WO2017071379A1 - Vaccin contre le cancer indiqué pour le traitement du cancer de l'estomac et son procédé de préparation - Google Patents

Vaccin contre le cancer indiqué pour le traitement du cancer de l'estomac et son procédé de préparation Download PDF

Info

Publication number
WO2017071379A1
WO2017071379A1 PCT/CN2016/096035 CN2016096035W WO2017071379A1 WO 2017071379 A1 WO2017071379 A1 WO 2017071379A1 CN 2016096035 W CN2016096035 W CN 2016096035W WO 2017071379 A1 WO2017071379 A1 WO 2017071379A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
gastric cancer
exosomes
tumor
saha
Prior art date
Application number
PCT/CN2016/096035
Other languages
English (en)
Chinese (zh)
Inventor
李淑兰
Original Assignee
李淑兰
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 李淑兰 filed Critical 李淑兰
Priority to CN201680035650.7A priority Critical patent/CN107708728A/zh
Publication of WO2017071379A1 publication Critical patent/WO2017071379A1/fr

Links

Classifications

    • 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
    • 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

Definitions

  • the invention belongs to the field of tumor vaccines, and particularly relates to a tumor vaccine for treating gastric cancer, and a preparation method of the gastric cancer vaccine.
  • Gastric cancer is the second most common cancer in the world. In the past 50 years, although the incidence and mortality of gastric cancer have declined in the global scale, especially in some developed countries, there are still about 1 million new cases every year. Due to the lack of early diagnosis, most patients with symptomatic gastric cancer are advanced at the time of diagnosis. At present, the traditional treatment methods are surgery, chemotherapy and radiotherapy. These treatments are invasive or toxic and side effects, and they can not effectively solve the problems of tumor metastasis and recurrence. Multidrug resistance often occurs after chemotherapy. In short, under traditional treatment, the prognosis of gastric cancer is still poor. The production of malignant tumors is not only due to the malignant transformation of cells, but also because tumor cells can escape the surveillance of the host immune system.
  • Tumor immunotherapy aims to control tumors by regulating the patient's immune system and exerting its own immune surveillance and defense functions.
  • special emphasis has been placed on the development and application of tumor vaccines, which have broad application prospects due to their induction of active specific immune responses.
  • the body's immune system specifically kills tumor cells by identifying antigenic targets of tumor cells.
  • Exosomes are a class of bilayer membranous vesicles that originate in the endocytic system and are excreted outside the cell, between 40 and 100 nm in diameter. Exosomes can be secreted by a variety of cells including dendritic cells, tumor cells, etc., containing a large number of proteins and lipid components closely related to its source and function, as an important carrier of information transmission between cells, participating in a variety of pathophysiological processes. . Tumor cell-derived exosomes contain important immune molecules such as tumor common antigen and thermophilin, which can exhibit anti-tumor effects through various pathways, and as a new type of tumor vaccine, it has obvious advantages over DC vaccine.
  • tumor-derived exosomes can inhibit or even destroy immune cells that play a role in tumors, such as down-regulating the expression of some NK receptors, affecting the activation of some innate immune cells in tumor immunity.
  • Others can significantly inhibit IL-2 and inhibit the proliferation of human lymphocytes, thus playing a negative role in the immunotherapy of tumors.
  • These tumor-derived exosomes may be the key factors for tumor tissue to escape the immune system clearance, which brings many difficulties and challenges to tumor immunotherapy. Therefore, how to improve the immunostimulatory ability of tumor cells derived from exosomes, and reduce its immunosuppressive ability has great practical significance in tumor immunotherapy.
  • a gastric cancer vaccine with improved therapeutic effect comprising an exosomes body secreted by gastric cancer cells, wherein the gastric cancer cells are treated by a combination of Salpichrolide U and SAHA, and the molar ratio of Salpichrolide U to SAHA is 0.8 to 1.2:1. .
  • gastric cancer vaccine with improved therapeutic effect further includes an adjuvant.
  • the adjuvant is an aluminum adjuvant.
