WO2023078279A1 - 免疫原性组合物及其用途 - Google Patents

免疫原性组合物及其用途 Download PDF

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WO2023078279A1
WO2023078279A1 PCT/CN2022/129153 CN2022129153W WO2023078279A1 WO 2023078279 A1 WO2023078279 A1 WO 2023078279A1 CN 2022129153 W CN2022129153 W CN 2022129153W WO 2023078279 A1 WO2023078279 A1 WO 2023078279A1
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immunogen
cell
immunogenic composition
oral vaccine
tumor
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陈志铭
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澄交生物科技股份有限公司
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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
    • A61K39/02—Bacterial 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

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  • the present invention relates to an immunogenic composition, which comprises a glycosylated chaperonin 60 (glycosylated chaperonin 60) derived from Lactobacillus reuteri and an immunogen.
  • the present invention also relates to an oral vaccine (oral vaccine) comprising the immunogenic composition, and the use of the oral vaccine in anticancer.
  • Heat shock protein is a kind of molecular chaperone (molecular chaperone) produced by cells (eg, eukaryotic and prokaryotic cells) in response to environmental stress. HSPs are divided into different families according to their molecular weights, including HSP10, HSP40, HSP60, HSP70, HSP90 and HSP100. HSP60 [also known as GroEL or chaperonin 60 (chaperonin 60, Cpn60) is one of the most studied HSPs, it is usually expressed on the surface of pathogenic bacteria, and when the pathogen invades the host cell, it will cause The expression of HSP60 was significantly increased. HSP60 is known to act as a bacterial immunogen that induces both innate and adaptive immune responses.
  • HSP60 is known to act as a bacterial immunogen that induces both innate and adaptive immune responses.
  • Bansal A. et al. (2010), Mol. Cell Biochem., 337:213-221 Bansal A. et al. disclose the use of HSP60 derived from Salmonella typhi to vaccinate mice Vaccination is effective in stimulating immune responses and providing protective immunity against lethal S. typhi Ty2 infection.
  • Yamaguchi H. et al. revealed the use of HSP60 derived from Helicobacter pylori to target specific pathogen-free bacteria Vaccination of specific pathogen free (SPF) mice can effectively inhibit the colonization of Helicobacter pylori.
  • SPF pathogen free mice
  • these vaccinated mice were observed to have severe postimmunization gastritis with destruction of glandular structure, surface erosion and marked inflammatory cell infiltration caused by gastritis infiltration).
  • glycosylated HSP60 can be isolated from probiotic bacteria.
  • Tytgat H.L.et al. (2016), J.Mol.Microbiol.Biotechnol., 26:345-358
  • Tytgat H.L. et al. isolated from Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG, LGG) thalline
  • Various glycosylated proteins including glycosylated GroEL.
  • glycosylated HSP60 derived from probiotics can be used in vaccines.
  • the present invention provides an immunogenic composition (immunogenic composition), which comprises a glycosylated chaperonin 60 (glycosylated chaperonin60, Cpn60) derived from Lactobacillus reuteri ) and an immunogen (immunogen).
  • immunogenic composition which comprises a glycosylated chaperonin 60 (glycosylated chaperonin60, Cpn60) derived from Lactobacillus reuteri ) and an immunogen (immunogen).
  • the present invention provides an oral vaccine comprising a particle comprising:
  • the present invention provides the use of an immunogenic composition as described above for the manufacture of a medicament for vaccination.
  • the present invention provides a use of the immunogenic composition as described above for the preparation of a medicament for combating liver cancer.
  • the present invention provides a method of combating liver cancer comprising administering to a subject an oral vaccine as described above.
  • Fig. 1 shows the relative OD value measured for IgA in the serum of each group of mice of embodiment 2;
  • Figure 2 shows the relative OD values measured for IgG1 in the serum of each group of mice in Example 2.
  • the present invention provides an immunogenic composition (immunogenic composition), which comprises a glycosylated chaperonin 60 (glycosylated chaperonin 60, Cpn60) derived from Lactobacillus reuteri (Lactobacillus reuteri) and an immunogen (immunogen ).
  • immunogenic composition which comprises a glycosylated chaperonin 60 (glycosylated chaperonin 60, Cpn60) derived from Lactobacillus reuteri (Lactobacillus reuteri) and an immunogen (immunogen ).
  • immunogen and “antigen” are used interchangeably to mean a molecule comprising one or more epitopes capable of inducing a humoral response in a subject.
  • the immunogen can be an attenuated (attenuated), inactivated (inactivated) or modified (modified) live pathogen (pathogen) [for example, bacteria (bacteria), virus (virus), fungus ( fungi, parasites or other microbes].
  • pathogen for example, bacteria (bacteria), virus (virus), fungus ( fungi, parasites or other microbes].
  • the immunogen can also be a component or molecule derived from a pathogen, cell, tissue or organ, for example, an isolated, purified or recombinant protein ( protein), peptide (peptide), nucleic acid (nucleic acid), lipid (lipid) or carbohydrate (carbohydrate).
  • the immunogen may be a tumor-associated immunogen.
  • tumor-associated immunogen refers to proteins or polypeptides expressed by tumor cells, for example, surface proteins or polypeptides located on the cell membrane of tumor cells, nucleoproteins, and glycoproteins.
  • the tumor is selected from the group consisting of hepatocellular carcinoma (HCC), cholangiocarcinoma, hepatoblastoma, lymphoma, sarcoma ( sarcoma), and combinations thereof.
