Methods for preparing nanoparticle formulations

Abstract

The present invention relates to methods of preparing lipid nanoparticle compositions that include a polypeptide, a cation, compound capable of coordinating cations, and a population of encapsulating lipids. The methods afford lipid nanoparticles after a single operation and have favorable properties for the delivery of therapeutic agents.

Description

[0001] PATENT

[0002] ATTORNEY-DOCKET NO.: 51663-008WO2

[0003] METHODS FOR PREPARING NANOPARTICLE FORMULATIONS

[0004] BACKGROUND OF THE INVENTION

[0005] This invention relates to lipid nanoparticle compositions and methods for their preparation. There is a need to develop shorter and less expensive methods for making lipid nanoparticles. Understanding the formation of lipid nanoparticles and their properties is useful for improving the delivery of therapeutic agents to treat diverse human diseases.

[0006] SUMMARY OF THE INVENTION

[0007] The present disclosure provides lipid nanoparticle compositions and methods for their preparation.

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

[0009] (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 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;

[0010] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0011] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0012] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and

[0013] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition. 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.

[0014] 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-methylPATENT

[0015] ATTORNEY-DOCKET NO.: 51663-008WO2

[0016] ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues.

[0017] 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-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.

[0018] In some embodiments, all 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.

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

[0020] In some embodiments, the polypeptide comprises from 10 to 25 histidine residues.

[0021] In some embodiments, the polypeptide comprises from 10 to 12 histidine residues.

[0022] In some embodiments, the polypeptide comprises 11 histidine residues.

[0023] 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.

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

[0025] In some embodiments, the STING agonist is cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,Rp), c-di-

[0026] GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP,

[0027]

[0028] <

[0029]

[0030] ( ), 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.

[0031] In some embodiments, the compound capable of coordinating cations is a TLR agonist.PATENT

[0032] ATTORNEY-DOCKET NO.: 51663-008WO2

[0033] 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.

[0034] 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.

[0035] In some embodiments, the plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

[0036] 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).

[0037] 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).

[0038] In some embodiments, the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) are provided as solutions.

[0039] In some embodiments, the solutions are warmed to from 30 °C to 40 °C prior to the combining step (b).

[0040] In some embodiments, the combining step (c) comprises combining from two to seven portions of water with the mixture.

[0041] In some embodiments, the combining step (c) comprises vortexing, agitating, sonicating, homogenizing, dispersing, emulsifying, or microfluidic mixing.

[0042] In some embodiments, the water and mixture of step (c) is covered with a moisture-resistant film while combining.

[0043] In some embodiments, the reducing step (d) comprises passing the lipid particulate mixture through a high-pressure homogenizer.

[0044] In some embodiments, the passing is at from 40 psi to 60 psi.

[0045] In some embodiments, the purifying of step (e) comprises dialysis, centrifugation, ultrafiltration, tangential flow filtration, size-exclusion chromatography, liquid-liquid extraction, or field flow fractionation.

[0046] In some embodiments, the compound capable of coordinating a cation is combined last in the combining step (b).

[0047] DEFINITIONS

[0048] 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.PATENT

[0049] ATTORNEY-DOCKET NO.: 51663-008WO2

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

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.PATENT

[0061] ATTORNEY-DOCKET NO.: 51663-008WO2

[0062] 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.

[0063] 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.

[0064] 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.”

[0065] 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.

[0066] 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.

[0067] 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.

[0068] As used herein, the term “lipidated” refers to a compound that has been covalently conjugated to a lipid (such as an encapsulating lipid).

[0069] 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.

[0070] As used herein, the term “microfluidic mixing” refers to the precise method of mixing using smallchannel tubes and mixing done in small-scale channels, such as on a chip. The orientation of channels combined with the external forces applied achieves a rapid and thorough mixing of multiple samples as they flow together through the specially-designed channels of the microfluidic device.PATENT

[0071] ATTORNEY-DOCKET NO.: 51663-008WO2

[0072] 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.

[0073] 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 benef it / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G.

[0074] Wermuth), Wiley-VCH, 2008. These salts may be acid addition salts involving inorganic or organic acids. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable acid. Methods for preparation of the appropriate salts are well-established in the art. In the context of the disclosure, lipids may be provided as pharmaceutically acceptable salts, e.g., the sodium or ammonium salt of a phospholipid.

