WO2016034094A1 - 制备dc-ctl的试剂盒及其应用 - Google Patents

制备dc-ctl的试剂盒及其应用 Download PDF

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WO2016034094A1
WO2016034094A1 PCT/CN2015/088648 CN2015088648W WO2016034094A1 WO 2016034094 A1 WO2016034094 A1 WO 2016034094A1 CN 2015088648 W CN2015088648 W CN 2015088648W WO 2016034094 A1 WO2016034094 A1 WO 2016034094A1
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ctl
monoclonal antibody
preparing
kit
agent
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French (fr)
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姜舒
张芸
罗朝霞
纪惜銮
杨顺
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深圳市茵冠生物科技有限公司
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  • the invention belongs to the field of tumor treatment kits, and in particular relates to a kit for preparing DC-CTL and an application thereof.
  • Immune cell therapy refers to the input of immune cells with anti-tumor activity in tumor patients, directly killing tumor cells or stimulating the body's anti-tumor immune response, thereby achieving the purpose of treating tumors.
  • immune cell therapy has become an important auxiliary means of cancer treatment. It overcomes the limitations of clinical routine treatment, can effectively eliminate small residual cancer, inhibit tumor recurrence, and improve patient immunity. It has broad prospects in the field of clinical treatment of tumors.
  • DC Dendritic Cell
  • DC tumor vaccines such as Sipuleucel-T (Dendreon, USA), CreaVaxRCC (CreaGene, Korea) and Hybricell (Genoa Biotechnologia, Brazil) were successively approved for marketing.
  • the DC tumor vaccine has progressed from the laboratory to the clinic, and its feasibility and safety as well as the effectiveness of some patients have been confirmed.
  • Current DC vaccine preparation methods include two methods: antigen-loaded sensitized DCs and genetically modified DCs.
  • Antigen-loaded sensitized DC The method of antigen-loading sensitized DC mainly includes tumor cell whole antigen sensitization and tumor antigen polypeptide sensitization. Studies have shown that in view of the fact that many tumor antigens are still unclear, the use of whole tumor antigens to induce T cells to produce a wider range of immune responses, a variety of different tumor antigen components simultaneously stimulate DC, induce specific CTLs for different antigenic determinants, the killing range is even more Wide, can avoid immune escape of tumor cells, however, because the whole tumor antigen component is not clear, it may induce autoimmune response, there is a safety hazard. At present, there are synthetic tumor antigen polypeptides on the market, which have clear components and high safety.
  • the CTL killing target activated by DCs loaded with synthetic tumor antigen polypeptides is clear and specific. However, a single antigen polypeptide is loaded with DC, and the stimulation-induced CTL can only specifically kill tumor cells expressing a single antigen polypeptide, and the killing range is limited.
  • DC Genetically modified DC.
  • tumor antigen genes costimulatory molecules, cytokines, chemokines and the like are commonly used to modify DCs, and the methods include viral vectors and physical and chemical methods. Studies have shown that physicochemical methods are not only less efficient than viral vectors, but may also cause loss of DC death and surface markers.
  • the viral vector has high transfection efficiency and can load large gene fragments, which have effects on both quiescent and mitotic cells, and the expression of foreign genes is more efficient.
  • the efficiency of lentivirus transfection is high, but it will be exogenous.
  • the gene is inserted into the host cell genome with a high degree of risk, and the adenovirus is relatively safe, but the transfection efficiency is not ideal.
  • Cytotoxic T lymphocyte is an effector T cell that specifically recognizes antigens presented by MHC molecules, thereby specifically killing tumor cells and cells infected by microorganisms. CTL can kill target cells efficiently and specifically without damaging normal tissues.
  • the mechanism of CTL killing tumor cells includes: 1) release of granzyme and perforin directly induce tumor cell death; 2) induction of tumor cell apoptosis by Fas/FasL pathway; 3) secretion of cytokines such as IFN- ⁇ and TNF- ⁇ to kill tumor Cells or inhibit tumor cell growth.
  • the sensitized DC When a specific tumor antigen is loaded with DC, the sensitized DC is induced to secrete a large amount of IL-12 after maturation, thereby effectively stimulating initial T cell activation and proliferation, and IL-2 secreted by T cell activation is further stimulated by autocrine. By its own proliferation, a large number of proliferating T cells eventually differentiate into effector T cells, exerting a targeted anti-tumor effect.
  • the tumor antigen-specific CTL induced by sensitized DC is mainly CD8+ T cells, and some are CD4+ T cells. Make For effector T cells, CTL can efficiently kill tumor cells expressing specific tumor antigens.
  • DC vaccine preparation technology has defects:
  • the DC vaccine is prepared by using a synthetic single tumor antigen polypeptide-loaded DC, and the composition is clear, but the amount of the loaded tumor antigen polypeptide is single, and the therapeutic effect is limited.
  • DC-activated CTLs loaded with a certain tumor antigen polypeptide can only effectively kill tumor cells expressing the tumor antigen.
  • the genetically modified DC method is not perfect, and the transfection efficiency is low or the safety index is low.
  • the antigen and TNF- ⁇ are generally co-cultured with immature DC to induce DC maturation and achieve antigen presentation of DC, however, antigen presentation efficiency of DC is not high, and activated CTL kills. Not active.
  • the current conventional method for preparing CTL is to sort CD8+ T cells by flow cytometry or immunomagnetic beads, and then use the combination of cytokines for cell expansion.
  • the commonly used cytokines include IL-2 and IFN- ⁇ . , IL-7 and PHA.
  • the way of sorting cells by flow cytometry itself will have a certain impact on cell viability, and if the flow cytometer is to sort out a certain number of target cells, the proportion of positive cells must be high, otherwise the time will be longer. It has a great influence on cell activity. If the method of increasing the initial density of cells is adopted, one may block the tube, and the second will increase the misjudgment and waste of sorting.
  • One of the objects of the present invention is to provide a kit for preparing a DC-CTL, the kit for preparing a DC-CTL comprising: an agent for isolating a single nuclear cell;
  • Tumor antigen active epitope peptide of sensitized DC Tumor antigen active epitope peptide of sensitized DC
  • an agent for promoting CTL amplification and activation is one or more of anti-CD3 monoclonal antibody, IFN- ⁇ , IL-1 ⁇ , PHA, IL-2, IL-7 Combination with anti-CD28 monoclonal antibody, anti-ICOS monoclonal antibody.
