Nanoencapsulated pharmaceutical compositions and uses thereof

Abstract

The present disclosure relates to a pharmaceutical composition for treating or preventing a disease. The pharmaceutical composition may include a polymer-drug nanoaggregate having a polymer and at least one bioactive agent, which may include a STING polypeptide, a nucleic acid encoding the STING polypeptide, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulating molecule, or a combination thereof. The pharmaceutical composition may be a vaccine or a vaccine adjuvant. The present disclosure also relates to methods for treating or preventing diseases using the pharmaceutical compositions. The diseases may include infectious diseases caused by viruses or other pathogens, such as influenza, rabies, or respiratory diseases, such as severe acute respiratory syndrome (SARS) caused by coronavirus, such as MERS-CoV, SARS-CoV, and 2019 coronavirus disease (COVID-19) caused by the virus SARS-CoV-2 and variants thereof.

Description

[0001] References to Electronic Sequence Listings

[0002] This application contains a sequence listing in the following specification, and an electronic version thereof is submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy was created on February 4, 2023, is named "010816-16093001.xml", and is 33,108 bytes in size. The sequence listing contained in the .XML file submitted at the same time is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to a pharmaceutical composition and a method for treating or preventing a disease in a patient in need thereof. The pharmaceutical composition may be a vaccine or an adjuvant. Background Art

[0004] Synthetic polymers have been shown to have important applications as effective delivery vehicles or as excipients in drug formulations.

[0005] Symmetrically branched polymers (SBPs), such as dendrimers, including starburst dendrimers (or dense star polymers) and combburst dendrigrafts (or supercomb branched polymers), are some examples. Those polymers generally have: (a) a well-defined core, (b) at least two concentric dendritic layers (generations) with symmetrical (equal length) branches and branch attachment points, and (c) external surface groups, such as polyamidoamine (PAMAM)-based branched polymers and dendrimers described in U.S. Pat. Nos. 4,435,548; 4,507,466; 4,568,737; 4,587,329; 5,338,532; 5,527,524 and 5,714,166. Other examples include polyethyleneimine (PEI) dendrimers, such as those disclosed in U.S. Pat. No. 4,631,337; polypropyleneimine (PPI) dendrimers, such as those disclosed in U.S. Pat. Nos. 5,530,092; 5,610,268 and 5,698,662; Frechet-type polyether and polyester dendrimers, core shell tectodendrimers, and other dendrimers, such as those described in "Dendritic Molecules," edited by Newkome et al., VCH Weinheim, 1996, "Dendrimers and Other Dendritic Polymers," edited by Frechet & Tomalia, John Wiley & Sons, Ltd., 2001; and U.S. Pat. No. 7,754,500.

[0006] Combburst dendrimers are constructed by a stepwise synthesis process with a core molecule and concentric layers with symmetrical branches. In contrast to dendrimers, combburst dendrimers or polymers are generated with monodisperse linear polymer building blocks (U.S. Pat. Nos. 5,773,527, 5,631,329, and 5,919,442). In addition, the branching pattern is different from that of dendrimers. For example, combburst dendrimers form branching junctions along the polymer backbone (chain branches), while starburst dendrimers are usually branched at the ends (terminal branches). Due to the living polymerization technology used, the molecular weight distribution (M) of these polymers (core and branches) is w / Mn ) is usually very narrow. Therefore, the comb-on-graft dendritic grafts produced by the graft-on-graft process are clearly defined as M w / M n Closer to 1 than usual.

[0007] SBPs (e.g., dendrimers) are primarily produced by divergent or convergent synthetic methods through repeated protection and deprotection procedures. Since dendrimers utilize small molecules as building blocks for the core and branches, the molecular weight distribution of dendrimers is usually limited. In the case of lower generations, dendrimers of a single molecular weight are usually obtained. Dendrimers generally use small molecule monomers as building blocks, while dendrimer grafts use linear polymers as building blocks.

[0008] In addition to dendrimers and dendritic grafts, other SBPs may include symmetrical star-shaped or comb-shaped polymers, such as symmetrical star-shaped and comb-shaped polyethylene oxide (PEO), polyethylene glycol (PEG), polyethyleneimine (PEI), polypropyleneimine (PPI), polyoxazoline (POX), polymethyloxazoline (PMOX), polyethyloxazoline (PEOX), polypropyloxazoline (PPOX), polystyrene, polymethyl methacrylate (PMMA) or polydimethylsiloxane.

[0009] Asymmetric branched polymers (ABPs) can be of two different types: regular ABPs and random ABPs. Asymmetric branched dendrimers or regular ABPs (reg-ABPs) typically have a core, controlled and well-defined asymmetric (unequal length) branches and asymmetric branch junctions, as described in U.S. Patent Nos. 4,289,872; 4,360,646 and 4,410,688. On the other hand, random ABPs (ran-ABPs) have: a) no core, b) external and internal functional groups, c) random / variable branch lengths and patterns (i.e., terminal branches and chain branches), and d) unevenly distributed interior void space.

[0010] The synthesis and mechanism of random ABPs (such as those prepared from PEI) are reported in Jones et al., J. Org. Chem. 9, 125 (1944), Jones et al., J. Org. Chem. 30, 1994 (1965) and Dick et al., J. Macromol. Sci. Chem., A4 (6), 1301-1314, (1970). Random ABPs, such as those prepared from POX, poly(2-oxazoline), poly(2-methyloxazoline) (PMOX), and poly(2-ethyloxazoline) (PEOX), are reported by Litt (J. Macromol. Sci. Chem. A9(5), 703-727 (1975)) and Warakomski (J. Polym. Sci. Polym. Chem. 28, 3551 (1990). The synthesis of random ABPs generally involves either a one-pot divergent process or a one-pot convergent process.

[0011] A polymer may also be a homopolymer or a copolymer. A copolymer is a polymer or polymer backbone formed by polymerization of different monomers or repeating units of different monomers. A homopolymer may refer to a polymer or polymer backbone composed of the same repeating units, i.e., a homopolymer is formed from the same monomers. A monomer may be a simple compound or a complex or collection of compounds, where the collection or complex is a repeating unit in a homopolymer.

[0012] Although branched polymers (including SBPs and ABPs) have been used for drug delivery, these attempts have mainly focused on chemically linking drugs to polymers, or physically encapsulating these drugs inside by unimolecular encapsulation (e.g., those described in U.S. Pat. Nos. 5,773,527; 5,631,329; 5,919,442; and 6,716,450). For example, dendrimers and dendritic grafts are considered to physically capture bioactive molecules using unimolecular encapsulation methods, such as those described in U.S. Pat. Nos. 5,338,532, 5,527,524, and 5,714,166 for dense star polymers, and U.S. Pat. No. 5,919,442 for supercomb branched polymers. Similarly, unimolecular encapsulation of various drugs using SBP to form "dendrimer cassettes" is reported in Tomalia et al., Angew. Chem. Int. Ed. Engl., 1990, 29, 138 and in "Dendrimers and Other Dendritic Polymers", edited by Frechet & Tomalia, John Wiley & Sons, Ltd., 2001, pp. 387-424.

[0013] Branched core-shell polymers with hydrophobic core and hydrophilic shell can be used for capturing poorly water-soluble (poorly water soluble) drugs by molecular encapsulation. Random branching and hyperbranched core-shell structures with hydrophilic core and hydrophobic shell are also used for carrying drugs by unimolecular encapsulation and preformed (pre-formed) nano micelles (U.S. Patent number 6,716,450, and Liu et al., Biomaterials 2010,10,1334-1341). However, these unimolecular and preformed micellar structures are generated in the absence of drugs.

[0014] Observe block copolymers, such as miscellaneous arm polymers (miktoarm polymers) (i.e., Y-shaped / AB2-type star polymers) and linear (A)-dendritic (B) block copolymers form stereocomplexes with paclitaxel (Nederberg et al., Biomacromolecules 2009, 10, 1460-1468 and Luo et al., Bioconjugate Chem. 2010, 21, 1216). These block copolymers are very similar to traditional lipids or AB-type linear block copolymers, which are well-known surfactants for generating micelles. However, such branched block copolymers are difficult to prepare and are therefore not suitable for large-scale production.

[0015] Water insoluble or poorly water soluble bioactive agents are difficult to formulate. Typically, multiple surfactants, detergents and other materials or complex high energy emulsification processes may be required. Large biomolecules (e.g., albumin) have been used in some formulations of water insoluble paclitaxel, such as Celgene and Bristol-Myers Squibb under their respective trademarks. However, the availability and large-scale production of such biomolecules faces major challenges.

[0016] Vaccines help the body recognize and destroy certain targets, such as microorganisms or other pathogens that cause infection. Adjuvants are often used to modify, enhance, or increase the potency or effectiveness of vaccines to provide better immunity against a specific disease. Aluminum-containing adjuvants have been used in vaccines since the 1930s. Adding small amounts of aluminum helps the body build stronger immunity to microorganisms. Monophosphoryl lipid A (MPL) (also known as "AS04") is used in vaccines in the United States. It has an immune-enhancing effect. MF59 is an adjuvant based on an oil-in-water emulsion that contains squalene (a naturally occurring oil found in many plant and animal cells as well as in the human body). MF59 adjuvant has been used in Europe since 1997 and in the United States since 2016. (An influenza vaccine licensed for use in adults 65 years of age or older). Another adjuvant, AS01B, is an adjuvant suspension used with the antigen component of the Shingrix vaccine. AS01B is made from monophosphoryl lipid A (MPL) and QS-21, a natural compound extracted from the Chilean soapbark tree (Quillaja saponaria Molina). AS01B is also a component of vaccines currently being tested in clinical trials, including malaria and HIV vaccines.

[0017] There is a continuing need for new drug formulations that can deliver drugs more effectively or improve immunogenicity (eg, vaccine efficacy) by stimulating better immunity. Summary of the invention

[0018] In some cases, the present invention relates to a pharmaceutical composition comprising: a nanoaggregate comprising a polymer and at least one bioactive agent, such as at least one stimulator of interferon genes (STING) polypeptide or a portion thereof, a nucleic acid encoding a STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof; and optionally a pharmaceutically suitable carrier; wherein the pharmaceutical composition is soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution; wherein the polymer is water soluble; and wherein the polymer comprises: a first polymer comprising at least one first terminal group modified with an H or hydrophobic portion and a second terminal group modified with a hydrophilic portion (the second The invention relates to a polymer comprising a first terminal group, wherein the first terminal group comprises 1% to 100% H and 0% to 99% hydrophobic moiety, wherein the hydrophobic moiety comprises a saturated or unsaturated aliphatic hydrocarbon, an aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons; and the second terminal group comprises a group modified by an amine, an amide, an imine, an imide, a carboxyl group, a hydroxyl group, an ester, an ether, an acetate, a phosphate, a ketone, an aldehyde, a sulfonate, or a combination thereof; a second polymer; or a combination thereof.

[0019] In some cases, the pharmaceutical composition can be a medicine for treating or preventing a disease selected from an immune disease (immunedisorder), an infectious disease and a combination thereof. In some cases, the pharmaceutical composition can be an adjuvant (Nano-Adjuvant), to enhance an immune response (e.g., for a vaccine). In some cases, the pharmaceutical composition can be a preventive vaccine, a therapeutic vaccine, or a combination thereof, wherein the pharmaceutical composition includes an adjuvant (Nano-Adjuvant) and further includes at least one immunizing agent for stimulating an immune response of a subject in need thereof.

[0020] In some cases, the present invention relates to a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject an effective dose of a pharmaceutical composition disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A-Figure 1D Examples of SBP include ( Figure 1A ) dendrimers, ( Figure 1B ) dendritic grafts, ( Figure 1C ) regular comb-branched polymers and ( Figure 1D ) Star-branched polymers. They all have a core, which is spherical or linear.

[0022] Figure 2A and Figure 2B Examples of chemical structures of symmetrically branched polypropyleneimine (PPI) dendrimers. Figure 2A : A dendrimer containing 4-PPI. Figure 2B : A dendrimer containing additional 8-PPI.

[0023] Figure 3 Examples of chemical modification reactions of symmetrically branched PPI dendrimers. The numbers 8, 16, 32, 64, 128, etc. indicate the number of reactive groups at the surface of the dendrimer.

[0024] Figure 4A and Figure 4B Random ( Figure 4A ) and regularization ( Figure 4B ) Schematic example of an asymmetrically branched polymer (ABP).

[0025] Figure 5 Examples of chemical structures of randomly asymmetrically branched PEI homopolymers.

[0026] Figure 6A-6C Examples of synthetic schemes. Fig. 6A: Chemical modification reaction of random asymmetric branched PEI homopolymer. Figure 6B : Example of a one-pot synthesis of hydrophobically modified randomly branched poly(2-ethyloxazoline) with primary amino groups at the focal point of the polymer. The initiator / surface group (I) is a brominated hydrocarbon. The reaction opens the oxazoline ring. Figure 6C : Non-limiting examples of polymers having different first and second end groups.

[0027] Fig. 7A and Figure 7B In branched polymers ( Fig. 7A )SBP and ( Figure 7B ) are illustrative schematic examples of drugs loaded in or at a surface domain or region of a hGH polypeptide. In this and other figures, R represents a surface group and the solid circles represent a bioactive agent, such as a drug of interest.

[0028] Figure 8 Schematic illustration of an example of a nanoparticle containing both a drug molecule (solid circles) and a branched polymer with surface groups (R).

[0029] Fig.9A and Fig. 9B In branched polymers ( Fig.9A )SBP and / or( Fig. 9B ) Schematic examples of water-insoluble or poorly water-soluble drugs loaded at the hydrophobic surface groups of ABP. In this figure and other figures, a thin wavy line represents a hydrophobic surface group.

[0030] Fig. 10A and Fig. 10B Various including SBP ( Fig. 10A ) and including ABP( Fig. 10B ) is a schematic example of a drug-containing nanoparticle that also carries at least one targeting group or portion, such as an antibody, represented by "Y" in this figure and other figures.

[0031] Figures 11A-11D Structural formulas of examples of STING agonists. Fig.11A :Formula (1)-(6). Fig. 11B :Formula (7)-(12). Fig. 11C :Formula (13)-(18). Fig.11D :Formula (19)-(24). Fig.11E :Formula (25)-(29).

[0032] Fig.12 Examples of receptor binding domain (RBD) antigen constructs with three RBD sequences. Linkers are not shown in AG1-AG6.

[0033] Figures 13A-13F Representative schematic illustration of examples of expression cassettes and corresponding RBD antigen fusion proteins. Fig.13A : Phylogenetic tree of some related viruses, such as coronaviruses (CoV). Fig. 13B : Examples of RBD-antigen fusion proteins. Fig. 13C : Example of an antigen with 3 RBDs with an L15 linker. Fig.13D : Example of an antigen with 3 RBDs with an L20 linker. Fig.13E : Example of an antigen with 2 RBDs. Fig.13F : Another example of an antigen with 2 RBDs.

[0034] Fig.14 Example of a representative sequence alignment of RBD sequences from various betacoronaviruses.

[0035] Figures 15A-15D Example of immune response in mice. Fig.15A : Cytokine mRNA levels 6 hours after nasal vaccination. Fig. 15B : Serum IgG levels specific to SARS-CoV-2RBD after two doses of vaccination. Fig. 15C : Serum IgG levels specific for SARS-CoV RBD after two doses of vaccination. Fig.15D : Serum IgG levels specific for Middle East Respiratory Syndrome (MERS)-CoV RBD after two doses of vaccination.

[0036] Figures 16A-16D Example of immune response of mice at day 63 after first immunization (3 doses). Fig.16A : Serum IgG levels specific to SARS-CoV-2RBD after three doses of vaccination. Fig. 16B : Serum IgA levels specific to SARS-CoV-2RBD after three doses of vaccination. Fig. 16C : BALIgA levels specific to SARS-CoV-2RBD after three doses of vaccination. Fig.16D : Levels of neutralizing antibodies specifically against SARS-CoV-2 (D614G) RBD after three doses of vaccination with different adjuvants.

[0037] Figures 17A-17D Examples of cellular immune responses in the lung and spleen following intranasal (IN) immunization. Fig.17A : Splenic IFN-γ data. Fig. 17B : Lung IFN-γ data. Fig. 17C: Splenic IL-4 data. Fig.17D : Lung IL-4 data.

[0038] Fig.18A - Figure 18D Example of comparison of dose levels and IgG immune responses for intranasal (IN) and intramuscular (IM) immunizations. Fig.18A : Serum IgG specific for SARS-CoV-2RBD. Fig.18B : Serum IgG specific for SARS-CoV RBD. Fig.18C : Serum IgG specific for MERS-CoV RBD.

[0039] Figures 19A-19D Examples of neutralizing antibodies from intranasal (IN) immunization. Fig.19A : Levels of neutralizing antibodies specific to SARS-CoV-2 (D614G strain). Fig.19B : Levels of neutralizing antibodies specific to SARS-CoV-2 (BA.5 strain). Fig.19C : Levels of neutralizing antibodies specific to SARS-CoV. Fig.19D : Levels of neutralizing antibodies specific to MERS-CoV.

[0040] Figure 20A-20C Example of measurement data of IgG immune response in monkeys (rhesus monkey, Macaca mulatta). Fig. 20A : IgG levels in serum. Fig. 20B : IgG levels in BAL. Fig. 20C : IgG levels in NAL.

[0041] Figure 21A-Figure 21C Example of measurement data of neutralizing antibodies in monkeys (rhesus macaques). Fig.21A : Levels of neutralizing antibodies specific to SARS-CoV-2 (D614G strain). Fig. 21B : Levels of neutralizing antibodies specific to SARS-CoV-2 (Delta strain). Fig. 21C : Levels of neutralizing antibodies specific to SARS-CoV-2 (BA.5 strain). Fig.21D : Levels of neutralizing antibodies specific to SARS-CoV-2 (BA.2.2 strain). Fig.21E : Levels of neutralizing antibodies specific to SARS-CoV. Fig.21F : Levels of neutralizing antibodies specific to MERS-CoV. DETAILED DESCRIPTION

[0042] By reading the following specific embodiments, it will be easier for those of ordinary skill in the art to understand the features and advantages of the present invention. It should be understood that certain features of the present invention described above and below in the context of a separate embodiment may also be provided in combination in a single embodiment. On the contrary, for the sake of brevity, the various features of the present invention described in the context of a single embodiment may also be provided separately or in any combination or sub-combination. In addition, unless the context clearly specifies otherwise, references in the singular may also include plural forms (e.g., "a" and "an" may refer to one, or one or more).

[0043] Unless expressly stated otherwise, the use of numerical values ​​within the various ranges specified in this application are expressed as approximations, as if the minimum and maximum values ​​within the ranges were modified by the word "about". In this way, slight variations above and below the stated ranges can be used to achieve substantially the same results as the values ​​within the ranges. In addition, the disclosure of ranges is intended as a continuous range including every value between and including the minimum and maximum values ​​recited.

[0044] Drug solubility in the present disclosure is defined as <30 (soluble), 30-100 (poorly soluble), and >100 (insoluble) relative to the fraction of solvent required to dissolve a portion of the bioactive agent or drug. Water solubility is defined herein as <30 (water soluble), 30-100 (poorly soluble), and >100 (water insoluble) relative to the fraction of water required to dissolve a portion of the bioactive agent or drug.

[0045] For the purposes of this disclosure, despite the presence of branches of different lengths and the random presence of the branches, randomly branched PEI is considered a homopolymer because the branched polymer consists of a single monomer, ethyleneimine or aziridine repeating units. Since A and B in the (AB) repeating unit are different monomers, polymers with a "(AB)-(AB)-(AB)..." structure can also be considered a homopolymer. Homopolymers can be linear or branched. In addition, one or more monomer or complex monomer components can be modified, substituted, derivatized, etc., for example, modified to carry functional groups. For the purposes of this disclosure, such molecules are homopolymers because the polymer backbone consists of a single type of simple monomer or complex monomer.

[0046] The term "polymer" refers to any polymer applicable to the present invention as defined above and below. In an example, the polymer may include polyoxazoline or modified polyoxazoline disclosed herein. In a further example, the polymer may include modified polyoxazoline, which may include one or more second end groups, such as -NH2, -NH, -NH3. + , other basic groups, or combinations thereof, provided that 0.01% to 100% of the second end groups do not contain primary amines. In some cases, 0.01% to 100%, 0.1% to 100%, or 1% to 100% of the second end groups do not contain primary amines. In some cases, 1% to 100% of the second end groups may include hydroxyl groups. All percentages are based on the total number of second end groups.

[0047] The term "bioactive agent" refers to a molecule, compound, complex of one or more compounds or molecules, or a combination thereof that can provide biological activity in vivo, in vitro, or in combination thereof. Pharmaceutical compositions can include one or more bioactive agents, for example, pharmaceutically active agents (PAAs) or active pharmaceutical ingredients (APIs), and other biologically active or inert compounds, which can include emollients, antioxidants (e.g., astaxanthin), bleaching agents, antiperspirants, drugs, moisturizers, fragrances, colorants, adjuvants, pigments, dyes, antioxidants, oils, fatty acids, lipids, inorganic salts, organic molecules, sunscreens, vitamins, drugs, keratolytic agents, UV blocking agents, tanning accelerators, decolorants, deodorants, perfumes, insect repellents, or a combination thereof. Some examples of bioactive agents are described in detail in the present disclosure. In some cases, the term "biologically active agent" can include a STING polypeptide, a nucleic acid encoding the STING polypeptide, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof.

[0048] The terms "pharmaceutical suitable carrier, pharmaceuticallysuitable carriers" and "pharmaceutically suitable carrier, pharmaceutically suitable carriers" refer to one or more inactive ingredients in an approved drug product. Inactive ingredients listed in the "Inactive Ingredients in Approved Drug Products" database maintained and updated by the U.S. Food and Drug Administration (FDA) may be suitable. In some cases, a pharmaceutically suitable carrier may also be referred to as an excipient.

[0049] The term "subject" used in the present disclosure refers to an animal, a human being or a human patient. The term "animal" refers to wild animals, captured or zoo-raised animals, and domesticated animals, including livestock, farm animals, pets, experimental animals, such as horses, cattle, pigs, donkeys, mules, camels, goats, sheep, monkeys, rabbits, dogs, cats, mice, rats, etc. Warm-blooded animals are suitable. The term "human" refers to a human patient suffering from one or more diseases in need of treatment, a person suffering from one or more diseases unrelated to treatment, or a healthy person. In some cases, the subject can be a human patient or a healthy person.

