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.: 51663-005WO2

[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: i. a polypeptide 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, and the polypeptide is not covalently conjugated to a lipid;

[0009] ii. a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+;

[0010] Hi. a compound capable of coordinating cations; and

[0011] iv. a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound.

[0012] 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, or a polyethylene glycol (PEG) phospholipid.

[0013] In some embodiments, the plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

[0014] In some embodiments, the phosphatidylcholine is 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the steroid is cholesterol, and the PEG phospholipid is 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG-5000-PE).

[0015] 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).PATENT

[0016] ATTORNEY-DOCKET NO.: 51663-005WO2

[0017] 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.

[0018] 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

[0019]

[0020] PATENT

[0021] ATTORNEY-DOCKET NO.: 51663-005WO2

[0022]

[0023] 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)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.

[0024] In some embodiments, the compound capable of coordinating cations is a TLR agonist. 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.

[0025] In some embodiments, at least 66% of the amino acid residues in the polypeptide 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.

[0026] In some embodiments, at least 85% of the amino acid residues in the polypeptide 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.

[0027] In some embodiments, at least 95% of the amino acid residues in the polypeptide 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-PATENT

[0028] ATTORNEY-DOCKET NO.: 51663-005WO2

[0029] 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.

[0030] In some embodiments, all of the amino acid residues in the polypeptide 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.

[0031] In some embodiments, the polypeptide comprises from 5 to 33 histidine residues.

[0032] In some embodiments, the polypeptide comprises from 10 to 25 histidine residues.

[0033] In some embodiments, the polypeptide comprises from 10 to 12 histidine residues.

[0034] In some embodiments, the polypeptide comprises 11 histidine residues. In some embodiments, the polypeptide comprises 6 histidine residues. In some embodiments, the polypeptide comprises 33 histidine residues.

[0035] In some embodiments, the average particle size of the lipid nanoparticle is from 20 to 500 nm. In some embodiments, the average particle size of the lipid nanoparticle is from 50 to 500 nm. In some embodiments, the average particle size of the lipid nanoparticle is from 75 to 250 nm. In some embodiments, the average particle size of the lipid nanoparticle is from 40 to 120 nm. In some embodiments, the lipid nanoparticle has a polydispersity index from 0.1 to 0.3.

[0036] In some embodiments, the lipid nanoparticle has a zeta potential from -30 mV to +30 mV. 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.

[0037] In some embodiments, the subject has an autoimmune disorder.

[0038] In some embodiments, the subject has cancer.

[0039] In some embodiments, the method further comprises administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

[0040] In another aspect, the disclosure provides a method of preparing any of the above lipid nanoparticle compositions comprising:

[0041] (a) providing a polypeptide 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, and the polypeptide is not covalently conjugated to a lipid; a cation, wherein the cation is Oo2+, Oo3+, Ou+, Ou2+, Mn2+, or Zn2+; a compound capable of coordinatingPATENT

[0042] ATTORNEY-DOCKET NO.: 51663-005WO2

[0043] cations; and a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound;

[0044] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0045] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0046] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and

[0047] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

[0048] DEFINITIONS

[0049] 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.

[0050] 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.

[0051] 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 a subject. In some embodiments, lipid nanoparticle compositions are provided via subcutaneous administration.

[0052] 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.

[0053] As used herein, the term “average” refers to the mean value for a parameter.

[0054] 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.

[0055] As used herein, the term “CRYSTAL” refers to a lipid nanoparticle composition of the invention including a polypeptide of from 3 to 35 amino acid residues, a cation, a compound capable of coordinating cations, and a plurality of encapsulating lipids that encapsulate the polypeptide, cation, and compound.

[0056] 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 aPATENT

[0057] ATTORNEY-DOCKET NO.: 51663-005WO2

[0058] 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.

[0059] 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. The terms “nanoassembly” and “nanoassemblies” are interchangeably used with “core” and “core material” and similarly comprise polypeptides, cations, or compounds capable of coordinating cations of the invention, either alone or in any combination.

[0060] 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 CXCL 10 transcripts serves as a surrogate measure for immunostimulatory activity; heightened production indicates an activation of pro-inflammatory responses.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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, 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.

[0065] 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.PATENT

[0066] ATTORNEY-DOCKET NO.: 51663-005WO2

[0067] 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.”

[0068] 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.

[0069] As used herein, the terms “isoglutamic acid” and “isoaspartic acid” refer to amino acid residues glutamic acid and aspartic acid that are incorporated into a polypeptide and form connecting peptide bonds to adjacent residues through the side chain carboxylic acid as opposed to the C-terminus carboxylic acid. The term “isopeptide bond” may be used to refer to such connectivity.

