WO2021160038A1 - 一种嵌合抗原受体及应用 - Google Patents

一种嵌合抗原受体及应用 Download PDF

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WO2021160038A1
WO2021160038A1 PCT/CN2021/075544 CN2021075544W WO2021160038A1 WO 2021160038 A1 WO2021160038 A1 WO 2021160038A1 CN 2021075544 W CN2021075544 W CN 2021075544W WO 2021160038 A1 WO2021160038 A1 WO 2021160038A1
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car
cells
domain
antigen receptor
dectin
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French (fr)
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王玮
魏于全
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四川大学
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Priority to EP21753498.1A priority patent/EP4105240A4/en
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Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a chimeric antigen receptor and its application.
  • Chimeric Antigen Receptor (CAR) T cell immunotherapy has undergone a series of evolutionary processes.
  • the first generation of CAR-mediated T cell activation was accomplished through the tyrosine activation motif on CD3 ⁇ or FceRIg.
  • the anti-tumor activity of the first-generation CAR-modified T cells is limited in the body.
  • the decrease in T cell proliferation eventually leads to T cell apoptosis.
  • the second generation CAR adds a new costimulatory signal in the cell, such as 4-1BB or CD28.
  • the second-generation CAR Compared with the first-generation CAR, the second-generation CAR has the same antigen specificity, increased T cell proliferation, increased cytokine secretion, increased secretion of anti-apoptotic proteins, and delayed cell death.
  • the existing CAR-T cell immunotherapy has only made significant progress in the treatment of hematological malignancies, and its success in the treatment of solid tumors is limited.
  • the progress of CAR-T cell immunotherapy in the treatment of hematological malignancies includes non-Hodgkin’s lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) . So far, the objective response rate (ORR) of clinical trials of CD19-specific or BCMA-specific CAR-T is 48%-95%.
  • US Food and Drug Administration US FDA approved two CAR-T cell products for B-cell lymphoma, Axicabtagene Ciloleucel (KTE-C19, Kite Pharma) and Tisagenlecleuce (CTL019, Novartis).
  • Dectin-1 is a new subgroup of C-type lectin receptors (CLRs). Dectin-1 includes extracellular domain, transmembrane domain and intracellular domain. The extracellular part of Dectin-1 has been used as a scFv targeting fungal CAR-T cells. Dectin-1 is mainly expressed not only on myeloid cells including neutrophils, monocytes, dendritic cells and macrophages, but also on certain subgroups of human T cells and B cells . Dectin-1 plays an important role in tumor growth and metastasis by activating NK cells. Dectin-1 can also regulate various cellular responses, such as DC maturation, antigen presentation, and the production of cytokines and chemokines. In addition, in theory, Dectin-1 can directly induce innate immune memory and affect the development of CD8, CD4, T cells and B cells.
  • CLRs C-type lectin receptors
  • the experimental team of the present invention designed to incorporate Dectin-1 into the second-generation CAR structure as a costimulatory signal domain to evaluate the function and anti-tumor activity of its modified second-generation CAR-T cells.
  • one of the objectives of the present invention is to provide a novel second-generation CAR structure.
  • a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the transmembrane domain and the intracellular domain of the chimeric antigen receptor form a costimulatory signal structure Domain, the costimulatory signal domain includes the full length or fragment of amino acid encoding reverse dectin-1.
  • the extracellular domain includes a single chain antibody targeting CD19 or HER2.
  • the chimeric antigen receptor also includes a CD8 ⁇ hinge region, a reverse dectin-1 transmembrane domain, a reverse dectin-1 intracellular signal domain, and a CD3 ⁇ intracellular signal domain, which are sequentially connected.
  • sequence encoding the reverse dectin-1 amino acid is shown in SEQ ID NO.3.
  • amino acid sequence encoding the reverse dectin-1 transmembrane domain is shown in SEQ ID NO.2.
  • amino acid sequence encoding the intracellular signal domain of the reverse dectin-1 is shown in SEQ ID NO.1.
  • the lentiviral vector includes pCLK, psPAX2 or pMD2.0G.
  • the purpose of the present invention is also to provide a CAR-T cell modified according to the above-mentioned novel second-generation CAR and its application.
  • a CAR-T cell expressing the above chimeric antigen receptor A CAR-T cell expressing the above chimeric antigen receptor.
  • An anti-tumor drug containing the above CAR-T cells and pharmaceutically acceptable excipients and/or adjuvants.
  • the tumor includes hematological tumors and solid tumors.
  • the tumor includes large B-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, myeloid leukemia, lymphocytic leukemia, breast cancer, gastric cancer, esophageal cancer or ovarian cancer.
  • the CAR-T cells can stimulate the secretion of effector cytokines.
  • effector cytokines include IFN- ⁇ , TNF- ⁇ and IL-6.
  • the CAR-T cells can stimulate the secretion of central memory T cells.
  • the tumor includes hematological tumors and solid tumors.
  • the tumor includes large B-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, myeloid leukemia, lymphocytic leukemia, breast cancer, gastric cancer, esophageal cancer or ovarian cancer.
  • the object of the present invention is also to provide a method for synthesizing the above-mentioned chimeric antigen receptor.
  • the method for synthesizing the above-mentioned chimeric antigen receptor is characterized in that it comprises the following steps: (1) synthesizing the gene sequence of the reverse dectin-1 transmembrane domain-the reverse dectin-1 intracellular domain or the reverse dectin -1 Transmembrane domain-CD3 ⁇ intracellular signal domain gene sequence; (2) Synthesize primers according to the target, and then synthesize the chimeric antigen receptor gene sequence by overlapping PCR method.
  • the step (2) specifically includes: first amplifying the gene sequence bound by the extracellular domain and the CD8 ⁇ hinge region with primers F1 and R1, and then amplifying the reverse dectin with primers F2 and R2 -1 transmembrane domain-CD3 ⁇ intracellular signal domain gene sequence, and finally the gene sequence combined with the extracellular domain and CD8 ⁇ hinge region and the reverse dectin-1 transmembrane domain-CD3 ⁇ intracellular signal domain
  • the gene sequence of is used as a template, and F1 and R2 are used as primers to synthesize the gene sequence of the chimeric antigen receptor.
  • primer F1 is shown in SEQ No. 7
  • R1 is shown in SEQ No. 8
  • F2 is shown in SEQ No. 9
  • R2 is shown in SEQ No. 10.
  • the method for synthesizing the gene sequence of the reverse dectin-1 transmembrane domain-reverse dectin-1 intracellular domain is consistent.
  • the present invention provides a novel second-generation CAR structure using reverse Dectin-1 as a costimulatory signal domain and CAR-T cells prepared therefrom.
  • the in vivo and in vitro experimental results of the present invention show that the new CAR design affects T cell function through Dectin-1 costimulation, and enhances the secretion and lysis ability of a variety of cytokines (including IFN- ⁇ , TNF- ⁇ and IL-6) , Reduce exhaustion, increase cell expansion and significant anti-tumor activity.
  • the experiment of the novel CAR-T cell provided by the present invention proves to be effective against a variety of solid tumors and hematological malignancies.
  • Figure 1 is a CAR construct and expression map: (A) a schematic diagram of a chimeric receptor, which contains a single-chain fragment that binds to HER2 or CD19, and is different in the transmembrane domain and the intracellular domain; (B) hH8 -BBz CAR and hHD-Dz CAR, h198-BBz CAR and h19D-Dz CAR are expressed on the surface of human T cells;
  • Figure 2 shows the in vitro cytokine production and cytotoxicity production of CAR-T cells.
  • the supernatants from four different CAR-T cells or control T cells were quantitatively analyzed by ELISA.
  • Figure 3 shows the phenotype, failure marker expression and CAR expression of HER2-specific CAR-T cells:
  • A Flow cytometry density chart of the phenotypic characteristics of each CAR-T cell: with CD3 or CD4 or CD8 or initial ( TN) (CD45RA-/CD62L-) central memory T cells (T CM ) (CD45RO+/CD62L+) or effector memory T cells (T EM ) (CD45RO+/CD62L-);
  • each CAR-T cell inhibitory molecule Flow cytometry density chart: cells with PD-1 or LAG3 or CTLA-4 or TIM3;
  • C hH8-BBz CAR-T cells and hHD-Dz CAR-T cells measured by flow cytometry over time CAR expression;
  • FIG. 5 is a schematic diagram of the second-generation chimeric antigen receptor (RD-1) targeting HER2;
  • Figure 6 is a diagram showing the expression detection of human T cells transfected with the traditional second-generation CAR gene
  • FIG. 7 is a diagram showing the expression detection of human T cells transfected with the new second-generation CAR (RD-1) gene
  • Figure 8 is a detection diagram of CAR-T cells (RD-1) targeting HER2-positive tumor cells SKOV3 and HER2-negative tumor cells MDA-MB-468 to secrete IFN- ⁇ .
