Nanoparticle formulations

Abstract

The present invention relates to lipid nanoparticle compositions that include a polypeptide, a cation, a compound capable of coordinating cations, and a plurality of encapsulating lipids, as well as methods of using the lipid nanoparticle compositions in the treatment of disease, and methods for preparing the lipid nanoparticle compositions. The lipid nanoparticle compositions may be used for treating diseases in a subject, such as autoimmune diseases or cancer.

Description

[0001] PATENT

[0002] ATTORNEY-DOCKET NO.: 51663-007WO2

[0003] NANOPARTICLE FORMULATIONS

[0004] BACKGROUND OF THE INVENTION

[0005] This invention relates to lipid nanoparticle compositions having immunostimulatory activity. The immune system has many pathways that may become dysregulated in a subject suffering from disorders such as autoimmune disease or cancer. The innate immune system may become overactive and control over the immune response is critical to mediate the damage to tissues that frequently coincides with these disorders. Understanding the pathways involved in innate immunity and mechanisms to modulate the immune response are useful for developing therapeutic approaches for diverse human diseases, including cancer and autoimmune diseases.

[0006] SUMMARY OF THE INVENTION

[0007] The present disclosure provides lipid nanoparticle compositions, methods of their use in the treatment of diseases, such as autoimmune disease and cancer, and methods for their preparation.

[0008] In the first aspect, the disclosure provides a lipid nanoparticle composition comprising:

[0009] i) a polypeptide having the structure of Formula (la) or Formula (lb):

[0010] A BFormula (la) orB— AFormula (lb)

[0011] wherein A is a tethering domain comprising from 3 to 12 amino acid residues, wherein each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan; and B is a coordinating domain comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues; ii) a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+;

[0012] iii) a compound capable of coordinating cations; and

[0013] iv) a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound.

[0014] In some embodiments, the tethering domain comprises from 5 to 10 amino acid residues.

[0015] In some embodiments, the tethering domain consists of leucine residues.

[0016] In some embodiments, the tethering domain comprises a leucine residue.

[0017] In some embodiments, the tethering domain consists of phenylalanine residues.

[0018] In some embodiments, the tethering domain comprises a phenylalanine residue.

[0019] In some embodiments, the coordinating domain comprises from 5 to 30 histidine residues.

[0020] In some embodiments, the coordinating domain comprises from 10 to 25 histidine residues. In some embodiments, the coordinating domain comprises from 10 to 12 histidine residues. In some embodiments, the coordinating domain comprises 11 histidine residues.

[0021] In some embodiments, the plurality of encapsulating lipids comprises a phosphatidic acid, a phosphatidylcholine, a phosphatidylethanolamine, a phosphatidylglycerol, a phosphatidylinositide, aPATENT

[0022] ATTORNEY-DOCKET NO.: 51663-007WO2

[0023] phosphatidylserine, a sphingomyelin, a cephalin, a cardiolipin, a steroid, a cerebroside, or a polyethylene glycol (PEG) phospholipid.

[0024] In some embodiments, the plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

[0025] In some embodiments, the phosphatidylcholine is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the steroid is cholesterol, and the PEG phospholipid is PEG-5000-PE.

[0026] In some embodiments, the phosphatidic acid is 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or 1,2-dioleoyl-sn- glycero-3-phosphate (DOPA) (18:1 PA).

[0027] In some embodiments, the compound capable of coordinating cations is a STING agonist, purine containing or purine derived agent, Toll-Like receptor (TLR) agonist, NOD-Like receptor (NLR) agonist, RIG-I-Like receptor (RLR) agonist, cytosolic DNA sensor (CDS) agonist, C-type lectin receptor (CLR) agonist, or inflammasome inducer.

[0028] In some embodiments, the compound capable of coordinating cations is a STING agonist.

[0029] In some embodiments, the STING agonist is cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp, Rp), c-di-

[0030]

[0031] PATENT ATTORNEY-DOCKET NO.: 51663-007WO2

[0032] o

[0033] OH, Gemcitabine (0H F), STING-agonist-C11 (

[0034] Mol. Wt.: 382.44

[0035] ), STING agonist-1 ( ), STING agonist G10

[0036]

[0037] ( ), cGAM(PS)2, 2’3’-cGAM(PS)2(Rp / Sp), 2’2’-cGAM(PS)2, 2’3’-cGAM(PS)2, cGAMP Fluorinated, 2'3'-cGAMP Fluorinated, 2'2'-cGAMP Fluorinated, 2’3’-cdAMP, 2’2’-cdAMP, 3’3’- cdAMP, c-di-AM(PS)2, 2’2’-c-di-AM(PS)2, 3’3’-c-di-AM(PS)2, 2’3’-cdAMP Fluorinated, 2’2’-cdAMP Fluorinated, 3’3’-cdAMP Fluorinated, cdGMP, 2’3’-cdGMP, 2’2’-cdGMP, 3’3’-cdGMP, c-di-GM(PS)2, 2’3’- c-di-GM(PS)2, 2’2’-c-di-GM(PS)2, 3’3’-c-di-GM(PS)2, cdGMP Fluorinated, 2’3’-cdGMP Fluorinated, 2’2’- cdGMP Fluorinated, 3’3’-cdGMP Fluorinated, 2’3’-cAIMP, 2’2’-cAIMP, 3’3’-cAIMP, cAIMP Difluor (3'3'- cAIMP Fluorinated, 2'3'-cAIMP Fluorinated, 2'2'-cAIMP Fluorinated, cAIM(PS)2 Difluor, 3’3’-cAIM(PS)2 Difluor (Rp / Sp), 2’3’-cAIM(PS)2 Difluor, 2’2’-cAIM(PS)2 Difluor, 2’3’-cdlMP, 2’2’-cdlMP, 3’3’-cdlMP, c-di- IM(PS)2, 2’3’-c-di-IM(PS)2, 2’2’-c-di-IM(PS)2, 3’3’-c-di-IM(PS)2, c-di-IMP Fluorinated, 2’3’-cdlMP Fluorinated, 2’2’-cdlMP Fluorinated, 3’3’-cdlMP Fluorinated, or an amidobenzimidazole (ABZI)-based compound.

[0038] In some embodiments, the compound capable of coordinating cations is a TLR agonist.

[0039] In some embodiments, the TLR agonist is a TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-7 agonist, TLR-8 agonist, or TLR-9 agonist.

[0040] In some embodiments, the average particle size of the nanoparticle is from 20 to 500 nm.

[0041] In some embodiments, the average particle size of the nanoparticle is from 50 to 500 nm.

[0042] In some embodiments, the average particle size of the nanoparticle is from 75 to 250 nm.

[0043] In another aspect, the disclosure provides a method for stimulating an innate immune response in a subject comprising administering to the subject an effective amount of any of the above lipid nanoparticle compositions.PATENT

[0044] ATTORNEY-DOCKET NO.: 51663-007WO2

[0045] In some embodiments, the subject has an autoimmune disorder.

[0046] In some embodiments, the subject has cancer.

[0047] In some embodiments, the method further comprises administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

[0048] In another aspect, the disclosure provides a method of preparing any of the above lipid nanoparticle compositions comprising:

[0049] (a) providing a polypeptide having the structure of Formula (la) or Formula (lb):

[0050] A BFormula (la) orB— AFormula (lb)

[0051] wherein A is a tethering domain comprising from 3 to 12 amino acid residues, wherein each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan; and

[0052] B is a coordinating domain comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues; a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+; a compound capable of coordinating cations; and a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound;

[0053] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0054] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0055] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and

[0056] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

[0057] DEFINITIONS

[0058] To facilitate the understanding of the present disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the disclosure. Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps. The terminology herein is used to describe specific embodiments of the disclosure, but their usage does not limit the disclosure, except as outlined in the claims.

[0059] As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.

[0060] As used herein, the terms “administer” and “administering” are used to indicate the process of providing a therapeutic, pharmaceutical, housing compartment, medication, or the like thereof to aPATENT

[0061] ATTORNEY-DOCKET NO.: 51663-007WO2

[0062] subject. In some embodiments, lipid nanoparticle compositions are provided via subcutaneous administration.

[0063] As used herein, the term “agitating” refers to a technique for mixing components together in a vessel, where the components in the mixture are forcibly moved by induced movement. The mixture is mechanically moved by a rotor, propeller, or other mechanical part to create a flow pattern that further mixes the components. Shaking is one form of agitation which may be, for example, orbital shaking or multi-axis rotational shaking.

