WO2018188235A1 - 一种乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群及其应用 - Google Patents

一种乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群及其应用 Download PDF

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WO2018188235A1
WO2018188235A1 PCT/CN2017/096512 CN2017096512W WO2018188235A1 WO 2018188235 A1 WO2018188235 A1 WO 2018188235A1 CN 2017096512 W CN2017096512 W CN 2017096512W WO 2018188235 A1 WO2018188235 A1 WO 2018188235A1
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antigen
liver cancer
hepatitis
cells
virus
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黄月华
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黄月华
璞晞（广州）生物免疫技术有限公司
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    • A—HUMAN NECESSITIES
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/53—Liver
    • 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
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
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    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of specific cellular immunotherapy of hepatitis B virus-associated liver cancer, in particular to an antigen presenting signal group of hepatitis B virus antigen peptide combined with liver cancer cell antigen information and application thereof, and the specific application is to activate dendritic cells Induction of T cell application.
  • liver cancer In stark contrast to the global mortality rate of cancer, the incidence and mortality of liver cancer are increasing year by year.
  • the latest epidemiological data show that the annual incidence of liver cancer is 782,000, the death toll is 745,000, and the average 5-year survival rate is less than 5%.
  • the overall survival time is only 10 months; China accounts for 55% of the world's liver cancer, becoming the country with the highest incidence of liver cancer and death, more than 85% of patients have a history of HBV infection, almost all cancer cells contain HBV gene.
  • Common methods include surgery (tumor resection and transplantation) and local treatment (radiofrequency, ablation, microwave, cryotherapy, absolute alcohol injection, transcatheter arterial chemoembolization, TACE), 50-60%.
  • the patient has reached the middle and late stage at the time of initial diagnosis. There is no chance of surgery.
  • the local treatment effect is up to 14% compared with the untreated patients.
  • the 2-year survival rate is up to 14%, and there are certain side effects.
  • liver failure is caused. Therefore, liver cancer has become a heavy economy in China. Burden and people's health killers.
  • Anti-tumor immunotherapy can avoid this problem to a certain extent.
  • tumor-specific T cells have the most potential, and T cells can pass through the blood.
  • the patrol inspection "find the tumor for attack.
  • cancer T cell immunotherapy can continue to alleviate the overall survival rate of some blood tumors; however, the low content of T cells in the tumor restricts the successful expansion of immunotherapy.
  • DCs dendritic cells
  • T cell maturation, differentiation and proliferation are key promoters of T cell activation and proliferation, and play an important role in the stages of T cell maturation, differentiation and proliferation, and activate DC energy. Effectively activates the anti-tumor immune effect of T cells, and therefore, enhances T cell function by restoring DC.
  • vaccines to treat advanced tumors, including in vitro DC-loaded tumor antigens for reinfusion or induction of intratumoral DCs to present endogenous antigens, suggesting that there is potential and prospect for improving antigen presentation to promote functional T cells.
  • HBV-HCC hepatitis B virus-associated liver cancer
  • HBV-HCC has highly heterogeneous tumor antigens with diversity, complexity and variability, which can help cancer cells easily escape the monitoring of the immune system
  • HBV-HCC The immunosuppressive microenvironment formed inside makes it difficult for immune cells to break through their "protection net.”
  • the present invention aims to solve the main technical difficulties in the current immunotherapy of solid tumors represented by liver cancer, including the high heterogeneity, diversity, complexity, and variability of antigens of solid tumors, and provides a group of hepatitis B virus.
  • DC cells induced by this signal group become DC vaccines (COST-DC) carrying antigenic signals of multi-specific hepatitis B virus-associated liver cancer, which can activate the proliferation and function of tumor-specific T cells, thereby enhancing their resistance to hepatitis B liver cancer. effect.
  • Another object of the present invention is to provide a DC vaccine carrying an antigenic signal of a multi-specific hepatitis B virus-associated liver cancer.
  • a set of antigen-presenting signal groups of hepatitis B virus antigen peptide combined with liver cancer cell antigen information consisting of HBV core antigen 18-27 peptide, HBV pre-S2 antigen 44-53 peptide and human hepatoma cell line HepG2 lysate, HBV core antigen
  • the mass ratio of the 18-27 peptide, the HBV pre-S2 antigen 44-53 peptide and the human liver cancer cell line HepG2 lysate was 0.5 to 1:0.5 to 1:1 to 2.
  • HBV core antigen 18-27 peptide is abbreviated as HBcAg
  • its amino acid sequence is FLPSDFFPSV
  • HBV pre-S2 antigen 44-53 peptide is abbreviated as HBPreS2
  • its amino acid sequence is SILSKTGDPV
  • human hepatoma cell line HepG2 lysate is abbreviated as HepG2-Lys.
  • Hepatitis B virus-associated liver cancer can also be called hepatitis B liver cancer.
