WO2014180306A1 - 编码gpc3嵌合抗原受体蛋白的核酸及表达gpc3嵌合抗原受体蛋白的t淋巴细胞 - Google Patents

编码gpc3嵌合抗原受体蛋白的核酸及表达gpc3嵌合抗原受体蛋白的t淋巴细胞 Download PDF

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WO2014180306A1
WO2014180306A1 PCT/CN2014/076913 CN2014076913W WO2014180306A1 WO 2014180306 A1 WO2014180306 A1 WO 2014180306A1 CN 2014076913 W CN2014076913 W CN 2014076913W WO 2014180306 A1 WO2014180306 A1 WO 2014180306A1
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gpc3
chimeric antigen
antigen receptor
nucleic acid
cells
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PCT/CN2014/076913
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English (en)
French (fr)
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李宗海
高慧萍
蒋华
石必枝
王华茂
李克桑
王红阳
杨胜利
顾健人
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上海益杰生物技术有限公司
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Priority to KR1020157034790A priority Critical patent/KR101857229B1/ko
Priority to EP14794936.6A priority patent/EP2995682A4/en
Priority to US14/889,778 priority patent/US10731127B2/en
Priority to JP2016512212A priority patent/JP6426713B2/ja
Publication of WO2014180306A1 publication Critical patent/WO2014180306A1/zh

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Definitions

  • the present invention relates to the field of tumor cell therapy and, more particularly, to the field of therapeutic treatment of transgenic T lymphocytes which specifically express epithelial-derived tumors of GPC3.
  • Glypican-3 Glypican-3, GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS: SGB, SGBS or SGBS1
  • Glypican-3 GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS: SGB, SGBS or SGBS1
  • Glypican-3 GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS: SGB, SGBS or SGBS1
  • SDYS SGB
  • SGBS SGBS1
  • the GPC3 gene encodes a 70-kDa precursor core protein that can be cleaved by furin to produce a soluble 40-kDa amino-terminal (N-terminal) peptide that can enter the bloodstream 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 tissues 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, hepatic metastasis, 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, and neuroblastoma. Considering that GPC3 is specifically expressed in hepatocellular carcinoma, melanoma and other tumors, it is considered to be a candidate for tumor immunotherapy.
  • Antibody-dependent (ADCC) or complement dependent (CDC) cytotoxicity studies using anti-GPC3 antibodies for liver cancer detection and anti-GPC3 antibodies have been reported.
  • Antibodies commonly used for therapeutic recognition 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, continuous administration and/or increased dosing is required for tumor antibody therapy, which results in an increased cost of treatment for the patient and, in some cases, even termination of treatment.
  • therapeutic antibodies, as heterologous proteins may also have an allergic reaction in the body and a risk of neutralizing anti-antibodies against the therapeutic antibody.
  • 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 [Grupp SA, et Al. Adoptive cellular therapy. Curr Top Microbiol Immunol., .2011;344: 149-72].
  • TCR T Cell Receptor
  • the scFv of antibodies against tumor cell-associated antigens is fused to the intracellular signal activation motifs such as CD3 ⁇ or F CS RI Y of T lymphocyte receptors.
  • CAR Chimeric antigen receptor
  • 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 FCSRIY.
  • ITAM immunoreceptor tyrosine activation motif
  • FCSRIY FCSRIY
  • the intracellular signal region 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 secretion of cytokines is relatively small, and can not stimulate 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): 11029-1 1 1036.].
  • CAR T lymphocytes were added to the intracellular signaling region of CD28 or CD 137 (aka 4- 1BB), in which the chimeric antigen receptor portions were joined as follows: scFv-TM-CD28 -ITAM or scFv-TM-/CD 137-ITAM.
  • the B 7/CD28 or 4- 1 BBL/CD 137 co-stimulatory action in the intracellular signal region causes sustained proliferation of T lymphocytes and can increase the levels of cytokines such as IL-2 and IFN- ⁇ secreted by T lymphocytes.
  • the survival cycle and anti-tumor effect of CAR T in vivo are improved [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-CD 137-ITAM or scFv-TM-CD28-CD 134-ITAM, further improved
  • the survival cycle of CAR T 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 CD 137 domains. PNAS, 2009, 106(9): 3360- 3365. ] 0
  • CAR T lymphocytes have attractive prospects in tumor immunotherapy, some potential risks are also needed.
  • specific antigens recognized by low expression of CAR in certain/normal tissues may cause damage to 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.
  • CAR conjugate signaling in CAR reduces the threshold required for effector cell activation, allowing genetically modified T lymphocytes to be activated under low-level or no antigen-triggered conditions, resulting in a large number of cells.
  • the release of the factor may lead to the so-called "cytokine storm”.
  • This singnal leakage can lead to off-target cytotoxicity, resulting in non-specific tissue damage.
  • the so-called "cytokine storm” is triggered by the low expression of Her2 in normal lung tissue to the death of the patient [ 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.].
  • a first aspect of the invention relates to a nucleic acid encoding a GPC3 chimeric antigen receptor protein expressed on the surface of a T lymphocyte, the chimeric antigen receptor protein expressed thereby allowing T lymphocytes expressing the receptor to be highly expressed against GPC3 Tumor cells have a highly specific cytotoxic effect.
  • the GPC3 chimeric antigen receptor protein comprises a sequence-ligated extracellular binding region, a transmembrane region and an intracellular signaling region, wherein the extracellular binding region comprises a single-chain antibody scFv that specifically recognizes a C-terminal epitope of GPC3 ( GPC3).
  • the extracellular binding region of the chimeric antigen receptor protein is linked to the transmembrane region of CD8 or CD28 via the CD8 hinge region, and the intracellular signal region is followed by the transmembrane region.
  • the nucleic acid sequence of the invention may be in the form of DNA or in the form of 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 encoding the corresponding amino acid Codons 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 which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80%, optimally at least 90% or at least 95% identity between the two sequences. .
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C ; or (2) hybridization Adding a denaturant such as 50% / formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or only between the two sequences is at least 90% identical, more preferably 95% or more Hybridization occurs only when it occurs.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of one of SEQ ID NOS: 22-25.
  • a monoclonal antibody that specifically recognizes the C-terminal epitope of human GPC3 has been disclosed, for example, in Chinese Patent Publication No. CN101186650A, a Chinese pharmaceutical company; and, in addition, the Advances in Liver Cancer Antibody Therapies: A Focus on Glypican-3 and Mesothelin, BioDrugs. 201 1 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, which are located at the C-terminus 524- against the GPC3 epitope. 563 amino acid residues, and also reported monoclonal antibodies such as GPC3-C02 and 1G12.
  • monoclonal antibodies can be used to prepare a single-chain antibody portion of a chimeric antigen receptor protein encoded by the nucleic acid of the present invention.
  • Other monoclonal antibodies recognizing the C-terminal epitope of GPC3 can also be used in the present invention in a suitable manner.
  • 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) NY. 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 an antibody” in the present invention includes, for example, when a derivative of the antibody is obtained by phage display technology, and can be used, for example, in a BIAcore system.
  • Surface plasmon resonance technology to increase the efficiency of phage antibodies binding to GPC3 epitopes (Schier, Human Antibody Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the term "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 target antigen.
  • the degree of specificity can be judged by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.
  • the specific recognition is preferably determined by flow cytometry, and the criteria for specific recognition in specific cases can be judged by those skilled in the art based on the common knowledge in the art.
  • the transmembrane region of the chimeric antigen 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 CD8a or CD28.
  • the CD8 alpha hinge region is a flexible region, therefore, CD8 or CD28 and the transmembrane region plus the hinge region are used to target the recognition domain of the chimeric antigen receptor CAR s cF V and The inner signal areas are connected.
  • the intracellular signal region can be selected from the group consisting of CD3 ⁇ , FcsRIy, CD28, CD137, the intracellular signal region of the CD134 protein, and combinations 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 important signal transduction regions in the TCR-CD3 complex.
  • CD3 ⁇ Z (hereinafter abbreviated as DZ) is a truncated CD3 ⁇ sequence having no ⁇ motif, and is generally constructed as a negative control in the practice of the present invention.
  • F CS RI Y is mainly distributed on the surface of mast cells and basophils, and it contains an ITAM motif similar in structure, distribution and function to CD3.
  • CD28, CD137, and CD134 are conjugated signaling molecules, and the co-stimulatory action of intracellular signal segments after binding to their respective ligands causes sustained proliferation of T lymphocytes and can enhance T lymphocytes.
  • the cells secrete levels of cytokines such as IL-2 and IFN- ⁇ , while increasing the survival cycle and anti-tumor effect of CAR T lymphocytes in vivo.
  • the GPC3 chimeric antigen receptor protein encoded by the nucleic acid of the present invention may be selected from a chimeric antigen receptor protein which is sequentially linked as follows:
  • CD28a in the relevant chimeric antigen receptor protein represents the transmembrane region of CD28 molecule
  • CD28b generation Table The intracellular signal region of the CD28 molecule.
  • the various chimeric antigen receptors targeting GPC3 described above are collectively referred to as SC Fv(GPC3)-CAR.
  • the nucleic acid of the invention has the sequence set forth in SEQ ID NOs: 18-21.
  • the nucleic acid of the invention is a nucleic acid encoding a chimeric antigen receptor protein having one of SEQ ID NOs: 22-25.
  • a second aspect of the invention includes a vector comprising the above nucleic acid encoding a chimeric antigen receptor protein expressed on the surface of a T lymphocyte.
  • the vector used in the present invention is a lentiviral plasmid vector pPWT-eGFP o.
  • the plasmid belongs to a third generation auto-inactivated lentiviral vector system, and the system has three plasmids, the encoded protein Gag/Pol, The packaging plasmid psPAX2 encoding the Rev protein; the envelope plasmid PMD2.G encoding the VSV-G protein; and the empty vector pPWT-eGFP, which can be used for recombinant introduction of the nucleic acid sequence of interest, ie the nucleic acid sequence encoding CAR.
  • the empty vector pPWT-eGFP (which itself is a mock in subsequent experiments) regulates enhanced green fluorescent protein (eGFP) by an elongation factor-1 alpha (elongation factor-1 ⁇ , EF-1 ⁇ ) promoter.
  • the recombinant expression vector pWPT-eGFP-F2A-CAR which contains the nucleic acid sequence of interest encoding CAR, is obtained by ribosomal skipping sequence 2A (F2A) derived from food-and-mouth disease virus (FMDV). ) to achieve co-expression of eGFP and CAR.
  • F2A ribosomal skipping sequence 2A
  • FMDV food-and-mouth disease virus
  • the vector of the invention has a nucleic acid sequence as set forth in one of SEQ ID NOs: 27-30.
  • a third aspect of the invention includes a virus comprising the above vector.
  • the virus of the present invention includes an infectious virus after packaging, and also includes a virus to be packaged containing a component which is packaged 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 (i.e., containing scFv (GPC3)-CAR).
  • a fourth aspect of the invention comprises a transgenic T lymphocyte transduced with a nucleic acid of the invention or transduced with the above-described recombinant plasmid comprising the nucleic acid of the invention, or a virus comprising the plasmid.
  • 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 Sleeping Beauty system or PiggyBac transposon is much higher than that of ordinary electroporation, and the nucleofector transfection apparatus is combined with the Sleeping Beauty transposing subsystem. It has been reported [Davies JK., et al. Combining CD 19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OFl-10.] The method and the method have 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 culture of the tau lymphocytes to reach the clinical level in vitro.
  • the transduced nucleic acid is expressed on its surface by transcription and translation.
  • the in vitro cytotoxicity assay of various cultured tumor cells demonstrates that the transgenic T lymphocytes expressing the chimeric antigen receptor on the surface of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity).
  • the nucleic acid encoding the chimeric antigen receptor protein of the present invention the plasmid comprising the nucleic acid, the virus comprising the plasmid, and the transgenic tau lymphocytes transduced with the nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.
  • the tau lymphocytes of the invention express a chimeric antigen receptor on their surface, and the chimeric antigen receptor is expressed by a nucleic acid encoding one of SEQ ID NOS: 18-21.
