WO2019220441A1 - Compositions and methods for treating cancer resistant to an anti-cancer agent - Google Patents

Compositions and methods for treating cancer resistant to an anti-cancer agent Download PDF

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Publication number
WO2019220441A1
WO2019220441A1 PCT/IL2019/050552 IL2019050552W WO2019220441A1 WO 2019220441 A1 WO2019220441 A1 WO 2019220441A1 IL 2019050552 W IL2019050552 W IL 2019050552W WO 2019220441 A1 WO2019220441 A1 WO 2019220441A1
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Prior art keywords
composition
cancer
tlivin
nps
agent
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PCT/IL2019/050552
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French (fr)
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WO2019220441A8 (en
Inventor
Dina Ben Yehuda
Ihab Abd-Elrahman
Riki Perlman
Boaz Nachmias
Simon Benita
Taher Nassar
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Hadasit Medical Research Services And Development Ltd.
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Publication of WO2019220441A1 publication Critical patent/WO2019220441A1/en
Publication of WO2019220441A8 publication Critical patent/WO2019220441A8/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Abstract

Compositions and methods for treating cancer resistant to an anti-cancer agent are provided. Accordingly, there is provided a method of treating cancer exhibiting a resistance to an anti-cancer agent in a subject comprising administering to the subject a therapeutically effective amount of t Livin. Also provided are compositions comprising t Livin attached to or encapsulated in a nanoparticle comprising poly(lactide-co-glycolide) and compositions comprising t Livin and a targeting moiety, wherein the t Livin is attached to or encapsulated in a cell penetrating agent and/or a stabilizing agent.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCER RESISTANT
TO AN ANTI-CANCER AGENT
RELATED APPLICATIQN/S
This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/671,454 filed on May 15, 2018, the contents of which are incorporated herein by reference in their entirety.
SEQUENCE LISTING STATEMENT
The ASCII file, entitled 77314 Sequence Listing.txt, created on May 13, 2019, comprising 10,928 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to compositions and methods for treating cancer resistant to an anti-cancer agent.
Considerable progress has been made in the development of new effective regimens for the treatment of different types of cancers including colon cancer, pancreatic cancer, lung cancer, melanoma, lymphoma, leukemia and the like. Unfortunately, increased response rates to current chemotherapeutic and targeted therapy regimens have not been translated into marked improvements in survival since, in many instances, durations of response rates have been short term. The development of drug resistance is a major obstacle to successful cancer therapy. Great efforts have focused on the underlying mechanisms that turn promising targeted therapies which induce initial tumor shrinkage ineffective following few months, resulting in refractory or untreatable cancers.
The Inhibitor of Apoptosis Protein (IAP) family of proteins has been shown to inhibit apoptosis induced by a variety of stimuli mainly by binding and inhibiting specific caspases. Eight human IAPs have been identified to date: c-IAPl, C-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2 and Livin.
Livin, also known as baculoviral IAP repeat-containing 7; BIRC7, KIAP, ML- IAP and Livin inhibitor-of-apoptosis, contains a single baculovirus IAP repeats (BIR) domain at the N- terminus and a carboxy-terminal RING domain [Ashhab Y et al. FEBS Lett. 200l;495(l-2):56- 60]. The BIR domain was shown to play a role in the anti-apoptotic function of IAPs [Chai J et al. Cell. 2000;l04(5):769-780; Huang Y, et al. Cell. 2000; l04(5):781-790]. Livin encodes two highly similar splicing variants, termed Livin a and b that differ only in 18 amino acids located between the BIR and the RING domains, which are present in the a but not in the b isoform. Following apoptotic stimuli, both Livin isoforms a and b undergo a specific proteolytic cleavage that trims the 52 amino acids at the N-terminus of Livin (i.e. at the Asp52 residue). From each isoform a truncated C-terminal Livin is thus produced, of approximately 30 kDa (also termed p30 also termed tLivina) and 28 kDa (also termed p28 also termed tLivh^), respectively, containing the full BIR and RING domains [Ashhab Y et al. FEBS Lett. 200l;495(l-2):56-60]. These truncated forms of Livin are collectively referred to as tLivin. tLivin is not only devoid of Livin anti- apoptotic activity but also acquires a pro-apoptotic effect [Nachmias B et al. Cancer Res. 2003;63(l9):6340-634; Abd-Elrahman I et al. Cancer Research. 2009;69(l3):5475-5480] and was shown to promote more than one form of cell death in the same cell type (Shiloach et al PLoS One. 2014 Jun 24;9(6):el0l075). A 70 amino acids derivative of tLivin [denoted herein as mini-tLivin (mtLivin)] was identified which remarkably demonstrates a pro-apoptotic activity as potent as tLivin [Nachmias B et al. apoptosis. 2007; 12(7): 1129- 1142].
U.S Patent No. 7,517,949 discloses Livin-derived peptides with pro-apoptotic activity. Specifically provided are peptides p30-Livin a and p28-Livin b, derived from Livin a and b truncation, respectively, as well as compositions thereof. These peptides display pro-apoptotic activity and as such are used for the enhancement and/or induction of apoptosis, as well as for the treatment of cancer.
Additional background art includes:
US Patent Application Publication no. US20150125430;
International Patent Application Publication no. W02007034479;
International Patent Application Publication no. WO2013042125;
International Patent Application Publication no. W02012101638;
International Patent Application Publication no. W02012101639;
Karra & Benita. Curr Drug Metab. (2012) 13:22-41;
Karra et al Curr Cancer Drug Targets. (2013) 13: 11-29; and
Karra et al. Small (2013) 9(24): 4221-36.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of treating cancer exhibiting a resistance to an anti-cancer agent in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of tLivin, thereby treating the cancer exhibiting resistance to the anti-cancer agent in the subject.
According to some embodiments of the invention, the method comprising administering to the subject at least one anti-cancer agent.
According to some embodiments of the invention, the at least one anti-cancer agent comprises the anti-cancer agent.
According to an aspect of some embodiments of the present invention there is provided a composition comprising tLivin for use in the treatment of cancer resistant to an anti-cancer agent.
According to some embodiments of the invention, the composition comprising at least one anti-cancer agent.
According to some embodiments of the invention, the at least one anti-cancer agent comprises the anti-cancer agent.
According to some embodiments of the invention, the anti-cancer agent is not an apoptotic agent.
According to some embodiments of the invention, the anti-cancer agent is an immunodulatory molecule.
According to some embodiments of the invention, the anti-cancer agent is selected from the group consisting of IMiDs (e.g. Revlimid, Thalidomide, Pomalidomide), proteasome inhibitors (e.g. Velcade, Carfilzomib), Rituximab, fludarabine and Bendamustine.
According to some embodiments of the invention, the tLivin is administered in a formulation comprising a targeting moiety.
According to some embodiments of the invention, the tLivin is administered in a formulation comprising a cell penetrating agent and/or a stabilizing agent.
According to an aspect of some embodiments of the present invention there is provided a composition comprising tLivin attached to or encapsulated in a nanoparticle comprising poly(lactide-co-glycolide).
According to an aspect of some embodiments of the present invention there is provided a composition comprising tLivin and a targeting moiety, wherein the tLivin is attached to or encapsulated in a cell penetrating agent and/or a stabilizing agent.
According to some embodiments of the invention, the cell penetrating agent and/or the stabilizing agent is selected from the group consisting of a nanoparticle, a liposome, a viral vector, a cell penetrating peptide and poly(alkylene) glycols. According to some embodiments of the invention, the penetrating agent and/or a stabilizing agent is a nanoparticle.
According to some embodiments of the invention, the tLivin is attached to an outer surface of the nanoparticle.
According to some embodiments of the invention, the tLivin is attached to the nanoparticle via a linker.
According to some embodiments of the invention, the linker comprises an oleyl cysteineamide (OCA).
According to some embodiments of the invention, the nanoparticle comprises poly(lactide-co-glycolide), polylactide (PLA), polyglycolide, polylactide-polyglycolide, and/or polyethylene glycol-co-lactide (PEG-PLA).
According to some embodiments of the invention, the nanoparticle comprises poly(lactide-co-glycolide).
According to some embodiments of the invention, the concentration of the poly(lactide- co-glycolide) is 0.05 to 5 mg / ml.
According to some embodiments of the invention, the targeting moiety comprises an immunomodulatory molecule.
According to some embodiments of the invention, the targeting moiety is a CD40 and/or PD-l binding molecule.
According to some embodiments of the invention, the targeting moiety is a CD40 binding molecule.
According to some embodiments of the invention, the targeting moiety is selected from the group consisting of CD19, CD20, CD38, CD138, EGFR, Her-2 and PMSA binding molecule.
According to an aspect of some embodiments of the present invention there is provided a composition comprising tLivin and a CD40 binding molecule.
According to some embodiments of the invention, the CD40 binding molecule comprises a CD40L polypeptide and/or an anti-CD40 antibody.
According to some embodiments of the invention, the CD40 binding molecule comprises a CD40L polypeptide.
According to some embodiments of the invention, the tLivin is in a concentration of
0.25-0.5 mg / ml.
According to some embodiments of the invention, the targeting moiety is in a concentration of 0.25-0.5 mg / ml. According to some embodiments of the invention, the nanoparticle has a diameter of 50 - 250 nm.
According to some embodiments of the invention, the composition is lyophilized.
According to some embodiments of the invention, the composition comprises a cryo- protectant.
According to some embodiments of the invention, the cryo-protectant comprises trehalose and/or b-cyclodextrine.
According to some embodiments of the invention, the composition comprises an anti cancer agent.
According to an aspect of some embodiments of the invention there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition, thereby treating the cancer in the subject.
According to an aspect of some embodiments of the invention there is provided the composition, for use in the treatment of cancer.
According to an aspect of some embodiments of the invention there is provided a method of increasing an amount of platelets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition, thereby increasing the amount of platelets in the subject.
According to an aspect of some embodiments of the invention there is provided a method of treating thrombocytopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition, thereby treating thrombocytopenia in the subject.
According to an aspect of some embodiments of the invention there is provided the composition, for use in the treatment of thrombocytopenia.
According to some embodiments of the invention, the subject has cancer.
According to some embodiments of the invention, the cancer exhibits a resistance to an anti-cancer agent.
According to some embodiments of the invention, the resistance is acquired resistance.
According to some embodiments of the invention, the cancer is selected from the group consisting of melanoma, lymphoma, multiple myeloma, chronic lymphocytic leukemia (CLL), lung cancer and prostate cancer.
According to some embodiments of the invention, the tLivin is p30-Livin a. According to some embodiments of the invention, the tLivin comprises an amino acid sequence comprising SEQ ID NO: 2.
According to some embodiments of the invention, the tLivin is p28-Livin b.
According to some embodiments of the invention, the tLivin comprises an amino acid sequence comprising SEQ ID NO: 4.
According to some embodiments of the invention, the tLivin is mtLivin.
According to some embodiments of the invention, the mtLivin comprises an amino acid sequence comprising SEQ ID NO: 6.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
BRIEL DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIGs. 1A-B demonstrate mtLivin expression and purification from C43(DE3) bacteria cells. Figure 1A is a schematic representation demonstrating expression of mtLivin with a His- tag and a TEV cleavage sequence (SEQ ID NO: 8) and purification by chromatography using Ni- NTA agarose and gel filtration chromatography. Figure 1B shows purified mtLivin (SEQ ID NO: 6) and inactive mtLivin (i.e. prior to TEV cleavage), as determined by SDS-PAGE and Coomassie staining.
FIG. 2 demonstrates mtLivin peptide (SEQ ID NO: 6) purified from C43(DE3) bacteria cells contains only minor contaminations, as determined by SDS-PAGE and Coomassie staining. FIG. 3 is a bar graph demonstrating cell death of 293T cells following 24 hours incubation with the indicated concentrations of mtLivin peptide, as determined by PI staining and flow cytometry analysis. Results are expressed as the percentage of dead cells.
FIG. 4 demonstrates mtLivin peptide conjugated to PLGA nanoparticles (mtLivin-NPs), as determined by coomassie staining (left image) and western blot using anti-Livin antibody (right image).
