WO2016149264A1 - Compositions et procédés pour sensibiliser les cellules à l'apoptose induite par trail - Google Patents

Compositions et procédés pour sensibiliser les cellules à l'apoptose induite par trail Download PDF

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WO2016149264A1
WO2016149264A1 PCT/US2016/022462 US2016022462W WO2016149264A1 WO 2016149264 A1 WO2016149264 A1 WO 2016149264A1 US 2016022462 W US2016022462 W US 2016022462W WO 2016149264 A1 WO2016149264 A1 WO 2016149264A1
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trail
dox
trailpeg
cells
cancer
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PCT/US2016/022462
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Seulki Lee
Martin G. Pomper
Magdalena Swierczewska
Yumin OH
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The Johns Hopkins University
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Priority to US15/559,108 priority Critical patent/US20180050090A1/en
Publication of WO2016149264A1 publication Critical patent/WO2016149264A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • rhTRAIL human tumor necrosis factor-related apoptosis inducing ligand
  • rhTRAIL selectively transduces apoptotic signals by binding to death receptors (DRs) that are widely expressed in most cancers, TRAIL-R1/DR4 and TRAIL-R2/DR5, while sparing normal cells.
  • DRs death receptors
  • This high tumor specificity along with broad applicability across multiple cancer types and proven safety in humans make TRAIL an ideal candidate for cancer therapy.
  • recent clinical trials of rhTRAIL, e.g. dulanermin, or humanized DR agonistic monoclonal antibodies tested as either a monotherapy or combined with anticancer agents have failed to demonstrate benefits in cancer patients compared with historical controls. The disappointing results raise concerns for the therapeutic implications of rhTRAIL.
  • the primary challenge in TRAIL-based therapy is natural resistance.
  • the majority of primary cancer cells are TRAIL-resistant.
  • Mechanisms of TRAIL resistance are distinct among cancer cell types; however, they commonly comprise of: reduced cell surface DR expression, inhibited caspase-8 activation - the initiator caspase, up-regulated anti-apoptotic molecules such as Bcl-2 and the inhibitors of apoptosis (IAP) family proteins, and reduced expression of pro-apoptotic markers like Bax/Bak.
  • IAP apoptosis
  • rhTRAIL In addition to TRAIL-resistance, rhTRAIL has an extremely short half-life in physiological conditions, 3-5 min in rodents and less than 30 minutes in humans. It is widely accepted that wild-type proteins with short half-lives do not exhibit similar biological potency in physiological conditions as those tested in vitro. Use of a more stable form of rhTRAIL with an extended half-life would be expected to improve TRAIL action in physiological conditions, particularly for a biologic with an exceptionally short half-life like TRAIL.
  • TRAILPEG long-acting PEGylated TRAILs
  • iLZ-TRAIL isoleucine-zipper-tagged TRAIL
  • TRAILPEG has increased stability over rhTRAIL with a significantly longer circulation half-life in rats.
  • TRAILPEG demonstrated superior in vivo anticancer potencies in xenografts bearing TRAIL-sensitive HCT116 colon cancer tumors over iLZ-TRAIL.
  • increasing circulation time of TRAIL cannot be a solution for targeting primary tumors associated with TRAIL resistance at the molecular level.
  • TRAIL can have clinical efficacy in cancer by simultaneously addressing two key limitations, TRAIL resistance and its short half-life.
  • a TRAIL sensitizer was selected in TRAIL-resistant colon cancer cells through cell- based screening and TRAIL and apoptotic signals were explored at the molecular level.
  • the selected TRAIL sensitizer alone or formulated with tumor-homing polymer nanoparticles were systemically administered to xenografts bearing TRAIL-resistant tumors followed by TRAILPEG administration to investigate a synergistic effect on TRAIL-induced apoptosis in vivo.
  • the present invention provides a method for treating cancer in a subject comprising: 1) identification of the tumor's TRAIL sensitivity; administering to the subject a nanoparticle comprising an effective amount of one or more TRAIL sensitizing compounds; and administering to the subj ect an effective amount of a pegylated TRAIL peptide to induce apotosis in the cancer of the subject.
  • the present invention provides a composition comprising pegylated TRAIL peptide.
  • the present invention provides composition comprising a nanoparticle comprising an effective amount of one or more TRAIL sensitizing compounds.
  • FIGS 1A-1E PEGylation extends the biological half-life of TRAIL, but does not enhance apoptosis in TRAIL-resistant cancer cell lines.
  • B HCT116 xenografts were established and mice were intravenously treated when the tumor was palpable with four rounds of saline, iLZ -TRAIL (200 ⁇ g) or TRAILPEG (200 ⁇ g, protein-based).
  • animals were sacrificed and tumors were processed and analyzed for TUNEL staining. Fluorescence images were acquired under a confocal microscope and overlaid with Hoechst 33258 staining.
  • D Human tumor cell lines: colon (HT-29, SW620, HCT116), prostate (PC3), breast (MDA-MB-231, MCF7) and lung (A549) and normal human cell line: kidney (HEK293T) were collected and examined for their sensitivities to iLZ -TRAIL and TRAILPEG by cell death assay.
  • Figures 2A-2F Doxorubicin (DOX) initiates a caspase cascade and induces apoptosis when combined with TRAILPEG in TRAIL-resistant cancer cell lines.
  • DOX Doxorubicin
  • A DNA damaging agents sensitize TRAIL-induced apoptosis in HT-29 cells.
  • HT-29 cells were treated with sublethal doses of doxorubicin (DOX, 2 ⁇ g/mL), 5-fluorouracil (5-FU, 10 ⁇ g/mL), cisplatin (CIS, 2 ⁇ g/mL) and irinotecan (IRINO, 2.9 ⁇ g/mL) for 24 h and further incubated with TRAILPEG (1 ⁇ g/mL) for an additional 24 h.
  • C A combination of TRAILPEG and DOX but not drug alone sensitizes TRAIL-induced apoptosis in various TRAIL-resistant cells, HT-29 (colon), MDA-MB-231 (breast), A549 (lung), and PC3 (prostate), as in TRAIL-sensitive HCT116 (colon) cancer cells.
  • HT-29 cells were left untreated or stimulated with 500 ng/ml of TRAILFlag for 1 h.
  • the lysates were immunoprecipitated with FLAG (M2) and analyzed by Western blotting using DR4 and DR5 antibodies.
