WO2017024440A1 - 含Toll样受体胞内结构域的嵌合抗原受体 - Google Patents

含Toll样受体胞内结构域的嵌合抗原受体 Download PDF

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WO2017024440A1
WO2017024440A1 PCT/CN2015/086352 CN2015086352W WO2017024440A1 WO 2017024440 A1 WO2017024440 A1 WO 2017024440A1 CN 2015086352 W CN2015086352 W CN 2015086352W WO 2017024440 A1 WO2017024440 A1 WO 2017024440A1
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receptor
intracellular domain
cells
toll
cell
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PCT/CN2015/086352
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French (fr)
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李鹏
赖允鑫
林思妙
姚瑶
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深圳市体内生物医药科技有限公司
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Priority to CN201580000296.XA priority Critical patent/CN107207613B/zh
Priority to AU2015376656A priority patent/AU2015376656B2/en
Priority to NZ72207615A priority patent/NZ722076A/en
Priority to EP15900639.4A priority patent/EP3202792B1/en
Priority to PCT/CN2015/086352 priority patent/WO2017024440A1/zh
Priority to US15/112,619 priority patent/US10093717B2/en
Publication of WO2017024440A1 publication Critical patent/WO2017024440A1/zh

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Definitions

  • the present invention relates to the field of cellular immunotherapy technology for tumors, and in particular to a chimeric antigen receptor, a nucleic acid encoding the same, and a cell expressing the same, and use thereof in the preparation of a medicament for treating tumor.
  • CAR T cells are T cells that express a chimeric receptor that recognizes a specific antigen and can transmit signals [1].
  • CAR T cells play an important role in anti-tumor by expressing chimeric antigen receptor (CAR) molecules, which usually include extracellular, transmembrane and intracellular segments: the extracellular segment is composed of antibody heavy and light chains
  • the variable region is a single-chain variable region (ScFv) formed by ligation of a peptide segment; the intracellular segment is an intracellular segment chimera of various signaling molecules, including CD3zeta, CD28, OX-40, 4-1BB, etc.
  • the transmembrane region is derived from the transmembrane region of other molecules such as CD8, CD4, CD28 and CD3zeta.
  • the gene of the single-chain variable fragment portion is isolated, for example, from a hybridoma that produces a monoclonal antibody that recognizes the target antigen.
  • the T cell expressing the CAR molecule directly recognizes the tumor cell surface antigen independently of the expression of the major histocompatibility antigen type I on the tumor cell, and simultaneously activates the T cell, and thus the T cell expressing the CAR can effectively kill the tumor cell.
  • CAR T cells recognize specific molecules on the surface of tumor cells through antigen-antibody recognition patterns, and then activate, proliferate and exert cell killing functions through their intracellular signaling.
  • CAR T mainly focus on blood tumors, such as lymphoma, chronic lymphocytic leukemia, and acute lymphoid leukemia.
  • blood tumors such as lymphoma, chronic lymphocytic leukemia, and acute lymphoid leukemia.
  • CAR T treatment in other types of tumors, including: anti-5T4 CAR T for solid tumors, anti-ROR-1 CAR T for solid tumors, anti-PCSA CAR T for solid tumors, and anti-Mesothelin CAR T treatment.
  • chimeric antigen receptor T cell immunotherapy has been proven It can effectively treat a variety of tumors, including chemotherapy-resistant and relapsed leukemia.
  • problems in the treatment of chimeric antigen receptor T cells such as the killing function of chimeric antigen receptor T cells in patients, persistence, and the formation of memory chimeric antigen receptor T cells.
  • the structural design of CAR molecules has undergone multiple generations of research and development.
  • the structure of the first generation CAR molecule comprises a single-chain variable fragment (scFv) that recognizes a tumor cell surface antigen, a transmembrane domain, and an intracellular domain of a TCR complex CD3 ⁇ that activates T cells. Since the intracellular segment of the first generation of CAR has only the CD3 ⁇ signaling region and no co-stimulatory signal, the function of the first generation of CAR T cells is greatly deficient, and its expansion, persistence and effect function in the patient are all Showing a low level. In order to enhance the ability of first-generation CAR to activate T cells, a second-generation CAR has been developed.
  • the second-generation CAR has intracellularly added costimulatory molecules (such as CD28, CD134 (OX-40), CD137 (4-1BB). ), etc.)
  • costimulatory molecules such as CD28, CD134 (OX-40), CD137 (4-1BB). ), etc.
  • the intracellular molecular signaling domain of the source Clinical trials have shown that second-generation CAR T cells show better proliferation, persistence and effector function in patients. Most of the clinical trials of second-generation CAR T cells are anti-CD19 CAR T treatment of B-cell leukemia. Although CAR T cell clinical trials have achieved efficacy, there is room for further improvement.
  • the third generation of CAR was developed to further enhance the efficacy of CAR T cell therapy.
  • the intracellular segment of the third generation of CAR introduces a signal transduction region of two costimulatory molecules.
  • one costimulatory signal is the intracellular region of CD28 and the other is the intracellular signaling region of CD134, CD137 or ICOS.
  • Different combinations of costimulatory signals may affect the function and efficacy of CAR T cells. Studies have shown that not all third-generation CARs are better than the second generation. It can be seen that the CAR structure design in the prior art is not very mature, and it is necessary to further improve the CAR molecule in terms of activation of T cell activity, elimination of regulatory T cell immunosuppression, and formation of memory T cells.
  • TLRs Toll-like receptors
  • PRR Recognition receptor
  • TLRs Toll-like receptors
  • TLRs are PRRs that specifically recognize molecules from different microorganisms that have a conserved structure and activate an intrinsic immune response. Toll-like receptors are an important class of protein molecules involved in innate immunity and a bridge between non-specific immunity and specific immunity. There are 10 human TLRs that have been found, respectively TLR1-10.
  • TLR2 can form dimers with TLR1 and TLR6, respectively, and recognize microbial exogenous molecules and host endogenous molecules. More and more studies have shown that TLR signaling plays an important role in tumor development and treatment. Because some TLRs are widely expressed on the cell surface of immune cells, different TLRs play different roles in these cells. The researchers found that TCR-activated human T cells express TLR2 and do not express TLR4, and TLR2 provides a costimulatory signal for T cell function activation and memory T cell maintenance [2]. Effect of TLR2 on CD8-positive T cells: Down-regulation of the TCR signal intensity threshold required for T cell activation, leading to the formation of memory T cells under low TCR signaling [7].
  • TLR1/TLR2 agonists can effectively abolish regulatory T cell function by down-regulating Foxp3 expression [3, 4, 5]. Further studies have found that TLR1/TLR2 agonists can achieve therapeutic effects in general tumors by reducing regulatory T cells and upregulating the function of cytotoxic T cells [6].
  • TLR2 is expressed on activated T cells as a costimulatory receptor.Proc.Natl.Acad.Sci.USA 101:3029 -3034.
  • TLR1/TLR2agonist induces catalyst regression by reciprocal modulation of effector And regulatory T cells. Journal of immunology 186, 1963-1969.
  • TLR2 engagement on CD8 T cells enables generation of functional memory cells in response to a suboptimal TCR signal .Journal of immunology 182,1860-1867.
