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WO2017031760A1 - 干涉片段及其应用 - Google Patents

干涉片段及其应用 Download PDF

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WO2017031760A1
WO2017031760A1 PCT/CN2015/088286 CN2015088286W WO2017031760A1 WO 2017031760 A1 WO2017031760 A1 WO 2017031760A1 CN 2015088286 W CN2015088286 W CN 2015088286W WO 2017031760 A1 WO2017031760 A1 WO 2017031760A1
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杨光华
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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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention relates to the field of immunobiology technology, in particular to interference fragments and their applications.
  • Malignant tumors are the main type of fatal disease in humans and the mortality rate remains high.
  • the latest statistics from the Ministry of Health indicate that malignant tumors rank first in the top ten causes of death among Chinese residents.
  • the annual medical expenses for patients with malignant tumors account for about 20% of the total health expenditure, which is the largest medical burden for the whole society.
  • the current treatments are still very limited in achieving the goals of curing, prolonging the survival of patients with malignant tumors and improving the quality of life.
  • metastasis whole body Sexual or advanced malignancies are more difficult to cure.
  • systemic treatment includes traditional chemotherapy, molecular targeted drug therapy, biological therapy (including stem cell transplantation therapy and immune cell therapy), and gene therapy.
  • traditional chemotherapy has made some progress in the treatment of certain tumors, it still contributes little to prolonging the survival of cancer patients.
  • the natural resistance of tumor cells to chemotherapeutic drugs and acquired multidrug resistance and chemotherapy toxicity have seriously hindered the development of chemotherapy.
  • Clinical practice over the years has proven that it is not an advantage program for systemic therapy.
  • Immune cell therapy has been developed for nearly two decades. Because of its wide therapeutic range (can be used for solid tumors and leukemia), it is especially effective for small tumor lesions (including metastasis, recurrence, cancer cells in the blood). Side effects, but also applicable to all stages of the tumor (such as late release, chemotherapy can not be used), so it is an advantage of systemic treatment. Immune cell therapy has a significant effect on improving quality of life and prolonging survival. It is highly praised at home and abroad and is one of the best methods for systemic therapy at present. The clinical potential is huge.
  • PD-1 belongs to costimulatory molecule 28 (CD28)/cytotoxic lymphocyte associated antigen (Cytotoxic lymphocyte associated antigen). An inhibitory receptor of the CTLA-4) family. PD-1 is not only a marker of programmed cell death, but also expressed on the surface of B lymphocytes, T lymphocytes, NK cells, NKT cells, and macrophages. In recent years, it has been found that the surface of T lymphocytes activated by antigens transiently expresses PD-1, and the sustained expression of PD-1 may cause T cell dysfunction. The extensive expression and immunosuppressive function of PD-1 has made it the focus of immunotherapy in recent years.
  • PD-1 plays an important role in the treatment of diseases such as cancer, autoimmune diseases, and chronic viral infections.
  • diseases such as cancer, autoimmune diseases, and chronic viral infections.
  • the main application of PD-1 is mainly to develop antibodies that specifically block PD-1 signaling, and then to use such antibody drugs for the treatment of advanced cancer and metastatic cancer.
  • the therapeutic effect is acceptable, it is often Drug-related adverse events occur.
  • Antibody drugs that specifically block PD-1 signaling are used in the treatment of advanced cancer and metastatic cancer. Although the therapeutic effect is acceptable, the total incidence of drug-related adverse events (AEs) is 41%, 3/4. The grade of serious drug AEs is 5%, including: skin (16%), gastrointestinal reactions (12%), and lungs (7%). The incidence of drug-related pneumonia was (6%), and the incidence of grade 3/grade pneumonia was 2% (2/129). Two patients died of pneumonia in the early stage of the trial.
  • Antibodies that block the PD-1 signaling pathway do not act directly on tumor tissue but act by activating anti-tumor immunity. Due to the complexity of the immune response and individual differences, the therapeutic effects of these antibodies vary widely among different types of cancer patients and between individuals. As mentioned earlier, the side effects of PD-1 inhibitors (antibodies) after intravenous reinfusion are present, and patients with systemic immune disease should not use this drug because PD-1 inhibitors can enhance the killing ability of immune cells. Increase the immune response and worsen the condition. There are also some patients who have incomplete response after antibody treatment.
  • an object of the present invention is to provide a means for effectively inhibiting the expression of PD-1 in an immune cell, thereby inhibiting the expression of PD-1 in an immune cell, thereby further suppressing the obtained immune cell of PD-1 expression. It is used for preparing immunotherapeutic drugs and is used for immunotherapy of tumors, thereby effectively solving the above problems and improving the application range and application effect of PD-1 in immunotherapy.
  • the present invention successfully constructs a lentiviral vector targeting shRNA of PD-1 by using genetic engineering technology, and screens a target sequence with an interference efficiency of more than 90%; and then, based on the interference obtained by screening
  • the target sequence is transferred to the immune cells, T lymphocytes (including CTL, TIL, CD8+T, NKT cells, etc.) by the pd-1 shRNA lentiviral vector carrying the corresponding interference fragment.
  • the expression of PD-1 is inhibited for a long period of time, and the immune cells inhibiting the expression of PD-1 are obtained.
  • the expression of the proliferating cell PD1 obtained after the cells enter the human body is also inhibited; further, the inventors return the modified T cells. Human or tumor local, and found that It can effectively improve the effect of immunotherapy and minimize side effects.
  • the invention provides an interference segment.
  • the target of the interference segment is a nucleotide sequence as shown below:
  • the interference fragment of the present invention has good targeting properties against the above-mentioned target, and the interference suppressing effect on PD-1 is outstanding.
  • transferring the above interference fragment into an immune cell T lymphocytes (for example, CTL, TIL, CD8+T, NKT cells, etc.), can cause long-term suppression of PD-1 expression in the immune cell.
  • the immune cells inhibiting the expression of PD-1 are obtained, and the expression of the proliferating cell PD1 obtained after the cells enter the human body is also inhibited, whereby the obtained immune cells inhibiting the expression of PD-1 can be effectively used for the preparation of immunotherapeutic drugs. Furthermore, it is used for immunotherapy of tumors, effectively improving the effect of immunotherapy and minimizing side effects.
  • interference segment according to the above embodiment of the present invention may also have the following additional technical features:
  • the interference segment consists of a sense strand and an antisense strand having the nucleotide sequence:
  • Antisense strand 5'-AATTCAAAAAAGCCTGTGTTCTCTGTGGACTATCTCTTGAATAGTCCAC AGAGAACACAGGCG-3' (SEQ ID NO: 7).
  • the present invention also provides a method of inhibiting PD-1 expression in an immune cell.
  • the method comprises: transferring the interference segment described above into the immune cell.
  • the method can effectively inhibit the expression of PD-1 in immune cells and obtain immune cells with inhibition of PD-1 expression. Furthermore, the obtained immune cells inhibiting the expression of PD-1 can be effectively used for the preparation of immunotherapeutic drugs, thereby obtaining an immunotherapeutic agent having a good therapeutic effect and low side effects; in addition, the immune cells inhibiting the expression of PD-1 can also be directly used. It is used to return to the human body or the tumor, so that it can effectively treat or prevent tumors.
  • the method of transferring the interference fragment into the immune cell is not particularly limited.
