EA042663B1 - P-ETOXY NUCLEIC ACIDS FOR STAT3 INHIBITION - Google Patents

Description

This application claims priority over U.S. Provisional Application No. 62/487,292, filed April 19, 2017, the contents of which are incorporated herein by reference.

State of the art

Field of invention

This invention as a whole, as a rule, relates to the field of medicine. More specifically, it relates to liposomal formulations of P-ethoxy oligonucleotides that hybridize to a polynucleotide product of the STAT3 gene, and methods for the preparation and use of such formulations in medicine, especially in the treatment of solid and hematological cancers that have high expression or increased activity of the STAT3 gene. .

Description of the Prior Art

Many genes are involved in the sustained proliferation and survival of cancer cells in premalignant tumors and advanced refractory tumors. Members of the signal transducers and activators of transcription (STAT) signaling pathways are known to play an important role in this property. STAT3 is a transcription factor that is activated in about 70% of all solid tumors and blood cancers. Upon activation, STAT3 translocates from the cytosol to the nucleus to direct the expression of a large number of genes, including Survivin, Bcl- _XL , Mcl-1, c-Myc, cyclin D1, p21, cyclin E, matrix metalloproteinase-9 and -2, and VEGF, to regulate a wide range of cellular processes such as cell survival, growth, migration, invasion, metastasis, and angiogenesis (Wu et al., 2011; Hardee et al., 2013). In addition, STAT3 regulates several important signaling pathways involved in cancer progression, including the IL-3, IL-6, and NF-kB cascades, and also activates feedback pathways, enhancing its own activation (Hardee et al., 2013).

While STAT3 is normally expressed at low levels in the cytosol of normal cells, it is often overexpressed and upregulated (phosphorylated and localized to the nucleus) in cancers, including diffuse large B-cell lymphoma (DLBCL). In DLBCL, it is most often activated in the ABC subtype of the disease and less frequently in the GC subtype of DLBCL (Wu et al., 2011; Hardee et al., 2013; Scuto et al., 2011). Importantly, STAT3 overexpression, hyperphosphorylation, and nuclear localization correlate with worse overall prognosis in these tumors compared to tumors expressing lower levels of STAT3. In addition, tumors harboring overactivated STAT3 also show increased NF-κB signaling in the ABC subtype of DLBCL, indicating that STAT3 signaling plays a role in the progression of DLBCL.

Due to the nature of the STAT3 protein, designing small molecules to directly target its signaling activity is difficult, making it an ideal target for antisense and other genetic knockdown approaches. Given the central role that STAT3 plays in many forms of cancer and other diseases, several attempts have been made to develop peptide, oligonucleotide decoy, and small molecule compounds to block STAT3 activation, dimerization, DNA binding, or to accelerate enzyme inactivation (Miklossy et al., 2013). As an alternative approach, several groups are working on the development of Janus kinase (JAK) inhibitors to block STAT3 phosphorylation and activation in LLS (lymphocytic lymphosarcoma) and other diseases, with varying success (Amin et al., 2003; Fagard et al., 2013).

Approaches that target STAT3 specifically include the use of oligonucleotides to block translation of STAT3 mRNA into protein or to induce RNA degradation via RNase H mechanisms through antisense oligonucleotide (ASO) technology. These strategies offer a way to reduce toxicity and are less adapted to resistance mutations. Several attempts at this approach for STAT3 were unsuccessful. Thus, there is a need for improved STAT3-targeting antisense compositions for use in the treatment of diseases.

The essence of the invention

This document provides compositions and methods that induce growth inhibition and/or apoptosis in a broad set of STAT3 controlled cancer cells. Expression of the STAT3 protein is prevented by a non-toxic nuclease-resistant oligonucleotide that targets STAT3-encoded polynucleotides in combination with a neutral liposome, thereby eliminating the pool of available STAT3 protein.

In one embodiment, compositions are provided comprising populations of oligonucleotides that hybridize to a polynucleotide product of the STAT3 gene. In some aspects, the population oligonucleotides are composed of nucleoside molecules linked to each other by backbone phosphate bonds, wherein at least one of the backbone phosphate bonds in each oligonucleotide is a P-ethoxy backbone bond, and no more than 80% of the backbone phosphate bonds in each oligonucleotide are P-ethoxy backbone bonds. In some aspects, at least one of the backbone phosphate bonds in each oligonucleotide is a backbone phosphodiester bond. In some aspects, the oligonucleotides of the population contain the sequence according to any of SEQ ID NO: 1-4. In some aspects, the population oligonucleotides contain the sequence

- 1 042663 according to SEQ ID NO: 1. In some aspects, the population oligonucleotides contain the sequence according to SEQ ID NO: 2. In some aspects, the population oligonucleotides contain the sequence according to SEQ ID NO: 3. In some aspects, the population oligonucleotides contain the sequence according to SEQ ID NO: : 4. In various aspects, population oligonucleotides inhibit STAT3 expression. In some aspects, the composition is lyophilized.

In some aspects, from 10 to 80% of the backbone phosphate bonds are P-ethoxy backbone bonds; 20 to 80% of the backbone phosphate bonds are P-ethoxy backbone bonds; 30 to 80% of the backbone phosphate bonds are P-ethoxy backbone bonds;

40 to 80% of the backbone phosphate bonds are P-ethoxy backbone bonds;

50 to 80% of the backbone phosphate bonds are P-ethoxy backbone bonds or 60 to 70% of the backbone phosphate bonds are P-ethoxy backbone bonds and any range derived from them. In some aspects, from 20 to 90% of the backbone phosphate bonds are backbone phosphodiester bonds; 20 to 80% of the backbone phosphate bonds are backbone phosphodiester bonds; 20 to 70% of the backbone phosphate bonds are backbone phosphodiester bonds; 20 to 60% of the backbone phosphate bonds are backbone phosphodiester bonds; 20 to 50% of the backbone phosphate bonds are ostley phosphodiester bonds, or 30 to 40% of the backbone phosphate bonds are backbone phosphodiester bonds, or any range derived from them.

In various aspects, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or whichever -any value in between, the backbone phosphate bonds are P-ethoxy backbone bonds. In various aspects, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any value between them, the backbone phosphate bonds are phosphodiester backbone bonds. In certain aspects, the backbone phosphodiester linkages are distributed throughout the oligonucleotides. As such, oligonucleotides are not chimeric molecules. In some aspects, the oligonucleotides do not contain a backbone phosphothioate bond.

In some aspects, the population oligonucleotides are in the range of 7 to 30 nucleotides in size. In certain aspects, the population oligonucleotides are in the range of 12 to 25 nucleotides in size. In various aspects, the population oligonucleotides are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29 or 30 nucleotides. The size range may be the average size of the oligonucleotides in the population.

In some aspects, the population oligonucleotides have an average size of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, not more than 5, 6, 7, 8, 8, 9, 10, 11, 11, 12, 13, 14, 15, 15, 16, 17, 18, 19, 20, 20 , 21, 22, 23, or 24, respectively, the backbone phosphate bonds in each oligonucleotide are the P-ethoxy backbone bond. In some aspects, the population oligonucleotides have an average size of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides and at least 2, 2, 2, 2, 3, 3, 3, 3, 4, 4, 4, 4, 4, 5, 5, 5, 5, 5, 5, 6, The 6, 6, 6, or 6, respectively, backbone phosphate bonds in each oligonucleotide represent the backbone phosphodiester bond. For example, oligonucleotides in a population may have an average size of 18 nucleotides, with no more than 14 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond; oligonucleotides in a population may have an average size of 20 nucleotides, with no more than 16 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond; oligonucleotides in a population may have an average size of 25 nucleotides, with no more than 20 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond; or the oligonucleotides in the population may have an average size of 30 nucleotides, with no more than 24 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond.

In some aspects, the population of oligonucleotides contains one kind of oligonucleotides. In other aspects, the population of oligonucleotides contains at least two kinds of oligonucleotides. One kind of oligonucleotide may have the same nucleotide sequence but may or may not have P-ethoxy bonds at different positions in the molecule. Thus, a population may be homogeneous in nucleotide sequence and heterogeneous in the distribution of backbone phosphodiester bonds among the population's oligonucleotides. In addition, the population may be heterogeneous in the number of P-ethoxy backbone bonds and backbone phosphodiester bonds among the oligonucleotides in the population. As a non-limiting example, a first portion of the oligonucleotides in a population may have 70% P-ethoxy bonds and 30% phosphodiester bonds, while a second portion of the oligonucleotides in a population may have 60% P-ethoxy bonds and 40% phosphodiester bonds. In some aspects, the oligonucleotide population comprises antisense oligonucleotides, short interfering RNAs (siRNAs), microRNAs (miRNAs), or piwiRNAs (piRNAs).

In various aspects, the composition further comprises phospholipids. In some aspects, the phospholipids and oligonucleotides are present in a molar ratio of from about 5:1 to about 100:1. In some aspects, the oligonucleotides and phospholipids form an oligonucleotide-lipid complex, such as, for example, a liposome complex. In some aspects, at least 75, 76, 77, 78, 79,

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80, 81, 82, 83, 84, 85, 86, 87, 88, 89 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the liposomes are less than 5 µm in diameter. In some aspects, at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

88, 89 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the liposomes are less than 4 µm in diameter. In some aspects, the population of oligonucleotides is included in the population of liposomes.

In some aspects, the phospholipids are uncharged or have a neutral charge at physiological pH. In some aspects, the phospholipids are neutral phospholipids. In certain aspects, the neutral phospholipids are phosphatidylcholines. In certain aspects, the neutral phospholipids are dioleoylphosphatidylcholine. In some aspects, phospholipids essentially do not contain cholesterol.

In one embodiment, pharmaceutical compositions are provided comprising the oligonucleotide and phospholipid composition of these embodiments and a pharmaceutically acceptable carrier. In some aspects, the composition further comprises a chemotherapeutic agent.

In one embodiment, methods are provided for delivering a therapeutically effective amount of an oligonucleotide to a cell, comprising contacting the cell with a pharmaceutical composition according to these embodiments. In some aspects, the method is a method of treating hyperplasia, cancer, an autoimmune disease, or an infectious disease.

In one embodiment, methods for treating a subject with cancer, an autoimmune disease, or an infectious disease are provided, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to these embodiments. In some aspects, the subject is a human. In some aspects, the cancer is bladder, blood, lymphoma, pancreas, bone, bone marrow, brain, breast, colon, esophagus, stomach, head and neck, kidney, liver, lung, prostate, skin, testis, tongue, ovary or uterus. In various aspects, the cancer is lung adenocarcinoma, leukemia, lymphoma such as, for example, diffuse large B cell lymphoma, or pancreatic ductal adenocarcinoma. In some aspects, the autoimmune disease is systemic lupus erythematosus, spondyloarthropathy, Sjögren's disease, Crohn's disease, diabetes mellitus, multiple sclerosis, or rheumatoid arthritis. In some aspects, the infectious disease is a bacterial infection, a fungal infection, a viral infection, or a parasitic infection. In some aspects, the composition is administered subcutaneously, intravenously, or intraperitoneally. In some aspects, the method further includes administering to the subject at least a second anti-cancer therapy. In some aspects, the second cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, or cytokine therapy. In some aspects, immunotherapy is a checkpoint blockade therapy. In some aspects, the introduction of the composition reduces the expression of the STAT3 protein in the patient.