  • WCL Whole cell lysates.
  • E and F DR5 induction in HT-29 cells by DOX.
  • HT-29 cells were treated with DOX for 24 h and the cell extracts were examined for mRNA levels of DR4 and DR5 using gene-specific primers by qRT-PCR analysis.
  • Figures 3A-3F When combined with TRAILPEG, DOX synergizes TRAIL- induced apoptosis in HT-29 cells through DR5 upregulation and partially by JNK-mediated apoptosis.
  • A Western blotting analysis of HT-29 cells treated with TRAILPEG (1 ⁇ g/mL) and DOX (2 ⁇ g/mL) alone or in combination with different incubation times. The cell extracts were prepared and the levels of DR4, DR5 and cleaved caspase-8 (CI. Casp-8) and caspase-3 (CI. Casp-3) were examined.
  • FIGS 4A-4E HAC/DOX but not free DOX accumulates in tumors for a sustained period of time and potentiates caspase cascade when combined with TRAILPEG.
  • A Upper; schematic diagram of HAC/DOX, hyaluronic acid-based conjugate (HAC) carrying doxorubicin in the core and FITC dye molecules labeled on the surface for fluorescence microscopy. Lower; a chemical structure of HAC, hyaluronic acid chemically conjugated with cholanic acid.
  • HAC hyaluronic acid-based conjugate
  • B Cancer cells were examined for their CD44 expression. The cell extracts were prepared and the levels of CD44 were examined by western blotting.
  • C HAC/DOX rapidly internalizes and releases DOX in HT-29 cells. HT-29 cells were incubated with HAC/DOX (2 ⁇ g/mL, doxorubicin-based) for 10 min and 60 min.
  • FIGS 5A-5E Simultaneous treatment of TRAILPEG and HAC/DOX initiates apoptosis and reduces tumor growth in TRAIL-resistant tumors in vivo.
  • A Ly sates of HT- 29 tumors from mice treated with TRAILPEG (200 ⁇ g per mouse) and HAC/DOX (7 mg/kg, DOX-based) alone or in combination were western blotted for death receptors (DR5, DR4), cleaved caspases and ⁇ -actin (loading control) expression analysis.
  • B, C The relative fold increase of DR and caspase expressions.
  • DOX doxorubicin
  • DOX 5-fluorouracil
  • CIS 0.5 ⁇ g/mL
  • IRINO irinotecan
  • doxorubicin DOX, 2 ⁇ g/mL
  • TRAILPEG 1 ⁇ g/mL, protein-based
  • Figures 7A-7B (A) Chemical structures, RP-HPLC and MALDI-TOF mass spectra of DR5 specific binding peptide, DR5-A and (B) FITC-labeled DR5-A, FITC-DR5- A.
  • Figures 8A-8C (A) Characterization of FITC-DR5-A.
  • HT-29 cells were treated with FITC-DR5-A for 10 min or pretreated with anti-DR5 antibody for 60 min followed by FITC-DR5-A treatment and captured under a confocal microscope.
  • B HCT116 cells were treated with DR5-A followed by TRAILPEG for 3 h.
  • the cell lysates were examined for cellular apoptosis by western blotting for indicated antibodies.
  • CI. cleaved.
  • C HCT116 cells were treated with DR5 antagonistic peptide followed by TRAILPEG or DR5 agonistic antibody for 3 h.
  • the cell lysates were examined by Western blotting for the apoptosis marker, cleaved PARP-1 (CI. PARP-1), the caspase-3 substrate.
  • FIGS 9A-9C (A) Diameter measurements of HAC and HAC/DOX, DOX loaded HAC, as measured by a Malvern Zetasizer Nano Z. (B) FACS analysis to determine the levels of HAC internalization after HT-29 cells were treated with HAC/DOX or
  • HAC/FITC for 1 h. Samples were analyzed using a flow cytometer.
  • C Intracellular staining of HT-29 cells after being treated with HAC/DOX or HAC/FITC at indicated times and captured under a confocal microscope.
  • FIG. lOA-lOC (A) Quantification of the accumulated doxorubicin after HAC/DOX injection in Figure 4E. (B) When HT-29 xenografts tumors reached a diameter of 200 mm3, mice were intravenously treated with DOX (low: 2 mg/kg, or high: 7 mg/kg) and HAC/DOX (2 or 7 mg/kg, DOX-based) followed by TRAILPEG. The tissue extracts were prepared and the activation of caspases (Casp-8, -9, and -3) were examined by western blotting. (B) Quantification of the cleaved caspases in (B).
  • the present invention provides a method for treating cancer in a subject comprising utilizing rhTRAIL with an extended half-life and effectively sensitizing TRAIL-resistant tumors through tumor-homing TRAIL sensitizers.
  • the present invention provides a method of treating cancer in a subject comprising administering to the subject a TRAIL peptide that is pegylatated and a composition comprising a TRAIL sensitizing agent and a nanoparticle.
  • the present invention provides a method for treating cancer in a subject comprising: 1) identification of the tumor's TRAIL sensitivity; administering to the subject a nanoparticle comprising an effective amount of one or more TRAIL sensitizing compounds; and administering to the subject an effective amount of a pegylated TRAIL peptide to induce apotosis in the cancer of the subject.
  • the present invention provides a composition comprising pegylated TRAIL peptide.
  • the present invention provides composition comprising a nanoparticle comprising an effective amount of one or more TRAIL sensitizing compounds.
  • TRAIL receptor agonists TRAs
  • DRAs death receptor agonist
  • the treatment strategy can apply a diverse set of carriers, TRAIL sensitizers and TRAs (recombinant human TRAIL variants, antibodies, DR peptide, etc.).
  • TRAIL sensitizers and TRAs recombinant human TRAIL variants, antibodies, DR peptide, etc.
  • a biomaterial nanoparticle, a common chemotherapeutic, and a PEGylated TRAIL protein is utilized as the carrier, sensitizer and TRA, respectively.
  • a TRA long-acting no more than one dose per day, induces effective trimerization, and is stable in vivo.
  • TRAIL binds two proapoptotic death receptors (DRs), TRAIL-Rl and -R2 (TNFRSFIOA and 10B), as well as two other membrane receptors that do not induce death and instead may act as decoys for death signaling.