  • a DC vaccine carrying an antigenic signal of a multi-specific hepatitis B virus-associated liver cancer specifically, an antigen presenting signal group combining the hepatitis B virus antigen peptide and the liver cancer cell antigen information as described above, and being added to the peripheral blood for isolation and culture In mature moDC cells, a vaccine can be obtained by culturing.
  • a DC vaccine carrying an antigenic signal of a multi-specific hepatitis B virus-associated liver cancer is prepared by adding the antigen presenting signal group (COST) of the hepatitis B virus antigen peptide and the liver cancer cell antigen information as described above to the AIM.
  • COST antigen presenting signal group
  • the -V culture solution was cultured to the mature moDC cells on the 6th day, and the final concentration of COST was 24 ug/ml.
  • the AIM-V culture solution was supplemented the next day, and the vaccine was recovered after 2 days of culture.
  • a hepatitis B liver cancer killing T cell the specific step is: the above-prepared multi-specific hepatitis B virus-related Hepatitis B killer T cells (COST-CTL) can be obtained by DC vaccine of antigenic signal of hepatocarcinoma and adoptive culture of T cells obtained from peripheral blood.
  • COST-CTL multi-specific hepatitis B virus-related Hepatitis B killer T cells
  • a hepatitis B liver cancer killing T cell the specific step is: taking a cell number of 2 ⁇ 5x10 6 DC vaccine carrying multi-specific hepatitis B virus-associated liver cancer antigen signal, and separating and amplifying from the peripheral blood on the 8th day
  • the T cells were cultured in a row, and the number of T cells was 2 to 5 ⁇ 10 7 .
  • the cells were co-cultured for 2 days at 37 ° C in a 5% CO 2 incubator, that is, on the 10th day; the AIM-V culture solution was supplemented with 5 to 10 ml, and the culture solution contained the concentration.
  • IL-2 It is 300 IU/ml of IL-2 and 15% by volume of albumin with a mass concentration of 20%; Day 12: 5-10 ml of AIM-V broth supplemented with IL-2 at a concentration of 300 IU/ml and quality The volume concentration was 20% albumin 1.5 ml; Day 14: obtaining viral tumor antigen co-activated DC induced specific T cells, ie, hepatitis B liver cancer killer T cells (COST-CTL) were obtained.
  • COST-CTL hepatitis B liver cancer killer T cells
  • a COST-DC-CTL cell preparation for treating hepatitis B liver cancer is prepared by the following method: the hepatitis B liver cancer killer T cell obtained as above, and the DC vaccine of the antigen signal carrying the multiple specific hepatitis B virus-associated liver cancer prepared as above The mixture was mixed and the mixture was transferred to physiological saline containing 1% human albumin to prepare a COST-DC-CTL cell preparation for treating hepatitis B liver cancer.
  • a COST-DC-CTL cell preparation for treating hepatitis B liver cancer wherein the DC vaccine of the hepatitis B liver cancer killer T cells obtained above and the antigen signal carrying the multiple specific hepatitis B virus-associated liver cancer is in a ratio of 8 to 18:1 cells ( That is, 8 to 18 COST-CTL cells were mixed with one COST-DC cell to obtain "multi-weapon combination antigen presenting signal group DC vaccine-induced specific tumor killer T cells (COST-DC-CTL)", and moved in. 100 ml of physiological saline containing 1% human albumin was prepared as a cell preparation for treatment.
  • COST-DC-CTL multi-weapon combination antigen presenting signal group DC vaccine-induced specific tumor killer T cells
  • the ratio of the mixed cells of the hepatitis B killer T cells to the DC vaccine carrying the antigenic signal of the multiple specific hepatitis B virus-associated liver cancer is 10:1.
  • the specific COST-DC-CTL cell preparation prepared by the invention is used in the treatment of liver cancer patients, and the specific method of use is directly into the tumor through perfusion of the hepatic artery or through the peripheral vein.
  • the patient's tumor microenvironment can be improved 2 days before the reinfusion by intravenous infusion of cyclophosphamide (CY) 250 mg/m 2 ;
  • Cellular reinfusion can be administered systemically by intravenous infusion, or localization of the hepatic artery into the patient.
  • the invention provides a method for treating hepatitis B virus-associated liver cancer, specifically for enhancing HBV-HCC specific immunity: 1 in vitro multiple antigen loading DC: inducing peripheral blood monocyte-derived dendritic cells (moDC), using two groups HBV antigen peptide (HBcAg and PreS2) and human hepatoma cell line (HepG2) lysate (containing various liver cancer antigens: AFP, GPC3, PCA) co-stimulated moDC, and activated activated DC cells were induced to induce maturation.
  • moDC peripheral blood monocyte-derived dendritic cells
  • HepG2 human hepatoma cell line
  • the present invention has the following beneficial effects:
  • the present invention is the first to invent a group of antigen presenting signal groups (COST) of hepatitis B virus antigen peptide combined with liver cancer cell antigen information.
