WO2017161950A1 - 皮氨钙联合佐剂及含有皮氨钙联合佐剂的疫苗 - Google Patents

皮氨钙联合佐剂及含有皮氨钙联合佐剂的疫苗 Download PDF

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WO2017161950A1
WO2017161950A1 PCT/CN2017/000208 CN2017000208W WO2017161950A1 WO 2017161950 A1 WO2017161950 A1 WO 2017161950A1 CN 2017000208 W CN2017000208 W CN 2017000208W WO 2017161950 A1 WO2017161950 A1 WO 2017161950A1
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adjuvant
calcium
combination
combined
cells
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林海祥
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林海祥
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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response

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  • the present invention relates to the field of medical immunology, and in particular to vaccine adjuvants and vaccines containing the same.
  • cross-linking agent such as polyethyleneimine or polyethylene glycol
  • other immunostimulating agents sodium arginine combined adjuvant
  • the adjuvant promotes non-specific immunity and specific immunity of the body.
  • the combination of arginine and calcium adjuvant can not only further improve the immunogenicity of the antigen, but also further transform the immune type, and convert the preventive vaccine into a therapeutic vaccine to further improve the therapeutic vaccine against various intracellular conditions under safe conditions. Infection (bacteria, intracellular infection of bacteria, parasites, etc.), anti-tumor effect.
  • the various components of the arginine combined adjuvant are compounded by chemical bonds such as hydrogen bonds, and combined with other immune promoters, the synergistic stimulation of the body's immune system through different mechanisms can further enhance the effect of immune healing.
  • Toll-like receptor agonists include a prophylactic or therapeutic, human or veterinary vaccine with further anti-infection against various intracellular infections (bacteria, parasite
  • the effect also includes an anti-tumor vesicle vaccine combined with a calcium sulphate adjuvant.
  • the arginine combined adjuvant itself has an immunostimulating effect and can be non-specifically resistant to various intracellular infections (bacteria, parasites, etc. of viruses and intracellular infections) and to various tumors, and can be separately prepared into a solid, half. Solid, liquid and gaseous dosage forms such as water injections, sprays and various ointments for anti-tumor and anti-infective applications.
  • FCA complete Freund's adjuvant
  • MPL immunomodulator
  • MDP immunomodulator
  • QuiA immunomodulatory molecules
  • MPL immunomodulatory molecules
  • QS21 immunomodulatory molecules
  • the combination of CPG and aluminum adjuvant has a synergistic effect to promote humoral immunity of recombinant hepatitis C antigen, and the aluminum adjuvant acts as a delivery system for CPG to fully exert the high-efficiency immune effect of CPG.
  • the formulation of the adjuvant should be to use the adjuvant in combination with the immunomodulator and the carrier to optimize the immunological activity, and select different immunostimulating agents according to the mechanism of the disease and the mechanism of action of the adjuvant to enhance the body's needs.
  • Immune response The immune promoter contains a variety of substances, and promotes the body's immune response through different mechanisms and channels. The adjuvant effect is more pronounced.
  • TLR receptor agonists are potent vaccine adjuvants, 13 species of Toll-like receptors are found in mice, and 11 species are found in humans.
  • Human Toll-like receptors 1, 2, 4, 5, and 6 recognize microbial components that are extracellular and expressed on the cell surface, such as Toll-like receptors 2, 4, which recognize bacterial lipoproteins and lipopolysaccharides.
  • Toll-like receptors 3, 7, 8, and 9 recognize nucleic acids of viruses or bacteria and are in endosomes and lysosomes.
  • Toll-like receptors can recognize a variety of different microbial components, and through their important signaling, first cause a non-specific immune response, and then launch a specific immune response, producing cytokines and chemokines to differentiate the initial T cells.
  • Toll-like receptors also produces inflammatory tissues that activate nuclear factor KB (NF-kB) and interferon regulatory factors in B cells, and induce immune, inflammatory genes to release inflammatory chemokines and type 1 interferons ( IFN1).
  • Toll-like receptor signaling is through both pathways dependent on and independent of myeloid differentiation factor (MyD88).
