WO2016086813A1 - 双靶向gpc3和asgpr1的转基因免疫效应细胞及其应用 - Google Patents

双靶向gpc3和asgpr1的转基因免疫效应细胞及其应用 Download PDF

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WO2016086813A1
WO2016086813A1 PCT/CN2015/095938 CN2015095938W WO2016086813A1 WO 2016086813 A1 WO2016086813 A1 WO 2016086813A1 CN 2015095938 W CN2015095938 W CN 2015095938W WO 2016086813 A1 WO2016086813 A1 WO 2016086813A1
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gpc3
chimeric antigen
asgpr1
antigen receptor
cells
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French (fr)
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王华茂
蔡秀梅
赵红霞
宋波
陈远梅
朱寅玉
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科济生物医药（上海）有限公司
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Priority to EP15865161.2A priority Critical patent/EP3228702A4/en
Priority to US15/532,965 priority patent/US11453860B2/en
Publication of WO2016086813A1 publication Critical patent/WO2016086813A1/zh

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Definitions

  • the present invention relates to the field of tumor immunotherapy, and more particularly to transgenic immune effector cells that dually target GPC3 and ASGPR1 and uses thereof.
  • T lymphocytes The role of T lymphocytes in tumor immune responses is receiving increasing attention.
  • Adoptive immunotherapy based on T lymphocytes has achieved certain effects in some tumors, and this immunotherapy method can overcome the above defects of antibody treatment, but the therapeutic effect in most tumors is still unsatisfactory.
  • the specificity of recognition of target cells by CTL depends on the discovery of T cell receptor (TCR), scFv of antibodies against tumor cell-associated antigens, CD3 ⁇ or Fc ⁇ RI ⁇ of T lymphocyte receptors, etc.
  • TCR T cell receptor
  • scFv antibodies against tumor cell-associated antigens
  • CD3 ⁇ or Fc ⁇ RI ⁇ of T lymphocyte receptors
  • the intracellular signal activation motif is fused to a chimeric antigen receptor (CAR) and genetically modified on the surface of T lymphocytes by, for example, lentivirus infection.
  • CAR chimeric antigen receptor
  • CAR T lymphocytes are capable of selectively targeting T lymphocytes to tumor cells and specifically killing tumors in a non-limiting manner in a major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • Chimeric antigen receptors include extracellular binding regions, transmembrane regions, and intracellular signaling regions.
  • the extracellular region contains a scFv capable of recognizing a tumor-associated antigen
  • a transmembrane region adopts a transmembrane region of a molecule such as CD8, CD28
  • the intracellular signal region adopts an immunoreceptor tyrosine activation motif (ITAM) CD3 ⁇ or Fc ⁇ RI ⁇ and co-stimulation.
  • ITAM immunoreceptor tyrosine activation motif
  • the intracellular signal region of the signaling molecules CD28, CD137, CD134, and the like.
  • the intracellular signal domain contains only ITAM as the first generation of CAR T lymphocytes, wherein the chimeric antigen receptor portions are linked as follows: scFv-TM-ITAM.
  • This kind of CAR T can stimulate the anti-tumor cytotoxic effect, but the cytokine secretion is relatively small, and can not stimulate the long-lasting anti-tumor effect in vivo [Zhang T. et al. Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth In a manner involving multiple cytokines and cytotoxic pathways, Can Res 2007, 67(22): 11029–11036.].
  • CD28 or CD137 aka 4-1BB
  • the chimeric antigen receptor portions were joined as follows: scFv-TM-CD28-ITAM or scFv -TM-CD137-ITAM.
  • Co-stimulation of B7/CD28 or 4-1BBL/CD137 in the intracellular signal domain causes sustained proliferation of T lymphocytes, and can increase the secretion of cytokines such as IL-2 and IFN- ⁇ by T lymphocytes, and increase CAR T Survival cycle and anti-tumor effect in vivo [Dotti G. et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients. J Clin Invest, 2011, 121(5): 1822-1826.].
  • CAR T lymphocytes developed in recent years, in which the chimeric antigen receptor parts are linked as follows: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further enhancing CAR T is at The survival cycle in vivo and its anti-tumor effect [Carpenito C., et al. Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. PNAS, 2009, 106(9): 3360-3365.].
  • CAR T lymphocytes have attractive prospects in tumor immunotherapy, some potential risks need to be considered.
  • specific antigens that can be recognized by the low expression of CAR in certain normal tissues may cause damage to the normal tissues of CAR T lymphocytes expressing the corresponding antigen.
  • CAIX antigen carbonic anhydrase IX
  • the antigen carbonic anhydrase IX (CAIX) expressed on tumor cells of patients with renal cell carcinoma is the first case for the clinical treatment of CAR T lymphocytes. It is also the first case to report the off-target effect of CAR cells. . Patients developed grade 2-4 hepatotoxicity after multiple injections of CAR T lymphocytes.
  • liver bile duct epithelial cells have low expression of CAIX, and the original clinical trial was interrupted while excluding any evaluation of the patient's therapeutic effect [Stoter G. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX : first clinical experience. J clin oncol, 2006, 24(13): e20-e22.; Ngo MC., et al. Ex vivo gene transfer for improved adoptive immunotherapy of cancer. Human Molecular Genetics, 2011, R1 - R7].
  • Phosphatidylinositol-3 (Glypican-3, GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) is a cell surface protein belonging to heparan sulfate proteoglycan family.
  • the GPC3 gene encodes a 70-kDa precursor core protein that can be cleaved by furin to produce a soluble 40-kDa-capable amino-terminal (N-terminal) peptide and 30- A membrane-bound carboxy-terminal (C-terminal) peptide containing two heparan sulfate (HS) sugar chains around kDa.
  • the GPC3 protein is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • GPC3 is highly expressed in the fetal liver, but not in the liver tissue of normal adults, but it is restored in hepatocellular carcinoma, which is closely related to the occurrence and development of liver cancer, not only in the early detection of liver cancer, but also in the early detection rate. And with the development of liver cancer, its detection rate has also increased.
  • the expression of GPC3 was not detected in hepatic adenocarcinoma, cholangiocarcinoma, liver metastases, and 12 common solid tumors and 21 non-hepatoma cell lines.
  • GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma, and the like.
  • Detection of liver cancer using anti-GPC3 antibodies and antibody-dependent (ADCC) or complement-dependent anti-GPC3 antibodies The Lai's (CDC) cytotoxicity research protocol has been reported.
  • Antibodies commonly used for treatment recognize the C-terminus of the GPC3 protein.
  • antibody therapy has a limitation on the half-life of antibodies in the blood circulation in the body. Generally, the half-life is mostly within 23 days. Thus, sustained administration and/or increased dosing is required for tumor antibody therapy, which results in an increase in the cost of treatment for the patient and, in some cases, even termination of treatment.
  • therapeutic antibodies may also have an allergic reaction in the body and a risk of neutralizing anti-antibodies against the therapeutic antibody.
  • the asialoglycoprotein receptor (ASGPR1, or ASGR1), also known as galactose receptor, is a transmembrane protein with a relative molecular mass of about 41,000 and consists of two structurally distinct subunits, H1 and H2. H1 is the main component of the receptor.
  • the extracellular domain of ASGPR1 contains a carbohydrate recognition domain (CRD) that recognizes and binds to galactose residues and N-acetylgalactosamine residues. Receptor-mediated endocytosis occurs when CRD binds to a specific sugar residue.
  • CRD carbohydrate recognition domain
  • ASGPR1 The main function of ASGPR1 is to eliminate glycoproteins, lipoproteins and apoptotic cells that lose terminal sialic acid in the peripheral blood circulation and expose galactose residues or acetylgalactosamine residues, and also mediate hepadnaviruses (such as hepatitis B virus and hepatitis C virus). ) Binding and uptake with hepatocytes.
  • ASGPR1 is mainly expressed on the surface of hepatic parenchyma cells in the hepatic sinusoids of mammals, and has a high density of up to 500,000 receptors per cell surface. The number and function of ASGPR1 expression decreased in the development of liver diseases such as hepatocellular carcinoma, hepatitis, and cirrhosis.
  • a dual-targeted chimeric antigen receptor (CAR) immune effector cell the cell expressing a chimeric receptor: a chimeric antigen receptor that specifically recognizes GPC3; and specificity The chimeric antigen receptor of ASGPR1 is recognized.
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor that specifically recognizes GPC3 comprises: a single-chain antibody against anti-GPC3 (scfv), a T cell stimulation signaling molecule.
  • the T cell stimulation signal molecule is selected from the group consisting of: CD3 ⁇ or Fc ⁇ RI ⁇ ; more preferably, the chimeric antigen receptor that specifically recognizes GPC3 comprises (preferably 5' ⁇ 3') : anti-GPC3 single-chain antibody (scfv), CD8 transmembrane region, CD3 ⁇ .
  • the chimeric antigen receptor that specifically recognizes GPC3 comprises (preferably 5' ⁇ 3') : anti-GPC3 single-chain antibody (scfv), CD8 transmembrane region, CD3 ⁇ .
  • reporter gene 1 such as eGFP
  • F2A F2A
  • reporter gene 1 is linked to a single-chain antibody against GPC3 by F2A.
  • Reporter 1 presents the expression of the chimeric antigen receptor that specifically recognizes GPC3 in the cell.
  • the chimeric antigen receptor that specifically recognizes GPC3 has the amino acid sequence set forth in SEQ ID NO: 17.
  • the chimeric antigen receptor that specifically recognizes GPC3 is expressed by a viral vector; preferably a lentiviral vector, such as pWPT.
  • the chimeric antigen receptor that specifically recognizes ASGPR1 comprises: a single chain antibody against ASGPR1 (scfv), a T cell activated costimulatory signaling molecule.
  • the T cell activated costimulatory signal molecule is selected from the group consisting of: CD27, CD28, CD137, CD134, an intracellular signal region of an ICOS protein, or a combination thereof; more preferably, the specificity
  • the chimeric antigen receptor recognizing ASGPR1 includes (preferably 5' ⁇ 3'): a single-chain antibody against ASGPR1 (scfv), a transmembrane region of CD28 (CD28a), and an intracellular signal region of CD28 (CD28b) And CD137 intracellular signal region.
  • the reporter gene 2 (eg, mCherry), F2A is further included at the 5' end of the anti-ASGPR1 single-chain antibody (scfv), and the reporter gene 2 is linked to the anti-ASGPR1 single-chain antibody by F2A, Reporter 2 presents the expression of the chimeric antigen receptor that specifically recognizes ASGPR1 in the cell.
  • the chimeric antigen receptor that specifically recognizes ASGPR1 has the amino acid sequence set forth in SEQ ID NO: 24.
  • the chimeric antigen receptor that specifically recognizes ASGPR1 is expressed by a viral vector; preferably a lentiviral vector, such as pWPT.
  • the immune effector cells are selected from the group consisting of: T lymphocytes, NK cells or NKT cells.
  • the use of the chimeric antigen receptor immune effector cell for the preparation of a tumor-treating kit is a GPC3 and ASGPR1 double positive tumor; preferably said The tumor is liver cancer.
  • kits for treating a tumor comprising: the chimeric antigen receptor immune effector cell; the tumor is a GPC3 and ASGPR1 double positive tumor
  • the tumor is liver cancer.
  • kits for preparing the chimeric antigen receptor immune effector cell comprising:
  • an expression construct a comprising an expression cassette (which can be expressed in immune cells) that specifically recognizes a chimeric antigen receptor of GPC3;
  • Expression construct b which comprises an expression cassette (which can be expressed in immune cells) that specifically recognizes the chimeric antigen receptor of ASGPR1.
  • the expression cassette for a chimeric antigen receptor that specifically recognizes GPC3 comprises a construct (expression vector) expressing a single-chain antibody against GPC3 and a T cell-stimulating signal molecule; preferably, it has SEQ ID NO: The nucleotide sequence shown in 17.
  • the expression cassette for specifically recognizing the chimeric antigen receptor of ASGPR1 comprises a construct (expression vector) expressing a single-chain antibody against ASGPR1 and a T cell-activated costimulatory signal molecule; It has the nucleotide sequence shown by SEQ ID NO: 23.
  • the chimeric antigen receptor that specifically recognizes GPC3 comprises: a single chain antibody against GPC3, a T cell stimulation signal molecule; preferably, the T cell stimulation signal molecule is selected from the group consisting of: CD3 ⁇ or Fc ⁇ RI ⁇ ;
  • the chimeric antigen receptor for specifically recognizing ASGPR1 comprises: a single chain antibody against ASGPR1, a T cell activated costimulatory signal molecule; preferably, the T cell activated costimulatory signal molecule is selected from the group consisting of: CD27 , CD28, CD137, CD134, the intracellular signal region of the ICOS protein, or a combination thereof.
  • FIG. 1 Western-blot results of expression levels of ASGPR1 and GPC3 in various liver cancer cell lines.
  • Figure 2 Flow cytometric results of expression levels of ASGPR1 and GPC3 in various liver cancer cell lines.
  • FIG. 1 Western-blot detection of GPC3 and ASGPR1 expression in MHCC-97H-G, MHCC-97H-A, and MHCC-97H-GA stably transfected cell lines.
  • FIG. 1 Western blot analysis of the expression of the target gene of infected T cells.
  • T cells expressing different chimeric antigen receptors were co-cultured with target cells expressing different antigens, and T cell proliferation was detected on day 7.
  • Figure 6 The ratio of Mock T, GCAR T, ACCR T, and GZ+28BB T lymphocytes to MHCC-97H-GA cells positive for ASGPR1 and GPC3 expression, or MHCC-97H negatively expressed by both cells. After incubation for 24 hours, T lymphocytes were collected and the expression of BCL-XL X L was detected.
  • Figure 7 In vivo anti-tumor activity assay of MHCC-97H-A, MHCC-97H-G, and MHCC-97H-GA cells.
  • FIG. 1 The number of T cells in peripheral blood was measured 1 week after adoptive transfer of genetically modified T lymphocytes.
  • the inventors have intensively studied for the first time to disclose a genetically modified immune effector cell capable of simultaneously recognizing GPC3 and ASGPR1, which can be applied to the treatment of GPC3 and ASGPR1 double positive tumors (such as liver cancer).
  • chimeric antigen receptor (CAR) immune effector cells is well known in the art and is an immune effector cell that expresses a tumor-specific chimeric antigen receptor using genetic engineering techniques and exhibits certain in vitro and clinical trials. Targeting, killing activity and persistence are adoptive cell immunotherapy methods.