  • the term “approximately” is typically expressed as +/-5% of the stated value, more typically +/-4% of the stated value, and more typically ++ of the stated value +/-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.
  • RD-1 is the reverse Dectin-1 (ReverseDection-1), and its amino acid sequence is "GLVVAIVLIVLCLIGLIVAILRWPPSAACSGKESVVAIRTNSQSDFHLQTYGDEDLNELDPHYEM” as shown in SEQ NO. 3; and the amino acid sequence of Dectin-1 is "MEYHPDLENLDEDGYTQLHFVIDSQSNTAVVAVIVAVINOSEQAVNOVAVIVAVINOGYTQLHFVIDSQSNTAVGSCALVVAVINOGYTQLHFV Shown.
  • RD-1 intracellular domain is "RWPPSAACSGKESVVAIRTNSQSDFHLQTYGDEDLNELDPHYEM”, as shown in SEQ NO.1; the amino acid sequence of "RD-1 transmembrane domain” is “GLVVAIVLIVLCLIGLIVAIL”, as shown in SEQ NO.2.
  • Dectin-1 in the CAR structure refers to the reverse Dectin-1.
  • Cell lines All cell lines are derived from ATCC. K562 cells (myeloid leukemia) and NALM6 cells (lymphocytic leukemia) were combined with heat-inactivated 10% fetal bovine serum (FBS) (PAN, Germany.Cat: ST30-3302), penicillin (100U/ mL) (Gibco, Thermo Fisher, Waltham, MA. Cat: SV30010) and streptomycin (100ug/mL) (Gibco, Thermo Fisher, Waltham, MA. Cat: SV30010).
  • FBS fetal bovine serum
  • penicillin 100U/ mL
  • streptomycin 100ug/mL
  • SK-OV-3 cystadenocarcinoma of the ovary
  • MDA-MB-468 breast cancer
  • FBS penicillin
  • streptomycin 100ug/mL Cultured in DMEM.
  • SK-OV-3 was also engineered to express luciferase.
  • Anti-CD19 or anti-HER2 CARs include single-chain variable fragments (scFv) specific for CD19 (clone FMC63) or HER2 (clone 4D5).
  • the scFv is followed by the human CD8 ⁇ hinge region, followed by the human CD8 ⁇ transmembrane domain (TM), 4-1BB or CD3 ⁇ intracellular domains (ICDs).
  • Overlapping PCR was used to form the CARs sequence.
  • a single lentiviral plasmid encoding each CAR sequence was constructed by double digestion with PCLK lentiviral vector.
  • the 4 CARs sequences constructed are as follows:
  • HEK-293T cells embryonic kidney cells
  • FBS fetal bovine serum
  • penicillin 100U/mL
  • chain in DMEM Cultivate together with mycin (100ug/mL) (Gibco, Thermo Fisher, Waltham, MA. Cat: SV30010).
  • HEK-293T cells To produce the supernatant-containing lentivirus, first transfect HEK-293T cells with the plasmids detailed above: use the appropriate CAR encoding plasmids psPAX2 and pMD2.0G (Invitrogen). Change the medium 12 hours after transfection. Collect the supernatant and centrifuge to remove cell debris. The supernatant was filtered and concentrated by ultracentrifugation at 19,700 rpm for 2 hours. Discard the supernatant. The lentivirus particles were dissolved in PBS medium, and the concentrated lentivirus was stored at -80°C. The concentrated lentivirus titer was measured by quantitative real-time polymerase chain reaction (Q-RT-PCR).
  • Q-RT-PCR quantitative real-time polymerase chain reaction
  • PBMCs peripheral blood mononuclear cells
  • X-VIVO 15 medium PBMC cultured in Sigma-Aldrich, Cat: 10771. Stimulate PBMC with anti-CD3/CD28 magnetic beads (Gibco, Thermo Fisher, Waltham, MA. Cat: 11131D). After 24 hours, the T cells were cultured with a lentivirus with a multiplicity of infection (MOI) of 5 for 48 hours, and then the cells were washed and cultured in T cell culture medium. The transduction efficiency was determined by the expression of CAR measured by flow cytometry.
  • MOI multiplicity of infection
  • anti-CD3-FITC (clone: HIT3a, Cat: 300306, Biolegend), anti-CD8-APC (clone: HIT8a, Cat: 300912, Biolegen), anti-CD4-PE (clone: RPA-T4, Cat: 300508, Biolegend), anti-CD45RO-PE (BD Biosciences, clone: UCHL1, Cat: 555493), anti-CD62L-APC (BD Biosciences, clone: DREG-56, Cat: 559772), anti-PD-1-APC (clone :EH12.2H7,Cat:329908,Biolegend), anti-CTLA-4-APC (Cat:369612,Biolegen), anti-LAG3-APC (Cat:369212,Biolegend), anti-TIM3-APC (Cat:345012, Biolegend).
  • Target cells (NALM6, K562, SK-OV-3 or MDA-MB-468, 1 ⁇ 10 5 cells/well) were seeded into 96-well plates and incubated overnight at 37°C in 5% CO 2. After that, CAR-T cells were added at an effect/target ratio (E:T) of 5 or 10. The number of CAR-T cells was normalized by transduction efficiency. The supernatant was collected after 24 hours of co-cultivation with target cells.
  • E:T effect/target ratio
  • IFN- ⁇ Cat: 88-7316-88, TNF- ⁇ Cat: 88-7346-88, and IL-6Cat: 88-7066-88 Use Invitrogen's ELISA kit for cytokine determination (IFN- ⁇ Cat: 88-7316-88, TNF- ⁇ Cat: 88-7346-88, and IL-6Cat: 88-7066-88) to quantitatively detect IFN - ⁇ , TNF- ⁇ and IL-6.
  • RTCA Real-time cytotoxicity assays
  • the cytotoxicity of CAR-T cells was measured by a real-time cytotoxicity assay (ACEA Bioscience, Inc. xCELLigence RTCA SP).
  • the SK-OV-3 or MDA-MB-468 cells were cultured in the E-plate 96 detection plate at a rate of 1 ⁇ 10 4 cells/well for about 24 hours.
  • mice 6-week-old female B-NSG mice (NOD-PrkdcscidIL2rgtm1/Bcgen) were used for this study. Each mouse received an intraperitoneal injection with 2 ⁇ 10 6 SK-OV-3-luc cells.
  • SK-OV-3-luc cells are engineered from SK-OV-3 cells, which can express luciferase, and use fluorescent signals to show tumor growth in subsequent tumor bioluminescence imaging experiments.
  • the tumors received intraperitoneal injection again, with 1 ⁇ 10 7 CAR-T cells (hH8-BBz or hHD-Dz or control). After another 3 days, the CAR-T cells were injected intraperitoneally into each mouse again.
  • the bioluminescence imaging (BLI) of the tumor was performed by IVIS (in vivo imaging system) on the planned 3rd, 10th, 17th, 24th, 31st and 55th days. Tumor flux (photons/s/cm 2 /steradian) is quantified by measuring the photon signal in the region of interest (ROI) around the tumor.
  • the BLI data was demonstrated using live image software (v2.50, Xenogen; Caliper Life Sciences). The survival analysis data was established at the death of each mouse.
  • the log-rank (Mantel-Cox) test was used to analyze Kaplan-Meier survival data. Transform the data when the variance needs to be standardized. The symbols indicate statistical significance as follows: *P ⁇ 0.05; **P ⁇ 0.01 and ***P ⁇ 0.001.
  • the scFv domain targeting CD19 or HER2 epitope is combined with Dectin-1TM, Dectin-1 and CD3 ⁇ ICD to design a new second-generation CAR construct (Figure 1A).
  • Two other CAR structures were generated using parts of the most popular second-generation CAR structure (human CD8 ⁇ TM with 4-1BB and CD3 ⁇ ICDs). All constructed CARs were expressed on the surface of T cells ( Figure 1B).
  • the expression rate of hH8-BBz (4-1BB) CAR was 49.61%
  • the expression rate of hHD-Dz (Dectin-1) CAR was 46.17%.
  • the expression rate of h198-BBz (4-1BB) CAR is 92.95%, and the expression rate of h19D-Dz (Dectin-1) CAR is 95.07%.
  • the expression of anti-HER2 CAR is lower than that of anti-CD19 CAR, there is no significant difference in the expression level of anti-HER2 or anti-CD19 CAR containing the same costimulatory signal domain.
  • the subsequent effector functions of CAR-T cells can be evaluated by the secretion of cytokines. Therefore, after exposure to tumor cells expressing HER2 or CD19, the effect of Dectin-1 as a costimulatory signal molecule on the release of cytokines from new CAR-T cells was evaluated.
  • hH8-BBz CAR-T cells secreted more TNF- ⁇ ( Figures 2A, 2B and 2C).
  • the levels of IFN- ⁇ and TNF- ⁇ produced by h19D-Dz CAR-T cells are similar to h198-BBz CAR-T cells ( Figures 2A, 2B and 2C).
