKT细胞的方法还包括：将上述培养后获得的慢病毒感染的NKT细胞用IL-2的终浓度为800-1200U/mL的GT-T551培养液进行体外诱导，待细胞的密度为80-90％时将细胞转入细胞培养袋中，隔1.5-2.5天加入IL-2的终浓度为800-1200U/mL的新鲜GT-T551培养液进行扩增培养并将细胞扩增至总量为1×109-2×109个细胞。
嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞表达的嵌合抗原受体的成熟蛋白氨基酸序列如SEQID NO.1所示。其中，本领域技术人员应该理解的是，嵌合抗原受体前体蛋白包括串联的信号肽、CD30ScFv、CD8的hinge区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域，蛋白质翻译后在细胞内粗面型内质网切除信号肽后成为成熟嵌合抗原受体蛋白，分泌输出后并定位于NKT细胞的细胞膜上。该嵌合抗原受体的成熟蛋白氨基酸序列对应的基因编码序列如SEQID NO.2所示。该嵌合抗原受体以基因CD8的hinge区和跨膜区及CD137和CD3ζ的胞内信号结构域串联而成的结构为信号传导结构域，其氨基酸序列如SEQID NO.9所示，对应的基因编码序列如SEQID NO.10所示。
本发明还提供了上述方法制备得到的工程化CD30靶向性的NKT细胞。
本发明还提供了工程化CD30靶向性的NKT细胞在制备用于治疗肿瘤的制剂中的应用。优选情况下，肿瘤是CD30阳性的淋巴瘤。所述淋巴瘤可以为霍奇金淋巴瘤或非霍奇金淋巴瘤，进一步优选地，所述淋巴瘤为霍奇金淋巴瘤。
本发明提供了治疗癌症等疾病的组合物和方法。该癌症可为实体瘤、原发肿瘤或转移性肿瘤或血液学恶性肿瘤。利用本发明的组合物和方法可治疗的其他疾病包括病毒、细菌和寄生虫感染以及自身免疫疾病。
NKT细胞
自然杀伤细胞(NKT)是一种特殊类型的T淋巴细胞亚群，具有T细胞和NK细胞两重性质。NKT细胞能表达T细胞的TCR与NK细胞的NKR-P1两种受体，在TCR和NKR介导下，NKT细胞能够产生大量的IL-4及INFγ，对肿瘤细胞发挥细胞杀伤作用。NKT细胞通过自身表面的CD16与特异性抗体的Fc段结合，发挥ADCC(antibody-dependent cell-mediated cytotoxicity)作用。
NKT细胞的功能主要包括免疫调节和细胞毒作用，NKT细胞受到刺激后，可以分泌大量的IL-4,IFN-γ，GM-CSF,IL-13和其它细胞因子和趋化因子，发挥免疫调节作用，NKT细胞是联系固有免疫和获得性免疫的桥梁之一。NKT细胞活化后具有NK细胞样细胞毒活性，可溶解NK细胞敏感的靶细胞，主要效应分子为穿孔素，Fas配体以及IFN-γ。
但在抗体依赖的细胞介导的杀伤作用过程中，由于抗体能与靶细胞上的相应 抗原表位特异性结合，NKT细胞可杀伤任何已与抗体结合的靶细胞，因此抗体与靶细胞上的抗原结合是特异性的，但NKT细胞对靶细胞的杀伤作用是非特异性的。
NKT细胞的制备方法
本领域中已存在已知的NKT细胞、T细胞的制备方法，以及活化和扩增方法，例如可以分别参考文献：Motohashi,S.,Nagato,K.,Kunii,N.,Yamamoto,H.,Yamasaki,K.,Okita,K.,Hanaoka,H.,Shimizu,N.,Suzuki,M.,Yoshino,I.,Taniguchi,M.,Fujisawa,T.and Nakayama,T.,A phase I-II study of alpha-galactosylceramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patients with advanced and recurrent non-small cell lung cancer.J Immunol2009.182:2492-2501；D.L.Porter,B.L.Levine,M.Kalos,A.Bagg,C.H.June,Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia,The New England journal of medicine,2011.365:725-733.
