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WO2012004410A1 - B7-h3 antagonists and taxanes - Google Patents

B7-h3 antagonists and taxanes Download PDF

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Publication number
WO2012004410A1
WO2012004410A1 PCT/EP2011/061750 EP2011061750W WO2012004410A1 WO 2012004410 A1 WO2012004410 A1 WO 2012004410A1 EP 2011061750 W EP2011061750 W EP 2011061750W WO 2012004410 A1 WO2012004410 A1 WO 2012004410A1
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Prior art keywords
antagonist
cancer
cells
cell
taxane
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French (fr)
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Øystein FODSTAD
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Oslo Universitetssykehus hf
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Oslo Universitetssykehus hf
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the present invention relates to the use of a combination dosage comprising a B7- H3 antagonist and a taxane for the preparation of a medicament for preventing the progression of cancer in a human.
  • An aspect of the present invention relates to a B7-H3 antagonist for sensitizing a cancer cell to a taxane.
  • Another aspect of the present invention relates to a method for preventing the progression of cancer comprising providing an individual in need thereof, and administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane.
  • Breast cancer is an epithelial cancer and is the most common type of cancer as well as the most common cause of cancer-related death in women. Once breast cancer becomes metastatic, it is difficult to cure because it is often resistant to commonly used chemotherapeutics, such as a taxane-based regime, which is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Hence, if the sensitivity to taxane-based chemotherapy could be increased, it would represent an important step in improving the clinical management of breast cancer.
  • B7-H3 a transmembrane protein with immunoglobulin-like structure, is known to have immunoregulatory properties with both inhibitory and stimulatory effects on the activation of T-cells. It has also been shown that B7-H3 expressed at the cell surface of neuroblastoma cells can inhibit natural killer-mediated lysis. The B7-H3 protein is found in many human tissues, and it is observed to be overexpressed in a wide range of human cancers.
  • B7-H3 structurally is a transmembrane protein, it is also found in the cytoplasm of tumor cells. Furthermore, a soluble form of the protein has been found in serum of patients with NSCLC, and elevated levels of circulating B7-H3 correlated with poor clinical outcome. The same group has reported the release of soluble B7-H3 from monocytes, dendritic cells, activated T-cells and some cancer cell lines.
  • Stat3 signal transducer and activator of transcription
  • Stat3 The activation of Stat3 is through phosphorylation by various tyrosine kinases, such as Janus activated kinases (Jak) and Src family kinases, which are associated with and activated by cytokine and growth factor receptors.
  • Jak Janus activated kinases
  • Src family kinases which are associated with and activated by cytokine and growth factor receptors.
  • the tyrosine phosphorylation leads to Stat dimerization, followed by nuclear translocation, and activation of the expression of several genes.
  • Stat3 constitutive activation of Stat3 occurs frequently in breast tumors and has been confirmed in a wide range of breast cancer cell lines, possibly through over-expression or constitutive activation of upstream receptors such as EGFR and HER2 or non- receptor tyrosine kinases. High levels of Stat3 activity have been shown to predict intrinsic chemotherapeutic drug resistance due to the upregulation of the antiapoptotic factors Bcl-xL, Bcl-2, cl-1 and Survivin.
  • An aspect of the present invention relates to the use of a combination dosage comprising a B7-H3 antagonist and a taxane for the preparation of a medicament for preventing the progression of cancer in a human.
  • Another aspect of the present invention relates to the use of a combination of a B7-H3 antagonist and a taxane for the manufacture of a medicament for the treatment of cancer in a subject, wherein the administration pattern of said medicament comprises:
  • the cancer an epithelial cancer. Substances
  • Another aspect of the present invention relates to a B7-H3 antagonist for use in a method of treatment comprising sensitizing a cancer cell to a taxane.
  • Another aspect of the present invention relates to a method for preventing the progression of cancer comprising providing an individual in need thereof, and administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane.
  • the B7-H3 antagonist selected from the group consisting of an siRNA directed to the mRNA encoding B7-H3, an shRNA directed to the mRNA encoding B7-H3, a miRNA directed to the mRNA encoding B7-H3, a polyclonal antibody, a monoclonal antibody, an aptamer, a soluble B7- H3 receptor that binds directly to B7-H3, an RNase H inducing antisense
  • oligonucleotide directed to the mRNA encoding B7-H3, and a ribozyme directed to the mRNA encoding B7-H3.
  • the taxane selected from the group consisting of paclitaxel and docetaxel.
  • cancer selected from the group consisting of breast cancer, ovarian cancer, clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer and pancreatic cancer.
  • NSCLC nonsmall-cell lung cancer
  • A Stable cell transfectants of the parental MDA-MB-231 and MDA-MB-435 human breast cancer cell lines expressing either vector control TR33 or B7-H3shRNA were generated. Western blot results showed the protein level of B7-H3 of MDA-MB-231 (left) and MDAMB-435 cells (right), expressing either vector control TR33 or shB7- H3 and their corresponding parental cells, ⁇ -actin was probed as a loading control.
  • B Cell viability of 231, 231-TR33, 231-shB7-H3 (left), 435, 435-TR33, and 435- shB7-H3 (right) following treatment with paclitaxel at various concentrations for 72 h was assessed using MTS assay.
  • Annexin V-FITC stained cells was increased in B7-H3 knockdown cells.
  • 231-TR33 and 231-shB7-H3 were treated with 20 nmol/L paclitaxel for 0, 48, and 72 h, respectively, and apoptosis was examined by Annexin V-FITC staining and flow cytometry.
  • TUNEL-positive cells The percentage of TUNEL-positive cells was increased in B7-H3 knockdown cells.
  • Cells were treated with 0, 10 and 20 nmol/L paclitaxel for 72 h, and apoptosis was examined by TUNEL assay and flow cytometry.
  • B7-H3 overexpressing MDA-MB-231 cell varaiants were more resistance to pacilitaxelinduced apoptosis.
  • B7-H3 overexpressing 231-B7-H3 cells expressed higher level of B7-H3, compared to the 231-EGFP control cells as detected by Western blot.
  • Middle; B7-H3 overexpressing cells showed decreased level of cleaved-PARP compared to the control cells upon treatment with 20nmol/L paclitaxel for 72 h.
  • apoptosis-specific ELISA detection revealed that the level of cytoplasmic histoneassociated DNA fragments in paclitaxel treated 231- B7H3 cells was much lower than in control 231-EGFP cells.
  • FIG. 3 B7-H3 silencing suppressed Stat3 phosphorylation in breast cancer cells.
  • A Cells were treated with 20 nmol/L paclitaxel or left untreated. Whole cell lysates were made 72 h following treatment and probed for phospho- and total Stat3 with ⁇ -actin as a loading control.
  • B and C B7-H3 silencing downregulated Mcl-1 and Survivin expression. Paclitaxel treatment and Western blotting were performed as described above, and ⁇ - actin or a-tublin was used as loading controls.
  • Figure 4. B7-H3 regulated the phosphorylation of Stat3 at least partially through Jak2.
  • A The levels of phosphorylated Jak2 were decreased in B7-H3 knockdown cells. Cells as indicated were treated with 20 nmol/L paclitaxel or left untreated. Whole cell lysates were made 72 h following treatment and then probed for phospho- and total Jak2 with ⁇ -atefcin as a loading control. B, AG490 treatment almost abolished the phosphorylation level of Jak2 and lead to reduced levels of tyrosine phosphorylation of Stat3 in both B7-H3 knockdown cells and control cells.
  • MDA MB-231 and MDA-MB-435 cell variants 22 were treated with 50 pmol/L or 100 pmol/L AG490, respectively, for 24 h and Western blot analysis was performed to examine the protein levels of phospho- and total Jak2 and Stat3 with ⁇ -actin as a loading control.
  • FIG. 1 Growth curves of breast cancer xenografts in nude mice established by s.c injection of 5x106 MDA-MB-435-shB7-H3 or MDA-MB-435-TR33 cells in both flanks of nude mice with or without paclitaxel treatment. Each group consisted of 8 animals.
  • Paclitaxel (10 mg/kg) was injected i.v. into the tail vein at day 0 when the average tumor diameter was 5-6 mm. The tumor diameter was measured 1-2 times per week. The data is presented as mean of two independent experiments ⁇ standard error of the mean (SEM).
  • FIG. 6 Melanom cell line: FEMX-I TR33 treated with Docetaxel for 72 hours. Results shown as % Growth relative to untreated cells.
  • An aspect of the present invention relates to the use of a combination dosage comprising a B7-H3 antagonist and a taxane for the preparation of a medicament for preventing the progression of cancer in a subject.
  • Another aspect of the present invention relates to the combination of a B7-H3 antagonist and a taxane for the manufacture of a medicament for the treatment of cancer in a subject, wherein the administration patternsof said medicament comprises:
  • Another aspect of the present invention relates to a combination dosage comprising a B7-H3 antagonist and a taxane for use in preventing the progression of cancer in a human.
  • combination and combinational dosage use interchangeably and refers to the uses and methods of the present invention, wherein the B7-H3 antagonist and a taxane are administered in combination i.e. as a combinational dosage.
  • a B7-H3 antagonist and a taxane is administered separately.
  • the combination dosage of a B7-H3 antagonist and a taxane is administered simultaneously with the same release rates.
  • the combination dosage of a B7-H3 antagonist and a taxane is administered simultaneously with different release rates.
  • the B7-H3 antagonist is the B7-H3 antagonist and the taxane is administered separately at time points selected from the group selected from 1 hour, 2 hours, 4 rhcrars, 10 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 108 hours and 120 hours.
  • Another aspect of the present invention relates to a method for preventing the progression of cancer comprising providing an individual in need thereof, and administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane.
  • prevention of progression of cancer also to inhibition of progression, treatment of cancer and for prevention of relapse or treatment of relapse of cancer.
  • prevention of the progression of cancer is the prevention of the progression of cancer a treatment of said cancer.
  • In another embodiment of the present invention is the prevention of the progression of cancer a prevention of relapse and/or treatment of relapse of said cancer.
  • the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a nonsmall-cell lung cancer (NSCLC) cell, a colorectal cancer cell, a pancreatic cancer cell and a cell from all other types of carcinomas.
  • NSCLC nonsmall-cell lung cancer
  • the cancer cell a breast cancer cell.
  • the carreer is selected from the group consisting of breast cancer, ovarian cancer, clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer and all other types of carcinomas.
  • the cancer is a breast cancer.
  • a “carcinoma” is tumor tissue derived from putative epithelial cells whose genome has become altered or damaged to such an extent that the cells become transformed, and begin to exhibit abnormal malignant properties.
  • Epithelial cancers are therefore known as carcinomas.
  • the uses and methods of the B7-H3 antagonists described herein are efficient in the treatment of epithelial cancers.
  • the cancer type an epithelial cancer.