The oligonucleotide includes an antisense nucleic acid molecule that specifically hybridizes to a nucleic acid molecule encoding a target protein or regulating the expression of a target protein. Specific hybridization means that an antisense nucleic acid molecule hybridizes with a target nucleic acid molecule and regulates its expression. Preferably, specific hybridization also means that no other genes or transcripts are affected. The oligonucleotide may be a single stranded nucleic acid and may contain 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleic bases. In specific aspects, the oligonucleotide may contain 15 to 30, 19 to 25, 20 to 23, or 21 contiguous nucleobases. In certain embodiments, the oligonucleotide inhibits translation of a gene that stimulates the growth of a cancerous or precancerous or hyperplastic mammalian (eg, human) cell. The oligonucleotide may induce apoptosis in the cell and/or inhibit translation of the oncogene or other target gene. In certain embodiments, the implementation of the oligonucleotide component contains one kind of oligonucleotide. In other embodiments, the implementation of the oligonucleotide component contains 2, 3, 4 or more kinds of oligonucleotides that target 1, 2, 3, 4 or more genes. The composition may further comprise a chemotherapeutic or other anti-cancer agent, which may or may not be included in the lipid component or liposome of the invention. In additional embodiments, the implementation of the oligonucleotide component is included in the liposome or lipid component.

Capture, encapsulate, and include refer to a lipid or liposome that forms an obstacle to free diffusion into solution by association with or around an agent of interest, for example, a liposome can encapsulate the agent in a lipid layer or within a water-containing compartment within or between lipid layers. In some embodiments, the composition is contained in a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be prepared for administration to a human or patient.

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In some embodiments, the implementation of the lipid component is essentially neutral charge, because it contains a neutral phospholipid, or net neutral charge. In some aspects, the neutral phospholipid may be a phosphatidylcholine, such as DOPC, egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylhofsphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, 1,2-diaracidoyl-sn- glycero-3-phosphocholine (DAPC), 1,2-dieucosenoyl-snglycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1- palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-oleoyl-2-palmitoyl phosphatidylcholine (OPPC), or lysophosphatidylcholine. In other aspects, the neutral phospholipid may be a phosphatidylethanolamine, such as dioleoylphosphatidylethanolamine (DOPE), disteraoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), or lysophosphatidylethanolamine.

In certain embodiments, the implementation of the phospholipid component may contain 1, 2, 3, 4, 5, 6, 7, 8 or more species or types of neutral phospholipids. In other embodiments, the implementation of the phospholipid component may contain 2, 3, 4, 5, 6 or more species or types of neutral phospholipids.

In some embodiments, the implementation of the lipid component may be essentially neutral charge, because it contains a positively charged lipid and a negatively charged lipid. The lipid component may further comprise neutrally charged lipid(s) or phospholipid(s). The positively charged lipid may be a positively charged phospholipid. The negatively charged lipid may be a negatively charged phospholipid. The negatively charged phospholipid may be a phosphatidylserine such as dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS) or brain phosphatidylserine (BPS). The negatively charged phospholipid may be a phosphatidylglycerol such as dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), or dioleylphosphatidylglycerol (DOPG). In some embodiments, the composition further comprises cholesterol or polyethylene glycol (PEG). In other embodiments, the implementation of the composition essentially does not contain cholesterol. In some embodiments, the implementation of the phospholipid is a naturally occurring phospholipid. In other embodiments, the phospholipid is a synthetic phospholipid.

Liposomes can be prepared from one or more phospholipids, provided that the lipid material is substantially uncharged. It is important that the composition is substantially free of anionic and cationic phospholipids and cholesterol. Suitable phospholipids include phosphatidylcholines and others that are well known to those skilled in the art.

Another aspect of the invention includes methods for delivering an oligonucleotide to a cell, comprising contacting the cell with a neutral lipid composition of the invention. The methods will provide the composition of the invention in an effective amount. An effective amount is an amount of a therapeutic agent that attenuates, retards, reduces, or eliminates a cell, pathology, or disease state in a subject. The cell may be contained in a subject or patient, such as a human. The method may further include a method of treating cancer or other hyperplastic pathology. Cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lymph node, lung, nasopharynx, neck, prostate, skin, stomach , testis, tongue, ovary or uterus. In some embodiments, the method further includes a method of treating a non-malignant disease or hyperplastic pathology. The cell may be precancerous or cancerous. In some embodiments, the compositions and methods inhibit cell growth, induce apoptosis in a cell, and/or inhibit translation of an oncogene. The oligonucleotide can inhibit the translation of a gene that is overexpressed in the cancer cell.

In some embodiments, the methods of the invention further comprise administering additional therapy to a subject. Additional therapy may include the administration of a chemotherapeutic agent (eg, paclitaxel or docetaxel), surgery, radiation therapy, and/or gene therapy. In some aspects, the chemotherapy is docetaxel, paclitaxel, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, etoposide mitomycin (VP16, etoposide mitomycin ), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatin, 5-fluorouracil, vincristine, vinblastine, methotrexate, or combinations thereof. In some embodiments, the chemotherapy is a taxane such

- 4 042663 as docetaxal or paclitaxel. Chemotherapy may be given before, during, after, or a combination thereof, with respect to the neutral lipid composition of the invention. Chemotherapy may be administered within 0, 1, 5, 10, 12, 20, 24, 30, 48, or 72 hours or more of the neutral lipid composition. The neutral lipid composition, the second anti-cancer therapy, or both the neutral lipid composition and the anti-cancer therapy may be administered intratumorally, intravenously, intraperitoneally, subcutaneously, orally, or various combinations thereof.

It is contemplated that any embodiment discussed herein can be implemented with any method or composition of the invention, and vice versa. In addition, the compositions according to the invention can be used to achieve the methods according to the invention.

As used herein, substantially free with respect to the designated component is used herein to indicate that none of the specified components has been intentionally included in the composition and/or is present only as a contaminant or in trace amounts. Thus, the total amount of said component as a result of any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the designated component can be detected by standard analytical methods.

As used herein, a singular noun can mean one or more. As used herein in a formula, when used in combination with a singular noun, it can mean one or more.

The use of the term or in a claim is to be understood as and/or unless it is expressly stated that it refers only to alternatives or alternatives are mutually exclusive, although the disclosure supports a definition that refers only to alternatives and/or. As used herein, other can mean at least one more or more.

Throughout this document, the term about is used to indicate that a value includes the variant of the error inherent in the device, the method used to determine the value, or the variant that exists among the subjects of the study.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicative of preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to a person skilled in the art from this detailed description. .

Brief description of graphic materials

The following drawings form part of this specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

Fig. 1. Inhibition of STAT3 expression by a liposomal antisense STAT3 agent. The ability of a liposomal antisense STAT3 agent to inhibit STAT3 expression was tested in two lung adenocarcinoma cell lines, H1975 and HCC 827. A liposomal antisense STAT3 agent corresponding to SEQ ID NO: 4 (180 μg/ml) or an equivalent amount of empty liposomes was incubated with each cell line within four days.

Fig. 2A-2C. Viability suppression of non-small cell lung cancer cells with a liposomal antisense STAT3 agent. The ability of a liposomal antisense STAT3 agent to suppress the viability of non-small cell lung cancer cells was tested on three lung adenocarcinoma cell lines: H1975 (Fig. 2A), HCC 827 (Fig. 2B) and H358 (Fig. 2C).

A liposomal antisense STAT3 agent corresponding to one of SEQ ID NOs: 1-4 was incubated with each cell line for four days.

Fig. 3A, 3B. Viability suppression of leukemia and lymphoma cells with a liposomal antisense STAT3 agent. The ability of the liposomal antisense STAT3 agent to inhibit leukemia cell growth was tested in three human leukemia cell lines (K-562, MV4-11 and Kasumi-1) (FIG. 3A) and five human lymphoma cell lines (DOHH-2). (SU-DHL-4, SU-DHL-6, SU-DHL-10 and RL) (FIG. 3B). The liposomal antisense STAT3 agent corresponding to SEQ ID NO: 4 was incubated with each cell line for four days.

Description of exemplary embodiments

The present invention provides compositions and methods for delivering an anti-STAT3 oligonucleotide (eg, a gene expression inhibitor) into a cell with a lipid composition, in some aspects a lipid composition with a net charge of about zero, i. neutral lipid

- 5 042663 compositions. In some embodiments, the lipid composition is an uncharged liposome. These methods can be effectively used to treat cancer or an autoimmune disease.

lipids and liposomes.

The term liposomes is used herein to refer to lipid-containing vesicles having a lipid bilayer, as well as other lipid carrier particles that can capture or contain antisense oligonucleotides. As such, liposome is a general term encompassing a variety of single-layer, multi-layer and multi-purpose lipid carriers formed by the creation of closed lipid bilayers or aggregates. In addition, liposomes may have an indefinite lamellar structure. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilayer liposomes have many lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of an aqueous solution. The lipid components self-rearrange to form closed structures and trap water and solutes between lipid bilayers (Ghosh and Bachhawat, 1991). However, this invention also encompasses compositions that have other structures in solution than the normal vesicular structure. For example, lipids may have a micellar structure or simply exist as heterogeneous aggregates of lipid molecules.

Liposomes are a form of nanoparticles that are carriers for delivering various drugs to diseased tissue. The optimal size of liposomes depends on the target tissue. In tumor tissue, the vasculature is discontinuous and pore sizes vary from 100 to 780 nm (Siwak et al., 2002). In comparison, the pore size in normal vascular endothelium is <2 nm in most tissues and 6 nm in postcapillary venules. It is believed that negatively charged liposomes are more rapidly removed from the circulation than neutral or positively charged liposomes; however, recent studies have shown that the type of negatively charged lipid affects the rate of uptake of liposomes by the reticuloendothelial system (RES). For example, liposomes containing negatively charged lipids that are not sterically shielded (phosphatidylserine, phosphatidic acid, and phosphatidylglycerol) clear faster than neutral liposomes. Interestingly, cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) and cation-liposome-DNA complexes are more actively bound and taken up by endothelial cells of angiogenic blood vessels via endocytosis than anionic, neutral, or sterically stabilized neutral liposomes. (Thurston et al., 1998; Krasnici et al., 2003). Cationic liposomes may not be ideal delivery vehicles for tumor cells because surface interactions with tumor cells create a barrier effect to the binding site through electrostatic interactions, inhibiting further association of delivery systems with tumor spheroids (Kostarelos et al., 2004). However, neutral liposomes appear to be better able to penetrate into the tumor. The toxicity of specific liposome preparations is also a concern. Cationic liposomes cause dose-dependent toxicity and inflammation of the lungs by promoting the release of intermediate reactive oxygen species, and this effect is more pronounced for multivalent cationic liposomes than for monovalent cationic liposomes such as DOTAP (Dokka et al., 2000). Neutral and negative liposomes do not appear to exhibit pulmonary toxicity (Guitierrez-Puente et al., 1999). For cationic liposomes, despite effective capture of nucleic acids, there are few examples of successful gene silencing in vivo, perhaps due to their stable intracellular nature and, as a result, the inability to release the content - nucleic acids. Charge-neutral lipids or charge-neutralized lipid compositions, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), are used herein because of their neutral properties and success in delivering antisense oligonucleotides in vivo.

The present invention provides methods and compositions for combining an oligonucleotide, such as an antisense oligonucleotide, with a lipid and/or liposome. The oligonucleotide can be incorporated into the aqueous interior of the liposome, embedded in the lipid bilayer of the liposome, attached to the liposome via a binding molecule that is associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, suspended in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Compositions combined with liposomes or liposomes/oligonucleotides provided herein are not limited to any particular structure in solution. For example, they can be presented as a bilayer structure, as micelles, or as a compressed structure. They may also simply be dispersed in solution, possibly forming aggregates that are neither uniform in size nor shape.

Lipids.