  • COST antigen presenting signal groups
  • DC cells induced by this signal group become DC vaccines (COST-DC) carrying antigenic signals of multi-specific hepatitis B virus-associated liver cancer, which can activate the proliferation and function of tumor-specific T cells, thereby enhancing their resistance to hepatitis B liver cancer. effect.
  • the upper panel in Figure 1 is the mature DC cells (flow cytometry results); the lower panel shows the DC mature marker expression rate.
  • Figure 2 shows that mature DCs can simultaneously express multiple liver cancer protein antigens and hepatitis B virus genes.
  • Figure 3 shows ELISPOT detection of T cell culture in vitro to expand and secrete high levels of cytokines.
  • Figure 4 shows the inhibitory effect of cyclophosphamide (CY) on Trges.
  • Figure 5 shows DC cells in which normal DC and peripheral blood derived mature DCs activate T cells more than cancer tissue sources.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • the medium contained IL-4 (interleukin 4) at a concentration of 1000 IU/ml and GM-CSF (colony stimulating factor) at a concentration of 1000 IU/ml.
  • IL-4 interleukin 4
  • GM-CSF colony stimulating factor
  • peripheral blood mononuclear cells using mcs+ portable blood cell harvesting machine, according to the procedure set in the manual, peripheral blood circulation 2000 ⁇ 2500ml, collecting peripheral blood mononuclear cells 50ml, 5810R centrifuge 500G centrifugation for 20 minutes, collecting upper plasma Dispense into a 1.5 ml centrifuge tube and freeze as a routine biochemical assay.
  • the lower cells were diluted 1:1 with physiological saline, transferred to a 50 ml centrifuge tube containing 25 ml of lymphocyte separation solution, and centrifuged for 30 minutes using a 5810R centrifuge at 450 G.
  • the intermediate white cell layer was carefully aspirated by a disposable pipette and transferred to a 50 ml centrifuge containing 40 ml of physiological saline. Tube, gently pipet evenly, centrifuge at 250G for 15 minutes, discard the supernatant, add 45ml of normal saline, gently pipette evenly, centrifuge at 250G for 15 minutes, discard the supernatant; repeat the washing 3 times, add AIM-V medium, and count the cells.
  • T25 flasks were separately added to 4 ml of AIM-V medium, and the isolated monocytes were evenly dispensed into culture flasks. The cell concentration was controlled at 2 ⁇ 10 6 cells/ml, 37 ° C, 5%. Incubate for 120 min in a CO 2 saturated humidity incubator. The suspended cells are T cells, and the remaining adherent cells are monocytes (immature DC).
  • Monocyte-induced dendritic cell (moDC) maturation culture 4 ml of AIM-V medium containing 1000 IU/ml of IL-4 and a concentration of 1000 IU/ml was added to the adherent cells. GM-CSF; 3 days later, 2 ml of AIM-V medium was supplemented, and cultured on day 6 at 37 ° C in a 50% CO 2 incubator to mature into monocyte-induced dendritic cells (moDC). See Figure 1: Top panel: Flow cytometry to confirm DC maturation markers; bottom panel: Confirmation that DC successfully induced and expressed multiple epitopes.
  • T cell expansion culture the suspension cells, ie, T cells, were transferred to a T75 flask containing 4 ml of AIM-V culture medium, and washed 3 times with AIM-V medium, and the amount of washing liquid was 2 ml/time; Then add OKT3 at a final concentration of 1 ⁇ g/ml and anti-CD28 at a final concentration of 1 ⁇ g/ml, and incubate in a 37 ° C, 50% CO 2 incubator; Day 2: T cells supplemented with AIM-V medium 5- 10 ml, the culture solution contains IL-2 at a concentration of 300 IU/ml and 1.5 ml of albumin at a mass concentration of 20%; on the fourth day: T cells are supplemented with 5-10 ml of AIM-V culture solution, and the culture solution contains 300 IU. /ml of IL-2 and 1.5 ml of albumin having a mass concentration of 20% were continuously cultured in a 37 ° C, 50% CO 2 incubator until day 8,
  • hepatitis B core antigen (HBcAg) 18-27 peptide (amino acid sequence: FLPSDFFPSV) and 1 mg of hepatitis B surface pre-S2 antigen (HBPreS2) peptide 44-53 (amino acid sequence: SILSKTGDPV Australia,) were taken from a -80 ° C low temperature refrigerator.
  • Mimotopes Pty Ltd powder, high-speed centrifugation to the bottom of the tube, 25ul DMSO per tube, diluted to 40mg / ml of preservation solution, stored in -80 ° C refrigerator, which is HBcAg stimulating stock solution and HBPreS2 stimulating stock solution.