FIG. 5 shows bar graphs demonstrating cell death of 293T, MEWO and 721.221 / Livin b cells following 24 hours incubation with the indicated concentrations (pg / ml) of mtLivin-NPs, as determined by PI staining and flow cytometry analysis. Results are expressed as the percentage of dead cells.
FIG. 6 shows coomassie staining of PLGA NPs loaded with increasing amounts of purified mtLivin peptide.
FIGs. 7A-B demonstrate the morphology of blank PLGA NPs (Figure 7 A) and PLGA NPs conjugated with 0.25 mg / ml mtLivin peptide (Figure 7B). Shown are TEM micrographs of uranyl acetate negatively stained NPs at different areas of the grid, scale bar = 200 nm.
FIGs. 8A-B demonstrate the biological effect of PLGA NPs loaded with different concentration of mtLivin (MTV) peptide. Figure 8A shows coomassie staining of increasing amounts of purified mtLivin peptide conjugated to PLGA NPs in comparison to increasing amounts of BSA. Figure 8B is a graph demonstrating cell death induced by incubating 721.221 cells with the indicated concentrations of mtLivin peptide conjugated to PLGA NPs, as determined by PI staining and flow cytometry analysis.
FIGs. 9A-B demonstrate the effect of lyophilization of free mtLivin (MTV) and mtLivin conjugated to PLGA NPs (MTV NPS). Shown is peptide stability as determined by coomassie staining (Figure 9A) and activity as determined by induction of 293T cells death (Figure 9B) of mtLivin and mtLivin-NPs lyophilized with the indicated cryoprotectants following reconstitution with water. CD - b-cyclodextrin; S - sucrose; T - trehalose.
FIGs. 10A-C demonstrate cell death and cell cycle analysis of L428 Hodgkin lymphoma cells following incubation with mtLivin PLGA NPs. Figure 10A demonstrates quantitation of mtLivin PLGA NPs, as determined by coomassie staining. Figures 10B-C demonstrate cell death (Figure 10B) and cell cycle analysis (Figure 10C) following 24 hours incubation with mtLivin PLGA NPs, as determined by PI staining and flow cytometry analysis.
FIG. 11 demonstrates cell cycle analysis of LY19 DLBCL cells following 24 hours incubation with CD40L-mtLivin (MTV) NPs as compared to the indicated controls, as determined by PI staining and flow cytometry analysis. Shown representative flow cytometry histograms and a summarizing bar graph.
FIG. 12 demonstrates cell death and fluorescence of LY 19 DLBCL cells stably expressing GLuc following incubation with mtLivin-NPs. Cells were treated with PLGA blank NPs (PLGA) or PLGA NPs loaded with mtLivin (MTVNPS) at 2 concentrations (0.25 mg and 0.5 mg). Shown is cell death as determined by Annexin V staining and flow cytometry analysis; and luminescence, indicating living cells, as determined by a Tecan reader. Untreated cells present the highest luminescence and luminescence decreases with the increase in apoptosis.
FIG. 13 is a calibration curve of doxorubicin as determined by UV detector at 475 nm; the calibration curve ranged between 5 to 500 pg/ml of water : acetonitrile (1:1).
FIG. 14 demonstrates the effect of doxorubicin on cell death and fluorescence of LY19 DLBCL cells stably expressing GLuc. Cells were treated with PLGA NPs (PLGA) or PLGA NPs loaded with mtLivin (MTVNPS) at 2 concentrations (0.25 mg and 0.5 mg) with or without doxorubicin (DOX, 4pg). Shown is cells death as determined by Annexin V staining and flow cytometry analysis (top); and luminescence as determined by a Tecan reader (bottom). Untreated cells present the highest luminescence and luminescence decreases with the increase in apoptosis.
FIGs. 15A-E demonstrate the establishment and analysis of the in-vivo LY19Gluc DLBLC xenograft mouse model. Figure 15A is a bar graph demonstrating percent of mice with tumors. Figure 15B is a graph demonstrating tumor development over time as determined by measurement with a caliper. Figures 15C-D show images of the sub-cutaneous xenograft tumors and metastases as visualized in IVIS. Figure 15E is a bar graph demonstrating GLuc activity in mice urine.
FIG. 16 is a bar graph demonstrating cell death of LY 19 DLBCL cells following 48 hours incubation with CD40L-mtLivin (MTV) NPs as compared to the indicated controls, as determined by Annexin V staining and flow cytometry analysis.
FIG. 17A-D demonstrate the effect on tumor growth following treatment with the indicated NPs in the in-vivo LYl9Gluc DLBLC xenograft mouse model as compared to untreated control. Shown is average tumor size over time, as determined with a caliper (Figures 17A-B), mouse survival (Figure 17C) and tumor cell death, as determined by caspase-3 activity assay (Figure 17D).
FIG. 18 demonstrates the establishment and analysis of the in-vivo disseminated LYl9Gluc DLBLC xenograft mouse model. Shown are photon flux signal from representative animals (top) and a bar graph summarizing tumor cell photon flux that was measured using photon flux imaging indicating tumor growth (bottom) at days 2, 22 and 46 at the points that mice were scarified. Heat map key: bright red, highest photon emission; cool blue, lowest photon emission.
FIG. 19 shows immunohistochemistry analysis using ant-CD20 staining of mice organs (spleen, brain, spinal cord BM, lungs and liver) at different days post IV injection of LYl9Gluc DLBLC cells demonstrating diffuse infiltration of lymphoma cells to these organs.
FIG. 20 is a bar graph demonstrating the effect on body weight following treatment with the indicated NPs in the in-vivo disseminated LYl9Gluc DLBLC xenograft mouse model as compared to untreated control. Body weight was measured at the indicated time points. All mice of control groups were sacrificed between days 28-36 and mt-Livin-NPs and CD40L+mtLivin-NPs -treated groups at day 39.
FIG. 21 shows Kaplan-Maier survival curves demonstrating the effect of survival following treatment with the indicated NPs in the in-vivo disseminated LYl9Gluc DLBLC xenograft mouse model as compared to untreated control. The log-rank test was used to compare the percent animal survival between treatment groups.
FIGs. 22A-C demonstrate the effect on tumor growth following treatment with the indicated NPs in the in-vivo disseminated LYl9Gluc DLBLC xenograft mouse model as compared to untreated and vehicle controls. Tumor burden was monitored by quantification of tumor-derived gaussia lucif erase- activity. Figure 22A shows representative luminescent images showing amount and location of lymphoma cells in control and treated mice. Figure 22B is a bar quantitating Glue activity as a measurement of lymphoma burden. Figure 22C demonstrate lymphoma burden in mice as determined by Gaussia luciferase (Glue) activity in 5 pl of serum.
FIG. 23 demonstrates the effect on BM infiltration of LY l9Gluc cells following treatment with the indicated NPs in the in-vivo disseminated LYl9Gluc DLBLC xenograft mouse model as compared to untreated and vehicle controls. Shown are bar graphs demonstrating quantitation of Glue activity in the BM (left) and flow cytometry analysis of CD 19 and CD20 expression in bone marrow cells collected from mice femur (right).
FIG. 24 shows immunohistochemistry analysis using anti-CD20 staining of mice spinal cord and brain tissues demonstrating the extent of infiltration of lymphoma cells to the CNS following treatment with the indicated NPs in the in-vivo disseminated LYl9Gluc DLBLC xenograft mouse model as compared to untreated controls.
FIG. 25 demonstrates that conjugation of CD40L to PLGA and MTV-NPs maintains CD40L biological activity. OCI-Lyl9 cells were stably transfected with a luciferase reporter construct joined to tandem repeats of the NF-kB response element (OCI-Lyl9-NFKB-Luc). These cells were treated for 24 hours with NPs (PLGA), NPs conjugated to CD40L (CD40L- NPs) or CD40L and mtLivin (MTV-CD40L-NPs), or free CD40L (0.25 pg / ml). NF-KB luciferase activity was determined by Tecan reader. CD40L, both free and conjugated to NPs, increased luciferase activity compared to controls.
FIG. 26 is a bar graph demonstrating cell death of LY 19 DLBCL cells following 24 hours incubation with CD40L-mtLivin (MTV) NPs as compared to the indicated controls, as determined by Annexin V staining and flow cytometry analysis.
FIG. 27 is a bar graph demonstrating IFNy levels in the serum of mice 17 days following IV injection of lymphoma cells and treatment with the indicated NPs, as determined by ELISA.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to compositions and methods for treating cancer resistant to an anti-cancer agent.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
The development of drug resistance is a major obstacle to successful cancer therapy. Great efforts have focused on the underlying mechanisms that turn promising targeted therapies which induce initial tumor shrinkage ineffective following few months, resulting in refractory or unbeatable cancers.
The Inhibitor of Apoptosis Protein (IAP) family of proteins has been shown to inhibit apoptosis induced by a variety of stimuli mainly by binding and inhibiting specific caspases. Eight human IAPs have been identified to date: c-IAPl, C-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2 and Livin. Livin, a member in the IAP family of proteins, is trimmed following apoptotic stimuli to form a truncated C-terminal Livin referred to as tLivin which is not only devoid of Livin anti- apoptotic activity but also acquires a pro-apoptotic effect. A 70 amino acids derivative of tLivin [denoted herein as mini-tLivin (mtLivin)] was identified which remarkably demonstrates a pro-apoptotic activity as potent as tLivin [Nachmias B et al. apoptosis. 2007; 12(7): 1129- 1142]
As is illustrated hereinunder and in the Examples section, which follows, the present inventors show that free mtLivin or mtLivin conjugated to PLGA nanoparticles induced tumor cells death in-vitro (Examples 1, Figures 1A-5, 10A-C and 12). Following, the inventors optimized the PFGA nanoparticles loaded with mtFivin to obtain nanoparticles with increased activity and stability (Examples 2-3, Figures 6-9B). Moreover, PFGA nanoparticles loaded with mtFivin in combination with a targeting moiety (e.g. CD40F) or in combination with doxorubicin had an improved effect on tumor cells (DFBCF) death/growth in-vitro and in-vivo as compared to a single component treatment (Examples 4-5, Figures 11 and 13-27).
In addition, the present inventors show that administration of mtFivin conjugated to PFGA nanoparticles increased the amount of platelets in-vivo (Example 6).
Thus, according to a first aspect of the present invention, there is provided a method of treating cancer exhibiting a resistance to an anti-cancer agent in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of tFivin, thereby treating the cancer exhibiting resistance to the anti-cancer agent in the subject.
According to an additional or an alternative aspect of the present invention, there is provided a composition comprising tFivin for use in the treatment of cancer resistant to an anti cancer agent.
The term“treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition e.g. cancer) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
As used herein, the term“preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
As used herein, the term“subject” includes mammals, preferably human beings at any age which suffer from the pathology. In a specific embodiment, this term encompasses individuals who are at risk to develop the pathology.
According to specific embodiments, the subject is diagnosed with thrombocytopenia.
According to specific embodiments, the subject suffers from or is at a risk of platelet reduction associated with exposure to radiation or drug or chemical e.g. chemotherapy.
According to specific embodiments, the subject has cancer.