Lipids are fatty substances that can be natural or synthetic. For example, lipids include fat droplets that naturally occur in the cytoplasm, as well as a class of compounds that are well known to those skilled in the art that contain long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes. An example is the lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

Lipid compositions according to this invention may contain phospholipids. In some embodiments, one kind or type of phospholipid can be used in the creation of lipid compositions such as liposomes. In other embodiments, more than one species or type of phospholipid may be used.

Phospholipids include glycerophospholipids and some sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine (DOPC), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylhofsphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, 1,2-diaracidoyl- sn-glycero-3-phosphocholine (DAPC), 1,2-dieucosenoylsn-glycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), palmitoyloleoyl phosphatidylcholine (POPC), 1-oleoyl-2-palmitoyl phosphatidylcholine (OPPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG) ), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylglycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA), dioleoyl phosphatidic acid (DOPA), dimyristoyl phosphatidylethanolamine (DMPE) , dipalmitoyl phosphatidylethanolamine (DPPE), distearoliphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylethanolamine (POPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), brain phosphatidylserine (BPS), distearoyl sphingomyelin (DSSP), brain sphingomyelin ( BSP), dipalmitoyl sphingomyelin (DPSP), lysophosphatidylcholine and lysophosphatidylethanolamine.

Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols and phosphatidylethanolamines; since phosphatidylethanolamines and phosphatidylcholines are not charged under physiological conditions (ie, at a pH of about 7), these compounds may be particularly useful for the production of neutral liposomes. In some embodiments, the implementation of the DOPC phospholipid is used to obtain uncharged liposomes or lipid compositions. In some embodiments, a lipid that is not a phospholipid (eg, cholesterol) can also be used.

Phospholipids may be from natural or synthetic sources. However, phospholipids from natural sources, such as egg or soy phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin, and plant or bacterial phosphatidylethanolamine, are not used in some embodiments as the primary phosphatidine (i.e., is 50 % or more of the total phosphatide composition), since this can lead to instability and leakage of the resulting liposomes.

neutral liposomes.

Neutral liposomes or lipid composition or uncharged liposomes or lipid composition, as used herein, are defined as liposomes or lipid compositions having one or more lipids that give a substantially neutral net charge (essentially uncharged). In some embodiments, neutral liposomes or lipid compositions may contain primarily lipids and/or phospholipids that are themselves neutral. In some embodiments, amphipathic lipids can be included in or used to create neutral liposomes or lipid compositions. For example, a neutral liposome can be created by combining positively and negatively charged lipids so that these charges essentially cancel each other out, resulting in a substantially neutral net charge. By substantially neutral or substantially uncharged is meant that few, if any, lipids in a given population (e.g., populations of liposomes) contain a charge that is not offset by the opposite charge of another component (e.g., less than 10% of the components contain an uncompensated charge, more preferably less than 5% and most preferably less than 1%). In some embodiments of this invention, a composition can be prepared in which the lipid component of the composition is essentially neutral, but not in the form of liposomes.

The size of liposomes varies depending on the method of synthesis. A liposome suspended in an aqueous solution is generally in the form of a spherical vesicle and may have one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel array of molecules represented by the formula XY, where X is a hydrophilic moiety and Y is a hydrophobic moiety. In an aqueous suspension, the concentric layers are arranged such that the hydrophilic moieties tend to remain in contact with the aqueous phase and the hydrophobic regions tend to self-assembly. For example, when aqueous phases are present within a liposome, lipid molecules can form a bilayer known as a lamella with an XY-YX arrangement. Lipid aggregates can form when the hydrophilic and hydrophobic portions of more than one lipid molecule become associated with each other. The size and shape of these aggregates will depend on many different variables such as the nature of the solvent and the presence of other compounds in solution.

Liposomes within the scope of this invention can be prepared in accordance with known laboratory techniques, such as, for example, the method of Bangham et al. (1965), the contents of which are incorporated herein by reference; the method of Gregoriadis (1979), the contents of which are incorporated herein by reference; the method of Deamer and Uster (1983), the contents of which are incorporated by reference; and the reverse phase evaporation method described by Szoka and Papahadjopoulos (1978). The above methods differ in their respective abilities to capture aqueous material, and their respective ratios of water space to lipid.

In some embodiments, a neutral liposome may be used to deliver an oligonucleotide, such as an antisense oligonucleotide. The neutral liposome may contain one kind of oligonucleotides that target the translation of one gene, or the neutral liposome can contain several kinds of oligonucleotides that target the translation of multiple genes. In addition, the neutral liposome may also contain a chemotherapeutic agent in addition to the oligonucleotide; thus, in some embodiments, the chemotherapeutic agent and the oligonucleotide can be delivered to a cell (eg, a cancer cell in a human patient) in the same or separate compositions.

Dried lipids or lyophilized liposomes can be dehydrated and reconstituted with an appropriate concentration of a suitable solvent (eg DPBS buffer or Hepes). The mixture can then be actively mixed in a vortex mixer. Liposomes can be resuspended at an appropriate concentration of total phospholipid (eg, about 10-200 mM). The unencapsulated oligonucleotide can be removed by centrifugation at 29,000 g and the liposomal pellets washed. Alternatively, unencapsulated oligonucleotides can be removed by dialysis against excess solvent. The amount of encapsulated oligonucleotide can be determined using standard methods.

Inhibition of gene expression.

An inhibitory oligonucleotide can inhibit transcription or translation of a gene in a cell. The length of the oligonucleotide may be from 5 to 50 or more nucleotides, and in some embodiments from 7 to 30 nucleotides. In some embodiments, the oligonucleotide may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 in length, 28, 29 or 30 nucleotides. The oligonucleotide may contain a nucleic acid and/or a nucleic acid analog. Typically, an inhibitory oligonucleotide will inhibit the translation of a single gene in a cell; however, in some embodiments, an inhibitory oligonucleotide may inhibit translation of more than one gene within a cell.

Within an oligonucleotide, the components of an oligonucleotide need not be of the same type or homogeneous throughout (eg, an oligonucleotide may contain a nucleotide and a nucleic acid or nucleotide analog). In some embodiments of this invention, the oligonucleotide may contain only one nucleic acid or nucleic acid analog. The inhibitory oligonucleotide may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more contiguous nucleobases, including all ranges in between. , which hybridize with a complementary nucleic acid to form a double-stranded structure.

Nucleic acids.

The present invention provides methods and compositions for delivering an oligonucleotide using neutral liposomes. Since an oligonucleotide is composed of a nucleic acid, methods relating to nucleic acids (eg, nucleic acid production, nucleic acid modification, etc.) can also be used on the oligonucleotide.

The term nucleic acid is well known in the art. The term nucleic acid, as used herein, generally refers to a molecule (ie, strand) of DNA, RNA, or a derivative or analogue thereof that contains a nucleic base. These definitions refer to a single-stranded or double-stranded nucleic acid. Double-stranded nucleic acids can be formed by fully complementary binding; however, in some embodiments, the implementation of the double-stranded nucleic acid can be formed by partial or sufficient complementary binding. As used herein, the term single-stranded nucleic acid may be denoted by the prefix oc and double-stranded nucleic acid by the prefix ds.

Nucleic bases.

As used herein, the term nucleobase refers to a heterocyclic base, such as, for example, a naturally occurring nucleobase (i.e., A, T, G, C, or U) found in at least one naturally occurring nucleic acid (ie, DNA and RNA), as well as naturally occurring or non-naturally occurring derivatives and analogues of such a nucleic base. A nucleobase can typically form one or more hydrogen bonds (i.e., anneal or hybridize) with at least one naturally occurring nucleobase in a manner that can replace naturally occurring nucleobase pairs (i.e., hydrogen bonding between A and T, G and C and A and U). The nucleobase can be incorporated into a nucleoside or nucleotide using any of the chemical or natural synthesis methods described herein or known to those skilled in the art.

Purine and/or pyrimidine nucleobase(s) encompasses naturally occurring purine and/or pyrimidine nucleobases, as well as derivatives and analogs thereof, including, but not limited to, a purine or pyrimidine substituted with one or more alkyl, carboxyalkyl, amino, hydroxyl, halogen (ie fluoro, chloro, bromo or iodo), thiol or alkylthiol moieties. Preferred alkyl (eg, alkyl, caboxyalkyl, etc.) moieties contain from about 1, about 2, about 3, about 4, about 5 to about 6 carbon atoms. Other non-limiting examples of a purine or pyrimidine include deazapurine, 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine, 8-bromoguanine, 8-chloroguanine, bromtyline, 8-aminoguanine, 8-hydroxyguanine, 8-methylguanine, 8-thioguanine , azaguanine, 2-aminopurine, 5-ethylcytosine, 5-methylcyosine, 5-bromouracil, 5-ethyluracil, 5-ioduracil, 5-chlorouracil, 5-propyluracil, thiouracil, 2-methyladenine, methylthioadenine, K, K-dimethyladenine, azaadenines , 8-bromadenine, 8-hydroxyadenine, 6-hydroxyaminopurine, 6-thiopurine, 4-(6-aminohexyl/cytosine) and the like.

Purine and pyrimidine derivatives or analogs include, but are not limited to (abbreviation/description of modified base):

ac4c/4-acetylcytidine, Mam5s2u/5-methoxyaminomethyl-2-thiouridine,

Chm5u/5-(carboxyhydroxylmethyl)uridine, Man q/Beta, D-mannosylqueosin, Cm/2'-O-methylcytidine, Mcm5s2u/5-methoxycarbonylmethyl-2-thiouridine, Cmnm5s2u/5-carboxymethylamine-methyl-2-thiouridine, Mcm5u /5-methoxycarbonylmethyluridine, Cmnm5u/5-carboxymethylaminomethyluridine, Mo5u/5-methoxyuridine,

D/Dihydrouridine, Ms2i6a, 2-methylthio-K6-isopentenyladenosine, Fm/2'-O-methylpseudouridine, Ms2t6a/N-((9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbomoyl)threonine,

Gal q/Beta, D-galactosylqueosin,

Mt6a/N-((9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbomoyl)threonine, Gm/2'-O-methylguanosine, Mv/Uridine-5-hydroxyacetic acid methyl ester, I/Inosine, o5u /uridine-5-hydroxyacetic acid (v), I6a/N6-isopentenyladenosine, Osyw/Vibutoxosine, m1a/1-methyladenosine, P/Pseudouridine, m1f/1-methylpseudouridine, Q/Queosine, m1g/1-methylguanosine, s2c/2 -thiocytidine, m1I/1-methylinosine, s2t/5-methyl-2-thiouridine, m22g/2,2-dimethylguanosine, s2u/2-thiouridine, m2a/2-methyladenosine, s4u/4-thiouridine, m2g/2-methylguanosine , T/5-methyluridine, m3c/3-methylcytidine, t6a/N-((9-beta-D-ribofuranosylpurin-6-yl)carbamoyl)threonine, m5c/5-methylcytidine, Tm/2'-O-methyl- 5-methyluridine, m6a/N6-methyladenosine, Um/2'-O-methyluridine, m7g/7-methylguanosine, Yw/Vibutosine, Mam5u/5-methylaminomethyluridine, or X/3-(3-amino-3-carboxypropyl)uridine, (acr3)p. Nucleosides.

As used herein, a nucleoside refers to a single chemical entity containing a nucleobase covalently attached to a linker moiety of the nucleobase. A non-limiting example of a nucleic linker moiety is a sugar containing 5 carbon atoms (i.e., a 5-carbon sugar), including, but not limited to, deoxyribose, ribose, arabinose, or a 5-carbon sugar derivative or analog. Non-limiting examples of a 5-carbon sugar derivative or analog include 2'-fluoro-2'-deoxyribose or carbocyclic sugar, where the carbon is replaced by an oxygen atom in the sugar ring. As used herein, a fragment generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure.