[0050] The terms "antibody," "antibodies," or "fragment of an antibody" may include natural or synthetic antibodies that selectively bind to an antigen. The terms include polyclonal and monoclonal antibodies produced from animals, cells (including eukaryotic or prokaryotic cells), cell-free systems, or chemical synthesis. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of those immunoglobulin molecules, as well as human or humanized versions of immunoglobulin molecules that selectively bind to a target antigen.

[0051] The term "aqueous solution" as used in the present disclosure refers to a solution comprising 80% to 100% water, the percentage being based on the total non-solid weight of the aqueous solution. The aqueous solution may further include additional components, such as salts, acids, bases, buffers, solvents, organic solvents, granules, emulsions, solids or non-solids, detergents, small molecules, macromolecules, other ingredients, or combinations thereof. The term "non-solid weight" refers to the weight of the solid content of the aqueous solution after drying (e.g., by removing all water or other liquids).

[0052] The term "infectious disease" refers to a disease caused by a harmful organism (pathogen), such as bacteria, viruses, fungi, protozoa, worms, parasites, prions, parts thereof, or combinations thereof. Infectious diseases can be spread from person to person through contact with animals, insects, or through contaminated food, water, or soil. Some examples of infectious diseases can include Chickenpox (Varicella), Coronavirus, Dengue, Diphtheria, Ebola, Influenza, Hepatitis, Hib Disease, HIV / AIDS, HPV (Human Papillomavirus), Japanese Encephalitis, Measles, Meningococcal Disease, Monkeypox, Mumps, Norovirus, Pneumococcal Disease, Polio, Rabies, Respiratory Syncytial Virus (RSV), Rotavirus, Rubella (German Measles), Herpes Zoster, Tetanus (Lockjaw), Whooping Cough (Pertussis), Zika, and other known diseases, or diseases that have not yet appeared or have not yet been identified.

[0053] The term "vaccine" refers to a substance or a group of substances that are intended to cause the immune system of a subject (e.g., a human or an animal) to respond to a microorganism (e.g., a bacterium, a virus, a fungus, a protozoa, a worm, a parasite, a prion, or other harmful organism (pathogen)). Vaccines can help the body recognize and destroy microorganisms or other pathogen cells. In some cases, vaccines may include proteins from microorganisms or other pathogen cells, nucleic acids encoding the proteins, toxins, nucleic acids, oligonucleotides; DNA; RNA; mRNA; siRNA; single guide RNA (sgRNA); or a combination thereof. In some cases, vaccines may include modified proteins, nucleic acids encoding the modified proteins, toxins, nucleic acids, modified nucleic acids, oligonucleotides, or modified oligonucleotides; DNA; RNA; mRNA; siRNA; sgRNA; or a combination thereof that are intended to cause the immune system to respond to microorganisms or other pathogens. Modified or synthetic DNA, RNA, mRNA, siRNA, sgRNA, or a combination thereof may also be suitable.

[0054] The term "adjuvant" or "adjuvants" refers to drugs, substances, biologically active agents, agents, or combinations thereof, which are used to modify, enhance or increase the potency or effectiveness of an immunogen (e.g., a vaccine) to provide better immunity against a specific antigen (e.g., a specific disease). Adjuvants may include one or more organic molecules; antigenic molecules that can mimic specific pathogen-associated molecular patterns, including liposomes, lipopolysaccharides, molecular cages for antigens, bacterial cell wall components, and endocytosed nucleic acids, such as RNA, double-stranded RNA (dsRNA), DNA, single-stranded DNA (ssDNA), DNA containing methylated or unmethylated CpG dinucleotides; inorganic compounds, such as potassium alum, aluminum hydroxide, aluminum phosphate, hydroxyapatite; oils, such as paraffin oil, propolis, peanut oil; bacterial products, such as inactivated bacteria; plant products, such as those from soybeans or other plants; cytokines, such as IL-1, IL-2, or IL-12; or combinations thereof.

[0055] The terms "isomer" and "isomers" refer to molecules that share the same chemical formula but whose atoms are connected in different ways or arranged in space in different ways, including structural isomers in which the atoms are bonded together in different orders, geometric isomers in which the atoms are bonded in the same order but with different configurations around the bonds, such as cis-isomers or trans-isomers, and enantiomers that have the same chemical structure but the three-dimensional arrangement of atoms around the asymmetric carbon is different so that they are mirror images of each other.

[0056] The terms "admix, ad-mix, admlxed, admixing," "co-admix, co-admlxed, co-admixing," or grammatical variations thereof, refer to the mixing of two or more different drug products prior to administration so that the two or more drug products are administered only once, e.g., by injection. The mixing can typically be performed in a secondary container, in which the two or more drug products can be mixed together and typically can be stored for a limited time (usually less than a few days, e.g., less than 1-3 days, less than 24 hours, less than 8 hours, or less than 4 hours) prior to administration.

[0057] The term "coformulation, coformulate, coformulating, co-formulating, co-formulated, co-formulate, co-formulation" or grammatical variations thereof refers to two or more drug substances formulated together in the same primary packaging (pills for oral dosage, e.g., vials for injection). Since the two or more drug substances are already mixed, there is no need to mix them. The two or more drug substances formulated together can be stored for a long time, e.g., 6 months, 12 months, 24 months or longer. One or more pharmaceutically acceptable excipients can be used in the coformulation.

[0058] The terms “pre-mix”, “pre-mixed”, “premix”, “premixed” may mean “admix”, “admixing”, “admixed”, “coformulate”, “coformulated”, “coformulating”, “co-formulate”, “co-formulated”, or a combination thereof.

[0059] The terms "variant", "variants thereof", "variants thereof", "variants thereof", "variants thereof", "variants of, for example, a variant or non-identical form of a pathogen or a portion thereof, are used throughout this disclosure. In some cases, the term may refer to a variant protein, DNA, or RNA of a pathogen. In some cases, the term refers to a variant of a coronavirus. In some cases, a variant may include a variant of the spike glycoprotein (S protein) or a portion thereof, a variant of the spike glycoprotein receptor binding domain (RBD) polypeptide or a portion thereof, a variant of a membrane protein (M protein) or a portion thereof, or a variant of an envelope protein (E protein) or a portion thereof of a variant coronavirus. In some cases, variants can include the variant discovered in the UK (B.1.1.7) (SARS-CoV-2α with N501Y mutation), the variant discovered in South Africa (B.1.351) (SARS-CoV-2β with K417N, E484K, and N501Y mutations), the variant discovered in Japan / Brazil (P.1) (SARS-CoV-2γ with K417T, E484K, and N501Y mutations), the variant discovered in India (SARS-CoV-2δ), the variant discovered in the United States (California) (B.1.427), the variant discovered in the United States (California) (B.1.429), the B.1.525 variant (first discovered in the UK) , B.1.526 (first found in the United States (New York)), B.1.526.1, B.1.617 (first found in India, with L452R, E484Q, D614G mutations), B.1.617.1 (first found in India, with at least 154K mutation and mutations in other positions), B.1.617.3 (first found in India, with T19R, G142D, L452R, E484Q, D614G, P681R, D950N mutations), P.2 (first found in Brazil, with at least E484K, D614G, V1176F mutations), or other variants of coronavirus derived from SARS-CoV-2, announced by the US CDC (US Centers for Disease Control and Prevention) (https: / / www.cdc.gov / coronavirus / 2019-ncov / variants / variant-info.html). In some cases, the variant may include a mutation, deletion, or combination thereof in the S protein, N protein, E protein, or M protein. In some cases, the variant may include one or more mutations at one or more of positions 417, 484, 501, 677, or 681 of the S protein, position 85 of the M protein, position 377 of the N protein, or a combination thereof. In some cases, the variant may include E501Y, E484K, or a combination thereof of the S protein.

[0060] In some instances, the disclosure relates to a pharmaceutical composition comprising:

[0061] A nanoaggregate comprising a polymer and at least one bioactive agent, wherein the bioactive agent comprises at least one STING polypeptide or a portion thereof, a nucleic acid encoding a STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof; and

[0062] Optionally, a pharmaceutically suitable carrier;

[0063] wherein the pharmaceutical composition is soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution;

[0064] wherein the polymer is water soluble; and

[0065] The polymer comprises:

[0066] A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% hydrophobic moiety, wherein the hydrophobic moiety comprises a saturated or unsaturated aliphatic hydrocarbon, aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, amide, imine, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate, or a combination thereof;

[0067] a second polymer; or

[0068] Its combination.

[0069] In some cases, the polymer may include a first polymer, as disclosed herein, comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% hydrophobic moiety, the hydrophobic moiety may include a saturated or unsaturated aliphatic hydrocarbon, aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, amide, imide, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate, or a combination thereof.

[0070] In some cases, the polymer can be composed of a first polymer, as disclosed herein, which includes at least one first end group modified with H or a hydrophobic portion and a second end group modified with a hydrophilic portion, wherein the first end group includes 1% to 100% H and 0% to 99% hydrophobic portion, the hydrophobic portion can include saturated or unsaturated aliphatic hydrocarbons, aromatic hydrocarbons, or combinations thereof having 1 to about 22 carbons, and the second end group includes a group modified by an amine, amide, imide, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate, or a combination thereof. In some cases, the pharmaceutical composition may be free of a polymer selected from: one or more hydroxy dendrimers (HD); ethylene diamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated generation-4,5,6,7,8,9,10 dendrimers, or a combination thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); poly(caprolactone) (PCL); (PPO-PEO); poly (γ-L-glutamic acid) (PGA); poly (L-phenylalanine ethyl ester) (PAE); poly (L-lysine) (PLL); methyl PEG (mPEG); poly (aspartic acid) (poly (aspartamic acid)) (PasP); poly (L-histidine) (PLH); poly (vinylamine) (PEI); poly (N-vinylpyrrolidone) (poly (N-vinylpyrrolidone), PVP); poly (L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly (hydroxybutyrate) (PHB); poly (ethylene oxide) (PEO); poly (L-glutamic acid-γ-benzyl ester) (PBLG); phosphatidylserine (PS); poly (isohexyl cyanoacrylate) (PIHCA); poly (allylamine hydrochloride) (poly (allylamine hydrochlorine), PAH); poly (γ-propargyl) (PP); and combinations thereof. In some embodiments, the first end group includes 1% to 100% H and 0% to 99% hydrophobic moiety.

[0071] In some cases, the polymer may include a second polymer comprising one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers, or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); poly(caprolactone) (PCL); (PPO-PEO); poly (γ-L-glutamic acid) (PGA); poly (L-phenylalanine ethyl ester) (PAE); poly (L-lysine) (PLL); methyl PEG (mPEG); poly (aspartic acid) (PasP); poly (L-histidine) (PLH); poly (vinylamine) (PEI); poly (N-vinyl pyrrolidone) (PVP); poly (L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly (hydroxybutyrate) (PHB); poly (ethylene oxide) (PEO); poly (L-glutamic acid-γ-benzyl ester) (PBLG); phosphatidylserine (PS); poly (isohexyl cyanoacrylate) (PIHCA); poly (allylamine hydrochloride) (PAH); poly (γ-propargyl) (PP); or a combination thereof.

[0072] In some cases, the polymer may be composed of: one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); poly(caprolactone) (PCL); (PPO-PEO); poly (γ-L-glutamic acid) (PGA); poly (L-phenylalanine ethyl ester) (PAE); poly (L-lysine) (PLL); methyl PEG (mPEG); poly (aspartic acid) (PasP); poly (L-histidine) (PLH); poly (vinylamine) (PEI); poly (N-vinylpyrrolidone) (PVP); poly (L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); polyhydroxybutyrate (PHB); poly (ethylene oxide) (PEO); poly (L-glutamic acid-γ-benzyl ester) (PBLG); phosphatidylserine (PS); poly (isohexyl cyanoacrylate) (PIHCA); poly (allylamine hydrochloride) (PAH); poly (γ-propargyl) (PP); and combinations thereof. In some cases, the polymer may include only the polymers listed above, and exclude the above-mentioned polymer including at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety.

[0073] In some cases, the polymer may include a first polymer and one or more subsequent polymers (also referred to as "second polymers") selected from one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); poly(caprolactone) (PCL); (PPO-PEO); poly (γ-L-glutamic acid) (PGA); poly (L-phenylalanine ethyl ester) (PAE); poly (L-lysine) (PLL); methyl PEG (mPEG); poly (aspartic acid) (PasP); poly (L-histidine) (PLH); poly (vinylamine) (PEI); poly (N-vinyl pyrrolidone) (PVP); poly (L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly (hydroxybutyrate) (PHB); poly (ethylene oxide) (PEO); poly (L-glutamic acid-γ-benzyl ester) (PBLG); phosphatidylserine (PS); poly (isohexyl cyanoacrylate) (PIHCA); poly (allylamine hydrochloride) (PAH); poly (γ-propargyl) (PP); and combinations thereof.

[0074] In some cases, polymer can include polyoxazoline (POX), it includes linear part, branched part or its combination, and wherein said polyoxazoline (POX) can include poly-(2-methyloxazoline), poly-(2-ethyloxazoline) and poly-(2-propyloxazoline), or its combination.In some cases, polyoxazoline can be poly-(2-ethyloxazoline) (PEOX).

[0075] In some cases, the polyoxazoline can include a molar ratio of monomer to initiator in a range of 50:1 to 80:1.

[0076] In some cases, 1% to 100% of the second end groups do not contain primary amines. In some cases, in the pharmaceutical compositions disclosed herein, 1% to 100% of the second end groups may include hydroxyl groups. All percentages are based on the total number of second end groups.

[0077] In some cases, the first end group includes 1% to 100% H and 0% to 99% hydrophobic moiety, 1% to 99% H and 1% to 99% hydrophobic moiety, 1% to 90% H and 10% to 99% hydrophobic moiety, 1% to 85% H and 15% to 99% hydrophobic moiety, 1% to 80% H and 20% to 99% hydrophobic moiety, 1% to 75% H and 25% to 99% hydrophobic moiety, 1% to 70% H and 30% to 99% hydrophobic moiety, 1% to 65% H and 35% to 99% hydrophobic moiety, 1% to 60% H and 40% to 99% hydrophobic moiety, 1% to 55% H and 45% to 99% hydrophobic moiety, 1% to 50% H and 50% to 99% hydrophobic moiety. The invention also provides a range of H and 99% hydrophobic moieties, including all percentages within the stated ranges, wherein the percentages are based on the total number of first end groups in the polymer. In some cases, the first end group includes 1% to 50% H and 50% to 99% hydrophobic moieties, 1% to 40% H and 60% to 99% hydrophobic moieties, 1% to 30% H and 70% to 99% hydrophobic moieties, 1% to 20% H and 80% to 99% hydrophobic moieties, 1% to 10% H and 90% to 99% hydrophobic moieties, 1% to 5% H and 95% to 99% hydrophobic moieties, or 1% to 2% H and 98% to 99% hydrophobic moieties, including all percentages within the range, the percentages are based on the total number of first end groups in the polymer. In some cases, the percentages are based on the moles of first end groups in the polymer.

[0078] Alternatively, the polymer can be described using a ratio of H to hydrocarbon groups ("hydrocarbons"), such as H:hydrocarbons = 0.01:1 to 100: 1. In some cases, the first end group includes a ratio of H to hydrophobic moieties in the range of 0.01:1 to 100:1, including all ratios within the range. In some cases, the first end group comprises a ratio of H: hydrophobic moiety in the range of 0.01:1 to 100:1, 0.1:1 to 100:1, 0.2:1 to 100:1, 0.5:1 to 100:1, 0.7:1 to 100:1, 1:1 to 100:1, 2.0:1 to 100:1, 5:1 to 100:1, 10:1 to 100:1, 20:1 to 100:1, 30:1 to 100:1, 40:1 to 100:1, 50:1 to 100:1, 60:1 to 100:1, 70:1 to 100:1, 80:1 to 100:1, 90:1 to 100:1, 95:1 to 100:1, including all ratios within the ranges. In some cases, the first end group comprises a ratio of H: hydrophobic moiety in the range of 0.01:1 to 10:1, 0.1:1 to 10:1, 0.1:1 to 10:1, 0.2:1 to 10:1, 0.5:1 to 10:1, 0.7:1 to 10:1, 1:1 to 10:1, 2.0:1 to 10:1, 5:1 to 10:1, 10:1, 20:1 to 10:1, 30:1 to 10:1, 40:1 to 10:1, 50:1 to 10:1, 60:1 to 10:1, 70:1 to 10:1, 80:1 to 10:1, 90:1 to 10:1, 95:1 to 10:1, including all ratios within the ranges. In some cases, the first end group includes a ratio of H to hydrophobic moiety in the range of 0.01:1 to 5:1, 0.1:1 to 5:1, 0.1:1 to 5:1, 0.2:1 to 5:1, 0.5:1 to 5:1, 0.7:1 to 5:1, 1:1 to 5:1, 2.0:1 to 5:1, 5:1, 10:1, 20:1 to 5:1, 30:1 to 5:1, 40:1 to 5:1, 50:1 to 5:1, 60:1 to 5:1, 70:1 to 5:1, 80:1 to 5:1, 90:1 to 5:1, 95:1 to 5:1, including all ratios within the range. In some cases, the first end group includes a ratio of H: hydrophobic moiety selected from 0.01: 1, 0.1: 1, 0.2: 1, 0.5: 1, 0.7: 1, 1: 1, 2.0: 1, 3.0: 1, 4.0: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 95: 1 and 100: 1, including ratios about those ratios. The ratio can be based on the molar ratio of H to the hydrocarbon group.

[0079] Percentages and ratios can be converted using conventional methods, for example, a ratio of 0.01:1 can be converted to approximately 1%, 0.2:1 can be converted to approximately 17%, 0.5:1 can be converted to approximately 33%, 1:1 can be converted to approximately 50%, 1.5:1 can be converted to approximately 60%, 2:1 can be converted to approximately 67%, 5:1 can be converted to approximately 83%, 10:1 can be converted to approximately 90%, 20:1 can be converted to approximately 95%, and 100:1 can be converted to approximately 99%.

[0080] The percentage or ratio of the hydrogen-modified first end group and the hydrocarbon-modified first end group can be measured using HPLC known to those skilled in the art.

[0081] In the pharmaceutical compositions disclosed herein, the first end group may include H or a hydrophobic portion, which may include a saturated or unsaturated aliphatic hydrocarbon, an aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group may include a group modified by an amine, an amide, an imine, an imide, a carboxyl group, a hydroxyl group, an ester, an ether, an acetate, a phosphate, a ketone, an aldehyde, a sulfonate, or a combination thereof.

[0082] In one example, the first end group may include hydrogen (H), in one example, a hydrocarbon having 2 to 22 carbons, in another example, 4 to 22 carbons, in yet another example, 6 to 22 carbons, in yet another example, 7 to 22 carbons, in yet another example, 8 to 22 carbons, in yet another example, 10 to 22 carbons, in yet another example, 12 to 22 carbons, in yet another example, 14 to 22 carbons, in yet another example, 16 to 22 carbons, and in a further example, 18 to 22 carbons. In a specific example, the first end group may include 18 carbons, for example, (CH3(CH2) 17 )-group. In some cases, the first end group may include a hydrocarbon having 7 to 22 carbons. In some cases, the first end group may include H. In some cases, the first end group may include 1% to 100% H and 0% to 99% of a hydrophobic portion, the hydrophobic portion may include a saturated or unsaturated aliphatic hydrocarbon, an aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons. In some embodiments, the first end group includes 1% to 100% H and 0% to 99% of a hydrophobic portion. The first end group may be modified by selecting various initiators. In some cases, p-toluenesulfonic acid, trifluoroacetic acid, methyl toluenesulfonate, HCl, HBr, HI, H-Br, hydrocarbon-Br, such as C1 to C 22 -Br, or a combination thereof may be used as an initiator. The polymers prepared herein may be mixed together in a predetermined ratio.

[0083] Initiator can include hydrophobic electrophilic molecules, including hydrocarbons, aliphatic hydrocarbons, aromatic hydrocarbons or combinations thereof, and halide functional groups, such as alkyl halides, aralkyl halides, acyl halides or combinations thereof. The example of such compounds can include monofunctional initiators, such as hydrocarbons containing 1 to about 22 carbons with saturated or unsaturated chemical bonds, such as methyl iodide / bromide / chloride, ethyl iodide / bromide / chloride, 1-iodine / bromine / chlorobutane, 1-iodine / bromine / chlorohexane, 1-iodine / bromine / chlorododecane, 1-iodine / bromine / chlorooctadecane, benzyl iodide / bromide / chloride etc. Other initiators can include allyl bromide / chloride. Acyl halides can also be used, such as acyl bromides / acyl chlorides, benzoyl bromides / chlorides and compounds containing tosyl, such as p-toluenesulfonic acid (p-toluenesulfonic acid), methyl toluenesulfonate (methyl tosylate) and other toluenesulfonic acid esters. Any one or more initiators can be used in combination. In some cases, the initiator can also include a hydrophilic portion including a proton / H-containing molecule, such as p-toluenesulfonic acid, trifluoroacetic acid, methyl toluenesulfonate, HCl, HBr, HI, or a combination thereof.

[0084] During the polymerization process, an initiator may be used to start the polymerization. When used, various molar ratios of monomer to initiator may be used to obtain a particular polymer. A particular polymer may have different properties, such as molecular weight, size of branching, and other properties, including those unexpectedly discovered by the applicant as disclosed herein. In some cases, a suitable molar ratio of monomer to initiator may be from 20:1 to 100:1, including any and all ratios within the range, for example, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, and 100:1, including 20; 1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49: 1, 50:1, 51:1, 52:1, 53:1, 54; 1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1. 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78: 1,79:1,80:1,81:1,82:1,83:1,84:1,85:1,86:1,87:1,88:1,89:1,90:1,91:1,92:1,93:1,94:1,95:1,96:1,97:1,98:1,99:1,100:1 etc., this means to use the mol ratio of the monomer in the above-mentioned scope and initiator to prepare selected polymer.In some cases, poly-oxazoline disclosed herein can be included in the mol ratio of the monomer in the scope of 50:1 to 80:1 and initiator, this means that the mol ratio (comprising any and all ratios in the described scope) of the monomer in the scope of 50:1 to 80:1 and initiator can be used for producing selected polymer.