  • TLR1, 2, 4, 5, 6, 7, 8, 9 are myeloid-dependent differentiation factor (MyD88) pathway;
  • TLR3, 4 are non-dependent myeloid differentiation factor (MyD88) pathway or TRIF (containing inducible interferon-beta linker) Toll-like receptor region) pathway.
  • TIR Toll/Interleukin-1 receptor domain containing a linker protein, such as MyD88, TRIF, TIRAP (Toll/Interleukin-1 receptor containing a linker protein), and downstream of TRAM (related to a TRIF linker molecule) Signaling plays a leading role in inducing specific immunity.
  • Adjuvants or immunostimulants that use only a single target for some vaccines do not induce an effective protective immune response. Based on this, a large number of TLR receptor agonists as described above are being used in combination with other kinds of immunostimulating agents in preclinical and clinical trials.
  • the depletion of T cells and immunosuppression of tumors are removed by adding PD-1/PD-L1 antibody, and CTLA4 such as activated T cells is added to promote the transfection of cells.
  • Agents such as PEI (polyethyleneimine), PEG (polyethylene glycol) and other combined adjuvants can achieve the target.
  • PEI polyethyleneimine
  • PEG polyethylene glycol
  • An amino compound containing polyinosinic acid-polycytidine (poly-cell), calcium ion and amino-based antibiotic or grafting with a crosslinking agent such as polyethyleneimine or polyethylene glycol The preparation of the immunoadjuvant in the preparation of the immunoadjuvant alone or in combination with other immunostimulating agents synergistically enhances the body's immunostimulating substance, dermal calcium sulphate combined adjuvant, as an application in the preparation of an immunoadjuvant.
  • a vaccine comprising a combination of a calcium sulphate adjuvant according to claim 1. It is characterized in that the adjuvant is a combination containing a calcium sulphate adjuvant, and the vaccine includes various anti-intracellular infections (bacteria, parasites, etc. of viruses and intracellular infections) and various anti-tumor vaccines.
  • Exogenous immune stem cells stimulated with an immunostimulating agent containing a sulphate adjuvant according to claim 1, such as in vitro stimulation of proliferation and maturation of DC-CIK cells and CAR-T cells and T cells and reinfusion into the body to enhance anti-tumor The application of anti-intracellular infection immunity. It is characterized in that the immunostimulant contains a calcium sulphate combined adjuvant.
  • the action mechanism of the vaccine of the present invention and the vaccine containing leucocalcium combined adjuvant and the further immune effect of the vaccine using the sulphate adjuvant and the vaccine thereof will be described below with reference to the examples, and the present invention is not limited to these examples.
  • the combination of arginine calcium and adjuvants and their vaccines helps to understand.
  • sulphate adjuvant is a double-stranded RNA adjuvant and is an agonist of TLR3 receptor. Because non-antibiotic amino compounds replace kanamycin, it is more than PICKCa. Safer and more effective.
  • CPG is a CPG oligonucleotide and is a ligand for TLR9. It is classified into A, B, and C types according to the nucleotide sequence and length, and activated B cells (lysosomal fractions) and plasma cell-like DCs of type A and C. IFN- ⁇ . Class B activates B cells (endosomal parts) and matures DCs.
  • spleen cells were stimulated with CPG2395 and lysine alone or in combination to detect cytokine secretion and cell proliferative ability; in vivo, CPG2395 and lysine were used to immunize mice alone or in combination as adjuvants for MUCI polypeptide antigens.
  • ELISA method to detect antibody titer in immune serum CTL killing assay to detect cytotoxicity of killer T cells, flow cytometry analysis of changes in cell subsets before and after immunization; in tumor prevention experiments, through tumor inhibition rate and lung
  • the number of metastatic nodules and other indicators were used to evaluate the effect of both as adjuvants on tumor prevention in MUCI-immunized mice.
  • TLR3 receptor ligand picocalcium phosphate and the TLR7/8 receptor ligand R848 are combined and combined with the carcinoembryonic antigen CEA526-533 peptide to promote dendritic cell (DC) maturation, and the use of calcium sulphate alone. Comparison can significantly enhance the anti-cancer effect.