Various types of covalent attachment(s) of a nucleic base to a linker fragment of a nucleic base are known in the art. As a non-limiting example, a nucleoside containing a purine (ie, A or G) or 7-deazapurine nucleobase typically contains a covalent attachment of the 9 position of the purine or 7-deazapurine to the 1' position of the 5-carbon sugar. In another non-limiting example, a nucleoside containing a pyrimidine nucleobase (ie, C, T, or U) typically contains the covalent attachment of the 1 position of the pyrimidine to the 1' position of a 5 carbon sugar (Kornberg and Baker, 1992).

- 9 042663

Nucleotides.

As used herein, a nucleotide refers to a nucleoside further comprising a backbone bond. A backbone bond generally covalently attaches a nucleotide to another molecule containing the nucleotide or to another nucleotide to form a nucleic acid. The backbone bond of naturally occurring nucleotides typically contains a phosphate group (eg, a backbone phosphodiester bond) that is covalently attached to a 5-carbon sugar. Attachment of the backbone fragment usually occurs at the 3' or 5' position of the 5-carbon sugar. However, other types of attachments are known in the art, in particular where the nucleotide contains derivatives or analogues of a naturally occurring 5-carbon sugar or phosphate moiety.

Nucleic acid analogues.

The nucleic acid may contain or consist entirely of a derivative or analog of the nucleic base, a linker portion of the nucleic base and/or a backbone bond, which may be present in a naturally occurring nucleic acid. As used herein, a derivative refers to a chemically modified or altered form of a naturally occurring molecule, while the terms mimic or analog refer to a molecule that may or may not resemble a naturally occurring molecule or fragment structurally, but has similar functions. . Nucleic, nucleoside and nucleotide analogs or derivatives are well known in the art.

Non-limiting examples of nucleosides, nucleotides, or nucleic acids containing a 5-carbon sugar and/or backbone-linked derivatives or analogs include those in US Pat. No. 5,681,947, which describes oligonucleotides containing purine derivatives that form triple helices with and /or prevent the expression of dsDNA;

in US Pat.

in US patent No. 5614617, which describes analogues of oligonucleotides with substitutions on pyrimidine rings, which have increased nuclease stability;

in US Pat.

US Pat. No. 5,446,137, which describes oligonucleotides containing at least one 5-carbon sugar moiety substituted at the 4' position with a non-hydrogen substituent that can be used in hybridization assays;

US Pat. No. 5,886,165, which describes oligonucleotides with both 3'-5' backbone deoxyribonucleotides and 2'-5' backbone ribonucleotides;

US Pat. No. 5,714,606, which describes a modified backbone bond wherein the oxygen at the 3' position of the backbone bond is replaced by a carbon atom to increase the resistance of nucleic acids to nucleases;

in US patent No. 5672697, which describes oligonucleotides containing one or more 5'-methylenephosphonate backbone bonds, which increase resistance to nucleases;

in US Pat.

US Pat. No. 5,223,618, which describes oligonucleotide analogs with 2 or 3 backbone carbon bonds linking the 4' position and the 3' position of an adjacent 5-carbon sugar moiety to increase cellular uptake, nuclease resistance, and hybridization to an RNA target;

US Pat. No. 5,470,967, which describes oligonucleotides containing at least one backbone sulfamate or sulfamide bond that are useful as nucleic acid hybridization probes;

US Pat.

US Pat. No. 5,858,988, which describes a hydrophobic carrier agent attached to the 2'-O position of oligonucleotides to enhance their membrane permeability and stability;

in US patent No. 5214136, which describes oligonucleotides conjugated with an anthraquinone at the 5'-end, which have enhanced hybridization with DNA or RNA; increased resistance to nucleases;

US Pat. No. 5,700,922, which describes PNA-DNA-PNA chimeras in which the DNA contains 2'-deoxy-erythro-pentofuranosyl nucleotides to increase nuclease resistance, binding affinity, and the ability to activate RNase H;

in US Pat. No. 5,708,154 which describes RNA coupled to DNA to form a hybrid

DNA-RNA;

in US patent No. 5908845, which describes polyester nucleic acids in which one or more nucleic bases are connected to chiral carbon atoms in the backbone of the polyester;

US Pat. Nos. 5,786,461, 5,891,625, 5,786,461, 5,773,571; 5,766,855; , contain one or more nucleotides or nucleosides that contain a nucleobase moiety, a nucleolinker moiety that is not a 5-carbon sugar (e.g. aza nitrogens, amido and/or ureido compounds), and/or a backbone bond that is not is a backbone phosphate bond (eg, aminoethylglycine, polyamide, polyethylene, polythioamide, polysulfinamide or polysulfonamide backbone bond); and US Pat. No. 5,855,911, which describes a hydrophobic, nuclease-resistant P-ethoxy backbone bond.

Other modifications and uses of nucleic acid analogs are known in the art, and it is expected that these methods and types of nucleic acid analogs can be used with this invention.

Preparation of nucleic acids.

The nucleic acid can be obtained by any method known to the person skilled in the art, such as chemical synthesis, enzymatic or biological production. Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide) include a nucleic acid prepared by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase methods, such as those described in EP 266032, which is incorporated herein by reference, or using deoxynucleoside H-phosphonate intermediates as described by Froehler et al. (1986) and US Pat. No. 5,705,629, each of which is incorporated herein by reference. One or more kinds of oligonucleotides can be used in the methods of this invention. Various mechanisms for the synthesis of oligonucleotides have been disclosed in, for example, US Pat.

Purification of nucleic acids.

Nucleic acid can be purified on polyacrylamide gels, cesium chloride gradient centrifugation, or by any other means known to those skilled in the art (see, for example, Sambrook et al. (2001), incorporated herein by reference).

In some embodiments, this invention provides a nucleic acid that is an isolated nucleic acid. As used herein, the term isolated nucleic acid refers to a nucleic acid molecule (e.g., an RNA or DNA molecule) that has been separated from, or otherwise purified from, the bulk of genomic and transcribed nucleic acids from one or more cells. In some embodiments, an isolated nucleic acid refers to a nucleic acid that has been separated from or otherwise purified from the bulk of cellular components or reactive components in vitro, such as, for example, macromolecules such as lipids or proteins, small biological molecules, etc. P.

Hybridization.

As used herein, hybridizing, hybridizing, or capable of hybridizing refers to the formation of a two- or three-stranded molecule, or a molecule with a partial two- or three-stranded nature. As used herein, annealing is synonymous with hybridize.

As used herein, stringent conditions or high stringency are those that allow hybridization between or within one or more nucleic acid strands containing complementary sequence(s) but exclude hybridization of random sequences. Stringent conditions allow for a slight mismatch between the nucleic acid and the target strand, if any. Such conditions are well known to those skilled in the art and are preferred for applications requiring high selectivity.

Stringent conditions may include low salt and/or high temperature conditions, such as providing about 0.02 to about 0.15 M NaCl at temperatures of about 50 to about 70°C. It is understood that the temperature and ionic strength of the desired stringency is determined in part by the length of the particular nucleic acid(s), the length and nucleotide composition of the target sequence(s), the charge composition of the nucleic acid(s), and the presence or concentration of formamide,

- 11 042663 tetramethylammonium chloride or other solvent(s) in the hybridization mixture.

It is also understood that these ranges, compositions and conditions for hybridization are only mentioned as non-limiting examples and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison with one or more positive or negative controls. Depending on the intended application, it is preferable to use different hybridization conditions to achieve different degrees of nucleic acid selectivity for the target sequence. In a non-limiting example, the identification or isolation of a related target nucleic acid that does not hybridize to the nucleic acid under stringent conditions can be achieved by hybridization at low temperature and/or high ionic strength. Such conditions are referred to as low stringency or low stringency conditions, and non-limiting examples of low stringency include hybridization conducted at about 0.15 to about 0.9 M NaCl over a temperature range of about 20 to about 50°C. Of course, one skilled in the art can further modify the low or high stringency conditions to suit a particular application.

Method for producing a liposomal P-ethoxy antisense drug product.

Antisense oligonucleotides (oligos) complementary to certain regions of the target mRNA were used to inhibit the expression of endogenous genes. When antisense oligonucleotides bind to a target mRNA, a DNA-RNA hybrid is formed. This hybrid construct inhibits translation of the mRNA and thus the expression of the encoded protein. If a protein is essential for cell survival, inhibition of its expression can lead to cell death. Therefore, antisense oligonucleotides may be useful tools in anticancer and antiviral therapy.

The main barriers to using antisense oligonucleotides to inhibit gene expression are cell instability, low cellular uptake, and poor intercellular delivery. Natural phosphodiesters are not resistant to nuclease hydrolysis; thus, high concentrations of antisense oligonucleotides are required before any inhibitory effect is observed. Modified phosphodiester analogues such as P-ethoxy have been made to overcome this problem of nuclease hydrolysis, but have not provided a satisfactory solution.

Cellular uptake of antisense oligonucleotides is low. To overcome this problem, physical methods such as calcium phosphate precipitation, DEAE-dextran mediation, or electroporation have been used to increase cellular uptake of oligonucleotides. These methods are difficult to reproduce and are not applicable in vivo. Cationic lipids such as lipofectin are also used to deliver oligonucleotides. An electrostatic interaction is formed between cationic lipids and negatively charged oligonucleotides, leading to the formation of a complex, which is then taken up by target cells. Because these cationic lipids do not protect oligonucleotides from nuclease cleavage and are harmful to the cell membrane, they are only useful for delivering nuclease-resistant phosphorothioates, but not nuclease-cleavable phosphodiesters.

Another modified phosphodiester analogue that has been prepared is Pethoxy. The P-ethoxy antisense backbone does not adversely affect bleeding and complement activation, which are some of the toxic effects that have been reported for other antisense analogs. Modifications to P-ethoxy oligonucleotides are carried out in the phosphate backbone so that the modification will not interfere with the binding of these oligonucleotides to the target mRNA. P-ethoxy oligonucleotides are prepared by adding an ethyl group to the non-bridging oxygen atom of the phosphate backbone, making these oligonucleotides uncharged compounds. Despite their resistance to nucleases, cellular uptake and intracellular delivery of P-ethoxy oligonucleotides is poor because upon internalization, these oligonucleotides remain locked inside endosomal/lysosomal vacuoles, making it difficult for them to access the target mRNA.

P-ethoxy antisense drug.

The liposomal P-ethoxy antisense drug product consists of two cGMP products, both of which have an FDA-required Certificate of Analysis with FDA-approved release criteria. Raw materials, solvents and the final medicinal product are described in this document. When manufactured, the drug product is an amber or white lyophilized crystal or powder that contains the following materials: an oligonucleotide (eg, antisense P-ethoxy drug), neutral lipids (eg, DOPC), and a surfactant (eg, polysorbate 20). In preparation for administration to a patient, a saline solution is added to the tube, and at this time liposomes are formed with a P-ethoxy antisense agent, which is placed in the interior of the liposome.

P-ethoxy antisense drug.

Specific physical properties (such as solubility and hydrophobicity, which are then influenced

solubility of the drug product in saline, oligo incorporation into liposomes, and liposomal particle size) of the finished product can be determined using a predetermined mixture of raw material P-ethoxy and phosphodiester amidite, in the production of P-ethoxy antisense drug substance. Although the loss of the P-ethoxy backbone group occurs randomly during the preparation of oligonucleotides, resulting in phosphodiester linkages in these units, this loss may not create the desired ratio of P-ethoxy:Phosphodiester backbone linkages within the oligonucleotide. In this case, the raw material mixture of P-ethoxy and phosphodiester amidite complements the expected amount of deletions of the P-ethoxy backbone, thus creating an oligonucleotide with the desired ratio. An increase in the number of P-ethoxy molecules in the oligonucleotide backbone results in the molecule becoming more hydrophobic (resulting in larger liposomal particles; Table 1), less polar, and less soluble (Table 2). Methods for testing a charge-neutral hydrophobic P-ethoxy drug include mass spectrometry to determine the length distribution of oligonucleotides, and assays to determine drug solubility, which for practical solubility purposes is visual inspection of a drug product reconstituted in saline. As the oligonucleotide becomes less soluble due to more P-ethoxy backbone bonds, the reconstituted solution becomes whiter while particles are being formed as the hydrophobicity becomes too high.