  • the immune effector cells include, for example, T cells, NK cells.
  • chimeric antigen receptor immune effector cells Conventional methods for preparing "chimeric antigen receptor immune effector cells" are known to those skilled in the art, including allowing them to express extracellular antigen (eg, tumor associated antigen) binding regions; and intracellular costimulation of intracellular costimulatory cells. Domains such as CD28 (preferably including CD28a, CD28b), CD137, CD27, CD3 ⁇ (preferably CD3) Intracellular domain), one or more of CD8, CD19, CD134, CD20, FcR ⁇ . By binding to the corresponding ligands, the second signal of the immune effector cells is activated, the proliferation ability of the immune cells and the secretory function of the cytokines are enhanced, and the survival time of the activated immune cells is prolonged.
  • CD28 preferably including CD28a, CD28b
  • CD137 CD27
  • CD3 ⁇ preferably CD3 Intracellular domain
  • the tumor refers to a tumor that is double positive for GPC3 and ASGPR1, and the tumor includes, for example, liver cancer.
  • the present inventors have unexpectedly discovered in the study that phosphatidylinositol-3 (Glypican-3, GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) and saliva
  • the acid-protein receptor ASGPR1, or ASGR1 is co-expressed in liver cancer cells, which has not been reported so far.
  • the present inventors designed dual-targeted immune effector cells for these two proteins, which simultaneously contain a chimeric antigen receptor capable of recognizing GPC3 ( Chimeric antigen receptor recognizing GPC3, GCAR) and chimeric costimulatory receptor recognizing ASGPR1 (ACCR).
  • a chimeric antigen receptor capable of recognizing GPC3 Chimeric antigen receptor recognizing GPC3, GCAR
  • chimeric costimulatory receptor recognizing ASGPR1 chimeric costimulatory receptor recognizing ASGPR1
  • a chimeric receptor extracellular region comprises a polypeptide composition recognizing GPC3, and the intracellular region has a T cell stimulation signal (such as an ITAM motif of CD3 ⁇ or Fc ⁇ RI ⁇ ), which is called GCAR;
  • the extracellular region of the chimeric receptor contains a polypeptide that recognizes ASGPR, and the intracellular region contains a T cell-activated costimulatory signal segment (which may be an intracellular signal segment of CD28, CD137, or other costimulatory signaling molecule), ACCR.
  • the chimeric receptors each contain a transmembrane region.
  • the polypeptide recognizing GPC3 or ASGPR may be a ligand protein, a small molecule polypeptide, a single chain antibody, a single domain antibody or other antibody fragment.
  • the nucleic acid sequence encoding the chimeric receptor of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the nucleic acid codons encoding the amino acid sequences of the chimeric antigen receptor proteins of the present invention may be degenerate, that is, a plurality of degenerate nucleic acid sequences encoding the same amino acid sequence are included in the scope of the present invention. Degenerate nucleic acid codons encoding corresponding amino acids are well known in the art.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • a monoclonal antibody that specifically recognizes the C-terminal epitope of human GPC3 has been disclosed, for example, in Chinese Patent Publication No. CN101186650A, and has been disclosed in the literature, Advances in Liver Cancer Antibody Therapies: A Focus on Glypican-3 and Mesothelin, BioDrugs. .2011 October 1;25(5):275–284
  • Other monoclonal antibodies known to specifically recognize C-terminal epitopes, including GC33 and hGC33 have also been reported, respectively, for the epitope of GPC3 located at C-terminal 524-563 Amino acid residues, as well as monoclonal antibodies such as GPC3-C02 and 1G12, have also been reported.
  • monoclonal antibodies can be used to prepare the invention A single-chain antibody portion of a chimeric antigen receptor encoded by a nucleic acid.
  • Other monoclonal antibodies that recognize the C-terminal epitope of GPC3 can also be used in the present invention in a suitable manner.
  • monoclonal antibodies against the heparan sulfate chain of GPC3 (Gao W, Kim H, Feng M, Phung Y, Xavier CP, Rubin JS, Ho M. Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate Chains of glypican-3 for liver cancer therapy. Hepatology. 2014 Aug; 60(2): 576-87).
  • a single-chain antibody (scfv) that specifically recognizes human GPC3 is used.
  • the single-chain antibody scFv (GPC3) can be produced by a genetic engineering method or a chemical synthesis method according to the sequence of the GPC3 monoclonal antibody disclosed above.
  • the term "single-chain antibody (scFv) fragment" as used in the present invention refers to an antibody fragment defined by a heavy chain variable region (VH) and a light chain variable region which are linked by a linker ( The recombinant protein of VL), the linker associates these two domains to ultimately form an antigen binding site.
  • the size of scFv is typically 1/6 of that of an intact antibody.
  • the single chain antibody is preferably a sequence of one amino acid strand encoded by one nucleotide chain.
  • the single-chain antibodies used in the present invention may be further modified, either singly or in combination, using conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations, and/or other modifications. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. The indicated modifications are preferably carried out at the nucleic acid level.
  • the above single chain antibodies may also include derivatives thereof.
  • the "derivative of antibody” in the present invention includes, for example, when a derivative of the antibody is obtained by phage display technology, surface plasmon resonance technology such as used in the BIAcore system can be used to increase the efficiency of phage antibody binding to the GPC3 epitope. (Schier, Human Antibody Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • specific recognition in the present invention means that the bispecific antibody of the present invention does not or substantially does not cross-react with any polypeptide other than the antigen of interest.
  • the degree of specificity can be judged by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.
  • specific recognition is preferably determined by flow cytometry, and the criteria for specific recognition in specific cases can be judged by one of ordinary skill in the art based on the common knowledge in the art.
  • the transmembrane region of the chimeric receptor may be selected from the transmembrane region of a protein such as CD8 or CD28.
  • CD8 or CD28 is a natural marker on the surface of T lymphocytes.
  • the human CD8 protein is a heterodimer composed of two chains, ⁇ or ⁇ .
  • the transmembrane region is selected from the transmembrane region of CD8 alpha or CD28.
  • the CD8 ⁇ hinge region is a flexible region, and therefore, CD8 or CD28 and the transmembrane region plus the hinge region are used to link the target recognition domain of the chimeric receptor CAR or CCR with the intracellular signal region. .
  • the intracellular signal region of GCAR may be selected from CD3 or Fc ⁇ RI ⁇ ; the intracellular signal region of ACCR may be selected from the group consisting of CD27, CD28, CD137, CD134, the intracellular signal region of the ICOS protein, or a combination thereof.
  • the CD3 molecule consists of five subunits, of which the CD3 ⁇ subunit (also known as CD3 zeta, abbreviated as Z) contains three ITAM motifs, which are TCR-CD3 An important signal transduction region in the complex.
  • CD3 ⁇ Z is a truncated CD3 ⁇ sequence that does not have an ITAM motif and is generally constructed as a negative control in the practice of the present invention.
  • Fc ⁇ RI ⁇ is mainly distributed on the surface of mast cells and basophils, which contains an ITAM motif similar in structure, distribution and function to CD3 ⁇ .
  • CD27, CD28, CD137, CD134, and ICOS are costimulatory signaling molecules, and the co-stimulatory effect of intracellular signal segments after binding to their respective ligands causes sustained proliferation of T lymphocytes and can be improved.
  • T lymphocytes secrete levels of cytokines such as IL-2 and IFN- ⁇ , while increasing the survival cycle and antitumor effect of the corresponding T lymphocytes in vivo.
  • the anti-GPC3 chimeric receptor protein GCAR encoded by the nucleic acid of the present invention may be selected from the group consisting of scFv(GPC3)-CD8-CD3 ⁇ ; wherein scFv (GPC3) represents a single-chain antibody recognizing GPC3, and CD8 represents CD8. Transmembrane zone.
  • the anti-ASGPR1 chimeric receptor protein ACCR encoded by the nucleic acid of the present invention may be selected from the group consisting of Fv(ASGPR)-CD28a-CD28b-CD137; wherein Fv(ASGPR1) represents a recognition in the relevant chimeric antigen receptor protein.
  • Fv(ASGPR1) represents a recognition in the relevant chimeric antigen receptor protein.
  • Single domain antibody of ASGPR1; CD28a represents the transmembrane region of CD28 molecule, CD28b represents the intracellular signal region of CD28 molecule, and CD137 represents the intracellular signal region of CD137 molecule.
  • the invention further encompasses a nucleic acid construct or vector encoding a chimeric antigen receptor protein expressed on the surface of a T lymphocyte as described above.
  • the vector used in the invention is a lentiviral plasmid vector pWPT-eGFP. This plasmid belongs to the third generation auto-inactivated lentiviral vector system.
  • the system has three plasmids, namely, the coding protein Gag/Pol, the packaging plasmid psPAX2 encoding Rev protein, and the envelope plasmid PMD2.G encoding VSV-G protein;
  • the vector pWPT-eGFP which can be used for recombinant introduction of a nucleic acid sequence of interest, ie a nucleic acid sequence encoding a CAR.
  • the empty vector pPWT-eGFP (which itself is a mock in subsequent experiments) regulates the expression of enhanced green fluorescent protein (eGFP) by the elongation factor-1 ⁇ (elongation factor-1 ⁇ , EF-1 ⁇ ) promoter.
  • the recombinant expression vector pWPT-eGFP-F2A-CAR comprising the nucleic acid sequence of interest encoding CAR was obtained by ribosomal skipping sequence 2A (F2A). ) to achieve co-expression of eGFP and CAR.
  • the invention also includes viruses comprising the vectors described above.
  • the virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious virus.
  • Other viruses transducing T lymphocytes known in the art and their corresponding plasmid vectors can also be used in the present invention.
  • the virus is a lentivirus comprising the above pWPT-eGFP-F2A-CAR recombinant vector (ie containing scFv (GPC3)-CAR).
  • the present invention also encompasses a genetically modified T lymphocyte which is transduced with a nucleic acid of the present invention or which is transduced with the above-described recombinant plasmid containing the nucleic acid of the present invention, or a virus comprising the same.
  • Conventional nucleic acid transduction methods including non-viral and viral transduction methods, can be used in the present invention.
  • Non-viral based transduction methods include electroporation and transposon methods.
  • the nucleofector nuclear transfection device developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of the target gene.
  • the transduction efficiency of the transposon system based on the Sleeping Beauty system or the PiggyBac transposon is much higher than that of ordinary electroporation.
  • the nucleofector transfection apparatus has been reported in combination with the Sleeping Beauty transposition system [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OF1-10.], the method has high transduction efficiency and can achieve targeted integration of the target gene.
  • the transduction method for achieving chimeric antigen receptor gene-modified T lymphocytes is based on a transduction method of a virus such as a retrovirus or a lentivirus.
  • the method has the advantages of high transduction efficiency, stable expression of the foreign gene, and shortening the time for the T lymphocytes to reach the clinical level in vitro.
  • the transduced nucleic acid is expressed on its surface by transcription and translation.
  • the anti-GPC3 chimeric antigen receptor gene-modified T lymphocytes of the present invention have a highly specific tumor cell killing effect (also called cytotoxicity) by in vitro cytotoxicity experiments on various cultured tumor cells.
  • the nucleic acid encoding the chimeric antigen receptor protein of the present invention, the plasmid containing the nucleic acid, the virus comprising the plasmid, and the transgenic T lymphocyte transduced with the above nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.
  • the invention also relates to a kit comprising the dual targeted immune effector cells.
  • the invention also relates to kits for the preparation of the dual targeting immune effector cells of the invention. After reading the present invention, those skilled in the art will know how to prepare the kit or kit. Instructions for use of the instructions may also be included in the kit or kit.
  • Example 1 Detection of expression levels of ASGPR1 and GPC3 in various hepatoma cell lines and establishment of stably expressing hepatoma cell lines
  • the present inventors first examined the expression of ASGPR1 and GPC3 in various liver cancer cell lines.
  • mouse anti-human GPC3 monoclonal antibody (Shanghai Ruijin Biotechnology Co., Ltd.) and mouse anti-human ASGPR1 monoclonal antibody (Abcam) were added after blocking for 2 hours with 5% skim milk. °C overnight. The next day, 0.5% PBST was washed 3 times, then HRP-goat anti-mouse antibody (Shanghai Ruijin Biotechnology Co., Ltd.) was added, and the cells were incubated for 1 hour at room temperature, washed 3 times with 0.5% PBST, and then added to the substrate for reaction, exposure and development.
  • HRP-goat anti-mouse antibody (Shanghai Ruijin Biotechnology Co., Ltd.) was added, and the cells were incubated for 1 hour at room temperature, washed 3 times with 0.5% PBST, and then added to the substrate for reaction, exposure and development.
  • Flow cytometry HepG2, Huh7, Hep3B, PLC/PRF/5, MHCC-97H, SK-HEP-1, and SMMC-7721 cells with good growth status were treated with 10 mM EDTA cells.
  • centrifuge at 3000 to 4000 rpm for 5 min. Wash 2 times with 2 mL of 1% NCS PBS.
  • Mouse anti-human GPC3 monoclonal antibody (Shanghai Ruijin Biotechnology Co., Ltd.) and mouse anti-human ASGPR1 monoclonal antibody (Abcam, final concentration of 5 ⁇ g/mL, ice bath for 45 min; followed by washing with 2 mL of 1% NCS PBS) After 2 times, goat anti-mouse IgG-FITC (Shanghai Ruijin Biotechnology Co., Ltd.) was added, ice bath for 45 min; 1% NCS PBS was washed twice, and then 200-500 ⁇ L of 1% NPBS was added to resuspend the cells, and the cells were detected.