  • DRs proapoptotic death receptors
  • TRAIL-Rl and -R2 TNFRSFIOA and 10B
  • TRAIL binding to its cognate DRs induces formation of a death-inducing signaling complex, ultimately leading to caspase activation and initiation of apoptosis.
  • TRAs can be: TRAIL peptide; or an agonistic TRAIL receptor binding fragment or variant thereof.
  • a nucleic acid and amino acid sequence for human TRAIL are known in the art (UniProtKB database accession no. P50591).
  • the TRAIL is a soluble TRAIL
  • soluble TRAIL means, for example, a fragment of full-length
  • the TRAIL of the present invention can have 50, 60, 70, 75,
  • TRAIL can encompass a functional fragment that can agonize signaling through TRAIL-Rl and/or TRAIL-R2.
  • the TRAIL peptide can include amino acids 39-281, 41-281, 91 -281, 92-281 , 95-281 , and 114-281 , or a functional fragments or variants thereof.
  • the functional fragments of TRAIL include amino acids 132-281, amino acids 95-281, or amino acids 1 14-281 to include C-terminal of TRAIL that includes receptor binding domain.
  • variants can have one or more substitutions, deletions, or additions, or any combination.
  • TRAIL ligands or agonists can form, a multimer, preferably a trimer.
  • the trimer can be a homotrimer, or a heterotrimer, for example.
  • a TRAIL analogue, or an agonistic TRAIL receptor can include a binding fragment or variant.
  • TRAIL can be a recombinant or native TRAIL
  • TRAIL can include increased affinity or specificity for one or more agonistic TRAIL receptors (e.g., TRAIL-Rl (DR4) and/or TRAIL-R2 (DR5)), reduced affinity or specificity for one or more antagonistic or decoy TRAIL receptors (e.g., receptors DcRl and DcR2) or a combination compared to wildtype or endogenous TRAIL
  • agonistic TRAIL receptors e.g., TRAIL-Rl (DR4) and/or TRAIL-R2 (DR5)
  • antagonistic or decoy TRAIL receptors e.g., receptors DcRl and DcR2
  • TRAIL peptides or proteins can include TRAIL fusion proteins.
  • the TRAIL fusion proteins may contain a domain that functions to dimerize or multimerize two or more fusion proteins.
  • the TRAIL peptides or proteins form dimers or multimers that are formed can be homodimeric/homomultimeric or heterodimeric/heteromultimeric.
  • TRAIL peptides of the present invention can also comprise one or more linker domains that can either be a separate domain, or altematively can be contained within one of the other domains (TRAIL polypeptide or second polypeptide) of a fusion protein.
  • linker domains can either be a separate domain, or altematively can be contained within one of the other domains (TRAIL polypeptide or second polypeptide) of a fusion protein.
  • TRAIL-mimic compositions can include three TRAIL- protomer subsequences combined in one polypeptide chain, termed the single-chain TRAIL- receptor-binding domain (scTRAIL-RBD).
  • TRAIL fusion proteins have a multimerization domain, such as a dimerization or trimerization domain, or a combination thereof that can lead to, for example, dimeric, trimeric, or hexameric molecule.
  • the fusion protein that facilitates trimer formation includes a receptor binding fragment of TRAIL amino-terminally fused to a trimerizing leucine or isoleucine zipper domain.
  • the present methods can use an antibody that specifically binds to a TRAIL receptor, including, for example, recombinant antibodies, fragments of antibodies, single-chain antibodies, monovalent antibodies, single-chain antibody variable fragments, divalent single-chain variable fragments, diabodies, triabodies, tetrabodies, human or humanized antibodies, hybrid antibodies/chimeric antibodies, TRA conjugates or complexes, conjugate molecules linked to the TRA, including polymers or copolymers, and polyalkylene oxides (e.g. PEG).
  • a TRAIL receptor including, for example, recombinant antibodies, fragments of antibodies, single-chain antibodies, monovalent antibodies, single-chain antibody variable fragments, divalent single-chain variable fragments, diabodies, triabodies, tetrabodies, human or humanized antibodies, hybrid antibodies/chimeric antibodies, TRA conjugates or complexes, conjugate molecules linked to the TRA, including polymers or copolymers, and polyalkylene oxides (e.g. PEG).
  • derivatives of PEG include, but are not limited to, methoxypoly ethylene glycol succinimidyl propionate, methoxypoly ethylene glycol N- hydroxysuccinimide, methoxypoly ethylene glycol aldehyde, methoxypoly ethylene glycol maleimide and multiple-branched polyethylene glycol.
  • the PEG molecular weight in the invention can be within about, 1 - 100 kDa, can be linear or branched, can comprise biopolymers, polypeptides, hyaluronic acid, chitosan, albumin, chondroitin sulfate, and XTEN technology (Versartis).
  • the TRAILPEG complex can be complexed with a carrier (e.g. to form nanoparticle).
  • a carrier e.g. to form nanoparticle.
  • compositions of the present invention can be combined with targeting moieties, such as, for example, antibodies, small molecules, peptides, conjugates to improve purification, Tag-removal, facilitate small molecule attachment, solubility or a combination thereof, elastin-like polypeptides and the Sortase A (SrtA) transpeptidase, SUMO tags, His tags, FLAG tags and MYC tags.
  • targeting moieties such as, for example, antibodies, small molecules, peptides, conjugates to improve purification, Tag-removal, facilitate small molecule attachment, solubility or a combination thereof, elastin-like polypeptides and the Sortase A (SrtA) transpeptidase, SUMO tags, His tags, FLAG tags and MYC tags.
  • the expression or solubility enhancing amino acid sequence can be manipulated using one or more of the following: maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO
  • linkers or spacers e.g. peptides to link domains, regions or sequences to each other.
  • the present invention provides a specific TRA sensitizer: upregulates death receptors (commercially available or novel). These sensitizers can increase targeting to or accumulation of TRA to site of interest, and are preferably chemotherapeutic agents on their own.
  • TRAIL is capable of inducing apoptosis in tumor cells of diverse origin, a majority of tumor cells are resistant to the apoptotic effects of TRAIL, suggesting that TRAIL alone may be ineffective for the treatment of these cancers. Furthermore, several studies have shown that chemotherapeutic drugs ⁇ e.g.