[0085] The polymers can be prepared using the monomers and initiators described herein and in PCT Publication No. WO 2014 / 123791, the entire contents of which are incorporated herein by reference.

[0086] A hydrogen-modified randomly branched PEOX polymer having a certain monomer to initiator molar ratio in the range of 20:1 to 100:1 can be prepared as described above using an initiator selected from a hydrophilic portion including a proton / H containing molecule, such as p-toluenesulfonic acid, trifluoroacetic acid, methyl toluenesulfonate, HCl, HBr, HI, or a combination thereof.

[0087] Hydrocarbons C1 to (CH3(CH2)2 can be prepared using initiators as described above with a monomer to initiator molar ratio ranging from 20:1 to 100:1. 21 )-modified randomly branched PEOX polymer, wherein the initiator is selected from CH3-Br, (CH3(CH2))-Br, (CH3(CH2)2)-Br, (CH3(CH2)3)-Br, (CH3(CH2)4)-Br, (CH3(CH2)5)-Br, (CH3(CH2)6)-Br, (CH3(CH2)7)-Br, (CH3(CH2)8)-Br, (CH3(CH2)9)-Br, (CH3(CH2) 10 )-Br, (CH3(CH2) 12 )-Br, (CH3(CH2) 12 )-Br, (CH3(CH2) 13 )-Br, (CH3(CH2) 14 )-Br, (CH3(CH2) 15 )-Br, (CH3(CH2) 16 )-Br, (CH3(CH2) 17 )-Br, (CH3(CH2) 18 )-Br, (CH3(CH2) 19 )-Br, (CH3(CH2) 20 )-Br and (CH3(CH2) 21 )-Br. Mixtures of initiators may also be suitable.

[0088] In some cases, the hydrogen-modified randomly branched PEOX polymer and the hydrocarbon C1 to (CH3(CH2) prepared above can be mixed in a predetermined ratio. 21 )-modified randomly branched PEOX polymers to prepare hydrocarbon mixtures (e.g., C1 to (CH3(CH2) 21 )-modified first end group and H modified first end group). In some cases, the polymer may include 1% to 99% hydrocarbons, such as C1 to (CH3(CH2) 21 )-modified first end groups, and 1% to 99% H-modified first end groups.

[0089] In some cases, the first end group may include H and C1 to C2 in a ratio ranging from 0.01:1 to 100:1. 22 The hydrophobic part of a hydrocarbon (e.g. (CH3(CH2) 17 In some cases, the first end group may include H and C1 to C2 in a ratio ranging from 0.1:1 to 5:1. 22 The hydrophobic part of a hydrocarbon (e.g. (CH3(CH2) 17 )-).

[0090] Hydrocarbons including PEOX CH3(CH2) 17 A polymer having a mixture of a )-modified first terminal and a H-modified first terminal may be referred to as a "H / C 18 Polymers with specific initiator molar ratios (e.g., 60:1, 70:1, 80:1, etc.) can be referred to as "H / C 18 PEOXABP60", "H / C 18 PEOXABP70","H / C 18 PEOXABP80” etc.

[0091] The polymers disclosed above and below may be suitable and may include linear polymers, branched polymers, symmetrically branched polymers, asymmetrically branched polymers, dendritic polymers, dendritic graft polymers, comb-branched polymers, star-branched polymers, or combinations thereof. The polymers are water soluble. In an example, the polymers may be dissolved in water to produce an aqueous solution of 12% by weight or more.

[0092] The second end group can include ammonia, ammonia derivatives, ethylenediamine (EDA), ethylenediamine derivatives, piperazine, piperazine derivatives, tris (2-aminoethyl) amine, 4- (aminomethyl) piperidine, 1,3-diaminopropane, 2,2'- (ethylenedioxy) bis (ethylamine), diethylenetriamine, 1,4,7,10- tetraazacyclododecane, hexamethylenediamine, triethylenetetramine, 1,8- diaminooctane or a group modified by its combination. In another example, the second end group can include a group modified by ethylenediamine (EDA), ethylenediamine derivatives or a group modified by its combination. Any derivative of ethylenediamine disclosed herein is suitable. The polymer can have a reaction challenge molar ratio (reaction challenge molar ratio) (for example, polyoxazoline reactive chain end (reactive chain end) and EDA) in the range of 1:1 to 1:100. In one example, the polymer can have the reaction excitation mol ratio of the reactive chain ends of polyoxazoline and EDA in the range of 1:1 to 1:100, 1:2 to 1:100 in another example, 1:2 to 1:50 in another example, 1:2 to 1:40 in another example, 1:2 to 1:30 in a further example, 1:2 to 1:20 in another example, 1:2 to 1:15 in another example, and 1:5 to 1:15 in a further example. In a further example, polymer can have the reaction excitation mol ratio of the reactive chain ends of polyoxazoline and EDA of about 1:10. The polyoxazoline modified by EDA disclosed herein can provide the functional group that the polymer charge as disclosed herein has pH dependency variation. In some cases, pharmaceutical composition disclosed herein can include polymer, and the polymer can have the mol ratio of the reactive chain ends of polyoxazoline and EDA of about 1:10. In some cases, 1% to 99%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 1% to 5%, 1% to 4%, 1% to 3%, 1% to 2% of the second end groups may include EDA-modified groups. In some cases, 1% to 99%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 1% to 5%, 1% to 4%, 1% to 3%, 1% to 2% of the second end groups may include primary amines.

[0093] In some cases, the second end group can include a group modified by an amine, amide, imine, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate, or a combination thereof, provided that 0.01% to 100%, 0.1% to 100%, 1% to 100%, 5% to 100%, 10% to 100%, 15% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 99% to 100% of the second end groups may be free of primary amines. In some cases, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the second end groups may not contain primary amines. In some cases, about 50% to 100% of the second end groups may not contain primary amines. In some cases, about 75% to 100% of the second end groups may not contain primary amines. In some cases, about 90% to 100% of the second end groups may not contain primary amines.

[0094] In some cases, 100% of the second end groups of the polymer may include a hydroxyl-modified group. In some cases, as disclosed herein, CH3(CH2) 17 -Br can be used as the initiator for the polymerization of 2-ethyloxazoline by the cationic ring-opening process, to generate randomly branched polymers, then, for example, the randomly branched polymers are dissolved in water, to produce the second end modified by hydroxyl. In some cases, initiator is selected from the initiator comprising a hydrophilic part (it comprises a molecule containing proton / H), for example tosic acid, trifluoroacetic acid, methyl toluenesulfonate, HCl, HBr, HI or its combination, and the initiator can be used as the initiator for the polymerization of 2-ethyloxazoline by the cationic ring-opening process, to generate randomly branched polymers, subsequently, for example, the randomly branched polymers are dissolved in water, to produce the second end modified by hydroxyl. In some cases, about 100% of the second end group can include hydroxyl. In some cases, about 100% of the second end group can not contain primary amine.

[0095] The pharmaceutical composition may have a pH value in the range of about 3.0 to about 10.0. In some cases, the pharmaceutical composition may have a pH value in the range of about 7.0 to about 9.0. In some cases, the pharmaceutical composition may have a pH value in the range of about 7.0 to about 8.0. In some cases, the pharmaceutical composition may have a pH value in the range of about 7.0 to about 7.5. In some cases, the pharmaceutical composition may have a pH value in the range of about 3.0 to about 6.9, or about 4.0 to about 6.9. In some cases, the pharmaceutical composition may have a pH value in the range of about 4.0 to about 7.0. In some cases, the pharmaceutical composition may have a pH value in the range of about 4.0 to about 7.5. In some cases, the pharmaceutical composition may have a pH value in the range of about 5.6 to about 6.9. The pharmaceutical composition may be adjusted with an acid or a base to reach the desired pH range. An acid (e.g., HCl or other acid) may be suitable. A base (e.g., NaOH or other base) may be suitable.

[0096] In some cases, the pharmaceutical composition may have a pH value in the range of about 3.0 to about 10.0, and wherein 1% to 100% of the second end groups do not contain primary amines. In some cases, the pharmaceutical composition may have a pH value in the range of about 5.6 to about 6.9, and wherein about 100% of the second end groups do not contain primary amines, i.e., 0% of the second end groups contain primary amines. In some cases, 1% to 100% of the second end groups may include hydroxyl groups, the percentage being based on the total number of second end groups.

[0097] Polymer H / C having a hydroxyl group as the second end group 18 PEOXABP can be referred to as H / C 18 PEOXABP-OH. Polymers with specific initiator molar ratios (e.g., 60:1, 70:1, 80:1, etc.) can be referred to as "H / C 18 PEOXABP60-OH", "H / C 18 PEOXABP70-OH", "H / C 18 PEOXABP80-OH" etc. Polymers having an amine group as a second end group H / C 18 PEOXABP can be referred to as H / C 18 PEOXABP-NH2. Polymers with specific initiator molar ratios (e.g., 60:1, 70:1, 80:1, etc.) can be referred to as "H / C 18 PEOXABP60-NH2", "H / C 18 PEOXABP70-NH2", "H / C 18 PEOXABP80-NH2", etc. In some cases, mixtures of the polymers disclosed herein may be suitable.

[0098] In any of the pharmaceutical compositions disclosed above and below, the polymer may include a polyoxazoline (POX) comprising a linear portion, a branched portion, or a combination thereof. In one example, the polymer may include a plurality of linear portions linked together, in another example, one or more linear portions are linked to one or more branched portions, in yet another example, one or more branched portions are linked together, for example, Figure 1A-Figure 10B Each linear portion can independently have various lengths, modifications, or combinations thereof. Each branched portion can independently have various lengths, number of branches, modifications, or combinations thereof.

[0099] Polyoxazoline (POX) can include poly-(2-oxazoline), poly-(2-substituted oxazoline), it includes poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline), poly-(isopropyloxazoline) (PiPOX), or its combination. In an example, POX can include poly-(2-methyloxazoline) (PMOX), in another example, include poly-(2-ethyloxazoline) (PEOX), in another example, include poly-(2-propyloxazoline) (PPOX), in another example, include poly-(isopropyloxazoline) (PiPOX), or in further example, include the combination of two or more poly-(2-substituted oxazoline), wherein the two or more poly-(2-substituted oxazoline) can be the repeating unit in the polyoxazoline polymer, also referred to as composite monomer. Polyoxazoline (POX) is hydrophilic. The polyoxazoline (POX) may be free of monomers having hydrophobic side chains, whether simple or complex, such as monomers having 4 or more carbons (C4 and above).

[0100] Figure 1A-Figure 1D and Figure 2A-2B Some examples of symmetrically branched polymers (SBPs) with symmetrical branches are schematically depicted in , where all homopolymers of interest have one core and present symmetrical branching junctions consisting of terminal branches or chain branches throughout the homopolymer. The functional groups are primarily present on the exterior of the polymer.

[0101] Modified SBPs can be obtained, for example, by chemically attaching functional groups to, for example, symmetrically branched PAMAM or PPI dendrimers (commercially available from Aldrich), polyether dendrimers, polyester dendrimers, comb-branched / star-branched polymers, for example, those containing PEO, PEG, PMOX or PEOX; polystyrene, and comb-branched dendritic grafts, for example, those containing PEOX, PMOX or PEI. Synthetic procedures for preparing such SBPs / dendrimers are known, as described above and below.

[0102] In some cases, the higher branching density of SBPs can make the polymers compact at the molecular level with well-defined internal void spaces, which makes such molecules suitable as carriers for water-insoluble or poorly water-soluble drugs, for example, one or more bioactive agents entrapped or encapsulated therein.

[0103] Surface modification can enhance the performance and use of the resulting modified SBP. For example, a water-insoluble SBP can be rendered water-soluble by appropriate modification, while an SBP with a high charge density can be modified to carry a low charge or no charge on or on the polymer surface. On the other hand, a water-soluble SBP can be modified with hydrophobic surface groups to enhance the ability to dissolve water-insoluble or weakly water-soluble drugs on or in its surface. Modification can occur at any position of the polymer, for example, at the end, side chain, backbone residue, etc.

[0104] In one embodiment of the present disclosure, SBPs (e.g., symmetrically branched PEI dendrimers, PPI dendrimers, PAMAM dendrimers, or symmetrically branched PEI dendritic grafts) can be modified by using different kinds of, for example, primary amine groups, for example, by Michael addition or by adding acrylates to the amine groups of the homopolymers. Thus, for example, by Michael addition, methyl acrylate can be introduced to the primary and / or secondary amino groups of PEI, PPI, and polylysine (PLL) homopolymers. The ester groups can then be further derivatized, for example, by an amidation reaction. Thus, such an amidation reaction using, for example, ethylenediamine (EDA) can achieve the addition of amino groups at the ends of the newly formed branches. Other modifications of the homopolymers can be made using known chemicals, for example, as provided in "Poly (amines) and Poly (ammonium salts), "in "Handbook of Polymer Synthesis," (Part A), Kricheldorf ed., New York, Marcel Dekker, 1994; and "Dendrimers and Other Dendritic Polymers" Frechet & Tomalia, eds., John Wiley & Sons, Ltd., 2001. Derivatives of EDA can also be used and include any molecular entity including reactive EDA, substituted EDA, or other members of the polyethyleneamine family, such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, etc., including polyethyleneamine, tetramethylethylenediamine, etc. Amidation reactions using, for example, ethylenediamine (EDA) can also modify the polymer charge density at the ends of the newly formed branches. As disclosed herein, the charge of polymers having such amidation groups can have a pH-dependent change, resulting in a pH-dependent change in polymer charge density.

[0105] In certain embodiments, the modification may include a portion that contributes to or enhances the hydrophobicity of a polymer or portion thereof. For example, a hydrophobic functional group, such as an aliphatic chain comprising a hydrocarbon chain of 1 to about 22 carbons (which may be saturated or unsaturated, linear, cyclic or branched, an aromatic structure (e.g., containing one or more aromatic rings, which may be fused rings), or a combination thereof, may be used as a modifier and added to the polymer by implementing the chemical methods provided herein as taught herein. By such addition, a modified SBP, such as a modified PEI, PPI, PAMAM dendrimer, or PEI dendrimer graft may be formed. An example of a PAMAM modified PPI dendrimer is Figure 3 As shown. As an extension of SBP (such as PPI and PEI), the modified SBP thus obtained is also symmetrically branched. Depending on the solvent environment (i.e. pH or polarity), the surface functional groups can carry different charges and / or charge densities, and / or hydrophobic groups. Then, the molecular shape and surface functional group positions (i.e. surface functional group backfolding) can be further adjusted based on these characteristic properties.

[0106] In another embodiment of the present disclosure, any of a variety of synthetic procedures can be used to prepare modified SBP, such as a synthetic scheme known to be easy to react with a suitable site on a homopolymer. In addition, any of a variety of reagents can be used in the selected synthetic scheme to achieve any of the various modifications or additions to the homopolymer backbone. Therefore, for example, in the case of the Michael addition reaction of the above-mentioned amine, any of the additions of various substituents can be used, for example, in the alkylation stage, for example, using any of a variety of acrylate reagents, for example, acrylates including hydrocarbon substituents, for example, including saturated or unsaturated hydrocarbons of 1 to about 22 carbons, which can be substituted, aliphatic, aromatic, cyclic, saturated at one or more bonds, or a combination thereof. Therefore, suitable reactants include methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate, octyl acrylate, nonyl acrylate, decyl acrylate, undecyl acrylate, dodecyl acrylate, etc., and mixtures thereof. Similarly, in the amidation stage in the examples illustrated above, any of a variety of amines can be used. For example, EDA, monoethanolamine, tris(hydroxymethyl)aminomethane, alkylamines, allylamines, or any amino-modified polymer, including those comprising PEG, PEO, perfluoropolymers, polystyrene, polyethylene, polydimethylsiloxane, polyacrylates, polymethylmethacrylate, etc., and mixtures thereof, may be used.

[0107] This synthetic strategy not only allows for the symmetrical growth of the molecule (where more branches with different chemical compositions can be introduced), but also allows for the addition of multiple functional groups to the exterior of the polymer structure. The precursor homopolymer can be modified successively using the same or different synthetic methods until the desired SBP with the appropriate molecular weight and functional groups is obtained. In addition, the hydrophobic and hydrophilic properties and charge density of such polymers can be customized to suit specific application requirements using suitable monomers and suitable modification reactions used to construct the homopolymer.

[0108] In another embodiment of the present disclosure, if a divergent synthesis method is used, the chain ends of symmetrical star-branched or comb-branched homopolymers (e.g., poly(2-oxazoline) or poly(2-substituted oxazolines), including, for example, poly(2-methyloxazoline), poly(2-ethyloxazoline), poly(2-propyloxazoline), and poly(2-butyloxazoline, etc.), PEI, PEO / ethylene glycol, polyvinylpyrrolidone (PVP), polyphosphate, polyvinyl alcohol (PVA) or polystyrene chain ends) can be modified using another small molecule or polymer to generate various functional groups at the ends of the homopolymer chains, including primary, secondary or tertiary amines, carboxylates, hydroxyls, aliphatics (e.g., hydrocarbon chains), aromatics, fluoroalkyls, aryls, PEGs, PEOs, acetates, amides and / or esters. Alternatively, if a convergent synthesis approach is employed, various initiators can be used to introduce the same type of functional groups at the chain ends ("Dendritic Molecules," Newkome et al., eds., VCH, Weinheim, 1996; "Dendrimers and Other Dendritic Polymers," Frechet & Tomalia, eds., John Wiley & Sons, Ltd., 2001; and J. Macromol. Sci. Chem. A9(5), pp. 703-727 (1975)).

[0109] Some examples of asymmetrically branched polymers (ABPs) having asymmetrical branches are described in Figure 4A-4BSchematically depicted in , some of the target polymers lack a core and present asymmetric branch junctions consisting of chain branches and terminal branches throughout the homopolymer. Junctional groups are typically present both externally and internally. However, when larger functional groups (e.g., larger hydrophobic or hydrophilic groups) are used, the functional groups may typically be preferentially and possibly necessarily attached to the exterior of the ABP, for example, possibly due to steric effects. Therefore, such surface-modified branched polymers (MBPs) can be used to solubilize water-insoluble or weakly water-soluble drugs or to form nanoaggregates therewith.

[0110] For example, functional groups can be chemically attached to a regular ABP such as polylysine (e.g., branched PLL), a random ABP such as PEI (available from Aldrich, Polysciences, or BASF under the trade name Modified ABPs can be obtained by using polyoxazolines (commercially available) or polyoxazolines, which can be prepared according to the procedure of Litt (J. Macromol. Sci. Chem. A9 (5), pp. 703-727 (1975)). Other ABPs can include, but are not limited to, polyacrylamide, polyphosphate, PVP, PVA, etc. Random asymmetric branched PEI can be prepared mainly by cationic ring-opening polymerization of ring-strained cyclicimine monomers (e.g., aziridine (ethyleneimine) and azetidine (propyleneimine)), with Lewis or Bronsted acid as initiator (Dernier et al., "Ethylenediamine and Other Aziridines," Academic Press, New York, (1969); and Pell, J. Chem. Soc. 71 (1959)). Since many methods are essentially one-pot processes, large amounts of random ABPs can be produced.

[0111] The synthetic methods for preparing ABPs generally generate various branching junctions within the macromolecule. In other words, the terminal and chain branching junctions are mixed and distributed throughout the molecular structure. Compared with dendritic polymers and dendritic grafts, random ABPs can have a lower branching density and a more open molecular structure. Although the branching pattern is random, the average ratio of primary, secondary, and tertiary amine groups can be relatively consistent with a ratio of about 1:2:1, such as described in: Dick et al., J. Macromol. Sci. Chem., A4 (6), 1301-1314 (1970) and Lukovkin, Eur. Polym. J. 9, 559 (1973). In one example, the polymers disclosed herein can include primary, secondary, and tertiary amine groups in a ratio of about 1:2:1.

[0112] The presence of branching junctions can make random ABPs (e.g., asymmetrically branched PEI) form macromolecules with possible spherical, ovoid, or similar configurations. In the spherical structure, there are pockets of various sizes formed by incomplete branching junctions inside the macromolecule. Unlike dendritic polymers and dendritic grafts, whose internal pockets are always located around the central core of the molecule, the pockets of random ABPs are unevenly distributed throughout the molecule. Therefore, random ABPs have both external functional groups and unevenly distributed internal functional groups, which can further react with various molecules to form new macromolecular structures-a kind of modified random ABP.

[0113] Although there is a core, the functional groups of regular ABPs can also be distributed on the outside and inside, which is very similar to random ABPs. One such homopolymer is PLL, which can be prepared as described in the following documents: U.S. Patent Nos. 4,289,872; 4,360,646 and 4,410,688. As taught herein and known in the art, such homopolymers can also be modified in a manner similar to random ABPs.

[0114] In one embodiment of the present disclosure, an ABP (e.g., a randomly asymmetrically branched PEI or a regularly asymmetrically branched PLL) is modified by, for example, adding acrylates to the amine groups of a polymer (e.g., PEI and PLL homopolymers) using different types of primary and / or secondary amines, for example, by Michael addition. The ester groups can then be further derivatized, for example, by an amidation reaction. Thus, for example, such an amidation reaction using, for example, EDA can produce the addition of amino groups at the ends of the newly formed branches. Other modifications of the polymer can be made using known chemistries, for example, as provided in the above-mentioned "Poly (amines) and Poly (ammonium salts)". By such additions, a modified ABP, such as a modified PEI or PLL homopolymer, is formed. As an extension of an ABP (e.g., PEI and PLL), the resulting modified ABP is also asymmetrically branched. Depending on the solvent environment (i.e., pH or polarity), the surface functional groups can carry different charges and charge densities. The molecular shape and functional group positions (i.e., functional group inverse folding) can then be further adjusted based on these characteristic properties.

