WO2018207153A1

WO2018207153A1 - Toll-like receptor agonist modified particles and their use in lung cancer

Info

Publication number: WO2018207153A1
Application number: PCT/IB2018/053314
Authority: WO
Inventors: Joseph M. Desimone; Jillian PERRY; Chintan KAPADIA; Shaomin TIAN; James Christopher LUFT; Tojan RAHHAL
Original assignee: University of North Carolina at Chapel Hill
Current assignee: University of North Carolina at Chapel Hill
Priority date: 2017-05-11
Filing date: 2018-05-11
Publication date: 2018-11-15
Anticipated expiration: 2019-11-11

Abstract

The subject matter described herein is directed to molded nanoparticles that are covalently linked to a toll-like receptor agonist molecule, methods of treating lung cancer with the particles, methods of preparing the particles, and compositions comprising the particles.

Description

TOLL-LIKE RECEPTOR AGONIST MODIFIED PARTICLES AND THEIR USE

IN LUNG CANCER

Reference to a Sequence Listing Submitted as a Text File This application includes an electronic sequence listing in a file named

"514324SequenceListing_ST25," created on May 10, 2018, and containing 626 bytes, which is hereby incorporated by reference in its entirety for all purposes.

Government Support

This invention was made with government support under Grant Number CA198999 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

The subject matter described herein is directed to molded nanoparticles that are covalently linked to a toll- like receptor agonist molecule, and methods of treating lung cancer with the particles, and methods of preparing the particles and compositions comprising the particles.

Background

Lung cancer is the leading cause of cancer death and the second most common cancer among both men and women in the United States. Lung cancers are grouped into two main types: small cell and non-small cell, with non-small cell lung cancer (NSCLC) accounting for 80-85% of all lung cancers. Though there have been many advances in early detection and surgical resection methodologies, as well as new radiation and chemotherapeutic treatments, the 5-year survival rates for NCSLC remain poor, as majority of patients are diagnosed with advanced stage disease.

What is therefore needed is a means for treating lung cancer, in particular, NSCLC.

The subject matter described herein addresses this shortcoming in the art.

Brief Summary

In embodiments, the subject matter described herein is directed to engineered hydrogel particles having toll- like receptor molecule agonist molecules covalently linked to the surface of the particles.

In embodiments, the subject matter described herein is directed to methods of treating lung cancer by administering a composition comprising engineered hydrogel particles having toll- like receptor molecule agonist molecules covalently linked to the surface of the particles or by administering engineered hydrogel particles having toll- like receptor molecule agonist molecules covalently linked to the surface of the particles.

In embodiments, the subject matter described herein is directed to methods of preparing engineered hydrogel particles having toll-like receptor molecule agonist molecules covalently linked to the surface of the particles.

Other embodiments are also described.

Brief Description of the Figures

Fig. 1 depicts IVIS live animal imaging of luminescent 344SQ tumor bearing mice. Arrows indicate a cytosine-phosphate-guanine (CpG) nucleotide treatment (20 μg of instilled CpG).

Figs. 2A-2B depicts percent change in mouse body weight after tumor cell inoculation and percent survival. Black triangles indicate CpG treatment.

Fig. 3 depicts surviving mice challenged with orthotopic 344SQ tumor cells, indicated by the arrow.

Fig. 4 depicts in vivo CpG release from nanoparticles in Gambles solution, artificial lysosomal fluid (ALF), and 1 OX PBS.

Fig. 5 depicts histology images of mouse lungs (inoculated with 344SQ tumors) 24 and 48 hours post-treatment with either NP-CpG or CPG (green).

Fig. 6 depicts histology images of mouse lungs (inoculated with 344SQ tumors) 24 and 48 hours post-treatment with NP-CpG (20 μg CpG).

Fig. 7 depicts histology images of mouse lungs (inoculated with 344SQ tumors) 24 and 48 hours post-treatment with NP-CpG (20 μg CpG). Yellow indicates NP-CpG, indicated macrophages (F4/80 stain).

Fig. 8 depicts flow analysis of mouse lungs inoculated with 344SQ cells and treated with NP-CpG (20 μg CpG) through instillation.

Fig. 9 depicts the survival curve for mice inoculated with 344SQ in the lung and treated with clodrosome to deplete macrophages.