  • HepG2 cell line culture HepG2 cell line HepG2 was purchased from the Shanghai Cell Bank of Chinese Academy of Sciences, and cultured in 10% FBS DMEM in a 37 ° C, 5% CO 2 cell culture incubator to observe cell growth to the area of the culture flask. The next experiment can be carried out at around 90%.
  • Protein concentration of cell protein lysate by BCA method The protein standard solution (2 mg/ml) was diluted with PBS to a concentration of 0, 125, 250, 500, 1000, 2000 ng/ul. According to the number of samples to be tested, calculate the total amount of the test solution. The sample to be tested should pay attention to setting the double hole, and prepare the test solution in an accurate ratio: A liquid 1X, B liquid 50X. Add the assay solution to a 96-well plate at 200 ul per well. The protein quantification standard and the protein sample solution were then separately added to the wells at 10 ul per well. The added 96-well plate was placed in an incubator at 37 ° C for 30 minutes and then detected in a microplate reader.
  • COST multi-weapon combination antigen presenting signal group
  • DC vaccine (COST-DC) preparation with multi-weapon combination antigen presenting signal group The above COST was added to the matured moDC cells on the 6th day, the final concentration was 24ug/ml, and the next day was supplemented with AIM-V. The culture medium was cultured on the 8th day after 2 days of culture, and the COST-DC vaccine was obtained. The expression of CD83, CD86 and HLA-DR was detected by flow cytometry, and some were used for the preparation of specific killer T cells (CTL) of hepatitis B liver cancer. Nitrogen is stored frozen.
  • CTL specific killer T cells
  • Patients with liver cancer in any state can be used, and the course of treatment is individualized, including:
  • TACE chemoembolization
  • Prohibited disease patients with autoimmune diseases
  • a single dose of cyclophosphamide can be administered intravenously 2 days before the treatment of the immune cells in an amount of 250 mg/m 2 .
  • the role is to improve the internal environment of the tumor, especially the level of Tregs, which helps COST-DC-CTL to exert optimal antiviral efficacy.
  • Figure 4 Optimization of the optimal dose of cyclophosphamide (CY) is effective in reducing the inhibitory factors of T cell activation.