  • the reagent for isolating the mononuclear cells is a Ficoll lymphocyte separation solution or a percoll cell separation solution.
  • Reagents for inducing DC differentiation and maturation 400-600 U/mL IL-4, 800-1200 U/mL GM-CSF, 800-1200 U/mL TNF- ⁇ (preferably median)
  • the tumor antigen active epitope peptide of the sensitized DC (selecting a dipeptide or a combination of peptides according to the tumor antigen expressed by the tumor): including but not limited to MAGE 1: 61-169, MAGE-A 12: 170-178, AIM -2: 14-23, TRP-2: 180-188, gp100: 209-217, HER2: 369-377, HER2: 342-350, IL-13R ⁇ 2: 345-354, PSA: 248-257, PAP213-221, PAP112-120, PSMA441-450, PSMA624-632, ESO-1: 161-180, CEA652-660, survivin 96-104, EGFR800-809, MRP3: 503-511, SART2: 93-101.
  • the reagent for promoting CTL amplification and activation 50-80 ng/mL anti-CD3 monoclonal antibody (preferably 50), 1000-1200 U/mL IFN- ⁇ (preferably 1000), 80-150 U/mL IL-1 ⁇ (preferably 100) ), 50-100 ng/mL PHA (preferably 100), 300-500 U/mL IL-2 (preferably 300), 20-30 ng/mL IL-7 (preferably 30), 20-50 ng/mL anti-CD28 monoclonal antibody ( Preferably 30), 20-40 ng/mL anti-ICOS monoclonal antibody (preferably 30).
  • 50-80 ng/mL anti-CD3 monoclonal antibody preferably 50
  • 1000-1200 U/mL IFN- ⁇ preferably 1000
  • 80-150 U/mL IL-1 ⁇ preferably 100
  • 50-100 ng/mL PHA preferably 100
  • 300-500 U/mL IL-2 preferably 300
  • 20-30 ng/mL IL-7 preferably 30
  • amino acid sequence of gp100:209-217 is shown as SEQ ID NO: 1
  • HER2:369-377 amino acid sequence is shown as SEQ ID NO: 2.
  • the concentration of the various tumor antigen active epitope peptides is from 10 ⁇ g/ml to 20 ⁇ g/ml.
  • the DC-CTL kit also includes components:
  • Another object of the present invention is to prepare a DC-CTL by using the aforementioned kit for preparing DC-CTL, comprising the steps of:
  • T lymphocytes in peripheral blood mononuclear cells using an agent that promotes CTL amplification and activation, and inducing T lymphocytes to differentiate and proliferate to CTL.
  • the order of adding the tumor antigen active epitope peptide and TNF- ⁇ in the step C) is: first adding the tumor antigen active epitope peptide, and adding TNF- ⁇ after 1-2 hours.
  • the order of adding the anti-CD28 monoclonal antibody and the anti-ICOS monoclonal antibody in the step D) is: adding the anti-CD28 monoclonal antibody for 1.5-3 h (preferably 2 h) and then adding the anti-ICOS monoclonal antibody.
  • the method for sensitizing DC by using two or more tumor antigen active epitope peptides is to first add a tumor antigen active epitope peptide, and after the DC uptake of the tumor antigen active epitope peptide, the inflammatory medium TNF- ⁇ is added, Fully stimulate DC maturation, better play antigen presentation, and more strongly induce stimulation of CTL production.
  • the agent for promoting CTL amplification and activation includes an anti-CD28 monoclonal antibody and an anti-ICOS monoclonal antibody, which can stimulate T lymphocyte proliferation, produce various cytokines, and differentiate into CTL.
  • the anti-CD28 monoclonal antibody of the invention is added 1.5-3h (preferably 2h) and then the anti-ICOS monoclonal antibody is added, and the anti-ICOS monoclonal antibody provides a costimulatory signal to better promote the proliferation of T lymphocytes and regulate T lymphocytes. Differentiation and maintenance of the effects and functions of activated T lymphocytes (including memory T lymphocytes) play an important role in the immune response and maintenance effect phase.
  • the cytokine combination used in the CTL culture of the invention can promote the activation and proliferation of T lymphocytes, increase the number of T lymphocyte clones, and has high purity, and the prepared DC vaccine can significantly improve the cytotoxicity of the induced CTL.
  • the preparation of CTL of the invention does not require the use of flow cytometry or immunomagnetic beads to sort CD8+ T cells from peripheral blood mononuclear cells, and the CTL prepared by the method of the invention has high purity, strong proliferation, and a large number of tumor cells. High killing activity.
  • Figure 1 shows the results of DC flow cytometry.
  • Figure 2 is a schematic diagram showing the secretion of IL-12 by DC.
  • Figure 3 is a schematic diagram showing the results of CTL cell counting.
  • Figure 4 is a schematic diagram showing the results of CTL flow cytometry.
  • Figure 5 is a schematic diagram showing the secretion of IFN- ⁇ by CTL cells.
  • Figure 6 is a schematic diagram showing the results of the cytotoxic killing experiment.
  • Tumor antigen active epitope peptide synthesized by standard Fmoc scheme, purified and purified by high performance liquid chromatography, identified by mass spectrometry and molecular weight determination. The results showed that the purity of the above-mentioned tumor antigen active epitope peptide was higher than 95%, and the molecular weight was in agreement with the theoretical value.
  • the above tumor antigen active epitope peptides were each dissolved in sterile double distilled water at a concentration of 10 ug/mL, and stored at -80 ° C in portions.
  • Peripheral blood was collected 70-80 mL under aseptic conditions, and blood samples were sent to the laboratory for separation of peripheral blood mononuclear cells.
  • the blood was transferred to two 50 mL centrifuge tubes, centrifuged at 2000 rpm for 10 min, and the upper autologous plasma was transferred to a 50 mL centrifuge tube, inactivated in a 56 ° C water bath for 30 min, and stored at 4 ° C for use.
  • the remaining blood was blown evenly with 0.01 mol/L PBS solution in a 1:1 dilution.
  • the cell separation solution, the ratio of the lymphocyte separation solution to the diluted blood volume is 2:1, and the lymphocyte separation solution is slowly added to the upper part of the diluted blood along the centrifuge tube wall to keep the liquid interface clear, 2000 rpm, 30 min.