  • epithelial cancers/carcinomas Several types of epithelial cancers/carcinomas are known :
  • Adenocarcinoma refers to a carcinoma featuring microscopic glandular-related tissue cytology, tissue architecture, and/or gland-related molecular products, e.g., mucin.
  • Squamous cell carcinoma refers to a carcinoma with observable features and characteristics indicative of squamous differentiation (intercellular bridges, keratinization, squamous pearls).
  • Anaplastic carcinoma refers to a heterogeneous group of high-grade carcinomas that feature cells lacking distinct histological or cytological evidence of any of the more specifically differentiated neoplasms. These tumors are referred to as Anaplastic or Undifferentiated carcinomas. Large cell carcinomas composed of large, monotonous rounded or overtly polygonal-shaped cells with abundant cytoplasm.
  • Small cell carcinomas cells are usually round and are less than approximately 3 times the diameter of a resting lymphocyte and little evident cytoplasm.
  • epithelial cancers selected from the group consisting of adenocarcinomas, squamous cell carcinomas, adenosquamous carcinomas, anaplastic carcinomas, anaplastic carcinomas, large cell carcinomas and small cell carcinomas.
  • epithelial cancer selected from the group consisting of breast cancer, ovarian cancer, clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer and all other types of carcinomas.
  • NSCLC nonsmall-cell lung cancer
  • “Grading” of carcinomas refers to the employment of criteria intended to semi- quantify the degree of cellular and tissue maturity seen in the transformed cells relative to the appearance of the normal "parent" epithelial tissue from which the carcinoma derives.
  • Grade 1 or "Well Differentiated”: Indicates there is a close, or very close, resemblance to the normal parent tissue, and that the tumor cells are easily identified and classified as a particular malignant histological entity.
  • Grade 2 or “Moderately Differentiated”: Indicates there is considerable resemblance to the parent cells and tissues, but that abnormalities can commonly be seen and the more complex features are not particularly well-formed.
  • Grade 3 or "Poorly Differentiated”: Indicates that there is very little resemblance between the malignant tissue and the normal parent tissue, with abnormalities evident and the more complex architectural features are usually rudimentary or primitive.
  • Grade 4 Indicates that these carcinomas bear no significant resemblance to the corresponding parent cells and tissues, with no visible formation of glands, ducts, bridges, stratified layers, keratin pearls, or other notable characteristics consistent with a more highly differentiated neoplasm.
  • the "grading" of the carcinoma selected from the group consisting of grade 1, grade 2, grade 3 and grade 4.
  • Administration pattern is the "grading" of the carcinoma selected from the group consisting of grade 1, grade 2, grade 3 and grade 4.
  • the sensitizing periods will depend on the B7-H3 antagonists selected for sensitization and the type of cancer.
  • the first sensitizing period selected from the group consisting of 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 108 hours and 120 hours.
  • the subsequent sensitizing periods of different length as the first sensitizing period are the subsequent sensitizing periods of different length as the first sensitizing period.
  • the therapeutical periods will also depend on the taxane selected for the treatment of cancer and on the type of cancer.
  • the first therapeutical period selected from the group consisting of 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 108 hours and 120 hours.
  • the therapeutically effective amounts and the sensitizing effective amounts will also depend of the B7-H3 antagonist and taxane selected for sensitization and treatment.
  • An aspect of the present invention relates to a B7-H3 antagonist for use in a method of treatment comprising sensitizing a cancer cell to a taxane.
  • B7-H3 antagonists for use in a method of treatment comprising sensitizing a cancer cell to a taxane.
  • the B7-H3 antagonist of the present invention may function through several different mechanisms, but the hallmark of the B7-H3 antagonist of the present invention is the ability to minimize the function and/or expression of B7-H3 mRNA transcripts or protein.
  • Such mechanisms are known in the art and can be either direct antagonists that function directly on the B7-H3 gene, mRNA or protein or alternatively through an indirectly through an antagonistic or agonistic pathway where one or more proteins other than B7-H3 that interact directly or indirectly with B7-H3 is/are regulated by expression and/or function.
  • the antagonists of the present invention can be competitive antagonists (also known as surmountable antagonists) that reversibly bind to receptors at the same binding site (active site) as the endogenous ligand or agonist, but without activating the target.
  • competitive antagonists also known as surmountable antagonists
  • Agonists and antagonists "compete" for the same binding site on the target, and once bound, an antagonist will block agonist binding.
  • the level of activity of the target will be determined by the relative affinity of each molecule for the site and their relative concentrations. High concentrations of a competitive agonist will increase the proportion of receptors that the agonist occupies and higher concentrations of the antagonist will be required to obtain the same degree of binding site occupancy.
  • the antagonists of the present invention can also be non-competitive antagonists (sometimes called non-surmountable antagonists) which are allosteric
  • the antagonists of the present invention can also be uncompetitive antagonists which differ from non-competitive antagonists in that they require target activation by an agonist before they can bind to a separate allosteric binding site.
  • This type of antagonism produces a kinetic profile in which the same amount of antagonist blocks higher concentrations of agonist better than lower
  • the antagonists of the present invention can also be partial agonists which are defined as drugs that, at a given target, might differ in the amplitude of the functional response that they elicit after maximal target occupancy.
  • partial agonists can act as a competitive antagonist in the presence of a full agonist, as it competes with the full agonist for target occupancy, thereby producing a net decrease in the target activation as compared to that observed with the full agonist alone.
  • the antagonists of tha present invention also be an inverse agonist that can have effects similar to those of an antagonist, but causes a distinct set of downstream biological responses.
  • Constitutively active targets that exhibit intrinsic or basal activity can have inverse agonists, which not only block the effects of binding agonists like a classical antagonist but also inhibit the basal activity of the target.
  • the B7-H3 antagonist selected from the group consisting of a competitive antagonist, a non-competitive antagonist, an uncompetitive antagonist, a partial agonist and an inverse agonist.
  • the B7-H3 antagonist an RNAi antagonist mediated through an RNAi pathway.
  • the RNAi antagonist selected from the group consisting of an siRNA directed to the mRNA encoding B7-H3, an shRNA directed to the mRNA encoding B7-H3 and a miRNA directed to the mRNA encoding B7-H3.
  • the B7-H3 antagonist selected from the group consisting of an siRNA directed to the mRNA encoding B7-H3, an shRNA directed to the mRNA encoding B7-H3, a miRNA directed to the mRNA encoding B7-H3, a polyclonal antibody, a monoclonal antibody, an aptamer, a soluble B7-H3 receptor that binds directly to B7-H3, an RNase H inducing antisense oligonucleotide directed to the mRNA encoding B7-H3, and a ribozyme directed to the mRNA encoding B7-H3.
  • the B7-H3 antagonist of the present invention targeting an upstream and/or downstream event that has a direct and/or indirect effect on B7-H3.
  • tafee nucleic acid based B7-H3 antagonists may individual nucleic adds- optionally be substituted by the nucleic acid analogues selected from the group consisting of PNA, LIMA, TINA, and Morpholino (PMO). Taxanes
  • taxanes relate to diterpenes produced by the plants of the genus Taxus (yews). They were first derived from natural sources, but some have been synthesized artificially. Taxanes have been used to produce various chemotherapy drugs and the primary mechanism of the taxane class of drugs is the disruption of microtubule function. Thus, taxanes are essentially mitotic inhibitors.
  • paclitaxel which is better known under its trademark Taxol.
  • paclitaxel is dissolved in Cremophor EL and ethanol, as a delivery agent.
  • Cremophor EL Cremophor EL
  • ethanol a delivery agent.
  • paclitaxel sold under the trademark Abraxane.
  • Taxotere Another well known taxane is docetaxel which is better known under its trademark Taxotere.
  • the taxane selected from the group consisting of paclitaxel and docetaxel. Sensitizing
  • the B7-H3 antagonists of the present invention have the ability to sensitize cancer cells to taxanes.
  • the cancer types that the B7-H3 antagonists target can be divided into three groups.
  • cancer types where taxanes presently are used in treatment but where the B7-H3 antagonist causes a bonus effect.
  • the cancers types sensitize by the B7-H3 antagonists of the present invention selected from the group consisting of cancer types that until now are resistant to taxanes, cancer types where taxanes until now only have had limited success and cancer types where taxanes presently are used in treatment, but where the B7-H3 antagonist causes a bonus effect.
  • An important aspect of the present invention pertains to a composition comprising the taxane and/or the B7-H3 antagonist of the present invention.
  • composition in order to ensure optimum performance of such composition it is preferred that it comprises an immunologically and pharmacally acceptable carrier, excipient, vehicle and/or adjuvant.
  • an immunologically and pharmacally acceptable carrier, excipient, vehicle or adjuvant is usually classified by application location (or exposition). However, routes of administration can also basically be classified whether the effect is local (in topical administration) or systemic (in enteral or parenteral administration).
  • Topical administration refers to a local effect where the substance is applied directly where its action is desired. Sometimes, however, the term topical is defined as applied to a localized area of the body or to the surface of a body part, without necessarily involving target effect of the substance, making the
  • the classification rather a variant of the classification based on application location.
  • the route of administration is selected from the group consisting of intradermal application, epicutaneous application and nasal application.
  • Enteral administration refers to where a desired effect is systemic (non-local), and the substance is given via the digestive tract.
  • Parenteral administration refers to where a desired effect is systemic, and the substance is given by routes other than the digestive tract.
  • Example 1 - B7-H3 silencing increases paclitaxel sensitivity by abrogating
  • B7-H3 A correlation between high expression of the immunoregulatory protein B7-H3 and poor prognosis has been observed in many human cancers. However, the role and mechanism of B7-H3 in cancer cell chemoresistance is unknown. We hypothesized that B7-H3 might affect drug resistance in a non-immunological manner and studied paclitaxel activity in two breast cancer cell lines both in vitro and in vivo. The overall objective was to elucidate the function of B7-H3 in human cancer.
  • B7-H3 expression was silenced by shRNA, resulting in significantly increased sensitivity to paclitaxel, mediated via increased susceptibility to apoptosis. Furthermore, B7-H3 knock down cells had reduced levels of activated Stat3.
  • the sensitization to paclitaxel seen in vitro was confirmed in a xenograft model in nude mice. The results are translational as they demonstrate the role of B7-H3 in paclitaxel resistance, and suggest that manipulating the expression of B7-H3 and/or its signalling pathway partners may represent a novel approach to increase clinical sensitivity to paclitaxel, and potentially also to other chemotherapeutic drugs.
  • breast cancer is the most common type of cancer as well as the most common cause of cancer-related death in women. Once breast cancer becomes metastatic, it is difficult to cure because it is often resistant to commonly used
  • chemotherapeutics such as a taxane-based regime, which is one of the most effective chemotherapeutic agents in the treatment of breast cancer.
  • a taxane-based regime which is one of the most effective chemotherapeutic agents in the treatment of breast cancer.