  • the interference fragment is transferred into the immune cell using a lentiviral vector system. Thereby, the interference fragment is easily transferred into the immune cell, thereby effectively improving the interference efficiency and realizing the inhibition of PD-1 expression in the immune cell.
  • the immune cell is a lymphocyte, preferably a T cell, a B cell, an NK cell, an NKT cell, more preferably a tumor infiltrating T lymphocyte or a CTL cell.
  • the immune cells inhibiting the expression of PD-1 can be efficiently obtained, and further, the obtained immune cells inhibiting the expression of PD-1 can be used for the preparation of an immunotherapeutic drug, and an immunotherapeutic agent having a good therapeutic effect and a low side effect can be effectively obtained. It can be directly used; the immune cells inhibited by PD-1 expression can also be directly used for reintroduction of human or tumor parts, thereby effectively treating or preventing tumors.
  • immune cells mainly refers to lymphocytes in human blood, such as T lymphocytes, B lymphocytes, NK lymphocytes, and NKT lymphocytes.
  • T lymphocytes may be tumor infiltrating T lymphocytes, and various T cells, such as CTL cells, cultured or induced in vitro.
  • CTL cell refers to a cytotoxic T lymphocyte that is stimulated by autologous lymphocytes by DC cells loaded with tumor antigen and mixed with autologous lymphocytes.
  • the present invention also provides the use of the aforementioned interference fragment for the preparation of an immunotherapeutic drug.
  • the drug comprises an immune cell in which PD-1 expression is inhibited. Therefore, the administration of the drug to a tumor patient can effectively inhibit the expression of PD-1 in the tumor cells of the patient, thereby effectively treating the tumor; in addition, the drug can be effectively used for the prevention of the tumor, and the effect of treating or preventing the tumor is good, and the side effect is good. low.
  • the immune cell in which PD-1 expression is inhibited is obtained by transferring the interference fragment described above into the immune cell to obtain an immune cell in which PD-1 expression is inhibited.
  • immune cells in which PD-1 expression is inhibited can be efficiently obtained.
  • the interference fragment is transferred into the immune cell using a lentiviral vector system.
  • the interference fragment is easily transferred into the immune cell, the interference efficiency is high, and the inhibitory effect on the expression of PD-1 in the immune cell is good.
  • the immune cell is a lymphocyte, preferably a T cell, a B cell, an NK cell, an NKT cell, more preferably a tumor infiltrating T lymphocyte or a CTL cell.
  • a lymphocyte preferably a T cell, a B cell, an NK cell, an NKT cell, more preferably a tumor infiltrating T lymphocyte or a CTL cell.
  • the present invention also provides an immunotherapeutic drug.
  • the immunotherapeutic drug comprises immune cells inhibited by PD-1 expression, and the immune cells inhibited by the PD-1 expression inhibit the immune cells by the method of inhibiting PD-1 expression in the immune cells as described above. Obtained in PD-1 expression. Therefore, the administration of the immunotherapeutic agent of the present invention to a tumor patient can effectively inhibit the expression of PD-1 in the tumor cells of the patient, thereby effectively treating the tumor; in addition, the medicament can be effectively used for the prevention of tumors, and for treating or preventing tumors. Good effect and low side effects.
  • FIG. 1 shows a schematic structural view of an LV3 carrier according to an embodiment of the present invention
  • Figure 2 shows the results of western detection of PD-1 protein in cells of interest transfected with each shRNA lentiviral vector, according to one embodiment of the present invention
  • Figure 3 shows the results of flow cytometry analysis of the number of PD-1 positive cells in the cells of interest transfected with each shRNA lentiviral vector, in accordance with one embodiment of the present invention.
  • a control RNA (negative control) was set up, and three PD-1 shRNA lentiviral vectors were constructed as follows:
  • DNA oligo was designed using Designer3.0 (Genepharma) software, and the synthetic primers were completed by Shanghai Jima Pharmaceutical Technology Co., Ltd.
  • the loop structure in the LV3-shRNA template selected TTCAAGAGA to avoid the formation of a termination signal; the 5' end of the sense strand template added GATCC, which is complementary to the sticky end formed by BamHI digestion; the antisense strand template AATTC was added to the 5' end to complement the sticky ends formed by EcoRI digestion, as follows:
  • shUN the specific information of shUN and three PD-1 shRNA lentiviral vectors are as follows:
  • control shNC The specific information of the control shNC is:
  • Target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 1);
  • Antisense strand sequence
  • Plasmid information of three PD-1 shRNA lentiviral vectors: PDCD1-Homo-694, PDCD1-Homo-791 and PDCD1-Homo-238 are shown in Table 1-3, respectively.
  • the DNA oligo was dissolved with TE (pH 8.0) at a concentration of 100 ⁇ M.
  • TE pH 8.0
  • the corresponding sense chain and antisense strand oligo solution were taken, and the annealing reaction system was configured according to the following ratio:
  • Annealing was carried out on a PCR machine according to the following procedure: 95 ° C for 5 min; 85 ° C for 5 min; 75 ° C for 5 min; 70 ° C for 5 min; 4 ° C for storage. After annealing, a shRNA template was obtained at a concentration of 10 ⁇ M. The resulting template solution was diluted 50-fold to a final concentration of 200 nM for the ligation reaction.
  • the ligation reaction of the vector was carried out according to the following system:
  • Competent cells are prepared by the calcium chloride method, and the specific steps are as follows:
  • the bacteria were transferred to a sterile, single-use, ice-cold 50 ml polypropylene tube, placed on ice for 10 minutes, and the culture was allowed to cool to 0 °C.
  • the cells were recovered by centrifugation at 4000 rpm for 10 minutes at 4 °C.
  • the cells were divided into small portions (100 ⁇ l/branch) and stored frozen at -70 °C.
  • the plate was poured into a 37 ° C incubator and cultured for 16 hours.
  • the positive clones were screened by the following steps and verified by sequencing:
  • the correct sequencing strains were extracted using a high-purity plasmid medium extraction kit, and the resulting plasmids can be used in conventional molecular biology experiments and cytological experiments. If the cytotoxicity is large when used for cell transfection, re-transform into E. coli Top10, and then prepare a higher purity plasmid using the kit or CsCl ultracentrifugation.
  • Lentiviral packaging cell transfection The 293T cells in the logarithmic growth phase were digested with trypsin, plated in 6-well plates, cultured in a 50 mL/L CO 2 incubator at 37 ° C, and the cell density was 60% to 70%. Dyeing. Wash cells twice with PBS before transfection.
  • the cells isolated from 100 ml of peripheral blood collected from 1 AFP-positive liver cancer patient were transferred to a 50 ml centrifuge tube for centrifugation (1800 rpm, 10 min), the supernatant was aspirated, and the supernatant was slowly added to the lymphocyte separation. On a liquid Ficoll solution (GE), the volume ratio is 1:1, centrifuged (2000 rpm, 20 min);
  • the white floc cells of the collection interface were added to the PBS, gently pipetted and mixed, and centrifuged (1500 rpm, 10 min);
  • the collected cells were gently pipetted and mixed, and 20 ml of the basal medium was added and uniformly blown, transferred to a 75 cm 2 culture flask, and cultured at 37 ° C in a 5% CO 2 incubator for 2 hours.
  • the remaining 2 ml of the remaining solution in the original vial (75 cm 2 flask) will be used to induce differentiation of monocytes into DC cells.
