WO2021123320A1 - Composition et son utilisation pour une thérapie universelle contre les cellules tumorales - Google Patents

Composition et son utilisation pour une thérapie universelle contre les cellules tumorales Download PDF

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WO2021123320A1
WO2021123320A1 PCT/EP2020/087239 EP2020087239W WO2021123320A1 WO 2021123320 A1 WO2021123320 A1 WO 2021123320A1 EP 2020087239 W EP2020087239 W EP 2020087239W WO 2021123320 A1 WO2021123320 A1 WO 2021123320A1
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seq
fragment
protein
sequence
cells
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PCT/EP2020/087239
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Behnam NADERI-KALALI
Walter MIEDL
Amrollah MOSTAFAZADEH-MALEH
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Dr. Kalali Prof. Mostafazadeh Dipl. Ing. Miedl (Kmm) Gesellschaft Bürgerlichen Rechts
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Publication of WO2021123320A1 publication Critical patent/WO2021123320A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1754—Insulin-like growth factor binding proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4743—Insulin-like growth factor binding protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24024—Gelatinase A (3.4.24.24), i.e. matrix metalloproteinase 2 or MMP2

Definitions

  • the present invention relates to a composition, a cell supernatant comprising the same, a pharmaceutical composition comprising the same, and a method of preparing the same.
  • Tumorigenesis is a (multi) complex biological process in which different types of cells contribute in an orchestral manner (Cao, et al, 2017).
  • This cellular network consists of malignant cells, endothelial cells, immune cells, and fibroblasts, among of which non-tumor cells play different roles but orchestra to angiogenesis and tumor metastasis, and fibroblasts of stromal cells play an important role in angiogenesis (cancer-associated fibroblast) (Cao et al, 2017; Kalluri, et al, 2016).
  • fibroblasts In addition to collagen synthesis, fibroblasts have secretory phenotype releasing different compounds into the extracellular matrix, each of which can have important roles at all stages of cancer progression, wound healing and regeneration (Kalluri, et al, 2016). Cancer stem cells are among a few cancer cells surviving during different steps of metastasis and have high potential in initiating and promoting metastasis (Zhang et al, 2018). Moreover, serum starvation has been recently considered as a model for a biological study on tumor (Tian et al, 2017; Netherlands et al, 2017).
  • Tissue specific stem cells are main stem cells in tissues hemostasis. These undifferentiated cells characterized by self-renewing, rendering them a prolonged lifespan which make these parent cells prone or susceptible to a variety set of intrinsic and extrinsic mutagenic factors transforming these cells to a special type of tumorigenic stem cells. Accumulating data show that these cells are resistance to radiotherapy and chemotherapy, causing the relapse of cancer related diseases after treatment with such current available cancer therapy.
  • CSCs cancer stem cells
  • Hedgehog, and Wnt signaling pathways that may be dysregulated in normal breast stem cells and consequently differentiated into CSCs (Chatterjee & Sil, 2019; Munoz, lliou, & Esteller, 2012), some transcriptional factors including octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), the homeobox domain transcription factor Nanog and C-myc also play role in this differentiation (Wan, 2019; Yamanaka, 2008).
  • Oct4 octamer-binding transcription factor 4
  • Sox2 sex determining region Y-box 2
  • Nanog sex determining region Y-box 2
  • C-myc also play role in this differentiation (Wan, 2019; Yamanaka, 2008).
  • Oct4 and Sox2 have a key role in maintaining and inducing pluripotency (Esch et al., 2013; Yilmaz & Benvenisty, 2019), and also contribute in tumor growth and metastasis (Munro, Wickremesekera, Peng, Tan, & Itinteang, 2018; Sarkar & Hochedlinger, 2013).
  • Nanog is considered as a key regulator of embryonic development and cellular reprogramming, and takes part in self-renewal of CSCs (Das et al., 2019; Jeter, Yang, Wang, Chao, & Tang, 2015), C-myc is an oncoprotein with various roles in apoptosis, division and differentiation of cell (van Schaijik, Davis, Wickremesekera, Tan, & Itinteang, 2018). Previous studies showed that the expression of Yamanaka factors and Nanog are associated with CSCs so that the decrease in this expression leads to the differentiation of the CSCs to the cancer cells (Leis et al., 2012; Xiang et al., 2011; You, Guo, & Huang, 2018).
  • Downregulation of pluripotency transcription factors can be carried out through different signaling pathways including the Wnt/ -catenin, Shh (sonic hedgehog), Notch/y-secretase/Jagged and BMP (Czerwinska and Kaminska, 2015).
  • Shh sonic hedgehog
  • Notch/y-secretase/Jagged BMP (Czerwinska and Kaminska, 2015).
  • this suppression is carried out through DNA methylation and histone modeification in the promotor region of these genes.
  • overexpression of C-myc can be replaced by Nanog and be dispensable.
  • Oct4, Sox2, and Nanog are regarded as the main regulators of pluripotency. Injection of 16SFS into the tumor region causes reduction of sternness and cell growth and subsequently induces differentiation, apoptosis and necrosis.
  • targeting CSCs in sense of causing apoptosis or promoting differentiation would provide an approach for treating tumors and/or cancers.
  • the replicative/ functionally active cells e.g. neonatal fibroblasts or human PBMCs
  • PBMCs reprogrammed their metabolism to aerobic glycolysis under serum starvation conditions (Warburg effect).
  • these cells in this circumstance produce proteins which are able to induce differentiation of CSCs and cause apoptosis as well as necrosis in tumor cells and particularly in CSCs.
  • the present invention provides a composition which comprises Pigment epithelium-derived factor protein having a sequence of SEQ ID NO: 1 or a fragment thereof, Thrombospondin-2 protein having a sequence of SEQ ID NO: 2 or a fragment thereof, and/or Metalloproteinase inhibitor 2 protein having a sequence of SEQ ID NO: 3 or a fragment thereof, and/or Insulin-like growth factor-binding protein 4 having a sequence of SEQ ID NO: 4 or a fragment thereof.
  • Said composition can be obtained under certain starvation condition which acts against tumor cells especially CSCs.
  • said composition further comprises Uveal autoantigen protein with coiled-coil domains and ankyrin repeats having a sequence of SEQ ID NO: 5 or a fragment thereof, and/or Insulin-like growth factor-binding protein 5 having a sequence of SEQ ID NO: 6 or a fragment thereof.
