WO2015168102A2 - Stabilisation de protéine prolongée par le resvératrol - Google Patents

Stabilisation de protéine prolongée par le resvératrol Download PDF

Info

Publication number
WO2015168102A2
WO2015168102A2 PCT/US2015/027960 US2015027960W WO2015168102A2 WO 2015168102 A2 WO2015168102 A2 WO 2015168102A2 US 2015027960 W US2015027960 W US 2015027960W WO 2015168102 A2 WO2015168102 A2 WO 2015168102A2
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
protein
therapeutic
polypeptide
resveratrol
Prior art date
Application number
PCT/US2015/027960
Other languages
English (en)
Other versions
WO2015168102A9 (fr
WO2015168102A3 (fr
Inventor
Mien-Chie Hung
Chun-Te Chen
Original Assignee
Board Of Regents, The University Of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Publication of WO2015168102A2 publication Critical patent/WO2015168102A2/fr
Publication of WO2015168102A3 publication Critical patent/WO2015168102A3/fr
Publication of WO2015168102A9 publication Critical patent/WO2015168102A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • 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
    • 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
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the fields of cancer biology and medicine. More particularly, it concerns methods of increasing the in vivo half-life of a therapeutic protein and use of such methods in treating disease.
  • Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer-related deaths in the United States with a 5 -year survival of less than 6% (Raimondi et al., 2009; Erkan et al., 2012).
  • Kras mutation has been shown to drive uncontrolled proliferation and activation of survival signaling pathways in pancreatic, colorectal, and lung cancers, with over 95% of PDAC harboring oncogenic Kras (Bryant et al., 2014).
  • Genetically engineered mouse model (GEMM) studies further provided evidence to support mutant Kras G12D as the major driver in PDAC tumor initiation (Ying et al., 2012). Nonetheless, there are currently no effective therapies targeting Kras mutant cancers.
  • compositions comprising a therapeutic polypeptide and a stilbenoid (e.g., resveratrol, pterostilbene, piceid), wherein the therapeutic polypeptide comprises an endostatin polypeptide fused to a cytosine deaminase polypeptide.
  • the polypeptide is comprised in a liposome.
  • the polypeptide further comprises a protein transduction domain.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a composition of the present embodiments.
  • the composition is administered systemically, intravenously, intraarterially, intratumorally, intramuscularly, orally, or by infusion.
  • kits for treating cancer in a patient in need thereof comprising (a) administering to the patient a therapeutically effective amount of a polypeptide comprising an endostatin polypeptide fused to a cytosine deaminase polypeptide; and (b) administering to the patient an amount of a stilbenoid (e.g., resveratrol, pterostilbene, piceid) effective to increase the in vivo half-life of the polypeptide administering in step (a).
  • a stilbenoid e.g., resveratrol, pterostilbene, piceid
  • the method further comprises administering 5-fluorocytosine to the patient.
  • the patient is a human patient.
  • the cancer is pancreatic cancer, head and neck cancer, ovarian cancer, thyroid cancer, oral cancer, prostate cancer, melanoma, colon cancer, breast cancer, angioma, sarcoma, lung cancer, brain cancer, pancreatic cancer, liver cancer, bladder cancer, or gastrointestinal cancer.
  • the cancer is a pancreatic ductal adenocarcinoma.
  • the cancer is metastatic, recurrent, or multi-drug resistant.
  • the patient is treated at least a second time. In some aspects of the present methods, the patient is treated over a period of 1 week to 6 months.
  • kits for increasing the in vivo half-life of a therapeutic protein comprising (a) administering to the patient the therapeutic protein; and (b) administering to the patient an amount of a stilbenoid (e.g., resveratrol, pterostilbene, piceid)effective to increase the in vivo half-life of the therapeutic protein.
  • a stilbenoid e.g., resveratrol, pterostilbene, piceid
  • the polypeptide is administered systemically, intravenously, intraarterially, intratumorally, intramuscularly, orally, or by infusion.
  • the resveratrol is administered systemically, intravenously, intraarterially, intratumorally, intramuscularly, orally, or by infusion.
  • the polypeptide and the stilbenoid are administered by the same route. In some aspects of the present methods, the polypeptide and the stilbenoid are administered by distinct routes.
  • the polypeptide is administered prior to administration of the stilbenoid. In some aspects of the present methods, the polypeptide is administered following administration of the stilbenoid. In some aspects of the present methods, the stilbenoid is administered prior to and following administration of the polypeptide. In some aspects of the present methods, the polypeptide and the stilbenoid are administered simultaneously.
  • a therapeutic polypeptide comprising an endostatin polypeptide fused to a cytosine deaminase polypeptide is characterized as having a certain percentage of identity as compared to a reference polypeptide.
  • the percentage of identity may be about, at most, or at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) between the therapeutic polypeptide and the reference polypeptide. It is also contemplated that percentage of identify may relate to a particular modified region of a polypeptide as compared to that same region of the reference polypeptide.
  • a therapeutic polypeptide, for example, characterized as having at least 90% identity to a reference polypeptide means that 90% of the amino acids in that therapeutic polypeptide are identical to the amino acids in the reference polypeptide.
  • the therapeutic polypeptide has a sequence that is about, at most, or at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) identical to the sequence of SEQ ID NO: 1 or a fragment thereof.
  • "essentially free,” in terms of a specified component is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • FIGS. 1A-B EndoCD targets pancreatic tumor vessels. Tumor-bearing mice were anesthetized, and fluorescent-labeled EndoCD was injected via tail vein. Mice were visualized by two-photon microscopy (Carl Zeiss upright multiple photon microscopy, LSM 7MP). Representative images shown were from the same mouse.
  • FIG. 1 A human pancreatic cancer model AsPCl .
  • FIG. IB mouse stromal-enriched PDAC model MP1070. Vessels were visualized by FITC-dextran and EndoCD by Qdot 605 -EndoCD. Bar, 50 ⁇ . Time-lapse videos were also obtained.
  • FIGS. 2A-E Resveratrol prolongs protein stability of EndoCD and sensitizes its cell killing effect by inhibiting chymotrypsin-like protease activity.
  • FIG. 2A pancreatic cancer cell lines were treated with different doses of EndoCD/5-FC and with or without 10 ⁇ of resveratrol. Cell viability was determined by MTT assay. The cell viability of 5-FC alone group was set as 100%. Solid black circle, EndoCD/5-FC; open black circle, EndoCD/5-FC/Resv.
  • FIG. 2B EndoCD was injected into mice and resveratrol (Resv) was orally administered daily. Protein concentration was detected in sera by ELISA and normalized to the concentration measured on day 0.
  • FIG. 2C mice sera were incubated with EndoCD, resveratrol, or the combination for 5 days and then treated in pancreatic cancer cells. The final treatment dose of EndoCD was 1.25 ⁇ and resveratrol was 10 ⁇ , as calculated by day 0. The cell viability of 5-FC alone group was set as 100%.
  • FIG. 2D trypsin-, chymotrypsin-, and elastase-like activities detected from mice sera treated with or without 10 ⁇ of resveratrol after 48 hours.
  • FIG. 2E chymotrypsin A protein activity with different doses of resveratrol. Synergistic effect (CI ⁇ 1) indicated by combination index (CI) (Chou and Talalay, 1984).
  • FIGS. 3A-E Therapeutic efficacy of EndoCD/5-FC/resveratrol in a various PDAC models.
  • FIG. 3 A MP1070/luc cell was inoculated into pancreas to generate stromal- enriched PDAC.
  • One week after tumor inoculation mice were intravenously injected with 7.5 mg/kg of EndoCD twice per week.
  • 5-FC prodrug was intraperitoneally injected 1 h after protein treatment and resveratrol (40 mg/kg) orally administered five times per week. Arrows represent protein treatment.
  • FIG. 3B survival curve of mice-bearing stromal-enriched PDAC.
  • FIG. 3A survival curve of mice-bearing stromal-enriched PDAC.
  • FIG. 3C AsPcl/luc cells were inoculated into the pancreas to generate PDAC.
  • FIG. 3D survival curve of mice-bearing human PDAC.
  • FIG. 3E inducible Kras genetically engineered mouse model. Survival of iKrasG12Dp53L/+ were treated with drugs after doxycycline induced 8 weeks throughout their life.
  • N 10.
  • FIGS. 4A-N EndoCD/5-FC/resveratrol reduces PDAC stroma formation. Mice bearing stromal-enriched PDAC were treated with EndoCD/5-FC, resveratrol, or the combination. Blood flow parameters and percentage of tumor vessel density were calculated by high frequency ultrasound.
  • FIG. 4A time to peak.
  • FIG. 4B rise time.
  • FIG. 4C wash-in rate.
  • Absorbance Units (A.U.) are shown on y-axis.
  • FIG. 4D vessel density. Representative images of immunostaining of tumor tissues are shown.
  • FIG. 4E trichrome staining.
  • FIG. 4F collagen intensity.
  • FIG. 4G a-SMA antibody and TUNEL staining.
  • FIG. 4G a-SMA antibody and TUNEL staining.
  • FIG. 4H a-SMA intensity.
  • FIG. 41 CD31 antibody and TUNEL staining.