  • hHD-Dz and hH8-BBz CAR-T cells can effectively lyse SK-OV-3 tumors cell.
  • hHD-Dz CAR-T cells showed much stronger lytic cytotoxicity than hH8-BBz CAR-T cells ( Figure 2D).
  • hHD-Dz, hH8-BBz CAR-T and control T cells showed similar percentages of CD4+ or CD8 + T cells (Figure 3A). Although a considerable proportion of effector memory T (TEM, CD45RO+CD62L-) was observed in hHD-Dz and hH8-BBz CAR-T cells, hHD-Dz CAR-T cells showed a higher rate than hH8-BBz CAR-T cells. Many central memory T cells (TCM, CD45RO+CD62L+) ( Figure 3A).
  • the present invention also evaluated the expression pattern of the inhibitory receptors of anti-HER2 CAR-T cells (Figure 3B), including PD-1, CTLA-4, TIM3 and LAG3.
  • the present invention further explores the time course of anti-HER2 CAR expression. Although CAR expression decreased within 96 hours, CAR expression reached more than 90% after another 48 hours (Figure 3C).
  • the above results indicate that the Dectin-1 signaling domain in the new CAR-T cells may lead to the expression of obvious phenotypes and exhaustion markers, and increase the proliferation potential of T cells.
  • the present invention further studied the anti-tumor activity of anti-HER2 CAR-T cells (hH8-BBz or hHD-Dz CAR-T cells) in vivo.
  • the overall survival and tumor volume were evaluated ( Figure 4).
  • anti-HER2 CAR-T cell therapy delayed tumor progression (Figure 4A), and the median survival time of tumor-bearing mice in the anti-HER2 CAR-T cell group at least doubled ( Figure 4A). 4B).
  • the log-rank (Mantel-Cox) test proved that there is a statistically significant difference in the survival rate between the anti-HER2 CAR-T cell group and the control T cell group.
  • 100% of the mice in the hHD-Dz CAR-T cell group survived on day 55, while the hH8-BBz CAR-T group showed a longer overall survival period ( Figure 4B).
  • This embodiment also provides a costimulatory signal domain of a chimeric antigen receptor, the costimulatory signal domain comprising an intracellular region of RD-1.
  • the costimulatory signal domain includes CD3 ⁇ .
  • the intracellular region of RD-1 comprises an amino acid sequence containing SEQ No. 1 or an amino acid sequence having no less than 90% identity with said SEQ No. 1.
  • the costimulatory signal domain further comprises CD3, CD4, CD8, FcR, DAP10, DAP12, CD27, CD28, CD137, CD134, ICOS, OX40, CD30, CD40, PD-1, LFA-1, CD2, CD7 , LIGHI, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (IGHTR), SLAMF7, NKp80 KLRE1, CD160, CD19, CD83, IL-2R ⁇ , IL-R ⁇ , IL-7R ⁇ , ITGA4, VLA1, CD49 ⁇ , ITGA4, IA4, CD49D, ITGA6, LA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11 ⁇ , LFA-1, ITGAM, CDIIB, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1.ITGB7 TAFR
  • This embodiment also provides a chimeric antigen receptor containing the aforementioned costimulatory signal domain.
  • the chimeric antigen receptor includes an antigen recognition domain, a CD8 ⁇ hinge region, a transmembrane region, an RD-1 intracellular region, and CD3 ⁇ which are sequentially connected.
  • amino acid sequence contained in the transmembrane region can anchor the chimeric antigen receptor on the cell membrane.
  • the transmembrane region includes an RD-1 transmembrane region.
  • the RD-1 transmembrane region comprises an amino acid sequence containing SEQ No. 2 or an amino acid sequence that is not less than 90% identical to SEQ No. 2.
  • the antigen recognition domain is a HER2 binding domain.
  • the chimeric antigen receptor comprises an amino acid sequence containing SEQ No. 5.
  • the antigen recognition domain is a CD19 binding domain.
  • the chimeric antigen receptor comprises an amino acid sequence containing SEQ No. 6.
  • This embodiment also provides a method for synthesizing the aforementioned chimeric antigen receptor, which includes the following steps: step (1) synthesizing the RD-1-CD3 ⁇ gene sequence; step (2) synthesizing the chimeric antigen receptor gene sequence .
  • the step (2) specifically includes: first amplifying the gene sequence combining the antigen recognition domain and the CD8 ⁇ hinge region with primers F1 and R1, and then amplifying the RD-1-CD3 ⁇ gene sequence with primers F2 and R2, Finally, the gene sequence of the antigen recognition domain and the CD8 ⁇ hinge region and the RD-1-CD3 ⁇ gene sequence are used as templates, and F1 and R2 are used as primers to synthesize the gene sequence of the chimeric antigen receptor.
  • primer F1 is shown in SEQ No. 7
  • R1 is shown in SEQ No. 8
  • F2 is shown in SEQ No. 9
  • R2 is shown in SEQ No. 10.
  • the synthetic amino acid sequence of (anti-HER2 scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic)-(CD3 ⁇ ) is SEQ No. 5.
  • This embodiment also provides a recombinant plasmid vector constructed by the aforementioned chimeric antigen receptor and expression vector.
  • the expression vector is a pCLK vector.
  • This embodiment also provides the construction method of the aforementioned recombinant plasmid vector.
  • the construction method includes using Mlu I and Spe I as restriction sites, connecting the antigen receptor gene and the expression vector to obtain the recombinant plasmid vector. Plasmid vector.
  • This embodiment also provides an immune cell modified by the aforementioned chimeric antigen receptor.
  • the immune cells are T lymphocytes.
  • the immune cells also include one or more of B lymphocytes, K lymphocytes and NK lymphocytes.
  • This embodiment also provides a method for obtaining the aforementioned immune cells.
  • the method includes the following steps: using Mlu I and Spe I as restriction sites, connecting the chimeric antigen receptor gene and the expression vector to obtain recombination Plasmid vector; the recombinant plasmid vector and the packaging plasmid are jointly transferred into cultured cells and cultured to obtain recombinant virus particles; then the recombinant virus particles are used to modify immune cells.
  • the modification method includes using a viral vector or a non-viral vector system.
  • the viral vector system includes one or more of retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors.
  • non-viral vector system includes one or more of a transposon system, a CRISPR gene editing system, a TALEN system, a liposome transfection system, and an electrotransfection system.
  • the purpose of the present invention is to also provide an application of the aforementioned immune cells in the preparation of anti-tumor drugs.
  • the anti-tumor drug composition is characterized in that it also includes a chemical drug.
  • chemical drugs include cyclophosphamide and/or fludarabine.
  • the anti-tumor drug composition is an anti-breast cancer and/or anti-ovarian cancer drug.
  • This embodiment also provides a composition comprising the aforementioned chimeric antigen receptor, the composition further comprising ⁇ -glucan receptor, Syk, CR3, and CD11b that bind to the costimulatory signal domain One or more.
  • the primers used to obtain the CAR gene in this example are shown in Table 1.
  • the CAR gene whose full-length antigen is HER2 is obtained by PCR:
  • the first step, gene synthesis synthesize the full length of the RD-1 (TM+cytoplasmic)-CD3 ⁇ fragment.
  • the second step is to synthesize primers and use overlapping PCR to obtain (anti-HER2 scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic)-(CD3 ⁇ ) fusion fragment, which specifically includes the following two steps:
  • the synthetic amino acid sequence of (anti-HER2 scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic)-(CD3 ⁇ ) is SEQ NO.5.
  • the first step, gene synthesis synthesize the full length of the RD-1 (TM+cytoplasmic)-CD3 ⁇ fragment.
  • the second step is to design primers and use overlap PCR to obtain (anti-HER2 scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic)-(CD3 ⁇ ) fusion fragment, which specifically includes the following two steps:
  • the new second-generation CAR gene with chimeric antigen receptor gene (anti-HER2 scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic)-(CD3 ⁇ ) (the structure diagram is shown in Figure 5)
  • the traditional two Generation CAR gene (anti-HER2 scFV)-(CD8 ⁇ hinge+TM)-(4-1BB)-(CD3 ⁇ ) (the schematic diagram is shown in Figure 5) are respectively loaded into the lentiviral vector PCLK, and combined with the two helper vectors psPAX2 and pMD2.G is co-transfected into T cells, packaged separately to obtain virus particles, and concentrated by centrifugation to obtain high-concentration lentiviral vectors.
  • lymphocytes Use density gradient centrifugation to separate lymphocytes, and stimulate the lymphocytes with CD3 antibody (1ug/ml) and IL-2 (100IU/ml). One day later, lymphocytes were collected for virus transfection, and lymphocytes were cultured for 48 hours, and the transfected lymphocytes were collected.
  • the collected lymphocytes that have been transfected with the virus are subjected to flow cytometric detection with a specific antibody that recognizes the Fab fragment of the antibody.