Figs. lOA-lOC depicts bio luminescence IVIS images of mice inoculated with 344SQ luciferance expressing tumor cells, treated with NP-CpG and (A) clodrosome to deplete macrophages, (B) anti-Asialo GM1 to deplete NK cells, and (C) anti-CD8 to deplete CD8 T cells. Detailed Description

The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

A strategy emerging to treat NSCLC is immunotherapy, which utilizes medicines to stimulate the immune system to better find, fight, and destroy cancer. Patients with specific genetic mutations may benefit from targeted therapies such as the epidermal growth factor receptor (EGFR) blockers erlotinib (Tarceva®), afatinib (Gilotrif®), and gefitinib (Iressa®). These drugs block the signal from EGFR that tells cells to grow. They can be used to treat patients with certain mutations in the EGFR gene, which are more common in women and people who have not smoked. Immunotherapies may offer significant benefit to lung cancer patients, including those for whom other treatments are ineffective. Bevacizumab (Avastin®) is a monoclonal antibody that targets vascular endothelial growth factor (VEGF), a protein that helps new blood vessels grow. By preventing tumors from growing new blood vessels, a process called angiogenesis, Avastin starves the tumor of nutrients. Ramucirumab (Cyramza®) is another angiogenesis inhibitor that can be used to treat NSCLC. In 2015, two new immunotherapy drugs, nivolumab (Opdivo®) and pembrolizumab (Keytruda®), both are check-point inhibitors, were approved by the FDA for the treatment of lung cancer.

Multiple immunotherapies are currently under investigation, including other checkpoint inhibitors, therapeutic cancer vaccines, adoptive T cell transfers, and toll-like receptor (TLR) agonists. TLR-9 agonists, such as CpG oligonucleotides, are currently being investigated for the treatment of lung cancer with mixed results.^1"2 These inconsistent clinical findings may be a function of lack of appropriate administration/accumulation of CpG.^3"6 However, as shown herein, engineered hydrogel particles having covalently linked TLR agonists improved survival nearly three-fold.

The TLR-9 agonist CpG when administered locally has multiple desirable features as related to the treatment of cancer due to its potent stimulation of innate and adaptive immunity, specifically the activation of dendritic cells (DCs), induction of T-helper (TH) 1 cytokines, and reduction of Tregs^{1 8}. CpG stimulates an immune response after binding to the TLR-9 receptors in the endosome of antigen presenting cells such as DCs and macrophages¹. Since nanoparticles are naturally cleared by macrophages, DCs, and other APC's, they are excellent carriers of CpG delivery to immune cells. Furthermore, by attaching or encapsulating CpG to or within nanoparticles, systemic release of proinflammatory cytokines can be reduced and therefore attenuate the systemic side effects associated with soluble CpG.

Particles and Methods of Treatment

As described herein, Particle Replication in Non- wetting Templates (PRINT) hydrogel particles locally deliver oligonucleotides, e.g., CpG to orthotopic 344SQ murine lung tumors (a model of human NSCLC) via orotracheal instillation. These nanoparticles having a covalently linked oligonucleotide (NP-OLI, specifically, NP-CpG) promoted tumor regression and culminated in 100% survival of mice with otherwise fatal 344SQ lung cancer, as compared to only 30% survival of mice treated with soluble CpG. Furthermore, surviving mice remained cancer free when re-challenged with orthotopic tumors months after cessation of CpG therapy.

As used herein, the term "particle" or "particles" is intended to mean one or more molded particles. The particles can comprise a polymer matrix. In embodiments, the particles are hydrogel particles comprising PEG. The methods and materials for fabricating the particles described herein are further described and disclosed in patent applications, each of which are incorporated herein by reference in their entirety: U.S. Pat. Nos. 9,340,001; 9,214,590; 9,205,594; 8,992,992; 8,945,441; 8,662,878; 8,518,316;

8,444,907; 8,444,899; 8,439,666; 8,420,124; 8,268,446; 8,263,129; 8,158,728; 8,128,393; 7,976,759; U.S. Pat. Application Publications Nos. 2016-0236379, 2016-0059473, 2015- 0283079, 2014-0027948, 2013-0209564, 2013-0228950, 2013-0011618, 2011-151015, 2010-0003291, 2009-0165320; and PCT Publication No. WO2015/073831. While certain embodiments described herein employ 80nm x 320 nm particles, the particles are scalable to other sizes including but not limited to 55 nm x 70 nm, 80 nm x 180 nm, Ιμιτι x Ιμιτι , and additional micron sized particles.

Described herein are engineered and manufactured PRINT particles covalently linked to one or more toll-like receptor (TLR) agonists. To achieve CpG modified NPs, the amine groups on NP surface were reacted with the succinimidyl ester of the SMCC (succinimidyl 4-(N-maleimido methyl) eye lohexane-l-carboxy late) linker, forming an amide bond. After modification with the linkers, NPs were then incubated overnight with thiol-containing CpG, resulting in CpG conjugation to the NPs via Michael addition.