[0064] As used herein, the term “average” refers to the mean value for a parameter.

[0065] As used herein, the term “cation” refers to any molecule which carries a net positive charge. As used herein, the term “centrifugation” refers to the process of using centrifugal forces to separate particles in a solution.

[0066] As used herein, the term “coordinating” refers to the action of molecule which donates electrons to a metal ion (e.g. a cation). A functional group on the molecule has a lone pair that forms a coordinative bond and acts as a link between the functional group and the cation. An exemplary coordinating group of the disclosure is the nitrogen atom of a histidine residue, which donates electrons to the cation and coordinates to form a cation-polypeptide complex and serves as a part of the core material of the lipid nanoparticle compositions.

[0067] As used herein, the terms “core” and “core material” refer to inner components of the lipid nanoparticle that may be surrounded by encapsulating lipids. The core or core material comprises the polypeptides, cations, or compounds capable of coordinating cations of the invention, either alone or in any combination. The core material is surrounded by the encapsulating lipids during nanoparticle synthesis.

[0068] As used herein, the term “CXCL10” refers to the C-X-C motif chemokine ligand 10 (CXCL10) or interferon gamma-induced protein 10 (IP10), a small human cytokine produced by the CXCL10 gene. The detection of CXCL10 protein or CXCL10 transcripts serves as a surrogate measure for immunostimulatory activity; heightened production indicates an activation of pro-inflammatory responses.

[0069] As used herein, the term “dialysis” refers to a purification method that uses a semi-permeable membrane to selectively remove components from a mixture. Dialysis may be used to separate small molecule components from a macromolecular structure and can use size-exclusion filters of a given molecular weight cutoff to isolate high molecular weight entities.

[0070] As used herein, the term “dispersing” refers to the use of very high force and mixing speeds to mechanically split aggregates apart. The process drives a rotary component, such as a blade, at high speeds such that the shear force along the bottom and top of the blade breaks (e.g. disperses) the particles into smaller variants as they are ejected from the outer edges of the device.

[0071] As used here, the term “emulsifying” refers to a high shear method of reducing the size of particles in a mixture by using a rotor to draw material into a mixer. The shear forces introduced between the gap of the rotor and the stationary pieces of the device causes a breakdown of the particles.

[0072] As used herein, the terms “encapsulating” and “encapsulate” refers to the act of enclosing an object within a shell of a material. The lipid nanoparticle compositions of the disclosure comprise a core material that is surrounded by lipids that encapsulate and protect the core material by forming a spherical shell of lipid molecules. The material that is encapsulated does not necessarily need to be fully enclosed,PATENT

[0073] ATTORNEY-DOCKET NO.: 51663-007WO2

[0074] and a polypeptide, cation, compound, or other lipid of the invention may be associated on the surface of a lipid nanoparticle or protrude outward from the encapsulating lipid shell.

[0075] As used herein, the term “field flow fractionation” refers to the separation of high molecular weight substances from impurities using a flowthrough separation platform but does not include a stationary phase. Separation is achieved by applying an external field or cross-flow. The components of the mixture will respond and aggregate on one accumulation wall of the separation vessel based on the type of field applied and the components to be separated.

[0076] As used herein, “high-pressure homogenization” refers to the process by which the particles of a mixture may be reduced in average particle size by applying the mixture through a small opening at increased pressure. Forcing the mixture through the small opening under pressure causes a jettison of particles, which upon impact are disrupted, yielding the same material but having a reduced particle size. The technique may be used to combine individual components to form a composition and may simply be referred to as “homogenization.”

[0077] As used herein, the terms “improve” and “improving,” in reference to recovery from a disease or condition refers to an enhancement of recovery in one or more parameters measuring or quantifying the severity of the disorder relative to the recovery in these parameters in or prior to treatment with the compositions described herein. Alternatively, improvement may be measured with respect to a reference subject having the same diagnosis as the subject but that did not receive treatment with a compound or composition of the disclosure.

[0078] As used herein, the term “lipid” refers to refers to molecules of synthetic or biological origin that are soluble in organic solvents (e.g., chloroform) but show little to no solubility in water.

[0079] As used herein, the term “lipidated” refers to a compound that has been covalently conjugated to a lipid (such as an encapsulating lipid).

[0080] As used herein, the term “liquid-liquid extraction” refers to a method of purifying a compound which involves partitioning the mixture between two liquids which are not miscible with one another. Some components of the crude mixture may be more soluble in one liquid than the other, and following agitation, these components will separate and remain in the solvent that they are more soluble in. Even if the separation is only partial, the extraction can be repeated numerous times with the same or different immiscible solvent mixtures and thereby improve product purity.

[0081] As used herein, the term “microfluidic mixing” refers to the precise method of mixing using smallchannel tubes and mixing done in small-scale channels, such as on a chip. The orientation of channels combined with the external forces applied achieves a rapid and thorough mixing of multiple samples as they flow together through the specially-designed channels of the microfluidic device.

[0082] As used herein, the term “moisture-resistant film” refers to a flexible and self-sealing material used to cover samples that is easily removed, but impermeable to liquids such as water. A nonlimiting example of a moisture-resistant film is parafilm.

[0083] As used herein, the term “pharmaceutically acceptable salt” represents those salts of the compounds in the compositions described that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G.PATENT

[0084] ATTORNEY-DOCKET NO.: 51663-007WO2

[0085] Wermuth), Wiley-VCH, 2008. These salts may be acid addition salts involving inorganic or organic acids. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable acid. Methods for preparation of the appropriate salts are well-established in the art. In the context of the disclosure, lipids may be provided as pharmaceutically acceptable salts, e.g., the sodium or ammonium salt of a phospholipid.

[0086] As used herein, the term “polydispersity index” or “PDI” refers to homogeneity of the size of nanoparticles in a population, with a smaller PDI reflecting a narrower particle size distribution among the nanoparticles. Lipid nanoparticle compositions of the disclosure may have a polydispersity index from 0 to 0.3, such as 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30. In some embodiments, the lipid nanoparticle composition has a polydispersity index from 0.1 to 0.3.

[0087] As used herein, the term “polypeptide” refers to a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides for inclusion in the lipid nanoparticle compositions of the disclosure may be from 3 to 35 residues in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 residues in length. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or a multi-molecular complex such as a dimer, trimer, or tetramer. They can also include single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0088] As used herein, the term “purifying” refers to the act of removing undesirable components from a mixture. In the context of the disclosure, the lipid nanoparticle compositions may be purified by removing free components that do not get encapsulated within a nanoparticle when following the preparatory protocols. Excess components may be purified by size-exclusion techniques, such as dialysis against a membrane having a molecular weight cutoff.

[0089] As used herein, the term “size-exclusion chromatography” refers to a separation method which uses a solid stationary phase to absorb a solution mobile phase that is passed through. The design of the stationary phase is such that particles are separated based on molecular weight or size. The stationary phase, typically porous beads, absorbs the smaller molecules of the eluent, and the flowthrough solution will contain the higher molecular weight species that did not absorb as well on the stationary phase.

[0090] As used herein, “sonicating” refers to the application of ultrasonic waves to a mixture to fragment aggregated molecules or components.

[0091] As used herein, the term “STING agonist” refers to a compound or molecule that activates stimulator of interferon gene (STING) receptors and stimulates the transcription of pro-inflammatory cytokines.

[0092] As used herein, the term “subject” refers to a human, non-human primate, or other mammal, such as but not limited to dog, cat, horse, cow, pig, goat, monkey, rat, mouse, and sheep. In preferred embodiments, the subject is a human.PATENT

[0093] ATTORNEY-DOCKET NO.: 51663-007WO2

[0094] As used herein, the term “tangential flow filtration” or “cross-flow filtration” refers to a separation technique where the fluid to be filtered is passed in parallel to the filter itself. The technique uses a cycling loop to repeatedly filter a solution in a continuous process.

[0095] As used herein, the term “tethering peptide” refers to a polypeptide of the invention that has both a tethering domain and a coordinating domain. The coordinating domain includes amino acid residues that might stabilize a cation by donating electrons. The tethering domain includes lipophilic amino acid residues that serve as a substitute for a lipid and serves to anchor the polypeptide into the shell of encapsulating lipids.

[0096] As used herein, the term “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, a “therapeutically effective amount” depends upon the context in which it is being applied. For example, in the context of administering the lipid nanoparticle compositions disclosed herein to treat a disorder, a therapeutically effective amount of a composition is, for example, an amount sufficient to reverse or alleviate the symptoms of the disorder.