  • goat anti-mouse IgG-FITC (Shanghai Ruijin Biotechnology Co., Ltd.) was added, ice bath for 45 min; 1% NCS PBS was washed twice, and then 200-500 ⁇ L of 1% NPBS was added to resuspend the cells, and the cells were detected.
  • ASGPR1 and GPC3 were expressed in different degrees except for MHCC-97H, SK-HEP-1 and SMMC-7721 cells, which were consistent with Western-blot results.
  • Hepatoma cell line MHCC-97H is a GPC3/ASGPR1 double-negative cell.
  • the double-negative cell line MHCC-97H was selected to construct GPC3+, ASGPR1+ and GPC3+/ASGPR1+ overexpressing cell lines, and MHCC-97H-G and MHCC-97H-A were established.
  • Three cell lines of MHCC-97H-GA represent MHCC-97H cell line expressing GPC3, MHCC-97H cell line expressing ASGPR1, and MHCC-97H cell line expressing GPC3 and ASGPR1, respectively.
  • pWPT-GPC3 plasmid construction using Hep 3B cell cDNA as template, upstream primer 5'-agcttacgccccctagcgccccccgg tcgccaccatggccgggaccgtgcgcacc-3' (SEQ ID NO: 1) and downstream primer 5'-CGAGGTCGA CCTATCAGTGCACCAGGAAGAAGAAGCAC-3' (SEQ ID NO: 2)
  • the full-length ORF nucleic acid fragment of GPC3 was amplified, and the PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation at 94 ° C for 40 s, annealing at 56 ° C for 40 s, extension at 68 ° C for 50 s, for 30 cycles; then total extension at 68 ° C for 10 min.
  • the amplified product was confirmed by agarose gel electrophoresis. After double digestion with MluI and SalI, it was inserted into the lentiviral vector pWPT-eGFP which was digested with the same double restriction enzyme. The positive clone was picked and sequenced to confirm that the sequence was correct and the full-length GPC3 was obtained.
  • the coding sequence of the protein (SEQ ID NO: 4) (SEQ ID NO: 3) expresses the plasmid pWPT-GPC3 plasmid.
  • pWPT-ASGPR1 plasmid construction a plasmid containing human ASGPR1 full-length ORF as a template, upstream primer 5'-gcttacgcccctagcgctaccggtcgccaccatgaccaaggagtatcaagacc-3' (SEQ ID NO: 5) and downstream primer 5'-CGAGGTCGACCTATTAAAGGAGAGGTGGCTCCT GGCT-3' (SEQ ID NO: 6)
  • Amplification of ASGPR full-length ORF nucleic acid fragment PCR amplification conditions: pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 56 ° C, 40 s; extension: 68 ° C, 50 s, 30 The cycle was followed by a total extension of 68 ° C for 10 min.
  • the amplified product was confirmed by agarose gel electrophoresis. After double digestion with MluI and SalI, it was inserted into the pWPT-eGFP vector which was digested with the same restriction enzyme, and the positive clone was picked and identified.
  • the expression plasmid pWPT-ASGPR1 plasmid containing the coding sequence (SEQ ID NO: 7) of the full-length ASGPR protein (SEQ ID NO: 8) was obtained correctly.
  • Viral infection 1 mL of virus supernatant was taken, and a final concentration of 0.6 ⁇ g/mL polybrene was added, 5 ⁇ 10 4 cells were resuspended, 2200 rpm, RT, and centrifuged at room temperature for 30 min to suspend infection. After resuspending the cell bundle after centrifugation, the cells were suspended, and 1 mL of fresh medium was added and plated in 6-well. After the cells were almost full, the cells were collected, and Western-blot was used to detect the expression of foreign proteins in the mixed clones.
  • Example 2 Establishment of GCAR T cells (or GZ T cells), ACCR T cells (or 28BB T cells), and GZ+28BB T cells
  • the amplification of the scFv (GPC3) sequence uses the single-stranded bifunctional antibody nucleotide GPC3/CD3 as a template, and the sequence of the template is shown in SEQ ID NO: 9 of Chinese Patent Application No. 201210480326.
  • the primer pair used for amplification was the upstream primer 5'-gatgttgtgatgactcagtctc-3' (SEQ ID NO: 9) and the downstream primer 5'-gcgctggcgtcgtggttgaggagacggtgaccag-3' (SEQ ID NO: 10) for amplification of scFv (GPC3) ( SEQ ID NO: 11, which encodes SEQ ID NO: 12); the amplified strand of interest is 746 bp in size.
  • the PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 58 ° C, 40 s; extension: 68 ° C, 40 s; 30 cycles, then total extension 68 ° C, 10 min.
  • the PCR amplified bands were confirmed by agarose gel electrophoresis to match the expected fragment size.
  • the nucleic acid sequence of the GPC3 chimeric antigen receptor protein other than scFv was obtained by PCR using the sequence SEQ ID NO: 1 disclosed in Patent Application No. 201310108532.2 as a template.
  • CD8-CD3 ⁇ (Z) is templated with scFv(EFGR)-CD8-CD3 ⁇ (SEQ ID NO:1 in patent application 201310108532.2), using the upstream primer 5'-accacgacgccagcgccgcgaccac-3' (SEQ ID NO: 13) and downstream Primer 5'-gaggtcgacctagcgagggggcaggcctgcatgtgaag-3' (SEQ ID NO: 14) amplification, PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation 94 ° C 40 s, annealing 60 ° C 40 s, extension 68 ° C 40 s, 25 cycles ; After the total
  • the target bands were 549 bp, respectively, and the PCR amplified bands were confirmed by agarose gel electrophoresis to meet the expected fragment size.
  • the eGFP nucleic acid fragment carrying the F2A and CD8 signal peptides at the 3' end was amplified with the upstream primer 5'-cttacgcccctagcgctaccggtcgccacca tggtgagcaagggcgaggag-3' (SEQ ID NO: 15) and the downstream primer 5'-cggcctggcggggtggagcag-3' (SEQ ID NO: 16
  • the PCR amplification conditions were pre-den
  • fragment CD8-CD3 ⁇ amplified as in the preceding paragraph, the eGFP nucleic acid fragment (having a mass of about 50 ng) with the F2A and CD8 signal peptide at the 3' end and the equimolar scFv (GPC3) (about 50 ng) were respectively Fragment splicing and PCR, splicing conditions: pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: 68 ° C, 140 s, 5 cycles, then total extension 68 ° C, 10 min , supplemented with DNA polymerase and upstream primer 5'-cttacgccccccccccccccccgtcgccaccatggtgagc aagggcgaggag-3' (SEQ ID NO: 15) and downstream primer 5'-gaggtcgacctagc
  • the sequence containing the full-length eGFP-F2A-GPC3-CD3 ORF (SEQ ID NO: 17, encoding SEQ ID NO: 18) was obtained by amplification, and the theoretical size was 2161 bp, respectively.
  • the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the pWPT-eGFP vector was digested by MluI and SalI, and the positive clones were ligated and transformed. The positive clones were identified and sequenced to confirm that the sequence was correct to obtain pWPT-eGFP-F2A-GPC3-CD3 ⁇ .
  • the single domain antibody sequence against ASGPR is SEQ ID NO: 20, obtained by whole gene sequence synthesis (SEQ ID NO: 19, which encodes SEQ ID NO: 20).
  • the mCherry nucleic acid fragment sequence carrying the F2A and CD8 signal peptides at the 3' end was obtained by whole gene sequence synthesis.
  • the nucleic acid sequence of the ASGPR-CD28a-CD28b-CD137 chimeric antigen receptor protein other than the anti-ASGPR single domain antibody was obtained by PCR using the sequence SEQ ID NO: 3 disclosed in Patent Application No. 201310108532.2, respectively.
  • upstream primer 5'-accacgacgccagcgccgcgaccac-3' (SEQ ID NO: 13) and the downstream primer 5'GAGGTCGACCTACAGTTCACATCCTCCTTCT-3 were used as scFv(EFGR)-CD8-28BBZ (SEQ ID NO: 3 in patent application 201310108532.2) template.
  • PCR amplification conditions were pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: 68 ° C, 40 s; for 25 cycles, Then always extend 68 ° C, 10 min.
  • the PCR amplified band was confirmed by agarose gel electrophoresis to conform to the expected fragment size (477 bp).
  • Amplification revealed a full-length ORF sequence comprising mCherry-F2A-ASGPR-CD28a-CD28b-CD137 (SEQ ID NO: 23, encoding SEQ ID NO: 24) with a theoretical size of 1711 bp, respectively.
  • the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the pWPT-eGFP vector was digested by MluI and SalI, and the pWPT-eGFP vector was ligated and transformed. The positive clone was identified and sequenced to confirm that the sequence was correct to obtain pWPT-mCherry-F2A-ASGPR-CD28a-CD28b-CD137.
  • PBMC Peripheral blood mononuclear cells
  • the Quantum 007 lymphocyte culture medium was added at a density of about 1 ⁇ 10 6 /mL, and magnetic beads coated with anti-CD3 and CD28 antibodies (Invitrogen) were added at a cell:magnetic bead ratio of 1:1.
  • the culture was incubated with recombinant human IL-2 at a final concentration of 100 U/mL for 24 h.
  • CTL cells were then infected with the recombinant lentivirus constructed above as MOI ⁇ 5.
  • the infected cells were passaged every other day at a density of 5 ⁇ 10 5 /mL, and a recombinant human IL-2 having a final concentration of 100 U/mL was added to the lymphocyte culture solution.
  • Infected CTL cells were tested for expression of different chimeric antigen receptors by flow cytometry on day 8 of culture. Since eGFP was co-expressed with CAR and mCherry was co-expressed with CCR, eGFP or mCherry-positive cells were detected to express chimeric antigen receptors. Positive cells. The positive rate of virus-infected CTL cells expressing different chimeric antigen receptors using uninfected T lymphocytes as a negative control is shown in Table 1.
  • GCAR T chimeric antigen receptor T cell
  • ACCR T cell chimeric costimulatory receptor T cell
  • Infected Mock T cells, GCAR T cells, ACCR T cells, and GZ+28BB T cells were collected by centrifugation, and then lysed by adding cell lysate to extract cell proteins, and BCA kit (Thermo) was used for protein quantification.
  • the above collected samples were electrophoresed on a 12% SDS-PAGE, and 20 ⁇ g of total protein was separately loaded.
  • SDS-PAGE electrophoresis the mice were blocked with 5% skim milk for 2 hours, and then mouse anti-human CD3 ⁇ monoclonal antibody (Sigma) and mouse anti-human CD28 antibody (Abeam) were added, and overnight at 4 °C.
  • HRP-goat anti-mouse secondary antibody was added to the next day after 0.5% PBST washing, and incubated for 1 hour at room temperature. After washing with 0.5% PBST, the substrate was added for color development and exposed.
  • Figure 4A shows that only endogenous CD3 expression was detected in Mock T and ACCR T cells, while endogenous CD3 expression was detected in both GCAR T and GZ+28BB T cells. Expression of exogenously transfected CD3.
  • Figure 4B shows that only endogenous CD28 expression was detected in Mock T and GCAR T cells, while endogenous CD28 expression and exogenous transfection of CD28 were detected in both ACCR T and GZ+28BB T cells. expression. Cell lines indicating stable transfection of chimeric antigen receptors were successfully constructed.
  • Target cells HepG2 cells (expressing endogenous GPC3 and ASGPR1, purchased from ATCC), MHCC-97H, and stably transfected hepatoma cell line MHCC-97H-G (high expression GPC3), MHCC-97H-A (high expression) ASGPR1), MHCC-97H-GA (simultaneous expression of GPC3 and ASGPR1).
  • Effector cells CACS cells positive for chimeric antigen receptor expression were detected by in vitro cultured FACS as demonstrated in Example 2: GCAR T cells, ACCR T cells, and GZ+28BB T cells.
  • the effective target ratio is 3:1, and the number of target cells is 10000/well, which corresponds to effector cells according to different target-to-target ratios. Five replicate wells were set for each group, and the average of five replicate wells was taken. The detection time is 18h.
  • Each experimental group each target cell + T cells expressing different chimeric antigen receptors
  • Control group 1 maximum release of LDH from target cells
  • Control group 2 spontaneous release of LDH from target cells
  • Control group 3 Effector cells spontaneously released LDH.
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. This method is based on the colorimetric detection method and can replace the 51 Cr release method.
  • the assay quantitatively measures lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • LDH is a stable cytoplasmic enzyme that is released when cells are lysed and released in much the same way as 51 Cr is released in radioactive analysis.
  • the released LDH medium supernatant can be detected by a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan.
  • INT tetrazolium salt
  • the amount of red product produced is directly proportional to the number of cells lysed. Refer specifically to the instructions for the CytoTox 96 non-radioactive cytotoxicity test kit.
  • the cytotoxicity calculation formula is:
  • T cells were unable to express ASGPR1 - GPC3 - (MHCC-97H) (MHCC-97H cells, not expressing ASGPR1 and GPC3 genes) regardless of whether or not the chimeric antigen receptor of the present invention was expressed.
  • ASGPR1 + GPC3 - (MHCC-97H-A) (MHCC-97H-A cells, which express only ASGPR1 but not GPC3 gene) produce specific cytotoxic effects.
  • the chimeric antigen receptor-expressing GCAR T cells and GZ+28BB T cells of the present invention are directed against ASGPR1 - GPC3 + (MHCC-97H-G) cells (MHCC-97H-G cells, which express only GPC3 but not ASGPR1 gene).
  • ASGPR1 + GPC3 + MHCC-97H-GA or HepG2
  • ASGPR1 and GPC3 genes are expressed
  • the cytotoxicity of GZ+28BB double-infected T cells to MHCC-97H-GA and HepG2 hepatoma cells at 5:1 was 55% and 57%, respectively, and its cytotoxicity was 35% compared with GCAR T (35% and 36%) slightly stronger.
  • T cells transfected with a virus containing a mock plasmid did not produce cytotoxic effects on the above five cells.
  • Target cells MHCC-97H-G, MHCC-97H-A, MHCC-97H-GA cells were added to mitomycin (final concentration of 20 ⁇ g/mL) and incubated for 2 h at 37 °C. After washing twice with PBS, the residual mitomycin was removed. Trypsinize and suspend into a cell suspension. CTL cells were infected with a virus containing different chimeric antigen receptors, mixed with target cells at a cell density of 5 ⁇ 10 5 /ml, subcultured, counted, and passaged cell culture medium every other day. Additional IL-2 (final concentration 100 U/ml) was counted once a week for a total of three weeks.
  • T cells expressing different chimeric antigen receptors were co-cultured with target cells expressing different antigens, and about 6 to 20-fold amplification was performed on day 7 (see Fig. 5), compared with GZ+28BB T cells.
  • Other T cells including GCAR T, ACCR T, Mock T) have greater amplification capacity under the stimulation of double antigens.
  • Example 6 ASGPR1/GPC3 protein stimulation up-regulates the expression of BCL-XL in GCAR T, ACCR T and GZ+28BB T lymphocytes
  • the tumor growth curve shown in Figure 7 was 38 days after tumor cell inoculation.
  • the volume of transplanted tumors in the GZ+28BB T cell treatment group was significantly smaller than that in the other treatment groups (GZ+28BB T vs No T cell, inhibition rate 72%, ***P ⁇ 0.001; GZ+28BB T vs ACCR T, inhibition)
  • the rate was 70%, ***P ⁇ 0.001; GZ+28BB T vs GCAR T, inhibition rate was 64%, ***P ⁇ 0.001), however, in the implantation of ASGPR + GPC3 - and ASGPR + GPC3 - GZ+28BB T cell treatment group could not significantly inhibit tumor growth, indicating that GZ+28BB T cells can significantly inhibit tumor growth only when both antigens are present.
  • cell number / ⁇ L number of cells positive for FITC or PE ⁇ total number of beads / number of beads / 50 ⁇ L.