[0070] 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.

[0071] As used herein, the term “I ipidated” refers to a compound that has been covalently conjugated to a lipid (such as an encapsulating lipid).

[0072] 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.

[0073] As used herein, the term “microfluidic mixing” refers to the precise method of mixing using small-channel 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.

[0074] 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.

[0075] 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.

[0076] 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. Wermuth), Wiley-VCH, 2008. These salts may be acid addition saltsPATENT

[0077] ATTORNEY-DOCKET NO.: 51663-005WO2

[0078] 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.

[0079] 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 O 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] As used herein, “sonicating” refers to the application of ultrasonic waves to a mixture to fragment aggregated molecules or components.

[0084] 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.PATENT

[0085] ATTORNEY-DOCKET NO.: 51663-005WO2

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a prior art multi-step synthetic process for preparing lipid nanoparticles that include a lipidated polypeptide.PATENT

[0095] ATTORNEY-DOCKET NO.: 51663-005WO2

[0096] FIG. 2 shows a single-step synthetic process for preparing lipid nanoparticles that include an unconjugated polypeptide and lipid.

[0097] FIG. 3 shows the STING activation in RAW-Dual murine report cells for unconjugated polypeptide formulations using various length polyhistidine peptides and compared to the CMP formulation.

[0098] FIG. 4A shows Transmission Electron Microscopy (TEM) images of nanoassemblies of CDA and Mn2+(CDA / Mn).

[0099] FIG. 4B shows STEM-EDX analysis of CDA / Mn nanoassemblies. N, P, O, and Mn are representative elements in CDA / Mn nanoassemblies.

[0100] FIG. 5A shows TEM images of nanoassemblies of CDA / Mn / Hise, CDA / Mn / Hisu, CDA / Mn / HiS33, and CDA / Mn / d-Hisn.

[0101] FIG. 5B shows a schematic illustration of different states of nanoassemblies and photographs of the assembling process. The addition of Hisu results in a self-assembly effect. The addition of Mn2+led to precipitations; upon brief sonication, the states are reversible.

[0102] FIG. 5C shows Zeta potential values of as prepared and washed CDA / Mn and CDA / Mn / His11 nanoassemblies.

[0103] FIG. 6 shows BMDCs were incubated with 10 pg / mL of He-CRYSTAL, d-Hn-CRYSTAL, H33-CRYSTAL, and Hn-CRYSTAL for 20 hours and the supernatant was tested for levels (pg / ml_) of IFN-p, TNF-a, IL-6, and CXCL10.

[0104] FIG. 7A shows an investigation of antitumor efficacy of CRYSTALS, including He-CRYSTAL, H11-CRYSTAL, H33-CRYSTAL, and d-Hn-CRYSTAL in B16F10 tumor-bearing C57BL / 6 mice.

[0105] Treatment with the indicated dosage was performed on day 0, 4, and 8 via intravenous (IV) administration. Tumor size and survival were monitored.

[0106] FIG. 7B shows C57BL / 6 mice serum IFN-p, IFN-a, IL-6, TNF-a, and CXCL10 measured via ELISA 4 hours after IV administration of CRYSTALS on day 0. The data represents the mean ± standard error of the mean (s.e.m.), from two independent experiments with n = 4-10.

[0107] FIG. 8A shows pharmacokinetic profiles of Hn-CRYSTAL and Hn-DOPE-CRYSTAL in B16F10 tumor-bearing C57BL / 6 mice. Doses of 10 pg Hn-CRYSTAL and 10 pg Hn-DOPE-CRYSTAL were injected intravenously and CDA concentration in plasma was measured at the indicated timepoints.

[0108] FIG. 8B shows the antitumor efficacy of Hn-CRYSTAL and Hn-DOPE-CRYSTAL in B16F10 tumor-bearing C57BL / 6 mice. Treatment with the indicated dosage was performed on day 0, 4, and 8 via IV administration. Tumor size and survival were monitored. The data represent the mean ± s.e.m., from a representative experiment with n = 5.

[0109] FIG. 9 shows the antitumor efficacy of Hn-CRYSTAL (Organic) and Hn-CRYSTAL (Aqueous) in B16F10 tumor-bearing C57BL / 6 mice. Treatment with the indicated dosage was performed on day 0 via IV administration. Tumor size and survival were monitored. The data represent the mean ± s.e.m., from a representative experiment with n = 5.