In embodiments, the subject matter described herein is directed to engineered particles having covalently linked TLR agonist on the surface of the particle. In particular, TLR-9 agonists induce activation and maturation of plasmacytoid dendritic cells and enhance differentiation of B cells into antibody- secreting plasma cells. In embodiments, the TLR agonist is an oligonucleotide. In particular, the agonist is a cytosine-phosphate- guanine (CpG) nucleotide, such as a di- nucleotide or oligonucleotide. In embodiments, the CpG oligonucleotide has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to a 5' to 3' sequence: TCCATGACGTCCTGACGTT. In embodiments, the CpG oligonucleotide is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a 5' to 3' sequence:

TCCATGACGTCCTGACGTT. In a further embodiment, a homologous CpG

oligonucleotide can have a sequence identity with a 5' to 3' sequence:

TCCATGACGTCCTGACGTT, for example, of greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%. Also useful are any known synthetic oligodeoxynucleotide (ODN) agonists since they can be covalently linked as described herein to the particles. A "covalent" bond is a bond which involves the sharing of pairs of electrons. As used herein, a "conjugate" refers to atoms and/or molecules that are covalently bound to one another. For example, a particle covalently bound to a CpG can be referred to as a CpG conjugated particle.

In embodiments, the subject matter described herein is directed to methods of treating lung cancer comprising administering to a subject in need thereof an engineered particle, e.g., a nanoparticle, having a covalently linked TLR agonist(s) on the particle surface. Described herein in an embodiment is a method to treat an orthotopic model of human NSCLC with CpG covalently attached to nanoparticle (80x320 nm) hydrogel PRINT particles, delivered to the lung through orotracheal instillation. The Particle Replication In Non-wetting Templates (PRINT®) process allows for the molding of shape- specific particles on the order of tens of nanometers to several microns in size and enables the simultaneous and independent control over numerous physical parameters (size, shape, composition, cargo, and surface properties). PRINT particles have previously been assessed for lung delivery through instillation⁷. After instillation, particles remained in the lungs for up to 7 days without clearance or triggering of host immunity, suggesting their potential for sustained and localized delivery of therapeutics to the lungs⁷.

In embodiments, the subject matter described herein is directed to methods of treating a lung cancer in a subject in need thereof by administering a pharmaceutical composition comprising engineered particles having a TLR agonist(s) covalently linked to the surface of the particle. A subject "in need of or "in need thereof the methods disclosed herein may be a subject that is experiencing a disease state and/or is anticipated to experience a disease state, and the methods, particles, and/or compositions of the presently disclosed subject matter are used for therapeutic and/or prophylactic treatment of the disease state. As used herein, the term "disease state" refers to any abnormal condition that interferes with a physiological process. Non-limiting disease states are listed herein and more than one disease state (i.e., multiple disease states) may be present in a subject at the same time.

In embodiments, the particles and methods provide about 3x improved survival over soluble TLR-9 agonists.

In embodiments, the particles and methods provide about 70% increase in survival of a lung cancer subject. In embodiments, the particles and methods provide a decrease in side effects as compared to soluble TLR-9 agonists.

In embodiments, the particles and methods provide a targeted delivery of TLR-9 agonists to immune cells and a significant improvement in survival in an established lung cancer model.

In embodiments, the subject matter described herein is directed to methods of preparing engineered particles having a TLR agonist covalently linked to the surface of the particle.

As described herein, in embodiments, is a method of making a pharmaceutical composition, comprising: contacting a PEG hydrogel particle having amine groups on a surface with succinimidyl ester of a succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate linker to form an amide bond on the PEG hydrogel particle; and

incubating the amide bond modified PEG hydrogel particle with thiol-containing TLR agonist, whereby the thiol-containing TLR agonist conjugates to the amide bond modified PEG hydrogel particle.

As in any embodiment above, a method wherein the TLR agonist comprises TLR-

9.

As in any embodiment above, a method wherein the TLR agonist comprises an oligonucleotide.

As in any embodiment above, a method wherein the TLR agonist comprises a cytosine-phosphate-guanine (CpG) oligonucleotide.

As in any embodiment above, a method wherein the TLR agonist comprises a synthetic oligodeoxynucleotide (ODN) agonist.

As in any embodiment above, a method wherein the pharmaceutical composition is administered through inhalation delivery.