  • HCC hepatocellular carcinoma
  • cholangiocarcinoma cholangiocarcinoma
  • hepatoblastoma lymphoma
  • sarcoma sarcoma
  • the tumor is hepatocellular carcinoma.
  • the tumor can also be carcinoma in situ in a cancer patient.
  • the immunogen is selected from the group consisting of hepatocellular carcinoma cell-associated immunogen, cholangiocarcinoma cell-associated immunogen, cholangiocarcinoma cell-associated immunogen immunogen), hepatoblastoma cell-associated immunogen, lymphoma cell-associated immunogen, sarcoma cell-associated immunogen ), and their combinations.
  • the immunogen is an H22 cell-associated immunogen.
  • oral vaccine which comprises a particle (particle), which contains:
  • the resistant starch inner shell and the calcium alginate outer shell can be formed using techniques well known and commonly used by those skilled in the art.
  • the resistant starch may contain 15-35 wt% of amylose and 65-85 wt% of amylopectin. In some embodiments, the resistant starch contains 17-24wt% amylose and 76-83wt% amylopectin.
  • the resistant starch may be a plant-derived resistant starch type 2 (resistant starch type 2, RS2).
  • Such plants may include, but are not limited to: bananas, potatoes, sweet potatoes, corn, sorghum, cassava, wheat, and rice.
  • the present invention provides the use of an immunogenic composition as described above for the preparation of a medicament for vaccination.
  • the present invention also provides a use of the above-mentioned immunogenic composition for preparing a medicine for fighting liver cancer.
  • the term "vaccination” means active immunization, that is, the induction of a specific immune response by administering an immunogen to an individual.
  • the medicine can be manufactured into a dosage form (dosage form) suitable for oral administration (oral administration), which includes, but is not limited to: sterile powder, Tablet, troche, lozenge, pellet, capsule, dispersible powder or granule, solution, suspension , emulsion, syrup, elixir, slurry and the like.
  • oral administration includes, but is not limited to: sterile powder, Tablet, troche, lozenge, pellet, capsule, dispersible powder or granule, solution, suspension , emulsion, syrup, elixir, slurry and the like.
  • the medicinal product may be an oral vaccine.
  • the medicine may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) which is widely used in pharmaceutical manufacturing technology.
  • the pharmaceutically acceptable carrier may contain one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, disintegrating agent ( decomposer), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent ), gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like.
  • the pharmaceutically acceptable carrier may contain one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, disintegrating agent ( decomposer), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent ), gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like.
  • the medicinal product may further comprise an adjuvant selected from the group consisting of saponin, trehalose dimycolate, cholera toxin , monophosphoryl lipid A (monophosphoryl lipid A), lipopolysaccharides (lipopolysaccharides), cytokines (eg, interferon (IFN) or granulocyte-macrophage colony-stimulating factor, GM-CSF)], sex hormone dehydroepiandrosterone (dehydroepiandrosterone, DHEA), Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum gel [eg, aluminum hydroxide, aluminum phosphate, or potassium aluminum sulfate], oily adjuvants (water-in-oil-in-water, W/O/W type) ), water-in-oil (W/O) emulsion, oil-in-water (O/W) emulsion, concanavalin A (Concana
  • the present invention further provides a method for combating liver cancer, which comprises administering an oral vaccine as described above to an individual.
  • administration and “administration” are used interchangeably and mean introducing, providing, or delivering ( delivering) an intended active ingredient to perform its intended function.
  • the term "individual” means any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs ), goats, dogs, cats, mice, and rats.
  • the dose and frequency of administration of the oral vaccine will vary depending on the route of administration and the age, physical condition and response of the individual.
  • the oral vaccine can be administered orally in a single dose or divided into several doses.
  • BCRC bioresource collection and research center
  • FIRDI Food Industry Research and Development Institute
  • BCRC Bioresource Collection and Research Center
  • the 60-kDa protein band was excised from the gel by in-gel trypsin digestion combined with liquid chromatography-tandem mass spectrometry method (liquid chromatography-tandem mass spectrometry, LC-MS/MS) for identification.
  • the LC-MS/MS analysis instrument used is UltiMate TM 3000RSLCnano LC system (Thermo Fisher Scientific) and 6600 system (Applied Biosystems Sciex), the analysis software is Mascot version 2.7, and the obtained data is compared with the bacterial database of NCBI and SWISS-PROT. The applicant believed that the protein was glycosylated Cpn60 based on the results of LC-MS/MS analysis.
  • the banana resistant starch used in the following experiments was prepared according to the method described in TW 202210086A, and belongs to the second type of resistant starch (resistant starch type 2, RS2).
  • mice Male BALB/c mice (8 to 10 weeks old) used in the following experiments were purchased from the Experimental Animal Center (Building G, No. 99, Lane 130, Section 1, Yuanyuan Road, Nangang District, Taipei City, Taiwan, China). All experimental animals were kept in an animal room with 12 hours of light and 12 hours of darkness respectively, the room temperature was maintained at 20-26° C. and the relative humidity was maintained at 30-70%, and water and feed were adequately supplied. The handling of experimental animals and all experimental procedures were carried out in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of the Experimental Animal Center.
