WO2016197570A1 - 嵌合抗原受体修饰的t细胞及其用途 - Google Patents

嵌合抗原受体修饰的t细胞及其用途 Download PDF

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WO2016197570A1
WO2016197570A1 PCT/CN2015/096775 CN2015096775W WO2016197570A1 WO 2016197570 A1 WO2016197570 A1 WO 2016197570A1 CN 2015096775 W CN2015096775 W CN 2015096775W WO 2016197570 A1 WO2016197570 A1 WO 2016197570A1
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cells
region
cell
car
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齐菲菲
鲁薪安
何霆
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北京艺妙神州医疗科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present invention relates to the field of immunology and molecular biology, and more particularly to chimeric antigen receptor modified T cells and uses thereof.
  • CLL Chronic Lymphocytic Leukemia
  • One method of treating these patients is to genetically modify T cells to target antigens expressed on tumor cells by expression of a Chimeric Antigen Receptor (CAR).
  • CAR is an antigen receptor designed to recognize cell surface antigens in a human leukocyte antigen-independent manner. Attempts to treat these types of patients with genetically modified T cells expressing CAR have been somewhat successful (Molecular Therapy, 2010, 18: 4, 666-668; Blood, 2008, 112: 2261-2271).
  • CAR-T Chimeric Antigen Receptor-T cell
  • the first generation of CAR-T cells are activated by the extracellular binding region - single-chain fragment variable (scFV), transmembrane region (TM) and intracellular signal region - immunoreceptor tyrosine
  • the immunooreceptor tyrosine-based activation motif (ITAM) consists in which the chimeric antigen receptor portions are linked as follows: scFv-TM-CD3 ⁇ .
  • This kind of CAR-T cells can stimulate anti-tumor cytotoxic effects, but cytokine secretion is less, 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, Cancer Res 2007, 67(22): 11029-11036.].
  • CAR-T cells incorporates the intracellular signaling region of CD28 or CD137 (aka 4-1BB), in which the chimeric antigen receptor portions are joined as follows: scFv-TM-CD28-ITAM or scFv -TM-CD137-ITAM.
  • Co-stimulation of B7/CD28 or 4-1BBL/CD137 in the intracellular signaling region causes sustained proliferation of T cells and increases the levels of cytokines such as IL-2 and IFN- ⁇ secreted by T cells, while increasing CAR-T Life 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 cells have attractive prospects for tumor immunotherapy, some potential risks need to be considered.
  • specific antigens that can be recognized by low expression of CAR in certain/normal tissues may cause damage to normal tissues of CAR-T cells expressing the corresponding antigen.
  • excessive costimulatory signals in CAR reduce the threshold required for effector cell activation, allowing genetically modified T cells to be activated under conditions of low or no antigen triggering, resulting in the release of large numbers of cytokines. It may trigger a so-called "cytokine storm.” This signal leakage can lead to off-target cytotoxicity, resulting in non-specific tissue damage.
  • compositions and methods for treating human cancer are disclosed in Patent Application No. 201180067173.X, which includes the administration of genetically modified T cells to express a CAR, wherein the CAR comprises an antigen binding region (ie, an extracellular binding region), Transmembrane region, costimulatory signaling region, and CD3 ⁇ signaling region.
  • the antigen-binding domain scFv on the surface of this CAR-T cell is non-humanized (mouse-derived), which results in the CAR-T cell itself being immunogenic.
  • a first aspect of the invention provides a nucleic acid molecule encoding a chimeric antigen receptor expressed on the surface of a T cell, the chimeric antigen receptor comprising a sequentially linked signal peptide, an extracellular binding region, an optional hinge region (hinge) A region, a transmembrane region, and an intracellular signal region, wherein the nucleotide sequence of the extracellular binding region is as set forth in SEQ ID No: 4 or SEQ ID No: 5.
  • a second aspect of the invention provides a vector comprising the nucleic acid molecule of the chimeric antigen receptor expressed on the surface of a T cell of the first aspect of the invention.
  • a third aspect of the invention provides a virus comprising the vector of the second aspect of the invention, comprising a virus capable of infecting after packaging, and a virus to be packaged which is packaged as an essential component of an infectious virus.
  • a fourth aspect of the invention provides a genetically modified T cell comprising the nucleic acid sequence of the first aspect of the invention or the vector of the second aspect of the invention or the virus of the third aspect of the invention.
  • a fifth aspect of the invention provides a medicament comprising the T cell of the fourth aspect of the invention.
  • a sixth aspect of the invention provides the use of a T cell of the fourth aspect of the invention for the preparation of a medicament for treating a tumor.
  • the antigen-binding region scFv on the surface of the CAR-T cell of the present invention is humanized and thus has high immunogenicity. Greatly reduced.
  • Figure 1 shows the results of detection of chimeric antigen receptor-modified T cells.
  • Figure 2 shows the results of labeling CAR-T19 cells with a carbocyanine dye Did.
  • Figure 3 shows the results of labeling Daudi cells with a carbocyanine dye Did.
  • Figure 4 shows the results of detecting the killing effect of CAR-T19 cells on Daudi cells by flow cytometry.
  • Figure 5 shows the results of comparison of the killing effect of T cells and CAR-T19 cells on target cells Daudi.
  • Figure 6 shows the results of comparison of the killing effect of CAR-T19 containing different scFv regions on target cells Daudi.
  • Figure 7 shows the results of in vitro detection of the killing effect of different CAR-T19 on cells of B lymphocyte leukemia patients.
  • a first aspect of the invention provides a nucleic acid molecule encoding a chimeric antigen receptor expressed on the surface of a T cell, the chimeric antigen receptor comprising a sequentially linked signal peptide, an extracellular binding region, optionally A hinge region, a transmembrane region, and an intracellular signal region, wherein the nucleotide sequence of the extracellular binding region is as set forth in SEQ ID No: 4 or SEQ ID No: 5.