As in any embodiment above, a method wherein the pharmaceutical composition is administered in an amount of about 1 μg/kg to 50 mg/kg.

As in any embodiment above, a method wherein the PEG hydrogel particle comprises an engineered molded non-spherical shape, in cross-section, comprising about 80 nm by 320 nm, about 55 nm by 70 nm, about 80 nm by 180 nm, or about 1 μιη by 1 μιη.

As in any embodiment above, a method wherein the pharmaceutical composition is for treating non-small cell lung cancer (NSCLC).

As in any embodiment above, a method wherein the pharmaceutical composition further comprises a pharmaceutical excipient.

In embodiments, as described herein, is a method for reducing toxicity effects associated with a soluble treatment of TLR-9 agonists comprising:

administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a plurality of particles, wherein each particle of the plurality comprises:

an engineered molded non-spherical shape,

a hydrogel, and

a TLR-9 agonist covalently attached to a surface of the particle. As in any embodiment above, a method wherein the pharmaceutical composition is administered in an amount of about 1 μg/kg to 50 mg/kg.

As in any embodiment above, a method wherein the therapeutically effective amount comprises about 1 mg to about 1,500 mg TLR agonist.

As in any embodiment above, a method wherein the reduction in toxicity effects results in increasing the survival rate of the first plurality of subjects administered an effective amount of the plurality of particles by three times when compared to the survival rate of a second plurality of subjects administered an equivalent amount of a soluble TLR- 9 agonist.

As in any embodiment above, a method wherein the TLR-9 agonist comprises a cytosine-phosphate-guanine (CpG) oligonucleotide.

As in any embodiment above, a method wherein the hydrogel particle comprises an engineered molded non-spherical shape, in cross-section, comprising about 80 nm by 320 nm, about 55 nm by 70 nm, about 80 nm by 180 nm, or about 1 μιη by 1 μιη.

As in any embodiment above, a method wherein the reduction in toxicity effects results in reduction of systemic release of pro-inflammatory cytokines.

In addition to the TLR agonist present on the surface of the particles, particles described herein can comprise an active agent dispersed in a biocompatible matrix. The matrix is in particular embodiments a hydrogel containing polyethylene glycol (PEG). The biocompatible matrix is formed from one or more materials that are compatible with living tissue or a living system and are preferably not toxic, injurious or physically adversely reactive. Particles of the subject matter described herein are also compatible with living tissue or a living system. However, some degree of toxicity, injury, or physical adversity may be tolerated, such as that necessary to diagnose, cure, mitigate, treat, or prevent a disease in an organism. In some embodiments, the biocompatible matrix is comprised of one or more polymers. The polymer may be a homopolymer, or a hetero- or co-polymer, such as an alternating or block copolymer. Preferably, such polymeric matrix is biocompatible, biodegradable, bioresorbable, biodissolvable, and/or bioclearable in or from the human body. Suitable biocompatible polymers include polymers selected from the group consisting of a polyester, a polyanhydride, a polyamide, a phosphorous-based polymer, a poly(cyanoacrylate), a polyorthoester, a polyurethane, a polyorthoester, a polyether, a carbohydrate, a polypeptide, a hydro xypropylcellulose, a poly(ethylene glycol), a wax, a hydrogel, a phosphatidylcholine, a polydihydropyran, a polyacetal, a biodegradable polymer, and combinations thereof. In some embodiments, the polyester is selected from the group consisting of polylactic acid, polylactide, polyglycolic acid,

poly(hydroxybutyrate), poly(8-caprolactone), poly(P-L-malic acid), polydioxanone, poly(lactide-co-glycolide) polymer, and polyhydroxyalkanoate. In some embodiments, the polyanhydride is selected from the group consisting of poly(sebacic acid), poly(adipic acid), and poly(terephthalic acid). In some embodiments, the polyamide is selected from the group consisting of poly(imino carbonates) and polyamino acids. In some

embodiments, the phosphorous-based polymer is selected from the group consisting of polyphosphate, a polyphosphonate, and a polyphosphazene. In embodiments, the polymer is a polyester. In preferred embodiments, the polymer is a poly(lactide-co-glycolide) polymer.

In some embodiments, the particles are highly uniform with respect to shape, size and/or composition. One way in which such particles may be fabricated is using PRINT™ Technology (Liquidia Technologies, Inc.), which is a method capable of forming particles that: (i) are monodisperse in size and uniform shape, (ii) can be molded into any shape, (iii) can be comprised of essentially any matrix material, in particular biocompatible materials, (iv) can be formed under mild conditions (compatible with delicate cargoes), (v) are amenable to post-functionalization chemistry to covalently link a TLR agonist on the particle surface, and (vi) which initially fabricates particles in an addressable 2D array.