  • CAR third generation chimeric antigen receptor
  • the chimeric antigen receptors of the present invention have significant advantages over the prior art in terms of T cell activation, elimination of regulatory T cell immunosuppression, and formation of memory T cells.
  • the invention provides a chimeric antigen receptor comprising an extracellular domain capable of binding an antigen, a transmembrane domain and at least one intracellular domain.
  • intracellular domain refers to any oligopeptide or polypeptide known to function in a cell as a transmitting signal to cause activation or inhibition of a biological process.
  • at least one intracellular domain refers to a Toll-like receptor 1 and/or a Toll-like receptor 2 intracellular domain, or a Toll-like receptor 1 and/or a Toll-like receptor 2 intracellular domain in tandem with other Signal transduction regions such as the intracellular domain of CD3 ⁇ , CD28, 41BB, and the like.
  • the Toll-like receptors (TLR) 1 or Toll-like receptor 2 intracellular domain is an intracellular signaling region, also known as a TIR domain.
  • Toll-like receptors are type I transmembrane proteins that recognize microbes that invade the body and activate immune cell responses. It is believed to play a key role in the innate immune system.
  • Toll-like receptors are a class of pattern recognition receptors (PRRs) that recognize pathogen molecules that are different from the host. These molecules are collectively referred to as pathogen-associated molecular patterns (PAMP) and are part of the most conservative immune system.
  • PRRs pattern recognition receptors
  • PAMP pathogen-associated molecular patterns
  • the antigen may be a tumor antigen
  • the tumor antigen includes, for example, 5T4, ⁇ 5 ⁇ 1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, ⁇ -catenin/ m, Bcr-abl, MN/C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/ m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201- R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE,
  • the extracellular domain capable of binding an antigen refers to a single-chain variable fragment that binds to an antibody that targets the antigen.
  • the above CAR molecule may have only the Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain as its intracellular domain, and may also contain Toll-like receptor 1 and/or Toll-like One or more (eg, 2 or 3) other intracellular domains outside of the receptor 2 intracellular domain.
  • the intracellular domain in addition to the Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain, the intracellular domain further comprises a CD3 sputum intracellular domain; further preferably, said The Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain is arranged on the C-terminal side of the CD3 cell intracellular domain.
  • the intracellular domain in addition to the Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain, the CD3 sputum intracellular domain, the intracellular domain also includes a CD28 intracellular domain.
  • the intracellular domain is a CD28 intracellular domain, a CD3 sputum intracellular domain, and a Toll-like receptor 1 and/or Toll-like receptor 2 intracellularly linked from the N-terminal side. Domain.
  • the chimeric antigen receptors of the invention also encompass the case where the intracellular domain comprises two or more intracellular domains joined in tandem; and, alternatively, the Toll-like receptor 1 and / Or the Toll-like receptor 2 intracellular domain can be arranged on the N-terminal side of the intracellular domain of the chimeric antigen receptor.
  • the chimeric antigen receptor comprises, in sequence from the N-terminal side, a single-chain variable region of an anti-tumor antigen antibody as an extracellular domain, a transmembrane domain and an intracellular domain of a CD28 molecule. , CD3 ⁇ intracellular domain, Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain.
  • the invention provides a nucleic acid encoding a chimeric antigen receptor according to the first aspect.
  • the present invention provides a chimeric antigen receptor-expressing cell, wherein the nucleic acid according to the second aspect is introduced; preferably, the cell is a T cell or a cell population containing T cells.
  • the present invention provides a method of producing a chimeric antigen receptor-expressing cell according to the third aspect, comprising the step of introducing a nucleic acid according to the second aspect into a cell; preferably, the cell It is a T cell or a cell population containing T cells.
  • the present invention provides a chimeric antigen receptor according to the first aspect, the nucleic acid of the second aspect or the chimeric antigen receptor expression cell of the third aspect, in the preparation of a tumor for treatment Use of the drug.
  • the tumor is a solid tumor or a hematoma.
  • the tumor is B-ALL or lung cancer.
  • the CAR of the present invention is characterized in that it comprises the Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain as its intracellular domain.
  • the intracellular domain of the Toll-like receptor 1 and/or Toll-like receptor 2 includes variants thereof having the same function.
  • variant refers to a substitution or deficiency that contains one or several to many amino acids. Any variant that is lost or added, provided that the variant substantially retains the same function as the original sequence.
  • the present invention promotes the proliferation and killing effect of CAR T cells and the formation of memory CAR T cells by introducing Toll-like receptor 1 and/or Toll-like receptor 2 intracellular signaling domains in CAR molecules, thereby improving tumor immunity. Effects and therapeutic effects.
  • the CAR molecules of the present invention have the following advantages: 1) promoting anti-apoptosis and proliferation of CAR T cells in vitro and in vivo; 2) significantly improving the anti-tumor function of CAR T cells; 3) It can reduce the immunosuppressive effect of regulatory T cells; 4) It is beneficial to form memory CAR T cells and inhibit tumor recurrence; in view of the above advantages, the CAR molecules of the invention have good clinical application prospects in anti-tumor therapy.
  • Figure 1 shows the in vitro killing effect of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells on K562-GL cells that do not express CD19.
  • Figure 2 shows the in vitro killing effect of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells on CD19-expressing K562-CD19-GL cells.
  • Figure 3 shows the in vitro killing effect of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells on CD19-expressing NALM6-GL cells.
  • Figure 4 shows the in vitro killing effect of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells on CD19-expressing REH-GL cells.
  • Figure 5 shows the secretion levels of IL-2 in supernatants after co-culture of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells with K562GL or K562-CD19-GL cells for 18 h, respectively.
  • Figure 6 shows the efficiency of CARMeso and CARMesoT1 transfection of T cells by lentivirus.
  • Figure 7 shows the in vitro killing effect of the lentivirus-transfected T cells of Figure 6 on A549 GL cells; wild-type T cells, and CAR T cells against CD22 molecules as controls.
  • Figure 8 shows the efficiency of transfection of T cells by GFP, CAR19, CAR19T1, and CAR19T2 by lentivirus.
  • Figure 9 shows the effect of the lentivirus-transfected T cells of Figure 8 on the size of tumors formed by NALM6 cells in immunodeficient mice;
  • Figure 10 shows the effect of the lentiviral transfected T cells of Figure 8 on the tumor weight formed by NALM6 cells in immunodeficient mice.
  • Figure 11 shows the effect of Lentiviral transfected T cells of Figure 6 on the formation of tumor weight in A549-GL cells in immunodeficient mice.
  • the TLR1 and/or TLR2 signaling domain sequences were inserted into the segment to construct four novel chimeric antigen receptors: anti-CD19 ScFv-CD28-CD3 ⁇ -TLR1/TLR2 and anti-Mesothelin ScFv-CD28-CD3 ⁇ -TLR1/TLR2, respectively
  • the four chimeric antigen receptors were abbreviated as: CAR19T1, CAR19T2, CARMesoT1, CARMesoT2, and the proliferation function, tumor killing function and memory CAR T formation of CAR T cells expressing the above CAR molecules were verified by in vitro and in vivo experiments.