  • the transgenic T lymphocytes of the invention express a chimeric antigen receptor on the surface, the amino acid sequence of the chimeric antigen receptor being selected from one of SEQ ID NOs: 22-25.
  • Fig. 1 shows a schematic structural view of a lentiviral vector pWPT-eGFP-F2A-CAR comprising the CAR sequence of the present invention as an example.
  • Figure 2 is a schematic diagram showing the connection relationship between different regions of the CAR of the present invention contained in a lentiviral vector as an example, wherein eGFP and svFv (GPC3)-specific chimeric antigens linked by the ribosomal hopping sequence F2 A Receptor.
  • eGFP and svFv GPC3
  • Figure 3 shows the nucleic acid electrophoresis pattern of the lentiviral plasmid of Example 1 identified by Mlul and Sail double digestion.
  • Ml is the DS2000 molecular weight marker (Guangzhou Dongsheng Biotechnology Co., Ltd.); M2 is Hind ///marker (Guangzhou Dongsheng Biotechnology Co., Ltd.).
  • Lanes 1-5 are respectively
  • Fig. 4 is a view showing the results of flow cytometry analysis of eGFP expressed by cells infected with CTL (cytotoxic T lymphocytes;) of Example 2 of the present invention.
  • FIG. 5 shows the in vitro growth of CTL cells expressing different chimeric antigen receptors (CAR+) according to Example 2 of the present invention.
  • the figure shows that CTL cells expressing different chimeric antigen receptors were expanded 200-fold in vitro on day 14 after viral infection.
  • Figure 6A shows the flow cytometry to detect the expression level of GPC3 on each hepatocellular carcinoma cell line, and GPC3 is fine.
  • the expression on the cell is expressed by Mean Fluorescence Intensity (MFI);
  • Fig. 6B shows the results of western blot on the expression level of GPC3 on each of the above cell lines, wherein GAPDH is a sample internal control.
  • MFI Mean Fluorescence Intensity
  • Figure 7A shows the percentage of tumor-free mice in each group as a function of time after treatment with Huh-7 xenografts using CAR T cells targeting GPC3;
  • Figure 7B shows that each group of 6 mice was sacrificed after each The size of the tumor in the body.
  • Fig. 8A is a comparison result of the volume of residual tumor of each group of mice of Example 5;
  • Fig. 8B is a comparison result of the weight of residual tumor of each group of mice of Example 5;
  • Fig. 8C shows the succession of Fig. 8C
  • the number of peripheral blood T cells surviving in each group of mice of Example 5 was measured 1 week after infusion of T lymphocytes;
  • Fig. 8D shows the tumors shown after the execution of each group of mice of Example 5;
  • Fig. 8E shows It is the tumor regression of mice after GPC3-28BBZ CAR T lymphocyte treatment in Example 5 (compared with Mock group).
  • Fig. 9 is a view showing the results of immunohistochemical staining of the aggregation of the T-cells of Example 5 in the transplanted tumor tissues of each group of mice.
  • Figure 10A shows the xenograft volume of each group of mice of Example 6.
  • Fig. 10B shows the results of weighing the tumor tissue after the end of the experiment in each group of mice of Example 6.
  • Figure 10C shows the tumor tissue of each group of mice of Example 6;
  • Figure 10D shows the results of detecting the number of T cells in the peripheral blood of each group of mice of Example 6 after one week of the last adoptive transfer of T cells. .
  • Figures 11A and 11B show the bioluminescence intensities and actual imaging images of tumors of the mice of each group of Example 7, respectively.
  • Fig. 11C and D show the abdominal tumor-bearing condition (in vitro) and the anatomical tumor condition of each group of mice of Example 7.
  • Fig. 11E shows the results of measuring the number of T cells in peripheral blood of each group of mice after 1 week of the last adoptive transfer of T cells.
  • Figure 11F shows the results of measuring the number of CAR-positive T cells in peripheral blood of each group of mice after 1 week of the last adoptive transfer of T cells.
  • Fig. 1 1G shows the survival rate of each group of mice of Example 7 after tumor inoculation. detailed description
  • Table 1 illustrates the order of ligation of the various portions of the chimeric antigen receptor of the present invention, which can also be seen in Figure 2.
  • Amplification of the scFv (GPC3) sequence is based on the single-stranded bifunctional antibody nucleotide GPC3/CD3 constructed in our laboratory.
  • the sequence of the template can be found in Chinese Patent Application No. 201210480326.x. SEQ ID NO: 9.
  • the primer pair used for amplification is the upstream primer 5'-gatgttgtgatgactcagtctc-3, (SEQ ID ⁇ : 1) and the downstream primer 5'-gcgctggcgtcgtggttgaggagacggtgaccag-3' (SEQ ID NO: 2) for amplification of scFv (GPC3);
  • the amplified bands were all 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; 25 cycles, then a total extension of 68 ° C, 10min.
  • the PCR amplified bands were confirmed by agarose gel electrophoresis to match the expected fragment size.
  • the nucleic acid sequences of the GPC3 chimeric antigen receptor protein other than scFv were obtained by PCR using the sequences SEQ ID NO: 1 to 4 disclosed in Patent Application No. 201310108532.2, respectively.
  • the CAR portion of the following GPC3 chimeric antigen receptor proteins other than scFv (GPC3), wherein CD8-CD3 ⁇ ( ⁇ ) and CD28a-CD28b-CD137-CD3 ⁇ (28 ⁇ ), respectively, are scFv (EFGR) - CD8-CD3 ⁇ (SEQ ID NO: 1 in Patent Application 201310108532.2) and SCP scFv (EFGR)-CD28a-CD28b-CD137-CD3 ⁇ (SEQ ID NO: 2 in Patent Application 201310108532.2) is a template, using upstream primer 5' -accacgacgccagcgccgcgaccac-3, (SEQ ID NO: 3) and downstream of the 5'-
  • CD8-CD3 delta ⁇ (delta Z, ⁇ ⁇ for short), CD8-CD137-CD3 ⁇ ( ⁇ ) and CD28a-CD28b-CD3 ⁇ (28Z) are obtained as follows:
  • the CD137 intracellular domain was obtained by amplification of the upstream primer 5'-aaacggggcagaaagaaactc-3' (SEQ ID NO: 10) and the downstream primer 5'-cagttcacatcctccttc-3' (SEQ ID ⁇ : 11), and the PCR amplification conditions were the same as above.
  • the theoretical size of the band is 126 bp, and the amplified product is confirmed by agarose gel electrophoresis to be consistent with the theoretical size.
  • the region and Z were spliced by the same splicing and PCR amplification to obtain the target fragment: CD8a hinge region-CD28 transmembrane region-28Z intracellular region, ie CD28a-CD28b-CD3 ⁇ , splicing and PCR amplification conditions are the same as above.
  • the theoretical size of the band is 706 bp, and the amplified product is confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the eGFP nucleic acid fragment carrying the F2A and CD8 ⁇ signal peptides at the 3' end is obtained by the upstream gene 5 ' -cttacgcccctagcgcccccgtggcccccatcatggtgagcaagggcgaggag-3 ' (SEQ ID ⁇ 0:16) and the downstream primer 5' ⁇ £ ; £ 3 ⁇ 4 ⁇ 0 ⁇ 0 ⁇ 1 ⁇ & ⁇ 0& ⁇ -3' (SEQ ID NO: 17)
  • Amplification of eGFP nucleic acid fragments with F2A and CD8 ⁇ signal peptides at the 3' end of a lentiviral vector for patent application
  • the pWPT-eGFP-F2A-806-Z disclosed in 201310108532.2 is a template, and the PCR amplification conditions are pre-denaturation: 94 ° C, 4 min; denaturation: 94 ° C
  • nucleic acid fragments respectively, a fragment amplified by CD8-CD3 ⁇ , CD8-CD137-CD3 ⁇ , CD28a-CD28b-CD3 ⁇ in the above paragraph "(2) Nucleic acid sequence of other portions of chimeric antigen receptor” , CD28a-CD28b-CD137-CD3 ⁇ with the equimolar portion of the aforementioned paragraph "(3) nucleic acid sequence of other portions of the chimeric antigen receptor" amplified with the 3' end
  • eGFP nucleic acid fragment of F2A and CD8a signal peptide (mass approximately 50 ng) and equimolar scFv (GPC3) (mass approximately 50 ng)
  • GPC3 equimolar scFv
  • the splicing conditions were: pre-denaturation: 94 ° C, 4 min ; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: 68 ° C, 140s, for 5 cycles, then a total extension of 68 °C, 10 min, supplemented with DNA polymerase ⁇ every upstream and upstream 5 '-cttacgccccctagcgcccccgtggccaccatggtgagcaagggggaggag-3 ' (SEQ ID NO: 16) and downstream primer 5' - gaggtcgacctagcgagggggggggcctgcatg -3 ' (SEQ ID NO: 13)
  • eGFP-GPC3-Z The theoretical sizes of eGFP-GPC3-Z, eGFP-GPC3-BBZ, eGFP-GPC3-28Z and eGFP-GPC3-28BBZ were 2161 bp, 2278 bp, 2302 bp and 2428 bp, respectively.
  • the amplified product was confirmed by agarose gel electrophoresis to be in agreement with the theoretical size.
  • the upstream primer 5 '-gatgttgtgatgactcagtctc-3 ' (SEQ ID NO: 1) and the downstream primer 5'-gaggtcgacctagcgagggggcagggccggcatg -3 ' (SEQ ID NO: 13) were used without adding the 3' end.
  • An eGFP nucleic acid fragment (having a mass of about 50 ng) carrying the F2A and CD8 a signal peptide, a nucleic acid GPC3-Z (SEQ ID NO: 18), GPC3-BBZ (SEQ ID NO:) encoding a GPC3 chimeric antigen receptor protein can be obtained.
  • GPC3-28Z SEQ ID NO: 20
  • GPC3-28BBZ SEQ ID NO: 21
  • GPC3-Z SEQ. ID NO: 22
  • GPC3-BBZ SEQ ID NO: 23
  • GPC3-28Z SEQ ID NO: 24
  • GPC3-28BBZ SEQ ID NO: 25
  • fragment CD8-CD3delta e fragment amplified in the above-mentioned paragraph "(2) Nucleic acid sequence of other portions of the chimeric antigen receptor" and the equimolar paragraph “(3) chimeric antigen receptor other parts, respectively, are equimolar.
  • the amplified fragment and the equimolar scFv use the upstream primer 5'-cttacgcccctagcgcccccggtcgccaccatggtgagcaagggcgaggag-3 ' (SEQ ID NO: 16) and the downstream bow
  • the same conditions were used for splicing and PCR, and eGFP-GPC3- ⁇ ⁇ (SEQ ID ⁇ : 31) was obtained by amplification, and the theoretical size was 1858 bp.
  • the amplified product was confirmed by agarose gel electrophoresis to a theoretical size of 1.
  • the reverse transcription of the RT-PCR Kit was used to synthesize the first strand of cDNA. Using the first strand of cDNA as a template, respectively, with Heavy
  • Primers ⁇ Light Primer Mix is a primer (primer purchased from Shanghai Ruijin Biotechnology Co., Ltd.;), amplifying VH, VL Gene, PCR conditions: predenaturation at 94 °C for 4 min, denaturation at 94 °C for 40 s, annealing at 55 °C for 40 s, extension at 68 °C for 40 s, extension at 68 °C for 7 min after 30 cycles.
  • the PCR products were detected by agarose gel electrophoresis, and the VH and VL fragments were recovered by the gel recovery kit.
  • VH and VL fragments were used as templates, and the Linker-Primer Mix was used as a primer (primer was purchased from Shanghai Ruijin Biotechnology Co., Ltd.).
  • the overlapping PCR method was used to splicing VH and VL fragments into scFv.
  • PCR conditions denaturation at 94 °C for 1 min, 63 Annealing at °C for 4 min for a total of 7 cycles. After 7 cycles, Linker-Primer Mix, polymerase buffer and double distilled water were added to the 50 ⁇ l reaction system to continue the PCR.