  • IACUC Institutional Animal Care and Use Committee
  • the mouse hepatocellular carcinoma cell line H22 used in the following experiments was purchased from Elabscience (Catalog No: EP-CL-0341). H22 cells were inoculated in RPMI 1640 medium [supplemented with 10% fetal bovine serum (FBS)], and cultured in an incubator with the culture conditions set at 37°C and 5% CO 2 . Thereafter, replace with fresh medium approximately every 2 days. When the cell density reached about 80-90% confluence, the cells were subcultured.
  • RPMI 1640 medium supplied with 10% fetal bovine serum (FBS)
  • H22 cell-associated immunogen H22cell-associated immunogen
  • H22 cells were homogenized by adding an appropriate amount of saline, and then centrifuged at 10,000 rpm for 10 minutes at 4°C, and the resulting supernatant was used as an H22 cell-associated immunogen and stored at -20°C for later use.
  • Embodiment 1 Preparation of oral vaccine (oral vaccine)
  • two comparative oral vaccines (comparative oral vaccines 1 and 2) and one control oral vaccine were prepared according to the above-mentioned steps, the difference being:
  • the comparative oral vaccine 1 only added H22 cell-associated immunogen
  • the comparative oral vaccine 2 only added glycosylated Cpn60 derived from Lactobacillus reuteri, and the comparative oral vaccine did not The immunogenic composition is added.
  • Each of the obtained oral vaccines was freeze-dried for 24 hours, and the freeze-dried powder thus formed was stored at -20°C until use.
  • Embodiment 2 In vivo immunogenicity test (in vivo Immunogenicity assay) of oral vaccine of the present invention
  • phosphate buffered saline phosphate buffered saline
  • mice in each group were administered with booster doses by tube feeding, that is, the mice in the comparison groups 1 and 2 were administered with 200 ⁇ L of comparative oral vaccines 1 and 2 (mixed in PBS), the mice in the experimental group were administered with 200 ⁇ L of the oral vaccine of the present invention (mixed in PBS), and the mice of the control group were administered with 200 ⁇ L of Control oral vaccine (in PBS).
  • the mice in each group were dosed once a day for a total of 4 days.
  • Blood samples were collected from the tail arteries of mice in each group by arterial puncture before the start of dosing (ie, day 0) and at the end of days 15 and 30 after the start of dosing. Thereafter, the obtained blood sample was centrifuged at 12,000 rpm for 5 minutes at 4° C., and then the supernatant was collected as a serum sample.
  • each diluted serum sample was added to each well of the 96-well culture plate for reaction, and then the biotinylated detection antibody (biotinylated detection antibody) and HRP conjugate provided in each kit were used to react (HRP conjugate) for detection and 3,3',5,5'-tetramethylbenzidine (TMB) for coloration. Finally, the absorbance value (OD 450 ) of each well was measured with an ELISA reader at a wavelength of 450 nm.
  • a 96-well culture plate was coated and blocked according to the above method, but no biotinylated detection antibody and HRP conjugate were added, and the resulting 96-well culture plate was used for The same absorbance value (OD 450 ) was measured, and the measured absorbance value (OD 450 ) was used as the background value.
  • the relative OD value (relative OD value) is calculated by substituting the measured OD450 value into the following formula (1):
  • Figure 1 shows the relative OD values measured for IgA in the sera of mice of each group at the end of day 30 after the start of administration. It can be seen from Figure 1 that the relative OD values measured by the experimental group and the comparative group 2 were significantly higher than those measured by the control group, but there was no significant difference between the comparative group 1 and the control group.
  • Figure 2 shows the relative OD values measured for IgG1 in the sera of mice of each group at the end of day 30 after the start of administration. It can be seen from Figure 1 that the relative OD values measured in the experimental group were significantly higher than those measured in the control group, while there was no significant difference between the comparison group 1, comparison group 2 and the control group.
  • the relative OD value measured for IgG1 at the end of the 30th day after the start of administration was higher than that measured at the end of the 0th day and at the end of the 15th day after the start of administration. recipients (data not shown). This means that the oral vaccine of the present invention can effectively induce IgG1 production, and the IgG1 content will increase significantly with time.
  • the use of H22 cell-associated immunogen alone cannot induce antibody production, while the use of glycosylated Cpn60 derived from Lactobacillus reuteri alone can only induce a small amount of antibody production.
  • the immunogenic composition according to the present invention is able to effectively induce high production of IgA and IgG1 specific for H22 cell-associated immunogen.
  • the glycosylated Cpn60 derived from Lactobacillus reuteri can enhance the immunogenicity (immunogenicity) of an immunogen (for example, an immunogen related to liver tumors), so when it is combined with an immunogen When used in combination, they can successfully elicit an immune response and have excellent development potential in the vaccine industry.