  • above composition or above preferred composition further comprises one or more of proteins or fragments thereof selected from a group consisting of: Stromelysin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 7 or
  • Metalloproteinase inhibitor 1 protein having a sequence of SEQ ID NO: 9 or a fragment thereof, Pentraxin-related protein PTX3 having a sequence of SEQ ID NO: 10 or a fragment thereof, Cathepsin D protein having a sequence of SEQ ID NO: 11 or a fragment thereof, Follistatin- related protein 1 having a sequence of SEQ ID NO: 12 or a fragment thereof, Fibulin-1 protein having a sequence of SEQ ID NO: 13 or a fragment thereof, Endosialin protein having a sequence of SEQ ID NO: 14 or a fragment thereof, Inter-alpha-trypsin inhibitor heavy chain H2 protein having a sequence of SEQ ID NO:
  • SPARC protein having a sequence of SEQ ID NO: 16 or a fragment thereof,
  • Growth-regulated alpha protein having a sequence of SEQ ID NO: 17 or a fragment thereof, Insulin-like growth factor-binding protein 6 having a sequence of SEQ ID NO: 18 or a fragment thereof, Protein NOV homolog having a sequence of SEQ ID NO: 19 or a fragment thereof, Follistatin protein having a sequence of SEQ ID NO: 20 or a fragment thereof, Alpha-enolase protein having a sequence of SEQ ID NO: 17 or a fragment thereof, Insulin-like growth factor-binding protein 6 having a sequence of SEQ ID NO: 18 or a fragment thereof, Protein NOV homolog having a sequence of SEQ ID NO: 19 or a fragment thereof, Follistatin protein having a sequence of SEQ ID NO: 20 or a fragment thereof, Alpha-enolase protein having a sequence of SEQ ID NO: 17 or a fragment thereof, Insulin-like growth factor-binding protein 6 having a sequence of SEQ ID NO: 18 or a fragment thereof, Protein NOV homolog having a sequence of SEQ ID
  • Tumor necrosis factor-inducible gene 6 protein having a sequence of SEQ ID NO: 21 or a fragment thereof, Tumor necrosis factor-inducible gene 6 protein having a sequence of SEQ ID NO: 21 or a fragment thereof, Tumor necrosis factor-inducible gene 6 protein having a sequence of SEQ ID NO: 21 or a fragment thereof, Tumor necrosis factor-inducible gene 6 protein having a sequence of SEQ
  • the composition of the present invention not only induces differentiation and down regulates the stem cell (sternness marker) but also induces apoptosis-dependent necrosis in tumor cells. Moreover, the composition of the present invention inhibits tumor cells migration (metastasis) and their growth.
  • the gene expression levels of stem cell markers including Oct4, Sox2, Nanog, and C- myc in breast CSCs (LA7) were evaluated during 24 h, 72 h and 7 days of in vitro exposure to FF, P14, and 16h-SFS; with the expectation of decreasing the expression of these markers and consequently increasing the differentiation of CSCs.
  • cell migration and cell growth was investigated in vitro. The in vivo effect has been investigated in Rats and ex vivo experimental proof of concept has been demonstrated using human breast cancer cells isolated from human tumors compared to healthy cells.
  • the term “comprise”, “comprises” or “comprising”, should be understood as the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step.
  • the term “consisting of” is a particular embodiment of the term “comprise”, indicating that any other non-stated member, integer or step is excluded.
  • the term “comprise” encompasses the term “consist of”.
  • the term “comprising” encompasses “including” as well as “consisting”, for example, a composition “comprising” X may consist exclusively of X or may include something additional, for example, X + Y.
  • the first aspect of the present invention relates to a composition
  • a composition comprising: Pigment epithelium- derived factor protein having a sequence of SEQ ID NO: 1 or a fragment thereof, Thrombospondin-2 protein having a sequence of SEQ ID NO: 2 or a fragment thereof, and/or Metalloproteinase inhibitor 2 protein having a sequence of SEQ ID NO: 3 or a fragment thereof, and/or Insulin-like growth factor binding protein 4 having a sequence of SEQ ID NO: 4 or a fragment thereof.
  • the composition in the present invention comprises Pigment epithelium-derived factor protein having a sequence of SEQ ID NO: 1 or a fragment thereof, Thrombospondin-2 protein having a sequence of SEQ ID NO: 2 or a fragment thereof, Metalloproteinase inhibitor 2 protein having a sequence of SEQ ID NO: 3 or a fragment thereof, and Insulin-like growth factor-binding protein 4 having a sequence of SEQ ID NO: 4 or a fragment thereof.
  • composition in the present invention disclosed above further comprises: Uveal autoantigen protein with coiled-coil domains and ankyrin repeats having a sequence of SEQ ID NO: 5 or a fragment thereof, and/or Insulin-like growth factor-binding protein 5 having a sequence of SEQ ID NO: 6 or a fragment thereof.
  • composition in the present invention disclosed above further comprises one or more of proteins or fragments thereof selected from a group consisting of: Stromelysin-1 protein having a sequence of SEQ ID NO: 7 or a fragment thereof, Thrombospondin-1 protein having a sequence of SEQ ID NO: 8 or a fragment thereof, Metalloproteinase inhibitor 1 protein having a sequence of SEQ ID NO:
  • Pentraxin-related protein PTX3 having a sequence of SEQ ID NO: 10 or a fragment thereof
  • Cathepsin D protein having a sequence of SEQ ID NO: 11 or a fragment thereof
  • Follistatin-related protein 1 having a sequence of SEQ ID NO: 12 or a fragment thereof, Fibulin-1 protein having a sequence of SEQ ID NO: 13 or a fragment thereof, Endosialin protein having a sequence of SEQ ID NO: 14 or a fragment thereof, Inter-alpha-trypsin inhibitor heavy chain H2 protein having a sequence of SEQ ID NO: 15 or a fragment thereof, SPARC protein having a sequence of SEQ ID NO: 16 or a fragment thereof, Growth-regulated alpha protein having a sequence of SEQ ID NO: 17 or a fragment thereof, Insulin-like growth factor-binding protein 6 having a sequence of SEQ ID NO: 18 or a fragment thereof, Protein NOV homolog having a sequence of SEQ ID NO: 19 or a fragment thereof, Fol listatin protein having a sequence of SEQ ID NO: 20 or a fragment thereof, Alpha-enolase protein having a sequence of SEQ ID NO: 21 or a fragment thereof, Tumor necrosis factor-inducible gene 6 protein
  • fragment refers to an analogue of the corresponding protein.
  • the above-disclosed fragments comprise an amino acid sequence which shows an amino acid identity of more than 70%, preferably more than 75%, more preferably more than 80% to the full-length of the corresponding protein, or shows an amino acid identity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the full- length of the corresponding protein.
  • fragment refers to a polypeptide comprising the one or more of the conserved domains of the corresponding protein.
  • fragment refers to a polypeptide comprising at least a region which shows an amino acid identity of more than 70%, preferably more than 75%, more preferably more than 80% to one of the conserved domains of the corresponding protein, or shows an amino acid identity of 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of the conserved domains of the corresponding protein.
  • one or more of proteins or fragments thereof included therein have an origin of vertebrate, e.g. human, nonhuman primates, rabbit, mouse, rat, cow, bird, sheep, goat, cat, dog, pig, or horse cells, preferably mammal, more preferably human.
  • vertebrate e.g. human, nonhuman primates, rabbit, mouse, rat, cow, bird, sheep, goat, cat, dog, pig, or horse cells, preferably mammal, more preferably human.
  • composition disclosed above are for use as a medicament, optionally for use in the treatment of tumor and/or cancer, further optionally for use in the treatment of breast tumor and/or cancer.
  • tumors included in the present invention comprise sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the antibodies of the disclosure may be used to delay development of a disease or disorder or to slow the progression of a disease or disorder.
  • treating refers to improving, alleviating, and/or decreasing the severity of one or more symptoms of a condition being treated.
  • treating cancer refers to improving the patient's condition, alleviating, delaying or slowing progression or onset, decreasing the severity of one or more symptoms of cancer.
  • treating cancer includes any one or more of: decreasing tumor size, decreasing rate of tumor size increase, halting increase in size, decreasing the number of metastases, decreasing pain, increasing survival, and increasing progression free survival.
  • a therapeutically effective amount of the above-disclosed composition may be administered orally or non-orally e.g. by injection to a subject or a patient.
  • Oral administration includes but is not limited to: topical administration, or parenteral administration, or combinations thereof.
  • Administration by injection includes but is not limited to: via the intramuscular or intraperitoneal route, or the composition of the present invention can be injected directly into the target tumor.
  • a "subject”, “individual” or “patient” may be a vertebrate, such as a human. The vertebrate may be a mammal.