  • cisplatin carboplatin, etoposide, camptothecin, paclitaxel, vincristine, and vinblastine, doxorubicin, gemcitabine and 5-fluorouracil
  • chemotherapeutic drugs not only induce death receptors in vitro, but also in tumor xenografts in nude mice, suggesting that these conventional chemotherapeutic drugs might enhance the cytotoxicity of TRAIL in humans.
  • TRAIL Several breast and prostate cancer cells are resistant to apoptosis by TRAIL, and chemotherapeutic drugs sensitize TRAIL-resistant cells to undergo apoptosis by up-regulating DR4 and/or DR5 and activating caspase.
  • the chemotherapeutic drugs synergize with TRAIL in reducing tumor growth, inducing tumor-cell apoptosis and enhancing survival of tumor- bearing mice. Furthermore, it has been shown that
  • chemotherapeutic drugs such as cisplatin, carboplatin, etoposide, camptothecin, doxorubicin, gemcitabine, 5- fluorouracil, paclitaxel, vincristine, and vinblastine can be used with TRAIL to kill TRAIL- sensitive and -resistant breast cancer cells.
  • Sensitizing agents can include, for example, chemopreventative drugs, curcumin, and phytochemicals, naturally occurring antioxidant compounds (e.g. resveratrol)
  • the methods of the present invention can include, but are not limited to, immunotherapies, gene therapies, anti- angiogenic agents, and
  • chemotherapeutic agents such as, for example, adriamycin, doxorubicin, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, thiotepa, docetaxel, busulfan, cytoxin, taxol, paclitaxel, methotrexate, gemcitabine, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins, melphalan and other related nitrogen mustards.
  • chemotherapeutic agents such as, for example, adriamycin, doxorubicin, 5-fluorouracil, cytosine arabinoside, cycl
  • the methods of the present invention can include radiation therapies, biologies for cancer therapy, including, HERCEPTINTM (trastuzumab), which may be used to treat breast cancer and other forms of cancer; RITUXANTM (rituximab),
  • ZEVALINTM ibritumomab tiuxetan
  • LYMPHOCIDETM epratuzumab
  • GLEEVECTM imatinib mesylate
  • BEXXARTM tositumomab
  • Certain exemplary antibodies also include ERBITUXTM; VECTIBIXTM, IMC- C225; IRESSATM (gefitinib); TARCEVATM (ertinolib); KDR (kinase domain receptor) inhibitors; anti VEGF antibodies and antagonists (e.g., AVASTINTM and VEGF-traps); anti- VEGF (vascular endothelial growth factor) receptor antibodies, peptibodies, and antigen binding regions; anti-Ang-1 and Ang-2 antibodies, peptibodies (e.g., AMG 386, Amgen Inc), and antigen binding regions; antibodies to Tie-2 and other Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies against Tie-2 kinase inhibitors; and CAMPATHTM, (alemtuzumab).
  • VEGF antibodies and antagonists e.g., AVASTINTM and VEGF-traps
  • anti- VEGF vascular endothelial growth factor
  • compositions and methods include use of HDAC inhibitors; anti -inflammatory agents; inhibitors of COX-2 and/or iNOS.
  • the sensitizers can be administered prior to and/or subsequent to (collectively, "sequential treatment"), and/or simultaneously with (“concurrent treatment”) a specific binding agent of the present invention.
  • Sequential treatment such as pretreatment, post-treatment, or overlapping treatment
  • combination also includes regimens in which the drugs are alternated, or wherein one component is administered long-term and the other(s) are administered intermittently.
  • Components of the combination may be administered in the same or in separate
  • compositions and by the same or different routes of administration.
  • tumor-homing carrier of a TRA sensitizer targets tumors by specific ligands or enhanced permeability effect and delivers active sensitizer.
  • Carriers may covalently or non-covalently bind the TRA sensitizer.
  • TRA sensitizer may be opro-drug.
  • Preferably carries would be biodegradable.
  • Products currently in development for tumor-homing or tumor targeted approaches include: microspheres;
  • virosomes engineered nanoparticles (e.g. AccurinsTM by Bind Therapeutics); dendrimers; nanocrystals; block copolymer micelles; polymeric nanoparticles; albumin-bound (e.g. Abraxane ®); PLGA nanoparticles; chitosan analog nanoparticles; PEG nanoparticles;
  • targeting moieties can be peptides, antibodies, proteins and others compounds listed above.
  • the compositions and methods can be used to trean various cancer indications.
  • the present invention may be used to treat individual that has cancer, such as brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cell, bone, colon, stomach, breast, endometrium, prostate, testicle, ovary, central nervous system, skin, head and neck, esophagus, or bone marrow cancer.
  • the cancer is mesothelioma.
  • said cancer is leukemia.
  • the cancer is epithelial cancer.
  • the bone marrow cancer is multiple myeloma.
  • the individual has been identified as having a high risk for the development of cancer (see, for example, WO2008/094319 A2).
  • the cancers which can be treated by the methods of the invention include, but are not limited to, liver cancer, brain cancer, renal cancer, breast cancer, pancreatic cancer (adenocarcinoma), colorectal cancer, lung cancer (small cell lung cancer and non-small-cell lung cancer), spleen cancer, cancer of the thymus or blood cells (i.e., leukemia), prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric carcinoma, head and neck squamous cell carcinoma, melanoma, and lymphoma.
  • the cancer is non-small cell lung cancer (NSCLC) (see. WO2013/148877 Al).
  • Treating includes reducing the likelihood of a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing any level of regression of the disease; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, even if the underlying pathophysiology is not affected or other symptoms remain at the same level.
  • prophylactic or therapeutic treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered.
  • physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Polymer is used to refer to molecules composed of repeating monomer units, including homopolymers, block copolymers, heteropolymers, random copolymers, graft copolymers and so on. "Polymers” also include linear polymers as well as branched polymers, with branched polymers including highly branched, dendritic, and star polymers.
  • a monomer is the basic repeating unit in a polymer.
  • a monomer may itself be a monomer or may be dimer or oligomer of at least two different monomers, and each dimer or oligomer is repeated in a polymer.
  • incorporad is art-recognized when used in reference to a therapeutic agent, dye, or other material and a polymeric composition, such as a composition of the present invention. In certain embodiments, these terms include incorporating, formulating or otherwise including such agent into a composition that allows for sustained release of such agent in the desired application.