Cancers which may be treated by some embodiments of the invention can be any solid or non-solid cancer and/or cancer metastasis. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but not limited to, tumors of the gastrointestinal tract (colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small and/or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors), endometrial carcinoma, dermatofibro sarcoma protuberans, gallbladder carcinoma, Biliary tract tumors, prostate cancer, prostate adenocarcinoma, renal cancer (e.g., Wilms’ tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cells tumor, immature teratoma of ovary, uterine, epithelial ovarian, sacrococcygeal tumor, choriocarcinoma, placental site trophoblastic tumor, epithelial adult tumor, ovarian carcinoma, serous ovarian cancer, ovarian sex cord tumors, cervical carcinoma, uterine cervix carcinoma, small-cell and non- small cell lung carcinoma, nasopharyngeal, breast carcinoma (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic ; breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer- 1, breast cancer-3; breast-ovarian cancer), squamous cell carcinoma (e.g., in head and neck), neurogenic tumor, astrocytoma, ganglioblastoma, neuroblastoma, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, B cell, Burkitt, cutaneous T cell, histiocytic, lymphoblastic, T cell, thymic), gliomas, adenocarcinoma, adrenal tumor, hereditary adrenocortical carcinoma, brain malignancy (tumor), various other carcinomas (e.g., bronchogenic large cell, ductal, Ehrlich-Lettre ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinocellular, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependimoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblast), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., multiforme, astrocytoma), glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), hypernephroma, insulinoma, islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphatic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute - megakaryoblastic, monocytic, acute myelogenous, acute myeloid, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, Friend, granulocytic or myelocytic, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic- macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition to myeloid malignancy, acute nonlymphocytic leukemia), lymphosarcoma, melanoma, mammary tumor, mastocytoma, medulloblastoma, mesothelioma, metastatic tumor, monocyte tumor, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's, histiocytic cell, Jensen, osteogenic, reticulum cell), schwannoma, subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma, testicular tumor, thymoma and trichoepithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme; multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, lynch cancer family syndrome II, male germ cell tumor, mast cell leukemia, medullary thyroid, multiple meningioma, endocrine neoplasia myxosarcoma, paraganglioma, familial nonchromaffin, pilomatricoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, familial, rhabdoid tumors, soft tissue sarcoma, and Turcot syndrome with glioblastoma.
According to specific embodiments, the cancer is selected from the group consisting of melanoma, lymphoma, multiple myeloma, chronic lymphocytic leukemia (CLL), lung cancer and prostate cancer.
According to a specific embodiment, the cancer is diffused large B-cell lymphoma (DLBCL).
According to specific embodiments, the cancer exhibits resistance to an anti-cancer agent.
As used herein, the phrase“resistance to an anti-cancer agent” or“resistant to an anti cancer agent” refers to non-responsiveness to an anti-cancer treatment as may be manifested by tumor size, in-vitro activity assays and/or patient survival.
According to a specific embodiment, resistance refers to no amelioration in disease symptoms or progression according to a regulatory agency guidelines (e.g., FDA) for the specific anti-cancer agent used. Resistance to treatment can be primary resistance or acquired resistance.
As used herein the term“acquired resistance” refers to progression of resistance following initial positive response to therapy.
According to specific embodiments, the method comprising administering to the subject at least one anti-cancer agent. According to specific embodiments, the anti-cancer agent administered to the subject is the agent which the cancer exhibits resistance to.
As used herein, the phrase“anti-cancer agent” refers to any therapeutic agent that has an anti-tumor effect including, but not limited to, chemotherapy, small molecules, biological drugs, hormonal therapy, antibodies and targeted therapy.
Non-limiting examples of such anti-cancer agents include, but are not limited to: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- I a; Interferon Gamma- I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).
Non-limiting examples for anti-cancer approved drugs include: abarelix, aldesleukin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, AZD4547, AZD2281, bevacuzimab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dactinomycin, actinomycin D, Darbepoetin alfa, Darbepoetin alfa, daunorubicin liposomal, daunorubicin, decitabine, Denileukin diftitox, dexrazoxane, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, Elliott's B Solution, epirubicin, Epoetin alfa, erlotinib, estramustine, etoposide, exemestane, Filgrastim, floxuridine, fludarabine, fluorouracil 5-FU, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, hydroxyurea, Ibritumomab Tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, Interferon alfa- 2b, irinotecan, lenalidomide, letrozole, leucovorin, Leuprolide Acetate, levamisole, lomustine, CCNU, meclorethamine, nitrogen mustard, megestrol acetate, melphalan, L-PAM, mercaptopurine 6-MP, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, Nofetumomab, Oprelvekin, Oprelvekin, oxaliplatin, paclitaxel, palbociclib palifermin, pamidronate, pegademase, pegaspargase, Pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin mithramycin, porfimer sodium, procarbazine, quinacrine, Rasburicase, Rituximab, sargramostim, sorafenib, streptozocin, sunitinib maleate, tamoxifen, temozolomide, teniposide VM-26, testolactone, thioguanine 6-TG, thiotepa, thiotepa, topotecan, toremifene, Tositumomab, Trametinib, Trastuzumab, tretinoin ATRA, Uracil Mustard, valrubicin, vinblastine, vinorelbine, zoledronate and zoledronic acid.
According to specific embodiments, the anti-cancer agent is selected from the group consisting of Gefitinib, Lapatinib, Afatinib, BGJ398, CH5183284, Linsitinib, PHA665752, Crizotinib, Sunitinib, Pazopanib, Imatinib, Ruxolitinib, Dasatinib, BEZ235, Pictilisib, Everolimus, MK-2206, Trametinib / AZD6244, Vemurafinib / Dabrafenib, CCT196969 / CCT241161, Barasertib, VX-680, Nutlin3, Palbociclib, BI 2536, Bardoxolone, Vorinostat, Navitoclax (ABT263), Bortezomib, Vismodegib, Olaparib (AZD2281), Simvastatin, 5- Fluorouricil, Irinotecan, Epirubicin, Cisplatin and Oxaliplatin.
According to specific embodiments, the anti-cancer agent is selected from the group consisting of IMiDs (e.g. Revlimid, Thalidomide, Pomalidomide), proteasome inhibitors (e.g. Velcade, Carfilzomib), Rituximab, fludarabine and Bendamustine.
According to specific embodiments, the anti-cancer agent is selected from the group consisting of DAC, Doxorubicin, Dexamethasone, Etoposide, Mechloretamine, Methotrexate, Ibrutinib, R-CHOP (e.g. rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin [vincristine], prednisone), EPOCH-R (e.g. Rituximab, etoposide, prednisolone, oncovin, [vincristine], cyclophosphamide, hydroxydaunorubicin), Lenalidomide (inhibiting NF-KB signaling), inhibitors of the PI3K pathway and BCL2 family antagonists (e.g. navitoclax, ABT- 199).
According to specific embodiments, the anti-cancer agent is doxorubicin.
According to specific embodiments, the anti-cancer agent is lipophilic.
Thus, anti-cancer agents which can be used with specific embodiments of the present invention can be, but not limited to, a lipophilic derivative of any of the anti-cancer agents described herein.
According to specific embodiments, the anti-cancer agent is an apoptotic agent.
According to other specific embodiments, the anti-cancer agent is not an apoptotic agent.
As used herein the term“apoptotic agent” refers to an agent which activates a signal transduction or induces a cellular damage leading to apoptosis of the cell it binds to. Methods of detecting apoptosis are well known in the art and includes e.g. Annexin V staining, TUNEL assay, caspase activity assays, mitochondrial viability assays and/or expression of apoptotic genes e.g. BAX, Bcl2, caspases.
According to specific embodiments, the anti-cancer agent is an immunomodulatory molecule. As used herein the term“immunomodulatory molecule" refers to a molecule that binds an immune-check point protein. Such molecules include, but are not limited to antibodies, small molecules, polypeptides and the like.
According to specific embodiments, the immunomodulatory molecule modulates the activity of one or more immune-check point proteins in an agonistic or antagonistic manner resulting in recruitment of an immune cell to elicit an immune activity against a cancer cell.
As used herein the term "immune-check point protein" refers to an antigen independent protein that modulates an immune cell response (i.e. activation or function). Immune-check point proteins can be either co- stimulatory proteins [i.e. positively regulating an immune cell activation or function by transmitting a co-stimulatory secondary signal resulting in activation of an immune cell] or inhibitory proteins (i.e. negatively regulating an immune cell activation or function by transmitting an inhibitory signal resulting in suppressing activity of an immune cell). According to specific embodiments, the immune-check point protein regulates activation or function of a T cell. Numerous checkpoint proteins are known in the art and include, but not limited to, PD1, PDL-l, CTLA-4, CD80, LAG-3, TIM-3, KIR, IDO, 0X40, OX40L, CD 137 (4- 1BB), 4-1BBL, CD27, CD70, CD40, CD40L, GITR, CD28, CD86, and ICOS (CD278), ICOSL.
Non-limiting examples of clinically approved immunomodulation agents which can be used with specific embodiments of the present invention include ipiliumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda) and atezolizumab (Tecentriq).
According to specific embodiments, the immune-check point protein is CD40 protein.
According to a specific embodiment the CD40 protein refers to the human protein, such as provided in the following GenBank Number NP_00l24l.
According to a specific embodiment, the molecule which binds CD40 is the naturally occurring ligand (e.g. CD40L) or a functional derivative or variant thereof which retains the ability to specifically bind to CD40.
According to one embodiment CD40L is human CD40L.
According to specific embodiments the CD40 binding molecule comprises a CD40L polypeptide.
According to specific embodiments, CD40L polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 9.
According to specific embodiments, CD40L polypeptide is a functional CD40L homologue (e.g. fragment or derivative thereof), which exhibit the desired activity {i.e., binding CD40). Such homologues can be, for example, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NO: 9.
According to specific embodiments, the CD40L polypeptide is a soluble polypeptide.
According to specific embodiments, the CD40L polypeptide is a recombinant human polypeptide. Such recombinant polypeptides can be commercially obtained from e.g. Peprotech.
According to specific embodiments, the CD40 binding molecule comprises an anti-CD40 antibody.
The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VF) that are capable of binding to an epitope of an antigen.
Methods of producing polyclonal and monoclonal antibodies, human and humanized antibodies, as well as fragments thereof are well known in the art (See for example, Harlow and Fane, Antibodies: A Faboratory Manual, Cold Spring Harbor Faboratory, New York, 1988, incorporated herein by reference).
Numerous anti-CD40 antibodies are known in the art and include, but not limited to CP- 870,893 (Pfizer), SGN-40 (Seattle Genetics), ENZ-ABS 148-0100 (Enzo Fife Sciences, Inc), 334303(BioFegend), 3072 (BioVision), 130-094-133 (Miltenyi Biotec), MA5-15535 (Invitrogen Antibodies).
Fivin (also known as baculoviral IAP repeat-containing 7; BIRC7, KIAP, MF-IAP and Fivin inhibitor-of-apoptosis) encoded by the BIRC7 gene, is a member in the anti-apoptotic IAP family of proteins. Fivin contains a single baculovirus IAP repeats (BIR) domain at the N- terminus and a carboxy-terminal RING domain. Human Fivin encodes two highly similar splicing variants, termed Fivin a and b that differ only in 18 amino acids located between the BIR and the RING domains, which are present in the a but not in the b isoform. Following apoptotic stimuli, both Fivin isoforms a and b undergo a specific proteolytic cleavage that trims the 52 amino acids at the N-terminus of Fivin thereby forming a truncated form of Fivin which are collectively referred to as tFivin.
As used herein“tFivin” refers to p30-Fivin a and/or p28-Fivin b polypeptide or a polynucleotide encoding same; or functional homologs e.g., functional fragments thereof. A functional derivative or fragment of p30-Fivin a and/or p28-Fivin b polypeptide is able to induce apoptosis. Assay for testing apoptosis are well known in the art and include e.g. Annexin V staining, TUNEL assay, propidium iodide (PI) and/or caspase expression or activity.
According to one embodiment tLivin is human tLivin.
According to specific embodiments tLivin is p30-livin a, SEQ ID NO: 1 or 2 disclosed e.g. in U.S Patent No. 7,517,949.
According to yet other specific embodiments tLivin can be p28-Livin b, SEQ ID NO: 3 or 4 disclosed e.g. in U.S Patent No. 7,517,949.
The term“tLivin” also refers to functional tLivin homologues (naturally occurring or synthetically/recombinantly produced), which exhibit the desired activity {i.e., induction of apoptosis). Such homologues can be, for example, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NOs: 2 and/or 4 or 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical to the polynucleotide sequence encoding same.
Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, and MUSCLE.
According to specific embodiments, tLivin is mtLivin, SEQ ID NO: 5 or 6.