  • B7-H3 a transmembrane protein with immunoglobulin-like structure , is known to have immunoregulatory properties with both inhibitory and stimulatory effects on the activation of T-cells. It has also been shown that B7-H3 expressed at the cell surface of neuroblastoma cells can inhibit natural killer-mediated lysis. The B7-H3 protein is found in many human tissues, and it is observed to be overexpressed in a wide range of human cancers. Notably, immunohistochemical reports have shown a correlation between high expression of B7-H3 and poor outcome in patients with clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), and pancreatic cancer.
  • NSCLC nonsmall-cell lung cancer
  • B7-H3 structurally is a transmembrane protein, it is also found in the cytoplasm of tumor cells. Furthermore, a soluble form of the protein has been found in serum of patients with NSCLC, and elevated levels of circulating B7-H3 correlated with poor clinical outcome. The same group has reported the release of soluble B7-H3 from monocytes, dendritic cells, activated T-cells and some cancer cell lines.
  • Stat3 signal transducer and activator of transcription
  • Stat3 The activation of Stat3 is through phosphorylation by various tyrosine kinases, such as Janus activated kinases (Jak) and Src family kinases, which are associated with and activated by cytokine and growth factor receptors.
  • Jak Janus activated kinases
  • Src family kinases which are associated with and activated by cytokine and growth factor receptors.
  • Constitutive activation of Stat3 occurs frequently in breast tumors and has been confirmed in a wide range of breast cancer cell lines, possibly through over-expression or constitutive activation of upstream receptors such as EGFR and HER2 or nonreceptor tyrosine kinases.
  • High levels of Stat3 activity have been shown to predict intrinsic cbeaiotherapeutic drug resistance due to the upregulation of the cteesr--- antiapoptotic factors Bcl-xL, Bcl-2, Mcl-1 and Survivin.
  • Anti-human B7-H3 antibody and anti-Survivin was purchased from R&D Systems, Inc.
  • Antibodies against Stat3, p-Stat3Tyr705, p-Stat3Ser727, Jak2, p-Jakl, p- Jak2, p-Src, Mcl-1 and PARP were from Cell Signaling Technology.
  • Antibody against ⁇ -actin and Tryphostins AG490 were from Sigma-Aldrich Chemical Co.
  • the horseradish peroxidise conjugated secondary anti-mouse, anti-rabbit, and anti- goat antibodies were from Bio-Rad Laboratories, Inc. Paclitaxel was from Mead Johnson Inc. (Princeton, NJ).
  • Cell lines MDA-MB-231 and MDA-MB-435 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). The cells were cultured in DMEM/F12 medium (Mediatech, Inc.) supplemented with a 10% fetal bovine serum (FBS, Atlanta Biologicals Inc., Lawrenceville, GA) and 1% penicillin-streptomycin (Gibco, Paisley, UK) at 37°C under an atmosphere of 5% C02.
  • ATCC American Type Culture Collection
  • HuSH 29mer shRNA constructs against B7-H3 (shB7-H3) and control plasmid pRS non effective TR30003 (TR33) were purchased from Origene Technologies, Inc.
  • shRNA construct or control vector were transfected into MDA-MB-231 and MDA- MB-435 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocols, followed by selection with 1 pg/ml puromycin for 2 weeks. Antiboitic-resistant clones were isolated in medium with 0.5 pg/ml puromycin. RT-PCR and immunoblotting were performed to confirm the
  • NM_001024736 were amplified by PCR using the following primer sequences: Forward ( 5 '-TCACTCG AG CCCTG AGTCCCAG AGTCG G C- 3 ') (SEQ ID NO: 3); Reverse ( 5 '- ACTG AATTCGGTTGTG G GTG GTCTGTTC AT- 3 ') (SEQ ID NO:4); and then the full length cDNA was cloned into xho I and EcoR I linearized plasmid vector pIRES2- EGFP (Clonetech, USA).
  • the control vector pIRES2-EGFP and human B7-H3 expression vector pIRES2-B7-H3 were transfected in MDA-MB-231 cells respectively using Lipofectamine 2000. Stable clones were selected in medium containing 1.8 mg/mL Geneticin (Invitrogen, USA). The expression of B7-H3 was detected by Western blot. In vitro growth inhibition
  • Cells (1 x 104 cells) were initially plated in triplicate in 96-well culture plates. Twenty-four hours later, the medium was replaced with fresh medium with or without paclitaxel and incubated for indicated time. Cell viability was determined using cellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, USA).
  • 3 x 105 cells were grown in triplicate in 60 mm dishes with addition of 20 nmol/L paclitaxel for 0, 48 h, and 72 h respectively. And then cells were harvested and processed as described in the Annexin V-FITC Apoptosis Detection Kit I manual (BD Transduction Laboratories, BD Biosciences) and analyzed by flow cytometry (BD LSR II).
  • TUNEL assay s ⁇ " ⁇ ' ⁇ ' Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay was performed using recombinant terminal transferase (TdT) and biotin-16-dUTP (Roche Diagnostics GmbH, Mannheim, Germany). After treated with with with 0, 10 and 20 nmol/L paclitaxel for 72 h, cells were processed following manufacturer's protocol and then analyzed by flow cytometer (BD Biosciences, San Jose, CA). Each experiment was repeated three times.
  • phenylmethylsulfonyl fluoride 1 mmol/L Na3V04, and protease inhibitor cocktail (Sigma-Aldrich ) for 20 min on ice.
  • the proteins were separated by SDS-PAGE and then electrotransferred onto nitrocellulose membrane (Bio-rad). Membranes were probed with indicated antibodies following the manufacturer's protocol.
  • mice Four groups of seven female Balb/c nude mice, bred at the nude rodent facility at the Norwegian Radium Hospital were used. The animals were maintained under specific pathogen-free conditions, and food and water were supplied ad libitum. Animal experiments were performed according to protocols approved by the animal care and use committee and were in compliance with the guidelines on animal welfare of the Norwegian National Committee for Animal Experiments. When the animals were 6-8 weeks of age, 5x106 cells (MDA-MB-435 shB7-H3 or MDA-MB-435 TR33 cells) in 0.2 ml PBS were injected s.c. in both flanks of nude mice. Animals bearing tumors with diameters ⁇ 4 mm or > 8 mm at 20 days after injection were excluded.
  • 5x106 cells MDA-MB-435 shB7-H3 or MDA-MB-435 TR33 cells
  • paclitaxel for therapy experiments, a stock solution of paclitaxel in ethanol (6 mg/ml) was dissolved in PBS and a single dose of 10 mg/kg of the drug was injected i.v. into the tail vein when the mean tumor diameter was 5-6 mm (day 0). The control mice received only the solvent. Tumor diameters were measured one to two times per week. Tumor volume was calculated by the formula 0.5 x length x width2 and growth curves constructed, and the data presented as mean of two independent experiments ⁇ standard error of the mean (SEM).
  • Paclitaxel is known to exert its cytotoxic effect through induction of apoptosis (25), and hence we investigated whether the
  • the extent of apoptosis was investigated by checking the amount of Annexin-5 stained cells, a marker for early stage apoptosis in MDA-MB-231 and MDA-MB-435 cells, and in MDA-MB-231 cells also for the amount of TUNEL positive cells, which reflects late stage apoptosis.
  • the response to 20 nmol/L paclitaxel was time dependent with an increase in the amount Annexin-V positive cells detected at 48 and 72 h (Fig. 2A).
  • MDA-MB-231 B7-H3 knockdown cells were more sensitive to paclitaxel-induced apoptosis, with the percentage of apoptotic cells in 231-shB7-H3 cells about two fold that in 231- TR33 cells at both 48 h and 72 h. Similar results were observed in MDA-MB-435 cell variants (Fig. S1A). The late stage apoptotic response was also dose
  • TUNEL positive cells were 11% versus 2% at 48 h and 18% versus 6% at 72 h, respectively. These results were confirmed by investigating the
  • paclitaxel had a marginal (not statistically significant) effect on tumor growth rate of MDA-MB-435 TR33 tumors, whereas the drug showed a relatively strong antitumor effect in mice carrying MDAMB-435 shB7-H3 xenografts.
  • the drug showed a relatively strong antitumor effect in mice carrying MDAMB-435 shB7-H3 xenografts.
  • the effect became significant from day 22 (all the p-values at each time point after day 22 were lower than 0.03).
  • the growth rate of MDA-MB-435 TR33 cells was faster than that of B7-H3 knockdown cells. This does not, however, explain the differences in paclitaxel response between the two groups.
  • the in vivo data strongly confirms the effects observed in the in vitro cellular cytotoxic assay that B7-H3 plays a critical role in paclitaxel responsiveness of breast cancer cells.
  • taxanes have significantly improved the overall and disease-free patient survival (28, 29).
  • the taxanes exert a response rate between 25% and 69% used as first-line treatment.
  • inherited and acquired resistance to taxanes represents a major obstacle in improving the overall response and survival. Therefore, the development of new therapeutic interventions to overcome taxane resistance becomes an important task in cancer research.
  • MDA-MB-435 The human MDA-MB-435 cell line was originally described as of breast cancer origin, whereas gene expression array studies indicated the cells to originate from malignant melanoma. However, subsequent evidence suggests that MDA-MB-435 is in fact a breast cancer cell line. In our study, the results obtained with MDA-MB- 435 cells were closely similar to those with MDA-MB-231 breast cancer cells. Importantly, our promising in vitro results demonstrating that B7-H3 seem to play a critical role in paclitaxel resistance, were confirmed in an animal model using the MDAMB-435 xenografts in which the growth rate of established B7-H3 knock down xenografts was significantly reduced whereas the growth of transfection control tumors were only marginally affected.
  • MDA-MB-435 vector control cells were faster than that of B7-H3 knockdown cells. This cannot, however, explain the differences in sensitivity to paclitaxel, as it is well known that fast-growing tumors generally respond better to chemotherapy than more slowly growing counterparts.
  • B7-H3 confers resistance to paclitaxel by reducing the sensitivity of the tumor cells to apoptosis, mediated via the Jak2/Stat3 pathway.
  • B7-H3 and its pathway partners are potential targets for developing novel therapeutic approaches.
  • our data demonstrates that B7-H3 plays an important role in determining the resistance to paclitaxel via non-immune mechanisms. Whether this effect of B7-H3 may extend to other drugs and tumor types is not yet determined, but preliminary results in our laboratory are promising and will be further explored.
  • Figures 6 and 7 shown experiments similar to the experiments shown in example 1 but with docetaxel as the active anti-cancer drug.

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Abstract

The present invention relates to the use of a combination dosage comprising a B7-H3 antagonist and a taxane for the preparation of a medicament for preventing the progression of cancer in a human. An aspect of the present invention relates to a B7-H3 antagonist for sensitizing a cancer cell to a taxane. Another aspect of the present invention relates to a method for preventing the progression of cancer comprising providing an individual in need thereof, and administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane.