  • the selective combination of ligands of TLR 2, 3, 4, 7, 8, and 9 can further induce dendritic cell (DC) maturation, resulting in a series of positive effects expected by tumor immunity.
  • DC dendritic cell
  • TLR-DC 20 ug/ml calcium sulphate and 1.5 ug/ml R848 were added on the last two days of culture.
  • TLR-DC+CEA 20ug/ml calcium sulphate and 1.5 ug/ml R848 were added on the last two days of culture, then 40 ug/ml peptide CEA526 was added. -533, placed in a cell culture incubator. After 3-4 hours, the excess unbound peptide was washed away to obtain a DC that was combined with TLR and loaded with CEA.
  • CEA526-533 peptide group DC Conventional culture load CEA526-533 peptide group DC (conventional DC+CEA): Add 40ug/ml peptide CEA526-533, place in cell culture incubator, take out after 3-4 hours, wash away excess unbound The peptide, that is, the DC loaded with CEA526-533.
  • the IL-12 secretion of DC in the combined TLR group was significantly higher than that in the conventional group, which were 948.67 ⁇ 104.36pg and 215.24 ⁇ 19.3spg, respectively, P ⁇ 0.01.
  • the effector cells obtained after co-culture with DC in the combined TLR group have a certain killing effect on the ratio of MC38-CEA intestinal cancer cells in the higher effector cell: target cell (E:T) ratio, which is significantly better than the conventional group ( P ⁇ 0.05).
  • mice in the DC vaccine group activated with TLR and loaded with CEA peptide were significantly smaller than the other groups (P ⁇ 0.01), and the survival period was Significantly better than the other groups (P ⁇ 0.01).
  • the DC vaccine in the combined TLR group can secrete more IL-12 than the conventional DC vaccine.
  • the DC vaccine-induced antigen-specific CTLs in the combined TLR group can efficiently produce a specific immune response against mouse MC38-CEA intestinal cancer cells.
  • the combined TLR group and CEA-loaded DC vaccine have significant anti-tumor effect on colon cancer-bearing mice and can prolong the survival of mice.
  • TLR3, RIG-1, MDA-5 and IFN- ⁇ Upregulation of expression of cellular receptors TLR3, RIG-1, MDA-5 and IFN- ⁇ :
  • the picocalcium adjuvant is a ligand for TLR3, RIG-1 and MDA-5.
  • the expression levels of TLR3, RIG-1, MDA-5 and IFN- ⁇ mRNA were detected by fluorescence quantitative PCR after 3 and 6 hours of treatment with PHA and transfection reagent PEI at appropriate ratios.
  • Calcium adjuvant and transfection reagent PEI group significantly up-regulated the expression of TLR3, RIG-1, MDA-5 and IFN- ⁇ mRNA, and at 6 hours, with other control groups (PBS group), separate skins of calcium sulphate adjuvant, alone The transfection reagent PEI group had significant difference P ⁇ 0.01.