  • nucleic acid 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 invention also relates to variants of the nucleic acid encoding fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of the nucleic acid can be naturally occurring allelic variants or non-naturally occurring variants. These nucleic acid variants include substitution variants, deletion variants, and insertion variants.
  • allelic variant is an alternative form of polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter its coding.
  • the function of the peptide is well known to those of ordinary skill in the art.
  • the signal peptide is a GMCSF signal peptide having a nucleotide sequence as set forth in SEQ ID No: 1.
  • the signal peptide is a CD8 signal peptide, the nucleotide sequence of which is set forth in SEQ ID No: 2.
  • the hinge region can be selected from a hinge region of a protein such as CD8 or CD28.
  • the CD8 or CD28 is a natural marker on the surface of T cells.
  • the hinge region is a CD8 hinge region (CD8-hinge) having a nucleotide sequence of SEQ ID No: 6.
  • the hinge region is a CD28 hinge region (CD28-hinge), the nucleotide sequence of which is set forth in SEQ ID No: 7.
  • the transmembrane region may be selected from a transmembrane region of a protein such as CD8 or CD28.
  • the transmembrane region is a CD8 transmembrane region (CD8-TM), the nucleotide sequence of which is set forth in SEQ ID No: 8.
  • the transmembrane region is a CD28 transmembrane region (CD28-TM), the nucleotide sequence of which is set forth in SEQ ID No: 9.
  • the "extracellular binding region” comprises an scFv (anti-CD19 scFv) that specifically recognizes a human CD19 epitope.
  • scFv refers to an antibody fragment which comprises a variable region of heavy chain (VH) and a variable region of light linked by a linker.
  • VH variable region of heavy chain
  • VL variable region of light linked by a linker.
  • the recombinant protein of chain, 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 scFv is preferably an amino acid sequence encoded by a single nucleotide chain.
  • the scFv 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 modification methods.
  • specific recognition means that the bispecific antibody of the 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.
  • the intracellular signal region can be selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD28, CD137, an intracellular signal region of a CD134 protein, and combinations thereof.
  • the CD3 molecule consists of five subunits, of which the CD3 ⁇ subunit (also known as CD3zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex.
  • CD3 ⁇ Z is a mutated CD3 ⁇ sequence that does not have an ITAM motif, and is generally used as a construction component of a negative control in the examples 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 ⁇ .
  • CD28, CD137, and CD134 are costimulatory signal molecules, and the co-stimulatory action of intracellular signal segments after binding to their respective ligands causes sustained proliferation of T cells and enhances IL-secretion of T cells.
  • cytokine levels such as IFN- ⁇ , while increasing CAR-T The survival cycle and anti-tumor effect of cells in the body.
  • the intracellular signal regions used in the present invention are various combinations comprising a signal region selected from the group consisting of:
  • CD28 signal region having a nucleotide sequence as shown in SEQ ID No: 10;
  • CD137 signal region (CD137-signal) having a nucleotide sequence as shown in SEQ ID No: 11;
  • the CD3 ⁇ signal region (CD3 ⁇ -signal) has a nucleotide sequence as shown in SEQ ID No: 12.
  • the chimeric antigen receptor protein expressed on the surface of a T cell of the invention is selected from the group consisting of a signal peptide comprising a ligated sequence, an extracellular binding region, an optional hinge region, a transmembrane region, and a cell.
  • Chimeric antigen receptor protein in the inner signal region is selected from the group consisting of a signal peptide comprising a ligated sequence, an extracellular binding region, an optional hinge region, a transmembrane region, and a cell.
  • GMCSF-scFv-S1-CD8-hinge-CD8-TM-CD137-signal-CD3 ⁇ -signal the nucleic acid sequence of which is shown in SEQ ID No:13;
  • GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD137-signal-CD3 ⁇ -signal the nucleic acid sequence of which is shown in SEQ ID No: 14.
  • GMCSF-scFv-S1-CD28-TM-CD28-signal-CD3 ⁇ -signal the nucleic acid sequence of which is shown in SEQ ID No: 15;
  • GMCSF-scFv-S2-CD28-TM-CD28-signal-CD3 ⁇ -signal the nucleic acid sequence of which is shown in SEQ ID No:16;
  • GMCSF-scFv-S1-CD8-hinge-CD8-TM-CD28-signal-CD137-signal-CD3 ⁇ -signal the nucleic acid sequence of which is shown in SEQ ID No:17;
  • GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD28-signal-CD137-signal-CD3 ⁇ -signal the nucleic acid sequence of which is shown in SEQ ID No:18;
  • the second aspect of the present invention provides a vector comprising the nucleic acid molecule of the chimeric antigen receptor expressed on the surface of a T cell of the first aspect of the present invention.
  • the vector used in the invention is a lentiviral plasmid vector pLenti-CMV-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 the Rev protein, the envelope plasmid pVSVG encoding the VSV-G protein, and the empty vector pLenti.
  • - CMV-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 pLenti-CMV-eGFP (which itself is a mock in subsequent experiments) regulates enhanced green fluorescent protein (eGFP) by an elongation factor-1 ⁇ (elongation factor-1 ⁇ , EF-1 ⁇ ) promoter. expression.
  • the recombinant expression vector pLenti-CMV-eGFP comprising the nucleic acid sequence of interest encoding CAR is passed by a ribosome hopping sequence from food and mouth vires disease (FMDV). (ribosomal skipping sequence 2A) (referred to as F2A) to achieve co-expression of eGFP and CAR.
  • FMDV food and mouth vires disease
  • the vector used in the present invention is a retroviral vector, pMSCV-Ubc-GFP, which is used to overexpress a nucleic acid sequence of interest, ie, a nucleic acid sequence encoding a CAR.
  • the third aspect of the present invention provides the virus comprising the vector of the second aspect of the present invention, which comprises a packaged infectious virus, and a package to be packaged as an essential component of an infectious virus. virus.
  • the virus is a lentivirus comprising the pLenti-CMV-eGFP-F2A-CAR recombinant vector of the second aspect of the invention.