  • the COST-DC-CTL cell preparation can be administered locally to the hepatic artery; otherwise, intravenous reinfusion is given. Routine examination before and after immunotherapy: blood routine, biochemistry, liver and kidney function; and observation and recording of vital signs.

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Abstract

一种乙肝病毒(HBV)抗原肽联合肝癌细胞抗原信息的抗原提呈信号群及其应用，信号群由HBV核心抗原18-27肽段、HBV前S2抗原44-53肽段和人肝癌细胞株HepG2裂解液组成，HBV核心抗原18-27肽段、HBV前S2抗原44-53肽段和人肝癌细胞株HepG2裂解液三者的质量比为0.5～1：0.5～1：1～2。通过该信号群所诱导的DC细胞成为携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗，可激活肿瘤特异性T细胞的增殖和功能，从而加强其抗击乙肝肝癌的作用。

Description

一种乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群及其应用 技术领域
本发明涉及乙肝病毒相关性肝癌的特异性细胞免疫治疗技术领域，具体地，涉及一种乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群及其应用，具体应用为活化树突状细胞诱导T细胞的应用。
背景技术
与全球癌症总体死亡率持续下降成鲜明对比，肝癌发病率和死亡率逐年上升，最新流行病学数据显示，肝癌年发病为78.2万人，死亡病例74.5万，平均5年生存率低于5％，总体生存时间只有10个月；我国占了全球肝癌55％，成为肝癌发病和死亡人数最多的国家，其中85％以上患者有HBV感染病史，几乎所有癌细胞都含有HBV基因。然而，治疗手段在20年进步不大，常用方法有手术(肿瘤切除和移植)和局部治疗(射频、消融、微波、冷冻、无水酒精注射、肝动脉化疗栓塞，TACE)，50～60％患者初诊时已经到了中晚期，没有手术机会，局部治疗疗效与无治疗患者比较2年生存率最多增加14％，且有一定的毒副作用，严重者引发肝衰竭，因此，肝癌成为我国沉重的经济负担和人民健康杀手。
传统抗癌药在肿瘤耐药复发和进展时基本无效，且细胞毒性大，抗肿瘤免疫治疗能够在一定程度避免该问题，其中肿瘤特异性T细胞最具潜力，T细胞能通过在血液中“游走巡查”找到肿瘤进行攻击，近期研究证实，癌症T细胞免疫疗法能持续缓解某些血液肿瘤改善总生存率；然而，肿瘤内T细胞低含量制约着免疫疗法的成功扩展。肿瘤免疫学核心理论指出，担负人体最大抗原递呈功能的树突状细胞(DCs)是T细胞活化和增殖的关键启动者，在T细胞成熟、分化、增殖各阶段发挥重要作用，活化DC能有效激活T细胞的抗肿瘤免疫效应，因此，通过恢复DC对T细胞功能有增强作用。近年来一些临床研究利用疫苗治疗中晚期肿瘤，包括体外DC负载肿瘤抗原进行回输或诱导肿瘤内DC以递呈内源性抗原，提示改善抗原递呈促进功能性T细胞具有一定潜力和前景。
根据慢性HBV感染的核心理论为机体对病毒的免疫耐受以及乙肝肝癌中存在大量的淋巴细胞浸润，提示控制肿瘤进展和攻击/消灭癌细胞依赖于数量足够功能强大的特异性T细胞，通过产生或增强免疫反应治疗乙肝病毒相关性肝癌(HBV-HCC)拥有巨大的潜能。目前围绕某些癌症进行免疫治疗的临床研究疗效初步呈现，但由于肿瘤细胞和免疫细胞相互作用、彼 此制衡十分明显，诱导产生的特异性T细胞数量和功能不全，未能较好地在实体肿瘤中发挥抑制作用。其中主要的技术难点在于：(1)HBV-HCC带有高度异质性的肿瘤抗原，具有多样性、复杂性、变异性，能帮助癌细胞轻易逃避免疫系统的监测；(2)HBV-HCC内部形成的免疫抑制性微环境，使免疫细胞难以攻破其“保护网”。
发明内容
本发明为了解决目前免疫治疗以肝癌为代表的实体肿瘤疗效欠佳的主要技术难点，包括实体肿瘤所具有的高度异质性、多样性、复杂性、变异性的抗原特点，提供一组乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群(COST)。通过该信号群所诱导的DC细胞成为携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗(COST-DC)，可激活肿瘤特异性T细胞的增殖和功能，从而加强其抗击乙肝肝癌的作用。
本发明的另一个目的是提供一种携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗。
本发明的再一个目的是提供一种乙肝肝癌杀伤性T细胞。
本发明的再一个目的是提供一种治疗乙肝肝癌的COST-DC-CTL细胞制剂。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
一组乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群，由HBV核心抗原18-27肽段、HBV前S2抗原44-53肽段和人肝癌细胞株HepG2裂解液组成，HBV核心抗原18-27肽段、HBV前S2抗原44-53肽段和人肝癌细胞株HepG2裂解液三者的质量比为0.5～1：0.5～1：1～2。
