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WO2013170364A9 - Polypeptide sparc et matrice de collagène iv pour sensibiliser les cellules cancéreuses à un agent chimiothérapeutique - Google Patents

Polypeptide sparc et matrice de collagène iv pour sensibiliser les cellules cancéreuses à un agent chimiothérapeutique Download PDF

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Publication number
WO2013170364A9
WO2013170364A9 PCT/CA2013/000485 CA2013000485W WO2013170364A9 WO 2013170364 A9 WO2013170364 A9 WO 2013170364A9 CA 2013000485 W CA2013000485 W CA 2013000485W WO 2013170364 A9 WO2013170364 A9 WO 2013170364A9
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polypeptide
collagen
sparc
cancer cell
composition
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PCT/CA2013/000485
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WO2013170364A1 (fr
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Isabella T. Tai
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The University Of British Columbia
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    • 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
    • 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
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • This invention relates to the field of cancer therapies.
  • the invention relates to the sensitization of cancer cells through the administration of SPARC polypeptide and collagen-IV matrix.
  • Cancer is one of the leading causes of death in humans and while standard chemotherapy, radiotherapy and surgical intervention successfully reduce tumor load in many cases, resistance to chemotherapeutic intervention is not uncommon, especially in solid tumors. Resistance may develop following exposure to a chemotherapeutic agent and can further impede tumor regression. It is this chemotherapy resistance, which leads to treatment failure and subsequently accounts for the high mortality rates in cancer.
  • the molecular basis of chemotherapy resistance is largely genetic, and can take many forms. Many mutations responsible for the initial development of tumors may also contribute to drug resistance. For example, loss of DNA mismatch repair (MMR) gene function has been associated with a more rapid emergence of clinical drug resistance in some cancers and mutations in the K- ras gene are associated with an increased relapse rate, mortality and a poor chemotherapeutic response. Aberrant expression and dysregulation of proteins involved in the normally tightly regulated cell replication cycle may also be protective of tumors. These proteins may be loosely referred to as 'oncogenes'. Gene products p21 and p27, for example, have been shown to protect tumors from undergoing apoptosis elicited by various anticancer agents. Adhesion molecules, such as E-cadherin, may also confer resistance to cells exposed to chemotherapeutic agents. The mechanisms involved in therapeutic resistance are varied and may be very complex.
  • Chemosensitizers may act in concert with the chemotherapeutic agent, or may serve to counteract resistance mechanisms in the cell.
  • Existing chemosensitizers include small molecule drugs such as photosensitizers or drug efflux pump inhibitors, and more recently, antisense oligonucleotides.
  • New compounds with chemosensitizing activity include US 5,776,925 and WO 02/00164, which provide examples of novel chemical compounds that enhance cytotoxicity of therapeutic agents.
  • Antisense sequences with chemosensitizing activity - often specifically targeting oncogenes - are varied and may be found for almost any target.
  • survivin is a protein that modulates apoptosis and is frequently overexpressed in cancer cells.
  • Antisense survivin oligonucleotides have been demonstrated to downregulate expression of Survivin, and sensitize cells to chemotherapeutic agents such as docetaxel and etopotide.
  • cancer therapy sensitizers may act in concert with cancer therapeutic agents, (for example, radiotherapy or chemotherapy), or may serve to counteract resistance mechanisms in the cell to the cancer therapeutic agent.
  • cancer therapeutic agents for example, radiotherapy or chemotherapy
  • SPARC Secreted protein acidic and rich in cysteine
  • SPARC is one example of a gene with significantly decreased expression in multidrug resistant cell lines in vitro, with a possible tumor suppressor role (Tai, LT. et al. 2005. J. Clin Invest. 115:1492-1502).
  • SPARC also known as osteonectin, belongs to a family of matricellular proteins having counter-adhesive properties, disruptive of cell-matrix interactions (Bornstein P. 1995. J. Cell Biol 130:503-6; Sage E. H. and Bornstein P. 1991. J Biol Chem; 266: 14831- 4).
  • SPARC has been demonstrated to play a role in bone mineralization, tissue remodeling, endothelial cell migration, morphogenesis and angiogenesis. Additional studies suggest that cleavage of SPARC by MMP-3 results in peptides that affect angiogenesis (Sage et al. 2003 J. Biol Chem 287: 37849-37857). SPARC also has a role in malignancy, as variable gene and protein expression of SPARC have been linked to cancer progression in a number of tumors.
  • the SPARC protein is under expressed in several types of cancer, including colorectal cancer (CRC), and has been shown to enhance chemosensitivity and inhibit tumor growth in vivo.
  • CRC colorectal cancer
  • SPARC is also known to bind collagen-IV, a component of the extracellular matrix (ECM), in a calcium-dependent manner in vitro.
  • ECM extracellular matrix
  • SPARC protein as a chemosensitizer is described by WO 2004/064785.
  • SPARC polypeptides are described as chemotherapeutic sensitizers by WO 2008/000079.