[0075] As used herein, the term “polydispersity index” or “PDI” refers to homogeneity of the size of nanoparticles in a population, with a smaller PDI reflecting a narrower particle size distribution among the nanoparticles. Lipid nanoparticle compositions of the disclosure may have a polydispersity index from 0 to 0.3, such as 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30. In some embodiments, the lipid nanoparticle composition has a polydispersity index from 0.1 to 0.3.

[0076] 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.

[0077] 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.

[0078] 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.PATENT

[0079] ATTORNEY-DOCKET NO.: 51663-008WO2

[0080] As used herein, “sonicating” refers to the application of ultrasonic waves to a mixture to fragment aggregated molecules or components.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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 of 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.

[0089] 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.PATENT

[0090] ATTORNEY-DOCKET NO.: 51663-008WO2

[0091] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a prior art multi-step synthetic process for preparing lipid nanoparticles that include a lipidated polypeptide.

[0092] FIG. 2 shows a single-step synthetic process for preparing lipid nanoparticles that include an unconjugated polypeptide and lipid.

[0093] 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.

[0094] DETAILED DESCRIPTION OF THE INVENTION

[0095] 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.

[0096] Lipid nanoparticle compositions

[0097] 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.

[0098] Polypeptides

[0099] 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, dimethyllysine, trimethyllysine, acetyllysine, ornithine, methylornithine, dimethylornithine, trimethylornithine,PATENT

[0100] ATTORNEY-DOCKET NO.: 51663-008WO2

[0101] 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-N,N-dimethylated) or to a single w-nitrogen (being asymmetrically w-A / -dimethylated).

[0102] 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, 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 histidinePATENT

[0103] ATTORNEY-DOCKET NO.: 51663-008WO2

[0104] 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.

[0105] 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.

[0106] Cations

[0107] 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.

[0108] Cations of the lipid nanoparticle compositions of the disclosure are Co2+, Co3+, Cu+, Cu2+, Mn2+, and Zn2+.

[0109] Compounds capable of coordinating cations

[0110] 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.

[0111] In some embodiments, the compound capable of coordinating cations is a STING agonist. In some embodiments, the STING agonist is cGAMP, cdiAMP, cdiGMP, cAIMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, cAIMP Difluor, cAIM(PS)2, Difluor (Rp / Sp), 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2 (Rp,Rp), c-di-

[0112] GMP Fluorinated, 2’3’-c-di-GMP, c-di-IMP,

[0113]

[0114] <

[0115]

[0116] ( ), 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.

[0117] In some embodiments, the compound capable of coordinating cations is a TLR agonist.PATENT

[0118] ATTORNEY-DOCKET NO.: 51663-008WO2

[0119] 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.

[0120] Encapsulating lipids

[0121] 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. Lysophospholipids are lipids in which hydrolysis has caused one of the acyl groups of the lipid (in a phospholipid) to be removed.

[0122] / '. phosphatidic acids

[0123] 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-dili noleoyl-sn-glycero-3-phosphate, 1 ,2-diarachidonoyl-sn-glycero-3-phosphate, and 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphate.

[0124] / ' / . phosphatidylcholines

[0125] 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-PATENT

[0126] ATTORNEY-DOCKET NO.: 51663-008WO2

[0127] 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-d imyristoleoyl-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.

[0128] Hi. phosphatidylethanolamines

[0129] Phosphatidylethanolamines (PEs) are highly prevalent phospholipids in eukaryotes, featuring a 2-amino-ethanol polar head group, which may be protonated and positively charged at physiological pH. PEs may be zwitterionic (having both positive and negative charge) depending on the pH of the solution. Exemplary phosphatidylethanolamines for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1 ,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioctanoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-didecanoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilauroyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dielaidoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, and 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine.

[0130] Cephalins may also be considered PEs and are applicable as lipids for the lipid nanoparticle compositions of the disclosure.

[0131] iv. phosphatidylglycerols

[0132] 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-(T-rac-glycerol), 1 ,2-dimyristoyl-sn-glycero-3-phospho-(1 ’-rac-glycerol), 1 ,2-dipentadecanoyl-sn-PATENT

[0133] ATTORNEY-DOCKET NO.: 51663-008WO2

[0134] 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)].

[0135] v. phosphatidylinositides

[0136] 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.