在本发明的一个较佳的实施方式中，改良的制备NKT细胞的方法如下。
(1)取人静脉血于含肝素的真空管中。采用淋巴细胞分离液，通过密度梯度离心方法分离获得单个核细胞(PBMCs)。
(2)PBMCs洗三次后，采用含有0.6体积％的人自体血清的NKT细胞培养基GT-T551调整细胞终浓度为2×106个细胞/mL；将细胞接种于预先经过终浓度为10μg/mL的retronectin包被的75cm2细胞培养瓶中。然后在培养基里加入终浓度为500U/mL的重组人白介素2，50ng/ml CD3单克隆抗体，50ng/mL的重组人白介素-15，在37℃、饱和湿度为5％的CO2培养箱中培养。
(3)培养第4天，将细胞转移至未包被的培养瓶中，并每2-3天按照细胞生长状况加入培养液，及人重组IL-2浓度为500U/ml；第12-16天，得到NKT细胞，流式细胞术对NKT细胞表型进行分析。
采用本发明方法制得的NKT细胞群中，CD3+CD8+NKT细胞/CD3+CD4+NKT细胞的比率为10/1～4/1(优选为9/1～6/1)；并且CD3+CD56+NKT细胞/CD3+CD8+NKT细胞的比率为1/18～10/18(优选为5/18～10/18)。
并且NKT细胞群中，CD3+细胞比率≥95％；CD3+CD8+细胞比率≥90.99％；CD3+CD56+细胞比率≥15％(优选地，≥18％；更优选地≥20％；最优选地≥22％，如≥24％、≥26％、≥28％、≥30％)；CD8+CD56+细胞比率≥15％(优选地，≥18％；更优选地≥20％；最优选地≥22％，如≥24％、≥26％、≥28％、≥30％)。。
NKT细胞的细胞表型为CD3+和CD56+，表达T细胞受体(TCR)的可变区基因和天然杀伤(NK)细胞表面标记NK1·1(NKR-P1C)，兼具有T淋巴细胞强大的抗瘤活性和NK细胞的非MHC限制性杀瘤优点。在杀伤发面，弥补了T细胞MHC限制性的局限性，杀瘤谱、杀伤能力都大大优于T细胞。
与传统T细胞不同，NKT细胞是一群兼具NK功能和T细胞特点的特殊免疫细胞。NKT细胞具有高度保守的TCR表型(TCRVα24/Vβ11-人),同时共表达NK细胞特有标志CD161，它不识别由经典的MHC-Ⅰ或Ⅱ类分子递呈的肽类抗原,而是识别由非经典的MHC-Ⅰ类分子呈递的糖脂类分子抗原。NKT细胞识别由CD1d分子提呈的特异糖脂分子,并能够分泌大量细胞因子,参与机体的先天性免疫和获得性免疫反应。目前，多项研究证实NKT细胞在抗肿瘤免疫、抗感染免疫、自身 免疫病、免疫调节中发挥了重要作用，许多生理病理过程均有NKT细胞的参与。
传统的T细胞在体外存活时间比较短暂，而根据本发明的NKT细胞在体外存活时间较久，能够贴壁感染，感染率高。而T细胞感染效率低，而且感染后的T细胞死亡率也高。
嵌合抗原受体
本发明提供了包括细胞外结构域、跨膜结构域、和细胞内结构域的嵌合抗原受体(CAR)。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供了经过基因工程改造以表达CAR的细胞(例如，T细胞、NKT细胞)，其显示显著的抗肿瘤性质。本发明的CAR还可以包括胞外结构域，所述胞外结构域具有融合至T细胞抗原受体复合物ζ链(例如，CD3ζ)的细胞内信号传导结构域的抗原结合结构域。本发明的CAR当在NKT细胞中表达时，能够基于抗原结合特异性改变抗原识别。示例性抗原为CD30，因为该抗原在肺癌细胞上表达。
在一个实施方式中，本发明的CAR包括包含本发明特定信号传导结构域(CD8的hinge区和跨膜区、CD137和CD3ζ的胞内信号结构域串联而成)。，与其他方式的CAR相比，本发明的信号传导结构域显著增加了抗肿瘤活性和CART细胞的体内持久性。
抗原结合结构域
在一个实施方式中，本发明的CAR包括被称为抗原结合结构域的靶-特异性结合元件。抗原结合结构域的选择取决于限定靶细胞表面的配体的类型和数目。例如，可选择抗原结合结构域，以识别用作与具体疾病状态相关的靶细胞上的细胞表面标记的配体。因此，细胞表面标记的例子包括与病毒、细菌和寄生虫感染，自身免疫疾病和癌细胞相关的那些标记。
在本发明的内容中，“肿瘤抗原”指由引起免疫应答特别是T-细胞介导的免疫应答的肿瘤细胞产生的蛋白质。本发明的CAR的抗原结合结构域靶向CD30。优选地，本发明的CAR中的抗原结合结构域为抗-CD30scFV(single—chain antibody fragment，scFv)，其中抗-CD30scFV的核酸序列包括SEQ ID NO:7中提出的序列。在一个实施方式中，抗-CD30scFV包括编码SEQ ID NO:20的氨基酸序列的核酸序列。在另一个实施方式中，本发明的CAR的抗-CD30scFV如SEQ ID NO:20所示。
Figure PCTCN2016071371-appb-000001
Figure PCTCN2016071371-appb-000002
绞链区和跨膜区
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
跨膜结构域可源于天然来源或合成来源。在天然来源中，该结构域可源于任何膜结合蛋白或跨膜蛋白。具体用于本发明的跨膜区可源于T-细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154(即至少包括上述中的跨膜区(一个或多个))。
优选地，本发明的CAR中的绞链区和跨膜区为CD8的绞链区和跨膜区。在一个实施方式中，编码CD8的绞链区和跨膜区的核苷酸序列包括SEQ ID NO:3的核酸序列。在本发明的一个优选的实施方式中，CD8的绞链区和跨膜区包括以下氨基酸序列：