  • a therapeutic agent or other material is incorporated into a polymer matrix, including, for example, attached to a monomer of such polymer (by covalent or other binding interaction) and having such monomer be part of the polymerization to give a polymeric formulation, distributed throughout the polymeric matrix, appended to the surface of the polymeric matrix (by covalent or other binding interactions), encapsulated inside the polymeric matrix, etc.
  • co-incorporation or “co-encapsulation” refers to the incorporation of a therapeutic agent or other material and at least one other therapeutic agent or other material in a subject composition.
  • any therapeutic agent or other material is encapsulated in polymers
  • a therapeutic agent or other material may be first encapsulated in a microsphere and then combined with the polymer in such a way that at least a portion of the microsphere structure is maintained.
  • a therapeutic agent or other material may be sufficiently immiscible in the polymer of the invention that it is dispersed as small droplets, rather than being dissolved in the polymer. Any form of encapsulation or incorporation is contemplated by the present invention, in so much as the sustained release of any encapsulated therapeutic agent or other material determines whether the form of encapsulation is sufficiently acceptable for any particular use.
  • Pharmaceutically acceptable salts are art-recognized, and include relatively nontoxic, inorganic and organic acid addition salts of compositions of the present invention, including without limitation, therapeutic agents, excipients, other materials and the like.
  • pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.
  • suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts.
  • the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine,
  • dimethylamine, and triethylamine mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine
  • amino acids such as arginine and lysine
  • guanidine N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenthylamine; (trihydroxymethyl) aminoethane; and the like, see, for example, J. Pharm. Sci., 66: 1-19 (1977).
  • substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with the permitted valency of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction.
  • a composition of this invention may further contain one or more adjuvant substances or the like.
  • additional materials may affect the characteristics of the resulting composition.
  • fillers such as bovine serum albumin (BSA) or mouse serum albumin (MSA)
  • BSA bovine serum albumin
  • MSA mouse serum albumin
  • the amount of filler may range from about 0.1 to about 50% or more by weight of the composition. Incorporation of such fillers may affect the sustained release rate of any encapsulated substance.
  • carbohydrates may be used in certain embodiments in the present invention.
  • Buffers, acids and bases may be incorporated in the compositions to adjust pH.
  • Agents to increase the diffusion distance of agents released from the composition may also be included.
  • the charge, lipophilicity or hydrophilicity of a composition may be modified by employing an additive.
  • surfactants may be used to enhance miscibility of poorly miscible liquids.
  • suitable surfactants include dextran, polysorbates and sodium lauryl sulfate.
  • surfactants are used in low concentrations, generally less than about 5%.
  • Therapeutic formulations of the product may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the product having the desired degree of purity with optional pharmaceutically acceptable carriers, diluents, excipients or stabilizers typically employed in the art, i.e., buffering agents, stabilizing agents,
  • additives are generally nontoxic to the recipients at the dosages and concentrations employed, hence, the excipients, diluents, carriers and so on are pharmaceutically acceptable.
  • compositions can take the form of solutions, suspensions, emulsions, powders, sustained-release formulations, depots and the like.
  • suitable carriers are described in "Remington's Pharmaceutical Sciences,” Martin.
  • Such compositions will contain an effective amount of the biopolymer of interest, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation will be constructed to suit the mode of administration.
  • Buffering agents help to maintain the pH in the range which approximates physiological conditions. Buffers are preferably present at a concentration ranging from about 2 mM to about 50 mM.
  • Suitable buffering agents for use with the instant invention include both organic and inorganic acids, and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture etc.), succinate buffers (e.g., succinic acid monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid- potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-mon
  • Preservatives may be added to retard microbial growth, and may be added in amounts ranging from 0.2%-l % (w/v).
  • Suitable preservatives for use with the present invention include phenol, benzyl alcohol, m-cresol, octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (e.g., chloride, bromide and iodide), hexamethonium chloride, alkyl parabens, such as, methyl or propyl paraben, catechol, resorcinol,
  • compositions of the instant invention and include polhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Polyhydric alcohols can be present in an amount of between about 0.1 % to about 25%, by weight, preferably 1 % to 5% taking into account the relative amounts of the other ingredients.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine etc.
  • organic sugars or sugar alcohols such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., ⁇ 10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, saccharides, monosaccharides, such as x
  • Stabilizers can be present in the range from 0.1 to 10,000 w/w per part of biopolymer.
  • Additional miscellaneous excipients include bulking agents, (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine or vitamin E) and cosolvents.
  • bulking agents e.g., starch
  • chelating agents e.g., EDTA
  • antioxidants e.g., ascorbic acid, methionine or vitamin E
  • cosolvents e.g., ascorbic acid, methionine or vitamin E
  • Non-ionic surfactants or detergents may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein.
  • Suitable non-ionic surfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188 etc.), Pluronic® polyols and polyoxyethylene sorbitan monoethers (TWEEN-20®, TWEEN-80® etc.).
  • Non-ionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.
  • the present invention provides liquid formulations of a biopolymer having a pH ranging from about 5.0 to about 7.0, or about 5.5 to about 6.5, or about 5.8 to about 6.2, or about 6.0, or about 6.0 to about 7.5, or about 6.5 to about 7.0.
  • the incubation of the amine-reacting proteoglycan with blood or tissue product can be carried out a specific pH in order to achieve desired properties.
  • the incubation can be carried out at between a pH of 7.0 and 10.0 (e.g., 7.5, 8.0, 8.5, 9.0, and 9.5).
  • the incubation can be carried out for varying lengths of time in order to achieve the desired properties.
  • iLZ-TRAIL and TRAILPEG were prepared as described previously (Mol. Cancer Ther. 9(6): 1719-1729 (2010)) and generously provided by Theraly Pharmaceuticals Inc.
  • the PK of proteins were measured in cynomolgus monkeys.
  • Male cynomolgus monkeys (4-5 kg, Korea Research Institute of Chemical Technology (KRICT), Daejeon, Korea) were fasted for 12 h before drug administration.
  • KRICT Korean Research Institute of Chemical Technology
  • iLZ-TRAIL and TRAILPEG (12.5 ⁇ g/kg, protein-based) were i.v.