  • PCR conditions pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 40 s, annealing at 58 °C for 40 s, elongation at 68 °C for 1 min, extension at 68 °C for 7 min after 30 cycles.
  • the PCR product was detected by agarose electrophoresis, and the scFv fragment was recovered by a gel recovery kit.
  • the cells were washed three times with 0.1 M phosphate buffer (PBS), and the above-mentioned medium-inducible culture supernatant was added to a 96-well plate at 50 ⁇ l per well.
  • PBS phosphate buffer
  • the mass is about 50 ng.
  • the three fragments are spliced and PCR according to the pattern shown in Figure 2.
  • the splicing conditions are: pre-denaturation: 94 ° C, 4 min ; denaturation: 94 ° C, 40 s; annealing: 60 ° C, 40 s; extension: 68 ° C, 140s, for 5 cycles, then total extension 68 °C, 10min, supplement DNA polymerization ⁇ every and upstream of the 5'-cttacgcccctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 ' (SEQ ID
  • the vector system used to construct the lentiviral plasmid vector of the present invention is exemplified by a third generation auto-inactivated lentiviral vector system, which has three plasmids, the protein Gag/Pol, the packaging plasmid psPAX2 encoding the Rev protein; and the coding VSV.
  • F2A is a core sequence of 2A (or "self-cleaving polypeptide 2A") from foot-and-mouth disease virus. It has a "self-shearing" function of 2A, which enables co-expression of upstream and downstream genes.
  • 2A has an effective and feasible strategy for constructing gene therapy polycistronic vectors due to its high shear efficiency, high balance of upstream and downstream gene expression and short self-sequence.
  • the sequence is used to achieve co-expression of the target gene with GFP or eGFP, and the expression of CAR can be detected indirectly by detecting GFP or eGFP.
  • a lentiviral expression vector in which eGFP linked to F2A is co-expressed with a specific CAR is constructed, and is collectively referred to as pWPT-eGFP-F2A-CAR.
  • the target gene eGFP-F2A-CAR obtained in the above step 2 is digested with Mlul and Sail restriction enzymes, and ligated into the same double-digested pWPT vector to construct a lentiviral vector expressing each chimeric antigen receptor. .
  • the successfully constructed vector was identified by Mlul and Sail digestion (Fig. 3) and the sequence was determined correctly, and it was ready for lentiviral packaging.
  • eGFP-F2A-CAR is transcribed into one mRNA, but is finally translated into two peptide chains, eGFP and anti-GPC3 chimeric antigen receptor, wherein the anti-GPC3 chimeric antigen receptor will be guided by the CD8 a signal peptide.
  • the eGFP-GPC3- ⁇ used as a negative control expresses a GPC3 chimeric antigen receptor (GPC3- ⁇ ) containing a truncated ⁇ ⁇ on the cell membrane surface after insertion into a lentiviral plasmid vector, the amino acid sequence of which is SEQ ID NO :32.
  • the obtained full nucleic acid sequence of the vector containing each of the CARs of interest is as follows: pWPT-eGFP-F2A-GPC3- ⁇ Z (SEQ ID NO: 26); pWPT-eGFP-F2A-GPC3-Z (SEQ ID NO: 27); pWPT -eGFP-F2A-GPC3-BBZ (SEQ ID NO: 28); pWPT-eGFP-F2A-GPC3-28Z (SEQ ID NO: 29); pWPT-eGFP-F2 A-GPC3 -28BBZ (SEQ ID NO: 30) .
  • Plasmid transfection of 293T packaging lentivirus 293T cells (ATCC: CRL-11268) cultured to a 6th to 10th passage were inoculated at a density of 6 ⁇ 10 6 and cultured overnight in a 10 cm culture dish at 37° C., 5% CO 2 for transfection.
  • the medium was DMEM (PAA) containing 10% fetal calf serum (PAA), and the next day, the medium was changed to serum-free DMEM about 2 hours before transfection.
  • the transfection efficiency (ie, the proportion of cells showing green fluorescence) was observed on the next day of transfection, and the positive transfection efficiency of ⁇ 80% was successful in the transfection experiment.
  • the virus was collected by filtration using a 0.45 ⁇ m filter (Millipore), and then centrifuged at 28000 rpm for 2 hours at 4 ° C using a Beckman Optima L-100XP ultracentrifuge, the centrifugation supernatant was discarded, and the resulting precipitate was centrifuged. 10 1/50 stock volume of Quantum 007 medium (PAA) was resuspended and stored at -80 °C in 100 ⁇ L/tube for virus titration or infection of T lymphocytes.
  • PPAA Quantum 007 medium
  • the titer (U/mL) positive rate X dilution factor X 100X 10 4 was calculated.
  • the titer of the above virus containing mock (ie empty vector) control and each eGFP-F2 A-CAR packaged by the calcium phosphate transfection method was at a level of about 0.5 to 2 X 10 6 U/mL, and was measured after concentration. The virus titer is approximately 2X 10 7 U/mL.
  • Human peripheral blood mononuclear cells were obtained from peripheral blood of healthy people by density gradient centrifugation (Shanghai Blood Center For the peripheral blood mononuclear cells, CTL was obtained by negative sorting method of CTL cell magnetic beads (Stem Cell Technologies). The sorted CTL cells were used for flow cytometry to detect the purity of CTL cells, and the positive rate of CTL cells was 95%. It is advisable to proceed to the next step.
  • the Quantum 007 lymphocyte culture medium (PAA) was added at a density of about IX 10 6 /mL and the magnetic beads (Invitrogen) coated with anti-CD3 and CD28 antibodies were added at a ratio of cells: magnetic beads of 1:1.
  • Recombinant human IL-2 (Shanghai Huaxin Biotech Co., Ltd.;) with a final concentration of 100 U/mL was cultured for 24 h. CTL cells were then infected with MOI 5 with the above recombinant lentivirus. The infected cells were passaged every other day at a density of 5 X 10 5 /mL, and a recombinant human IL-2 at a final concentration of 100 U/mL was supplemented in 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, positive cells detecting eGFP were positive cells expressing chimeric antigen receptors (Fig. 4). .
  • the positive rate of CTL cells infected with virus expressing different chimeric antigen receptors using uninfected T lymphocytes as a negative control is shown in the following table. The positive rate results indicate that a certain positive rate of CAR+ CTL cells can be obtained by the method of lentivirus infection.
  • CTL cells were subcultured, counted, and supplemented with IL-2 at a cell density of 5 ⁇ 10 5 /ml, respectively, after infection with a virus containing different chimeric antigen receptors.
  • the final concentration was 100U/ml), about 200-fold amplification on day 14 of culture (see Figure 5), indicating that CTL cells expressing different chimeric antigen receptors can be expanded in vitro for a number of subsequent in vitro toxicity tests and in vivo. The test provides a guarantee.
  • Example 3 In vitro toxic effect test of chimeric antigen receptor cells
  • the target cells used in the in vitro toxicity test were the hepatocellular carcinoma cell lines shown in Table 3 below, and the effector cells were the FACS-detected positive cells expressing chimeric antigen receptors expressed in FACS as demonstrated in Example 2 for 12 days.
  • Flow cytometry and western blot were used to detect the expression levels of GPC3 on hepatocellular carcinoma cell lines HepG2, Huh-7, Hep3B, PLC/PRF/5 and SK-HEP-1.
  • the flow results are shown in Figure 6A, and GPC3 is in the cells.
  • the expression above is expressed by Mean Fluorescence Intensity (MFI).
  • MFI Mean Fluorescence Intensity
  • the expression level of GPC3 in each cell line was further verified by western blot.
  • the results are shown in Fig. 6B, wherein GAPDH is a sample internal control.
  • the results showed that GPC3 was expressed at different levels in four hepatocellular carcinoma cell lines HepG2, Huh-7 ⁇ Hep3B and PPLC/PRF/5, and no expression of GPC3 was detected in SK-HEP-1.
  • Each experimental group each target cell + CTL expressing different chimeric antigen receptors
  • Control group 1 The maximum release of LDH from target cells
  • Control group 2 Target cells spontaneously release LDH
  • Control group 3 Effector cells spontaneously release LDH.
  • CytoT OX 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 CytoT OX 96® assay quantitatively measures lactate dehydrogenase (LDH). 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. 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.
  • INT tetrazolium salt
  • the effective target ratio is 3: 1, 1 : 1 and P l : 3, and the target cell number is 10000 / well C5 ( ⁇ L 2x l0 5 /mL;), corresponding effector cells according to different target ratios.
  • Five replicate wells were set for each group, and the average of five replicate wells was taken. The detection time is 18h.
  • Control group 1 - control group 2 experimental results show that: Specifically, as shown in Table 4, the CTL expressing the chimeric antigen receptor GPC3-Z CAR+ and the CTL of GPC3-28BBZ CAR+ of the present invention are GPC3-positive hepatocellular carcinoma cell lines HepG2, Huh-7, Hep3B and PLC/PRF. /5 cells all showed very significant specific cytotoxicity, and showed a higher effective cytotoxic effect than the gradient-dependent immediate target ratio, while the GPC3-negative hepatocellular carcinoma cell line SK-HEP-1 No specific cytotoxic effects.
  • Table 4 the CTL expressing the chimeric antigen receptor GPC3-Z CAR+ and the CTL of GPC3-28BBZ CAR+ of the present invention are GPC3-positive hepatocellular carcinoma cell lines HepG2, Huh-7, Hep3B and PLC/PRF. /5 cells all showed very significant specific cytotoxicity, and showed a higher effective cytotoxic effect than the gradient-dependent immediate
  • GPC3-28BBZ has a cytotoxicity of 97% against GPC3-positive hepatoma cell HepG2 at a target-to-target ratio of 3:1, and a cytotoxicity of up to 84% against GPC3-positive liver cancer cell PLC/PRF/5.
  • the data of the ratio-dependent ratio further shows the specific cytotoxic effect of the CTL expressing the chimeric antigen receptor targeting GPC3 in the present invention on GPC-positive liver cancer cells.
  • the CTL expressing the chimeric antigen receptor (GPC3-Z and GPC3-28BBZ) targeting GPC3 in the present invention showed specific toxic effects on GPC3-positive liver cancer cells, and the expression carries the co-stimulatory molecule CD28 and The GPC3-28BBZ CAR T cells in the signal region of CD137 have a stronger toxic effect than GPC3-Z CAR T cells without both signal regions.
  • Table 4
  • GPC3-Z CAR T lymphocyte group GPC3-28BBZ CAR T lymphocyte group with different target ratio (1:1 and 1 :2)
  • the control group was GPC3-5Z CAR T lymphocyte, mock CAR T lymphocyte Control group and saline control group.
  • cyclophosphamide 200 mg/kg was intraperitoneally injected.
  • Huh7 cells (2 10 6 /piece) were subcutaneously inoculated into the right temporal part of the mouse, respectively.
  • Huh-7 cells in the logarithmic growth phase were harvested, and the cell density was adjusted to 1 ⁇ 10 7 /ml with saline, and the injection volume was 20 ( ⁇ L ( 2> ⁇ 10 6 /only).
  • Tumor inoculation The diary is the 0th day.
  • Adoptive transfer of T cells When the tumor volume of the mice is 200-300 mm 3 , the cyclophosphamide (200 mg/kg) is injected intraperitoneally on the 13th day after the tumor is inoculated, and the experimental group and the control group are injected 8 ⁇ in the tail vein on the 14th day. 10 6 / genetically modified T lymphocytes (positive transfection efficiency is about 50%) or saline only.
  • Grouping 30 6-8 week old NOD/SCID mice were randomly divided into 4 groups, 6 in each group.
  • the experimental group was: GPC3-28BBZ CAR T lymphocyte treatment group, control group: 2D3-28BBZ CAR T lymphocytes The control group, the Mock gene-modified CAR T lymphocyte control group, and the saline control group.
  • mice in the control group were terminated with a tumor volume of 2000 mm 3 .
  • 3/6 mice had tumor regression, and the residual tumor volume (Fig. 8A) and weight (Fig. 8B) were significant compared with the three control groups. Difference (***P ⁇ 0.001).