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Abstract

提供了一种双靶向GPC3和ASGPR1的转基因免疫效应细胞及其应用，该细胞是一种能同时识别GPC3和ASGPR1的基因修饰的免疫效应细胞，其可应用于GPC3和ASGPR1双阳性肿瘤如肝癌的治疗。

Description

双靶向GPC3和ASGPR1的转基因免疫效应细胞及其应用 技术领域
本发明涉及肿瘤免疫治疗领域，更具体地，本发明涉及双靶向GPC3和ASGPR1的转基因免疫效应细胞及其应用。
背景技术
T淋巴细胞在肿瘤免疫应答中的作用日益受到重视。基于T淋巴细胞的过继性免疫治疗在部分肿瘤中取得了一定的效果，并且该种免疫治疗方法可以克服抗体治疗的上述缺陷，但在大多数肿瘤的疗效仍不能令人满意。近年来，根据CTL对靶细胞的识别特异性依赖于T淋巴细胞受体(T Cell Receptor，TCR)的发现，将针对肿瘤细胞相关抗原的抗体的scFv与T淋巴细胞受体的CD3ζ或FcεRIγ等胞内信号激活基序融合成嵌合抗原受体(Chimeric antigen receptor，CAR)，并将其通过如慢病毒感染等方式基因修饰在T淋巴细胞表面。这种CAR T淋巴细胞能够以主要组织相容性复合物(Major Histocompatibility Complex，MHC)非限制性方式选择性地将T淋巴细胞定向到肿瘤细胞并特异性地杀伤肿瘤。CAR T淋巴细胞是肿瘤免疫治疗领域的一个新的免疫治疗策略。
嵌合抗原受体(CAR)包括胞外结合区，跨膜区和胞内信号区。通常胞外区包含能够识别肿瘤相关抗原的scFv，跨膜区采用CD8，CD28等分子的跨膜区，胞内信号区采用免疫受体酪氨酸活化基序(ITAM)CD3ζ或FcεRIγ及共刺激信号分子CD28、CD137、CD134等的胞内信号区。
胞内信号区仅包含ITAM的为第一代CAR T淋巴细胞，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-ITAM。该种CAR T可以激发抗肿瘤的细胞毒性效应，但是细胞因子分泌比较少，并且在体内不能激发持久的抗肿瘤效应[Zhang T.et al.Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways，Can Res 2007，67(22):11029–11036.]。
随后发展的第二代CAR T淋巴细胞加入了CD28或CD137(又名4-1BB)的胞内信号区，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28-ITAM或scFv-TM-CD137-ITAM。胞内信号区发生的B7/CD28或4-1BBL/CD137共刺激作用引起T淋巴细胞的持续增殖，并能够提高T淋巴细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR T在体内的存活周期和抗肿瘤效果[Dotti G.et al.CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients.J Clin Invest，2011，121(5):1822-1826.]。
近些年发展的第三代CAR T淋巴细胞，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28-CD137-ITAM或scFv-TM-CD28-CD134-ITAM，进一步提高了CAR T在 体内的存活周期和其抗肿瘤效果[Carpenito C.，et al.Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains.PNAS，2009，106(9):3360–3365.]。
尽管CAR T淋巴细胞在肿瘤免疫治疗中具有诱人的前景，但一些潜在的风险亦需要考虑。比如，由于某些/种正常组织低表达CAR所能识别的特异性抗原可能造成CAR T淋巴细胞对表达相应抗原的正常组织的损伤。如，针对肾细胞癌患者肿瘤细胞上表达的抗原碳酸酐酶IX(CAIX)是第一个用于临床的CAR T淋巴细胞过继治疗的案例，也是第一个报道含CAR细胞的脱靶效应的案例。病人在多次输入CAR T淋巴细胞后出现2-4级肝毒性。分析原因为肝胆管上皮细胞低表达CAIX，原临床试验被迫中断同时排除了病人治疗效果的任何评价[Stoter G.et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX:first clinical experience.J clin oncol，2006，24(13):e20-e22.；Ngo MC.，et al.Ex vivo gene transfer for improved adoptive immunotherapy of cancer.Human Molecular Genetics，2011，R1–R7]。另外，CAR中过多的共刺激信号会降低效应细胞激活所需的阈值，使得基因修饰的T淋巴细胞在低水平抗原或没有抗原触发的条件下也可能会被活化，导致大量细胞因子的释放以致可能引发所谓的“细胞因子风暴”。这种信号外漏(singnal leakage)会导致脱靶细胞毒性，从而产生非特异性的组织损伤。例如，在采用针对Her2的第三代CAR临床治疗一个具有肝和肺转移的晚期结肠癌患者的过程中由于正常肺组织中低表达Her2而引发所谓的“细胞因子风暴”致病人猝死[Morgan RA.，et al.Report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing Erbb2.Molecular Therapy，2010，18(4):843–851.]。因此，本领域迫切需要研究化解CAR T细胞疗效低及存在潜在风险的问题的方法，开发出更有效杀伤肿瘤细胞的方法。
磷脂酰肌醇蛋白多糖-3(Glypican-3，GPC3，又称DGSX，GTR2-2，MXR7，OCI-5，SDYS，SGB，SGBS或SGBS1)是一种细胞表面蛋白，属于硫酸乙酰肝素蛋白多糖家族。GPC3基因编码产生70-kDa左右的前体核心蛋白，该前体蛋白能够被弗林蛋白酶(furin)剪切产生40-kDa左右的可溶性的能够进入血液的氨基端(N末端)肽和30-kDa左右含有2个硫酸乙酰肝素(HS)糖链的膜结合的羧基端(C末端)肽。GPC3蛋白通过糖基磷脂酰肌醇(GPI)锚依附在细胞膜上。
GPC3高度表达于胎儿肝脏，而不表达于正常成年人的肝组织，但在肝细胞肝癌中恢复表达，与肝癌的发生发展有十分密切的关系，不仅在肝癌发生的早期检出率较高，而且随着肝癌的发展，其检出率也随之增高。而GPC3的表达在肝腺癌，胆管细胞癌，肝转移癌和12种常见实体瘤和21种非肝癌细胞系中均未检测出。此外，GPC3也在例如黑色素瘤，卵巢透明细胞癌、卵黄囊瘤、神经母细胞瘤等肿瘤中表达。
利用抗GPC3抗体进行肝癌检测和利用抗GPC3抗体的抗体依赖的(ADCC)或补体依 赖的(CDC)细胞毒性研究方案已有报道。一般用于治疗的抗体识别的是GPC3蛋白的C末端。然而，抗体治疗存在着抗体在体内血液循环中半衰期的限制，一般来说，半衰期大多在23天以内。因此，持续给药和/或增大给药剂量是肿瘤抗体治疗所要求的，这导致患者治疗成本的增加，以及某些情况下，甚至不得已终结治疗。另外，治疗性抗体作为异源蛋白，还有可能在体内产生过敏反应及针对该治疗性抗体的中和性抗抗体的风险。
本发明人的已有研究表明针对GPC3的CAR T细胞对GPC3阳性的肝癌细胞具有非常好的杀伤作用(CN201310164725.x)。但是，GPC3虽然在肝脏等大部分正常器官没有表达，但是在胃腺体(3/7[43％])、肾小管((9/17[53％])和睾丸生殖细胞中有表达。需要考虑单独靶向GPC3的CAR T细胞就有可能对这些正常组织(胃腺体，肾小管和睾丸生殖细胞)产生损伤。
去唾液酸糖蛋白受体(asialoglycoprotein receptor，ASGPR1，或称ASGR1)，又称半乳糖受体，是一种跨膜蛋白，相对分子质量约41000，由H1和H2两个结构不同的亚基组成，H1是受体的主要组分。ASGPR1的胞外结构域含有糖识别结构域(carbohydrate recognition domain，CRD)，能识别和结合半乳糖残基和N乙酰半乳糖胺残基。当CRD与特异的糖残基结合后，即发生受体介导的胞吞作用。ASGPR1的主要功能是清除外周血循环中失去末端唾液酸而暴露半乳糖残基或乙酰半乳糖胺残基的糖蛋白、脂蛋白和凋亡细胞，还介导嗜肝病毒(如乙肝病毒和丙肝病毒)与肝细胞的结合及摄取。ASGPR1主要表达于哺乳动物肝窦状隙的肝实质细胞表面，密度很高，每个细胞表面可多达500000个受体。在发生肝细胞肝癌、肝炎、肝硬化等肝脏疾病时，ASGPR1表达数量及功能有所下降。
目前现有技术中，从来没有关于ASGPR1作为免疫细胞治疗的靶点，更没有将GPC3、ASGPR1与CAR免疫效应细胞联合应用的报导。
发明内容
本发明的目的在于提供一种双靶向GPC3和ASGPR1的转基因免疫效应细胞及其应用。
在本发明的第一方面，提供一种双靶向的嵌合抗原受体(CAR)免疫效应细胞，该细胞表达如下嵌合受体：特异性识别GPC3的嵌合抗原受体；和特异性识别ASGPR1的嵌合抗原受体。
在一个优选例中，所述的特异性识别GPC3的嵌合抗原受体包括：抗GPC3的单链抗体(scfv)，T细胞刺激信号分子。
在另一优选例中，所述的T细胞刺激信号分子选自：CD3ζ或FcεRIγ；更佳地，所述的特异性识别GPC3的嵌合抗原受体包括(较佳地5’→3’)：抗GPC3的单链抗体(scfv)，CD8跨膜区，CD3ζ。
在另一优选例中，在抗GPC3的单链抗体(scfv)的5’端，还包括报告基因1(如eGFP)、F2A，藉由F2A将报告基因1与抗GPC3的单链抗体相连，藉由报告基因1呈现细胞内所述特异性识别GPC3的嵌合抗原受体的表达情况。
在另一优选例中，所述的特异性识别GPC3的嵌合抗原受体具有SEQ ID NO:17所示的氨基酸序列。
在另一优选例中，所述的特异性识别GPC3的嵌合抗原受体由病毒载体表达；较佳地为慢病毒载体，如pWPT。
在另一优选例中，所述的特异性识别ASGPR1的嵌合抗原受体包括：抗ASGPR1的单链抗体(scfv)，T细胞激活的共刺激信号分子。
在另一优选例中，所述的T细胞激活的共刺激信号分子选自：CD27，CD28，CD137，CD134，ICOS蛋白的胞内信号区，或其组合；更佳地，所述的特异性识别ASGPR1的嵌合抗原受体包括(较佳地5’→3’)：抗ASGPR1的单链抗体(scfv)，CD28分子的跨膜区(CD28a)，CD28分子的胞内信号区(CD28b)和CD137胞内信号区。
在另一优选例中，在抗ASGPR1的单链抗体(scfv)的5’端，还包括报告基因2(如mCherry)、F2A，藉由F2A将报告基因2与抗ASGPR1的单链抗体相连，藉由报告基因2呈现细胞内所述特异性识别ASGPR1的嵌合抗原受体的表达情况。
在另一优选例中，所述的特异性识别ASGPR1的嵌合抗原受体具有SEQ ID NO:24所示的氨基酸序列。
在另一优选例中，所述的特异性识别ASGPR1的嵌合抗原受体由病毒载体表达；较佳地为慢病毒载体，如pWPT。
在另一优选例中，所述的免疫效应细胞选自：T淋巴细胞，NK细胞或NKT细胞。
在本发明的另一方面，提供所述的嵌合抗原受体免疫效应细胞的用途，用于制备治疗肿瘤的药盒；所述的肿瘤是GPC3和ASGPR1双阳性肿瘤；较佳地所述的肿瘤是肝癌。
在本发明的另一方面，提供一种用于治疗肿瘤的药盒，所述的药盒中包括：所述的嵌合抗原受体免疫效应细胞；所述的肿瘤是GPC3和ASGPR1双阳性肿瘤；较佳地所述的肿瘤是肝癌。
在本发明的另一方面，提供一种用于制备所述的嵌合抗原受体免疫效应细胞的试剂盒，所述的试剂盒中包括：
(a)表达构建物a，其包括特异性识别GPC3的嵌合抗原受体的表达盒(能在免疫细胞中表达)；和
(b)表达构建物b，其包括特异性识别ASGPR1的嵌合抗原受体的表达盒(能在免疫细胞中表达)。
在一个优选例中，所述的特异性识别GPC3的嵌合抗原受体的表达盒包括表达抗GPC3的单链抗体和T细胞刺激信号分子的构建物(表达载体)；较佳地其具有SEQ ID NO: 17所示的核苷酸序列。
在另一优选例中，所述的特异性识别ASGPR1的嵌合抗原受体的表达盒包括表达抗ASGPR1的单链抗体和T细胞激活的共刺激信号分子的构建物(表达载体)；较佳地其具有SEQ ID NO: 23所示的核苷酸序列。
在另一优选例中，所述的特异性识别GPC3的嵌合抗原受体包括：抗GPC3的单链抗体，T细胞刺激信号分子；较佳地，所述的T细胞刺激信号分子选自：CD3ζ或FcεRIγ；
所述的特异性识别ASGPR1的嵌合抗原受体包括：抗ASGPR1的单链抗体，T细胞激活的共刺激信号分子；较佳地，所述的T细胞激活的共刺激信号分子选自：CD27，CD28，CD137，CD134，ICOS蛋白的胞内信号区，或其组合。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、ASGPR1和GPC3在各种肝癌细胞系的表达水平的Western-blot检测结果。
图2、ASGPR1和GPC3在各种肝癌细胞系的表达水平的流式细胞检测结果。
图3、Western-blot检测MHCC-97H-G、MHCC-97H-A、MHCC-97H-GA稳转细胞系中GPC3、ASGPR1表达情况。
图4、Western blot检测感染的T细胞目的基因的表达。
图5、表达不同嵌合抗原受体的T细胞与表达不同抗原的靶细胞共培养后，在第7天检测T细胞的增殖情况。
图6、将Mock T、GCAR T、ACCR T以及GZ+28BB T淋巴细胞与ASGPR1和GPC3表达阳性的MHCC-97H-GA细胞或两者阴性表达的MHCC-97H的细胞按效靶比1:1共孵育24h，收集T淋巴细胞，检测BCL-XL XL的表达情况。
图7、MHCC-97H-A、MHCC-97H-G、MHCC-97H-GA细胞体内抗肿瘤活性检测。
图8、过继转移基因修饰的T淋巴细胞后1周，检测外周血内T细胞的数量。
具体实施方式
本发明人经过深入的研究，首次揭示了一种能同时识别GPC3和ASGPR1的基因修饰的免疫效应细胞，其可应用于GPC3和ASGPR1双阳性肿瘤(如肝癌)的治疗。
术语“嵌合抗原受体(CAR)免疫效应细胞”是本领域公知的，其是利用基因改造技术表达肿瘤特异性嵌合抗原受体的免疫效应细胞，在体外和临床试验中显示出一定的靶向性、杀伤活性和持久性，为过继性细胞免疫治疗方法。所述的免疫效应细胞例如包括T细胞，NK细胞。
常规的制备“嵌合抗原受体免疫效应细胞”的方法是本领域技术人员已知的，包括让其表达胞外抗原(如肿瘤相关抗原)结合区；以及胞内共刺激细胞分子胞内结构域，例如CD28(较佳地包括CD28a，CD28b)，CD137，CD27，CD3ζ(较佳地为CD3ζ细 胞内域)，CD8，CD19，CD134，CD20，FcRγ中的一种或多种。通过它们与相应配体结合，激活免疫效应细胞的第二信号，增强免疫细胞的增殖能力及细胞因子的分泌功能，延长活化免疫细胞的存活时间。
本发明中，除非另外说明，所述的肿瘤是指GPC3和ASGPR1双阳性的肿瘤，例如所述的肿瘤包括：肝癌。
本发明人在研究中意外地发现，磷脂酰肌醇蛋白多糖-3(Glypican-3，GPC3，又称DGSX，GTR2-2，MXR7，OCI-5，SDYS，SGB，SGBS或SGBS1)和去唾液酸糖蛋白受体(asialoglycoprotein receptor，ASGPR1，或称ASGR1)在肝癌细胞中共表达，这是至今没有文献报导的。
基于GPC3和ASGPR1的共表达这一新发现以及本发明人长期研究经验，本发明人针对这两个蛋白设计了双靶向的免疫效应细胞，其同时含有能识别GPC3的嵌合抗原受体(Chimeric antigen receptor recognizing GPC3，GCAR)以及能识别ASGPR1的嵌合共刺激受体(Chimeric costimulatory receptor recognizing ASGPR1，ACCR)。
本发明的免疫效应细胞，其中一种嵌合受体胞外区含有一段识别GPC3的多肽组成，胞内区具有T细胞刺激信号(如CD3ζ或FcεRIγ的ITAM基序)，称为GCAR；另一个嵌合受体的胞外区含有一段识别ASGPR的多肽，胞内区含有T细胞激活的共刺激信号区段(可以是CD28，CD137或者其它共刺激信号分子的胞内信号区段)，称为ACCR。所述嵌合受体均含有一个跨膜区。所述识别GPC3或ASGPR的多肽可以是配体蛋白、小分子多肽、单链抗体、单结构域抗体或其它抗体片段。
本发明的编码嵌合受体的核酸序列可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明的编码嵌合抗原受体蛋白氨基酸序列的核酸密码子可以是简并的，即编码同一氨基酸序列的多种简并核酸序列都包含在本发明的范围之中。编码对应氨基酸的简并核酸密码子是本领域公知的。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
特异性识别人GPC3的C末端表位的单克隆抗体已被公开在例如，中国专利文献CN101186650A中外制药株式会社)，此外据文献Advances in Liver Cancer Antibody Therapies:A Focus on Glypican-3 and Mesothelin，BioDrugs.2011 October 1；25(5):275–284其他已知特异性识别C末端表位的单克隆抗体包括GC33和hGC33也分别被报道，其针对GPC3的抗原决定簇位于C端524-563位氨基酸残基，同时被报道的还包括GPC3-C02和1G12等单克隆抗体。这些被公开的单克隆抗体可以用于制备本发明的 核酸所编码的嵌合抗原受体中的单链抗体部分。其他识别GPC3的C末端表位的单克隆抗体也可以合适的方式运用于本发明。还有针对GPC3的硫酸乙酰肝素链的单克隆抗体也有报道(Gao W，Kim H，Feng M，Phung Y，Xavier CP，Rubin JS，Ho M.Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy.Hepatology.2014 Aug；60(2):576-87)。