  • CTL1 medium preparation methods of CTL1 medium, CTL2 medium and CTL3 medium are:
  • CTL1 medium Take 1 tube of CTL1 factor from -20 degrees, melt at room temperature, add to 20ml of basal medium, And add 10% of the customer's serum, which is filtered using a 0.22um needle filter.
  • CTL2 medium One tube of CTL2 factor was taken out from -20 degrees, melted at room temperature, and added to 20 ml of basal medium, and a 0.22 um needle filter was filtered and used.
  • CTL3 medium One tube of CTL3 factor was taken out from -20 degrees, melted at room temperature, and added to 500 ml of basic medium, wherein a 0.22 um needle filter was used for filtration, and three tubes were used for the entire culture process.
  • CTL1 factor (including gamma interferon, 1000 U/ml): 1 ml, long-term storage at -20 °C, and stored at 4 °C for 2 weeks.
  • CTL2 factor including IL-1, 1000 U/ml; IL-2, 1000 U/ml; CD3 antibody, 100 ng/ml; CD28 antibody, 100 ng/ml: 1 ml, long-term storage at -20 ° C, and stored at 4 ° C for 2 weeks.
  • CTL3 factor (including IL-2, 1000 U/ml; IL-7, 20 ng/ml; IL-15, 20 ng/ml): 1 ml, long-term storage at -20 ° C, and stored at 4 ° C for 2 weeks.
  • the target cells are obtained.
  • the cells of interest were divided into 6-well plates, 10 6 cells per well, and used.
  • the collected virus supernatant was added to the target cells obtained by the above preparation, and an equal volume of fresh medium (CTL3 medium) was added to culture the cells of interest (total 3 ml).
  • the preparation method is as follows:
  • Glycine 2.9g; Tris 5.8g; SDS 0.37g; methanol 200ml; add ddH2O to 1000ml.
  • Blocking solution TBST containing 5% skim milk powder.
  • Electrophoresis An electrophoresis gel was prepared and subjected to SDS-PAGE.
  • the PVDF film was covered with glue, coated with filter paper and sponge, and rolled with a test tube to remove air bubbles.
  • Horseradish peroxidase-conjugated secondary antibody (rat anti-human) (diluted with TBST) was added and shaken smoothly for 2 hr at room temperature.
  • Interference efficiency of PDCD1-Homo-791 (target sequence SEQ ID NO: 10: GCCACCATTGTCTTTCCTAGC): 78%;
  • Interference efficiency of PDCD1-Homo-238 (target sequence SEQ ID NO: 15: GCTAAACTGGTACCGCATGAG): 8%;
  • Interference efficiency of PDCD1-Homo-694 (target sequence SEQ ID NO: 5: GCCTGTGTTCTCTGTGGACTA): 88%.
  • PD791 GCCACCATTGTCTTTCCTAGC (SEQ ID NO: 10),
  • PD694 GCCTGTGTTCTCTGTGGACTA (SEQ ID NO: 5),
  • the PD238 has no interference effect on PD1.
  • Two important PD1 interference targets are obtained in the present invention.
  • the interference fragment carried by the PD694 lentiviral vector was transferred into an immune cell to prepare an immune cell which inhibited the expression of PD-1, and the specific steps were as follows:
  • the cells isolated from 100 ml of peripheral blood collected from 1 AFP-positive liver cancer patient were transferred to a 50 ml centrifuge tube for centrifugation (1800 rpm, 10 min), the supernatant was aspirated, and the supernatant was slowly added to the lymphocyte separation. On a liquid Ficoll solution (GE), the volume ratio is 1:1, centrifuged (2000 rpm, 20 min);
  • the white floc cells of the collection interface were added to the PBS, gently pipetted and mixed, and centrifuged (1500 rpm, 10 min);
  • the collected cells were gently pipetted and mixed, and 20 ml of the basal medium was added and uniformly blown, transferred to a 75 cm 2 culture flask, and cultured at 37 ° C in a 5% CO 2 incubator for 2 hours.
  • the remaining 2 ml of the remaining solution in the original vial (75 cm 2 flask) will be used to induce differentiation of monocytes into DC cells.
  • the cell culture medium was aspirated, washed once with PBS, fresh 15 ml of CTL3 medium was added, and 5*10 8 PD694 lentiviral vector was added to the culture flask for mixing, and the solution was changed after 24 hours.
  • PEI (1 ⁇ g / ⁇ l) Polysciences (CAT#23966-2 formulated into a stock solution) was prepared according to the following steps: First, the endotoxin-free sterile water was heated to about 80 ° C to dissolve the PEI, and cooled to room temperature; Adjust the pH to 7.0, filter and sterilize with a 0.22 ⁇ m filter, store at -20 °C after dispensing, and store the working solution at 4 °C for later use.
  • the centrifuge tube was taken out, and the cell suspension was taken out into a culture flask (75 cm 2 ) with a pipette, and DC medium was added thereto, and the culture was continued for 3 days to obtain mature DC cells.
  • the mature DC cells were mixed with the aforementioned activated cultured lymphocytes at a volume ratio of 1:20.
  • Co-culture with CTL3 medium for seven days induced the production of CTL cells and other killer cells.
  • the immune cell population (PD694-CTL cells) prepared in this example contained not only a large number of CTL cells, but also various immune cells such as NK and CIK cells.
  • the present invention not only prepares AFP-specific CTL cells (PD694-CTL cells) in vitro, but also NKT cells. These lymphocytes have been modified by PD694's lentiviral vector prior to antigen stimulation, and their PD1 has been inhibited by more than 90%.
  • the PD694-CTL cells prepared in Example 1 were returned to the patient, and the reinfusion effect was observed as follows:
  • the interference fragment of the present invention has good targeting property against its target (SEQ ID NO: 5), has outstanding interference inhibition effect on PD-1, and can be effectively used for inhibiting PD-1 expression of immune cells, thereby obtaining PD- 1
  • the expression-inhibited immune cells can be effectively used for the preparation of immunotherapeutic drugs.