  • Figure 6 shows the expression detection of traditional second-generation CAR gene transfected human T cells.
  • the primary antibody is a biotin-labeled goat anti-mouse Fab monoclonal antibody
  • the second antibody is a PE-labeled anti-streptavidin flow cytometry antibody.
  • the first lane is T cells transfected with empty virus
  • the second lane is T cells transfected with CAR gene.
  • hH8-BBz is the traditional second-generation CAR gene (anti-HER2 scFV)-(CD8 ⁇ hinge+TM)-(4-1BB)-(CD3 ⁇ );).
  • Figure 7 shows the expression detection of the new second-generation CAR gene transfected into human T cells.
  • the primary antibody is a biotin-labeled goat anti-mouse Fab monoclonal antibody
  • the second antibody is a PE-labeled anti-streptavidin flow cytometry antibody.
  • the first lane is T cells transfected with empty virus
  • the second lane is T cells transfected with CAR gene.
  • hHD-Dz is the new second-generation CAR gene (anti-HER2 scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic)-(CD3 ⁇ )).
  • New second-generation CAR-T cells (anti-HER2 scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic)-(CD3 ⁇ )-T cells
  • RTCA Real Time Cellular Analysis, real-time label-free cell analysis technology
  • hH8-BBz is the traditional second-generation CAR gene (anti -HER2 scFV)-(CD8 ⁇ hinge+TM)-(4-1BB)-(CD3 ⁇ ); hHD-Dz is the new second-generation CAR gene (anti-HER2scFV)-(CD8 ⁇ hinge)-(RD-1TM+Cytoplasmic) -(CD3 ⁇ )).
  • a typical CAR mainly consists of three key parts, including antigen-recognizing single-chain variable fragments (scFv), hinge and transmembrane domains (TM), such as CD3, CD28 or CD8 proteins; and intracellular signaling domains (ICDs) , Such as CD3 ⁇ or FcR ⁇ .
  • CAR contains one or more intracellular costimulatory signal domains, such as CD28, 4-1BB, CD27, OX40, ICOS, DAP10, IL-15R ⁇ , MyD88/CD40 and TLR2 to transmit activation signals.
  • our experimental team couples the scFv domain targeting CD19 or HER2 with 4-1BB or dectin-1 signaling ICD to construct four different second-generation CARs.
  • Experimental data shows that in both in vitro and in vivo experiments, the new CAR design affects T cell function through Dectin-1 costimulation, such as enhancing the secretion and lysis of cytokines, reducing failure, increasing cell expansion and significantly The anti-tumor activity.
  • T CM cells are far more important than T EM cells.
  • T CM and significant depletion marker expression in hHD-Dz CAR-T cells may indicate that new CAR-T cells can be transduced by Dectin-1 costimulatory signals in solid tumors. It is less affected by the tumor immunosuppressive microenvironment.
  • Li D Li D, Li X, Zhou WL, Huang Y, Liang X, Jiang L, et al.Genetically engineered T cells for cancer immunotherapy.Signal transduction and targeted therapy 2019, 4:35.
  • Brown GD Crocker PR.Lectin Receptors Expressed on Myeloid Cells.Microbiologyspectrum 2016, 4(5).

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Abstract

提供一种嵌合抗原受体，包括胞外结构域、跨膜结构域和胞内结构域组成的共刺激信号结构域，该共刺激信号结构域包括编码反向dectin-1的氨基酸全长或片段，可治疗多种实体肿瘤和血液恶性肿瘤。

Description

一种嵌合抗原受体及应用
优先权申请
本申请要求2020年2月13提交的中国发明专利申请【CN2020100907495】、名称为“一种嵌合抗原受体的共刺激信号结构域及其应用”的优先权，该优先权发明专利申请以引用方式全文并入。
技术领域
本发明属于生物医药领域，具体涉及一种嵌合抗原受体及应用。
背景技术
嵌合抗原受体(Chimeric Antigen Receptor，CAR)T细胞免疫疗法，作为一种非常有前景的肿瘤治疗策略，经历了一系列演化过程。第一代CAR介导的T细胞激活是通过CD3ζ或FceRIg上的酪氨酸激活基序完成的。但第一代CAR改造T细胞的抗肿瘤活性在体内受到了限制，因T细胞增殖减少最终导致T细胞的凋亡。第二代CAR在胞内增加了一个新的共刺激信号，例如4-1BB或CD28。第二代CAR与第一代CAR相比，抗原特异性不变，T细胞增殖、细胞因子分泌增加，抗细胞凋亡蛋白分泌增加，细胞死亡延迟。然而，现有的CAR-T细胞免疫疗法目前仅在治疗血液恶性肿瘤方面取得了显著进展，而在治疗实体瘤领域的成功有限。
CAR-T细胞免疫疗法在治疗血液恶性肿瘤方面的进展包括非霍奇金淋巴瘤(NHL)，B细胞急性淋巴细胞白血病(ALL)，多发性骨髓瘤(MM)和慢性淋巴细胞白血病(CLL)。到目前为止，CD19特异性或BCMA特异性CAR-T的临床试验的客观缓解率(ORR)为48％～95％。2017年，美国食品药品监督 管理局(US FDA)批准了两种针对B细胞淋巴瘤的CAR-T细胞产品，AxicabtageneCiloleucel(KTE-C19,Kite Pharma)和Tisagenlecleuce(CTL019,Novartis)。在Axicabtageneciloleucel的1/2期临床试验中，对108名难治性大B细胞淋巴瘤患者进行了为期2年的随访。结果显示，83％的患者达到客观缓解(OR)，58％的患者达到完全缓解(CR)，平均随访期为15.4个月(IQR 13.7-17.3)。这表明CAR-T细胞治疗可以维持长期缓解。但在实体瘤治疗中，CAR-T细胞疗法仍然面临许多挑战。例如缺乏适当的肿瘤特异性抗原，肿瘤微环境抑制以及CAR-T细胞定位和持久性不足。另外，持续的抗原暴露会导致CAR-T细胞衰竭，进而损害CAR-T细胞对肿瘤的有效性。因此，需要设计新的CAR来用于实体瘤的治疗。