  • the present application is based in part on the discovery that collagen-IV matrix in the presence of SPARC polypeptide sensitizes cancer cells to chemotherapeutic treatments. Furthermore, it was also found that there is a synergism between SPARC and collagen-IV matrix in promoting cell death and/or growth inhibition of cancer.
  • a method of sensitizing a cancer cell including: contacting the cancer cell with a SPARC polypeptide and a collagen-IV polypeptide.
  • the method may further include contacting of the cancer cell with a chemotherapeutic agent.
  • the contacting of the cancer cell may be ex vivo.
  • the contacting of the cancer cell may be in vivo.
  • a composition including a SPARC polypeptide and a collagen-IV polypeptide, wherein the composition sensitizes a cancer cell to a chemotherapeutic agent.
  • the composition may further include a chemotherapeutic agent.
  • the composition may be formulated as a gel or paste.
  • the composition may further comprise a chemotherapeutic agent.
  • a pharmaceutical composition for treating colorectal cancer including a SPARC polypeptide and a collagen-IV polypeptide.
  • the pharmaceutical composition may be formulated as a gel or paste.
  • the pharmaceutical composition may further comprise a chemotherapeutic agent.
  • composition including a SPARC polypeptide and a collagen-IV polypeptide for sensitizing a cancer cell.
  • composition including a SPARC polypeptide and a collagen-IV polypeptide and a pharmaceutically acceptable carrier for sensitizing a cancer cell.
  • a SPARC polypeptide and a collagen-IV polypeptide in the manufacture of a medicament for sensitizing a cancer cell.
  • a commercial package including (a) a SPARC polypeptide and collagen-IV polypeptide; and (b) instructions for the use thereof for sensitizing a cancer cell.
  • the commercial package may further include a
  • a commercial package including (a) a pharmaceutical composition including a SPARC polypeptide, a collagen-IV polypeptide and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for sensitizing a cancer cell.
  • a commercial package comprising (a) the composition as described herein; and (b) instructions for the use thereof for sensitizing a cancer cell.
  • the commercial package may further include a pharmaceutically acceptable carrier.
  • the vector may include a SPARC polypeptide and a collagen-IV polypeptide encoding polynucleotides.
  • an apparatus for sensitizing cancer cells including a collagen-IV polypeptide and a SPARC polypeptide, wherein the apparatus is operable to bring cells to be sensitized in contact with the collagen-IV polypeptide and the SPARC polypeptide.
  • the apparatus may include a collagen-IV lined chamber.
  • the chamber may be a flow through chamber.
  • the flow through chamber may be a column.
  • the apparatus may be further operable to bring cells to be sensitized in contact with a chemotherapeutic agent.
  • the apparatus may be suitable for colorectal cancer cells.
  • the apparatus may be used in hemodialysis-like or plasmapheresis-like procedure, whereby the blood from a subject is removed for passage through the apparatus and then returned to the subject following exposure to the collagen-IV polypeptide and the SPARC polypeptide.
  • the apparatus may be used to filter/expose other cells which are to be returned to the subject (for example bone marrow cells).
  • a method of sensitizing a cancer cell including contacting the cancer cell with a SPARC polypeptide and an agent that increases the level of ITGA6 protein in said cancer cells.
  • an anti-cancer in accordance with another aspect of the invention, there is provided an anti-cancer
  • composition including a nucleic acid vector, which directs the expression of a SPARC polypeptide and the expression of ITGA6.
  • a vector including a SPARC polypeptide and an ITGA6 polypeptide encoding polynucleotides.
  • the SPARC polypeptide may be provided via gene therapy.
  • the collagen-IV polypeptide may be provided via gene therapy.
  • the SPARC polypeptide may be provided in a pharmaceutical composition.
  • the collagen-IV polypeptide may be provided in a pharmaceutical composition.
  • the SPARC polypeptide may include the full-length protein.
  • the SPARC polypeptide may include a sensitizing fragment or variant.
  • the SPARC polypeptide and the collagen-IV polypeptide form a pharmaceutical composition.
  • the SPARC polypeptide, the collagen-IV polypeptide, and the chemotherapeutic agent may form a pharmaceutical composition.
  • the cancer cell may be a colorectal cancer cell.
  • the colorectal cancer cell may be chemotherapy resistant.
  • the cancer cell may be a human cancer cell.
  • the collagen-IV polypeptide may be a collagen-IV polypeptide matrix.