  • the preparation method of the gastric cancer vaccine comprises the following steps: gastric cancer cells are cultured in a CO 2 incubator containing DMEM high-sugar medium containing fetal bovine serum, and the cells are monolayer adherent growth, and passaged once every 3 to 4 days. After the cells were grown to log phase, the cells were inoculated. After 24 hours of inoculation, the cells were treated with Salpichrolide U and SAHA. After 24 hours of culture, the culture supernatant was collected and stored at low temperature. The collected culture supernatant of gastric cancer cells was removed by centrifugation.
  • the cells are taken, and the supernatant is removed; the supernatant is removed by centrifugation, the supernatant is collected, concentrated by ultrafiltration, and the concentrate is centrifuged to transfer the separated and purified concentrate to a centrifuge tube, and ultracentrifuged at a low temperature to obtain an exosomes. Small body.
  • the preparation method comprises the steps of: gastric cancer cells are cultured in a DMEM high glucose medium containing 100 ml/L fetal bovine serum in a 50 ml/L CO 2 incubator at 37 ° C, and the cells are monolayer adherent growth. Passage once every 3 to 4 days, inoculate 3 ⁇ 10 6 /100 ml when cells are grown to log phase; use 1 ⁇ 10 -6 mol/L Salpichrolide U and 1 ⁇ 10 -6 mol/L after 24 hours of inoculation The cells were treated with SAHA. After 24 hours of culture, the culture supernatant was collected and stored at 4 ° C.
  • the collected gastric cancer cell culture supernatant was centrifuged for 10 min to remove the cells, and the supernatant was taken.
  • the cell debris was removed by centrifugation at 1500 g for 30 min.
  • the supernatant was concentrated by ultrafiltration through a 100 kU MWCO Centriplus centrifugal ultrafiltration tube, and concentrated at 1500 g for 30 min to obtain a concentrate.
  • the separated and purified concentrate was transferred to a centrifuge tube, and centrifuged at 100 kg for 60 min at a horizontal angle of 4 ° C.
  • the precipitate contains the exosomes body.
  • the gastric cancer vaccine is used in the preparation of a medicament for preventing gastric cancer.
  • nano-sized small vesicle exosomes secreted by untreated gastric cancer cells and soluble immune molecules thereof have significant inhibitory effects on lymphocyte proliferation function.
  • the present invention creatively uses Salpichrolide U and SAHA to treat gastric cancer cells, and the results show that the exosomes secreted by the treated gastric cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition.
  • the invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.
  • MFC cell line was cultured in a culture medium containing 10% fetal bovine serum, penicillin 100 IU/mL, streptomycin 100 ⁇ g/mL (DMEM high sugar), and cultured in a 37 ° C 5% CO 2 incubator. When in the logarithmic phase, inoculate at 3 ⁇ 10 6 /100 ml.
  • Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
  • Experimental grouping The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experiment Group 3 (Salpichrolide U and SAHA combined dosing).
  • control group no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1 ⁇ 10 -6 mol/L 24 h after inoculation, after 24 h of dosing
  • the culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1 ⁇ 10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation.
  • 1 ⁇ 10 -6 mol/L of Salpichrolide U and 1 ⁇ 10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
  • Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
  • Experimental group The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Salpichrolide U and SAHA combined dosing).
  • control group no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1 ⁇ 10 -6 mol/L 24 h after inoculation, after 24 h of dosing
  • the culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1 ⁇ 10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation.
  • 0.8 ⁇ 10 -6 mol/L of Salpichrolide U and 1 ⁇ 10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
  • Salpichrolide U self-made, the preparation method is the same as the literature (Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. origanifolia and S. tristis var. lehmannii, J. Nat. Prod., 2013, 76, 2219-2225) , identified as Salpichrolide U; SAHA was purchased from sigma.
  • Experimental group The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Salpichrolide U and SAHA combined dosing).
  • control group no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was inoculated with Salpichrolide U at a concentration of 1 ⁇ 10 -6 mol/L 24 h after inoculation, after 24 h of dosing
  • the culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1 ⁇ 10 -6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation.
  • 1.2 ⁇ 10 -6 mol/L of Salpichrolide U and 1 ⁇ 10 -6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.
  • HMAC-CP7OG cryogenic ultracentrifuge 100 KU MW COMilliporeAmicon high recovery high flow tangential flow ultrafiltration centrifuge tube (Millipore).
  • Example 7 Detection of proliferation of PBMC cells by H3-TdR incorporation assay