  • FIG. 4 J CD31 intensity.
  • FIG. 4K CD45 antibody and TUNEL staining.
  • FIG. 4L CD45 intensity.
  • FIG. 4M TUNEL intensity.
  • FIG. 5 Collagen levels in human AsPCl and mouse stromal-enriched MP 1070 pancreatic cancer.
  • MP1070/luc and AsPCl/luc PDAC cells were injected into the pancreas of mice, and tumor tissues were subjected to trichrome staining.
  • Three mice per groups were orthotopically inoculated with the same amount of MP1070/luc and AsPCl/luc PDAC cells in pancreas. Tumors were harvested the same day to determine collagen level.
  • FIGS. 6A-B Therapeutic efficacy of EndoCD/5-FC in an orthotopic mouse model of human PDAC.
  • FIG. 6A cell killing effect was assessed in AsPCl/luc cells treated with 200 ⁇ g/ml of 5-FC and various concentrations of EndoCD. The cell viability of 5-FC alone group was set as 100%.
  • FIG. 6B EndoCD proteins (7.5 mg/kg and 60 mg/kg) were injected intravenously twice per week. An hour after protein treatment, mice were given 500 mg/kg of 5-FC by intraperitoneal injection. Arrows represent protein treatment. Solid black diamond, Cont; Solid black square, 7.5 mg/kg EndoCD/5-FC; Open black circle, 60 mg/kg EndoCD/5-FC.
  • FIG. 7 Cell killing effect of resveratrol. Pan02/luc, Capanl, and AsPCl/luc cells were treated with various concentrations of resveratrol. Resveratrol at the concentration of 10 ⁇ was selected for combination treatment with EndoCD/5-FC. [0030] FIG. 8. Levels of caspase-6 after resveratro 1/5 -FU treatment. AsPCl cells (1 x 10 6 ) were treated with resveratrol (0, 10, 50, 200 ⁇ ) or in combination with 5-FU (IC 50 ) for 72 h. Cell lysates were subjected to Western blot analysis with anti-caspase-6 antibody.
  • FIGS. 9A-D Resveratrol prolongs the protein stability of Endo, EndoCD fusion protein, INFy, and trastuzumab.
  • FIG. 9A EndoCD protein (12.5 ⁇ ) or FIG. 9B, INFy (2 ⁇ ) were mixed with mouse sera with or without resveratrol, and at the indicated time points, an equal amount of protein was subjected to SDS-PAGE for Western blot analysis using anti-His-tag antibody or anti-F Fy gamma antibody.
  • FIG. 9C Mouse was administered trastuzumab (2 mg) by intravenous injection, and at the indicated time point mice serum was collected. An equal amount of serum was subjected to dot blot analysis for anti-human secondary antibody.
  • FIG. 9D Endo protein (12.5 ⁇ ) was mixed with mouse sera with or without resveratrol, and at the indicated time points, an equal amount of protein was subjected to SDS-PAGE for Western blot analysis using anti-His tag antibody. Protein band intensity was normalized to the intensity on day 0.
  • FIG. 9E Endo or EndoCD, each at a concentration of 12.5 ⁇ , was mixed with different doses of resveratrol in mouse serum, and at day 3, equimolar amounts of protein were subjected to SDS-PAGE for Western blot analysis using anti-His tag antibody. Solid black square, protein alone; Black open circle, protein/Resv.
  • FIGS. 10A-B The effect of EndoCD/5-FC/resveratrol on stroma microenvironment by ultrasound imaging. Mice bearing stromal-enriched PDAC were treated with EndoCD/5-FC, resveratrol (Resv), or EndoCD/5-FC/Resv. Representative images showing blood flow parameters with the indicated treatment.
  • FIG. 10A time to peak.
  • FIG. 12 Therapeutic efficacy of EndoCD/5-FC/resveratrol in a stromal- enriched PDAC models by MRI.
  • MP1070/luc cell was inoculated into pancreas to generate stromal-enriched PDAC.
  • FIGS. 13A-B Stilbenoids inhibit chymotrypsin-like protease activity.
  • the inhibitory effect of pterostilbene and piceid against chymotrypsin A proteinase activity towards its substrate N-succinyl-L-phenylalanine-p-nitroanilide was testing in the presence of different doses of each compound.
  • FIG. 13 A chymotrypsin A protein activity with different doses of pterostilbene.
  • FIG. 13B chymotrypsin A protein activity with different doses of piceid.
  • DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • Proteins with short half-lives may not be as effective and may need to be administered through repeated or higher doses than if their half-life in the presence of serum were longer.
  • a stilbenoid e.g., resveratrol, pterostilbene, piceid
  • resveratrol can prolong the stability of multiple proteins, including endostatin (Endo) and Endostatin-Cytosine Deaminase Uracil Phosphoribosyltransferase (EndoCD).
  • the protein stabilities of Endostatin and EndoCD in mouse serum are half a day and three days, respectively. However, when administered with resveratrol, the protein stabilities in mouse serum was prolonged for each of the two proteins to least seven days.
  • the protein stabilizing effect of stilbenoids may benefit protein therapy for various diseases and may allow for a reduction in the dosing schedule for various diseases.
  • resveratrol has been shown to have anticancer activities
  • the combination of resveratrol with an anti-cancer drug may show a synergistic effect. Indeed, resveratrol enhanced the killing effect of EndoCD and the efficacy of an antibody therapeutic.
  • resveratrol (or related stilbenoids) in combination with an anti-cancer protein may also enhance the effectiveness of the anti-cancer protein.
  • EndoCD in PDAC was found to be substantially enhanced by resveratrol, a stilbene phytoalexin found in the skin of grapes (Shankar et al., 2007) that exhibits anti-cancer activities, including sensitizing tumor cells to multiple chemotherapeutic agents, such as 5-FU, gemcitabine, paclitaxel, and cisplatin (Gupta et al, 2011).
  • Resveratrol also increases the protein stability of EndoCD, a function that was not previously known, by reducing the activity of chymotrypsin-like protease.
  • Pancreatic ductal adenocarcinoma is an extremely aggressive disease with poor prognosis for which there are currently no effective treatment strategies.
  • Endostatin an angiogenesis inhibitor that targets proliferating endothelial cells
  • Cytosine deaminase linked to uracil phosphoribosyltransferase converts 5-FC prodrug to chemodrug 5-FU and has a greater cancer cell killing effect than cytosine deaminase alone.
  • CD exerts off-target effects, as it does not specifically target tumors (Chung-Faye et al., 2001; Ramnaraine et al., 2003).
  • an EndoCD fusion protein effectively reduced tumor growth in mouse models of breast and colorectal cancers.
  • Administration of EndoCD via intravenous delivery in tumor-bearing mice converted nontoxic prodrug 5-FC to cytotoxic chemo-drug 5-FU, which accumulated at the tumor site with a concentration 70-fold higher than the clinically equivalent dose.
  • EndoCD associates with dual anti-angiogenesis and tumor-targeting chemotherapy activities that induce a bystander killing effect of endothelial and tumor cells surrounding the vessels (Chen et al., 2011), which is an ideal strategy to target the tumor microenvironment.
  • Administration of the naturally occurring compound resveratrol increases the protein stability of EndoCD by reducing chymotrypsin-like protease activity (an effect also seen with other stilbenoids) and sensitizes cells to EndoCD/5-FC- induced cell killing, which more effectively reduces tumor growth and extends survival than EndoCD/5-FC and resveratrol alone in various pancreatic cancer mouse models.
  • EndoCD/5-FC/resveratrol decreases intratumoral vascular density and stroma formation and enhances apoptosis in tumors as well as in surrounding endothelial and stromal composite cells.
  • the EndoCD/5-FC/stilbenoid combination may be an effective treatment option for PDAC.
  • FOLFIRINOX bolus and infusional 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin
  • FOLFIRINOX bolus and infusional 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin
  • EndoCD converts non-cytotoxic 5-FC to chemo-drug 5-FU at a level 70 times higher than the clinical equivalent dose of 5-FU predominantly at the tumor site (Chen et al, 2011).
  • EndoCD/5-FC selectively binds to pancreatic tumor vessels but not normal vessels, resulting in tumor- specific conversion of 5- FC to 5-FU that effectively inhibits pancreatic cancer growth.
  • the therapeutic efficacy of EndoCD/5-FC was enhanced by the addition of resveratrol, which increased the protein stability of EndoCD by reducing chymotrypsin-like proteinase activity and sensitized its cancer cell killing effect.
  • EndoCD/5-FC/resveratrol also induced apoptosis in other cell types, including cancer cells, activated PSCs, and leukocytes, which led to the depletion of collagen formation and reduction of vessel density, thereby exerting both anti-tumor and anti-fibrotic effects.
  • Tumor angiogenesis plays a critical role in tumor growth and metastasis.
  • the combination of bevacizumab (anti-angiogenic agent) and gemcitabine (chemotherapeutic drug) has failed to extend the survival benefit in PDAC patients (Kindler et al, 2010) most likely due to the cytostatic nature of anti-angiogenic effect in which cancer cells that are not killed by these drugs later develop an even more malignant phenotype, resulting in tumor invasion and metastasis (Loges et al., 2009).
  • the difference in the rate at which gemcitabine and bevacizumab inhibit tumor growth may produce antagonistic effect due to the decrease in chemodrug delivery caused by inhibition of angiogenesis.
  • resveratrol sensitizes cells to 5-FU (Gupta et al., 2011) to induce a bystander effect in the tumor microenvironment before tumor vessels are destroyed. Therefore, this strategy may be able to overcome the low tumor-specific chemotherapeutic effect and the lack of stromal depletion activity, among others, that are associated with the gemcitabine plus bevacizumab combination in PD AC treatment.