[0137] Phosphoinositides are a minority component in cellular membranes. Phosphoinositides for use in the lipid nanoparticle compositions of the disclosure include 1 ,2-dihexanoyl-sn-glycero-3-phospho-(1 ’-myoinositol), 1 ,2-dioctanoyl-sn-glycero-3-phospho-(1 ’-myo-inositol), 1 ,2-dipalmitoyl-sn-glycero-3-phospho-(1 '-myo-inositol), 1 ,2-distearoyl-sn-glycero-3-phosphoinositol, 1 ,2-dioleoyl-sn-glycero-3-phospho-(1 ’-myoinositol), and phosphorylated derivatives thereof.

[0138] vi. phosphatidylserines

[0139] 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.

[0140] v / 7. sphingolipids

[0141] 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.

[0142] viii. cardiolipins

[0143] Cardiolipins are phospholipids comprising two phosphatidyl glycerol backbones linked together through another glycerol head group. Cardiolipins for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1 ',3'-bis[ 1 ,2-dimyristoyl-sn-glycero-3-phospho]-glycerol, 1 ',3'-bis[1 ,2-dipalmitoyl-sn-glycero-3-phospho]-glycerol, 1 ' ,3'-bis[1 ,2-distearoyl-sn-glycero-3-phospho]-glycerol,PATENT

[0144] ATTORNEY-DOCKET NO.: 51663-008WO2

[0145] 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 -palm itoy l-2-oleoyl-sn-glycero-3-phospho]-glycerol.

[0146] ix. steroids

[0147] 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.

[0148] x. polyethylene glycol (PEG) phospholipids

[0149] Polyethylene glycol (PEG) phospholipids feature a phosphoethanolamine structure where the amine of the head group has been amidated to link a hydrophilic PEG moiety to the lipid. Incorporation of PEG phospholipids as a polymer for the nanoparticle lipid mixture can improve the properties of the nanoparticle for drug delivery. The length of the PEG group is characterized by the molecular weight, such that, for example, the compound 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350] represents a PEG phospholipid having a molecular weight of 350 Daltons. PEG phospholipids for use in the lipid nanoparticle compositions of the disclosure include, but are not limited to, 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350] , 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] , 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] , 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] , 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene g lycol)- 1000], 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene g lycol)- 1000], 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene g lycol)- 1000], 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene g lycol)- 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-PATENT

[0150] ATTORNEY-DOCKET NO.: 51663-008WO2

[0151] 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].

[0152] Methods of stimulating an immune response

[0153] In another aspect, the disclosure provides methods of stimulating immune response in a subject to treat diseases or conditions. The lipid nanoparticle compositions of the disclosure may be used to deliver therapeutic drugs as part of the core material, or the core materials may separately activate the immune system and improve the response to a drug that is encapsulated within the nanoparticle or administered to the subject exogenously. Thus, there is presented a method for stimulating an innate immune response in a subject that comprises administering to the subject an effective amount of any of the above lipid nanoparticle compositions. In some embodiments, the subject has an autoimmune disorder. In some embodiments, the subject has cancer. In some embodiments, the method further comprises administering one or more additional therapeutic agents for treating an autoimmune disorder to the subject.

[0154] Methods of preparing lipid nanoparticles

[0155] In another aspect, the disclosure provides a method of preparing any of the above lipid nanoparticle compositions comprising:

[0156] (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 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;

[0157] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0158] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0159] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and

[0160] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

[0161] EXAMPLES

[0162] 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.PATENT

[0163] ATTORNEY-DOCKET NO.: 51663-008WO2

[0164] Example 1. Preparation of lipid nanoparticle compositions

[0165] A study was performed to prepare lipid nanoparticle compositions having formulations of lowered complexity for the purpose of reducing production costs and simplifying the composition. Accordingly, lipid nanoparticles of the invention may be prepared by multiple methods, either by multi-step or single-step processes.

[0166] Materials and methods

[0167] Chemicals and reagents

[0168] 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).

[0169] 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).

[0170] Reagent preparation

[0171] 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.

[0172] Polypeptide solutions were prepared at 10 mg / mL in EtOH spiked with 0.5% trifluoroacetic acid (TFA).

[0173] Multi-step synthesis protocol

[0174] 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).

[0175] One-step synthesis protocol

[0176] 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.

[0177] 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 parafilm and vortexed slowly. A 5 mL portion of the solution was passed through a high-pressure homogenizer (Avestin B15) forPATENT

[0178] ATTORNEY-DOCKET NO.: 51663-008WO2

[0179] 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.

[0180] Example 2. Size characteristics of unconjugated polyhistidine nanoparticles

[0181] Analytical Methods

[0182] 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).

[0183] 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.