  • a “therapeutically effective amount” of the composition in the present invention is variable according to factors such as age, sex, the disease state, weight of the subject, and the ability of the substance/molecule, to elicit a desired response in the subject.
  • a therapeutically effective amount may encompass an amount in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • the second aspect of the present invention relates to a cell supernatant comprising the composition disclosed above.
  • the cells from which the supernatant is derived in the present invention can be primary cells or secondary cells.
  • the cells from which the supernatant is derived in the present invention may include but not be limited to: keratinocytes, fibroblasts, adipocytes, epithelial cells (including but not limited to: intestinal epithelial cells, mammary epithelial cells), neural cells, glial cells, endothelial cells, muscle cells, hepatocytes, buccal mucosa cells, hepatic cells, blood cells and immune cells (including but not limited to: bone marrow cells, cells in mononuclear cord blood, and peripheral blood mononuclear cells including lymphocytes, T-cells, B-Cells, and NK cells), stem cells including induced pluripotent stem cells (iPS cells), precursors of these cell types, or even HeLa, MCF-7, or other cancer cells.
  • epithelial cells including but not limited to: intestinal epithelial cells, mammary epithelial cells
  • neural cells including but not limited to: intestinal epithelial cells, mammary epitheli
  • the cells from which the supernatant is derived in the present invention preferably has an origin of vertebrate, e.g. human, nonhuman primates, rabbit, mouse, rat, cow, bird, sheep, goat, cat, dog, pig, horse, preferably mammal, more preferably human.
  • vertebrate e.g. human, nonhuman primates, rabbit, mouse, rat, cow, bird, sheep, goat, cat, dog, pig, horse, preferably mammal, more preferably human.
  • the cells from which the supernatant is derived in the present invention can be primary cells isolated from a vertebrate, e.g. human, nonhuman primates, rabbit, mouse, rat, cow, bird, sheep, goat, cat, dog, pig, horse, preferably from a mammal, more preferably from a human, or can be secondary cells derived from said primary cells
  • the cells from which the supernatant is derived in the present invention are fibroblast cells, more preferably are human neonatal fibroblast cells.
  • the cells from which the supernatant is derived in the present invention are peripheral blood mononuclear cells, more preferably are lymphocytes, even more preferably are human lymphocytes.
  • the supernatant of the present invention is obtained after the cell undergoes a starvation treatment.
  • the time period for starvation can be 8-112 hours, preferably 16-96 hours, more preferably 24-88 hours, even more preferably 32, 40, 48, 56, 64, 72, 80 or 104 hours.
  • the inventors found that the starvation time is correlated with the desired effects of the present invention, and a starvation period of 16-96 hours is connected with a remarkable anti-cancer effect of the cell supernatant.
  • the cells can be sub-cultured for 2 to 20 times, preferably 3 to 18 times, or 4-18 times, more preferably 3 to 16 times, or 6-16 times, even more preferably 3 to 5 times, and also can be 8-14 times, or 9, 10, 11, 12, 13, or 15 times.
  • the time of said sub-culture can be determined by investigating the growth speed of cells. The inventors found that once the growth speed of cells is significantly slowed down, then the cells are ready for undergoing starvation treatment. The times of sub-culture is also correlated with the desired effects of the present invention. The inventors found that with 3 to 5 times of passage, the anti-cancer effect of the cell supernatant is even better.
  • the cell supernatant of the present invention can be used as a medicament.
  • the cell supernatant of the present invention is for use in the treatment of tumor and/or cancer, including (but not limited thereto) sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocar
  • the cell supernatant in the present invention is for use in the treatment of breast tumor and/or cancer.
  • the third aspect of the present invention is to provide a pharmaceutical composition comprising the composition disclosed above, or the cell supernatant disclosed above.
  • the pharmaceutical composition in the present invention contains pharmaceutically acceptable carriers and/or excipients including but not being limited to: stabilizing agents, surface-active agents, salts, buffers, coloring agents etc.
  • the pharmaceutical composition in the present invention is in the form of a kit of parts.
  • the pharmaceutical composition of the present invention can be for use in the treatment of tumor and/or cancer which can be selected from the list disclosed above with regard to the composition for use or the cell supernatant for use.
  • the pharmaceutical composition is for use in the treatment of breast tumor and/or cancer.
  • the fourth aspect of the present invention is to provide an ex vivo method of preparing the composition of the present invention, comprising starving cells in culture for a determined time period, wherein said composition is contained in the cell supernatant after said starvation treatment.
  • the cells can be primary cells or secondary cells.
  • the cells may include but not be limited to: keratinocytes, fibroblasts, adipocytes, epithelial cells (including but not limited to: intestinal epithelial cells, mammary epithelial cells), neural cells, glial cells, endothelial cells, muscle cells, hepatocytes, buccal mucosa cells, hepatic cells, blood cells (including but not limited to: lymphocytes, bone marrow cells, cells in mononuclear cord blood, peripheral blood mononuclear cells), immune cells (including but not limited to: T-cells, B-Cells), stem cells including induced pluripotent stem cells (iPS cells), or precursors of these cell types.
  • epithelial cells including but not limited to: intestinal epithelial cells, mammary epithelial cells
  • neural cells including but not limited to: intestinal epithelial cells, mammary epithelial cells
  • glial cells including endothelial cells
  • muscle cells glial cells
  • the cells preferably have an origin from a vertebrate, e.g. human, nonhuman primates, rabbit, mouse, rat, cow, bird, sheep, goat, cat, dog, pig, horse, preferably from a mammal, more preferably from a human.
  • the cells can be primary cells isolated from a vertebrate, e.g. human, nonhuman primates, rabbit, mouse, rat, cow, bird, sheep, goat, cat, dog, pig, horse, preferably from a mammal, more preferably from a human, or can be secondary cells derived from said primary cells.
  • the cells are fibroblast cells, more preferably are human neonatal fibroblast cells.
  • the time period can be 8-112 hours, preferably 16-96 hours, more preferably 24-88 hours, even more preferably 32, 40, 48, 56, 64, 72, 80 or 104 hours.
  • the cells Before undergoing starvation treatment, the cells can be sub-cultured for 2 to 20 times, preferably 4 to 18 times, more preferably 6 to 16 times, even more preferably 3 to 5 times, and also can be , 8 to 14 times, 9, 10, 11, 12, 13, or 15 times.
  • the time of said sub-culture can be determined by investigating the growth speed of cells. The inventors found that once the growth speed of cells is significantly slowed down, then the cells are ready for undergoing starvation treatment. The times of sub-culture is also correlated with the desired effects of the present invention. The inventors found that with 8 to 14 times of passage, the anti-cancer effect of the cell supernatant is good. In this regard, the inventors found that with 3 to 5 times of passage, the anti-cancer effect of the cell supernatant is even better.
  • the fifth aspect of the present invention is to provide a cell supernatant obtained by the above disclosed ex vivo method.
  • Fig. 1 Morphology of fibroblasts in passages 3 (a) and 14 (b). Compared to fibroblasts at passage 3, the fibroblasts at passage 14 exhibited some morphological changes such as enlarged cytoplasm with increased levels of granules. Moreover, the nucleus appeared more obviously, together indicated that the cells entered to senescent phase based on the previous study (Lipetz & Cristofalo, 1972). Magnification: 100X and 200X for photomicrographs (a) and (b) respectively.
  • Fig. 2 Cytotoxicity evaluation of the three biological fluids including 16h-starved fibroblasts supernatant (16h-SFS), senescent fibroblast fluid (P14) and ovarian follicular fluid (FF) on LA7 cell line using MTT assay after 24 h (A), 48 h (B) and 72 h (C). These fluids were diluted in DMEM before treatment. The dilution factors were 1/1, 1/2, 1/4, 1/8, 1/16 and 1/32 Data are shown as mean ⁇ SD (three replicates). *: indicates P ⁇ 0.05 and, ***: indicates P ⁇ 0.001. P ⁇ 0.05 was considered as significant level statistically.