[0097] As used herein, the term “TLR agonist” refers to synthetic or natural compounds that activate the Toll-like receptors (TLRs) on immune cells. TLRs are pattern recognition receptors that play critical roles in innate immunity.

[0098] As used herein, the terms “treat” and “treating” refer to a therapeutic treatment of a disorder in a subject. The effect of treatment can include reversing, alleviating, reducing severity of, inhibiting the progression of, reducing the likelihood of recurrence of the disorder or one or more symptoms or manifestations of the disorder, stabilizing (i.e., not worsening) the state of the disorder as compared to the state and / or the condition of the disease or disorder in the absence of the therapeutic treatment.

[0099] As used herein, the term “vortexing” refers to the mixing of a solution by applying a rapid oscillating force to forcibly mix a container’s contents.

[0100] As used herein, the term “Z-average” or “Z-ave” refers to the average particle size of a collection of lipid nanoparticles as determined by dynamic light scattering. Z-ave is a hydrodynamic parameter representing the mean size of an ensemble of nanoparticles and is applicable to particles when in a dispersion or in solution. The “average particle size” similarly refers to the Z-average, a weighted mean value, when discussing the size of the lipid nanoparticles of the invention. The average size a lipid nanoparticle describes a representative value for a population of nanoparticles and is an intensity-weighted mean value of nanoparticle size that is calculated and provided as an output following dynamic light scattering (DLS) measurements.

[0101] As used herein, the term “zeta potential” refers to the electrokinetic potential of the lipid nanoparticle composition. Zeta potential describes the charge at the surface of a nanoparticle and is provided in millivolts (mV). The charge properties of a lipid nanoparticle may be important to tune in order to control undesirable interactions with cells or tissues. In some embodiments, the lipid nanoparticle composition has a zeta potential from -30.0 mV to +30.0 mV.

[0102] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a single-step synthetic process for preparing lipid nanoparticles that a tethering peptide and lipid composition 1.

[0103] FIG. 2 shows a single-step synthetic process for preparing lipid nanoparticles that a tethering peptide and lipid composition 2.PATENT

[0104] ATTORNEY-DOCKET NO.: 51663-007WO2

[0105] FIG. 3A shows the measured STING activation using Quanti-Luc luminescence in RAW-Dual murine reporter cells using a Leu6-His11 tethering peptide formulation with either lipid formulation 1 or 2.

[0106] FIG. 3B shows the levels of CXCL10 transcripts in RAW-Dual murine report cells determined by BOSTER Mouse CXCL10 Elisa kit PicoKine using a Leu6-His11 tethering peptide formulation with either lipid formulation 1 or 2.

[0107] FIG. 4 shows the measured STING activation using Quanti-Luc luminescence in RAW-Dual murine reporter cells using formulations SMP-C and SMP-K.

[0108] FIG. 5 shows the levels of CXCL10 transcripts in RAW-Dual murine report cells determined by BOSTER Mouse CXCL10 Elisa kit PicoKine for formulations SMP-C and SMP-K.

[0109] FIG. 6 shows the measured STING activation using Quanti-Luc luminescence in RAW-Dual murine reporter cells using formulations with tethering domains of varied length.

[0110] DETAILED DESCRIPTION OF THE INVENTION

[0111] The present disclosure provides lipid nanoparticle compositions (e.g., any of SMP-Ato SMP-N) that include a polypeptide, cation, a compound capable of coordinating a cation, and a plurality of lipids that encapsulate the aforementioned core components, as well as use and preparation methods thereof. The lipid nanoparticle compositions of the invention may be used to treat diseases or disorders, such as autoimmune disease or cancer, in a subject. The sections that follow describe exemplary components and combinations for the lipid nanoparticle compositions, methods of treating utilizing the lipid nanoparticles, and preparation methods therefor.

[0112] Lipid nanoparticle compositions

[0113] The present disclosure provides compositions that include a plurality of lipids that encapsulate a solid core material, which may comprise a polypeptide having the structure of Formula (la) or Formula (lb):

[0114] A BFormula (la) orB— AFormula (lb),

[0115] where A is a tethering domain comprising from 3 to 12 amino acid residues and each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan, and B is a coordinating domain comprising from 3 to 35 amino acid residues and at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues. The core material also includes a cation (e.g. a metal cation) and a compound capable of coordinating cations. The lipid nanoparticle compositions of the disclosure assist in the delivery of a payload which subsequently stimulates the immune system via delivery of the metal cation and any coordinated compound that is part of the lipid nanoparticle core material. The lipid nanoparticles of the invention have immunostimulatory activity and may be used in the treatment of subjects with cancer or autoimmune diseases.PATENT

[0116] ATTORNEY-DOCKET NO.: 51663-007WO2

[0117] Polypeptides

[0118] Polypeptides of the lipid nanoparticles of the invention have a tethering domain ranging in length from 3 to 12 residues, e.g. from 4 to 11, from 5 to 10, or from 6 to 9 residues (e.g. the length of the tethering domain may be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 residues in length). Each residue of the tethering domain is independently selected from leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan. In some embodiments, the tethering domain consists of leucine residues. In some embodiments, the tethering domain comprises a leucine residue. In some embodiments, the tethering domain consists of phenylalanine residues. In some embodiments, the tethering domain comprises a phenylalanine residue.

[0119] Polypeptides of the lipid nanoparticles of the invention also have a coordinating domain ranging in length from 3 to 35 residues, e.g. from 10 to 25, from 10 to 12, or 11 residues (e.g. the length of coordinating domain may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 residues in length). At least 50%, e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or 98% of the coordinating domain residues are histidine, 1-methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues. Any proteinogenic amino acid residue may be present in the polypeptide, such as alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp, e.g. aspartate), cysteine (Cys), glutamic acid (Glu, e.g. glutamate), glutamine (Gin), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Vai), or pyrrolysine (Pyl). Methylated and acetylated versions of the amino acid residues indicate attachment of these groups to the side chain of the amino acid residues. For instance, methylarginine refers to co- / V-methylated arginine, and the asymmetric and symmetric versions of dimethyl arginine refer to methylation at either both w-nitrogens of arginine (being symmetrically co- / V, / V-dimethylated) or to a single co- nitrogen (being asymmetrically co- / V-dimethylated).

[0120] In a coordinating domain comprising 30 amino acid residues, at least 15 (preferably 20) of the 30 residues would therefore be histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues. Accordingly, in some embodiments, at least 50% (e.g. at least 66%, 85%, or 95%) of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, orPATENT

[0121] ATTORNEY-DOCKET NO.: 51663-007WO2

[0122] valine residues. In some embodiments, all (e.g. 100%) ofthe amino acid residues are histidine, 1-methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues. In some embodiments, at least 50%, e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or 98% ofthe amino acid residues ofthe coordinating domain are histidine residues. In some embodiments, at least 66% of the amino acid residues ofthe coordinating domain are histidine residues. In some embodiments, the coordinating domain consists of histidine residues.

[0123] It is understood that all natural and non-natural analogs ofthe above amino acids may be used in accordance with the invention. Reference to an amino acid residue is meant to include both L- and D-amino acid residues, as well as any isotopes thereof (e.g. amino acid residues incorporating higher weight isotopes than that most prevalent for individual atoms such as deuterium, tritium,13C, or15N). Modification ofthe amide backbone ofthe polypeptide is also considered, such as N-terminus or C-terminus modifications, as well as modification of a / p peptide bond nitrogen (such as tertiary amides of the polypeptide backbone with / V-alkylated peptide linkages, e.g. / V-methyl). Isomers of amino acid residues may also be considered within the scope ofthe invention, e.g., the isoaspartic acid residue which forms a connecting peptide bond through the side chain of aspartic acid instead ofthe C-terminal carboxylate.

[0124] Cations

[0125] Cations ofthe lipid nanoparticle compositions have immunostimulatory activity which, when administered to a subject alongside a compound that can coordinate the cation, provides an amplification to both the signaling cascade and production of cytokines that is associated with the compound.

[0126] Cations ofthe lipid nanoparticle compositions of the disclosure are Co2+, Co3+, Cu+, Cu2+, Mn2+, and Zn2+.

[0127] Compounds capable of coordinating cations

[0128] In some embodiments, the compound capable of coordinating cations is a STING agonist, purine containing or purine derived agent, Toll-Like receptor (TLR) agonist, NOD-Like receptor (NLR) agonist, RIG-I-Like receptor (RLR) agonist, cytosolic DNA sensor (CDS) agonist, C-type lectin receptor (CLR) agonist, or inflammasome inducer.