Dectin-1，即CLEC-7A，是C型凝集素受体(CLRs)的一个新的亚组。Dectin-1包括细胞外结构域，跨膜结构域和细胞内结构域。Dectin-1的细胞外部分已被用作靶向真菌的CAR-T细胞的scFv。Dectin-1不仅主要在包括中性粒细胞，单核细胞，树突状细胞和巨噬细胞在内的髓样细胞上表达，而且在人的T细胞和B细胞的某些亚群上也表达。Dectin-1通过激活NK细胞在肿瘤生长和转移中起重要作用。Dectin-1还可以调节各种细胞反应，例如DC成熟，抗原呈递以及细胞因子和趋化因子的产生。此外，理论上，Dectin-1可以直接诱导先天免疫记忆，并影响CD8、CD4、T细胞和B细胞的发育。
综上，本发明实验团队设计将Dectin-1并入二代CAR结构中作为共刺激信号域，评估其改造的新型二代CAR-T细胞的功能和抗肿瘤活性。以解决上述问题中的至少一个。
发明内容
有鉴于此，本发明的目的之一在于提供一种新型的二代CAR结构。
一种嵌合抗原受体，包括胞外结构域、跨膜结构域和胞内结构域，所述嵌合抗原受体的所述跨膜结构域和所述胞内结构域组成共刺激信号结构域，所述共刺激信号结构域包括编码反向dectin-1的氨基酸全长或片段。
进一步，所述胞外结构域包括靶向CD19或靶向HER2的单链抗体。
进一步，所述嵌合抗原受体还包括依次连接的CD8α铰链区、反向dectin-1跨膜结构域、反向dectin-1胞内信号域和CD3ζ胞内信号域。
进一步，编码所述反向dectin-1氨基酸的序列如SEQ ID NO.3所示。
进一步，编码所述反向dectin-1跨膜结构域的氨基酸的序列如SEQ ID NO.2所示。
进一步，编码所述反向dectin-1胞内信号域的氨基酸序列如SEQ ID NO.1所示。
包含上述任一项所述的嵌合抗原受体的慢病毒载体。
进一步，所述慢病毒载体包括pCLK、psPAX2或pMD2.0G。
本发明的目的还在于提供一种根据上述新型的二代CAR改造的CAR-T细胞及其应用。
一种CAR-T细胞，表达上述嵌合抗原受体。
一种抗肿瘤药，含有上述CAR-T细胞和药学上允许添加的辅料和/或助剂。
进一步，所述肿瘤包括血液肿瘤和实体肿瘤。
进一步，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
上述CAR-T细胞在制备抗肿瘤药物中的应用。
进一步，所述CAR-T细胞可以刺激效应细胞因子分泌。
进一步，所述效应细胞因子包括IFN-γ，TNF-α和IL-6。
进一步，所述CAR-T细胞可以刺激细中央记忆型T细胞分泌。
进一步，所述肿瘤包括血液肿瘤和实体肿瘤。
进一步，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
本发明的目的还在于提供一种合成上述嵌合抗原受体的方法。
上述嵌合抗原受体的合成方法，其特征在于，包括以下步骤：(1)合成所述反向dectin-1跨膜结构域-反向dectin-1胞内结构域的基因序列或反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列；(2)根据靶点合成引物，再利用重叠PCR法合成所述嵌合抗原受体基因序列。
进一步，所述步骤(2)具体包括：先用引物F1、R1扩增所述胞外结构域和所述CD8α铰链区结合的基因序列，然后再用引物F2、R2扩增所述反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列，最后以所述胞外结构域和CD8α铰链区结合的基因序列和所述反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列为模板，以F1、R2为引物，合成所述嵌合抗原受体的基因序列。
进一步，所述引物F1如SEQ No.7所示，R1如SEQ No.8所示，F2如SEQ No.9所示，R2如SEQ No.10所示。
合成所述反向dectin-1跨膜结构域-反向dectin-1胞内结构域的基因序列的方法一致。
有益效果
本发明提供了一种以反向Dectin-1作为共刺激信号域的新型二代CAR结构以及其制备得到的CAR-T细胞。本发明的体内体外实验结果显示，新的CAR设计通过Dectin-1共刺激影响了T细胞功能，增强了多种细胞因子(包括IFN-γ，TNF-α和IL-6)的分泌和裂解能力，降低了衰竭力，增加了细胞扩增和显著的抗肿瘤活性。本发明提供的新型CAR-T细胞实验证明对多种实体肿瘤和血液恶性肿瘤有效。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。
图1是CAR构建体和表达图：(A)嵌合受体的示意图，该受体包含与HER2或CD19结合的单链片段，并且在跨膜结构域和胞内域不同；(B)hH8-BBz CAR和hHD-Dz CAR以及h198-BBz CAR和h19D-Dz CAR在人T细胞的表面表达；
图2为CAR-T细胞的体外细胞因子产生和细胞毒性产生图，通过ELISA定量分析了来自四个不同CAR-T细胞或对照T细胞的上清液，细胞与目标阳性或阴性肿瘤细胞共培养过夜(E：T＝10：1或5：1)(n＝3/组)；通过单向方差分析进行统计分析，然后进行Tukey的事后分析；显着性认为P＜0.05：(A)IFN-γ,(B)IL-6和(C)TNF-α,(D)RTCA分析显示T细胞溶解能力(n＝4/组)；
图3为HER2特异性CAR-T细胞的表型、衰竭标记表达和CAR表达：(A)每个CAR-T细胞表型特征的流式细胞仪密度图：具有CD3或CD4或CD8或初 始(TN)(CD45RA-/CD62L-)中央记忆T细胞(T CM)(CD45RO+/CD62L+)或效应记忆T细胞(T EM)(CD45RO+/CD62L-)；(B)每个CAR-T细胞抑制分子的流式细胞仪密度图：具有PD-1或LAG3或CTLA-4或TIM3的细胞；(C)通过流式细胞仪测量的hH8-BBz CAR-T细胞和hHD-Dz CAR-T细胞随时间的CAR表达；
图4为HER2特异性CAR-T细胞的体内抗肿瘤活性：(A)SK-OV-3-luc荷瘤模型的体内实时成像(n＝4/组)；(B)每组总体生存率的Kaplan-Meier分析；
图5为靶向HER2的二代嵌合抗原受体(RD-1)的结构示意图；
图6为传统二代CAR基因转染人的T细胞的表达检测图；
图7为新二代CAR(RD-1)基因转染人的T细胞的表达检测图；
图8为CAR-T细胞(RD-1)靶向HER2阳性肿瘤细胞SKOV3和HER2阴性肿瘤细胞MDA-MB-468分泌IFN-γ的检测图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。
在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围
Figure PCTCN2021075544-appb-000001
的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。
“RD-1”即为反向Dectin-1(Reverse Dection-1),其氨基酸序列为“GLVVAIVLIVLCLIGLIVAILRWPPSAACSGKESVVAIRTNSQSDFHLQTYGDEDLNELDPHYEM”如SEQ NO.3所示；而Dectin-1的氨基酸序列为“MEYHPDLENLDEDGYTQLHFDSQSNTRTAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLG”如SEQ NO.4所示。“RD-1胞内结构域”氨基酸序列为“RWPPSAACSGKESVVAIRTNSQSDFHLQTYGDEDLNELDPHYEM”，如SEQ  NO.1所示；“RD-1跨膜结构域”氨基酸序列为“GLVVAIVLIVLCLIGLIVAIL”，如SEQ NO.2所示。发明人研究发现，在制备新型二代CAR时，采用反向Dectin-1作为共刺激结构域能高效地产生和保持Dectin-1的活性和功能(即能影响T细胞功能，增强多种细胞因子(包括IFN-γ，TNF-α和IL-6)的分泌和裂解能力，降低衰竭力，增加细胞扩增和显著的抗肿瘤活性)。因此在本文中，如无特别说明，在CAR结构中Dectin-1均是指的反向Dectin-1。
实施例一
1.材料与方法
细胞系：所有细胞系来源于ATCC。将K562细胞(髓细胞白血病)和NALM6细胞(淋巴细胞白血病)在RPMI-1640中与热灭活的10％胎牛血清(FBS)(PAN,Germany.Cat:ST30-3302)、青霉素(100U/mL)(Gibco,Thermo Fisher,Waltham,MA.Cat:SV30010)和链霉素(100ug/mL)(Gibco,Thermo Fisher,Waltham,MA.Cat:SV30010)一起培养。人癌细胞系SK-OV-3(卵巢囊腺癌)和MDA-MB-468(乳腺癌)在含有热灭活的10％FBS、青霉素(100U/mL)和链霉素(100ug/mL)的DMEM中培养。还工程化SK-OV-3使其表达荧光素酶。