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Abstract

一种含聚肌苷酸-聚胞苷酸、钙离子和除氨基类抗生素之外的氨基化合物或与聚乙烯亚胺、聚乙二醇等交联剂形成纳米颗粒单独或联合其他免疫促进剂在制备免疫佐剂中的应用。

Description

皮氨钙联合佐剂及含有皮氨钙联合佐剂的疫苗
本发明涉及医学免疫学领域，特别是涉及疫苗佐剂及含有该佐剂的疫苗。新佐剂皮氨钙联合佐剂的组成：聚肌胞-氯化钙-除氨基类抗生素之外的氨基化合物或与聚乙烯亚胺、聚乙二醇等交联剂形成接枝物纳米颗粒联合其他免疫促进剂(皮氨钙联合佐剂)作为在制备免疫佐剂中的应用，共同促进机体的非特异免疫和特异性免疫的佐剂。皮氨钙联合佐剂不仅能进一步提高抗原的免疫原性，更具有进一步转变免疫类型的作用，使预防性疫苗转变成治疗性疫苗，以在安全前提下进一步提高治疗性疫苗抗各种细胞内感染(病毒和细胞内感染的细菌、寄生虫等)、抗各种肿瘤的效果。皮氨钙联合佐剂中各种成分是通过氢键等化学键复合在一起，与其它免疫促进剂联合使用是通过不同机理协同刺激机体的免疫系统达到进一步提高免疫治愈疾病的效果。
联合其他免疫促进剂包括各种Toll样受体激动剂、矿物盐如铝佐剂、悬浮微粒清洁剂如MF59、ASO3、AFO3、乳化剂，皂化剂QS21，ISCOM，GLA-SE，颗粒释放剂如nanoparticles、PLGA、VLP、Liposomes，Virosomes，人细胞因子，趋化因子，活化配体，DC共刺激分子，IL-12，IL-15，GM-CSF，MCP-1，CD40L，DEC-205、细菌外毒素CT，LT(modified)以及PD1/PD-L1、TIM-3阻断性抗体，激活T细胞的CTLA4和促进细胞转染制剂如PEI、PEG等；皮氨钙联合佐剂的疫苗是指含有皮氨钙联合佐剂的疫苗包括预防用或治疗用，人用或兽用疫苗而具有进一步提高抗各种细胞内感染(病毒和细胞内感染的细菌、寄生虫等)抗各种肿瘤的效果，也包括皮氨钙联合佐剂抗肿瘤囊泡疫苗。皮氨钙联合佐剂本身即具有免疫促进作用能够非特异性地抗各种细胞内感染(病毒和细胞内感染的细菌、寄生虫等)和抗各种肿瘤的效果，能单独制备成固体、半固体、液体和气体剂型如水针剂、喷雾剂和各种膏剂在抗肿瘤抗感染中的应用。
为了获得理想的免疫应答联合佐剂应用的例子是很多的，如完全弗氏佐剂(FCA)就是将免疫调节剂结核杆菌与W/O乳剂联合应用导致Th1和Th2应答。鲨烯和MOD联合应用成功；脂质体可以和各种免疫调节分子MPL、MDP、QuiA联合；MPL和QS21联合。CPG和铝佐剂联合应用具有协同作用，促进重组丙型肝炎抗原的体液免疫，铝佐剂作为CPG的运载系统使CPG的高效免疫效应充分发挥。
佐剂的配方应该是将佐剂与免疫调节剂和运载体联合使用，使免疫活性达到最优，并根据疾病的发生机理和佐剂的作用机理选择不同的免疫促进剂联合应用以增强机体所需的免疫应答。免疫促进剂包含多种，通过不同机理和渠道促进机体的免疫反应，适当的联合应用使 佐剂效果更加明显。大量的临床前试验和临床试验数据表明TLR受体激动剂是强效的疫苗佐剂，Toll样受体在小白鼠中发现13种，在人体中发现11种。人Toll样受体1、2、4、5、6识别细胞外和表达在细胞表面的微生物成分，如：Toll样受体2、4识别细菌的脂蛋白和脂多糖。Toll样受体3、7、8、9识别病毒或细菌的核酸并在核内体和溶酶体。Toll样受体可以识别各种不同微生物成分，经过其重要的信号传导作用，首先引起非特异性免疫反应，继而发动特异性免疫反应，产生细胞因子和趋化因子使初始T细胞分化。