  • the centrifuge tube was taken out and the liquid in the tube was divided into 4 layers, from top to bottom: plasma, white cloud-like thin layer (single nuclear cell layer), lymphocyte separation solution, red blood cells and granulocytes.
  • a single nuclear cell layer was aspirated, and the cells were washed with 3-4 volumes of PBS, 1500 rpm, 10 min, and washed again by adding 4-5 volumes of PBS to the cell pellet, 1000 rpm, 10 min.
  • the cell pellet was resuspended in 30-40 mL of GTT551 serum-free medium containing 10% autologous plasma, placed in a 37 ° C, 5% CO 2 incubator for 2 h, and the adherent cells and suspension cells were harvested for differentiation into DC and T lymphocyte culture.
  • GTT551 serum-free medium containing 10% autologous plasma was supplemented according to cell growth, and a final concentration of 500 U/mL IL-4, 1000 U/mL GM-CSF was added, and placed in a 37 ° C, 5% CO 2 incubator. The culture is continued, and the above medium and cytokines are added depending on the growth of the cells.
  • the cells were harvested on the 6th day, and the cell density was adjusted to 1 ⁇ 10 6 /mL, and 10 ug/mL of the tumor antigen active epitope peptide was added, respectively. After 2 hours, 1000 U/mL TNF- ⁇ was added to stimulate DC maturation and exert antigen presentation. A DC vaccine loaded with a tumor antigen active epitope peptide was harvested on day 7.
  • Immature DC imDC
  • mature DC mature DC
  • the imDC on day 3 of DC culture and the mDC on day 7 were taken for detection.
  • the plate was capped and reacted at 37 ° C for 90 min. After the reaction, the liquid in the microplate was removed by an automatic washer.
  • the biotin anti-human IL-12 antibody working solution was sequentially added in 0.1 mL per well (except TMB blank coloring well), reacted at 37 ° C for 60 min, and washed three times with 0.01 M PBS.
  • the ABC working solution was sequentially added in 0.1 mL per well (except for the TMB blank coloring hole), reacted at 37 ° C for 30 min, and washed 5 times with 0.01 M PBS.
  • TMB coloring solution was added in order of 90 ⁇ L per well, and the reaction was stopped at 20 ° C for 20-25 min at 37 ° C.
  • TMB stop solution was added in order of 0.1 mL per well.
  • the blue color turned yellow, and the OD value was measured at 450 nm with a microplate reader.
  • the OD value of the sample was subtracted from the OD value of the blank well, and the OD value and the standard concentration were plotted on the XY axis.
  • the IL-12 concentration was found on the standard curve and multiplied by the dilution factor to calculate the IL-12 concentration in the sample.
  • the suspension cell density was adjusted to 1 ⁇ 10 6 /mL with GTT551 serum-free medium containing 10% autologous plasma, and placed in a culture flask pre-coated with 50 ng/mL anti-CD3 monoclonal antibody, and the final concentration was 100 ng/mL PHA, 1000 U. /mL IFN- ⁇ was placed in a 37 ° C, 5% CO 2 incubator for further culture.
  • GTT551 serum-free medium containing 10% autologous plasma was supplemented according to cell growth, and the final concentration was 300 U/mL IL-2, 30 ng/mL IL-7, 100 ng/mL PHA, and placed at 37 ° C, 5 Continue cultivation in the %C0 2 incubator.
  • the DC vaccine (the ratio of DC vaccine to T lymphocytes was 1:10) was co-cultured, and the first round of CTL-induced amplification was performed.
  • the final concentration of the medium was 300 U/mL IL-2, 30 ng/mL.
  • IL-7 and 100 ng/mL PHA a final concentration of 30 ng/mL anti-CD28 monoclonal antibody was also added.
  • a final concentration of 30 ng/mL anti-ICOS monoclonal antibody was added, and GTT551 containing 10% autologous plasma was added every other day.
  • Serum-free medium the above cytokines were added to maintain the optimal concentration of cytokines, and the culture was continued at 37 ° C in a 5% CO 2 incubator.
  • the DC vaccine (the ratio of DC vaccine to T lymphocyte was 1:10) was added again to co-culture, and the T lymphocytes were induced to differentiate into CTL, and the second round of CTL induction was performed.
  • the final concentration of the medium was 300 U/mL IL-2, 30 ng/mL IL-7, 100 ng/mL PHA, 30 ng/mL anti-CD28 monoclonal antibody, and added a final concentration of 30 ng/mL anti-ICOS monoclonal antibody after 2 h, supplemented with 10% every other day
  • Autologous plasma GTT551 serum-free medium was added to the above cytokines, and culture was continued at 37 ° C in a 5% CO 2 incubator.
  • a conventional culture method group was established as a control group, and a conventional culture method group was cultured using a conventional cytokine combination, including IL-2, IFN- ⁇ , IL-7, and PHA.
  • the anti-CD28 monoclonal antibody and the anti-ICOS monoclonal antibody added by the invention can stimulate the proliferation of T lymphocytes, produce various cytokines, and differentiate into CTL, preferably by adding Anti-ICOS monoclonal antibody was added after adding anti-CD28 monoclonal antibody for about 2 h (1.5-3 h).
  • the cytokine combination used in the CTL culture of the invention can promote the activation and proliferation of T lymphocytes, increase the number of T lymphocyte clones, and has high purity, and the prepared DC vaccine can significantly improve the cytotoxicity of the induced CTL.
  • the number of CTLs obtained by the conventional culture method group was (1.2 ⁇ 0.4) ⁇ 10 9
  • the number of CTLs obtained by the anti-CD28 monoclonal antibody group (conventional culture method group + anti-CD28 monoclonal antibody) was (1.9 ⁇ 0.5) ⁇ 10 9
  • the number of CTLs obtained by the anti-ICOS monoclonal antibody group (conventional culture method group + anti-ICOS monoclonal antibody) was (1.6 ⁇ 0.4) ⁇ 10 9
  • the number of CTLs obtained by the novel culture method of the present invention was (2.8 ⁇ 0.9) ⁇ 10. 9.
  • the number of CTLs obtained by the new culture method was significantly different from other culture methods (P ⁇ 0.05).