  • protein and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.
  • fusion protein refers to a chimeric protein containing proteins or protein fragments operably linked in a non-native way.
  • half-life (1 ⁇ 2-life) refers to the time that would be required for the concentration of a polypeptide thereof to fall by half in vitro or in vivo, for example, after injection in a mammal.
  • operable combination refers to a linkage wherein the components so described are in a relationship permitting them to function in their intended manner, for example, a linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of desired protein molecule, or a linkage of amino acid sequences in such a manner so that a fusion protein is produced.
  • linker is meant to refer to a compound or moiety that acts as a molecular bridge to operably link two different molecules, wherein one portion of the linker is operably linked to a first molecule, and wherein another portion of the linker is operably linked to a second molecule.
  • PEGylated refers to conjugation with polyethylene glycol (PEG), which has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification.
  • PEG polyethylene glycol
  • PEG can be coupled ⁇ e.g., covalently linked) to active agents through the hydroxy groups at the end of the PEG chain via chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids have been explored as novel biomaterial that would retain the biocompatibility of PEG, but that would have the added advantage of numerous attachment points per molecule (thus providing greater drug loading), and that can be synthetically designed to suit a variety of applications.
  • the term "gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof.
  • the polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so as the desired enzymatic activity is retained.
  • native refers to the typical form of a gene, a gene product, or a characteristic of that gene or gene product when isolated from a naturally occurring source.
  • a native form is that which is most frequently observed in a natural population and is thus arbitrarily designated the normal or wild-type form.
  • engineered refers to a gene or gene product that displays modification in sequence and functional properties (i.e., altered characteristics) when compared to the native gene or gene product.
  • vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • expression vector refers to any type of genetic construct comprising a nucleic acid coding for an R A capable of being transcribed.
  • R A molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell.
  • control sequences refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
  • terapéuticaally effective amount refers to an amount of a therapeutic composition (such as a therapeutic polynucleotide and/or therapeutic polypeptide) that is employed in methods to achieve a therapeutic effect.
  • therapeutic benefit or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • treatment of cancer may involve, for example, inhibition of angiogenesis of a cancer cell and/or tissue, inhibition or retardation of cell growth, facilitation of cell death, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis.
  • Treatment of cancer may also refer to prolonging survival of a subject with cancer.
  • a therapeutically effective amount of a compound is not required to cure a disease but will provide a treatment for a disease.
  • Treatment and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • a treatment may include administration of a pharmaceutically effective amount of a therapeutic protein.
  • Subject and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.
  • Stilbene refers to stilbenes, bibenzyls (7,8- dihydrostilbenes) and phenyldihydroisocoumarins together with a number of nitrogen free phenathrenols, which are thought to be products of the same metabolic pathway that leads to stilbenes (Gorham, 1980).
  • Stilbenes (7,8-dihydrostilbenes) generally have two stereoisomeric forms, a trans- or a cis-skeleton.
  • Stilbenoids are a class of plant phenolics with a 1 ,2-diphenylethylene or 1 ,2-diphenylethane nucleus in their structure.
  • stilbenes and bibenzyls are hydroxy and/or methoxy substituted at the 3,3',4,4',5, and 5' positions.
  • Some naturally occurring stilbenes and bibenzyuls include pinosylvin (3,5-dihydroxy stilbene), piceatannol (3,3',4,5'-tetrahydroxlystilbene), piceid (3,4',5-trihydroxystilbene-3-0-P-D-glucopyranoside), and resveratrol (3,4'5- trihydroxystilbene).
  • Stilbenoids include resveratrol, pterostilbene, and piceid, pinosylvin, compounds disclosed in U.S. Patent Appln. Publn. 2012/0165281. Resveratrol is found in the skin of red grapes and in other fruits as well as in the roots of Japanese knotweed ⁇ Polygonum cuspidatum).
  • Pterostilbene is chemically related to resveratrol and is found in blueberries and grapes.
  • Piceid is a stilbenoid glucoside and is a resveratrol derivative found in grape juice as well as in Japanese knotweed. III. Therapeutic proteins
  • compositions of the present invention comprise a therapeutic or diagnostic protein or polypeptide, or nucleic acids encoding therefore. Any such therapeutic or diagnostic protein or polypeptide, or nucleic acid encoding such may be used in the present invention, whether presently know to those of skill or discovered after the filing of this application.
  • therapeutic proteins or polypeptides may include, but not be limited to, suicide proteins, toxin proteins, pro-apoptotic proteins, cytokine proteins, and/or anti-angiogenic proteins.
  • the therapeutic polypeptide or protein is a "suicide protein" that causes cell death by itself or in the presence of other compounds.
  • a suicide protein is thymidine kinase of the herpes simplex virus.
  • Additional examples include thymidine kinase of varicella zoster virus, the bacterial gene cytosine deaminase (which converts 5-fluorocytosine to the highly toxic compound 5-fluorouracil), p450 oxidoreductase, carboxypeptidase G2, ⁇ -glucuronidase, penicillin- V-amidase, penicillin-G-amidase, ⁇ -lactamase, nitroreductase, carboxypeptidase A, linamarase (also referred to as ⁇ -glucosidase), the E. coli gpt gene, and the E. coli Deo gene, although others are known in the art.
  • cytosine deaminase which converts 5-fluorocytosine to the highly toxic compound 5-fluorouracil
  • p450 oxidoreductase carboxypeptidase G2
  • ⁇ -glucuronidase penicillin- V-amidase
  • the suicide protein converts a prodrug into a toxic compound.
  • prodrug means any compound useful in the methods of the present invention that can be converted to a toxic product, i.e. toxic to tumor cells. The prodrug is converted to a toxic product by the suicide protein.
  • prodrugs include: ganciclovir, acyclovir, and FIAU (l-(2-deoxy-2-fluoro-P-D- arabinofuranosyl)-5-iodouracil) for thymidine kinase; ifosfamide for oxidoreductase; 6- methoxypurine arabinoside for VZV-TK; 5-fluorocytosine for cytosine deaminase; doxorubicin for ⁇ -glucuronidase; CB1954 and nitrofurazone for nitroreductase; and N- (Cyanoacetyl)-L-phenylalanine or N-(3-chloropropionyl)-L-phenylalanine for carboxypeptidase A.
  • the prodrug may be administered readily by a person having ordinary skill in this art. A person with ordinary skill would readily be able to determine the most appropriate dose and route for the administration of the prodrug.
  • the prodrug is administered in a dose of from about 1-20 mg/day/kg body weight, from about 1- 50 mg/day/kg body weight, or about 1-100 mg/day/kg body weight.
  • a therapeutic protein or polypeptide is a cancer suppressor, for example p53 or Rb, or a nucleic acid encoding such a protein or polypeptide.
  • a cancer suppressor for example p53 or Rb
  • a nucleic acid encoding such a protein or polypeptide is known.
  • those of skill know of a wide variety of such cancer suppressors and how to obtain them and/or the nucleic acids encoding them.
  • pro-apoptotic therapeutic proteins and polypeptides include pro-apoptotic therapeutic proteins and polypeptides, for example, pl5, pl6, or p21 WAF 1 .
  • pro-apototic proteins or polypeptides which are wild-type Bik or mutant Bik comprising similar or greater activity compared to wild-type Bik.
  • the Bik mutant comprises a substitution at Thr 33 , Ser 35 , or both Thr 33 and Ser 35.
  • the substitution is with Asp.
  • U.S. Patent No. 8,304,526 which is incorporated by reference herein in its entirety, describes Bik, mutant Biks, and nucleic acid sequences encoding them.
  • Cytokines, and nucleic acid encoding them may also be used as therapeutic proteins and polypeptided.
  • Examples include: GM-CSF (granulocyte macrophage colony stimulating factor); TNFa (Tumor necrosis factor a); Interferons including, but not limited to, IFN a and IFN ⁇ ; and Interleukins including, but not limited to, Interleukin-1 (IL1), Inter leukin-B eta (IL-beta), Interleukin-2 (IL2), Interleukin-4 (IL4), Interleukin-5 (IL5), Interleukin-6 (IL6), Interleukin-8 (IL8), Interleukin-10 (IL10), Interleukin-12 (IL12), Interleukin-13 (IL13), Interleukin-14 (IL14), Interleukin-15 (IL15), Interleukin-16 (IL16), Interleukin-18 (IL18), Interleukin-23 (IL23), Interleukin-24 (IL24), although other embodiments