作为本发明的优选方式，采用特异性识别人GPC3的单链抗体(scfv)。单链抗体scFv(GPC3)可以根据上述公开的GPC3单克隆抗体的序列通过基因工程方法或化学合成方法制备。本发明中使用的术语“单链抗体(scFv)片段”指的是通过如下定义的抗体片段，其是包含通过接头(linker)连接的重链可变区(VH)和轻链可变区(VL)的重组蛋白，接头使得这两个结构域相关联，以最终形成抗原结合位点。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。本发明使用的单链抗体可单独或联合使用本领域已知的常规技术，例如氨基酸缺失、插入、取代、增加、和/或重组以及/或其他修饰方法作进一步修饰。根据一种抗体的氨基酸序列在其DNA序列中引入这种修饰的方法对本领域技术人员来说是众所周知的；见例如，Sambrook，分子克隆：实验手册，Cold Spring Harbor Laboratory(1989)N.Y.。所指的修饰优选在核酸水平上进行。上述单链抗体还可以包括其衍生物。本发明中“抗体的衍生物”包括例如当通过噬菌体展示技术获得所述抗体的衍生物时，可使用如BIAcore系统中使用的表面等离子共振技术来增加与GPC3抗原表位结合的噬菌体抗体的效率(Schier，人抗体杂交瘤7(1996)，97-105；Malmborg,免疫学方法杂志183(1995)，7-13)。还包括，例如WO 89/09622中描述的嵌合抗体的产生的方法，EP-A10239400和WO90/07861中描述的人源化抗体产生的方法，WO91/10741,WO94/02602和WO96/33735中有描述的产生异种抗体例如小鼠中的人抗体的方法所产生的抗体的衍生物。
本发明的术语“特异性识别”的意思是本发明的双特异性抗体不与或基本上不与目标抗原以外的任意多肽交叉反应。其特异性的程度可以通过免疫学技术来判断，包括但不限于免疫印迹，免疫亲和层析，流式细胞分析等。在本发明中，特异性识别优选通过流式细胞技术来确定，而具体情况下特异性识别的标准可由本领域一般技术人员根据其掌握的本领域常识来判断。
嵌合受体的跨膜区可以选自CD8或CD28等蛋白的跨膜区。CD8或CD28是T淋巴细胞表面的天然标记物。人CD8蛋白是个异二聚体，由αβ或者γδ两条链组成。在本发明的一个实施方案中，跨膜区选自CD8α或者CD28的跨膜区。此外，CD8α铰链区(hinge)是一个柔性区域，因此，CD8或CD28和跨膜区加上铰链区被用于将嵌合受体CAR或CCR的靶点识别结构域和胞内信号区连接起来。
GCAR的胞内信号区可以选自CD3或FcεRIγ；ACCR的胞内信号区可以选自CD27，CD28，CD137，CD134，ICOS蛋白的胞内信号区，或其组合。CD3分子由五个亚单位组成，其中CD3ζ亚单位(又称CD3 zeta，简称Z)含有3个ITAM基序，该基序是TCR-CD3 复合体中重要的信号转导区。CD3δZ是截短的不具有ITAM基序的CD3ζ序列，在本发明实践中一般作为阴性对照的构建。FcεRIγ主要分布在肥大细胞和嗜碱性粒细胞表面，其含有一个ITAM基序，在结构、分布及功能上与CD3ζ类似。此外如前所述，CD27、CD28、CD137、CD134、ICOS是共刺激信号分子，在与各自配体结合后其胞内信号区段产生的共刺激作用引起T淋巴细胞的持续增殖，并能够提高T淋巴细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高存在相应T淋巴细胞在体内的存活周期和抗肿瘤效果。
本发明的核酸所编码的抗GPC3嵌合受体蛋白GCAR可以选自按如下方式顺序连接：scFv(GPC3)-CD8-CD3ζ；其中scFv(GPC3)代表识别GPC3的单链抗体，CD8代表CD8的跨膜区。
本发明的核酸所编码的抗ASGPR1嵌合受体蛋白ACCR可以选自按如下方式顺序连接：Fv(ASGPR)-CD28a-CD28b-CD137；其中相关嵌合抗原受体蛋白中Fv(ASGPR1)代表识别ASGPR1的单结构域抗体；CD28a代表CD28分子的跨膜区，CD28b代表CD28分子的胞内信号区，CD137代表CD137分子的胞内信号区。
本发明还包含上述编码表达于T淋巴细胞表面的嵌合抗原受体蛋白的核酸构建物或载体。在一个具体实施方案中，本发明使用的载体是一种慢病毒质粒载体pWPT-eGFP。该质粒属于第三代自灭活慢病毒载体系统，该系统共有三个质粒即编码蛋白Gag/Pol、编码Rev蛋白的包装质粒psPAX2；编码VSV-G蛋白的包膜质粒PMD2.G；及空载体pWPT-eGFP，其可以用于重组引入目的核酸序列，即编码CAR的核酸序列。空载体pPWT-eGFP(其本身为后续试验中的mock)中由延长因子-1α(elongation factor-1α，EF-1α)启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，eGFP)的表达。而包含编码CAR的目的核酸序列的重组表达载体pWPT-eGFP-F2A-CAR是通过由来自口蹄疫病毒(food-and-mouth disease virus，FMDV)的核糖体跳跃序列(ribosomal skipping sequence 2A)(简称F2A)实现eGFP与CAR的共表达的。
本发明还包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其他转导T淋巴细胞的病毒及其对应的质粒载体也可用于本发明。
在本发明的一个实施方案中，所述病毒是包含上述pWPT-eGFP-F2A-CAR重组载体(即含有scFv(GPC3)-CAR)的慢病毒。
本发明还包括一种基因修饰的T淋巴细胞，其被转导有本发明的核酸或被转导有本发明的上述包含所述含有该核酸的重组质粒，或包含该质粒的病毒。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。基于非病毒的转导方法包括电穿孔法和转座子法。近期Amaxa公司研发的nucleofector核转染仪能够直接将外源基因导入细胞核获得目的基因的高效转导。另外，基于睡美人转座子(Sleeping Beauty system)或PiggyBac转座子等转座子系统的转导效率较普通电穿孔有较大提高，将 nucleofector转染仪与睡美人转座子系统联合应用已有报道[Davies JK.，et al.Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res，2010，70(10):OF1-10.]，该方法既具有较高的转导效率又能够实现目的基因的定点整合。在本发明的一个实施方案中，实现嵌合抗原受体基因修饰的T淋巴细胞的转导方法是基于病毒如逆转录病毒或慢病毒的转导方法。该方法具有转导效率高，外源基因能够稳定表达，且可以缩短体外培养T淋巴细胞到达临床级数量的时间等优点。在该转基因T淋巴细胞表面，转导的核酸通过转录、翻译表达在其表面。通过对各种不同的培养的肿瘤细胞进行体外细胞毒实验证明，本发明的抗GPC3嵌合抗原受体基因修饰的T淋巴细胞具有高度特异性的肿瘤细胞杀伤效果(亦称细胞毒性)。因此本发明的编码嵌合抗原受体蛋白的核酸，包含该核酸的质粒，包含该质粒的病毒和转导有上述核酸，质粒或病毒的转基因T淋巴细胞可以有效地用于肿瘤的免疫治疗。
本发明还涉及包含所述双靶向的免疫效应细胞的药盒。本发明还涉及应用于制备本发明的双靶向的免疫效应细胞的试剂盒。在阅读了本发明后，本领域技术人员了解如何制备所述的药盒或试剂盒。所述的药盒或试剂盒中中还可包含说明用法的使用说明书。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、ASGPR1和GPC3在各种肝癌细胞系的表达水平的检测以及稳定表达的肝癌细胞系的建立
1、ASGPR1和GPC3在各种肝癌细胞系的表达水平的检测
本发明人首先检测了ASGPR1和GPC3在各种肝癌细胞系的表达。
Western-blot检测方法：将生长状态良好的肝癌细胞HepG2、Huh-7、Hep3B、PLC/PRF/5、MHCC-97H、SK-HEP-1以及SMMC-7721细胞分别使用D-PBS洗2次后，加T-REP组织细胞裂解液，冰上裂解1h后，12000rpm离心10min，收集上清。采用BCA法定量按照BCA测定试剂盒说明书操作，紫外分光光度计检测570nm波长的吸光度。之后用12％SDS-PAGE电泳上述收集的样品，分别上样20μg总蛋白。SDS-PAGE电泳后，使用5％的脱脂牛奶封闭2小时后分别加入小鼠抗人GPC3单克隆抗体(上海锐劲生物技术有限公司)以及小鼠抗人ASGPR1单克隆抗体(Abcam公司)，4℃过夜。次日，0.5％PBST洗涤3次后加入HRP-羊抗鼠抗体(上海锐劲生物技术有限公司)，室温孵育1小时，0.5％PBST洗涤3次后加入底物反应，曝光、显影。
Western-blot检测结果如图1所示，ASGPR1以及GPC3蛋白除了在MHCC-97H、 SK-HEP-1以及SMMC-7721细胞不表达外，在其余细胞中均有不同程度的表达。
流式细胞检测：将生长状态良好的肝癌细胞HepG2、Huh7、Hep3B、PLC/PRF/5、MHCC-97H、SK-HEP-1以及SMMC-7721细胞用10mM的EDTA消化处理细胞吹散细胞于流式管中，3000～4000rpm离心5min。2mL 1％NCS PBS洗涤2次。加入小鼠抗人GPC3单克隆抗体(上海锐劲生物技术有限公司)以及小鼠抗人ASGPR1单克隆抗体(Abcam公司，终浓度为5μg/mL，冰浴45min；之后用2mL 1％NCS PBS洗涤2次后，加入羊抗鼠IgG-FITC(上海锐劲生物技术有限公司)，冰浴45min；1％NCS PBS洗涤2次后加入200～500μL 1％NPBS重悬细胞，上机检测。
流式结果如图2所示，ASGPR1以及GPC3除了在MHCC-97H、SK-HEP-1以及SMMC-7721细胞不表达外，在其余细胞中均有不同程度的表达，与Western-blot结果一致。
2、ASGPR1和GPC3稳定表达肝癌细胞系的构建及鉴定
肝癌细胞MHCC-97H是GPC3/ASGPR1双阴性细胞，选择双阴性的细胞系MHCC-97H来构建GPC3+，ASGPR1+及GPC3+/ASGPR1+的过表达细胞系，建立MHCC-97H-G、MHCC-97H-A、MHCC-97H-GA三个细胞系，分别代表表达GPC3的MHCC-97H细胞系、表达ASGPR1的MHCC-97H细胞系、同时表达GPC3和ASGPR1的MHCC-97H细胞系。
pWPT-GPC3质粒构建：以Hep 3B细胞cDNA为模板，以上游引物5’-agcttacgcgtcctagcgctaccgg tcgccaccatggccgggaccgtgcgcacc-3’(SEQ ID NO:1)和下游引物5’-CGAGGTCGA CCTATCAGTGCACCAGGAAGAAGAAGCAC-3’(SEQ ID NO:2)扩增获得GPC3全长ORF核酸片段，PCR扩增条件为预变性：94℃，4min；变性94℃ 40s，退火56℃ 40s，延伸68℃ 50s，进行30个循环；然后总延伸68℃ 10min。扩增产物经琼脂糖电泳确认，经过MluI和SalI双酶切后插入到经同样双酶切的慢病毒载体pWPT-eGFP连接转化，挑取阳性克隆鉴定并测序确认序列正确即获得含有全长GPC3蛋白(SEQ ID NO:4)的编码序列(SEQ ID NO:3)表达质粒pWPT-GPC3质粒。
pWPT-ASGPR1质粒构建：以含有人的ASGPR1全长ORF的质粒为模板，以上游引物5’-gcttacgcgtcctagcgctaccggtcgccaccatgaccaaggagtatcaagacc-3’(SEQ ID NO:5)和下游引物5’-CGAGGTCGACCTATTAAAGGAGAGGTGGCTCCT GGCT-3’(SEQ ID NO:6)扩增ASGPR全长ORF核酸片段，PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：56℃，40s；延伸：68℃，50s，进行30个循环，然后总延伸68℃，10min，扩增产物经琼脂糖电泳确认，经过MluI和SalI双酶切后插入到经同样酶切的pWPT-eGFP载体连接转化，挑取阳性克隆鉴定并测序确认序列正确即获得含有全长ASGPR蛋白(SEQ ID NO:8)的编码序列(SEQ ID NO:7)的表达质粒pWPT-ASGPR1质粒。
GPC3和ASGPR1慢病毒包装：
(1)以6×106的密度接种培养至第6～10代的293T细胞于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染，培养基为含10％胎牛血清(PAA)的DMEM。
(2)A液配制：分别将目的基因质粒pWPT-eGFP(即为Mock)、pWPT-GPC3以及pWPT-ASGPR1各10μg，分别与包装质粒pPAX2：7.5μg和包膜质粒pMD2.G：3μg，将质粒溶解于800μl的无血清DMEM培养液中，轻轻混匀。
(3)B液配制：将60μg PEI(1μg/μl)溶解于800μl的无血清DMEM培养液中，轻轻混匀，室温孵育5min。
(4)将A液加入B液中轻轻混合，室温下孵育20min。然后将转染复合物1.6ml滴加入10cm培养皿中。
(5)4-5h小时后，用2％FBS的DMEM培基给转染的293T细胞换液。
(6)37℃孵育72h，收集病毒液上清。
病毒感染：取1mL病毒上清，加入终浓度为0.6μg/mL polybrene，重悬5×104细胞，2200rpm，RT，室温离心30min，悬浮感染。离心结束后重悬细胞束后，细胞悬起，补加1mL新鲜培基，铺于6-well中。待细胞基本长满，收集细胞，Western-blot检测混合克隆中细胞中外源蛋白的表达情况。
结果如图3所示，在MHCC-97H-G、MHCC-97H-A、MHCC-97H-GA三个稳转细胞系中分别检测到GPC3、ASGPR1的表达以及GPC3和ASGPR1蛋白的共表达。表明ASGPR1和GPC3稳定转染的肝癌细胞系构建成功。
实施例2、GCAR T细胞(或称GZ T细胞)，ACCR T细胞(或称28BB T细胞)以及GZ+28BB T细胞的建立
1、pWPT-eGFP-F2A-GPC3-CD3ζ构建
scFv(GPC3)序列的扩增以单链双功能抗体核苷酸GPC3/CD3为模板，模板的序列参见中国专利申请201210480326.x中的SEQ ID NO:9。扩增所采用的引物对为上游引物5’-gatgttgtgatgactcagtctc-3’(SEQ ID NO:9) 和下游引物5’-gcgctggcgtcgtggttgaggagacggtgaccag-3’(SEQ ID NO:10)用于扩增scFv(GPC3)(SEQ ID NO:11，其编码SEQ ID NO:12)；目的扩增条带大小为746bp。PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：58℃，40s；延伸：68℃，40s；进行30个循环，然后总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。
GPC3嵌合抗原受体蛋白的除scFv(GPC3)外其他部分的核酸序列分别以专利申请号为201310108532.2中公开的序列SEQ ID NO:1为模板通过PCR方式获得。具体地，CD8-CD3ζ(Z)以scFv(EFGR)-CD8-CD3ζ(专利申请201310108532.2中SEQ ID NO:1)模板，采用上游引物5’-accacgacgccagcgccgcgaccac-3’(SEQ ID NO:13)和下游引物5’-gaggtcgacctagcgagggggcagggcctgcatgtgaag-3’(SEQ ID NO:14)扩增，PCR扩增条件为预变性：94℃，4min；变性94℃ 40s，退火60℃ 40s，延伸68℃ 40s，进行25个循环；然 后总延伸68℃ 10min。目的条带分别为549bp，PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小。3’端带有F2A及CD8信号肽的eGFP核酸片段扩增以上游引物5’-cttacgcgtcctagcgctaccggtcgccacca tggtgagcaagggcgaggag-3’(SEQ ID NO:15)和下游引物5’-cggcctggcggcgtggagcag-3’(SEQ ID NO:16)扩增慢病毒载体中3’端带有F2A及CD8信号肽的eGFP核酸片段，以专利申请201310108532.2中公开的以scFv(EGFR)-CD8-CD3ζ(专利申请201310108532.2中SEQ ID NO:1)模板为模板，PCR扩增条件为预变性：94℃ 4min；变性94℃ 40s，退火56℃ 40s，延伸68℃ 50s，进行25个循环；然后总延伸68℃，10min，理论大小为883bp，扩增产物经琼脂糖电泳确认与理论大小一致。
分别将如前述段落中扩增获得的片段CD8-CD3ζ，3’端带有F2A及CD8信号肽的eGFP核酸片段(质量约为50ng)及等摩尔的scFv(GPC3)(质量约为50ng)三片段拼接并PCR，拼接条件为：预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行5个循环，然后总延伸68℃，10min，补充DNA聚合酶及上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggtgagc aagggcgaggag-3’(SEQ ID NO:15)和下游引物5’-gaggtcgacctagcgaggg ggcagggcctgcatg-3’(SEQ ID NO:14)后PCR扩增25个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行20个循环，然后总延伸68℃，10min。扩增获得包含全长eGFP-F2A-GPC3-CD3ζORF(SEQ ID NO:17，编码SEQ ID NO:18)序列，理论大小分别为2161bp，扩增产物经琼脂糖电泳确认与理论大小一致。经过MluI和SalI双酶切经同样酶切的pWPT-eGFP载体连接转化，挑取阳性克隆鉴定并测序确认序列正确即获得pWPT-eGFP-F2A-GPC3-CD3ζ。
2、pWPT-mCherry-F2A-ASGPR-CD28a-CD28b-CD137质粒构建