[0129] In some embodiments, the compound capable of coordinating cations is a STING agonist. In some embodiments, the STING agonist is cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp, Rp), c-di-PATENT

[0130] ATTORNEY-DOCKET NO.: 51663-007WO2

[0131] GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP,

[0132] ), STING-agonist-C11 (

[0133] MN S. f >-NH 0'^ a I; a ■■ j 'yH] Mot WV: 382.44 ), STING agonist-1 ( ), STING agonist G10

[0134]

[0135] ), cGAM(PS)2, 2’3’-cGAM(PS)2(Rp / Sp), 2’2’-cGAM(PS)2, 2’3’-cGAM(PS)2, cGAMP Fluorinated, 2'3'-cGAMP Fluorinated, 2'2'-cGAMP Fluorinated, 2’3’-cdAMP, 2’2’-cdAMP, 3’3’- cdAMP, c-di-AM(PS)2, 2’2’-c-di-AM(PS)2, 3’3’-c-di-AM(PS)2, 2’3’-cdAMP Fluorinated, 2’2’-cdAMPPATENT

[0136] ATTORNEY-DOCKET NO.: 51663-007WO2

[0137] Fluorinated, 3’3’-cdAMP Fluorinated, cdGMP, 2’3’-cdGMP, 2’2’-cdGMP, 3’3’-cdGMP, c-di-GM(PS)2, 2’3’-c-di-GM(PS)2, 2’2’-c-di-GM(PS)2, 3’3’-c-di-GM(PS)2, cdGMP Fluorinated, 2’3’-cdGMP Fluorinated, 2’2’-cdGMP Fluorinated, 3’3’-cdGMP Fluorinated, 2’3’-cAIMP, 2’2’-cAIMP, 3’3’-cAIMP, cAIMP Difluor (3'3'-cAIMP Fluorinated, 2'3'-cAIMP Fluorinated, 2'2'-cAIMP Fluorinated, cAIM(PS)2 Difluor, 3’3’-cAIM(PS)2 Difluor (Rp / Sp), 2’3’-cAIM(PS)2 Difluor, 2’2’-cAIM(PS)2 Difluor, 2’3’-cdlMP, 2’2’-cdlMP, 3’3’-cdlMP, c-di-IM(PS)2, 2’3’-c-di-IM(PS)2, 2’2’-c-di-IM(PS)2, 3’3’-c-di-IM(PS)2, c-di-IMP Fluorinated, 2’3’-cdlMP Fluorinated, 2’2’-cdlMP Fluorinated, 3’3’-cdlMP Fluorinated, or an amidobenzimidazole (ABZI)-based compound.

[0138] In some embodiments, the compound capable of coordinating cations is a TLR agonist.

[0139] In some embodiments, the TLR agonist is a TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-7 agonist, TLR-8 agonist, or TLR-9 agonist.

[0140] Encapsulating lipids

[0141] Lipids used in the lipid nanoparticle compositions of the disclosure provide an encapsulating shell and protective structure for the core material of the lipid nanoparticle. Lipids are hydrophobic materials that are insoluble in water and have many possible structures; lipids of the disclosure serve as structural lipids and encapsulate the core material (the polypeptide, the cation, and compound capable of coordinating a cation) within a lipid membrane. A mixture of lipids (e.g. two or more lipids of different chemical structure) can be ideal for encapsulating the core material to improve the nanoparticle properties or loading of the solid core components into the nanoparticle. The encapsulating lipids need not necessarily encapsulate every individual cation, compound, or polypeptide of the nanoparticle, and the cation, compound, or polypeptide may be non-covalently associated with the encapsulating lipids at the surface of the lipid nanoparticles and not fully enclosed. In some embodiments, the plurality of encapsulating lipids comprises a phosphatidic acid, a phosphatidylcholine, a phosphatidylethanolamine, a phosphatidylglycerol, a phosphatidylinositide, a phosphatidylserine, a sphingomyelin, a cephalin, a cardiolipin, a steroid, a cerebroside, ora polyethylene glycol (PEG) phospholipid. In some embodiments, the encapsulating lipids of the disclosure are lysophospholipids, and the lipid nanoparticle compositions may therefore include phospholipids having partially hydrolyzed lipid tails and having a free hydroxyl group. Lysophospholipids are lipids in which hydrolysis has caused one of the acyl groups of the lipid (in a phospholipid) to be removed.

[0142] / . phosphatidic acids

[0143] Phosphatidic acids (PAs) are anionic (negatively charged) and bioactive lipids involved in several cellular processes. PAs have a phosphorylated hydroxyl group that is not protonated to carry the negative charge of the molecule. Phosphatidic acids for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1,2-dihexanoyl-sn-glycero-3-phosphate, 1,2-dioctanoyl-sn-glycero-3-phosphate, 1,2-didecanoyl-sn-glycero-3-phosphate, 1,2-dilauroyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphate, 1,2-diheptadecanoyl-sn-glycero-3-phosphate, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-d ilinoleoyl-sn-glycero-3-phosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphate, and 1,2-didocosahexaenoyl-sn-glycero-3-phosphate.PATENT

[0144] ATTORNEY-DOCKET NO.: 51663-007WO2

[0145] / / . phosphatidylcholines

[0146] Phosphatidylcholines (PCs) are phospholipids that are highly abundant in cells and are involved in the transport of cholesterol. Phosphatidylcholines have a positive charge on the polar head group due to the methylated ammonium ion that is present in the choline structure. PCs may be zwitterionic (having both positive and negative charge but remaining net neutral) depending on the pH of the solution. Some saturated phosphatidylcholines for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1,2-dipropionyl-sn-glycero-3-phosphocholine, 1,2-dibutyryl-sn-glycero-3-phosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine, 1,2-dioctanoyl-sn-glycero-3-phosphocholine, 1,2-dinonanoyl-sn-glycero-3-phosphocholine, 1,2-didecanoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-ditridecanoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, 1,3-dipalmitoyl-rac-glycero-2-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine, 1,2-diarachidoyl-sn-glycero-3-phosphocholine, 1,2-diheneicosanoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1,2-ditricosanoyl-sn-glycero-3-phosphocholine, or 1,2-dilignoceroyl-sn-glycero-3-phosphocholine. Unsaturated PCs may also be used and include, but are not limited to, 1,2-divaccenoyl-sn-glycero-3-phosphocholine, 1,2-di[(8Z)octadecenoyl]-sn-glycero-3-phosphocholine, 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitelaidoyl-sn-glycero-3-phosphocholine, 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dielaidoyl-sn-glycero-3-phosphocholine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dieicosenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-dierucoyl-sn-glycero-3-phosphocholine, 1,2-dinervonoyl-sn-glycero-3-phosphocholine, and 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine.

[0147] Hi. phosphatidylethanolamines

[0148] Phosphatidylethanolamines (PEs) are highly prevalent phospholipids in eukaryotes, featuring a 2-amino-ethanol polar head group, which may be protonated and positively charged at physiological pH. PEs may be zwitterionic (having both positive and negative charge) depending on the pH of the solution. Exemplary phosphatidylethanolamines for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine, 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, and 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine.PATENT

[0149] ATTORNEY-DOCKET NO.: 51663-007WO2

[0150] Cephalins may also be considered PEs and are applicable as lipids for the lipid nanoparticle compositions of the disclosure.

[0151] iv. phosphatidylglycerols

[0152] Phosphatidylglycerols (PGs) are anionic phospholipids that are frequently found as a membrane component in eukaryotes and microorganisms. PGs furthermore serve as precursors to cardiolipins. Phosphatidylglycerols for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dioctanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-didecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dilauroyl-sn-glycero-3-phospho-(l'-rac-glycerol), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipentadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1,2-distearoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1,2-dielaidoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1,2-dilinolenoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], and 1,2-didocosahexaenoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)].

[0153] v. phosphatidylinositides

[0154] Phosphatidylinositides (Pls, e.g. phosphoinositides) are phospholipids having a myo-inositol polar head group which may be multiply phosphorylated at any of the inositol hydroxyl groups.

[0155] Phosphoinositides are a minority component in cellular membranes. Phosphoinositides for use in the lipid nanoparticle compositions of the disclosure include 1,2-dihexanoyl-sn-glycero-3-phospho-(1 '-myoinositol), 1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo- inositol), 1,2-distearoyl-sn-glycero-3-phosphoinositol, 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol), and phosphorylated derivatives thereof.

