Nanoparticle PRR agonist compositions and use to treat solid tumor cancer

Abstract

Disclosed are pharmaceutical compositions formulated from immunomodulators formed from glycogen nanoparticles, such as monodisperse phytoglycogen nanoparticles, complexed with a pattern recognition receptor agonist, such as a TLR agonist which can be poly I:C. Also disclosed are methods of treating solid tumor cancers using the described immunomodulators, and uses of the immunomodulators to treat solid tumor cancers and to manufacture medicaments to treat solid tumor cancers.

Description

NANOPARTICLE PRR AGONIST COMPOSITIONS AND USE TO TREAT SOLIDTUMOR CANCERFIELD OF THE INVENTION

[0001] The present invention relates to immune-therapeutic compositions and methods and, in particular, nanoparticle delivery of a pattern recognition receptor agonist, such as a Toll-like Receptor (TLR) agonist, to treat solid tumor cancer, such as an ovarian cancer.BACKGROUND

[0002] Solid tumor cancers include breast, ovarian, lung, colon, bladder, pancreatic and prostrate cancers, and are conventionally treated with therapies such as surgery, chemotherapy and radiation therapy, as well as newer therapies such as targeted therapies and immunotherapies.

[0003] Immunotherapies use various aspects of a patient’s immune system to fight the cancer. An immune response is a complex process involving many interacting components within both the innate and adaptive arms of the immune system. Proper activation of the innate immune system is not only required to control infection and cancer but is also imperative for the initiation and engagement of the adaptive immune response. Mechanistically, the magnitude and characteristics of innate immune responses are, in part, driven by engagement of a highly conserved family of pattern recognition receptor (PRR) proteins termed Toll-like Receptors (TLRs). One of the primary roles of TLRs is to recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) and engagement of TLRs by their agonists results in rapid induction of the innate immune response. Other pattern recognition receptors such as RIG-I and MDA5 which result in induction of the innate immune response can also play a role.1WSLEGAL\095576\00010\42816987v7

[0004] There are 10 defined TLRs in humans and are widely expressed by many cell types including subsets of immune cells such as Dendritic Cells (DCs) which are antigen-presenting cells that play an important role in the induction and maintenance of innate and adaptive immune responses. TLRs are also expressed on non-immune cells such as epithelial cells and fibroblasts. TLRs 1, 2, 4, 5, 6, and 10 are expressed on the cell surface, whereas the nucleic acid sensing TLRs 3, 7, 8, and 9 are sequestered in endosomes. Each TLR has specific agonists which activate immune signaling; the cell surface-expressed TLRs tend to respond to bacterial cell components such as peptidoglycans (TLR 1 and 2), lipoproteins (TLR 1 and 6), lipopolysaccharides (TLR 4), flagella (TLR 5) and di / triacylated lipopeptides (TLR 10). Endosomal TLRs respond to viral and bacterial nucleic acid-based agonists such as double-stranded RNA (TLR 3), single-stranded RNA (TLR 7 and 8), and CpGDNA (TLR 9).

[0005] Given TLRs’ ability to modulate the immune response, compositions and formulations engaging TLRs have been developed for the treatment and prevention of infection (viral, bacterial, parasitic), cancer, and immunodeficiencies. There are indications that engagement of TLRs by their cognate agonists can induce anti-tumor immune responses in laboratory animals. However, in humans, a relatively small therapeutic window for these agonists has been observed as the efficacy has been limited by low circulation time, poor solubility, agonist poly dispersity and systemic toxicity. Toxic manifestations after parenteral administration in humans include transient fever, minor liver enzyme change and a slight decrease in circulating leukocyte numbers. Toxicity -related adverse events in clinical trials of TLR agonists, notably dangerous inflammatory responses arising from agonists penetrating into the cytoplasm of cells, especially when delivered intravenously, have prevented further clinical development.

[0006] Intravenous delivery of TLR agonists also faces the challenge of rapid metabolism by serum enzymes which results in a short drug-half life. Modified versions of TLR agonists with longer2WSLEGAL\095576\00010\42816987v7circulation times have been created (see, e.g., U.S. Patent No. 5532130) but these tend to prolong the toxic side-effects, which are mostly attributed to nonspecific distribution that results in immune activation in healthy tissue while delivering sub-therapeutic concentrations of the drug to the intended target tissue.

[0007] Other formulations that incorporate TLR agonists into lipid nanoparticles (e.g. US Patent No. 8357374) and synthetic polymeric devices (eg. PCT application WO2013106852A1) have been created in an effort to improve tissue distribution. However, inherent toxicity (lipid nanoparticles, liposomes, polyethylene imine) and poor scalability (synthetic nanoparticles) of these nanoparticle systems have limited further clinical development. Conjugation strategies that employ TLR agonist formulations containing targeting molecules (e.g. homing peptides) have also failed to progress into clinical trials, largely because they must be administered intratumorally or by intraperitoneal injection.

[0008] Glycogen-based compositions have been developed which allow for activation of TLRs and may provide improved stability and lower toxicity, as described in PCT International Application PCT / CA2019 / 051167, the entire contents of which are incorporated herein by reference. The glycogen / phytoglycogen nanoparticles did not have any cytotoxic effects in fish and mammalian cells, and when linked to a TLR agonist, demonstrated protection from viral infection in HEL-299 cells and HFF-1 cells, and induced a more robust innate immune system response in both immune and non- immune cells, when compared to unbound agonist.

[0009] There remains a need in the art for immunotherapy methods for solid cancers using glycogenbased compositions and pattern recognition receptor agonists.3WSLEGAL\095576\00010\42816987v7SUMMARY OF THE INVENTION

[0010] Generally, disclosed are pharmaceutical compositions formulated with immunomodulators formed from glycogen nanoparticles, such as monodisperse phytoglycogen nanoparticles, complexed with a pattern recognition receptor agonist. Also disclosed are methods of treating solid tumor cancers with such immunomodulators, and uses of the immunomodulators to treat solid tumor cancers and to manufacture medicaments to treat solid tumor cancers. Pattern recognition receptors can include but not limited to, a TLR, RIG-I or MDA5, each of which can stimulate an innate multifaceted immune response which attacks the tumor.

[0011] In one aspect, disclosed are compositions comprising a monodisperse immunomodulator comprising a glycogen nanoparticle covalently or non-covalently linked to a pattern recognition receptor (PRR) agonist, such as an agonist which activates at least one of a TLR, RIG-I or MDA5. Surprisingly, the compositions described herein are less toxic than the PRR agonist by itself, despite producing a substantially enhanced innate immunity response, despite being more stable in serum, and despite concentrating in the liver and in the tumor. Furthermore, when monodisperse glycogen nanoparticles are combined with polydisperse PRR agonists, the resulting immunomodulator is unexpectedly monodisperse.

[0012] In some embodiments, the PRR agonist is a TLR agonist, such as a TLR3 agonist. In some embodiments, the PRR agonist comprises a nucleic acid, such as a double- stranded RNA, doublestranded DNA, single-stranded RNA, single-stranded DNA, or any synthetic analogs thereof. Suitable TLR agonists include poly I:C, variations or derivatives of poly I:C, unmethylated cytidine phosphate guanosine (CpG), CpG oligodeoxynucleotides (ODN), or a locked nucleic acid (LNA).4WSLEGAL\095576\00010\42816987v7

[0013] In some embodiments, an immunomodulator described herein is formulated as a pharmaceutically acceptable composition comprising monodisperse immunomodulators and a pharmaceutically acceptable carrier, such as water, a saline solution or a phosphate-buffered saline (PBS) solution.