Description

B7 - H3 ANTAGONI STS AND TAXANES
Technical field of the invention
The present invention relates to the use of a combination dosage comprising a B7- H3 antagonist and a taxane for the preparation of a medicament for preventing the progression of cancer in a human. An aspect of the present invention relates to a B7-H3 antagonist for sensitizing a cancer cell to a taxane. Another aspect of the present invention relates to a method for preventing the progression of cancer comprising providing an individual in need thereof, and administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane.
Background of the invention
Breast cancer is an epithelial cancer and is the most common type of cancer as well as the most common cause of cancer-related death in women. Once breast cancer becomes metastatic, it is difficult to cure because it is often resistant to commonly used chemotherapeutics, such as a taxane-based regime, which is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Hence, if the sensitivity to taxane-based chemotherapy could be increased, it would represent an important step in improving the clinical management of breast cancer.
B7-H3, a transmembrane protein with immunoglobulin-like structure, is known to have immunoregulatory properties with both inhibitory and stimulatory effects on the activation of T-cells. It has also been shown that B7-H3 expressed at the cell surface of neuroblastoma cells can inhibit natural killer-mediated lysis. The B7-H3 protein is found in many human tissues, and it is observed to be overexpressed in a wide range of human cancers.
Notably, immunohistochemical reports have shown a correlation between high expression of B7-H3 and poor outcome in patients with clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer and pancreatic cancer. In all cases, the overexpression of B7-H3 was correlated to lymph node metastasis and tumor infiltration, and the effects were ascribed to the putative immune-inhibitory function of B7-H3. Although B7-H3 structurally is a transmembrane protein, it is also found in the cytoplasm of tumor cells. Furthermore, a soluble form of the protein has been found in serum of patients with NSCLC, and elevated levels of circulating B7-H3 correlated with poor clinical outcome. The same group has reported the release of soluble B7-H3 from monocytes, dendritic cells, activated T-cells and some cancer cell lines.
By using immunoprecipitation and mass spectrometry we have not been able to confirm this in our cell models, possibly suggesting that not all cancer cells expressing membrane bound B7-H3 release a soluble form of the protein.
Whereas previous studies have focused on the immune-regulatory function of B7- H3 and linked this to tumor progression and poor prognosis, our studies in melanoma cells, with no immunological factors present, demonstrated for the first time that the knockdown of B7-H3 expression resulted in a significant inhibition of cell migration and invasion, indicating that B7-H3 is involved in cancer cell invasion and metastasis via a non-immunological mechanism.
Since our results indicated a direct association between B7-H3 expression and metastasis, and since metastasis and chemoresistance are closely related processes, we focused our present study on the putative role of B7-H3 in the sensitivity of metastatic breast cancer cells to paclitaxel and the possible underlying mechanisms. Stat3 (signal transducer and activator of transcription) is a cytoplasmic transcription factor that regulates cellular differentiation,
proliferation, and survival in response to numerous cytokines and growth factors and hereby plays a key role in malignant processes. The activation of Stat3 is through phosphorylation by various tyrosine kinases, such as Janus activated kinases (Jak) and Src family kinases, which are associated with and activated by cytokine and growth factor receptors. The tyrosine phosphorylation leads to Stat dimerization, followed by nuclear translocation, and activation of the expression of several genes. Constitutive activation of Stat3 occurs frequently in breast tumors and has been confirmed in a wide range of breast cancer cell lines, possibly through over-expression or constitutive activation of upstream receptors such as EGFR and HER2 or non- receptor tyrosine kinases. High levels of Stat3 activity have been shown to predict intrinsic chemotherapeutic drug resistance due to the upregulation of the antiapoptotic factors Bcl-xL, Bcl-2, cl-1 and Survivin.
Summary of the invention Use of a B7-H3 antagonist and a taxane
An aspect of the present invention relates to the use of a combination dosage comprising a B7-H3 antagonist and a taxane for the preparation of a medicament for preventing the progression of cancer in a human. Another aspect of the present invention relates to the use of a combination of a B7-H3 antagonist and a taxane for the manufacture of a medicament for the treatment of cancer in a subject, wherein the administration pattern of said medicament comprises:
(i) administering to said subject a first sensitizing effective amount of a B7- H3 antagonist during a first sensitizing period; and
(ii) after completion of said first sensitizing period, administering to said subject a first therapeutically effective amount of a taxane during a first therapeutical period; and
(iii) optionally continue sensitizing and treatment according to (i) and (ii).
In an embodiment of the present invention is the cancer an epithelial cancer. Substances
Another aspect of the present invention relates to a B7-H3 antagonist for use in a method of treatment comprising sensitizing a cancer cell to a taxane.
Method of treatment
Another aspect of the present invention relates to a method for preventing the progression of cancer comprising providing an individual in need thereof, and administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane. In an embodiment ofethe present invention is the B7-H3 antagonist selected from the group consisting of an siRNA directed to the mRNA encoding B7-H3, an shRNA directed to the mRNA encoding B7-H3, a miRNA directed to the mRNA encoding B7-H3, a polyclonal antibody, a monoclonal antibody, an aptamer, a soluble B7- H3 receptor that binds directly to B7-H3, an RNase H inducing antisense
oligonucleotide directed to the mRNA encoding B7-H3, and a ribozyme directed to the mRNA encoding B7-H3.
In another embodiment of the present invention is the taxane selected from the group consisting of paclitaxel and docetaxel.
In yet another embodiment of the present invention is the cancer selected from the group consisting of breast cancer, ovarian cancer, clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer and pancreatic cancer.
Brief description of the figures
Figure 1. B7-H3 silencing increased the sensitivity to paclitaxel in breast cancer cells.
A, Stable cell transfectants of the parental MDA-MB-231 and MDA-MB-435 human breast cancer cell lines expressing either vector control TR33 or B7-H3shRNA were generated. Western blot results showed the protein level of B7-H3 of MDA-MB-231 (left) and MDAMB-435 cells (right), expressing either vector control TR33 or shB7- H3 and their corresponding parental cells, β-actin was probed as a loading control. B, Cell viability of 231, 231-TR33, 231-shB7-H3 (left), 435, 435-TR33, and 435- shB7-H3 (right) following treatment with paclitaxel at various concentrations for 72 h was assessed using MTS assay. Data are presented as the percentage of cell growth inhibition measured in cells treated without taxol. Columns, mean of three independent experiments performed in triplicates; bars, S.E.; ** p< 0.01; *** p < 0.001. Figure 2. B7-H3 silencing sensitizes breast cancer cells.rto paclitaxel-induced apoptosis.
A, The percentage of Annexin V- FITC stained cells was increased in B7-H3 knockdown cells. 231-TR33 and 231-shB7-H3 were treated with 20 nmol/L paclitaxel for 0, 48, and 72 h, respectively, and apoptosis was examined by Annexin V-FITC staining and flow cytometry.
B, The percentage of TUNEL-positive cells was increased in B7-H3 knockdown cells. Cells were treated with 0, 10 and 20 nmol/L paclitaxel for 72 h, and apoptosis was examined by TUNEL assay and flow cytometry.
C, PARP cleavage was increased in B7-H3 knockdown cells, as detected by
Western blotting following treatment with 20 nmol/L paclitaxel for 72 h. β-actin was used as a loading control.
D, 21 B7-H3 overexpressing MDA-MB-231 cell varaiants were more resistance to pacilitaxelinduced apoptosis. Left; B7-H3 overexpressing 231-B7-H3 cells expressed higher level of B7-H3, compared to the 231-EGFP control cells as detected by Western blot. Middle; B7-H3 overexpressing cells showed decreased level of cleaved-PARP compared to the control cells upon treatment with 20nmol/L paclitaxel for 72 h. Right; apoptosis-specific ELISA detection revealed that the level of cytoplasmic histoneassociated DNA fragments in paclitaxel treated 231- B7H3 cells was much lower than in control 231-EGFP cells.
Figure 3. B7-H3 silencing suppressed Stat3 phosphorylation in breast cancer cells. A, Cells were treated with 20 nmol/L paclitaxel or left untreated. Whole cell lysates were made 72 h following treatment and probed for phospho- and total Stat3 with β-actin as a loading control. B and C, B7-H3 silencing downregulated Mcl-1 and Survivin expression. Paclitaxel treatment and Western blotting were performed as described above, and β- actin or a-tublin was used as loading controls. Figure 4. B7-H3 regulated the phosphorylation of Stat3 at least partially through Jak2.
A, The levels of phosphorylated Jak2 were decreased in B7-H3 knockdown cells. Cells as indicated were treated with 20 nmol/L paclitaxel or left untreated. Whole cell lysates were made 72 h following treatment and then probed for phospho- and total Jak2 with β-atefcin as a loading control. B, AG490 treatment almost abolished the phosphorylation level of Jak2 and lead to reduced levels of tyrosine phosphorylation of Stat3 in both B7-H3 knockdown cells and control cells. MDA MB-231 and MDA-MB-435 cell variants 22 were treated with 50 pmol/L or 100 pmol/L AG490, respectively, for 24 h and Western blot analysis was performed to examine the protein levels of phospho- and total Jak2 and Stat3 with β-actin as a loading control.
Figure 5. Growth curves of breast cancer xenografts in nude mice established by s.c injection of 5x106 MDA-MB-435-shB7-H3 or MDA-MB-435-TR33 cells in both flanks of nude mice with or without paclitaxel treatment. Each group consisted of 8 animals.
Paclitaxel (10 mg/kg) was injected i.v. into the tail vein at day 0 when the average tumor diameter was 5-6 mm. The tumor diameter was measured 1-2 times per week. The data is presented as mean of two independent experiments ± standard error of the mean (SEM).
Figure 6: Melanom cell line: FEMX-I TR33 treated with Docetaxel for 72 hours. Results shown as % Growth relative to untreated cells.
Figure 7: Melanom cell lines: FEMX-I TR33 and MDA MB 435 treated with
Docetaxel for 72 hours.
Detailed description of the invention
Use
An aspect of the present invention relates to the use of a combination dosage comprising a B7-H3 antagonist and a taxane for the preparation of a medicament for preventing the progression of cancer in a subject.
Another aspect of the present invention relates to the combination of a B7-H3 antagonist and a taxane for the manufacture of a medicament for the treatment of cancer in a subject, wherein the administration patternsof said medicament comprises:
(i) administering to said subject a first sensitizing effective amount of a B7- H3 antagonist during a first sensitizing period; and
(ii) after completion of said first sensitizing period, administering to said subject a first therapeutically effective amount of a taxane during a first therapeutical period; and
(iii) optionally continue sensitizing and treatment by administering to said subject subsequent sensitizing effective amounts of a B7-H3 antagonists during subsequent sensitizing periods and subsequent therapeutically effective amounts of a taxanes during subsequent therapeutical periods.
Another aspect of the present invention relates to a combination dosage comprising a B7-H3 antagonist and a taxane for use in preventing the progression of cancer in a human.