[0115] In another embodiment, any of a variety of synthetic schemes can be used to prepare the modified ABP, for example, a synthetic scheme known to readily react with suitable sites on a homopolymer. In addition, any of a variety of reagents can be used in the selected synthetic scheme to achieve any of a variety of modifications or additions to the polymer backbone. Thus, for example, in the case of the Michael addition reaction of the amine described above, the addition of any of a variety of substituents can be used in the alkylation stage, as provided above, for example, using an acrylate, which can include a saturated or unsaturated hydrocarbon, such as a hydrocarbon comprising 1 carbon to about 22 carbons, which can be aliphatic, branched, saturated, aromatic, cyclic, or a combination thereof. In one example, the hydrocarbon can have 2 to 22 carbons, in another example 4 to 22 carbons, in yet another example 6 to 22 carbons, in yet another example 7 to 22 carbons, in yet another example 8 to 22 carbons, in yet another example 10 to 22 carbons, in yet another example 12 to 22 carbons, in yet another example 14 to 22 carbons, in yet another example 16 to 22 carbons, in a further example 18 to 22 carbons, and in yet another example 20 to 22 carbons. In a specific example, the first end group can include 18 carbons, such as (CH3(CH2) 17)-group. Suitable reactants include methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate, octyl acrylate, nonyl acrylate, decyl acrylate, undecyl acrylate, dodecyl acrylate, etc., and mixtures thereof. Similarly, in the amidation stage in the examples exemplified above, any of the various amines can be used in the methods provided herein and known in the art. For example, EDA, monoethanolamine, tris(hydroxymethyl)aminomethane, alkylamine, allylamine or any amino-modified polymer can be used, including PEG, perfluoropolymers, polystyrene, polyethylene, polydimethylsiloxane, polyacrylate, polymethyl methacrylate, etc., and mixtures thereof. In addition, hydrophobic groups (including aliphatic groups (e.g., C1 to about C1) and hydrophobic groups (including aliphatic groups (e.g., C1 to about C1)) can be used. 22 The attachment of modified ABPs to the modified ABPs may be achieved by using reactions such as epoxidation, amidation, Michael addition (including reaction with maleimide using -SH or -NH2 groups), aldehyde / keto-amine / hydrazide coupling, iodine / iodoacetyl-SH coupling, hydroxylamine-aldehyde / ketone coupling, etc. Such synthetic strategies not only allow asymmetric growth of the molecule (introducing more pockets), but also allow the addition of multiple functional groups inside and outside the structure. The homopolymers may be further modified using the same or different synthetic methods until the desired ABPs with appropriate molecular weight and functional groups are obtained. In addition, the hydrophobic and hydrophilic properties and charge density of such homopolymers may be tailored to suit specific application requirements using suitable monomers and suitable modification reactions used to construct the homopolymers. An example of a modified ABP is Figure 5 A modified hyperbranched PEI such as Fig. 6A shown.

[0116] In another embodiment of the present disclosure, the focus of random ABP (e.g., POX) (merged from various reactive chain ends during convergent synthesis) can be capped or reacted with another small molecule to generate various functional groups at the homopolymer chain ends, including primary, secondary or tertiary amines, carboxylates, hydroxyls, alkyls, fluoroalkyls, aryls, PEGs, acetates, amides and / or esters. Alternatively, various initiators can be used to introduce the same type of functional groups at the surface groups where polymerization begins during convergent synthesis (J. Macromol. Sci. Chem. A9 (5), pp. 703-727 (1975)).

[0117] The Litt and Warakomski procedure described above can be used to prepare randomly branched poly(2-ethyloxazoline) surface-modified with alkyl groups having primary amine groups at the focal point of the branched polymer. For example, CH3(CH2) 17 -Br can be used as an initiator for the polymerization of 2-ethyloxazoline via a cationic ring-opening process to generate randomly branched polymers, which are then quenched with N-tert-butyloxycarbonylpiperazine (N-Boc-piperazine) or EDA. End-capping with a large excess of EDA can allow the hydrophobically modified branched poly(2-ethyloxazoline) polymers to be functionalized with primary amine groups at the focal point ( Figure 6B ). Alternatively, the N-Boc-piperazine-terminated hydrophobically modified branched poly(2-ethyloxazoline) polymer can also be deprotected to generate free amino groups at the focal point. In some cases, the polymer can include a modified branched poly(2-ethyloxazoline) functionalized with a primary, secondary or tertiary amine, carboxylate, hydroxyl, alkyl, fluoroalkyl, aryl, PEG, acetate, amide or ester group at the focal point of the polymer, wherein two or more reactive chain ends are combined during convergent synthesis.

[0118] In some cases, the alkyl surface-modified randomly branched poly(2-ethyloxazoline) can have hydroxyl groups at the focal point of the branched polymer. For example, the focal point of the polymer can be hydrolyzed to, for example, hydroxyl groups when dissolved in water (e.g., containing, for example, 1N Na2CO3).

[0119] In some cases, the focal point of a polymer described herein can include a second end group modified with a hydrophilic moiety.

[0120] The introduction of primary amine groups into hydrophobically modified branched poly(2-oxazoline) homopolymers can enhance drug solubility and generate bioactive agent-induced nanoaggregates (e.g. Figure 7A-7B , Figure 8 , Figure 9A-9B Primary amine groups also allow for the attachment of various targeting groups (e.g., antibodies, antigen-binding portions thereof, antigens, or members of binding pairs) to, for example, hydrophobically modified branched poly(2-oxazoline) polymers ( Figure 10A-10B ). This may be particularly useful before mixing the polymer and the bioactive agent. Such nanoaggregates or nanoparticles containing such targeting groups and modifications thereof can provide targeting capabilities on the nanoaggregates with the bioactive agent and enable the bioactive agent to be released preferentially or only at the desired therapeutic location. As described above, when the polymer is used to mix with the bioactive agent to produce the pharmaceutical composition of the present disclosure, it is preferred that the polymer has 1% to 100% of the second end groups without primary amines.

[0121] As disclosed herein, modified branched polymers (MBPs) (e.g., hydrophobically modified homopolymers, including SBPs, ABPs, or combinations thereof) can be used to generate encapsulating polymers or nanocapsules for dissolving water-insoluble bioactive agents. In an organic solvent environment, the hydrophilic or amphiphilic interior can be poly(2-oxazoline), poly(2-substituted oxazolines), wherein the poly(2-substituted oxazolines) can include poly(2-methyloxazoline), poly(2-ethyloxazoline), poly(2-propyloxazoline), poly(isopropyloxazoline) (PiPOX), or combinations thereof, PEG, PEO, polyphosphonates, and the like. The hydrophobic exterior can include aliphatic hydrocarbons (e.g., from C1 to about C1 22 ), aromatic hydrocarbons, polyethylene polymers, polystyrene polymers, perfluoropolymers, polydimethylsiloxanes, polyacrylates, polymethyl methacrylates, etc. In an aqueous environment, the situation is just the opposite. In drug-induced nanoaggregates in an aqueous environment, drug molecules (e.g., water-insoluble bioactive agents) can associate with the hydrophobic groups / domains of MBP ( Figure 9A-9B ). The branching density (e.g., from low generation (e.g., star-shaped and comb-shaped homopolymers) to high generation dendrimers and dendritic grafts), and the amount of hydrophobic surface group coverage of the branched homopolymer (e.g., 0% to 100% coverage) will affect the homopolymer solubility, which in turn will affect the ability to dissolve or adsorb / absorb bioactive agents. For example, an increase in branching density and hydrophobe coverage will make the homopolymer more compatible with the bioactive agent.

[0122] In further examples, ABPs and SBPs having about 0.1 to about 30% or more by weight of surface hydrophobic components are effective in dissolving or dispersing poorly water-soluble or water-insoluble bioactive agents. In addition, the branched homopolymers used (e.g., POX, PMOX, PEOX, PPOX, PEO / PEG, polyacrylamide, polyphosphates, PVP, and PVA) are soluble in water and various organic solvents, thereby promoting the formation of nanoparticles or nanoaggregates containing bioactive agents. Good water solubility and good hydrophobic drug miscibility in aqueous solution (with or without other organic solvents) make such homopolymers useful for improving the solubility of poorly water-soluble bioactive agents. For example, by reducing the need for formulation steps, processing time, and the use of complex and expensive equipment currently used in the pharmaceutical industry, the target homopolymers simplify the manufacturing process and reduce production costs. If additional branching density is desired, the SBP or ABP can first be modified using additional groups described herein and then, for example, attached to additional hydrophobic functional groups to enhance the solubility of the bioactive agent.

[0123] In one example, the polymer is configured to have an effective branching density, an amount of hydrophobic groups at the polymer surface, or a combination thereof, for encapsulating a water-insoluble form of a bioactive agent in nanoaggregates. The effective branching density, the amount of hydrophobic groups at the polymer surface, or a combination thereof can be altered as described above and below.

[0124] In one example, the polymer may have a hydrophobic group (including aliphatic (e.g., C1 to about C 22 The POX polymer may be a homopolymer of a repeating unit comprising a single monomer or a repeating unit comprising two or more monomers in each repeating unit.

[0125] In some cases, the polymer may comprise an asymmetrically branched polymer (ABP) or a dendritic asymmetrically branched polymer, such as an asymmetrically branched PEOX formed from an initiator and monomers in the ratios disclosed herein. In some cases, the polymer may comprise a randomly branched poly(2-ethyloxazoline) having one or more first end groups (e.g., a hydrophobic moiety disclosed herein) and a second end group located at the focal point of the branched polymer, such as by using, for example, Figure 6B The chain transfer polymerization illustrated herein converges on the synthesis of a reactive linear PEOX polymer to form a modified randomly branched PEOX.

[0126] In some cases, the polymers may have different first end groups and different second end groups. Some examples are Figure 6C As shown: polymers (1) to (4) have -OH as the second terminal group, polymers (5) to (8) have -NH2 as the second terminal group, polymers (1) and (5) have H as the first terminal group, polymers (2) and (6) have -CH3 as the first terminal group, polymers (3) and (7) have C 12 As the first terminal group, polymers (4) and (8) have C 18As the first end group.Polyoxazoline (POX) polymer can be a linear polymer, a branched polymer or a polymer with a combination of one or more linear parts and one or more branched parts.Polyoxazoline (POX) can include poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline), poly-(isopropyloxazoline) (PiPOX), or a combination thereof.Although the above text describes the specific first end group and the second end group, other first and second groups disclosed herein can be suitable.In some cases, the second end group can include a group modified by amine, amides, imines, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate or a combination thereof.The first end group and the second end group can be modified according to methods and processes well known to those skilled in the art.If desired, one or more reagents well known to those skilled in the art, joints or intermediates can be used.

[0127] In any of the pharmaceutical compositions disclosed above and below, the polyoxazoline may comprise a molar ratio of monomer to initiator in the range of 50:1 to 80:1.

[0128] The pharmaceutical composition may include additional polymers selected from ABP, ABP, MBP, such as symmetrically branched PAMAM or PPI dendrimers, polyether dendrimers, polyester dendrimers, comb-branched / star-branched polymers (e.g., those comprising PEO, PEG, PMOX or PEOX, polystyrene), and comb-branched dendritic grafts (e.g., those comprising PEOX, PMOX, PEI, polylysine (e.g., branched PLL), polyacrylamide, polyphosphate, PVP, PVA, or a combination thereof). Random asymmetrically branched PEI can be prepared primarily by cationic ring-opening polymerization of cyclic imine monomers having ring strain (e.g., aziridine (ethyleneimine) and azetidine (propyleneimine), or a combination thereof). Additional polymers can be mixed with the nanoaggregates disclosed herein. In one example, the nanoaggregates can be mixed with one or more additional polymers after they are formed.

[0129] As used herein, the pharmaceutical compositions, processes, methods and uses disclosed herein throughout the present disclosure, the term "biologically active agent" refers to a substance that can be, as disclosed herein, a drug based on a natural or synthetic small molecule, an inorganic-based drug, a biopharmaceutical, a drug based on a natural or synthetic macromolecule, a modification and / or derivative thereof, or a combination thereof. The biologically active agent may include a drug based on a natural or synthetic small molecule, an inorganic-based drug, a biopharmaceutical, a drug based on a natural or synthetic macromolecule, a modification and / or derivative thereof, or a combination thereof, wherein at least one of the drugs is poorly water soluble or water insoluble. The drug of interest may be a small molecule, a salt thereof, in which the molecule is modified to be water insoluble or poorly water soluble, or may be a biomolecule modified to be water insoluble or poorly water soluble, especially when the water insoluble or poorly water soluble form of the drug has improved properties, such as improved bioavailability, lower toxicity, better pharmacokinetics, or a combination thereof. Suitable examples may include drugs that are poorly soluble or insoluble in water, or drugs that have been modified to be water insoluble or poorly soluble in water to have improved properties. Bioactive agents may include growth agents, AIDS adjunct agents, alcohol abuse products (e.g., agents used to treat dependence or withdrawal), Alzheimer's disease therapeutics, amyotrophic lateral sclerosis therapeutics, analgesics, anesthetics, anticonvulsants, antidiabetics, antidotes, antifibrotic therapeutics, antihistamines, antioxidants (e.g., dyes and pigments, such as astaxanthin), anti-infectives (e.g., antibiotics, antivirals, antifungals, amebicides, anthelmintics, antimalarials, antileprosy drugs, etc.), antitumor drugs, antiparkinsonian drugs, antirheumatic drugs, appetite stimulants, biological response modifiers, biological preparations, blood regulators (e.g., anticoagulants, colony stimulating factors, hemostatics, plasma volume expanders, thrombin inhibitors, etc.), bone metabolism regulators, cardioprotective agents, cardiovascular drugs (e.g., adrenergic blockers, adrenergic stimulants, angiotensin converting enzyme (ACE) inhibitors, antiarrhythmic drugs, antilipidemic drugs, calcium channel blockers, diuretics, pressors, etc.), central nervous system (CNS) stimulants, cholinesterase inhibitors, contraceptives, fertility treatment drugs, ovulation stimulants, cystic fibrosis management agents (management agents), detoxifying agents, diagnostic agents, dietary supplements, dopamine receptor agonists, endometriosis treatments, enzymes, erectile dysfunction treatments, foot care products, gastrointestinal (GI) treatments (e.g., antacids, antidiarrheals, antiemetics, antiflatulence, bowel emptying drugs, digestive enzymes, histamine receptor agonists, laxatives, proton pump inhibitors, prostaglandins, etc.), Gaucher'sDisease treatments, gout treatments, homeopathic medicines, skin treatments, vitamins, nutrients, hormones, hypercalcemia treatments, hypocalcemia treatments, immunomodulators, immunosuppressants, L-carnitine deficiency treatments, mast cell stabilizers, migraine treatments, motion sickness treatments (e.g., benadryl and phenergan), decongestants, antihistamines, cough suppressants, multiple sclerosis treatments, muscle relaxants, nasal treatments (e.g., anti-inflammatory drugs), smoking cessation aids aid), appetite suppressants, nucleoside analogs, obesity treatments, ophthalmic products (e.g., antibiotics, antiglaucoma agents, artificial tears, lubricants, etc.), sexual aids, osteoporosis treatments, ear products (e.g., anti-infectives and cerumenolytics), minerals, oxytocin, parasympathetic blockers, parasympathomimetics, patent ductus arteriosus drugs, phosphate binders, porphyria drugs, prostaglandins, psychotherapeutics, contrast media, respiratory drugs (e.g., anti-inflammatory drugs, antitussives, bronchodilators Biologically active agents include, but are not limited to, sedatives, decongestants, expectorants, leukotriene antagonists, surfactants, etc.), salt substitutes, sedatives, hypnotics, skin and mucosal treatments (e.g., acne treatments), anorectal treatments (e.g., hemorrhoid treatments and enemas), antiperspirants, antipruritics, antipsoriatics, antiseborrheic agents, burn treatments, cleansers, depigmenting agents, emollients, hair growth retardants, hair growth stimulants, keratolytics, hair problem treatments, mouth and throat problem treatments, photosensitizers, wart treatments, wound care treatments, or combinations thereof. Biologically active agents may also include over-the-counter drugs and products (e.g., deodorants), Tourette's syndrome drugs, tremor treatments, urinary tract drugs (e.g., acidifiers, alkalizers), antispasmodics, benign prostatic hyperplasia treatments, calcium oxalate stone preventives, enuresis treatments, vaginal preparations (e.g., anti-infectives, hormones, etc.), vasodilators, vertigo treatments, Wilson's disease treatments, etc.