[0014] Also disclosed herein is a method of treating a solid tumor cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an immunomodulator or a composition comprising the immunomodulator. In some embodiments, the immunomodulator or a composition comprising the immunomodulator is administered in combination with an additional therapeutic agent.

[0015] Also disclosed herein is the use of an immunomodulator described herein to treat a solid tumor cancer in a patient in need thereof, where the immunomodulator is administered to the patient in a therapeutically effective amount. The immunomodulator may be administered in combination with another therapeutic agent.

[0016] Also disclosed herein is the use of an immunomodulator described herein in the manufacture of a medicament for treating a solid tumor cancer.

[0017] In some embodiments, the solid tumor cancer to be treated is selected from the group consisting of ovarian cancer, breast cancer, colorectal or small intestine cancers, renal pelvis cancer, pancreatic cancer, bladder cancer, prostrate or lung cancer.BRIEF DESCRIPTION OF THE FIGURES

[0018] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, examples of embodiments and / or features.5WSLEGAL\095576\00010\42816987v7

[0019] Fig. 1 shows results of ELISA measurement of expression of CXCL10 in cancer cell lines following treatment with naked poly I:C and poly I:C complexed with phytoglycogen nanoparticles (labeled in Fig 1 as NDX).

[0020] Fig. 2 shows photographs of the abdominal cavity of mice after ovarian tumor induction, with a control group treated with PBS (left) and a group treated with GLY100 (right).

[0021] Fig. 3 is a graph showing Metastatic tumor score of the control group (PBS) and the group treated with GLY100. The symbol *** indicates a statistically significant difference via paired T-test @ 95% significance.

[0022] Fig. 4 is a graph showing average tumor weight of the control group (PBS) and the group treated with GLY100. The symbol **** indicates a statistically significant difference via paired T- test @ 95% significance.

[0023] Fig. 5 shows IFIT1 expression levels of a cancer cell line, comparing a control group (Mock) to cells exposed to either naked poly I:C (PIC) or poly I:C complexed with phytoglycogen nanoparticles (NDX PIC) in a 2:1 weight ratio - where the cells are incubated with no serum present, pre-incubated with serum and then treated with the composition, and where the cells are treated with the composition in serum. (Basal = media that does not have antibiotics or fetal bovine serum; FBS = fetal bovine serum; Full = media that contains both antibiotics and fetal bovine serum)

[0024] Fig. 6 shows nucleic acid PAGE analysis showing non-degraded dsRNA and degradation products, with naked poly I:C and poly I:C complexed with phytoglycogen nanoparticles (GLY100). The data shows that in FBS (fetal bovine serum) neither degraded nor non-degraded dsRNA (poly I:C) are present when poly I:C is complexed with the phytoglycogen nanoparticle (“+”). Naked dsRNA (i.e. not attached to the nanoparticle) is fully degraded in 5% FBS in about 60 min.6WSLEGAL\095576\00010\42816987v7

[0025] Fig. 7A shows tumor weight and tumor score from a mouse model of epithelial ovarian cancer, and treated with GLY100 at 3 treatment levels: 2.4 ug, 12 ug and 60 ug of GLY100 (2 weeks post treatment initiation). The above treatment levels are equivalent to 0.12, 0.6 and 3 mg / kg (as poly I:C) or 0.36, 1.8 and 9 mg / kg as GLY100. The symbol * indicates a statistically significant difference via paired T-test @ 95% significance.

[0026] Fig. 7B shows tumor weight and tumor score from a mouse model of epithelial ovarian cancer, and treated with GLY100 at 3 treatment levels: 2.4 ug, 12 ug and 60 ug of GLY100 (4 weeks post treatment initiation). The above treatment levels are equivalent to 0.12, 0.6 and 3 mg / kg (as poly I:C) or 0.36, 1.8 and 9 mg / kg as GLY100. The symbol * indicates a statistically significant difference via paired T-test @ 95% significance.DETAILED DESCRIPTION

[0027] Generally, disclosed are immunomodulator compositions comprising glycogen nanoparticles complexed with a pattern recognition receptor (PRR) agonist. Also disclosed are methods of using an immunomodulator comprising a glycogen nanoparticle complexed with a PRR agonist, in a method to treat a solid tumor cancer, and in some embodiments, in a method to treat ovarian cancer. As used herein, a “solid tumor cancer” is a cancer characterized by a solid mass of cancer cells which can occur in many different body parts or organs. A non-limiting list of examples of solid tumor cancers include ovarian cancer, breast cancer, colorectal or small intestine cancers, renal pelvis cancer, pancreatic cancer, bladder cancer, prostrate or lung cancer. A solid tumor cancer may also include a cancer which has metastasized from another location in the body. “Ovarian cancer” includes any cancer which begins in an ovary, including epithelial ovarian carcinomas, germ cell7WSLEGAL\095576\00010\42816987v7tumors, and stromal cell tumors, and further including recurrent or relapsed ovarian cancer which may or may not return to its original location or may be found elsewhere in the body.

[0028] Solid tumor cells which are susceptible to treatments as described herein trigger an immune response, therefore, without restriction to a theory, any solid tumor in which an immune response is triggered with a pattern recognition receptor agonist may be treated with the compositions and methods described herein. The following description describes the use of TLR agonists, however, one skilled in the art may readily envision and implement the use of another pattern recognition receptor agonist, such as a RIG-I or a MDA5 agonist.

[0029] In general terms, provided herein is a composition comprising an immunomodulator which comprises a glycogen nanoparticle complexed with a pattern recognition receptor (PRR) agonist. The PRR agonist may activate at least one of a TLR (such as TLR3), RIG-I or MDA5. In some embodiments, the glycogen nanoparticle comprises phytoglycogen nanoparticles (NPs), and the PRR agonist is a TLR3 agonist such as polyinosinic-polycytidylic acid (poly I:C).

[0030] In some embodiments, the immunomodulator composition is monodisperse as a result of using monodisperse glycogen nanoparticles.Glycogen Nanoparticles

[0031] “Glycogen” is a multi-branched polysaccharide of glucose and includes both animal glycogen and phytoglycogen which is plant-based glycogen. Glycogen includes both products derived from natural sources and synthetic products, including synthetic phytoglycogen i.e. glycogen-like products prepared using enzymatic processes on substrates that include plant-derived material e.g. starch. Glycogen and phytoglycogen are substantially identical and are composed of molecules of a- D glucose chains having an average chain length of 11-12, with 1-4 linkage and branching point 8WSLEGAL\095576\00010\42816987v7occurring at 1-6 and with a branching degree of about 6% to about 13%. Phytoglycogen NPs are typically slightly larger than animal glycogen NPs. Animal glycogen typically has shorter branches than phytoglycogen. Glycogen and phytoglycogen molecules may be modified as described further below or as is known in the art, and such modified forms are included in the scope of the term "glycogen".