These additional embodiments are disclosed herein:

1. A particle comprising an engineered hydrogel having a to 11- like receptor agonist covalently linked to the surface of said particle.

2. The particle of embodiment 1, wherein the to 11- like receptor agonist is a TLR-9 receptor agonist. 3. The particle of embodiment 2, wherein the TLR-9 receptor agonist is a CpG nucleotide molecule.

4. The particle of embodiment 3, wherein the oligonucleotide has at least 70% homology to a 5' to 3' sequence: TCC ATG ACGTCCTGACGTT .

5. The particle of embodiment 1, wherein the to 11- like receptor antagonist is linked via a group comprising a thiophosphate.

6. The particle of embodiment 1, wherein the hydrogel comprises PEG.

7. A pharmaceutical composition comprising a plurality of particles of embodiment 1 and a pharmaceutical excipient.

8. A method of treating lung cancer comprising, administering a therapeutically effective amount of the pharmaceutical composition of embodiment 7 or the particle of embodiment 1 to a subject in need thereof.

9. The method of embodiment 8, wherein administration is via instillation or intravenously.

10. A method of preparing a particle of embodiment 1 comprising,

i. preparing an engineered hydrogel particle having surface amine groups;

ii. contacting the engineered hydrogel particle having surface amine groups with a carboxylate to form a particle having surface amide groups;

iii. contacting the particle having surface amide groups with a thiol- containing reactant having a toll- like receptor molecule moiety,

wherein the particle is prepared.

As used herein, the terms "contact", "contacting", and the like, refer to two or more substances in close proximity so that an effect may occur. In embodiments, the effect is formation of a chemical bond such as, but not limited to, a covalent bond.

As referred to herein, an amount, value or shape that is the "same," "substantially the same" or "substantially similar" is one that does not vary in a significant way from a given reference point or value. With regard to particles formed by the present methods, the shapes and dimensions of the particles are reproducible and a plurality of particles is substantially the same in shape, size, and composition. A plurality of particles means at least two particles. Scanning electron micrography can be used to evidence the substantially similar nature of the particles even at nanometer resolution.

As used herein, the term "substantially mimicking" means a molded particle that has a shape that is predetermined from the mold used to prepare the particle. This term includes variance in the shape, size, volume, etc. of the particle from the mold itself.

However, the particles shape, size, volume etc. cannot be random since they are prepared from molds and substantially mimic the mold's shape, size, volume, etc. The term

"amorphous" refers to a shape that is not engineered. A shape that is not prepared from a mold can be amorphous. Amorphous shapes by definition cannot be systematically reproducible. This is in contrast to molded shapes.

According to some embodiments, the composition can further include a plurality of particles, where the particles have a substantially uniform mass, are substantially monodisperse, are substantially monodisperse in size or shape, or are substantially monodisperse in surface area. In some embodiments, the plurality of particles have a normalized size distribution of between about 0.80 and about 1.20, between about 0.90 and about 1.10, between about 0.95 and about 1.05, between about 0.99 and about 1.01, between about 0.999 and about 1.001. According to some embodiments, the normalized size distribution is selected from the group of a linear size, a volume, a three dimensional shape, surface area, mass, and shape. In yet other embodiments, the plurality of particles includes particles that are monodisperse in surface area, volume, mass, three-dimensional shape, or a broadest linear dimension.

Particle characteristics used to describe the shapes examined include: a) the shape diameter (SD); it is the minimum diameter of a circumscribed circle around the particle; b) the minimum feature size (MFS); it is the diameter of the smallest distinct geometry of the shape; and c) the volume of the shape. All of these characteristics can be readily determined by one of skill in the art using the information disclosed herein and

information known in the art. In embodiments where the shape of the particle is essentially a rod, the particles can have aspect ratios calculated by the width x height. Aspect ratio refers to the ratio of the longest axis to the shortest axis of a particle. Aspect ratios for rod shapes will be > 1 : 1. In embodiments, the aspect ratio is 2: 1 ; 3 : 1 ; 4: 1 ; 5 ; 1 ; 6: 1; 7: 1, 8: 1; 9: 1; 10: 1 and so on.

