WO2010124498A1 - Cellule-souche tumorale dont la résistance a été criblée, sa composition antigénique, le chargement de cellules dendritiques antitumorales avec lesdits antigènes, leurs méthodes de préparation, utilisations et kits ainsi qu'un vaccin à base de cellules dendritiques - Google Patents

Cellule-souche tumorale dont la résistance a été criblée, sa composition antigénique, le chargement de cellules dendritiques antitumorales avec lesdits antigènes, leurs méthodes de préparation, utilisations et kits ainsi qu'un vaccin à base de cellules dendritiques Download PDF

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WO2010124498A1
WO2010124498A1 PCT/CN2009/075307 CN2009075307W WO2010124498A1 WO 2010124498 A1 WO2010124498 A1 WO 2010124498A1 CN 2009075307 W CN2009075307 W CN 2009075307W WO 2010124498 A1 WO2010124498 A1 WO 2010124498A1
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WIPO (PCT)
Prior art keywords
stem cells
tumor
tumor stem
cells
ranges
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PCT/CN2009/075307
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English (en)
Inventor
Li Shen
Limin Hao
Yajun Wang
Yanling Ma
Qiming Huang
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Beijing Cellonis Biotechnology Co., Ltd
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Priority claimed from CNA2009100830853A external-priority patent/CN101560496A/zh
Priority claimed from CN200910082778A external-priority patent/CN101538554A/zh
Application filed by Beijing Cellonis Biotechnology Co., Ltd filed Critical Beijing Cellonis Biotechnology Co., Ltd
Publication of WO2010124498A1 publication Critical patent/WO2010124498A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells

Definitions

  • the present invention relates to a dendritic cell with said antigens obtained by pulsing normal dendritic cells with said antigen compositions of said resistance-screened tumor stem cells, its preparation method, use of such dendritic cells for the manufacturing of an anti-tumor medicament, a kit for preparing such dendritic cells, and a vaccine of such dendritic cells.
  • the related art only discloses how to get the related tumor stem cells, but it does not teach any extra operation of the obtained tumor stem cells. Moreover, the above therapies of the related art target tumor stem cells, but they still exist the problems of highly frequent emergences of treatment-resistant, recurrence and metastasis after radiotherapy and chemotherapy.
  • this application also provides a dendritic cell vaccine, which comprises dendritic cells, characterized in that said dendritic cells are the anti-tumor dendritic cells of the present invention.
  • the present invention may be applicable to all kinds of tumors which may be obtained tumor stem cells from, such as, but not limited to: breast cancer, glioma, lung cancer, brain tumor, nasopharyngeal carcinoma, hepatocellular carcinoma, carcinoma ventriculi, colon cancer, melanoma, osteosarcoma, renal cell carcinoma, prostate cancer, ovarian carcinoma, acute myelogenous leukemia, multiple myeloma, pancreatic cancer and metastatic cancer.
  • said metastatic cancer may be selected from metastatic brain cancer, metastatic lung cancer, metastatic liver cancer and metastatic neck cancer.
  • this application also provides a resistance-screened tumor stem cell(s) obtained by any of the above methods.
  • the proteins expressed by tumor stem cells after being screened by any of the methods of this invention are different from the proteins expressed without screening.
  • said screened tumor stem cells specifically express the proteins selected from the group consisting of epithelial growth factor receptor, P-glucoprotein, multidrug-associated protein, protein coded by ataxia-telangiectasia mutation gene (ATM), ataxia-telangiectasia Rad3 related protein and hypoxia inducible factor.
  • the step of the screening may be monitored by determining the specially expressed proteins.
  • Tumor stem cells share the biological properties of normal stem cells of the same type (such as the same phenotypes and the markers), for example, CD34+/CD38- is an antigen present in tumor stem cells obtained from leukemia, CD44+/CD24- is an antigen present in tumor stem cells obtained from breast cancer, CD133+ is an antigen present in tumor stem cells obtained from neuroglioma and melanoma.
  • the kit mentioned above may further comprise unnecessary animal serum albumin.
  • an animal serum or sera
  • said anti-tumor dendritic cells Before the preparation for a dendritic cell vaccine, said anti-tumor dendritic cells may be washed and re-suspended in saline.
  • the number of times for washing said anti-tumor dendritic cells with saline may be decided by determining residual components of medium in washing solution.
  • the amount of residual components of medium is deemed to be suitable to the extent that: when a dendritic cell vaccine of the present invention is administered (injected), residual components of the medium and a chemotherapeutic agent(s) shall not result in any indisposition for patient.
  • the final concentration of basic fibroblast growth factor reaches about 5ng/ml to about 100ng/ml when it is added to the basic medium for tumor stem cells(for example DMEM/F12).
  • the final concentration of insulin reaches about lmg/ml to about 50mg/ml when it is added to the basic medium for tumor stem cells (for example DMEM/F12).
  • the kit further comprises human serum albumin, wherein based on the total volume of said antigen composition, the content of human serum albumin ranges from about 1 percent weight in volume to about 5 percent weight in volume, preferably, from about 2 percent weight in volume to about 3 percent weight in volume.
  • CT, NMR and type-B ultrasonic test they may detect that: the size of the tumor tissue reduces.
  • the supernatant was discarded and the precipitate (pellet) was resuspended in the DMEM/F12 lml/g tissue. Then the resuspended tissue was squeezed through a mesh (BD) with a mesh size of 40 mm. The filtration of the 40-mm mesh can intercept large cell debris and non-digestion cell agglomerates, and leach cell suspension.
  • the passage cells were transferred to a new flask with DMEM/F12 supplemented with 8ng/ml B27, 30ng/ml EGF, 30ng/ml bEGF, and 20mg/ml insulin (The passage cells obtained from one flask may be transferred into several flasks. And the final concentration of the passage cells transferred into a new flask reached 2x 10 5 cells/ml.). According to the passage condition, the cells were passed further 4 th generation and obtained cells of neural sphere.
  • One flask of the tumor stem cells at 5 generation was digested by accutase. After being centrifuged, the cells' precipitate was resuspended in ImI IxPBS. Herein the cells were counted under inverted microscope and resuspended to 2x 10 6 cells/ml in PBS. 50 ⁇ l of the diluted cells suspension was transferred into a centrifuge tube containing FITC-CD44 and 20 ⁇ L PE-CD24, stained for 30mins at 4°C, then washed 3 times in ImI PBS, and then resuspended in 0.5ml PBS. The resultant cells were detected their cytotypes by FACSCalibur Flow cytometry. The result showed that CD44+ and CD24- breast stem cells were 50% of the resuspended cells.
  • the resistant-drug glioma stem cells may be obtained, and grow in the tarceva to the final concentration of 10mg/ml steadily.
  • Example 8 loading DCs with antigen compositions of resistance-screened tumor stem cells
  • 3 xlO 6 immature dendritic cells which were in the medium RPM 11640 supplemented with 5% bovine serum, were pulsed with the antigen composition of tumor stem cells (lysed from Ix IO 6 Of the tumor stem cells, which were not screened or were resistance-screened in example 6), incubated at 37°C in 5% CO 2 incubater for 4 hours.
  • the dendritic cells loaded with antigens were obtained by centrifugation at lOOOrpm for 10 min.
  • the DCs loaded with antigens were washed 3 times with 0.9% saline, were resuspended to I xIO 6 cells/ml, and were stored at 4°C until it was needed.
  • Results represent the mean ⁇ SD of DC treatment group, **P « 0.01 versus control group and # P ⁇ 0.01 versus comparative group.

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  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne des cellules souches dont la résistance a été criblée, leur méthode de préparation, l'utilisation de telles cellules souches tumorales pour la fabrication de médicaments antitumoraux et un kit de préparation desdites cellules souches tumorales. L'invention concerne également une composition antigénique desdites cellules souches tumorales, sa méthode de préparation, l'utilisation d'une telle composition antigénique pour fabriquer des médicaments antitumoraux et un kit de préparation de ladite composition antigénique. L'invention concerne en outre le chargement de cellules dendritiques avec lesdits antigènes obtenus par la pulsion de cellules dendritiques normales avec ladite composition antigénique desdites cellules souches tumorales, sa méthode de préparation, l'utilisation de telles cellules dendritiques pour fabriquer des médicaments antitumoraux, un kit de préparation de telles cellules dendritiques et un vaccin de telles cellules dendritiques. Les médicaments préparés à partir des cellules souches tumorales dont la résistance a été criblée, la composition antigénique de telles cellules souches tumorales et le chargement de cellules dendritiques avec lesdits antigènes procurés par l'invention, peuvent induire une réponse immunitaire in vitro et in vivo pour tuer les cellules souches tumorales qui provoquent la récurrence des tumeurs, ce qui permet de surmonter une résistance tumorale et de guérir radicalement une récurrence tumorale et des métastases.
PCT/CN2009/075307 2009-04-30 2009-12-04 Cellule-souche tumorale dont la résistance a été criblée, sa composition antigénique, le chargement de cellules dendritiques antitumorales avec lesdits antigènes, leurs méthodes de préparation, utilisations et kits ainsi qu'un vaccin à base de cellules dendritiques WO2010124498A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CNA2009100830853A CN101560496A (zh) 2009-04-30 2009-04-30 经耐受性筛选的肿瘤干细胞抗原负载的树突状细胞，其制法、应用、试剂盒及包括其的疫苗
CN200910083085.3 2009-04-30
CN200910082778A CN101538554A (zh) 2009-04-30 2009-04-30 经耐受性筛选的肿瘤干细胞、其制法、应用和试剂盒及其抗原组合物、制法、应用和试剂盒
CN200910082778.0 2009-04-30