  • the cell pellet obtained by centrifugation was washed twice with a pre-cooled 0.01 l mol/L PBS solution at 4 ° C, 1000 rpm, 10 min, and the cell pellet was resuspended in 200 ul PBS to adjust the cell density to 1 ⁇ 10 6 /mL, and added to CD3 in the dark.
  • CD4, CD8 antibody incubation at 4 ° C for 30 min in the dark, add 2 ml PBS, l000 rpm, l0 min, wash the cells to remove unbound antibody, remove the supernatant, resuspend the cell pellet in PBS, send at 4 ° C, upflow cells Instrument detection.
  • the T cell group, the conventional culture method group and the new culture method group were established, wherein the T cell group was a T lymphocyte which was not co-cultured with the DC carrying the tumor antigen active epitope peptide, and the conventional culture method group was prepared by conventional culture method and loaded with tumor.
  • the CTL of DC co-culture of the antigen-active epitope peptide, the new culture method group is a CTL for co-cultivation of DC with the tumor antigen-loaded active epitope peptide prepared by the culture method of the present invention.
  • the antibody-labeled plate was taken out, and the TMB blank color-developing well was set, and 0.1 mL of the standard diluted with a certain number and the sample diluted with the sample diluent were sequentially added.
  • the plate was capped and reacted at 37 ° C for 90 min. After the reaction, the liquid in the microplate was removed by an automatic washer.
  • the biotin anti-human IFN- ⁇ antibody working solution was sequentially added in 0.1 mL per well (except TMB blank coloring well), reacted at 37 ° C for 60 min, and washed three times with 0.01 M PBS.
  • the ABC working solution was sequentially added in 0.1 mL per well (except for the TMB blank coloring hole), reacted at 37 ° C for 30 min, and washed 5 times with 0.01 M PBS.
  • TMB coloring solution was added in order of 90 ⁇ L per well, and the reaction was protected from light for 20-25 min at 37 ° C.
  • TMB stop solution was added in order of 0.1 mL per well. The color turned yellow and the OD value was measured at 450 nm using a microplate reader. The OD value of the sample was subtracted from the OD value of the blank well, and the OD value and the standard concentration were plotted on the XY axis.
  • the IFN- ⁇ concentration was found on the standard curve and multiplied by the dilution factor to calculate the IFN- ⁇ concentration in the sample.
  • the control group, the conventional culture method group and the new culture method group were set up, and each group was provided with 3 duplicate wells.
  • CTL effector cells
  • human glioma cells U251 target cells
  • the effector cell suspension and the target cell suspension were in a total volume of 200 uL. After being cultured for 48 hours in a 5% CO 2 incubator at 37 ° C, 20 uL of CCK-8 was added to each well, and the incubation was continued for 2 hours, and then detected by a microplate reader at 450 nm.