  • Figure 8D shows the tumor tissue of each group shown after sacrifice of the mice.
  • Figure 8E shows GPC3-28BBZ CAR T lymphocyte therapy Post-mouse tumor regression (compared with Mock group). The results showed that GPC3-28BBZ CAR T lymphocytes significantly inhibited the growth of Huh-7 xenografts.
  • each group of Huh-7 xenografts was sectioned and immunostained with anti-human CD3e antibody.
  • the results of immunohistochemical staining are shown in Fig. 9.
  • the results showed that the human CD3-positive T cells in the subcutaneous xenografts of the GPC3-28BBZ CAR T cell treatment group were significantly more than the transduced 2D3-28BBZ and mock T cell treatment groups, while the saline group did not detect human T cells.
  • GPC3-28BBZ CAR T cells can accumulate at the tumor site to exercise their function of killing tumor cells.
  • GPC3-28BBZ CAR T cells can partially eliminate Huh-7 subcutaneous xenograft tumors with high expression of GPC3.
  • Example 6 Treatment of subcutaneous xenografts of PLC/PRF/5 with low expression of GPC3 by GPC3-28BBZ CAR T cells
  • Inoculation of tumor PLC/PRF/5 cells in logarithmic growth phase and well-preserved, adjusted to a cell density of 2.5 x 10 7 /mL with saline, injection volume of 200 ⁇ (5 > ⁇ 10 6 /only), tumor inoculation
  • the diary is the 0th day.
  • Adoptive transfer of sputum cells When the tumor volume of the mice reached 150 mm 3 , that is, on the 21st day, cyclophosphamide (200 mg/kg) was intraperitoneally injected. On the 22nd and 30th day, the experimental group and the control group were respectively in the tail vein. Injection of 8 > ⁇ 10 6 / genetically modified T cells (positive transfection efficiency of about 50%) or saline only.
  • Grouping 30 6-8 week old NOD/SCID mice were randomly divided into 5 groups, 6 in each group.
  • the experimental group was: GPC3-28BBZ CAR T lymphocyte treatment group, control group: 2D3-28BBZ CAR T lymphocytes The control group, the Mock gene-modified CAR T lymphocyte control group, and the saline control group.
  • mice When the average tumor volume of mice reached 150 mm 3 (day 21), the mice were randomly divided into 4 groups and injected intraperitoneally with 200 mg/kg cyclophosphamide, on days 22 and 30, respectively.
  • the tail vein adoptively transferred 8 G 10 6 GPC3-28BBZ, 2D3-28BBZ or mock gene-modified T lymphocytes (positive rate 50%, respectively) or only saline.
  • the experimental mice ended the experiment when the average tumor volume was as long as 1500 mm 3 .
  • Fig. 10A shows the tumor growth curve.
  • the volume of transplanted tumors of the GPC3-28BBZ CAR T lymphocyte treatment group was significantly smaller than that of the respective control groups (*P ⁇ 0.05).
  • Figure 10C shows the tumor tissue of each group shown in the mice at the end of the experiment, showing that the GPC3-28BBZ CAR T lymphocyte treatment group has a significant inhibitory effect on PLC/PRF/5 cells with low expression of GPC3.
  • Inoculation of tumors Collect good Huh-7 (Luc+) in logarithmic growth phase, adjust cell density to 1 ⁇ 10 8 /ml with saline, mix 25 ⁇ cell suspension with 25 ⁇ metrigel Matrigel (on ice), and none The bacteria were cleaned and the intraperitoneal operation was performed. The cells were mixed and inoculated into the right liver (2.5 ⁇ 10 6 /) of 40 6-8 weeks old NOD/SCID mice. The day of tumor inoculation was the 0th day.
  • mice tumor inoculation From the day of mouse tumor inoculation, weekly imaging, and 2 weeks later, intraperitoneal injection of cyclophosphamide (200 mg/kg), and randomly divided into 4 groups, 7 in each group, the experimental group was: GPC3-28BBZ
  • the CAR T lymphocyte treatment group and the control group were: 2D3-28BBZ CAR T lymphocyte control group, Mock gene modified CAR T lymphocyte control group, and saline control group.
  • T lymphocytes Adoptive transfer of T lymphocytes: On the 14th and 21st day after tumor inoculation, 5 ⁇ 10 6 / gene-modified T lymphocytes were infused into the tail vein of mice (positive transfection efficiency was about 50%, injection volume was 200 ⁇ ). Or only 200 ⁇ saline.
  • the tumors in the GPC3-28BBZ CAR T lymphocyte treatment group were significantly smaller than those in the control group and GPC3- was found in the subsequent 2nd and 3rd weeks of imaging.
  • the tumors of the 28BBZ CAR T lymphocyte treatment group were significantly smaller than those of the control group (Fig. 1 1A and Fig. 1 1B).
  • three control groups were found to be 2D3-28BBZ CAR T.
  • the mice in the lymphocyte treatment group, the mock T cell treatment group, and the saline group were significantly elevated in the abdomen, while the mice in the GPC3-28BBZ CAR T lymphocyte treatment group were normal in the abdomen (Fig.
  • mice 1 1C
  • mice the control mice were found to be in the control group.
  • the liver has a huge tumor occupying space, and No tumor growth was observed in the liver of mice in the GPC3-28BBZ CAR T lymphocyte treatment group (Fig. 11D).
  • Fig. 11D One week after the last infusion of T lymphocytes in mice, blood was collected from the eyelids of mice to detect the survival of infused T cells in mice. The results showed that the lymphocytes in the GPC3-28BBZ CAR T lymphocyte treated group were compared.
  • the median survival of the control group was 34 days in the mock control group, 39 days in the 2D3-28BBZ control group, and 33 days in the saline group (Fig. 11G). At this time point, the GPC3-28BBZ CAR T cell treatment group still remained. Survival is good, no abdomen is seen in the abdomen.
  • GPC3-28BBZ CAR T cells have a significant effect on Huh-7 liver orthotopic xenografts, and GPC3-28BBZ CAR T cells can survive effectively in Huh-7 orthotopic transplanted mice.
  • the chimeric antigen receptor-modified T cells targeting GPC3 protected by the present patent specifically recognize and kill GPC3-positive liver cancer cells, but have no effect on GPC3-negative liver cancer cells, and have potential clinical application value.
  • Table 5 Sequences in the present invention specifically recognize and kill GPC3-positive liver cancer cells, but have no effect on GPC3-negative liver cancer cells, and have potential clinical application value.

Abstract