  • the virus is a retrovirus comprising a pMSCV-Ubc-GFP-CAR recombinant vector of the second aspect of the invention. It will be appreciated that other T cell transfected viruses known in the art and their corresponding plasmid vectors can also be used in the present invention.
  • the fourth aspect of the invention provides a genetically modified T cell, wherein the nucleic acid sequence of the first aspect of the invention or the vector of the second aspect of the invention or the virus of the third aspect of the invention is transformed.
  • Nucleic acid transformation methods conventional in the art can be used in the present invention.
  • Non-viral based transformation 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 transformation of the target gene.
  • the transformation 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 SB Sleeping Beauty transposition subsystem. It has been reported [Davies JK., et al.
  • the method of transforming a chimeric antigen receptor gene-modified T lymphocyte is based on a transformation method of a virus such as a retrovirus or a lentivirus.
  • the method has the advantages of high transformation efficiency, stable expression of the foreign gene, and shortening the time for the T lymphocytes to reach the clinical level in vitro.
  • the transformed nucleic acid is expressed thereon by transcription and translation.
  • In vitro cytotoxicity experiments on various cultured tumor cells demonstrated 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 molecule encoding the chimeric antigen receptor of the present invention, the plasmid comprising the nucleic acid, the virus comprising the plasmid, and the transgenic T lymphocyte transformed with the above nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.
  • the fifth aspect of the invention provides a medicament comprising the T cell of the fourth aspect of the invention.
  • the medicament further comprises a pharmaceutically acceptable diluent, excipient or carrier, and the like.
  • the sixth aspect of the present invention provides the T cell of the fourth aspect of the present invention for use in the preparation of a tumor for treatment The use of the drug.
  • the CAR molecule of the invention comprises a signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region, and an intracellular signal region.
  • the nucleotide sequence of the extracellular binding region is the antigen binding region of the anti-CD19 chimeric antigen receptor (here named anti-CD19 scFv-S0, abbreviated as scFv-S0, which is derived from mouse, see J Immunother.2009 September; 32(7): 689-702.)
  • the nucleotide sequence (SEQ ID No: 3) was obtained by humanization.
  • the principle of humanization of antibodies is to maximize the binding of the framework region (FM) to a human sequence while ensuring antibody affinity, thereby reducing the immunogenicity of the antibody.
  • the antigen recognition region in SEQ ID No: 3 is kept unchanged, the remaining sequences are correspondingly changed, more than 40 humanized designs are performed, and these are synthesized by gene synthesis. Sequences, other parts of the CAR molecule were cloned from a human cDNA library by PCR, and then ligated, and finally the nucleotide sequence of the CAR molecule was prepared.
  • the humanized extracellular binding region sequence - the killing rate of CAR-T19 containing these two humanized transformation sequences is slightly better or not different than the killing rate of CAR-T19 containing scFv-S0 ( See Figure 6) in Example 4, which are referred to as anti-CD19 scFv-S1 (abbreviated as scFv-S1, nucleotide sequence as shown in SEQ ID No: 4) and anti-CD19 scFv-S2 (abbreviated as Is scFv-S2, the nucleotide sequence of which is shown in SEQ ID No: 5).
  • the preparation steps of the nucleotide sequence of the CAR molecule are specifically described below by taking SEQ ID No: 15 and SEQ ID No: 16
  • Primer design was first performed, and the primer sequences used in this example were as follows:
  • the corresponding CAR molecules were cloned by PCR using 1-1 and 1-2, 2-1 and 2-2, 3-1 and 3-2 as primers, respectively, which were GMCSF, CD28-TM+CD28-signal (the two parts are connected) and CD3 ⁇ -signal.
  • the GMCSF+scFv fragment was obtained by bridging primers 4-1 and 4-2, and the CD28-TM+CD28-signal+CD3 ⁇ -signal fragment was obtained by bridging primers 5-1 and 5-2, followed by bridging primers 6-1 and 6.
  • -2 Obtain the nucleotide sequence of the complete CAR molecule, and the restriction sites are SpeI and MluI.
  • Example 2 Construction of a viral vector comprising a nucleic acid sequence of a CAR molecule
  • the nucleotide sequence of the CAR molecule prepared in Example 1 was digested with SpeI (Fermentas) and MluI (Fermentas), and ligated into the SpeI-MluI site of the lentiviral pLenti-CMV-eGFP vector by T4 ligase (Fermentas). Point, transformed into competent E. coli (DH5 ⁇ ), after sequencing, the plasmid was extracted and purified using Qrigene's plasmid purification kit for subsequent lentiviral packaging experiments.
  • the recombinant viral vector prepared in Example 2 was first packaged into a virus, which was then infected with T cells to effect chimeric antigen receptor-modified T cells.
  • the present invention employs a transformation method based on a virus such as a retrovirus or a lentivirus. The method has the advantages of high transformation efficiency, stable expression of the foreign gene, and shortening the time for the T cells to reach the clinical level in vitro. Specific steps are as follows:
  • Lentiviral packaging Day 1: Inoculate 293T cells into 10cm culture dishes to ensure cell confluence of 80-90% on the next day of transfection; Day 2: Transfection, replace 293T medium 1 h before transfection 5 mL serum-free DMEM high glucose medium; 500 ⁇ L DMEM was added to the EP tube, followed by addition of 4 ⁇ g pR8.74, 400 ng pVSVG, 4 ⁇ g pLenti-CMV-eGFP-F2A-CAR vortex for 8 s; the plasmid was added (pR8.
  • 74+pLenti-CMV-eGFP-F2A-CAR 3 times volume of PEI (1 ⁇ g/ ⁇ L, total 25.2 ⁇ L), vortexed for 20s, and allowed to stand at room temperature for 15min; the mixed solution was added dropwise to 293T cells. After 6 h of transfection, the medium was changed to whole medium containing 10% FBS. After transfection for 48 hours, the cell culture supernatant was collected, centrifuged at 3000 rpm for 15 min at 4 ° C, and the supernatant was taken. The virus solution was filtered through a 0.45 ⁇ m PVDF filter, dispensed in an EP tube, and stored at -80 ° C to be infected with T cells.