[0110] FIG. 10 shows the antitumor efficacy of Hn-CRYSTAL in late-stage B16F10 tumor-bearing C57BL / 6 mice. Treatment with the indicated dosage was performed on day 0, 4, and 8 via IVPATENT

[0111] ATTORNEY-DOCKET NO.: 51663-005WO2

[0112] administration. Tumor size and survival were monitored. The data represent the mean ± s.e.m., from a representative experiment n = 4.

[0113] FIG. 11 shows the antitumor efficacy of intratumoral treatment of Hn-CRYSTAL in B16F10 tumor-bearing C57BL / 6 mice. Treatment with the indicated dosage was performed on day 0, 4, 8 via intratumoral administration. Tumor size and survival were monitored. The data represent the mean ± s.e.m., from a representative experiment with n = 5.

[0114] DETAILED DESCRIPTION OF THE INVENTION

[0115] The present disclosure provides lipid nanoparticle compositions 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.

[0116] Lipid nanoparticle compositions

[0117] The present disclosure provides compositions that include a plurality of lipids that encapsulate a solid core material, which may comprise a polypeptide comprising from 3 to 35 amino acid residues, having at least 50% of the amino acid residues 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, and the polypeptide is furthermore not covalently conjugated to a lipid (e.g. not lipidated). 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.

[0118] Polypeptides

[0119] Polypeptides of the lipid nanoparticles of the invention range 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 polypeptide 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 polypeptide residues are histidine, 1 -methylhistidine, 3-methylhistidine, arginine, methylarginine, asymmetric dimethylarginine, symmetric dimethylarginine, lysine, methyllysine,PATENT

[0120] ATTORNEY-DOCKET NO.: 51663-005WO2

[0121] 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 (lie), 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 w-A / -methylated arginine, and the asymmetric and symmetric versions of dimethyl arginine refer to methylation at either both w-nitrogens of arginine (being symmetrically w-A / ,A / -dimethylated) or to a single w-nitrogen (being asymmetrically w-A / -dimethylated).

[0122] In a polypeptide 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, the polypeptide comprises 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, and the polypeptide is not covalently conjugated to a lipid. 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, or valine residues. In some embodiments, all (e.g. 100%) 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,PATENT

[0123] ATTORNEY-DOCKET NO.: 51663-005WO2

[0124] 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% of the amino acid residues of the polypeptide are histidine residues. In some embodiments, at least 66% of the amino acid residues of the polypeptide are histidine residues. In some embodiments, the polypeptide consists of histidine residues. In some embodiments, the polypeptide consists of L-histidine residues. In some embodiments, the polypeptide consists of D-histidine residues.

[0125] It is understood that all natural and non-natural analogs of the 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 of the amide backbone of the polypeptide is also considered, such as N-terminus or C-terminus modifications, as well as modification of a peptide bond nitrogen (such as tertiary amides of the polypeptide backbone with A / -alkylated peptide linkages, e.g. / V-methyl). Isomers of amino acid residues may also be considered within the scope of the invention, e.g., the isoglutamic acid or isoaspartic acid residues which form a connecting peptide bond through the side chain carboxylic acid instead of the C-terminus carboxylic acid.

[0126] Cations

[0127] Cations of the 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.

[0128] Cations of the lipid nanoparticle compositions of the disclosure are Co2+, Co3+, Cu+, Cu2+, Mn2+, and Zn2+.

[0129] Compounds capable of coordinating cations

[0130] 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.

[0131] 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)2PATENT

[0132] ATTORNEY-DOCKET NO.: 51663-005WO2

[0133]

[0134] PATENT

[0135] ATTORNEY-DOCKET NO.: 51663-005WO2

[0136] " <

[0137]

[0138] STING agonist-1 ( ), STING agonist G10 (

[0139] 7), 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.

[0140] In some embodiments, the compound capable of coordinating cations is a TLR agonist. 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.

[0141] Encapsulating lipids

[0142] 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, or a 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.PATENT

[0143] ATTORNEY-DOCKET NO.: 51663-005WO2

[0144] Lysophospholipids are lipids in which hydrolysis has caused one of the acyl groups of the lipid (in a phospholipid) to be removed.

[0145] / '. phosphatidic acids

[0146] 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-dilinoleoyl-sn-glycero-3-phosphate, 1 ,2-diarachidonoyl-sn-glycero-3-phosphate, and 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphate.

[0147] / ' / . phosphatidylcholines

[0148] 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.PATENT

[0149] ATTORNEY-DOCKET NO.: 51663-005WO2

[0150] Hi. phosphatidylethanolamines

[0151] 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-d ilinoleoyl-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. Cephalins may also be considered PEs and are applicable as lipids for the lipid nanoparticle compositions of the disclosure.