  • an immunogen for example, an immunogen related to liver tumors

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Abstract

提供了免疫原性组合物及其用途。提供了一种免疫原性组合物，其包含有一源自于罗伊氏乳酸杆菌的糖基化伴侣蛋白60以及一免疫原，和包含有该免疫原性组合物的口服疫苗，以及该口服疫苗在抗癌上的用途。

Description

免疫原性组合物及其用途 技术领域
本发明是有关于一种免疫原性组合物(immunogenic composition)，其包含有一源自于罗伊氏乳酸杆菌(Lactobacillus reuteri)的糖基化伴侣蛋白60(glycosylated chaperonin 60)以及一免疫原。本发明也有关于一种包含有该免疫原性组合物的口服疫苗(oral vaccine)，以及该口服疫苗在抗癌上的用途。
背景技术
热休克蛋白(heat shock protein,HSP)是一种细胞(例如，真核与原核细胞)因应环境的压力所产生的分子伴侣(molecular chaperone)。HSP依据其分子量的大小而被区分为不同的家族，包括HSP10、HSP40、HSP60、HSP70、HSP90以及HSP100。HSP60[又被称作GroEL或伴侣蛋白60(chaperonin 60,Cpn60)是目前被研究最多的HSP之一，它通常会被表达于病原菌菌体的表面上，并且当该病原菌侵入宿主细胞时会使HSP60的表达量明显增加。HSP60已被知晓可做为一种细菌性免疫原(bacterial immunogen)，其能诱发先天性与适应性免疫反应。
目前已有研究显示衍生自病原菌的HSP能有效地对抗病原菌感染。例如，在Bansal A.et al.(2010),Mol.Cell Biochem.,337:213-221中，Bansal A.等人揭示使用衍生自伤寒沙门杆菌(Salmonella typhi)的HSP60来对小鼠进行疫苗接种(vaccination)能够有效地刺激免疫反应以及提供对抗致命性伤寒沙门杆菌Ty2感染(lethal S.typhi Ty2infection)的保护性免疫(protective immunity)。
在Yamaguchi H.et al.(2003),Clin.Diagn.Lab.Immunol.,10:808-812中，Yamaguchi H.等人揭示使用衍生自幽门螺旋杆菌(Helicobacter pylori)的HSP60来对无特定病原菌(specific pathogen free,SPF)小鼠进行疫苗接种能够有效地抑制幽门螺旋杆菌的定殖(colonization)。但是，这些经接种的小鼠被观察到有严重的免疫后胃炎(postimmunization gastritis)以及胃炎所造成的腺体结构(glandular structure)破坏、表面糜烂(surface erosion)以及显著的发炎细胞浸润 (inflammatory cells infiltration)。
已有研究报导，糖基化HSP60(glycosylated HSP60)可从益生菌菌体中被分离出来。例如，在Tytgat H.L.et al.(2016),J.Mol.Microbiol.Biotechnol.,26:345-358中，Tytgat H.L.等人从鼠李糖乳酸杆菌GG(Lactobacillus rhamnosus GG,LGG)菌体中分离出各种的糖基化蛋白(glycosylated proteins)，其中包括糖基化GroEL。
就申请人所知，迄今尚未有研究报导衍生自益生菌的糖基化HSP60可被应用在疫苗上。
发明内容
于是，在第一个方面，本发明提供一种免疫原性组合物(immunogenic composition)，其包含有一源自于罗伊氏乳酸杆菌(Lactobacillus reuteri)的糖基化伴侣蛋白60(glycosylated chaperonin60,Cpn60)以及一免疫原(immunogen)。
在第二个方面，本发明提供一种口服疫苗(oral vaccine)，其包含有一粒子(particle)，该粒子含有：
一如上所述的免疫原性组合物作为核心(core)；
一包覆该免疫原性组合物的抗性淀粉内壳(resistant starch inner shell)；以及
一包覆该抗性淀粉内壳的海藻酸钙外壳(calcium alginate outer shell)。
在第三个方面，本发明提供一种如上所述的免疫原性组合物供应用于制备一用来疫苗接种(vaccination)的医药品的用途。
在第四个方面，本发明提供一种如上所述的免疫原性组合物供应用于制备一用来对抗肝癌的医药品的用途。
在第五个方面，本发明提供一种对抗肝癌的方法，其包括对一个体投予一如上所述的口服疫苗。
附图说明
图1显示实施例2的各组小鼠的血清中针对IgA所测得的相对OD值；以及
图2显示实施例2的各组小鼠的血清中针对IgG1所测得的相对 OD值。
具体实施方式
为了本说明书的目的，将被清楚地了解的是：文字“包含有(comprising)”意指“包含但不限于”，以及文字“包括(comprises)”具有一对应的意义。
除非另外有所定义，在本文中所使用的所有技术性与科学术语具有本领域技术人员所共同了解的意义。一本领域技术人员会认知到许多与那些被描述于本文中者相似或等效的方法和材料，它们可被用于实施本发明。当然，本发明决不受到所描述的方法和材料的限制。
本发明提供一种免疫原性组合物(immunogenic composition)，其包含有一源自于罗伊氏乳酸杆菌(Lactobacillus reuteri)的糖基化伴侣蛋白60(glycosylated chaperonin 60,Cpn60)以及一免疫原(immunogen)。
如本文所使用的，术语“免疫原”与“抗原(antigen)”可被交替地使用，意指包含一或多个表位(epitope)的分子，其能够诱发一个体(subject)的体液性免疫反应(humoral immune response)和/或细胞免疫反应(cellular immune response)。