[0156] vi. phosphatidylserines

[0157] Phosphatidylserines (PSs) are another lipid component found in some tissues and are lipids featuring a serine-conjugate as the polar head group. In the present invention, phosphoserines may be either naturally-occurring L-serine lipids, or in some instances D-serine phosphoserines may be used. Phosphatidylserines for use in the lipid nanoparticle compositions of the disclosure may include 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine, 1,2-dioctanoyl-sn-glycero-3-phospho-L-serine, 1,2-didecanoyl-sn-glycero-3-phospho-L-serine, 1,2-dilauroyl-sn-glycero-3-phospho-L-serine, 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine, 1,2-distearoyl-sn-glycero-3-phospho-L-serine, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine, and 1,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine.

[0158] v / 7. sphingolipids

[0159] Sphingolipids are a structural component of animal cell membranes and may be utilized in the lipid nanoparticle compositions of the invention. Sphingomyelins have a sphingosine (2-amino-4-trans-octadecene-1,3-diol) core, which has a phosphocholine polar head group and a lipidated amino group.PATENT

[0160] ATTORNEY-DOCKET NO.: 51663-007WO2

[0161] Sphingomyelins for incorporation within the lipid nanoparticles of the invention may be egg, brain, or milk sphingomyelins. Cerebrosides are a derivative of sphingolipids which are instead linked to carbohydrates instead of phosphocholine and may also be considered for inclusion as a lipid nanoparticle encapsulating lipid.

[0162] v / 77. cardiolipins

[0163] Cardiolipins are phospholipids comprising two phosphatidyl glycerol backbones linked together through another glycerol head group. Cardiolipins for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-glycerol, 1',3'-bis[1,2-dipalmitoyl-sn-glycero-3-phospho]-glycerol, 1 ', 3'-bis[1,2-distearoyl-sn-glycero-3-phospho]-glycerol, 1 ',3'-bis[1,2-dimyristoleoyl-sn-glycero-3-phospho]-glycerol, 1 ', 3'-bis[1,2-dipalmitoleoyl-sn-glycero-3-phospho]-glycerol, 1 ', 3'- bis [ 1,2-dioleoyl-sn-glycero-3-phospho]-glycerol, and 1 ', 3'-bis [1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol.

[0164] lx. steroids

[0165] Steroids are multicyclic structures that are found in many membranes and contribute to the fluidity of the membrane structure. Membrane steroids for use in the lipid nanoparticles of the disclosure include cholesterol, sitosterol, or modified versions thereof.

[0166] x. polyethylene glycol (PEG) phospholipids

[0167] Polyethylene glycol (PEG) phospholipids feature a phosphoethanolamine structure where the amine of the head group has been amidated to link a hydrophilic PEG moiety to the lipid. Incorporation of PEG phospholipids as a polymer for the nanoparticle lipid mixture can improve the properties of the nanoparticle for drug delivery. The length of the PEG group is characterized by the molecular weight, such that, for example, the compound 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350] represents a PEG phospholipid having a molecular weight of 350 Daltons. PEG phospholipids for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dioleoyl-sn-glycero-3-PATENT

[0168] ATTORNEY-DOCKET NO.: 51663-007WO2

[0169] phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000], 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000].

[0170] Methods of stimulating an immune response

[0171] In another aspect, the disclosure provides methods of stimulating immune response in a subject to treat diseases or conditions. The lipid nanoparticle compositions of the disclosure may be used to deliver therapeutic drugs as part of the core material, or the core materials may separately activate the immune system and improve the response to a drug that is encapsulated within the nanoparticle or administered to the subject exogenously. Thus, there is presented a method for stimulating an innate immune response in a subject that comprises administering to the subject an effective amount of any of the above lipid nanoparticle compositions. In some embodiments, the subject has an autoimmune disorder. In some embodiments, the subject has cancer. In some embodiments, the method further comprises administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

[0172] Methods of preparing lipid nanoparticles

[0173] In another aspect, the disclosure provides a method of preparing any of the above lipid nanoparticle compositions comprising:

[0174] (a) providing a polypeptide having the structure of Formula (la) or Formula (lb):

[0175] A BFormula (la) orB— AFormula (lb)

[0176] wherein A is a tethering domain comprising from 3 to 12 amino acid residues, wherein each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan; and

[0177] B is a coordinating domain comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, orPATENT

[0178] ATTORNEY-DOCKET NO.: 51663-007WO2

[0179] valine residues; a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+; a compound capable of coordinating cations; and a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound;

[0180] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0181] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0182] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and

[0183] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

[0184] EXAMPLES

[0185] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0186] Example 1. Preparation of lipid nanoparticle compositions

[0187] A study was performed to prepare lipid nanoparticle compositions having formulations of lowered complexity for the purpose of reducing production costs and simplifying the composition. Accordingly, lipid nanoparticles of the invention may be prepared by multiple methods, either by multi-step or single-step processes.

[0188] Materials and methods

[0189] Chemicals and reagents

[0190] Lipids, including but not limited to 1,2-dioleolyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphocholine were purchased from Avanti (DOPE, Avanti Lipids Cat# 850725P; DOPC, Avanti Lipids Cat# 850375P). Polypeptides and tether peptides were purchased from GenScript (made to order, 85% minimum purity). Cyclic-di-adenosine was purchased from MedChem Express (Cat# HY12326A). Cholesterol and other steroidal lipids were purchased from Avanti Lipids (cholesterol, Cat# 700000P). Polyethylene glycol (PEG) phospholipids, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], were purchased from Avanti Lipids (PEG5000-PE, Cat# 880220P). Phosphatidic acids, e.g. 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), were purchased from Avanti Lipids (14:0 PA Cat# 830845P). Manganese (II) dichloride (MnCL) was purchased from Thermo Fisher Scientific (Cat# 036526.22).

[0191] Reagent preparation

[0192] Lipid stocks were prepared in glass vials to minimize lipids adsorbing to the preparation vessel. The lipids DOPE, DOPC, cholesterol, and PEG5000-PE were prepared as 10 mg / mL solutions in ethanol (EtOH). The phosphatidic acid 14:0 PA was prepared as a 1 mg / mL solution in EtOH. CDA was prepared as a 1 mg / mL solution in methanol (MeOH). MnCL was prepared as a 100 mM solution in MeOH.

[0193] Polypeptide solutions were prepared at 10 mg / mL in EtOH spiked with 0.5% trifluoroacetic acid (TFA).PATENT

[0194] ATTORNEY-DOCKET NO.: 51663-007WO2

[0195] Synthesis of control material

[0196] A multi-step process for the preparation of lipid nanoparticles which includes a lipidated polyhistidine that is covalently-attached to 1,2-dioleolyl-sn-glycero-3-phosphoethanolamine (DOPE) has been described previously in (Sun et al. Nature Nanotechnology, 2021, 16, 1260 - 1270). The material produced by these protocols uses a combination of cyclic-di-adenosine (CDA) and cation to create the core material and were previously dubbed CDA metal nanoparticles (CMP).

[0197] A process variant of the above protocol was developed. The core material was prepared by combining 5 mL of CDA at 1 mg / mL in MeOH with 2 mL of DOPE-His11 at 10 mg / mL in EtOH with 0.5 % TFA. Then, 0.34 mL of 100 mM MnCL was added, followed by 5 mL of EtOH. The mixture was vortexed for 10 seconds and incubated on a shaker for 30 minutes. The mixture of lipids was prepared by combining 1 mL of DOPC, 0.5 mL of cholesterol, and 0.64 of the PEG5000-PE in a glass vial covered with parafilm. The core material mixture was centrifuged for 30 minutes at 18k RCF to form a pellet. The supernatant was removed, and the mixture of lipids was dispensed over the core material pellet; the combined mixture was vortexed well until the pellet dispersed. A 5 mL volume of water was added in individual 1 mL portions, and the mixture was vortexed for 10 seconds. The resulting solution was passed through a high-pressure homogenizer once at 50 psi, and 2 mL of the resulting solution was dialyzed against 45 mL of 10% sucrose using a 20 kD molecular weight cutoff overnight to obtain the lipid nanoparticles (SMP).