[0152] iv. phosphatidylglycerols

[0153] 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-(1 ’-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)].

[0154] v. phosphatidylinositides

[0155] 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.

[0156] 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-(T-myo-inositol), 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.PATENT

[0157] ATTORNEY-DOCKET NO.: 51663-005WO2

[0158] vi. phosphatidylserines

[0159] 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 / .-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.

[0160] v / 7. sphingolipids

[0161] 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. 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] viii. 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-phosphoj-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] ix. 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.

[0168] Incorporation of PEG phospholipids as a polymer for the nanoparticle lipid mixture can improve thePATENT

[0169] ATTORNEY-DOCKET NO.: 51663-005WO2

[0170] 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-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].

[0171] Methods of stimulating an immune response

[0172] 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 forPATENT

[0173] ATTORNEY-DOCKET NO.: 51663-005WO2

[0174] 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.

[0175] Methods of preparing lipid nanoparticles

[0176] In another aspect, the disclosure provides a method of preparing any of the above lipid nanoparticle compositions comprising:

[0177] (a) providing a polypeptide comprising from 3 to 35 amino acid residues, wherein at least 50% of the amino acid residues are histidine, 1 -methylhistidine, 3-methylhistidi ne, 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, and the polypeptide is not covalently conjugated to a lipid; 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;

[0178] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0179] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0180] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and

[0181] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

[0182] EXAMPLES

[0183] 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.

[0184] Example 1. Preparation of lipid nanoparticle compositions

[0185] 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.PATENT

[0186] ATTORNEY-DOCKET NO.: 51663-005WO2

[0187] 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 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 (MnCh) 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). MnCIz was prepared as a 100 mM solution in MeOH. Polypeptide solutions were prepared at 10 mg / mL in EtOH spiked with 0.5% trifluoroacetic acid (TFA).

[0193] Multi-step synthesis protocol

[0194] 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 synthesis of nanoparticles using the multi-step route is outlined in FIG. 1. 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).

[0195] One-step synthesis protocol

[0196] A scalable lipid nanoparticle synthesis method was developed utilizing a polypeptide of 11 histidine residues (Histidine 11 , Hisu) that was not conjugated to DOPE, and the polypeptide and lipid were each instead added in an unconjugated form. The synthesis of nanoparticles using the one-step protocol is outlined in FIG. 2.

[0197] 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, 0.26 mL of DOPE, 0.14 mL of Hisu and 68 pL of MnCIz. The mixture was swirled. Sequentially, 6 mL of H2O was added in six portions, and the mixture covered with parafilmPATENT

[0198] ATTORNEY-DOCKET NO.: 51663-005WO2

[0199] 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, SMP-C, was taken for further characterization and in vitro assessment of STING immunostimulatory activity.

[0200] Example 2. Size characteristics of unconjugated polyhistidine nanoparticles

[0201] Analytical Methods

[0202] 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).

[0203] 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.

[0204] Results

[0205] Lipid nanoparticles made using the two different synthesis methods, having either lipidated (SMP-B, from the multi-step protocol) or free polyhistidine (SMP-C, from the one-step protocol), result in similarly-sized nanoparticles that also have a comparable size distribution. There is a noteworthy difference in surface charge, and lipidated-polyhistidine nanoparticles have a higher positive surface charge than the unconjugated formulation. The results are shown below in Table 1.

[0206] Table 1. Characterization of lipid nanoparticles having lipidated or free polypeptide.

[0207]

[0208] Example 3. In vitro potency of lipid nanoparticle formulations

[0209] The lipid nanoparticle formulations of the previous examples were tested in vitro for potency using a STING activation assay and a CXCL10 production assay.

[0210] 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).

[0211] 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 SMPPATENT

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[0213] formulation, 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).

[0214] Results

[0215] The results of the lipid nanoparticle formulation SMP-C on STING activation and CXCL10 production are presented below in Tables 2 and 3.

[0216] Table 2. Effect of unconjugated polypeptide formulation on STING activation in RAW-Dual murine cells.

[0217]

[0218] Table 3. Effect of unconjugated polypeptide formulation on CXCL10 expression in RAW-Dual murine cells.