According to specific embodiments, tLivin comprises a RING domain [i.e. a domain having a consensus sequence of C-X2-C-X[9-39]-C-X[i-3]-H-X[2-3]-C-X2-C-X[4 8]-C-X2-C; where: C is a conserved cysteine residue involved zinc coordination, H is a conserved histidine involved in zinc coordination, Zn is zinc atom, and X is any amino acid residue] .
According to specific embodiments, tLivin is a tLivin polynucleotide.
As used herein the term“polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
Exemplary nucleic acid sequences encoding Livin which can be used in accordance with the present teachings include, but are not limited to, SEQ ID NOs: 1, 3, 5, or 7.
To express exogenous Livin in mammalian cells, the polynucleotide sequence encoding Livin is preferably ligated into a nucleic acid construct suitable for mammalian cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
The nucleic acid construct (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this expression vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
According to a specific embodiment, the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed. Examples of cell type-specific promoters include promoters such as GPl lb (also known as CD41, Itga2b) promoter that is specifically expressed in MKs (Denarier, E. et al. (1993) Biochem. Biophys. Res. Commun. 30; 195(3): 1360-4), WASP and CD45 promoters that are specifically expressed in hematopoietic cells (Franco, A. Ballabio, et al. (1998) Blood, 91: 4554- 4560, and J F DiMartino, et al. (1994) International Immunology, 6(8): 1279-83, respectively), and CD34 promoter that is specifically expressed in hematopoietic stem cells and progenitors (Bum TC et al. (1992) Blood, 80(l2):305l-9).
In addition to the elements already described, the expression vector of some embodiments of the invention may contain enhancer elements, Polyadenylation sequences, eukaryotic replicon or other specialized elements.
The expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
Examples for mammalian expression vectors include, but are not limited to pcDNA3, pcDNA3.l(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.l, pSinRep5, DH26S, DHBB, pNMTl, pNMT4l, pNMT8l, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide.
The Livin polynucleotide of some embodiments of the invention can be introduced into cells by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et ah, [Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992)]; Ausubel et ah, [Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989)]; Chang et ah, [Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995)]; Vega et ah, [Gene Targeting, CRC Press, Ann Arbor MI (1995)]; Vectors [A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988)] and Gilboa et al. [Biotechniques 4 (6): 504-512 (1986)] and include, for example, stable or transient transfection, electroporation and infection with recombinant viral vectors.
According to a specific embodiment, the Livin polynucleotide is expressed from a viral vector in which case the cells are infected with the virus, as further described hereinbelow. Examples for viral vector include, but are not limited to pWZL-blast which is available, for example, from Addgene.
Alternatively or additionally, tLivin is a tLivin polypeptide.
The term "polypeptide" as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.
Further details in this respect are provided hereinunder.
Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N- methylated amide bonds (-N(CH3)-CO-), ester bonds (-C(=0)-0-), ketomethylene bonds (-CO- CH2-), sulfinylmethylene bonds (-S(=0)-CH2-), a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl (e.g., methyl), amine bonds (-CH2-NH-), sulfide bonds (-CH2-S-), ethylene bonds (- CH2-CH2-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), fluorinated olefinic double bonds (-CF=CH-), retro amide bonds (- NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally present on the carbon atom.
These modifications can occur at any of the bonds along the polypeptide chain and even at several (2-3) bonds at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted by non-natural aromatic amino acids such as l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O- methyl-Tyr.
The polypeptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
The term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.
Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with the present invention.
Table 1
Figure imgf000023_0001
Figure imgf000024_0001
Table 2
Figure imgf000024_0002
Figure imgf000025_0001
Figure imgf000026_0001
Since the present polypeptides are preferably utilized in therapeutics which require the peptides to be in soluble form, the polypeptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
The tLivin polypeptides of some embodiments of the invention may be synthesized by any techniques known to those skilled in the art of peptide synthesis, for example but not limited to recombinant DNA techniques or solid phase peptide synthesis.
According to specific embodiments, tLivin is administered in a formulation comprising a targeting moiety, as further described hereinbelow.
According to some embodiments, tLivin and/or the targeting moiety is attached to or encapsulated in a cell penetrating agent and/or a stabilizing agent.
Thus, according to an aspect of the present invention, there is provided a composition comprising tLivin and a targeting moiety, wherein said tLivin is attached to or encapsulated in a cell penetrating agent and/or a stabilizing agent. Attaching or encapsulating tLivin and/or the targeting moiety to or into a cell penetrating agent and/or a stabilizing agent may be effected by any of the methods known in the art e.g. the methods disclosed in Liposome Technology, Vol. II, Incorporation of Drugs, Proteins, and Genetic Material, CRC Press; Monkkonen, J. el al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145; Lasic D D., Liposomes Technology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M, Lasic D D, Chem Phys Lipids, 1993 September;64(l-3):35-43; International Patent Application Publication nos. W02007034479, WO2013042125, W02012101638 and W02012101639. According to specific embodiments, the cell penetrating agent and/or the stabilizing agent is selected from the group consisting of a nanoparticle, a liposome, a viral vector, a cell penetrating peptide and poly(alkylene) glycols.
According to specific embodiments, the cell penetrating agent and/or the stabilizing agent is a nanoparticle.
As used herein, the term "nanoparticle" refers to a particle or particles having an intermediate size between individual atoms and macroscopic bulk solids. Generally, a nanoparticle has a characteristic size (e.g., diameter for generally spherical nanoparticles, or length for generally elongated nanoparticles) in the sub-micrometer range, e.g., from about 1 nm to about 1000 nm, from about 1 nm to about 500 nm, or from about 1 nm to about 200 nm, or of the order of 10 nm, e.g., from about 1 nm to about 100 nm.
According to specific embodiments, the nanoparticle has a diameter of 50 - 250 nm
According to specific embodiments, the nanoparticle has a diameter of 10 - 100 nm.
The nanoparticles may be of any shape, including, without limitation, elongated particle shapes, such as nanowires, or irregular shapes, in addition to more regular shapes, such as generally spherical, hexagonal and cubic nanoparticles. According to one embodiment, the nanoparticles are generally spherical.
The particles of this aspect of the present invention may have a charged surface (i.e., positively charged or negatively charged) or a neutral surface.
Agents which are used to fabricate the particles may be selected according to the desired charge required on the outer surface of the particles.
According to specific embodiments, the nanoparticles comprise polymers such as, but not limited to polyethylene glycol (PEG), polysialic acid, polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), poly-(lactic-co-glycolic)poly-(vinyl-alcohol), polyvinylpyrrolidone, polyethyloxazoline, polyllydroxyetlyloxazolille, solyhydroxypryloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
According to specific embodiments, the nanoparticle comprises poly(lactide-co- glycolide), polylactide (PLA), polyglycolide, polylactide-polyglycolide, and/or polyethylene glycol-co-lactide (PEG-PLA).
According to specific embodiments, the nanoparticle comprises poly(lactide-co- glycolide). Hence, according to an aspect of the present invention, the is provided a composition comprising tLivin attached to or encapsulated in a nanoparticle comprising poly(lactide-co- glycolide).
As used herein, the term“poly(lactide-co-glycolide)” describes Poly(D,L-lactide-co- glycolide) PLGA, CAS NO. 26780-50-7.
According to specific embodiments, the concentration of the poly(lactide-co-glycolide) is at least 0.01, at least 0.02, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 2 mg ml.
According to specific embodiments, the concentration of the poly(lactide-co-glycolide) is up to 10, up to 8, up to 7, up to 6, up to 5 mg / ml.
According to specific embodiments, the concentration of the poly(lactide-co-glycolide) is 0.05 to 5 mg / ml.
The nanoparticles may also include other components. Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or cholesterol esters or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the biologically active lipid into the lipid assembly. Examples of sterols include cholesterol, cholesterol hemisuccinate, cholesterol sulfate, or any other derivatives of cholesterol. Preferred lipid assemblies according the invention include either those which form a micelle (typically when the assembly is absent from a lipid matrix) or those which form a liposome (typically, when a lipid matrix is present).
The nanoparticles of the present invention may be modified to enhance their circulatory half-life (e.g. by PEGylation) to reduce their clearance, to prolong their scavenging time-frame and to allow antibody binding. The PEG which is incorporated into the particles may be characterized by of any of various combinations of chemical composition and/or molecular weight, depending on the application and purpose.
According to specific embodiments, the nanoparticles are prepared as described in International Patent Application Publication nos. W02007034479, WO2013042125,
W02012101638; Karra & Benita. Curr Drug Metab. (2012) 13:22-41; Karra et al Curr Cancer Drug Targets. (2013) 13: 11-29; and/or Karra et al. Small (2013) 9(24): 4221-36, the contents of which are fully incorporated herein by reference.
According to specific embodiments, the cell penetrating agent and/or the stabilizing agent is a liposome.
Liposomes include any synthetic (i.e., not naturally occurring) structure composed of lipid bilayers, which enclose a volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be of different sizes, may contain a low or a high pH and may be of different charge. The liposomes may be prepared by any of the known methods in the art [Monkkonen, J. el al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145; Lasic D D., Liposomes Technology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M, Lasic D D, Chem Phys Lipids, 1993 September;64(l-3):35-43]. The liposomes may be positively charged, neutral or negatively charged.
The liposomes may be a single lipid layer or may be multilamellar.
Suitable liposomes in accordance with the invention are non-toxic liposomes such as, for example, those prepared from phosphatidyl-choline phosphoglycerol, and cholesterol. The diameter of the liposomes used can range from 0.1-1.0 microns. However, other size ranges may also be used. For sizing liposomes, homogenization may be used, which relies on shearing energy to fragment large liposomes into smaller ones. Homogenizers which may be conveniently used include microfluidizers produced by Microfluidics of Boston, MA. In a typical homogenization procedure, liposomes are recirculated through a standard emulsion homogenizer until selected liposomes sizes are observed. The particle size distribution can be monitored by conventional laser beam particle size discrimination. Extrusion of liposomes through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is an effective method for reducing liposome sizes to a relatively well defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller pore membranes to achieve a gradual reduction in liposome size.
According to specific embodiments, the cell penetrating agent and/or the stabilizing agent is a viral vector.
Viral vectors offer several advantages including higher efficiency of transformation, and targeting to, and propagation in, specific cell types. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through specific cell receptors.
Retroviral vectors represent one class of vectors suitable for use with some embodiments of the invention. Protocols for producing recombinant retroviruses and for infecting cells in- vitro or in-vivo with such viruses can be found in, for example, Ausubel et al., [eds, Current Protocols in Molecular Biology, Greene Publishing Associates, (1989)]. Other suitable expression vectors may be an adenovirus, a lentivirus, a Herpes simplex I virus or adeno- associated virus (AAV). Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.
According to specific embodiments, the cell penetrating agent and/or the stabilizing agent is a cell penetrating peptide.
Cell-Penetrating Peptides (CPPs) are short peptides (<40 amino acids), with the ability to gain access to the interior of almost any cell. They are highly cationic and usually rich in arginine and lysine amino acids. They have the exceptional property of carrying into the cells a wide variety of covalently and noncovalently conjugated cargoes such as proteins, oligonucleotides, and even 200 nm liposomes. Therefore, according to additional exemplary embodiment CPPs can be used to transport tLivin to the interior of cells.
TAT (transcription activator from HIV-l), pAntp (also named penetratin, Drosophila antennapedia homeodomain transcription factor) and VP22 (from Herpes Simplex virus) are examples of CPPs that can enter cells in a non-toxic and efficient manner and may be suitable for use with some embodiments of the invention. Protocols for producing CPPs-cargos conjugates and for infecting cells with such conjugates can be found, for example L Theodore et al. [The Journal of Neuroscience, (1995) 15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994) 91:664-668], and Jing Bian et al. [Circulation Research. (2007) 100: 1626- 1633]
According to specific embodiments, the cell penetrating agent and/or the stabilizing agent is poly(alkylene) glycols.