编码表达于人T淋巴细胞表面的嵌合抗原受体蛋白的核酸，所述嵌合抗原受体蛋白包含顺序连接的胞外结合区，跨膜区和胞内信号区，其中所述胞外结合区包含特异性识别GPC3的C末端表位的单链抗体scFv(GPC3)。一种转基因T淋巴细胞，其表面表达一种嵌合抗原受体，所述嵌合抗原受体由前述核酸编码表达。

Description

编码 GPC3嵌合抗原受体蛋白的核酸及表达 GPC3嵌合抗原受体蛋白的 T淋巴细胞 技术领域
本发明涉及肿瘤细胞治疗领域， 更具体地， 涉及对特异性表达 GPC3的上皮来源的肿 瘤的转基因 T淋巴细胞治疗领域。 背景技术 说 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， 属于书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。 考虑到 GPC3在肝细胞肝 癌， 黑色素瘤等肿瘤中特异性的高表达， 其被认为是肿瘤免疫治疗的一个候选靶标。
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。
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 [Grupp SA, et al. Adoptive cellular therapy. Curr Top Microbiol Immunol. ,.2011;344: 149-72 ]。 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 CD3 ζ或 FCSRIY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 (Maj or 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 [Schmitz M, et al. Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer. J Biomed Biotechnol, 2010, doi : 10. 1155/2010/956304.]。
嵌合抗原受体包括胞外结合区， 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) CD3 ζ或 FCSRIY及共剌激信号分子 CD28、 CD 137、 CD 134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- 1 1036.]。
随后发展的第二代 CAR T淋巴细胞加入了 CD28或 CD 137(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-/CD 137-ITAM。 胞内信号区发生的 B 7/CD28或 4- 1 BBL/CD 137共剌激作用引起 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-CD 137-ITAM或 scFv-TM-CD28-CD 134-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 CD 137 domains. PNAS, 2009, 106(9): 3360-3365. ] 0
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]。 另夕卜， CAR 中过多的共剌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" 至夂病人粹死 [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， 本领域存在着对克服上述缺陷的编码 GPC3嵌合抗原受体蛋白的淋巴细胞肿瘤 治疗方案的强烈需求。 发明内容
本发明的第一方面涉及编码一种表达于 T淋巴细胞表面的 GPC3嵌合抗原受体蛋白的 核酸， 其表达的嵌合抗原受体蛋白使得表达该受体的 T淋巴细胞针对高表达 GPC3的肿瘤 细胞具有高度特异性的细胞毒性作用。所述 GPC3嵌合抗原受体蛋白包含顺序连接的胞外 结合区， 跨膜区和胞内信号区， 其中所述胞外结合区包含特异性识别 GPC3的 C末端表位 的单链抗体 scFv(GPC3)。上述嵌合抗原受体蛋白的胞外结合区通过 CD8铰链区与 CD8或者 CD28的跨膜区相连接， 跨膜区后紧接胞内信号区。
本发明的核酸序列可以是 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。
本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%， 较佳地至少 70%， 更 佳地至少 80%， 最佳地至少 90%或至少 95%相同性的多核苷酸。 本发明特别涉及在严格条 件下与本发明所述多核苷酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指： （1)在 较低离子强度和较高温度下的杂交和洗脱， 如 0.2 X SSC, 0.1%SDS， 60 °C ; 或 (2)杂交时 加有变性剂， 如 50% / 甲酰胺， 0.1%小牛血清 /0.1% Ficoll, 42°C等； 或 仅在两条序 列之间的相同性至少在 90%以上,更好是 95%以上时才发生杂交。 并且， 可杂交的多核苷 酸编码的多肽与 SEQ ID NO:22-25之一所示的成熟多肽有相同的生物学功能和活性。
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. 201 1 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蛋白中的单链抗体部分。 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。
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 W096/33735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。
嵌合抗原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 α β或者 γ δ两条链组成。 在本发 明的一个实施方案中，跨膜区选自 CD8 a或者 CD28的跨膜区。此夕卜， CD8 α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抗原受体 CAR的 靶点识别结构域 s cF V和胞内信号区连接起来。
胞内信号区可以选自 CD3 ζ， FcsRIy, CD28， CD137, CD134蛋白的胞内信号区， 及其组合。 CD3分子由五个亚单位组成， 其中 CD3 ζ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。 CD3 δ Z (后文简称 DZ) 是截短的不具有 ΙΤΑΜ基序的 CD3 ζ序列， 在本发明实践中一般作为阴性对照的构建。 FCSRIY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 CD3 类似。 此外如前所述， CD28， CD137, CD134是共剌激信号分子， 在 与各自配体结合后其胞内信号区段产生的共剌激作用引起 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 GPC3嵌合抗原受体蛋白可以选自按如下方式顺序连接的嵌 合抗原受体蛋白：
scFv(GPC3)-CD8-CD3 ζ，
scFv(GPC3)-CD8-CD137-CD3 ζ，
scFv(GPC3)-CD28a-CD28b-CD3 ζ，
scFv(GPC3)-CD28a-CD28b-CD137-CD3 ζ，
及其组合， 其中相关嵌合抗原受体蛋白中 CD28a代表 CD28分子的跨膜区， CD28b代 表 CD28分子的胞内信号区。上述各种靶向 GPC3的嵌合抗原受体统称为 SCFv(GPC3)-CAR。 在本发明的一个实施方案中， 本发明的核酸具有如 SEQ ID NO: 18~21所述的序列。在 本发明的另一个实施方案中， 本发明的核酸是编码具有如 SEQ ID NO: 22-25之一的嵌合 抗原受体蛋白的核酸。
本发明的第二方面包括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 核酸的载体。 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 pPWT-eGFP o 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 pPWT-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 -1 α ( elongation factor- 1 α , EF-1 α ) 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。 在一个具体实施方案中， 本发明 的载体具有如 SEQ ID NO: 27-30之一的核酸序列。
本发明的第三方面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。
本发明的第四方面包括一种转基因 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 CD 19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OFl-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 τ淋巴细胞到达临 床级数量的时间等优点。 在该转基因 τ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， 本发明的表面表 达嵌合抗原受体的转基因 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 τ淋巴细胞可以有效地用于肿瘤的免疫治疗。
在一个实施方案中， 本发明的 τ淋巴细胞， 其表面表达一种嵌合抗原受体， 所述嵌合 抗原受体由 SEQ ID NO: 18-21之一的核酸编码表达。 在另一个实施方案中， 本发明的转 基因 T淋巴细胞表面表达一种嵌合抗原受体， 所述嵌合抗原受体的氨基酸序列选自 SEQ ID NO:22-25之一。 附图说明
图 1 显示的是作为示例的本发明 的包含编码 CAR序列的慢病毒载体 pWPT-eGFP-F2A-CAR的结构示意图。
图 2 显示的是作为示例的包含在慢病毒载体中的本发明的 CAR的不同区之间连接关 系的示意图， 其中由核糖体跳跃序列 F2 A连接的 eGFP和 svFv(GPC3)特异性嵌合抗原受 体。
图 3显示的是 Mlul 和 Sail 双酶切鉴定实施例 1的慢病毒质粒的核酸电泳图。 其中 Ml 是 DS2000分子量标记物 （广州东盛生物科技有限公司）； M2是 Hind ///标记物 （广州东 盛生物科技有限公司）。 泳道 1-5分别是
1 : pWPT-eGFP-F2A-GPC3- δ Ζ;
2: pWPT-eGFP-F2A-GPC3-Z;
3 : pWPT-eGFP-F2A-GPC3-BBZ;
4: pWPT-eGFP-F2A-GPC3-28Z;
5 : pWPT-eGFP-F2A-GPC3-28BBZ。
图 4显示的是本发明实施例 2的病毒感染 CTL (细胞毒性 T淋巴细胞；)后细胞表达的 eGFP的流式细胞技术检测结果。
图 5 显示的是本发明实施例 2的表达不同嵌合抗原受体 （CAR+) 的 CTL细胞的体外 生长情况。 图中显示在病毒感染后第 14天， 表达不同嵌合抗原受体的 CTL细胞体外扩增 200倍。
图 6A显示的是流式细胞仪检测各肝细胞肝癌细胞系上 GPC3的表达水平， GPC3在细 胞上的表达以平均荧光强度 （Mean Fluorescence Intensity, MFI) 表示； 图 6B显示的是上 述各细胞系上 GPC3的表达水平的 western blot结果， 其中 GAPDH为上样内参对照。
图 7A显示的是在采用靶向 GPC3的 CAR T细胞对 Huh-7移植瘤治疗后随时间变化的各 组内无瘤小鼠的百分比； 图 7B显示的是每组 6只小鼠处死后各自体内肿瘤体积大小。
图 8A显示的是实施例 5的各组小鼠的残余肿瘤的体积的对比结果； 图 8B显示的是实 施例 5的各组小鼠的残余肿瘤的重量的对比结果； 图 8C显示的是过继输注 T淋巴细胞 1周 后检测实施例 5的各组小鼠外周血 T细胞存活数量； 图 8 D显示的是实施例 5的各组小鼠处 死后所示的肿瘤情况； 图 8E显示的是实施例 5中 GPC3-28BBZ CAR T淋巴细胞治疗后小鼠 肿瘤消退情况 （与 Mock组对比） 。
图 9显示是对实施例 5的过继输注的 T细胞在各组小鼠的移植瘤组织中聚集的免疫组 织化学染色检测结果。
图 10 A显示的是实施例 6的各组小鼠的移植瘤体积。 图 10 B显示的是实施例 6的各组小 鼠在实验结束后取瘤组织称重的结果。 图 10C显示的是实施例 6的各组小鼠的肿瘤组织； 图 10D显示的是实施例 6的各组小鼠在最后一次过继转移 T细胞 1周后检测外周血中 T细胞 的数量的结果。
图 11A和 11B分别显示的是实施例 7的各组小鼠的肿瘤的生物荧光强度和肿瘤的实际 成像图片。 图 11C和 D分别显示的实施例 7的各组小鼠的腹部荷瘤情况 （体外） 和解剖所 见的肿瘤情况。 图 11E显示的是最后一次过继转移 T细胞 1周后检测各组小鼠外周血中 T细 胞的数量的结果。 图 11F显示的是最后一次过继转移 T细胞 1周后检测各组小鼠外周血中 CAR阳性的 T细胞的数量的结果。 图 1 1G显示的是实施例 7的各组小鼠的在肿瘤接种后随 时间的存活率。 具体实施方式
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 而不能理解为用于限制本发明的范围。 下面实施例中如未注明具体条件的实验方法， 则 按照常规条件如 Sambrook等， "分子克隆： 实验手册 （New York: Cold Spring Harbor laboratory Press, 1989) " 中所述的条件， 而在实施例中明确说明有试剂公司说明书时， 则按照说明书所建议的条件进行。 实施例 1. 表达本发明核酸编码的嵌合抗原受体蛋白的慢病毒质粒的构建及病毒包 装 下表 1解释了本发明示例的嵌合抗原受体各部分的连接顺序， 该连接还可参见图 2中 所示。