[0219]

[0220] Example 4. Effect of polyhistidine length on formulation

[0221] A study was performed to determine the effect of varying polypeptide length on the in vitro activity of the lipid nanoparticle compositions. Decreasing the length of the polyhistidine below 11 decreases the potency of the formulation. Previous results suggest that longer polyhistidine chains are not beneficial to the CMP formulation. Here, the formulations were tested with free polyhistidine of lengths 11 , 9, 7, and 5; the obtained data shows that the optimal length is 11 residues and shorter polyhistidine chains have lower activity (FIG. 3).PATENT

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[0223] Example 5. Synthesis and Characterization of His(n)-CRYSTAL

[0224] A study was performed to prepare and characterize lipid nanoparticle compositions having polypeptides not conjugated to a lipid that were prepared by a multi-step synthetic protocol.

[0225] Materials and methods

[0226] Synthesis and characterization of CDA / Mn and CDA / Mn / (His)n nanoassemblies

[0227] To prepare CDA / Mn2+nanoassemblies, CDA (1 mg / mL, methanol (MeOH)) and MnCIz (100 mM, MeOH) were mixed with 100:7 volume ratios, followed by vortexing at room temperature for 2 hours with sporadic water bath (37Hz, 100W) sonication. After centrifugation at 20,000 g for 10 min, the pellets were resuspended in deionized (DI) water via water bath sonication. 10 pL of suspensions containing CDA / Mn were added onto a transition electron microscopy (TEM) grid and air dried for TEM and Scanning Transmission Electron Microscopy-Energy Dispersive X-ray (STEM-EDX) analysis.

[0228] To form CDA / Mn / Hisu nanoassemblies, CDA (1 mg / mL, MeOH) and Hisu (10 mg / mL) (100: 40 volume ratio) were rapidly mixed together to form the first nanoassembly. The self-assembling condition was kept at pH < 5 to protonate Hisu. After self-assembly was completed, MnCIz (100 mM, MeOH, 100: 40: 7 volume ratio) was added with vortexing to form the intermetallic nanoassembly. CDA / Mn / Hisu was vortexed at room temperature for 2 hours with sporadic water bath (37Hz, 100W) sonication. After centrifugation at 20,000 g for 10 min, pellets were resuspended in DI water with water bath sonication. 10 pL of suspension containing CDA / Mn / Hisu was added onto a TEM grid and air-dried for TEM and STEM-EDX analysis. For AFM analysis, 10 pL of suspension containing CDA / Mn / Hisu was added onto a Mica discs (Fisher Scientific, NC1535937). To form CDA / Mn / Hise, CDA / Mn / HiS33, and CDA / Mn / d-Hisu, Hisu was replaced with Hise (Histidine 6), HiS33 (Histidine 33), and d-Hisu (d-Histidine 11) (10 mg / mL) with the same synthesis and characterization protocol as above.

[0229] Synthesis and characterization of His(n)-CRYSTAL

[0230] Hn-CRYSTAL was prepared by first forming CDA / Mn / Hisu nanoassemblies and then encapsulating the nanoassemblies within lipid layers. To form CDA / Mn / Hisu nanoassembly, CDA (1 mg / mL, MeOH) and Hisu (10 mg / mL, ethanol (EtOH) with 0.5% TFA) (100: 40 volume ratio) were rapidly mixed together to form the 1st nanoassembly. The self-assembling condition was kept at pH < 5 to protonate Hisu. After complete self-assembling, MnCIz (100 mM, MeOH, 100: 40: 7 volume ratio) was added rapidly with vortexing to form the intermetallic nanoassembly. CDA / Mn / His11 was vortexed at room temperature for 2 hours with sporadic water bath (37Hz, 100W) sonication. After centrifugation at 20,000 g for 10 min, the intermetallic nanoassembly (containing 100 pg CDA) was resuspended in lipid mixtures containing 20 pL DOPC (Avanti Polar Lipids: 850375C-25mg, 10 mg / mL in EtOH), 10 pL cholesterol (Avanti Polar Lipids: 700000P-500mg, 10 mg / mL in EtOH), and 12.8 pL 18:0 PEG5000 PE (Avanti Polar Lipids: 880220P-25mg, 10 mg / mL in EtOH). After brief water bath sonication (37Hz, 100W), 128 pL water was added to the clear CDA / Mn / His11@encapsulating-lipid mixture for CRYSTAL formation. To prepare He-CRYSTAL, H33-CRYSTAL, and d-Hn-CRYSTAL,PATENT

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[0232] Hisi 1 was replaced with Hise, HiS33, and d-Hisu in otherwise identical synthesis and characterization protocols. Unless specified, all Hn-CRYSTALs were synthesized with this protocol. To make Hn-CRYSTAL (Aqueous), a CDA / Mn / Hisu nanoassembly was synthesized in DI water by dissolving CDA, MnCIz, and Hisu in DI water followed by the remainder of the synthesis protocol, above. To prepare Hn-DOPE-CRYSTAL, Hisu was conjugated to DOPE-N-hydroxy-succinimide (NHS) lipid and employed in the above synthesis protocol replacing the Hisu with DOPE-Hisu conjugate.