  • TRAIL concentration of TRAIL was determined by Human TRAIL/TNF SF 10 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) and analyzed using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA) based on a four-parameter logistic standard curve derived from iLZ-TRAIL and TRALPEG, respectively. PK parameters were obtained by non-compartmental analysis from WinNonlin (Pharsight Corporation, Mountain View, CA).
  • the cell lines were purchased from American Type Culture Collection (Manassas, VA).
  • HT-29, SW620, HCT116, and MDA-MB-231 cells were maintained in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Life Technology, Carlsbad, CA), 1% penicillin, and 1% streptomycin (Life Technology).
  • FBS fetal bovine serum
  • PC-3 and A549 cells were maintained in F-12K medium (Sigma) supplemented with 10% FBS, 1% penicillin, and 1% streptomycin.
  • HEK293T cells were cultured in Modified Eagles Medium (MEM) (Sigma) supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. Typically, 2 ⁇ 10 5 cells per well were plated in 6-well plates for treatment of agents.
  • MEM Modified Eagles Medium
  • a total of 1 ⁇ 10 4 cells were plated in 0.1 mL in 96-well flat bottom plates and incubated for 24h before being exposed to various stimuli. After incubation for the indicated times, 5 ⁇ g/mL MTT solution was added to each well and incubated for 1 h. After removal of the medium, 200 of DMSO was added to each well to dissolve the formazan crystals. The absorbance at 540 nm was determined using a microplate reader (Bio-Tek Instruments, Inc, Winooski, VT). Triplicate wells were assayed for each condition.
  • TUNEL assay In situ DNA Strand Break Labeling (TUNEL assay) [0100] Tumor tissues were recovered from euthanized animals. Sections (5 ⁇ ) were cut from 10% neutral buffered, formalin-fixed, paraffin- embedded tissue blocks. Apoptotic cell death in tumor tissues was visualized by performing TdT-mediated dUTP nick end labeling (TU EL) assays according to the manufacturer instructions (Roche Mannheim, Germany).
  • HT-29 cells grown on coverslips in 12-well plates were treated with indicated agents.
  • the cells were fixed in 4% paraformaldehyde for 5 min and then washed with ice- cold PBS (pH 8.0) three times. Finally, the cells were mounted on slides for visualization under a Fluoview FVlOi-DOC confocal microscope (Olympus Optical, Tokyo, Japan).
  • Anti-caspase-8 (Cell Signaling Technology, Danvers, MA, #9746), anti-cleaved PARP-1 (Cell Signaling Technology, #5625), anti-cleaved caspase-3 (Cell Signaling Technology, #9664), anti-cleaved caspase-9 (Cell Signaling Technology, #7237), anti-CD44 (Cell Signaling Technology, #5640), anti-p-JNK (Cell Signaling Technology, #4668), anti-p- p53 (Serl5 Cell Signaling Technology, #9284), anti-BCl-2 (Cell Signaling Technology, #2870), anti-p-BCL-2 (Cell Signaling Technology, #2875 ), anti-BCL-XL (Cell Signaling Technology, #2764), anti-DR4 (Abeam, Cambridge, MA, #13890), anti-DR5 (Abeam, #47179), anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA, sc-1694) , or anti-
  • cells were lysed and sonicated briefly in ice-cold PBS buffer (1 mM PMSF, and 1 ⁇ g/ml each of aprotinin, leupeptin, and pepstatin A).
  • Cell lysates were clarified by centrifugation, resolved by SDS- PAGE, and proteins on gels were transferred to nitrocellulose (Bio-Rad, Hercules, CA) using a semidry blotter (Bio-Rad).
  • the membrane was blocked with 3% BSA in TBST (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) and incubated overnight at 4°C with primary antibodies. Immunoblots were visualized by an enhanced chemiluminescence method.
  • HT-29 cells were cultured in 6 well plates for 24 h and the cells were transfected with DR5 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, sc-40237) or control siRNA for 48 h. Transfection was carried out using Lipofectamine 2000 reagent (Invitrogen) following to the manufacturer's instructions.
  • Quantitative RT (reverse transcription)-PCR Total cellular RNA was purified from HT-29 cells using Trizol reagent (Life Technology) and subjected to amplification with Superscript One-Step RT-PCR system (Life Technology). Real-time PCR was carried out using a StepOneTM Real-Time PCR System according to the manufacturer instructions (Life Technology). The mean cycle threshold value (Ct) from triplicate samples was used to calculate the gene expression, ⁇ -actin was used as an internal control to normalize the variability in expression. Experiment was repeated three times with identical results.
  • DR4 forward 5'-TGT GAC TTT GGT TGT TCC GTT GC-3' (SEQ ID NO: 1) and reverse 5'-ACC TGA GCC GAT GCA ACA ACA G-3' (SEQ ID NO: 2); DR5, forward 5'- AAG ACC CTT GTG CTC GTT GT-3' (SEQ ID NO: 3) and reverse 5'-AGG TGG ACA CAA TCC CTC TG-3' (SEQ ID NO: 4); actin, forward 5'- TCC CTG GAG AAG AGC TAC GA-3' (SEQ ID NO: 5) and reverse 5'-AGC ACT GTG TTG GCG TAC AG-3' (SEQ ID NO: 6).
  • mice bearing HT-29 xenograft tumors were intravenously administered with DOX (7 mg/kg) and HAC/DOX (containing 7 mg/kg equivalent doxorubicin) when tumors reached 300 mm 3 .
  • DOX 7 mg/kg
  • HAC/DOX 7 mg/kg equivalent doxorubicin
  • 3 mice in one group were euthanized by cervical dislocation.
  • Whole blood was collected via cardiac puncture with a heparinized syringe. Tumors were dissected out and frozen at -70 °C immediately. Plasma samples were isolated from whole blood by centrifugation at 3000g for 5 min.
  • HT-29 cells achieved 80% confluence, the cells were pretreated with doxorubicin for 24 h and then incubated with 500 ng/mL Flag-TRAIL (Enzo Life Sciences, Farmington, NY) for 30 min at 37 °C.
  • the cells were lysed with DISC IP lysis buffer (30 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100 with 1 mM PMSF, and 1 ⁇ g/mL each of aprotinin, leupeptin, and pepstatin A).
  • Cell lysates were incubated with Flag (M2) beads (Sigma) overnight. The beads were subsequently washed three times with cold PBS, resolved onto SDS-PAGE gels and subjected to Western blot analysis.