  • Experimental group blank control group (plant lectin and PBMC cells only), exosomes control group (no drug group), exosomes experimental group 1 (dosing Salpichrolide U), exosomes experimental group 2 (dosing SAHA), exosomes experiment Group 3 (Salpichrolide U and SAHA combined dosing), exosomes extracted supernatant control group (no drug group), exosomes extraction supernatant group 1 (dosing Salpichrolide U), exosomes extraction supernatant group 2 ( Dosing SAHA), exosomes extraction supernatant group 3 (Salpichrolide U and SAHA combined dosing), each group of three holes.
  • PBMC cells were added to 5 ml of fresh RPMI 1640 medium containing 10% fetal bovine serum. After cell counting, the dilution was 5000/50 ⁇ l. According to the previous grouping, 50 ⁇ l/well was added to the 96-well plate. At the same time, a mixture of penicillin 100 IU/mL and streptomycin 100 ⁇ g/mL was added in a ratio of 1:1000, 50 ⁇ l of the sample was further added, and finally 100 ⁇ l of IPHA was added.
  • the drug-treated supernatant extracted by exosomes and the PBMC cells co-cultured had little effect on cell proliferation, and there was no statistically significant difference compared with the blank control group (P>0.05), without drug treatment.
  • the supernatant had a significant effect on lymphocyte proliferation, and there was a statistically significant difference compared with the blank control group or the supernatant group (P ⁇ 0.05).
  • the medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.
  • mice healthy Kunming mice, MFC gastric cancer mice, mouse gastric cancer cells (MFC), DMEM high sugar medium (purchased from GIBCO), fetal bovine serum.
  • Example 2 Mouse gastric cancer cell (MFC) culture was carried out according to the above Example 1; the cultured cells were divided into 4 groups, the first group was not administered, the second group was administered Salpichrolide U, the third group The drug SAHA was added, and the fourth group of Salpichrolide U and SAHA was combined.
  • the specific drug treatment was referred to Example 2; the collected cell culture supernatants were prepared according to Example 5 to prepare exosomes tumor vaccine.
  • the tumor inhibition rate (%) (control group tumor size - treatment group tumor size) / Control group tumor size ⁇ 100%.
  • the growth of gastric cancer xenografts was inhibited after treatment with exosomes tumor vaccine.
  • the tumor masses of exosomes group 1, exosomes group 2 and exosomes group 3 were (1.76 ⁇ 0.24) g, (1.67 ⁇ 0.26) g, (0.84 ⁇ 0.17), respectively. g, compared with the blank control group (3.37 ⁇ 0.30) g and the exosomes control group (2.74 ⁇ 0.17) g, the difference was significant (P ⁇ 0.05).
  • the combination of Salpichrolide U and SAHA was the strongest inhibitory effect, and the tumor quality was (0.84 ⁇ 0.17) g, the tumor inhibition rate reached 75.07%.
  • the medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.
  • Adjuvants generally refer to a class of substances that specifically bind to an antigen by physical or chemical means to enhance their specific immunity.
  • the most widely used adjuvant in the world is the aluminum adjuvant.
  • An adsorptive vaccine is formed in a mixture of alumina and aluminum phosphate or in alumina.
  • the aluminum phosphate adjuvant and the aluminum hydroxide adjuvant are positively charged at physiological pH, and the negatively charged protein is adsorbed in the lattice structure of the aluminum salt, and can be used for the treatment of the gastric cancer vaccine in the preparation of the gastric cancer vaccine of the present invention. effect.
  • Liposomes can also be used as adjuvants for the gastric cancer vaccine of the present invention to improve the therapeutic effect of gastric cancer vaccines.
  • the present invention creatively uses Salpichrolide U and SAHA to treat gastric cancer cells, and the results show that the exosomes secreted by the treated gastric cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition.
  • the invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un vaccin contre le cancer indiqué pour le traitement du cancer de l'estomac et son procédé de préparation, le vaccin contre le cancer comprenant des corpuscules exosomes de sécrétions de cellules cancéreuse, lesquelles cellules cancéreuses subissent une multithérapie mettant en oeuvre un salpichrolide U et un SAHA, la concentration molaire entre le salprichrolide U et le SAHA étant de 0,8-1,2 : 1.
PCT/CN2016/096035 2015-10-28 2016-08-19 Vaccin contre le cancer indiqué pour le traitement du cancer de l'estomac et son procédé de préparation WO2017071379A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201680035650.7A CN107708728A (zh) 2015-10-28 2016-08-19 一种用于治疗胃癌的肿瘤疫苗及其制备方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510711700.6A CN105169379A (zh) 2015-10-28 2015-10-28 一种用于治疗胃癌的肿瘤疫苗及其制备方法
CN201510711700.6 2015-10-28