Combination dosage
In the present context is the wording combination and combinational dosage use interchangeably and refers to the uses and methods of the present invention, wherein the B7-H3 antagonist and a taxane are administered in combination i.e. as a combinational dosage.
In an embodiment of the present invention is the combination dosage of a B7-H3 antagonist and a taxane is administered separately.
In another embodiment of the present invention is the combination dosage of a B7-H3 antagonist and a taxane is administered simultaneously with the same release rates. In yet another embodiment of the present invention is the combination dosage of a B7-H3 antagonist and a taxane is administered simultaneously with different release rates.
In an embodiment of the present invention is the B7-H3 antagonist and the taxane is administered separately at time points selected from the group selected from 1 hour, 2 hours, 4 rhcrars, 10 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 108 hours and 120 hours.
Treatment of cancer
Another aspect of the present invention relates to a method for preventing the progression of cancer comprising providing an individual in need thereof, and administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane.
In the present context refers prevention of progression of cancer also to inhibition of progression, treatment of cancer and for prevention of relapse or treatment of relapse of cancer. In an embodiment of the present invention is the prevention of the progression of cancer a treatment of said cancer.
In another embodiment of the present invention is the prevention of the progression of cancer a prevention of relapse and/or treatment of relapse of said cancer.
Cancers
Several types of cancers and cancer cells are targeted by the antagonists and taxanes of the present invention and through the uses and methods of the present invention.
In an embodiment of the present invention is the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a nonsmall-cell lung cancer (NSCLC) cell, a colorectal cancer cell, a pancreatic cancer cell and a cell from all other types of carcinomas.
In a preferred embodiment of the present invention is the cancer cell a breast cancer cell. In yet another embodiment of the present invention is the carreer is selected from the group consisting of breast cancer, ovarian cancer, clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer and all other types of carcinomas.
In another embodiment of the present invention is the cancer is a breast cancer. Epithelial cancers and carcinomas
A "carcinoma" is tumor tissue derived from putative epithelial cells whose genome has become altered or damaged to such an extent that the cells become transformed, and begin to exhibit abnormal malignant properties.
Epithelial cancers are therefore known as carcinomas. The uses and methods of the B7-H3 antagonists described herein are efficient in the treatment of epithelial cancers.
The efficiency of the antagonists is substantiated in the examples of the present application.
Thus, in one aspect of the present invention is the cancer type an epithelial cancer.
Several types of epithelial cancers/carcinomas are known :
Adenocarcinoma refers to a carcinoma featuring microscopic glandular-related tissue cytology, tissue architecture, and/or gland-related molecular products, e.g., mucin. Squamous cell carcinoma refers to a carcinoma with observable features and characteristics indicative of squamous differentiation (intercellular bridges, keratinization, squamous pearls). Adenosquamous carcinomarfiefers to a mixed tumor containing both
adenocarcinoma and squamous cell carcinoma, wherein each of these cell types comprise at least 10% of the tumor volume. Anaplastic carcinoma refers to a heterogeneous group of high-grade carcinomas that feature cells lacking distinct histological or cytological evidence of any of the more specifically differentiated neoplasms. These tumors are referred to as Anaplastic or Undifferentiated carcinomas. Large cell carcinomas composed of large, monotonous rounded or overtly polygonal-shaped cells with abundant cytoplasm.
Small cell carcinomas cells are usually round and are less than approximately 3 times the diameter of a resting lymphocyte and little evident cytoplasm.
Thus relates one embodiment of the present invention to epithelial cancers selected from the group consisting of adenocarcinomas, squamous cell carcinomas, adenosquamous carcinomas, anaplastic carcinomas, anaplastic carcinomas, large cell carcinomas and small cell carcinomas.
In another embodiment of the present invention is the epithelial cancer selected from the group consisting of breast cancer, ovarian cancer, clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer and all other types of carcinomas.
"Grading" of carcinomas refers to the employment of criteria intended to semi- quantify the degree of cellular and tissue maturity seen in the transformed cells relative to the appearance of the normal "parent" epithelial tissue from which the carcinoma derives.
Grade 1, or "Well Differentiated": Indicates there is a close, or very close, resemblance to the normal parent tissue, and that the tumor cells are easily identified and classified as a particular malignant histological entity. Grade 2, or "Moderately Differentiated": Indicates there is considerable resemblance to the parent cells and tissues, but that abnormalities can commonly be seen and the more complex features are not particularly well-formed. Grade 3, or "Poorly Differentiated": Indicates that there is very little resemblance between the malignant tissue and the normal parent tissue, with abnormalities evident and the more complex architectural features are usually rudimentary or primitive. Grade 4, or "Undifferentiated Carcinoma": Indicates that these carcinomas bear no significant resemblance to the corresponding parent cells and tissues, with no visible formation of glands, ducts, bridges, stratified layers, keratin pearls, or other notable characteristics consistent with a more highly differentiated neoplasm.
Thus, in one embodiment of the present invention is the "grading" of the carcinoma selected from the group consisting of grade 1, grade 2, grade 3 and grade 4. Administration pattern
The sensitizing periods will depend on the B7-H3 antagonists selected for sensitization and the type of cancer.
Thus, in one embodiment of the present invention is the first sensitizing period selected from the group consisting of 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 108 hours and 120 hours.
In another embodiment of the present invention are the subsequent sensitizing periods of the same length as the first sensitizing period.
In another embodiment of the present invention are the subsequent sensitizing periods of different length as the first sensitizing period.
The therapeutical periods will also depend on the taxane selected for the treatment of cancer and on the type of cancer. Thus, in one embodiment of the present invention is the first therapeutical period selected from the group consisting of 1 hour, 2 hours, 4 hours, 10 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 108 hours and 120 hours.
In another embodiment of the present invention are the subsequent therapeutical periods of the same length as the first therapeutical period.
In another embodiment of the present invention are the subsequent therapeutical periods of different length as first therapeutical period.
The therapeutically effective amounts and the sensitizing effective amounts will also depend of the B7-H3 antagonist and taxane selected for sensitization and treatment.
Substances
An aspect of the present invention relates to a B7-H3 antagonist for use in a method of treatment comprising sensitizing a cancer cell to a taxane. B7-H3 antagonists
The B7-H3 antagonist of the present invention may function through several different mechanisms, but the hallmark of the B7-H3 antagonist of the present invention is the ability to minimize the function and/or expression of B7-H3 mRNA transcripts or protein.
Such mechanisms are known in the art and can be either direct antagonists that function directly on the B7-H3 gene, mRNA or protein or alternatively through an indirectly through an antagonistic or agonistic pathway where one or more proteins other than B7-H3 that interact directly or indirectly with B7-H3 is/are regulated by expression and/or function.
The antagonists of the present invention can be competitive antagonists (also known as surmountable antagonists) that reversibly bind to receptors at the same binding site (active site) as the endogenous ligand or agonist, but without activating the target.
Agonists and antagonists "compete" for the same binding site on the target, and once bound, an antagonist will block agonist binding. The level of activity of the target will be determined by the relative affinity of each molecule for the site and their relative concentrations. High concentrations of a competitive agonist will increase the proportion of receptors that the agonist occupies and higher concentrations of the antagonist will be required to obtain the same degree of binding site occupancy.
The antagonists of the present invention can also be non-competitive antagonists (sometimes called non-surmountable antagonists) which are allosteric
antagonists.
These antagonists bind to a distinctly separate binding site from the agonist, exerting their action to that receptor via the other binding site.
The antagonists of the present invention can also be uncompetitive antagonists which differ from non-competitive antagonists in that they require target activation by an agonist before they can bind to a separate allosteric binding site.
This type of antagonism produces a kinetic profile in which the same amount of antagonist blocks higher concentrations of agonist better than lower
concentrations of agonist.
The antagonists of the present invention can also be partial agonists which are defined as drugs that, at a given target, might differ in the amplitude of the functional response that they elicit after maximal target occupancy.
Although they are agonists, partial agonists can act as a competitive antagonist in the presence of a full agonist, as it competes with the full agonist for target occupancy, thereby producing a net decrease in the target activation as compared to that observed with the full agonist alone. Finally can the antagonists of tha present invention also be an inverse agonist that can have effects similar to those of an antagonist, but causes a distinct set of downstream biological responses. Constitutively active targets that exhibit intrinsic or basal activity can have inverse agonists, which not only block the effects of binding agonists like a classical antagonist but also inhibit the basal activity of the target.
In an embodiment of the present invention is the B7-H3 antagonist selected from the group consisting of a competitive antagonist, a non-competitive antagonist, an uncompetitive antagonist, a partial agonist and an inverse agonist.
In another embodiment of the present invention is the B7-H3 antagonist an RNAi antagonist mediated through an RNAi pathway. In an another embodiment is the RNAi antagonist selected from the group consisting of an siRNA directed to the mRNA encoding B7-H3, an shRNA directed to the mRNA encoding B7-H3 and a miRNA directed to the mRNA encoding B7-H3.
In another embodiment of the present invention is the B7-H3 antagonist selected from the group consisting of an siRNA directed to the mRNA encoding B7-H3, an shRNA directed to the mRNA encoding B7-H3, a miRNA directed to the mRNA encoding B7-H3, a polyclonal antibody, a monoclonal antibody, an aptamer, a soluble B7-H3 receptor that binds directly to B7-H3, an RNase H inducing antisense oligonucleotide directed to the mRNA encoding B7-H3, and a ribozyme directed to the mRNA encoding B7-H3.
In another embodiment of the present invention is the B7-H3 antagonist of the present invention targeting an upstream and/or downstream event that has a direct and/or indirect effect on B7-H3.
In yet another embodiment of the present invention is the B7-H3 antagonist targeting a receptor selected from the group consisting of EGFR, HER2 and nonreceptor tyrosine kinases. tafee nucleic acid based B7-H3 antagonists may individual nucleic adds- optionally be substituted by the nucleic acid analogues selected from the group consisting of PNA, LIMA, TINA, and Morpholino (PMO). Taxanes
The term taxanes relates to diterpenes produced by the plants of the genus Taxus (yews). They were first derived from natural sources, but some have been synthesized artificially. Taxanes have been used to produce various chemotherapy drugs and the primary mechanism of the taxane class of drugs is the disruption of microtubule function. Thus, taxanes are essentially mitotic inhibitors.
The most well known taxane is paclitaxel which is better known under its trademark Taxol. In this formulation, paclitaxel is dissolved in Cremophor EL and ethanol, as a delivery agent. I another formulation, in which paclitaxel is bound to albumin, is paclitaxel sold under the trademark Abraxane.
Another well known taxane is docetaxel which is better known under its trademark Taxotere.
Thus, in an embodiment of the present invention is the taxane selected from the group consisting of paclitaxel and docetaxel. Sensitizing
The successes of combination anticancer therapy have suggested that all cancers can be treated provided that the correct combination of drugs at the correct doses and correct intervals are established. However, with time, tumor cells develop mechanisms of resistance to apoptosis and no longer respond to the majority of therapies.