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Abstract

提供了干涉片段及其应用，其中该干涉片段的作用靶点为如下所示的核苷酸序列：5'-GCCTGTGTTCTCTGTGGACTA-3'(SEQ ID NO：5)。

Description

干涉片段及其应用 技术领域
本发明涉及免疫生物学技术领域，具体涉及干涉片段及其应用。
背景技术
恶性肿瘤是人类主要的致死性疾病类型，死亡率居高不下。卫生部最新统计数据表明恶性肿瘤居我国居民前十位死因首位。2010年中国恶性肿瘤发病患者268万人，死亡197万人；目前全国共有恶性肿瘤患者600余万人。每年用于恶性肿瘤病人的医疗费用约占卫生总费用的20％，是全社会最大的医疗负担。目前的治疗手段对于达到治愈、延长恶性肿瘤患者生存期以及改善生活质量等目标的作用仍然十分有限，尤其对于某些类型的恶性肿瘤即使是早期诊断也难以获得满意的疗效，至于转移性、全身性或进展期的恶性肿瘤则更难以治愈。
目前的肿瘤治疗策略中，手术和放疗等局部治疗对局限性肿瘤有较好的疗效，而对于全身性、转移性或局部治疗后的微小残留病灶则主要依靠化疗或免疫疗法进行系统性治疗。肿瘤发病是多步骤、多基因作用的结果，肿瘤细胞表现出生长、分化与凋亡的失控。临床上病人症状的多样性和个体遗传异质性，以及肿瘤远处转移病灶的出现，使肿瘤治疗必须以全身性疾病的观点，采用个体化的综合治疗方案。即根据每个肿瘤病人的特点，采用局部治疗结合全身治疗；不仅要消除局部病灶的肿瘤，还要控制肿瘤的复发、转移生长及肿瘤对重要脏器的侵袭，才能真正有效地提高肿瘤病人的治愈率和生存期。通常肿瘤局部病灶的治疗可采取手术和物理治疗(包括射频消融、热疗、放疗、冷冻、超声波及激光治疗等)方法消除，然而对于影像学不能发现的肿瘤微小病灶(如转移复发灶、局部治疗后的残留病灶、血液中的癌细胞)，以上局部治疗手段则无能为力，必须通过全身性治疗手段加以解决。由于肿瘤的转移、复发、扩散更为致命，因此全身性治疗显得尤为重要和紧迫。目前全身性治疗有传统化学治疗、分子靶向药物治疗、生物治疗(包括干细胞移植治疗和免疫细胞治疗)以及基因治疗等。传统化疗虽然在某些肿瘤治疗方面取得了一些进展，但对延长肿瘤病人生存期方面仍然贡献不大。而且肿瘤细胞对化疗药的天然耐药和获得性多药耐药以及化疗毒性，使化学治疗的发展受到了严重的妨碍。多年来的临床实践证明它并非是全身治疗的优势项目。
免疫细胞治疗是近二十年发展起来的，由于它治疗范围广(可用于实体肿瘤及白血病)，特别对肿瘤微小病灶(包括转移、复发灶、血液中的癌细胞)更为有效，无毒副作用，而且适用于肿瘤各阶段(如晚期放、化疗不能使用)，因此它是全身治疗的一个优势项目。免疫细胞治疗对提高生命质量、延长生存期有显著的效果，倍受国内外推崇，是目前全身治疗的最佳手段之一，临床潜力巨大。
程序性死亡因子1(programmed cell death-1,PD-1)是属于共刺激分子28(costimulatory molecule28,CD28)/细胞毒性T淋巴细胞相关抗原4(cytotoxic lymphocyte associated antigen, CTLA-4)家族的抑制性受体。PD-1不但是程序性死亡细胞的标记，同时表达于B淋巴细胞、T淋巴细胞、NK细胞、NKT细胞以及巨噬细胞表面。近年来研究更是发现，被抗原激活的T淋巴细胞表面会短暂地表达PD-1，而PD-1的持续表达可能会导致T细胞功能紊乱。PD-1表达的广泛性和免疫抑制功能，使其成为近年来免疫治疗的焦点。在癌症、自身免疫病、慢性病毒感染等疾病的治疗中，PD-1均起到了重要的作用。目前，主要PD-1的应用主要表现为开发特异性封闭PD-1信号的抗体，然后将此类抗体药物用于晚期癌症及转移性癌症的治疗，虽然治疗效果尚可，但也经常会有药物相关的不良事件发生。
因而，目前PD-1在免疫治疗方面的应用仍有待改进。
发明内容
首先，需要说明的是，本发明是基于发明人的下列发现而完成的：
将特异性封闭PD-1信号的抗体药物用于晚期癌症及转移性癌症的治疗，虽然治疗效果尚可，但药物相关的不良事件(adverse events,AEs)总发生率为41％，3/4级的严重药物AEs为5％，主要包括：皮肤(16％)、胃肠道反应(12％)、肺部(7％)。药物相关的肺炎发生率为(6％)，3级/4级肺炎发生率为2％(2/129)，试验早期有2例患者因肺炎死亡。
并且，部分病人对于抗体治疗有应答，也有部分病人在接受抗体治疗后其各项指标无明显变化。有的病人在接受抗体治疗后应答不完全，例如一位72岁多器官癌变的男性病患，在经过一个疗程的治疗后，胰脏的癌变转移性明显降低，但是其他位点癌变无明显变化。有的病人对于抗体治疗应答完全，发生肿瘤退化。此外，有的病例对于此抗体治疗应答情况复杂，例如一位52岁的多处淋巴结核肝脏癌变的女性患者，在接受了抗体治疗后，多处癌变衰退但是有一处淋巴结癌变增强。
封闭PD-1信号通路的抗体不是直接作用于肿瘤组织而是通过激活抗肿瘤免疫发挥作用。由于免疫应答的复杂性以及个体差异，这些抗体的治疗效果在不同类型的癌症患者以及不同个体之间差异较大。如前所述，PD-1抑制剂(抗体)经过静脉回输后产生的副作用是存在的，同时系统性免疫疾病的病人不宜用该药，因为PD-1抑制剂可以增强免疫细胞的杀伤能力，增加免疫反应，病情加重。也有部分病人抗体治疗后应答不完全。
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种能够有效抑制免疫细胞PD-1表达的手段，由此，通过抑制免疫细胞中PD-1的表达，进而将获得的PD-1表达抑制的免疫细胞用于制备免疫治疗药物，并将该药物用于肿瘤的免疫治疗，从而能够有效解决上述问题，提高PD-1在免疫治疗中的应用范围和应用效果。
具体地，为了解决上述问题，本发明利用基因工程技术，成功构建了针对PD-1的shRNA的慢病毒载体，并筛选获得了干扰效率超过90％的靶点序列；然后，基于筛选获得的干扰靶点序列，通过携带相应干涉片段的pd-1shRNA慢病毒载体，将筛选的相应干涉片段转入免疫细胞——T淋巴细胞(包括：CTL，TIL，CD8+T，NKT细胞等)，使其PD-1的表达受到长期抑制，得到PD-1表达抑制的免疫细胞，其中该细胞进入人体后获得的增殖细胞PD1的表达也被抑制；进而，发明人将该类经过修饰的T细胞回输人体或肿瘤局部，结果发现， 能够有效提高免疫治疗效果，最大程度上降低了副作用。
因而，根据本发明的一个方面，本发明提供了一种干涉片段。根据本发明的实施例，该干涉片段的作用靶点为如下所示的核苷酸序列：
5′-GCCTGTGTTCTCTGTGGACTA-3′(SEQ ID NO：5)。
发明人惊奇地发现，本发明的干涉片段针对上述靶点靶向性好，对PD-1的干涉抑制效果突出。并且，根据本发明的实施例，将上述干涉片段转入免疫细胞——T淋巴细胞(例如CTL，TIL，CD8+T，NKT细胞等)，能够使该免疫细胞PD-1的表达受到长期抑制，得到PD-1表达抑制的免疫细胞，并且该细胞进入人体后获得的增殖细胞PD1的表达也被抑制，从而，该获得的PD-1表达抑制的免疫细胞能够有效用于制备免疫治疗药物，进而用于肿瘤的免疫治疗，有效提高免疫治疗效果，最大程度上降低了副作用。
另外，根据本发明上述实施例的干涉片段还可以具有如下附加的技术特征：
根据本发明的实施例，所述干涉片段由具有下述核苷酸序列的正义链和反义链组成：