[0184] Results

[0185] Lipid nanoparticles made using the two different synthesis methods, having either lipidated (SMP-B) or free polyhistidine (SMP-C), result in similarly-sized nanoparticles that also have a comparable size distribution. SMP-B was prepared according to the multi-step synthesis protocol, and SMP-C according to the one step synthesis protocol. There is a noteworthy difference in surface charge, and lipidated-polyhistidine nanoparticles have a higher positive surface charge than the unconjugated formulation. The Z-average, polydispersity index (PDI), and zeta potential in mV for the SMP-B and SMP-C formulations is shown below in Table 1.

[0186] Table 1. Characterization of lipid nanoparticles having lipidated or free polypeptide.

[0187]

[0188] Example 3. In vitro potency of lipid nanoparticle formulations

[0189] The lipid nanoparticle formulations of the previous examples were tested in vitro for potency using a STING activation assay and a CXCL10 production assay.

[0190] 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).

[0191] In vitro potency based on production of CXCL10 was performed using RAW-Dual murine reporter cells (Invivogen) that were plated at 200K per well. Cells were incubated with the SMP formulation and the supernatant was collected after a 24-hour time-period. CXCL10 quantification was done using thePATENT

[0192] ATTORNEY-DOCKET NO.: 51663-008WO2

[0193] 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).

[0194] Results

[0195] The results of the lipid nanoparticle formulation SMP-C on STING activation and CXCL10 production are presented below in Tables 2 and 3.

[0196] Table 2. Effect of unconjugated polypeptide formulation on STING activation in RAW-Dual murine cells.

[0197]

[0198] Table 3. Effect of unconjugated polypeptide formulation on CXCL10 expression in RAW-Dual murine cells.

[0199]

[0200] Example 4. Effect of polyhistidine length on formulation

[0201] 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 (FIG. 3).

[0202] ENUMERATED EMBODIMENTS

[0203] E1. A method of preparing a lipid nanoparticle composition comprising:

[0204] (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 isPATENT

[0205] ATTORNEY-DOCKET NO.: 51663-008WO2

[0206] 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;

[0207] (b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;

[0208] (c) combining water with the mixture of step (b) to form a lipid particulate mixture;

[0209] (d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and

[0210] (e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition. E2. The method of embodiment 1 , 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.

[0211] E3. The method of embodiment 2, 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.

[0212] E4. The method of embodiment 3, 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.

[0213] E5. The method of embodiment 4, wherein all 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.

[0214] E6. The method of any one of embodiments 1 to 5, wherein the polypeptide comprises from 5 to 30 histidine residues.

[0215] E7. The method of embodiment 6, wherein the polypeptide comprises from 10 to 25 histidine residues.PATENT

[0216] ATTORNEY-DOCKET NO.: 51663-008WO2

[0217] E8. The method of embodiment 7, wherein the polypeptide comprises from 10 to 12 histidine residues.

[0218] E9. The method of embodiment 8, wherein the polypeptide comprises 11 histidine residues.

[0219] E10. The method of any one of embodiments 1 to 9, 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.

[0220] E11. The method of embodiment 10, wherein the compound capable of coordinating cations is a STING agonist.

[0221] E12. The method of embodiment 11 , 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-

[0222]

[0223] PATENT

[0224] ATTORNEY-DOCKET NO.: 51663-008WO2

[0225] ><

[0226] ""

[0227]

[0228] agonist-C11 ( ), STING agonist-1 ( ), STING agonist

[0229]

[0230] ol Wt:: 433.88

[0231] 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.

[0232] E13. The method of embodiment 10, wherein the compound capable of coordinating cations is a TLR agonist.

[0233] E14. The method of embodiment 13, 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.

[0234] E15. The method of any one of embodiments 1 to 14, 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.

[0235] E16. The method of embodiment 15, wherein the plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

[0236] E17. The method of embodiment 18, 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).

[0237] E18. The method of embodiment 16 or 17, 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).PATENT

[0238] ATTORNEY-DOCKET NO.: 51663-008WO2

[0239] E19. The method of any one of embodiments 1 to 18, wherein the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) are provided as solutions. E20. The method of embodiment 19, wherein the solutions are warmed to from 30 °C to 40 °C prior to the combining step (b).

[0240] E21. The method of any one of embodiments 1 to 20, wherein the combining step (c) comprises combining from two to seven portions of water with the mixture.