  • the target cell selected for the in vitro cell model is Daudi cells.
  • the Daudi cell line is derived from a Burkitt's lymphoma patient and is a typical B lymphoblastoid cell that is widely used in basic and preclinical trials for mechanisms such as leukemia production and therapy.
  • Daudi cells highly express the B cell antigen CD19, which can be recognized and killed by CAR-T19 (CAR19 targeting CD19) cells. Therefore, we used Daudi cells as target cells (T), CAR-T19 cells as effector cells (E), and T cells as control cells (C) to construct a cell model for in vitro killing. This model mimics to a high extent the therapeutic effects of CAR-T19 cells or T cells on B lymphocytic leukemia in vivo.
  • the T cell recognizes the target cell, it causes apoptosis and eventually cleaves.
  • the late apoptosis process and the lysis process of the cells are coincident but not identical. Therefore, we established two models for detecting cell killing effects in vitro for apoptosis and lysis.
  • Calcein-AM is a cell staining reagent that can fluorescently label living cells. Because it enhances hydrophobicity based on Calcein, it can easily penetrate living cell membranes. When it enters the cytoplasm, it is hydrolyzed by the esterase to Calcein and remains in the cell, thereby emitting strong green fluorescence.
  • CAR-T19 cells including GMCSF–scFv-S1–CD28-TM–CD28-signal–CD3 ⁇ -signal or GMCSF–scFv-S2–CD28-TM–CD28 -signal–CD3 ⁇ -signal CAR-T19 cells, abbreviated as CAR-T19 (S1), CAR-T19 (S2), respectively, and the killing effect of unmodified T cells on target cells Daudi.
  • the detection procedure is the same as that described in 1).
  • the killing effect of CAR-T19 cells is increased by at least 20-fold (the killing rate of T cells is set to 1, CAR-T19 (S1)
  • the killing rates with CAR-T19 (S2) were 20.5 and 22, respectively.
  • CAR-T19s of the present invention containing the humanized extracellular binding region scFv (scFv-s1 and scFv-s2) (CAR-T19 (S1), CAR- as described above) T19(S2)) and CAR-T19 containing the unhumanized extracellular binding region scFv (scFv-s0) (including GMCSF-scFv-S0-CD28-TM-CD28-signal-CD3 ⁇ -signal (its nucleic acid)
  • the CAR-T19 sequence of SEQ ID No: 19, abbreviated as CAR-T19 (S0) compares the killing ability of target cells.
  • the specific detection procedure is as described in 1).
  • the humanized modification of scFv did not affect the killing efficiency of CAR-T19 on target cells.
  • the cells of the B lymphocyte leukemia patient are first isolated, and are mainly divided into two major steps: First, PBMC (peripheral blood mononuclear cells) are isolated from the patient's bone marrow (this step is a person skilled in the art) Known); In the second step, B cells are isolated from PBMC.
  • PBMC peripheral blood mononuclear cells
  • lymphocytes are mixed with sheep red blood cells (SRBC) treated with bromine diamino isothiohydride (AET), wherein all T lymphocytes can adsorb AET-SRBC to form a firm
  • SRBC sheep red blood cells
  • AET bromine diamino isothiohydride
  • all T lymphocytes can adsorb AET-SRBC to form a firm
  • the stable and large E-garland the percentage of the E-garland is higher than the percentage of the E-garland formed by the T lymphocytes and the normal untreated SRBC, and its formation is rapid, not easy to fall off, and the repeatability is good.
  • the lymphocyte stratification solution is used for separation.
  • the AET-E-garland is easy to sink to the bottom of the tube, and the B lymphocytes can be directly taken from the interface of the stratified liquid.
  • This example examined the killing effect of different CAR-T19s in the present invention on cells of B lymphocyte leukemia patients in vitro. The results are shown in Figure 7.
  • the different CAR-T19 (represented by different CAR molecules in the figure) of the present invention significantly enhanced the killing ability of cells of B lymphocyte leukemia patients compared to unmodified T cells.