[0032] "Nanoparticles" or "NPs" are particles which have at least one dimension in the nano-scale, such as less than about 500 nm, 100 nm, 50 nm, or 20 nm. As used herein “glycogen nanoparticles” is used to refer to both glycogen and phytoglycogen NPs.

[0033] In some embodiments, the glycogen NPs are phytoglycogen NPs. Use of phytoglycogen eliminates the risk of contamination with prions or endotoxins, which could be associated with animal or bacterial sources.

[0034] Each glycogen nanoparticle comprises a single homopolymer molecule, made of polymerized, highly branched glucose units characterized by very high molecular weight (e.g. up to about 107Da). These NPs are approximately spherical and can be manufactured with different sizes, for example in the range of 20 to 150 nm in diameter by varying the starting material and processing steps. In one embodiment, individual (non-agglomerated) glycogen NPs have an average particle diameter of between about 10 nm and about 150 nm, in one embodiment about 30 nm to about 150 nm, in one embodiment about 60 nm to about 110 nm.

[0035] The high density of surface groups on the glycogen particles results in a variety of unique properties of glycogen nanoparticles, such as fast dissolution in water, low viscosity and shear thinning effects for aqueous solutions at high concentrations of glycogen nanoparticles. This contrasts with high viscosity and poor solubility of linear and low-branched polysaccharides of comparable9WSLEGAL\095576\00010\42816987v7molecular weight. Furthermore, it allows formulation of highly concentrated (up to 30%) stable dispersions in water or DMSO.

[0036] In some embodiments, the phytoglycogen NPs described herein have several properties that make them suitable for use in pharmaceutical compositions. These phytoglycogen NPs are non-toxic, have no known allergenicity, and can be degraded by glycogenolytic enzymes (e.g. amylases and phosphorylases) of the human body. The products of enzymatic degradation are non-toxic molecules or by-products of glucose. Glycogen nanoparticles are generally photostable and stable over a wide range of pH, and electrolyte concentrations. Further, their high molecular weight (typically 106- 107Da) is believed to be associated with longer intravascular retention time. Also, nanoparticles in this size range can concentrate in tumors via the EPR (enhanced permeability and retention) effect, where nano-sized drugs and macromolecules leak out of leaky tumor blood vessels (enhanced permeability) and get trapped in the tumor tissue due to poor lymphatic drainage (retention), leading to accumulation in the tumor.

[0037] Monodisperse glycogen NPs can have properties that address a number of requirements for materials used in pharmaceutical and biomedical applications: for example, predictable biodistribution in different tissues and associated pharmacokinetics; hydrophilicity; biodegradability; and nontoxicity. In some embodiments, glycogen NPs comprise nanoparticles manufactured according to methods described herein. In some embodiments, the described methods enable production of substantially spherical, substantially monodisperse NPs, each of which is a single glycogen molecule.

[0038] United States Patent Application 20100272639 Al, the disclosure of which is incorporated by reference in its entirety, describes a process for the production of glycogen NPs from bacterial and shellfish biomass. The processes disclosed generally include the steps of mechanical or chemical cell10WSLEGAL\095576\00010\42816987v7disintegration; separation of insoluble cell components by centrifugation; elimination of proteins and nucleic acids from cell lysate by enzymatic treatment followed by dialysis which produces an extract containing crude polysaccharides, lipids, and lipopolysaccharides (LPS) or, alternatively, phenol- water extraction; elimination of LPS by weak acid hydrolysis, or by treatment with salts of multivalent cations, which results in the precipitation of insoluble LPS products; and purification of the glycogen enriched fraction by ultrafiltration and / or size exclusion chromatography; and precipitation of glycogen with a suitable organic solvent or a concentrated glycogen solution can be obtained by ultrafiltration or by ultracentrifugation; and freeze drying to produce a glycogen powder. Glycogen NPs produced from bacterial biomass were characterized by a MWt 5.3-12.7 x 106Da, a particle size of 35-40 nm in diameter and were monodisperse.

[0039] Methods of producing monodisperse compositions of phytoglycogen are described in the US Patent No. 9,737,608, entitled “Phytoglycogen Nanoparticles and Methods of Manufacture Thereof, the disclosure of which is incorporated by reference in its entirety. In one embodiment, the described methods of producing monodisperse phytoglycogen nanoparticles include: a. immersing disintegrated phytoglycogen-containing plant material in water at a temperature between about 0° and about 50°C; b. subjecting the product of step a. to a solid-liquid separation to obtain an aqueous extract; c. passing the aqueous extract of step b. through a microfiltration material having a maximum average pore size of between about 0.05 um and about 0.15 um; and d. subjecting the filtrate from step c. to ultrafiltration to remove impurities having a molecular weight of less than about 300 kDa or less than about 500 kDa, to obtain an aqueous composition comprising monodisperse phytoglycogen nanoparticles. In one embodiment of the method, the phytoglycogen-containing plant material is a cereal or a mixture of cereals, in one embodiment, corn. In one embodiment, step c. comprises passing the aqueous extract of step b. through (c. 1) a first microfiltration material having a maximum average11WSLEGAL\095576\00010\42816987v7pore size between about 10 um and about 40 um; (c.2) a second microfiltration material having a maximum average pore size between about 0.5 um and about 2.0 um, and (c.3) a third microfiltration material having a maximum average pore size between about 0.05 and 0.15 um. The method can further include a step (e.) of subjecting the aqueous composition comprising monodisperse phytoglycogen nanoparticles to enzymatic treatment using amylosucrose, glycosyltransferase, branching enzymes or any combination thereof. The method avoids the use of chemical, enzymatic or thermal treatments that can degrade the phytoglycogen material. The aqueous composition can further be dried.

[0040] In some embodiments, the glycogen NPs can be surface modified to introduce desired functional groups, such as to facilitate electrostatic linking with the PRR agonist [8], For example, the NPs can be cationized by amination or with a quaternary ammonium, for example, glycidyl trimethyl ammonium chloride. In another example, the NPs can be anionized using methods well known in the art [7],

[0041] In some embodiments, the phytoglycogen is obtained from sweet com (Zea mays var. saccharata and Zea mays var. rugosa . In some embodiments, the sweet com is of standard (su) type or sugary enhanced (se) type. In some embodiments, the composition is obtained from dent stage or milk stage kernels of sweet com.Monodispersity

[0042] In one embodiment, monodisperse glycogen nanoparticles are used, which are preferably monodisperse phytoglycogen nanoparticles.

[0043] The poly dispersity index (PDI) of a composition of NPs can be determined by the dynamic light scattering (DLS) technique, where PDI is determined as the square of the ratio of standard12WSLEGAL\095576\00010\42816987v7deviation to mean diameter. A polydiversity index (PDI*) can also be expressed through the distribution of the molecular weight of polymer where PDI* equals the ratio of the weight-average molar mass (Mw) and the number-average molar mass (Mn). In the first case, a completely monodisperse material would have a PDI of zero (0.0) and in the second case the PDI* would be 1.0. PDI* can be measured by size-exclusion chromatography with multi-angle static light scattering (SEC-MALS).