In addition, some type of cancer may successfully be treated by some anticancer therapies that others might not succeed in treating. The B7-H3 antagonists of the present invention have the ability to sensitize cancer cells to taxanes. The cancer types that the B7-H3 antagonists target can be divided into three groups.
First are cancer types that until now are resistant to taxanes. Second are cancer types where taxanes until now only have had limited success.
Third are cancer types where taxanes presently are used in treatment, but where the B7-H3 antagonist causes a bonus effect. Thus in an embodiment of the present invention are the cancers types sensitize by the B7-H3 antagonists of the present invention selected from the group consisting of cancer types that until now are resistant to taxanes, cancer types where taxanes until now only have had limited success and cancer types where taxanes presently are used in treatment, but where the B7-H3 antagonist causes a bonus effect.
Delivery
An important aspect of the present invention pertains to a composition comprising the taxane and/or the B7-H3 antagonist of the present invention.
In order to ensure optimum performance of such composition it is preferred that it comprises an immunologically and pharmacally acceptable carrier, excipient, vehicle and/or adjuvant. Thus in an embodiment of the present invention is the taxane formulated in a composition with an immunologically and pharmacally acceptable carrier, excipient, vehicle or adjuvant. Admtmstration route -fvmss Routes of administration are usually classified by application location (or exposition). However, routes of administration can also basically be classified whether the effect is local (in topical administration) or systemic (in enteral or parenteral administration).
Topical administration refers to a local effect where the substance is applied directly where its action is desired. Sometimes, however, the term topical is defined as applied to a localized area of the body or to the surface of a body part, without necessarily involving target effect of the substance, making the
classification rather a variant of the classification based on application location. Thus relates one embodiment of the present application of the B7-H3 antagonist or taxes, wherein the route of administration is selected from the group consisting of intradermal application, epicutaneous application and nasal application.
Enteral administration refers to where a desired effect is systemic (non-local), and the substance is given via the digestive tract.
Thus relates another embodiment of the present application of the B7-H3 antagonist or taxes, wherein the route of administration is oral administration that involves the gastrointestinal tract.
Parenteral administration refers to where a desired effect is systemic, and the substance is given by routes other than the digestive tract.
Thus relates another embodiment of the present application of the B7-H3 antagonist or taxes, wherein the route of administration is selected from the group consisting of intravenous application, intramuscular application,
intraosseous infusion, intraperitoneal application and transmucosal application. Examples
Example 1 - B7-H3 silencing increases paclitaxel sensitivity by abrogating
Jak2/Stat3 phosphorylation. Abstract
Purpose: A correlation between high expression of the immunoregulatory protein B7-H3 and poor prognosis has been observed in many human cancers. However, the role and mechanism of B7-H3 in cancer cell chemoresistance is unknown. We hypothesized that B7-H3 might affect drug resistance in a non-immunological manner and studied paclitaxel activity in two breast cancer cell lines both in vitro and in vivo. The overall objective was to elucidate the function of B7-H3 in human cancer.
Experimental Design : MDA-MB-231 and MDA-MB-435 cancer cells and
variants transfected with shRNA against B7-H3 as well as transfection control cells were exposed to paclitaxel and the effects of B7-H3 knock down on cell survival, apoptosis, and on molecules in the Jak/Stat pathway were examined. Further, the efficacy of paclitaxel to inhibit the growth of MDA-MB-435 xenografts established from the cell variants was examined.
Results: Silencing of B7-H3 increased the sensitivity of paclitaxel in both tumor cell types as a result of enhanced drug-induced apoptosis. B7-H3 knock down decreased the level of Stat3 Tyr705 phosphorylation and its direct downstream targets Mcl-l and Survivin and the phosphorylation of Jak2, an upstream molecule of Stat3, was also significantly decreased. Paclitaxel treatment in vivo was significantly more effective in B7-H3 knockdown xenografts compared to controls. Conclusion: Our data demonstrates that B7-H3 causes anti-apoptosis and induces paclitaxel resistance in our cell lines, at least partially by interfering with the Jak2/Stat3 pathway in cancer cells. These novel findings may have important implications for the design of new approaches targeting B7-H3 overexpressing tumors. Translational relevance asifcr The expression of the B7-H3 protein correlates with poor prognosis in several tumor types, and is also associated with the ability of tumor cells to metastasize. On this basis, we hypothesized that B7-H3 could confer resistance to
chemotherapy. In MDA-MB-231 and MDA-MB-435 cancer cells B7-H3 expression was silenced by shRNA, resulting in significantly increased sensitivity to paclitaxel, mediated via increased susceptibility to apoptosis. Furthermore, B7-H3 knock down cells had reduced levels of activated Stat3. The sensitization to paclitaxel seen in vitro was confirmed in a xenograft model in nude mice. The results are translational as they demonstrate the role of B7-H3 in paclitaxel resistance, and suggest that manipulating the expression of B7-H3 and/or its signalling pathway partners may represent a novel approach to increase clinical sensitivity to paclitaxel, and potentially also to other chemotherapeutic drugs. Introduction
Breast cancer is the most common type of cancer as well as the most common cause of cancer-related death in women. Once breast cancer becomes metastatic, it is difficult to cure because it is often resistant to commonly used
chemotherapeutics, such as a taxane-based regime, which is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Hence, if the sensitivity to taxane-based chemotherapy could be increased, it would represent an important step in improving the clinical management of breast cancer.
B7-H3, a transmembrane protein with immunoglobulin-like structure , is known to have immunoregulatory properties with both inhibitory and stimulatory effects on the activation of T-cells. It has also been shown that B7-H3 expressed at the cell surface of neuroblastoma cells can inhibit natural killer-mediated lysis. The B7-H3 protein is found in many human tissues, and it is observed to be overexpressed in a wide range of human cancers. Notably, immunohistochemical reports have shown a correlation between high expression of B7-H3 and poor outcome in patients with clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), and pancreatic cancer. In all cases, the overexpression of B7-H3 was correlated to lymph node metastasis and tumor infiltration, and the effects were ascribed to the putative immune-inhibitory function of B7-H3. Although B7-H3 structurally is a transmembrane protein, it is also found in the cytoplasm of tumor cells. Furthermore, a soluble form of the protein has been found in serum of patients with NSCLC, and elevated levels of circulating B7-H3 correlated with poor clinical outcome. The same group has reported the release of soluble B7-H3 from monocytes, dendritic cells, activated T-cells and some cancer cell lines.
By using immunoprecipitation and mass spectrometry we have not been able to confirm this in our cell models, possibly suggesting that not all cancer cells expressing membrane bound B7-H3 release a soluble form of the protein.
Whereas previous studies have focused on the immune-regulatory function of B7- H3 and linked this to tumor progression and poor prognosis, our studies in melanoma cells, with no immunological factors present, demonstrated for the first time that the knockdown of B7-H3 expression resulted in a significant inhibition of cell migration and invasion, indicating that B7-H3 is involved in cancer cell invasion and metastasis via a non-immunological mechanism.
Since our results indicated a direct association between B7-H3 expression and metastasis, and since metastasis and chemoresistance are closely related processes, we focused our present study on the putative role of B7-H3 in the sensitivity of metastatic breast cancer cells to paclitaxel and the possible underlying mechanisms. Stat3 (signal transducer and activator of transcription) is a cytoplasmic transcription factor that regulates cellular differentiation,
proliferation, and survival in response to numerous cytokines and growth factors and hereby plays a key role in malignant processes. The activation of Stat3 is through phosphorylation by various tyrosine kinases, such as Janus activated kinases (Jak) and Src family kinases, which are associated with and activated by cytokine and growth factor receptors.
The tyrosine phosphorylation leads to Stat dimerization, followed by nuclear translocation, and activation of the expression of several genes. Constitutive activation of Stat3 occurs frequently in breast tumors and has been confirmed in a wide range of breast cancer cell lines, possibly through over-expression or constitutive activation of upstream receptors such as EGFR and HER2 or nonreceptor tyrosine kinases. High levels of Stat3 activity have been shown to predict intrinsic cbeaiotherapeutic drug resistance due to the upregulation of the cteesr--- antiapoptotic factors Bcl-xL, Bcl-2, Mcl-1 and Survivin.
In the present study, we found that silencing of B7-H3 sensitized the breast cancer cells MDA-MB-231 and MDA-MB-435 to paclitaxel both in vitro and in vivo. Moreover, we discovered that knock down of B7-H3 affected the phosphorylation status of Stat3, possibly through activation of Jak2, and lead also to
downregulation of the direct target genes of Stat3; Survivin and Mcl-1. The interference of B7-H3 with the Jak2/Stat3/Survivin/Mcl-1 pathway may explain the observed increased sensitivity of breast cancer cells to paclitaxel lacking the B7-H3 protein. This novel finding may have important implications for the therapeutic approach of paclitaxel- refractory breast cancers.
Materials and Methods
Reagents
Anti-human B7-H3 antibody and anti-Survivin was purchased from R&D Systems, Inc. Antibodies against Stat3, p-Stat3Tyr705, p-Stat3Ser727, Jak2, p-Jakl, p- Jak2, p-Src, Mcl-1 and PARP were from Cell Signaling Technology. Antibody against β-actin and Tryphostins AG490 were from Sigma-Aldrich Chemical Co. The horseradish peroxidise conjugated secondary anti-mouse, anti-rabbit, and anti- goat antibodies were from Bio-Rad Laboratories, Inc. Paclitaxel was from Mead Johnson Inc. (Princeton, NJ).
Cells and cell culture
Cell lines MDA-MB-231 and MDA-MB-435 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). The cells were cultured in DMEM/F12 medium (Mediatech, Inc.) supplemented with a 10% fetal bovine serum (FBS, Atlanta Biologicals Inc., Lawrenceville, GA) and 1% penicillin-streptomycin (Gibco, Paisley, UK) at 37°C under an atmosphere of 5% C02.