Figure PCTCN2016071371-appb-000003
胞内结构域
本发明的CAR的胞内结构域或另外的细胞内信号传导结构域是造成其中已放置CAR的免疫细胞的至少一种正常效应子功能的活化的原因。术语“效应子功能”指的是细胞的专有功能。例如，T细胞的效应子功能可为包括细胞因子分泌的细胞溶解活性或辅助活性。因此术语“细胞内信号传导结构域”指的是转导效应子功能信号并指导细胞实施专有功能的蛋白部分。尽管通常可使用整个细胞内信号传导结构域，但在很多例子中，不必使用整个链。就使用细胞内信号传导结构域的截短部分而言，这种截短部分可用于代替完整的链，只要它转导效应子功能信号。术语细胞内信号传导结构域因此指包括足以转导效应子功能信号的细胞内信号传导结构域的任何截短部分。
用于本发明的CAR的细胞内信号传导结构域的优选例子包括T细胞受体(TCR)的胞浆序列和协同行动以在抗原受体结合后开始信号转导的共受体，以及这些序列的任何衍生物或变体和具有相同的功能能力的任何合成序列。
已知通过TCR单独产生的信号不足以完全活化T细胞，并且也需要次级或共刺激信号。因此，T细胞活化可被认为由两个不同类的胞浆信号传导序列介导：通过TCR(初级胞浆信号传导序列)开始抗原-依赖性初级活化的那些和以抗原-非依赖性方式起作用以提供次级或共刺激信号(次级胞浆信号传导序列)的那些。
初级胞浆信号传导序列以刺激方式或以抑制方式调节TCR复合物的初级活 化。以刺激方式起作用的初级胞浆信号传导序列可包含信号传导基序，其已知为基于免疫受体酪氨酸的活化基序或ITAM。
包含在本发明中具有具体用途的初级胞浆信号传导序列的ITAM的例子包括源于TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。特别优选地，本发明的CAR中的胞浆信号传导分子包括源于CD3ζ的胞浆信号传导序列。
在优选的实施方式中，CAR的胞浆结构域可被设计以本身包括CD3-ζ信号传导结构域，或可与在本发明的CAR的内容中有用的任何其他期望的胞浆结构域(一个或多个)联合。例如，CAR的胞浆结构域可包括CD3ζ链部分和共刺激信号传导区。共刺激信号传导区指的是包括共刺激分子的细胞内结构域的一部分CAR。共刺激分子是淋巴细胞对抗原的有效应答所需的细胞表面分子，而不是抗原受体或它们的配体。这种分子的例子包括CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、
NKG2C、B7-H3和与CD83特异性结合的配体等等。因此，尽管本发明主要以4-1BB作为共刺激信号传导元件的例子，但其他共刺激元件也位于本发明的范围内。
本发明的CAR的胞浆信号传导部分内的胞浆信号传导序列可以随机或以规定的顺序相互连接。任选地，短的寡肽或多肽连接体，优选长度在2和10个氨基酸，可形成该连接。甘氨酸-丝氨酸双联体提供了特别合适的连接体。
在一个实施方式中，胞浆结构域被设计以包括CD3ζ的信号传导结构域和CD28的信号传导结构域。在另一个实施方式中，胞浆结构域被设计以包括CD3ζ的信号传导结构域和CD137的信号传导结构域。
在一个实施方式中，本发明的CAR中的胞浆结构域被设计以包括CD137的信号传导结构域和CD3ζ的信号传导结构域，其中CD137的信号传导结构域包括SEQ ID NO:4中提出的核酸序列和CD3-ζ的信号传导结构域包括SEQ ID NO:5中提出的核酸序列。
在一个实施方式中，本发明的CAR中的胞浆结构域被设计以包括CD137的信号传导结构域和CD3ζ的信号传导结构域，其中CD137的信号传导结构域包括编码SEQ ID NO:18的氨基酸序列的核酸序列，和CD3ζ的信号传导结构域包括编码SEQ ID NO:19的氨基酸序列的核酸序列。
在一个实施方式中，本发明的CAR中的胞浆结构域被设计以包括CD137的信号传导结构域和CD3ζ的信号传导结构域，其中CD137的信号传导结构域包括SEQ ID NO:18中提出的氨基酸序列，和CD3ζ的信号传导结构域包括SEQ ID NO:19中提出的氨基酸序列。
优选地，CD137的胞内信号结构域包含如下氨基酸序列：
Figure PCTCN2016071371-appb-000004
优选地，CD3ζ的胞内信号结构域包含如下氨基酸序列：
Figure PCTCN2016071371-appb-000005
载体
本发明包括包含CAR序列的DNA构建体，其中该序列包括可操作地连接至信号传导结构域的核酸序列的抗原结合结构域的核酸序列。可用于本发明的CAR的示例性的信号传导结构域包括抗-CD30scFv、CD8铰链和跨膜区、和CD137和CD3ζ胞内信号传导结构域。在一个实施方式中，本发明的CAR包括SEQ ID NO:10中提出的核酸序列。在另一个实施方式中，本发明的CAR包括编码SEQ ID NO:9的氨基酸序列的核酸序列。在另一个实施方式中，本发明的CAR包括SEQ ID NO:9中提出的氨基酸序列。
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DNA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，通常通过可操作地连接编码CAR多肽或其部分的核酸至启动子，并将构建体并入表达载体，实现编码CAR的天然或合成核酸的表达。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。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核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。