[0198] Synthesis of lipid nanoparticles with tethering peptides

[0199] A method preparing lipid nanoparticle compositions having polypeptides that include a domain of 11 histidine residues (Histidine 11, His 11) conjugated to a domain of lipophilic amino acid residues was developed. One of the components that introduces complexity in nanoparticle manufacture is the DOPE-His11 conjugate, where DOPE and His11 are covalently linked using a chemical reaction. This compound must be separately conjugated and purified prior to SMP preparation and requires additional characterization which compounds costs. We hypothesized that polypeptides with lipophilic domains, replacing the DOPE with a peptide sequence that has strong hydrophobic character such as series of leucine or phenylalanine residues, would be capable of tethering the core material to the lipid bilayer in a similar manner as DOPE-His11 but without the need for chemical conjugation and the related process steps. For this purpose, peptides comprised of a coordinating sequence and a hydrophobic tethering sequence, e.g., polyleucine or poly phenylalanine, were synthesized as single peptide molecules and used to prepare lipid nanoparticles. Two different mixtures of lipids were tested for combining with the tethering peptides.

[0200] Lipid Composition 1

[0201] To a glass vial was added 83 pL of DOPC, 41 pL cholesterol, 53 pL PEG-5000-PE and 12 pL 14:0 PA, 1 mL of CDA, 0.4 mL of tethering peptide, and 68 pL of MnCh. The mixture was swirled.

[0202] Sequentially, 6 mL of H2O was added in six portions, and the mixture covered with parafilm and vortexed slowly. A 5 mL portion of the solution was passed through a high-pressure homogenizer (Avestin B15) for 1 pass at 50 psi. A 2 mL portion of the remaining solution was transferred to a dialysis cup having a 20 kD molecular weight cutoff and dialyzed against 45 mL of 10% sucrose (ThermoFisher Scientific, Cat# 88405). The solution was dialyzed overnight. The resulting formulation, lipid composition 1, was taken for further characterization and in vitro assessment of STING immunostimulatory activity.PATENT

[0203] ATTORNEY-DOCKET NO.: 51663-007WO2

[0204] Lipid Composition 2

[0205] The protocol for preparing lipid composition 1 was repeated, including 0.12 mL of DOPE solution when combining the components with the novel peptide to generate lipid composition 2.

[0206] Example 2. Size characteristics of lipid nanoparticles with tethering peptides

[0207] Analytical Methods

[0208] Size distribution measurements were performed by dynamic light scattering (DLS). The parameters measured are average size by intensity (Z-ave (nm)), polydispersity index (PDI), and average surface charge given by zeta potential (mV).

[0209] Cyclic-di-adenosine (CDA) concentration was determined by absorbance at 260 nm wavelength light using 60 mM ammonium acetate in isopropyl alcohol (IPA). The concentration was measured by dissolving 20 pL of the lipid nanoparticle solution in 180 pL 60 mM ammonium acetate in IPA and measuring the absorbance at 260 nM with a UV clear 96-well plate.

[0210] Results

[0211] Lipid nanoparticles prepared with tethering peptides provide similarly-sized nanoparticles to control formulations and have a comparable size distribution. The results are shown below in Table 1.

[0212] Table 1. Characterization of lipid nanoparticles that include tethering peptides.

[0213] Zeta CDA Structure Z-ave Polydispersity

[0214] Formulation Potential (pg / mL) (Lipid composition) (nm) Index (PDI)

[0215] (mV)

[0216] 180.1 0.15 33.16 136 SMP-A DOPE-His11 conjugate

[0217] DOPE + His11 174.8 0.22 15.27 89 SMP-B

[0218] unconjugated

[0219] Leu3-His11 165.1 0.31 16.69 56 SMP-C

[0220] Lipid composition 1

[0221] Leu6-His11 130.5 0.29 26.58 70 SMP-D

[0222] Lipid composition 1

[0223] Leu9-His11 291.3 0.46 -1.14 68 SMP-E

[0224] Lipid composition 1

[0225] Leu12-His11 178.3 0.15 27.8 84 SMP-F

[0226] Lipid composition 1

[0227] Leu3-His11 122.1 0.13 10.03 78 SMP-G

[0228] Lipid composition 2

[0229] Leu6-His11 128 0.18 13.36 78 SMP-H

[0230] Lipid composition 2

[0231] Leu9-His11 301.3 0.37 22.37 87 SMP-I

[0232] Lipid composition 2

[0233] Leu12-His11 207.9 0.211 24.84 85 SMP-J

[0234] Lipid composition 2

[0235] Phe3-His11 134.7 0.22 11.18 51 SMP-K

[0236] Lipid composition 1

[0237] Phe6-His11 148 0.21 11.7 77 SMP-L

[0238] Lipid composition 1

[0239] Phe3-His11 140.2 0.14 7.49 63 SMP-M

[0240] Lipid composition 2

[0241] Phe6-His11 157.2 0.11 10.3 75 SMP-N

[0242]

[0243] Lipid composition 2PATENT

[0244] ATTORNEY-DOCKET NO.: 51663-007WO2

[0245] Example 3. In vitro potency of lipid nanoparticle formulations with tethering peptides

[0246] Lipid nanoparticle formulations prepared by the methods of the previous examples were evaluated in vitro by STING activation and CXCL10 production assays.

[0247] In vitro potency by STING activation was performed using RAW-Dual murine reporter cells (Invivogen) that were plated at 200K per well. Cells were incubated with the SMP formulation and the supernatant was collected after a 24-hour time-period. STING activation was read by measuring luminescence after addition of the Quanti-Luc reagent (Invivogen) to the supernatant. This assay has been previously described (Riera-Tur et al Nucleic Acid Ther. 2024, 34, 257–271).

[0248] In vitro potency based on production of CXCL10 was performed using RAW-Dual murine reporter cells (Invivogen) that were plated at 200K per well. Cells were incubated with the SMP formulation at the indicated concentrations and the supernatant was collected after a 24-hour time-period. CXCL10 quantification was done using the BOSTER Mouse CXCL10 Elisa kit PicoKine according to the manufacturer’s instructions. This assay has been previously described (Zheng et al. Translational Research, 2023, 252, 79 – 90).

[0249] Results

[0250] The results of the lipid nanoparticle formulation SMP-D on STING activation and CXCL10 production are presented below in Tables 2 and 3. The tested lipid nanoparticle formulation included the tethering peptide Leu6-His11.

[0251] Table 2. Effect of tethering peptide formulation on STING activation in RAW-Dual murine cells.

[0252] STING Activation (Luminescence)

[0253] CDA Negative concentration SMP-A SMP-D CMP control (pg / mL)

[0254] 0.04 10673.33 8833.33 - - 0.2 12633.33 11200 - 1 50266.67 27433.33 - -

[0255]

[0256] 5 193333.30 133000 19200 82733.33

[0257] Table 3. Effect of tethering peptide formulation on CXCL10 expression in RAW-Dual murine cells.

[0258] CXCL10 Quantification (pg / mL)

[0259] CDA concentration SMP-A SMP-D CMP

[0260] (pg / mL)

[0261] 0.2 56.08 79.93 - 1 580.64 654.22 -

[0262]

[0263] 5 1894.78 2255.70 96.84

[0264] Example 4. Effect of lipid composition on in vitro activity

[0265] A study was performed to determine the effect of varying the lipid composition on the in vitro activity of the lipid nanoparticle compositions. The formulations tested were the Leu6-His11 tethering peptide with either lipid composition 1 or lipid composition 2. Here, the simpler formulation does not include DOPE has higher STING activation and CXCL10 quantification (FIG. 3A and FIG. 3B). The results of these studies show that either lipid composition with the tethering peptide outperforms the CMPPATENT

[0266] ATTORNEY-DOCKET NO.: 51663-007WO2

[0267] reference formulation in both the STING activation and CXCL10 production assays. The results of these experiments are shown in Tables 4 and 5.

[0268] Table 4. Effect of lipid composition on lipid nanoparticle-induced STING activation in RAW-Dual murine cells.

[0269] STING Activation (Luminescence)

[0270] CDA Negative concentration SMP-D SMP-H CMP control (pg / mL)

[0271] 0.04 8833.33 8476.66 - - 0.2 11200 9543.33 ■

[0272] 1 27433.33 15300 - -10 133000 51833.33 19200 8273.33

[0273]

[0274] 10 33000 51833.33

[0275] Table 5. Effect of lipid composition on lipid nanoparticle-induced CXCL10 expression in RAW-Dual murine cells.