Toll样受体与致病因子结合也产生炎性组织使B细胞核内因子kB(NF-kB)和干扰素调节因子活化，诱发免疫、炎性基因释放炎性趋化因子和1型干扰素(IFN1)。Toll样受体的信号传导通过依赖和非依赖髓样分化因子(MyD88)两种通路。TLR1、2、4、5、6、7、8、9是依赖髓样分化因子(MyD88)通路；TLR3、4是非依赖髓样分化因子(MyD88)通路或者TRIF(含有诱生干扰素-β接头的Toll样受体区域)通路。含有接头蛋白的TIR(Toll/白细胞介素-1受体)域，如：MyD88、TRIF、TIRAP(含有接头蛋白的Toll/白细胞介素-1受体)、TRAM(有关TRIF接头分子)的下游信号传导对诱生特异性免疫起着主导作用。
部分TLR及其配体
Figure PCTCN2017000208-appb-000001
Figure PCTCN2017000208-appb-000002
对一些疫苗只使用单一靶点的佐剂或免疫促进剂并不能诱导产生有效的保护性免疫反应。基于这一点，如上所述大量的TLR受体激动剂与其他种类的免疫促进剂联合使用正在进行临床前实验和临床试验。另外根据疾病机理和皮氨钙佐剂作用机理，加入如PD-1/PD-L1抗体去除对T细胞的耗竭和对肿瘤的免疫抑制，加入如激活T细胞的CTLA4以及促进细胞转染的促进剂如PEI(聚乙烯亚胺)、PEG(聚乙二醇)等组成联合佐剂，更能达到目标目的。为此本发明提供的技术方案是：
1.一种含聚肌苷酸-聚胞苷酸(聚肌胞)、钙离子和除氨基类抗生素之外的氨基化合物或与聚乙烯亚胺、聚乙二醇等交联剂形成接枝物在制备免疫佐剂中形成纳米颗粒单独或以其为核心联合其他免疫促进剂协同增强机体免疫刺激物质------皮氨钙联合佐剂作为在制备免疫佐剂中的应用。
2.根据权利1所述的一种含有皮氨钙联合佐剂的疫苗。其特征在于所述的佐剂是含有皮氨钙联合佐剂，所述的疫苗包括各种抗细胞内感染(病毒和细胞内感染的细菌、寄生虫等)疫苗和各种抗肿瘤疫苗。
3.一种含有皮氨钙联合佐剂在制备用于哺乳动物或人的预防性和治疗性疫苗中的应用。
4.根据权利1所述用含有皮氨钙佐剂的免疫刺激物体外刺激免疫干细胞，如体外刺激DC-CIK细胞和CAR-T细胞及T细胞的增殖成熟并回输体内以达到增强抗肿瘤、抗细胞内感染免疫效果的应用。其特征在于所述的免疫刺激物含有皮氨钙联合佐剂。
5.根据权利1所述用皮氨钙联合佐剂本身作为非特异性促进机体免疫力的应用，包括制备成固体、半固体、液体和气体剂型的应用，如制备成水针剂、喷雾剂和各种膏剂的应用。其特征在于所述的免疫增强剂或免疫促进剂含有皮氨钙联合佐剂。
下面结合实例阐述本发明皮氨钙联合佐剂及含有皮氨钙联合佐剂的疫苗作用机理和较单独使用皮氨钙佐剂及其疫苗的进一步的免疫效果，本发明并非仅限定这些举例的皮氨钙联合佐剂及其疫苗而在帮助理解。
一.皮氨钙佐剂与TLR9配体CPG联合应用：皮氨钙佐剂属于双链RNA佐剂，是TLR3受体的激动剂，由于非抗生素类氨基化合物取代了卡那霉素较PICKCa更安全更有效。CPG是CPG寡核苷酸，是TLR9的配体，根据核苷酸序列和长度分为A、B、C类，A和C类活化B细胞(溶酶体部分)及浆细胞样DC，产生IFN-α。B类活化B细胞(内体部分)并使DC成熟。与皮氨钙佐剂联合应用就具有进一步提高免疫的效果。用合成蛋白MUC作为肿瘤疫苗联合应用CPG2395和皮氨钙，较单独使用更能有效地增强机体抗肿瘤免疫应答，CD4+T细胞 和CD8+T细胞都较单独使用高，对肺癌小白鼠的预防和转移灶的减少二者联合应用也比单独使用好。