As used herein, the term“alkylene glycol” describes a -[0-(CR’R”)z]y- group, with R’ and R” being as defined herein (and/or as defined herein for Ri and R2), and with z being an integer of from 1 to 10, preferably, from 2 to 6, more preferably 2 or 3, and y being an integer of 1 or more. Preferably R’ and R” are both hydrogen. When z is 2 and y is 1, this group is ethylene glycol. When z is 3 and y is 1, this group is propylene glycol. When y is greater than 1, this group is also referred to herein as“alkylene glycol chain”.
When y is greater than 4, the alkylene glycol chain is also referred to herein as poly(alkylene glycol) moiety. In some embodiments of the present invention, a poly(alkylene glycol) moiety can have from 2 to 10 alkylene glycol groups, such that y is, for example, 2 to 10, or from 2 to 8, or from 2 to 6, or from 3 to 4.
According to specific embodiments, the poly( alkylene) glycol is polyethylene glycol
(PEG). According to specific embodiments, the tLivin is attached to an inner surface of the cell penetrating agent and/or the stabilizing agent (e.g. nanoparticle).
According to other specific embodiments, the tLivin is attached to an outer surface of the cell penetrating agent and/or the stabilizing agent (e.g. nanoparticle).
According to other specific embodiments, the targeting moiety is attached to an outer surface of the cell penetrating agent and/or the stabilizing agent (e.g. nanoparticle).
The attachment can be covalent or non-covalent attachment.
Conjugation methods which can be used in accordance with some embodiments of the present invention can be divided to direct binding or indirect binding. Some methods are provided hereinbelow and are summarized in a review by Karra N and Benita S, Curr Drug Metab. (2012) 13:22-4.
Thus, for example, non-covalent approaches include adsorption of the ligand/ Ab to the surface of the NPs, and Biotin- Avidin complexes.
Non-limiting examples of covalent approaches include amide linkage- activation of the end groups of carboxyl terminated PLA and PLGA by a carbodiimide that will result in an active ester intermediate that can be coupled to the amine functional groups of an antibody by carbodiimide chemistry, and thioether linkage- The reaction between thiol functional groups and maleimide groups is highly efficient and leads to stable thioether bonds. Such a linkage may be formed between e.g. maleimide-bearing NPs and thiol bearing antibodies and ligands. Alternatively, thiol-surface activated NPs may also react with maleimide-activated antibodies.
According to specific embodiments, the attachment is covalent attachment.
According to specific embodiments, the tLivin is directly attached to the cell penetrating agent and/or the stabilizing agent (e.g. nanoparticle).
According to other specific embodiments, the tLivin is attached to the cell penetrating agent and/or the stabilizing agent (e.g. nanoparticle) via a linker.
Any linker known in the art can be used with some embodiments of the invention, such as, but not limited to, OCA, SMCC, Antibody-drug conjugation; Sulfo-KMUS, Antibody- liposome conjugation.
According to specific embodiments, the linker comprises an oleyl cysteineamide (OCA).
As used herein, the term“oleyl cysteineamide (OCA)”, CAS NO. 67603-51-4. describes a derivative of oleic acid functionalized with a polar thiol group contributed by cysteine, Molecular Formula C21H39NO3S. Loading of the particle with tLivin, the anti-cancer agent or the targeting moiety can be effected concomitant with, or following particle assembly.
According to specific embodiments, attaching tLivin and/or the targeting moiety to the cell penetrating agent and/or the stabilizing agent is effected by the methods disclosed in the Examples section which follows which serve as an integral part of the specifications.
According to specific embodiments, the tLivin is in a concentration of 0.25-0.5 mg / ml.
According to specific embodiments, the concentration of tLivin is 0.001 - 10 mg, 0.01 - 10 mg, 0.05 - 1 mg or 0.05 - 1 mg conjugated to PLGA NPs via an oleyl cysteineamide (OCA) linker / ml.
According to specific embodiments, the concentration of poly(lactide-co-glycolide) is 0.001 - 10 mg / ml, 0.01 - 10 mg, 0.05 to 5 mg / ml or 0.05 - 1 mg /ml.
According to specific embodiments, the ratio between tLivin and poly(lactide-co- glycolide) is 0.05 - 1 mg tLivin and 0.1 - 2.5 mg poly(lactide-co-glycolide).
According to specific embodiments, the concentration of the linker (e.g. OCA) is 0.001 - 10 mg / ml, 0.01 - 10 mg, 0.05 -5 mg/ml or 0.05 - 1 mg /ml.
According to specific embodiments, the ratio between the linker and tLivin is 0.1 : 1 to 1:0.1 w / w.
According to specific embodiments, the nanoparticle has a diameter of 50 - 800 nm, 50 - 400 nm or 50 - 250 nm.
According to specific embodiments, the concentration of the targeting moiety is 0.01 - 20 mg / ml, 0.01 - 10 mg / ml, 0.05 - 5 mg / ml or 0.05 - 2 mg / ml.
According to specific embodiments, the targeting moiety is in a concentration of 0.25-0.5 mg / ml.
According to specific embodiments, the ratio between the tLivin and the targeting moiety is from 0.01:1 to 1: 0.01.
According to specific embodiments, the composition comprising tLivin disclosed herein comprises an anti-cancer agent. Non-limiting examples of anti-cancer agents that can be used with specific embodiments are disclosed hereinabove. According to specific embodiments, the concentration of the anti-cancer agent is 0.01 - 100 mg, 0.01 - 10 mg or 0.05 - 5 mg.
According to specific embodiments, the composition comprising tLivin disclosed herein comprises a cryo-protectant.
Non-limiting examples of cryo-protectants that can be used with some embodiments of the present invention include, trehalose, b-cyclodextrine, sucrose, mannitol, xylitol and fructose. According to specific embodiments, the cryo -protectant comprises trehalose and/or b- cyclodex trine.
According to specific embodiments, the cryo-protectant is at a concentration of 0.1 - 100 mg / ml, 0.5 - 50 mg/ ml, 1 - 25 mg / ml or 2 - 20 mg /ml.
The term“targeting moiety”, as used herein, relates to a functional group which serves to target or direct the tLivin or the composition comprising same described herein to a cancer cell. Such targeting moieties include, but are not limited to antibodies, cell surface receptor, ligands, hormones, lipids, sugars and dextrans.
According to specific embodiments, the targeting moiety induces internalization of tLivin into the cancer cell.
Non-limiting examples for known cancer antigens which the targeting moiety can bind include MAGE-AI, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A7, MAGE-AS, MAGE-A9, MAGE-AIO, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-l, RAGE- 1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE- C1/CT7, MAGE-C2, NY-ES0-1, LAGE-l, SSX-l, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX- 5, SCP-l and XAGE, melanocyte differentiation antigens, p53, ras, CEA, MUCI, HER2 peptide, PMSA, PSA, tyrosinase, Melan-A, MART-I, gplOO, gp75, alphaactinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-l, dek-can fusion protein, EF2, ETV6- AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomerase, GnTV, Herv-K-mel, NA-88, SP17, and TRP2- 2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE- 5, MAGE-6, pl85erbB2, plSOerbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, alpha.-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29YBCAA), CA 195, CA 242, CA-50, CAM43, CD68\KPl, CO-029, FGF-5, 0250, Ga733 (EpCAM), HTgp-l75, M344, MA-50, MG7-Ag, MOV18, NBU70K, NYCO-I, RCASI, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, tyrosinase related proteins, TRP-l, or TRP-2.
According to specific embodiments, the targeting moiety is selected from the group consisting of CD19, CD20, CD38, CD138, EGFR, Her-2 and PMSA binding molecule. According to specific embodiments, the cancer antigen is an immune check-point molecule.
Thus, according to specific embodiments, the targeting moiety is an immunomodulatory molecule.
Examples of immune check-point molecules and their binding immunomodulatory molecules that can serve as a targeting moiety are provided hereinabove.
According to specific embodiments, the targeting moiety is a CD40 and/or PD-l binding molecule.
According to specific embodiments, the targeting moiety is a CD40 binding molecule.
Thus, according to an aspect of the present invention, there is provided a composition comprising tLivin and a CD40 binding molecule.
Alternatively, or additionally, a tumor antigen may be identified using cancer cells obtained from the subject by e.g. biopsy. For example, a method as described herein may comprise the step of identifying a tumor antigen which is displayed by one or more cancer cells in a sample obtained from the subject.
As is illustrated in the Examples section, which follows, the present inventors show that compositions comprising mtLivin had in-vitro and in-vivo anti-tumor effects and also increased the number or platelets in-vivo (Examples 1-6).
Thus, according to an aspect of the present invention, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition comprising tLivin disclosed herein, thereby treating the cancer in the subject.
According to an alternative or an additional aspect of the present invention, there is provided the composition comprising tLivin disclosed herein for use in the treatment of cancer.
According to an alternative or an additional aspect of the present invention, there is provided a method of increasing an amount of platelets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition comprising tLivin disclosed herein, thereby increasing the amount of platelets in the subject.
According to an alternative or an additional aspect of the present invention, there is provided a method of treating thrombocytopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition comprising tLivin disclosed herein, thereby treating thrombocytopenia in the subject. According to an alternative or an additional aspect of the present invention, there is provided the composition comprising tLivin disclosed herein for use in the treatment of thrombocytopenia.The tLivin, compositions comprising same and the anti-cancer agents of the present invention can be administered to the subject per se or as a part of a pharmaceutical composition.
The tLivin or the compositions comprising same of some embodiments of the invention can be administered to a subject in combination with other established (e.g. gold standard) or experimental therapeutic regimen for the treatment of e.g. cancer including, but not limited to chemotherapy, radiotherapy, phototherapy and photodynamic therapy, surgery, nutritional therapy, ablative therapy, combined radiotherapy and chemotherapy, brachiotherapy, proton beam therapy, immunotherapy, cellular therapy and photon beam radiosurgical therapy, hormonal therapy and targeted therapy.
According to specific embodiments, the tLivin and/or the compositions comprising same of the present invention are not administered to the subject with an additional anti-cancer agent. According to specific embodiments, the tLivin and/or the compositions comprising same of the present invention are not administered to the subject with doxorubicin.
According to specific embodiments, the composition comprising tLivin of the present invention do not comprise (e.g. encapsulate) an anti-cancer agent (e.g. doxorubicin).
The tLivin or the compositions comprising same of some embodiments of the invention can be administered to a subject in combination with an anti-thrombocytopenia therapy e.g. platelet production stimulating factor e.g. thrombopoietin (TPO), TPO agonist, stem cell factor (SCF) and/or Phorbol myristate acetate (PMA).
As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term "active ingredient" refers to any one of tLivin, compositions comprising same, targeting moieties and anti-cancer agents, accountable for the biological effect.
According to specific embodiments, the tLivin and/or the targeting moiety are the only active agents in the composition.
Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include cyclodextrins, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in“Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections. According to specific embodiments, the active agent is applied topically (e.g. to the skin and/or eye) or by the pulmonary route of administration using appropriate spray devices either in a lyophilized powder form or in an aqueous dispersion.
Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a subop timal delivery method. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
According to specific embodiments, the composition is lyophilized.
Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in a lyophilized powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (tLivin, composition comprising same, anti-cancer agent) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer e.g., cancer exhibiting resistance to an anti-cancer agent) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1
P-1)·
Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
As used herein the term“about” refers to ± 10 %
The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".
The term“consisting of’ means“including and limited to”.
The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases“ranging/ranges between” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);“Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. MATERIALS AND METHODS
Production and Purification of mtLivin peptide - mtLivin peptide (also denoted herein as MTV) was recombinantly expressed in a bacterial expression system [C43 (DE3) E coli bacteria strain] with a His-tag and a TEV cleavage sequence (Figures 1A-B, SEQ ID NOs: 7-8).
The bacteria were propagated in a bioreactor to a high mass density. Following, mtLivin was extracted using bacterial pellet lysis and purified by chromatography using Ni- NTA agarose, Tev protease cleavage and gel filtration chromatography. The purified peptide was immediately lyophilized. Presence of contaminants was evaluated by SDS-PAGE following by Coomassie staining.