  • Fig. 3 Doubling time alterations in LA7 treated for 72 h with control medium and three biological fluids. All fluids increased the LA7 doubling time value significantly. ***: indicates P ⁇ 0.001. Cont; control (medium + 10% FBS), 16h-SFS; 16h starved fibroblasts culture supernatant, P14; supernatant of fibroblasts at passage 14, FF; ovarian follicular fluid.
  • Fig. 4 LA7 acridine orange / ethidium bromide staining.
  • the cells were treated for 24 h with; A and Al) control medium (DMEM+10%FBS), B and Bl) FF (ovarian follicular fluid), C and Cl) P14 (fibroblasts culture supernatant at passage 14), D and Dl) 16h-SFS (16 h starved fibroblasts culture supernatant).
  • A- D are LA7 cells before staining and Al-Dl are those cells after staining. All treated cells exhibited some degrees of both apoptosis and necrosis. Magnification; 200 X for A-D and 100X for Al-Dl.
  • Fig. 5 Annexin V-PE/7-AAD staining and flow cytometry analysis.
  • A) Forward scatter -height (FSC-H) and side scatter-height (SSC-H) of LA7 cells and the gated cells.
  • DMEM+10%FBS control medium
  • P14 fibroblasts culture supernatant at passage 14
  • FF ovarian follicular fluid
  • 16h-SFS (16 h starved fibroblasts culture supernatant
  • Fig. 6 LA7 scratch test results. Analysis of the migration potential in LA7 cells exposed to the three biological fluids in time 0, 24 h and 48 h. C; control (medium + 10% FBS), FF; ovarian follicular fluid, P14; supernatant of fibroblast at passage 14, 16h-SFS;16 h starved fibroblasts culture supernatant, all indicated fluids were able to suppress the cells migration after 48 h. Magnification:40X.
  • Fig. 7 Expression of C-myc, Nanog, Oct4, and Sox2 in LA7 cells exposed to 16h-SFS with dilutions of 1, 1/16 and 1/32 for 24 h, 72h and 7 days. *: indicates p ⁇ 0.05.
  • Fig. 8 Expression of C-myc, Nanog, Oct4, and Sox2 in LA7 cells exposed to senescent fibroblast fluid with dilutions 1, 1/8, and 1/16 for 24 h, 72 h and 7 days of exposure. *: indicates p ⁇ 0.05.
  • Fig. 9 Expression of C-myc, Nanog, Oct4, and Sox2 in LA7 cells exposed to FF with dilutions 1, 1/2 and 1/16 for 24h, 72h and 7 days of exposure. *: indicates p ⁇ 0.05.
  • Fig. 10 Viability of MCF10A and MCF7 cells.
  • Tumorigenic and non-tumorigenic human breast cell lines (MCF7 and MCF10A) were cultured to a confluency of around 80%. Subsequently, cells were treated with medium, 16SFS and 16SFS complemented with FCS. MTT assay was performed to analyze the effect of treatment on viability of the cells.
  • Fig. 11 Tumors size and animals weight in test and control groups (a) The volume of tumor after injection of LA7 and different solutions, (b) Alteration of rat's weight between three groups after injection of LA7 and different solutions. The different letters show significant difference between groups (p ⁇ 0.05).
  • Fig. 12 Tumors X-ray imaging. Alteration in the volume of tumors was confirmed by X-ray images, a) Normal saline group, b) DMEM group c) 16h-SFS group. Maximum size was measured in DMEM and minimum size in 16h-SFS groups.
  • Fig. 13 Flistology of breast cancer in rats, a): Low mitosis (arrow), b) High mitosis (arrow), c) Low vascularity (arrow), d) High vascularity (arrow), e) Monomorphic nuclear cells (MNs). f) Polymorphic nuclear cells (PMNSs) (arrow), g) Necrosis (arrow). The magnification of a-d and g is x400 and e-f is xlOOO. H& E staining was used.
  • Fig. 14 Histology of breast cancer in rat. a): Cytokeratin positive, b) Estrogen receptor- negative (arrow), c) Mast cells. The magnification of a-b is x400 and c is xlOOO. Immunohistochemical staining was used in images of a-b and Giemsa staining used in image of c.
  • Fig. 15 Effects of different solutions on angiogenesis and mitosis in LA7 induced tumors
  • HPF High-power field.
  • Fig. 16 Alterations in the expression of Nanog, C-myc, Oct-4, and Sox2 in the breast tumors from rat exposed to normal saline or DMEM or 16h-SFS. Different letters show significant difference in each group (P ⁇ 0.05).
  • Fig. 17 Alterations in the rat weight in different groups at the first day of experiment to the 60th day. Apart from 8th day, the weights of rats increased significantly. Data are shown as meaniSD.
  • Fig. 18 Histopathology of the intestine stained with H&E in the different groups, (a) (Normal saline): EP (Epithelium), LG (Lieberkuhn's gland), M (muscle), SM (submucosal), (b) (Normal saline + LA7): EL (epithelium loss),), (c) (DMEM): EL (epithelium loss), (H (villous edema), (d) (LA7+DMEM):, EL (epithelium loss), H (villous edema), (e) (LA7+16SFS):, loss of villi, (f) (16h-SFS): EL (epithelium loss), H (villious edema, Scale: 2oo pm, x29. The most abundant of damages was observed in 16h- SFS group.
  • Fig. 19 The average length of intestinal villi in different groups measured by Motic images Plus 2.0 ML. The most abundant change in the length of villi compared to normal saline group was observed in normal saline + LA7 group. Data are shown as mean ⁇ SD.
  • Fig. 20 Histopathology of the liver stained with H&E and Masson's trichrome in the different groups, (a) (DMEM +LA7; Masson's trichrome): p (pyknosis), v (vacuoles), bv (congestion), (b) (16h-SFS +LA7; Masson's trichrome): p (pyknosis), f (fibrosis), b (congestion in and around the central vein, (c) (16h-SFS; H&E): i (inflammation), bv (hyperemia), (d) (16h-SFS; Masson's trichrome):, (e) (Normal saline; H&E): without damage, (f) (Normal saline; Masson's trichrome): without damage.
  • DMEM +LA7; Masson's trichrome p (pyknosis), v (vacuoles), bv (
  • FIG. 21 Histopathology of the lung stained with H&E and Masson's trichrome in the different groups, (a) (DMEM+La7; H&E): at (alveolar wall thickening), fc (foam cell), ia (inflammation of alveolar wall), ba (congestion in alveolar vessels), (b) (16h-SFS; H&E): ta (thickening of alveolar wall), bv (venous congestion), iv (sever inflammation around the vein), (c) (normal saline +LA7; H&E): at (alveolar wall thickening), i (inflammation), dbc (degeneration of bronchial wall), ibc (inflammatory cells in bronchial secretion), (d) (normal saline +LA7; Masson's
  • Fig. 22 Blood biochemical and cellular alterations in the different groups after 2 months treatment with indicated solutions. The different hematological and biochemical characteristics measured, the glucose, percentage of eosinophil and triglyceride had significant differences among groups. In comparison to vehicle medium (DMEM), 16h-SFS solution was able to increase glucose level in LA7 injected rats. The different letters show that there was significant difference between groups, with p ⁇ 0.05 was considered as significant.
  • DMEM vehicle medium
  • FIG. 23 Effects of different fractions of 16-SFS solution on MCF-7 cells survival.