  • FIGURE 1 shows a series of graphs of the effect of collagen-IV matrix on cell viability (MTT assay) following 5FU administration for several colorectal cancer (CRC) cell lines where each cell line has variable levels of SPARC expression (MIP101 ; RKO; HCT1 16; MIP/SP (overexpressing); and MIP/Zeo (empty vector control);
  • MIP101 variable levels of SPARC expression
  • RKO variable levels of SPARC expression
  • HCT1 16 overexpressing
  • MIP/SP overexpressing
  • MIP/Zeo empty vector control
  • FIGURE 2 shows two graphs comparing caspase 3/7 activity (i.e. apoptosis) in CRC cells cultured on collagen-IV matrix and without collagen-IV matrix, following 5FU (5 ⁇ ) addition after 24 hours of cell seeding, where caspase 3/7 activity was measured at 12, 24, 48 and 72 hour intervals;
  • FIGURE 3 shows graphs of gene expression analysis for integrins by qRT-PCR for alfa3 (A), alfa9 (B) and alfa6 (C) integrins and immuiioblots (D) of integrin alfa6 (ITGA6) comparing membrane and cytoplasm expressionby cell type, in the presence of collagen-IV matrix and 5FU;
  • FIGURE 4 shows two bar graphs of the effect of ITGA6 knockdown on cell viability (MTT assay) in two cell lines having no SPARC expression (MIP/ZEO) and high SPARC expression (MIP/SP), where cells were transiently transfected with ITGA6 shRNA (optimized for 80% knockdown) and subsequently treated with 5FU 24 hours later;
  • FIGURE 5A shows a series of immunoblots comparing the effect of ITG6A knockdown (transfected with ITGA6 siRNA or scramble siRNA) on PTEN/Akt expression in two cell lines having no SPARC expression (MIP/ZEO) and high SPARC expression (MIP/SP), where the cells were grown with and without collagen-IV matrix and exposed to 5FU for different time intervals;
  • FIGURE 5B shows a bar graph of densitometry values to determine the phosphorylation levels of PTEN
  • FIGURE 5C shows a bar graph of densitometry values to determine the phosphorylation levels of pAKT (Thr308).
  • FIGURE 5D shows a bar graph of densitometry values to determine the phosphorylation levels of pAKT (Ser473).
  • SPARC protein is encoded by a 912 bp mRNA (for example, GenBank Accession:
  • the Homo sapiens SPARC is on chromosome 5 (NCBI Reference Sequence: NC 000005.9).
  • Human SPARC encodes a 303 amino acid protein (see for example, GenBank: CAG33080.1).
  • a 'SPARC polypeptide' as used herein refers to the full length 303 amino acid SPARC protein sequence and to any fragment or variant thereof, known in the art, that retains chemo-sensitzing activity.
  • Rahman M. et al. PLOS ONE 10.1371/journal.pone.0026390 Published: 1 November 201 1
  • TABLE1 shows fragments that were tested in WO/2008/000079.
  • Collagen-IV is encoded by the COL4A1 gene (for example, Homo sapiens collagen, type IV, alpha 1 (COL4A1), mRNA is encoded by NCBI Reference Sequence: NM_001845.4).
  • the Homo sapiens collagen, type IV, alpha 1 (COL4A1), is on chromosome 13 (NCBI Reference Sequence: NG_01 1544.1).
  • Human collagen-IV encodes a 1678 amino acid protein (see for example, GenBank: BAA04809.1).
  • Collagen-IV is a major component of basement membranes and provides major structural support for the basement membrane matrix. When the collagen-IV meshwork is assembled, it provides a scaffold for the assembly of other basement membrane components through interactions with laminin, entactin and proteoglycan. Collagen-IV is often used as a substrate for epithelial, endothelial, muscle and nerve cells growth.
  • BD Biosciences (San Jose, CA) offers human (354245) and mouse (354233 or 356233) collagen-IV.
  • 'integrin alpha-6' or 'ITGA6' is a protein that in humans is encoded by the ITGA6 gene (NCBI Reference Sequence: NM 001079818.1 encodes the transcript variant 1 , mRNA (5680 bp); and NCBI Reference Sequence: NM_000210.2 encodes the transcript variant 2, mRNA (5810 bp)).
  • the human ITGA6 protein is 1 130 amino acids (for example,
  • the integrins are cell-surface proteins that have both an a chain and a ⁇ chain, which can form different integrins depending on the binding partner.
  • the integrins as a group are involved in cell adhesion and cell-surface signaling.
  • a 'patient or a 'subject' are used interchangeably.
  • a subject may be human, or a non-human animal, such as a rodent or transgenic mouse.
  • a 'composition' may include small organic or inorganic molecules with distinct molecular composition made synthetically, found in nature, or of partial synthetic origin. Included in this group are nucleotides, nucleic acids, amino acids, peptides, polypeptides, proteins, peptide nucleic acids or complexes comprising at least one of these entities.
  • a composition may be comprised of the effective composition alone (i.e. a pharmacologically effective amount) or in combination with a pharmaceutically acceptable excipient.
  • a 'pharmaceutically acceptable excipient' includes any and all solvents, dispersion media, coatings, antibacterial, antimicrobial or antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the excipient may be suitable for intravenous, intraperitoneal, intramuscular, intrathecal or oral administration.
  • the excipient may include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersion. Use of such media for preparation of medicaments is known in the art.
  • a 'pharmacologically effective amount' of a medicament refers to using an amount of a medicament present in such a concentration to result in a therapeutic level of drug delivered over the term that the drug is used. This may be dependent on the mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the medicament. The determination of what dose is a 'pharmacologically effective amount' requires routine optimization, which is within the capabilities of one of ordinary skill in the art.