Cells - 293T cells (ATCC CRL-3216), MEOW (ATCC HTB-65) (Melanoma cell line expressing endogenously the Livin), 721.221 (ATCC CRL- 1855)/ b (B cells ectopically expressing Livin b), OCTLY19 (DSMZ ACC 528)(diffuse large B cell lymphoma), L428 (DSMZ ACC-197) (Hodgkin lymphoma), 721.221 (ATCC CRL-1855) cells (EBV-immortalized human lymphoblastoid cells), L428 Hodgkin's lymphoma cells and LY19 (diffused large B-cell lymphoma, DLBCL) cells. Mononuclear cells were isolated using ficoll from patients’ bone marrow.
Expression of GLuc in LY19 DLBCL cells -GLuc (Bakhos A Tannous Nature Protocols, 2009, (4) 582-591) was introduced to OCI-Lyl9 cells by electroporation as described in Deepak Rai et al. PNAS, 2010. 107 (7) 3111-3116. Positive expressing cells were selected by neomycin selection. For in-vitro Glue activity measurement, 500,000 OCTLyl9-Gluc cells were plated in a 24-wells plate and incubated overnight with / without treatment. A total of 50 pl conditioned medium was harvested from cells and Glue activity was measured by adding 20 mM coelenterazine (Biovision). Prior to addition to the sample, the substrate was incubated at room temperature for 30 minutes for stabilization. Photon counts were determined over 10 seconds in a luminometer (Tecan, lifesciences). For tumor monitoring viability and proliferation, blood was withdrawn once a week over the experiment and aliquots of whole blood or serum were assayed for Glue activity. 5 mΐ of serum were assayed for the reporter activity. For in-vivo Glue bioluminescence imaging, Coelenterazine was injected IV at a dose of 4 mg / kg body weight. 200 mΐ of coelenterazine with cold PBS were injected IV immediately prior to imaging. Using Glue as a tumor marker, the signal was localized in the animal using in vivo bioluminescence imaging once a week.
NLkB-Lucif erase activity - OCTLyl9 under the control of the NF-kB response elements were generated by stable transfection with pNL3.2 NF-kB -RE [NlucP NF-kB-RE Hygro] (Promega, Madison). Positive expressing cells were selected by neomycin. For in-vitro Glue and Nano-Glu activity measurement, 5xl05 cells were plated in a 24-wells plate and incubated overnight with or without treatment. A total of 50 pl conditioned medium was harvested from cells and luciferase activity was measured according to the manufacturer's instructions. Prior to addition to the sample, the substrate was incubated at room temperature for 30 minutes for stabilization. Photon counts were determined over 10 seconds in a luminometer (Tecan, lifesciences).
SDS-PAGE and Western blot -was effected as described in Abd-Elrahman I et al. Cancer Research. 2009;69(l3):5475-5480.
Coomassie staining - was effected as described in Badescu G et al Bioconjug Chem. 20l4;25(3):460-469
Cell death analysis - Cell death was analyzed by annexin V / propidium iodide (PI) staining and flow cytometry. In all, 0.5 x 106 cells were cultured with various concentrations of treatments (NPs, chemotherapy) for 48 hours. Cells were then stained using 1 pg / ml annexin V-FITC, washed once in annexin -V-binding buffer and stained with 0.5 pg / ml PI, and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA).
Preparation of P LG A nanoparticles (NPs) - Polymeric nanospheres, designated here as nanoparticles (NPs) were prepared using a well-established interfacial deposition method [H. Fessi, F. Puisieux, J-Ph. Devissaguet, N. Ammoury and S. Benita Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int. J. Pharm., 55, pp. R1-R4 (1989)]. Briefly, the organic phase contained 150 mg of the polymer PLGA MW 50,000 Dalton, the cross-linker OCA (oleyl cysteineamide, synthesized and characterized according to the Karra et al, and suppk, 2013) (5 mg) were dissolved in 25 ml acetone prior to NPs formation. For the preparation of nanoparticles, the organic phase was added to 50 ml of an aqueous solution containing 50mg Solutol® HS 15. The suspension was stirred at 900 rpm for 30 minutes and subsequently, the acetone fraction was evaporated by a rotor evaporator. The formulations were adjusted to pH 6.5-7.
A fluorescence formulation was prepared by adding a Fluorescein isothiocyanate-labeled PLGA-NPs at 1 % (w/w) concentration of the total PLGA.
A doxorubicin (DOX) loaded formulation was prepared by adding 200 mΐ trimethylamine and 100 mg of DOX-HC to the organic phase in order to render the DOX into the base form and be able to be incorporated in the formed NPs. The suspension was stirred at 900 rpm for 30 minutes and subsequently, the acetone fraction was evaporated by a rotor evaporator. Following encapsulation, the non-capsulated DOX HC1 was separated, by vivaspin filters (300k) at 4500 rpm centrifugation in three cycles of washings. The yield of the encapsulation was determined using UV detection method at wavelength of 475 nm.
Preparation of PLGA-NPs conjugated to macromolecules - NPs loaded with macromolecules (e.g. peptides, antibodies) were prepared using the approach of interacting the activated maleimide - macromolecules with the NP surface bearing SH -groups thanks to the anchoring of the amphiphilic OCA linker molecules at the interface of the NPs enabling thiol surface functionalization which can react with maleimide- activated monoclonal antibodies, proteins and peptides (as described in Karra et al 2013), resulting in the conjugation of the macromolecule to the surface of the NPs via the formation of the thioether covalent bond. Briefly, the PLGA-NPs conjugated with macromolecules [mtLivin peptide (SEQ ID NO: 6), CD40L, Peprotech; Mabthera, Roche] were prepared as follows: Specifically, NPs were prepared with OCA (SH-group) anchored at the interface of the NPs. The macromolecules (e.g. mtLivin SEQ ID NO: 6, antibodies) were activated by conjugation of maleimide group (sulfo- SMCC) at a molar ratio of 1 : 5, respectively. The conjugation process duration lasted 3 - 4 hours. Following conjugation, the untreated sulfo-SMCC (cross-linker) was separated, by vivaspin filters (3k) at 4500 rpm centrifugation (lOOOxg). The activated macromolecules were mixed with the surface activated OCA NPs and stirred at 900 rpm (60xg) overnight. Following, the suspension was filtered using 300k vivaspin filters centrifuged at 4500 rpm (lOOOxg).
Preparation of lyophilized peptide-loaded PLGA NPs - Several batches of PLGA-NPs loaded with mtLivin peptide were prepared as described above. Five types of sugars were evaluated as cryo- and lyoprotectants for the freeze-drying process of OPA NPs: sucrose, (+) trehalose, D (-) mannitol, xylitol, and the macrocyclic oligosugar h y dro x y p o p y 1 - b -c y c 1 ode x l i n (HPpCD). Different sugar concentrations were utilized to find the most suitable concentration for the drug-loaded NPs lyophilization. The NP suspension was freeze-dried using an Epsilon 2- 6d Martin Christ lyophilizer (Gef., Germany) to obtain a dry powder. The conditions used in the freeze-drying process are shown in Table 3 hereinbelow.
Table 3: Freeze drying conditions for PLGA NP.
Figure imgf000045_0001
Figure imgf000046_0001
Particle size determination - Mean particle size was determined by photon correlation spectroscopy using a Zetasizer 5000 (Malvern Instruments Ltd, UK). Each NP batch was appropriately diluted immediately following preparation with double-distilled and filtered water (0.22 pm Millipore filter). Particle size distribution was measured in triplicates for each batch.
Transmission Electron Microscopy (TEM) - Morphological evaluation of the NPs was performed on carbon-coated Cu grids (300-mesh), using an electron microscope (CM12 TEM, Philips) at lOOkV, following negative staining using 2% uranyl acetate.
Protein content - Protein content on NPs was determined using electrophoresis. For example, Figure 6 shows Coomassie staining of increasing amounts of purified mtLivin peptide conjugated to PLGA NPs.
In-vivo DLBCL subcutaneous xenograft model - All experiments were approved by the institute animal care ethics committee. Male NOD/SCID mice 7-8 weeks old were maintained under specific pathogen-free conditions. Mice were injected sub-cutaneously (SC) with tumorigenic LY 19 Glue cells in the right flank. Weight and tumor volume were measured every 2-3 days throughout the experiment and urine was collected for Gaussia Luciferase Assay. Tumors were measured with a caliper and under the IVIS. Mice were sacrificed when one dimensional tumor diameter reached 1-2 cm (according to the guidelines of the animal core facilities). Blood, serum, tumors and organs were collected for analysis at the time of sacrifice. Mice were randomly divided into treatments groups with free access to food and water. Each group received formulation in the form of MTV-loaded PLGA NPs, CD40L-loaded PLGA NPs CD40L-MT V -loaded PLGA NPs or PLGA NPs. The formulations (0.2 mg MTV / 0.5 mg PLGA, 1.25 mg DOX, 0.5 mg CD40L, 200 pL) were injected at days 4, 11, 20 post tumor cell injection, through mouse tail vein. Following 4 injection of mtLivin-NPs (15 days of treatment), platelets were counted in a MACSQuant Analayzer 10 (Miltenyi Biotech).
In-vivo disseminated DLBCL xenograft model - OCI-LY19- GLuc cells were injected into the tail vein of NOD/SCID mice (Shi JQ et al. Mol Cancer Ther. 2012 11(9):2045-53). The OCI-LY19- GLuc cells constitutively express a gaussia luciferase reporter (Glue), allowing photon flux detection (following coelenterazine, Glue substrate, injection) with IVIS imaging. All mice were observed with at least twice a week physical exams for signs and symptoms of disease and weight checks, complete blood count (CBC). Mice that exhibited ruffled fur, hunched posture, weight loss or severe CBC abnormalities were considered moribund and were scarified. Throughout the study, animals were imaged to determine tumor burden. The mice were scarified at days 2, 22 and 46 cells injection and CD20 expression was determined by immunohistochemistry.
Mice were randomly divided into treatments groups with free access to food and water. Each group received formulation in the form of dox-loaded PLGA NPs, MTV-loaded PLGA NPs, CD40L-loaded PLGA NPs, CD40L-MTV-loaded PLGA NPs or PLGA NPs. The formulations (0.2 mg MTV / 0.5 mg PLGA, 0.5 mg CD40L, 200 pL) were injected at days 2, 8, 15, 24, 31 post tumor cell injection, through mouse tail vein.
Caspase-3 activity assay - Subcutaneous tumor samples were homogenized in caspase assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1 % CHAPS, 1 mM EDTA, 10 % glycerol and 10 mM DTT) (JIA JIA el al. 2015 ,11(3): 1623-1628) Caspase-3 activity in 50 pg of protein fractions was determined by CaspACE™ Assay System, Colorimetric, according to manufacturers’ instructions.
GLu activity and Flow cytometry analysis of femoral bone marrow cells - Lollowing sacrifice mice femurs (from both hind limbs) were purged with 1 mL PBS to obtain bone marrow cells. The suspension was passed through a 70 pm LalconTM cell strainer (BD Biosciences), followed by centrifugation (5 minutes at 1500 rpm) and washed in cold PBS. Equal number of BM cells was assayed for the Glue activity. Anti-human CD 19 and CD20 antibodies were added to cells. Cells were incubated for 30 minutes at RT and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA).
Immunohistochemistry - Following sacrifice, mice spleen, lungs, liver, spinal cord and brain were collected, fixed, embedded in paraffin and sectioned. OCI-OCTLyl9 lymphoma cells dissemination was evaluated by immunohistochemistry with anti-CD20 antibody (Cell Marque).
IFN ELISA - Blood was collected form mice 17 days following IV injection of lymphoma cells, as described above. IFNy levels were determined in the serum by ELISA (Murine IFN-gamma TMB ELISA Development Kit, PeproTech Asia 900-T98), according to manufacturer’s instructions. EXAMPLE 1
FREE mtLIVIN OR mtLIVIN CONJUGATED TO NANOPARTICLES INDUCES
TUMOR CELLS DEATH IN-VITRO
A system for production of large quantities of the potent mtLivin (SEQ ID NO: 6) was set up in C43(DE3) bacteria cells. SDS-PAGE and Coomassie staining of demonstrated that only minor contaminations were present in the purified fractions (Figures 1A-B and 2).