  • the CAR molecule includes an extracellular region, a transmembrane region, and an intracellular region, and therefore, the construction steps of the CAR plasmid used in the following examples include:
  • the gene DNA of each gene required for the CAR plasmid is obtained by gene synthesis, including: variable region sequence of anti-CD19 antibody immunoglobulin heavy and light chain, anti-Mesothelin antibody immunoglobulin heavy chain and light chain variable Regional sequence, CD28 transmembrane signal region sequence, TLR1 signal transduction region sequence, TLR2 signal transduction region sequence, CD3 ⁇ signal transmission region sequence;
  • novel gene molecules of the present invention are obtained by ligating the respective gene sequences of the above-mentioned synthesis by a step of enzymatic cleavage, ligation, and the like, and the sequence structures of the novel CAR molecules of the present invention are as follows:
  • CAR19T1 variable region (extracellular region) of anti-CD19 antibody immunoglobulin heavy and light chain, CD28 transmembrane signal region (transmembrane region), CD3 ⁇ signaling region + TLR1 signaling region;
  • CAR19T2 variable region (extracellular region) of anti-CD19 antibody immunoglobulin heavy and light chains, CD28 transmembrane signal region (transmembrane region), CD3 ⁇ signaling region + TLR2 signaling region;
  • CAR19 control group of CAR19T1/T2: variable region (extracellular region) of anti-CD19 antibody immunoglobulin heavy and light chain, CD28 transmembrane signal region (transmembrane region), CD3 ⁇ signaling region;
  • CARMesoT1 anti-Mesothelin antibody immunoglobulin variable and light chain variable region (extracellular region), CD28 transmembrane signal region (transmembrane region), CD3 ⁇ signaling region + TLR1 signaling region;
  • CARMesoT2 variable region of the anti-Mesothelin antibody immunoglobulin heavy and light chain (extracellular region), CD28 transmembrane signal region (transmembrane region), CD3 ⁇ signaling region + TLR1 signaling region;
  • CARMeso control group of CARMesoT1/T2: variable region (extracellular region) of anti-Mesothelin antibody immunoglobulin heavy and light chains, CD28 transmembrane signal region (transmembrane region), CD3 ⁇ signaling region.
  • the above six CAR molecular nucleic acid sequences were inserted into the second generation lentiviral expression vector pWPXLd-GFP, respectively, to construct pWPXLd-CAR19T1-GFP, pWPXLd-CAR19T2-GFP, pWPXLd-CAR19-GFP, pWPXLd-CARMesoT1-GFP, pWPXLd- CARMesoT2-GFP and pWPXLd-CARMeso-GFP plasmids.
  • the chimeric antigen receptor group carrying the intracellular domain of TLR1 or/and TLR2 of the present invention is prepared as follows. Plasmid:
  • the plasmid pUC57-CAR19 containing the CAR19 gene (SEQ ID NO. 1) was obtained by gene synthesis, molecular cloning, etc., and the CAR19 gene comprises an anti-CD19 monoclonal antibody ScFv (SEQ ID NO. 15), a CD28 transmembrane region and a cell. Inner zone, CD3 intracellular zone, ie CD19ScFv28Z.
  • TIR1 The intracellular signaling domains of TLR1 and TLR2 are referred to as TIR1 (the nucleotide sequence thereof is shown in SEQ ID NO. 11) and TIR2 (the nucleotide sequence of which is shown in SEQ ID NO. 12), respectively, and TIR1 is TLR1.
  • the protein has the most C-terminal 162 amino acids (aa625-786, the sequence is shown in SEQ ID NO. 13), and TIR2 is the most C-terminal 159 amino acids of the TLR2 protein (aa626-784, sequence is shown in SEQ ID NO. .
  • the Mesothelin mAb scFv domain sequence (SEQ ID NO. 17) was obtained by gene synthesis, and the pspXLd-CAR19T1-GFP and pWPXLd-CAR19T2-GFP were replaced with the Mesothelin mAb scFv domain by restriction endonuclease PmeI and NotI, respectively.
  • the CD19 mAb scFv domain was obtained as pWPXLd-CARMesoT1-GFP or pWPXLd-CARMesoT2-GFP, respectively.
  • CARMesoT1/T2 is the CAR22 plasmid
  • its plasmid structure is: lentiviral expression vector pWPXLd-anti-CD22 antibody immunoglobulin heavy and light chain variable region (extracellular region)-CD28 transmembrane signal region ( Transmembrane region)-CD3 ⁇ signaling region-GFP; prepared by gene synthesis of anti-CD22 antibody immunoglobulin heavy and light chain variable regions (ie SEQ ID NO. 16), and by enzyme digestion
  • the CAR22 plasmid pWPXLd-CAR22-GFP
  • CAR plasmid of the present invention prepared in Examples 1 and 2 and the relevant control plasmid, respectively, by over-expression of GFP (blank control), CAR19T1-GFP, CAR19T2-GFP, CAR19-GFP (control), CARMesoTLR1- Eight lentiviruses of GFP, CARMesoTLR2-GFP, CARMeso-GFP (control), CAR22-GFP (positive control).
  • the CAR-containing plasmid was uniformly described as a pWPXLd-CAR-GFP plasmid
  • the Lentivirus overexpressing CAR was uniformly described as a CAR lentivirus.
  • the medium is: DMEM high sugar medium + 10% FBS (fetal calf serum) + 1% double antibody (100 ⁇ penicillin-streptomycin mixed solution);
  • the six plasmids of pWPXLd-CAR-GFP were separately separated by PEI (ie, containing CAR19T1, CAR19T2, CAR19, CARMesoT1, CARMesoT2, CARMeso, respectively).
  • the blank control plasmid pWPXLd-GFP was introduced into the 293T cells together with the lentiviral packaging helper plasmids pMD2.G and psPAX2, and the reagents and dosages were as follows:
  • the culture supernatant was collected at 24, 48 and 72 hours after transformation, respectively, and fresh medium (DMEM high glucose medium + 1% FBS + 1% double antibody) was added;
  • the collected virus solution was dispensed into a PCR tube and stored frozen at -80 ° C until use.
  • T cells Stimulate T cells by magnetic beads coated with CD2, CD3, CD28 antibody (product source: German scorpio), ie, coated magnetic beads and T cells mixed in a ratio of 1:2, the final density of T cells should be 5 ⁇ 10 6 / ml / cm2. After mixing, the cells were incubated at 37 ° C in a 5% CO 2 incubator for 48 hours.
  • 5CAR T cell expansion The CAR T cell density was maintained at about 1 ⁇ 10 6 /ml, and a half-quantity change was performed every 2-3 days. After two weeks, the number of CAR T cells can be amplified by a factor of 100.
  • the GFP-positive cells were successfully transfected, and the GFP-positive ratio was detected by flow cytometry, that is, 6 CAR T cells were obtained (CAR19T1-GFP, CAR19T2-GFP, CAR19-GFP, CARMesoTLR1-GFP, CARMesoTLR2-GFP, respectively).
  • the GFP T (blank control), CAR19T1 T, CAR19T2T and CAR19 T (control) prepared in Example 4, or GFP T, CARMesoT1 T, CARMesoT2 T and CARMeso T (control) cells were separately treated with 1 ⁇ 10 4 tumor cells in different ratios. The cells were mixed and added to a 96-well U-shaped plate. Each group was set up with 3 duplicate wells, and the tumor cells alone were added as a positive control. After centrifugation at 250 g for 5 min, they were placed in a 37-degree 5% CO 2 incubator for 18 h.