[0276] CXCL10 Quantification (pg / mL)

[0277] CDA concentration SMP-D SMP-H CMP

[0278] (pg / mL)

[0279] 0.2 79.93 39.93 - 1 654.22 187.77 -

[0280]

[0281] 5 2255.70 1256.64 96.84

[0282] Example 5. Effect of short tethering peptides on lipid nanoparticle activity

[0283] A study was performed to determine if short tethering peptides, such as Leu3 or Phe3, might serve as effective lipophilic domains to anchor the coordinating peptide in the nanoparticle compositions (FIG. 4 and FIG. 5). Both tethering peptides Leu3-His11 and Phe3-His11 were prepared using lipid composition 1. The resulting lipid nanoparticles were evaluated fortheir activation of STING and CXCL10 production and those data are shown below in Table 6 and Table 7.

[0284] Table 6. Activity of lipid nanoparticles with short tethering peptides on STING activation in RAW- Dual murine cells.

[0285] STING Activation (Luminescence)

[0286] CDA Negative concentration SMP-C SMP-K CMP control (pg / mL)

[0287] 0.04 8570 8026.66 - - 0.2 9056.66 9533.33 - - 1 11266.67 10060 - - 10 19233.33 21600 19200 8273.33PATENT

[0288] ATTORNEY-DOCKET NO.: 51663-007WO2

[0289] Table 7. Activity of lipid nanoparticles with short tethering peptides on CXCL10 expression in RAW-Dual murine cells.

[0290] CXCL10 Quantification (pg / mL)

[0291] CDA concentration SMP-C SMP-K CMP

[0292] (pg / mL)

[0293] 0.2 20.42 33.7 - 1 36.37 55.14 -

[0294]

[0295] 5 141.42 159.09 96.84

[0296] Example 6. Effect of tethering domain length on lipid nanoparticle STING activation in RAW-Dual murine cells

[0297] A study was performed to determine the effect of varying the length of the tethering domain on the in vitro STING activation. The formulations tested were the Leu3-His11, Leu6-His11, Leu9-His11, and Leu12-His11 tethering peptides with lipid composition 2 (FIG. 6). These results show that by only having a tethering domain of 6 or more leucine residues, the tethering peptide nanoparticle formulations outperform the reference CMP formulation in a STING activation assay and removes the need for conjugation to DOPE. The results of this study are shown in Table 8.

[0298] Table 8. Activity of lipid nanoparticles with tethering domain length peptides on STING activation in RAW-Dual murine cells.

[0299] STING Activation (Luminescence)

[0300] CDA Negative concentration SMP-G SMP-H SMP-I SMP-J CMP

[0301] control (pg / mL)

[0302] 0.04 8670 8476.66 - - - - 0.2 8943.33 9543.33 16600 21033.33 ■ ■■ 1 11466.67 15300 39466.67 63233.33 - - 10 17166.67 51833.33 60066.67 101100 19200 8273.33

[0303] ENUMERATED EMBODIMENTS

[0304] E1. A lipid nanoparticle composition comprising:

[0305] i) a polypeptide having the structure of Formula (la) or Formula (lb):

[0306] A BFormula (la) orB— AFormula (lb)

[0307] wherein A is a tethering domain comprising from 3 to 12 amino acid residues, wherein each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan; and

[0308] B is a coordinating domain comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues;PATENT

[0309] ATTORNEY-DOCKET NO.: 51663-007WO2

[0310] ii) a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+;

[0311] iii) a compound capable of coordinating cations; and

[0312] iv) a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound.

[0313] E2. The lipid nanoparticle composition of embodiment 1, wherein the tethering domain comprises from 5 to 10 amino acid residues.

[0314] E3. The lipid nanoparticle composition of embodiment 2, wherein the tethering domain consists of leucine residues.

[0315] E4. The lipid nanoparticle composition of embodiment 2, wherein the tethering domain comprises a leucine residue.

[0316] E5. The lipid nanoparticle composition of embodiment 2, wherein the tethering domain consists of phenylalanine residues.

[0317] E6. The lipid nanoparticle composition of embodiment 2, wherein the tethering domain comprises a phenylalanine residue.

[0318] E7. The lipid nanoparticle composition of any one of embodiments 1 to 6, wherein the coordinating domain comprises from 5 to 30 histidine residues.

[0319] E8. The lipid nanoparticle composition of embodiment 7, wherein the coordinating domain comprises from 10 to 25 histidine residues.

[0320] E9. The lipid nanoparticle composition of embodiment 8, wherein the coordinating domain comprises from 10 to 12 histidine residues.

[0321] E10. The lipid nanoparticle composition of embodiment 9, wherein the coordinating domain comprises 11 histidine residues.

[0322] E11. The lipid nanoparticle composition of any one of embodiments 1 to 10, wherein the plurality of encapsulating lipids comprises a phosphatidic acid, a phosphatidylcholine, a phosphatidylethanolamine, a phosphatidylglycerol, a phosphatidylinositide, a phosphatidylserine, a sphingomyelin, a cephalin, a cardiolipin, a steroid, a cerebroside, or a polyethylene glycol (PEG) phospholipid.

[0323] E12. The lipid nanoparticle composition of embodiment 11, wherein plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

[0324] E13. The lipid nanoparticle composition of embodiment 12, wherein the phosphatidylcholine is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the steroid is cholesterol, and the PEG phospholipid is PEG-5000-PE.

[0325] E14. The lipid nanoparticle composition of embodiment 12, wherein the phosphatidic acid is 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or 1,2-dioleoyl-sn- glycero-3-phosphate (DOPA) (18:1 PA).

[0326] E15. The lipid nanoparticle composition of any one of embodiments 1 to 14, wherein the compound capable of coordinating cations is a STING agonist, purine containing or purine derived agent, Toll-Like receptor (TLR) agonist, NOD-Like receptor (NLR) agonist, RIG-I-Like receptor (RLR) agonist, cytosolic DNA sensor (CDS) agonist, C-type lectin receptor (CLR) agonist, or inflammasome inducer.

[0327] E16. The lipid nanoparticle composition of embodiment 15, wherein the compound capable of coordinating cations is a STING agonist.

[0328] E17. The lipid nanoparticle composition of embodiment 16, wherein the STING agonist is cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP Difluor, cAIM(PS)2,PATENT

[0329] ATTORNEY-DOCKET NO.: 51663-007WO2

[0330] Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp. Rp), c-di-GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP,

[0331]

[0332] STING agonist-1 ( ), STING agonist G10 ( ), cGAM(PS)2, 2’3’-cGAM(PS)2(Rp / Sp), 2’2’-cGAM(PS)2, 2’3’-cGAM(PS)2, cGAMP Fluorinated, 2'3'-cGAMP Fluorinated, 2'2'-cGAMP Fluorinated, 2’3’-cdAMP, 2’2’-cdAMP, 3’3’-cdAMP, c-di-AM(PS)2, 2’2’-c-di-AM(PS)2, 3’3’-c-di-AM(PS)2, 2’3’-cdAMP Fluorinated, 2’2’-cdAMP Fluorinated, 3’3’-cdAMP Fluorinated, cdGMP, 2’3’-cdGMP, 2’2’-cdGMP, 3’3’-cdGMP, c-di-GM(PS)2, 2’3’-c-di-GM(PS)2, 2’2’-c-di-GM(PS)2, 3’3’-c-di-GM(PS)2, cdGMP Fluorinated, 2’3’-cdGMP Fluorinated, 2’2’-cdGMP Fluorinated, 3’3’-cdGMP Fluorinated, 2’3’-cAIMP, 2’2’-cAIMP, 3’3’-cAIMP, cAIMP Difluor (3'3'-cAIMP Fluorinated, 2'3'-cAIMP Fluorinated, 2'2'-cAIMP Fluorinated, cAIM(PS)2 Difluor, 3’3’-cAIM(PS)2 Difluor (Rp / Sp), 2’3’-cAIM(PS)2PATENT

[0333] ATTORNEY-DOCKET NO.: 51663-007WO2

[0334] Difluor, 2’2’-cAIM(PS)2 Difluor, 2,3’-cdlMP, 2,2’-cdlMP, 3’3’-cdlMP, c-di-IM(PS)2, 2’3’-c-di-IM(PS)2, 2’2’-c-di-IM(PS)2, 3’3’-c-di-IM(PS)2, c-di-IMP Fluorinated, 2’3’-cdlMP Fluorinated, 2’2’-cdlMP Fluorinated, 3’3’-cdlMP Fluorinated, or an amidobenzimidazole (ABZI)-based compound.

[0335] E18. The lipid nanoparticle composition of embodiment 15, wherein the compound capable of coordinating cations is a TLR agonist.

[0336] E19. The lipid nanoparticle composition of embodiment 18, wherein the TLR agonist is a TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-7 agonist, TLR-8 agonist, or TLR-9 agonist.