The formulation should use a particle size of which 90% is less than 5000 nm and is soluble as a function of the nucleotide composition. For example, if the oligonucleotide is 18-20 nucleotides in length, then at least five backbone phosphate bonds must be backbone phosphodiester bonds. This is confirmed by experiments 7-10, shown in table. 1, which shows data on 18-mer oligonucleotides. However, if the length of the oligonucleotide is 25 nucleotides, then at least six phosphate bonds of the backbone must be phosphodiester bonds of the backbone.

Table 1

Liposomal particle size variability with antisense agent backbone composition

Ethyl backbone removal after production Particle size characteristics: Cumulative distribution function EXPERI -ment Constructed frame antisense agent Main Peak ¹ Deletion of mixture ^e 90% value (nm) ** 50% value (nm) 300 nm value (%) 1 3-ex amidite substitution -6 -5.67 2130 911 15.30 2 3-ex -6 -5.67 2420 1004 15.50

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amidite substitution 3 3-ex amidite substitution -6 -6.12 3682 +943 15.50 4 3-ex amidite substitution -7 -6.66 3805 +978 14.60 5 100% R- ethoxy -5 -5.66 3924 +976 16.00 6 2-uh amidite substitution -5 -5.32 4387 1888 11.60 7 ^a 100% Retoxy -4 -4.22 5057 1131 17.70 8 100% R- ethoxy -4 -4.52 5659 1359 10.00 9 ^b 100% Retoxy -4 -4.38 7571 1909 2.60 ^10th 100% R- ethoxy -4 -4.38 7994 1653 14.40

** The drug release criterion for 90% liposome particles must be less than or equal to 5000 nm.

^a This batch was discarded due to poor solubility; in particular, antisense particles in reconstituted solution.

^b This batch had a lower volume of DMSO and tBA with 2 mg antisense agent in a 20 ml vial, which added an additional component to increase liposomes.

^c This batch was not released because it did not meet the particle size release specification.

^d The main peak represents the most common number of P-ethoxy deletions in the oligonucleotide population.

^e Mixture deletion is the average number of P-ethoxy deletions in the oligonucleotide population.

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table 2

Solubility of Liposomal Particles with Antisense Agent Backbone Composition

Ethyl backbone removal after production Solubility of the drug agent Experiment The engineered backbone of the antisense agent Basic peak Deletion of mixture ^e visual observation ** Solubility assessment 1 3-amidite substitution -6 -5.67 the solution looks like skimmed milk good 2 3-amidite substitution -6 -5.67 the solution looks like skimmed milk good 3 3-amidite substitution -6 -6.12 the solution looks like skimmed milk good 4 3-amidite substitution -7 -6.66 the solution looks like skimmed milk good 5 100% P-ethoxy -5 -5.66 the solution looks like skimmed milk good 6 2-amidite substitution -5 -5.32 the solution looks like skimmed milk good 7 100% P-ethoxy -4 -4.52 white solution satisfy spruce 8 ^b 100% P-ethoxy -4 -4.38 white satisfy solution spruce ^9th 100% P-ethoxy -4 -4.38 white solution satisfy spruce 10 ^a 100% P-ethoxy -4 -4.22 particles of white solution not suitable

** If there are particles in the drug sample, the lot will be rejected.

^a This batch was discarded due to poor solubility; in particular, antisense particles in reconstituted solution.

^b This batch had a lower volume of DMSO and tBA with 2 mg antisense agent in a 20 ml vial, which added an additional component to increase liposomes.

^c This batch was not released because it did not meet the particle size release specification.

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Preparation, filtration and lyophilization of a liposomal P-ethoxy antisense drug product.

g of oligo pE is dissolved in DMSO at a ratio of 10 mg of oligonucleotide per 1 ml of DMSO. DOPC is then added to tert-butyl alcohol in a ratio of 1 g DOPC per 1719 ml tert-butyl alcohol. Oligo and DOPC are combined and mixed in a ratio of 1 g of oligonucleotide to 2.67 g of DOPC. Then, 20 ml of a 0.835% (v/v) solution of polysorbate 20 was added to the mixture, resulting in a final concentration of 0.039 mg/ml. The solution is passed through a sterile filter before filling into glass vials for lyophilization.

The effect of surfactant on liposome particle size was determined by titrating the amount of surfactant (Table 3). In the absence of polysorbate 20, only 2.8% of the particles had a diameter of 300 nm or less. In the presence of 1x polysorbate 20, 12.5% of the particles had a diameter of 300 nm or less. With the addition of 3x-10x polysorbate 20, about 20% of the particles had a diameter of 300 nm or less. Thus, increasing the amount of surfactant from 1x to 3x results in a decrease in particle size.

Table 3

Liposomal Particle Size Variability with Surfactants

Particle size specifications: Cumulative distribution function Experiment Amount of surfactant 50% value 90% meaning ** 300 nm value 1 Oh 5301 nm 10719 nm 2.8% 2 1x 1053 nm 4054 nm 12.5% 3 Zx 785 nm 2926 nm 19.1% 4 5x 721 nm 2691 nm 21.9% 5 yuh 734 nm 2937 nm 21.4%

** The drug release criterion for 90% liposome particles must be less than or equal to 5000 nm.

Preparation of a liposomal P-ethoxy antisense drug product for administration.

The lyophilized preparation was hydrated with normal saline (0.9%/10 mM NaCl) to a final oligo concentration of 10-5000 μM. Liposomal-P-ethoxy-oligos were mixed by hand shaking.

Methods for testing a liposomal P-ethoxy antisense drug product.

Visual inspection of the manufactured medicinal product.

After manufacturing, a trial vial containing the medicinal product is selected and a visual inspection is carried out. The absence of liquid is mandatory and amber crystals at the bottom of the vial are then acceptable, and acceptability increases towards a white, flaky powder or appearance, which is the best result. The white appearance indicates a better drying process with a high surface area to mass ratio, which is very conducive to recovery for use.

Visual Inspection of Patient Ready Reconstituted Drug IV: Normal saline is added to the ampoule containing the manufactured liposome antisense P-ethoxy drug product and shaken to reconstitute as a solution with a completely dissolved drug crystal or powder. There are three main observations:

1) so that the crystal or powder is completely dissolved,

2) there are no white clots of undissolved material and

3) Appearance is milky white or looks like skimmed milk.

The bluer the reconstituted liquid looks, the better, as it signals a smaller liposomal particle size that reflects light in the blue spectrum.

Mass spectrometry: Mass spectrometry (mass spectra) is used to display the profile of various masses in a sample. When an antisense P-ethoxy material is produced, a mass spectrometry analysis is performed on the sample. The result shows peaks of material present on the grid, which has increasing mass on the x-axis to the right, and relative abundance on the y-axis increasing upward. The profile from the sample is analyzed to determine the relative number of P-ethoxy backbones in the P-ethoxy sample, recognizing that the peak profile is (from the far right) full length material with all backbones consisting of a P-ethoxy bond, the next peak on the left is full length with one backbone with a deletion

- 16 042663

P-ethoxy (and hence the ethyl removed, resulting in a normal backbone phosphodiester bond), etc. The mass spectrum pattern shifted to the right represents a P-ethoxy sample having more P-ethoxy backbones and therefore having the properties of being more hydrophobic and less soluble; and similarly, shifting to the left has opposite effects. Checking the mass spectrometry plot of a sample can also be used to determine if filtration during manufacture has any adverse effect on the composition of the oligonucleotides present in the filtered drug product.

UV Testing: UV light testing is used to determine the mass of an oligonucleotide present in a sample. Oligonucleotides absorb light in the 260 nm range. As a result, UV testing of the finished reconstituted drug product has come to be used as a method for determining the amount of oligonucleotide drug substance in a drug vial. In terms of manufacturing development and innovation, UV testing has been used to determine if there are problems during filtration in manufacturing or poor solubility of the P-ethoxy antisense drug, resulting in fewer oligonucleotides in solution and therefore more low UV readings. The method will be tested and will probably be part of the final testing of the product.

Liposomal particle size: The vial of the finished drug product is diluted to the original concentration and tested for liposomal particle size. The result is often a coarse normal distribution having a center point, tails and means, or a coarse normal distribution of most particles and smaller secondary peaks of smaller liposome particles resulting from second order particle formation effects. It is important that the liposome particles are not too large as they can cause side effects in patients (eg, create blood flow problems in small blood vessels in the lungs). As a result, the criteria for drug product release include particle size testing demonstrating that 90% of the liposomes are 5 µm or smaller. In addition, smaller liposomes are preferred because they will be better taken up by cells, and secondly, smaller liposomes can penetrate into the pores of vessels, thereby allowing liposomes to enter the interior of tumors, increasing the effectiveness of liposome P-ethoxy antisense drug treatment. product.

Methods of treatment.

According to certain aspects of the present invention, an oligonucleotide-lipid complex (eg, an oligonucleotide incorporated in an uncharged liposome) is provided for the treatment of diseases such as cancer, an autoimmune disease, or an infectious disease. In particular, the oligonucleotide may have a sequence that allows base pairing with a human nucleotide sequence and thus can inhibit the expression of a protein encoded by the human nucleotide sequence.

Treating and treating refer to the administration or application to a subject of a therapeutic agent, or the performance of a procedure or method of treatment for a subject to obtain a therapeutic benefit for a health-related disease or pathology. For example, treatment may include administering a pharmaceutically effective amount of the oligonucleotide-lipid complex.

Subject and patient refer to human or non-human, such as primate, mammal, and vertebrates. In specific embodiments, the subject is a human.

The term therapeutic benefit or therapeutically effective, as used throughout this specification, refers to anything that promotes or improves a subject's well-being in the treatment of such a pathology. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, cancer treatment may include, for example, reducing the size of the tumor, reducing the invasiveness of the tumor, reducing the rate of cancer growth, or preventing metastasis. Treatment of cancer may also refer to prolonging the survival of a subject with cancer. Treatment of an autoimmune disease may include, for example, reducing the expression of a self-antigen against which an unwanted immune response occurs, induction of tolerance to a self-antigen against which there is an unwanted immune response, or inhibition of an immune response against a self-antigen. Treatment of an infectious disease may include, for example, eliminating the infectious agent, reducing the level of the infectious agent, or maintaining the level of the infectious agent at a certain level.

Tumors for which these treatments are useful include any type of malignant cell, such as those found in solid tumor, hematologic tumor, metastatic cancer, or non-metastatic cancer. Illustrative solid tumors may include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, esophagus, gastrointestinal tract, gingiva, liver, skin, stomach, testis, tongue , uterus, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, bone, lung, bladder, melanoma, prostate, and breast. Typical hematologic tumors include bone marrow tumors, T- or B-lymphocyte malignancies, leukemias, lymphomas such as, for example, diffuse large B-cell lymphoma, blastomas,

- 17 042663 myeloma, etc. Additional examples of cancers that can be treated using the methods provided herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell lung cancer), abdominal cancer, hepatocellular carcinoma, gastric or gastric cancer (including gastrointestinal cancer and cancer of the gastrointestinal stroma), pancreatic cancer (including pancreatic ductal adenocarcinoma ), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, renal or kidney cancer, prostate cancer, vulvar cancer , thyroid cancer, various types of head and neck cancer, melanoma, superficially spreading melanoma, lentigo malignant melanoma, peripheral macular melanoma, nodular melanoma, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small cell lymphocytic (ML) NHL; intermediate grade/follicular NHL; intermediate degree diffuse NHL; high degree immunoblastic NHL; high degree lymphoblastic NHL; a high degree of small-celled with non-cleaved nucleus of NHL cells; NHL with massive lymphadenopathy; diffuse large B-cell lymphoma; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloid leukemia.