进一步地，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)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些 实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
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，以起动转录。
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。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
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)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体，更优选地为pWPT-GFP慢病毒载体。本发明人研究证实，使用该慢病毒载体构建本发明CAR，对NKT细胞的转染效率较高，且具有高度的可重复性。
在本发明的一个优选地实施方式中，所述载体中还包括信号肽序列。优选地，所述信号肽序列连接在所述抗原结核结构域核酸序列的上游。
优选地，所述信号肽为大鼠生长激素信号肽；更优选的，所述大鼠生长激素信号肽的核酸序列如SEQ ID NO.:6所示。本发明人在研究中，意外地发现，在本发明的载体为慢病毒载体时，使用大鼠生长激素信号肽能够显著提高包含本发明CAR的慢病毒载体对NKT细胞的转染效率。
治疗性应用
本发明包括用慢病毒载体(LV)转导的细胞(例如，NKT细胞、T细胞)。例如，LV编码将特异性抗体的抗原结合结构域与CD3-ζ、CD137组合的细胞内结构域联 合的CAR。因此，在一些例子中，转导的NKT细胞可引起CAR-介导的NKT-细胞应答。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的NKT细胞-介导的免疫应答的方法，其包括以下步骤：施用给哺乳动物表达CAR的NKT细胞，其中CAR包括特异性地与预定标靶相互作用的结合部分，包括例如人CD3ζ的细胞内结构域的ζ链部分，和共刺激信号传导区。
在一个实施方式中，本发明包括一类细胞疗法，其中NKT细胞被基因修饰以表达CAR，和CAR-NKT细胞被注入需要其的接受者中。注入的细胞能够杀死接受者的肿瘤细胞。不像抗体疗法，CAR-NKT细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-NKT细胞可经历稳固的体内NKT细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰NKT细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗CD30CAR-NKT细胞引起抗表达CD30的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括抗-CD30scFv、CD8铰链和跨膜区、和CD137和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、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、霍奇金氏疾病、非霍奇金氏淋巴瘤(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、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。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)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌、。
在一个实施方式中，本发明的CAR的抗原结合结构域被设计以治疗具体的癌症。例如，被设计以靶向CD30的CAR可用于治疗癌症和紊乱，包括但不限于肺癌等。
在一个实施方式中，针对癌症和紊乱可利用靶向CD19、CD20、CD22和ROR1的CAR的组合进行治疗。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩展细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
在此通过引用并入的在美国专利号5,199,942中描述的造血干细胞和祖细胞的离体扩展程序，可被应用至本发明的细胞。其他合适的方法在本领域中是已知的，因此本发明不限于细胞离体扩展的任何具体方法。简单地说，T细胞的离体培养和扩展包括：(1)从外周血收获物或骨髓外植体收集来自哺乳动物的CD34+造血干细胞和祖细胞；和(2)离体扩展这样的细胞。除了美国专利号5,199,942中描述的细胞生长因子，其他因子诸如flt3-L、IL-1、IL-3和c-kit配体，也可用于培养和扩展细胞。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