  • MCF-7 cells human breast cancer cell line
  • viability was measured using MTT assay following the cells treatment with DMEM (Dulbecco's Modified Eagle Medium )+5% FBS (fetal bovine serum), R100 ,R100 +FBS , R50 , R50+FBS , R30 , R30+FBS, R3, R3+FBS, DMEM for 24h.
  • the cell viability percentage was calculated based on the absorbance ratio obtained for each indicated treatment compared to DMEM+10% FBS control and multiplied by 100 (% of control).
  • LA7 cells rat mammary gland cancer stem cells
  • MTT assay following the cells treatment with DMEM (Dulbecco's Modified Eagle Medium) +5% FBS (fetal bovine serum), DMEM, R100, R50, R30, R3, R100+FBS, R50+FBS for 24h.
  • the cell viability percentage was calculated based on the absorbance ratio obtained for each indicated treatment compared to DMEM+10% FBS control and multiplied by 100 (% of control).
  • A The result indicated that R100, R50, R3 inhibited the growth of LA7 cells as negative control (DMEM).
  • Figure 25 Reduction in tumor size and Synergistic effect of 16-SFS solution and bone marrow derived mesenchymal cells (BMMCs) in up-regulation of Cxcl9 and CxcllO chemokine genes expression.
  • BMMCs bone marrow derived mesenchymal cells
  • the size of tumor in these animals was compared to the size of the tumor in rats received BMMCs only (MSC group) and 16-SFS only, as well as compared to the PBS and DMEM groups as the vehicle controls (six rats in each group). As it can be seen, the tumor size was reduced in MSC and 16-SFS group significantly (P ⁇ 0.05) when compared to their related controls, PBS and DMEM respectively. The tumor size was the lowest level in the group received both treatments. (B and C).
  • Figure 26 Effects of 96-SPS solution on Spr and Gchl genes expression and lactate production in rat LA7 induced tumors.
  • 96-SPS solution was evaluated the expression of two important genes in cells metabolisms, i.e. Spr and Gchlin those cells.
  • Spr gene expression was significantly down-regulated in 96-SPS treated tumors compared to RPMI and normal saline controls (P ⁇ 0.01 and P ⁇ 0.05 respectively).
  • B compare to the control the 96-SPS solution was able to down-regulate the Gchl gene expression (P ⁇ 0.001).
  • C The 96-SPS treated tumors exhibited a lower level of lactate compared to normal saline control (P ⁇ 0.05). This reduction was not statistically significant in RPMI treated group (P>0.05).
  • D A significant reduction in FT ions (increased pH value) was observed in 96-SPS treated group compared to normal saline control (P ⁇ 0.05).
  • RPMI group showed no significant changes in pH value (P>0.05). All data was expressed as mean ⁇ SD (standard deviation) and p>0.05 was considered statistically significant. Error bars show SD.
  • Figure 27 Sox2 and Oct4 sternness genes expression in 96-SPS treated LA7 induced tumors and controls.
  • Figure 28 Histopathologic findings in 96-SPS treated LA7 induced tumors.
  • B A tumor isolated from 96-SPS treated rat. After removing the tumor from the body, it was dissected by scalpel in the middle, the pus like liquid was run off and the hallow space remained.
  • C Circles indicate the degree of necrosis in different groups of tumors.
  • the 96h-SPS treated tumors exhibited the highest extent of necrosis in H and E staining.
  • Figure 29 Effects of 16SFS solution on growth of normal and tumor derived cells survival. To estimate the therapeutic index for 16SFS solution we evaluated the effects of this solution on the viability of MCF-
  • Example 1 Method of production of the mixture with human neonatal fibroblasts
  • Fibroblasts were isolated from fresh foreskin of children (1 to 1.5 months old) by enzymatic method using dispase and collagenase enzymes (Pandamooz et al., 2012), and cultured in Dulbecco's modified eagle's medium (DMEM, Biowest, France), supplemented with 10% fetal bovine serum (FBS, Sigma, Germany), 100 pg/mL streptomycin and 100 lU/mL Penicillin. The cells were incubated at 37 e C with 5% C02 saturation, and were sub-cultured after reaching 70% confluency. The cells have been sub-cultured for fourteen times until their growth was severely slowed down.
  • DMEM Dulbecco's modified eagle's medium
  • FBS fetal bovine serum
  • Penicillin 100 pg/mL
  • the cells were incubated at 37 e C with 5% C02 saturation, and were sub-cultured after reaching 70% confluency. The cells have been sub-culture
  • those cells culture supernatant was harvested (P14 solution) and stored -20 °C until use.
  • Fibroblasts were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. When the cells reached 70% confluency, the cells culture supernatant was removed and washed three times with phosphate buffered saline (PBS). Then, the cells were starved in serum-free DMEM for 16 h at 37 °C. At the end of this period, the starved fibroblasts culture supernatant (16h-SFS) was collected and stored at -20 °C for further analysis.
  • PBS phosphate buffered saline
  • Example 2 Method of production of the mixture with human PBMCs:
  • PBMCs peripheral blood mononuclear cells
  • PBMCs-count and viability was determined by trypane staining and using Neubauer Chamber. About 7.5xl0 6 PBMCs were cultured in 75 cm 2 cell culture flasks in glucose- containing serum-free RPMI-1640 with 100 U/mL penicillin and lOOpg/mL streptomycin for 96 hours. Then after, the cells culture supernatant was harvested by centrifugation for 5 minutes at 2000 g.
  • each sample (approximately 20 pg protein) were transferred into 2-ml LoBind Eppendorf tubes with pierced lids and frozen for 66 h at -80°C. Subsequently the frozen samples were vacuum dried in a lyophile overnight. The dried samples were each resolved in 50 mI buffer (8 M urea/0.4 M NFI4FIC03, pH 8,5).
  • the sample solution was diluted to 2 M urea / 0.1 M NFI4FIC03 with H PLC grade water (VWR). Digest with mass-spec grade trypsin (Serva, porcine) was performed at 37°C overnight. Enzymatic digestion was performed based on the valid version of the SOP H-VED-001 "Enzymatische Spaltung”. The sample was acidified to 1% TFA and 10 mI of the digest was used for the nano-LCESIMS/MS analysis.
  • HPLC separation was done using an EASY-nLClOOO (Thermo Scientific) system with the following columns and chromatographic settings:
  • the peptides were applied to a C18 column (Acclaim ® PepMap 100 pre column, C18, 3 pm, 2cm x 75 pm Nanoviper, Thermo Scientific) and subsequently separated using an analytical column (EASY-Spray column, 50 cm x 75 pm ID, PepMap C18 2 pm particles, 100 A pore size, Thermo Scientific) by applying a linear gradient (A: 0.1% formic acid in water, B: 0.1% formic acid in 100% ACN) at a flow rate of 280 nl/min.
  • A 0.1% formic acid in water
  • B 0.1% formic acid in 100% ACN
  • composition of the mixture was analyzed as follows:
  • LC-ESI-MS/MS data were used for a database search with the software Mascot (Matrix Science) using human sequences from SwissProt database (20 259 sequences).
  • Peptide mass tolerance was set to 50 ppm
  • fragment mass tolerance was set to 0.6 Da
  • a significance threshold p ⁇ 0.05 was used.
  • Oxidation at methionine was set as variable modification for the searches.
  • Peptides with up to 1 missed cleavage site were searched.
  • An ion score cut off of >26 (individual ion score greater than 26 indicate identity or extensive homology) was applied for the peptide identification.
  • the detailed results of the database searches are supplied in the appendix, including the proteins identified starting with the highest scores and the peptides identified by MS/MS for each protein.