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Abstract

提供了嵌合抗原受体修饰的T细胞及其用途，所述嵌合抗原受体包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区，其中所述胞外结合区的核苷酸序列如SEQ ID No:4或SEQ ID No:5所示。

Description

嵌合抗原受体修饰的T细胞及其用途 技术领域
本发明涉及免疫学和分子生物学领域，更具体地涉及嵌合抗原受体修饰的T细胞及其用途。
背景技术
大部分具有B细胞恶性肿瘤—包括慢性淋巴细胞白血病(Chronic Lymphocytic Leukemia，CLL)的患者，使用化疗、靶向治疗的治愈率以及预后都很差。治疗这些患者的一个方法是基因修饰T细胞以通过嵌合抗原受体(Chimeric Antigen Receptor，CAR)的表达，靶向在肿瘤细胞上表达的抗原。CAR是被设计为以人白细胞抗原-非依赖性的方式识别细胞表面抗原的抗原受体。利用表达CAR的基因修饰的T细胞治疗这些类型患者的尝试已经得到一定程度的成功(Molecular Therapy,2010,18:4,666-668；Blood,2008,112:2261-2271)。
随着嵌合抗原受体T细胞(Chimeric Antigen Receptor-T cell，CAR-T)技术的不断发展，目前CAR-T主要可划分为三代。
第一代CAR-T细胞由胞外结合区——单链抗体(single-chain fragment variable,scFV)、跨膜区(transmembrane region,TM)和胞内信号区——免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif,ITAM)组成，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD3ζ。该种CAR-T细胞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,Cancer Res2007,67(22):11029-11036.]。
随后发展的第二代CAR-T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细胞的持续增殖，并能够提高T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细胞对表达相应抗原的正常组织的损伤。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(signal leakage)会导致脱靶细胞毒性，从而产生非特异性的组织损伤。
目前在专利申请号201180067173.X中公开了治疗人癌症的组合物和方法，该发明包括涉及施用基因修饰的T细胞，以表达CAR，其中CAR包括抗原结合区(即，胞外结合区)、跨膜区域、共刺激信号传导区和CD3ζ信号传导区。但是该CAR-T细胞表面的抗原结合区scFv是非人源化的(小鼠来源的)，这导致CAR-T细胞本身具有免疫原性。
综上可知，本领域存在着对克服上述缺陷的CAR-T细胞的强烈需求。
发明内容
本发明的第一方面提供了编码表达于T细胞表面的嵌合抗原受体的核酸分子，所述嵌合抗原受体包含顺序连接的信号肽、胞外结合区、任选的铰链区(hinge region)、跨膜区和胞内信号区(signal region)，其中所述胞外结合区的核苷酸序列如SEQ ID No:4或SEQ ID No:5所示。
本发明的第二方面提供了包含本发明第一方面的表达于T细胞表面的嵌合抗原受体的核酸分子的载体。
本发明的第三方面提供了包含本发明第二方面的载体的病毒，其包括包装后的具有感染能力的病毒，也包括包装为具有感染力的病毒所必需成分的待包装的病毒。
本发明的第四方面提供了基因修饰的T细胞，其包含本发明第一方面的核酸序列或本发明第二方面的载体或本发明第三方面的病毒。
本发明的第五方面提供了包含本发明第四方面的T细胞的药物。
本发明的第六方面提供了本发明第四方面的T细胞用于制备治疗肿瘤的药物的用途。
本发明的CAR-T细胞表面的抗原结合区scFv是经人源化改造的，因此其免疫原性大 大降低。
附图说明
根据以下参照附图进行的详细描述，本发明的上述和其他方面、特征和优点会变得更加清楚。
图1示出了表达嵌合抗原受体修饰的T细胞的检测结果。
图2示出了用羰花青染料Did对CAR-T19细胞进行标记的结果。
图3示出了用羰花青染料Did对Daudi细胞进行标记的结果。
图4示出了通过流式细胞术检测CAR-T19细胞对Daudi细胞杀伤作用的结果。
图5示出了T细胞和CAR-T19细胞对靶细胞Daudi的杀伤效果的比较结果。
图6示出了含有不同scFv区域的CAR-T19对靶细胞Daudi的杀伤效果的比较结果。
图7示出了体外检测不同的CAR-T19对B淋巴细胞白血病患者细胞的杀伤作用的结果。
具体实施方式
如上所述，本发明的第一方面提供了编码表达于T细胞表面的嵌合抗原受体的核酸分子，所述嵌合抗原受体包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区，其中所述胞外结合区的核苷酸序列如SEQ ID No:4或SEQ ID No:5所示。
本发明的术语“核酸”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。本发明还涉及所述核酸的变异体,其编码与本发明具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。所述核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。如本领域普通技术人员所熟知的，等位变异体是一种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。
在一个实施方案中，所述信号肽为GMCSF信号肽，其核苷酸序列如SEQ ID No:1所示
在一个实施方案中，所述信号肽为CD8信号肽，其核苷酸序列如SEQ ID No:2所示。
在一个实施方案中，所述铰链区可以选自CD8或CD28等蛋白的铰链区。所述CD8或CD28是T细胞表面的天然标记物。
在一个具体的实施方案中，所述铰链区为CD8铰链区(CD8-hinge)，其核苷酸序列如SEQ ID No:6。
在一个具体的实施方案中，所述铰链区为CD28铰链区(CD28-hinge)，其核苷酸序列如SEQ ID No:7所示。
在一个实施方案中，所述跨膜区可以选自CD8或CD28等蛋白的跨膜区。
在一个具体的实施方案中，所述跨膜区为CD8跨膜区(CD8-TM)，其核苷酸序列如SEQ ID No:8所示。
在一个具体的实施方案中，所述跨膜区为CD28跨膜区(CD28-TM)，其核苷酸序列如SEQ ID No:9所示。
所述“胞外结合区”包含特异性识别人CD19表位的scFv(anti-CD19 scFv)。