通常地，如本文所述活化和扩展的细胞可用于治疗和预防无免疫应答的个体中产生的疾病。特别地，本发明的CAR-修饰的T细胞用于治疗CCL。在某些实施方式中，本发明的细胞用于治疗处于形成CCL风险中的患者。因此，本发明提供了治疗或预防CCL的方法，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。
本发明的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。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以104至109个细胞/kg体重的剂量，优选105至106个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，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NKT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NKT细胞组合物优选通过i.v.注射施用。NKT细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将NKT细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的NKT细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
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个本发明经修饰的NKT细胞(如，CARCD30-NKT细胞)，通过例如静脉回输的方式，施用于患者。
本发明的优点包括：
1、采用本发明的嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞治疗CD30阳性的霍奇金淋巴瘤及非霍奇金淋巴瘤时，对淋巴瘤细胞具有很好的特异杀伤活性，且对已经过多次治疗(如采用CD30单克隆抗体结合细胞毒素药物或者放射性同位素治疗等)但均无明显疗效的CD30阳性的霍奇金淋巴瘤患者有显著地治疗效果；
2、本发明首次构建了一种对CD30阳性肿瘤具有显著杀伤效果的表达嵌合抗原受体的NKT细胞，并且该NKT细胞具有调节Car-T细胞群内的免疫平衡状态的功能；
3、传统的表达嵌合抗原受体的T细胞在体外存活时间比较短暂，而本发明的表达嵌合抗原受体的NKT细胞在体外存活时间较久，而且能够贴壁感染，感染效率高。而T细胞感染效率低，而且感染后的T细胞死亡率也高。
4、本发明的嵌合抗原受体，以CD8的绞链区和跨膜区及CD137和CD3ζ的胞内信号结构域串联而成的结构为信号传导结构域，能够保持对NKT细胞的高效转染，并且在NKT细胞内能够高效表达；
5、与传统的表达嵌合抗原受体的T细胞相比，本发明的表达嵌合抗原受体的 NKT细胞具有显著更高的体外溶瘤活性；
6、本发明的表达嵌合抗原受体的NKT细胞回输至患者体内，在外周血和骨髓中可以大量扩增，并且可以不依赖于白介素2而持续长久的存在。
实施例
以下的实施例将对本发明作进一步的说明，但并不因此限制本发明。
以下实施例中的实验方法，如无特殊说明，均为本领域常规方法。下述实施例中所用的实验材料，如无特殊说明，均为自常规生化试剂商店购买得到，其中：
NKT细胞培养基GT-T551购自TaKaRa公司。
淋巴细胞分离液购自TBD公司。
CD3单克隆抗体、重组纤维连接蛋白(retronectin)均购自TaKaRa公司。
重组人蛋白干扰素-γ、重组人白介素2购自protech公司。
总RNA提取试剂盒RNAiso Reagent、高保真DNA聚合酶(
Figure PCTCN2016071371-appb-000006
HS DNA Polymerase)、T4DNA连接酶购自TaKaRa公司。
RevertAidTM First Strand cDNA Synthesis Kit购自Fermentas公司。
BglⅡ、EcoRI、MluI、BamHI、SalI购自Fermentas公司。
琼脂糖凝胶DNA回收试剂盒、普通DNA产物纯化试剂盒、质粒小提试剂盒均购自天根生化科技有限公司。
pWPT-GFP、psPAX2、pMD2.G均购自Addgene公司。
pGSI购自北京天一辉远生物科技有限公司。
Trans1-T1Phage Resistant化学感受态细胞购自北京全式金生物技术有限公司。
LipofectamineTM 2000 Transfection Reagent转染试剂购自Invitrogen公司。
293T包装细胞购自美国ATCC。
PEG6000-NaCl中PEG6000终浓度为25.5质量％，NaCl终浓度为1.2M，PEG6000和NaCl均购自上海索莱宝生物科技有限公司。
胎牛血清购自德国PAA公司。
CD30阳性的淋巴瘤细胞系karpas299及L428由南方医科大学提供。
5-羧基荧光素琥珀酰亚胺酯购自上海谱振生物科技有限公司。
膜联蛋白V-RPE试剂盒购自美国BD公司。
所有引物均由北京天一辉远生物科技有限公司合成。
实施例1 NKT细胞的制备
(1)取人静脉血于含肝素的真空管中。采用淋巴细胞分离液，通过密度梯度离心方法分离获得单个核细胞(PBMCs)。