  • An exemplary, but not limiting or comprehensive list of therapeutic proteins or polypeptides includes (followed in some cases by the a GenBank Accession No. for a nucleic acid encoding them): Herpes simplex virus type 1 (mutant KG111) thymidine kinase (J04327); Herpes simplex virus type 2 (strain 9637) thymidine kinase (tk) (M29941); Varicella zoster thymidine kinase (M36160); Escherichia coli cytosine deaminase (S56903); p450 oxidoreductase (D17571); carboxypeptidase G2 (M12599); ⁇ -glucuronidase (M15182); penicillin- V-amidase (M15660); penicillin-G-amidase (AF161313); ⁇ -lactamase (AY029068); nitroreductase
  • coli gpt (X00221); E. coli Deo (X03224); p53 (AF307851); Rb (XM 053409); pl5 (U19796); pl6 [(U12818) (U12819) and (U12820)]; p21 WAF"1 (AF497972); GM-CSF (Ml 0663); TNF a (AY066019); IFN a (M34913); IFN a (J00219); Interferon gamma; Interferon beta; IL1 (M28983); IL-beta; IL2 (K02056); IL3 (M14743); IL4 (M23442); IL5; IL6 (M29150); IL7 (J04156); IL8; IL10 (U16720); IL12A (NM 000882); IL12B (NM 002187); IL13; IL14; IL15 (U14407); IL16;
  • Tumor Necrosis Factor SuperFamily member 11 TNF-related activation-induced cytokine; soluble form (isoform 2, sODF, sRANKL).
  • Tumor Necrosis Factor SuperFamily member 11 TNF-related activation-induced cytokine; soluble form (isoform 2, sODF, sRANKL).
  • Tumor Necrosis Factor SuperFamily member 11 TNF-related activation-induced cytokine; soluble form (isoform 2, sODF, sRANKL).
  • Tumor Necrosis Factor SuperFamily member 11 TNF-related activation-induced cytokine
  • soluble form isoform 2, sODF, sRANKL
  • Tumor Necrosis Factor SuperFamily member 11 TNF-related activation-induced cytokine
  • soluble form isoform 2, sODF, sRANKL
  • Tumor Necrosis Factor SuperFamily member 11 TNF
  • diagnostic proteins include Green fluorescent protein (M62653), Luciferase, or a combination thereof.
  • Any anti-angiogenic protein, polypeptide, or peptide may be utilized for the angiogenesis inhibitor coupled to a therapeutic or diagnostic agent, such as the exemplary targeting fusion gene products of the present invention.
  • Angiogenic inhibitors also referred to as antiangiogenic compounds are those that inhibit, reduce, halt, retard, impede, prevent, deter, slow down, reverse, or hinder angiogenesis.
  • the angiogenesis in a tissue imparts deleterious effects on the tissue in which the blood vessels are generating, such as to a tumor or a retina.
  • Exemplary embodiments include Angiopoietin-2, Angiostatin, AntiThrombin III (AT3), Amino-terminal fragment of Urokinase, Calreticulin, Endostatin, VEGF Receptor 2 (soluble fragment) (prepared by removal of a transmembrane region), VEGF Receptor 1 (soluble fragment) (prepared by removal of a transmembrane region), Interferon-alpha Inducible Protein 10, the 5 Kringle domains of plasminogen, Kringle-5 domain of plasminogen, Mammary serine protease inhibitor, Monokine -induced by Interferon-gamma, Angiostatic chemokines Fusion Gene, Pigment Epithelium-Derived Factor, C-term hemopexin domain of MMP-2, Platelet Factor 4 (CXCL4), Proliferin-Related Protein, Endothelium-specific receptor tyrosine kinase, Tissue inhibitor of metalloproteina
  • antiangiogenic sequences include (accompanied, where appropriate, with their GenBank sequence): endostatin (AF333247); angiostatin; tumstatin (AF258351) and thrombospondin (M81339).
  • a nucleic acid segment encoding a therapeutic or diagnostic protein or polypeptide is comprised in a vector, such as a nonviral vector, a viral vector, or a combination thereof.
  • the viral vector may be an adenoviral vector, a retroviral vector, or an adeno-associated viral vector.
  • the nonviral vector may be a plasmid or a liposome.
  • the nucleic acid segment may also be comprised in a pharmaceutical composition.
  • Any combinations of the therapeutic or diagnostic fusion nucleic acids could be delivered as gene therapy reagents to use against cancer or angiogenesis-dependent diseases as one component along with an antiangiogenic gene product listed herein or known in the art.
  • fusion proteins encoded by these fusion genes could be expressed and purified for protein therapy targeting cancer and other angiogenesis dependent diseases.
  • a therapeutic sequence may also serve as a diagnostic sequence, and vice versa.
  • an angiogenesis inhibitor coupled to a diagnostic agent is used prior to angiogenesis inhibitor coupled to a therapeutic agent.
  • the fusion proteins will be expressed from a nucleic acid sequence encoding an anti-angiogenesis polypeptide region linked to a therapeutic protein or polypeptide region or a diagnostic protein or polypeptide region.
  • nucleic acid sequence encoding for example, an anti- angiogenesis polypeptide region could be placed at either the 5' or the 3' end of the nucleic acid sequence encoding the therapeutic protein or polypeptide region or a diagnostic protein or polypeptide region.
  • the invention is not restricted in regard to how nucleic acids encoding these fusion proteins should be constructed into an expression vector.
  • the anti-angiogenic nucleic acid and the therapeutic or diagnostic nucleic acid may be constructed into a vector in separate construction steps. Alternatively, they may be first fused, then constructed into an expression vector.
  • the invention also relates to nucleic acids encoding a fusion protein comprising an anti-angiogenesis polypeptide region linked to a therapeutic protein or polypeptide region or a diagnostic protein or polypeptide region. Such nucleic acids may be comprised in a vector, complexed with a lipid, and/or comprised in a pharmaceutically acceptable excipient.
  • fusion of two full-length coding sequences can be achieved by methods well known in the art of molecular biology. It is preferred that a fusion polynucleotide contain only the AUG translation initiation codon at the 5' end of the first coding sequence without the initiation codon of the second coding sequence to avoid the production of two separate encoded products.
  • a leader sequence may be placed at the 5' end of the polynucleotide in order to target the expressed product to a specific site or compartment within a host cell to facilitate secretion or subsequent purification after gene expression.
  • the two coding sequences can be fused directly without any linker or by using a linker.
  • a linker for connecting the antiangiogenic and therapeutic/diagnostic proteins comprises either VPGVG (elastm Val-Pro-Gly-Val-Gly) or Gly-Gly-Gly-Ser-Gly.
  • VPGVG elastm Val-Pro-Gly-Val-Gly
  • Gly-Gly-Gly-Ser-Gly Other linkers are known to those of skill in the art, such as are described in WO 99/16889, which is incorporated by reference herein in its entirety.
  • a polynucleotide that encodes a fusion protein may be used to generate recombinant DNA molecules that direct the expression of a fusion protein, fusion peptide fragments, or a functional equivalent thereof, in appropriate cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention of the cloning and expression of the fusion protein. Such DNA sequences include those capable of hybridizing to the fusion sequences or their complementary sequences under stringent conditions, which are well known to a skilled artisan.
  • Altered DNA sequences that may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent fusion gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues within a fusion sequence, which result in a silent change thus producing a functionally equivalent fusion protein.
  • Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and or the amphipathic nature of the residues involved, which is well known to a skilled artisan.
  • the nucleotide sequence coding for a chimeric protein, or a functional equivalent is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, as described elsewherein in greater detail.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, as described elsewherein in greater detail.
  • the fusion gene products as well as host cells or cell lines transfected or transformed with recombinant fusion expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies ⁇ i.e., monoclonal or polyclonal) that bind to epitopes of the proteins to facilitate their purification.
  • a variety of host-expression vector systems may be utilized to express the fusion protein coding sequence, and these are well known in the art.
  • targeting fusion gene product and “targeting fusion” refer to proteins or polypeptides having amino acid sequences that comprise at least one anti-angiogenesis component and at least one therapeutic and/or diagnostic component in said fusion and that are biologically active in that they are capable of performing similar activities to at least one of their native components, and in some embodiments both components are capable of performing activities similar to the native separate components.
  • they are preferably capable of anti-angiogenesis activity, pro-apoptotic activity, anti-cell proliferative activity, anti-tumor activity and/or cross-reactive antibody activity with an anti-targeting fusion antibody raised against at least one component of the targeting fusion gene product.
  • targeting fusion gene product includes analogs of targeting fusion molecules that exhibit at least some biological activity in common with native targeting fusion. Furthermore, those skilled in the art of mutagenesis will appreciate that other analogs, as yet undisclosed or undiscovered, may be used to construct targeting fusion analogs.
  • substantially identical when used to define either a targeting fusion amino acid sequence or targeting fusion nucleic acid sequence, means that a particular subject sequence, for example, a mutant sequence, varies from the sequence of its separate components by, for example, one or more substitutions, deletions, additions, or a combination thereof, the net effect of which is to retain at least some biological activity of at least part of the respective component part.