  • the protein significance threshold was set to p ⁇ 0.05 and an ion score cut off of >26 (individual ion score greater than 26 indicate identity or extensive homology) was applied for the peptide identification.
  • composition disclosed in the present invention may further comprise one or more proteins selected from the table below.
  • proteins disclosed in the above Table are particularly supposed to have a synergic anti cancer effect.
  • the table below shows a short list of high score functional candidates expressed under described starvation conditions by neonatal fibroblasts (16SFS mixture). Marked proteins are very important proteins in the list. These possibly more important candidates are selected based on their known/described functions and expression levels in the supernatant (protein score) identified by mass spectroscopy.
  • LA7 cells rat mammary gland tumor cells
  • DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/ streptomycin) at 37 °C with 5% C02.
  • the cytotoxic effect of the above mentioned supernatants (protein mixture) on LA7 cells were examined using the MTT (3-(4, 5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay (Denizot & Lang, 1986).
  • the cells (lxlO 4 ) were seeded into each well and treated with different dilution of the fluids as 1/10, 1/2, 1/4, 1/8, 1/16, 1/32 in DMEM solution for 24, 48 and 72 h. Then after, the plate was washed with PBS and incubated with the medium (50 pL) containing 5 mg/mL of MTT dissolved in PBS for 4 h at 37°C. The supernatants were then discarded and 150 pL of Dimethylsulfoxide (DMSO) was added and mixed thoroughly to dissolve dark blue formazan crystals. Then, the optical density (OD) was recorded at 570 nm.
  • DMSO Dimethylsulfoxide
  • the viability percentage was calculated by the following formula: (OD treated)/ (OD control) xl00, and consequently 50% viability was considered as the half maximal inhibitory concentration (IC 5 o). All experiments were performed in triplicate. As mentioned above, the P14 was prepared from fibroblasts at passage 14. At this passage, some morphological changes such as enlarged cytoplasm with increased levels of granules were appeared. Also the nucleus in those cells was more obviously seen in comparison to the same cells at passage 3 (fig.
  • LA7 cells (5xl0 4 ) were cultured in 24-well plate, and the three biological fluids were added after 24 h. Trypsinization was performed after 48 h and cell numbers were determined (fig. 3). Doubling time was calculated for each fluid as the following formula (Roth V. 2006):
  • LA7 cells (2.5 xlO 6 ) were cultured in 6-well plate to reach 70% confluency and then incubated with three biological fluids for 24 h. After trypsinization, the cells were stained with Annexin V/phycoeritrin/ 7- Amino-Actinomycin (Annexin V-PE /7-AAD) according to the manufacturer's protocol (BD Bioscience, Germany) and analyzed by flow cytometry (BD FACSCALIBUR, USA) (Fig. 4)
  • LA7 cells (lxlO 5 ) were cultured in 12-well plate, scratched vertically using a 100 mI_ micro pipette tip after reaching 100% confluency (Fig. 6). The three biological fluids were added into the plates, and the potential of the migration was evaluated after 24 h and 48h, finally the results were analyzed with image J software (NIH USA).
  • stem-loop RT-qPCR assay was used to quantify the gene expression, according to the previously described method (Fattahi, Amirbozorgi, Lotfi, & Amini Navaei, 2018). Briefly, cDNA was synthesized via mRNA specific stem loop-RT primer and RevertAid Reverse Transcriptase (Fermentas, Lithuania). The primers of the four genes were designed using AllelelD 6.0 software (Table 1). RT-qPCR assay was performed with universal Taqman probe using ABI 7300 real-time PCR apparatus (Applied Biosystems, USA). Amplification was programmed as an initial denaturation at 95°C for 5 min, followed by 45 cycles of 95 °C for 30 s and 60 °C for 1 min.
  • Inter- and intra-assay coefficients of variation were calculated for Gapdh,C-myc, Sox2, Nanog, and Oct4 genes expression.
  • NM_001009178.2 Oct4 Specific forward primer (SEQ ID NO: 35): 225
  • CAGCAGCGACTCTGAAGAAGAAC RT-PCR primer SEQ ID NO: 40
  • Table 2 Inter- and intra-assay coefficients of variation (CV3 ⁇ 4, for Gapdh, C-mvc, Sox2, Nanoa, and Oct4 using stem-loop (S. D primers
  • MCF7 cells (a breast cancer cell line isolated in 1970 from a 69-year-old Caucasian woman) as well as MCF10A cells (a non-tumorigenic epithelial breast cell line) were cultured in a 96-well culture plate containing DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/ streptomycin) at 37 °C with 5% C02.
  • the cytotoxic effect of the above mentioned supernatants (protein mixture) on these cells were examined using the MTT (3-(4, 5- dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide) assay (Denizot & Lang, 1986).
  • the cells (lxlO 4 ) were seeded into each well and treated with 16SFS solution for 24h. Then after, the plate was washed with PBS and incubated with the medium (50pL) containing 5 mg/mL of MTT dissolved in PBS for 4 h at 37°C. The supernatants were then discarded and 150 pL of Dimethylsulfoxide (DMSO) was added and mixed thoroughly to dissolve dark blue formazan crystals. Then, the optical density (OD) was recorded at 570 nm and the viability was calculated accordingly.
  • DMSO Dimethylsulfoxide
  • Example 6 In vivo experiment with 16SFS (16 hourse starved fibroblasts)
  • Sprague Dawley rats (187 ⁇ 3 g) were obtained from the Laboratory Animal Facility at the Department of Medicine, Medical University of Babol, Iran. The procedure of maintaining the animals was according to the instructions of Ethics Committee of the Babol University of Medical Sciences.
  • the rats (5 rats per cage) were divided into 3 groups (A-C), and DMEM (A), 16h-SFS (B), and normal saline (C) were subcutaneously injected into the right mammary gland pad of the animals in each group (300 pL once daily for 6 days). The weight of rats was measured every two days in all groups.
  • LA7 cell suspension containing 6 xlO 6 cells in 300 pL phosphate buffered saline (PBS) was injected subcutaneously into the right flank mammary fat pad of each rat through surgery, and then the injected point was stitched. Then, the indicated medium for each group was injected into the rats once daily for 7 days.
  • PBS phosphate buffered saline
  • the tumor size was daily measured by using calipers, and blood samples were obtained from the tail of the rats for biochemical analysis including glucose, transglutaminase, cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and albumin using commercially available kits (Pars Azmoon Co., Tehran, Iran) and by an Auto-analyzer (Response 910, Dia Sys, Holzheim, Germany).
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • albumin albumin
  • the stained sections were then evaluated, and images were captured using light microscopy (Olympus BX51, Tokyo, Japan).
  • the type of tumor was identified using Ki67 (Dako, Denmark, Clone MIB-1), cytokeratin (Dako, Denmark, Clone AE1/AE3), CD45 (Diagnostic BioSystem, Clone PD7/26+2B11), and Estrogen receptors(ER) markers (Dako, Denmark, Clone 1D5).
  • Ki67 DermataTajhiz Azam, Tehran, Iran
  • cytokeratin Dako, Denmark, Clone AE1/AE3
  • CD45 Diagnostic BioSystem, Clone PD7/26+2B11
  • Estrogen receptors(ER) markers Dako, Denmark, Clone 1D5
  • the samples were placed in RNA later solution (YektaTajhiz Azam, Tehran, Iran) and stored at -80 °C.