(2)PBMCs洗三次后，采用含有0.6体积％的胎牛血清的NKT细胞培养基GT-T551调整细胞终浓度为2×106个细胞/mL；将细胞接种于预先经过终浓度为5μg/mL CD3单克隆抗体及终浓度为10μg/mL的retronectin包被的75cm2细胞培养瓶中。然后在培养基里加入终浓度为1000U/mL的重组人蛋白干扰素-γ和1000U/mL的重组人白介素2，在37℃、饱和湿度为5％的CO2培养箱中培养。
(3)第四天，向培养瓶中补加100mL含有0.6体积％的胎牛血清的NKT细胞培养基GT-T551，并加入终浓度为1000U/mL的重组人白介素2。于37℃、饱和湿度为5％的CO2培养箱中培养4天，得到NKT细胞，流式细胞术对NKT细胞表型进行分析。结果见图1，其中CD3+:49.64％；CD3+CD4+:27.71％；CD3+CD8+:21.93％；CD3+CD56+:2.95％；CD8+CD56+:2.86％。
实施例2  慢病毒表达载体pWPT-CD30ScFv-CD8-CD137-CD3ζ的构建
(1)NKT细胞cDNA的制备
离心沉淀实施例1培养得到的NKT细胞，用总RNA提取试剂盒RNAiso Reagent提取细胞的总RNA，-80℃保存备用。提取的总RNA用逆转录试剂盒RevertAidTM First Strand cDNA Synthesis Kit逆转录得NKT细胞cDNA，-20℃保存备用。
(2)慢病毒质粒pWPT-CD8-CD137-CD3ζ的制备
设计并合成如下引物序列(其中，下划线标记为保护碱基，方框为酶切位点)：
Figure PCTCN2016071371-appb-000007
以步骤(1)中NKT细胞cDNA为模板，用引物P1和P2进行PCR扩增，得到长287bp的CD8的hinge区和跨膜区，核苷酸序列如SEQID NO.3所示，两端分别含有MluI和BglⅡ酶切位点和保护碱基；用引物P3和P4进行PCR扩增，得到长146bp的CD137胞内信号结构域，核苷酸序列如SEQID NO.4所示，两端分别含有BglⅡ和EcoRI酶切位点及保护碱基；用引物P5和P6进行PCR扩增，得到长359bp的CD3ζ的胞内信号结构域，核苷酸序列如SEQID NO.5所示，两端分别含有EcoRI和SalI酶切位点及保护碱基。各步PCR扩增反应体系相同，以扩增CD137胞内信号结构域为例，进行PCR扩增，PCR反应条件参照
Figure PCTCN2016071371-appb-000008
HS DNA Polymerase的说明书，反应体系(50μL)如下：
双蒸水：32.5μL
5×反应buffer：10μL
dNTP混合物(每种2.5mM)：4μL
P3(10mM):1μL
P4(10mM):1μL
NKT细胞cDNA(200ng/ul)：1μL
Figure PCTCN2016071371-appb-000009
HS DNA Polymerase：0.5μL
将上述PCR产物用1％的琼脂糖凝胶进行分离，用琼脂糖凝胶DNA回收试剂盒进行DNA片段回收。得到片段后分别进行双酶切反应，酶切产物用普通DNA产物纯化试剂盒回收备用。
慢病毒表达载体pWPT-GFP用MluI/SalI双酶切，酶切产物经1％的琼脂糖凝胶进行分离，用琼脂糖凝胶DNA回收试剂盒回收大的载体片段，然后与之前回收的CD8、CD137、CD3ζ片段通过T4DNA连接酶连接，连接产物转化Trans1-T1Phage Resistant化学感受态细胞，37℃培养16h后挑取单克隆，37℃，250rpm培养12h后用质粒小提试剂盒提取质粒。提取的质粒经限制性内切酶MluI和SalI双酶切鉴定，鉴定电泳图见图2，其中，1泳道：DNA分子量标记D2000；2泳道：质粒pWPT-GFP的酶切片段(835bp)；3泳道：质粒pWPT-CD8-CD137-CD3ζ的酶切片段(756bp)。将鉴定正确的质粒送北京天一辉远生物科技有限公司对插入的融合基因片段进行测序。将测序结果正确的重组质粒命名为pWPT-CD8-CD137-CD3ζ。
(3)慢病毒质粒pWPT-CD30ScFv-CD8-CD137-CD3ζ的制备
全基因合成编码大鼠生长激素信号肽和CD30ScFv融合基因的核苷酸序列，序列如SEQID NO.8所示，由北京天一辉远生物科技有限公司合成，其5’端含有BamHI、kozak序列，3’端含有MluI酶切位点，将前述融合基因克隆在质粒pGSI中，命名为pGSI-CD30ScFv。质粒经BamHI/MluI双酶切，酶切产物经1％琼脂糖凝胶进行分离，用琼脂糖凝胶DNA回收试剂盒回收目的片段备用。
pWPT-CD8-CD137-CD3ζ质粒经限制性内切酶BamHI/MluI酶切，酶切产物经1％琼脂糖凝胶进行分离，用琼脂糖凝胶DNA回收试剂盒回收载体片段备用。然后与回收的含有大鼠生长激素信号肽和CD30ScFv的DNA片段通过T4DNA连接酶进行连接，具体方法见说明书。将连接产物转化Trans1-T1Phage Resistant化学感受态细胞，37℃培养16h后挑取单克隆，37℃，250rpm培养12h后，用质粒小提试剂盒提取质粒。提取的质粒经限制性内切酶BamHI/SalI双酶切鉴定，鉴定结果如图3所示，其中，M1：DNA分子量标记D2000；1泳道：质粒pWPT-CD30ScFv-CD8-CD137-CD3ζ未酶切片段(10232bp)；2泳道：质粒pWPT-CD30ScFv-CD8-CD137-CD3ζ的酶切片段；M2：DNA分子量标记D15000。将鉴定正确的质粒送北京天一辉远生物科技有限公司对插入的融合基因片段进行测序。将测序结果正确的重组质粒命名为pWPT-CD30ScFv-CD8-CD137-CD3ζ，其结构示意图如图4所示，其中包括大鼠生长激素信号肽(核苷酸序列如SEQID NO.6所示)、抗CD30单链抗体(核苷酸序列如SEQID NO.7所示)、CD8的hinge区和跨膜区及CD137的胞内信号结构域和CD3ζ的胞内信号结构域，其中，该嵌合抗原受体以基因CD8的hinge区和跨膜区及CD137和CD3ζ的胞内信号结构域串联而成的结构为信号传导结构域，其氨基酸序列如SEQID NO.9所示，对应的基因编码序列如SEQID NO.10所示。