[0130] Further examples of bioactive agents may include unmodified or other forms of the drug that may be modified, for example, into salts, ionized forms, or hydrophilic forms that may be modified to remove such functional groups, modifications, etc. to produce poorly water soluble or water insoluble bioactive agents. If the pharmaceutical composition comprises two or more bioactive agents, at least one of the bioactive agents may be modified or has been modified to be water insoluble or poorly water soluble. Examples of such bioactive agents may include analgesics / antipyretics (e.g., aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine hydrochloride, propoxyphene hydrochloride, propoxyphene napsylate, meperidine hydrochloride, hydromorphone hydrochloride, morphine sulfate, oxycodone hydrochloride, codeine phosphate, dihydrocodeine bitartrate, pentazocine hydrochloride, hydrocodone bitartrate, levorphanol tartrate, diflunisal, triethanolamine salicylate, nalbuphine hydrochloride, mefenamic acid, butorphanol tartrate, choline salicylate, butalbital, phenyltoloxamine citrate, diphenhydramine citrate, methoxythiazine, cinnamedrine hydrochloride, hydrochloride), meprobamate, etc.); anesthetics (e.g., cyclopropane, enflurane, halothane, isoflurane, methoxyflurane, nitrous oxide, propofol, etc.); antiasthmatics (e.g., azelastine, ketotifen, traxanox, amlexanox, sodium cromoglycate, ibudilast, montelukast, nedocromil, oxamol, pranlukast, serotonin, supelalast toluene, thiamide, zafirlukast, zileuton, beclomethasone, budesonide, dexamethasone, flunisolide, triamcinolone acetonide, etc.); antibiotics (e.g., neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline, etc.); quinolones, fluoroquinolones, antidepressants (e.g., nefopam, oxypredine, hydrochloride Doxepin, amoxapine, trazodone hydrochloride, amitriptyline hydrochloride, maprotiline hydrochloride, phenelzine sulfate, desipramine hydrochloride, nortriptyline hydrochloride, phenylcypromine sulfate, fluoxetine hydrochloride, doxepin hydrochloride, imipramine hydrochloride, imipramine pamoate, nortriptyline, amitriptyline hydrochloride, isocarboxazid, trimipramine maleate, protriptyline hydrochloride, etc.); antidiabetic drugs (such as biguanides, hormones, sulfonylurea derivatives, etc.); antifungal drugs (such as griseofulvin, ketoconazole, amphotericin B, nystatin, candidiasis, etc.); antihypertensive drugs (such as propranolol, propafenone, oxprenolol, nifedipine, reserpine, trimethaphan camsylate), phenoxybenzamine hydrochloride, pargyline hydrochloride, deserpidine, diazoxide, guanethidine monosulfate, minoxidil, rescinnamine, sodium nitroprusside, Rauvolfiaserpentina, Rauvolfiaserpentina mixed base, phentolamine mesylate, reserpine, etc.);Anti-inflammatory drugs (e.g., nonsteroidal compounds, such as indomethacin, naproxen, ibuprofen, ramifenazone, piroxicam, etc.; steroidal compounds, such as cortisone, dexamethasone, fluazacort, hydrocortisone, prednisolone, prednisone, etc.); antineoplastic drugs (e.g., doxorubicin, cyclophosphamide, dactinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, gemcitabine, mitomycin, methotrexate, fluorouracil, carboplatin, nitrogen mustard β-chloronitrosourea (carmustine, BCNU), 1-chloroethyl- 3-cyclohexyl-1-nitrosourea (lomustine, CCNU), methyl CCNU, cisplatin, etoposide, interferon, camptothecin and its derivatives, phenesterine, Taxol and its derivatives, Taxotere and its derivatives, vinblastine, vincristine, tamoxifen, etoposide, piposulfan, etc.); antianxiety drugs (e.g. lorazepam, buspirone hydrochloride, prazepam, chlordiazepoxide hydrochloride, oxazepam, clorazepate potassium (clorazepate dipotassium), diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol, halazepam, chlormezazone, dantrolene, etc.); immunosuppressants (e.g. cyclosporine, azathioprine, mizoribine, FK506 (tacrolimus), rapamycin (sirolimus), etc.); anti-migraine drugs (e.g. ergotamine tartrate, propranolol hydrochloride, isometheptenemucate, dichloralphenazone, etc.); azone, etc.); sedatives / hypnotics (e.g., barbiturates (e.g., pentobarbital, sodium pentobarbital, sodium secobarbital, etc.) or benzodiazepines (e.g., flurazepam hydrochloride, triazolam, tomazepam, midazolam hydrochloride, etc.); antianginal drugs (e.g., beta-adrenergic blockers, calcium channel blockers (e.g., nifedipine, diltiazem hydrochloride, etc.) and nitrates (e.g., nitroglycerin, isosorbide dinitrate, pentaerythritol tetranitrate, erythrityl tetranitrate, etc.); tetranitrate, etc.); antipsychotics (e.g., haloperidol, loxapine succinate, loxapine hydrochloride, thioridazine, thioridazine hydrochloride, thiothixene, fluphenazine hydrochloride, fluphenazine decanoate, fluphenazine enanthate, trifluoperazine hydrochloride, chlorpromazine hydrochloride, perphenazine, lithium citrate, prochlorperazine, etc.); antimanic drugs (e.g., lithium carbonate);Antiarrhythmic drugs (e.g., bretylium tosylate, esmolol hydrochloride, verapamil hydrochloride, amiodarone, encamide hydrochloride, digoxin, digitoxin, mexiletine hydrochloride, disopyramide phosphate, procainamide hydrochloride, quinidine sulfate, quinidine gluconate, quinidine polygalacturonate, flecainide acetate, tocamide hydrochloride) hydrochloride, lidocaine hydrochloride, etc.); antiarthritis drugs (such as phenylbutazone, sulindac, penicillamine, salsalate, piroxicam, azathioprine, indomethacin, meclofenamic acid sodium, gold sodium thiomalate, ketoprofen, auranofin, aurothioglucose, tolmetin sodium, etc.); antigout drugs (such as colchicine, allopurinol, etc.); anticoagulants (such as heparin, heparin sodium, warfarin sodium, etc.); thrombolytic drugs (such as urokinase, streptokinase, altoplase, etc.); antifibrinolytic drugs (such as ammonia hexanoic acid); blood activators (e.g., pentoxifylline); antiplatelet agents (e.g., aspirin, empirin, ascriptin, etc.); anticonvulsants (e.g., valproic acid, divalproex sodium, phenytoin, phenytoin sodium, clonazepam, primidone, phenobarbital, phenobarbital sodium, carbamazepine, amobarbital sodium, methsuximide, metharbital, mephenytoin, phensuximide, methyphenytoin, ethotoin, phenylacetylurea, secobarbital sodium sodium), clorazepate, trimethadione, etc.); anti-parkinsonian drugs (e.g., ethosuximide, etc.); antihistamines / antipruritics (e.g., hydroxyzine hydrochloride, diphenhydramine hydrochloride, chlorpheniramine maleate, brompheniramine maleate, cyproheptadine hydrochloride, terfenadine, clemastine fumarate, triprolidine hydrochloride, carbinoxamine maleate, diphenylpyralidone hydrochloride, phenindamine tartrate, azatadine maleate, tripeliramine hydrochloride, dexchlorpheniramine maleate, maleate), medimethazine hydrochloride, isobutrazol tartrate, etc.); agents that can be used for calcium regulation (e.g., calcitonin, parathyroid hormone, etc.); antibacterial drugs (e.g., amikacin sulfate, aztreonam, chloramphenicol, chloramphenicol palmitate, chloramphenicol sodium succinate, ciprofloxacin hydrochloride, clindamycin hydrochloride, clindamycin palmitate, clindamycin phosphate, metronidazole, metronidazole hydrochloride, gentamicin sulfate, lincomycin hydrochloride, tobramycin sulfate, vancomycin hydrochloride, polymyxin B sulfate, polymyxin E sodium mesylate, colistin sulfate, etc.); antiviral drugs (e.g., interferon gamma, zidovudine, amantadine hydrochloride, ribavirin, acyclovir, etc.);Antimicrobial drugs (such as cephalosporins (such as cefazolin sodium, cephradine, cefaclor, cefpirin sodium, ceftizoxime sodium, cefoperazone sodium, cefotetan disodium, cefuroxime axetil, ceftriaxone sodium, cefotaxime sodium, cefadroxil monohydrate, ceftazidime, cephalexin, ceftriaxone sodium, cefonic acid sodium, cefuroxime hydrochloride monohydrate, cefmandolin sodium, cefoxitin sodium, cefonic acid sodium, cefuroxime sodium, cefuroxime sodium, cefuroxime sodium, cefuroxime sodium, etc.), penicillins (such as ampicillin, amoxicillin, benzathine penicillin G, cyclacillin, ampicillin sodium, penicillin GK, penicillin VK, piperacillin sodium, oxacillin sodium, baclofen hydrochloride The invention relates to the treatment of inflammatory inflammatory diseases such as inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, and inflammatory bowel disease. The invention relates to the treatment of inflammatory inflammatory diseases such as inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, inflammatory bowel disease, sulfate), albuterol, bitolterol, isoproterenol mesylate hydrochloride, terbutaline sulfate, epinephrine bitartrate, orproterenol sulfate, epinephrine, epinephrine bitartrate); anticholinergics (e.g. ipratropium bromide); xanthines (e.g. aminophylline, diprophylline, orproterenol sulfate, aminophylline); mast cell stabilizers (e.g. sodium cromoglycate); inhaled corticosteroids (e.g. flunisolide, beclomethasone dipropionate monohydrate, etc.), salbutamol, beclomethasone dipropionate (BDP), ipratropium bromide, budesonide, ketotifen, salmeterol xinafoate, terbutaline sulfate, triamcinolone, theophylline, nedocromil sodium, orproterenol sulfate, salbutamol, flunisolide, etc.);Hormones (e.g. androgens (e.g. danazol, testosterone cypionate, fluoxymesterone, ethyltostosterone, testosterone enanthate, methyltestosterone, fluoxymesterone, testosterone cypionate, etc.), estrogens (e.g. estradiol, estrone sulfate piperazine (estropipate), conjugated estrogens, etc.), progestins (e.g. medroxyprogesterone acetate, norethisterone acetate, etc.), corticosteroids (e.g. triamcinolone , betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, prednisone, methylprednisolone acetate suspension, triamcinolone acetonide, methylprednisolone, prednisolone sodium phosphate, methylprednisolone sodium succinate, hydrocortisone sodium succinate, methylprednisolone sodium succinate, hydroxyflurane, hydrocortisone, hydrocortisone cypionate, prednisolone, fludrocortisone acetate, paramethasone acetate, prednisolone tert-butyl acetate (prednisolone tebulate), prednisolone acetate, prednisolone sodium phosphate, hydrocortisone sodium succinate, etc.), thyroid hormones (e.g., levothyroxine sodium); etc.; hypoglycemic drugs (e.g., human insulin, purified bovine insulin, purified porcine insulin, glyburide, chlorpropamide, glipizide, tolbutamide, tolazamide, etc.); hypolipidemic drugs (e.g., clofibrate, dextrothyroxine sodium, probucol, lovastatin, niacin, etc.); proteins (e.g., DNA enzymes, alginase, superoxide dismutase, lipase, etc.); nucleic acids (e.g., sense nucleic acids or antisense nucleic acids encoding any therapeutically useful protein (including any protein described herein), etc.); drugs that can be used for erythropoiesis (e.g., erythropoietin); anti-ulcer or anti-retroviral Flow agents (such as famotidine, cimetidine, ranitidine hydrochloride, etc.); antinauseants or antiemetics (such as chlorpheniramine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, thiethylperazine, scopolamine, etc.); oil-soluble vitamins (such as vitamin A, D, E, K, etc.); mitotane, visadine, halogenated nitrosoureas, anthracyclines, ellipticine, etc.; STING inhibitors such as C-176, C-170 and C-171; STING activators, such as 3',3'-cGAMP (3',3'-cyclic GMP-AMP, cyclic GMP-AMP, cGAMP); STING agonists, such as SR-717 lithium, α-mangostin or diABZI (aminobenzimidazole);STING agonist (diABZI STING agonist-1, compound 3), STING agonist-1 (G10), CF501 (formula (1)), CF502 (formula (5)), CF504 (formula (7)), CF505 (formula (8)), CF508 (formula (4)), CF509 (formula (6)), CF510 (formula (2)), CF511 (formula (9)) (Liu, et al. al., Cell Research, 1-19, 2022. https: / / doi.org / 10.1038 / s41422-022-00612-2) or MSA-2 (Tocris, Minneapolis, MN); STING antagonists, such as SN-011 (GUN35901) or H-151; indoleamine dioxygenase (IDO) inhibitors or IDO1 inhibitors, such as Epacadostat (INCB24360), BMS-986205, PF-0684003, Navoximod, Indoximod, NLG802 (indoximod prodrug) or LY3381916; and combinations thereof. ;

[0131] In some cases, the bioactive agent may comprise any one of the bioactive agents listed above and below. In some cases, the bioactive agent may comprise two or more of the bioactive agents listed above and below.

[0132] In some cases, the bioactive agent may comprise an immunoglobulin, such as IgG, IgM, or one or more molecules disclosed and prepared according to the processes and methods described in US Pat. No. 10,688,048, which is incorporated herein by reference.

[0133] In some cases, the biologically active agent may comprise at least one STING polypeptide or a portion thereof, a nucleic acid encoding a STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulator, an IDO inhibitor, an IDO1 inhibitor, or a combination thereof. The biologically active agent may comprise RNA, mRNA, siRNA, sgRNA, DNA, oligonucleotides, or a combination thereof, each of which encodes one of the above-mentioned STING polypeptides or portions thereof, STING inhibitors, STING activators, STING agonists, STING antagonists, STING modulators, IDO inhibitors, or IDO1 inhibitors.

[0134] In some cases, the bioactive agent may include a STING (stimulator of interferon genes) protein, a STING agonist, a STING activator, a STING inhibitor, a STING antagonist, or a combination thereof. In some cases, the bioactive agent may include one or more IDO or IDO1 inhibitors. Any STING protein, STING agonist, STING activator, STING inhibitor, STING antagonist, IDO inhibitor or IDO1 inhibitor disclosed herein or found thereafter may be suitable. In some cases, the bioactive agent may include a STING regulatory molecule, such as a benzimidazole compound disclosed in patent publications WO2017175156A1 and WO2020156363 by Liu et al. (Cell Research, 1-19, 2022), a pyridyl imidazole compound disclosed in patent publications WO2019134705, WO2020010451 and US20200031825, or a combination thereof.

[0135] In some cases, the bioactive agent may include one or more STING agonists. In some cases, the STING agonist may include one or more compounds having formula (1)-(29): Figures 11A-11E ),

[0136]

[0137]

[0138]

[0139]

[0140] or its corresponding salt, solvate, prodrug, isomer, or combination thereof.

[0141] In some cases, the bioactive agent may comprise a compound having the formula:

[0142] Formula (1)

[0143] or

[0144] Formula (4)

[0145]

[0146] A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

[0147] In some cases, the nanoaggregates are water soluble and may comprise a polymer and at least one bioactive agent comprising at least one STING polypeptide or portion thereof, a nucleic acid encoding the STING polypeptide or portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof. The pharmaceutical composition comprising the nanoaggregates is soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution. In some cases, the pharmaceutical composition comprising the nanoaggregates is soluble in an aqueous solution to produce at least 2 mg / mL of the bioactive agent disclosed herein, or a combination thereof, dissolved in the aqueous solution. In some cases, a pharmaceutical composition comprising nanoaggregates is soluble in an aqueous solution to produce at least 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, or more of a bioactive agent disclosed herein, or a combination thereof, dissolved in the aqueous solution.

[0148] Any polymer disclosed herein can be suitable.In some instances, polymer can include poly-oxazoline (POX), it comprises linear part, branched part or its combination, and wherein poly-oxazoline (POX) includes poly-(2-oxazoline), poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline), poly-(isopropyloxazoline) (PiPOX) or its combination.In some instances, poly-oxazoline can be poly-(2-ethyloxazoline).

[0149] As used throughout the present disclosure, the size of the nanoaggregate before lyophilization may be less than 150nm. In some cases, the size of the nanoaggregate before lyophilization may be less than 120nm. "Less than" means that the size may be less than the defined size (nm) and may be about 0nm, i.e., the nanoaggregate solution may be a clear solution without measurable particles or aggregates. For example, "less than 150nm" refers to within the range of 0nm to 150nm, and "less than 120nm" refers to within the range of 0nm to 120nm. In some cases, the size of the nanoaggregate or nanoparticle before lyophilization may be from about 0.01nm to about 100nm. In some cases, the size of the nanoaggregate or nanoparticle before lyophilization may be from about 0.01nm to about 120nm. In some cases, the size of the nanoaggregate or nanoparticle before lyophilization may be from about 0.01nm to about 150nm. In some cases, the size of the nanoaggregate or nanoparticle before lyophilization may be from about 50 to about 100nm. In some cases, the size of the nanoaggregates or nanoparticles before lyophilization can be about 50 to about 120 nm. Particle size can be measured by light scattering.

[0150] In some cases, the nanoaggregates may be free of human serum albumin, organic solvents, detergents, or oils. In some cases, the nanoaggregates may be free of human serum albumin, organic solvents, detergents, oils, or free acids. In some cases, the nanoaggregates may be free of human serum albumin. In some cases, the nanoaggregates may be free of organic solvents. In some cases, the nanoaggregates may be free of detergents. In some cases, the nanoaggregates may be free of oils. In some cases, the nanoaggregates may be free of free acids. In some cases, the nanoaggregates may be free of substances selected from human serum albumin, organic solvents, detergents, oils, free acids, and combinations thereof.

[0151] In some cases, the pharmaceutical composition may be free of human serum albumin, organic solvents, detergents, or oils. In some cases, the pharmaceutical composition may be free of human serum albumin, organic solvents, detergents, oils, or free acids. In some cases, the pharmaceutical composition may be free of human serum albumin. In some cases, the pharmaceutical composition may be free of organic solvents. In some cases, the pharmaceutical composition may be free of detergents. In some cases, the pharmaceutical composition may be free of oils. In some cases, the pharmaceutical composition may be free of free acids. In some cases, the pharmaceutical composition may be free of substances selected from human serum albumin, organic solvents, detergents, oils, free acids, and combinations thereof.

[0152] The pharmaceutical composition of the present disclosure may be a drug for treating or preventing a disease selected from an immune disease, an infectious disease, and a combination thereof. Immune diseases may include immunodeficiency diseases, overactive immune diseases, autoimmune diseases, and other diseases or symptoms with abnormal immune systems. In some cases, immune diseases may be various autoinflammatory, autoimmune, and degenerative diseases, such as those associated with the STING (interferon gene stimulator) signaling pathway or the IDO pathway. In some cases, immune diseases may be associated with inflammation, cell stress, and tissue damage mediated by STING in infection. In some cases, the pharmaceutical composition of the present disclosure may include STING proteins, STING agonists, STING activators, STING inhibitors, STING antagonists, IDO inhibitors, IDO1 inhibitors, or combinations thereof. In some cases, the pharmaceutical composition of the present disclosure may include one or more STING inhibitors. In some cases, the pharmaceutical composition of the present disclosure may include one or more STING activators.

[0153] The pharmaceutical composition of the present disclosure may be a medicament for treating or preventing one or more of the diseases disclosed herein.

[0154] In some cases, the pharmaceutical composition may include two or more bioactive agents, wherein at least one of the two or more bioactive agents is water-insoluble or poorly water-soluble. In some cases, at least one of the two or more bioactive agents is at least one STING polypeptide or portion thereof, a nucleic acid encoding the STING polypeptide or portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING regulatory molecule, or a combination thereof. In some cases, the pharmaceutical composition may include at least one water-insoluble or poorly water-soluble bioactive agent in a nanoaggregate, and one or more additional bioactive agents (which are contained in the nanoaggregate or not contained in the nanoaggregate). The pharmaceutical composition may include a nanoaggregate, wherein the nanoaggregate includes a polymer and two or more bioactive agents, each of which is water-insoluble or poorly water-soluble. The pharmaceutical composition may comprise a nanoaggregate comprising a polymer and at least one biologically active agent (comprising at least one STING polypeptide or portion thereof, a nucleic acid encoding the STING polypeptide or portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof), and one or more additional biologically active agents, each of which is water-soluble.

[0155] The term "combination thereof" used for the combinations of bioactive agents disclosed above refers to a combination of two or more bioactive agents, wherein such combination does not have an unwanted effect, such as an unwanted interaction between or among the bioactive agents. It should be understood that some combinations of bioactive agents may be inappropriate, or may be undesirable, such as those with unwanted interactions. For example, a combination of theophylline and ciprofloxacin or warfarin and diflunisal may be inappropriate. Thus these combinations or any combination determined to be inappropriate by appropriate guidelines or regulations are excluded.

[0156] As described above, the size of the nano aggregates before freeze drying may be less than 150nm. In some cases, the size of the nano aggregates before freeze drying may be less than 120nm. In some cases, the size of the nano aggregates before freeze drying may be in the range of about 0.01nm or about 0nm to about 150nm. The size of the nano aggregates before freeze drying may be in the range of about 50nm to about 150nm. In some cases, the size of the nano aggregates or nanoparticles before freeze drying may be about 50 to about 100nm. In some cases, the size of the nano aggregates or nanoparticles before freeze drying may be about 50 to about 120nm. In another example, the size of the nano aggregates before freeze drying may be in the range of about 70 to 90nm. The particle size may be measured by light scattering.

[0157] In any of the pharmaceutical compositions disclosed above and below, the nanoaggregates may have a weight ratio of polymer to bioactive agent in the range of about 2: 1 to about 200: 1. The nanoaggregates may have a weight ratio of polymer to bioactive agent in the range of about 2: 1 to about 200: 1 in one example, about 2: 1 to about 150: 1 in another example, about 2: 1 to about 120: 1 in yet another example, about 2: 1 to about 100: 1 in yet another example, about 2: 1 to about 80: 1 in yet another example, about 2: 1 to about 60: 1 in yet another example, about 2: 1 to about 40: 1 in yet another example, about 2: 1 to about 30: 1 in yet another example, and about 2: 1 to about 40: 1 in yet another example. In one example, about 2:1 to about 20:1, in another example, about 2:1 to about 15:1, in another example, about 2:1 to about 10:1, in another example, about 2:1 to about 8:1, in another example, about 5:1 to about 10:1, in another example, about 5:1 to about 8:1, in another example, about 5:1, in another example, about 6:1 to about 8:1, in another example, about 6:1, in another example, about 7:1, in another example, about 7.5:1, and in another example, about 8: 1. When the pharmaceutical composition comprises two or more bioactive agents, the ratio of the polymer to the bioactive agent can be based on the total weight of the polymer and the bioactive agent.

[0158] In some cases, the pharmaceutical composition may be an adjuvant (e.g., of a vaccine). In some cases, the pharmaceutical composition may be an adjuvant for a vaccine for treating or preventing an infectious disease, and the infectious disease is selected from varicella, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika, and combinations thereof. In some cases, the pharmaceutical composition may be an adjuvant for a vaccine for treating or preventing an infectious disease, and the infectious disease is caused by 229Eα-coronavirus, NL63α-coronavirus, OC43β-coronavirus, HKU1β-coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, its variants, or a combination thereof.

[0159] In some cases, the pharmaceutical composition may be a prophylactic vaccine, a therapeutic vaccine, or a combination thereof, wherein the pharmaceutical composition may comprise an adjuvant, such as any of the adjuvant formulations disclosed herein, and further comprises at least one immunizing agent for stimulating an immune response in a subject in need thereof. In some cases, the adjuvant may comprise at least one STING agonist. In some cases, the adjuvant may comprise at least one compound of formula (1)-formula (29), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof. In some cases, the adjuvant may be any one of the adjuvant formulations selected from adjuvant formulations 1 to adjuvant formulations 6 as disclosed below. The immunizing agent may comprise an inactive microbe selected from bacteria, viruses, fungi, protozoa, worms, parasites, prions, parts thereof, or combinations thereof; toxins, nucleic acids encoding toxins; proteins; nucleic acids encoding proteins; oligonucleotides; DNA; RNA; mRNA; siRNA; sgRNA; fragments thereof; or combinations thereof.

[0160] In some cases, pharmaceutical composition can be formulated for treating or preventing at least one infectious disease. In some cases, pharmaceutical composition can be formulated for treating or preventing at least one infectious disease, it is selected from chickenpox, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika and its combination. In some cases, pharmaceutical composition can be formulated for treating or preventing at least one infectious disease, it is caused by 229Eα-coronavirus, NL63α-coronavirus, OC43β-coronavirus, HKU1β-coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, its variant or its combination.

[0161] In some cases, the pharmaceutical composition may have a pH in the range of 3.0 to 6.9. The pH may be measured in an aqueous solution of the pharmaceutical composition.

[0162] In some cases, a pharmaceutical composition may comprise one or more bioactive agents disclosed herein, derivatives thereof, or combinations thereof, wherein 1% to 100% of the second end groups do not contain primary amines, and wherein 1% to 100% of the second end groups comprise hydroxyl groups.

[0163] In some cases, the pharmaceutical composition may further comprise one or more subsequent biologically active agents selected from proteins, peptides, antibodies, fragments of antibodies, compounds, small molecule drugs, one or more chemotherapeutic drugs, and combinations thereof.

[0164] The pharmaceutical compositions disclosed above and below may further comprise an additional bioactive agent that is formulated without a polymer, in particular a polymer disclosed herein. The phrase "an additional bioactive agent formulated without a polymer" refers to a bioactive agent formulation comprising a bioactive agent and without a polymer disclosed herein, wherein the additional bioactive agent may be a salt, a base, a bioactive agent formulated with an organic solvent, a detergent, an oil or a free acid, a protein, a lipid, or a combination thereof. In some cases, the pharmaceutical composition may comprise one or more bioactive agents formulated with an aluminum salt, an organic adjuvant, or a combination thereof.

[0165] The term "soluble in aqueous solution" means that the solution contains no detectable particles or has a particle size that can be filtered through a 0.22 μm filter (filtration rating (R f) in the range of 50% to 100%), wherein the filtration rate refers to the volume percentage of the filter that passes through the filter before the filter is clogged resulting in filtration obstruction. As used throughout this disclosure, the term "0.22 μm filter" refers to a filter device having a filtration pore size of 0.22 μm. The term "0.8 μm filter" refers to a filter device having a filtration pore size of 0.8 μm. A combination of a 0.8 μm filter and a 0.22 μm filter may be suitable.

[0166] Pharmaceutical compositions disclosed herein can be formulated for parenteral, oral, nasal, transdermal (topical), transmucosal, rectal administration, vaginal administration or its combination, and can include one or more pharmaceutically suitable carriers. In some cases, pharmaceutical compositions disclosed herein can be formulated for intravenous (IV), intradermal (ID), subcutaneous (SC), oral, transdermal (topical), transmucosal, rectal administration or its combination. In some cases, pharmaceutical compositions disclosed herein can be formulated for intravenous (IV), intradermal (ID), subcutaneous (SC), transdermal (topical), nasal spray, inhalation, oral tablets, eye drops, rectal immunity, vaginal immunity, transmucosal administration or its combination. In some cases, pharmaceutical compositions disclosed herein can be formulated for oral administration, such as tablets, capsules, oral sprays, solutions or suspensions. In some cases, pharmaceutical compositions disclosed herein can be formulated for nasal administration, such as nasal sprays. Pharmaceutically suitable carriers disclosed herein can be suitable.

[0167] In some cases, the immunizing agent may comprise at least a polypeptide of a coronavirus spike (S) glycoprotein, a DNA encoding the spike (S) glycoprotein, an RNA encoding the spike (S) glycoprotein, a receptor binding domain (RBD) of the spike (S) glycoprotein, a DNA encoding the RBD, an RNA encoding the RBD, a portion thereof, or a combination thereof.