To investigate the effect of the produced mtLivin on living cells, 293T cells were incubated with various concentrations of the peptide. Free mtLivin elicited rapid (within 4 hours), dose-dependent cell death, whereas the addition of an inactive control peptide, had no significant effect on cell viability (Figure 3).
To improve the delivery and efficacy of mtLivin for the treatment of tumors, mtLivin was conjugated onto poly(lactide-co-glycolide) (PLGA) surface activated nanoparticles (NPs) (Figure 4).
Incubation of 293T, MEOW (Melanoma cell line expressing endogenously Livin) and 721.221/ b (B cells ectopically expressing Livin b) with the mtLivin NPs elicited rapid, dose- dependent cell death, whereas no significant effect on cells viability was observed upon exposure to empty, control NP (CTR NP) (Figure 5). Importantly, lower amounts of mtLivin were required for inducing cell death when encapsulated in NPs (1.5 pg / pl mtLivin encapsulated in NPs compared to 46 pg / mΐ free mtLivin) probably due to the enhanced cell penetration of the NPs via endocytosis.
Further evaluation of the effect of mtLivin NPs on survival and cell cycle of L428 Hodgkin lymphoma cells demonstrated that incubation with mtLivin-NPs not only induced cell death of L428 cells but also virtually destroyed the cell cycle of these cells (Figures 10A-C).
Gaussia Luciferase (GLuc) is a recently discovered, naturally secreted protein from the deep sea copepod, Gaussia princeps. It is the smallest known luciferase and it is one of the brightest known. It is also stable at elevated temperatures. The secretion signal of GLuc is functional in mammalian cells. In vivo imaging using Glue demonstrated much higher sensitivity than the Firefly or Renilla Luciferases.
GLuc was introduced to the OCTLY19 DLBCL cells and the luminescence in the cells and the correlation with cell death were evaluated. As shown in Figure 12, cell death induced by the mtLivin-NPs was almost maximal at a dose of 0.25 mg mtLivin. EXAMPLE 2
OPTIMIZATION OF PLGA NPs LOADED WITH mtLIVIN
Various PLGA nanoparticles (NPs) formulations loaded with different polypeptide at different concentrations were prepared. Table 4 below describes the composition of the successful formulations and respective physicochemical properties. Several batches from each formulation were also prepared (data not shown).
Table 4: Physicochemical properties of the various peptide-conjugated PLGA NPs formulations and toxicity of blank PLGA NPs
Figure imgf000049_0001
* Shown are toxicity results in 293T, LY19 and L428 cells.
The results presented in Table 4 hereinabove indicate that high concentration of PLGA elicited toxic side effects to all three cell lines tested [293T (kidney cancer cells), OCTLY19 (diffuse large B cell lymphoma), and L428 (Hodgkin lymphoma)]. Indeed, blank NPs or NPs conjugated to mtLivin were toxic to all the cells when prepared with 2.5 mg / ml PLGA indicating that the cells were sensitive to PLGA concentration, i.e. the number of NPs incubated with the cells irrespective of the presence of mtLivin. However, when the concentration of PLGA was decreased from 2.5 to 0.5 mg / ml, no toxicity sign was observed. All the remaining formulations were considered safe irrespective of the type of polypeptide or peptide density conjugated to the NPs.
It should be noted that when only mtLivin peptide was conjugated to the NPs, the mean diameter remained in the order of 100 nm. When a second polypeptide (either an entire monoclonal antibody or a fragment) was conjugated to the NPs, the mean diameter increased up to 200 nm.
Finally, all the NP formulations exhibited a negatively surface charge potential around 35-40 mV.
Evaluation of NPs’ morphology using TEM indicated that blank NPs and conjugated NPs exhibit a spherical morphology structure with average diameters of 83 nm and 82 nm, respectively (Figures 7A-B and Table 5 hereinbelow). All the NP formulations exhibited a negatively surface charge potential around 36-41 mV (Table 5 hereinbelow).
In view of these results, 0.5 mg / ml PLGA was selected for mtLivin-NPs formulation. To optimize the protein concentration in the PLGA NPs, increasing amounts of mtLivin were conjugated to 0.5 mg / ml PLGA. Following, various cell lines [including 293T cells, 721.221 cells (EBV-immortalized human lymphoblastoid cells), L428 Hodgkin's lymphoma cells and OCI-LY19 (diffused large B-cell lymphoma) cells] were used to evaluate the effect of mtLivin- NPs on cell survival. mtLivin-NPs -induced cell death was determined by Propidium Iodide (PI) staining or by the subGl fraction in cell cycle analysis, using flow cytometry. Figure 8B shows that maximal cell death was achieved with a concentration of 0.2 mg mtLivin.
Table 5: Physicochemical properties of the various protein-loaded PLGA nanoparticles formulations
Figure imgf000050_0001
EXAMPLE 3
STABILITY OF FREE mtLIVIN AND mtLIVIN CONJUGATED TO PLGA NPs
In order to enhance the long-term stability of the formulation, a study on lyophilization and stability of free mtLivin peptide and mtLivin peptide conjugated to NPs was effected. Consequently, free mtLivin can remain stable over two weeks up to one month only following lyophilization. The peptide can be reconstituted in PBS buffer prior to being conjugated to the PLGA NPs. A formulation PLGA (0.5 mg / ml) loaded with mtLivin peptide (0.25 mg / ml) and OCA (0.5 mg / ml) was selected and considered the most optimized formulation in solution.
A common limitation of using polymeric NPs in aqueous suspension is due to their poor chemical and physical stability when conserved over long time storage. Therefore, lyophilization of these colloidal systems is an alternative method to achieve long-term stability. NPs have thin and fragile shell structure, which may not resist to the stress of such process.
Freezing is considered to be the most aggressive and critical step during the lyophilization. This step can cause aggregation or destruction of the NPs. Therefore, several cryo-protectants including three disaccharides [sucrose (S), trehalose (T) and a cyclic oligosaccharide HR-b-cyclodextrin (CD)] were tested at different concentrations compared to the PLGA content in the selected formulation (Table 6 hereinbelow).
Following reconstitution with water, 293T cells were incubated with the mtLivin- conjugated NPs, and cell death was determined by PI (Table 6 hereinbelow and Figure 9B).
The results in Table 6 demonstrate that the addition of cryoprotectant [specifically trehalose (at a ratio of 1:25 and 1:50) and HR-b-cyclodextrin (1:5 and 1:10 concentration)] was necessary for NPs stabilization and for mtLivin activity. It should be pointed that the free form of mtLivin could not be stabilized easily and only one single cryo -protectant at a specific ratio (1:10 of HR-b-cyclodextrin) succeeded in stabilizing the free mtLivin lyophilized peptide (Table 6 hereinbelow).
Table 6: Stability and biological activity of free mtLivin peptide and mtLivin peptide conjugated to PLGA NPs following lyophilization
Figure imgf000051_0001
Figure imgf000052_0001
EXAMPLE 4
PLGA NPs LOADED WITH mtLIVIN IN COMBINATION WITH A TARGETING MOIETY HAVE AN IMPROVED EFFECT ON TUMOR CELL DEATH IN- VITRO
Bi-functional CD40L-mtLivin-NPs
CD40 is a type-l transmembrane protein and is expressed in more than 90 % of B-cell malignancies. Thus, the biological activity of targeted, bifunctional mtLivin-CD40L-NPs was evaluated. As shown in Table 5 hereinabove, when CD40L was conjugated the mean diameter of the NPs increased up to 200 nm.
In the first step, the behavior of CD40L-NPs conjugation on binding with CD40 receptor was evaluated by testing the ability of CD40L to induce NF-kB activity in target cells. NF-KB- dependent gene transcription was measured in OCI-Lyl9 stably expressing NF-xB-dependent lucif erase reporter (OCI-Lyl9-NF-KB RE)(l6) (Figure 25). In the absence of exogenous stimuli or in the control NPs, low levels of NF-kB binding activity were detectable in OCI-Lyl9-NF- KB RE cells. Stimulation with free rhCD40L resulted in a potent induction of NF-kB binding activity. In-vitro the CD40L conjugated to NPs (both CD40L-NPS and CD40L-MTV-NPS) maintained its ability to bind to target cells and to induce NF- KB activity as well as free rhCD40L (Figure 25). In-vitro targeting of the NPs using CD40L-mtLivin-NPs elicited significant cell death of LY19 DLBCL cells (Figures 16 and 26). In addition, as seen in Figure 11, while the respective mono-functional mtLivin-NPs and CD40L-NPs exhibited a minor effect on the cell cycle of LY19 cells, targeting the mtLivin-NPs to the cell using CD40L (bi-functional NPs), dramatically affected the cell cycle such that there were no cycling cells (i.e. no cells in S or M phases of the cell cycle).
NPs loaded with mtLivin and doxorubicin
To enhance the efficacy of the delivery system the incorporation of doxorubicin within the NPs was investigated. First, the content and the encapsulation yield of doxorubicin (DOX) in the nanoparticles was determined (Figure 13). The encapsulation yield of doxorubicin in PLGA NPs was calculated following washing by the vivaspin filters using the calibration curve shown in Figure 13: Encapsulation yield = 45 % : Final DOX-loaded NPs concentration = 0.9 mg / ml. The physicochemical properties of the NPs formulations with DOX are shown in Table 7. Table 7: Physicochemical properties of the various protein-loaded PLGA nanoparticles formulations
Figure imgf000053_0001
The results show clearly the feasibility to encapsulate DOX in PLGA NPs. The encapsulation yield is low (45 %) due the hydrophilic nature of the doxorubicin, and the physicochemical properties as depicted in Table 7 show no change in zeta potential in the various NPs formulations. However, the size of the NPs increased.
Following, cell death induced by DOX encapsulated in NPs was evaluated in LY19 DLBCL cells stably expressing GLuc. Treatment by mtLivin-NPs with or without DOX was compared (Figure 14). As shown, cell death was induced by mtLivin-NPs and was maximal at 0.25 mg mtLivin concentration and DOX further increased cell death.
EXAMPLE 5
PLGA NPs LOADED WITH mtLIVIN IN COMBINATION WITH A TARGETING MOIETY HAVE AN IMPROVED EFFECT ON TUMOR GROWTH IN-VIVO
A LY19 Glue DLBCL xenograft subcutaneous model
A DLBCL xenograft subcutaneous model was established in NOD/SCID mice using LY19 Glue cells. Mice were injected sub-cutaneously with tumorigenic cells in the right flank. Establishment of the model and methods of analysis are shown in Figs. 15A-E. As shown, Lyl9 GLuc cells were tumorigenic in NOD-SCID mice: 90 % of the mice developed tumors and tumor development can be monitored by measuring tumor volume, IVIS and by measuring GLuc activity in the urine of mice.
Following, DLBCL sub-cutaneous tumors were induced in 50 mice, divided to 5 groups, and the following treatments were tested: Control (untreated), NPs, DOX, CD40L-NPs and CD40L+mtLivin-NPs. The experiment lasted 30 days and treatments were given 3 times on days 4, 11 and 20. All formulations were tested in-vitro prior to the experiment in mice (not shown).
As shown in Figures 17A-C, the CD40L+mtLivin-NPs significantly (p<0.008) reduced tumor volume and increased mouse survival as compared to untreated control, NPs or CD40L- NPs. Further, the highest level of apoptosis was found in tumors from mice that were treated with CD40L+mtLivin-NPs demonstrating the ability of the nanoparticles to target the tumors and to induce tumor cell death (Figure 17D). In addition, treatment with CD40L+mtLivin-NPs was more effective than treatment with DOX, which it the conventional treatment to lymphoma (Figure 17 A).