Cancer can specifically be of the following histological type, although not limited to these: neoplasm, malignant; carcinoma; undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; a combination of hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; lymphoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial colipolyposis; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; acidophilic carcinoma; oxyphilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; carcinoma of the adrenal cortex; endometrial carcinoma; carcinoma of the skin appendages; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; cricoid cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary glands; acinar cell carcinoma; glandular squamous cell carcinoma; adenocarcinoma/squamous metaplasia; thymoma, malignant; ovarian stroma tumor, malignant; thecoma, malignant; folliculoma, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; paraganglioma outside the mammary gland, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; pigmentless melanoma; superficially spreading melanoma; malignant melanoma in a giant pigmented nevus; melanoma of epithelioid cells; blue blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchioma, malignant; Brenner's tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonic carcinoma; teratoma, malignant; thyroid teratoma, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of the bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblast fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillar astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodentroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; neurogenic tumor of the olfactory organs; meningioma, malignant; neurofibrosarcoma; neuroma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease;

- 18 042663 paragranuloma; malignant lymphoma, small cell lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; fungal mycosis; other specific non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative disease of the small intestine; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

Autoimmune diseases for which these treatments are useful include, but are not limited to, systemic lupus erythematosus, spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis, diabetes mellitus, celiac disease, autoimmune thyroid disease, autoimmune liver disease, Addison's disease, graft rejection, graft-versus-host disease, host-versus-graft disease, ulcerative colitis, Crohn's disease, irritable bowel disease, inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis, familial Mediterranean fever, amyotrophic lateral sclerosis, Sjögren's syndrome, early arthritis , viral arthritis, multiple sclerosis or psoriasis. Diagnosis and treatment of these diseases are well documented in the literature.

Infectious diseases for which these treatments are useful include, without limitation, bacterial infections, viral infections, fungal infections, and parasitic infections. Exemplary viral infections include infections with hepatitis B virus, hepatitis C virus, human immunodeficiency virus 1, human immunodeficiency virus 2, human papillomavirus, herpes simplex virus 1, herpes simplex virus 2, herpes zoster, varicella, Coxsackie A16 virus, cytomegalovirus , ebola virus, enterovirus, Epstein-Barr virus, hanatavirus, hendravirus, viral meningitis, respiratory syncytial virus, rotavirus, West Nile virus, adenovirus, and influenza virus. Illustrative bacterial infections include those of Chlamydia trachomatis, Listeria monocytogenes, Helicobacter pylori, Escherichia coli, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intraceliuiar e, M. kansaii, M. gordonae ), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Streptococcus pyogenes (group A streptococci), Streptococcus agalactiae (group B streptococci), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic species Campylobacter, видами Enterococcus, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, видами Corynebacterium, Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, видами Bacteroides, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, Actinomyces israelli, Shigella spp. (eg S. flexneri, S. sonnei, S. dysenteriae), and Salmonella spp. Exemplary fungal infections include Candida albicans, Candida glabrata, Aspergillus fumigatus, Aspergillus terreus, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, and Chlamydia irachomatis infections.

The oligonucleotide-lipid complex can be used herein as an antitumor, antiviral, antibacterial, antifungal, antiparasitic or anti-autoimmune agent in various forms. In a specific embodiment, the invention provides methods of using an oligonucleotide-lipid complex, comprising contacting a population of diseased cells with a therapeutically effective amount of the oligonucleotide-lipid complex for a period of time sufficient to inhibit or reverse the disease.

In one embodiment, in vivo contacting is accomplished by administering to a patient an intravenous, intraperitoneal, subcutaneous, or intratumoral injection of a therapeutically effective amount of a physiologically tolerable composition comprising an oligonucleotide-lipid complex of the present invention. The oligonucleotide-lipid complex can be administered parenterally by injection or by gradual infusion over time.

Therapeutic compositions containing the oligonucleotide-lipid complex are typically administered intravenously or subcutaneously, for example by unit dose injection. The term dosage unit, when used in relation to a therapeutic composition, refers to physically discrete units suitable as unit doses for a subject, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in combination with the desired diluent, i. carrier or carrier.

The compositions are administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount to be administered depends on the subject being treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The exact amounts of active ingredient required for administration depend on the judgment of the practitioner and are individual to each individual. However, suitable

- 19 042663 dose ranges for systemic use are disclosed in this document and depend on the route of administration. Suitable regimens for initial and booster administration are also contemplated and are illustrated by primary administration followed by repeat doses one or more hours apart followed by injection or another administration. Exemplary multiple administrations are described herein and are particularly preferred to maintain continuously high serum and tissue levels of the polypeptide. Alternatively, a continuous intravenous infusion sufficient to maintain blood concentrations within the ranges indicated for in vivo therapy is contemplated.

It is contemplated that the oligonucleotide of the invention may be administered systemically or locally to treat a disease, such as to inhibit the growth of tumor cells, or to kill cancer cells in cancer patients with locally advanced or metastatic cancers. They can be administered intravenously, intrathecally, subcutaneously and/or intraperitoneally. They may be administered alone or in combination with antiproliferative drugs. In one embodiment, they are administered to reduce a patient's cancer burden prior to surgery or other procedures. Alternatively, they may be administered after surgery to ensure that any remaining cancer (eg, cancer that has not been removed by surgery) will not survive.

A therapeutically effective amount of an oligonucleotide is a predetermined amount calculated to achieve the desired effect, i. to inhibit the expression of the target protein. Thus, the dosage ranges for administering the oligonucleotides of this invention are large enough to achieve the desired effect. The dose should not be so high as to cause side effects such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, neurological effects, and the like. In general, the dosage will generally vary with the age, condition, sex, and degree of disease of the patient, and may be determined by one of skill in the art. The dose may be adjusted by the attending physician in case of any complications.

The composition according to this invention is preferably administered to the patient parenterally, for example by intravenous, intra-arterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural or intrathecal injection, or may be used ex vivo. Preferred doses are 5 to 25 mg/kg. Administration is preferably repeated on a schedule until the cancer has disappeared or regressed, and may be combined with other forms of therapy.

Pharmaceuticals.

A pharmaceutical composition containing liposomes typically includes a sterile pharmaceutically acceptable carrier or diluent such as dextrose or saline.

Where an uncharged lipid component (eg, in the form of a liposome) containing an oligonucleotide is used clinically, it is generally useful to provide the lipid complex as a pharmaceutical composition suitable for the intended use. Typically, this will entail the preparation of a pharmaceutical composition that is substantially free of pyrogens as well as any other impurities that may be harmful to humans or animals. Appropriate buffers may also be used to render the complex stable and to allow uptake by target cells.

The phrases pharmaceutically or pharmacologically acceptable refer to molecular substances and compositions that do not cause an adverse, allergic or other adverse reaction when administered to an animal, such as a human, as the case may be. The preparation of a pharmaceutical composition that contains at least one uncharged lipid component containing an oligonucleotide or additional active ingredient will be known to those skilled in the art in light of this disclosure, exemplified by Remington: The Science and Practice of Pharmacy, 21 ^st , 2005 , is incorporated herein by reference. In addition, for administration to animals (eg, humans), it should be understood that formulations must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA's Office of Biological Standards.

As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, fillers, disintegrants, lubricants, sweeteners, flavors, colorants, such like materials, and combinations thereof, as known to the person skilled in the art. A pharmaceutically acceptable carrier is preferably formulated for human administration, although in some embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but which would not be acceptable (e.g., due to government regulation) for human administration. Unless any conventional carrier is incompatible with the active ingredient, it is intended to be used in therapeutic or pharmaceutical compositions.

The actual amount of dose of a composition of this invention administered to a patient or subject may be determined by physical and physiological factors such as body weight, severity of pathology, type of disease being treated, previous or concomitant therapeutic interventions, patient characteristics, and route of administration. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

In some embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% active compound. In other embodiments, the active compound may comprise, for example, from about 2% to about 75% by weight of the dose unit, or, for example, from about 25% to about 60%, and any range derived therefrom. In other non-limiting examples, the dose may also be from about 1 µg/kg/body weight, about 5 µg/kg/body weight, about 10 µg/kg/body weight, about 50 µg/kg/body weight, about 100 µg/ kg/body weight, about 200 mcg/kg/body weight, about 350 mcg/kg/body weight, about 500 mcg/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg /body weight up to about 1000 mg/kg/body weight or more per administration and any range derived from them. In non-limiting examples of the range obtained from the numbers listed herein, a range of about 5 μg/kg/body weight to about 1000 mg/kg/body weight, from about 5 μg/kg/body weight to about 500 mg/ kg/body weight, etc.

The oligonucleotide according to these embodiments can be administered at a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 μg or more nucleic acid per dose. Each dose may have a volume of 1, 10, 50, 100, 200, 500, 1000 or more μl or ml.

Solutions of the therapeutic compositions may be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The therapeutic compositions according to this invention are preferably administered in the form of injectable compositions in the form of liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid prior to injection may also be prepared. These preparations can also be emulsified. A typical composition for this purpose contains a pharmaceutically acceptable carrier. For example, the composition may contain 10, 25, 50, or up to 100 mg of human serum albumin per ml of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers, and the like.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antioxidants, chelating agents and inert gases. The pH and the precise concentration of the various components of the pharmaceutical composition are adjusted according to well known parameters.

Therapeutic compositions according to this invention may contain classical pharmaceutical preparations. The introduction of therapeutic compositions according to this invention will be by any conventional route, if the target tissue is available through this route. This includes oral, nasal, buccal, rectal, vaginal, or topical. Topical administration may be particularly useful for the treatment of skin cancer, for the prevention of chemotherapy-induced alopecia or other hyperproliferative skin disorder. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions are usually administered as pharmaceutically acceptable compositions which contain physiologically acceptable carriers, buffers or other excipients. Aerosol delivery can be used to treat lung pathologies. The volume of the aerosol is from about 0.01 to 0.5 ml.

An effective amount of the therapeutic composition is determined based on the intended purpose. The term dose unit or dose refers to physically discrete units suitable for use by a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired responses discussed above in connection with its administration, i.e. in an appropriate way and according to the treatment regimen. The amount to be administered, both as a function of the number of treatments and the unit of dose, depends on the desired protection or effect.

The exact amounts of the therapeutic composition also depend on the judgment of the practitioner.

- 21 042663 and are individual for each person. Factors influencing dose include the physical and clinical condition of the patient, the route of administration, the intended goal of treatment (eg, symptomatic relief versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent.

Combined treatment.

In some embodiments, the implementation of the compositions and methods according to this invention include an inhibitory oligonucleotide or an oligonucleotide capable of expressing an inhibitor of gene expression, in combination with a second or additional therapy. The methods and compositions, including combination therapy, enhance the therapeutic or protective effect and/or enhance the therapeutic effect of another anti-cancer or anti-proliferative therapy. The therapeutic and prophylactic methods and compositions may be provided in a combined amount effective to achieve the desired effect, such as killing the cancer cell and/or inhibiting cell hyperproliferation. This process may include contacting cells with an inhibitor of gene expression and a second therapy. The tissue, tumor, or cell may be contacted with one or more compositions or pharmaceutical formulations containing one or more agents (i.e., a gene expression inhibitor or anticancer agent), or by contacting the tissue, tumor, and/ or cages with two or more different compositions or formulations, with one composition providing:

1) inhibitory oligonucleotide;

2) an anti-cancer agent or

3) both an inhibitory oligonucleotide and an anticancer agent.