表 1
Figure imgf000011_0001
1.核酸片段的扩增
(1) scFv(GPC3)序列的扩增 scFv(GPC3)序列的扩增以本实验室构建的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 ΝΟ:1 ) 和 下 游 引 物 5 ' -gcgctggcgtcgtggttgaggagacggtgaccag-3 ' (SEQ IDNO:2) 用于扩增 scFv(GPC3); 目的扩 增条带大小均为 746bp。 PCR扩增条件为预变性： 94°C， 4min; 变性： 94°C， 40s; 退火： 58°C， 40s; 延伸： 68°C， 40s; 进行 25个循环， 然后总延伸 68°C， 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。
(2)嵌合抗原受体其他部分的核酸序列
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: 1~4为模板通过 PCR方式获得。 具体地， 其 中如下各种 GPC3嵌合抗原受体蛋白除 scFv (GPC3)外的 CAR部分， 其中 CD8-CD3 ζ (Ζ) 和 CD28a-CD28b-CD137-CD3 ζ (28ΒΒΖ)， 分别以 scFv(EFGR)-CD8-CD3 ζ (专利申请 201310108532.2中 SEQ ID NO: 1) 禾口 scFv(EFGR)-CD28a-CD28b-CD137-CD3 ζ (专利申请 201310108532.2中 SEQ IDNO:2) 为模板， 采用上游引物 5'-accacgacgccagcgccgcgaccac-3， (SEQ ID NO :3) 禾口下游弓 I物 5'-gaggtcgacctagcgagggggcagggcctgcatgtgaag-3' (SEQ ID NO:4) 扩增， PCR扩增条件为预变性： 94°C， 4min; 变性： 94°C， 40s; 退火： 60°C， 40s; 延伸： 68°C， 40s; 进行 25个循环， 然后总延伸 68°C， 10min。 目的条带分别为 549bp 和 816bp， 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。
另夕卜， CD8-CD3 delta ζ (delta Z， 简称 δΖ)， CD8-CD137-CD3 ζ (ΒΒΖ)和 CD28a-CD28b-CD3 ζ (28Z)分别通过如下方式获得：
Α) 加 lml Trizol ( Invitrogen公司） 于 1 X 107健康人外周血单个核细胞 （上海市血液 中心提供） 中裂解细胞后， 采用酚-氯仿法抽提总 RNA， 采用 ImProm-IFM 逆转录试剂盒 ( promaga公司） 逆转录制备 cDNA。 以上述制备的 cDNA为模板， 分别：
( i ) 以上游引物 5 ' -gtcaccgtctcctcaaccacgacgccagcg-3 ' ( SEQ ID NO:5 ) 和下游引物 5 ' -ggtgataaccagtgacaggag-3 ' ( SEQ ID NO:6) 扩增获得 CD8 α铰链区-跨膜区， PCR 扩增 条件为预变性： 94°C， 4min; 变性： 94°C， 30s; 退火： 58°C， 30s; 延伸： 68°C， 30s; 进行 25个循环， 然后总延伸 68°C， lOmino 条带理论大小为 198bp， 扩增产物经琼脂糖电 泳确认与理论大小一致。
( ii ) 以上游引物 5 ' -gtcaccgtctcctcaaccacgacgccagcg-3 ' ( SEQ ID NO： 5 )和下游引物 5 ' -gaggtcgacctacgcgggggcgtctgcgctcctgctgaacttcactctggtgataaccagtg-3 ' ( SEQ ID NO :7) 扩 增获得 CD8 a铰链区-跨膜区 -delta Z (即 CD8-CD3 delta ζ )， PCR 扩增条件同上。 条带理 论大小为 261bp， 扩增产物经琼脂糖电泳确认与理论大小一致。
( iii ) 以上游引物 5 ' -ttttgggtgctggtggtggttgg-3 ' ( SEQ ID NO:8 ) 和下游引物 5 ' -gctgaacttcactctggagcgataggctgcgaag-3 ' ( SEQ ID NO:9) 扩增获得 CD28跨膜区-胞内信 号区片段， PCR 扩增条件同上， 条带理论大小为 219bp， 扩增产物经琼脂糖电泳确认与 理论大小一致。
( iiii ) 以上游引物 5 ' -aaacggggcagaaagaaactc-3 ' ( SEQ ID NO: 10 ) 和下游引物 5'-cagttcacatcctccttc-3 ' ( SEQ ID ΝΟ: 11 )扩增获得 CD137胞内区， PCR扩增条件同上， 条 带理论大小为 126bp， 扩增产物经琼脂糖电泳确认与理论大小一致。
( iiiii ) 以上游引物 5'-cactggttatcaccagagtgaagttcagcaggagc-3' ( SEQ ID NO: 12) 禾口下 游引物 5'-gaggtcgacctagcgagggggcagggcctgcatg-3， ( SEQ ID NO: 13 ) 扩增获得 CD3 zeta 信 号区， PCR 扩增条件同上， 条带理论大小为 339bp， 扩增产物经琼脂糖电泳确认与理论 大小一致。
B) 核酸片段的拼接
( i ) 以上游引物 5'-accacgacgccagcgccg-3， ( SEQ ID NO: 14 ) 和下游引物
5 ' -cacccagaaaataataaag-3 ' ( SEQ ID NO: 15 ) 拼接获得 CD8 α铰链区 -CD28跨膜区， 拼接 条件： CD8 a铰链区 （50ng) + CD28跨膜区 （50ng) 预变性： 94°C， 4min; 变性： 94°C，
30s; 退火： 60°C， 30s; 延伸： 68°C， 30s, 进行 5个循环， 然后总延伸 68°C， 10min， 补 充 DNA聚合酶及上下游引物后 PCR扩增 25个循环， 扩增条件为预变性： 94V， 4min; 变 性： 94°C， 30s; 退火： 60°C， 30s; 延伸： 68°C， 30s, 进行 25个循环， 然后总延伸 68°C， lOmino 理论大小为 216bp。 扩增产物经琼脂糖电泳确认与理论大小一致。
(ii) 以上游引物 5'-aaacggggcagaaagaaactc-3， ( SEQ ID NO: 10) 和下游引物 5'- gaggtcgacctagcgagggggcagggcctgcatg-3' ( SEQ ID NO:13) 拼接扩增获得 CD137-CD3 ζ， 即为 ΒΒΖ， 拼接和 PCR 扩增条件同上。 条带理论大小为 478bp， 扩增产物经琼脂糖电泳 确认与理论大小一致。
(iii) 以上游引物 5'-gtcaccgtctcctcaaccacgacgccagcg-3， (SEQ ID NO:5) 和下游引物 5 ' -gaggtcgacctagcgagggggcagggcctgcatg-3 ' (SEQ ID NO:13) 将 CD8 α铰链区-跨膜区与 BBZ拼接及 PCR获得目的片段： CD8a铰链区-跨膜区 -BBZ胞内区， SP CD8-CD137-CD3 ζ， 拼接和 PCR扩增条件同上。 理论大小为 691bp。 扩增产物经琼脂糖电泳确认与理论大 小一致。
(iiii) 以上游引物 5'-gtcaccgtctcctcaaccacgacgccagcg-3， (SEQ ID NO:5)和下游引物 5 ' -gaggtcgacctagcgagggggcagggcctgcatg-3 ' (SEQ ID NO:13) 将 CD8 α铰链区 -CD28跨膜 区-胞内区与 Z采用同上的拼接和 PCR扩增获得目的片段： CD8a铰链区 -CD28跨膜区 -28Z 胞内区， 即 CD28a-CD28b-CD3 ζ， 拼接和 PCR扩增条件同上。 条带理论大小为 706bp， 扩 增产物经琼脂糖电泳确认与理论大小一致。
(3) 3' 端带有 F2A及 CD8 α信号肽的 eGFP核酸片段的获得 以上游弓 I物 5 ' -cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 ' (SEQ ID ^^0:16)和下游引物5' §§£；£¾§0§§0§1§§&§0&§ -3' (SEQ ID NO: 17) 扩增慢病毒载体中 3' 端 带有 F2A及 CD8 α 信号肽的 eGFP核酸片段， 以专利申请 201310108532.2中公开的 pWPT-eGFP-F2A-806-Z为模板， PCR扩增条件为预变性： 94°C， 4min; 变性： 94°C， 40s; 退火： 56°C， 40s; 延伸： 68°C， 50s, 进行 25个循环， 然后总延伸 68°C， 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。
2.核酸片段的拼接 分别将如前述段落 "(2) 嵌合抗原受体其他部分的核酸序列" 中扩增获得的片段 CD8-CD3 ζ， CD8-CD137-CD3 ζ， CD28a-CD28b-CD3 ζ， CD28a-CD28b-CD137-CD3 ζ 与等摩尔的前述段落 "（3) 嵌合抗原受体其他部分的核酸序列" 中扩增获得的 3' 端带有
F2A及 CD8a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) 三片段按图 2所示模式拼接并 PCR， 拼接条件为： 预变性： 94°C， 4min; 变性： 94°C， 40s; 退火： 60°C， 40s; 延伸： 68 °C， 140s, 进行 5个循环， 然后总延伸 68 °C， 10min， 补充 DNA聚合酉每及上游弓 I物 5 '-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 ' (SEQ ID NO: 16)和下游引物 5' - gaggtcgacctagcgagggggcagggcctgcatg -3 ' ( SEQ ID NO: 13 ) 后 PCR扩增 25个循环， 扩增条件为预变性： 94°C， 4min; 变性： 94°C， 40s; 退火： 60°C， 40s;延伸： 68 °C， 140s,进行 25个循环，然后总延伸 68 °C， lOmin。扩增获得 eGFP-GPC3-Z、 eGFP-GPC3-BBZ、 eGFP-GPC3-28Z及 eGFP-GPC3-28BBZ理论大小分别为 2161bp， 2278bp, 2302bp和 2428bp。 扩增产物经琼脂糖电泳确认与理论大小一致。
在与同上相同的实验条件下， 采用上游引物 5 '-gatgttgtgatgactcagtctc-3 ' ( SEQ ID NO： 1 )和下游引物 5 ' - gaggtcgacctagcgagggggcagggcctgcatg -3 ' ( SEQ ID NO： 13 )， 在不添 加 3 ' 端带有 F2A及 CD8 a信号肽的 eGFP核酸片段 （质量约为 50ng)， 可以获得编码 GPC3 嵌合抗原受体蛋白的核酸 GPC3-Z(SEQ ID NO: 18)、 GPC3-BBZ(SEQ ID NO: 19)、 GPC3-28Z(SEQ ID NO: 20)及 GPC3-28BBZ(SEQ ID NO: 21)序列， 上述各核酸序列分别编 码具有如下氨基酸序列的 GPC3嵌合抗原受体蛋白， GPC3-Z(SEQ ID NO: 22)、 GPC3-BBZ(SEQ ID NO: 23)、 GPC3-28Z(SEQ ID NO: 24)及 GPC3-28BBZ(SEQ ID NO: 25) 。
此外， 分别将如前述段落" (2) 嵌合抗原受体其他部分的核酸序列 "中扩增获得的片 段 CD8-CD3delta e片段与等摩尔的前述段落 "（3 ) 嵌合抗原受体其他部分的核酸序列" 中 扩 增 获 得 的 片 段 及 等 摩 尔 的 scFv ( GPC3 ) 采 用 上 游 引 物 5'- cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 ' (SEQ ID NO: 16)禾口下游弓 |物 5 ' -gaggtcgacctacgcgggggcgtctgcgctcctgctgaacttcactctggtgataaccagtg-3 ' ( SEQ ID NO :7 ) 同 上条件进行拼接并 PCR，扩增获得 eGFP-GPC3- δ Ζ ( SEQ ID ΝΟ:31 )，理论大小为 1858bp， 扩增产物经琼脂糖电泳确认与理论大小一。
2-1. 编码无关对照 2D3-28BBZ CAR的慢病毒载体的构建 （即
pWPT-eGFP-F2A-2D3-28BBZ )
A. 抗 EGFR新型异构体 EGFRvA的胞内区多肽 （WTE) 的 2D3单链抗体核酸片段的获 得 以抗 WTE多肽的杂交瘤 2D3细胞株（由上海锐劲生物科技有限公司制备) mRNA为模 板， 用 RT-PCR Kit反转录， 合成 cDNA第一链。 以 cDNA 第一链为模板， 分别以 Heavy
Primers ^ Light Primer Mix为引物 （引物购自上海锐劲生物科技有限公司；)， 扩增 VH、 VL 基因， PCR条件： 94°C预变性 4 min， 94°C变性 40 s， 55 °C退火 40 s， 68 °C延伸 40 s， 30个循环后 68 °C延伸 7min。琼脂糖凝胶电泳检测 PCR产物，胶回收试剂盒分别回收 VH、 VL片段。
再以 VH、 VL片段互为模板， Linker-Primer Mix为引物 (引物购自上海锐劲生物科技有 限公司）， 重叠 PCR法拼接 VH和 VL片段成 scFv， PCR条件： 94°C变性 lmin， 63 °C退火延 伸 4min， 共进行 7个循环。 7个循环后在 50μ1反应体系中补加 Linker-Primer Mix、 聚合酶 缓冲液和双蒸水， 继续 PCR。 PCR条件： 94°C预变性 4min， 94°C变性 40 s， 58 °C退火 40 s, 68 °C延伸 lmin， 30个循环后 68 °C延伸 7min。 琼脂糖电泳检测 PCR产物， 胶回收 试剂盒回收 scFv片段。