  • Percent similarity may be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group.
  • the GAP program utilizes the alignment method of Needleman et al., 1970, as revised by Smith et al., 1981. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • a polynucleotide or polynucleotide region has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" or "homology" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al, eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • the invention concerns the use of targeting fusion nucleic acids, genes and gene products, or the corresponding protein, polypeptide, or peptide.
  • a sequence essentially as targeting fusion means that the sequence substantially corresponds to at least a portion of the targeting fusion gene and has relatively few bases or amino acids (whether DNA or protein) that are not identical to those of targeting fusion (or a biologically functional equivalent thereof, when referring to proteins).
  • biologically functional equivalent is well understood in the art and is further defined in detail herein.
  • sequences that have between about 70%> and about 80%>; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99% of amino acids that are identical or functionally equivalent to at least part of the amino acids of targeting fusion will be sequences that are "essentially the same.”
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of activity. Since, in many embodiments, it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even countervailing properties (e.g., antagonistic vs. agonistic). It is thus contemplated by the inventors that various changes may be made in at least part of the sequence of the targeting fusion proteins or peptides (or underlying DNA) without appreciable loss of their desired biological utility or activity.
  • Bioly functional equivalent protein or peptide is the concept that there is a limit to the number of changes that maybe made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity.
  • Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted.
  • a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.
  • residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e.g., residues in active sites, such residues may not generally be exchanged.
  • Amino acid substitutions such as those that might be employed in modifying targeting fusion, are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents. [0090] In making such changes, the hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (- 3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+.1); glutamate (+3.0+.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • nucleic acid sequences encoding a therapeutic protein or fusion protein are disclosed.
  • nucleic acid sequences can be selected based on conventional methods.
  • an open reading frame encoding a protein may be codon optimized for expression in specific organisms.
  • Various vectors may be also used to express the protein of interest, such as EndoCD.
  • Exemplary vectors include, but are not limited, plasmid vectors, viral vectors, transposon, or liposome-based vectors.
  • Host cells may be any that may be transformed to allow the expression and secretion of a therapeutic protein and conjugates thereof.
  • the host cells may be bacteria, mammalian cells, yeast, or filamentous fungi.
  • bacteria include Escherichia and Bacillus.
  • Yeasts belonging to the genera Saccharomyces, Kiuyveromyces, Hansenula, or Pichia would find use as an appropriate host cell.
  • Various species of filamentous fungi may be used as expression hosts, including the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, and Pyricularia.
  • Examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCCCRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
  • CHO-K1 Chinese hamster ovary cells
  • GH1 rat pituitary cells
  • ATCC CCL2.2 HeLa S3 cells
  • H-4-II-E rat hepatoma cells
  • COS-1 SV40-transformed monkey kidney cells
  • NIH-3T3 ATCC CRL 1658
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the homogenization and crude fractionation of the cells, tissue, or organ to polypeptide and non-polypeptide fractions.
  • the protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) unless otherwise specified.
  • Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing.
  • a particularly efficient method of purifying peptides is fast-performance liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC).
  • a purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • An isolated or purified protein or peptide therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%>, about 80%), about 90%o, about 95%, or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation- exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • a protein or peptide may be isolated or purified.
  • a His tag or an affinity epitope may be comprised in such a protein or peptide to facilitate purification.
  • Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind. This is a receptor-ligand type of interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g. , altered pH, ionic strength, temperature, etc.).
  • the matrix should be a substance that does not adsorb molecules to any significant extent and that has a broad range of chemical, physical, and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. It should be possible to elute the substance without destroying the sample or the ligand.
  • Size exclusion chromatography is a chromatographic method in which molecules in solution are separated based on their size, or in more technical terms, their hydrodynamic volume. It is usually applied to large molecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase.
  • the underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size.
  • Each size exclusion column has a range of molecular weights that can be separated.
  • the exclusion limit defines the molecular weight at the upper end of this range and is where molecules are too large to be trapped in the stationary phase.
  • the permeation limit defines the molecular weight at the lower end of the range of separation and is where molecules of a small enough size can penetrate into the pores of the stationary phase completely and all molecules below this molecular mass are so small that they elute as a single band.
  • High-performance liquid chromatography or high-pressure liquid chromatography, HPLC is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
  • HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used.
  • compositions of the present invention comprise an effective amount of one or more forms of a therapeutic or diagnostic protein or polypeptide, such as an anti-angiogenesis targeting fusion and/or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one therapeutic or diagnostic protein or polypeptide or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, ' flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
  • the therapeutic or diagnostic protein or polypeptide composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, topically, locally, using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. , liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • lipid compositions e.g. , liposomes
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • a dose may also comprise from about 1 microgram/kg body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 :microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein, hi non- limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight, a range of
  • the composition may comprise various antioxidants to retard oxidation of one or more component.
  • the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the therapeutic or diagnostic protein or polypeptide may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • nasal solutions or sprays, aerosols or inhalants are generally designed to be compatible with the target tissue type.
  • nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.
  • Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained.
  • the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5.
  • the therapeutic or diagnostic protein or polypeptide is prepared for administration by such routes as oral ingestion.
  • the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof.
  • Oral compositions may be incorporated directly with the food of the diet.
  • Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof, hi other aspects of the invention, the oral composition may be prepared as a syrup or elixir.
  • a syrup or elixir and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
  • an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof.
  • a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, com starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.;
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
  • the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • the therapeutic or diagnostic protein or polypeptides may be used for the treatment of diseases, including cancers.
  • the invention at least in part, discloses treatment methods using EndoCD in conjunction with a stilbenoid (e.g. , resveratrol, pterostilbene, piceid).
  • Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor.
  • Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.
  • Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like.
  • cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung
  • cancer of the peritoneum gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer)