[0044] In one embodiment, the pharmaceutical composition comprises monodisperse glycogen nanoparticles having a PDI of less than about 0.3, less than about 0.2, less than about 0.15, or less than about 0.10 as measured by DLS. In one embodiment, the pharmaceutical composition comprises monodisperse glycogen nanoparticles having a PDI* of less than about 1.3, less than about 1.2, less than about 1.15, or less than about 1.10, as measured by SEC-MALS.

[0045] In some embodiments, the monodisperse glycogen NP composition is substantially pure, meaning that impurities such as peptides and lipids are substantially absent or below the analytical detection limits. For example, <0.1% proteins and <0.09% lipids cannot be detected by standard analytical methods.Immunomodulator Complexes

[0046] Compositions described herein comprise immunomodulators, each comprising a glycogen NP complexed or linked to PRR agonist molecules. In some embodiments, the glycogen nanoparticles are covalently linked to PRR agonists. In other embodiments, glycogen nanoparticles are non- covalently linked to PRR agonists. As used herein, “covalently linked” refers to a link via a covalent bond, whether directly or via a linker. The term “non-covalently linked” refers to all non-covalent13WSLEGAL\095576\00010\42816987v7interactions including electrostatic interactions, hydrophobic interactions, physical entanglement, Van der Waals forces and combinations thereof.

[0047] In some embodiments, the NPs are treated by methods known in the art to introduce positive charges to the surface of the nanoparticles, such as with a quaternary ammonium salt, for example, glycidyl trimethyl ammonium chloride (GTAC). In some examples, up to about 50% of the cationizable sites of the glucose molecules are cationized in this process. The maximum degree of substitution is 3, and preferably the nanoparticle uses a degree of substitution (DoS) from about 0.1 to about 1.5. The DoS may be varied to any value in the preferred range using well-known techniques.

[0048] In one embodiment the PRR agonist is a macromolecule, for example, a nucleic acid, a peptide, a peptidoglycan or a lipopolysaccharide. If a nucleic acid is used, the size of the nucleic acid is not particularly restricted, however in some embodiments, size variation is preferably minimized. In some embodiments, the nucleic acid may have up to about 10,000 base pairs. In one embodiment, the nucleic acid is between 10 and 10,000 nucleotides in length, and preferably between 1000 and 10,000 nucleotides in length.

[0049] In some embodiments, the PRR agonist may comprise a TLR agonist which is a nucleic acid such as double-stranded (ds) RNA, dsDNA or single-stranded (ss) RNA, ssDNA, or a synthetic analog or mimic of any of the foregoing nucleic acids. In some embodiments, the TLR agonist is a TLR3 agonist such as poly I:C (commercially available Invivogen™), or a variant or derivative of poly I:C, such as poly-IC12U (Ampligen™) or poly-ICLC (Hiltonol™).

[0050] In some embodiments, the TLR agonist may comprise synthetic dsRNA mimics such as RGC100 [1] or ARNAX [2, 3], In some other embodiments, the TLR agonist may comprise in vitro transcribed double stranded RNA [4, 5, 6],14WSLEGAL\095576\00010\42816987v7

[0051] In some embodiments, the PRR agonist comprises poly I:C, which is non-covalently linked to cationically-modified glycogen nanoparticles by electrostatic interaction. The poly I:C can be complexed with the glycogen NPs by dissolving cationic phytoglycogen in a standard PBS solution, and allowed to equilibrate, for example by sitting at room temperature for about 24 hours. HMW or LMW Poly I:C (Invivogen™) is then mixed in solution with the phytoglycogen solution to adhere electrostatically to the cationic phytoglycogen. In some embodiments, a solution of the TLR3 agonist is added to the phytoglycogen solution and then mixed.

[0052] The weight ratio of glycogen nanoparticles to the PRR agonist may be varied. Ratios from about 1 :20 to about 4: 1 (nanoparticle:PRR agonist) have been found to induce a more robust immune response compared to agonist alone. Preferred ratios may be between about 1 : 10 - 2:1, dependent on the nanoparticle preparation and the source of the glycogen. Without restriction to a theory, the molecular weight of the poly I:C can also affect preferred ratios of NP:PRR agonist.

[0053] One known issue with many nucleic acid PRR agonists such as poly I:C, is that it is very polydisperse, and it is difficult to control batch to batch variations. Poly I:C shows multi-modal size distribution - typically 3 peaks in the molecular size chart. Surprisingly, the immunomodulator compositions described herein can mitigate that poly dispersity by the use of monodisperse glycogen NPs. Thus, in some embodiments, the described composition comprises monodisperse immunomodulator complexes comprising monodisperse glycogen NPs and polydisperse poly I:C.

[0054] In some embodiments, the immunomodulator composition comprises an average immunomodulator particle size in the range of about 50 to about 500 nm, preferably in the range of about 100 to about 300 nm, more preferably in the range of about 150 nm to about 250 nm, with a PDI of less than about 0.30, preferably less than about 0.20, and more preferably less than about 0.15.15WSLEGAL\095576\00010\42816987v7Pharmaceutical Compositions

[0055] In one embodiment, there is provided a pharmaceutical composition that comprises, consists essentially of, or consists of a composition of immunomodulators described herein.

[0056] A “pharmaceutical composition” is a composition suitable for administration to a human being in a medical setting. Preferably a pharmaceutical composition is sterile and produced according to GMP guidelines. Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing a disclosed compound (the “active ingredient” or in this case the immunomodulators) into association with a carrier and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit. Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0057] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as16WSLEGAL\095576\00010\42816987v7Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances, and the like that do not deleteriously interact with the immunomodulators of this disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0058] The novel immunomodulators of the invention may in some embodiments be admixed, encapsulated, or otherwise associated with other molecules, molecule structures or mixtures of compounds and may be combined with any pharmaceutically acceptable carrier or excipient.

[0059] For the purposes of formulating pharmaceutical compositions, monodisperse glycogen nanoparticles prepared as taught herein, may be provided in a dried parti culate / powder form or may be dissolved e.g. in an aqueous solution. Where a low viscosity is desired, the glycogen nanoparticles may suitably be used in formulations in a concentration of up to about 20% w / w. In applications where a higher viscosity is desirable, the nanoparticles may be used in formulations in concentrations above about 20% w / w. In applications where a gel or semi-solid is desirable, concentrations up to about 35% w / w can be used, or the nanoparticles can be used in combination with viscosity builders or gelling agent.

[0060] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Without limiting the generality of the foregoing, in some embodiments, the formulation is administered by intravenous injection, which permits treatment of all tumors with a vascular supply,17WSLEGAL\095576\00010\42816987v7even if their location is not known, or is inaccessible. Alternatively, the route of administration may be topical, e.g. administration to the skin or by inhalation or in the form of ophthalmic or optic compositions; enteral, such as orally (including, although not limited to in the form of tablets, capsules or drops) or in the form of a suppository; or parenteral, including e.g. subcutaneous, intravenous, intraarterial or intra-muscular. In some embodiments, the composition may be injected intra-tum orally.