  • cancer refers to a proliferative disorder caused or characterized by the proliferation of cells, which have lost susceptibility to normal growth control.
  • cancer includes tumors and any other proliferative disorders. Cancers of the same tissue type usually originate in the same tissue, and may be divided into different subtypes based on their biological characteristics. Four general categories of cancers are carcinoma (epithelial tissue derived), sarcoma (connective tissue or mesodermal derived), leukemia (blood-forming tissue derived) and lymphoma (lymph tissue derived). Over 200 different types of cancers are known, and every organ and tissue of the body may be affected.
  • organs and tissues that may be affected by various cancers include pancreas, breast, thyroid, ovary, uterus, testis, prostate, thyroid, pituitary gland, adrenal gland, kidney, stomach, esophagus, colon or rectum, head and neck, bone, nervous system, skin, blood, nasopharyngeal tissue, lung, urinary tract, cervix, vagina, exocrine glands and endocrine glands.
  • a cancer may be multicentric or of unknown primary site (CUPS).
  • a 'cancerous cell' refers to a cell that has undergone a transformation event and whose growth is no longer regulated to the same extent as before the transformation event.
  • a tumor refers to a collection of cancerous cells, often found as a solid or semi-solid lump in or on the tissue or in a subject.
  • a cancer or cancerous cell may be described as 'sensitive to' or 'resistant to' a given therapeutic regimen or chemotherapeutic agent based on the ability of the regimen to kill cancer cells or decrease tumor size, reduce overall cancer growth (i.e. through reduction of angiogenesis), and/or inhibit metastasis. Cancer cells that are resistant to a therapeutic regimen may not respond to the regimen and may continue to proliferate. Cancer cells that are sensitive to a therapeutic regimen may respond to the regimen resulting in cell death, a reduction in tumor size, reduced overall growth (tumor burden), or inhibition of metastasis.
  • this may manifest itself in a reduction in tumor size, overall growth/tumor burden, or the incidence of metastasis of about 10% or more, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or more, to about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold or more.
  • Monitoring of a response may be accomplished by numerous pathological, clinical and imaging methods as known to persons of skill in the art.
  • a common theme for a chemotherapeutic agent or combination of agents is to induce death of the cancerous cells.
  • DNA adducts such as nitrosoureas, busulfan, thiotepa, chlorambucil, cisplatin, mitomycin, procarbazine, or dacacarbazine, slow the growth of the cancerous cell by forcing the replicating cell to repair the damaged DNA before the M-phase of the cell cycle, or may themselves cause sufficient damage to trigger apoptosis of the cancerous cell.
  • chemotherapeutic agents may enable the cancerous cell to be killed by aspects of the patient or test subject's humoral or acquired immune system, for example, the complement cascade or lymphocyte attack.
  • a 'chemotherapeutic regimen' or 'chemotherapy' refers to the administration of at least one chemotherapy agent, to treat cancerous cells.
  • Chemotherapy agents may be administered to a subject in a single bolus dose, or may be administered in smaller doses over time.
  • a single chemotherapeutic agent may be used (single-agent therapy) or more than one agent may be used in combination (combination therapy).
  • Chemotherapy may be used alone to treat some types of cancer.
  • chemotherapy may be used in combination with other types of treatment, for example, radiotherapy or alternative therapies (for example immunotherapy) as described herein.
  • a chemosensitizer may be administered as a combination therapy with a chemotherapy agent, radiotherapy, or alternative therapies.
  • a 'chemotherapeutic agent' refers to a medicament that may be used to treat cancer, and generally has the ability to kill cancerous cells directly.
  • chemotherapeutic agents include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous agents. Examples of alternate names are indicated in brackets.
  • alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis antagonists such as estramustine phosphate; and triazines such as dacarbazine (DTIC, dimethyl- triazenoimidazolecarboxamide) and temozolomide.
  • nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil
  • antimetabolites include folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin (5- fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine and fludarabine; and topoisomerase inhibitors such as amsacrine.
  • folic acid analogs such as methotrexate (amethopterin)
  • pyrimidine analogs such as fluorouracin (5- fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytos
  • Examples of natural products include vinca alkaloids such as vinblastine (VLB) and vincristine; taxanes such as paclitaxel and docetaxel (Taxotere); epipodophyllotoxins such as etoposide and teniposide; camptothecins such as topotecan and irinotecan; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin, and tunicamycin (Tm); enzymes such as L- asparaginase; and biological response modifiers such as interferon alpha and interlelukin 2.
  • VLB vinblastine
  • Taxanes such as paclitaxel and docetaxel (Taxotere)
  • epipodophyllotoxins such as etoposide and teniposide
  • hormones and antagonists include luteinising releasing hormone agonists such as buserelin; adrenocorticosteroids such as prednisone and related preparations; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgens such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs such as leuprolide.
  • releasing hormone agonists such as buserelin
  • adrenocorticosteroids such as prednisone and related preparations
  • progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol
  • miscellaneous agents include thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants such as mitotane ( ⁇ , ⁇ '-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as imatinib.
  • platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin
  • anthracenediones such as mitoxantrone
  • substituted ureas such as hydroxyurea
  • methylhydrazine derivatives such as procarbazine (N-methylhydr
  • chemotherapeutic agents for use in accordance with the invention include, without limitation, nanoparticle albumin-bound paclitaxels.
  • the term 'radiotherapeutic regimen' or 'radiotherapy' refers to the administration of radiation to kill cancerous cells. Radiation interacts with various molecules within the cell, but the primary target, which results in cell death is the deoxyribonucleic acid (DNA). However, radiotherapy often also results in damage to the cellular and nuclear membranes and other organelles. DNA damage usually involves single and double strand breaks in the sugar- phosphate backbone. Furthermore, there can be cross-linking of DNA and proteins, which can disrupt cell function. Depending on the radiation type, the mechanism of DNA damage may vary as does the relative biologic effectiveness. For example, heavy particles (i.e. protons, neutrons) damage DNA directly and have a greater relative biologic effectiveness.
  • heavy particles i.e. protons, neutrons
  • Electromagnetic radiation results in indirect ionization acting through short-lived, hydroxyl free radicals produced primarily by the ionization of cellular water.
  • Clinical applications of radiation consist of external beam radiation (from an outside source) and brachytherapy (using a source of radiation implanted or inserted into the patient).
  • External beam radiation consists of X-rays and/or gamma rays
  • brachytherapy employs radioactive nuclei that decay and emit alpha particles, or beta particles along with a gamma ray.
  • Radiotherapy may further be used in combination chemotherapy, with the chemotherapeutic agent acting as a radiosensitizer.
  • the choice of radiotherapy may be suited to an individual subject as determined by a skilled person at the point of care, taking into consideration the tissue and stage of the cancer. Examples of radiotherapy approaches to various cancers may be found in, for example "Clinical Oncology", 3rd edition. Churchill Livingstone/ Elsevier Press, 2004. ABELOFF, MD. editor.
  • the term 'alternative therapeutic regimen' or 'alternative therapy' may include for example, biologic response modifiers (including polypeptide-, carbohydrate-, and lipid- biologic response modifiers), toxins, lectins, antiangiogenic agents, receptor tyrosine kinase inhibitors (for example IressaTM (gefitinib), TarcevaTM (erlotinib), ErbituxTM (cetuximab), imatinib mesilate (GleevecTM), proteosome inhibitors (for example bortezomib, VelcadeTM); VEGFR2 inhibitors such as PTK787 (ZK222584), aurora kinase inhibitors (for example ZM447439); mammalian target of rapamycin (mTOR) inhibitors, cyclooxygenase-2 (COX-2) inhibitors, rapamycin inhibitors (for example sirolimus, RapamuneTM); farnesyltransferase inhibitors
  • an immunotherapeutic agent would also be considered an alternative therapeutic regimen.
  • examples include chemokines, chemotaxins, cytokines, interleukins, or tissue factor.
  • Suitable immunotherapeutic agents also include serum or gamma globulin containing preformed antibodies; nonspecific immunostimulating adjuvants; active specific immunotherapy; and adoptive immunotherapy.
  • alternative therapies may include other biological-based chemical entities such as polynucleotides, including antisense molecules, polypeptides, antibodies, gene therapy vectors and the like. Such alternative therapeutics may be administered alone or in combination, or in combination with other therapeutic regimens described herein.
  • suitable alternative therapeutic regimens include, without limitation, antibodies to molecules on the surface of cancer cells such as antibodies to Her2 (e.g., Trastuzumab), EGF or EGF Receptors, VEGF (e.g., Bevacizumab) or VEGF Receptors, CD20, and the like.
  • the therapeutic agent may further comprise any antibody or antibody fragment which mediates one or more of complement activation, cell mediated cytotoxicity, inducing apoptosis, inducing cell death, and opsinization.
  • an antibody fragment may be a complete or partial Fc domain.
  • a 'chemosensitizer' or 'sensitizer' is a medicament that may enhance the therapeutic effect of a chemotherapeutic agent, radiotherapy treatment or alternative therapeutic regimen, and therefore improve efficacy of such treatment or agent.
  • Chemosensitizers may be used to overcome a resistant phenotype or to allow for a lower dose of a chemotherapeutic agent, radiotherapy treatment or alternative therapeutic regimen (and with the lower dose, reduced side effects).
  • the sensitivity or resistance of a tumor or cancerous cell to treatment may also be measured in an animal, such as a human or rodent, by, e.g., measuring the tumor size, tumor burden or incidence of metastases over a period of time.
  • a composition or a method of treatment may sensitize a tumor or cancerous cell's response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is about 10% or more, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or more, to about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15- fold, about 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method.
  • the determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of a person versed in the art.
  • 'peptide,' 'polypeptide,' and 'protein' may be used interchangeably, and refer to a compound comprised of at least two amino acid residues covalently linked by peptide bonds or modified peptide bonds, for example peptide isosteres (modified peptide bonds) that may provide additional desired properties to the peptide, such as increased half-life.