在体外，用CPG2395和皮氨钙单独或联合刺激脾细胞，进行细胞因子分泌情况以及细胞增殖能力的检测；在体内CPG2395和皮氨钙单独或者联合作为MUCI多肽抗原的佐剂免疫小鼠，通过ELISA方法检测免疫血清中的抗体滴度、CTL杀伤实验检测杀伤性T细胞的细胞毒作用、流式细胞技术分析免疫前后各细胞亚群的变化；在肿瘤预防实验中，通过抑瘤率以及肺转移结节数等指标评价二者作为佐剂对MUCI免疫小鼠肿瘤预防效果的影响。
结果表明体外用CPG2395、皮氨钙以及二者联合与正常小鼠的脾细胞混合培养24小时后收集细胞培养上清，检测到CPG2395与皮氨钙联合与单独应用相比能更好地诱导IL-12、TNF-a以及IFN-Y的产生。对免疫后小鼠脾细胞进行流式细胞术分型，联合组小鼠CD4+T细胞及CDS+细胞数量均较单用组升高。CPG2395与皮氨钙联合作为佐剂与MUCI联合免疫C57BL/6小鼠后接种Lewis肺癌后，对肺癌的预防效果优于CPG2395或皮氨钙单独作为佐剂的平行组。而且肺转移灶的数量也明显较少。具体结果是不同佐剂分别与MUCI疫苗复合后接种荷瘤小白鼠后33天处死，剥离瘤体称重，解剖取肺脏，观察并记录肺部转移灶的数量与大小。统计并计算各组肿瘤的平均重量。结果表明对照组中平均瘤重达到6.14g±1.749，CPG2395佐剂组平均瘤重为3.79g±2.789，P＝0.282。皮氨钙佐剂组平均瘤重为3.19g±2.199，P＝0.141，联合组肿瘤最小，平均瘤重仅为2.5g±2.479，P＝0.016。表明CPG2395、皮氨钙与MUCI联合免疫的佐剂效果优于单用组，能够更好的激活机体的免疫应答，能够更好的抑制肿瘤的发生发展。
二.TLR3受体的配体皮氨钙和TLR7/8受体的配体R848联合应用并结合癌胚抗原CEA526-533肽段后促进树突状细胞(DC)成熟，与单独使用皮氨钙比较能显著增强抗癌效果。
Toll样受体(TLR)2、3、4、7、8、9的配体选择性地联合应用可以进一步诱导树突状细胞(DC)成熟，产生一系列肿瘤免疫所期望的正向效应，如上调CCR7、CD4O、CD8O、CD8O的表达，上调Th1，，下调Th2反应，介导IFN-Y、IL-12、TNF、IL-6等的分泌，以及在一定程度上阻断Treg的负向调节作用。
1、DC分组及具体操作方法：
A.联合TLR组DC(TLR-DC)：在培养的最后两天加入20ug/ml皮氨钙和1.5ug/ml R848。
B.联合TLR及荷载CEA526-533肽段组DC(TLR-DC+CEA)：在培养的最后两天加入20ug/ml皮氨钙和1.5ug/ml R848，然后加入40ug/ml的肽段CEA526-533，置于细胞培养箱中。3-4小时后取出，洗去多余未结合的肽段，即得到联合TLR并荷载CEA的DC。
C.常规培养的荷载CEA526-533肽段组DC(常规DC+CEA)：加入40ug/ml的肽段CEA526-533，置于细胞培养箱中，3-4小时后取出，洗去多余未结合的肽段，即得到荷载CEA526-533的DC。
D.常规培养DC。
2、结果：
(1).联合TLR组DC的IL-12分泌量显著高于常规组，分别为948.67±104.36pg和215.24±19.3spg，P＜0.01。
(2).与联合TLR组DC共培养后获得的效应细胞对MC38-CEA肠癌细胞在较高效应细胞∶靶细胞(E∶T)比值时具有一定的杀伤作用，显著优于常规组(P＜0.05)。
(3).对于MC38细胞与联合TLR组以及常规组DC共培养的效应细胞均无明显杀伤效应，两组无明显差异(P＞0.05)。
(4).MC38-CEA肠癌细胞建立的小鼠肿瘤模型中，联合TLR激活并且荷载CEA肽段的DC疫苗组的小鼠肿瘤平均体积明显小于其余各组(P＜0.01)，并且存活期明显优于其余各组(P＜0.01)。