[0168] Coronaviruses are a class of RNA viruses that cause disease in mammals and birds, including humans. Coronaviruses may include (1) the genus alphacoronavirus, which includes the following species: alphacoronavirus 1 (TGEV, feline coronavirus and canine coronavirus), human coronavirus 229E, human coronavirus NL63, Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2, and Scotophilus bat coronavirus 512; (2) the genus betacoronavirus, which includes the following species: betacoronavirus 1 (bovine coronavirus and human coronavirus OC43), hedgehog coronavirus 1, human Coronavirus HKU1, Middle East respiratory syndrome-related coronavirus, mouse coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, severe acute respiratory syndrome-related coronavirus (SARS-CoV and SARS-CoV-2), and Tylonycteris bat coronavirus HKU4; (3) the genus Gammacoronavirus, which includes the following species: avian coronavirus and beluga coronavirus SW1; and (4) the genus Deltacoronavirus, which includes the following species: nightingale coronavirus HKU11 and porcine coronavirus HKU15. An example of a phylogenetic tree of some viruses is shown in Figure 1. Fig.13A shown.

[0169] The coronavirus may include 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (beta coronavirus that causes Middle East Respiratory Syndrome (or MERS)), SARS-CoV (beta coronavirus that causes severe acute respiratory syndrome (or SARS)), SARS-CoV-2 (the new coronavirus that causes coronavirus disease 2019 (or COVID-19)), variants thereof, or combinations thereof.

[0170] In some cases, the coronavirus may include 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, variants thereof, or combinations thereof.

[0171] In some cases, the coronavirus can be selected from 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, variants thereof, and combinations thereof.

[0172] In some cases, the immunizing agent may include at least one polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or a combination thereof. In some cases, the immunizing agent may include two or more of the RBD sequences disclosed therein. In some cases, the immunizing agent may include three or more of the RBD sequences disclosed therein.

[0173] In some cases, the immunizing agent may comprise at least one polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some cases, the immunizing agent may comprise an immunizing agent selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and combinations thereof.

[0174] In some cases, the weight ratio of the immunizing agent: adjuvant may be in the range of 1:50 to 50:1, wherein the weight ratio is based on the weight of the immunizing agent and the bioactive agent as an adjuvant. In some cases, the pharmaceutical composition may include a weight ratio of the RBD antigen fusion protein: bioactive agent in the range of 1:50 to 50:1, wherein the bioactive agent may be selected from Formula (1)-Formula (29) and combinations thereof. In some cases, the pharmaceutical composition may include a weight ratio of the RBD antigen fusion protein: bioactive agent in the range of 1:50 to 50:1, wherein the bioactive agent may be selected from Formula (1), Formula (4) and combinations thereof.

[0175] The pharmaceutical composition may further include one or more subsequent bioactive agents selected from proteins, peptides, antibodies, fragments of antibodies, compounds, small molecule drugs, one or more chemotherapeutic drugs and combinations thereof. In some cases, the subsequent bioactive agent may be a vaccine identical or different from the pharmaceutical composition disclosed herein. In some cases, the pharmaceutical composition may be a vaccine for treating or preventing a disease, and the disease is caused by chickenpox, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika or a combination thereof, and the subsequent bioactive agent may be the same or different vaccine. In some cases, the pharmaceutical composition may be a vaccine for treating or preventing a disease caused by a coronavirus, and the subsequent biologically active agent may be a vaccine or a vaccine for treating or preventing chickenpox, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), shingles, tetanus, pertussis, Zika, or a combination thereof.

[0176] In some cases, the present disclosure also relates to a method for treating or preventing a disease in a subject in need thereof, which method may include administering to the subject an effective dose of a pharmaceutical composition comprising:

[0177] A nanoaggregate comprising a polymer and at least one bioactive agent comprising at least one STING polypeptide or portion thereof, a nucleic acid encoding the STING polypeptide or portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof; and

[0178] Optional pharmaceutically suitable carrier;

[0179] wherein the pharmaceutical composition is soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution;

[0180] wherein the polymer is water soluble; and

[0181] The polymer may include:

[0182] A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% hydrophobic moiety, the hydrophobic moiety comprising a saturated or unsaturated aliphatic hydrocarbon, aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, amide, imine, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate, or a combination thereof; or

[0183] a second polymer comprising one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly(γ-L-glutamic acid) (PGA), poly(L-phenylalanine ethyl ester) (PAE), poly(L-lysine) (PLL), methyl PEG (mPEG), polyaspartic acid (PasP), poly(L-histidine) (PLH), poly(vinylamine) (PEI), poly(N-vinylpyrrolidone) (PVP), poly(L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly(hydroxybutyrate) (PHB), poly(ethylene oxide) (PEO), poly(γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly(isohexyl cyanoacrylate) (PIHCA), poly(allylamine hydrochloride) (PAH), poly(γ-propargyl) (PP); or

[0184] Its combination.

[0185] In some cases, the polymer can include a first polymer as disclosed herein.

[0186] In some cases, the polymer may be composed of a first polymer as disclosed herein. In some cases, the pharmaceutical composition may be free of a polymer selected from: one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), poly aspartic acid (PasP), poly (L-histidine) (PLH), poly (vinylamine) (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP), and combinations thereof.

[0187] In some cases, the polymer may include one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), poly aspartic acid (PasP), poly (L-histidine) (PLH), poly (vinylamine) (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP), or a combination thereof.

[0188] In some cases, the polymer may include a first polymer and one or more subsequent polymers (also referred to as "second polymers") selected from one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), poly aspartic acid (PasP), poly (L-histidine) (PLH), poly (vinylamine) (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP), and combinations thereof.

[0189] The bioactive agents disclosed herein may be suitable.

[0190] Any polymer disclosed herein can be suitable.In some cases, polymer can include poly-oxazoline (POX), it comprises linear part, branched part or its combination, and wherein poly-oxazoline (POX) includes poly-(2-oxazoline), poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline), poly-(isopropyloxazoline) or its combination.In some cases, poly-oxazoline is poly-(2-ethyloxazoline).

[0191] As described above, the size of the nano aggregates before freeze drying may be less than 150nm. In some cases, the size of the nano aggregates before freeze drying may be less than 120nm. In some cases, the size of the nano aggregates before freeze drying may be in the range of about 0.01nm or about 0nm to about 150nm. The size of the nano aggregates before freeze drying may be in the range of about 50nm to about 150nm. In some cases, the size of the nano aggregates or nanoparticles before freeze drying may be about 50 to about 100nm. In some cases, the size of the nano aggregates or nanoparticles before freeze drying may be about 50 to about 120nm. In another example, the size of the nano aggregates before freeze drying may be in the range of about 70 to 90nm. The particle size may be measured by light scattering.

[0192] In some cases, the nanoaggregates may have a weight ratio of polymer to bioactive agent in the range of about 2: 1 to about 20: 1. In some cases, the nanoaggregates may have a weight ratio of polymer to bioactive agent in the range of about 5: 1 to about 8:1.

[0193] In some cases, the nanoaggregates may be free of human serum albumin, organic solvents, detergents, or oils. In some cases, the nanoaggregates may be free of human serum albumin, organic solvents, detergents, oils, or free acids. In some cases, the nanoaggregates may be free of human serum albumin. In some cases, the nanoaggregates may be free of organic solvents. In some cases, the nanoaggregates may be free of detergents. In some cases, the nanoaggregates may be free of oils. In some cases, the nanoaggregates may be free of free acids. In some cases, the nanoaggregates may be free of substances selected from human serum albumin, organic solvents, detergents, oils, free acids, and combinations thereof.

[0194] In some cases, the pharmaceutical composition may be free of human serum albumin, organic solvents, detergents, or oils. In some cases, the pharmaceutical composition may be free of human serum albumin, organic solvents, detergents, oils, or free acids. In some cases, the pharmaceutical composition may be free of human serum albumin. In some cases, the pharmaceutical composition may be free of organic solvents. In some cases, the pharmaceutical composition may be free of detergents. In some cases, the pharmaceutical composition may be free of oils. In some cases, the pharmaceutical composition may be free of free acids. In some cases, the pharmaceutical composition may be free of substances selected from human serum albumin, organic solvents, detergents, oils, free acids, and combinations thereof.

[0195] In some cases of the methods disclosed herein, 1% to 100% of the second end groups do not contain primary amines. In some cases, 1% to 100% of the second end groups contain hydroxyl groups. All percentages are based on the total number of second end groups.

[0196] In some cases, the pharmaceutical composition may have a pH in the range of about 3.0 to 6.9.

[0197] In some cases, suitable for use in the method, the bioactive agent may include at least a compound having formula (1) to formula (29) ( Figures 11A-11E ), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

[0198] In some cases, the bioactive agent may comprise a compound having the formula

[0199] Formula (1)

[0200] or

[0201] Formula (4)

[0202] or

[0203] A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

[0204] In some cases, the disease can be selected from an immune disease, an infectious disease, and combinations thereof.

[0205] In some cases, immune disorders may include the aforementioned immunodeficiency disorders, overactive immune disorders, autoimmune diseases, and other diseases or conditions with an abnormal immune system.

[0206] The pharmaceutical composition can be administered to a subject by intravenous (IV) injection, subcutaneous (SC) injection, intramuscular (IM) injection, intradermal (ID) injection, nasal spray, inhalation, oral tablets, eye drops, rectal immunization, vaginal immunization, or a combination thereof.

[0207] In some cases, pharmaceutical composition can be (for example vaccine) adjuvant.In some cases, pharmaceutical composition is preventive vaccine, therapeutic vaccine or its combination, wherein pharmaceutical composition can include adjuvant, and can further include at least one immunizing agent for stimulating the immune response of the experimenter needing it.In some cases, pharmaceutical composition is selected for treating or preventing at least one infectious disease, it is selected from chickenpox, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika and combination thereof.

[0208] The immunizing agent may comprise at least a polypeptide of the coronavirus spike (S) glycoprotein, a DNA encoding the spike (S) glycoprotein, an RNA encoding the spike (S) glycoprotein, a receptor binding domain (RBD) of the spike (S) glycoprotein, a DNA encoding the RBD, an RNA encoding the RBD, a portion thereof, or a combination thereof.

[0209] In some cases, the immunizing agent may comprise a receptor binding domain (RBD) of the spike (S) glycoprotein of at least one 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, variants thereof, or a combination thereof.

[0210] In some cases, the immunizing agent may comprise at least one polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or a combination thereof. In some cases, the immunizing agent may comprise at least one polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0211] In some cases, the weight ratio of the immunizing agent: adjuvant may be in the range of 1:50 to 50:1, wherein the weight ratio is based on the weight of the immunizing agent and the bioactive agent as an adjuvant. In some cases, suitable for the method, the pharmaceutical composition may include a weight ratio of RBD antigen fusion protein: bioactive agent in the range of 1:50 to 50:1, wherein the bioactive agent can be selected from Formula (1)-Formula (29) and combinations thereof. In some cases, the pharmaceutical composition may include a weight ratio of RBD antigen fusion protein: bioactive agent in the range of 1:50 to 50:1, wherein the bioactive agent can be selected from Formula (1), Formula (4) and combinations thereof.

[0212] The method may further include the step of administering to the subject one or more subsequent bioactive agents, the subsequent bioactive agents being selected from proteins, peptides, antibodies, fragments of antibodies, compounds, small molecule drugs, one or more chemotherapeutic drugs, vaccines, and combinations thereof, wherein the one or more subsequent bioactive agents may each be administered to the subject before, simultaneously, or after the administration of the pharmaceutical composition. In some cases, the subsequent bioactive agent may be a vaccine different from the pharmaceutical composition. In some cases, the pharmaceutical composition may be a vaccine for treating or preventing a disease caused by coronavirus, and the subsequent bioactive agent may be a vaccine or a vaccine for treating or preventing chickenpox, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika, or a vaccine of a combination thereof. Commercially available vaccines and vaccines listed by the US Centers for Disease Control and Prevention (CDC) can be used as follow-up biologically active agents.

[0213] The subsequent bioactive agents described herein may include any chemical or small molecule drug, chemotherapeutic drug, inorganic-based drug, biological drug or macromolecular-based drug, modifications or derivatives thereof, and combinations thereof.

[0214] In some cases, the pharmaceutical composition may include an adjuvant and an immunizing agent disclosed herein, wherein the adjuvant is administered to a subject in need thereof before, at the same time, or after the immunizing agent is administered to the subject. In some cases, the pharmaceutical composition may include an adjuvant containing formula (1)-(29) or a combination thereof, wherein the adjuvant may be administered to a subject in need thereof before, together (mixed or co-formulated) or after the immunizing agent (e.g., commercially available vaccines, DNA, mRNA, or proteins, such as RBD antigen fusion proteins disclosed herein). In some cases, the adjuvant may be administered 1 minute to 1 day, 5 days, 10 days, 21 days, 30 days, 60 days, 90 days, or 120 days before the administration of the immunizing agent. In some cases, the adjuvant may be administered simultaneously with the immunizing agent. In some cases, the adjuvant may be administered 1 minute to 1 day, 5 days, 10 days, 21 days, 30 days, 60 days, 90 days, or 120 days after the administration of the immunizing agent.

[0215] In some cases, an adjuvant of the present disclosure comprising a polymer and a compound of formula (1)-(29) or a combination thereof can be administered to a subject in need thereof before, together (blended or co-formulated), or after administration of a vaccine for treating or preventing a disease selected from varicella, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika, and combinations thereof.

[0216] In some cases, a polymer, a compound of formula (1)-(29) or a combination thereof and an immunogenic agent (comprising at least SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 NO:21, or a combination thereof) can be administered to a subject in need thereof before, together (mixed or co-formulated) or after administration of a vaccine for treating or preventing a disease, the disease being selected from varicella, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika, and combinations thereof.

[0217] In some cases, the method may further include the step of administering one or more booster injections of a pharmaceutical composition (e.g., an adjuvant or vaccine disclosed herein) to the subject. In some cases, the method may include the step of administering one or more booster injections using an adjuvant. In some cases, the method may include the step of administering one or more booster injections using a vaccine (comprising an adjuvant and one or more immunizing agents). In some cases, the method may include the step of administering one or more booster injections using an adjuvant disclosed herein (comprising formula (1)-(29) or a combination thereof) and an RBD antigen fusion protein to the subject.

[0218] The subsequent biologically active composition can be administered by intravenous (IV), intramuscular (IM), subcutaneous (SC) or intradermal (ID) injection, orally, by inhalation, nasally, ocularly (e.g., using drops or ointments), transdermally (e.g., using patches), or a combination thereof. Combinations of any of the above routes of administration may also be suitable.

[0219] Suitable for use in the methods, the pharmaceutical composition may have a weight ratio of immunizing agent:adjuvant in the range of 1:50 to 50:1, wherein the weight ratio is based on the weight of the immunizing agent and the bioactive agent as adjuvant.

[0220] As disclosed above and below, the nanoaggregates can be linked to targeting moieties or groups to form conjugates, such that the targeting groups are incorporated with the desired nanocomposite particles. Figures 10A-10B ), the targeting moiety or group includes but is not limited to an antibody (or an antigen-binding portion thereof), an antigen, a cognate carbohydrate (e.g., sialic acid), a cell surface receptor ligand, a moiety that binds to a cell surface receptor (e.g., prostate-specific membrane antigen (PSMA)), a moiety that binds to a cell surface carbohydrate, an extracellular matrix ligand, a cytosolic receptor ligand, a growth factor, a cytokine, an incretin, a hormone, a lectin, a lectin target (e.g., galactose, a galactose derivative, N-acetylgalactosamine, mannose, a mannose derivative, etc.), a vitamin (e.g., folic acid, biotin, etc.), avidin, streptavidin, neutravidin, etc.

[0221] In some cases, the bioactive agent can be dissolved in methanol or ethanol in various amounts up to 40 mg / mL. The hydrocarbon (CH3(CH2) 17 ) modified randomly branched PEOX60 (molar ratio of monomer to initiator = 60:1) (referred to herein as C 18 PEOXABP60) and dissolved in methanol or ethanol at different concentrations up to 100 mg / mL. The two solutions can then be mixed in various volumes so that the final homopolymer and bioactive agent weight ratio in the mixture is 2:1 to 20:1 to form nanoaggregates. The nanoaggregates can be rotary evaporated to dryness to form dry nanoaggregates. The dry nanoaggregates can then be redissolved in water or saline, followed by sterile filtration with a 0.22 μm filter and freeze-dried for 20 to 72 hours, depending on the volume, to produce freeze-dried nanoaggregates as dry powders.

[0222] In some cases, the polymer mixture H / C 18PEOXABP60 may be suitable. Polymer mixture H / C 18 PEOXABP60 may include a polymer having a second end group modified with -OH, NH2, or a combination thereof.

[0223] Pharmaceutical compositions comprising the nanoaggregates disclosed herein can be formulated to be compatible with the intended route of administration and can include one or more pharmaceutically suitable carriers. Examples of routes of administration include parenteral administration, such as intravenous (IV), intradermal (ID), subcutaneous (SC), oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration can include one or more pharmaceutically suitable carriers, such as sterile diluents, such as water for injection, saline, oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as HCl or NaOH. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic as articles of manufacture. The pharmaceutical composition can be packaged in a container, pack or dispenser together with instructions for administration.

[0224] The pharmaceutical composition may be suitable for injection and may include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injections or dispersions. For intravenous administration, suitable carriers may include physiological saline, antibacterial water, E (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). The composition is sterile and fluid to the extent that there is injectability. The composition must be stable under the conditions of manufacture and storage, and must be preserved to avoid the contaminating effects of microorganisms (such as bacteria and fungi). The pharmaceutical composition may contain one or more solvents or dispersion media, including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid PEG, polysorbate, etc.) and suitable mixtures thereof. Some pharmaceutically suitable carriers can be used to maintain the appropriate fluidity of the composition, such as by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, using a thickener, and using a surfactant. Further pharmaceutically suitable carriers may include various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, ascorbic acid, etc.) to prevent or inhibit the growth or action of microorganisms; and isotonic agents may be included in the composition as pharmaceutically suitable carriers, such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride. Agents that delay absorption, such as aluminum monostearate or gelatin, can also be used as pharmaceutically suitable carriers.

[0225] In further embodiments, the pharmaceutical composition may include one or more pharmaceutically suitable carriers that will protect the compound from being rapidly cleared from the subject's body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations are apparent to those skilled in the art. Materials may also be commercially available from, for example, Alza Corporation and Nova Pharmaceuticals, Inc.

[0226] Sterile injectable solutions can be prepared in the following manner: the required amount of active compound is incorporated into an appropriate solvent containing one or a combination of ingredients listed above (as required), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle comprising a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof.

[0227] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier to obtain a syrup or liquid preparation, or used as a mouthwash, wherein the compound in the fluid carrier is administered orally, swished and expectorated or swallowed.

[0228] Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as mint, methyl salicylate, or orange flavoring.

[0229] For administration by inhalation, the compounds are delivered in the form of, for example, an aerosol spray from a pressured container or dispenser that contains a suitable propellant (eg, a gas such as carbon dioxide); a nebulizer; or a spray.

[0230] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream commonly known in the art. Another known penetrant is dimethyl sulfoxide.

[0231] The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0232] For ease of administration and uniformity of dosage, it is advantageous to formulate oral or parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as single doses for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic endpoint. The dosage, such as the preferred route of administration and amount, can be obtained by implementing methods known in the art based on empirical data obtained from preclinical and clinical studies. The dosage and delivery form may depend on and may be dependent on the characteristics of the bioactive agent, the characteristics of the polymer, the specific therapeutic effect to be achieved, the characteristics and conditions of the recipient, etc. For repeated administration over several days or longer, depending on the condition, treatment can be continued until the desired endpoint is reached.

[0233] In some cases, the present disclosure relates to a nanoaggregate comprising a polymer and at least one biologically active agent comprising at least one STING polypeptide or portion thereof, a nucleic acid encoding the STING polypeptide or portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING antagonist, a STING modulatory molecule, or a combination thereof;

[0234] wherein the nanoaggregates are soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution;

[0235] wherein the polymer is water soluble; and

[0236] The polymer includes:

[0237] A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% hydrophobic moiety, the hydrophobic moiety comprising a saturated or unsaturated aliphatic hydrocarbon, aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, amide, imine, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate, or a combination thereof; or

[0238] a second polymer comprising one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), poly aspartic acid (PasP), poly (L-histidine) (PLH), poly (vinylamine) (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP), or a combination thereof.

[0239] In some cases, about 1% to 100% of the second end groups may be free of primary amines. In some cases, 1% to 100% of the second end groups of the nanoaggregates disclosed herein may contain hydroxyl groups. All percentages are based on the total number of second end groups.

[0240] In some cases, the size of the nano aggregates can be less than 150nm before freeze-drying. In some cases, the size of the nano aggregates can be less than 120nm before freeze-drying. In some cases, as previously described, the size of the nano aggregates can be in the range of about 0.01nm or about 0nm to about 150nm before freeze-drying. In some cases, the size of the nano aggregates can be in the range of about 50nm to about 120nm before freeze-drying.

[0241] In some cases, the poly(2-ethyloxazoline) can comprise a molar ratio of monomer to initiator in a range of 50:1 to 80:1.

[0242] In some cases, the nanoaggregates may have a weight ratio of polymer to bioactive agent in a range of about 2: 1 to about 200: 1. In some cases, the nanoaggregates may have a weight ratio of polymer to bioactive agent in a range of about 5: 1 to about 8:1.

[0243] The nanoaggregates may be free of human serum albumin, organic solvents, detergents or oils. The nanoaggregates may be free of human serum albumin, organic solvents, detergents, oils or free acids.

[0244] In some cases, suitable for use in the nanoaggregates disclosed herein, the bioactive agent may include at least a compound having formula (1) to formula (29) ( Figures 11A-11E ), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

[0245] In some cases, the bioactive agent may comprise a compound having the formula

[0246] Formula (1)

[0247] or

[0248] Formula (4)

[0249] or

[0250] A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

[0251] The present invention further relates to the use of nanoaggregates containing a polymer and at least one bioactive agent and optionally a pharmaceutically suitable carrier for the preparation of a medicament for treating a disease, wherein the bioactive agent includes at least one STING polypeptide or a portion thereof, a nucleic acid encoding the STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof;

[0252] wherein the disease is selected from immune diseases, infectious diseases, and combinations thereof;

[0253] wherein the nanoaggregates are soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution;

[0254] wherein the polymer is water soluble; and

[0255] The polymer includes:

[0256] A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% hydrophobic moiety, the hydrophobic moiety comprising a saturated or unsaturated aliphatic hydrocarbon, aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, amide, imine, imide, carboxyl, hydroxyl, ester, ether, acetate, phosphate, ketone, aldehyde, sulfonate, or a combination thereof; or

[0257] a second polymer comprising one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), polyaspartic acid (PasP), poly (L-histidine) (PLH), poly (vinylamine) (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP); or a combination thereof.