实施例3  嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞的制备
(1)慢病毒的包装和浓缩
用分光光度计分别测定慢病毒表达质粒pWPT-CD30ScFv-CD8-CD137-CD3ζ和辅助质粒psPAX2、pMD2.G的浓度，三种质粒以4:2:1的质量比用LipofectamineTM 2000 Transfection Reagent转染试剂共转染293T包装细胞。分别在转染48h、72h时收集病毒上清于50mL EP管中，4℃，2000g离心10min，转移两次得到的上清至新EP管中，用4.5μm滤器过滤病毒上清；过滤的病毒上清与5×PEG6000-NaCl按照4:1的体积比混匀，4℃静置2h，然后4℃，10000g离心20min，弃上清，沉淀用1mL的4℃预冷的无菌PBS溶解，即得嵌合抗原受体的病毒浓缩液，按每管100μL进行分装，-80℃保存备用。
按照上述方法，利用慢病毒表达质粒pWPT-GFP和辅助质粒psPAX2、pMD2.G共转染293T包装细胞，收集病毒上清，浓缩，获得表达GFP绿色荧光蛋白的慢病毒浓缩液。
(2)慢病毒感染NKT细胞及感染后细胞的扩增培养
取实施例1的在75cm2培养瓶中培养的1×107个NKT细胞，弃掉旧的培养液，加入2mL新鲜NKT细胞培养基GT-T551、200μL步骤(1)得到的病毒浓缩液、2μL 1×10-6mg/mL鱼精蛋白，终浓度为1000U/mL的重组人白介素2，置于37℃、饱和湿度为5％的CO2培养箱中感染12小时后，弃培养液，得到的NKT细胞称为CAR30-NKT细胞。同时用表达GFP绿色荧光蛋白的慢病毒浓缩液对NKT细胞进行同步感染(得到的NKT细胞称为CART-GFP细胞)，用于计算该病毒的感染效率。将感染后的细胞转至未经CD3和retronectin包被的75cm2培养瓶中，加入20mL的NKT细胞培养基GT-T551，再加入终浓度为1000U/mL的重组人白介素2，于37℃、饱和湿度为5％的CO2培养箱中培养18h。用流式细胞术检测该病毒的感染效率，结果如图5所示，感染效率为36.38％。
(3)体外诱导扩增CAR30-NKT细胞群
将上述培养后的NKT细胞用重组人白介素2的终浓度为1000U/mL的NKT细胞培养基GT-T551进行体外诱导，待细胞的密度为85％时将细胞转入细胞培养袋中，隔2天加入重组人白介素2的终浓度为1000U/mL的新鲜NKT细胞培养基GT-T551进行扩增培养，待细胞扩增到总量为1.5×109个细胞左右后，采用流式细胞仪对感染的细胞群体进行鉴定，结果见图6，CD3+：89.99％；CD3+CD4+：58.18％；CD3+CD8+：31.81％；CD3+CD56+：6.38％；CD8+CD56+：5.37％。
实施例4  CAR30-NKT细胞对人霍奇金淋巴瘤细胞杀伤作用的细胞毒性分析
分别取实施例3中制备的CAR30-NKT细胞、CART-GFP细胞和实施例1中培养的NKT细胞接种于96孔板，用5-羧基荧光素琥珀酰亚胺酯(CFSE)进行染色，与CD30阳性的淋巴瘤细胞系karpas299及L428以效靶比(杀伤细 胞：靶细胞)1:1，5:1，10:1，20:1，40:1比率进行共培养，经过24小时的共培养后，将细胞用膜联蛋白V-RPE试剂盒染色，同时设置对照组分别为未加入免疫细胞杀伤处理的淋巴瘤细胞系karpas299及L428，并将细胞用膜联蛋白V-RPE试剂盒染色。流式细胞术对细胞凋亡进行检测，细胞凋亡的量根据下面的公式计算：凋亡率＝(对照-样品)/对照×100％，对照为未加免疫细胞杀伤处理的细胞存活数目；样品为加入相对应的效靶比(杀伤细胞：靶细胞)的免疫细胞杀伤处理的细胞存活数目，见图7。嵌合抗原受体CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞对CD30阳性淋巴瘤细胞具有特异杀伤活性，且CAR30-NKT细胞的特异杀伤活性明显优于NKT细胞。
实施例5  CAR30-NKT细胞对CD30阳性的霍奇金淋巴瘤患者的治疗效果
取5×108个CD30ScFv-CD8-CD137-CD3ζ修饰的NKT细胞(即CAR30-NKT细胞)，经过100ml生理盐水稀释后，连续三天静脉回输到下述CD30阳性的霍奇金淋巴瘤患者(在利用本发明的CAR30-NKT细胞进行靶向免疫治疗前，已经过多次治疗(包括采用CD30单克隆抗体结合细胞毒素药物或者放射性同位素治疗等)，但均无明显疗效)体内，回输后对患者的治疗状况进行评估。