2.构建编码CAR的质粒
抗CD19或抗HER2的CARs包括特异于CD19(克隆FMC63)或HER2(克隆4D5)的单链可变片段(scFv)。scFv之后是人CD8α铰链区，然后是人CD8α跨膜结构域(TM)，4-1BB或CD3ζ胞内结构域(ICDs)。使用重叠PCR形成CARs序列。编码每个CAR序列的单个慢病毒质粒是用PCLK慢病毒载体进行双酶切构建的。构建的4条CARs序列如下：
1)hH8-BBZ:HER2 scFv-CD8α(hinge+TM)-4-1BB-CD3ζ ICDs
2)hHD-DZ:HER2 scFv-CD8α hinge+Dectin-1(TM+cytoplasm)-CD3ζ ICDs
3)h198-BBZ:CD19 scFv-CD8α(hinge+TM)-4-1BB-CD3ζ ICDs
4)h19D-DZ:CD19 scFv-CD8α hinge+Dectin-1(TM+cytoplasm)-CD3ζ ICDs
3.生产慢病毒颗粒
将购于ATCC的HEK-293T细胞(胚胎肾细胞)在DMEM中与热灭活的10％胎牛血清(FBS)(PAN,Germany.Cat:ST30-3302)，青霉素(100U/mL)和链霉素(100ug/mL)(Gibco,Thermo Fisher,Waltham,MA.Cat:SV30010)一起培养。
为生产含有上清液的慢病毒，先用上述详述的质粒转染HEK-293T细胞：用适合的CAR编码质粒psPAX2和pMD2.0G(Invitrogen)。转染12小时后更换培养基。收集上清液并离心除去细胞碎片。过滤上清液，并通过在19,700rpm下超速离心2小时进行浓缩。弃去上清液。将所述慢病毒颗粒溶解在PBS培养基中，并将浓缩的慢病毒保存在-80℃。通过定量实时聚合酶链反应测量浓缩的慢病毒滴度(Q-RT-PCR)。
4.分离、转导、生产和扩增CAR-T细胞
用Ficoll-hypaque密度梯度法(Lonza,Cat:04-418Q)从健康献血者血液中分离外周血单个核细胞(PBMCs)，所有样本均经生物治疗国家重点实验室伦理委员会知情同意并批准后获得。
在含有5％人血清(Sigma-Aldrich,H4522)和100U/ml重组人IL-2(rhIL-2)(PeproTech,NJ,USA.Cat:200-02-10)的X-VIVO 15培养基(Sigma-Aldrich,Cat:10771)中培养的PBMC。用抗CD3/CD28磁珠(Gibco,Thermo Fisher,Waltham, MA.Cat:11131D)刺激PBMC。24小时后，将T细胞与感染复数(MOI)为5的慢病毒培养48小时，然后将细胞洗涤并在T细胞培养基中培养。通过流式细胞仪测定的CAR表达确定转导效率。
5.流式细胞检测
所有流式细胞仪检测均在新生细胞流式细胞仪(ACEA Biosicences,Inc.)上进行，并用新生细胞表达(ACEA Biosicences,Inc.)分析数据。
利用生物素-SP结合的亲和山羊抗鼠IgG(Cat:120962,Jackson Immune Research)，F(ab’)2片段与PE-链霉亲和素(Cat:405203,BD Biosciences)特异性结合，评价其转导效率和相关CAR蛋白的表达。
以下抗体用于分化表型和衰竭标记分析：
anti-CD3-FITC(clone:HIT3a,Cat:300306,Biolegend)、anti-CD8-APC(clone:HIT8a,Cat:300912,Biolegen)、anti-CD4-PE(clone:RPA-T4,Cat:300508,Biolegend)、anti-CD45RO-PE(BD Biosciences,clone:UCHL1,Cat:555493)、anti-CD62L-APC(BD Biosciences,clone:DREG-56,Cat:559772)、anti-PD-1-APC(clone:EH12.2H7,Cat:329908,Biolegend)、anti-CTLA-4-APC(Cat:369612,Biolegen)、anti-LAG3-APC(Cat:369212,Biolegend)、anti-TIM3-APC(Cat:345012,Biolegend)。
6.体外细胞因子实验
将靶细胞(NALM6，K562，SK-OV-3或MDA-MB-468，以1×10 5个细胞/孔)接种到96孔板中，并在5％CO 2中于37℃孵育过夜。之后，以效/靶比(E：T)为5或10的比例添加CAR-T细胞。通过转导效率将CAR-T细胞数量归一化(normalized)。与靶细胞共培养24小时后收集上清液。使用Invitrogen公司 用于细胞因子测定的ELISA试剂盒(IFN-γ Cat:88-7316-88,TNF-α Cat:88-7346-88,and IL-6Cat:88-7066-88)来定量检测IFN-γ，TNF-α和IL-6。
7.实时细胞毒性检测(Real-time cytotoxicity assays,RTCA)
通过实时细胞毒性测定法测量CAR-T细胞的细胞毒性作用(ACEA Bioscience,Inc.xCELLigence RTCA SP)。将SK-OV-3或MDA-MB-468细胞以1×10 4个细胞/孔的数量在E-plate 96检测板中培养约24小时。将CAR-T细胞(hH8-BBz和hHD-Dz)或对照T细胞(mock T cells)以10的效/靶比添加到平板中，根据制造商提供的实验方法采集和分析数据(ACEA 162 Bioscience,Inc.RTCA Software 2.1)。
8.体内异种移植研究
采用6周大的雌性B-NSG小鼠(NOD-PrkdcscidIL2rgtm1/Bcgen)用于本研究。每只小鼠接受一次腹腔注射，注射2×10 6个SK-OV-3-luc细胞。
SK-OV-3-luc细胞是由SK-OV-3细胞工程化设计得到，其可以表达荧光素酶，在后续的肿瘤生物发光成像实验中通过荧光信号显示肿瘤的生长情况。
肿瘤生长3天后再次接受腹腔注射，注射1×10 7个CAR-T细胞(hH8-BBz或hHD-Dz或对照)。再过3天后，再进行一次将CAR-T细胞通过腹腔注射给每只小鼠。肿瘤的生物发光成像(BLI)在计划的第3天、第10天、第17天、第24天、第31天和第55天通过IVIS(活体成像系统)进行。肿瘤通量(光子/s/cm 2/steradian)是通过测量肿瘤周围感兴趣区域(ROI)内的光子信号来量化的。利用活体图像软件(v2.50,Xenogen；Caliper Life Sciences)对BLI数据进行了演示。在每只小鼠死亡时建立生存分析数据。
9.数据
使用GraphPad Prism v6.01(GraphPad Software Inc.)和SPSS v17进行统计绘图和分析。数据表示为平均值±SD。单向方差分析用于在单一条件下3组的比较。
使用对数秩(Mantel-Cox)检验分析Kaplan-Meier生存数据。在需要标准化方差时对数据进行转换。符号表示统计显着性如下：*P<0.05；**P<0.01和***P<0.001。
实施例二
1.具有Dectin-1共刺激信号域的新型CAR结构
在本发明中，将靶向CD19或HER2表位的scFv域与Dectin-1TM，Dectin-1和CD3ζ ICD结合，设计了新型的第二代CAR构建体(图1A)。用最流行的第二代CAR结构(带有4-1BB和CD3ζ ICDs的人源CD8α TM)的部分生成了另外两个CAR结构。所有构建的CAR在T细胞表面表达(图1B)。关于抗HER2的CARs，hH8-BBz(4-1BB)CAR表达率为49.61％，hHD-Dz(Dectin-1)CAR表达率为46.17％。关于抗CD19的CARs，h198-BBz(4-1BB)CAR表达率是92.95％，h19D-Dz(Dectin-1)CAR表达率是95.07％。尽管抗HER2 CAR的表达低于抗CD19 CAR，但在包含相同共刺激信号域的抗HER2或抗CD19 CAR中，其表达水平上没有明显差异。
2.Dectin-1共刺激对新型CAR-T细胞效应功能的影响
通常，可以通过细胞因子的分泌来评估CAR-T细胞后来的效应子功能。因此，在暴露于表达HER2或CD19的肿瘤细胞后，评估Dectin-1作为共刺激信号分子对新CAR-T细胞释放细胞因子的影响。
在靶向阳性细胞系(SK-OV-3和NALM6)中吸收Dectin-1信号结构域后， 抗HER2或抗CD19 CAR-T细胞释放的效应细胞因子(包括IFN-γ，TNF-α和IL-6)显着增加(图2A，2B和2C)。本研究中的抗HER2 CAR-T细胞未显示出针对MD-MB-468(阴性细胞系)的细胞因子产生显着增加(图2A，2B和2C)。在SK-OV-3细胞系中，hHD-Dz CAR-T细胞比hH8-BBz CAR-T细胞显示更高的IFN-γ水平。相反，hH8-BBz CAR-T细胞分泌更多的TNF-α(图2A，2B和2C)。在NALM6细胞系中，h19D-Dz CAR-T细胞产生的IFN-γ和TNF-α水平与h198-BBz CAR-T细胞相似(图2A，2B和2C)。