依据本发明，该免疫原可以是一经减毒的(attenuated)、灭活的(inactivated)或经修饰的(modified)活病原体(pathogen)[例如，细菌(bacteria)、病毒(virus)、真菌(fungi)、寄生虫(parasite)或其他微生物(microbes)]。此外，该免疫原也可以是一衍生自病原体、细胞(cell)、组织(tissue)或器官(organ)的组分(component)或分子(molecule)，例如，经分离、纯化或重组的蛋白质(protein)、胜肽(peptide)、核酸(nucleic acid)、脂质(lipid)或糖类(carbohydrate)。
依据本发明，该免疫原可以是一种与肿瘤有关的免疫原(tumor-associated immunogen)。
如本文所使用的，术语“与肿瘤有关的免疫原”意指由肿瘤细胞所表达的蛋白质或多肽，例如，位于肿瘤细胞的细胞膜的表面蛋白质或多肽、核蛋白以及糖蛋白等。
依据本发明，该肿瘤是选自于由下列所构成的群组：肝细胞癌(hepatocellular carcinoma,HCC)、胆管癌(cholangiocarcinoma)、肝母细胞瘤(hepatoblastoma)、淋巴瘤(lymphoma)、肉瘤(sarcoma)，以及它 们的组合。
在本发明的一个较佳具体例中，该肿瘤是肝细胞癌。
在某些具体例中，该肿瘤也可以是癌症患者的原位癌(carcinoma in situ)。
依据本发明，该免疫原是选自于由下列所构成的群组：肝细胞癌细胞-关联性免疫原(hepatocellular carcinoma cell-associated immunogen)、胆管癌细胞-关联性免疫原(cholangiocarcinoma cell-associated immunogen)、肝母细胞瘤细胞-关联性免疫原(hepatoblastoma cell-associated immunogen)、淋巴瘤细胞-关联性免疫原(lymphoma cell-associated immunogen)、肉瘤细胞-关联性免疫原(sarcoma cell-associated immunogen)，以及它们的组合。
在本发明的一个较佳具体例中，该免疫原是H22细胞-关联性免疫原。
本发明也提供一种口服疫苗(oral vaccine)，其包含有一粒子(particle)，该粒子含有：
一如上所述的免疫原性组合物作为核心(core)；
一包覆该免疫原性组合物的抗性淀粉内壳(resistant starch inner shell)；以及
一包覆该抗性淀粉内壳的海藻酸钙外壳(calcium alginate outer shell)。
依据本发明，该抗性淀粉内壳与该海藻酸钙外壳可利用本领域技术人员所详知且惯用的技术来形成，在此方面，可参考，例如，TW202210086A、Zafarullah M.et al.(2017),FoodHydrocoll.,66:286-295、N H Zakaria et al.(2017),IOP Conf.Ser.:Mater.Sci.Eng.doi:10.1088/1757-899X/209/1/012087以及Carbinatto F.M.et al.(2012),Int.J.Pharm.,423:281-288。
依据本发明，以该抗性淀粉的总重量为计算基础，该抗性淀粉可包含有15-35wt％的直链淀粉(amylose)以及65-85wt％的支链淀粉(amylopectin)。在某些具体例中，该抗性淀粉包含有17-24wt％的直链淀粉以及76-83wt％的支链淀粉。
依据本发明，该抗性淀粉可以是源自于植物的第二型抗性淀粉 (resistant starch type 2,RS2)。该植物可包括，但不限于：香蕉、马铃薯、番薯、玉米、高粱、木薯、小麦以及稻米。
此外，本发明提供一种如上所述的免疫原性组合物供应用于制备一用来疫苗接种(vaccination)的医药品的用途。
本发明也提供一种如上所述的免疫原性组合物供应用于制备一用来对抗肝癌的医药品的用途。
如本文所使用的，术语“疫苗接种”意指主动免疫(active immunization)，也就是通过对一个体投予免疫原来诱发特异性免疫反应(specific immune response)。
依据本发明，该医药品可利用本领域技术人员所详知的技术而被制造成一适合于口服给药(oral administration)的剂型(dosage form)，这包括，但不限于：无菌的粉末、锭剂(tablet)、片剂(troche)、口含锭(lozenge)、丸剂(pellet)、胶囊(capsule)、分散性粉末(dispersible powder)或细颗粒(granule)、溶液、悬浮液(suspension)、乳剂(emulsion)、糖浆(syrup)、酏剂(elixir)、浓浆(slurry)以及类似物。
依据本发明，该医药品可以是一口服疫苗。
依据本发明，该医药品可进一步包含有一被广泛地使用于药物制造技术的药学上可接受的载剂(pharmaceutically acceptable carrier)。例如，该药学上可接受的载剂可包含一或多种选自于下列的试剂：溶剂(solvent)、缓冲液(buffer)、乳化剂(emulsifier)、悬浮剂(suspending agent)、分解剂(decomposer)、崩解剂(disintegrating agent)、分散剂(dispersing agent)、黏结剂(binding agent)、赋形剂(excipient)、稳定剂(stabilizing agent)、螯合剂(chelating agent)、稀释剂(diluent)、胶凝剂(gelling agent)、防腐剂(preservative)、润湿剂(wetting agent)、润滑剂(lubricant)、吸收延迟剂(absorption delaying agent)、脂质体(liposome)以及类似物。有关这些试剂的选用与数量是落在本领域技术人员的专业素养与例行技术范畴内。