  • the experiment was divided into blank control group, target cell group, effector group and experimental group (new culture method group, conventional culture method group), and the experimental group was composed of tumor antigen active epitope peptide gp100:209-217 (SEQ ID NO: 1).
  • the HER2: 369-377 (SEQ ID NO: 2) loaded DC induced preparation of CTL.

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Abstract

本发明公开了一种制备DC-CTL的试剂盒及其应用，所述制备DC-CTL的试剂盒包括：分离单个核细胞的试剂；诱导DC分化成熟的试剂；致敏DC的肿瘤抗原活性表位肽；促进CTL扩增和激活的试剂，所述促进CTL扩增和激活的试剂为抗CD3单克隆抗体、IFN-γ、IL-1α、PHA、IL-2、IL-7中的一种或者几种与抗CD28单克隆抗体、抗ICOS单克隆抗体的组合。

Description

制备DC-CTL的试剂盒及其应用 技术领域
本发明属于肿瘤治疗试剂盒领域，尤其是涉及一种制备DC-CTL的试剂盒及其应用。
背景技术
(一)已有技术描述
1.免疫细胞治疗
近几十年来，肿瘤治疗取得了较大的进展，但仍存在一些难题：手术治疗可有效地降低肿瘤负荷，但对残留的微小癌灶和转移癌灶效果欠佳；放化疗可减小肿瘤病灶并抑制肿瘤细胞的生长，但是不能有效区分正常细胞和肿瘤细胞，对正常组织毒副作用较大。随着细胞生物学及分子生物学的快速发展，靶向性强、毒副作用小的免疫细胞疗法受到人们极大关注。免疫细胞治疗指向肿瘤患者体内输入具有抗肿瘤活性的免疫细胞，直接杀伤肿瘤细胞或激发机体抗肿瘤免疫反应，从而达到治疗肿瘤的目的。作为一种新型的肿瘤治疗方式，免疫细胞治疗现已成为肿瘤治疗的重要辅助手段，其克服了临床常规治疗的局限性，可有效地消除微小残留癌灶，抑制肿瘤复发，提高患者免疫能力，在肿瘤临床治疗领域具有广阔的前景。
2. DC
树突状细胞(Dendritic Cell，DC)是由美国学者Steinman于1973 年发现的，是目前为止发现的抗原提呈功能最强的免疫细胞，在免疫反应的诱导和调控中发挥重要作用。DC能够显著刺激初始T细胞增殖，是机体适应性T细胞免疫应答的启动者，在适应性T细胞免疫应答的诱导中起着关键的"决策作用"，因此它是肿瘤免疫治疗中增强抗肿瘤抗原反应的理想治疗手段之一，在肿瘤疫苗领域已备受关注。自从1996年美国斯坦福大学医学中心Hsu等在Nature Medicine上报道了全球首项DC肿瘤疫苗临床研究以来，诱导肿瘤抗原特异性效应细胞的DC肿瘤疫苗相关研究持续升温，大量的临床研究相继开展。Sipuleucel-T(Dendreon，美国)、CreaVaxRCC(CreaGene，韩国)和Hybricell(Genoa Biotechnologia，巴西)等DC肿瘤疫苗相继获得上市批准。DC肿瘤疫苗已经由实验室走向临床，其可行性和安全性以及对部分患者的有效性已经得以证实。
目前DC疫苗制备方法包括两种方法：抗原负载致敏DC和基因修饰DC。
1)抗原负载致敏DC。抗原负载致敏DC的方法主要有肿瘤细胞全抗原致敏和肿瘤抗原多肽致敏。研究表明鉴于许多肿瘤抗原仍未明确，采用全肿瘤抗原诱导T细胞产生的免疫反应范围更广，多种不同肿瘤抗原成分同时刺激DC，诱导出针对不同抗原决定簇的特异性CTL，杀伤范围更广，可以避免肿瘤细胞发生免疫逃逸，然而因为全肿瘤抗原成分不明确，可能诱发自身免疫反应，存在安全隐患。目前市面上有人工合成肿瘤抗原多肽商品，成分明确，安全性高，用人工合成的肿瘤抗原多肽负载DC激活的CTL杀伤靶点明确，特异性强， 但是单一抗原多肽负载DC，刺激诱导的CTL只能特异性杀伤表达单一抗原多肽的肿瘤细胞，杀伤范围局限。
2)基因修饰DC。目前常用肿瘤抗原基因、共刺激分子、细胞因子、趋化因子等修饰DC，方法包括病毒载体和理化方法。研究表明理化方法较病毒载体不仅转染效率低下，而且可能引起DC死亡和表面标志的丧失。而病毒载体转染效率高，且能负载较大基因片段，对于静止期和分裂期细胞都有作用，外源基因表达也更高效，然而慢病毒转染效率虽高，但会将外源性基因插入宿主细胞基因组，危险程度高，腺病毒相对安全，但是转染效率不甚理想。
3. DC-CTL
细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte，CTL)，是效应T细胞，可特异性识别MHC分子提呈的抗原，进而特异性地杀伤肿瘤细胞及受微生物感染的细胞等。CTL可高效且特异地杀伤靶细胞，而不损害正常组织。CTL杀伤肿瘤细胞的机制包括：1)释放颗粒酶和穿孔素直接诱导肿瘤细胞死亡；2)通过Fas/FasL途径诱导肿瘤细胞凋亡；3)分泌IFN-γ和TNF-α等细胞因子杀伤肿瘤细胞或抑制肿瘤细胞生长。
用特定的肿瘤抗原负载DC，致敏的DC被诱导成熟后分泌大量的IL-12，进而有效地刺激初始T细胞活化、增殖，T细胞活化后分泌的IL-2通过自分泌的方式进一步刺激自身的增殖，大量增殖的T细胞最终分化为效应T细胞，发挥靶向抗肿瘤效应。致敏DC诱导的肿瘤抗原特异性CTL主要为CD8+T细胞，部分为CD4+T细胞。作 为效应T细胞，CTL可高效地杀伤表达特定肿瘤抗原的肿瘤细胞。
(二)已有技术问题及缺陷描述
1. DC疫苗制备技术存在缺陷：
1)自体肿瘤组织来源的全肿瘤抗原获取不易，且全肿瘤抗原成份众多，可能引发自身免疫反应，存在安全风险。
2)使用人工合成的单一肿瘤抗原多肽负载DC制备DC疫苗，成分明确，但负载的肿瘤抗原多肽数量单一，治疗效果有限。负载某一肿瘤抗原多肽的DC激活的CTL只能有效杀伤表达该肿瘤抗原的肿瘤细胞。
3)基因修饰DC方法尚不完善，转染效率低或者安全指数低。
2.现有技术制备DC疫苗时，一般是将抗原和TNF-α与未成熟DC共培养，以诱导DC成熟和实现DC的抗原呈递作用，然而DC的抗原呈递效率不高，激活的CTL杀伤活性不强。