  • the tumor cells were selected from NALM6-GL (GFP+Luciferase), REH-GL, K562-GL and K562-CD19-GL.
  • a luciferase-containing leukemia or lymphoma cell line in vitro, when GFP T, CARMeso T, CARMesoT1 T, and CARMesoT2 T cells are used to identify the killing function of solid tumors, the tumor cells are selected from A549-GL with luciferase expressing Mesothelin. Lung adenocarcinoma cell line.
  • Luciferase quantitative killing efficiency evaluation method 18 hours after CAR T cells were co-cultured with tumor cells (the experimental control group was cultured with tumor cells alone), 100 ⁇ l/well of fluorescein was added to a 96-well cell culture plate. The photozyme substrate (1 ⁇ ) was used to resuspend the cells, and the RLU (relative light unit) was immediately measured by a multi-function microplate reader, and the measurement time was set to 1 second. Killing ratio calculation formula: 100% ⁇ (control hole reading - experimental hole reading) / control hole reading (no blank reading of cells can be ignored); the results are shown in Figures 1-4.
  • IL-2 levels in supernatants of GFP T, CAR19 T, CAR19T1 T, and CAR19T2 T cells were co-cultured with K562GL or K562-CD19-GL cells for 18 hours, respectively.
  • the results in Figure 5 show that The level of IL-2 secreted by CAR19T2 T cells was higher than that of CAR19 T cells, suggesting that the addition of the intracellular domain of TLR2 can increase IL-2 secretion of CAR T cells, that is, promote T cell proliferation.
  • Example 6 CAR19T1/T2 T cells recognize killing tumors in vivo
  • CAR19 T can have a good killing effect on blood tumors, it is subcutaneous.
  • the killing effect of solid tumors is not good; and the addition of the intracellular domain of TLR1 or TLR2 can significantly improve the killing effect of second-generation CAR T cells on solid tumors.
  • Example 7 CARMesoT1/T2 T cells recognize killing tumors in vivo
  • mice To compare the effects of CAR22 T, CARMesoT1 T and CARMesoT2 T cells in killing tumors in vivo, an equal number (1 ⁇ 10 5 ) of A549-GL cells were transplanted to 12 NSI (NOD/SCID IL2rg -/- ) immunodeficiency The hind limbs of the mice were subcutaneously; the 7th day and the 14th day after NALM6 cell transplantation (day 0 of tumor cell transplantation), 2 ⁇ 10 6 and 1 ⁇ 10 6 T cells, respectively (three groups: CAR22 T, CARMesoT1 T and CARMesoT2 T, 4 mice in each group, the proportion of positive cells shown in Figure 6 were intravenously injected into NSI immunodeficient mice that had been transplanted with NALM6 cells; on day 68, all mice were euthanized and taken. The tumor was weighed and recorded.
  • NSI NOD/SCID IL2rg -/-
  • the TLR1 and TLR2 signaling domains were used to verify the tumor killing function of CAR T in vitro and in vivo.

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Abstract

本发明提供了一种嵌合抗原受体、其编码核酸和表达细胞，以及其在制备治疗肿瘤的药物中的用途。所述嵌合抗原受体的胞内结构域至少包括Toll样受体1和/或Toll样受体2的胞内结构域。

Description

含Toll样受体胞内结构域的嵌合抗原受体 技术领域
本发明涉及肿瘤的细胞免疫治疗技术领域，具体地，涉及一种嵌合抗原受体，编码其的核酸和表达其的细胞，以及其在制备治疗肿瘤的药物中的用途。
背景技术
嵌合抗原受体(CAR，Chimeric Antigen Receptors)T细胞是表面表达识别特定抗原并能传递信号的嵌合型受体的T细胞【1】。CAR T细胞通过表达嵌合抗原受体(CAR)分子而在抗肿瘤中发挥重要作用，CAR分子通常包括胞外段、跨膜区和胞内段：胞外段是由抗体重链和轻链可变区通过一条肽段相连接而形成的单链可变区(ScFv)；胞内段是各种信号传导分子的胞内段嵌合体，包括CD3zeta、CD28、OX-40、4-1BB等；跨膜区则来自其他分子(如CD8，CD4，CD28和CD3zeta)的跨膜区。单链可变片段部分的基因分离自例如产生识别靶抗原的单克隆抗体的杂交瘤。表达CAR分子的T细胞独立于肿瘤细胞上的主要组织相容性抗原I型的表达而直接识别肿瘤细胞表面抗原，并且在同时激活T细胞，并因此表达CAR的T细胞可以有效杀伤肿瘤细胞。简言之，CAR T细胞通过抗原-抗体识别模式对肿瘤细胞表面的特异分子进行识别，然后通过其胞内的信号传导进行激活、增殖并发挥细胞杀伤功能。
目前，研究人员对CAR T的临床研究成果主要集中于血液肿瘤，如淋巴瘤、慢性淋系白血病、急性淋系白血病等。而近期在其它种类的肿瘤治疗的CAR T也有了一些进展，其中包括：抗5T4 CAR T治疗实体瘤，抗ROR-1 CAR T治疗实体瘤，抗PCSA CAR T治疗实体瘤，抗Mesothelin CAR T治疗胰腺间皮瘤，抗EGFRvIII CAR T治疗胶质瘤、成胶质细胞瘤，抗CD22 CAR T治疗B细胞肿瘤，抗L1CAM CAR T治疗成神经细胞瘤，抗MUC16&IL6 CAR T治疗卵巢癌等。综上，嵌合抗原受体T细胞免疫疗法已被证明 能有效治疗各种肿瘤，包括化疗耐受和复发的白血病。但目前嵌合抗原受体T细胞治疗存在很多问题，如嵌合抗原受体T细胞在患者体内的杀伤功能，持久性，记忆性嵌合抗原受体T细胞的形成等。
CAR分子的结构设计经历多代的研究发展。第一代CAR分子的结构包含识别肿瘤细胞表面抗原的单链可变片段(scFv)、跨膜结构域和激活T细胞的TCR复合物CD3ζ的胞内结构域。由于第一代CAR的胞内段只有CD3ζ信号传递区域，没有共刺激信号，因而第一代CAR T细胞的功能存在很大缺陷，其在病人体内的扩增、持久性和效应功能等方面都表现出低水平。为了增强第一代CAR激活T细胞的能力的目的，已经开发出第二代CAR，第二代CAR在胞内加入了共刺激分子(如CD28、CD134(OX-40)、CD137(4-1BB)等)来源的胞内分子信号传递域。临床试验表明，第二代CAR T细胞在病人体内表现出较好的增殖、持久性和效应功能。第二代CAR T细胞的临床试验，大部分是抗CD19 CAR T对B细胞白血病的治疗。虽然CAR T细胞临床试验获得了疗效，但还存在进一步改善的空间。第三代CAR是为了进一步提高CAR T细胞疗法的疗效而开发的。第三代CAR的胞内段引入了两个共刺激分子的信号传递区域。通常情况下，一个共刺激信号是CD28胞内区域，另一个则是CD134、CD137或ICOS等的胞内信号传递区域。共刺激信号的不同组合可能影响CAR T细胞的功能和疗效，研究表明，并不是所有的第三代CAR都比第二代好。由此可知，现有技术中的CAR结构设计并非十分成熟，需要进一步针对T细胞活性激活、消除调节性T细胞免疫抑制作用和形成记忆性T细胞等方面，对CAR分子进行改良。
美国免疫学家Janeway提出了模式识别理论，将天然免疫针对主要靶分子信号称作病原相关的分子模式(Pathogen-associated molecular pattern，PAMP)；相对应的识别受体称为模式识别受体(pattern recognition receptor，PRR)。Toll样受体(TLR)是可特异性识别来源于不同微生物的、具有保守结构的分子，并激活内在免疫应答的PRR。 Toll样受体是参与固有免疫的一类重要蛋白质分子，也是连接非特异性免疫和特异性免疫的桥梁。已经发现的人TLR有10种，分别为TLR1-10。大部分的TLR都是单独发挥作用的，除了TLR2可以分别与TLR1、TLR6形成二聚体，识别微生物外源分子和宿主内源分子。越来越多的研究显示，TLR信号在肿瘤发育和治疗中发挥着重要功能。因为一些TLR广泛表达于免疫细胞的细胞表面，而不同的TLR在这些细胞中发挥着不同的作用。研究人员发现，TCR激活的人体T细胞表达TLR2，不表达TLR4，TLR2为T细胞功能激活和记忆性T细胞维持提供共刺激信号【2】。TLR2对CD8阳性T细胞的作用：下调T细胞激活所需的TCR信号强度阈值，促使低TCR信号下的记忆性T细胞形成【7】。TLR1/TLR2激动剂可通过下调Foxp3表达，有效消除调节性T细胞功能【3、4、5】。进一步研究发现，TLR1/TLR2激动剂可通过减少调节性T细胞和上调细胞毒性T细胞的功能，对一般肿瘤达到治疗的效果【6】。
引用文献
1.Restifo，N.P.，Dudley，M.E.，and Rosenberg，S.A.(2012).Adoptive immunotherapy for cancer：harnessing the T cell response.Nature reviews Immunology 12，269-281.