  • a peptide may comprise at least two amino acids.
  • the amino acids comprising a peptide or protein described herein may also be modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It is understood that the same type of modification may be present in the same or varying degrees at several sites in a given peptide.
  • modifications to peptides may include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • RNA RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), and modified linkages (e.g., alpha anomeric polynucleotides, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • pendent moieties e.g., polypeptides
  • modified linkages e.g., alpha anomeric polynucleotides
  • PNA 'Peptide nucleic acids'
  • PNA refers to modified nucleic acids in which the sugar phosphate skeleton of a nucleic acid has been converted to an N-(2-aminoethyl)-glycine skeleton.
  • the sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge under neutral conditions resulting in electrostatic repulsion between complementary chains, the backbone structure of PNA does not inherently have a charge. Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences.
  • PNAs are generally more robust than nucleic acids. PNAs may also be used in arrays and in other hybridization or other reactions as described above and herein for oligonucleotides.
  • the term 'vector' refers to a polynucleotide compound used for introducing exogenous or endogenous polynucleotide into host cells.
  • a vector comprises a nucleotide sequence, which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are non-limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated polynucleotide molecule.
  • nucleic acid constructs comprising control elements and a nucleic acid molecule described herein operatively linked to the control elements (e.g., a suitable promoter) for expression of a polypeptide or a polypeptide herein described.
  • control elements e.g., a suitable promoter
  • Protein expression is dependent on the level of RNA transcription, which is in turn regulated by DNA signals.
  • translation of mRNA requires, at the very least, an AUG initiation codon, which is usually located within about 10 to about 100 nucleotides of the 5' end of the message.
  • the invention provides plasmids encoding polypeptides wherein the vector is, e.g., pCDNA3.1 or a derivative thereof.
  • the nucleic acid molecules described herein may comprise a coding region operatively linked to a suitable promoter, which promoter is preferably functional in eukaryotic cells.
  • a suitable promoter such as, without limitation, the RSV promoter and the adenovirus major late promoter can be used in the invention.
  • Suitable non-viral promoters include, but are not limited to, the phosphoglycerokinase (PGK) promoter and the elongation factor la promoter.
  • PGK phosphoglycerokinase
  • Non-viral promoters are desirably human promoters.
  • Additional suitable genetic elements many of which are known in the art, also can be ligated to, attached to, or inserted into the inventive nucleic acid and constructs to provide additional functions, level of expression, or pattern of expression.
  • the native promoters for expression of the SPARC family genes also can be used, in which event they are preferably not used in the chromosome naturally encoding them unless modified by a process that substantially changes that chromosome.
  • Such substantially changed chromosomes can include chromosomes transfected and altered by a retroviral vector or similar process.
  • such substantially changed chromosomes can comprise an artificial chromosome such as a HAC, YAC, or BAC.
  • nucleic acid molecules described herein may be operatively linked to enhancers to facilitate transcription.
  • Enhancers are cis-acting elements of DNA that stimulate the transcription of adjacent genes. Examples of enhancers, which confer a high level of transcription on linked genes in a number of different cell types from many species include, without limitation, the enhancers from SV40 and the RSV-LTR. Such enhancers can be combined with other enhancers, which have cell type-specific effects, or any enhancer may be used alone.
  • the molecules may further comprise a polyadenylation site following the coding region of the nucleic acid molecule. Also, preferably all the proper transcription signals (and translation signals, where appropriate) will be correctly arranged such that the exogenous nucleic acid will be properly expressed in the cells into which it is introduced. If desired, the exogenous nucleic acid also can incorporate splice sites (i.e., splice acceptor and splice donor sites) to facilitate mR A production while maintaining an in frame, full-length transcript. Moreover, the inventive nucleic acid molecules can further comprise the appropriate sequences for processing, secretion, intracellular localization, and the like.
  • the nucleic acid molecules can be inserted into any suitable vector.
  • suitable vectors include, without limitation, viral vectors.
  • Suitable viral vectors include, without limitation, retroviral vectors, alpha viral, vaccinial, adenoviral, adeno-associated viral, herpes viral, and fowl pox viral vectors.
  • the vectors preferably have a native or engineered capacity to transform eukaryotic cells, e.g., CHO-Kl cells.
  • the vectors useful in the context of the invention can be 'naked' nucleic acid vectors (i.e., vectors having little or no proteins, sugars, and/or lipids encapsulating them) such as plasmids or episomes, or the vectors can be complexed with other molecules.
  • nucleic acid vectors i.e., vectors having little or no proteins, sugars, and/or lipids encapsulating them
  • plasmids or episomes such as plasmids or episomes
  • Other molecules that can be suitably combined with the inventive nucleic acids include without limitation viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.
  • Transfection vectors capable of expressing polynucleotides encoding fragments or the full length SPARC polypeptide sequence or fragments or variants thereof and/or the collagen-IV polypeptide sequence into cancer cells, may be used to transform cancer cells to sensitize them prior to or concomitantly with a chemotherapeutic agent.