HBV核心抗原18-27肽段简称为HBcAg，其氨基酸序列为FLPSDFFPSV，HBV前S2抗原44-53肽段简称为HBPreS2，其氨基酸序列为SILSKTGDPV，人肝癌细胞株HepG2裂解液简称为HepG2-Lys。乙肝病毒相关性肝癌也可以称为乙肝肝癌。
一种携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗，具体为将如上所述的乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群，加入到从外周血分离、培养的成熟moDC细胞中，培养即可获得疫苗。
一种携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗，由以下方法制备得到：将如上所述的乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群(COST)加入用AIM-V培养液培养到第6天成熟的moDC细胞中，COST的终浓度为24ug/ml，次日补充AIM-V培养液，培养2天后回收获取疫苗。
一种乙肝肝癌杀伤性T细胞，具体步骤为：将如上制备的携带多重特异性乙肝病毒相关 性肝癌的抗原信号的DC疫苗，与从外周血分离获得的T细胞过继培养，即可获得乙肝肝癌杀伤性T细胞(COST-CTL)。
一种乙肝肝癌杀伤性T细胞，具体步骤为：取细胞数量为2～5x106的携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗，与从外周血分离、扩增后第8天的T细胞过继培养，T细胞数量为2～5x107，在37℃、5％CO2培养箱中共培养2天，即第10天；补充AIM-V培养液5～10ml，该培养液含浓度为300IU/ml的IL-2以及质量体积浓度为20％的白蛋白1.5ml；第12天：补充AIM-V培养液5～10ml，该培养液含浓度为300IU/ml的IL-2以及质量体积浓度为20％白蛋白1.5ml；第14天：获得病毒肿瘤抗原共活化DC诱导特异性T细胞，即获得乙肝肝癌杀伤性T细胞(COST-CTL)。
一种治疗乙肝肝癌的COST-DC-CTL细胞制剂，由以下方法制备得到：将如上获得的乙肝肝癌杀伤性T细胞，与如上制备的携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗混合，混合物移入含有1％人血白蛋白的生理盐水中即可制得治疗乙肝肝癌的COST-DC-CTL细胞制剂。
一种治疗乙肝肝癌的COST-DC-CTL细胞制剂，将如上获得的乙肝肝癌杀伤性T细胞与携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗以8～18:1细胞数量比(即8～18个COST-CTL细胞配上1个COST-DC细胞)混合，得到“多武器组合抗原提呈信号群DC疫苗诱导特异性肿瘤杀伤性T细胞(COST-DC-CTL)”，移入含有1％人血白蛋白的生理盐水100ml，制备成用于治疗的细胞制剂。
更优选地，所述乙肝肝癌杀伤性T细胞与携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗的混合细胞数量比为10：1。
本发明制备的特异性COST-DC-CTL细胞制剂，在治疗肝癌病人时，其具体的使用方法是通过肝动脉途径灌注直接进入肿瘤内，或者通过外周静脉途径回输。
为了使COST-DC-CTL细胞制剂的治疗效果发挥最好，在回输前2天可以对患者肿瘤微环境进行改良，方法是用环磷酰胺(CY)250mg/m2静脉滴注1次；细胞回输可通过静脉输注的全身给予，或肝动脉局部输入患者体内。
本发明提供一种治疗乙肝病毒相关性肝癌的方案，具体为加强HBV-HCC特异性免疫：①体外多重抗原负载DC：诱导外周血单核细胞衍生树突状细胞(moDC)后，利用两组HBV抗原肽(HBcAg和PreS2)和人肝癌细胞株(HepG2)裂解液(含多种肝癌抗原：AFP、GPC3、PCA)共刺激moDC，获得活化的定向诱导成熟的DC细胞。②体内自身肿瘤抗原刺激DC：手术或局部治疗可促进患者自身肿瘤抗原释放，提供DC更多特异性免疫源；治疗产生DNA损伤和炎症扩大了抗原递呈。③联合环磷酰氨(cyclophosphamide，CY)治疗可改善肿瘤免 疫抑制性内环境。
与现有技术相比，本发明具有如下有益效果：
本发明首次发明一组乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群(COST)。通过该信号群所诱导的DC细胞成为携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗(COST-DC)，可激活肿瘤特异性T细胞的增殖和功能，从而加强其抗击乙肝肝癌的作用。
附图说明
图1中上面的图为成熟DC细胞(流式细胞检测结果)；下面的图为DC成熟标记表达率。
图2为成熟DCs可同时表达多种肝癌蛋白抗原和乙肝病毒基因。
图3为ELISPOT检测T细胞体外培养扩增和分泌高水平细胞因子。
图4为环磷酰氨(CY)对Trges的抑制作用。
图5为正常肝和外周血来源成熟DC激活T细胞效应高于癌组织来源的DC细胞。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
AIM-V培养基的配置：该培养基内含浓度为1000IU/ml的IL-4(白介素4)和浓度为1000IU/ml的GM-CSF(集落刺激因子)。
实施例1