[0061] In another embodiment, the pharmaceutical compositions of the present invention can be delivered in the form of an implant. Suitably, these implants may be biocompatible, meaning that they will have no significant adverse effects on cells, tissue or in vivo function. Suitably, these implants may be bioresorbable or biodegradable (in whole or in part). Examples of implants include, without being limited to, tissue engineering scaffolds.Methods of Treating Cancer

[0062] The immunomodulator compositions and pharmaceutical compositions comprising the immunomodulator compositions may be used to treat cancer, and in particular, solid tumor cancers such as ovarian cancer, when administered to a patient in a therapeutically effective amount. “Therapeutically effective amount” is intended to include an amount of the immunomodulators of the present invention alone or an amount of the immunomodulators of the present invention in combination with other active ingredients effective to act as an inhibitor or effective to treat or ameliorate cancer.

[0063] As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, intratumoral, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route,18WSLEGAL\095576\00010\42816987v7including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal) Parenteral administration includes, e.g., intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a compound or composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g., anti-cancer agent, chemotherapeutic, or immunotherapeutic agent). The compounds or compositions described herein can be administered alone or can be co-administered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compound or composition individually or in combination (more than one compound or agent). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).

[0064] As used herein, “treating” or “treatment” cover the treatment of a disease-state in a mammal, particularly in a human, and include: (a) inhibiting the disease- state, i.e., arresting its development; and / or (b) relieving the disease-state, i.e., causing regression of the disease state. In some rare instances, “treatment” may prevent the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it. In some embodiments, the treatment causes a patient to mount a multifaceted immune response against a solid tumor because the immunomodulator "makes cold tumors hot". The immune response kills cancerous cells, thereby causing tumor shrinkage.

[0065] “Patient” or “subject” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of the immunomodulator or pharmaceutical composition, as provided herein. Non-limiting examples include humans, other mammals including19WSLEGAL\095576\00010\42816987v7companion animals (dogs and cats), bovines, rats, mice, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the patient is human.

[0066] In some embodiments, the immunomodulator comprises monodisperse and substantially pure phytoglycogen NPs complexed with HMW poly I:C (referred to herein as "GLY100"), suspended in a suitable liquid medium, and is administered by intravenous injection to the patient. In some embodiments, in smaller mammals, such as rodents, a suitable dose may be in the range of 0.1 mg to about 20 mg of GLY100 per kg of body weight, and preferably is about 0.5 to 5 mg of GLY100 per kg body weight. In humans, in some embodiments, a suitable dose may be in the range of 0.1 mg to about 15 mg per kg of body weight, preferably in the range of about 1.0 to about 10 mg, and more preferably in the range of about 1.5 mg to about 5 mg per kg of body weight. In some embodiments, a suitable dose may be less than about 1.0 mg per kg, and even as low as less than 0.10 mg per kg of body weight.

[0067] Without restriction to a theory, it is believed that a biologically relevant induction of the innate immune response can be achieved at a much lower dose when the PPR agonists are linked to glycogen NPs, than when administered without glycogen NPs. Without restriction to a theory, this can be a function of the large amount of PPR agonist that can be transported, as well as the enhanced immune response associated with the nanoparticle delivery agent.

[0068] In some embodiments, the dosage may be expressed as the dose of poly I:C provided, as a fraction of the maximum tolerated dose (MTD) of naked poly I:C, which can be estimated, determined empirically or which may be found in the literature. We have found that an effective dose can be less than about 20% of the MTD, or less than 10% of the MTD of poly I:C. In some embodiments, the dose may be between about 2% to about 3% of the MTD of naked poly I:C.20WSLEGAL\095576\00010\42816987v7

[0069] These doses, whether determined by mass per body weight or as a percentage of MTD, may apply to or be adapted to other PRR agonists by those skilled in the art.

[0070] A therapeutically effective dose may be administered daily, weekly, biweekly, semimonthly or monthly, or on any regular or intermittent schedule, which may partly be dependent on the size of the dose.Examples

[0071] The following examples are intended solely to illustrate aspects or features associated with the described invention, and not to limit any claimed invention.

[0072] Example 1 - Manufacture of ultrapure phytoglycogen from sweet corn kernels

[0073] 5.5 kg of frozen sweet corn was thawed at 4 °C over about 48 hours, and then ground. The ground corn was added to 22 kg of water and heated to 60° C. The pH was adjusted to about 5.0 using 0.5M sulfuric acid and agitated for 20-30 min. After cooling to about 40° C, the mixture was strained and filtered through a cloth filter, with care taken not to squeeze the filter cake. The filtered juice was then centrifuged and the supernatant collected, with care taken not to collect the gel layer which forms on top of the solid pellet.

[0074] The liquid supernatant is then transferred to a filtration apparatus, with care not to incorporate any lipid layer which may have formed on the liquid, heated to 40° C and pH adjusted to about 12 with 4M NaOH, and filtered thorough 100 kDa membrane elements. The retentate was washed first with a NaOH pH 11.8 solution and then repeatedly with reverse osmosis water. The phytoglycogen is then precipitated with 95% w / w ethanol in a 1 : 1 volumetric ratio, in a fridge overnight. The precipitated phytoglycogen is then centrifuged and the solids recovered, and dried in21WSLEGAL\095576\00010\42816987v7a vacuum oven at 60 C and 10 mbar pressure over two days. The dried phytoglycogen was then crushed and blended to produce a powder product.

[0075] According to dynamic light scattering (DLS) measurements, the phytoglycogen nanoparticles produced had a Z-average particle size diameter of 83.0 nm and a poly dispersity index of 0.081.

[0076] Example 2. TEMPO oxidized phytoglycogen.

[0077] Phytoglycogen nanoparticles produced in Example 1 are dissolved in glycine buffer (0.05 M, pH 10.20, 33 mL / g phytoglycogen). The sample is placed in an ice bath. After 4 hours, a solution of TEMPO (0.04 g / g phytoglycogen) in glycine buffer (1.7 mL / g Phytoglycogen) is added to the reaction mixture. NaBr (0.60 g / g phytoglycogen) is then added. After an hour (reaction at 3 °C), NaClO solution (4.52% chlorine) is introduced over the course of 30 minutes (80 mL / g phytoglycogen per addition). The reaction mixture is stirred at 0-5 ° C for 72 hours, then quenched with anhydrous ethanol (13 mL / g Phytoglycogen). The mixture is dialyzed (12-14 kDa cut-off) against RO water for 6 cycles, and lyophilized to afford an off-white powder.

[0078] Example 3. Quaternary ammonium cationization of phytoglycogen.

[0079] Phytoglycogen nanoparticles produced in Example 1 are mixed with aqueous sodium hydroxide solution (1.5-60.0 mmol of NaOH dissolved in 1-5 ml of water / g phytoglycogen) and heated to 25 or 45 °C. Over the course of 10-120 minutes, 2,3-epoxypropyltrimethylammonium chloride in water (69% solution, 3.07 mL / g phytoglycogen) is added. Alternatively, 8.23 mmol of (3-chloro-2- hydroxy-prop-l-yl)dimethylalkyl ammonium chloride (alkyl= lauryl, cocoalkyl, stearyl) is stirred with 0.82 mL of 50% NaOH at 45 °C for 5 minutes, and 3.33 ml of 20 % aqueous solution of phytoglycogen is added. Each reaction is stirred for another 2-6 hours at 45 °C. Water (5- 9 mL / g phytoglycogen) is 22WSLEGAL\095576\00010\42816987v7then added, the mixture is cooled to room temperature and neutralized with 1 M HC1. The product is precipitated and washed in ethanol or hexanes (50-80 mL / g phytoglycogen), re-dissolved in water (20 mL / g Phytoglycogen) and / or saturated NaCl (10 mL) and further purified by dialysis. Freeze-drying affords the product as a white solid. Resulting products had a degree of substitution in the range of about 0.14 - to about 1.46, depending at least in part on the quantity of ammonium reactant used.