In some embodiments, the physical properties of the particle are varied to enhance cellular uptake. In some embodiments, the size (e.g., mass, volume, length or other geometric dimension) of the particle is varied to enhance cellular uptake. In some embodiments, the charge of the particle is varied to enhance cellular uptake. In some embodiments, the charge of the particle ligand is varied to enhance cellular uptake. In some embodiments, the shape of the particle is varied to enhance cellular uptake. In some embodiments, the physical properties of the particle are varied to enhance biodistribution. In some embodiments, the size (e.g., mass, volume, length or other geometric dimension) of the particle is varied to enhance biodistribution. In some embodiments, the charge of the particle matrix is varied to enhance biodistribution. In some embodiments, the charge of the particle ligand is varied to enhance biodistribution. In some embodiments, the shape of the particle is varied to enhance biodistribution. In some embodiments, the aspect ratio of the particles is varied to enhance biodistribution. In some embodiments, the physical properties of the particle are varied to enhance cellular adhesion. In some embodiments, the size (e.g., mass, volume, length or other geometric dimension) of the particle is varied to enhance cellular adhesion. In some embodiments, the charge of the particle matrix is varied to enhance cellular adhesion. In some embodiments, the charge of the particle ligand is varied to enhance cellular adhesion. In some embodiments, the shape of the particle is varied to enhance cellular adhesion.

The term "treating" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. Thus, for example, "treating" a patient involves prevention of a particular disorder or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting or causing regression of a disorder or disease. As used herein the terms "treating" includes "ameliorating," which refers to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the condition or symptoms and does not necessarily indicate a total elimination of the underlying condition. In embodiments, the term "ameliorating" and "dampening" refer to a lessening of the severity of a symptom and there are clinical assessments and markers that can be used to identify and quantify the lessening of symptoms. Also included in the amelioration of symptoms is the perception by the subject that the symptoms have lessened.

As used herein, the terms "reduce," "reduces," "reduced," "reduction", and "inhibit", refer to a decrease in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%.

The term "therapeutically effective amount" as used herein refers to an amount of the particles containing a TLR-9 agonist that is sufficient to achieve a certain outcome, such as to treat lung cancer. The effective amount and dosage of such TLR agonists required to be administered for effective treatment are known in the art or can be readily determined by those of skill in this field. Of course, the amount of TLR agonist administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active compound, and the like. Determination of an effective dosage is well within the capabilities of those skilled in the art coupled with the general and specific examples disclosed herein. Where TLR agonists do not have a known dosage for certain diseases, the effective amount of the TLR agonist and the amount of a particular dosage form required to be administered for effective treatment can be readily determined by those of skill in this field. Thus, the term

"therapeutically effective amount" can mean an amount of a particles or TLR on the surface of the particles that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. A "therapeutically effective amount" of a particles or active agent(s) within the particles also means a nontoxic but sufficient amount of the agent to provide the desired effect. Dosage will depend on the patient, the severity and course of the disease, whether particles are administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history, and the discretion of the attending physician.

The particles are suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of particles is an initial candidate dosage for administration to the subject, whether, for example, by one or more separate administrations. In some embodiments, the dosage of the particles will be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the subject. Such doses may be administered intermittently, e.g. every week or every three weeks.

Useful values of amounts of TLR agonists are from about 1 mg to about 1,500 mg TLR-9 agonist per dosage form of the pharmaceutical composition. Preferred values are from about 10 mg to about 800 mg.

The present particles are distinguishable from bottom-up approaches for fabrication, a fundamental process difference compared to PRINT. Carriers may encapsulate, adsorb, or conjugate (chemically bind) CpG, but the majority of these systems are employed for investigating vaccines where CpG is utilized as a vaccine adjuvant to boost the immune response to a specific antigen^9"10.

Certain research has been done utilizing NP-CpG as a monotherapy for treating cancer ^3-5 For example, two particle-CpG formulations utilized to address orthotopic cancers have been reported. One formulation used carbon nanotubes (CNT) bound to thiol- CpG through a cleavable disulfide linkage ⁴. CNT-CpG was injected intracranially to treat glio ma-bearing mice ⁴. Another formulation utilized polyketal (PK) nanoemulsions to encapsulate CpG, then spray dried to form microparticles ³. These PK-CpG particles were instilled in the lung to treat lewis lung carcinoma ³.