1、外周血DC和T细胞分离及扩增培养。
(1)获取外周血单核细胞：应用mcs+便携式血细胞采集机，按照说明书设定的程序操作，外周血循环2000～2500ml，采集外周血单个核细胞50ml，5810R离心机500G离心20分钟，收集上层血浆分装至1.5ml离心管，冻存作为常规生化检测。下层细胞用生理盐水1：1稀释后，移入含有25ml淋巴细胞分离液的50ml离心管，使用5810R离心机450G离心30分钟，一次性吸管小心吸取中间白色细胞层，移入含有40ml生理盐水的50ml离心管，轻柔吹打均匀，250G离心15分钟，弃上清，加入生理盐水45ml轻柔吹打均匀，250G离心15分钟，弃上清；重复洗涤3次后，加入AIM-V培养基，细胞计数。
(2)DC和T细胞分离：T25培养瓶分别加入AIM-V培养基4ml，分离后的单核细胞平均分装到培养瓶中，细胞浓度控制在2x106个/ml，37℃、5％CO2饱和湿度培养箱中孵育120min，悬浮的细胞即为T细胞，剩余的贴壁细胞即单核细胞(未成熟DC)。
(3)单核细胞诱导树突状细胞(moDC)成熟培养：在贴壁细胞中加入4ml AIM-V培养基，该培养基内含浓度为1000IU/ml的IL-4和浓度为1000IU/ml的GM-CSF；3天后，补充AIM-V培养液2ml，置于37℃、50％CO2培养箱中培养至第6天，使其成熟为单核细胞诱导树突状细胞(moDC)。参见附图1:上图：流式细胞检测技术证实DC成熟标记；下图：证实DC成功诱导并表达多种表位。
(4)T细胞扩增培养：把悬浮细胞，即T细胞，移至含4ml的AIM-V培养液的T75培养瓶，以AIM-V培养基洗涤3次，洗涤液量为2ml/次；然后加入终浓度为1μg/ml的OKT3和终浓度为1μg/ml的抗-CD28，置于37℃、50％CO2培养箱中培养；第2天：T细胞补充AIM-V培养液5-10ml，该培养液含浓度为300IU/ml的IL-2以及质量体积浓度为20％的白蛋白1.5ml；第4天：T细胞补充AIM-V培养液5-10ml，该培养液内含300IU/ml的IL-2以及质量体积浓度为20％的白蛋白1.5ml，继续在37℃、50％CO2培养箱中培养至第8天，获得增殖的T细胞。
2、双重乙肝病毒抗原肽共刺激原液的制备，所有操作在生物安全柜中进行。
(1)从-80℃低温冰箱取出1mg乙肝核心抗原(HBcAg)18-27肽段(氨基酸序列：FLPSDFFPSV)和1mg乙肝表面前S2抗原(HBPreS2)肽段44-53(氨基酸序列：SILSKTGDPV Australia,Mimotopes Pty Ltd)粉剂，高速离心将粉末甩至管底，每管加入25ul DMSO，稀释为40mg/ml的保存液，于-80℃冰箱中保存，即为HBcAg刺激原液和HBPreS2刺激原液。
(2)取出HBcAg刺激原液1.5ul和HBPreS2刺激原液1.5ul，混合，成为HBV共刺激原液；补充AIM-V至5ml，肽段工作浓度为24ug/ml，分装，4℃保存。
3、多重肝癌抗原肽刺激原液的制备，所有操作在生物安全柜中进行。
(1)HepG2细胞株培养：肝癌细胞株HepG2购于中国科学院上海细胞库，以10％FBS的DMEM于37℃、5％CO2的细胞培养箱中培养，观察细胞生长到占培养瓶面积的90％左右即可进行下一步实验。
(2)蛋白裂解液的配制：冰上操作，以一瓶25cm2的培养瓶计算，吸取250ul RIPA于1.5ML离心管中，并加入蛋白酶抑制剂cocktail，混合均匀，使cocktail的终浓度为0.4mg/ml。
(3)裂解细胞，提取细胞蛋白裂解液：倒掉培养瓶内的细胞上清，用预冷的HBSS清洗两次，吸干液体后加入250ul蛋白裂解液，于冰上平置15分钟。用枪头将细胞刮除，收集细胞蛋白裂解液于一无菌的1.5ML离心管中。将离心管置于预冷好的冷冻离心机中，配平，以 4℃、14000RPM离心15分钟，取出后可见管底有白色蛋白残渣沉淀。取上清到另一无菌的1.5ML离心管中，放于冰上静置。
(4)BCA法测细胞蛋白裂解液的蛋白浓度：取蛋白定量标准液(2mg/ml)用PBS逐级稀释其浓度为0、125、250、500、1000、2000ng/ul。根据要测的样品数，计算好检测液的总用量，对待测的样品要注意设置复孔，并以准确的比例配制好检测液：A液1X，B液50X。添加检测液于96孔板中，每孔200ul。然后将蛋白定量标准液和蛋白样品液分别加进孔中，每孔10ul。将添加好后的96孔板放于37℃的恒温箱中放置30分钟，然后在酶标仪中检测。
(5)蛋白样品的稀释与过滤：用配制好的蛋白裂解液将原液稀释，用5ML的注射器将蛋白裂解液抽吸出来，并用0.22um的滤器过滤，使终浓度为24ug/ml(HepG2-Lys)，分装，4℃保存。
4、多武器组合抗原提呈信号群(COST)的配置及COST-DC疫苗的制备，所有操作在生物安全柜中进行。
(1)多武器组合抗原提呈信号群(COST)配置：取HBcAg抗原肽原液和HBPreS2抗原肽原液，从原液浓度为40mg/ml稀释为工作浓度24ug/ml；从步骤4获得的HepG2-Lys原液，从原液浓度为100mg/ml稀释为24ug/ml；COST配方为：HBcAg+HBPreS2+HepG2-Lys；配伍为：HBcAg:PreS2:HepG2-Lys＝0.5-1:0.5-1:1-2；工作浓度为24ug/ml。
(2)携带多武器组合抗原提呈信号群的DC疫苗(COST-DC)制备：把上述COST加入培养到第6天成熟的moDC细胞中，终浓度为24ug/ml，次日补充AIM-V培养液，培养2天后于第8天回收，获取COST-DC疫苗，流式细胞仪检测CD83、CD86、HLA-DR表达，部分用于乙肝肝癌特异性杀伤性T细胞(CTL)制备，部分液氮冻存。图2:通过COST诱导成熟的DC，可成功同时携带乙肝病毒抗原和肝癌抗原。