[0233] Results

[0234] CDA and Mn2+self-assembled to form ribbon-like nanostructures (FIG. 4A). STEM-EDX analysis confirmed the co-presence of CDA (element P) and Mn2+within nanoribbons (FIG. 4B). However, the physical features of the CDA / Mn exhibited non-reversible aggregation behavior and suboptimal colloidal stability. In contrast, the addition of polyhistidine (Hisn) improved colloidal stability with reversible aggregates behavior through brief sonication. Additionally, the increase of the length of polyhistidine led to the increased colloidal stability, corresponding to the smaller nanoassembly formation with the increase in the length of polyhistidine (FIG. 5A). Strong interactions of the CDA / Mn were overcome by the addition of positively charged (His)n, leading to electrostatic repulsion and observed improvements to colloidal stability (FIG. 5B, FIG. 5C). Together, polyhistidine regulates the formation of nanoribbons and led to a library of CDA / Mn / HiSn nanoassemblies.

[0235] To enable the efficient delivery, we then encapsulated CDA / Mn / Hise, CDA / Mn / Hisu, CDA / Mn / HiS33, CDA / Mn / d-Hisu within lipid layers (DOPC:cholesterol:DS-PEG5K (PEG-5000-PE): 14:0 PA = 1 :1 :0.07:0.3) to form He-CRYSTAL, Hn-CRYSTAL, or H33-CRYSTAL. We also synthesized a control d-Hn-CRYSTAL, which utilized D-form of Hu peptide.

[0236] Example 6. CRYSTAL exerts robust anti-tumor efficacy

[0237] A study was performed to evaluate the anti-tumor efficacy of lipid nanoparticle compositions including polypeptides not conjugated to lipids, in vitro and in vivo.

[0238] Materials and Methods

[0239] Investigation of His(n)-CRYSTAL in vitro

[0240] To get bone marrow-derived dendritic cells (BMDCs), bone marrow from C57BL / 6 mice was harvested and cultured in bacteriological Petri dishes with GM-CSF-containing media. The media were refreshed on days 3, 6, and 8. After eight days, BMDCs were harvested. Mouse BMDC cells (1 x 105 / well) were seeded in a 96-well plate and incubated with Hn-CRYSTAL. After 24 hours, supernatants were collected for cytokine ELISA assay at the Cancer Center Immunology Core of the University of Michigan.

[0241] Investigation of His(n)-CRYSTAL in mice

[0242] For the B16F10 tumor model, C57BL / 6 mice (Jackson Laboratories or laboratory-bred) were inoculated subcutaneously (s.c.) with the indicated number of B16F10 cells (in 100 pL HBSS) on the right flank. Tumor size and survival were monitored every 3 or 4 days. Tumor size was calculatedPATENT

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[0244] based on equation: volume = length x width2x 0.5, and tumor-bearing mice were assigned to different treatment groups. For cancer cell culture, B16F10 cells were cultured in complete RPMI 1640 (10% FBS, 1% Pen / Strep). To analyze the systemic STING activation, Hn-CRYSTAL were injected intravenously (IV) at the indicated doses. The cytokine levels in serum were measured by ELISA assay in the Cancer Center Immunology Core of the University of Michigan. For the pharmacokinetic study, 10 pg Hn-DOPE-CRYSTAL or Hn-CRYSTAL were injected intravenously and plasma CDA were measured at the indicated timepoints.

[0245] Results

[0246] We first investigated the STING activating efficiency of He-CRYSTAL, Hn-CRYSTAL, H33-CRYSTAL, and d-Hn-CRYSTAL in vitro by incubating them with mouse BMDCs. All the CRYSTAL lipid nanoparticle compositions efficiently elevated the levels of IFN-p, TNF-a, IL-6, and CXCL10 compared to the untreated control (FIG. 6). H33-CRYSTAL elicited significantly higher levels of all tested cytokines compared to other candidates, likely due to more efficient delivery in vitro due to its smaller nanoassembly core.

[0247] To investigate whether the trends in vitro correlated with in vivo responses, the therapeutic efficacy in mice bearing B16F10 melanoma tumors (FIG. 7A) was determined. All CRYSTAL candidates elicited potent systemic STING activation, as evidenced by the elevation of IFN-p, IFN-a, IL-6, TNF-a, and CXCL10 in the serum four hours post IV injection (FIG. 7B). Hn-CRYSTAL outperformed in sustaining the anti-tumor efficacy as well as prolonging the survival in comparison to all the other candidates (FIG. 7A), including d-Hn-CRYSTAL formed with D-form of Hn.