进一步研究了抗HER2 CAR-T细胞的细胞毒性功能，以说明抗原结合(antigen engagement)和CAR-T细胞的活化。在将抗HER2 CAR-T细胞与SK-OV-3或MDA-MB-468肿瘤细胞系共培养后，观察到hHD-Dz和hH8-BBz CAR-T细胞均能有效裂解SK-OV-3肿瘤细胞。hHD-Dz CAR-T细胞显示出比hH8-BBz CAR-T细胞强得多的裂解细胞毒性功能(图2D)。
3.抗HER2 CAR-T细胞的表型和衰竭标记表达
在细胞扩增7天后，分析了抗HER2 CAR-T细胞和对照T细胞的表型和衰竭标记表达(图3)。
hHD-Dz，hH8-BBz CAR-T和对照T细胞显示出相似的CD4 +或CD8 +T细胞百分比(图3A)。尽管在hHD-Dz和hH8-BBz CAR-T细胞中观察到了相当比例的效应记忆T(TEM，CD45RO+CD62L-)，但hHD-Dz CAR-T细胞比hH8-BBz CAR-T细胞显示出更多的中央记忆T细胞(TCM，CD45RO+CD62L+)(图3A)。
本发明还评估了抗HER2 CAR-T细胞(图3B)抑制性受体的表达模式，包括PD-1、CTLA-4、TIM3和LAG3。
结果表明，hHD-Dz CAR-T细胞中的PD-1或LAG3阳性细胞比hH8-BBz CAR-T细胞中少约10％。就TIM3和CTLA-4阳性细胞而言，在hHD-Dz和hH8-BBz CAR-T细胞中观察到了相似的百分比(图3B)。
本发明还进一步探讨了抗HER2 CAR表达的时程。尽管在96小时内CAR表达下降，但在另外48小时后CAR表达达到90％以上(图3C)。
总结起来，以上结果表明，新型CAR-T细胞中的Dectin-1信号域可能导致明显的表型和衰竭标记表达，并提高T细胞的增殖潜能。
4.抗HER2 CAR-T细胞的体内抗肿瘤活性
使用携带异种移植SK-OV-3-luc肿瘤细胞的NSG小鼠，本发明进一步研究了抗HER2 CAR-T细胞(hH8-BBz或hHD-Dz CAR-T细胞)的体内抗肿瘤活性。评价了总生存期和肿瘤体积(图4)。与对照T细胞治疗的小鼠相比，抗HER2 CAR-T细胞治疗使肿瘤进展延迟(图4A)，抗HER2 CAR-T细胞组的荷瘤小鼠中位生存期至少翻了一番(图4B)。对数秩(Mantel-Cox)测试证明，抗HER2 CAR-T细胞组和对照T细胞组之间的存活率存在统计学上的显着差异。此外，hHD-Dz CAR-T细胞组中的100％小鼠在第55天还存活，而hH8-BBz CAR-T组显示出更长的总生存期(图4B)。
实施例三
本实施例还提供一种嵌合抗原受体的共刺激信号结构域，所述共刺激信号结构域包含RD-1胞内区。
进一步，所述共刺激信号结构域包含CD3ζ。
进一步，所述RD-1胞内区包含含有SEQ No.1的氨基酸序列或与所述SEQ No.1不低于90％同一性的氨基酸序列。
进一步，所述共刺激信号结构域还包含CD3,CD4,CD8,FcR,DAP10,DAP12,CD27,CD28,CD137,CD134,ICOS,OX40,CD30,CD40,PD-1,LFA-1,CD2,CD7,LIGHI,NKG2C,B7-H3,特异结合CD83的配体，CDS,ICAM-1,GITR,BAFFR,HVEM(IGHTR),SLAMF7,NKp80 KLRE1,CD160,CD19,CD83,IL-2Rβ,IL-Rγ,IL-7Rα,ITGA4,VLA1,CD49α,ITGA4,IA4,CD49D,ITGA6,LA-6,CD49f,ITGAD,CD11d,ITGAE,CD103,ITGAL,CD11α,LFA-1,ITGAM,CDIIB,ITGAX,CD11c,ITGB1,CD29,ITGB2,CD18,LFA-1.ITGB7 TAFR2,TRANCE/RANKL,DNAM1(CD226),SLAMF4(CD244,2B4),CD84,CD96(Tactile),CEACAM1 CRTAM,Ly9(CD229),D160(BY55),PSGL1,CD100(SEMA4D),CD69,SLAMF6(NTB-A,Ly108 SLAM(SLAMF1,CD150,IPO-3),BLAME(SLAME8),SELPLG(CD162),LTBR,LAT,GADS,SLP-76 PAG/Cbp,NKp4,NWKp30,NKp46,NKG2D中的一种或多种。
本实施例还提供一种包含前述的共刺激信号结构域的嵌合抗原受体。
进一步，所述嵌合抗原受体包括依次连接的抗原识别域、CD8α铰链区、跨膜区、RD-1胞内区和CD3ζ。
进一步，所述跨膜区包含的氨基酸序列能够将所述嵌合抗原受体锚定在细胞膜上。
进一步，所述跨膜区包含RD-1跨膜区。
进一步，所述RD-1跨膜区包含含有SEQ No.2氨基酸序列或与所述SEQ No.2不低于90％同一性的氨基酸序列。
优选地，所述抗原识别域是HER2结合域。
进一步，所述嵌合抗原受体包含含有SEQ No.5的氨基酸序列。
优选地，所述抗原识别域是CD19结合域。
进一步，所述的嵌合抗原受体包含含有SEQ No.6的氨基酸序列。
本实施例还提供一种前述的嵌合抗原受体的合成方法，所述方法包括以下步骤：步骤(1)合成RD-1-CD3ζ基因序列；步骤(2)合成嵌合抗原受体基因序列。
进一步，所述步骤(2)具体包括：先用引物F1、R1扩增抗原识别域和CD8α铰链区结合的基因序列，然后再用引物F2、R2扩增所述RD-1-CD3ζ基因序列，最后以所述抗原识别域和CD8α铰链区结合的基因序列和所述RD-1-CD3ζ基因序列为模板，以F1、R2为引物，合成所述嵌合抗原受体的基因序列。
进一步，所述引物F1如SEQ No.7所示，R1如SEQ No.8所示，F2如SEQ No.9所示，R2如SEQ No.10所示。
合成的(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ)的氨基酸序列为SEQ No.5。
本实施例还提供一种前述的嵌合抗原受体与表达载体构建的重组质粒载体。
进一步，所述表达载体为pCLK载体。
本实施例还提供了前述的重组质粒载体的构建方法，所述构建方法包括以Mlu I和Spe I为酶切位点，连接所述抗原受体基因与所述表达载体，从而获得所述重组质粒载体。
本实施例还提供了一种前述嵌合抗原受体修饰的免疫细胞。
进一步，所述免疫细胞为T淋巴细胞。
进一步，所述免疫细胞还包括B淋巴细胞、K淋巴细胞和NK淋巴细胞中 的一种或多种。
本实施例还提供了一种获取前述的免疫细胞的方法，所述方法包括以下步骤：以Mlu I和Spe I为酶切位点，连接所述嵌合抗原受体基因与表达载体，得重组质粒载体；将所述重组质粒载体与包装质粒共同转入培养细胞中培养，获得重组病毒颗粒；再用所述重组病毒颗粒对免疫细胞进行修饰。
进一步，所述修饰的方法包括采用病毒载体或非病毒载体系统。
进一步，所述病毒载体系统包括逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体和仙台病毒载体中的一种或多种。
进一步，所述非病毒载体系统包括转座子系统、CRISPR基因编辑系统、TALEN系统、脂质体转染系统和电转染系统中的一种或多种。
本发明目的在于还提供一种前述免疫细胞在制备抗肿瘤药物中的应用。
进一步，所述抗肿瘤药物组合物，其特征在于，还包括化学药物。
进一步，所述化学药物包括环磷酰胺和/或氟达拉滨。
进一步，所述抗肿瘤药物组合物为抗乳腺癌和/或抗卵巢癌药物。
本实施例还提供了一种包含前述嵌合抗原受体的组合物，所述组合物还包括与所述共刺激信号结构域结合的β-葡聚糖受体、Syk、CR3和CD11b中的一种或多种。
本实施例中获得CAR基因所用的引物情况如表1。
表1获得CAR基因所用的引物
Figure PCTCN2021075544-appb-000002
Figure PCTCN2021075544-appb-000003
实施例四
PCR获得全长抗原为HER2的CAR基因：
(1)第一步，基因合成：合成RD-1(TM+cytoplasmic)-CD3ζ片段全长。
(2)第二步，合成引物，并利用重叠PCR方法得到(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ)融合片段，其中具体包括以下两个步骤：
(2.1)利用引物F1、R1扩增(anti-HER2 scFV)-(CD8α hinge)；利用F2、R2扩增(RD-1TM+Cytoplasmic)-(CD3ζ)。
(2.2)再以(anti-HER2 scFV)-(CD8α hinge)和(RD-1TM+Cytoplasmic)-(CD3ζ)为模版，F1、R2为引物，通过PCR获得全长CAR基因。
合成的(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ)的氨基酸序列为SEQ NO.5.