Employing the technique of Particle Replication In No n- wetting Templates

(PRINT®), CpG-PRINT particles were fabricated and used to treat a model of human NSCLC. CpG was conjugated to particle through as stabile thioether bond. PRINT applies a top-down method of soft lithography with traditional polymerization to create micro- and nanoscale structures with calibration quality, tunable characteristics. PRINT-CpG particles were superior in promoting survival in 344SQ tumor bearing mice when comparted to treatments with soluble CpG. The fabrication process is amenable to scale-up, cGMP compliant, and offers a wide array of sizes, shapes, and incorporation of other TLR agonists; promoting the possibility for individualized, tailored therapy options. Pharmaceutical Formulations

The particles can be formulated into pharmaceutical compositions as described herein. In an embodiment, the subject matter disclosed herein is directed to a method of treating a subject comprising administering a particle as described herein. The particles can be administered in any appropriate pharmaceutical formulation.

The administration of the particles and compositions comprising the particles can be accomplished through instillation or intravenous delivery. The pharmaceutical composition of the subject matter described herein may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.

In particular, the particles of the subject matter described herein are instilled to deliver particles to the lungs of a patient. Suitable formulations for administration by insufflation include the finely comminuted particle powders of the subject matter described herein which may be delivered by means of an insufflator. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is delivered into the trachea or other airway of the patient. The present subject matter is further described herein by the following non- limiting examples which further illustrate the subject matter described herein, and are not intended, nor should they be interpreted to, limit the scope of the subject matter described herein.

Examples

Example 1

Mice were inoculated with 2,000 luciferase-expressing 344SQ tumor cells in the lung. Disease progression was monitored by bioluminescence, weight gain/loss, and overall health of mouse. NP and control formulations were delivered to the lungs of anesthetized mice through instillation in a 50-μί volume. Doses were 20 μg of CpG per instillation, mice were dosed every three days once tumors were present (3 days after inoculation) for a total of 4 doses, and monitored for bioluminescence 3x a week (Fig. 1).

Compared to soluble CpG, PRINT-CpG provided enhanced protection from CpG toxicity as well as enhanced anti-tumor effects, resulting in 100% survival versus 30% survival of mice treated with soluble CpG (Figs. 2A-2B). Since nanoparticles are naturally cleared by macrophages, DCs, and other APCs, they are excellent carriers of CpG delivery to immune cells. Furthermore, by attaching or encapsulating CpG to or within

nanoparticles, systemic release of pro-inflammatory cytokines can be reduced and therefore attenuate the systemic side effects associated with soluble CpG. Furthermore, surviving mice remained cancer free when re-challenged with orthotopic tumors months after cessation of NP-CpG therapy (Fig. 3).

Example 2

CpG release in vitro utilizing Gambles solution (to simulate the extracellular environment of the deep lung, pH 7.4), lOx PBS, and artificial lysosomal fluid (ALF, pH 4.5) was evaluated. Approximately 80% CpG was released after 10 days in the Gambles solution, with much slower release in ALF (approximately 10% after 10 days) and PBS (approximately 25% in 10 days) (Fig. 4).

Example 3

NP-CpG biodistribution in the lungs was evaluated in vivo in mice inoculated with 2,000 luciferase-expressing 344SQ tumor cells in the lung. NPs (labeled with Dylight 680) were modified with Cy3-labled CpG. Dye labeled NP-CpG, and soluble CpG were instilled into the lungs of mice 3 days post inoculation with 344SQ cells. Mice were sacrificed 24 hours and 48 hours post CpG/NP-CpG instillation, lungs harvested, fixed in formalin, embedded in paraffin, sectioned and evaluated for NP-CpG and CpG via immunofluorescence imaging. As displayed in Fig. 5, the fluorescent signal from soluble CpG in the lung is less than that observed from the NP-CpG formulation. Furthermore, following zooming in on the lungs treated with NP-CpG, clearly observed was that 48 hours after treatment, the CpG is still associated with the NPs in the lung (Fig. 6).

Utilizing immunofluorescence imaging, lung tissue was stained with F4/80 to determine if the NP-CpG was co-localized with macrophages (Fig. 7). Interestingly, an influx of macrophages into the lungs at 48 hours post treatment was observed, with the NP-CpG signal co-localized with the macrophages.

Further investigated were the lungs (inoculated with 344SQ cells) of mice treated with NP-CpG utilizing flow cytometry and sorting for monocytes (regular vs

inflammatory), neutrophils, macrophages, T cells (CD8 and CD4), and T_reg_S. Interestingly an increase in inflammatory monocytes (IM) and T_regs was seen, as well as a decrease in macrophages 48 hours after treatment with NP-CpG (Fig. 8).