Generation of stable cell lines
HuSH 29mer shRNA constructs against B7-H3 (shB7-H3) and control plasmid pRS non effective TR30003 (TR33) were purchased from Origene Technologies, Inc. The sequences of shB7-H3: shRNA-1, ( 5 '-TTC AG CCTG G C AC AG CTC AACCTCATCTG - 3') (SEQ ID NO: l); shRNA-2, (5'- TCGTGTGCTG G AG AAAG ATCAAACAG AG C- 3 ') (SEQ ID N0:2). Either B7-H3
shRNA construct or control vector were transfected into MDA-MB-231 and MDA- MB-435 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocols, followed by selection with 1 pg/ml puromycin for 2 weeks. Antiboitic-resistant clones were isolated in medium with 0.5 pg/ml puromycin. RT-PCR and immunoblotting were performed to confirm the
knockdown of mRNA and protein of B7-H3 in those transfectants. To generate B7- H3 overexpession stable cell lines, cDNAs for full-length human B7H3
(NM_001024736) were amplified by PCR using the following primer sequences: Forward ( 5 '-TCACTCG AG CCCTG AGTCCCAG AGTCG G C- 3 ') (SEQ ID NO: 3); Reverse ( 5 '- ACTG AATTCGGTTGTG G GTG GTCTGTTC AT- 3 ') (SEQ ID NO:4); and then the full length cDNA was cloned into xho I and EcoR I linearized plasmid vector pIRES2- EGFP (Clonetech, USA). The control vector pIRES2-EGFP and human B7-H3 expression vector pIRES2-B7-H3 were transfected in MDA-MB-231 cells respectively using Lipofectamine 2000. Stable clones were selected in medium containing 1.8 mg/mL Geneticin (Invitrogen, USA). The expression of B7-H3 was detected by Western blot. In vitro growth inhibition
Cells (1 x 104 cells) were initially plated in triplicate in 96-well culture plates. Twenty-four hours later, the medium was replaced with fresh medium with or without paclitaxel and incubated for indicated time. Cell viability was determined using cellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, USA).
Annexin V-FITC Staining
3 x 105 cells were grown in triplicate in 60 mm dishes with addition of 20 nmol/L paclitaxel for 0, 48 h, and 72 h respectively. And then cells were harvested and processed as described in the Annexin V-FITC Apoptosis Detection Kit I manual (BD Transduction Laboratories, BD Biosciences) and analyzed by flow cytometry (BD LSR II). TUNEL assay s ·∞"·' ·' Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay was performed using recombinant terminal transferase (TdT) and biotin-16-dUTP (Roche Diagnostics GmbH, Mannheim, Germany). After treated with with 0, 10 and 20 nmol/L paclitaxel for 72 h, cells were processed following manufacturer's protocol and then analyzed by flow cytometer (BD Biosciences, San Jose, CA). Each experiment was repeated three times.
Quantification of apoptosis by ELISA kit
An apoptosis ELISA kit (Roche Diagnostics Co.) was used to quantitatively measure cytoplasmic histone-associated DNA fragments. After treated with with 0 and 20 nmol/L paclitaxel for 72 h, cells were analyzed following manufacturer's protocol. Each experiment was repeated three times. Western blot analysis
Western blotting was performed on whole cell extracts prepared by lysing cells in 50 mmol/L Tris-HCI (pH8.0), 150 mmol/L NaCI, 1% Triton-X 100, 10 mmol/L EDTA, 5 mmol/L NaF, 5 mmol/L sodium pyrophosphate, 1 mmol/L
phenylmethylsulfonyl fluoride, 1 mmol/L Na3V04, and protease inhibitor cocktail (Sigma-Aldrich ) for 20 min on ice. The proteins were separated by SDS-PAGE and then electrotransferred onto nitrocellulose membrane (Bio-rad). Membranes were probed with indicated antibodies following the manufacturer's protocol.
Immunoreactive bands were visualized using ECL Western Blotting Substrate (Pierce Biotechnology, Inc.).
In vivo studies
Four groups of seven female Balb/c nude mice, bred at the nude rodent facility at the Norwegian Radium Hospital were used. The animals were maintained under specific pathogen-free conditions, and food and water were supplied ad libitum. Animal experiments were performed according to protocols approved by the animal care and use committee and were in compliance with the guidelines on animal welfare of the Norwegian National Committee for Animal Experiments. When the animals were 6-8 weeks of age, 5x106 cells (MDA-MB-435 shB7-H3 or MDA-MB-435 TR33 cells) in 0.2 ml PBS were injected s.c. in both flanks of nude mice. Animals bearing tumors with diameters < 4 mm or > 8 mm at 20 days after injection were excluded. For therapy experiments, a stock solution of paclitaxel in ethanol (6 mg/ml) was dissolved in PBS and a single dose of 10 mg/kg of the drug was injected i.v. into the tail vein when the mean tumor diameter was 5-6 mm (day 0). The control mice received only the solvent. Tumor diameters were measured one to two times per week. Tumor volume was calculated by the formula 0.5 x length x width2 and growth curves constructed, and the data presented as mean of two independent experiments ± standard error of the mean (SEM).
Statistical analysis
Statistical evaluation for experimental data analysis was determined with an unpaired student's t-test. All data were shown as mean ± standard error of the mean (SEM). A statistical difference of P < 0.05 was considered significant.
Results
Silencing of B7-H3 enhanced paclitaxel-induced cytotoxicity in breast cancer cells To study the possible role of B7-H3 in affecting the sensitivity of breast cancer cells to paclitaxel , we used shRNA to create two stable B7-H3 knockdown sublines derived from the human cell lines MDA-MB-231 and MDA-MB435.
Compared to the parental cells and transfection control cells MDA-MB-231-TR33 (231-TR33) and MDA-MB-435-TR33 (435-TR33), the B7-H3 knockdown cells MDA- MB-231-shB7-H3 (231-shB7-H3) and MDA-MB-435-shB7-H3 (435-shB7-H3) expressed very low levels of B7-H3, indicating that B7-H3 shRNA effectively knocked down the expression of the protein (Fig. 1A). After treatment with various concentrations of paclitaxel for 72 h a dose- dependent inhibition of cell growth was observed in both MDA-MB-231 and MDA-MB-435 cells, and the B7-H3 knockdown counterpart cells became about two-fold more sensitive to paclitaxel compared to parental and control cells. In MDA-MB-231 cells, the inhibition of cell growth was 52 % after exposure to 20 nmol/L paclitaxel in the B7-H3 knockdown cells versus 18 % and 25% in parental and control cells.
In the MDA-MB-435 cells, 15 nmol/L paclitaxel induced inhibition of cell growth by 55 %, 31% and 38% in the B7-H3 knockdown, parental and control cells, respectively. Statistical analysis shows that the differences between B7-H3 -ε¾ - knockdown and control cells were significant in both the MDA-MB-231 and MDA- MB-435 cell lines, and the difference between parental cells and control cells have no significance. These results suggest that B7-H3 plays an important role in tumor cell resistance to paclitaxel. As there were no clear differences between the
parental and vector control cells with respect to paclitaxel responsiveness, we excluded the parental cell lines in the further biochemical and molecular analyses.
In separate experiments we have tested the effect of cisplatin in MDA-MB 435 cells, and also for this drug the B7-H3 knock down cells showed clearly increased sensitivity (not shown).
Increased paclitaxel-induced cytotoxicity in B7-H3 knockdown cells was
associated with enhanced apoptosis Paclitaxel is known to exert its cytotoxic effect through induction of apoptosis (25), and hence we investigated whether the
increased paclitaxel cytotoxicity observed in MDAMB-231 and MDA-MB-435 B7-H3 knockdown cells could be related to effects on apoptosis.
The extent of apoptosis was investigated by checking the amount of Annexin-5 stained cells, a marker for early stage apoptosis in MDA-MB-231 and MDA-MB-435 cells, and in MDA-MB-231 cells also for the amount of TUNEL positive cells, which reflects late stage apoptosis. For the early stage apoptosis, the response to 20 nmol/L paclitaxel was time dependent with an increase in the amount Annexin-V positive cells detected at 48 and 72 h (Fig. 2A). Notably, the MDA-MB-231 B7-H3 knockdown cells were more sensitive to paclitaxel-induced apoptosis, with the percentage of apoptotic cells in 231-shB7-H3 cells about two fold that in 231- TR33 cells at both 48 h and 72 h. Similar results were observed in MDA-MB-435 cell variants (Fig. S1A). The late stage apoptotic response was also dose
dependent in MDA-MB-231 cells, showing increased DNA fragmentation with drug concentration, and the 231-shB7-H3 cells were significantly more susceptible to paclitaxel-induced apoptosis compared to the vector control cells.
The amount of TUNEL positive cells were 11% versus 2% at 48 h and 18% versus 6% at 72 h, respectively. These results were confirmed by investigating the
cleavage of poly(ADP-ribose) polymerase (PARP), another marker of apoptosis. Both MDA-MB-231 and MDA-MB-435 B7-H3 knockdown cells had increased PARP cleavage compared to the control cells upon treatment with 20 nmol/L paclitaxel for 72 h (Fig. 2C and Fig. SIB). To validate the specificity of the effects observed in B7-H3 knockdown cells, we confirmed the results with another stable cell clone 231-shl-h9 (Fig. SIC and SID), which is constructed using a different B7-H3-targeting sequence shRNA-1 (see M &M). Taken together, these results demonstrate that the silencing of the B7-H3 protein makes the cells more prone to undergo paclitaxel-induced apoptosis. To confirm the effect of B7-H3 on paclitaxel resistance, we generated stable B7-H3 overexpressing cells. As shown in Figure 2D left, B7-H3 treansfected 231-B7-H3 cells expressed much higher level of B7-H3, compared to the control cells 231-EGFP, and showed decreased level of cleaved-PARP compared to the control cells upon treatment with 20nmol/L paclitaxel for 72 h (Fig. 2D, middle). Moreover, apoptosis-specific ELISA detection revealed that the level of
cytoplasmic histone-associated DNA fragments in 231-B7-H3 cells was
significantly lower than in control 231-EGFP cells after treatment with paclitaxel (Fig. 2D, right). These results indicated that B7-H3 overexpression made the breast cancer cells more resistant to paclitaxel-induced apoptosis and further confirmed the role of B7-H3 in MDA-MB-231 and MDA-MB-435 cell responsiveness to paclitaxel.
Silencing of B7-H3 repressed the Jak2/Stat3 pathway and its downstream antiapoptotic molecules Stat3, a transcription factor often constitutively activated in breast cancer cells, has previously been reported to lead to drug resistance (22) through the upregulation of antiapoptotic factors such as Bcl-xL, Bcl-2, Mcl-1 and Survivin (26). Since we observed chemo-sensitization accompanied by an increase in apoptosis in paclitaxel treated B7-H3 knock down variants of MDA-MB- 231 and MDA-MB-435 cells, we studied whether the effects of B7-H3 could be related to molecules and signaling pathways known to be involved in the apoptotic response. As seen in Figure 3A, the silencing of B7-H3 induced a dramatic reduction in the phosphorylation level of Stat3, an indicator of Stat3
activation, both in untreated cells as well as in cells treated with 20 nmol/L paclitaxel for 72 h. Furthermore, in cells treated with paclitaxel, Mcl-1 and
Survivin, the direct downstream targets of Stat3, were both repressed in B7-H3 silenced cells witfctereduced Stat3 phosphorylation levels (Fig. 3B and 3C). To elucidate whether the effects of B7-H3 on Stat3 was direct or indirect, we examined whether the molecular targets upstream of Stat3, such as Jakl, Jak2 and Src, were affected by the silencing of B7-H3.