(5).MC38肠癌细胞建立的小鼠肿瘤模型中，各组肿瘤平均体积无明显差异(P＞0.05)，且各组小鼠存活期无明显差异(P＞0.05)。
3、结论
(1).联合TLR组的DC疫苗较常规DC疫苗能分泌更多的IL-12。
(2).联合TLR组的DC疫苗诱导的抗原特异性CTLs能有效地产生抗小鼠MC38-CEA肠癌细胞的特异性免疫反应。
(3).联合TLR组且荷载CEA的DC疫苗对结肠癌荷瘤小鼠具有显著的抑瘤效应并可以延长小鼠的生存期。
三.皮氨钙佐剂联合应用转染制剂作用
1、显著抑制肿瘤细胞生长：皮氨钙佐剂细胞外直接作用肺癌细胞株A549，对肺癌细胞抑制率低于30％，加入转染试剂PEI，在合适搭配比例下，通过MTT 570nm测定OD值，表明皮氨钙佐剂细胞内作用明显抑制A549的生长。
2、显著促进肺癌细胞凋亡：采用Annexin V/FITC-PI双染方法显示皮氨钙佐剂与转染试剂PEI在合适搭配比例下，作用24小时A549细胞出现明显凋亡，较对照组(PBS组)、单独皮氨钙佐剂组、单独转染试剂PEI组均有显著差异P＜0.05。
3、上调细胞受体TLR3、RIG-1、MDA-5和IFN-β的表达：皮氨钙佐剂是TLR3、RIG-1、MDA-5的配体。用皮氨钙佐剂与转染试剂PEI在合适搭配比例下作用于A549细胞3、6小时后使用荧光定量PCR检测TLR3、RIG-1、MDA-5和IFN-βmRNA表达水平，结果显示皮氨钙佐剂与转染试剂PEI组明显上调TLR3、RIG-1、MDA-5和IFN-βmRNA的表达，在6小时时，与其他对照组(PBS组)、单独皮氨钙佐剂组、单独转染试剂PEI组均有显著差异P＜0.01。
以上所述仅为本发明的相关实施例，这些例子并非因此限制本发明的专利范围，只是利用本发明举例说明和明确了皮氨钙联合佐剂和含有皮氨钙联合佐剂疫苗较单纯皮氨钙佐剂和含有皮氨钙佐剂疫苗有更加突出的促进免疫效果。凡是利用本发明举例说明皮氨钙联合佐剂配方、检定内容和皮氨钙联合佐剂疫苗配方、检定内容所作的数据或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (5)

  1. 一种含聚肌苷酸-聚胞苷酸(聚肌胞)、钙离子和除氨基类抗生素之外的氨基化合物或与聚乙烯亚胺、聚乙二醇等交联剂形成接枝物在制备免疫佐剂中形成纳米颗粒单独或以其为核心联合其他免疫促进剂协同增强机体免疫刺激物质------皮氨钙联合佐剂作为在制备免疫佐剂中的应用。
  2. 根据权利1所述的一种含有皮氨钙联合佐剂的疫苗。其特征在于所述的佐剂是含有皮氨钙联合佐剂，所述的疫苗包括各种抗细胞内感染(病毒和细胞内感染的细菌、寄生虫等)疫苗和各种抗肿瘤疫苗。
  3. 一种含有皮氨钙联合佐剂在制备用于哺乳动物或人的预防性和治疗性疫苗中的应用。
  4. 根据权利1所述用含有皮氨钙佐剂的免疫刺激物体外刺激免疫干细胞，如体外刺激DC-CIK细胞和CAR-T细胞及T细胞的增殖成熟并回输体内以达到增强抗肿瘤、抗细胞内感染免疫效果的应用。其特征在于所述的免疫刺激物含有皮氨钙联合佐剂。
  5. 根据权利1所述用皮氨钙联合佐剂本身作为非特异性促进机体免疫力的应用，包括制备成固体、半固体、液体和气体剂型的应用，如制备成水针剂、喷雾剂和各种膏剂的应用。其特征在于所述的免疫增强剂或免疫促进剂含有皮氨钙联合佐剂。
PCT/CN2017/000208 2016-03-23 2017-03-02 皮氨钙联合佐剂及含有皮氨钙联合佐剂的疫苗 WO2017161950A1 (zh)

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