[0248] Next, the efficacy of CRYSTAL formed with Hisn and lipidated Hisn (Hn-DOPE conjugate) was compared in vivo. Both Hn-CRYSTAL and Hn-DOPE-CRYSTAL exhibited comparable pharmacokinetic profile (FIG. 8A) and antitumor efficacy (FIG. 8B) in B16F10 bearing mice, indicating that a lipid-conjugated form of the polypeptide is not essential for the potency of CRYSTAL.

[0249] The best performing candidate Hn-CRYSTAL was then used to compare the effects of solvents used in nanoassembly formation. Hn-CRYSTAL (Aqueous) was formed using DI water as the solvent in combining CDA, Mn2+, and Hisn, and compared against Hn-CRYSTAL (Organic). Hn-CRYSTAL (Organic) outperformed the Hn-CRYSTAL (Aqueous) in sustaining the antitumor efficacy in B16F10 bearing mice (FIG. 9), indicating the preparation conditions for nanoassembly formation effect the therapeutic efficacy of the resulting lipid nanoparticle composition.

[0250] The lipid nanoparticle composition Hn-CRYSTAL was used in a late-stage B16F10 tumor model to assess the efficacy against a complex tumor microenvironment (TME). In the late stage of the B16F10 model, tumor-bearing C57BL / 6 mice were treated with three IV injections of 10 pg Hn-CRYSTAL at days 0, 4, and 8, and treatment led to rapid tumor regression and sustained tumor control (FIG. 10). The therapeutic efficacy of Hn-CRYSTAL via intratumoral injection was also evaluated (FIG. 11). Three intratumoral doses of 5 pg Hn-CRYSTAL in the late-stage B16F10 tumor model demonstrated regression of tumors and prolonged the survival of B16F10 tumor-bearing mice compared to untreated groups.PATENT

[0251] ATTORNEY-DOCKET NO.: 51663-005WO2

[0252] ENUMERATED EMBODIMENTS

[0253] E1 . A lipid nanoparticle composition comprising:

[0254] i. a polypeptide 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, and the polypeptide is not covalently conjugated to a lipid;

[0255] ii. a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+;

[0256] Hi. a compound capable of coordinating cations; and

[0257] iv. a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound.

[0258] E2. The lipid nanoparticle composition of embodiment 1 , 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.

[0259] E3. The lipid nanoparticle composition of embodiment 2, wherein the plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid. E4. The lipid nanoparticle composition of embodiment 3, wherein the phosphatidylcholine is 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the steroid is cholesterol, and the PEG phospholipid is 1 .2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG-5000-PE).

[0260] E5. The lipid nanoparticle composition of embodiment 3 or 4, 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).

[0261] E6. The lipid nanoparticle composition of any one of embodiments 1 to 5, 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. E7. The lipid nanoparticle composition of embodiment 6, wherein the compound capable of coordinating cations is a STING agonist.

[0262] E8. The lipid nanoparticle composition of embodiment 7, 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-AMP, 2’3’-c-di-AM(PS)2 (Rp,Rp), c-di-GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP,PATENT

[0263] ATTORNEY-DOCKET NO.: 51663-005WO2

[0264]

[0265] & 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'-cAIMPPATENT

[0266] ATTORNEY-DOCKET NO.: 51663-005WO2

[0267] 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.

[0268] E9. The lipid nanoparticle composition of embodiment 6, wherein the compound capable of coordinating cations is a TLR agonist.

[0269] E10. The lipid nanoparticle composition of embodiment 9, 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.

[0270] E11. The method of any one of embodiments 1 to 10, wherein at least 66% of the amino acid residues in the polypeptide 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.

[0271] E12. The method of embodiment 11 , wherein at least 85% of the amino acid residues in the polypeptide 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. E13. The method of embodiment 12, wherein at least 95% of the amino acid residues in the polypeptide 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. E14. The method of embodiment 13, wherein all of the amino acid residues in the polypeptide 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. E15. The lipid nanoparticle composition of any one of embodiments 1 to 14, wherein the polypeptide comprises from 5 to 33 histidine residues.PATENT

[0272] ATTORNEY-DOCKET NO.: 51663-005WO2

[0273] E16. The lipid nanoparticle composition of embodiment 15, wherein the polypeptide comprises from 10 to 25 histidine residues.