正义链：5′-GATCCGCCTGTGTTCTCTGTGGACTATTCAAGAGATAGTCCACAGAGA ACACAGGCTTTTTTG-3′(SEQ ID NO：6)，
反义链：5′-AATTCAAAAAAGCCTGTGTTCTCTGTGGACTATCTCTTGAATAGTCCAC AGAGAACACAGGCG-3′(SEQ ID NO：7)。
根据本发明的另一方面，本发明还提供了一种抑制免疫细胞中PD-1表达的方法。根据本发明的实施例，该方法包括：将前面所述的干涉片段转入所述免疫细胞。
发明人发现，利用该方法能够有效抑制免疫细胞中PD-1的表达，获得PD-1表达抑制的免疫细胞。并且，获得的PD-1表达抑制的免疫细胞能够有效用于免疫治疗药物的制备，进而能够获得治疗效果好，副作用低的免疫治疗药物；此外，该PD-1表达抑制的免疫细胞也可以直接被用于回输人体或肿瘤局部，从而能够有效治疗或预防肿瘤。
根据本发明的实施例，在本发明的方法中，将干涉片段转入免疫细胞的方法不受特别限制。根据本发明的一些具体示例，利用慢病毒载体系统将所述干涉片段转入所述免疫细胞。由此，干涉片段易于转入免疫细胞，从而能够有效提高干涉效率，实现对免疫细胞PD-1表达的抑制。
根据本发明的实施例，所述免疫细胞为淋巴细胞，优选T细胞、B细胞、NK细胞，NKT细胞，更优选肿瘤浸润T淋巴细胞或CTL细胞。由此，能够有效获得PD-1表达抑制的免疫细胞，进而，将获得的PD-1表达抑制的免疫细胞用于免疫治疗药物的制备，能够有效获得治疗效果好，副作用低的免疫治疗药物。能够直接用于；该PD-1表达抑制的免疫细胞也可以直接被用于回输人体或肿瘤局部，从而能够有效治疗或预防肿瘤。
其中，需要说明的是，在本文中所采用的术语“免疫细胞”主要是指人体血液里的淋巴细胞，例如T淋巴细胞、B淋巴细胞、NK淋巴细胞，NKT淋巴细胞。其中，T淋巴细胞可以为肿瘤浸润T淋巴细胞，以及经过体外培养或诱导的各种T细胞，例如CTL细胞。术语“CTL细胞”在本文中是指由DC细胞负载肿瘤抗原并与自体的淋巴细胞混合，刺激自体淋巴细胞产生的细胞毒性T淋巴细胞。
根据本发明的又一方面，本发明还提供了前面所述的干涉片段在制备免疫治疗药物中的用途。根据本发明的实施例，所述药物中包含PD-1表达抑制的免疫细胞。由此，对肿瘤患者给予该药物，能够有效抑制患者肿瘤细胞中PD-1的表达，从而有效治疗肿瘤；此外该药物还可以有效用于肿瘤的预防，且治疗或预防肿瘤的效果好，副作用低。
根据本发明的实施例，所述PD-1表达抑制的免疫细胞是通过以下步骤获得的：将前面所述的干涉片段转入所述免疫细胞，以便获得PD-1表达抑制的免疫细胞。由此，能够高效地获得PD-1表达抑制的免疫细胞。
根据本发明的实施例，利用慢病毒载体系统将所述干涉片段转入所述免疫细胞。由此，干涉片段易于转入免疫细胞，干涉效率高，对免疫细胞PD-1表达的抑制效果好。
根据本发明的实施例，所述免疫细胞为淋巴细胞，优选T细胞、B细胞、NK细胞，NKT细胞，更优选肿瘤浸润T淋巴细胞或CTL细胞。由此，能够有效获得PD-1表达抑制的免疫细胞，进而，该PD-1表达抑制的免疫细胞能够用于免疫治疗药物的制备。
根据本发明的另一方面，本发明还提供了一种免疫治疗药物。根据本发明的实施例，该免疫治疗药物包含PD-1表达抑制的免疫细胞，所述PD-1表达抑制的免疫细胞是通过前面所述的抑制免疫细胞中PD-1表达的方法抑制免疫细胞中PD-1表达获得的。由此，对肿瘤患者给予本发明的免疫治疗药物，能够有效抑制患者肿瘤细胞中PD-1的表达，从而有效治疗肿瘤；此外该药物还可以有效用于肿瘤的预防，且治疗或预防肿瘤的效果好，副作用低。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例，LV3载体的结构示意图；
图2显示了根据本发明一个实施例，各shRNA慢病毒载体转染的目的细胞的PD-1蛋白western检测结果；以及
图3显示了根据本发明一个实施例，各shRNA慢病毒载体转染的目的细胞中PD-1阳性细胞数量的流式细胞仪检测结果。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。
实施例1
一、质粒构建：
设置对照NC(阴性对照)，按照以下方法，构建三种PD-1的shRNA慢病毒载体：
(一)、实验材料
1、DNA oligo使用Designer3.0(Genepharma)软件进行设计，合成引物由上海吉玛制药技术有限公司完成
2、实验中使用的仪器列表：
Figure PCTCN2015088286-appb-000001
3、实验中使用的试剂列表：
Figure PCTCN2015088286-appb-000002
Figure PCTCN2015088286-appb-000003
(二)、构建流程
1、Oligo Designer3.0
发明人设计模板时，LV3-shRNA模板中的loop结构选用了TTCAAGAGA以避免形成终止信号；正义链模板的5’端添加了GATCC，与BamHI酶切后形成的粘端互补；反义链模板的5’端添加了AATTC，与EcoRI酶切后形成的粘端互补，具体如下：
正义链：
5’-GATCC-(GN18)-(TTCAAGAGA)-(N18C)-TTTTTTG-3’；
反义链：
3’-G(CN18)-(AAGTTCTCT)-(N18G)-AAAAAACTTAA-5’。
其中，对照shNC以及三种PD-1的shRNA慢病毒载体的具体信息如下：
(1)、对照shNC的具体信息为：
靶点序列：5’-TTCTCCGAACGTGTCACGT-3’(SEQ ID NO：1)；
正义链序列：
GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTG(SEQ ID NO：2)；
反义链序列：
AATTCAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAACG(SEQ ID NO：3)；
转录产物序列：
GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTT(SEQ ID NO：4)。
(2)三种PD-1的shRNA慢病毒载体：PDCD1-Homo-694、PDCD1-Homo-791和PDCD1-Homo-238的质粒信息分别如表1-3所示。
2、LV3-shDNA模板的退火
将DNA oligo分别用TE(pH8.0)溶解，浓度为100μM。取相应的正义链和反义链oligo溶液，按照如下配比配置退火反应体系：
组分 体积(μl)
10X shDNA退火缓冲液 5
正义链(100μM) 5
反义链(100μM) 5
ddH2O 35
总体积 50
在PCR仪上按照如下程序进行退火处理：95℃5min；85℃5min；75℃5min；70℃5min；4℃保存。退火处理后得到浓度为10μM的shRNA模板。将所得模板溶液稀释50倍，终浓度为200nM，用于连接反应。
3、LV3载体的线性化
取10μg LV3载体，按照如下体系进行酶切处理：
组分 体积(μl)
2×Buffer Tango 10
BamHI 5
EcoRI 5
LV3 10μg
ddH2O 补至100
总体积 100
37℃酶切1小时，琼脂糖电泳，使用Agarose Gel DNA Purification Kit Ver2.0回收，电泳检测估算浓度，稀释浓度至50ng/μl。
4、LV3-shRNA载体的构建