Publications (1)

Publication Number Publication Date
WO2017071379A1 true WO2017071379A1 (fr) 2017-05-04

Family

ID=54892126

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/096035 WO2017071379A1 (fr) 2015-10-28 2016-08-19 Vaccin contre le cancer indiqué pour le traitement du cancer de l'estomac et son procédé de préparation

Country Status (2)

Country Link
CN (2) CN105169379A (fr)
WO (1) WO2017071379A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105169379A (zh) * 2015-10-28 2015-12-23 崔长友 一种用于治疗胃癌的肿瘤疫苗及其制备方法
CN111000867B (zh) * 2020-01-07 2022-04-29 河北医科大学 一种化疗药物作用后的肿瘤细胞上清液的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103432144A (zh) * 2013-09-12 2013-12-11 山东大学 醉茄内酯类化合物在制备治疗或预防肿瘤、神经退行性疾病的药物或保健品中的应用
CN103816535A (zh) * 2014-03-04 2014-05-28 肖文华 一种肿瘤疫苗及其制备方法
CN105169379A (zh) * 2015-10-28 2015-12-23 崔长友 一种用于治疗胃癌的肿瘤疫苗及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103432144A (zh) * 2013-09-12 2013-12-11 山东大学 醉茄内酯类化合物在制备治疗或预防肿瘤、神经退行性疾病的药物或保健品中的应用
CN103816535A (zh) * 2014-03-04 2014-05-28 肖文华 一种肿瘤疫苗及其制备方法
CN105169379A (zh) * 2015-10-28 2015-12-23 崔长友 一种用于治疗胃癌的肿瘤疫苗及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NICOTRA, V.E. ET AL.: "Withanolides with Phytotoxic Activity from Two Species of the Genus Salpichroa: S. Origanifolia and S. Tristis Var. Lehmannii", JOURNAL OF NATURAL PRODUCTS, vol. 76, no. 12, 4 December 2013 (2013-12-04), pages 2219 - 2225, XP055378989 *

Also Published As

Publication number Publication date
CN107708728A (zh) 2018-02-16
CN105169379A (zh) 2015-12-23

Similar Documents

Publication Publication Date Title
WO2021174738A1 (fr) Nanoparticule bionique recouverte d'une membrane de cellules souches mésenchymateuses avec surface surexprimant la molécule pd-l1, sa préparation et son application
WO2018041261A1 (fr) Médicament thérapeutique antitumoral
WO2018120683A1 (fr) Liquide d'injection à composants multiples
US11154571B2 (en) Exosomes sourced from granulocytic myeloid-derived suppressor cells and application thereof
CN107488235A (zh) 一种新的增强型抗原联合多肽诱导肝癌特异性ctl细胞的制备及应用
WO2017071380A1 (fr) Vaccin contre le cancer indiqué pour le traitement du cancer du foie et son procédé de préparation
KR20100109099A (ko) 암 특이 면역반응 유도능을 갖는 수지상 세포의 제조 방법,이 수지상 세포를 포함하는 암의 예방, 치료, 또는 전이 억제용 약제학적 조성물 및 키트
WO2017071379A1 (fr) Vaccin contre le cancer indiqué pour le traitement du cancer de l'estomac et son procédé de préparation
CN103861107B (zh) 一种药物组合物及其用途
CN103816535B (zh) 一种肿瘤疫苗及其制备方法
WO2017147894A1 (fr) Composition améliorant la capacité à tuer des cellules anormales, et utilisation associée
WO2017079881A1 (fr) Procédé permettant d'améliorer la capacité de tuer des cellules anormales et composition pharmaceutique
AU2016390488A1 (en) Application of dimethylamino micheliolide
CN117511886A (zh) 冷冻休克处理的装载减毒沙门氏菌的单核细胞或巨噬细胞的制备方法及其应用
Ding et al. Photosensitive small extracellular vesicles regulate the immune microenvironment of triple negative breast cancer
CN116948901A (zh) 食窦魏斯氏菌d-2胞外多糖在抑制结肠癌细胞中的应用
CN103610699A (zh) 一种蛞蝓的提取方法及其抗肺癌应用
KR20210016995A (ko) 방사선 조사된 암 세포 유래 엑소좀, 이를 이용하여 획득된 성숙 수지상 세포를 포함하는 암 치료용 백신 및 이의 제조방법
CN105031631A (zh) 一种HLA-A0201限制性抗Sox2特异性CTL的制备方法及其应用
WO2014139456A1 (fr) Application de l'hydroxyde d'aluminium dans la préparation d'un médicament pour le traitement du cancer du foie
WO2021032179A1 (fr) Application d'un vaccin contre pseudomonas aeruginosa dans le traitement d'une infection associée à une lésion de brûlure ou d'échaudure
Cui et al. Killing three birds with one stone: Tumor-membrane-decorated Prussian blue nanovaccines for synergistic management of skin tumors, radiation dermatitis and wounds
CN113041345A (zh) 一种纳米类毒素疫苗及其用途
CN111407784A (zh) 一种从骆驼蓬中制备pd-1/pd-l1抑制剂的方法
CN111358859A (zh) 一种从红枸杞中制备pd-1/pd-l1抑制剂的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16858816

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16858816

Country of ref document: EP

Kind code of ref document: A1