[0337] E20. The lipid nanoparticle composition of any one of embodiments 1 to 19, wherein the average particle size of the nanoparticle is from 20 to 500 nm.

[0338] E21. The lipid nanoparticle composition of embodiment 20, wherein the average particle size of the nanoparticle is from 50 to 500 nm.

[0339] E22. The lipid nanoparticle composition of embodiment 21, wherein the average particle size of the nanoparticle is from 75 to 250 nm.

[0340] E23. A method for stimulating an innate immune response in a subject comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of embodiments 1 to 22. E24. The method of embodiment 23, wherein the subject has an autoimmune disorder.

[0341] E25. The method of embodiment 23, wherein the subject has cancer.

[0342] E26. The method of embodiment 24, further comprising administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

[0343] E27. A method of preparing the lipid nanoparticle composition of any one of embodiments 1 to 22 comprising:

[0344] (a) providing a polypeptide having the structure of Formula (la) or Formula (lb):

[0345] A BFormula (la) orB— AFormula (lb)

[0346] wherein A is a tethering domain comprising from 3 to 12 amino acid residues, wherein each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan; and

[0347] B is a coordinating domain comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues; a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+; a compound capable of coordinating cations; and a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound;

[0348] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0349] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0350] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; andPATENT

[0351] ATTORNEY-DOCKET NO.: 51663-007WO2

[0352] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

[0353] OTHER EMBODIMENTS

[0354] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the disclosure and including such departures from the invention that come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

PATENTATTORNEY-DOCKET NO.: 51663-007WO2CLAIMS1. A lipid nanoparticle composition comprising:i) a polypeptide having the structure of Formula (la) or Formula (lb):A BFormula (la) orB— AFormula (lb)wherein A is a tethering domain comprising from 3 to 12 amino acid residues, wherein each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan; andB is a coordinating domain comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues;ii) a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+;iii) a compound capable of coordinating cations; andiv) a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound.

2. The lipid nanoparticle composition of claim 1, wherein the tethering domain comprises from 5 to 10 amino acid residues.

3. The lipid nanoparticle composition of claim 2, wherein the tethering domain consists of leucine residues.

4. The lipid nanoparticle composition of claim 2, wherein the tethering domain comprises a leucine residue.

5. The lipid nanoparticle composition of claim 2, wherein the tethering domain consists of phenylalanine residues.

6. The lipid nanoparticle composition of claim 2, wherein the tethering domain comprises a phenylalanine residue.

7. The lipid nanoparticle composition of any one of claims 1 to 6, wherein the coordinating domain comprises from 5 to 30 histidine residues.

8. The lipid nanoparticle composition of claim 7, wherein the coordinating domain comprises from 10 to 25 histidine residues.PATENTATTORNEY-DOCKET NO.: 51663-007WO29. The lipid nanoparticle composition of claim 8, wherein the coordinating domain comprises from 10 to 12 histidine residues.

10. The lipid nanoparticle composition of claim 9, wherein the coordinating domain comprises 11 histidine residues.

11. The lipid nanoparticle composition of any one of claims 1 to 10, wherein the plurality of encapsulating lipids comprises a phosphatidic acid, a phosphatidylcholine, a phosphatidylethanolamine, a phosphatidylglycerol, a phosphatidylinositide, a phosphatidylserine, a sphingomyelin, a cephalin, a cardiolipin, a steroid, a cerebroside, ora polyethylene glycol (PEG) phospholipid.

12. The lipid nanoparticle composition of claim 11, wherein plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

13. The lipid nanoparticle composition of claim 12, wherein the phosphatidylcholine is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the steroid is cholesterol, and the PEG phospholipid is PEG-5000-PE.

14. The lipid nanoparticle composition of claim 12, wherein the phosphatidic acid is 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

15. The lipid nanoparticle composition of any one of claims 1 to 14, wherein the compound capable of coordinating cations is a STING agonist, purine containing or purine derived agent, Toll-Like receptor (TLR) agonist, NOD-Like receptor (NLR) agonist, RIG-I-Like receptor (RLR) agonist, cytosolic DNA sensor (CDS) agonist, C-type lectin receptor (CLR) agonist, or inflammasome inducer.

16. The lipid nanoparticle composition of claim 15, wherein the compound capable of coordinating cations is a STING agonist.

17. The lipid nanoparticle composition of claim 16, wherein the STING agonist is cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di- oAMP, 2’3’-c-di-AM(PS)2 (Rp. Rp), c-di-GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP,PATENT ATTORNEY-DOCKET NO.: 51663-007WO2SN ""q;, Xagonist-C11 ( ), STING agonist-1 ( ), STING agonistG10 ( ), cGAM(PS)2, 2’3’-cGAM(PS)2(Rp / Sp), 2’2’-cGAM(PS)2, 2’3’-cGAM(PS)2, cGAMP Fluorinated, 2'3’-cGAMP Fluorinated, 2'2'-cGAMP Fluorinated, 2’3’-cdAMP, 2’2’-cdAMP, 3’3’-cdAMP, c-di-AM(PS)2, 2’2’-c-di-AM(PS)2, 3’3’-c-di-AM(PS)2, 2’3’-cdAMP Fluorinated, 2’2’-cdAMP Fluorinated, 3’3’-cdAMP Fluorinated, cdGMP, 2’3’-cdGMP, 2’2’-cdGMP, 3’3’-cdGMP, c-di-GM(PS)2, 2’3’-c-di-GM(PS)2, 2’2’-c-di-GM(PS)2, 3’3’-c-di-GM(PS)2, cdGMP Fluorinated, 2’3’-cdGMP Fluorinated, 2’2’-cdGMP Fluorinated, 3’3’-cdGMP Fluorinated, 2’3’-cAIMP, 2’2’-cAIMP, 3’3’-cAIMP, cAIMP Difluor (3'3'-cAIMP Fluorinated, 2'3'-cAIMP Fluorinated, 2'2'-cAIMP Fluorinated, cAIM(PS)2 Difluor, 3’3’-cAIM(PS)2 Difluor (Rp / Sp), 2’3’-cAIM(PS)2 Difluor, 2’2’-cAIM(PS)2 Difluor, 2’3’-cdlMP, 2’2’-cdlMP, 3’3’-cdlMP, c-di-IM(PS)2, 2’3’-c-di-IM(PS)2, 2’2’-c-di-IM(PS)2, 3’3’-c-di-IM(PS)2, c-di-IMPPATENTATTORNEY-DOCKET NO.: 51663-007WO2Fluorinated, 2’3’-cdlMP Fluorinated, 2’2’-cdlMP Fluorinated, 3’3’-cdlMP Fluorinated, or an amidobenzimidazole (ABZI)-based compound.

18. The lipid nanoparticle composition of claim 15, wherein the compound capable of coordinating cations is a TLR agonist.

19. The lipid nanoparticle composition of claim 18, wherein the TLR agonist is a TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-7 agonist, TLR-8 agonist, or TLR-9 agonist.

20. The lipid nanoparticle composition of any one of claims 1 to 19, wherein the average particle size of the nanoparticle is from 20 to 500 nm.

21. The lipid nanoparticle composition of claim 20, wherein the average particle size of the nanoparticle is from 50 to 500 nm.

22. The lipid nanoparticle composition of claim 21, wherein the average particle size of the nanoparticle is from 75 to 250 nm.

23. A method for stimulating an innate immune response in a subject comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 1 to 22.

24. The method of claim 23, wherein the subject has an autoimmune disorder.

25. The method of claim 23, wherein the subject has cancer.

26. The method of claim 24, further comprising administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

27. A method of preparing the lipid nanoparticle composition of any one of claims 1 to 22 comprising:(a) providing a polypeptide having the structure of Formula (la) or Formula (lb):A BFormula (la) orB— AFormula (lb)wherein A is a tethering domain comprising from 3 to 12 amino acid residues, wherein each amino acid residue is independently leucine, isoleucine, valine, phenylalanine, tyrosine, or tryptophan; andB is a coordinating domain comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine, acetylornithine, tyrosine, O-methyl-tyrosine, phenylalanine, tryptophan, glutamic acid, isoglutamic acid, carboxyglutamic acid, glutamic acid 5-methyl ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, orPATENTATTORNEY-DOCKET NO.: 51663-007WO2valine residues; a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+; a compound capable of coordinating cations; and a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound;(b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;(c) combining water with the mixture of step (b) to form a lipid particulate mixture;(d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and(e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

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