抗原为HER2的CAR的重组质粒载体的构建：
(1)第一步，基因合成：合成RD-1(TM+cytoplasmic)-CD3ζ片段全长。
(2)第二步，设计引物，并利用重叠PCR方法得到(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ)融合片段，其中具体包括以下两个步骤：
(2.1)利用引物F1、R1扩增(anti-HER2 scFV)-(CD8α hinge)；利用F2、R2扩增(RD-1TM+Cytoplasmic)-(CD3ζ)；
(2.2)以(anti-HER2 scFV)-(CD8α hinge)和(RD-1TM+Cytoplasmic) -(CD3ζ)为模版，F1、R2为引物，PCR获得全长CAR基因。
(3)以Mlu I和Spe I为酶切位点，连接通过上述步骤合成的CAR基因及PCLK载体，获得CAR-PCLK质粒载体。
T淋巴细胞的病毒转染及目的基因的表达检测：
(1)将具有嵌合抗原受体基因新的二代CAR基因(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ)(结构示意图如图5)，传统二代CAR基因(anti-HER2 scFV)-(CD8α hinge+TM)-(4-1BB)-(CD3ζ)(结构示意图如图5)分别装入慢病毒载体PCLK中，并与两个辅助载体psPAX2和pMD2.G共同转染T细胞，分别包装获得病毒颗粒，经离心浓缩后获得高浓度的慢病毒载体。
(2)利用密度梯度离心法，分离得到淋巴细胞，以CD3抗体(1ug/ml)和IL-2(100IU/ml)刺激淋巴细胞。一天后收集淋巴细胞进行病毒转染，培养淋巴细胞48小时，收集转染的淋巴细胞。
(3)收集到的经过病毒转染的淋巴细胞，用识别抗体Fab片段的特异性抗体，进行流式检测。
如图6为传统二代CAR基因转染人T细胞的表达检测，一抗为生物素标记的山羊抗小鼠Fab单克隆抗体，二抗为PE标记的抗streptavidin流式抗体。第一泳道为转染空载病毒的T细胞，第二泳道为转染CAR基因的T细胞。(图2中hH8-BBz为传统二代CAR基因(anti-HER2 scFV)-(CD8α hinge+TM)-(4-1BB)-(CD3ζ)；)。
如图7为新二代CAR基因转染人T细胞的表达检测，一抗为生物素标记的山羊抗小鼠Fab单克隆抗体，二抗为PE标记的抗streptavidin流式抗体。第一 泳道为转染空载病毒的T细胞，第二泳道为转染CAR基因的T细胞。(图3中hHD-Dz为新二代CAR基因(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ))。
在相同MOI(感染复数)病毒转染效力下，经流式细胞仪检测证实，新的二代CAR分子和传统二代CAR分子在淋巴细胞表面均高效表达。
对表达HER2的肿瘤细胞的杀伤作用：
传统二代CAR-T细胞：(anti-HER2 scFV)-(CD8α hinge+TM)-(4-1BB)-(CD3ζ)-T细胞
新二代CAR-T细胞：(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ)-T细胞
(1)利用RTCA(RealTime Cellular Analysis,实时无标记细胞分析技术)分别检测两种T细胞对HER2阳性细胞SKOV3(人卵巢癌细胞)肿瘤细胞杀伤效力检测，其检测结果如图2D。(图2D中hH8-BBz为传统二代CAR-T细胞；hHD-Dz为新二代CAR-T细胞)。
(2)利用ELISA分别检测两种CAR T细胞向HER2阳性肿瘤细胞SKOV3(人卵巢癌细胞)和HER2阴性肿瘤细胞MDA-MB-468(人乳腺癌细胞)分泌细胞因子情况,其检测分析结果如图8。图8清晰显示，新二代CAR T比传统二代CAR T向HER2阳性肿瘤细胞SKOV3分泌的细胞因子((IFNγ)情况要更多。(图8中hH8-BBz为传统二代CAR基因(anti-HER2 scFV)-(CD8α hinge+TM)-(4-1BB)-(CD3ζ)；hHD-Dz为新二代CAR基因(anti-HER2 scFV)-(CD8α hinge)-(RD-1TM+Cytoplasmic)-(CD3ζ))。
总结
典型的CAR主要由三个关键部分组成，包括识别抗原的单链可变片段(scFv)，铰链和跨膜结构域(TM)，例如CD3，CD28或CD8蛋白；以及细胞内信号域(ICDs)，例如CD3ζ或FcRγ。CAR包含一个或多个细胞内共刺激信号域，例如CD28、4-1BB，CD27，OX40，ICOS，DAP10，IL-15Rα，MyD88/CD40和TLR2，以传输激活信号。
已经证明，不同的TM和/或ICDs会影响T细胞扩增，存续和其他功能。最近，已经在患有实体瘤(例如转移性结直肠癌和肉瘤)的患者中测试了几种第二代CAR。与治疗血液恶性肿瘤所获得的结果相比，这些试验的结果令人振奋。因此，探索不同的共刺激域可能提供一种提高实体瘤中CAR-T细胞抗肿瘤效力的新方法。
在本发明中，我们的实验团队将靶向CD19或HER2的scFv结构域与4-1BB或dectin-1信号ICD偶联，以构建四个不同的第二代CAR。实验数据显示，在体外和体内实验中，新的CAR设计均通过Dectin-1共刺激影响了T细胞功能，例如增强了细胞因子分泌和裂解能力，降低了衰竭力，增加了细胞扩增和显著的抗肿瘤活性。
在本发明中，我们证实了先前研究的结果。显示具有4-1BB共刺激信号域的CAR-T细胞功能增强。有趣的是，hHD-Dz CAR-T细胞的体外T细胞功能(例如增加的细胞因子产量)与基于4-1BB的细胞相当，并且两者均优于对照T细胞。至于HER2特异性CAR-T细胞，hHD-Dz CAR-T细胞的IFN-γ分泌高于hH8-BBz，提示可能是Th1表型。hH8-BBz CAR-T细胞释放更多的TNF-α，与Th1/Th2表型一致。抗CD19 CAR-T细胞的相似细胞因子产生模式表明，与采用的具体共刺激信号传导域无关，表型是具有可比性的。在RTCA(实时细胞毒 性检测)中，我们阐明了hHD-Dz CAR-T细胞优于hH8-BBz CAR-T细胞的细胞毒性能力。以上结果表明，Dectin-1协同刺激为治疗实体瘤的CAR-T细胞免疫疗法提供了一种新的机制。
由于免疫抵抗和T细胞耗竭，CAR-T细胞疗法治疗实体瘤的最大挑战之一是抑制肿瘤微环境。然而，研究已经证实，不同T细胞表型可能在抗肿瘤免疫中发挥重要作用，例如，在过继免疫疗法中，T CM细胞远比T EM细胞重要。在本发明中，我们发现hHD-Dz CAR-T细胞中更多的T CM和显著的耗竭标记物表达表达可能表明新的CAR-T细胞可通过实体瘤中的Dectin-1共刺激信号转导受到肿瘤免疫抑制微环境的影响较小。
此外，我们通过建立肿瘤异种移植模型中的Dectin-1或4-1BB共刺激证明了第二代CAR-T细胞显著的抗肿瘤作用。但是，此处具有不同共刺激信号域的CAR在体内存活率方面显示出离散的抗肿瘤活性趋势。hHD-Dz CAR-T细胞在早期时间点显示出增强的效应子功能，而hH8-BBz CAR-T细胞则显示出较晚的抗肿瘤活性。这些观察结果表明，不同的共刺激信号域可能导致不同的T细胞表型。。
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。
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Claims (21)

  1. 一种嵌合抗原受体，包括胞外结构域、跨膜结构域和胞内结构域，其特征在于，所述嵌合抗原受体的所述跨膜结构域和所述胞内结构域组成共刺激信号结构域，所述共刺激信号结构域包括编码反向dectin-1的氨基酸全长或片段。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，所述胞外结构域包括靶向CD19或靶向HER2的单链抗体。
  3. 如权利要求2所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包括依次连接的CD8α铰链区、反向dectin-1跨膜结构域、反向dectin-1胞内信号域和CD3ζ胞内信号域。
  4. 如权利要求1所述的嵌合抗原受体，其特征在于，编码所述反向dectin-1氨基酸的序列如SEQ ID NO.3所示。
  5. 如权利要求3所述的嵌合抗原受体，其特征在于，编码所述反向dectin-1跨膜结构域的氨基酸的序列如SEQ ID NO.2所示。
  6. 如权利要求3所述的嵌合抗原受体，其特征在于，编码所述反向dectin-1胞内信号域的氨基酸序列如SEQ ID NO.1所示。
  7. 包含权利要求1-6任一项所述的嵌合抗原受体的慢病毒载体。
  8. 如权利要求7所述的慢病毒载体，其特征在于，所述慢病毒载体包括pCLK、psPAX2或pMD2.0G。
  9. 一种CAR-T细胞，其特征在于，表达权利要求1-6任一项所述的嵌合抗原受体。
  10. 一种抗肿瘤药，其特征在于，含有权利要求9所述的CAR-T细胞和药学上允许添加的辅料和/或助剂。
  11. 如权利要求10所述的抗肿瘤药，其特征在于，所述肿瘤包括血液肿瘤和实体 肿瘤。
  12. 如权利要求10所述的抗肿瘤药，其特征在于，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
  13. 权利要求9所述的CAR-T细胞在制备抗肿瘤药物中的应用。
  14. 如权利要求13所述的应用，其特征在于，所述CAR-T细胞可以刺激效应细胞因子分泌。
  15. 如权利要求14所述的应用，其特征在于，所述效应细胞因子包括IFN-γ，TNF-α和IL-6。
  16. 如权利要求13所述的应用，其特征在于，所述CAR-T细胞可以刺激细中央记忆型T细胞分泌。
  17. 如权利要求13所述的应用，其特征在于，所述肿瘤包括血液肿瘤和实体肿瘤。
  18. 如权利要求13所述的应用，其特征在于，所述肿瘤包括大B细胞淋巴瘤、B细胞淋巴瘤、非霍奇金淋巴瘤、髓细胞白血病、淋巴细胞白血病、乳腺癌、胃癌、食管癌或卵巢癌。
  19. 权利要求1-6任一项所述的嵌合抗原受体的合成方法，其特征在于，包括以下步骤：(1)合成所述反向dectin-1跨膜结构域-反向dectin-1胞内结构域的基因序列或反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列；(2)根据靶点合成引物，再利用重叠PCR法合成所述嵌合抗原受体基因序列。
  20. 如权利要求19所述的合成方法，其特征在于，所述步骤(2)具体包括：先用引物F1、R1扩增所述胞外结构域和所述CD8α铰链区结合的基因序列，然后再用引物F2、R2扩增所述反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序 列，最后以所述胞外结构域和CD8α铰链区结合的基因序列和所述反向dectin-1跨膜结构域-CD3ζ胞内信号域的基因序列为模板，以F1、R2为引物，合成所述嵌合抗原受体的基因序列。
  21. 如权利要求20所述的合成方法，其特征在于，所述引物F1如SEQ No.7所示，R1如SEQ No.8所示，F2如SEQ No.9所示，R2如SEQ No.10所示。
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