  • Gene therapy is a medical intervention that involves modifying the genetic material of living cells to fight disease. Gene therapy is being studied in clinical trials (research studies with humans) for many different types of cancer and for other diseases. Accordingly, the invention further provides for an isolated nucleic acid molecule encoding a SPARC polypeptide suitable for use in 'gene therapy' (for example, Patil et al, AAPS J. 7(l):E61-77 (2005)). In general, a gene may be delivered to the cell using a 'vector' such as those disclosed herein. The most common types of vectors used in gene therapy are viruses. Viruses used as vectors in gene therapy are genetically disabled, whereby they are unable to reproduce themselves.
  • viruses used as vectors include adenoviruses, adeno-associated viruses, poxviruses, and the herpes virus. Suitable viral gene therapy vectors and modes of their administration in vivo and ex vivo are known in the art.
  • Gene therapy can be performed both ex vivo and in vivo.
  • cells from the subject's blood or bone marrow are removed and grown in the laboratory.
  • the cells are exposed to the virus that is carrying the desired gene.
  • the virus enters the cells, and the desired gene becomes part of the cells' DNA.
  • the cells grow in the laboratory and are then returned to the patient by injection into a vein.
  • vectors such as, e.g., viruses or liposomes may be used to deliver the desired gene to cells inside the patient's body.
  • the collagen-IV and SPARC genes may be delivered to a subject ⁇ in vivo) or to a subject's cells (ex vivo) either in the same vector or in separate vectors, such that both are produced by or near the cancer cells being targeted.
  • the vectors expressing SPARC described herein may be used to transform cells being grown on a collagen- IV matrix in a filter.
  • the filter may be similar to the 'Hemopurifier' produced by Aethlon Medical, which is used to filter exosomes from the blood of cancer patients before returning the blood to the patient's body.
  • any cancer cells found in a subject's blood could be sensitized by the SPARC expressing cells grown on a collagen-IV matrix.
  • the subject's blood could be treated with a chemo therapeutic agent either ex vivo or in vivo.
  • a subject's cells may be treated ex vivo to by exposing the cells to SPARC expressing cells grown on a collagen-IV matrix and a chemotherapeutic agent to treat the cells prior to returning the cells to the subject.
  • the subject's cells may be cultured on a collagen-IV matrix and exposed to SPARC
  • the collagen-IV matrix may be used in a pharmaceutical composition by combining the collagen with SPARC protein and/or a chemotherapeutic agent.
  • a chemotherapeutic agent As reviewed by Friess W. (European Journal of Pharmaceutics and Biopharmaceutics (1998) 45:1 13-136) collagens have been used for aqueous preparations and gel preparations with vinblastine, cisplatin, Tc, 5-FU, etc. (see for example, Sutton R, et al. Sel Cancer Ther. (1990) 6(l):35-49; Davidson BS, et al. J Surg Res. (1995) 58(6):618-24; Ning S, et al. Radiother Oncol.
  • the collagen-IV matrix may be combined with SPARC peptide/protein and administered to a tumor locally.
  • the administration may be directly to a tumor or to a surgical site following tumor resection.
  • the compositions described herein may also include one or more chemotherapeutic agent(s).
  • the compositions described herein may also be viscous, in the form of a gel or paste.
  • the compositions when administered may also have the added benefit that the SPARC/collagen-IV matrix and/or one or more
  • compositions may have a longer residence time at the desired site. Furthermore, such compositions may provide delayed release of the SPARC peptide/protein and the one or more chemotherapeutic agents. Accordingly, the effect of the composition may be extended beyond that of traditional systemic delivery and or topical administration with a less viscous
  • MIP-101 cells are a colon carcinoma cell line, which are sensitive to chemotherapeutic treatments.
  • SPARC over-expressing MIP101 cells MIP/SP
  • empty-vector control MIP/ZEO
  • HCT116 intrinsically high-SPARC-expressing cells
  • RKO low- SPARC-expressing cells
  • MIPlOl , RKO and HCTl 16 cell lines were maintained in Dulbecco's Modified Eagle's medium supplemented with 1% penicillin or streptomycin, 1% kanamycin (InvitrogenTM), and 10% newborn calf serum at 3TC and 5% C0 2 .
  • MIP/ZEO and MIP/SP cell lines were derived from MIP 101 cell lines by transfection of empty vectors and stable transfection with SPARC, respectively and were supplemented with DMEM, 10% NCS, 1% PSK and 0.1% zeocin.
  • 5FU 5 ⁇ was added after 24 hours of cell seeding and cells were treated for 12, 24, 48 and 72 hours. Cells were harvested at each time point (i.e. 12, 24, 48 and 72 hours).
  • Caspase 3/7 activity was measured using caspase glo substrate (PromegaTM) with 5 ⁇ g/uL protein. Caspase 3/7 activity was increased significantly in MIP/SP cells after 12 hour of 5FU treatment in presence of CIV.