依据本发明，该医药品可进一步包含有一选自于由下列所构成的群组的佐剂(adjuvant)：皂苷(saponin)、海藻糖二霉菌酸酯(trehalose dimycolate)、霍乱毒素(choleric toxin)、单磷酰脂质A(monophosphoryl lipid A)、脂多糖(lipopolysaccharides)、细胞激素[例如，干扰素 (interferon,IFN)或粒细胞－巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)]、性激素脱氢表雄固酮(dehydroepiandrosterone,DHEA)、弗伦氏完全佐剂(Freund’s complete adjuvant)、弗伦氏不完全佐剂(Freund’s incomplete adjuvant)、铝胶(aluminum gel)[例如，氢氧化铝(aluminum hydroxide)、磷酸铝(aluminum phosphate)或硫酸铝钾盐(potassium aluminum sulfate)]、油质佐剂(water-in-oil-in-water,W/O/W型)、油包水型(water-in-oil,W/O)乳剂、水包油型(oil-in-water,O/W)乳剂、伴刀豆球蛋白A(Concanavalin A,Con A)、β-葡聚糖(β-glucan)，以及它们的组合。
此外，本发明进一步提供一种对抗肝癌的方法，其包括对一个体投予(administering)一如上所述的口服疫苗。
如本文中所使用的，术语“投予”以及“给药(administration)”可被交换地使用，并且意指通过任何合适的途径来对一个体导入(introducing)、提供(providing)或递送(delivering)一预定的活性成分以执行其预期的效用。
如本文中所使用的，术语“个体”意指任何感兴趣的哺乳类动物，诸如人(humans)、猴子(monkeys)、牛(cows)、绵羊(sheep)、马(horses)、猪(pigs)、山羊(goats)、狗(dogs)、猫(cats)、小鼠(mice)以及大鼠(rats)。
依据本发明，该口服疫苗的给药剂量与给药次数会视下列因素而变化：给药途径以及个体的年龄、身体状况与反应。一般而言，该口服疫苗可呈单一剂量或是分成数个剂量的形式而被口服地给药。
本发明将就下面的实施例来做进一步说明，但应了解的是，该等实施例只是供例示说明用，而不应被解释为本发明的实施上的限制。＜实施例＞
一般实验材料：
1.源自于罗伊氏乳酸杆菌(Lactobacillus reuteri)的糖基化伴侣蛋白60
(glycosylated chaperonin 60,Cpn60)的制备：
首先，将购自于食品工业发展研究所(Food Industry Research and Development Institute,FIRDI)的生物资源保存及研究中心(Bioresource Collection and Research Center,BCRC)(300新竹市食品路331号，中 国台湾)的罗伊氏乳酸杆菌BCRC 14691接种至MRS(De Man,Rogosa and Sharpe)肉汤培养基(HiMedia)中，并于37℃下进行培养历时48小时。所得到的培养物以6,000rpm予以离心历时15分钟，然后收集上清液并使用
Figure PCTCN2022129153-appb-000001
advance离心装置(
Figure PCTCN2022129153-appb-000002
advance centrifugal devices)(PALL Corporation)来进行浓缩，而得到一蛋白质样品。接着，取50μg所得到的蛋白质样品并采用本领域技术人员所详知且惯用的技术来进行12％十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析[12％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis]，继而为过碘酸-希夫(PAS)染色[periodic acid-Schiff(PAS)staining]，借此确认该蛋白质样品中存在一具有分子量为60kDa的糖基化蛋白质。
之后，将60-kDa蛋白质带(60-kDa protein band)从凝胶上切下来，该60-kDa蛋白质带通过胶体内胰蛋白酶切割(in-gel trypsin digestion)与液相层析-串联式质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)来进行鉴定。所使用的LC-MS/MS分析仪器为UltiMate TM 3000RSLCnano LC系统(Thermo Fisher Scientific)以及
Figure PCTCN2022129153-appb-000003
6600系统(Applied Biosystems Sciex)，分析软件为Mascot第2.7版，所得到的数据是使用NCBI与SWISS-PROT的细菌数据库来进行比对。申请人经由LC-MS/MS分析的结果而认为该蛋白质为糖基化Cpn60。
2.下面实验中所使用的香蕉抗性淀粉(banana resistant starch)是依据TW 202210086A当中所述的方法而被制得，并且是属于第二型抗性淀粉(resistant starch type 2,RS2)。
3.下面实验中所使用的麦芽糊精(maltodextrin)以及海藻酸钠(sodium alginate)分别是购自于鑫陇兴业有限公司(Sinlong Food's Additive Inc.)以及Sigma-Aldrich。
4.实验动物：
[根据细则26改正24.11.2022]　