2.Komai-Koma，M.，L.Jones，G.S.Ogg，D.Xu，and F.Y.Liew.2004.TLR2 is expressed on activated T cells as a costimulatory receptor.Proc.Natl.Acad.Sci.USA 101：3029-3034.
3.Takeuchi，O.，S.Sato，T.Horiuchi，K.Hoshino，K.Takeda，Z.Dong，R.L.Modlin，and S.Akira.2002.Cutting edge：role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins.J.Immunol.169：10-14.
4.Liu，H.，M.Komai-Koma，D.Xu，and F.Y.Liew.2006.Toll-like receptor 2 signaling modulates the functions of CD4+ CD25+ regulatory T cells.Proc.Natl.Acad.Sci.USA 103：7048-7053.
5.Sutmuller，R.P.，M.H.den Brok，M.Kramer，E.J.Bennink，L.W.Toonen，B.J.Kullberg，L.A.Joosten，S.Akira，M.G.Netea，and G.J.Adema.2006.Toll-like receptor 2 controls expansion and function of regulatory T cells.J.Clin.Invest.116：485-494.
6.Zhang，Y.，Luo，F.，Cai，Y.，Liu，N.，Wang，L.，Xu，D.，and Chu，Y.2011.TLR1/TLR2agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells.Journal of immunology 186，1963-1969.
7.Mercier，B.C.，Cottalorda，A.，Coupet，C.A.，Marvel，J.，and Bonnefoy-Berard，N.(2009).TLR2 engagement on CD8 T cells enables generation of functional memory cells in response to a suboptimal TCR signal.Journal of immunology 182，1860-1867.
发明内容
本发明的目的在于提供一种新的第三代嵌合抗原受体(CAR)，以及编码其的核酸，表达其的细胞，其在制备治疗肿瘤的药物中的用途。本发明的嵌合抗原受体在T细胞活性激活、消除调节性T细胞免疫抑制作用和形成记忆性T细胞等方面均具有相比于现有技术的明显优势。
本发明通过以下技术方案实现上述目的：
第一方面，本发明提供了一种嵌合抗原受体，其包含能够结合抗原的胞外结构域、跨膜结构域和至少一个胞内结构域。其中，“胞内结构域”是指已知在细胞中作为传输信号以引起生物过程的活化或抑制的结构域起作用的任何寡肽或多肽。而所述的至少一个胞内结构域是指Toll样受体1和/或Toll样受体2胞内结构域，或Toll样受体1和/或Toll样受体2胞内结构域串联其它信号传递区域如CD3ζ、CD28、41BB等的胞内结构域。
所述Toll样受体(Toll-like receptors，简称TLR)1或Toll样受体2胞内结构域为胞内信号传递区域，也称为TIR结构域。Toll样受体是I型跨膜蛋白质，识别侵入体内的微生物进而激活免疫细胞的应答。被认为在先天性免疫系统中起关键作用。Toll样受体是模式识别受体(pattern recognition receptors，PRR)的一类，识别与宿主不同的病原体分子。这些分子被统称为病原相关分子模式(pathogen-associated molecular patterns，PAMP)，是最保守的免疫系统的组成部分。Toll样受体1和Toll样受体都是 Toll样受体家族的重要成员。
对于上述CAR分子，作为优选，所述抗原可以是肿瘤抗原，所述肿瘤抗原例如包括：5T4、α5β1-整联蛋白、707-AP、AFP、ART-4、B7H4、BAGE、β-联蛋白/m、Bcr-abl、MN/C IX 抗体、CA125、CAMEL、CAP-1、CASP-8、CD4、CD19、CD20、CD22、CD25、CDC27/m、CD30、CD33、CD52、CD56、CD80、CDK4/m、CEA、CT、Cyp-B、DAM、EGFR、ErbB3、ELF2M、EMMPRIN、EpCam、ETV6-AML1、G250、GAGE、GnT-V、Gp100、HAGE、HER-2/new、HLA-A*0201-R170I、HPV-E7、HSP70-2M、HST-2、hTERT(或hTRT)、iCE、IGF-1R、IL-2R、IL-5、KIAA0205、LAGE、LDLR/FUT、MAGE、MART-1/melan-A、MART-2/Ski、MC1R、Mesothelin、肌球蛋白/m、MUC1、MUM-1、MUM-2、MUM-3、NA88-A、PAP、蛋白酶-3、p190minor bcr-abl、Pml/RARα、PRAME、PSA、PSM、PSMA、RAGE、RU1或RU2、SAGE、SART-1或SART-3、生存蛋白、TEL/AML1、TGFβ、TPI/m、TRP-1、TRP-2、TRP-2/INT2、VEGF、WT1、NY-Eso-1或NY-Eso-B等等；进一步优选地，所述肿瘤抗原为CD19或Mesothelin。本发明专利所提抗原也可以是在自身免疫性疾病中出现的炎性细胞表面分子或导致自身免疫的TCR。
优选地，所述能够结合抗原的胞外结构域是指结合靶向抗原的抗体的单链可变片段。
在具体实施方案中，上述CAR分子可以仅以Toll样受体1和/或Toll样受体2胞内结构域作为其胞内结构域，还可以包含除了Toll样受体1和/或Toll样受体2胞内结构域之外的一个或多个(例如2个或3个)其它胞内结构域。
例如，在优选的实施方案中，除了Toll样受体1和/或Toll样受体2胞内结构域之外，所述胞内结构域还包括CD3ζ胞内结构域；进一步优选地，所述Toll样受体1和/或Toll样受体2胞内结构域配置在CD3ζ胞内结构域的C末端侧。
在进一步优选的实施方案中，除了Toll样受体1和/或Toll样受体2胞内结构域，CD3ζ胞内结构域之外，所述胞内结构域还包括CD28胞内结构域。
在一个具体实施方案中，所述胞内结构域为自N-末端侧开始依次连接的CD28胞内结构域、CD3ζ胞内结构域和Toll样受体1和/或Toll样受体2胞内结构域。
此外，本发明的嵌合抗原受体还涵盖其胞内结构域包括串联连接的两个或更多个胞内结构域的情形；以及，可供选择地，所述Toll样受体1和/或Toll样受体2胞内结构域可以被配置在嵌合抗原受体胞内结构域的N-末端侧。