B.抗 WTE多肽 2D3单链抗体的表达及活性检测
Sfi J Not 双酶切上述步骤中 scFv片段禾 PpCANTAB 5E载体 (购自 Pharmacia公司）， 胶回收酶切片段， 16 迪接过夜后转化至感受态 E. coli HB2151， 次日从转化平板上挑取
20个单克隆进行 30 培养， 培养至 OD600为 0.4〜0.6时， 加入终浓度为 0.05mmol /L IPTG 诱导表达过夜 (18h)。 离心取上清， ELISA分析培养上清中可溶性 scFv表达情况。 具体而 言， 抗原 WTE-BSA (由上海锐劲生物科技有限公司制备)分别以 50ng/孔 （lng/μΐ , 50μ1/孔) 包被 96孔板， 37 孵育 2h， 5%PBS 脱脂奶粉 （光明乳业股份有限公司） 37 ¾闭21， 用
0.1M 的磷酸缓冲液 （PBS) 洗涤 3次，将上述中诱导表达培养上清加入 96孔板，每孔 50μ1，
37 孵育 lh。 PBST(PBS+0.05%Tween20)洗涤 3次后， 以 HRP标记的 anti-E tag抗体 (购自上 海锐劲生物科技有限公司） 1 : 1000稀释， 50μ1/孔， 37 孵育 lh。 PBST洗涤 3次， 加入以 1：
1000稀释的羊抗鼠 IgG-HRP (购自 Santa Cruz公司）， 37 孵育 lh。 PBST洗涤 5次， 力 PABTS 显色液 100μΙ 孔， 37 ¾光显色 10min。 使用 Bio-Rad Model 680酶标仪， 在波长 405nm下 检测吸光度值， 比阴性对照孔吸光度值高 2倍以上判断为阳性。 取 OD值最高的克隆 2D3-3 测序后抽取质粒 pCANTAB 5E 2D3-3 scfv,作为本专利所用的无关对照 CAR所用的单链抗 体 2D3并分别与如前述段落 " (2) 嵌合抗原受体其他部分的核酸序列" 中扩增获得的片段
CD28a-CD28b-CD137-CD3 ζ与等摩尔的前述段落 " （3 ) 嵌合抗原受体其他部分的核酸 序列" 中扩增获得的 3 ' 端带有 F2A及 CD8 a信号肽的 eGFP核酸片段 （质量约为 50ng) 三 片段按图 2所示模式拼接并 PCR， 拼接条件为： 预变性： 94°C， 4min; 变性： 94°C， 40s; 退火： 60°C， 40s; 延伸： 68 °C， 140s, 进行 5个循环， 然后总延伸 68 °C， 10min， 补充 DNA 聚合酉每及上游弓 I物 5'-cttacgcgtcctagcgctaccggtcgccaccatggtgagcaagggcgaggag-3 ' (SEQ ID
NO: 16)和下游引物 5 '- gaggtcgacctagcgagggggcagggcctgcatg -3 ' ( SEQ ID NO: 13 ) 后 PCR 扩增 25个循环， 扩增条件为预变性： 94°C， 4min; 变性： 94°C， 40s; 退火： 60°C， 40s; 延伸： 68°C， 140s, 进行 25个循环， 然后总延伸 68°C， 10min。 扩增获得 eGFP-2D3-28BBZ 理论大小为 2443bp。 扩增产物经琼脂糖电泳确认与理论大小一致。
3.慢病毒质粒载体的构建
作为示例的构建本发明的慢病毒质粒载体使用的载体系统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及基于空载体 pPWT-eGFP的编码目的基因 CAR的重 组表达载体。
在空载体 pPWT-eGFP中， 延长因子 -1 α ( elongation factor- 1 α , EF-1 α ) 的启动子调 控增强型绿色荧光蛋白 （enhanced green fluorescent protein, eGFP) 的表达， 而在编码目 的基因 CAR的重组表达载体中通过来自口蹄疫病毒的核糖体跳跃序列 （food and mouth disease virus, FMDV, ribosomal skipping sequence, F2 A)实现 eGFP与目的基因 CAR的共 表达。 F2A是来自口蹄疫病毒的 2A (或称为 "自剪切多肽 2A")的一段核心序列， 具备 2A的 "自剪切" 功能， 可以实现上游和下游基因共表达。 2A由于其剪切效率高、 上下游基因 表达平衡性高及自身序列短小的优点为构建基因治疗多顺反子载体提供了一种有效的可 行策略。尤其在基于嵌合抗原受体基因修饰 T淋巴细胞的免疫治疗中多应用该序列实现目 的基因与 GFP或者 eGFP的共表达， 通过检测 GFP或者 eGFP即可间接检测 CAR的表达。
本实施例构建了由 F2A相连的 eGFP与特异性 CAR共表达的慢病毒表达载体， 统称为 pWPT-eGFP-F2A-CAR。上述步骤 2中获得的目的基因 eGFP-F2A-CAR通过 Mlul和 Sail限制 性内切酶双酶切， 连入同样双酶切的 pWPT载体中， 从而构建表达各嵌合抗原受体的慢病 毒载体。 构建成功的载体经 Mlul和 Sail酶切鉴定 （图 3 ) 及序列测定正确后， 可以准备用 于慢病毒包装。 如前所述， eGFP-F2A-CAR转录为一条 mRNA， 但最终翻译为 eGFP和抗 GPC3嵌合抗原受体两条肽链，其中在 CD8 a信号肽的引导下抗 GPC3嵌合抗原受体将定位 在细胞膜上。 用作阴性对照的 eGFP-GPC3- δ Ζ在插入到慢病毒质粒载体后在细胞膜表面 表达含有截短的 δ Ζ的 GPC3嵌合抗原受体 (GPC3- δ Ζ)， 其氨基酸序列为 SEQ ID NO:32。
得到的含有各目的 CAR的载体的核酸全序列如下： pWPT-eGFP-F2A-GPC3- δ Z(SEQ ID NO :26)； pWPT-eGFP-F2A-GPC3-Z(SEQ ID NO :27)； pWPT-eGFP-F2A-GPC3-BBZ(SEQ ID NO:28) ； pWPT-eGFP-F2A-GPC3-28Z(SEQ ID NO:29) ； pWPT-eGFP-F2 A-GPC3 -28BBZ(SEQ ID NO:30)。
4.质粒转染 293T包装慢病毒 以 6X106的密度接种培养至第 6〜10代的 293T细胞 （ATCC: CRL-11268) 于 10cm培 养皿中， 37°C， 5% C02培养过夜准备用于转染。 培养基为含 10%胎牛血清 （PAA公司） 的 DMEM (PAA公司） ， 次日， 在转染前约 2小时更换培养液为无血清 DMEM。
转染的步骤如下：
4.1 将 20yg空质粒 pWPT-eGFP (mock对照） 或 20 μ g的各目的基因质粒 pWPT-eGFP-F2A-CAR,分别与 15 μ g包装质粒 PAX2:和 6 μ g包膜质粒 pMD2.G: ， 溶入 500 LMillQ水中， 混匀，
4.2 I¾jmA62yL2.5MCaCl2 (Sigma 公司） ， 以 1200rpm/min vortex混匀， 4.3 最后逐滴加入 500 yL2 XHeBS (280mMNaCl, lOmMKCl, 1.5mMNa2HP04 •2H20, 12mM葡萄糖， 50mM Hepes ( Sigma 公司）， pH7.05, 0.22 μ M过滤除菌）， 1200rpm/min vortex混勾 10s,
4.4 立即逐滴加入培养皿中， 轻轻摇匀， 37°C， 5%C02， 培养 4〜6h后， 更换为 含 10%胎牛血清的 DMEM。
在转染次日观察转染效率（即呈绿色荧光的细胞比例）， 〜80%的阳性转染效率即为 转染实验成功。 在转染 48h或 72h后， 使用 0.45μιη滤器 （Millipore公司） 过滤收集病毒， 然后采用 Beckman Optima L-100XP超速离心机 28000rpm， 4°C离心 2小时， 弃离心上清， 离心所得沉淀用 1/10 1/50原液体积的 Quantum 007培养液 （PAA公司） 进行重悬， 以 100 μ L/管分装冻存于 -80°C， 以待病毒滴定或感染 T淋巴细胞。
5.测定包装有 mock或者 eGFP-F2A-CAR的慢病毒滴度
第一天， 以 lX105/mL接种 293T细胞于 96孔培养板， 100μΙ 孔， 37°C， 5%C02培养， 培养液为含 10%胎牛血清的 DMEM。 第二天， 弃 50μΙ 孔培养上清， 补加 50 μί/孔新鲜 上述培养液， 并含终浓度为 6wg/mL的 polybrene, 37°C， 5% C02孵育 30min。 力 Ρ10μΙ 孔的病毒原液或 1 WL/孔的病毒浓缩液， 5倍稀释， 4个梯度， 两个复孔， 37°C， 5% C02 培养。 感染 48h后， 流式细胞仪检测 eGFP， 以阳性率为 5〜20%的细胞数为宜， 计算滴度 (U/mL)=阳性率 X稀释倍数 X 100X 104。 磷酸钙转染法包装的上述包含 mock (即空载体） 对照和各 eGFP-F2 A-CAR的病毒的滴度均为约 0.5〜2 X 106U/mL的水平， 经浓缩后所测的 病毒滴度约为 2X 107U/mL。
实施例 2. 重组慢病毒感染 CTL细胞
由健康人外周血通过密度梯度离心法获得人外周血单个核细胞 （上海市血液中心提 供） ， 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 I X 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(上海华新生物高技术有限公司;)剌激培养 24h。 然 后以 MOI 5用上述重组慢病毒感染 CTL细胞。 感染后的细胞每隔一天采用 5 X 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。
感染的 CTL细胞在培养第 8天时通过流式细胞检测各不同嵌合抗原受体表达， 由于 eGFP与 CAR共表达， 检测 eGFP的阳性细胞即为表达嵌合抗原受体的阳性细胞 （图 4) 。 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 率如下表所示。 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 CAR+ CTL细胞。
表 2
Figure imgf000018_0001
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 X 105/ml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)， 培养第 14天约有 200倍的扩增 (见图 5 ) ， 表明表达不同嵌合抗原受体的 CTL细胞在体外能够进行一定数量 的扩增， 为后续体外毒性试验及体内试验提供了保证。 实施例 3. 表达嵌合抗原受体细胞的体外毒性效果实验
体外毒性实验使用的靶细胞为如下表 3所示的肝细胞癌细胞系， 效应细胞为如实施例 2所验证的体外培养 12天的 FACS检测嵌合抗原受体表达的阳性细胞记为嵌合抗原受体阳 性 （CAR+) 的 CTL。 表 3
Figure imgf000019_0002
流式细胞仪及 western blot检测肝细胞肝癌细胞系 HepG2、 Huh-7、 Hep3B、 PLC/PRF/5 和 SK-HEP-1上 GPC3的表达水平， 流式结果如图 6A所示， GPC3在细胞上的表达以平均荧 光强度 （Mean Fluorescence Intensity, MFI) 表示。 另外采用 western blot的方法进一步验 证各细胞系 GPC3的表达水平， 结果如图 6B所示， 其中 GAPDH为上样内参对照。 结果表 明 GPC3在 4种肝细胞肝癌细胞系 HepG2、 Huh-7 ^ Hep3B禾 PPLC/PRF/5上有不同水平的表 达， 在 SK-HEP- 1的没有检测到 GPC3的表达。
各实验组和各对照组包含如下材料：
各实验组： 各靶细胞 +表达不同嵌合抗原受体的 CTL，
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。 CytoTOX 96®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。
效靶比视情况分别为 3 : 1， 1 : 1禾 P l :3， 靶细胞数量为 10000/孔 C5(^L 2x l05/mL；)， 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。
细胞毒性计算公式为： 细胞毒性％ =
Figure imgf000019_0001
X 1 00%
对照组 1 -对照组 2 实验结果表明: 具体如表 4所示， 本发明的表达嵌合抗原受体 GPC3-Z CAR+的 CTL和 GPC3-28BBZ CAR+的 CTL对 GPC3阳性的肝细胞癌细胞系 HepG2、 Huh-7、 Hep3B禾口 PLC/PRF/5细胞均表 现出非常显著的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， 而对 GPC3阴性的肝细胞癌细胞系 SK-HEP-1没有特异性细胞毒性作用。 其中 GPC3-28BBZ在效靶比 3： 1时对对 GPC3阳性的肝癌细胞 HepG2的细胞毒性高达 97%， 对 GPC3阳性的肝癌细胞 PLC/PRF/5的细胞毒性高达 84%。
效靶比依赖性的数据进一步显示本发明中表达靶向 GPC3的嵌合抗原受体的 CTL对 GPC阳性的肝癌细胞的特异性细胞毒性作用。
相比较而言， 转导含有 mock质粒 （不携带 GPC3-CAR的空质粒载体 pPWT-eGFP ) 或 者无关对照 CAR 2D3-28BBZ的病毒转染的作为空白对照的 CTL显示对上述五种细胞的细 胞毒性作用均非常低， 显示出对 GPC3表达的不敏感。 另外， 转导含有截短形式的 CD3 ξ 的 GPC3 -δΖ的 CTL由于没有胞内信号区， 亦与转导 mock和 2D3 -28BBZ的 CTL细胞相似的 毒性作用。 综上， 本发明中表达靶向 GPC3的嵌合抗原受体 （GPC3-Z和 GPC3-28BBZ ) 的 CTL显示出对 GPC3阳性的肝癌细胞具有特异性毒性作用，并且表达携带共剌激分子 CD28 和 CD137的信号区的 GPC3-28BBZ CAR T细胞具有较无二者信号区的 GPC3-Z CAR T细胞 更强的毒性作用。 表 4
Figure imgf000020_0001
SK-HEP-1 12.±3.6 9.8±6.6 2.1±3. 13±3.9 9.8±6.6 3.9±2.8
4
16±8.9 7.7±4.7 1.3±2. 5.4±4. 0.5±2.9 0.05±2.