[0080] In another example, glycidyl trimethyl ammonium chloride (GT AC) is used. 1.0 g of phytoglycogen NPs produced in Example 1 was added in 4*20mL vials. Then, 4mL of deionized water were added to each vial by using a pipette. Each vial was mixed with a homogenizer (Ultra Turrax™ IKA T 18 basic) and kept in the fridge overnight.

[0081] The next morning, the vials were taken out and brought to room temperature. A stir bar was placed in each, and two assemblies were prepared comprised of hot / stir plate, heat block for vials and a probe thermometer. The vials were placed two by two on these assemblies (the stir was not efficient enough if the 4 vials were placed together on the same plate.) Then, 0.093g of powdered NaOH, previously crushed in a mortar, was put in each vial, and the parameters of the hot / stir plate were set to 45°C and 760 rotations per minute. After few minutes, the sodium hydroxide was entirely dissolved and GTAC was injected in each vial using a pipette, according to the following Table 1.Table 1: Volume and amount of GTAC added during the reactionVial Volume added Total volume GTAC amount Equivalent of every 6 minutes of 3.7M GTAC to(jiL) - for 6 GTAC added (mmol) phytoglycogen additions (mL)1 600 3.6 13.3 2.12 300 1.8 6.6 1.13 150 0.90 3.3 0.54 75 0.45 1.7 0.323WSLEGAL\095576\00010\42816987v7

[0082] After the six additions, the solutions were left stirring for 6 hours at ~45°C and at 760 rotations per minute. After 3 hours, it was observed that the solutions in Sample 1 and Sample 2 vials were viscous and the stir was not very efficient anymore. Therefore, every 20 minutes both vials were vortexed for 1 min at the maximum speed.

[0083] After 6 hours, the heating was stopped. ImL of deionized water was added to each vial. Each was vortexed for 1 minute at the maximum speed and replaced on the stir plate again. They were left stirring overnight at room temperature and at 800 rotations per minute.

[0084] The next morning, 3mL of deionized water was added to each vial. After stirring with a vortex for 30 seconds, each vial was left stirring for 30 min at room temperature and at 800 rotations per minute. Then, the pH of each solution was adjusted to 6.0-6.7 by dropwise addition of dilute HC1. Each solution was poured into a dialysis membrane (12-14kDa - SPECTRAPOR 25mmxl00) and these were placed in a large beaker full of deionized water for 3 days of dialysis (six water changes). Then, the samples were collected for freezing overnight. Finally, they were lyophilized for 5 days. The powders were then analyzed by NMR. For that purpose, samples were prepared by dissolving 30 mg in 750pL of deuterium oxide.

[0085] Example 4. Amination of Phytoglycogen - addition of 2-Bromoethylamine hydrobromide

[0086] 50mL of DMSO was poured into a round bottom flask (500mL). Then, about 10g of phytoglycogen nanoparticles was added to the DMSO. With another 50mL of DMSO, the weighing dish was rinsed and washed down into the round bottom flask. The mixture was left stirring overnight at room temperature and at 350 rpm. A septum was also placed in the flask’s mouth to seal it.

[0087] The next day, 12.50g of powdered sodium hydroxide was added to the reaction mixture.After few minutes, the liquid became dark yellow (due to sodium hydroxide) and more viscous. The24WSLEGAL\095576\00010\42816987v7solution was left stirring at room temperature for lh30 to dissolve the sodium hydroxide. In the meantime, a solution with 2-Bromoethylamine hydrobromide was prepared. For that purpose, 16.25g of this aminating agent was added in a glass Schott Bottle (lOOmL) and then suspended in 75mL of DMSO. The solution was stirred for 15 minutes. The 2-Bromoethylamine hydrobromide was entirely dissolved and the solution was clear. Finally, this last solution was poured in the round bottom flask (in which some NaOH powder was still not dissolved). The round bottom flask was left stirring overnight at room temperature and at 680 rpm. (The temperature had increased a bit (~30°C) because of the heat released by the stirring.)

[0088] The day after, the solution was less yellow (paler) and some white particles appeared in the solution. Then, the pH of the mixture was adjusted to 7.8 by adding dropwise diluted HC1 at 1.019M about 1 lOmL. The pH was measured with the pHmeter. Because of its high volume, the solution was concentrated by rotavapor; some white particles were left behind in the flask before evaporation. The rotavapor was set to P=60mbar, dP=5mbar, T(bath)=50°C, rotation=140rpm, for 1 hour. The concentrated solution was transferred to dialysis membranes (Spectra / Por 4 - MW: 12-14kDA) which were placed in a beaker full of overnight deionized water for 3 days of dialysing with 6 water changes.

[0089] After dialysis, the solutions were frozen overnight and were lyophilized for about 3 days. The solid obtained was white and fluffy. The DS was measured by conductimetric titration to be 0.3.

[0090] Example 5 - Production of GLY 100

[0091] GLY100 was prepared by first preparing a Img / ml GTAC cationic phytoglycogen NP solution in accordance with Example 3. The phytoglycogen NPs had a degree of substitution (DoS) of 1.14. A l mg / ml solution of high molecular weight (HMW) poly I:C (Invivogen™) was prepared as well as PBS (without Magnesium / Calcium).25WSLEGAL\095576\00010\42816987v7

[0092] All solutions were first brought to room temperature. The poly I:C solution was then heated in a water bath at 70°C for 30 minutes and mixed by repeated pipetting at 15 minute intervals. The solution was removed from heat and quickly centrifuged, and allowed to cool at room temperature for 30 minutes.

[0093] PBS, the phytoglycogen NP solution and the poly I:C solution were mixed in a tube (PBS added first, then Phytoglycogen, then poly I:C) to complex the NPs with the poly I:C. The desired mass ratios were achieved by mixing different volumes in a tube as follows:

[0094] Each tube thus had a final volume of 150 ul, with a poly I:C concentration of 333 ug / mL. The GLY100 product had an average particle size of 193 nm, with a PDI of 0.1295 and zeta potential of +27.23, which compares to the starting materials as follows:26WSLEGAL\095576\00010\42816987v7

[0095] Each tube was then mixed by gentle pipetting and then stood at RT for 30 minutes to allow complexation to finish. 4.85mL of 2% Dulbecco's Modified Eagle Medium (DMEM) was added into each tube (Total volume=5mL) and mixed by gentle pipetting. This solution was then used for in vitro cell tests.

[0096] Example 6 - CXCL10 and IFIT1 Production Boosted

[0097] As shown in Fig. 5, GLY100 boosts IFIT1 production in cancer cells over naked poly I:C (labeled as PIC). IFIT1 expression levels, measured through qPCR was significantly elevated when exposed to GLY100 over poly I:C alone and phytoglycogen alone (shown in the Figure as NDX). Mock negative control media having no poly I:C and no glycogen NPs showed no IFIT1 expression. Basal media did not include antibiotics or fetal bovine serum. FBS included 10% fetal bovine serum. Full media included 1% antibiotics and 10% fetal bovine serum.