  • Relative luminescence units (RLU) were quantified using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTekTM).
  • integrin-alpha-6 integrin-alpha-6, ITGA6
  • the primers used for RT-PCR for integrin - alpha6 were as follows: (forward) 5' tggatcacaaagtggaacga 3, and (reverse) 5' tgtgcaaaacaggagcctta 3'.
  • alfa3 (A), alfa9 (B) and alfa6 (C) integrin expression was also carried out by immunoblot assay.
  • Antibodies used in the collagen studies include the following: ITGA6, phospho PTEN, Total PTEN, Phospho AKT, Total AKT, Caspase 8 (Cell Signaling TechnologiesTM); and Actin (ABMTM).
  • ITGA6 shRNA was obtained from Applied Biological Materials Inc.TM (Cat#i01 1 149 (ITGA6 siRNA target sequences X 4 - in iLentiTM-GFP siRNA Expression Vector)). siRNA Knockdown of ITGA6
  • Both MIP/ZEO and MIP/SP cells were transiently transfected with ITGA6 siRNA or scramble siRNA, and the cells were seeded either on collagen-IV coated plates or on uncoated plates, before being exposed to 5FU for different time intervals followed by immunoblotting for different proteins (i.e. PTEN, pAKT (Thr308), and pAKT (Ser473)). Furthermore, densitometry readings were taken to determine the phosphorylation levels of PTEN (FIGURE 5B), pAKT (Thr308) (FIGURE 5C) and pAKT (Ser473) (FIGURE 5D).
  • Human ITGA_6 siRNA were obtained from QiagenTM (Cat#SI02654078 (sense strand 5'- GGGUAAUAAACUUAGGUAATT-3 ' and anti-sense strand 5'- UUACCUAAGUUUAUUACCCTG-3' - with DNA overhangs); and Human ITGA_7 siRNA was also obtained from QiagenTM (Cat#SI02654155 (sense strand 5'- GGCCUGUGAUUAAUAUUCATT-3' and anti-sense strand 5'- UGAAUAUUAAUCACAGGCCGG-3 ' - with DNA overhangs)).
  • EXAMPLE 1 Presence of a collagen-IV matrix decreases cell viability in SPARC overexpressing colorectal cancer cell lines
  • FIGURE 1 shows that MIP/SP cells showed greater sensitivity to chemotherapy (5FU) on a collagen-IV matrix. There was a reduction in cell viability of 20% when SPARC expressing cells were grown on a collagen-IV matrix when compared to cells that were not grown on a collagen-IV matrix. Cell viability in low SPARC expressing cells, MIP101/MIP/ZEO/RKO were not influenced by the presence of a collagen-IV matrix.
  • FIGURE 3 This assay identified ITGA6 as highly expressed in cells overexpressing SPARC (MIP/SP; and HCT 116) as compared to low-SPARC expressing cells (i.e. MIP/101 ; MIP/ZEO; and RKO).
  • EXAMPLE 4 Effect of ITGA6 knockdowns on SPARC overexpressing colorectal cancer cell lines
  • FIGURE 4 shows that in MIP/SP cells ITGA6 following shRNA knockdown abrogates the effect of collagen-IV on cell viability.
  • ITGA6 siRNA reversed the effects of 5FU on collagen-IV.
  • Immunoblotting analysis of MIP/SP cells following exposure to ITGA6 siRNA and 5FU, on collagen-IV matrix shows a decrease in cleaved-caspase-8 suggesting that ITGA6 is required for SPARC-collagen mediated induction of apoptosis (FIGURE 5).
  • p-AKT (Thr308) level gradually decreased in a time- dependent manner following 5FU treatment in only MIP/SP cells on collagen-IV matrix.
  • a greater response to chemotherapy is achieved in CRCs expressing high levels of SPARC in an environment rich in collagen-IV.
  • 5FU down-regulates the p-AKT (Thr 308) levels in SPARC overexpressing CRC cell lines in the presence of collagen-IV as compared to control cells.
  • Transient knockdown of ITGA6 diminishes the effects of collagen-IV in SPARC overexpressing CRC cells through its effect on p-AKT levels and apoptosis.

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Abstract

La présente invention concerne des compositions et des procédés d'utilisation de celles-ci pour la sensibilisation à un traitement contre le cancer. Ces compositions peuvent comprendre le polypeptide SPARC (protéine sécrétée acide et riche en cystéine) pleine longueur et/ou des fragments fonctionnels de ceux-ci et un polypeptide de matrice de collagène IV ou les séquences de nucléotides codant pour ceux-ci. Les compositions peuvent être utilisées en combinaison avec des agents chimiothérapeutiques existant pour contacter une cellule cancéreuse. La présente invention concerne en outre des utilisations, des compositions pharmaceutiques, et des conditionnements commerciaux associés.
PCT/CA2013/000485 2012-05-18 2013-05-17 Polypeptide sparc et matrice de collagène iv pour sensibiliser les cellules cancéreuses à un agent chimiothérapeutique WO2013170364A1 (fr)

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