图8为血象分析CAR30-NKT细胞治疗CD30阳性的霍奇金淋巴瘤患者过程中白细胞总数、淋巴细胞总数、体温变化及白介素6水平的趋势图(FC为患者治疗前采取的清髓预处理方案)。结果显示，霍奇金淋巴瘤患者治疗前表现为高热、炎症及骨痛症状(根据VAS疼痛评分标准为7级骨痛)，经CAR30-NKT细胞治疗后患者白细胞数目逐渐减少并趋于正常状态，淋巴细胞数目逐渐增加(表明在CD30阳性的霍奇金淋巴瘤患者体内CAR30-NKT细胞能够逐渐扩增)，同时患者体温及白介素6水平逐渐恢复至正常并能够维持在正常水平，患者骨痛症状得到缓解(根据VAS疼痛评分标准为1级骨痛)。说明CAR30-NKT细胞治疗CD30阳性的淋巴瘤患者是安全、可耐受的，而且CAR30-NKT细胞对CD30阳性的霍奇金淋巴瘤患者(经已有的多种治疗手段治疗后均无明显疗效)治疗后，对患者的疾病有一定的治疗效果。
图9为CT分析CD30阳性的霍奇金淋巴瘤肝转移患者，经CAR30-NKT细胞治疗(一个月)后，患者肝部位病灶变化。结果显示，霍奇金淋巴瘤患者治疗后肝病灶(参见箭头)明显减少，进一步说明CAR30-NKT细胞对CD30阳性的霍奇金淋巴瘤患者(经已有的多种治疗手段治疗后均无明显疗效)治疗后，对患者的疾病有明显的治疗效果。
取实施例3中制备的CAR30-NKT细胞接种于25厘米的细胞培养瓶进行培养，并持续收集细胞上清，采用ELISA试剂盒分析细胞分泌白介素2的水平。结果显示本发明的CAR30-NKT细胞在体外能持续分泌白介素2细胞因子，具有独特的非依赖IL-2性的扩增能力，避免了临床使用时体外补充IL-2。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (12)

  1. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体以CD8的绞链区和跨膜区及CD137和CD3ζ的胞内信号结构域串联而成的结构为信号传导结构域；并且所述嵌合抗原受体的胞外结构域为抗CD30的单链抗体。
  2. 如权利要求1嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体为CD30ScFv-CD8-CD137-CD3ζ，由CD30ScFv、CD8的铰链区和跨膜区、CD137的胞内信号结构域和CD3ζ的胞内信号结构域串联构成。
  3. 如权利要求1嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体的氨基酸序列如SEQ ID NO.1所示。
  4. 如权利要求1嵌合抗原受体(CAR)，其特征在于，所述CD8的铰链区和跨膜区选自下组：
    (A)具有SEQ ID NO:17所示氨基酸序列的多肽；
    (B)具有与SEQ ID NO:17所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性)的多肽；(C)将SEQ ID NO:17中任一所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的衍生多肽；和/或
    所述CD137的胞内信号结构域选自下组：
    (A)具有SEQ ID NO:18所示氨基酸序列的多肽；
    (B)具有与SEQ ID NO:18所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；优选地≥95％的同源性；最优选地，≥97％的同源性)，并且能够转导效应子功能信号的多肽；
    (C)将SEQ ID NO:18中任一所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的衍生多肽；和/或
    所述CD3ζ的胞内信号结构域选自下组：
    (A)具有SEQ ID NO:19所示氨基酸序列的多肽；
    (B)具有与SEQ ID NO:19所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性)，并且能够转导效应子功能信号的多肽；
    (C)将SEQ ID NO:19中任一所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的衍生多肽。
  5. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1所述的嵌合抗原受体(CAR)。
  6. 一种载体，其特征在于，所述的载体含有权利要求2中所述的核酸分子；优选地，所述载体为慢病毒载体。
  7. 一种细胞，其特征在于，所述的细胞中含有权利要求6中所述的载体或染色体中整合有外源的权利要求5中所述的核酸分子。
  8. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1中所述的嵌合抗原受体、权利要求5中所述的核酸分子、权利要求6中所述的载体、或权利要求7中所述的宿主细胞。
  9. 权利要求1中所述的嵌合抗原受体、权利要求5中所述的核酸分子、权利 要求6中所述的载体、或权利要求7中所述的宿主细胞的用途，其特征在于，用于制备治疗肿瘤的药物或制剂。
  10. 如权利要求9所述的用途，其特征在于，所述的肿瘤包括CD30阳性的淋巴瘤。
  11. 如权利要求10所述的用途，其特征在于，所述淋巴瘤为霍奇金淋巴瘤。
  12. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1中所述的嵌合抗原受体、权利要求5中所述的核酸分子、权利要求6中所述的载体、权利要求7中所述的细胞、或权利要求8中所述的药物组合物。
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