按照如下体系进行载体的连接反应：
组分 体积(μl)
10×T4连接缓冲液 2
LV3(BamHI+EcoRI) 1
shDNA模板(100nM) 1
T4DNA连接酶(5weissU/μl) 1
ddH2O 15
总体积 20
22℃1小时，转化至大肠杆菌Top10(transform to Top10competent cells)
5、感受态细胞的制备：(氯化钙法)
利用氯化钙法制备感受态细胞，具体步骤如下：
5.1从于37℃培养16小时的新鲜平板中挑取一个单菌落，转到一个含有100ml LB培养基的1L烧瓶中。于37℃剧烈振摇培养3小时(旋转摇床，300转/分)。
5.2在无菌条件下将细菌转移到一个无菌、一次性使用的、用冰预冷的50ml聚丙烯管中，在冰上放置10分钟，使培养物冷却至0℃。
5.3于4℃，以4000转/分离心10分钟，回收细胞。
5.4倒出培养液，将管倒置1分钟,使最后残留的痕量培养液流尽。
5.5以10ml用冰预冷的0.1mol/L CaCl2重悬每份沉淀，放置于冰浴上。
5.6于4℃，以4000转/分离心10分钟，回收细胞。
5.7倒出培养液，将管倒置1分钟，使最后残留的痕量培养液流尽。
5.8每50ml初始培养物用2ml用冰预冷的0.1mol/L CaCl2(含20％甘油)重悬每份细胞沉淀。
5.9将细胞分装成小份(100μl/支)，放于-70℃冻存。
6、连接产物的转化
按照以下步骤进行连接产物的转化：
6.1从-70℃中取出感受态细胞，将装有感受态细胞的离心管冰上放置4分钟，待感受态细胞解冻后，加入10μl连接产物，轻柔混匀内容物，在冰中放置30分钟。
6.2将离心管放到预加温到42℃的水浴锅中放好的试管架上，放置90秒，不要摇动离心管。
6.3快速将离心管转移到冰浴中，使细胞冷却3分钟。
6.4向每支离心管加入800μl LB培养基(不含抗生素)，然后将离心管转移到37℃摇床，250转/分钟，培育45分钟使细菌复苏。
6.5取200μl培育后的细胞均匀涂布于含50μg/ml Ampicillin LB平板上。
6.6等平板上液体被吸收后，将平板倒置于37℃培养箱中，培养16小时。
7、阳性克隆的鉴定与测序
按照以下步骤鉴定筛选阳性克隆，并对其进行测序验证：
7.1从每块平板上挑取5个菌落，接种到含50μg/ml Ampicillin(氨苄西林)的LB培养基中，37℃培养16小时。
7.2使用碱裂解法抽提质粒。
7.3所得质粒用EcoRI进行单酶切鉴定。酶切结果表明，被EcoRI切开的可能是阳性克隆，将每组克隆进行测序鉴定。测序结果见表1-3。
7.4将测序正确的菌株采用高纯度质粒中量抽提试剂盒抽提，所得质粒可以用于常规的分子生物学实验和细胞学实验。如果在用于细胞转染时细胞毒性较大，请重新转化至大肠杆菌Top10中，然后用试剂盒或CsCl超速离心法制备更高纯度质粒。
表1、PDCD1-Homo-694质粒信息
Figure PCTCN2015088286-appb-000004
Figure PCTCN2015088286-appb-000005
表2、PDCD1-Homo-791质粒信息
Figure PCTCN2015088286-appb-000006
Figure PCTCN2015088286-appb-000007
表3、PDCD1-Homo-238的质粒信息
Figure PCTCN2015088286-appb-000008
Figure PCTCN2015088286-appb-000009
二、靶点筛选
(一)慢病毒载体准备：
按照以下步骤进行慢病毒载体准备：
1、慢病毒包装细胞转染用胰蛋白酶消化对数生长期的293T细胞，铺到6孔板中，37℃，50mL/L CO2培养箱内培养，细胞密度达60％～70％时转染.转染前用PBS清洗细胞2遍。
2、制备慢病毒包装系统中3种质粒DNA溶液(VSVG 2μg，P8.7.4μg，目的基因10μg)。
3、无菌水定容至25μL，再加入CaCl2(2.5mol/L)溶液25μL，混匀，4度冰上放置20min。
4、逐滴加入2×HBS缓冲盐溶液50μL，常温放置30min。
5、将DNA磷酸钙混合液转移至含单层细胞的培养液中，混匀，培养16h后弃去含有转染混和物的培养液，用新鲜的培养液替代。
6、转染后的48小时收取细胞上清液(1.5ml)。
(二)目的细胞制备：
1、外周血单个核细胞的分离
将从1例AFP阳性的肝癌患者采集的100ml外周血中分离获得的细胞，转移到50ml离心管中离心(1800rpm，10min)，吸去上清，取上清后的液体缓慢加在淋巴细胞分离液Ficoll液(GE)上，体积比为1:1，离心(2000rpm，20min)；
接着，收集界面的白色絮状细胞加入PBS，轻轻吹打混匀，离心(1500rpm，10min)；
接下来，重复离心洗涤共3遍；
然后，收取细胞轻轻吹打混匀，加入基础培养基20ml吹打均匀，转移至75cm2培养瓶中，于37℃，5％CO2培养箱培养2h。
2、淋巴细胞和单核细胞的分离
取出75cm2培养瓶，轻轻将75cm2培养瓶放直立，吸取18ml培养液于新的175cm2瓶中，以便用于淋巴细胞的活化培养；
针对原瓶(75cm2培养瓶)中剩余的2ml残液，将用于将单核细胞诱导分化为DC细胞。
3、淋巴细胞的培养
在用于培养淋巴细胞的175cm2瓶中，加入20ml CTL1细胞培养基，24h后再加入20ml CTL2培养基培养，然后每隔2～3d用CTL3培养基等量补液。
其中，CTL1培养基、CTL2培养基和CTL3培养基的配制方法为：
CTL1培养基：将1管CTL1因子从-20度取出，常温融化，加到20ml基础培养基中， 并添加10％的客户的血清，其中将0.22um针头滤器过滤后使用。
CTL2培养基：将1管CTL2因子从-20度取出，常温融化，加到20ml基础培养基中，其中将0.22um针头滤器过滤后使用。
CTL3培养基：将1管CTL3因子从-20度取出，常温融化，加到500ml基础培养基中，其中将0.22um针头滤器过滤后使用，整个培养过程用3管。
CTL1因子(包括γ干扰素，1000U/ml)：1ml，-20℃长期保存，4℃保存2周。
CTL2因子(包括IL-1，1000U/ml；IL-2，1000U/ml；CD3抗体，100ng/ml；CD28抗体，100ng/ml)：1ml，-20℃长期保存，4℃保存2周。
CTL3因子(包括IL-2，1000U/ml；IL-7，20ng/ml；IL-15，20ng/ml)：1ml，-20℃长期保存，4℃保存2周。
由此，获得目的细胞。然后，将目的细胞分在6孔板中，每孔106细胞，备用。
(三)病毒载体感染：
1、将收集的病毒上清液加入上述制备获得的目的细胞中，同时加入同等体积的新鲜培养基(CTL3培养基)进行培养目的细胞(共3ml)。
2、24小时后换液，正常培养。
3、感染96小时后收取细胞，提蛋白。
(四)PD-1蛋白western鉴定：
1、试剂
1)0.01M PBS(pH7.4)：NaCl 8.0g；KCl 0.2g；Na2HPO41.44g；KH2PO40.24g；
加ddH2O至1000ml。
2)蛋白酶抑制剂：
Protease Inhibitor Cocktail：Calbiochem公司，货号539134
3)RIPA：
137mM NaCl,1.4mM KH2PO4,1.7mM KCl,
5.0mM EDTA,5.0mM EGTA,4.3mM Na2HPO4.