[0241] E22. The method of any one of embodiments 1 to 21 , wherein the combining step (c) comprises vortexing, agitating, sonicating, homogenizing, dispersing, emulsifying, or microfluidic mixing.

[0242] E23. The method of embodiment 22, wherein the water and mixture of step (c) is covered with a moisture-resistant film while combining.

[0243] E24. The method of any one of embodiments 1 to 23, wherein the reducing step (d) comprises passing the lipid particulate mixture through a high-pressure homogenizer.

[0244] E25. The method of embodiment 24, wherein the passing is at from 40 psi to 60 psi.

[0245] E26. The method of any one of embodiments 1 to 25, wherein the purifying of step (e) comprises dialysis, centrifugation, ultrafiltration, tangential flow filtration, size-exclusion chromatography, liquid-liquid extraction, or field flow fractionation.

[0246] E27. The method of any one of embodiments 1 to 26, wherein the compound capable of coordinating a cation is combined last in the combining step (b).

[0247] OTHER EMBODIMENTS

[0248] 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.: 51663-008WO2CLAIMS1. A method of preparing a lipid nanoparticle composition comprising:(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 Co2+, Co3+, Cu+, Cu2+, Mn2+, or Zn2+; a compound capable of coordinating cations; and a plurality of encapsulating lipids, wherein the plurality of encapsulating lipids encapsulate the polypeptide, cation, and compound;(b) combining the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) to form a mixture;(c) combining water with the mixture of step (b) to form a lipid particulate mixture;(d) reducing the particle size of the lipid particulate mixture of step (c) to form crude lipid nanoparticles; and(e) purifying the crude lipid nanoparticles of step (d) to obtain the lipid nanoparticle composition.

2. The method of claim 1 , 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.

3. The method of claim 2, 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.

4. The method of claim 3, 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-methylPATENTATTORNEY-DOCKET NO.: 51663-008WO2ester, glutamine, aspartic acid, isoaspartic acid, aspartic acid 4-methyl ester, asparagine, serine, threonine, methionine, S-methyl-cysteine, leucine, isoleucine, or valine residues.

5. The method of claim 4, wherein all 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.

6. The method of any one of claims 1 to 5, wherein the polypeptide comprises from 5 to 30 histidine residues.

7. The method of claim 6, wherein the polypeptide comprises from 10 to 25 histidine residues.

8. The method of claim 7, wherein the polypeptide comprises from 10 to 12 histidine residues.

9. The method of claim 8, wherein the polypeptide comprises 11 histidine residues.

10. The method of any one of claims 1 to 9, 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.

11. The method of claim 10, wherein the compound capable of coordinating cations is a STING agonist.

12. The method of claim 11 , 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, 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.PATENTATTORNEY-DOCKET NO.: 51663-008WO213. The method of claim 10, wherein the compound capable of coordinating cations is a TLR agonist.

14. The method of claim 13, 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.

15. The method of any one of claims 1 to 14, 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.

16. The method of claim 15, wherein the plurality of encapsulating lipids comprises a phosphatidic acid, a steroid, a phosphatidylcholine, and a PEG phospholipid.

17. The method of claim 18, 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).

18. The method of claim 16 or 17, 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).

19. The method of any one of claims 1 to 18, wherein the polypeptide, cation, compound capable of coordinating cations, and plurality of encapsulating lipids of step (a) are provided as solutions.

20. The method of claim 19, wherein the solutions are warmed to from 30 °C to 40 °C prior to the combining step (b).

21. The method of any one of claims 1 to 20, wherein the combining step (c) comprises combining from two to seven portions of water with the mixture.

22. The method of any one of claims 1 to 21 , wherein the combining step (c) comprises vortexing, agitating, sonicating, homogenizing, dispersing, emulsifying, or microfluidic mixing.

23. The method of claim 22, wherein the water and mixture of step (c) is covered with a moistureresistant film while combining.

24. The method of any one of claims 1 to 23, wherein the reducing step (d) comprises passing the lipid particulate mixture through a high-pressure homogenizer.

25. The method of claim 24, wherein the passing is at from 40 psi to 60 psi.PATENTATTORNEY-DOCKET NO.: 51663-008WO226. The method of any one of claims 1 to 25, wherein the purifying of step (e) comprises dialysis, centrifugation, ultrafiltration, tangential flow filtration, size-exclusion chromatography, liquid-liquid extraction, or field flow fractionation.

27. The method of any one of claims 1 to 26, wherein the compound capable of coordinating a cation is combined last in the combining step (b).

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