在优选的具体实施方案中，所述嵌合抗原受体自N末端侧开始依次包括抗肿瘤抗原抗体的单链可变区作为胞外结构域，CD28分子的跨膜结构域和胞内结构域，CD3ζ胞内结构域，Toll样受体1和/或Toll样受体2胞内结构域。
第二方面，本发明提供了一种编码如第一方面所述的嵌合抗原受体的核酸。
第三方面，本发明提供了一种嵌合抗原受体表达细胞，其中引入了如第二方面所述的核酸；优选地，所述细胞为T细胞或含有T细胞的细胞群。
第四方面，本发明提供了一种制备如第三方面所述的嵌合抗原受体表达细胞的方法，其包括将如第二方面所述的核酸引入细胞的步骤；优选地，所述细胞为T细胞或含有T细胞的细胞群。
第五方面，本发明提供了一种如第一方面所述的嵌合抗原受体、如第二方面所述的核酸或如第三方面所述的嵌合抗原受体表达细胞在制备治疗肿瘤的药物中的用途。
优选地，所述肿瘤为实体瘤或血液瘤。
在本发明提供的该用途的具体实施例中，所述肿瘤为B-ALL或肺癌。
值得注意的是，本发明的CAR特征在于它包含Toll样受体1和/或Toll样受体2胞内结构域作为其胞内结构域。所述Toll样受体1和/或Toll样受体2的胞内结构域包括具有相同功能的其变体。术语“变体”是指包含一个或几个至多个氨基酸的取代、缺 失或添加的任何变体，条件是所述变体基本上保留原始序列所具有的相同功能。
有益效果
本发明通过在CAR分子中引入Toll样受体1和/或Toll样受体2胞内信号传递结构域，促进CAR T细胞的增殖和杀伤效应以及记忆性CAR T细胞的形成，进而提高肿瘤免疫效应和治疗效果。与现有技术中的CAR分子相比，本发明的CAR分子具有以下优势：1)促进CAR T细胞体内外的抗凋亡和增殖；2)显著提高了CAR T细胞的抗肿瘤功能；3)可降低调节性T细胞的免疫抑制作用；4)利于形成记忆性的CAR T细胞，抑制肿瘤复发；鉴于上述优势，本发明的CAR分子在抗肿瘤治疗中具有良好的临床应用前景。
附图说明
图1显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对不表达CD19的K562-GL细胞的体外杀伤效应。
图2显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对表达CD19的K562-CD19-GL细胞的体外杀伤效应。
图3显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对表达CD19的NALM6-GL细胞的体外杀伤效应。
图4显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞对表达CD19的REH-GL细胞的体外杀伤效应。
图5显示GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞分别与K562GL或K562-CD19-GL细胞共培养18h后，上清中IL-2的分泌水平。
图6显示CARMeso和CARMesoT1经慢病毒转染T细胞的效率。
图7显示图6中的经慢病毒转染的T细胞对A549GL细胞的体外杀伤效应；野生型T细胞，以及针对CD22分子的CAR T细胞作为对照。
图8显示GFP、CAR19、CAR19T1、CAR19T2经慢病毒分别转染T细胞的效率。
图9显示图8中的经慢病毒转染的T细胞对NALM6细胞在免疫缺陷小鼠体内形成的肿瘤大小的影响；
图10显示图8中的经慢病毒转染的T细胞对NALM6细胞在免疫缺陷小鼠体内形成的肿瘤重量的影响。
图11显示图6中经慢病毒转染的T细胞对A549-GL细胞在免疫缺陷小鼠体内形成肿瘤重量的影响。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
一般性方法
总的来说，在分别用于治疗血液瘤(急性B淋系白血病，B-ALL)的抗人CD19嵌合抗原受体和实体瘤(肺癌)的抗人Mesothelin嵌合抗原受体的胞内段中插入TLR1和/或TLR2信号传递结构域序列，构建四种新型嵌合抗原受体：抗CD19 ScFv-CD28-CD3ζ-TLR1/TLR2和抗Mesothelin ScFv-CD28-CD3ζ-TLR1/TLR2，以下分别将四种嵌合抗原受体简称为：CAR19T1、CAR19T2、CARMesoT1、CARMesoT2，并通过体内、外实验验证表达上述CAR分子的CAR T细胞的增殖功能、肿瘤杀伤功能和记忆性CAR T的形成。
CAR质粒的构建
如上所述，CAR分子包括胞外区域、跨膜区域、胞内区域，因此，以下实施例所用CAR质粒的构建步骤包括：
首先，通过基因合成获得CAR质粒所需各基因DNA，包括：抗CD19抗体免疫球蛋白重链和轻链的可变区域序列、抗Mesothelin抗体免疫球蛋白重链和轻链的可变 区域序列、CD28跨膜信号区域序列、TLR1信号传递区域序列、TLR2信号传递区域序列、CD3ζ信号传递区域序列；
然后，通过酶切、连接等步骤将所需要的上述合成的各基因序列串联起来，即获得本发明的新型CAR分子，其序列结构分别如下：
CAR19T1：抗CD19抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR1信号传递区域；
CAR19T2：抗CD19抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR2信号传递区域；
CAR19(CAR19T1/T2的对照组)：抗CD19抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域；
CARMesoT1：抗Mesothelin抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR1信号传递区域；
CARMesoT2：抗Mesothelin抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域+TLR1信号传递区域；
CARMeso(CARMesoT1/T2的对照组)：抗Mesothelin抗体免疫球蛋白重链和轻链的可变区域(胞外区域)、CD28跨膜信号区域(跨膜区域)、CD3ζ信号传递区域。
将以上六种CAR分子核酸序列分别插入第二代慢病毒表达载体pWPXLd-GFP中，分别构建pWPXLd-CAR19T1-GFP、pWPXLd-CAR19T2-GFP、pWPXLd-CAR19-GFP、pWPXLd-CARMesoT1-GFP、pWPXLd-CARMesoT2-GFP和pWPXLd-CARMeso-GFP质粒。
具体实施例
实施例1 CAR19T1、CAR19T2质粒的制备
按如下步骤制备本发明的携带含TLR1或/和TLR2胞内结构域的嵌合抗原受体基 因的质粒：
(1)通过基因合成、分子克隆等手段得到含CAR19基因(SEQ ID NO.1)的质粒pUC57-CAR19，CAR19基因包含抗CD19单抗ScFv(SEQ ID NO.15)、CD28跨膜区和胞内区、CD3ζ胞内区，即CD19ScFv28Z。