A disseminated PCI LY-19- GLuc lymphoma xenograft model
To better mimic lymphoma disease in humans, a disseminated lymphoma model was used to evaluate the anti-tumor effect associated with targeted mtLivin-NPs treatment. To generate this model, OCI-LY19- GLuc cells were injected into the tail vein of NOD/SCID mice. Establishment of the model and methods of analysis are shown in Figures 18-19. The DLBCL cells expressed luciferase enabling monitoring tumor size and location. Luciferase signal was observed as early as day 2 following cell injection and increased at day 22 and at day 46. As shown, cancer hotspots were detected in femur, spine, and brain, thereby mimicking lymphoma disease in humans. Further, in this model, tumors developed mainly in bone marrow, lungs, spleen and brain. In addition, Immunohistochemistry of tumor cells with anti-CD20 showed infiltration of OCI-lyl9 cells in the spleen and brain close to the time of tumor cells injection (Day 2). At day 22, the lymphoma cells were detected in mice spleen, brain and bone marrow (BM). At day 46, mice were euthanized when they lost weight (20 % of weight) and exhibited hindlimb paralysis. Lymphoma infiltrations were detected in the brain, bone marrow, lungs, spleen and liver.
Manifestations in the spinal cord indicate the terminal phase of lymphoma. In the described model, hind-leg paralysis was developed by day 28 in all mice experiment, indicating an engraftment rate of 100 %.
As paralysis preceded death in every case, the appearance of hind-leg paralysis was set as the end point for further survival analyses.
Following, disseminated OCI LY-19- GLuc lymphoma xenografts were induced in 60 mice. At day 3 post-injection of the OCI-Lyl9 cells, the mice were divided to 6 groups and were treated approximately once a week with: Control (untreated), PLGA, PLGA-OCA, CD40L-NPs, CD40L+mtLivin-NPs and mtLivin-NPs. The experiment lasted 39 days and treatments were given 5 times. To monitor engraftment, animals underwent bioluminescent imaging at various time points. In addition, mice were monitored for clinical signs of lymphoma (hindlimb paralysis and weight loss) and survival. All untreated controls died of disseminated disease proceeding by progressive weight loss or were sacrificed due to hindlimb paralysis within 28 days following tumor cell inoculation. All vehicle-treated mice (PLGA, PLGA-OCA, CD40L- NPs) exhibited paralysis and were euthanized between days 28 and 36 (Figure 20). In contrast, 37.5 % of mice treated with mtLivin-NPs and 71.4 % of mice treated with CD40L+mtLivin-NPs exhibited complete pathological response, i.e. no tumor cells seen in pathology of mouse organs (IHC, Figure 24) on day 39.
All treatments in this study were well-tolerated with no body weight loss.
The tumor burden was monitored by quantification of tumor-derived gaussia luciferase- activity. As shown in Figures 22A-C, mice treated with either CD40L-mtLivin-NPs or mtLivin- NPs exhibited significantly smaller tumors compared to the untreated and vehicles groups. ().
Kaplan-Meier survival curves (Figure 21) summarized mouse survival throughout the study. All Median survival rates for mice treated with either CD40L-mtLivin-NPs or mtLivin- NPs were significantly longer than that in the untreated and vehicles groups. (Figure 21). Importantly, the CD40L-mtLivin-NPs had a combined improved effect on mice survival as compared to CD40L-NPs and mtLivin-NPs.
The effect of the different treatments on infiltration of tumor cells to the BM was shown most clearly in CD40L-mtLivi-NPs treated mice (Figure 23). As shown, flow cytometry analysis of BM cells collected from the femur of mice in the untreated and vehicle-treated control, demonstrated a large presence of human CD19+/CD20+ lymphoma cells, 10 %-25 % of cells collected from the BM (Figure 23). A similar analysis of BM cells from mice treated with CD40L+mtLivin-NPs, demonstrated a significant reduction (P=0.03) in engraftment of the human CD19+/CD20 positive lymphoma cells. Quantitation of Glue activity in BM cells collected from the femur mice confirmed the results from the FACS analysis (Figure 23). This analysis, as well, indicated a combined improved effect of the CD40L-mtLivin-NPs compared to CD40L-NPs and mtLivin-NPs.
Further, immunohistochemical analysis of mice spinal cord and brain tissues using anti- CD20 as human lymphoma marker showed that treatment with mtLivin-NPs and CD40L- mtLivin-NPs decreased the amount of lymphoma cells into the CNS as compared to untreated control and CD40L-NPs -treated mice (Figure 24). Remarkably, about 50 % of mice treated with mtLivin-NPs exhibited infiltration of lymphoma cells into the CNS; and no infiltration to the CNS was detected in any of the CD40L-mtLivin-NPs treated mice.
In addition, high levels of IFNy were detected in the serum of mice treated with targeted PLGA-oca and increased with the targeted-MTV-NPs compared with untreated and control NPs (Figure 27). These results indicate the activity of Natural killer (NK) cells. NKs play an important role in the immune response to viral and bacterial infections and to transformed cells. Upon stimulation through their activating receptors, NKs also produce pro-inflammatory cytokines, including IFN-g.
Interestingly, comparing the anti-tumor effect of CD40L-mtLivin-NPs in the subcutaneous model to the effect in the disseminated LYl9-Luc model, CD40L-mtLivin-NPs yielded a better anti-tumor activity in the disseminated model.
EXAMPLE 6
PLGA NPs LOADED WITH mtLIVIN INCREASE THE AMOUNT OF PLATELETS IN-
VIVO
To determine the effect of mtLivin-NPs on platelet recovery, platelet count was determined in the DLBCL xenograft subcutaneous model described in Example 5 hereinabove. The results show about 2-fold increase in platelet count (p=0.036) 15 days following tumor injection compared to untreated control mice (Table 8 hereinbelow).
Table 8:
Figure imgf000057_0001
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

Claims

WHAT IS CLAIMED IS:
1. A method of treating cancer exhibiting a resistance to an anti-cancer agent in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of tLivin, thereby treating the cancer exhibiting resistance to the anti-cancer agent in the subject.
2. The method of claim 1, comprising administering to said subject at least one anti cancer agent.
3. The method of claim 2, wherein said at least one anti-cancer agent comprises said anti-cancer agent.
4. A composition comprising tLivin for use in the treatment of cancer resistant to an anti-cancer agent.
5. The composition for use of claim 4, comprising at least one anti-cancer agent.
6. The composition for use of claim 5, wherein said at least one anti-cancer agent comprises said anti-cancer agent.
7. The method or the composition for use of any one of claims 1-6, wherein said anti-cancer agent is not an apoptotic agent.
8. The method or the composition for use of any one of claims 1-7, wherein said anti-cancer agent is an immunodulatory molecule.
9. The method or the composition for use of any one of claims 1-6, wherein said anti-cancer agent is selected from the group consisting of IMiDs (e.g. Revlimid, Thalidomide, Pomalidomide), proteasome inhibitors (e.g. Velcade, Carfilzomib), Rituximab, fludarabine and Bendamustine.
10. The method or the composition for use of any one of claims 1-9, wherein said tLivin is administered in a formulation comprising a targeting moiety.
11. The method or the composition for use of any one of claims 1-10, wherein said tLivin is administered in a formulation comprising a cell penetrating agent and/or a stabilizing agent.
12. A composition comprising tLivin attached to or encapsulated in a nanoparticle comprising poly(lactide-co-glycolide) .
13. A composition comprising tLivin and a targeting moiety, wherein said tLivin is attached to or encapsulated in a cell penetrating agent and/or a stabilizing agent.
14. The composition of any one of claims 12-13 or the method or the composition for use of claim 11, wherein said cell penetrating agent and/or said stabilizing agent is selected from the group consisting of a nanoparticle, a liposome, a viral vector, a cell penetrating peptide and poly(alkylene) glycols.
15. The composition of any one of claims 12-13 or the method or the composition for use of claim 11, wherein said cell penetrating agent and/or a stabilizing agent is a nanoparticle.
16. The composition, the method or the composition for use of any one of claims 14-
15, wherein said tLivin is attached to an outer surface of said nanoparticle.
17. The composition, the method or the composition for use of any one of claims 14-
16, wherein said tLivin is attached to said nanoparticle via a linker.
18. The composition, the method or the composition for use of claim 17, wherein said linker comprises an oleyl cysteineamide (OCA).
19. The composition, the method or the composition for use of any one of claims 14- 18, wherein said nanoparticle comprises poly(lactide-co-glycolide), polylactide (PLA), polyglycolide, polylactide-polyglycolide, and/or polyethylene glycol-co-lactide (PEG-PLA).
20. The composition, the method or the composition for use of any one of claims 14- 18, wherein said nanoparticle comprises poly(lactide-co-glycolide).
21. The composition, the method or the composition for use of any one of claims 19- 20, wherein said concentration of said poly(lactide-co-glycolide) is 0.05 to 5 mg / ml.
22. The composition, the method or the composition for use of any one of claims 10 and 13-21, wherein said targeting moiety comprises an immunomodulatory molecule.
23. The composition, the method or the composition for use of claim 22, wherein said targeting moiety is a CD40 and/or PD-l binding molecule.
24. The composition, the method or the composition for use of claim 23, wherein said targeting moiety is a CD40 binding molecule.
25. The composition, the method or the composition for use of any one of claims 10 and 13-21, wherein said targeting moiety is selected from the group consisting of CD19, CD20, CD38, CD138, EGFR, Her-2 and PMSA binding molecule.
26. A composition comprising tLivin and a CD40 binding molecule.
27. The composition, the method or the composition for use of any one of claims 23- 24 and 26, wherein said CD40 binding molecule comprises a CD40L polypeptide and/or an anti- CD40 antibody.
28. The composition, the method or the composition for use of any one of claims 23- 24 and 26, wherein said CD40 binding molecule comprises a CD40L polypeptide.
29. The composition of any one of claims 12-28, wherein said tLivin is in a concentration of 0.25-0.5 mg / ml.
30. The composition or the composition for use of any one of claims 10 and 13-29, wherein said targeting moiety is in a concentration of 0.25-0.5 mg / ml.
31. The composition of any one of claims 12, 14-25 and 27-30, wherein said nanoparticle has a diameter of 50 - 250 nm.
32. The composition of any one of claims 12-31, wherein said composition is lyophilized.
33. The composition of any one of claims 12-32, wherein said composition comprises a cryo-protectant.
34. The composition of claim 33, wherein said cryo-protectant comprises trehalose and/or b-cyclodextrine.
35. The composition of any one of claims 12-34, wherein said composition comprises an anti-cancer agent.
36. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 12-35, thereby treating the cancer in the subject.
37. The composition of any one of claims 12-35, for use in the treatment of cancer.
38. A method of increasing an amount of platelets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 12-35, thereby increasing the amount of platelets in the subject.
39. A method of treating thrombocytopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 12-35, thereby treating thrombocytopenia in the subject.
40. The composition of any one of claims 12-35, for use in the treatment of thrombocytopenia.
41. The method of any one of claims 38-39 or the composition for use of claim 40, wherein the subject has cancer.
42. The method of the composition for use of any one of claims 36-41, wherein said cancer exhibits a resistance to an anti-cancer agent.
43. The method or the composition for use of any one of claims 1-11, 14-25, 27-28 and 36-42, wherein said resistance is acquired resistance.
44. The method or the composition for use of any one of claims 1-11, 14-24, 27-28 and 36-43, wherein said cancer is selected from the group consisting of melanoma, lymphoma, multiple myeloma, chronic lymphocytic leukemia (CLL), lung cancer and prostate cancer.
45. The composition, the method or the composition for use of any one of claims 1- 44, wherein said tLivin is p30-Livin a.
46. The composition, the method or the composition for use of claim 45, wherein said tLivin comprises an amino acid sequence comprising SEQ ID NO: 2.
47. The composition, the method or the composition for use of any one of claims 1- 44, wherein said tLivin is p28-Livin b.
48. The composition, the method or the composition for use of claim 47, wherein said tLivin comprises an amino acid sequence comprising SEQ ID NO: 4.
49. The composition, the method or the composition for use of any one of claims 1- 44, wherein said tLivin is mtLivin.
50. The composition, the method or the composition for use of claim 49, wherein said mtLivin comprises an amino acid sequence comprising SEQ ID NO: 6.
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