本文使用的术语“scFv”是指这样的抗体片段——其是包含通过接头(linker)连接的重链可变区(variable region of heavy chain，VH)和轻链可变区(variable region of light chain，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。scFv优选是由一条核苷酸链编码的氨基酸序列。本发明使用的scFv可通过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公知的(参见例如，Sambrook分子克隆：实验手册，Cold Spring Harbor Laboratory(1989)N.Y.)。所述修饰优选在核酸水平上进行。上述scFv还可以包括其衍生物。
本文使用的术语“特异性识别”意指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。
在一个实施方案中，所述胞内信号区可以选自CD3ζ、FcεRIγ、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CD3ζ亚单位(又称CD3zeta，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化区。CD3δZ是突变的不具有ITAM基序的CD3ζ序列，在本发明的实施例中一般作为阴性对照的构建组分。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，CD28、CD137、CD134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细胞的持续增殖，并能够提高T细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR-T细 胞在体内的存活周期和抗肿瘤效果。
本发明使用的胞内信号区有多种组合，其包括选自如下的信号区或其组合：
CD28信号区(CD28-signal)，其核苷酸序列如SEQ ID No:10所示；
CD137信号区(CD137-signal)，其核苷酸序列如SEQ ID No:11所示；和
CD3ζ信号区(CD3ζ-signal)，其核苷酸序列如SEQ ID No:12所示。
在一个具体的实施方案中，本发明的表达于T细胞表面的嵌合抗原受体蛋白选自如下的包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区的嵌合抗原受体蛋白：
GMCSF–scFv-S1–CD8-hinge–CD8-TM–CD137-signal–CD3ζ-signal，其核酸序列如SEQ ID No:13所示；
GMCSF–scFv-S2–CD8-hinge–CD8-TM–CD137-signal–CD3ζ-signal，其核酸序列如SEQ ID No:14所示；
GMCSF–scFv-S1–CD28-TM–CD28-signal–CD3ζ-signal，其核酸序列如SEQ ID No:15所示；
GMCSF–scFv-S2–CD28-TM–CD28-signal–CD3ζ-signal，其核酸序列如SEQ ID No:16所示；
GMCSF–scFv-S1–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal，其核酸序列如SEQ ID No:17所示；
GMCSF–scFv-S2–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal，其核酸序列如SEQ ID No:18所示；
如上所述，本发明的第二方面提供了包含本发明第一方面的表达于T细胞表面的嵌合抗原受体的核酸分子的载体。
在一个具体的实施方案中，本发明使用的载体是一种慢病毒质粒载体pLenti-CMV-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pVSVG；及空载体pLenti-CMV-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pLenti-CMV-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pLenti-CMV-eGFP是通过由来自口蹄疫病毒(food and mouth vires disease,FMDV)的核糖体跳跃序列 (ribosomal skipping sequence2A)(简称F2A)实现eGFP与CAR的共表达的。
在另一个实施方案中，本发明使用的载体是一种逆转录病毒载体pMSCV-Ubc-GFP，该质粒用于过表达目的核酸序列，即编码CAR的核酸序列。
如上所述，本发明的第三方面提供了包含本发明第二方面的载体的病毒，其包括包装后的具有感染能力的病毒，也包括包装为具有感染力的病毒所必需成分的待包装的病毒。
在一个实施方案中，所述病毒是包含本发明第二方面的pLenti-CMV-eGFP-F2A-CAR重组载体的慢病毒。在另一个实施方案中，所述病毒是包含本发明第二方面的pMSCV-Ubc-GFP-CAR重组载体的逆转录病毒。应理解，本领域中已知的其他转染T细胞的病毒及其对应的质粒载体也可用于本发明。
如上所述，本发明的第四方面提供了基因修饰的T细胞，其中转化有本发明第一方面的核酸序列或本发明第二方面的载体或本发明第三方面的病毒。
本领域常规的核酸转化方法——包括非病毒和病毒的转化方法都可以用于本发明。基于非病毒的转化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化。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化效率较普通电穿孔有较大提高，将nucleofector转染仪与SB睡美人转座子系统联合应用已有报道[Davies JK.,et al.Combining CD19redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res,2010,70(10):OF1-10.]，该方法既能够具有较高的转化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淋巴细胞的转化方法是基于病毒如逆转录病毒或慢病毒的转化方法。该方法具有转化效率高，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，转化的核酸通过转录、翻译表达在其上。对各种不同的培养的肿瘤细胞进行的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的核酸分子、包含该核酸的质粒、包含该质粒的病毒和转化有上述核酸、质粒或病毒的转基因T淋巴细胞都可以有效地用于肿瘤的免疫治疗。
如上所述，本发明的第五方面提供了包含本发明第四方面的T细胞的药物。
在一个实施方案中，所述药物还包含可药用的稀释剂、赋形剂或载体等。
如上所述，本发明的第六方面提供了本发明第四方面的T细胞用于制备治疗肿瘤的 药物的用途。