抗ASGPR的单结构域抗体序列如SEQ ID NO:20，通过全基因序列合成获得(SEQ ID NO:19，其编码SEQ ID NO:20)。3’端带有F2A及CD8信号肽的mCherry核酸片段序列通过全基因序列合成获得。ASGPR-CD28a-CD28b-CD137嵌合抗原受体蛋白的除抗ASGPR单结构域抗体外其他部分的核酸序列分别以专利申请号为201310108532.2中公开的序列SEQ ID NO:3为模板通过PCR方式获得。具体地，以scFv(EFGR)-CD8-28BBZ(专利申请201310108532.2中SEQ ID NO:3)模板，采用上游引物5’-accacgacgccagcgccgcgaccac-3’(SEQ ID NO:13)和下游引物5’GAGGTCGACCTACAGTTCACATCCTCCTTCT-3’(SEQ ID NO:21)扩增，PCR扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，40s；进行25个循环，然后总延伸68℃，10min。PCR扩增条带通过琼脂糖凝胶电泳确认符合预计的片段大小(477bp)。
分别将如前述段落中扩增获得的片段：3’端带有F2A及CD8信号肽的mCherry核 酸片段及等摩尔的抗ASGPR的单结构域抗体，以及CD28a-CD28b-CD137三片段拼接并PCR，拼接条件为：预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行5个循环，然后总延伸68℃，10min，补充DNA聚合酶及上游引物5’-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3’(SEQ ID NO:15)和下游引物5’-GAGGTCGACCTACAGTTCACATCCTCCT-3’(SEQ ID NO:22)后PCR扩增25个循环，扩增条件为预变性：94℃，4min；变性：94℃，40s；退火：60℃，40s；延伸：68℃，140s，进行25个循环，然后总延伸68℃，10min。扩增获得包含mCherry-F2A-ASGPR-CD28a-CD28b-CD137(SEQ ID NO:23，其编码SEQ ID NO:24)全长ORF序列，理论大小分别为1711bp。扩增产物经琼脂糖电泳确认与理论大小一致。经过MluI和SalI双酶切经同样酶切的pWPT-eGFP载体连接转化，挑取阳性克隆鉴定并测序确认序列正确即获得pWPT-mCherry-F2A- ASGPR-CD28a-CD28b-CD137。
GCAR和ACCR慢病毒包装：
(1)以6×106的密度接种培养至第6～10代的293T细胞于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染，培养基为含10％胎牛血清(PAA)的DMEM。
(2)A液配制：分别将目的基因质粒pWPT-eGFP(即为Mock)、pWPT--eGFP-F2A-GPC3-CD3ζ以及pWPT-mCherry-F2A-ASGPR-CD28a-CD28b-CD137各10μg，分别与包装质粒pPAX2：7.5μg和包膜质粒pMD2.G：3μg，将质粒溶解于800μl的无血清DMEM培养液中，轻轻混匀。
(3)B液配制：将60μg PEI(1μg/μl)溶解于800μl的无血清DMEM培养液中，轻轻混匀，室温孵育5min。
(4)将A液加入B液中轻轻混合，室温下孵育20min。然后将转染复合物1.6ml滴加入10cm培养皿中。
(5)4-5h小时后，用2％FBS的DMEM培基给转染的293T细胞换液。
(6)37℃孵育72h，收集病毒液上清。
3、重组慢病毒感染CTL细胞
外周血单核细胞(PBMC)用Ficoll(来自Biochrom)密度梯度离心方法，按照标准步骤从健康人供主的血液中分离。离心后，用1×磷酸盐缓冲液(PBS)洗涤细胞，然后重悬于RPMI 1640完全培养基(Gibco)，将分离好的外周血单个核细胞通过CTL细胞磁珠(Stem Cell Technologies)负性分选方法获得CTL，分选后的CTL细胞进行流式细胞检测CTL细胞的纯度，以CTL细胞的阳性率≥95％为宜进行下一步操作。以约1×106/mL密度加入Quantum 007淋巴细胞培养基液(PAA公司)培养并以细胞：磁珠比例为1:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen公司)和终浓度100U/mL的重组人IL-2刺激培养24h。然后以MOI≈5用上述构建的重组慢病毒感染CTL细胞。感染后的细胞每隔一天采用5×105/mL的密度进行传代，同时在淋巴细胞培养液中补加终浓度100U/mL的 重组人IL-2。
4、流式细胞分析T细胞感染的阳性率
感染的CTL细胞在培养第8天时通过流式细胞检测不同嵌合抗原受体表达，由于eGFP与CAR共表达以及mCherry与CCR共表达，检测eGFP或mCherry阳性的细胞即为表达嵌合抗原受体的阳性细胞。以未感染的T淋巴细胞作为阴性对照，表达不同嵌合抗原受体的病毒感染CTL细胞其阳性率如表1所示。该阳性率结果表明，通过慢病毒感染的方法能够获得一定阳性率的能识别GPC3的嵌合抗原受体T细胞(GCAR T)，能识别ASGPR1的嵌合共刺激受体T细胞(ACCR T细胞)以及GZ+28BB T细胞。
表1
转染有下列CAR的CTL细胞 CTL细胞感染阳性率
Mock(空载体对照) 66％
GCAR T 61％
ACCR T 57％
GZ+28BB T 30％
5、Western blot检测感染的T细胞目的基因的表达
将感染的Mock T细胞，GCAR T细胞，ACCR T细胞以及GZ+28BB T细胞离心收集后，加入细胞裂解液进行裂解，抽提细胞蛋白，BCA试剂盒(Thermo)进行蛋白定量。采用12％SDS-PAGE电泳上述收集的样品，分别上样20μg的总蛋白。SDS-PAGE电泳后，使用5％的脱脂牛奶封闭2小时后加入小鼠抗人CD3ζ单克隆抗体(Sigma)以及小鼠抗人CD28抗体(Abcam)，4℃过夜。次日0.5％PBST洗涤后加入HRP-羊抗鼠二抗，室温孵育1小时，0.5％PBST洗涤后加入底物显色，曝光。
结果如图4所示，图4A显示在Mock T以及ACCR T细胞中只检测到内源性CD3的表达，而在GCAR T以及GZ+28BB T细胞中则同时检测到内源性CD3的表达和外源转染CD3的表达。图4B则显示了在Mock T以及GCAR T细胞中只检测到内源性CD28的表达，而在ACCR T以及GZ+28BB T细胞中均同时检测到内源性CD28的表达以及外源转染CD28的表达。表明稳定转染嵌合抗原受体的细胞系均构建成功。
实施例3、体外细胞毒性实验
靶细胞：使用HepG2细胞(表达内源性GPC3和ASGPR1，购自ATCC)、MHCC-97H以及稳定转染的肝癌细胞系MHCC-97H-G(高表达GPC3)、MHCC-97H-A(高表达ASGPR1)、MHCC-97H-GA(同时表达GPC3和ASGPR1)。
效应细胞：如实施例2所验证的体外培养的FACS检测嵌合抗原受体表达阳性的CTL细胞：GCAR T细胞，ACCR T细胞以及GZ+28BB T细胞。
效靶比为3:1，靶细胞数量为10000/孔，根据不同效靶比对应效应细胞。各组均设5个复孔，取5个复孔的平均值。检测时间为第18h。
其中各实验组和各对照组如下：
各实验组：各靶细胞+表达不同嵌合抗原受体的T细胞，
对照组1：靶细胞最大释放LDH；
对照组2：靶细胞自发释放LDH；
对照组3：效应细胞自发释放LDH。
检测方法：采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。该方法是基于比色法的检测方法，可替代51Cr释放法。
Figure PCTCN2015095938-appb-000001
检测定量地测量乳酸脱氢酶(LDH)。LDH是一种稳定的胞质酶，在细胞裂解时会释放出来，其释放方式与51Cr在放射性分析中的释放方式基本相同。释放出的LDH培养基上清中，可通过30分钟偶联的酶反应来检测，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
细胞毒性计算公式为：
Figure PCTCN2015095938-appb-000002
实验结果如表2所示，T细胞不管是否表达本发明所述的嵌合抗原受体，均无法对ASGPR1-GPC3-(MHCC-97H)(MHCC-97H细胞，不表达ASGPR1以及GPC3基因)和ASGPR1+GPC3-(MHCC-97H-A)(MHCC-97H-A细胞，只表达ASGPR1而不表达GPC3基因)肝癌细胞产生特异性细胞毒性作用。但是本发明的表达嵌合抗原受体的GCAR T细胞和GZ+28BB T细胞对ASGPR1-GPC3+(MHCC-97H-G)细胞(MHCC-97H-G细胞，只表达GPC3而不表达ASGPR1基因)和ASGPR1+GPC3+(MHCC-97H-GA或HepG2)(MHCC-97H-GA或HepG2细胞，且ASGPR1以及GPC3基因均表达)均能表现出较强特异性细胞毒性作用，并呈现效靶比梯度依赖性即效靶比越高细胞毒性作用越强。GZ+28BB双感染的T细胞在效靶比3：1时对MHCC-97H-GA以及HepG2肝癌细胞的细胞毒性分别为55％和57％，其细胞毒性作用比GCAR T(分别为35％和36％)略强。
相比较而言，被含有mock质粒(不携带GCAR/ACCR的空质粒载体)的病毒转染的作为空白对照的T细胞对上述五种细胞均未产生细胞毒性作用。
表2
Figure PCTCN2015095938-appb-000003
Figure PCTCN2015095938-appb-000004
实施例5、体外细胞扩增实验
将靶细胞MHCC-97H-G，MHCC-97H-A，MHCC-97H-GA细胞加入丝裂霉素(20μg/mL的终浓度)，37℃孵育2h。然后用PBS洗两遍后，除去残余的裂霉素。胰酶处理，悬起成细胞悬液。将CTL细胞在分别感染包装有不同嵌合抗原受体的病毒后，以细胞密度为5×105/ml与靶细胞1:1混合，隔天传代培养、计数、并对传代的细胞培养液补加IL-2(终浓度为100U/ml)，每周计数一次，共计数三周。结果如下：表达不同嵌合抗原受体的T细胞与表达不同抗原的靶细胞共培养后，在第7天约有6～20倍的扩增(见图5)，GZ+28BB T细胞相比其他的T细胞(包括GCAR T、ACCR T，Mock T)，在双抗原的刺激下具有更大的扩增能力。
实施例6、ASGPR1/GPC3蛋白刺激可上调GCAR T、ACCR T以及GZ+28BB T淋巴细胞BCL-XL的表达
将Mock T、GCAR T、ACCR T以及GZ+28BB T淋巴细胞与ASGPR1和GPC3表达阳性的MHCC-97H-GA细胞或两者阴性表达的MHCC-97H的细胞按效靶比1:1共孵育24h，收集T淋巴细胞，检测BCL-XL XL的表达情况，结果如图6，在ASGPR1和GPC3表达阳性的MHCC-97H-GA细胞刺激下，与其他转染的T淋巴细胞相比，GZ+28BB T细胞的BCL-XL的表达水平出现上调，说明在CAR和CCR信号区同时作用的情况下可以上调细胞内BCL-XL的表达，该部分结果进一步提示在双抗原(ASGPR1/GPC3)刺激的作用下，GZ+28BB双感染的T淋巴细胞可能在体内能够更好的存活。
实施例7、体内抗肿瘤活性
(1)接种肿瘤：收集处于对数生长期并生长良好的MHCC-97H-A、MHCC-97H-G、MHCC-97H-GA细胞，每只小鼠接种细胞数为3×106。
(2)分组：每个种植瘤模型中，分别将6-8周龄NOD/SCID小鼠随机分为4组，每组6只，实验分组为：No T cell对照组，ACCR T治疗组，GCAR T治疗组，以及GZ+28BB T治疗组。
(3)过继转移T细胞：待小鼠肿瘤体积约200mm3时，腹腔注射环磷酰胺(200mg/kg)，次日，于尾静脉注射6×106/只基因修饰的T细胞(效靶比为2:1)或仅生理盐水。
(4)测量肿瘤体积，观察基因修饰的T细胞对皮下移植瘤的治疗效果。
结果显示，双转染的GZ+28BB T基因修饰的T淋巴细胞显著抑制表达双抗原的 MHCC-97H-GA细胞移植瘤的生长，图7所示肿瘤生长曲线，在肿瘤细胞接种后第38天，GZ+28BB T细胞治疗组小鼠移植瘤的体积明显小于其他治疗组(GZ+28BB T vs No T cell，抑制率72％，***P<0.001；GZ+28BB T vs ACCR T，抑制率70％，***P<0.001；GZ+28BB T vs GCAR T，抑制率64％，***P<0.001)，然而，无论在ASGPR+GPC3-以及ASGPR+GPC3-的种植瘤中，GZ+28BB T细胞治疗组均不能明显抑制肿瘤生长，表明GZ+28BB T细胞只有在双抗原同时存在的情况下才能显著抑制肿瘤生长。
实施例8、体内细胞扩增实验
过继转移基因修饰的T淋巴细胞后1周，检测外周血内T细胞的数量。
(1)采用肝素抗凝管从小鼠眼眶采血≥50μL；
(2)采用防倒吸移液器取20μL CD4-FITC/CD8-PE/CD3-PerCP抗体加入BD TruCount tubes(含有已知数量的珠子)，切忌Tip接触到珠子；吸取50μL充分混匀的抗凝血，盖上管帽与抗体轻柔充分混匀；室温孵育15min(20-25℃)；
(3)加450μL 1×BD FACS裂解缓冲液，盖上管帽与抗体轻柔充分混匀；室温孵育15min(20-25℃)；
(4)上机检测，首先分别对PerCP阳性的细胞进行圈门及beads圈门，进而分别由FITC和PE分析阳性细胞，
(5)计数二者阳性细胞数
计算公式：细胞数/μL＝FITC或PE阳性的细胞数×总的beads数目/beads数目/50μL。
结果如图8所示，在接种MHCC-97H-GA细胞的种植瘤模型中，GZ+28BB T细胞治疗组小鼠体内的T细胞数量极其显著地高于其他治疗组，提示GZ+28BB T淋巴细胞在体内能够更好地存活。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种双靶向的嵌合抗原受体免疫效应细胞，其特征在于，该细胞表达如下嵌合受体：
    特异性识别GPC3的嵌合抗原受体；和
    特异性识别ASGPR1的嵌合抗原受体。
  2. 如权利要求1所述的嵌合抗原受体免疫效应细胞，其特征在于，所述的特异性识别GPC3的嵌合抗原受体包括：抗GPC3的单链抗体，T细胞刺激信号分子。
  3. 如权利要求2所述的嵌合抗原受体免疫效应细胞，其特征在于，所述的T细胞刺激信号分子选自：CD3ζ或FcεRIγ；更佳地，所述的特异性识别GPC3的嵌合抗原受体包括：抗GPC3的单链抗体，CD8跨膜区，CD3ζ。
  4. 如权利要求1所述的嵌合抗原受体免疫效应细胞，其特征在于，所述的特异性识别ASGPR1的嵌合抗原受体包括：抗ASGPR1的单链抗体，T细胞激活的共刺激信号分子。
  5. 如权利要求4所述的嵌合抗原受体免疫效应细胞，其特征在于，所述的T细胞激活的共刺激信号分子选自：CD27，CD28，CD137，CD134，ICOS蛋白的胞内信号区，或其组合；更佳地，所述的特异性识别ASGPR1的嵌合抗原受体包括：抗ASGPR1的单链抗体，CD28分子的跨膜区，CD28分子的胞内信号区和CD137胞内信号区。
  6. 如权利要求1所述的嵌合抗原受体免疫效应细胞，其特征在于，所述的免疫效应细胞选自：T淋巴细胞，NK细胞或NKT细胞。
  7. 权利要求1-6任一所述的嵌合抗原受体免疫效应细胞的用途，用于制备治疗肿瘤的药盒；所述的肿瘤是GPC3和ASGPR1双阳性肿瘤；较佳地所述的肿瘤是肝癌。
  8. 一种用于治疗肿瘤的药盒，其特征在于，所述的药盒中包括：权利要求1-6任一所述的嵌合抗原受体免疫效应细胞；
    所述的肿瘤是GPC3和ASGPR1双阳性肿瘤；较佳地所述的肿瘤是肝癌。
  9. 一种用于制备权利要求1-6任一所述的嵌合抗原受体免疫效应细胞的试剂盒，其特征在于，所述的试剂盒中包括：
    (a)表达构建物a，其包括特异性识别GPC3的嵌合抗原受体的表达盒；和
    (b)表达构建物b，其包括特异性识别ASGPR1的嵌合抗原受体的表达盒。
  10. 如权利要求9所述的试剂盒，其特征在于，所述的特异性识别GPC3的嵌合抗原受体包括：抗GPC3的单链抗体，T细胞刺激信号分子；较佳地，所述的T细胞刺激信号分子选自：CD3ζ或FcεRIγ；
    所述的特异性识别ASGPR1的嵌合抗原受体包括：抗ASGPR1的单链抗体，T细胞激活的共刺激信号分子；较佳地，所述的T细胞激活的共刺激信号分子选自：CD27，CD28，CD137，CD134，ICOS蛋白的胞内信号区，或其组合。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020072546A2 (en) 2018-10-01 2020-04-09 Adicet Bio, Inc. COMPOSITIONS AND METHODS REGARDING ENGINEERED AND NON-ENGINEERED γδ -T CELLS FOR TREATMENT OF SOLID TUMORS
CN112143700A (zh) * 2019-06-26 2020-12-29 上海细胞治疗集团有限公司 制备过表达外源基因的免疫效应细胞的方法
WO2022028623A1 (zh) 2020-08-07 2022-02-10 佧珐药业有限公司 工程化改造的细胞以及工程化改造细胞的方法
WO2022214089A1 (zh) 2021-04-08 2022-10-13 克莱格医学有限公司 细胞免疫治疗的应用
WO2023274303A1 (zh) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 调控细胞生理活动的嵌合多肽
WO2023091909A1 (en) 2021-11-16 2023-05-25 Sotio Biotech Inc. Treatment of myxoid/round cell liposarcoma patients
EP4442322A1 (en) * 2023-04-06 2024-10-09 Medizinische Hochschule Hannover Liver-specific car for use against immunologic rejection