(2)通过内切酶PmeI和SpeI对所得到的pUC57-CAR19质粒进行酶切，获得CAR19基因，然后将CAR19基因连接至慢病毒载体pWPXLd-GFP中，构建pWPXLd-CAR19-GFP。
(3)用内切酶NotI和SpeI对所得到的pWPXLd-CAR19-GFP质粒进行酶切，获得CAR19基因的胞内片段28Z。
(4)用28Z串联TLR1胞内信号传递结构域的cDNA作为模板，构建4条引物(SEQ ID NO.2-5)，通过重叠延伸PCR得到28ZT1(SEQ ID NO.9)；同样地，用28Z串联TLR2胞内信号传递区域的cDNA作为模板，构建另外4条引物(SEQ ID NO.2，6，7，8)，通过重叠延伸PCR得到28ZT2(SEQ ID NO.10)。
(5)通过NotI和SpeI酶切，用28ZT1或28ZT2替换pUC57-CAR19中的28Z，分别得到pUC57-CAR19T1或pUC57-CAR19T2质粒。
(6)最后，通过内切酶PmeI和SpeI酶切，用CAR19T1或CAR19T2替换pWPXLD-CAR19-GFP中的CAR19，分别得到pWPXLd-CAR19T1-GFP或pWPXLd-CAR19T2-GFP质粒。
TLR1和TLR2的胞内信号传递结构域分别称为TIR1(其核苷酸序列如SEQ ID NO.11所示)和TIR2(其核苷酸序列如SEQ ID NO.12所示)，TIR1为TLR1蛋白最C端的162个氨基酸(a.a.625-786，序列如SEQ ID NO.13所示)，TIR2为TLR2蛋白最C端的159个氨基酸(a.a.626-784，序列如SEQ ID NO.14所示)。
实施例2 CARMesoT1、CARMesoT2、CAR22质粒的制备
通过基因合成得到Mesothelin单抗scFv结构域序列(SEQ ID NO.17)，通过内切酶PmeI和NotI酶切，用Mesothelin单抗scFv结构域分别替换pWPXLd-CAR19T1-GFP和pWPXLd-CAR19T2-GFP中的CD19单抗scFv结构域，分别得到pWPXLd-CARMesoT1-GFP或pWPXLd-CARMesoT2-GFP。
此外，CARMesoT1/T2的阴性对照为CAR22质粒，其质粒结构为：慢病毒表达载体pWPXLd-抗CD22抗体免疫球蛋白重链和轻链的可变区域(胞外区域)-CD28跨膜信号区域(跨膜区域)-CD3ζ信号传递区域-GFP；其制备方法为：通过基因合成抗CD22抗体免疫球蛋白重链和轻链的可变区域(即SEQ ID NO.16)，并通过酶切黏贴替代CAR19质粒中的抗CD19抗体免疫球蛋白重链和轻链的可变区域，即得CAR22质粒(pWPXLd-CAR22-GFP)。
实施例3 CAR质粒的慢病毒包装
使用实施例1、2所制备的本发明CAR质粒以及相关对照质粒，通过慢病毒包装，获得分别过表达GFP(空白对照)、CAR19T1-GFP、CAR19T2-GFP、CAR19-GFP(对照)、CARMesoTLR1-GFP、CARMesoTLR2-GFP、CARMeso-GFP(对照)、CAR22-GFP(阳性对照)的8种慢病毒。本实施例3和4中，将含CAR的质粒统一描述为pWPXLd-CAR-GFP质粒，将过表达CAR的慢病毒统一描述为CAR慢病毒。
具体步骤如下：
①在10cm培养皿中培养293T细胞，培养基为：DMEM高糖培养基+10％FBS(胎牛血清)+1％双抗(100×青霉素-链霉素混合溶液)；
②待150mm培养皿中的293T细胞密度达80-90％时，更换培养基：DMEM高糖培养基+1％FBS+1％双抗；
③更换培养基培养2-6小时后，用PEI分别将pWPXLd-CAR-GFP六种质粒(即，分别包含CAR19T1、CAR19T2、CAR19、CARMesoT1、CARMesoT2、CARMeso) 或空白对照质粒pWPXLd-GFP分别与慢病毒包装辅助质粒pMD2.G、psPAX2共同转导入293T细胞，加入试剂及剂量如下：
Figure PCTCN2015086352-appb-000001
④分别于转化后24、48和72小时，收集培养基上清，并加入新鲜培养基(DMEM高糖培养基+1％FBS+1％双抗)；
⑤培养基上清收集完毕，将上清2500g离心0.5小时后；
⑥取离心上清，用0.45um过滤器过滤后，利用超高速离心机28000rpm离心1.5小时；
⑦超高速离心后，轻轻去除上清，加入200ul PBS，置于4度12-16小时溶解，即得6种CAR慢病毒或空白对照GFP慢病毒；
⑧病毒溶解后，收集病毒溶液分装于PCR管，冻存于-80℃待用。
实施例4使用包装的CAR慢病毒感染人体T细胞
①T细胞的分离纯化：通过Ficoll密度梯度法分离出血液中的单个核细胞，经红细胞裂解液裂解去除红细胞后，再通过MACS Pan-T磁珠分选出T细胞；
②分选出来的T细胞用培养基(AIM-V培养基+5％FBS+青霉素100U/ml+链霉素0.1mg/ml稀释至细胞浓度2.5×106个/ml待用；
③通过包被CD2、CD3、CD28抗体的磁珠(产品来源：德国美天旎)刺激T细胞，即包被磁珠与T细胞以1∶2比例混合，T细胞最终密度应为5×106个/ml/cm2。混 合后，置于37℃、5％CO2培养箱培养刺激48小时。
④慢病毒转染T细胞：将激活的T细胞-磁珠混合液中的磁珠通过磁场作用去除，300g离心5min，去上清，用新鲜培养基重悬，分别加入表达CAR和GFP(空白对照)慢病毒(病毒加入量为MOI＝10)后，加入8μg/ml的polybrene和300IU/ml IL-2。置于37℃，5％CO2培养箱培养24h后，300g离心5min，去上清，用含300IU/ml IL-2的新鲜培养基重悬，即得过表达(7种)CAR的T细胞。
⑤CAR T细胞扩增：将CAR T细胞密度维持在1×106个/ml左右，每2-3天进行一次半量换液。两周后，CAR T细胞数可扩增100倍。GFP阳性的细胞为转染成功的细胞，GFP阳性比例通过流式进行检测，即得到6种CAR T细胞(分别简称CAR19T1-GFP、CAR19T2-GFP、CAR19-GFP、CARMesoTLR1-GFP、CARMesoTLR2-GFP、CARMeso-GFP)或空白对照T细胞(GFP-T)的比例。
实施例5 CAR T细胞体外识别杀伤肿瘤的效应
将实施例4制备的GFP T(空白对照)、CAR19T1 T、CAR19T2T和CAR19 T(对照)，或GFP T、CARMesoT1 T、CARMesoT2 T和CARMeso T(对照)细胞以不同比例分别与1x104的肿瘤细胞混合，加入到96孔U型板中，每组设3个复孔，并设单独加肿瘤细胞组作为阳性对照，250g离心5min后，置于37度5％CO2培养箱共培养18h；
体外比较GFP T、CAR19 T、CAR19T1 T、CAR19T2 T细胞对血液瘤的识别杀伤功能时，肿瘤细胞选用NALM6-GL(GFP+Luciferase)、REH-GL、K562-GL和K562-CD19-GL四种带荧光素酶的白血病或淋巴瘤细胞系；体外验证GFP T、CARMeso T、CARMesoT1 T、CARMesoT2 T细胞对实体瘤的识别杀伤功能时，肿瘤细胞选用表达Mesothelin的A549-GL带荧光素酶的人肺腺癌细胞系。