3.目前制备CTL的常规方法为使用流式细胞仪或免疫磁珠分选出CD8+T细胞，然后使用细胞因子组合进行细胞的扩增，常规使用的细胞因子包括IL-2、IFN-γ、IL-7和PHA。然而，流式细胞仪分选细胞的方式本身就会对细胞活性造成一定的影响，而且流式细胞仪要分选出一定数量的目的细胞的话，阳性细胞比例一定要高，不然时间会比较长，对细胞活性影响较大，如果采用提高细胞初始密度的方法，一是可能堵管，二是会增加分选的误判和浪费，细胞太多读取分析的时间短，判断会受到影响。再者，现在国内进行流式细胞仪分选细胞的价格不菲。免疫磁珠分选细胞，能得到的目的细胞数量比较大，对 细胞活力影响小，纯度也能达到90％，但是磁性微珠不能自动脱离目的细胞群，难以完全洗脱而成为了非细胞杂质，对目的细胞的后续培养存在一定的影响。
此外，现有的细胞因子组合培养扩增的CTL数量不足，细胞活性不高，难以满足临床需求。
发明内容
本发明的目的之一在于提供制备DC-CTL的试剂盒，所述制备DC-CTL的试剂盒包括：分离单个核细胞的试剂；
诱导DC分化成熟的试剂；
致敏DC的肿瘤抗原活性表位肽；
促进CTL扩增和激活的试剂，所述促进CTL扩增和激活的试剂为抗CD3单克隆抗体、IFN-γ、IL-1α、PHA、IL-2、IL-7中的一种或者几种与抗CD28单克隆抗体、抗ICOS单克隆抗体的组合。
进一步的：所述分离单个核细胞的试剂为Ficoll淋巴细胞分离液，或者percoll细胞分离液。
诱导DC分化成熟的试剂：400-600U/mL IL-4、800-1200U/mL GM-CSF、800-1200U/mL TNF-α(优选中位值)
所述致敏DC的肿瘤抗原活性表位肽(根据肿瘤所表达的肿瘤抗原选择双肽或多种肽的组合)：包括但不限于MAGE1：161-169、MAGE-A12：170-178、AIM-2：14-23、TRP-2：180-188、gp100：209-217、HER2：369-377、HER2：342-350、IL-13Rα2：345-354、PSA：248-257、 PAP213-221、PAP112-120、PSMA441-450、PSMA624-632、ESO-1:161-180、CEA652-660、survivin96-104、EGFR800-809、MRP3：503-511、SART2：93-101。
所述促进CTL扩增和激活的试剂：50-80ng/mL抗CD3单克隆抗体(优选50)、1000-1200U/mL IFN-γ(优选1000)、80-150U/mL IL-1α(优选100)、50-100ng/mL PHA(优选100)、300-500U/mL IL-2(优选300)、20-30ng/mL IL-7(优选30)、20-50ng/mL抗CD28单克隆抗体(优选30)、20-40ng/mL抗ICOS单克隆抗体(优选30)。
进一步的，所述gp100:209-217的氨基酸序列如SEQ ID NO：1所示，所述HER2:369-377氨基酸序列如SEQ ID NO：2所示。
进一步的：所述各种肿瘤抗原活性表位肽的浓度为10μg/ml-20μg/ml。
所述DC-CTL试剂盒还包括组份：
E)PBS缓冲液；
F)无血清培养基。
本发明的另一目的在于应用前述的制备DC-CTL的试剂盒制备DC-CTL，包括步骤：
A)使用分离单个核细胞的试剂分离单个核细胞；
B)使用诱导DC分化的试剂诱导外周血单个核细胞向DC分化；
C)使用肿瘤抗原活性表位肽致敏DC，然后刺激DC成熟；
D)使用促进CTL扩增和激活的试剂扩增外周血单个核细胞中的T淋巴细胞，并诱导T淋巴细胞向CTL分化与增殖。
进一步的：所述步骤C)中添加肿瘤抗原活性表位肽及TNF-α的顺序为：先加入肿瘤抗原活性表位肽，1-2h后再添加TNF-α。
进一步的：所述步骤D)中添加抗CD28单克隆抗体以及添加抗ICOS单克隆抗体的顺序为：添加抗CD28单克隆抗体1.5-3h(优选为2h)后再添加抗ICOS单克隆抗体。
本发明使用两种或多种肿瘤抗原活性表位肽致敏DC的过程为先加入肿瘤抗原活性表位肽，待DC摄取肿瘤抗原活性表位肽后，再加入炎性介质TNF-α，以充分地刺激DC成熟，更好地发挥抗原提呈作用，更强有力地诱导刺激CTL生成。
本发明所述的试剂盒中，促进CTL扩增和激活的试剂包括抗CD28单克隆抗体和抗ICOS单克隆抗体，可刺激T淋巴细胞增殖，产生多种细胞因子，并分化为CTL。本发明添加抗CD28单克隆抗体1.5-3h(优选为2h)后再添加抗ICOS单克隆抗体，抗ICOS单克隆抗体提供的共刺激信号能更好地促进T淋巴细胞增殖，调节T淋巴细胞的分化，维持活化后T淋巴细胞(包括记忆性T淋巴细胞)的效应和功能，在免疫应答及维持效应阶段起重要作用。
本发明培养CTL使用的细胞因子组合可促进T淋巴细胞活化和增殖，提高T淋巴细胞克隆数，纯度高，且制备的DC疫苗可明显提高诱导的CTL的细胞毒性。
本发明制备CTL无需使用流式细胞仪或免疫磁珠从外周血单个核细胞中分选出CD8+T细胞，利用本发明方法制备的CTL纯度高，增殖力强，数量多，对肿瘤细胞的杀伤活性高。
附图说明
图1为DC流式细胞仪鉴定结果。
图2为DC分泌IL-12的情况示意图。
图3为CTL细胞计数结果示意图。
图4为CTL流式细胞仪鉴定结果示意图。
图5为CTL细胞分泌IFN-γ的情况示意图。
图6为细胞毒性杀伤实验结果示意图。
具体实施方式
下面结合附图和实施例对本发明做进一步详细说明。
1.肿瘤抗原活性表位肽：采用标准Fmoc方案合成，高效液相色谱法进行纯化和纯度分析，质谱法进行鉴定和分子量测定。结果显示上述肿瘤抗原活性表位肽的纯度高于95％，分子量与理论值相符。上述肿瘤抗原活性表位肽分别用无菌双蒸水充分溶解，浓度为10ug/mL，分装保存于-80℃。
2.分离外周血单个核细胞
无菌条件下采集外周血70-80mL，将血液标本送至实验室，进行外周血单个核细胞的分离。将血液转移至2个50mL离心管，2000rpm，l0min离心，转移上层自体血浆至50mL离心管内，56℃水浴灭活30min，4℃冷藏备用。
余下血液与0.01mol/L PBS液按1:1稀释吹打均匀，用吸管取淋 巴细胞分离液，淋巴细胞分离液与稀释后血液的体积比例为2：1，将淋巴细胞分离液沿离心管壁缓慢加入稀释后血液的上方，保持液体分界面清晰，2000rpm，30min。
取出离心管，管内液体分为4层，从上到下依次为：血浆、白色云雾状薄层(单个核细胞层)、淋巴细胞分离液、红细胞和粒细胞。吸取单个核细胞层,加入3-4倍体积的PBS洗涤细胞，1500rpm，10min，往细胞沉淀加入4-5倍体积的PBS再次洗涤，1000rpm，10min。
将细胞沉淀用30-40mL含10％自体血浆的GTT551无血清培养基重悬，放入37℃，5％CO2培养箱静置2h，收获贴壁细胞及悬浮细胞，分别用于诱导分化成DC和T淋巴细胞培养。