  • pancreatic cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • the invention contemplates methods of using a therapeutic protein as an antitumor agent, and therefore comprises contacting a population of tumor cells with a therapeutically effective amount of a therapeutic protein for a time period sufficient to inhibit tumor cell growth.
  • the contacting in vivo is accomplished by administering, by intravenous or intraperitoneal injection, a therapeutically effective amount of a physiologically tolerable composition comprising a therapeutic protein of this invention to a patient.
  • the contacting of a therapeutic protein can also be accomplished by administering the therapeutic protein into the tissue containing the tumor cells.
  • a therapeutically effective amount of a therapeutic protein is a predetermined amount calculated to achieve the desired effect, i.e., cause the tumor cells to stop dividing.
  • the dosage ranges for the administration of a therapeutic protein of the invention are those large enough to produce the desired effect in which the symptoms of tumor cell division and cell cycling are reduced.
  • the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
  • the dosage will vary with age of, condition of, sex of, and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.
  • a therapeutically effective amount of a therapeutic protein may be an amount such that when administered in a physiologically tolerable composition is sufficient to achieve a intravascular (plasma) or local concentration.
  • the therapeutic compositions containing a therapeutic protein are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle.
  • the compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial administration and booster shots are also contemplated and are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Exemplary multiple administrations are described herein and are particularly preferred to maintain continuously high serum and tissue levels of a therapeutic protein. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
  • compositions and methods of the present embodiments involve administration of a therapeutic protein in combination with a second or additional therapy.
  • Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve administering to the cells both a therapeutic protein and a second therapy.
  • a tissue, tumor, or cell can be exposed to one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., a therapeutic protein or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) a therapeutic protein, 2) an anti-cancer agent, or 3) both a therapeutic protein and an anticancer agent.
  • a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, hormone therapy, or immunotherapy.
  • contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
  • a therapeutic protein may be administered before, during, after, or in various combinations relative to an anti-cancer treatment.
  • the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
  • the therapeutic protein is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient.
  • a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered.
  • This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.
  • chemotherapeutic agents may be used in accordance with the present embodiments.
  • the term "chemotherapy” refers to the use of drugs to treat cancer.
  • a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); do
  • DNA damaging factors include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • immunotherapies may be used in combination or in conjunction with methods of the embodiments.
  • immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Rituximab (RITUXAN®) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pi 55.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
  • cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN
  • chemokines such as MIP-1, MCP-1, IL-8
  • growth factors such as FLT3 ligand.
  • Patents 5,830,880 and 5,846,945) ; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl85 (Hollander, 2012; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
  • kits such as therapeutic kits.
  • a kit may comprise one or more pharmaceutical composition as described herein and optionally instructions for their use. Kits may also comprise one or more devices for accomplishing administration of such compositions.
  • a subject kit may comprise a pharmaceutical composition and catheter for accomplishing direct intravenous injection of the composition into a cancerous tumor.
  • a subject kit may comprise pre-filled ampoules of a therapeutic protein, optionally formulated as a pharmaceutical, or lyophilized, for use with a delivery device.
  • Kits may comprise a container with a label.
  • Suitable containers include, for example, bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials, such as glass or plastic.
  • the container may hold a composition that includes a therapeutic protein that is effective for therapeutic or non-therapeutic applications, such as described above.
  • the label on the container may indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above.
  • the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. XII. Examples
  • endostatin is known to bind to integrin receptors that are usually expressed on tumor endothelial cells (Folkman, 2006)
  • EndoCD binds preferentially to tumor vessels over normal pancreas counterparts.
  • orthotopic mouse models of PDAC were established from human AsPCl or mouse stromal- enriched MP 1070 PDAC cells.
  • the enriched stroma in MP 1070 tumors was readily detectable by the increased collagen intensity (Apte et ah, 2004) (FIG. 5).
  • FITC- labeled dextran was injected to mark the vessels in mice by green fluorescence, followed by tail vein catheterization (Hedrich and Bullock, 2004) to systemically delivery Qdot605- labeled EndoCD for the duration of imaging.
  • the relative distribution of EndoCD in the vasculature of tumor and normal pancreatic tissues from the same mouse was monitored by two-photon microscopy.
  • time-lapse video of normal pancreatic vessels in the mouse stromal-enriched PDAC model were taken.
  • Qdot605 -labeled EndoCD bio-conjugates accumulated at the tumor vessels but not normal pancreatic vessels.
  • EndoCD selectively targets to tumor vessels but not normal vessels in both human PDAC and stromal-enriched mouse PDAC models. It appears that the vessel-targeting potential of the EndoCD protein is not hindered by the stromal-enriched PDAC microenvironment.
  • EndoCD/5-FC the cytotoxic effect of EndoCD/5-FC was determined in AsPCl/luc cells stably expressing luciferase. As shown in FIG. 6 A, EndoCD/5-FC decreased cell viability in a dose-dependent manner.
  • an AsPcl/luc orthotopic pancreatic mouse model was established to validate the therapeutic efficacy of EndoCD/5-FC in vivo.
  • EndoCD 7.5 mg/kg or 60 mg/kg
  • 5-FC 500 mg/kg
  • EndoCD/5-FC but not the vehicle control, inhibited tumor growth in a dose-dependent manner (FIG. 6B, P ⁇ 0.05). These results indicate that EndoCD/5-FC inhibits pancreatic cancer cell viability and suppresses tumor growth.
  • resveratrol was shown to sensitize cells to 5-FU-mediated apoptosis in colorectal cancer cells via caspase-6 activation (Chan et al., 2008) at a high concentration (200 ⁇ ) of resveratrol. Similar results were observed in pancreatic cancer cells (FIG. 8). Interestingly, the dose of resveratrol used in combination with EndoCD/5-FC that induced strong synergistic chemosensitization was 20 times lower. Thus, other mechanisms might exist in addition to activation of the apoptosis signaling pathway by resveratrol. Endo has a short half-life in serum, which is a major reason why Endo was not clinically viable.
  • EndoCD/5-FC The enhanced stability of EndoCD by resveratrol might also increase the therapeutic efficacy of EndoCD/5-FC.
  • the killing effect was determined by an ex vivo serum co-culture assay in which pancreatic cancer cells were subjected to resveratrol, EndoCD, or the combination pre-incubated in mouse sera for 5 days.
  • FIG. 2C only the EndoCD/resveratrol combination potently enhanced the cell killing effect of 5-FC, suggesting that resveratrol-mediated EndoCD stabilization may play a major role in the synergistic effect of EndoCD/5-FC/resveratrol combination treatment.
  • EndoCD/5-FC/resveratrol markedly inhibited tumor growth compared with either treatment alone or vehicle control. Specifically, tumor growth inhibition on Day 28 by EndoCD/5-FC, resveratrol, and EndoCD/5-FC/resveratrol was 27%, 19%, and 70%>, respectively (FIG. 3 A). In addition, EndoCD/5-FC/resveratrol further extended median survival time from 30 to 44 days (or by 46.7%) compared with vehicle control (FIG. 3B). In the human AsPCl/luc orthotopic mouse model, which has less stroma than the MP1070/luc PDAC model (FIG.
  • EndoCD/5-FC/resveratrol also inhibits Kras-mediated PDAC was investigated.
  • an inducible Kras GEMM iKras (G12D)/conditional p53 knockout, iKrasG12Dp53L/+
  • Mice were administered EndoCD/5-FC, resveratrol, or the combination after an 8-week doxycycline induction until death.
  • EndoCD/5- FC/resveratrol increased the survival time of the mice from 105 to 182 days, extending it by 73% compared with either treatment alone or vehicle control (FIG. 4E).
  • the EndoCD/5- FC/resveratrol combination therapy substantially improves the survival outcome of Kras- mediated PDAC in mice.
  • the PDAC stromal microenvironment constitutes a barrier to drug delivery and reduces chemotherapeutic response (Olive et al., 2009)