[0258] In some cases, the polymer can include a first polymer as disclosed herein.

[0259] In some cases, the polymer may be composed of a first polymer as disclosed herein. In some cases, the pharmaceutical composition may be free of a polymer selected from: one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), poly aspartic acid (PasP), poly (L-histidine) (PLH), poly (vinylamine) (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP), and combinations thereof.

[0260] In some cases, about 1% to 100% of the second end groups may be free of primary amines. In some cases, 1% to 100% of the second end groups of the nanoaggregates disclosed herein may contain hydroxyl groups. All percentages are based on the total number of second end groups.

[0261] In some cases, the polymer may include one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), polyaspartic acid (PasP), poly (L-histidine) (PLH), polyvinylamine (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP), or a combination thereof.

[0262] In some cases, the polymer may include a first polymer and one or more subsequent polymers (also referred to as "second polymers") selected from one or more hydroxy dendrimers (HD), ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof, poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(propylene oxide) (PPO), poly(caprolactone) (PCL), (PPO-PEO), poly (γ-L-glutamic acid) (PGA), poly (L-phenylalanine ethyl ester) (PAE), poly (L-lysine) (PLL), methyl PEG (mPEG), poly aspartic acid (PasP), poly (L-histidine) (PLH), poly (vinylamine) (PEI), poly (N-vinyl pyrrolidone) (PVP), poly (L-leucine) (PLLeu), deoxycholic acid (DOCA), hydroxypropyl methylcellulose (HPMC), poly (hydroxybutyrate) (PHB), poly (ethylene oxide) (PEO), poly (γ-benzyl L-glutamate) (PBLG), phosphatidylserine (PS), poly (isohexyl cyanoacrylate) (PIHCA), poly (allylamine hydrochloride) (PAH), poly (γ-propargyl) (PP), and combinations thereof.

[0263] The bioactive agent may include at least a compound of formula (1) to formula (29) ( Figures 11A-11E), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

[0264] In some cases, the bioactive agent may comprise a compound having the formula

[0265] Formula (1)

[0266] or

[0267] Formula (4)

[0268] or

[0269] A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

[0270] In some cases, the disease may include an immune disease, an infectious disease, and combinations thereof.

[0271] In some cases, the polymer may include a polyoxazoline (POX) comprising a linear portion, a branched portion, or a combination thereof, and wherein the polyoxazoline (POX) includes poly(2-oxazoline), poly(2-methyloxazoline), poly(2-ethyloxazoline), poly(2-propyloxazoline), poly(isopropyloxazoline), or a combination thereof.

[0272] Applicants have unexpectedly discovered that adjuvants comprising a polymer disclosed herein and a compound having formula (1)-(29) ("Nano-Adjuvant") significantly enhance the antigenicity of a variety of antigens, immunogens, immunizing agents, and the like. In some cases, Nano-Adjuvant can enhance immune responses compared to those generated by traditional aluminum-containing adjuvants. In some cases, Nano-Adjuvant can enhance the production of neutralizing antibodies ( Fig.16D ).

[0273] The present disclosure will now be illustrated in the following non-limiting examples.

[0274] Example

[0275] The present invention is further defined in the following examples. It should be understood that these examples, although representing preferred embodiments of the present invention, are given by way of illustration only. From the above discussion and examples, those skilled in the art will be able to determine the essential characteristics of the present invention and to make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from its spirit and scope.

[0276] Materials and measurements

[0277] polymer

[0278] As previously described in PCT Publication WO2014 / 123791 (incorporated herein by reference in its entirety), an initiator (CH3(CH2) 17 )-Br to prepare hydrocarbon (CH3(CH2) with a monomer and initiator molar ratio of 60:1 17 ) modified randomly branched PEOX polymer (hereinafter referred to as "C 18 PEOXABP60”).

[0279] Another hydrocarbon-modified randomly branched PEOX polymer having a monomer to initiator molar ratio = 60:1 was prepared as described above using the initiator (CH3(CH2)5)-Br.

[0280] As described above, the initiator (CH3(CH2) 11 )-Br Another hydrocarbon-modified randomly branched PEOX polymer having a monomer to initiator molar ratio of 60:1 was prepared.

[0281] Another hydrocarbon-modified randomly branched PEOX polymer having a monomer to initiator molar ratio = 60:1 was prepared as described above using the initiator methyl p-toluenesulfonate.

[0282] A hydrogen-modified randomly branched PEOX polymer (herein "H-PEOXABP60") having a monomer to initiator molar ratio = 60:1 was prepared as described above with p-toluenesulfonic acid as the initiator.

[0283] Another hydrogen-modified randomly branched PEOX polymer having a monomer to initiator molar ratio = 60:1 was prepared as described above using the initiator trifluoroacetic acid.

[0284] The hydrogen-modified randomly branched PEOX polymers having a monomer to initiator molar ratio of 60:1 are collectively referred to as "H-PEOXABP60".

[0285] Table 1 shows the results of the experiments with H(H-PEOXABP60) and C 18 Hydrocarbon (C 18 Some non-limiting examples of polymers with various first ends modified with PEOXABP60). H and C 18 The presence of hydrocarbon modified first end groups was measured by HPLC. 18 The molar ratio of hydrocarbons and the percentage of H.

[0286] Table 1. Examples of mixed polymers H-PEOXABP60 and C 18 H and C of PEOXABP60 18 The ratio of hydrocarbons and the percentage of H (molar ratio).

[0287]

[0288] Initiator molar ratio = 60:1 containing hydrocarbon (CH3 (CH2) 17 ) modified first terminal and H modified first terminal (hereinafter referred to as "H / C 18 PEOXABP60”) was used to generate nanoaggregates.

[0289] With H / C of about 0.3 18 Polymer H / C 18 PEOXABP60 is terminated with a hydroxyl group as a second end group in water (referred to herein as "Polymer A1"). The pH value of the aqueous solution of Polymer A1 is in the range of 4.0 to 6.9. If necessary, the aqueous solution of Polymer A1 can be adjusted to have a pH value in the range of 5.6 to 7.5 or 4.0 to 10 using HCl or NaOH.

[0290] With H / C of 0.4 18 Polymer H / C 18 PEOXABP60 is terminated with a hydroxyl group as a second end group in water (referred to herein as "polymer A2"). The aqueous solution of polymer A2 can be adjusted to have a pH value in the range of 4.0 to 6.9. Another aqueous solution of polymer A2 can be adjusted to have a pH value in the range of 7.0 to 10 or 5.6 to 7.5 using HCl or NaOH.

[0291] With H / C of about 0.7 18 Polymer H / C 18 PEOXABP60 is terminated with a hydroxyl group as a second end group in water (referred to herein as "polymer A3"). The aqueous solution of polymer A3 has a pH value in the range of 4.0 to 6.9. If necessary, HCl or NaOH can be used to adjust the aqueous solution of polymer A3 to have a pH value in the range of 7.0 to 10 or 5.6 to 7.5.

[0292] All H / C 18 PEOXABP60 polymer can be called H / C 18 PEOXABP60-OH.

[0293] With H / C of about 0.7 18 Polymer H / C 18PEOXABP60 is end-capped with EDA (the molar ratio of the polyoxazoline reactive chain end to EDA is about 1:10), producing a polymer having a second end group comprising a group modified with a primary amine (referred to herein as "polymer B1"). The aqueous solution of polymer B1 has a pH value in the range of 7.0 to 10. HCl or NaOH can be used to adjust the aqueous solution of another polymer B1 to have a pH value in the range of 8.9 to 9.7. The aqueous solution of another polymer B1 can be adjusted to have a pH value in the range of 4.0 to 6.9.

[0294] With H / C of about 0.4 18 Polymer H / C 18 PEOXABP60 is end-capped with EDA (the molar ratio of polyoxazoline reactive chain ends to EDA is about 1:10), resulting in a polymer having a second end group comprising a group modified with a primary amine (referred to herein as "Polymer B2"). An aqueous solution of Polymer B2 can be adjusted to have a pH in the range of 7.0 to 10. Another aqueous solution of Polymer B2 can be adjusted to have a pH in the range of 4.0 to 10 or 5.6 to 7.5 using HCl or NaOH.

[0295] With H / C of about 0.3 18 Polymer H / C 18 PEOXABP60 is end-capped with EDA (the molar ratio of polyoxazoline reactive chain ends to EDA is about 1:10), resulting in a polymer having a second end group comprising a group modified with a primary amine (referred to herein as "Polymer B3"). An aqueous solution of Polymer B3 can be adjusted to have a pH value in the range of 7.0 to 10. An aqueous solution of another Polymer B3 can be adjusted to have a pH value in the range of 4.0 to 10 or 5.6 to 7.5 using HCl or NaOH.

[0296] All H / C 18 PEOXABP60 polymer can be called H / C 18 PEOXABP60-NH2.

[0297] Nanoparticle measurements

[0298] The sizes of various polymers, polymer-only nanoaggregates, and drug-induced polymer-drug nanoaggregates were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS Zen 3600 particle size analyzer (Malvern Panalytical Inc., Westborough, MA 01581, USA).

[0299] Example 1-2

[0300] With H / C 18 PEOXABP60-A (polymer A1) polymer: nanoparticles of formula (1)

[0301] Adjuvant formulation 1: The polymer prepared in the above polymer section with a H / C 18 The polymer A1 (200 mg) of the present invention is dissolved in water to produce a 100 mg / g solution. The compound of formula (1) (8 mg) is added to the polymer solution and processed as described above to produce an aqueous solution of nanoaggregates of formula (1) containing 2 mg / mL with a weight ratio of polymer: formula (1) = 25:1. The solution is filtered with 0.8 and 0.2 μm filters and freeze-dried for 20-100 h to produce a freeze-dried dry powder. The vial is stoppered and the ready-to-use white powder is stored at room temperature.

[0302] Adjuvant formulation 2: The above-prepared 18 The polymer A1 (400 mg) of the present invention was dissolved in water to produce a 100 mg / g solution. The compound of formula (1) (8 mg) was added to the polymer solution and processed as described above to produce an aqueous solution of nanoaggregates of formula (1) containing 2 mg / mL with a weight ratio of polymer: formula (1) = 50:1. The solution was filtered with 0.8 and 0.2 μm filters and lyophilized for 20-100 h to produce a lyophilized dry powder. The vial was stoppered and the ready-to-use white powder was stored at room temperature.

[0303] Embodiment 3-4

[0304] With H / C 18 PEOXABP60-A (polymer A2) polymer: nanoparticles of formula (1)

[0305] Adjuvant formulation 3: The above-prepared 18 The polymer A2 (400 mg) of the invention was dissolved in water to produce a 100 mg / g solution. The compound of formula (1) (8 mg) was added to the polymer solution and processed as described above to produce an aqueous solution of nanoaggregates of formula (1) containing 2 mg / mL with a weight ratio of polymer:formula (1) = 50:1. The solution was filtered with a 0.8 / 0.2 μm filter set and lyophilized for 20-100 h to produce a lyophilized dry powder. The vial was stoppered and the ready-to-use white powder was stored at room temperature.

[0306] Adjuvant formulation 4: The above-prepared 18The polymer A3 (400 mg) of the invention was dissolved in water to produce a 100 mg / g solution. The compound of formula (1) (8 mg) was added to the polymer solution and processed as described above to produce an aqueous solution of nanoaggregates of formula (1) containing 2 mg / mL with a weight ratio of polymer:formula (1) = 50:1. The solution was filtered with 0.8 and 0.2 μm filters and lyophilized for 20-100 h to produce a lyophilized dry powder. The vial was stoppered and the ready-to-use white powder was stored at room temperature.

[0307] Embodiment 5-6

[0308] Adjuvant formulation 5: The above-prepared 18 The polymer B3 (400 mg) of the invention was dissolved in water to produce a 100 mg / g solution. The compound of formula (1) (8 mg) was added to the polymer solution and processed as described above to produce an aqueous solution of nanoaggregates of formula (1) containing 2 mg / mL with a weight ratio of polymer:formula (1) = 50:1. The solution was filtered with 0.8 and 0.2 μm filters and lyophilized for 20-100 h to produce a lyophilized dry powder. The vial was stoppered and the ready-to-use white powder was stored at room temperature.

[0309] Adjuvant formulation 6: The above-prepared 18 The polymer B3 (200 mg) of the present invention was dissolved in water to produce a 100 mg / g solution. The compound of formula (1) (8 mg) was added to the polymer solution and processed as described above to produce an aqueous solution of nanoaggregates of formula (1) containing 2 mg / mL with a weight ratio of polymer: formula (1) = 25:1. The solution was filtered with 0.8 and 0.2 μm filters and lyophilized for 20-100 h to produce a lyophilized dry powder. The vial was stoppered and the ready-to-use white powder was stored at room temperature.

[0310] Adjuvant preparations are collectively referred to as "Nano-Adjuvant" or "Nan-Ad".

[0311] Example 7

[0312] Antigen design, construction, expression and purification

[0313] 1. Construction of RBD antigen fusion protein

[0314] Two or more RBD (receptor binding domain) antigen sequences from SARS-CoV-2 (Delta) (SEQ ID NO: 1), SARS-CoV (SEQ ID NO: 2), MERS-CoV (SEQ ID NO: 3) are linked together in series via a linker sequence to form an RBD antigen construct. Examples of constructs with three RBD sequences AG1-AG6 are schematically shown in Fig.12 In. Linker 1 and linker 2 may be the same or different. The AG1 RBD antigen protein with SARS-CoV-2 (Delta) RBD-linker-SARS-CoV RBD-linker-MERS-CoV RBD comprising linker L20 (SEQ ID NO:4: GGGGSGGGGSGGGGSGGGGS) is listed in the sequence table below (SEQ ID NO:5). The nucleic acid construct of AG1 was cut with restriction enzymes Eco RI and Nco1 and cloned into the expression vector pFUSE-hIgG1-Fc2, so that the target sequence was fused with the human IgG1Fc on the vector to form a fusion expression cassette (also referred to as "RBD antigen fusion protein expression cassette"). The fusion protein expression cassette (also referred to as "RBD antigen fusion protein expression cassette") was named "S2S1ML20 cassette" (S2: SARS-CoV-2 (Delta), S1: SARS-CoV, M: MERS-CoV, L20: linker L20). The fusion protein expressed from the S2S1ML20 cassette (also referred to as "RBD antigen fusion protein") is referred to as S2S1ML20. The viral lineage is shown in Fig.13A Examples of expression cassettes and corresponding RBD antigen fusion proteins are given in Fig. 13B Examples of antibodies with fusion proteins are shown in Figure 13C-13F Schematically illustrated in the example of an antigen fusion protein with three RBDs and an L15 linker ( Fig. 13C ), an example of an antigen fusion protein having three RBDs and an L20 linker ( Fig.13D ), an example of an antigen fusion protein with two RBDs (SARS2 RBD and MERS RBD) ( Fig.13E ) and another example of an antigen fusion protein with 2 RBDs (SARS2 RBD and SARS RBD) ( Fig.13F ). The linker may be located between RBD sequences, such as Fig. 13B In the embodiment, the antigen fusion protein is in the form of an IgG-like immunoglobulin. Representative RBD antigen amino acid sequence alignments are shown in Fig.14 As used herein, the terms SARS or SARS-CoV refer to SARS-CoV and are used interchangeably. The terms SARS2 or SARS-CoV-2 refer to SARS-CoV-2. The terms MERS or MERS-CoV refer to MERS-CoV.

[0315] Additional fusion proteins were also generated and included: S2S1ML10 with SARS-CoV-2 (Delta) RBD-Linker L10-SARS-CoV RBD-Linker L10-MERS-CoV RBD (SEQ ID NO:6); S2S1ML15: SARS-CoV-2 (Delta) RBD-Linker L15-SARS-CoV RBD-Linker L15-MERS-CoV RBD (SEQ ID NO:7); S2ML15: SARS-CoV-2 (Delta) RBD-Linker L15-MERS-CoV RBD (SEQ ID NO:8); and S2SL15: SARS-CoV-2 (Delta) RBD-Linker L15-SARS-CoV RBD (SEQ ID NO:9).

[0316] The linkers are: linker L15 (SEQ ID NO: 10) and linker L10 (SEQ ID NO: 11).

[0317] Additional RBD sequences include GenBank Accession No. UJH58758.1, SARS-CoV-2 (WA1) RBD (SEQ ID NO: 12); GenBank Accession No. UNE80990.1, SARS-CoV-2 (BA.2) RBD (SEQ ID NO: 13); GenBank Accession No. MT040334.1, pangolin coronavirus RBD (SEQ ID NO: 14); GenBank Accession No. YP_009825051.1, SARS-CoV RBD (SEQ ID NO: 15); GenBank Accession No. AGZ48828.1, WIV1 RBD (SEQ ID NO: 16); GenBank Accession No. AGZ48818.1, Rs3367 RBD (SEQ ID NO: 17); GenBank Accession No. QJE50589.1, SHC014 RBD (SEQ ID NO: 18); NO:18); GenBank Accession No. AFS88936.1, MERS-CoV evolutionary branch A (SEQ ID NO:19); GenBank Accession No. AKJ80137.2, MERS-CoV evolutionary branch B (SEQ ID NO:20); and GenBank Accession No. AVN89387.1, MERS-CoV evolutionary branch C (SEQ ID NO:21), are also used as antigens or antigen fusion proteins.

[0318] 2. Expression and purification of RBD antigen fusion protein

[0319] 2.1. Grow Expi293F cells to a cell density of 1×106 / ml.

[0320] 2.2. Cells were transfected with a vector containing the S2S1ML20 cassette using the transfection agent EsayTrans (available from Life-iLab).

[0321] 2.3. Continue culturing the cells for 5 days and then collect the supernatant by centrifuging the cells at 3000 rpm.

[0322] 2.4. Expressed S2S1ML20 was purified using Protein A beads (available from GenScript) in a gravity column equilibrated with PBS (phosphate buffered saline) at 4°C for 2 hours. Wash with PBS. Elute the protein with glycine solution.

[0323] 2.5. Check the purity and size of the protein using SDS-PAGE.

[0324] Example 8

[0325] Nasal vaccination

[0326] Fifteen BALB / c mice (female, 8 weeks old, available from Vital River Laboratories) were randomly divided into 5 groups of 3 mice each. The mice were given vaccines with antigenic proteins and adjuvant formulations shown in Table 2 intranasally. The mice were killed according to the approved protocol 6 hours after administration of the nasal vaccine component (also referred to as "nasal vaccination"). Lung samples were homogenized with TRIzol and total RNA was extracted according to the method provided by the TRIzol manufacturer. The mRNA levels of cytokines IFN-β, CXCL-10, CXCL-9, CCL-2, IL-6, IB-1β, IFN-g, TNF-α, CD40, CD86, TGFB-1 and IL-12 in the samples were quantified using RT-qPCR (reverse transcription quantitative polymerase chain reaction) kits (available from Takara).

[0327] Representative results are shown in Fig.15A In the graphs, the group IDs are shown in Table 2 and the amounts of compounds having formula (1) are shown. The intensities shown in grayscale 1-4 (S: intensity scale) reflect the level of immune response in induced cytokine production, with darker gray representing higher mRNA levels.

[0328] The data show that the vaccine composition with RBD antigen protein and adjuvant formulation 3 was used for most tests. Other adjuvant formulations prepared in Examples 1-6 above showed similar stronger immune responses when measured by cytokine production and other methods.

[0329] Table 2. Nasal vaccine components

[0330] Group ID PBS Antigenic protein S2S1ML20 Nano-Adjuvant PBS 20μL - - 0 20μL 5μg - 1 20μL 5μg 1 μg 5 20μL 5μg 5μg 20 20μL 5μg 20 μg

[0331] Example 9

[0332] Induction of immune responses in betacoronavirus-specific IgG

[0333] Thirty BALB / c mice (available from Vital River Laboratories) were randomly divided into five groups, with six mice in each group. The mice were immunized with the vaccine components and administration routes shown in Table 3. Untreated group ( group) were not vaccinated.

[0334] Each mouse was immunized on days 0, 28, and 56. Serum samples were collected on days 35 and 63.

[0335] The collected serum samples were heat inactivated at 56°C for 30 min. The levels of IgG specific for the RBD of SARS-CoV-2, SARS-CoV, and MERS-CoV were measured by ELISA. Briefly, the RBD proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV were coated in the wells of the ELISA plate, incubated overnight at 4°C, and then blocked with PBS (5% BSA, bovine serum albumin) at 37°C for 2 hours. The serum samples were serially diluted and added to the wells of the blocked ELISA plate. The plate was incubated at 37°C for 30 min and then washed 5 times with PBST (PBS containing 0.05% Tween-20). HRP-labeled rabbit anti-mouse IgG antibody was then added to the wells and incubated at 37°C for 30 min. The wells were washed 5 times with PBST, and then the chromogenic substrate TMB was added to the wells. After terminating the color reaction with H2SO4, the plate was analyzed using an enzyme reader at A 450 The color intensity of the plate was measured.

[0336] Representative results are shown in Figure 15B-Figure 15D And in Tables 4-6, from mice receiving 2 doses of vaccine components. The data show that both intramuscular (IM) injection and intranasal (IN) vaccination induce a strong immune response, which is measured by IgG specific for SARS-CoV-2, SARS-CoV and MERS-CoV RBD proteins. All P values ​​are compared with the untreated group by one-way ANOVA. The amount of adjuvant is based on the amount of compound formula (1) in the formulation.