  • RT-qPCR assay was used, according to the previously described method. Briefly, cDNA was synthesized via mRNA specific stem loop-RT primer and RevertAid Reverse Transcriptase (Fermentas, Lithuania). The primers of the four genes were designed using AllelelD 6.0 software (Table 3). RT-qPCR assay was performed with universal Taqman probe using ABI 7300 real-time PCR apparatus (Applied Biosystems, USA). Amplification was programmed as an initial denaturation at 95 °C for 5 min, followed by 45 cycles of 95 °C for 30 s and 60 °C for 1 min.
  • CAGCAGCGACTCTGAAGAAGAAC RT-PCR primer SEQ ID NO: 50
  • Stem-loop sequence is underlined; target specific portion of mRNA is shown with different color.
  • the data were expressed as mean ⁇ SD. To examine normality of the data, Kolmogorov Smirnov test was used. For multiple comparisons, One-Way ANOVA along with Tukey test was used. Also, repeated measure ANOVA was used for analyzing data from one group with different variables. The significance level assigned was P ⁇ 0.05. SPSS ver. 24 software (IBM corp., USA) was used for statistical analysis.
  • Fig. 12 After 8 days of LA7 injection, the tumors were observed to be explanted (Fig. 12). In 16h-SFS treated animal, the tumor size was remarkably smaller than animals treated with DMEM or normal saline ( Fig.11a and Fig. 12). Flowever, this difference was not statistically significant.
  • the tumor histology showed some different alterations in mitosis, necrosis and inflammatory cells infiltration among groups.
  • the number of mitotic cells in normal saline group had significant difference with the two other groups (p ⁇ 0.05) (Fig.15b).
  • the level of necrosis in 16h-SFS treated tumors was higher than the two other groups.
  • the number of mast cells (based on Giemsa staining) in DMEM group, and the level of polymorphonuclear cells (PMNs) in normal saline group were slightly higher than that in the two other groups.
  • the markers used Karlin, AE1/AE3, CD45, and ER
  • Example 7 Systemic effects of starved fibroblast culture supernatant on immunosuppressed rats treated with cancer stem cells (LA7)
  • Tumorigenesis is a (multi) complex biological process in which different types of cells contribute in an orchestral manner.
  • This cellular network consists of malignant cells, endothelial cells, immune cells, and fibroblasts, among of which non-tumor cells play different roles but orchestra to angiogenesis and tumor metastasis, and fibroblasts of stromal cells play an important role in angiogenesis (cancer-associated fibroblast).
  • fibroblasts have secretory phenotype releasing different compounds into the extracellular matrix, each of which can have important roles at all stages of cancer progression, wound healing and regeneration.
  • Cancer stem cells are among a few cancer cells surviving during different steps of metastasis and have high potential in initiating and promoting metastasis.
  • LA7 with especially expressed markers shows high tumorigenicity characteristics and is considered as a reliable model for studying the development and differentiation of breast cancer.
  • Research about metastatic breast cancer usually needs the methods for transduction and the evolution of tumor in animal models. Different methods have been introduced in this case, and theinduction of breast cancer tumor by injection of LA7 cells cultured in the medium is a relatively new method applied in cancer research in vivo.
  • angiogenesis plays a pivotal role in tumor survival and promotion. Indeed, serum starvation nearly is considered as a model for a biological study on tumor.
  • the 16SFS solution induces a rate of proliferation in fibroblasts after return to normal culture system (with serum). We expect that these proteins should have some positive effect through angiogenesis on tumor development or progression and spreading (metastasis).
  • LA7 as a model to examine this hypothesis. Because LA7 originates from Sprague Dawley rat, we initially used dexamethasone (3mg/kg) to suppress the immune system and to implant and progress tumor to different organs like liver, intestine, and lung.
  • mice Thirty female Wistar rats (8-10 weeks old, 244 ⁇ 4.5 g) were provided from the Laboratory Animal Facility at the Department of Medicine, University of Babol, Iran. They were maintained at 25 ⁇ 3 °C with relative humidity, a cycle of 12 h-light and 12 h-dark, and standard food pellets and tap water. The rats were divided into 6 groups (A-F) and after acclimation (5 rats per cage), dexamethasone was intraperitoneally injected to the animals in all groups (3 mg/kg once daily for 6 days) except group F similarly receiving normal saline (table 1). The weight of rats was measured every 2 days.
  • LA7 rat mammary gland tumor cells
  • DMEM Dulbecco's modified eagle's medium
  • FBS fetal bovine serum
  • All flasks containing these cells were incubated at 37 e C with 5% C02 saturation. After reaching 90% confluence, the supernatant of cultured LA7 cells were removed from the medium, and the cells were washed with PBS.
  • the cells were detached from the culture flask by adding trypsin (EDTA), put into a falcon tube, centrifuged at 400g for 5 min at 4 °C and washed with PBS. The cells were then counted using a hemocytometer.
  • EDTA trypsin
  • 16 hours-starved fibroblast cell culture supernatant The isolation of fibroblasts from the fresh foreskin of children (1 to 1.5 months old) was carried out by an enzymatic method using dispase and collagenase enzymes (Pandamooz et a I., 2012). The isolated fibroblasts were incubated in a complete culture medium. After reaching 80% confluence, the fibroblasts were cultured in free-serum DMEM, and the supernatant was collected after 16 hours (16h - SFS) based on our previous report (Golpour, et al. 2014).
  • LA7 cell suspensions containing 5x106 cells in 300 mI_ PBS were injected subcutaneously into the right flank mammary fat pad of each rat from group A (DMEM+LA7), C (16h-SFS+ LA7) and E (Normal saline+IA7) using insulin syringe and a 21-gauge needle; while other groups received DMEM (group B), 16h-SFS (group D).
  • group B group A
  • 16h-SFS group D
  • Five rats which were considered as dexamethasone treated control received only normal saline for indicated days (group F).
  • the special medium for each group including DMEM (A and B), 16h-SFS (C and D) and normal saline (E and F), were injected into the rats once daily for 14 days (Table 5).
  • Table 5 Experimental design and arrangement of injections Histological and biochemical examinations: After 53 days of LA7 cell injection (60th day), the rats were dissected and the liver, small intestine, and lung were removed, fixed in formalin and embedded in paraffin. The blocks were sectioned (5 pm), and stained with Hematoxylin & Eosin (H & E) or Masson's trichrome. The stained sections were then evaluated, and images were captured using light microscopy (Olympus BX51, Tokyo, Japan). For biochemical examinations, the blood samples were taken from the tail of the rats after 60 days, centrifuged at 400 g for 10 min and obtained serum was stored at -80 °C for further use.
  • Histological findings We evaluated the histopathological changes in the lung and liver qualitatively and semi-quantitatively, and determined some hematological indices implying the bone marrow function.
  • Example 8 Further discovering the potential proteins in the 16-SFS solution with anti-cancer activity
  • the 16-SFS solution was initially fractionated and analyzed through the MTT assay to determine the effects of different fractions on various cell lines (including rat LA7 cells and MCF-7; a human breast cancer cell line).
  • the 16- SFS solution was sequentially fractionated using amicon ultra-filtration centrifugal devices with different cutoffs (molecular weights; cutoff 3K, 30K, 50K and 100k).
  • the concentrated portions were collected and named R (e.g. R50; which means fraction with molecules bigger than 50 KDa).
  • the flow through was subjected to the next filtration device with smaller size.
  • the R100, R50, R30and R3 obtained from 16-SFS fractionation were used for further analysis.
  • the R100 fraction shows positive effect on MCF-7human cancer cells.
  • the R50 fraction remarkably reduces the cells viability but in presence of serum lost its effect.
  • the R30 and R3 fractions strongly reduce the cell growth and keep this property even when the serum was added to the fractions.