  • TRAILPEG improves pharmacokinetics and reduces tumor growth in TRAIL- sensitive tumor xenografts but does not influence apoptosis in TRAIL-resistant tumors.
  • TRAILPEG engineered with a 20 kDa PEG molecule was synthesized as previously reported and used throughout the study.
  • iLZ- TRAIL cleared from the blood with a ti/2 less than an hour.
  • TRAILPEG showed a 17-fold increase in ti/2 and a 38-fold increase in area under the curve (AUC) over iLZ-TRAIL and lasted in the blood up to 144 h after dosing (data not shown).
  • AUC area under the curve
  • TRAIL variants were intravenously administered every 3 days for a total of 4 times in HCT116 xenografts when the tumor was palpable (50 mm3) (Fig. IB).
  • HCT116 is a human colon cancer cell line that is relatively sensitive to TRAIL-induced apoptosis.
  • TRAILPEG 200 ⁇ g, protein-based
  • TGI tumor growth inhibition
  • TRAILPEG clearly showed tumor cell apoptosis in vivo compared to marginal signs in the iLZ-TRAIL-treated group.
  • TRAIL-resistant human tumor cell lines including colon (HT-29, SW620), prostate (PC3), breast (MDA-MB-231 , MCF7) and lung (A549) as well as TRAIL-sensitive HCT1 16 and normal human kidney HEK293T cells were incubated with 1 ⁇ g/mL of iLZ-TRAIL or TRAILPEG for 3 h and 24 h in respective media.
  • TRAIL sensitivities were expressed as induced cell death (%), calculated as the percentage relative to the untreated cells, and measured by MTT assays (Fig. ID and Fig.
  • TRAILPEG provoked strong apoptosis only in TRAIL-sensitive HCT1 16 cells, like iLZ- TRAIL, as evidenced by upregulated cleavage of poly(ADP-ribose) polymerase 1 (PARP-1), a substrate of caspase-3 (Fig. IE).
  • PARP-1 poly(ADP-ribose) polymerase 1
  • Fig. IE substrate of caspase-3
  • DOX/TRAILPEG potentiates DR-mediated apoptosis in TRAIL-resistant tumor cells.
  • HT-29 cells were exposed to sub-lethal doses of DOX (2 ug/mL), 5-FU (10 ⁇ .), CIS (2 ⁇ g/mL), or IRINO (3 ⁇ g/mL) combined with TRAILPEG, enhanced TRAIL-induced apoptosis was observed compared to drug alone (Fig. 2A).
  • DOX/TRAILPEG combination clearly enhanced apoptosis through the proteolytic activation of caspase-8 (Casp-8) and caspase-9 (Casp-9) and consequently cleaved PARP-1 in HT-29 cells (Fig. 2B).
  • Treatment of DOX also led to the phosphorylation of p53 and the activation of c-jun, a downstream substrate of c-Jun N-terminal kinase (JNK).
  • TRAILPEG and DOX induced low levels of cleaved PARP-1 in TRAIL-resistant human tumor cell lines, including HT-29, MDA-MB-231 (breast), A549 (lung), and PC3 (prostate).
  • cleaved PARP-1 expression was significantly increased in all TRAIL-resistant and TRAIL-sensitive cell lines examined, (Fig. 2C) and such synergism was correlated in cell death assays (Fig. 6C).
  • TRAIL DISC immunoprecipitation IP was assessed in HT-29 cells after treatment of DOX, TRAIL or DOX/TRAIL followed by DR4 and DR5 Western blotting (Fig. 2D).
  • TRAIL DISC demonstrated the recruitment of DR5, but not DR4, on the cellular membrane after DOX/TRAIL treatment.
  • HT-29 cells that were transfected with DR5 siRNA and treated with DOX had attenuated expression of DR5 (Fig. 2E).
  • qPCR quantitative real-time PCR
  • DOX increased DR5 mRNA by 60% in HT-29 cells compared to untreated cells, whereas DR4 mRNA levels did not change (Fig. 2F).
  • DOX/TRAILPEG accelerates proteolytic activation of caspases through DR5 upregulation in HT-29 cells.
  • HT-29 cells are TRAIL-resistant because of low DR5 expression on the cellular membrane.
  • DOX has been demonstrated to sensitize TRAIL-induced apoptosis by affecting the cell surface localization of DR5 in colon cancer cells.
  • DOX and DOX/TRAILPEG enhance apoptosis, HT-29 cells were treated with DOX or DOX/TRAILPEG at different time points. Pretreatment of DOX (2 ⁇ g/mL) activated Casp-8 when treated alone and Casp-3 when TRAILPEG was co-treated for 24 h (Fig. 3 A and 3B).
  • TRAILPEG DOX upregulated DR5 expression (3 to 4- fold), but not DR4, at the protein level.
  • TRAIL intrinsically binds to both DR4 and DR5, but we have shown that only altered levels of DR5 in HT-29 cells play a critical role in TRAIL- induced apoptosis while DR4 levels remained unchanged.
  • DR5-A peptide-based dimeric DR5 antagonist
  • Blocking DR5 substantially decreased the proteolytic activation of Casp-8, Capse- 9 and PARP-1 cleavage in cells treated with DOX/TRAILPEG while showing no effect on BCL2/BCL-XL expression that was mainly reduced by DOX (Fig 3E).
  • Tumor-homing HAC/DOX but not free DOX accumulates DOX concentrations in tumor tissues in vivo.
  • select anticancer agents acting as TRAIL sensitizers in vitro were not fully validated in animal models and when in vivo efficacy was demonstrated, relatively high doses of drugs were needed to effectively sensitize TRAIL-resistant tumors in vivo.
  • chemotherapeutic agents as TRAIL sensitizers are not clinically practical.
  • One effective way to utilize such toxic agents as a sensitizer while minimizing systemic toxicity in vivo is using a tumor-homing drug delivery system.
  • HAC hyaluronic acid-based conjugate
  • the HAC structure can self-assemble into a nanocarrier sequestering the hydrophobic/amphiphilic molecules to the center of the particle. Because of HAC's abundant functional groups, the surface of HAC can be modified with fluorophores or other detectable moieties for tracking and imaging in cells and in vivo (Nano Lett 12(7): 3613- 3620 (2012)). The schematic diagram and chemical structure of HAC is described in Fig. 4A. CD44 expression and therefore HAC drug delivery is dependent on cell types. Among the tested cells, HT-29, HCT116, MDA-MB-213 and A549 tumor cells express CD44 whereas SW620 and HEK293T cells do not express high levels of CD44 (Fig. 4B).