It is also contemplated that such combination therapy may be used in combination with chemotherapy, radiation therapy, surgical therapy or immunotherapy.

The inhibitory oligonucleotide can be administered before, during, after, or in various combinations regarding anti-cancer treatment. The introductions can be carried out at intervals from simultaneously to several minutes, up to several days, up to several weeks. In embodiments in which the inhibitory oligonucleotide is provided to the patient separately from the anticancer agent, it is generally guaranteed that there will not be a significant time gap between the time of each delivery so that the two compounds will still be able to provide a beneficial combined effect on the patient. In such instances, it is contemplated that it is possible to provide the patient with inhibitory oligonucleotide therapy and anticancer therapy within about 12 to 24 or 72 hours of each other, and more preferably within about 6 to 12 hours of each other. In some situations, it may be desirable to significantly increase the treatment period, with between the respective administrations there is a gap from several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8).

In some embodiments, the course of treatment will last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more. It is contemplated that one agent may be administered on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,

53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89 and/or 90 or any combination thereof and other agent administered on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 ,

38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and/or 90 or any of them combination.

Within one day (24 hour period), the patient may be assigned one or more doses of the agent(s). In addition, after a course of treatment, it is assumed that there is a period of time during which anti-cancer treatment is not carried out. This time period may be 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as his prognosis, endurance, health, etc.

Various combinations may be used. For the example below, inhibitory oligonucleotide therapy is A and anti-cancer therapy or autoimmune therapy is B:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B

B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A

B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A

A/ A/B/A

The administration of any compound or therapy of the present invention to a patient will follow the general protocols for administering such compounds, taking into account the toxicity of the agents, if any. Therefore, in some embodiments, there is a toxicity monitoring step that is inherent in combination therapy. Treatment cycles are expected

- 22 042663 will be repeated as needed. It is also contemplated that various standard therapies as well as surgery may be used in combination with the described therapy.

In specific aspects, conventional therapy is contemplated to include chemotherapy, radiation therapy, immunotherapy, surgical therapy, or gene therapy, and may be used in combination with gene expression inhibitor therapy, cancer therapy, or both gene expression inhibitor therapy and cancer therapy, as described. in this document.

Chemotherapy.

In these embodiments, a wide variety of chemotherapeutic agents can be used. The term chemotherapy refers to the use of drugs to treat cancer. A chemotherapeutic agent is used to refer to a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified according to their mode of action in the cell, such as whether and at what stage they affect the cell cycle. Alternatively, an agent can be characterized based on its ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations to affect nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamin; acetogenins (especially bulatacin and bulatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CB1-TM1); eleuterobine; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterin, prednimustine, trofosfamide and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediine antibiotics (eg calicheamicin, especially calicheamicin gamma.P and calicheamicin omega.P); dinemycin, including dinemycin A; bisphosphonates such as clodronate; esperamycin; as well as the neocarcinostatin chromophore and related chromoprotein enediin antibiotic chromophores, aclacinomysins, actinomycin, athrarnacin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carsinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-norubicin including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnicin, olivomycins, peplomycin, potfiromycin, puromycin, streptonycin, quelamycin, rhodo-igrycidine ubenimex, zinostatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane and testolactone; antiadrenal agents such as mitotane and trilostane; a folic acid supplement such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elformitin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidanin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSC polysaccharide complex; razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; taxoids such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantron; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (eg CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyllornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transferplatin, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Radiotherapy.

Other factors that cause DNA damage and are widely used include so-called γ-rays, x-rays and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also considered, such as microwaves,

- 23 042663 proton irradiation (US patents No. 5760395 and 4870287) and UV irradiation. Most likely, all of these factors cause a wide range of DNA damage, affecting DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for x-rays varies from daily doses of 50 to 200 roentgens for extended periods of time (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and absorption by tumor cells.

The terms contacted and exposed in relation to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed directly adjacent to the target cell. For example, to achieve cell killing, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

Immunotherapy.

In the context of cancer treatment, immunotherapeutic drugs are generally based on the use of immune effector cells and molecules to target and kill cancer cells. Trastuzumab (Herceptin™) is such an example. The immune effector may be, for example, an antibody specific for a marker on the surface of the tumor cell. One antibody may serve as an effector of therapy, or may recruit other cells to actually induce cell death. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and merely serves as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the target tumor cell. Various effector cells include cytotoxic T lymphocytes and NK cells. The combination of therapeutic methods, ie direct cytotoxic activity and ErbB2 inhibition or reduction, would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

Other immunotherapy may also be used as part of the combination therapy with gene therapy discussed above. In one aspect of immunotherapy, the tumor cell must have some marker that can be targeted, ie. not present on most other cells. There are many tumor markers, and any of them may be suitable for targeting in the context of this invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, bladder tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialil Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erbB and c155. An alternative aspect of immunotherapy is the combination of antitumor effects with immunostimulatory effects. There are also immunostimulatory molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, IFN-gamma, chemokines such as MIP-1, MCP-1, IL-8, and factors growth, such as the FLT3 ligand. Combining immunostimulatory molecules, either as proteins or using gene delivery, in combination with a tumor suppressor has been shown to enhance antitumor effects. In addition, antibodies against any of these compounds can be used to target the anticancer agents discussed herein.

Examples of immunotherapies currently being researched or used are immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatics (US Pat. , for example interferons α, β and γ; gene therapy for IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998), e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Pat. Nos. 5,830,880 and 5,846,945) and monoclonal antibodies e.g. 1998; US patent No. 5824311). It is contemplated that one or more anti-cancer therapies may be used with the gene silencing therapies described herein.

In active immunotherapy, an antigenic peptide, polypeptide or protein, or autologous or allogeneic tumor cell composition or vaccine, is usually administered with a separate bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993).

In adaptive immunotherapy, the patient's circulating lymphocytes or tumor-infiltrated lymphocytes are isolated in vitro, activated with lymphokines such as IL-2, or transduced with tumor necrosis genes, and injected backwards (Rosenberg et al., 1988; 1989).

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either increase the signal (eg, costimulatory molecules) or decrease the signal. Inhibitory immune checkpoints,

- 24 042663 which may be the target of immune checkpoint blockade include A2A adenosine receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), Cytotoxic T-cell-associated protein 4 lymphocytes (CTLA-4, also known as CD152), indolamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death protein 1 (PD-1), immunoglobulin domain and mucin domain 3 (TIM-3) of T-lymphocytes; and the V-domain of Ig suppressor of T-lymphocyte activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 and/or CTLA-4 axis.

Immune checkpoint inhibitors can be drugs such as small molecules, recombinant forms of ligand or receptors, or in particular antibodies such as human antibodies (e.g. International Patent Publication No. WO2015016718; Pardoll, Nat. Rev. Cancer, 12 (4):252-64, 2012; both are incorporated herein by reference). Known immune checkpoint protein inhibitors, or analogues thereof, may be used, in particular chimeric, humanized or human forms of antibodies may be used. As the skilled artisan knows, alternative and/or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and/or equivalent names are interchangeable within the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner ligands. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Illustrative antibodies are described in US Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists are known in the art for use in the methods presented herein, such as those described in US Patent Publication Nos. 20140294898, 2014022021, and 20110008369, all incorporated herein by reference.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, a PD binding antagonist -1 is AMP-224 Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 and OPDIVO, is an anti-PD-1 antibody described in WO 2006/121168. also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA® and SCH-900475 is an anti-PD-1 antibody described in WO 2009/114335 CT-011 also known as hBAT or hBAT-1 is anti-PD-1 antibody described in WO 2009/101611 AMP-224, also known as B7-DCIg, is a PDL2-Fc soluble receptor fusion described in WO 2010/027827 and WO 2011/066342.

Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete human CTLA-4 cDNA sequence has GenBank accession number L15006. CTLA-4 is found on the surface of T lymphocytes and acts as a switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T lymphocytes and transmits an inhibitory signal to T lymphocytes. CTLA4 is similar to the T cell costimulatory protein, CD28, and both molecules bind to CD80 and CD86, also referred to as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T lymphocytes, while CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T lymphocytes and may be important for their function. Activation of T cells via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

Anti-human CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for

- 25 042663 use in these methods can be obtained using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the methods disclosed herein may use anti-CTLA-4 antibodies disclosed in US Pat. No. 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP 675206, also known as tremelimumab; formerly ticilimumab), US patent No. 6207156; Hurwitz et al. (1998), Proc. Natl. Acad. sci. USA, 95(17):10067-10071; Camacho et al. (2004), J. Clin. Oncology, 22(145): Abstract No. 2505 (CP-675206 antibody) and Mokyr et al. (1998), Cancer Res. 58:5301-5304. The methods of each of the above publications are hereby incorporated by reference. Antibodies that compete with any of these art-known antibodies for binding to CTLA-4 can also be used. For example, a humanized antibody to CTLA-4 is described in International Patent Application No. WO 01/014424, WO 00/037504 and US Patent No. 8017114; all are incorporated herein by reference.

An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101 and Yervoy®) or antigen-binding fragments and variants thereof (see, for example, WO 01/14424). In other embodiments, the antibody comprises the CDRs or VRs of the heavy and light chains of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the ipilimumab VH region, and the CDR1, CDR2, and CDR3 domains of the ipilimumab VL region. In another embodiment, the antibody competes for binding and/or binds to the same epitope on CTLA-4 as the above antibodies. In another embodiment, the antibody shares at least about 90% variable region amino acid sequence identity with the above antibodies (eg, at least about 90%, 95%, or 99% variable region identity with ipilimumab).

Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in US Pat. all are incorporated herein by reference, and immunoadhesins such as those described in US Pat. No. 8,329,867 are incorporated herein by reference.

In some embodiments, the implementation of immunotherapy may be adaptive immunotherapy, which includes the transfer of autologous antigen-specific T-lymphocytes obtained ex vivo. T-lymphocytes used for adaptive immunotherapy can be obtained either by expansion of antigen-specific T-lymphocytes or by genetically engineered retargeting of T-lymphocytes (Park, Rosenberg et al., 2011). Isolation and transfer of tumor-specific T-lymphocytes have been successful in the treatment of melanoma. New specificities in T-lymphocytes have been successfully obtained through genetic transfer of transgenic T-lymphocyte receptors or chimeric antigen receptors (XAP) (Jena, Dotti et al., 2010). CARs are synthetic receptors consisting of a targeting moiety that is linked to one or more signaling domains in a single fusion molecule. In general, a XAP binding fragment consists of a single chain antibody antigen-binding domain (scFv) containing the light and variable fragments of a monoclonal antibody connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used.

The signaling domains for first generation CAR are derived from the cytoplasmic region of CD3-zeta or Fc receptor gamma chains. CAR has successfully retargeted T-lymphocytes to antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors (Jena, Dotti et al., 2010).

In one embodiment, the invention provides a combination therapy for the treatment of cancer, the combination therapy comprising adaptive T-lymphocyte therapy and a checkpoint inhibitor. In one aspect, adaptive T-lymphocyte therapy includes autologous and/or allogeneic T-lymphocytes. In another aspect, autologous and/or allogeneic T lymphocytes target tumor antigens.

Surgery.