0.1％SDS,1％TritonX-100,1mMNaVO4,50mM NaF。
4)Bio-Rad公司Bradford染液，货号500－0006。
5)10％SDS-PAGE分离胶，浓缩胶，
其中，按照如下配方配制10ml分离胶：
ddH2O 4.0ml；30％丙烯酰胺3.3ml；1.5M Tris(pH8.8)2.5ml；
10％SDS 0.1ml；10％过硫酸铵0.1ml；TEMED 0.004ml。
按照如下配方配制浓缩胶2ml：
ddH2O 1.4ml；30％丙烯酰胺0.33ml；1M Tris(pH6.8)0.25ml；
10％SDS 0.02ml；10％过硫酸铵0.02ml；TEMED 0.002ml。
6)2X SDS－PAGE Loading Buffer：
Figure PCTCN2015088286-appb-000010
Figure PCTCN2015088286-appb-000011
7)蛋白marker，Bio-Rad公司，货号161－0324。
8)电泳液：
配5X Tris－Glycine Buffer储液，使用时稀释。
储液：0.125M Tris，1.25M Glycine，0.5％(W/V)SDS，
其配制方法如下：
称取Tris 15.1g；Glycine 94g；SDS 5.0g，置于1L的烧杯中，然后加入约800ml的去离子水，搅拌溶解，加入去离子水定容至1L后，室温保存。
9)PVDF膜，Millipore公司，货号IPVH00010。
10)转膜缓冲液(transfer buffer)：
甘氨酸2.9g；Tris 5.8g；SDS 0.37g；甲醇200ml；加ddH2O定容至1000ml。
11)TBST配制方法：
称NaCl 8g；KCl 0.2g；Tris 3g，溶于800ml蒸馏水中，加HCl调pH到7.4，定容到1升。然后加1ml Tween20，混匀。
12)封阻液：含有5％脱脂奶粉的TBST。
13)ECL反应底物，Pierce公司，supersignal west pico chemilumiscent sunstrate，货号prod#34080。
2、仪器
1)测定蛋白浓度，Eppendorf公司Biophotometer；
2)电泳及转膜用Bio-Rad powerpac 3000；
3、主要步骤
1)样品准备
PBS清洗细胞。去PBS后加含蛋白酶抑制剂(1:200)的总蛋白抽提试剂RIPA，冰上裂解30min。用细胞刮刮下细胞，吸取到1.5ml EP管中，然后4℃，13,000g离心15min。取上清液作为样品。Bradford比色法测定蛋白质浓度。
2)电泳：制备电泳凝胶，进行SDS-PAGE。
预先配制PAGE胶。
取相同质量的蛋白样品，加等体积的2×loading buffer，沸水浴中5min。
上样，以及蛋白marker，多余空用1×loading buffer补平。90V，20min，直至所有样品压成一线；120V，100min。
3)转膜
A、裁适当大小之PVDF膜，甲醇活化数秒，浸入transfer buffer中备用。其余相关器材，如海绵、滤纸、夹板等，事先也浸入transfer buffer中。
B、铺好夹板下层的海绵和滤纸，试管来回滚动去除气泡。
C、小心剥离电泳玻片上的PAGE胶，放置到滤纸上。将PVDF膜覆盖胶上，再覆以滤纸和海绵，用试管滚动去除气泡。
D、将此海绵/滤纸/凝胶按照厂家建议方法放入电泳装置中，凝胶面向阴极。
E、将上述装置放入缓冲液槽中，并灌满transfer buffer以淹没凝胶。
F、按照厂家所示接通电源，在冰盒开始转膜，100V，2hr。
4)免疫反应：
A、用TBST洗膜，5min。
B、加入封阻液，平稳摇动，室温1hr。
C、TBST洗膜，5min。
D、加入PDCD1人源一抗(用含3％脱脂奶粉的TBST稀释)，室温1hr或4℃放置12hr。
E、弃一抗，TBST洗膜，15min×3次。
F、加入辣根过氧化物酶偶联的二抗(鼠抗人)(用TBST稀释)，平稳摇动，室温2hr。
G、弃二抗，TBST洗膜，15min×3次。
5)ECL曝光
准备好曝光所需：底片，曝光，显影、定影液等。按照ECL产品说明书，等体积混合两组分，总体积1ml可供一张膜使用。暗室中曝光X光底片1～2min(视反应后光线强弱而定)，水洗后放入定影液中，直至底片完全透明。水洗后烘干底片。
各shRNA慢病毒载体转染的目的细胞的PD-1蛋白western检测结果见图2。
然后，基于western检测结果，计算三种shRNA慢病毒载体的干扰效率，结果如下：
PDCD1-Homo-791(靶点序列SEQ ID NO：10：GCCACCATTGTCTTTCCTAGC)的干扰效率：78％；
PDCD1-Homo-238(靶点序列SEQ ID NO：15：GCTAAACTGGTACCGCATGAG)的干扰效率：8％；
PDCD1-Homo-694(靶点序列SEQ ID NO：5：GCCTGTGTTCTCTGTGGACTA)的干扰效率：88％。
6)PD1阳性细胞的数量检测
同时对各shRNA慢病毒载体转染的目的细胞进行流氏细胞仪分析，检测PD1阳性细胞的数量，结果见图3。
由此，综合western和流式细胞仪检测结果，可知高效果干扰PD1的靶点为：
PD791：GCCACCATTGTCTTTCCTAGC(SEQ ID NO：10)，
PD694：GCCTGTGTTCTCTGTGGACTA(SEQ ID NO：5)，
而PD238则对PD1没有任何干扰效果。本发明中获得两个重要的PD1干扰靶点。
实施例2
以实施例1获得的PD694慢病毒载体为例，利用PD694慢病毒载体，将其携带的干涉片段转入免疫细胞，制备获得PD-1表达抑制的免疫细胞，具体步骤如下：
1外周血单个核细胞的分离
将从1例AFP阳性的肝癌患者采集的100ml外周血中分离获得的细胞，转移到50ml离心管中离心(1800rpm，10min)，吸去上清，取上清后的液体缓慢加在淋巴细胞分离液Ficoll液(GE)上，体积比为1:1，离心(2000rpm，20min)；
接着，收集界面的白色絮状细胞加入PBS，轻轻吹打混匀，离心(1500rpm，10min)；
接下来，重复离心洗涤共3遍；
然后，收取细胞轻轻吹打混匀，加入基础培养基20ml吹打均匀，转移至75cm2培养瓶中，于37℃，5％CO2培养箱培养2h。
2淋巴细胞和单核细胞的分离
取出75cm2培养瓶，轻轻将75cm2培养瓶放直立，吸取18ml培养液于新的175cm2瓶中，以便用于淋巴细胞的活化培养；
针对原瓶(75cm2培养瓶)中剩余的2ml残液，将用于将单核细胞诱导分化为DC细胞。
3淋巴细胞的培养
在用于培养淋巴细胞的175cm2瓶中，加入20ml CTL1细胞培养基，24h后再加入20ml CTL2培养基培养，然后每隔2～3d用CTL3培养基等量补液，进行活性活率检测验证是否合格。
吸出细胞培养基，用PBS清洗一遍后，换新鲜15ml CTL3培养基，并加入5*108PD694慢病毒载体至培养瓶中混合，24小时后换液。
4.单核细胞的处理