[0274] E17. The lipid nanoparticle composition of embodiment 16, wherein the polypeptide comprises from 10 to 12 histidine residues.

[0275] E18. The lipid nanoparticle composition of embodiment 17, wherein the polypeptide comprises 11 histidine residues.

[0276] E19. The lipid nanoparticle composition of any one of embodiments 1 to 18, wherein the average particle size of the lipid nanoparticle is from 20 to 500 nm.

[0277] E20. The lipid nanoparticle composition of embodiment 19, wherein the average particle size of the lipid nanoparticle is from 50 to 500 nm.

[0278] E21. The lipid nanoparticle composition of embodiment 20, wherein the average particle size of the lipid nanoparticle is from 75 to 250 nm.

[0279] E22. The lipid nanoparticle composition of any one of embodiments 1 to 21 , wherein the lipid nanoparticle has a polydispersity index from 0.1 to 0.3.

[0280] E23. The lipid nanoparticle composition of any one of embodiments 1 to 22, wherein the lipid nanoparticle has a zeta potential from -30 mV to +30 mV.

[0281] E24. 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 23.

[0282] E25. The method of embodiment 24, wherein the subject has an autoimmune disorder.

[0283] E26. The method of embodiment 24, wherein the subject has cancer.

[0284] E27. The method of embodiment 25, further comprising administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

[0285] OTHER EMBODIMENTS

[0286] 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.: 51663-005WO2CLAIMS1. A lipid nanoparticle composition comprising:i. a polypeptide 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, and the polypeptide is not covalently conjugated to a lipid;ii. a cation, wherein the cation is Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+;Hi. 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 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.

3. The lipid nanoparticle composition of claim 2, wherein the plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

4. The lipid nanoparticle composition of claim 3, wherein the phosphatidylcholine is 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the steroid is cholesterol, and the PEG phospholipid is 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG-5000-PE).

5. The lipid nanoparticle composition of claim 3 or 4, 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).

6. The lipid nanoparticle composition of any one of claims 1 to 5, 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.

7. The lipid nanoparticle composition of claim 6, wherein the compound capable of coordinating cations is a STING agonist.PATENTATTORNEY-DOCKET NO.: 51663-005WO28. The lipid nanoparticle composition of claim 7, 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-AMP, 2’3’-c-di-AM(PS)2 (Rp,Rp), c-di-GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP,> " "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,PATENTATTORNEY-DOCKET NO.: 51663-005WO22’2’-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.

9. The lipid nanoparticle composition of claim 6, wherein the compound capable of coordinating cations is a TLR agonist.

10. The lipid nanoparticle composition of claim 9, 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.

11. The method of any one of claims 1 to 10, wherein at least 66% of the amino acid residues in the polypeptide 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.

12. The method of claim 11 , wherein at least 85% of the amino acid residues in the polypeptide 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.

13. The method of claim 12, wherein at least 95% of the amino acid residues in the polypeptide 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.PATENTATTORNEY-DOCKET NO.: 51663-005WO214. The method of claim 13, wherein all of the amino acid residues in the polypeptide 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.

15. The lipid nanoparticle composition of any one of claims 1 to 14, wherein the polypeptide comprises from 5 to 33 histidine residues.

16. The lipid nanoparticle composition of claim 15, wherein the polypeptide comprises from 10 to 25 histidine residues.

17. The lipid nanoparticle composition of claim 16, wherein the polypeptide comprises from 10 to 12 histidine residues.

18. The lipid nanoparticle composition of claim 17, wherein the polypeptide comprises 11 histidine residues.

19. The lipid nanoparticle composition of any one of claims 1 to 18, wherein the average particle size of the lipid nanoparticle is from 20 to 500 nm.

20. The lipid nanoparticle composition of claim 19, wherein the average particle size of the lipid nanoparticle is from 50 to 500 nm.

21. The lipid nanoparticle composition of claim 20, wherein the average particle size of the lipid nanoparticle is from 75 to 250 nm.

22. The lipid nanoparticle composition of any one of claims 1 to 21 , wherein the lipid nanoparticle has a polydispersity index from 0.1 to 0.3.

23. The lipid nanoparticle composition of any one of claims 1 to 22, wherein the lipid nanoparticle has a zeta potential from -30 mV to +30 mV.

24. 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 23.

25. The method of claim 24, wherein the subject has an autoimmune disorder.PATENTATTORNEY-DOCKET NO.: 51663-005WO226. The method of claim 24, wherein the subject has cancer.

27. The method of claim 25, further comprising administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

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