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Cited By (8)

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AU2011332020B2 (en) * 2010-11-23 2014-03-13 The Rogosin Institute, Inc. Method for isolating a chemotherapeutic agent resistant cancer cell with stem cell properties
WO2015120198A1 (fr) * 2014-02-05 2015-08-13 Cedars-Sinai Medical Center Procédés et compositions pour le traitement du cancer et de maladies infectieuses
WO2016011347A1 (fr) * 2014-07-17 2016-01-21 The Trustees Of The University Of Pennsylvania Vaccins à base de cellules dendritiques multi-doses prêts à être injectés, et polythérapie anticancéreuse
US20160022789A1 (en) * 2012-08-15 2016-01-28 Neostem Oncology, Llc Individualized high purity colon carcinoma stem cells, methods and use of the same
US20160060599A1 (en) * 2014-08-29 2016-03-03 China Medical University Hospital Dendritic cell tumor vaccine and method for preparing the same
JP2016512423A (ja) * 2013-03-12 2016-04-28 ネオステム オンコロジー リミテッド ライビリティ カンパニー 能動的自家免疫療法のための高純度卵巣癌幹細胞
EP2911748A4 (fr) * 2012-10-24 2016-06-15 Univ Michigan Vaccination et traitement de cellule souche cancéreuse
US10039825B2 (en) 2010-04-20 2018-08-07 Cedars-Sinai Medical Center Combination therapy with CD4 lymphocyte depletion and MTOR inhibitors

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CN101538554A (zh) * 2009-04-30 2009-09-23 北京弘润天源生物技术有限公司 经耐受性筛选的肿瘤干细胞、其制法、应用和试剂盒及其抗原组合物、制法、应用和试剂盒
CN101560496A (zh) * 2009-04-30 2009-10-21 北京弘润天源生物技术有限公司 经耐受性筛选的肿瘤干细胞抗原负载的树突状细胞，其制法、应用、试剂盒及包括其的疫苗

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10039825B2 (en) 2010-04-20 2018-08-07 Cedars-Sinai Medical Center Combination therapy with CD4 lymphocyte depletion and MTOR inhibitors
AU2011332020B2 (en) * 2010-11-23 2014-03-13 The Rogosin Institute, Inc. Method for isolating a chemotherapeutic agent resistant cancer cell with stem cell properties
US20160022789A1 (en) * 2012-08-15 2016-01-28 Neostem Oncology, Llc Individualized high purity colon carcinoma stem cells, methods and use of the same
EP2911748A4 (fr) * 2012-10-24 2016-06-15 Univ Michigan Vaccination et traitement de cellule souche cancéreuse
JP2016512423A (ja) * 2013-03-12 2016-04-28 ネオステム オンコロジー リミテッド ライビリティ カンパニー 能動的自家免疫療法のための高純度卵巣癌幹細胞
WO2015120198A1 (fr) * 2014-02-05 2015-08-13 Cedars-Sinai Medical Center Procédés et compositions pour le traitement du cancer et de maladies infectieuses
US11213583B2 (en) 2014-02-05 2022-01-04 Cedars-Sinai Medical Center Methods and compositions for treating cancer and infectious diseases
WO2016011347A1 (fr) * 2014-07-17 2016-01-21 The Trustees Of The University Of Pennsylvania Vaccins à base de cellules dendritiques multi-doses prêts à être injectés, et polythérapie anticancéreuse
US20160060599A1 (en) * 2014-08-29 2016-03-03 China Medical University Hospital Dendritic cell tumor vaccine and method for preparing the same

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