荧光素酶(Luciferase)定量杀伤效率评估方法：CAR T细胞与肿瘤细胞共培养(实验对照组为肿瘤细胞单独培养)后18小时，在96孔细胞培养板中加入100μl/孔的荧 光素酶底物(1×)，将细胞重悬混匀，立即通过多功能酶标仪测定RLU(relative light unit)，测定时间设为1秒。杀伤比例计算公式：100％×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略)；其结果如图1-4所示。
结果表明，CAR19T1 T和CAR19T2 T细胞对表达CD19的肿瘤靶细胞的体外杀伤效率都显著高于CAR19 T细胞，在E：T(即Effector效应T细胞与Target靶细胞的比例)很小的情况下，即肿瘤靶细胞远大于效应T细胞，CAR19T1 T和CAR19T2 T细胞也能表现出很强的肿瘤杀伤活性(见图1-4)。
通过ELISA检测GFP T、CAR19 T、CAR19T1 T、CAR19T2 T细胞分别与K562GL或K562-CD19-GL细胞共培养18h后上清中的IL-2水平，结果如图5所示；图5结果显示，CAR19T2 T细胞分泌IL-2的水平高于CAR19 T细胞，这说明TLR2的胞内结构域的加入能提高CAR T细胞的IL-2分泌，即，可促进T细胞增殖。
另外，在CAR T细胞比例很低的情况下(如图6所示)，CARMesoT1 T细胞对表达Mesothelin的靶细胞的体外杀伤效率也显著高于CARMeso T细胞(见图7)。
实施例6 CAR19T1/T2 T细胞体内识别杀伤肿瘤
为比较GFP T、CAR19 T、CAR19T1 T和CAR19T2 T细胞的体内识别杀伤肿瘤的效应，将同等数量(2×105)的NALM6细胞分别通过皮下移植至16只NSI(NOD/SCID IL2rg-/-)免疫缺陷小鼠体内；NALM6细胞移植后第2天和第9天(肿瘤细胞移植当天为第0天)，将2×106个T细胞(四组：GFP T、CAR19 T、CAR19T1 T、CAR19T2，每组注射4只小鼠，阳性细胞比例如图8所示)静脉注射入已移植NALM6细胞的NSI免疫缺陷小鼠体内；第33天，将所有小鼠安乐处死，取肿瘤量大小并称重。
结果表明，CAR19T1 T和CAR19T2 T都能显著抑制皮下NALM6细胞的生长，而CAR19 T细胞组跟GFP T细胞组相比，肿瘤重量则没有显著变化(图9，10)。
另一方面，也说明CAR19 T虽然能对血液性肿瘤具有良好的杀伤效果，但对皮下 实体瘤的杀伤效果不佳；而加入TLR1或TLR2的胞内结构域后，则可显著改善第二代CAR T细胞对实体瘤的杀伤效果。
实施例7 CARMesoT1/T2 T细胞体内识别杀伤肿瘤
为比较CAR22 T、CARMesoT1 T和CARMesoT2 T细胞的体内识别杀伤肿瘤的效应，将同等数量(1×105)的A549-GL细胞分别移植至12只NSI(NOD/SCID IL2rg-/-)免疫缺陷小鼠后肢腹股沟处皮下；NALM6细胞移植后第7天和第14天(肿瘤细胞移植当天为第0天)，分别将2×106和1×106个T细胞(三组：CAR22 T、CARMesoT1 T和CARMesoT2 T，每组注射4只小鼠，阳性细胞比例如图6所示)静脉注射入已移植NALM6细胞的NSI免疫缺陷小鼠体内；第68天，将所有小鼠安乐处死，取肿瘤并称重记录。
结果表明，CARMeso T和CARMesoT1 T细胞均能抑制皮下肿瘤的生长，而CARMesoT1 T细胞比CARMeso T细胞的抑制效果更佳(图11)。
通过以上检测结果，对比实验组和对照组CAR T对肿瘤识别杀伤功能的强弱，验证了TLR1和TLR2信号传递结构域对CAR T体内、外肿瘤杀伤功能的改善。
申请人声明，本发明通过上述实施例来说明本发明的产品、用途及其使用方式，但本发明并不局限于上述详细用途和使用方式，即不意味着本发明必须依赖上述详细用途和使用方式才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种嵌合抗原受体，其包含能够结合抗原的胞外结构域、跨膜结构域和至少一个胞内结构域，其中，所述至少一个胞内结构域包括Toll样受体1和/或Toll样受体2胞内结构域。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述抗原是肿瘤抗原；
    优选地，所述能够结合抗原的胞外结构域是结合抗原的抗体的单链可变片段。
  3. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述胞内结构域还包括CD3ζ胞内结构域；
    优选地，所述Toll样受体1和/或Toll样受体2胞内结构域配置在CD3ζ胞内结构域的C末端侧。
  4. 根据权利要求3所述的嵌合抗原受体，其特征在于，所述胞内结构域进一步包括CD28胞内结构域；
    优选地，所述胞内结构域为自N-末端侧开始依次连接的CD28胞内结构域、CD3ζ胞内结构域和Toll样受体1和/或Toll样受体2胞内结构域；
    优选地，所述胞内结构域为自N-末端侧开始依次连接的CD3ζ胞内结构域、Toll样受体1和/或Toll样受体2胞内结构域和CD28胞内结构域。
  5. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述胞内结构域包括串联连接的两个或更多个胞内结构域；
    优选地，所述Toll样受体1和/或Toll样受体2胞内结构域配置在C-末端侧。
  6. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体自N末端侧开始依次包括抗肿瘤抗原抗体的单链可变区作为胞外结构域，CD28分子的跨膜结构域和胞内结构域，CD3ζ胞内结构域，Toll样受体1和/或Toll样受体2胞内结构域；
    优选地，所述肿瘤抗原为CD19或Mesothelin抗原。
  7. 编码如权利要求1-6任一项所述的嵌合抗原受体的核酸。
  8. 一种嵌合抗原受体表达细胞，其中引入了如权利要求7所述的核酸；优选地，所述细胞为T细胞或含有T细胞的细胞群。
  9. 制备如权利要求8所述的嵌合抗原受体表达细胞的方法，其包括将如权利要求7所述的核酸引入细胞的步骤；优选地，所述细胞为T细胞或含有T细胞的细胞群。
  10. 如权利要求1-6任一项所述的嵌合抗原受体、如权利要求7所述的核酸或如权利要求8所述的嵌合抗原受体表达细胞在制备治疗肿瘤的药物中的用途；
    优选地，所述肿瘤为实体瘤或血液瘤，更优选为B-ALL或肺癌。
PCT/CN2015/086352 2015-08-07 2015-08-07 含Toll样受体胞内结构域的嵌合抗原受体 WO2017024440A1 (zh)

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