[0098] The tests were conducted in the following cell lines, with substantially similar results: TOV1946, OV866(2), OV4485, TOV3133D, TOV30416.

[0099] In test animals, the above effect makes tumors visible to the animal’s immune system and CD8+ cells were seen to infiltrate the tumor. Without GLY100, CD8+ cells are excluded from the tumor.

[0100] Example 7 - Measurement of expression of CXCL10 in cancer cell lines

[0101] All cells were grown in 75cm2vented tissue culture flasks (Corning, North Carolina, USA) at 37°C with 5% CO2. Routine growth media was supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin (P / S) (Thermo Fisher Scientific, MA, USA). SKOV-3 cells (obtained from the ATCC, HTB-77) were cultured in McCoy's 5 A (Quality Biological, Maryland,27WSLEGAL\095576\00010\42816987v7USA) media. OVCAR-3 cells were cultured in RPMI-1640 (ATCC, VA, USA) media containing L- glutamine and supplemented with 0.01 mg / mL recombinant human insulin (Thermo Fisher Scientific). Cells were counted using trypan blue exclusion dye and the Thermo Fisher Countess II automated cell counter. After seeding at noted densities, all cells were allowed to attach overnight prior to experimental treatments. Treatments were completed in media that did contain any additives (P / S, FBS, or insulin), known as basal media.

[0102] Cells were seeded into 12 well plates at 1.5 xlO5cells / mL (1.5 x 105cells / well). Cells were then treated with 300pl basal media, or Ipg / mL of poly I:C alone, or Ipg / mL of GLY100 for 24h. The concentration used does not induce cell death after 24h but was sufficient to induce a measurable amount of CXCL10. The conditioned media was collected from the cells and centrifuged at 10,000xg for 5 min to remove debris. A human CXCL10 ELISA MAX deluxe set (439904; BioLegend, CA, USA) ELISA was used to quantify the level of CXCL10 in the supernatant following the manufacturers’ instructions. All samples were tested in technical triplicates. Concentrations were calculated using a standard curve analysis, with a limit of detection between 1000 pg / mL to 32 pg / mL. Values that fell below the limit of detection of <32pg / mL were given the value of 0. At least 3 independent experiments were run for each treatment group.

[0103] As can be seen in Fig. 1, GLY100 induced higher transcript expression and secretion of CXCL10 than poly (EC) alone. OVCAR-3, and SKOV-3 cells were treated with 1 pg / mL poly (EC), poly (EC)-Phytoglycogen (NDX) or control media. After 24 hours, CXCL10 transcript levels were measured using qRT-PCR (A, B) and secreted protein levels were measured in cell culture supernatant through ELISA (C, D). (A, B) Data are presented as relative to the mock treated cells and normalized to P-actin. One-way ANOVA analysis with Tukey's post-hoc test was performed on log2 transformed28WSLEGAL\095576\00010\42816987v7data. Significant differences, p < 0.05, are denoted with different letters. Mean ± SEM of 3 independent experiments.

[0104] Example 8 - Stability in Serum

[0105] GLY100 is more stable in serum than naked poly I:C. GLY100 and naked poly I:C were incubated in PBS with 5% FBS. As shown in Fig. 6, when naked poly I:C (GLY100 -) is incubated in 5% FBS, ds RNA is significantly degraded with lower molecular weight degradation products being detectable. When complexed with phytoglycogen NPs as GLY100, neither ds RNA (naked poly I:C) nor any degradation products may be detected.

[0106] IFIT1 expression levels also demonstrated stability of GLY100. As can be seen Fig. 5, immune stimulation in ovarian cancer cells treated in media containing 10% FBS was decreased greater than 75% for unbound dsRNA (labeled as PIC) indicating that naked poly I:C is degraded in serum. However, when nanoparticle bound dsRNA (GLY100+) is used, immune stimulation only decreases by 15% when cells are treated in FBS.

[0107] Example 9 - T oxi city

[0108] Poly I:C is known to trigger toxic effects and GLY100 presents a very high local concentration of poly I:C on the surface of the nanoparticles. Accordingly, one skilled in the art might expect that GLY100 can trigger toxic effects even though the bulk dosage is low. Surprisingly, GLY100 is non-toxic to animals at therapeutic dosages and is less toxic than an equivalent amount of naked poly I:C (in vitro). Without restriction to a theory, it appears that GLY100 concentrates in the tumor (specifically in the endosomes) and does not remain in circulation very long. Furthermore, without restriction to a theory, an effective therapeutic dose for GLY100 can be low enough to avoid significant toxic effects.29WSLEGAL\095576\00010\42816987v7

[0109] Example 10 - GLY100 Shrinks Tumors

[0110] Orthotopic, syngeneic mouse (average weight of 20 g) model of epithelial ovarian cancer were used to demonstrate that GLY100 produced significant primary tumor shrinkage after only 2 injections.

[0111] For tumor induction, mice were anesthetized with isofluorane inhalation, a dorsal midline incision was made and 1 x 106ID8 cells (originally obtained from Dr. Kathy Roby at Kansas State University) in 6ul PBS were injected under the ovarian bursa. The ovary was replaced and the incision was closed with two skin staples. Postsurgical monitoring was conducted according to animal care committee guidelines.

[0112] The day of surgery was considered day 1 for the experimental protocol. Mice progressed without intervention until day 64 post-tumor induction (PTI). At day 64 PTI, mice received 60ug GLY100 (2: 1) in 200ul PBS (n=7) or 200ul PBS alone (as control, n=9) intravenously through the retroorbital vein. Mice then received a second IV infusion of 60ug GLY100 or control one week later on day 71 PTI. At day 78 PTI, mice were euthanized, and tumors were collected and weighed.

[0113] All of the PBS mice had large numbers of metastatic tumors while GLY100 treatment reduced the observed numbers (Fig. 3). Tumor weight decreased with GLY100 treatment (Fig. 4). In Fig. 3, each point is a mouse having a tumor score corresponding to a scoring system where 0 (no visible tumors in the abdomen); + (less than 3 visible tumors in the abdomen); ++ (4-10 visible tumors in the abdomen); and +++ (greater than 10 visible tumors in the abdomen).

[0114] Thus, immune competent mice (syngeneic C57BL-6) with late-stage ovarian cancer, injected with GLY100 twice, 1 week apart, resulted in 70% shrinkage of the primary tumor and a substantial reduction in visible metastatic tumors, as may be seen in Fig. 2. At the doses provided, 30WSLEGAL\095576\00010\42816987v7GLY100 is non-toxic to tumor cells, thus the tumor shrinkage is the result of an immune response stimulated by GLY100.

[0115] Example 11 - GLY100 Effectiveness at Lower Doses

[0116] The testing of Example 10 was repeated with 2.4 ug GLY100 per dose and 12 ug GLY100 per dose (equivalent to 0.12, 0.6 and 3 mg / kg (as poly I:C) or 0.36, 1.8 and 9 mg / kg as GLY100). 12 mice were treated with the different doses and a negative control, and the mice sacrificed at day 78 PTI (2 weeks after start of therapy). Tumor score was determined as well as the presence of ascites. As can be seen in Figs. 7A, tumor shrinkage was significant even at the lowest dose, and number of tumors was somewhat reduced. Fig. 7B shows the results from 16 mice euthanized at Day 92 (4 weeks after start of therapy). Again, tumor shrinkage was significant even at the lowest dose.Interpretation.