a、在剩有2ml残液的原瓶(75cm2培养瓶)中加入15ml DC培养基继续培养4天，每2～3天半换液一次，以便将单核细胞诱导分化为未成熟的DC细胞。
b、PEI转染法进行抗原基因mRNA转染：第5天，用50ml离心管收集未成熟的DC细胞(离心1500rpm，10min)，弃掉上清后，将下层细胞转移到1.5ml离心管，然后直接加入100ul的试剂A(即AFP mRNA与PEI按照质量比1：3混合后获得的混合物)充分混匀底部细胞，盖好离心管，置于细胞培养箱中孵育30分钟，每隔10分钟取出上下倒置几次。
其中，AFP mRNA的制备方法请参见申请日为2015年5月5日，申请号为201510225512.2的专利申请的实施例1。
PEI(1μg/μl)Polysciences(CAT#23966-2配制成储液)是按照以下步骤配制获得的：首先，将无内毒素的无菌水加热至80℃左右溶解PEI，冷却到室温；然后，调整pH值至7.0，用0.22μm的滤器过滤消毒，分装后储存在-20℃，工作液可于4℃下保存备用。
c、取出离心管，用移液枪将细胞悬液取出加到培养瓶(75cm2)中，并加入DC培养基，继续培养3天，以便获得成熟的DC细胞。
5.共培养
第8天后，将成熟的DC细胞与前述经过活化培养的淋巴细胞按照体积比1:20混合， 用CTL3培养基共培养七天，诱导CTL细胞及其他杀伤性细胞的产生。
共培养七天后，对细胞进行流式检测，结果发现：本实施例制备获得的免疫细胞群(PD694-CTL细胞)中不仅含有大量的CTL细胞，而且还有NK及CIK细胞等多种免疫细胞：CTL细胞(CD8+，CD3+CD8+CD28+)、CIK细胞(CD3+CD56+)、NK细胞(CD3-CD56+)，即该免疫细胞群不仅具有强大杀伤特定肿瘤细胞的CTL细胞，还有CIK、NK等免疫细胞。
由此，发明人发现，本发明不仅在体外制备了AFP特异性CTL细胞(PD694-CTL细胞)，同时还有NKT细胞。这些淋巴细胞在接受抗原刺激之前已经过PD694的慢病毒载体的修饰，其PD1已经被抑制高达90％以上。
实施例3：临床实验
将实施例1制备获得的PD694-CTL细胞回输患者，并观察回输效果，具体如下：
将AFP阳性的肝癌患者20名分为2组(A组10名和B组10名)，其中，针对A组，按照实施例1中的方法在第14天和第15天获得足量CTL细胞后，给患者回输，每次回输细胞量为1～2*109；B组不回输细胞。采血一次连续回输2次为一个疗程。3个疗程结束后，进行观察。
结果发现：治疗三个疗程后，A组和B组有明显的区别。A组所有患者在回输后均感觉身体疲惫感减轻，食欲增加，疼痛减轻，体重增加，其中有8名患者AFP指标都有所下降；一名患者通过CT发现肝上3处原发兆消失，一名患者指标无进展。而B组所有患者各项症状均未改善，10名患者AFP指标均有所增加。可以明显看出，经过回输PD694-CTL细胞后，患者症状得到明显缓解。
工业实用性
本发明的干涉片段针对其靶点(SEQ ID NO：5)的靶向性好，对PD-1的干涉抑制效果突出，能够有效用于抑制免疫细胞的PD-1表达，进而获得的PD-1表达抑制的免疫细胞能够有效用于制备免疫治疗药物。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (10)

  1. 一种干涉片段，其作用靶点为如下所示的核苷酸序列：
    5′-GCCTGTGTTCTCTGTGGACTA-3′(SEQ ID NO：5)。
  2. 根据权利要求1所述的干涉片段，其特征在于，所述干涉片段由具有下述核苷酸序列的正义链和反义链组成：
    正义链：5′-GATCCGCCTGTGTTCTCTGTGGACTATTCAAGAGATAGTCCACAGAGAACACAGGCTTTTTTG-3′(SEQ ID NO：6)，
    反义链：5′-AATTCAAAAAAGCCTGTGTTCTCTGTGGACTATCTCTTGAATAGTCCACAGAGAACACAGGCG-3′(SEQ ID NO：7)。
  3. 一种抑制免疫细胞中PD-1表达的方法，其特征在于，包括：
    将权利要求1或2所述的干涉片段转入所述免疫细胞。
  4. 根据权利要求3所述的方法，其特征在于，利用慢病毒载体系统将所述干涉片段转入所述免疫细胞。
  5. 根据权利要求3所述的方法，其特征在于，所述免疫细胞为淋巴细胞，优选T细胞、B细胞、NK细胞，NKT细胞，更优选肿瘤浸润T淋巴细胞或CTL细胞。
  6. 权利要求1或2所述的干涉片段在制备免疫治疗药物中的用途，所述药物中包含PD-1表达抑制的免疫细胞。
  7. 根据权利要求6所述的用途，其特征在于，所述PD-1表达抑制的免疫细胞是通过以下步骤获得的：
    将权利要求1或2所述的干涉片段转入所述免疫细胞，以便获得PD-1表达抑制的免疫细胞。
  8. 根据权利要求7所述的用途，其特征在于，利用慢病毒载体系统将所述干涉片段转入所述免疫细胞。
  9. 根据权利要求7所述的用途，其特征在于，所述免疫细胞为淋巴细胞，优选T细胞、B细胞、NK细胞，NKT细胞，更优选肿瘤浸润T淋巴细胞或CTL细胞。
  10. 一种免疫治疗药物，其特征在于，包含PD-1表达抑制的免疫细胞，所述PD-1表达抑制的免疫细胞是通过权利要求3-5任一项所述的方法抑制免疫细胞中PD-1表达获得的。
PCT/CN2015/088286 2015-08-27 2015-08-27 干涉片段及其应用 Ceased WO2017031760A1 (zh)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN103820454A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA
CN103830725A (zh) * 2005-06-08 2014-06-04 达纳－法伯癌症研究院 通过抑制程序性细胞死亡1（pd-1）途经治疗持续性感染和癌症的方法及组合物
CA2897858A1 (en) * 2013-02-22 2014-08-28 Curevac Gmbh Combination of vaccination and inhibition of the pd-1 pathway

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN103830725A (zh) * 2005-06-08 2014-06-04 达纳－法伯癌症研究院 通过抑制程序性细胞死亡1（pd-1）途经治疗持续性感染和癌症的方法及组合物
CA2897858A1 (en) * 2013-02-22 2014-08-28 Curevac Gmbh Combination of vaccination and inhibition of the pd-1 pathway
CN103820454A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA

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