以下通过具体实施例来说明本发明的内容。应理解，所述具体实施例仅为说明目的，并不意味着本发明的内容仅限于具体实施例。
实施例1：CAR分子的核苷酸序列的制备
本发明的CAR分子包含信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区。所述胞外结合区的核苷酸序列是在抗CD19的嵌合抗原受体的抗原结合区(在此命名为anti-CD19 scFv-S0，简称为scFv-S0，其来源于小鼠，参见J Immunother.2009 September；32(7):689–702.)的核苷酸序列(SEQ ID No:3)的基础上进行人源化改造而获得的。应理解，抗体人源化改造的原则为在保证抗体亲和力的同时，最大限度地将骨架区(framework region,FM)改变为人源序列，以降低抗体的免疫原性。在该实施例中，将SEQ ID No:3中的抗原识别区保持不变，对其余的序列进行相应的改变，进行了超过40种的人源化设计，再通过基因合成的方法合成得到这些序列，CAR分子的其他部分都是利用PCR技术，分别从人cDNA文库中克隆得到，然后进行搭桥连接，最终制备得到CAR分子的核苷酸序列。将这些CAR分子转入T细胞，将包含含有这些经人源化改造序列的CAR分子的T细胞与包含含有scFv-S0的CAR分子的T细胞对靶细胞的杀伤能力进行比较，最终筛选得到两种经人源化改造的胞外结合区序列——包含这两种人源化改造序列的CAR-T19的杀伤率比包含scFv-S0的CAR-T19的杀伤率略好或与之没有差别(参见实施例4中的图6)，其分别称为anti-CD19 scFv-S1(简称为scFv-S1，其核苷酸序列如SEQ ID No:4所示)和anti-CD19 scFv-S2(简称为scFv-S2，其核苷酸序列如SEQ ID No:5所示)。下面以SEQ ID No:15和SEQ ID No:16为例，具体说明CAR分子的核苷酸序列的制备步骤。
首先进行引物设计，本实施例中使用的引物序列如下：
1-1：5’-ATGCTTCTCCTGGTGACAAG-3’
1-2：5’-TGGGATCAGGAGGAATGCTG-3’
2-1：5’-TACATCTGGGCGCCCTTGGCCGG-3’
2-2：5’-GGAGCGATAGGCTGCGAAGTCGCG-3’
3-1：5’-AGAGTGAAGTTCAGCAGGAGCG-3’
3-2：5’-TTAGCGAGGGGGCAGGGCCT-3’
4-1：5’-ATGCTTCTCCTGGTGACAAGCC-3’
4-2：5’-TGAGGAGACGGTGACTGAGGTTCCTTGG-3’
5-1：5’-GCGGCCGCAATTGAAGTTATGTA-3’
5-2：5’-TTAGCGAGGGGGCAGGGCCTGC-3’
6-1：5’-CTAGACTAGTATGCTTCTCCTGGTGACAAGCC-3’
6-2：5’-CGACGCGTTTAGCGAGGGGGCAGGGCCTGC-3’
以人的cDNA文库为模板，分别以1-1和1-2、2-1和2-2、3-1和3-2为引物，通过PCR克隆出相应的CAR分子部分，分别为GMCSF、CD28-TM+CD28-signal(这两部分是相连的)和CD3ζ-signal。再通过搭桥引物4-1和4-2获得GMCSF+scFv片段，通过搭桥引物5-1和5-2获得CD28-TM+CD28-signal+CD3ζ-signal片段，随后通过搭桥引物6-1和6-2获得完整的CAR分子的核苷酸序列，酶切位点为SpeI和MluI。
实施例2：包含CAR分子的核酸序列的病毒载体的构建
将实施例1中制备的CAR分子的核苷酸序列经SpeI(Fermentas)和MluI(Fermentas)双酶切、经T4连接酶(Fermentas)连接插入慢病毒pLenti-CMV-eGFP载体的SpeI-MluI位点，转化到感受态E.coli(DH5α)，经测序正确后，使用Qrigene公司的质粒纯化试剂盒提取并纯化质粒，用于后续的慢病毒包装实验。
实施例3：CAR-T细胞的构建
在该实施例中，首先使实施例2中制备的重组病毒载体包装成病毒，然后将该病毒感染T细胞以实现嵌合抗原受体修饰的T细胞。本发明采用了基于病毒如逆转录病毒或慢病毒的转化方法。该方法具有转化效率高，外源基因能够稳定表达，且可以缩短体外培养T细胞到达临床级数量的时间等优点。具体步骤如下：
慢病毒包装：第一天：接种293T细胞至10cm培养皿中，保证第二天转染时细胞汇合度为80-90％；第二天：转染，转染前1h把293T的培养基更换为5mL无血清DMEM高糖培养基；在EP管中加入500μL DMEM，随后加入4μg pR8.74、400ng pVSVG、4μg pLenti-CMV-eGFP-F2A-CAR涡旋振荡8s混匀；加入质粒(pR8.74+pLenti-CMV-eGFP-F2A-CAR)3倍体积的PEI(1μg/μL，共25.2μL)，涡旋振荡20s混匀，室温静置15min；将混合后的溶液逐滴加入293T细胞中；转染6h后，将培养基更换为含10％FBS的全培养基。转染48h后，收集细胞培养上清，4℃3000rpm离心15min，取上清，用0.45μm PVDF滤膜过滤病毒液，分装于EP管中，储存于-80℃，以待感染T细胞。
重组慢病毒感染T细胞：从健康人外周血中分离的CD8+T淋巴细胞，培养激活后以MOI＝5用上述重组慢病毒感染T细胞，感染后的细胞每隔一天采用2×105/mL的密度进行传代。感染的CD8+T淋巴细胞在培养第7天时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(结果如图1所示，左图为光镜下细胞代表图，右图为eGFP荧光表达图，左图右图为同一视野)。
实施例4：体外杀伤实验模型的构建
体外细胞模型所选用的靶细胞是Daudi细胞。Daudi细胞系来源于一名伯基特淋巴瘤(Burkitt's lymphoma)患者，是一种典型的B淋巴母细胞，广泛应用于基础和临床前试验中关于白血病生成和治疗等机理的研究。Daudi细胞高表达B细胞抗原CD19，可被CAR-T19(靶向CD19的CAR-T)细胞识别并杀伤。因此，我们利用Daudi细胞作为靶细胞(T)、CAR-T19细胞作为效应细胞(E)、T细胞作为对照细胞(C)，构建了体外杀伤的细胞模型。该模型在很高的程度上模拟了CAR-T19细胞或T细胞在体内对B淋巴细胞白血病的治疗作用。
1)建立两种监测细胞杀伤效果的模型
T细胞识别靶细胞之后，会使其发生凋亡，并最终裂解。其中细胞的晚期凋亡过程和裂解过程有重合但又不完全一样。因此我们针对细胞凋亡和裂解建立了两种体外检测细胞杀伤效果的模型。
1-1)流式细胞术检测靶细胞凋亡
首先，我们利用羰花青染料Did标记CAR-T19细胞，通过流式细胞术检测Did阳性的细胞达到99.17％，只有0.83％的CAR-T19细胞为Did阴性(图2)。
另外，在同样的检测指标下，99.94％的Daudi细胞为Did阴性(图3)。