下面实验中所使用的雄性BALB/c小鼠(8至10周大)是购自于实验动物中心(中国台湾台北市南港区研究院路一段130巷99号G栋)。所有的实验动物分别地被饲养于一个光照与黑暗各为12小时、室温维持在20-26℃以及相对湿度维持在30-70％的动物房中，而且水分与饲料被充分地 供给。有关实验动物的处理以及一切实验程序均是依据实验动物中心的实验动物照护及使用委员会(Institutional Animal Care and Use Committee,IACUC)的规范来进行。
5.小鼠肝细胞癌细胞系(mouse hepatocellular carcinoma cell line)H22的来源与培养：
下面实验中所使用的小鼠肝细胞癌细胞系H22是购自于Elabscience(Catalog No:EP-CL-0341)。H22细胞被接种在RPMI 1640培养基[添加有10％胎牛血清(fetal bovine serum,FBS)]中，并在培养条件设定为37℃、5％CO 2的培养箱中进行培养。之后，大约每隔2天更换新鲜的培养基。当细胞密度达到约80-90％汇合度(confluence)时，进行细胞继代培养(subculture)。
6.H22细胞-关联性免疫原(H22cell-associated immunogen)的制备：
对H22细胞加入适量的生理食盐水并予以均质化(homogenized)，继而于4℃下以10,000rpm进行离心历时10分钟，由此所形成的上清液被使用作为H22细胞-关联性免疫原并储存于-20℃下备用。
实施例1.口服疫苗(oral vaccine)的制备
首先，将200μL依据上面“一般实验材料”的第6项当中所得到的H22细胞-关联性免疫原与10μg依据上面“一般实验材料”的第1项当中所得到的源自于罗伊氏乳酸杆菌的糖基化Cpn60予以混合均匀，俾以得到一免疫原性组合物(immunogenic composition)。接着，将该免疫原性组合物混合以20μg的10％麦芽糊精以及40μg的20％香蕉抗性淀粉，借此香蕉抗性淀粉形成一内壳(inner shell)并包覆该免疫原性组合物以及麦芽糊精，而得到一初始粒子(initial particle)。
之后，对该初始粒子加入1mL的2％海藻酸钠以及2mL的0.1M氯化钙溶液(calcium chloride solution)并于室温下静置历时30分钟，借此海藻酸钠与氯化钙进行交联反应(cross-linking reaction)以形成一海藻酸钙外壳(calcium alginate outer shell)。该海藻酸钙外壳包覆该初始粒子，而得到一以抗性淀粉为基质的粒子(resistant starch-based particle)，其在下面实施例中被使用作为一试验口服疫苗(test oral vaccine)。
另外，为供比较，参照上面所述的步骤来制备2种比较口服疫苗 (comparative oral vaccine)(也就是比较口服疫苗1与2)以及1种对照口服疫苗(control oral vaccine)，不同处在于：在制备初始粒子时，该比较口服疫苗1仅添加H22细胞-关联性免疫原，而该比较口服疫苗2仅添加源自于罗伊氏乳酸杆菌的糖基化Cpn60，至于该对照口服疫苗则不添加该免疫原性组合物。
所得到的各个口服疫苗被进行冷冻干燥处理历时24小时，由此所形成的冷冻干燥粉末被储存于-20℃备用。
实施例2.本发明的口服疫苗的活体内免疫原性试验(in vivo Immunogenicity assay)
实验方法：
A.口服疫苗的给药(administration of oral vaccine)：
首先，将雄性BALB/c小鼠随机地分成4组(每组n＝5)，其中包括1个对照组、2个比较组(也就是比较组1与2)以及1个实验组。接着，通过管喂(oral gavage)的方式，对比较组1与2的小鼠给药以200μL的比较口服疫苗1与2[配于磷酸盐缓冲生理盐水(phosphate buffered saline,PBS)中]，对实验组的小鼠给药以200μL的本发明的口服疫苗(配于PBS中)，以及对对照组的小鼠给药以200μL的对照口服疫苗(配于PBS中)。各组的小鼠每天被给药一次，给药总共历时4天。
之后，在开始给药后的第20天时，通过管喂的方式，对各组的小鼠进行追加剂(booster doses)的给药，也就是，比较组1与2的小鼠被给药以200μL的比较口服疫苗1与2(配于PBS中)，实验组的小鼠被给药以200μL的本发明的口服疫苗(配于PBS中)，以及对照组的小鼠被给药以200μL的对照口服疫苗(配于PBS中)。各组的小鼠每天被给药一次，给药总共历时4天。
B.血清中IgA以及IgG1的含量分析：
在开始给药前(也就是第0天)以及在开始给药后的第15天与第30天结束时，通过动脉穿刺而从各组小鼠的尾部动脉来采集血液样品。之后，所得到的血液样品在4℃下以12,000rpm进行离心历时5分钟，继而收取上清液作为血清样品。
各个血清样品是分别使用Mouse IgG1 ELISA kit(Elabscience,Cat.No.E-EL-M3035)与Mouse IgA ELISA kit(Elabscience,Cat.No. E-EL-M0690)并依据制造商的操作指南来进行血清中IgG1与IgA含量的测定。简单来说，将50μg/mL H22细胞-关联性免疫原涂覆(coating)于一96-孔培养板的各孔中，继而使用封阻缓冲液(blocking buffer)[含有1％牛血清白蛋白(bovine serum albumin,BSA)的PBST]来进行封阻(blocking)。之后，将各个经稀释的血清样品添加至该96-孔培养板的各孔中来进行反应，继而使用各个试剂盒中所提供的生物素化侦测抗体(biotinylated detection antibody)以及HRP缀合物(HRP conjugate)来进行侦测(detection)，并且使用3,3',5,5'-四甲基联苯胺(3,3',5,5'-Tetramethylbenzidine,TMB)来进行呈色。最后，以一ELISA读取机(ELISA reader)在波长450nm下来测量各孔的吸光值(OD 450)。另外，参照上述方法对一96-孔培养板来进行涂覆及封阻处理，但是不添加生物素化侦测抗体以及HRP缀合物，由此所形成的96-孔培养板被拿来进行相同的吸光值(OD 450)测量，并将所测得的吸光值(OD 450)作为背景值。