Approximately 60% of cancer patients will undergo some type of surgery, which includes preventive, diagnostic or staging, curative and palliative surgery. Remedial surgery is a cancer treatment that can be used in combination with other therapies such as the treatments of the invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and/or alternative therapies.

Medical surgery involves resection, in which all or part of the cancerous tissue is physically removed, excised and/or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). It is further contemplated that the present invention may be used in combination with the removal of superficial cancers, precancerous lesions, or abnormal amounts of normal tissue.

- 26 042663

When some or all of the cancer cells, tissue, or tumor are removed, a cavity can form in the body. Treatment can be performed by perfusion, direct injection, or topical application to the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every

1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 11 or months. The doses of these treatments may also be modified.

other agents.

It is contemplated that other agents may be used in combination with certain aspects of these embodiments to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the activation of cell surface receptors and gap junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, agents that sensitize hyperproliferative cells to apoptotic inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will enhance the anti-hyperproliferative effects on the adjacent population of hyperproliferative cells. In other embodiments, cytostatic or differentiating agents may be used in combination with certain aspects of these embodiments to improve the anti-hyperproliferative activity of the treatments. Cell adhesion inhibitors are expected to improve the performance of these embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (CFA) inhibitors and lovastatin. Additionally, it is contemplated that other agents that sensitize a hyperproliferative cell to apoptosis, such as the c225 antibody, may be used in combination with certain aspects of these embodiments to improve treatment efficacy.

Kits and diagnostics.

In various aspects of the invention, a kit containing therapeutic agents and/or other therapeutic and delivery agents is provided. In some embodiments, this invention provides a kit for preparing and/or administering a therapy according to the invention. The kit may contain reagents that can be used to administer the active or effective agent(s) of this invention. Kit reagents may include at least one gene expression inhibitor (e.g., STAT3 oligonucleotides), one or more lipid components, one or more combination therapy anticancer components, and reagents for preparing, preparing, and/or administering the components according to of the invention, or perform one or more of the steps of the methods of the invention.

In some embodiments, the kit may also contain a suitable container means, which is a container that will not react with the components of the kit, such as an eppendorf tube, assay plate, syringe, vial, or test tube. The container may be made from sterilizable materials such as plastic or glass.

The kit may further contain instructions that describe the procedural steps of the methods and will correspond to substantially the same procedures as described herein or known to those skilled in the art.

Examples

The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will appreciate that the methods disclosed in the following examples represent methods discovered by the inventor for effective use in the practice of the invention, and thus may be considered as constituting preferred methods for its use. However, those skilled in the art, in light of this disclosure, will appreciate that numerous changes may be made to the specific embodiments disclosed and still obtain a similar or analogous result without departing from the spirit and scope of the invention.

Example 1 STAT3-targeted P-ethoxy oligonucleotides.

Oligonucleotides targeting STAT3 have been developed for use in a liposomal STAT3 antisense drug product to inhibit STAT3 expression. The contiguous STAT3 cDNA sequence is shown in SEQ ID NO: 5 and the STAT3 protein sequence is shown in SEQ ID NO: 6. The sequence of each of the oligonucleotides is shown in Table 1. 4.

- 27 042663

Table 4

STAT3 antisense sequences

SEQ GO NO: Subsequence 1 5’- САА AGT GGC ATG TGA ТТС -3’ 2 5’- GCT ATA CTG CTG GTC AAT -3’ 3 5’- CTT CGT AGA TTG TGC TGA -3’ 4 5’- CTG ATA ATT CAA CTC AGG -3’

Liposomal STAT3 antisense drug product was made in accordance with the methods described in this document. Mass spectrometric analysis showed that about 80% of the oligonucleotide drug substance had three to seven backbone phosphodiester bonds. Particle analysis showed that 90% of the liposomes had a particle diameter of 2319 nm or less, 50% of the liposomes had a particle diameter of 368 nm or less, and about 18% of the liposomes had a particle diameter of 300 nm or less.

Example 2 Inhibition of STAT3 expression by P-ethoxy oligonucleotides.

The ability of a liposomal STAT3 antisense agent to inhibit STAT3 expression was tested in two lung adenocarcinoma cell lines, H1975 and HCC 827. A liposomal STAT3 antisense agent corresponding to SEQ ID NO: 4 (180 μg/ml), or an equivalent amount of empty liposomes, was incubated with each cell line within four days. According to the data in FIG. 1 demonstrates that incubation with a liposomal STAT3 antisense agent, but not empty liposomes, reduces the expression level of STAT3 relative to β-actin.

Example 3 Inhibition of Cancer Cell Viability by Liposomal STAT3 Antisense Agent.

The ability of the liposomal STAT3 antisense agent to inhibit non-small cell lung cancer cell viability was tested in three lung adenocarcinoma cell lines: H1975, HCC 827 and H358. A liposomal antisense STAT3 agent corresponding to one of SEQ ID NOs: 1-4 was incubated with each cell line for four days. According to the data in FIG. 2A-2C demonstrate that incubation with a liposomal STAT3 antisense agent reduces cell viability of each cell line, with the liposomal STAT3 antisense agent according to SEQ ID NO: 4 being the most effective.

The ability of the liposomal STAT3 antisense agent to inhibit leukemia cell growth was analyzed in three human leukemia cell lines: K-562, MV4-11 and Kasumi-1. Liposomal STAT3 antisense agent corresponding to SEQ ID NO: 4 was incubated with each cell line for four days. According to the data in FIG. 3A demonstrates that incubation with a liposomal STAT3 antisense agent reduces cell viability of each cell line.

The ability of the liposomal STAT3 antisense agent to inhibit lymphoma cell growth was tested in five human lymphoma cell lines: DOHH-2, SU-DHL-4, SU-DHL-6, SU-DHL-10 and RL. Liposomal STAT3 antisense agent corresponding to SEQ ID NO: 4 was incubated with each cell line for four days. According to the data in FIG. 3B demonstrates that incubation with a liposomal STAT3 antisense agent reduces cell viability of each cell line.

All methods disclosed and claimed herein can be practiced and performed without undue experimentation in light of this disclosure. While the compositions and methods of this invention have been described in terms of the preferred embodiments, it should be apparent to those skilled in the art that changes may be applied to the methods and the steps or sequence of steps of the method described herein without departing from the concept essence and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can replace the agents described herein and at the same time the same or similar results will be obtained. All such analogous substitutions and modifications obvious to those skilled in the art are intended to be within the spirit, scope and concept of the invention as defined in the appended claims.

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Claims (36)

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Composition for lowering the expression level of the STAT3 protein in a cell, containing a population of oligonucleotides, wherein the oligonucleotides hybridize with the polynucleotide product of the STAT3 gene, while the oligonucleotides of the population consist of nucleoside molecules connected to each other by backbone phosphate bonds, with 60-75% of these phosphate bonds the backbones in each oligonucleotide are P-ethoxy backbone bonds, where 25-40% of the backbone phosphate bonds in each oligonucleotide are backbone phosphodiester bonds, where the population oligonucleotides contain a sequence according to any one of SEQ ID NOs: 1-4 each. 2. The composition according to claim 1, characterized in that the oligonucleotides of the population contain the sequence according to SEQ ID NO: 1. 3. The composition according to claim 1, characterized in that the oligonucleotides of the population contain the sequence according to SEQ ID NO: 2. 4. The composition according to claim 1, characterized in that the oligonucleotides of the population contain the sequence according to SEQ ID NO: 3. 5. The composition according to claim 1, characterized in that the oligonucleotides of the population contain the sequence according to SEQ ID NO: 4. 6. The composition according to claim 1, characterized in that the phosphodiester bonds of the backbone are distributed throughout each oligonucleotide. 7. A composition according to claim 1, characterized in that the backbone phosphodiester bonds are not clustered within a portion of each oligonucleotide. 8. The composition according to claim 1, characterized in that the population of oligonucleotides is heterogeneous in the number of P-ethoxy backbone bonds and phosphodiester backbone bonds present in the oligonucleotides of the population. 9. The composition according to claim 1, characterized in that the oligonucleotides of the population have a size in the range from 18 to 30 nucleotides. 10. The composition of claim 9, wherein the population oligonucleotides have an average size of 18 nucleotides, with no more than 14 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond. 11. The composition of claim 9, wherein the population oligonucleotides have an average size of 20 nucleotides, with no more than 16 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond. 12. The composition of claim 9, wherein the population oligonucleotides have an average size of 25 nucleotides, with no more than 20 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond. 13. The composition of claim 9, wherein the population oligonucleotides have an average size of 30 nucleotides, with no more than 24 backbone phosphate bonds in each oligonucleotide being a P-ethoxy backbone bond. - 33 042663 14. The composition according to claim 1, characterized in that the population of oligonucleotides contains one kind of oligonucleotides. 15. The composition according to claim 1, characterized in that the population of oligonucleotides contains at least two types of oligonucleotides. 16. The composition according to claim 1, characterized in that the population of oligonucleotides is heterogeneous in the distribution of backbone phosphodiester bonds among the oligonucleotides of the population. 17. The composition according to claim 1, characterized in that the oligonucleotides of the population inhibit the expression of the STAT3 protein. 18. The composition of claim 1 further comprising phospholipids, wherein the oligonucleotides and phospholipids form an oligonucleotide-lipid complex. 19. Composition according to claim 18, characterized in that the phospholipids are uncharged or have a neutral charge at physiological pH. 20. Composition according to claim 19, characterized in that the phospholipids are neutral phospholipids. 21. Composition according to claim 20, characterized in that the neutral phospholipids are phosphatidylcholines. 22. Composition according to claim 20, characterized in that the neutral phospholipids are dioleoylphosphatidylcholine. 23. Composition according to claim 18, characterized in that the phospholipids are substantially free of cholesterol. 24. The composition of claim 18, wherein the phospholipids and oligonucleotides are present in a molar ratio of from about 5:1 to about 100:1. 25. The composition of claim 18, wherein the oligonucleotide-lipid complex is further defined as a population of liposomes. 26. The composition of claim 25, further comprising a surfactant and characterized in that at least 90% of the liposomes are less than 5 µm in diameter. 27. The composition according to claim 25, characterized in that the population of oligonucleotides is contained in the population of liposomes. 28. Composition according to claim 1, characterized in that the composition is lyophilized. 29. A method for reducing the expression level of a STAT3 protein in a cell, which includes contacting the cell with the composition of claim 1. 30. Pharmaceutical composition for reducing the expression level of the STAT3 protein in a cell, containing the composition according to claim 18 and a pharmaceutically acceptable carrier. 31. A method of delivering a therapeutically effective amount of an oligonucleotide into a cell for the treatment of cancer or an autoimmune disease, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition according to claim 30. 32. The method of claim 31, wherein the subject is a human. 33. The method of claim 31, wherein the cancer is non-small cell lung cancer, pancreatic adenocarcinoma, breast cancer, prostate cancer, melanoma, colon cancer, leukemia, lymphoma, glioblastoma, osteosarcoma, oral cancer, ovarian cancer , uterine cancer, bone cancer, brain cancer, prostate cancer, kidney cancer, stomach cancer, esophageal cancer, rectal cancer, bladder cancer, testicular cancer, or liver cancer. 34. The method of claim 31, wherein the autoimmune disease is systemic lupus erythematosus, spondyloarthropathy, Sjögren's disease, Crohn's disease, diabetes mellitus, multiple sclerosis, or rheumatoid arthritis. 35. The method according to claim 31, characterized in that the composition is administered subcutaneously, intravenously or intraperitoneally. 36. The method of claim 31, further comprising administering to the subject at least a second cancer therapy, wherein the second cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, antiviral therapy, immunosuppressive therapy, antibiotic therapy, antiparasitic therapy, antifungal therapy or cytokine therapy. -