3.外周血单个核细胞向DC的诱导分化及肿瘤抗原活性表位肽致敏DC
1)将步骤2收获的贴壁细胞诱导分化为DC，具体方法如下：
往贴壁细胞培养瓶内加入20mL含10％自体血浆的GTT551无血清培养基,添加终浓度为500U/mL IL-4、1000U/mL GM-CSF，放入37℃，5％C02培养箱中继续培养。GM-CSF和IL-4共同作用可使单核细胞定向分化为未成熟DC，此时的DC具有较强的抗原摄取和加工能力，但抗原递呈能力很弱。
于第3天根据细胞生长情况补充含10％自体血浆的GTT551无血清培养基,添加终浓度为500U/mL IL-4、1000U/mL GM-CSF，放入37℃，5％C02培养箱中继续培养，根据细胞生长情况添加上述培养基和细胞因子。
于第6天收获细胞，调整细胞密度为1X106/mL，分别加入10ug/mL的肿瘤抗原活性表位肽。2h后添加1000U/mL TNF-α，刺激DC成熟，发挥抗原提呈作用。于第7天收获负载肿瘤抗原活性表位肽的DC疫苗。
2)DC的表型鉴定
分别取培养第3天的未成熟DC(immature DC，imDC)和第7天的成熟DC(mature DC，mDC)进行细胞表面标记物的检测。
加入0.25％胰酶消化DC，终止消化后1000rpm，10min，将细胞沉淀用4℃预冷的0.01mol/L PBS液洗涤2次，1000rpm，10min，用200ul PBS重悬细胞沉淀，调整细胞密度为1X106/ml,避光加入CD83、CD86、CD1a和HLA-DR荧光抗体，4℃避光孵育30min，加入2mL PBS，l000rpm，l0min，洗涤细胞以除去未结合的抗体，去上清，细胞沉淀用PBS液重悬，4℃送检，上流式细胞仪检测。
流式细胞仪检测结果显示第7天培养的mDC高表达CD83、CD86、CD1a和HLA-DR，表明成功诱导DC成熟。
表1 DC细胞表面标志物表达情况(
Figure PCTCN2015088648-appb-000001
％)
Figure PCTCN2015088648-appb-000002
*：与imDC组相比，P<0.05。
3)细胞因子检测(IL-12)
取DC培养第3天的imDC和第7天的mDC进行检测。取出已包被抗体的酶标板，设置TMB(3,3',5,5'-Tetra methylbenzidine)空白显色孔，依次加入0.1mL按一定倍数稀释的标准品和用样品稀释液稀释的样品。酶标板加上盖，37℃反应90min。反应后用自动洗板机吸去酶标板内的液体。将生物素抗人IL-12抗体工作液按每孔0.1mL依次加入(TMB空白显色孔除外)，37℃反应60min，0.01M PBS洗涤3次。将ABC工作液按每孔0.1mL依次加入(TMB空白显色孔除外)，37℃反应30min，0.01M PBS洗涤5次。按每孔90μL依次加入TMB显色液，37℃避光反应20-25min，按每孔0.1mL依次加入TMB终止液，此时蓝色立转黄色，用酶标仪在450nm测定OD值。样品OD值减去空白孔OD值后以OD值及标准品浓度为XY轴绘图，在标准曲线上查找IL-12浓度后乘以稀释倍数，计算样本中IL-12浓度。
结果表明mDC表达IL-12的水平明显高于imDC的表达水平，具有显著差异(P<0.05)。
4.外周血单个核细胞向CTL诱导增殖
1)将步骤2收获的悬浮细胞向CTL诱导增殖，具体方法如下：
用含10％自体血浆的GTT551无血清培养基调整悬浮细胞密度至1X106/mL，置入预先包被50ng/mL抗CD3单克隆抗体的培养瓶中，添加终浓度为100ng/mL PHA、1000U/mL IFN-γ，放入37℃，5％C02培养箱中继续培养。
于第2天添加终浓度为100U/mL IL-1α、300U/mL IL-2、30 ng/mL IL-7，放入37℃，5％C02培养箱中继续培养。
于第3天根据细胞生长情况补充含10％自体血浆的GTT551无血清培养基,添加终浓度为300U/mL IL-2、30ng/mL IL-7、100ng/mL PHA，放入37℃，5％C02培养箱中继续培养。
于第7天加入DC疫苗(DC疫苗与T淋巴细胞的比例为1：10)共培养，进行第一轮CTL诱导扩增，培养基除添加终浓度为300U/mL IL-2、30ng/mL IL-7、100ng/mL PHA外，还添加终浓度为30ng/mL抗CD28单克隆抗体，2h后再添加终浓度为30ng/mL抗ICOS单克隆抗体，隔天补充含10％自体血浆的GTT551无血清培养基，添加上述细胞因子，以保持细胞因子的最佳作用浓度，放入37℃，5％C02培养箱中继续培养。
于第14天再次加入DC疫苗(DC疫苗与T淋巴细胞的比例为1：10)共培养，强化T淋巴细胞向CTL的诱导分化，进行第二轮CTL诱导扩增，培养基添加终浓度为300U/mL IL-2、30ng/mL IL-7、100ng/mL PHA、30ng/mL抗CD28单克隆抗体，2h后再添加终浓度为30ng/mL抗ICOS单克隆抗体，隔天补充含10％自体血浆的GTT551无血清培养基，添加上述细胞因子，放入37℃，5％C02培养箱中继续培养。
于第21天收获CTL。设立常规培养法组作为对照组，常规培养法组采用常规的细胞因子组合培养，包括IL-2、IFN-γ、IL-7和PHA。
本发明添加的抗CD28单克隆抗体和抗ICOS单克隆抗体可刺激T淋巴细胞增殖，产生多种细胞因子，并分化为CTL，优选方式为添 加抗CD28单克隆抗体2h左右(1.5-3h)后再添加抗ICOS单克隆抗体。
本发明培养CTL使用的细胞因子组合可促进T淋巴细胞活化和增殖，提高T淋巴细胞克隆数，纯度高，且制备的DC疫苗可明显提高诱导的CTL的细胞毒性。
2)细胞计数
常规培养法组获得的CTL数量为(1.2±0.4)×109，抗CD28单克隆抗体组(常规培养法组+抗CD28单克隆抗体)获得的CTL数量为(1.9±0.5)×109，抗ICOS单克隆抗体组(常规培养法组+抗ICOS单克隆抗体)获得的CTL数量为(1.6±0.4)×109，采用本发明新培养方法获得的CTL数量为(2.8±0.9)×109，新培养方法获得的CTL数量与其他培养法相比，具有显著的统计学差异(P<0.05)。
3)CTL的表型鉴定
取第7天未与DC疫苗共培养的T淋巴细胞和第21天培养的CTL进行细胞表型检测：
将离心获得的细胞沉淀用4℃预冷的0.0l mol/L PBS液洗涤2次，1000rpm，10min，用200ul PBS重悬细胞沉淀，调整细胞密度为1×106/mL,避光加入CD3、CD4、CD8抗体，4℃避光孵育30min，加入2ml PBS，l000rpm，l0min，洗涤细胞以除去未结合的抗体，去上清，细胞沉淀用PBS液重悬，4℃送检，上流式细胞仪检测。设立T细胞组、常规培养法组和新培养法组，其中T细胞组为未与负载肿瘤抗原活性表位肽的DC共培养的T淋巴细胞，常规培养法组为 常规培养法制备与负载肿瘤抗原活性表位肽的DC共培养的CTL，新培养法组为本发明培养方法制备与负载肿瘤抗原活性表位肽的DC共培养的CTL。