相对OD值(relative OD value)是通过将所测得的OD 450数值代入下列公式(I)而被计算出：
其中：
Figure PCTCN2022129153-appb-000004
结果：
图1显示在开始给药后的第30天结束时，在各组小鼠的血清中针对IgA所测得的相对OD值。从图1可见，实验组与比较组2所测得的相对OD值皆明显高于对照组所测得者，而比较组1与对照组间则没有显著的差异性。
图2显示在开始给药后的第30天结束时，在各组小鼠的血清中针对IgG1所测得的相对OD值。从图1可见，实验组所测得的相对OD值明显高于对照组所测得者，而比较组1、比较组2与对照组间则没有显著的差异性。
此外，就实验组小鼠而言，在开始给药后的第30天结束时针对IgG1所测得的相对OD值高于在第0天以及在开始给药后的第15天 结束时所测得者(数据未显示)。这表示本发明的口服疫苗能有效地诱发IgG1生成，并且IgG1含量会随着时间的增加而明显升高。
综合以上的实验结果可知，单独使用H22细胞-关联性免疫原无法诱发抗体生成，而单独使用源自于罗伊氏乳酸杆菌的糖基化Cpn60仅能诱发生成少量的抗体。相反地，依据本发明的免疫原性组合物能够有效地诱发大量生成对于H22细胞-关联性免疫原具有特异性的IgA与IgG1。申请人据此而认为，源自于罗伊氏乳酸杆菌的糖基化Cpn60能够提升免疫原(例如，与肝肿瘤有关的免疫原)的免疫原性(immunogenicity)，因此当其与免疫原一起组合使用时能成功引起免疫反应，并且在疫苗产业上具有优异的发展潜力。
于本说明书中被引述的所有专利和文献以其整体被并入本案作为参考数据。若有所冲突时，本案详细说明(包含界定在内)将占上风。
虽然本发明已参考上述特定的具体例被描述，明显地在不背离本发明的范围和精神下可作出很多的修改和变化。因此意欲的是，本发明仅受如随文检附的权利要求书所示者的限制。

Claims (19)

  1. 一种免疫原性组合物(immunogenic composition)，其特征在于：该免疫原性组合物包含有一源自于罗伊氏乳酸杆菌(Lactobacillus reuteri)的糖基化伴侣蛋白60(chaperonin 60)以及一免疫原(immunogen)。
  2. 根据权利要求1所述的免疫原性组合物，其特征在于：该免疫原是一种与肿瘤有关的免疫原(tumor-associated immunogen)。
  3. 根据权利要求2所述的免疫原性组合物，其特征在于：该肿瘤是选自于由下列所构成的群组：肝细胞癌(hepatocellular carcinoma,HCC)、胆管癌(cholangiocarcinoma)、肝母细胞瘤(hepatoblastoma)、淋巴瘤(lymphoma)、肉瘤(sarcoma)，以及它们的组合。
  4. 根据权利要求3所述的免疫原性组合物，其特征在于：该肿瘤是肝细胞癌。
  5. 根据权利要求2所述的免疫原性组合物，其特征在于：该免疫原是选自于由下列所构成的群组：肝细胞癌细胞-关联性免疫原(hepatocellular carcinoma cell-associated immunogen)、胆管癌细胞-关联性免疫原(cholangiocarcinoma cell-associated immunogen)、肝母细胞瘤细胞-关联性免疫原(hepatoblastoma cell-associated immunogen)、淋巴瘤细胞-关联性免疫原(lymphoma cell-associated immunogen)、肉瘤细胞-关联性免疫原(sarcoma cell-associated immunogen)，以及它们的组合。
  6. 根据权利要求5所述的免疫原性组合物，其特征在于：该免疫原是H22细胞-关联性免疫原。
  7. 一种口服疫苗，其包含有一粒子(particle)，该粒子含有：
    一如权利要求1所述的免疫原性组合物作为核心(core)；
    一包覆该免疫原性组合物的抗性淀粉内壳(resistant starch inner shell)；以及
    一包覆该抗性淀粉内壳的海藻酸钙外壳(calcium alginate outer shell)。
  8. 根据权利要求7所述的口服疫苗，其特征在于：该免疫原是一种与肿瘤有关的免疫原。
  9. 根据权利要求8所述的口服疫苗，其特征在于：该肿瘤是选自于由下列所构成的群组：肝细胞癌、胆管癌、肝母细胞瘤、淋巴瘤、肉瘤，以及它们的组合。
  10. 根据权利要求9所述的口服疫苗，其特征在于：该肿瘤是肝细胞癌。
  11. 根据权利要求8所述的口服疫苗，其特征在于：该免疫原是选自于由下列所构成的群组：肝细胞癌细胞-关联性免疫原、胆管癌细胞-关联性免疫原、肝母细胞瘤细胞-关联性免疫原、淋巴瘤细胞-关联性免疫原、肉瘤细胞-关联性免疫原，以及它们的组合。
  12. 根据权利要求11所述的口服疫苗，其特征在于：该免疫原是H22细胞-关联性免疫原。
  13. 一种如权利要求1至6中任一项所述的免疫原性组合物供应用于制备一用来疫苗接种(vaccination)的医药品的用途。
  14. 根据权利要求13所述的用途，其特征在于：该医药品是一口服疫苗。
  15. 一种如权利要求4所述的免疫原性组合物供应用于制备一用来对抗肝癌的医药品的用途。
  16. 根据权利要求15所述的用途，其特征在于：该医药品是一口服疫苗。
  17. 根据权利要求15所述的用途，其特征在于：该免疫原是H22细胞-关联性免疫原。
  18. 一种对抗肝癌的方法，其包括对一个体投予一如权利要求10所述的口服疫苗。
  19. 一种对抗肝癌的方法，其包括对一个体投予一如权利要求12所述的口服疫苗。
PCT/CN2022/129153 2021-11-04 2022-11-02 免疫原性组合物及其用途 WO2023078279A1 (zh)

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