5、乙肝肝癌杀伤性T细胞的制备，所有操作在生物安全柜中进行：取COST-DC疫苗，细胞数量为2～5x106与扩增后第8天的T细胞，T细胞数量为2～5x107过继培养，在37℃、5％CO2培养箱中共培养2天，即第10天；补充AIM-V培养液5～10ml，该培养液含浓度为300IU/ml的IL-2以及质量体积浓度为20％的白蛋白1.5ml；第12天：补充AIM-V培养液5～10ml，该培养液含浓度为300IU/ml的IL-2以及质量体积浓度为20％白蛋白1.5ml；第14天：获得病毒肿瘤抗原共活化DC诱导特异性T细胞，即获得可应对肝癌细胞的多克隆T细胞(COST-CTL)。图3：制备成功的特异性肝癌细胞的多克隆T细胞可诱导抗肿瘤免疫因子IFN-r和针对癌细胞的CD8+T细胞。
6、COST-DC-CTL混合细胞制剂的配置：第13天，从液氮取出COST-DC疫苗0.5～1x107复苏，洗涤去掉细胞碎片，培养1天后，细胞数量控制在2x106；再和步骤5获得的数量为 0.5～1x108COST-CTL混合，细胞总数量控制在1～2x108；加入50ml离心管，生理盐水洗涤3～6次后，重悬于10ml的生理盐水中，同时加入20％白蛋白1.5ml以防止离子间的黏附，然后以注射器加入100ml生理盐水中，成为治疗用的COST-DC-CTL细胞制剂，在4℃中保存备用。图5:T细胞功能验证：肝癌患者外周血和正常肝组织来源中DC细胞经过COST活化后诱导出T细胞功能显著高于癌组织来源的DC细胞诱导的T细胞功能。
COST-DC-CTL细胞制剂的治疗适应症和禁忌症如下：
适用人群：
(1)任何状态下的肝癌患者都可以使用，方案疗程具有个体化，包括：
(2)初次诊断为早期小肝癌患者，可进行肿瘤切除手术的患者，我们在术后制定个体化特异性免疫细胞疗程，可预防肿瘤复发和转移。
(3)初次诊断已经为中晚期肝癌患者，无法进行手术切除。
(4)已经出现复发或/和转移的患者。
(5)局部消融术后患者。
(6)化疗栓塞(TACE)患者。
禁止症：伴有自身免疫性疾病患者
为了使COST-DC-CTL细胞制剂的治疗效果发挥最好，可以在免疫细胞治疗前2天，给予单一剂量环磷酰胺静脉滴注，用量为250mg/m2。作用是改善肿瘤内环境，尤其是Tregs的水平，有助于COST-DC-CTL发挥最佳的抗病毒效能。图4:优化出最佳剂量的环磷酰胺(CY)，可有效降低T细胞活化的抑制性因素。若病人无法手术切除病灶，但能够使用肝动脉化疗栓塞治疗，COST-DC-CTL细胞制剂可以肝动脉局部给予；否则，给予静脉回输。免疫细胞治疗前后常规检查：血常规、生化、肝肾功能；以及生命体征的观察和记录。
综上所述以上实施例不过是本发明的最佳实施方案，不可理解为对本发明保护范围的限定，对于该领域的技术工作人员根据本发明的实施例所做的不超出本发明技术方案的调整和改动，应认为落在本发明的保护范围内。

Claims (6)

  1. 一种乙肝病毒抗原肽库联合肝癌细胞抗原信息的抗原提呈信号群，其特征在于，由HBV核心抗原18-27肽段、HBV前S2抗原44-53肽段和人肝癌细胞株HepG2裂解液组成，HBV核心抗原18-27肽段、HBV前S2抗原44-53肽段和人肝癌细胞株HepG2裂解液三者的质量比为0.5～1：0.5～1：1～2。
  2. 一种携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗，其特征在于，将权利要求1所述的乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群加入到成熟的moDC细胞中，培养即可获得疫苗。
  3. 根据权利要求2所述的DC疫苗，其特征在于，由以下方法制备得到：将如上所述的乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群加入用AIM-V培养液培养到第6天成熟的moDC细胞中，终浓度为24ug/ml，次日补充AIM-V培养液，培养2天后回收获取疫苗。
  4. 一种乙肝肝癌杀伤性T细胞，其特征在于，由以下方法制备得到：将权利要求2或3制备的携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗，与从外周血分离获得的T细胞过继培养，即可获得乙肝肝癌杀伤性T细胞。
  5. 一种治疗乙肝肝癌的COST-DC-CTL细胞制剂，其特征在于，由以下方法制备得到：将权利要求4获得的乙肝肝癌杀伤性T细胞，与权利要求2或3制备的携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗混合，混合物移入含有1％人血白蛋白的生理盐水中即可制得治疗乙肝肝癌的COST-DC-CTL细胞制剂。
  6. 根据权利要求5所述的治疗乙肝肝癌的COST-DC-CTL细胞制剂，其特征在于，乙肝肝癌杀伤性T细胞与携带多重特异性乙肝病毒相关性肝癌的抗原信号的DC疫苗的细胞数量比为8～18:1。
PCT/CN2017/096512 2017-04-14 2017-08-08 一种乙肝病毒抗原肽联合肝癌细胞抗原信息的抗原提呈信号群及其应用 WO2018188235A1 (zh)

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