  • DOX is well- encapsulated in HAC (HAC/DOX) with high loading contents (21%, wt) and loading efficiency (71%) with mean diameter of 206 nm in PBS (10 mM, pH 7.4) (Fig. 9A).
  • HAC/DOX HAC labeled with fluorescein molecules and treated to HT-29 cells
  • HAC/DOX showed rapid cellular uptake after 10 min of incubation and saturated at 1 h (Fig. 4C and Fig. 9B).
  • HAC/DOX promptly burst releases the incorporated DOX inside the cell, as evidenced by the restored quenched fluorescence of DOX in microscopy and FACS data (Fig. 9C).
  • HAC was shown to be non-toxic in our previous studies.
  • DOX concentration in plasma and tumor tissues was quantified by fluorescence absorbance followed by an extraction process.
  • HAC/DOX and the same dose of DOX dissolved in saline was intravenously injected in HT-29 xenografts bearing approximately 300 mm 3 tumors, HAC/DOX delivered more DOX in the harvested tumor tissues than free DOX.
  • the concentration of DOX in the tumor region gradually decreased with time at 6 h post drug administration (Fig. 4D).
  • HAC/DOX markedly increased DOX accumulation in the tumor region from 6 h to 24 h and maintained accumulation 48 h post-injection.
  • HAC/DOX demonstrated 12-fold and 55-fold increased accumulation of DOX in isolated tumors at 24 h and 48 h, respectively, compared to that of DOX alone.
  • harvested tumor sections isolated at 48 h were stained with DAPI and visualized by confocal microscopy (Fig. 4E and Fig. 10A).
  • HAC/DOX treated tissues showed an obvious sign of DOX accumulation compared to the DOX treatment alone.
  • a tumor-homing HAC/DOX combined with long-acting TRAILPEG potentiates apoptosis in TRAIL-resistant tumor xenografts with reduced systemic toxicity.
  • mice were treated by HAC/DOX (5 mg/kg, DOX-based) 24 h before TRAILPEG treatment.
  • HAC/DOX 5 mg/kg, DOX-based
  • TRAILPEG the expression of DR5 and DR4 as well as Casp-8 and Casp-3 were analyzed in harvested tumor tissues.
  • HAC/DOX treatment increased the protein expression of DR5 in tumors by 70% in vivo while DR4 levels remained unchanged (Fig. 5A and 5B).
  • neither HAC/DOX nor TRAILPEG alone failed to initiate a caspase cascade.
  • Casp-8 and Casp-3 were strongly activated only when the
  • HAC/DOX and TRAILPEG were co-treated (Fig. 5C).
  • two TRAILPEG formulations with different DOX concentrations low (2 mg/kg, DOX-based, Doxiow) and high (7 mg/kg, close to maximum tolerated dose, Doxhigh), were injected in HT- 29 xenografts when tumor volumes reached 200 mm3 followed by TRAILPEG treatment.
  • Each tumor was harvested and analyzed by immunoblotting after 24 h of TRAILPEG treatment (Fig. 10B).
  • DOX at the low dose marginally increased the expression of cleaved Casp-9 and Casp-8 but showed some enhanced expression at the high DOX dose.
  • mice were intravenously treated with TRAILPEG alone or with DOX and HAC/DOX at a 7 mg/kg DOX dose every 3 days for a total of 3 times as indicated in Fig. 5D.
  • TRAILPEG alone marginally altered tumor growth.
  • the two TRAILPEG combinations suppressed tumor growth. TGI values were significantly decreased by the HAC/DOX and TRAILPEG combination throughout the study period (at day 28, for

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Abstract

La présente invention concerne des peptides TRAIL pégylés et leur utilisation conjointement à divers agents de sensibilisation TRAIL dans des formulations de nanoparticules de ciblage de tumeur pour une utilisation dans le traitement du cancer chez un sujet.
PCT/US2016/022462 2008-07-11 2016-03-15 Compositions et procédés pour sensibiliser les cellules à l'apoptose induite par trail WO2016149264A1 (fr)

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US10836999B2 (en) 2013-01-28 2020-11-17 St. Jude Children's Research Hospital, Inc. Chimeric receptor with NKG2D specificity for use in cell therapy against cancer and infectious disease
US11730833B2 (en) 2014-08-05 2023-08-22 Case Western University Coated plant virus imaging agents
US11730803B2 (en) 2014-11-07 2023-08-22 Case Western Reserve University Cancer immunotherapy using virus particles
US11617787B2 (en) 2014-11-07 2023-04-04 Case Western Reserve University Cancer immunotherapy using virus particles
US11883505B2 (en) 2015-06-29 2024-01-30 Case Western Reserve University Anticancer drug-containing plant virus particles
US11738090B2 (en) 2015-07-16 2023-08-29 Case Western Reserve University Plant virus particles for delivery of antimitotic agents
WO2017127495A1 (fr) * 2016-01-19 2017-07-27 The Johns Hopkins University Sensibilisation d'un cancer à des agonistes des récepteurs de mort cellulaire à l'aide d'inhibiteurs de kinases
US11654117B2 (en) 2016-11-03 2023-05-23 Case Western Reserve University Melt processed viral nanoparticle constructs
US11590183B2 (en) 2017-03-10 2023-02-28 Case Western Reserve University Cancer immunotherapy using virus particles
US11672840B2 (en) 2017-05-17 2023-06-13 Case Western Reserve University Anticancer trail-targeted plant virus particles
WO2018213587A1 (fr) * 2017-05-17 2018-11-22 Case Western Reserve University Particules virales végétales anticancéreuses ciblées sur trail
US11739301B2 (en) 2017-10-27 2023-08-29 Case Western Reserve University Tymovirus virus and virus-like particles as nanocarriers for imaging and therapeutic agents
US11896676B2 (en) 2019-08-07 2024-02-13 Case Western Reserve University Targeting cancer cells and tissue using filamentous plant virus particles
CN112494427A (zh) * 2020-12-02 2021-03-16 嘉兴学院 一种聚乳酸-多肽胶束及其应用

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