因此，我们先将CAR-T19细胞标记上Did，然后按照一定ET比与Daudi细胞混合孵育，进行杀伤；接着通过流式细胞仪检测Did阴性细胞的凋亡水平，所得结果能够反映出CAR-T19细胞对Daudi细胞的杀伤水平。我们利用Annexin V-FITC/PI检测细胞凋亡，FITC/PI双阴性的为正常细胞，FITC阳性、PI阴性为早期凋亡细胞，FITC/PI双阳性为晚 期凋亡细胞。按照1：2的ET比，在杀伤4小时之后检测Did阴性细胞的凋亡情况，结果如图4所示：早期凋亡(LR＝low right)为4.32％，晚期凋亡(UR＝up right)为4.55％，总体杀伤率为8.87％。
1-2)荧光法检测靶细胞裂解
Calcein-AM是一种可对活细胞进行荧光标记的细胞染色试剂，由于它在Calcein的基础上加强了疏水性,因此能够轻易穿透活细胞膜。当其进入到细胞质后，会被酯酶水解为Calcein而留在细胞内，从而发出强绿色荧光。我们先将靶细胞Daudi标记上Calcein-AM，然后利用CAR-T19细胞对其进行杀伤。Daudi细胞被CAR-T19杀伤并裂解之后，Calcein会释放到上清液中，检测上清的荧光强度(TEST RELEASE)，同时检测Daudi细胞自发凋亡裂解(SPONTANEOUS RELEASE)以及利用去垢剂(2％的Triton X-100)处理的Daudi细胞的最大裂解的荧光强度(MAXIMUM RELEASE)。按以下公式计算细胞裂解的百分比：[(test release-spontaneous release)/(maximum release–spontaneous release)]*100。
如表1所示，在ET为1：2的情况下，杀伤4小时之后Daudi细胞的裂解率为4.77％。所得结果基本与通过流式细胞术检测到的晚期凋亡的结果类似。
表1:荧光法检测CAR-T19细胞对Daudi细胞的裂解率
Figure PCTCN2015096775-appb-000001
2)T细胞与CAR-T19细胞的杀伤效果的比较
建立了体外细胞杀伤效果的模型之后，我们比较了构建的CAR-T19细胞(包含GMCSF–scFv-S1–CD28-TM–CD28-signal–CD3ζ-signal或GMCSF–scFv-S2–CD28-TM–CD28-signal–CD3ζ-signal的CAR-T19细胞，分别简称为CAR-T19(S1)、CAR-T19(S2))和没有改造的T细胞对靶细胞Daudi的杀伤效果。检测步骤同1)所述，结果如图5所示，在同样的条件下，CAR-T19细胞的杀伤效果提高了至少20倍(将T细胞的杀伤率设为1，CAR-T19(S1)和CAR-T19(S2)的杀伤率分别为20.5、22)。
3)含有不同scFv区域的CAR-T19的杀伤效果的比较
在本实施例中，将本发明的两种含有人源化改造的胞外结合区scFv(scFv-s1和scFv-s2)的CAR-T19(如上所述的CAR-T19(S1)、CAR-T19(S2))与含有未经人源化改造的胞外结合区scFv(scFv-s0)的CAR-T19(包含GMCSF–scFv-S0–CD28-TM–CD28-signal–CD3ζ-signal(其核酸序列如SEQ ID No:19所示)的CAR-T19，简称为CAR-T19(S0))对靶细胞的杀伤能力进行比较。具体检测步骤同1)所述，结果如图6所示，对scFv进行人源化修饰并没有影响CAR-T19对靶细胞的杀伤效率。
4)体外检测不同的CAR-T19对B淋巴细胞白血病患者细胞的杀伤作用
在该实施例中，首先分离出B淋巴细胞白血病患者的细胞，主要分为两大步：第一步，从患者骨髓中分离出PBMC(外周血单核细胞)(这一步是本领域技术人员公知的)；第二步，从PBMC中分离出B细胞。所述第二步的具体步骤如下：将淋巴细胞与用溴花二氨基异硫氢化物(简称AET)处理的绵羊红细胞(SRBC)混合，其中全部T淋巴细胞均能吸附AET—SRBC以形成牢固稳定而巨大的E—花环，该E—花环的百分比高于T淋巴细胞与正常未处理的SRBC形成的E—花环的百分比，并且其形成快速，不易脱落，重复性好。然后使用淋巴细胞分层液进行分离，此时AET—E—花环易沉于管底，而B淋巴细胞可直接取自分层液的界面。该实施例检测了本发明中不同的CAR-T19在体外对B淋巴细胞白血病患者细胞的杀伤作用。结果如图7所示：相比于未经改造的T细胞，本发明的不同的CAR-T19(图中以不同的CAR分子表示)对B淋巴细胞白血病患者细胞的杀伤能力都有显著增强。

Claims (10)

  1. 一种编码表达于T细胞表面的嵌合抗原受体的核酸分子，所述嵌合抗原受体包含顺序连接的信号肽、胞外结合区、跨膜区和胞内信号区，其中所述胞外结合区的核苷酸序列如SEQ ID No:4或SEQ ID No:5所示。
  2. 权利要求1的核酸分子，其中所述嵌合抗原受体在其胞外结合区和跨膜区之间还包含铰链区。
  3. 权利要求2的核酸分子，其中所述信号肽的核苷酸序列如SEQ ID No:1或SEQ ID No:2所示，所述铰链区的核苷酸序列如SEQ ID No:6或SEQ ID No:7所示，所述跨膜区的核苷酸序列如SEQ ID No:8或SEQ ID No:9所示，所述胞内信号区选自CD3ζ、FcεRIγ、CD28、CD137、CD134蛋白的胞内信号区，及其组合，优选所述胞内信号区的核苷酸序列由SEQ ID No:10和/或SEQ ID No:11和/或SEQ ID No:12组成。
  4. 权利要求1-3中任一项的核酸分子，其序列如SEQ ID No:13、SEQ ID No:14、SEQ ID No:15、SEQ ID No:16、SEQ ID No:17或SEQ ID No:18所示。
  5. 载体，其包含权利要求1-4中任一项的核酸分子。
  6. 权利要求5的载体，其为慢病毒质粒载体pLenti-CMV-eGFP或逆转录病毒载体pMSCV-Ubc-GFP。
  7. 病毒，其包含权利要求5或6的载体。
  8. T细胞，其中转化有权利要求1-4中任一项的核酸分子或权利要求5或6的载体或权利要求7的病毒。
  9. 包含权利要求8的T细胞的药物，优选所述药物还包含可药用的稀释剂、赋形剂或载体。
  10. 权利要求8的T细胞用于制备治疗肿瘤的药物的用途。
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CN111097043A (zh) * 2020-01-13 2020-05-05 广东昭泰体内生物医药科技有限公司 一种胃癌药物组合物及其应用
CN113584083A (zh) * 2020-04-30 2021-11-02 深圳市深研生物科技有限公司 用于逆转录病毒载体的生产细胞和包装细胞及其制备方法
CN112626029A (zh) * 2020-12-22 2021-04-09 深圳市赛欧细胞生物科技有限公司 一种转基因修饰的Daudi细胞及其制备方法、应用
CN112626029B (zh) * 2020-12-22 2022-12-20 深圳市赛欧细胞技术有限公司 一种转基因修饰的Daudi细胞及其制备方法、应用

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