结果显示常规培养法组和新培养法组CD3+细胞比例比T细胞组高，但无统计学意义，而常规培养法组和新培养法组的CD4+细胞比例下降，CD8+细胞比例升高，与T细胞组相比，具有统计学意义(P<0.05),且新培养法组比常规培养法组的CD8+细胞比例显著升高。
表2 CTL细胞表面标志物表达情况(
Figure PCTCN2015088648-appb-000003
％)
Figure PCTCN2015088648-appb-000004
4)细胞因子检测(IFN-γ)
取出已包被抗体的酶标板，设置TMB空白显色孔，依次加入0.1mL按一定倍数稀释的标准品和用样品稀释液稀释的样品。酶标板加上盖，37℃反应90min。反应后用自动洗板机吸去酶标板内的液体。将生物素抗人IFN-γ抗体工作液按每孔0.1mL依次加入(TMB空白显色孔除外)，37℃反应60min，0.01M PBS洗涤3次。将ABC工作液按每孔0.1mL依次加入(TMB空白显色孔除外)，37℃反应30min，0.01M PBS洗涤5次。按每孔90μL依次加入TMB显色液，37℃避光反应20-25min，按每孔0.1mL依次加入TMB终止液，此时蓝 色立转黄色，用酶标仪在450nm测定OD值。样品OD值减去空白孔OD值后以OD值及标准品浓度为XY轴绘图，在标准曲线上查找IFN-γ浓度后乘以稀释倍数，计算样本中IFN-γ浓度。设对照组、常规培养法组和新培养法组，每组设3个复孔。
结果显示，新培养法组分泌IFN-γ水平远远超过常规培养法组的分泌水平，具有显著差异(P<0.05)。
5.细胞杀伤实验
分别调整CTL(效应细胞)和人胶质瘤细胞U251(靶细胞)密度为1X106/mL和1X107/mL，按效靶比5：1、10：1、20：1往96孔板加入效应细胞悬液和靶细胞悬液，总体积为200uL，放入37℃，5％C02培养箱中培养48h后每孔加入20uL CCK-8，继续孵育2h后上酶标仪检测，于450nm读取OD值，杀伤率＝【1-(实验组OD值-效应细胞组OD值)/靶细胞组OD值】*100％。实验分为空白对照组、靶细胞组、效应细胞组和实验组(新培养法组、常规培养法组)，实验组均由肿瘤抗原活性表位肽gp100:209-217(SEQ ID NO：1)和HER2:369-377(SEQ ID NO:2)负载的DC诱导制备CTL。
结果表明，随着效靶比的增高，新培养法组和常规培养法组对人胶质瘤细胞U251的杀伤率逐渐升高，当效靶比为20:1的时候，对人胶质瘤细胞U251的杀伤性最强，杀伤率最高。并且，在同样的效靶比条件下，新培养法组对人胶质瘤细胞U251的杀伤率比常规培养法组杀伤率显著提高，具有统计学意义(P<0.05)。
Figure PCTCN2015088648-appb-000005

Claims (10)

  1. 一种制备DC-CTL的试剂盒，包括：
    分离单个核细胞的试剂；
    诱导DC分化成熟的试剂；
    致敏DC的肿瘤抗原活性表位肽；
    促进CTL扩增和激活的试剂，所述促进CTL扩增和激活的试剂为抗CD3单克隆抗体、IFN-γ、IL-1α、PHA、IL-2、IL-7中的一种或者几种与抗CD28单克隆抗体、抗ICOS单克隆抗体的组合。
  2. 如权利要求1所述的制备DC-CTL的试剂盒，其特征是：
    所述分离单个核细胞的试剂为Ficoll淋巴细胞分离液，或者percoll细胞分离液。
  3. 如权利要求1所述的制备DC-CTL的试剂盒，其特征是：所述诱导DC分化成熟的试剂为细胞因子IL-4、GM-CS、TNF-α中的一种或者几种；IL-4、GM-CSF、TNF-α的浓度分别为400-600U/mL、800-1200U/mL、800-1200U/mL。
  4. 如权利要求1所述的制备DC-CTL的试剂盒，其特征是：所述致敏DC的肿瘤抗原活性表位肽为MAGE1：161-169、MAGE-A12：170-178、AIM-2：14-23、TRP-2：180-188、gp100：209-217、HER2：369-377、HER2：342-350、IL-13Rα2：345-354、PSA：248-257、PAP213-221、PAP112-120、PSMA441-450、PSMA624-632、ESO-1:161-180、CEA652-660、survivin96-104、EGFR800-809、MRP3：503-511、SART2：93-101中的一种或者多种的组合。
  5. 如权利要求1所述的制备DC-CTL的试剂盒，其特征是：所述促进CTL扩增和激活的试剂中抗CD3单克隆抗体、IFN-γ、IL-1α、PHA、IL-2、IL-7的浓度分别为50-80ng/mL、1000-1200U/mL、80-150U/mL、50-100ng/mL、300-500U/mL、20-30ng/mL；所述抗CD28单克隆抗体、抗ICOS单克隆抗体的浓度分别为20-50ng/mL、20-40ng/mL。
  6. 如权利要求4所述的制备DC-CTL的试剂盒，其特征是：所述gp100:209-217的氨基酸序列如SEQ ID NO：1所示，所述HER2:369-377氨基酸序列如SEQ ID NO：2所示。
  7. 如权利要求4所述的制备DC-CTL的试剂盒，其特征是：所述各种肿瘤抗原活性表位肽的浓度为10μg/ml-20μg/ml。
  8. 如权利要求1-7任一项所述的制备DC-CTL的试剂盒，其特征是，还包括组分：
    E)PBS缓冲液；
    F)无血清培养基。
  9. 应用权利要求1-8任一项所述的制备DC-CTL的试剂盒制备DC-CTL的方法，包括步骤：
    A)使用分离单个核细胞的试剂分离单个核细胞；
    B)使用诱导DC分化的试剂诱导外周血单个核细胞向DC分化；
    C)使用肿瘤抗原活性表位肽致敏DC,然后刺激DC成熟；
    D)使用促进CTL扩增和激活的试剂扩增外周血单个核细胞中的T淋巴细胞，并诱导T淋巴细胞向CTL分化与增殖。
  10. 如权利要求9所述的制备DC-CTL的方法，其特征是：所述步骤C)在DC培养过程中先加入肿瘤抗原活性表位肽致敏，1-2h后再添加TNF-α促成熟；所述步骤D)中添加抗CD28单克隆抗体以及添加抗ICOS单克隆抗体的顺序为：添加抗CD28单克隆抗体1.5-3h后再添加抗ICOS单克隆抗体。
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