[0117] The forgoing description supplies specific details in order to provide a thorough understanding. Nevertheless, the skilled artisan would understand that the compositions, and associated methods of preparing or using the compositions can be implemented and used without employing these specific details. Indeed, the compositions and associated methods can be placed into practice by modifying the described compositions and associated methods and can be used in conjunction with any other compositions and techniques conventionally used in the industry.

[0118] The corresponding structures, materials, acts, and equivalents of all means or steps plus function elements in the claims appended to this specification are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed.31WSLEGAL\095576\00010\42816987v7

[0119] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such module, aspect, feature, structure, or characteristic with other embodiments, whether or not explicitly described. In other words, any module, element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility, or it is specifically excluded.

[0120] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation. The terms “preferably,” “preferred,” “prefer,” “optionally,” “may,” and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention. The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage.32WSLEGAL\095576\00010\42816987v7

[0121] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.

[0122] As will also be understood by one skilled in the art, all ranges described herein, and all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number(s) recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above.References

[0123] The following references are indicative of the level of skill of one skilled in the art, and are incorporated herein by reference, where permitted, in their entirety.1Naumann, Kai; Wehner, Rebekka; Schwarze, Anett; Petzold, Christiane; Schmitz, Marc; Rohayem, Jacques (2013). "Activation of Dendritic Cells by the Novel Toll-Like Receptor 3 Agonist RGC100". Clinical and Developmental Immunology. 2013 283649. doi: 10.1155 / 2013 / 2836492Seya, T., Takeda, Y., & Matsumoto, M. (2016). Tumor vaccines with dsRNA adjuvant ARNAX induces antigen-specific tumor shrinkage without cytokinemia. Oncolmmunology, 5(2). https: / / doi.org / 10.1080 / 2162402X.2015.10435Q63Komal, A., Noreen, M. & El-Kot, A.F. TLR3 agonists: RGC100, ARNAX, and poly-IC: a comparative review. Immunol Res 69, 312 322 (2021). https: / / doi.org / 10.1007 / sl2026-021-092Q3-4Mu, X. & Hur, S. Immunogenicity of In Vitro-Transcribed RNA. Acc. Chem. Res. 2021, 54, 21,33WSLEGAL\095576\00010\42816987v74012-4023;5Ko, K.H. et al. A novel defined TLR3 agonist as an effective vaccine adjuvant. Volume 14 - 2023 | https: / / doi.org / 10.3389 / fimmu.2023.1075291;6Le Naour, J., Thierry, S., Scuderi, S. A., Boucard-Jourdin, M., Liu, P., Bonnin, M., ... Werle, B. (2023). A Chemically Defined TLR3 Agonist with Anticancer Activity. Oncolmmunology, 12(1). https: / / doi.org / 10.1080 / 2162402X.2023.222751Q)7 Zhukouskaya et al. Anionically Functionalized Glycogen Encapsulates Melittin by Multivalent Interaction. Biomacromolecules 2022, 23, 8, 3371-33828 Pal et al. Synthesis, characterization and flocculation characteristics of cationic glycogen: A novel polymeric flocculant. Colloids and Surfaces A: Physicochemical and Engineering Aspect. Volume 289, Issues 1-3, 2006, Pages 193-199WSLEGAL\095576\00010\42816987v7

Claims

What is Claimed is:

1. A pharmaceutical composition comprising monodisperse immunomodulators comprising monodisperse glycogen nanoparticles complexed with a pattern recognition receptor agonist.

2. The composition of claim 1 wherein the immunomodulators have a PDI of less than about 0.2, or about 0.15.

3. The composition of claim 1 or 2 wherein the pattern recognition receptor agonist is a TLR agonist, such as a TLR3 agonist, a RIG-I agonist or a MDA5 agonist.

4. The composition of claim 3 wherein the TLR agonist is a TLR3 agonist.

5. The composition of claim 4 wherein the TLR3 agonist is poly I:C or a variant or derivative thereof, such as poly-IC12U or poly-ICLC.

6. The composition of any one of claims 1-5 wherein the glycogen nanoparticles comprise phytoglycogen nanoparticles.

7. The composition of any one of claims 1-6, wherein the glycogen nanoparticles have a poly dispersity index (PDI) of less than about 0.3, less than about 0.2, less than about 0.15, or less than about 0.10 as measured by DLS; or a polydiversity index (PDI*) of less than about 1.3, less than about 1.2, less than about 1.15, or less than about 1.10, as measured by SEC-MALS.

8. The composition of any one of claims 1 -7 wherein the glycogen nanoparticles and the PRR agonist are non-covalently linked.

9. The composition of any one of claims 1-8 comprising a mass ratio of glycogen nanoparticles to the PRR agonist in the range of about 1 :2 to about 2: 1.

10. The composition of any one of claims 1-9 wherein the glycogen nanoparticles are cationized before binding to the PPR agonist, with a degree of substitution between about 0.1 to about 1.5.35WSLEGAL\095576\00010\42816987v711. The composition of any one of claims 1-10 wherein the monodisperse immunomodulators are formed with substantially pure glycogen nanoparticles.

12. A method of treating a solid tumor cancer in a patient, wherein the patient is triggered to mount an immune response, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising immunomodulators comprising glycogen nanoparticles complexed with a pattern recognition receptor agonist.

13. The method of claim 12 wherein the pharmaceutical composition is the composition of any one of claims 1-11.

14. The method of claim 12 or 13 wherein solid tumor cancer is an ovarian cancer.

15. The method of claim 14 wherein the ovarian cancer is an epithelial ovarian carcinoma, a germ cell tumor, a stromal cell tumor and / or a recurrent or relapsed ovarian cancer.

16. The method of any one of claims 12-15, wherein the composition is administered intravenously, intraperitoneally, or intratumorally.

17. The method of any one of claims 12-16, wherein the patient is a mammal, preferably a human.

18. The method of any one of claims 12-17, wherein the immunomodulators are administered in a dose of 0.1 mg to about 15 mg per kg of body weight, about 1.0 to about 10 mg, or about 1.5 mg to about 5 mg per kg of body weight.

19. The method of any one of claims 12-17, wherein the immunomodulators are administered to provide a dose of PRR agonist which is a fraction of the maximum tolerated dose (MTD) of naked PRR agonist, such as less than about 20%, 10%, 5% or 3% of the MTD.

20. Use of an immunomodulator comprising a phytoglycogen nanoparticle bearing a TLR3 agonist comprising poly I:C, or a derivative or variant thereof, to treat a solid tumor cancer in a patient.36WSLEGAL\095576\00010\42816987v721. Use of an immunomodulator comprising a phytoglycogen nanoparticle bearing a TLR3 agonist comprising poly I:C, or a derivative or variant thereof, for the manufacture of a medicament for treating a solid tumor cancer in a patient.

22. The use of claim 20 or 21, wherein the solid tumor cancer is ovarian cancer.

23. The use of any one of claims 20-22 wherein the patient is a human.37WSLEGAL\095576\00010\42816987v7

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