  • those fractions are candidates for further analysis of their anti- cancer effects compare to the R100 fraction which shows pro-tumor activities. Inhibition of these proteins by specific antibodies or molecular based technologies (e.g. RNA silencing or gene therapy) in cancer cells may be beneficial in cancer therapy.
  • LC-ESI-mass spectrometry analysis of R3, R30, R50 and R100 fractions Using the nano-LC-ESI-MS/MS analysis of the whole 16-SFS solution and above mentioned fractions (SUPPLEMENTRAY) as well as according to the experimental analysis, we could propose the list of the following candidates having anti-cancer effect: Thrombospondin-1, Metalloproteinase inhibitor 1, Pentraxin-related protein PTX3, Follistatin-related protein 1, Fibulin-1, Pigment epithelium-derived factor, Inter-alpha-trypsin inhibitor heavy chain H2, SPARC, Insulin-like growth factor-binding protein 6, Metalloproteinase inhibitor 2, Insulin-like growth factor-binding protein 4, Follistatin, Liprin-beta-2 (Fragment), Uveal autoantigen with coiled-coil domains and ankyrin repeats, Transgelin-2 (Fragment), Stanniocalcin-2 (Fragment), Insulin-like growth factor-bind
  • Example 9 Co-administration of 16-SFS solution and bone marrow derived mesenchymal cells (BMMCs) enhances the inhibition of LA7 derived tumor development in rat in association with CXCL9, CXC10 chemokines gene expression
  • 16-SFS solution revealed the existence of stromal-cell derived factor 1(SDF1) in this solution
  • SDF1 stromal-cell derived factor 1
  • BMMCs were also able to decrease the size of the tumors when compared to the PBS and DMEM control groups (p ⁇ 0.01 and p ⁇ 0.001 respectively). Interestingly, in the in vitro study this was able to inhibit directly the LA7 proliferation (Figure 25D).
  • Example 10 96-SPS down-regulated the Spr and Gchl genes expression and reduced lactate levels in LA7-induced tumors
  • Lactate is one of the metabolic product described originally as Warburg effect which is a common metabolic reprogramming factor in solid tumors microenvironments.
  • the 96-SPS treated tumors exhibited a lower level of this ions compared to normal saline control (p ⁇ 0.05). This reduction was not statistically significant in RPMI treated group (p>0.05).
  • Figure 26C Interestingly, a significant reduction in FT ions (increased pH value) was also observed in this group compared to normal saline control (p ⁇ 0.05). RPMI group showed no significant changes (p>0.05).
  • the therapeutic index formula as the ratio of inhibition rate for malignant cells, (%99 for MCF7 in this experiment)/ratio of inhibition for healthy cells namely, (%11 in this study MCF10), the calculated therapeutic index of 16SFS solution is 9. Even in presence of serum this index is not changed remarkably.
  • dexamethasone had a significant effect on the weight of rat, there is no report to show histological damages of dexamethasone in the liver, lung, and intestine of rat after intraperitoneal exposure, and some studies introduced dexamethasone as a therapeutic and protective agent. Flowever, Kumar et al. showed that dexamethasone could cause hepatic steatosis in the rat. Nevertheless, the results of the Examples in the present application showed that pyknosis occurred in the liver cells of all groups treated with dexamethasone. Pyknosis shows stress condition (e.g., oxidative stress) and occurs in dying cells due to nuclear condensation.
  • stress condition e.g., oxidative stress
  • fibrosis is the disruption of extracellular matrix homeostasis due to the excessive production of some components especially collagen Tumor cells like LA7 can disrupt the homeostasis through activation of fibroblasts and some immune cells resulting in fibrosis; and this event leads to enhancing the potential of tumor cells to be colonized.
  • Foam cells are common histological damages in lung fibrosis and, however, our results did not show any relationship between fibrosis and foam cell formation, probably due to interactions of the other factors. Foam cell formation may be one reason for alteration of triglyceride concentration.
  • SEQ ID NO 3 Metalloproteinase inhibitor 2
  • SEQ ID NO 4 Insulin-like growth factor-binding protein 4
  • SEQ ID NO 5 Uveal autoantigen with coiled-coil domains and ankyrin repeats
  • MDEDVQEALLQIIQMRQGLVC SEQ ID NO 6 Insulin-like growth factor-binding protein 5
  • SEQ ID NO 9 Metalloproteinase inhibitor 1
  • SEQ ID NO 10 Pentraxin-related protein PTX3
  • SEQ ID NO 12 Follistatin-related protein 1
  • SEQ ID NO 15 Inter-alpha-trypsin inhibitor heavy chain H2
  • SEQ ID NO 17 Growth-regulated alpha protein
  • SEQ ID NO 18 Insulin-like growth factor-binding protein 6
  • SEQ ID NO 22 Tumor necrosis factor-inducible gene 6 protein
  • SEQ ID NO 23 Transforming growth factor-beta-induced protein ig-h3
  • SEQ ID NO 27 Stanniocalcin-2 (Fragment)
  • SEQ ID NO 29 Latent-transforming growth factor beta-binding protein 2
  • SEQ ID NO 30 Insulin-like growth factor-binding protein 7

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Abstract

La présente invention concerne une composition comprenant : une protéine de facteur dérivé de l'épithélium pigmentaire ayant une séquence de SEQ ID NO : 1 ou un fragment de celle-ci, la protéine thrombospondine-2 ayant une séquence de SEQ ID NO : 2 ou un fragment de celle-ci, et/ou la protéine d'inhibiteur de métalloprotéinase 2 ayant une séquence de SEQ ID NO : 3 ou un fragment de celle-ci, et/ou la protéine de liaison au facteur de croissance analogue à l'insuline 4 ayant une séquence de SEQ ID NO : 4 ou un fragment de celle-ci, une composition pharmaceutique la comprenant, et un procédé de préparation de celle-ci.
PCT/EP2020/087239 2019-12-20 2020-12-18 Composition et son utilisation pour une thérapie universelle contre les cellules tumorales WO2021123320A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011103472A1 (fr) * 2010-02-18 2011-08-25 Osiris Therapeutics, Inc. Procédés de fabrication de produits thérapeutiques comprenant des dispersions placentaires vitalisées
WO2015171142A1 (fr) * 2014-05-07 2015-11-12 Osiris Therapeutics, Inc. Compositions placentaires thérapeutiques, procédés de fabrication et méthodes d'utilisation
WO2020210248A1 (fr) * 2019-04-09 2020-10-15 Combangio, Inc. Procédés de fabrication et d'utilisation d'un sécrétome dérivé de cellules souches mésenchymateuses

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WO2011103472A1 (fr) * 2010-02-18 2011-08-25 Osiris Therapeutics, Inc. Procédés de fabrication de produits thérapeutiques comprenant des dispersions placentaires vitalisées
WO2015171142A1 (fr) * 2014-05-07 2015-11-12 Osiris Therapeutics, Inc. Compositions placentaires thérapeutiques, procédés de fabrication et méthodes d'utilisation
WO2020210248A1 (fr) * 2019-04-09 2020-10-15 Combangio, Inc. Procédés de fabrication et d'utilisation d'un sécrétome dérivé de cellules souches mésenchymateuses

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NYBERG PIA ET AL: "Endogenous Inhibitors of Angiogenesis", CANCER RESEARCH, vol. 65, no. 10, 15 May 2005 (2005-05-15), US, pages 3967 - 3979, XP055796917, ISSN: 0008-5472, Retrieved from the Internet <URL:https://cancerres.aacrjournals.org/content/canres/65/10/3967.full.pdf> DOI: 10.1158/0008-5472.CAN-04-2427 *
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