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104140974B (zh) * 2013-05-08 2017-09-29 科济生物医药（上海）有限公司 编码gpc‑3嵌合抗原受体蛋白的核酸及表达gpc‑3嵌合抗原受体蛋白的t淋巴细胞
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CN110123837A (zh) 2018-02-02 2019-08-16 科济生物医药（上海）有限公司 细胞免疫治疗的组合
EP3778649A4 (en) 2018-03-09 2022-05-04 CRAGE medical Co., Limited METHOD AND COMPOSITION FOR TREATMENT OF TUMOR
CN112074600A (zh) 2018-05-03 2020-12-11 科济生物医药（上海）有限公司 免疫效应细胞及其应用
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CN110577605A (zh) * 2018-06-11 2019-12-17 浙江启新生物技术有限公司 一种靶向多发性骨髓瘤多种抗原的嵌合抗原受体t（car-t）细胞的构建方法及其应用
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AU2019310855A1 (en) 2018-07-24 2021-03-11 Crage Medical Co., Limited Method for treating tumor using immune effector cell
EP3892333A4 (en) 2018-12-07 2022-11-30 CRAGE medical Co., Limited ANTI-TUMOR POLYIMMUNOTHERAPY
US20220056408A1 (en) * 2018-12-13 2022-02-24 Carsgen Therapeutics Co., Ltd. Immune effector cell targeting gpc3 and application thereof
JP2022524906A (ja) 2019-01-07 2022-05-11 クレージュ メディカル カンパニー，リミテッド 細胞免疫療法の組み合わせ
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GB201912515D0 (en) * 2019-08-30 2019-10-16 King S College London Engineered regulatory T cell
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CN111337681A (zh) * 2020-03-20 2020-06-26 上海东方肝胆外科医院 评估肝细胞癌预后的标志物、试剂盒及方法
CN114632089B (zh) * 2020-12-16 2024-08-13 北京瑞博开拓医药科技有限公司 含酰基糖胺基团化合物在制备治疗新型冠状病毒肺炎药物中的应用及疾病的治疗方法
CN118001381A (zh) * 2024-01-24 2024-05-10 首都医科大学附属北京地坛医院 一种药物组合物及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102803955A (zh) * 2009-05-21 2012-11-28 系统生物学研究所 肝损伤的新标志物
CN103833852A (zh) * 2012-11-23 2014-06-04 上海市肿瘤研究所 针对磷脂酰肌醇蛋白多糖-3和t细胞抗原的双特异性抗体
CN104140974A (zh) * 2013-05-08 2014-11-12 上海益杰生物技术有限公司 编码gpc-3嵌合抗原受体蛋白的核酸及表达gpc-3嵌合抗原受体蛋白的t淋巴细胞