We observed a decline in the phosphorylation of Jak2 in both 231- and 435-shB7- H3 cells (Fig. 4A). In contrast, no obvious activation of Jakl and Src was seen (Fig. S2A and S2B), indicating that the effect of B7-H3 on Stat3 occurs mainly through Jak2. To confirm this result, we treated the cells with the Jak2 selective inhibitor AG490 (Fig. 4B and 4C), at concentrations similar to what has been used in previous studies. As expected, the phosphorylation level of Jak2 was almost abolished followed by a dramatic inhibition of tyrosine phosphorylation of Stat3 in both B7-H3 knockdown cells and control cells after treatment with AG490 for 24 h. We observed similar results in 231-shl-h9 and 231-shl-glO cells (Fig. S3A). Taken together, these results show that the silencing of B7-H3 reduces the phosphorylation of Jak2, which leads to reduced phosphorylation of Stat3 and eventually leads to a decrease of the anti-apoptotic proteins, Mcl-1 and Survivin.
Silencing of B7-H3 enhanced the in vivo sensitivity to paclitaxel in xenograft models.
The in vitro experiments with the MDA-MB-231 and MDA-MB-435 cells showed that the cytotoxic effect of paclitaxel was enhanced in cells lacking the B7-H3 protein. Hence, we examined whether or not this could also be observed in vivo. After injecting MDAMB-435 B7-H3 knockdown and control cells s.c. into 8-week old female nude mice, we treated the animals with a single dose of paclitaxel when the tumors had reached a mean diameter of 5-6 mm. As shown in Figure 5, paclitaxel had a marginal (not statistically significant) effect on tumor growth rate of MDA-MB-435 TR33 tumors, whereas the drug showed a relatively strong antitumor effect in mice carrying MDAMB-435 shB7-H3 xenografts. When we performed a t-test on the statistical difference in tumor volume at each time point we found that, for MDA-MB-435 shB7-H3 tumors the effect became significant from day 22 (all the p-values at each time point after day 22 were lower than 0.03). The growth rate of MDA-MB-435 TR33 cells was faster than that of B7-H3 knockdown cells. This does not, however, explain the differences in paclitaxel response between the two groups. Clearly, the in vivo data strongly confirms the effects observed in the in vitro cellular cytotoxic assay that B7-H3 plays a critical role in paclitaxel responsiveness of breast cancer cells.
Discussion
The introduction of the taxanes, paclitaxel and docetaxel, in combination with anthracyclines and alkylating agents in the treatment of metastatic breast cancer, has significantly improved the overall and disease-free patient survival (28, 29). The taxanes exert a response rate between 25% and 69% used as first-line treatment. However, inherited and acquired resistance to taxanes represents a major obstacle in improving the overall response and survival. Therefore, the development of new therapeutic interventions to overcome taxane resistance becomes an important task in cancer research. In this study, we examined the role of B7-H3 in paclitaxel sensitivity in metastatic breast cancer cells and found that MDA-MB-231 and MDA-MB-435 cells lacking the B7-H3 protein were more sensitive to paclitaxel, compared to the control cells in vitro and in an animal model; whereas MDA-MB-231 B7-H3 overexpressing cells were less sensitive to paclitaxel-induced apoptosis. Our findings demonstrate that targeting B7-H3 could counteract cellular resistance to paclitaxel, and are therefore of potential clinical importance. We found that silencing of B7-H3 made the cells more sensitive to paclitaxel-induced apoptosis, possibly through suppressing the activity of the transcription factor Stat3.
This is in acordance with reports on induction of apoptosis following a blockade of Stat3 signaling in multiple malignancies, such as human head and neck squamous cell carcinoma, multiple myeloma, leukemic large granular lymphocytes, prostate cancer, melanoma, lung cancer, and lymphoma cells. Based on our finding of a link between the expression of B7-H3 and Stat3, we investigated the effects on candidate Stat3-regulated genes involved in apoptosis. Among the genes whose expression levels changed upon interference with the Stat3 pathway was Survivin, a member of the IAP family of antiapoptotic proteins. Overexpression of Survivin has been observed in several tumor types, including breast cancer where it has been associated with resistance to apoptosis. We found in our experiments that the expression of S-asswin was reduced upon silencing of B7-H3, and this might possibly be one of the mechanisms by which the cells are more prone to undergo apoptosis after paclitaxel treatment. In support of this, Lu et al report a direct link between Survivin expression and paclitaxel resistance in human breast cancer cells, while Gariboldi et al demonstrated that Survivin protein levels decreased concomitantly with phosphopho-Stat3 levels, thereby increasing doxorubicin sensitivity in metastatic breast cancer cells. It has also been shown that continuous activation of Stat3 signaling leads to induction of Survivin expression and results in resistance to apoptosis in breast cancer cells. Another antiapoptotic protein downstream of Stat3 and B7-H3 is Mcl-1, which is also a direct target gene transcriptionally activated by Stat3 (21, 43), and Mcl-1 downregulation has been shown to promote apoptosis in numerous human cancer cells. In our study, reduced expression of Mcl-1 correlated with increased apoptosis in MDA- MB-231 and MDA-MB-435 B7-H3 knockdown cells treated with paclitaxel. As mentioned above, our finding that the phosphorylation status of Stat3 was reduced upon knockdown of B7-H3 suggests that B7-H3 may regulate the expression of Survivin and Mcl-1 through its effect on Stat3.
When we investigated whether the observed sensitization of paclitaxel, could involve upstream activators of Stat3, like Jakl, Jak2, and Src, we observed reduced phosphorylation levels of Jak2 in B7-H3 knockdown cells. Blockage of phosphorylation of Jak2 by the Jak2 specific inhibitor AG490, resulted in reduced phosphorylation of Stat3, indicated that B7-H3 may mediate its effect on Stat3, at least in part via Jak2. This conclusion is supported by reports that the Src, Janus kinase, and epidermal growth factor receptor family tyrosine kinases are frequently activated in breast cancer cells and induce Stat3 activation (47, 48), and that blocking of these tyrosine kinase pathways with selective pharmacologic inhibitors results in decreased Stat3 activity and consequently enhanced apoptosis.
The human MDA-MB-435 cell line was originally described as of breast cancer origin, whereas gene expression array studies indicated the cells to originate from malignant melanoma. However, subsequent evidence suggests that MDA-MB-435 is in fact a breast cancer cell line. In our study, the results obtained with MDA-MB- 435 cells were closely similar to those with MDA-MB-231 breast cancer cells. Importantly, our promising in vitro results demonstrating that B7-H3 seem to play a critical role in paclitaxel resistance, were confirmed in an animal model using the MDAMB-435 xenografts in which the growth rate of established B7-H3 knock down xenografts was significantly reduced whereas the growth of transfection control tumors were only marginally affected. The growth rate of MDA-MB-435 vector control cells was faster than that of B7-H3 knockdown cells. This cannot, however, explain the differences in sensitivity to paclitaxel, as it is well known that fast-growing tumors generally respond better to chemotherapy than more slowly growing counterparts.
This study demonstrates that B7-H3 confers resistance to paclitaxel by reducing the sensitivity of the tumor cells to apoptosis, mediated via the Jak2/Stat3 pathway. B7-H3 and its pathway partners are potential targets for developing novel therapeutic approaches. Furthermore, in contrasts to previous reports focusing on its immunoregulatory effects, our data demonstrates that B7-H3 plays an important role in determining the resistance to paclitaxel via non-immune mechanisms. Whether this effect of B7-H3 may extend to other drugs and tumor types is not yet determined, but preliminary results in our laboratory are promising and will be further explored.
Example 2
Figures 6 and 7 shown experiments similar to the experiments shown in example 1 but with docetaxel as the active anti-cancer drug.

Claims

Claims
1. Use of the combination of: a) a B7-H3 antagonist; and b) a taxane for the manufacture of a medicament for the treatment of cancer in a subject, wherein the administration pattern of said medicament comprises:
(i) administering to said subject a first sensitizing effective amount of a B7- H3 antagonist during a first sensitizing period; and
(ii) after completion of said first sensitizing period, administering to said subject a first therapeutically effective amount of a taxane during a first therapeutical period; and
(iii) optionally continue sensitizing and treatment according to (i) and (ii).
2. A combination dosage comprising a B7-H3 antagonist and a taxane for use in preventing the progression of cancer in a human.
3. A method for preventing the progression of cancer comprising:
a) providing an individual in need thereof,
b) administration to said individual a combination dosage comprising a B7-H3 antagonist and a taxane.
4. A B7-H3 antagonist for use in a method of treatment comprising sensitizing a cancer cell to a taxane.
5. The use according to claims 1-2, the method according to claim 3 and the B7- H3 antagonist of claim 4, wherein the B7-H3 antagonist is selected from the group consisting of an siRNA directed to the mR A encoding B7-H3, an shRNA directed to the mRNA encoding B7-H3, a miRNA directed to the mRNA encoding B7-H3, a polyclonal antibody, a monoclonal antibody, an aptamer, a soluble B7-H3 receptor that binds directly to B7-H3, an RNase H inducing antisense oligonucleotide directed to the mRNA encoding B7-H3, and a ribozyme directed to the mRNA encoding B7-H3.
6. The use according to claims 1-2, the method according to claim 3 and the B7- H3 antagonist of claim 4, wherein the B7-H3 antagonist is an RNAi antagonist.
7. The use according to claims 1-2, the method according to claim 3 and the B7- H3 antagonist of claim 4, wherein the B7-H3 antagonist is an RNAi antagonist selected from the group consisting of an siRNA directed to the mRNA encoding B7- H3, an shRNA directed to the mRNA encoding B7-H3 and a miRNA directed to the mRNA encoding B7-H3.
8. The use according to claims 1-2, the method according to claim 3 and the B7- H3 antagonist of claim 4, wherein said taxane is selected from the group consisting of paclitaxel and docetaxel.
9. The use according to claims 1-2, and the method according to claim 3, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, clear cell renal cell carcinoma, urothelial cell carcinoma, prostate cancer, nonsmall-cell lung cancer (NSCLC), colorectal cancer and pancreatic cancer.
10. The use according to claims 1-2, and the method according to claim 3, wherein the cancer is a breast cancer.
11. The B7-H3 antagonist of claim 4, wherein the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a nonsmall-cell lung cancer (NSCLC) cell, a colorectal cancer cell, and a pancreatic cancer cell.
12. The B7-H3 antagonist of claim 4, wherein the cancer cell is a breast cancer cell.
13. The use according to claim 1, where in the first sensitizing period is 48 hours and the first therapeutical period is 48 hours.
14. The use according to etaim 2 and the method according to claim 3, wherein the combination dosage of a B7-H3 antagonist and a taxane is administered separately.
15. The use and the method according to claim 11, wherein the B7-H3 antagonist and the taxane is administered 48 hours apart.
PCT/EP2011/061750 2010-07-09 2011-07-11 B7-h3 antagonists and taxanes Ceased WO2012004410A1 (en)

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