  • the effect of EndoCD/5-FC/resveratrol on PDAC stromal microenvironment was examined, specifically the effect on tumor blood flow associated with angiogenesis, by using a high frequency ultrasound system (Fuchs et al., 2014).
  • Mice bearing stromal-enriched PDAC were treated with EndoCD/5-FC, resveratrol, or their combination. Prior to administration of the drugs, images were taken to ensure that all parameters were insignificantly different.
  • mice were re-imaged.
  • a contrast agent was injected into mice via tail vein catheterization, and signals were recorded by ultrasound.
  • Parameters representing the kinetics of blood flow in the tumor were calculated from time-intensity curves (Sboros et ah, 2011) using time-lapse video (Vevo 2100 system, Contrast Mode).
  • RT rise time
  • TTP time to peak
  • the stroma of PDAC comprises several cell types, including pancreatic stellate cells (PSC), endothelial cells, immune cells, and the extracellular matrix (Chu et al., 2007).
  • PSC pancreatic stellate cells
  • endothelial cells endothelial cells
  • immune cells extracellular matrix
  • extracellular matrix extracellular matrix
  • TUNEL assay to measure apoptosis
  • a-SMA staining activated pancreatic stellate cells marker
  • CD31 staining endothelial cell marker
  • CD45 staining leukocyte marker
  • trichrome staining to measure the amount of collagen in PDAC.
  • EndoCD/5-FC/resveratrol increased apoptosis (FIG. 4M), decreased the amount of collagen (FIGS. 4E and F), and reduced tumor vessel density as indicated by decreased CD31 (FIGS. 41 and J).
  • EndoCD/5 -FC/resveratrol also depleted stroma formation in the tumor microenvironment by reducing activated PSCs (decreased a-SMA intensity) (FIGS. 4G and H), leukocytes (decreased CD45 intensity) (FIGS. 4K and L), and collagen deposition (FIGS. 4E and F).
  • resveratrol enhances the protein stability of tumor-targeting EndoCD/5-FC and sensitizes tumors to its chemotherapeutic bystander effect, which penetrates the stroma to induce apoptosis of stromal composite cells, including endothelial cells, PSCs, leukocytes, and tumor cells, resulting in PDAC tumor shrinkage (FIG. 4N).
  • DMEM Dulbecco's modified Eagle's
  • FBS fetal bovine serum
  • MP1070 a primary murine pancreatic cancer cell line developed from a KrasG12Dp53L/+ conditional genomic modified mouse, was generated by Dr. Ronald DePinho's laboratory.
  • AsPCl/luc, Pan02/luc, and MP1070/luc with stable luciferase expression were maintained in either G418 or puromycin selection.
  • Cell lines were validated by short tandem repeats (STR) DNA fingerprinting by using the AmpFISTR Identifiler PCR Amplification Kit according to the manufacturer's instructions (Applied Biosystems; catalogue no. 4322288).
  • EndoCD protein was generated as previously described (Chen et ah, 2011) and dialyzed in either Tris-HCl or phosphate buffered saline (PBS) solution.
  • the sequence of the EndoCD protein is provided as SEQ ID NO: 1 (amino acids 1-184 are Endo; 185-186 are a linker; and 187-594 are CD:UPRT).
  • Cell Viability Assay Cell viability was evaluated by the tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Pancreatic cancer cells Capanl, AsPCl/luc, or pan02/luc cells were seeded (1 x 10 4 cells/well) in a 96-well plate. Different doses of resveratrol or EndoCD recombinant proteins with 200 ⁇ g/ml of 5-FC were added into each well.
  • MTT tetrazolium dye
  • MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
  • EndoCD/resveratrol was pre-mixed in fresh mice sera 5 days in 37°C incubator. Capanl, AsPCl/luc, or pan02/luc pancreatic cancer cells were then subjected to 1/10 of the pre- incubated mixture and cell viability was analyzed after treatment for 48 hr.
  • mice were inoculated with 1 x 106 AsPCl/luc human pancreatic cancer cells or MP1070/luc into the pancreas.
  • Mice received 40 mg/kg resveratrol (five times per weeks) by oral injection (Shankar et ah, 2011) and/or 7.5 mg/kg EndoCD (twice per week) by tail vein injection.
  • EndoCD 500 mg/kg 5-FC was given 1 hr after by intraperitoneal injection five times per week (Chung -F aye et ah, 2001).
  • Tumor volume was monitored by measuring luciferase signals using IVIS (In Vivo Imaging System; Xenogen, Alameda, CA). All animal procedures were conducted under regulations of Division of Laboratory Animal Medicine at The University of Texas MD Anderson Cancer Center. Animal protocols (Protocol Number 190511133) were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center.
  • AsPCl or mouse stromal-enriched PDAC MP 1070 cells were inoculated in mice pancreas. Once tumors reached approximately 5-10 mm in diameter, mice were anesthetized, tail vein catheterized, and identified and exteriorized pancreatic tumor through the laparotomy. Body temperature was kept at 37°C throughout the experiments using a heating pad. The upper midline laparotomy was made as short as possible ( ⁇ 15 mm). The pancreatic tumor was then transferred onto a soft tissue immobilization system (VueBio Technology, Pearland, TX). The organ stabilizer minimized the micro-vibration of the observed area caused by heart beat and respiratory movements.
  • Two-photo microscopy was performed using an upright multiphoton microscope 7MP (Carl Zeiss, LSM 7MP, NY) with coherent pulsed Ti:Sapphire laser (plus width 140 fs, repetition rate 80MHz; Coherent, Santa Clara, CA).
  • Anesthetized mouse was placed on the microscope stage.
  • the microscope with several lenses provides the long working distances required in vivo.
  • a mean laser power at the sample was between 10- 40mW, depending on the depth of imaging.
  • Microscope objective lenses used in this study were 10X, 20X plan Achromat (numerical aperture of 0.45 and 0.8, respectively). Images were acquired with 1024 x 1024 pixels spatial resolution.
  • Two-photon fluorescence signals were collected by an internal detector at an excitation wavelength at 910 nm, to enable the simultaneous acquisition of dextran-FITC signal and Qdot605-EndoCD signal.
  • the surface of the pancreatic tumor was initially screened at lower magnifications by setting out the X/Y plane and adjusting the Z axis manually to detect the optimal observation area containing blood vessels.
  • the imaging depth or imaging stack was determined arbitrarily to allow real- time visualization of the relative distribution of Qdot605 -labeled EndoCD in vivo. Data were analyzed using Zen 2011 software.
  • Benzoylarginyl-p-nitroanilide hydrochloride (BAPNA, Sigma- Aldrich), N-succinyl-L- phenylalanine-p-nitroanilide (SPNA, Sigma- Aldrich), N-succinyl-alanine-alanine-proline- leucine-p-nitroanilide (SAAPLPNA, Sigma- Aldrich) substrates were used to measure trypsin-, chymotrypsin-, and elastase-like enzyme activity, respectively. All small peptides were prepared as previously described to measure protease activity (Altshuler et ah, 2012).
  • MRI Small Animal Magnetic Resonance Imaging
  • Doppler and nonlinear contrast imaging studies were conducted twice on each mouse, once 10 days after tumor cell inoculation but before treatment initiation and once 18 days after cell inoculation and 8 days following treatment initiation.
  • a matching B- mode ultrasound frame was acquired for each acquired Doppler or nonlinear contrast imaging frame.
  • Tumor margins were manually segmented based on the B-mode data set for postprocessing.
  • Doppler Imaging The linear array was translated (0.152-mm step size) up to 15 mm in the elevation dimension to obtain a full volume acquisition of the tumor.
  • Nonlinear Contrast Imaging The transducer array was translated to an imaging slice near the midsection of the tumor that contained the largest tumor cross-section.
  • Fifty microliters of untargeted microbubble contrast (MicroMarkerTM, Bracco Group) were administered through a bolus tail vein injection at an infusion rate of 40 ⁇ /s. Imaging acquisition was initiated two seconds prior to the injection and continued for an additional 100 seconds.
  • Peak enhancement PE
  • area under the curve AUC
  • rise time RT
  • time to peak TTP
  • wash-in rate WiR, maximum slope
  • perfusion index PI, AUC/RT
  • Masson 's Trichrome Staining was purchased from Sigma. Tissue section was deparaffmized, rehydrated, and incubated in Bouin's Solution at room temperature overnight. Samples were washed in running tap water for 5 min to remove the yellow color from the section, stained in Weigert's Iron Hematoxylin Solution for 5 min, washed again in running tap water for 5 min, and then stained in Biebrich Scarlet-Acid Fuchsin for 5 min. Slides were then placed in phosphomolybdic/ phosphotungstic acid solution for 10 min, transferred to Aniline blue for 5 min, placed in 1% acetic acid solution for 3 min, and then rinsed in distilled water. Finally, the section was washed with 1% acetic acid for 1 minute and rinsed in distilled water.
  • the activated stroma index is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé de prolongation de la stabilité d'une protéine, qui peut être utilisé pour la stabilisation d'une protéine thérapeutique quelconque destinée à être utilisée dans le traitement de tout type de maladie. Par exemple, un stilbénoïde (par exemple, le resvératrol) peut être utilisé pour améliorer la stabilité d'une protéine EndoCD, qui peut sensibiliser des tumeurs aux effets anti-angiogéniques et chimiothérapeutiques ciblés de 5-FC, offrant ainsi une nouvelle approche thérapeutique pour le cancer.
PCT/US2015/027960 2014-04-28 2015-04-28 Stabilisation de protéine prolongée par le resvératrol WO2015168102A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201461985003P 2014-04-28 2014-04-28
US61/985,003 2014-04-28