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101302495A (zh) * 2008-07-03 2008-11-12 中国人民解放军第二军医大学 一种播散肝癌细胞的分离和鉴定方法
WO2011072290A2 (en) * 2009-12-11 2011-06-16 The Regents Of The University Of Michigan Targeted dendrimer-drug conjugates
WO2013126726A1 (en) * 2012-02-22 2013-08-29 The Trustees Of The University Of Pennsylvania Double transgenic t cells comprising a car and a tcr and their methods of use
US20130280220A1 (en) * 2012-04-20 2013-10-24 Nabil Ahmed Chimeric antigen receptor for bispecific activation and targeting of t lymphocytes
CN107058354A (zh) * 2013-04-01 2017-08-18 科济生物医药（上海）有限公司 编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的t淋巴细胞

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102803955A (zh) * 2009-05-21 2012-11-28 系统生物学研究所 肝损伤的新标志物
CN103833852A (zh) * 2012-11-23 2014-06-04 上海市肿瘤研究所 针对磷脂酰肌醇蛋白多糖-3和t细胞抗原的双特异性抗体
CN104140974A (zh) * 2013-05-08 2014-11-12 上海益杰生物技术有限公司 编码gpc-3嵌合抗原受体蛋白的核酸及表达gpc-3嵌合抗原受体蛋白的t淋巴细胞

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP3228702A4 *
WANG, WEI ET AL.: "Recent Development of Liver Targeted Drug Delivery System Based on Nanotechnology", POLYMER BULLETIN, no. 1, 31 January 2013 (2013-01-31), pages 137 - 154, ISSN: 1003-3726 *

Cited By (8)

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Publication number Priority date Publication date Assignee Title
WO2020072546A2 (en) 2018-10-01 2020-04-09 Adicet Bio, Inc. COMPOSITIONS AND METHODS REGARDING ENGINEERED AND NON-ENGINEERED γδ -T CELLS FOR TREATMENT OF SOLID TUMORS
CN112143700A (zh) * 2019-06-26 2020-12-29 上海细胞治疗集团有限公司 制备过表达外源基因的免疫效应细胞的方法
WO2022028623A1 (zh) 2020-08-07 2022-02-10 佧珐药业有限公司 工程化改造的细胞以及工程化改造细胞的方法
WO2022214089A1 (zh) 2021-04-08 2022-10-13 克莱格医学有限公司 细胞免疫治疗的应用
WO2023274303A1 (zh) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 调控细胞生理活动的嵌合多肽
WO2023091909A1 (en) 2021-11-16 2023-05-25 Sotio Biotech Inc. Treatment of myxoid/round cell liposarcoma patients
EP4442322A1 (en) * 2023-04-06 2024-10-09 Medizinische Hochschule Hannover Liver-specific car for use against immunologic rejection
WO2024208756A1 (en) * 2023-04-06 2024-10-10 Medizinische Hochschule Hannover Liver-specific car for use against immunologic rejection

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