HepG2
5 3 3
18±9.2 12.6±4. 4.8±3. 17±2.2 13±3.3 4.2±1.3
Huh-7
5 1 实施例 4靶向 GPC3的 CAR T细胞对髙表达 GPC3的 Huh-7移植瘤的早期治疗实验 动物分组： 40只 6~8周龄 NOD/SCID小鼠随机分为 6组， 每组 6~7只， 实验组分别为
GPC3-Z CAR T淋巴细胞组、 不同效靶比 （1 : 1禾卩 1 :2) 的 GPC3-28BBZ CAR T淋巴细胞组， 对照组分别为 GPC3-5Z CAR T淋巴细胞、 mock CAR T淋巴细胞对照组以及盐水对照组。
接种： 第 0天， 腹腔注射环磷酰胺 200mg/kg， 第 1天， 于小鼠右侧腋部皮下接种 Huh7 细胞 (2 106/只）分别与0?。3-2、 Mock、 GPC3-28BBZ ( 1 : 1和 1 :2) 组 CAR T淋巴细胞及 GPC3-5Z CAR T淋巴细胞与效应细胞 1 : 1的混合细胞， 盐水组仅接种 Huh7细胞 (2x 106/只）。
实验结果显示表达 GPC3-Z和 GPC3-28BBZ CAR T淋巴细胞能够特异性的抑制 GPC3阳 性的 Huh-7细胞的成瘤， 至对照组小鼠肿瘤长至 2000mm3结束实验 （移植瘤接种后第 28 天） ， GPC3-Z CAR T淋巴细胞治疗组， 2/6小鼠没有肿瘤生长， 而 GPC3-28BBZ CAR T 淋巴细胞治疗组在效靶比 1： 1及 1： 2组 6只小鼠均没有肿瘤生长， 而对照组如 Mock组和 GPC3-5Z CAR T淋巴细胞组及生理盐水组对小鼠成瘤没有影响 （图 7A) 。 图 7B为每组 6 只小鼠处死后所示各自体内肿瘤大小。 实施例 5靶向 GPC3的 CAR T细胞对髙表达 GPC3的 Huh-7皮下移植瘤的治疗实验
接种肿瘤： 收集处于对数生长期并生长良好的 Huh-7细胞， 采用生理盐水调整细胞密 度为 l x l07/ml， 注射体积为 20(^L ( 2>< 106/只） ， 肿瘤接种之日记为第 0天。
过继转移 T细胞： 待小鼠肿瘤体积至 200-300mm3时， 即接种肿瘤后第 13天腹腔注射环 磷酰胺 （200mg/kg) ， 实验组和对照组分别于第 14天于尾静脉注射 8χ 106/只基因修饰的 T 淋巴细胞 （阳性转染效率为 50%左右） 或仅生理盐水。
分组： 30只 6-8周龄 NOD/SCID小鼠随机分为 4组， 每组 6只， 实验组为： GPC3-28BBZ CAR T淋巴细胞治疗组、 对照组为： 2D3-28BBZ CAR T淋巴细胞对照组、 Mock基因修饰 的 CAR T淋巴细胞对照组、 以及盐水对照组。
在过继转移 T细胞 2周后 （第 28天） ， 对照组小鼠肿瘤体积长至 2000mm3时结束实验。 GPC3-28BBZ CAR T淋巴细胞治疗组 3/6只小鼠肿瘤出现了消退，小鼠残余肿瘤的体积（图 8A) 及重量 （图 8B ) 与 3个对照组分别相比， 结果都具有显著的差异 （***P < 0.001 ) 。 图 8D为处死小鼠后所示各组肿瘤组织情况。 图 8E显示 GPC3-28BBZ CAR T淋巴细胞治疗 后小鼠肿瘤消退情况 （与 Mock组对比） 。 结果显示 GPC3-28BBZ CAR T淋巴细胞能够显 著的抑制 Huh-7移植瘤的生长。
另外，在过继输注 T淋巴细胞 1周后检测小鼠外周血 T细胞存活数量，发现如图 8C所示， GPC3-28BBZ CAR T细胞治疗组小鼠体内的 T细胞数量明显高于 mock对照组和 2D3-28BBZ CAR T淋巴细胞治疗组 （GPC3-28BBZ vs mock, P = 0.001 1 ; GPC3-28BBZ vs 2D3-28BBZ, P = 0.0019； 2D3-28BBZ vs mock, P = 0.359)。图 9C所示结果表明 GPC3-28BBZ CAR T淋巴细胞在体内能够较好的存活。
实验结束时，对各组 Huh-7移植瘤组织进行组织切片并采用抗人 CD3e的抗体进行免疫 染色， 免疫组织化学染色结果故图 9所示。 结果显示， GPC3-28BBZ CAR T细胞治疗组小 鼠皮下移植瘤部位的人 CD3阳性的 T细胞明显多于转导 2D3-28BBZ和 mock T细胞治疗组， 而盐水组没有检测到人 T细胞， 提示 GPC3-28BBZ CAR T细胞能够在肿瘤部位聚集以行使 其杀伤肿瘤细胞的功能。
上述结果表明 GPC3-28BBZ CAR T细胞可部分地清除高表达 GPC3的 Huh-7皮下移植 瘤移植瘤。 实施例 6 GPC3-28BBZ CAR T 细胞对低表达 GPC3的 PLC/PRF/5移植皮下移植瘤的治 疗实验
接种肿瘤： 收处于对数生长期并生长良好的 PLC/PRF/5细胞， 采用生理盐水调整细胞 密度为 2.5 x l07/mL， 注射体积为 200μί ( 5 >< 106/只） ， 肿瘤接种之日记为第 0天。
过继转移 Τ细胞： 待小鼠肿瘤体积至 150mm3时， 即第 21天， 腹腔注射环磷酰胺 ( 200mg/kg ) ，于在第 22天及第 30天， 实验组和对照组分别于尾静脉注射 8 >< 106/只基因修 饰的 T细胞 （阳性转染效率为 50%左右） 或仅生理盐水。
分组： 30只 6-8周龄 NOD/SCID小鼠随机分为 5组， 每组 6只， 实验组为： GPC3-28BBZ CAR T淋巴细胞治疗组、 对照组为： 2D3-28BBZ CAR T淋巴细胞对照组、 Mock基因修饰 的 CAR T淋巴细胞对照组、 以及盐水对照组。
至小鼠肿瘤平均体积达 150mm3时 （第 21天） ， 将小鼠按过继转移的 T细胞随机分为 4 组并由腹腔注射 200mg/kg的环磷酰胺， 第 22和 30天， 分别经尾静脉过继转移 8 χ 106 GPC3-28BBZ、 2D3-28BBZ或 mock基因修饰的 T淋巴细胞 （阳性率分别为 50% ) 或仅生理 盐水进行治疗。 在第 46天， 对照组小鼠肿瘤平均体积长至 1500mm3时结束实验，
图 10A示肿瘤生长曲线， 在第 46天， GPC3-28BBZ CAR T淋巴细胞治疗组小鼠移植瘤 的体积明显小于各对照组（ *P < 0.05 )。图 10B示实验结束取瘤组织称重结果， GPC3-28BBZ CAR T淋巴细胞治疗组移植瘤与对照组移植瘤相比， 瘤重亦明显低于对照组 ( GPC3-28BBZ vs 盐水， P =0.0332； GPC3-28BBZ vs mock, P =0.021 1； GPC3-28BBZ vs 2D3-28BBZ , P =0.021 1 ) 。 图 10C为实验结束处死小鼠所示各组肿瘤组织情况， 显示 GPC3-28BBZ CAR T淋巴细胞治疗组对低表达 GPC3的 PLC/PRF/5细胞具有明显的抑制作 用。
为了检测 T细胞在小鼠体内的存活情况， 在最后一次过继转移 T细胞 1周后检测小鼠外 周血中 T细胞的数量。 结果如图 10D所示， 发现 GPC3-28BBZ CAR T淋巴细胞治疗组小鼠 体内的 T细胞数量明显高于对照组 （GPC3-28BBZ vs mock, P = 0.004; GPC3-28BBZ vs 2D3-28BBZ, P = 0.0097； 2D3-28BBZ vs mock, P = 0.0804 ) 提示 GPC3-28BBZ CAR T淋巴 细胞在体内能够较好的存活。
上述结果共同表明 GPC3-28BBZ CAR T淋巴细胞可显著抑制低表达 GPC3的 PLC/PRF/5移植瘤的生长。 实施例 7 GPC3-28BBZ CAR T 细胞对肝脏部位的 GPC3阳性的 Huh-7移植瘤的清除效 果实验
接种肿瘤： 收集处于对数生长期的良好的 Huh-7 ( Luc+) ， 采用生理盐水调整细胞密 度为 l x l08/ml， 25μί细胞悬液与 25μί的 metrigel基质胶混合 （冰上） ， 与无菌超净台中行 开腹腔手术， 分别将细胞混匀接种于 40只 6~8周龄 NOD/SCID小鼠肝脏右页 (2.5 χ 106/只）， 肿瘤接种之日记为第 0天。
分组：自小鼠肿瘤接种之日起，每周成像，至 2周后进行腹腔注射环磷酰胺（200mg/kg)， 并随机分为 4组， 每组 7只， 实验组为： GPC3-28BBZ CAR T淋巴细胞治疗组、 对照组为： 2D3-28BBZ CAR T淋巴细胞对照组、 Mock基因修饰的 CAR T淋巴细胞对照组、 以及盐水 对照组。
过继转移 T淋巴细胞： 在接种肿瘤后第 14天和 21天， 于小鼠尾静脉输注 5 χ 106/只基因 修饰的 T淋巴细胞 （阳性转染效率为 50%左右， 注射体积为 200μυ 或仅 200μί生理盐水。
在第一次输注转基因的 Τ淋巴细胞进行治疗后的第 1周， GPC3-28BBZ CAR T淋巴细胞 治疗组的小鼠肿瘤明显小于对照组并在随后的第 2和 3周成像均发现 GPC3-28BBZ CAR T 淋巴细胞治疗组的小鼠肿瘤明显小于对照组 （图 1 1A和图 1 1B ) ， 在最后一次输注基因修 饰的 T淋巴细胞一周后，发现 3个对照组即 2D3-28BBZ CAR T淋巴细胞治疗组、 mock T 细 胞治疗组和生理盐水组的小鼠腹部明显隆起， 而 GPC3-28BBZ CAR T淋巴细胞治疗组的 小鼠腹部正常 （图 1 1C ) ， 解剖小鼠发现对照组小鼠的肝脏部位有巨大的肿瘤占位， 而 GPC3-28BBZ CAR T淋巴细胞治疗组的小鼠肝脏部位未发现有肿瘤生长 （图 11D) 。 在小 鼠最后一次输注 T淋巴细胞一周后于小鼠眼眶采血以检测输注的 T细胞在小鼠体内的存活 情况， 结果显示 GPC3-28BBZ CAR T淋巴细胞治疗组小鼠体内的淋巴细胞较对照组 mock 和 2D3-28BBZ组多 (； GPC3-28BBZ vs mock, P = 0.0012; GPC3-28BBZ vs 2D3-28BBZ, P = 0.0156; 2D3-28BBZ vs mock, P = 0.355； 图 11E)， 就 CAR阳性的 T细胞而言， 外周血中亦 是 GPC3-28BBZ CAR T细胞治疗组最高 （GPC3-28BBZ vs mock, P = 0.0012; GPC3-28BBZ vs 2D3-28BBZ, P = 0.0015; 2D3-28BBZ vs mock, P = 0.22; 图 11F)。 实验在持续 4周后对照 组动物陆续死亡。 对照组中位生存期分别为 mock 对照组 34 天、 2D3-28BBZ对照组 39天 和生理盐水组 33天 (；图 11G)， 而在该时间点， GPC3-28BBZ CAR T细胞治疗组小鼠仍存 活良好， 腹部未见隆起。
综上所述， GPC3-28BBZ CAR T细胞对 Huh-7肝原位移植瘤具有显著的疗效， 并且 GPC3-28BBZ CAR T细胞在荷 Huh-7原位移植瘤小鼠体内能够有效的存活。
本专利申请保护的靶向 GPC3的嵌合抗原受体基因修饰的 T细胞可特异性地识别并杀 伤 GPC3阳性的肝癌细胞， 而对 GPC3阴性的肝癌细胞没有作用， 其具有潜在的临床应用 价值。 表 5 本发明中的序列
Figure imgf000024_0001

Claims

权 利 要 求 书
1. 编码表达于人 T淋巴细胞表面的抗 GPC3嵌合抗原受体蛋白的核酸，所述靶向 GPC3 的嵌合抗原受体蛋白包含顺序连接的胞外结合区， 跨膜区和胞内信号区， 其中所 述胞外结合区包含特异性识别 GPC3的 C末端表位的单链抗体 scFv(GPC3)。
2. 权利要求 1所述的核酸， 其中跨膜区选自包含 CD8或 CD28的跨膜区和铰链区的序 列。
3. 权利要求 1或 2的核酸，其中胞内信号区选自 CD3 ζ， FcsRIy, CD28， CD137, CD134 的胞内信号区序列， 及其组合。
4. 权利要求 3的核酸， 其中所述嵌合抗原受体蛋白是选自包含顺序连接的胞外结合 区， 跨膜区和胞内信号区的如下顺序连接的嵌合抗原受体蛋白：
scFv(GPC3)-CD8-CD3 ζ，
scFv(GPC3)-CD8-CD137-CD3 ζ，
scFv(GPC3)-CD28a-CD28b-CD3 ζ，
scFv(GPC3)-CD28a-CD28b-CD137-CD3 ζ，
及其组合， 其中相关嵌合抗原受体蛋白中 CD28a代表 CD28分子的跨膜区， CD28b 代表 CD28分子的胞内信号区。
5. 权利要求 1的核酸， 选自编码具有 SEQ ID NO:22〜25之一序列的嵌合抗原受体蛋 白的核酸。
6. 权利要求 1的核酸， 具有 SEQ ID NO: 18-21之一的序列。
7. 包含权利要求 1-6之一所述的核酸的载体。
8. 权利要求 7所述的载体，该载体来自慢病毒质粒 pWPT-eGFP，并具有如 SEQ ID NO:
27-30之一的核酸序列。
9. 包含权利要求 7或 8所述载体的病毒。
10.—种基因修饰的 T淋巴细胞， 其被转导有权利要求 1-6之一的核酸或权利要求 7或 8 所述的载体或权利要求 9所述的病毒。 一种转基因 T淋巴细胞， 其表面表达一种嵌合抗原受体， 所述嵌合抗原受体由 SEQ ID NO: 18-21之一的核酸编码表达。
—种基因修饰的 T淋巴细胞， 其表面表达一种嵌合抗原受体， 所述嵌合抗原受体的 氨基酸序列选自 SEQ ID NO:22-25之一。
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CN104140974B (zh) 2017-09-29
US10731127B2 (en) 2020-08-04
US20160215261A1 (en) 2016-07-28
CN104140974A (zh) 2014-11-12
EP2995682A4 (en) 2016-11-23
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CN107460201A (zh) 2017-12-12
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