A depletion study was conducted utilizing IV delivery of liposomal clodronate (clodrosome) to deplete macrophages prior to orthotopic inoculation with 344SQ cells. IV clodrosome was continued 2x weekly for the entire study, and mice were evaluated for survival. Depletion of macrophages resulted in a modest extension of survival (Fig. 9), indicating that perhaps depletion of macrophages could be one mechanism of the NP-CpG treatment. Further investigated was the in vivo depletions of macrophages (IV

clodrosome), CD8 T cells (IP anti-CD8), and NK Cells (IP anti-Asialo GM1), and their effect on NP-CpG therapeutic efficacy. Depletion of macrophages resulted in 2 out of 4 mice growing tumors, depletion of NK cells resulted in 2 out 4 mice growing tumors, and depletion of CD 8 T cells resulted in 2 out of 3 mice developing tumors (Figs. lOA-lOC). Toxicity was observed with the NP-CpG treatment and depletion treatments. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practicing the subject matter described herein. The present disclosure is in no way limited to just the methods and materials described.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs, and are consistent with: Singleton et al (1994) Dictionary of Microbiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York, NY; and Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York.

Throughout this specification and the claims, the words "comprise," "comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. It is understood that embodiments described herein include "consisting of and/or "consisting essentially of embodiments.

As used herein, the term "about," when referring to a value is meant to encompass variations of, in some embodiments + 50%, in some embodiments + 20%, in some embodiments + 10%, in some embodiments + 5%, in some embodiments + 1%, in some embodiments + 0.5%, and in some embodiments + 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of the range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these small ranges which may independently be included in the smaller rangers is also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

Many modifications and other embodiments set forth herein will come to mind to one skilled in the art to which this subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the subject matter is not to be limited to the specific

embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Bibliography

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Claims

THAT WHICH IS CLAIMED:

1. A method of making a pharmaceutical composition, comprising:

contacting a PEG hydrogel particle having amine groups on a surface with succinimidyl ester of a succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate linker to form an amide bond on the PEG hydrogel particle; and

2. The method of claim 1, wherein the TLR agonist comprises TLR-9.

3. The method of claim 1, wherein the TLR agonist comprises an oligonucleotide.

4. The method of claim 1, wherein the TLR agonist comprises a cytosine-phosphate- guanine (CpG) oligonucleotide.

5. The method of claim 1, wherein the TLR agonist comprises a synthetic oligodeoxynucleotide (ODN) agonist.

6. The method of claim 1, wherein the pharmaceutical composition is administered through inhalation delivery.

7. The method of claim 6, wherein the pharmaceutical composition is administered in an amount of about 1 μg/kg to 50 mg/kg.

8. The method of claim 1, wherein the PEG hydrogel particle comprises an engineered molded non-spherical shape, in cross-section, comprising about 80 nm by 320 nm, about 55 nm by 70 nm, about 80 nm by 180 nm, or about 1 μιη by 1 μιη.

9. The method of claim 1, wherein the pharmaceutical composition is for treating non-small cell lung cancer (NSCLC).

10. The method of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutical excipient.

11. A method for reducing toxicity effects associated with a soluble treatment of TLR- 9 agonists comprising:

an engineered molded non-spherical shape,

a hydrogel, and

a TLR-9 agonist covalently attached to a surface of the particle.

12. The method of claim 11, wherein the pharmaceutical composition is administered in an amount of about 1 μg/kg to 50 mg/kg.

13. The method of claim 11, wherein the therapeutically effective amount comprises about 1 mg to about 1,500 mg TLR agonist.

14. The method of claim 11, wherein the reduction in toxicity effects results in increasing the survival rate of the first plurality of subjects administered an effective amount of the plurality of particles by three times when compared to the survival rate of a second plurality of subjects administered an equivalent amount of a soluble TLR-9 agonist.

15. The method of claim 11, wherein the TLR-9 agonist comprises a cytosine- phosphate-guanine (CpG) oligonucleotide.

16. The method of claim 11, wherein the TLR agonist comprises a synthetic oligodeoxynucleotide (ODN) agonist.

17. The method of claim 11, wherein the pharmaceutical composition is administered through inhalation delivery.

18. The method of claim 11, wherein the hydrogel particle comprises an engineered molded non-spherical shape, in cross-section, comprising about 80 nm by 320 nm, about 55 nm by 70 nm, about 80 nm by 180 nm, or about 1 μιη by 1 μιη.

19. The method of claim 11, wherein the pharmaceutical composition is for treating non-small cell lung cancer (NSCLC).

20. The method of claim 11, wherein the pharmaceutical composition further comprises a pharmaceutical excipient.

21. The method of claim 11, wherein the reduction in toxicity effects results in reduction of systemic release of pro-inflammatory cytokines.