Publications (3)

Publication Number Publication Date
WO2015168102A2 true WO2015168102A2 (fr) 2015-11-05
WO2015168102A3 WO2015168102A3 (fr) 2016-03-17
WO2015168102A9 WO2015168102A9 (fr) 2016-05-12

Family

ID=54359482

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/027960 WO2015168102A2 (fr) 2014-04-28 2015-04-28 Stabilisation de protéine prolongée par le resvératrol

Country Status (1)

Country Link
WO (1) WO2015168102A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112789036A (zh) * 2018-08-02 2021-05-11 G·拉瓦尼安 包括白藜芦醇苷类和姜黄素类化合物的组合物

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109602730B (zh) * 2018-12-13 2021-10-01 山东师范大学 白藜芦醇联合双氢青蒿素在治疗肝癌和乳腺癌中的应用及其产品

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1501861A4 (fr) * 2002-05-06 2007-06-20 Univ Texas Ciblage de proteines pour l'apport de reactifs therapeutiques ou de diagnostic
EP1835929B8 (fr) * 2005-01-06 2016-07-27 Novo Nordisk A/S Procédés et traitements combinés anti-kir
WO2010045002A2 (fr) * 2008-09-26 2010-04-22 Tocagen Inc. Vecteurs de thérapie génique et cytosines déaminases
DK2443242T3 (en) * 2009-06-17 2017-01-30 Tocagen Inc Production cells for replicative retroviral vectors

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112789036A (zh) * 2018-08-02 2021-05-11 G·拉瓦尼安 包括白藜芦醇苷类和姜黄素类化合物的组合物

Also Published As

Publication number Publication date
WO2015168102A9 (fr) 2016-05-12
WO2015168102A3 (fr) 2016-03-17

Similar Documents

Publication Publication Date Title
US11534463B2 (en) Nucleic acids encoding kynurenine depleting enzymes
US10724026B2 (en) Engineered primate L-methioninase for therapeutic purposes
US11426464B2 (en) Engineered primate cystine/cysteine degrading enzymes as antineogenic agents
US20220275099A1 (en) Administration of kynurenine depleting enzymes for tumor therapy
US20230227796A1 (en) Enzyme-mediated depletion of adenosine and/or methylthioadenosine
WO2015168102A9 (fr) Stabilisation de protéine prolongée par le resvératrol

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15786760

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15786760

Country of ref document: EP

Kind code of ref document: A2