[0337] Table 3. Vaccine components

[0338] Group ID Drug administration Antigenic protein S2S1ML20 Nano-Adjuvant IM-20 Intramuscular (IM) 5μg 20 μg IN-20 Intranasal (IN) 5μg 20 μg IN-5 Intranasal (IN) 5μg 5μg IN-1 Intranasal (IN) 5μg 1 μg Unprocessed - - -

[0339] Table 4. SARS-CoV-2 RBD-specific IgG titers in mouse serum on day 35

[0340]

[0341] Table 5. SARS-CoV RBD-specific IgG titers in mouse serum on day 35

[0342]

[0343] Table 6. MERS-CoV RBD-specific IgG titers in mouse serum on day 35

[0344]

[0345] Representative results are shown in Fig.16A In Table 7, from mice receiving 3 doses of vaccine components (63 days after the first administration of vaccine components). The data show that both intramuscular (IM) injection and intranasal (IN) vaccination induce a stronger immune response, which is measured by the level of SARS-CoV-2RBD-specific IgG. All P values ​​were compared with the untreated group by one-way analysis of variance.

[0346] Table 7. SARS-CoV-2 RBD-specific IgG titers in mouse sera 63 days after the first administration of vaccine components

[0347]

[0348] Example 10

[0349] Induction of immune responses in SARS-CoV-2 RBD-specific IgA

[0350] The level of IgA specific to SARS-CoV-2 in the mice immunized above was measured by ELISA. Briefly, the RBD protein of SARS-CoV-2 was coated into the wells of the ELISA plate by incubation at 4°C overnight. In addition to using HRP-labeled rabbit anti-mouse IgA secondary antibody, the same method as above was followed.

[0351] Representative results are shown in Fig. 16B and are provided in Table 8. No SARS-CoV-2RBD-specific IgA was detected in mouse serum at 63 days after intramuscular (IM) injection. However, intranasal (IN) vaccination induced significant levels of SARS-CoV-2RBD-specific IgA in mouse serum.

[0352] Table 8. SARS-CoV-2 RBD-specific IgA titers in mouse sera 63 days after first administration of vaccine components

[0353]

[0354] Embodiment 11

[0355] Induction of immune responses in SARS-CoV-2 RBD-specific IgA in the lung

[0356] The level of IgA specific to SARS-CoV-2 in the lungs of the mice immunized above was measured by ELISA. In brief, at day 63, 1 mL of PBS was introduced into the lungs of mice and bronchoalveolar lavage (BAL) fluid was collected. SARS-CoV-2 specific IgA was measured by ELISA as described above.

[0357] Representative results are shown in Fig. 16C and Table 9. The data demonstrate the presence of SARS-CoV-2-specific IgA in mouse BAL fluid after intranasal (IN) inoculation.

[0358] Table 9. SARS-CoV-2 RBD-specific IgA titers in mouse sera 63 days after first administration of vaccine components

[0359] Group ID IN-20 IN-5 IN-1 Unprocessed RBD-specific titer ± SEM 270±0 210±37 270±0 5±0 P-value P<0.0001 P<0.0001 P<0.0001 /

[0360] Example 12

[0361] Neutralizing antibody titers in mice immunized with SARS-CoV-2 (D614G) and different adjuvants

[0362] Mice were immunized with 5 μg of the RBD antigen fusion protein S2S1ML20 formulated with the adjuvants listed in Table 10. Nano-Adjuvant (Adjuvant Formulation 3) comprising compound formula (1) was prepared as described above. Neutralizing antibodies specific for SARS-CoV-2 (D614G) were determined as described above. The data are shown in Tables 10 and Fig.16D The data showed that Nano-Adjuvant resulted in significantly higher (more than 10-fold) neutralizing antibody titers compared to traditional aluminum-containing adjuvants (available from Thermo Fisher). Intramuscular (IM) and intranasal (IN) injections had similar results.

[0363] Table 10. Titers of neutralizing antibodies from mice immunized with RBD antigen S2S1ML20 and different adjuvants

[0364]

[0365] Embodiment 13

[0366] Cellular immune responses in the lung after intranasal (IN) vaccination

[0367] The levels of IFN-γ and IL-4 (specific for SARS-CoV-2, SARS-CoV and MERS-CoV RBD) of mice immunized with intranasal (IN) vaccination (nasal immunization) (expressed as IN-1 above) were measured. In brief, mice were killed according to the approved protocol on the 63rd day after the administration of the nasal vaccine component. Lung samples were collected and treated with collagenase D and DNase I to produce a single lung cell suspension. Spleen samples were collected and homogenized to prepare a single spleen cell suspension. Red blood cells were removed with red blood cell lysate. The levels of IFN-γ and IL-4 specific for SARS-CoV-2, SARS-CoV and MERS-CoV RBD were measured with ELISPOT kits (available from Mabtech).

[0368] In short, about 2×10 5 Lung or spleen cells were added to the pre-coated wells of the ELISPOT plate. About 2 μg of SARS-CoV-2, SARS-CoV, and MERS-CoV proteins were added to stimulate the cells, and the plates were incubated for 48 hours without shaking. The wells were then washed 5 times with PBS. Biotinylated IFN-γ and IL-4 antibodies were added and incubated for 2 hours. After washing with PBS, streptavidin-labeled ALP (available from Mabtech) was added and the plates were incubated for 1 hour. A colorimetric phosphatase substrate was then added to the wells. The color sites on the cells were counted.

[0369] Representative results are shown in Figures 17A-17D The data showed that the vaccine components induced a strong cellular immune response in the lungs, as measured by SARS-CoV-2, SARS-CoV, and MERS-CoV RBD-specific IFN-γ and IL-4 after intranasal (IN) vaccination. The data also showed that there were SARS-CoV-2, SARS-CoV, and MERS-CoV-specific immune responses in the spleen.

[0370] Embodiment 14

[0371] Dose levels and IgG immune responses for intranasal (IN) and intramuscular (IM) immunization

[0372] The data presented above indicate that various doses of antigen and Nano-Adjuvant induce a broad immune response in mice. A dose of 2 μg Nano-Adjuvant (Adjuvant Formulation 3) was tested for intranasal (IN) immunization and a dose of 20 μg Nano-Adjuvant (Adjuvant Formulation 3) was tested for intramuscular (IM) immunization.

[0373] In brief, 24 BALB / c mice were randomly divided into 4 groups, 6 mice in each group. The mice of Group 1-Group 3 (G1-G3) were given the vaccine doses shown in Table 11 and the specified administration routes. Mice were immunized on Day 0 and Day 28. Group 4 (G4) mice were not vaccinated. On Day 35, serum was collected from each mouse. The level of IgG specific for SARS-CoV-2, SARS-CoV and MERS-CoV RBD proteins was measured as described above.

[0374] Representative examples are shown in Figures 18A-18C and Tables 12-14. The data show that for all tested viruses, intranasal (IN) immunization 2μg and intramuscular immunization 20μg Nano-Adjuvant induced similar IgG levels (G1 and G2). The response induced by the vaccine without adjuvant was weaker, as shown by the lower IgG levels. All G1-G3 had significantly higher IgG levels compared to the untreated Group 4 (G4).

[0375] Table 11. Vaccines and immunization administration

[0376]

[0377]

[0378] Table 12. SARS-CoV-2 specific IgG titers 35 days after first immunization

[0379]

[0380] Table 13. SARS-CoV specific IgG titers 35 days after first immunization

[0381]

[0382] Table 14. MERS-CoV specific IgG titers 35 days after first immunization

[0383]

[0384] Embodiment 15

[0385] Neutralizing antibody titers from intranasal (IN) immunization

[0386] Neutralizing antibodies induced by the vaccine prepared herein are tested by pseudovirus neutralization assay. In short, in the presence of serum from mice immunized with the vaccine described herein, pseudovirus containing trimeric transmembrane spike (S) glycoprotein of coronavirus and luciferase is interacted with target cells (Huh-7 cells) containing human angiotensin converting enzyme 2 (ACE2) (SARS-CoV-2 receptor). If serum contains neutralizing antibodies, luciferase activity will be reduced.

[0387] The mouse serum prepared from the above example was used.

[0388] 1. Preparation of pseudovirus containing trimeric transmembrane spike (S) glycoprotein of coronavirus:

[0389] 1.1. Grow HEK-293T cells to appropriate density for about 24 hours.

[0390] 1.2. HEK-293T cells were transfected with VigoFect transfection reagent (available from VigoFect, China) and co-transfected with plasmid PNL-4-3 (available from NIH AIDS Reagent Program, US, catalog number: 3418) and a plasmid containing the S glycoprotein of the indicated virus (pcDNA3.1-SARS-CoV-2, pcDNA3.1-SARS-CoV or pcDNA3.1-MERS-CoV).

[0391] 1.3. Replace the culture medium after 12 hours of culture.

[0392] 1.4. Collect the culture supernatant containing pseudovirus.

[0393] 2. Neutralization assay using pseudovirus:

[0394] 2.1. Huh-7 cells were plated at 10,000 cells per well in a 96-well plate.

[0395] 2.2. Serially dilute mouse serum with DMEM medium.

[0396] 2.3. Mix pseudovirus and serum and incubate at 37°C for 30 min.

[0397] 2.4. Add the pseudovirus-serum mixture to the wells containing Huh-7 cells.

[0398] 2.5. The light level generation was measured using a luciferase kit (available from Promega).

[0399] Representative results are shown in Figures 19A-19D and Tables 15-18. The data show that the vaccine with Nano-Adjuvant containing compound formula (1) induces strong broad-spectrum neutralizing antibodies against the viruses SARS-CoV-2 (D614G strain), SARS-CoV-2Omicron (BA.5 strain), SARS-CoV and MERS-CoV. No significant neutralizing antibodies were detected without Nano-Adjuvant. The P value is compared with the level of Group 4 (G4) in which mice were not immunized.

[0400] Table 15. Titers of SARS-CoV-2 (D614G strain) neutralizing antibodies 35 days after the first immunization

[0401] Group ID G1 G2 G3 G4 Neutralization titer ± SEM 13423±3139 7673±2056 50±0 50±0 P-value P<0.0001 P<0.0001 P>0.9999 /

[0402] Table 16. Titers of SARS-CoV-2 (BA.5 strain) neutralizing antibodies 35 days after the first immunization

[0403] Group ID G1 G2 G3 G4 Neutralization titer ± SEM 3375±1774 16654±11125 50±0 50±0 P-value P<0.0001 P<0.0001 P>0.9999 /

[0404] Table 17. Titers of SARS-CoV neutralizing antibodies 35 days after the first immunization

[0405] Group ID G1 G2 G3 G4 Neutralization titer ± SEM 59701±8000 30293±5495 493±370 50±0 P-value P<0.0001 P<0.0001 P＝0.07 /

[0406] Table 18. Titers of neutralizing antibodies against MERS-CoV at 35 days after the first immunization

[0407] Group ID G1 G2 G3 G4 Neutralization titer ± SEM 18804±2251 10022±1367 349±207 50±0 P-value P<0.0001 P<0.0001 P＝0.11 /

[0408] Example 16

[0409] IgG immune response in monkeys (rhesus macaques)

[0410] Three monkeys (Macaca mulatta available from Beijing Vafory Technology Co., Ltd.) were each immunized with a vaccine prepared as above comprising 100 μg of RBD antigen fusion protein S2S1ML20 and 400 μg of Nano-Adjuvant (adjuvant formulation 3) of compound formula (1). The monkeys were immunized by intramuscular (IM) injection on days 0 and 28. On days 14 and 35, serum samples, bronchoalveolar lavage (BAL) fluid (2 mL) and nasal wash (NAL) (2 mL) were collected from each monkey.

[0411] Monkey IgG specific for the viral SARS-CoV-2, SARS-CoV or MERS-CoV RBD was measured by ELISA. Briefly:

[0412] 1. Pre-coat the wells of an ELISA plate with SARS-CoV-2, SARS-CoV, or MERS-CoV RBD protein, respectively.

[0413] 2. The wells were blocked with PBS 5% BSA at 37°C for 2 hours.

[0414] 3. Serum samples, bronchoalveolar lavage (BAL) fluid and nasal wash (NAL) were serially diluted and then added to the wells. The plates were incubated at 37°C for 30 min.

[0415] 4. Wash the wells 5 times with PBST.

[0416] 5. Add HRP-labeled rabbit anti-monkey IgG antibody and incubate at 37°C for 30 min.

[0417] 6. Wash the wells 5 times with PBST.

[0418] 7. Add TMB substrate for color development.

[0419] 8. Terminate the reaction with H2SO4. 450 Measure the color level below.

[0420] Representative results are shown in Figure 20A-20C and Tables 19-27. The data show that the vaccine induces a strong immune response, as measured by IgG specific for SARS-CoV-2, SARS-CoV, and MERS-CoV RBD proteins. The data show that the vaccine induces a significant immune response in monkeys, as measured by the level of IgG specific for the RBD protein.

[0421] Table 19. SARS-CoV-2 RBD-specific IgG titers in monkey serum 35 days after first immunization

[0422] sample Day 0 Day 35 RBD-specific titer ± SEM 183±109 1638400±819200 P-value / P＝0.0003

[0423] Table 20. SARS-CoV RBD-specific IgG titers in monkey serum 35 days after first immunization

[0424] sample Day 0 Day 35 RBD-specific titer ± SEM 700±458 2457600±819200 P-value / P＝0.0008

[0425] Table 21. MERS-CoV RBD-specific IgG titers in monkey serum 35 days after first immunization

[0426] sample Day 0 Day 35 RBD-specific titer ± SEM 100±0 614400±204800 P-value / P<0.0001

[0427] Table 22. SARS-CoV-2 RBD-specific IgG titers in monkey BAL 35 days after first immunization

[0428] sample Day 0 Day 35 RBD-specific titer ± SEM 5±0 675±270 P-value / P＝0.0002

[0429] Table 23. SARS-CoV RBD-specific IgG titers in monkey BAL 35 days after first immunization

[0430] sample Day 0 Day 35 RBD-specific titer ± SEM 5±0 675±270 P-value / P＝0.0002

[0431] Table 24. Titers of MERS-CoV RBD-specific IgG in monkey BAL 35 days after first immunization

[0432]

[0433]

[0434] Table 25. SARS-CoV-2 RBD-specific IgG titers in monkey nasal wash (NAL) 35 days after first immunization

[0435] sample Day 0 Day 35 RBD-specific titer ± SEM 5±0 75±30 P-value / P＝0.0022

[0436] Table 26. SARS-CoV RBD-specific IgG titers in monkey nasal wash (NAL) 35 days after first immunization

[0437] sample Day 0 Day 35 RBD-specific titer ± SEM 5±0 495±360 P-value / P＝0.0053

[0438] Table 27. MERS-CoV RBD-specific IgG titers in monkey nasal wash (NAL) 35 days after first immunization

[0439] sample Day 0 Day 35 RBD-specific titer ± SEM 5±0 25±10 P-value / P＝0.0161

[0440] Embodiment 17

[0441] Neutralizing antibodies in monkeys (rhesus macaques)

[0442] Neutralizing antibody assays were performed as described previously. Representative results are shown in Figure 21A-Figure 21F and Tables 28-33. P values ​​are compared to samples on day 0. The data indicate that intramuscular (IM) immunization induces a robust immune response as measured by neutralizing antibodies against the viruses and variants tested, including SARS-CoV, MERS-CoV, and SARS-CoV-2 and variants.

[0443] Table 28. Titers of neutralizing antibodies specific to SARS-CoV-2 (D614G strain) in monkey serum 35 days after the first immunization

[0444]

[0445]

[0446] Table 29. Titers of neutralizing antibodies specific to SARS-CoV-2 (BA.5 strain) in monkey serum 35 days after the first immunization

[0447] sample Day 0 Day 14 Day 35 Neutralization titer ± SEM 50±0 81±15 2329±833 P-value / 0.375 P<0.0007

[0448] Table 30. Titers of neutralizing antibodies specific to SARS-CoV-2 (BA.2.2 strain) in monkey serum 35 days after the first immunization

[0449] sample Day 0 Day 14 Day 35 Neutralization titer ± SEM 50±0 60.33±10.33 2601±793 P-value / 0.8474 P<0.0001

[0450] Table 31. Titers of neutralizing antibodies specific to SARS-CoV-2 (Delta strain) in monkey serum 35 days after the first immunization

[0451] sample Day 0 Day 14 Day 35 Neutralization titer ± SEM 50±0 959.8±448 14899±7103 P-value / 0.375 P<0.0001

[0452] Table 32. Titers of neutralizing antibodies specific to SARS-CoV in monkey serum 35 days after the first immunization

[0453] sample Day 0 Day 14 Day 35 Neutralization titer ± SEM 205±6 709±107 21246±7070 P-value / 0.0239 P<0.0001

[0454] Table 33. Titers of neutralizing antibodies specific to MERS-CoV in monkey serum 35 days after the first immunization

[0455] sample Day 0 Day 14 Day 35 Neutralization titer ± SEM 50±0 405±54 3963±1157 P-value / 0.0006 P<0.0001

[0456] Sequence Listing

[0457] SEQ ID NO:1:SARS-CoV-2 (Delta)

[0458] GenBank:ON196573.1

[0459] SARS-CoV-2(Delta)RBD

[0460] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKL PDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN

[0465] SEQ ID NO:2:SARS-CoVGenBank:YP_009825051.1

[0466] SARS-CoV RBD

[0467] NITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLT

[0472] SEQ ID NO:3: MERS-CoV GenBank: AFS88936.1

[0473] MERS-CoV RBD

[0474] QAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL

[0479] SEQ ID NO:4: Linker L20

[0480] Artificial Sequence

[0481] GGGGSGGGGSGGGGSGGGGS

[0482] SEQ ID NO:5: Complete construct AG1 S2S1ML20 SARS-CoV-2(Delta)RBD-Linker-SARS-CoV RBD-Linker-MERS-CoV RBD( Fig.12 )(SEQ ID NO:5)

[0483] S2S1ML20

[0484] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGGGGSGGGGSGGGGSGGGGSNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTGGGGSGGGGSGGGGSGGGGSQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL SEQ ID NO:6: Complete construct S2S1ML10S2S1ML10

[0499] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGGGGSGGGGSNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTGGGGSGGGGSQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL

[0514] SEQ ID NO:7: Complete construct S2S1ML15S2S1ML15

[0515] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGGGGSGGGGSGGGGSNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTGGGGSGGGGSGGGGSQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL

[0530] SEQ ID NO:8: Complete construct S2ML15S2ML15

[0531] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGGGGSGGGGSGGGGSQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL

[0541] SEQ ID NO:9: Complete construct S2SL15S2S1L15

[0542] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGGGGSGGGGSGGGGSNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLT

[0552] SEQ ID NO:10: Linker L15

[0553] Artificial sequence

[0554] L15: GGGGSGGGGSGGGGS

[0555] SEQ ID NO:11: Linker L10

[0556] Artificial sequence

[0557] L10: GGGGSGGGGS

[0558] SEQ ID NO:12

[0559] GenBank: UJH58758.1

[0560] SARS-CoV-2 (WA1) RBD

[0561] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN

[0566] SEQ ID NO:13

[0567] GenBank: UNE80990.1

[0568] SARS-CoV-2 (BA.2) RBD

[0569] RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTN

[0574] SEQ ID NO:14

[0575] GenBank:MT040334.1

[0576] Pangolin coronavirus RBD

[0577] RVQPTISIVRFPNITNLCPFGEVFNASKFASVYAWNRKRISNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVVKGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNSVKQDALTGGNYLYRLFRKSKLKPFERDISTEIYQAGSTPCNGQVGLNCYYPLERYGFHPTTGVNYQPFRVVVLSFELLNGPATVCGPKLSTT

[0582] SEQ ID NO:15

[0583] GenBank:YP_009825051.1

[0584] SARS-CoV RBD

[0585] RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTD

[0590] SEQ ID NO:16

[0591] GenBank:AGZ48828.1

[0592] WIV1 RBD

[0593] RVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFTGCVLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIGYQPYRVVVLSFELLNAPATVCGPKLSTD

[0598] SEQ ID NO:17

[0599] GenBank:AGZ48818.1

[0600] Rs3367 RBD

[0601] RVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFTGCVLAWNTRNIDATQTGNYNYKYRSLRHGKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIGYQPYRVVVLSFELLNAPATVCGPKLSTD

[0606] SEQ ID NO:18

[0607] GenBank:QJE50589.1

[0608] SHC014 RBD

[0609] RVAPSKEVVRFPNITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFLGCVLAWNTNSKDSSTSGNYNYLYRWVRRSKLNPYERDLSNDIYSPGGQSCSAVGPNCYNPLRPYGFFTTAGVGHQPYRVVVLSFELLNAPATVCGPKLSTD

[0614] SEQ ID NO:19

[0615] GenBank:AFS88936.1

[0616] MERS-CoV clade A

[0617] QAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL

[0622] SEQ ID NO:20

[0623] GenBank:AKJ80137.2

[0624] MERS-CoV clade B

[0625] QAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL

[0630] SEQ ID NO:21

[0631] GenBank:AVN89387.1

[0632] MERS-CoV clade C

[0633] QAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPFKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKF

Claims

1. A pharmaceutical composition comprising: A nanoaggregate comprising a polymer and at least one bioactive agent, wherein the bioactive agent comprises at least one STING polypeptide or a portion thereof, a nucleic acid encoding the STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof; and Optionally, a pharmaceutically suitable carrier; wherein the pharmaceutical composition is soluble in an aqueous solution to produce at least 1 mg / mL of the at least one biologically active agent dissolved in the aqueous solution; wherein the polymer is water soluble; and The polymer comprises: A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% of the hydrophobic moiety, the hydrophobic moiety comprises a saturated or unsaturated aliphatic hydrocarbon having 1 to about 22 carbons, an aromatic hydrocarbon, or a combination thereof, and the second end group comprises a group modified with an amine, an amide, an imine, an imide, a carboxyl group, a hydroxyl group, an ester, an ether, an acetate, a phosphate, a ketone, an aldehyde, a sulfonate, or a combination thereof; a second polymer; or Its combination.

2. The pharmaceutical composition of claim 1, wherein the polymer comprises the first polymer.

3. The pharmaceutical composition of claim 1, wherein the second polymer comprises one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); polycaprolactone (PCL); (PPO-PEO); poly (γ-L-glutamic acid) (PGA); poly (L-phenylalanine ethyl ester) (PAE); poly (L-lysine) (PLL); methyl PEG (mPEG); poly (aspartic acid) (PasP); poly (L-histidine) (PLH); poly (vinylamine) (PEI); poly (N-vinyl pyrrolidone) (PVP); poly (L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly (hydroxybutyrate) (PHB); poly (ethylene oxide) (PEO); poly (L-glutamic acid-γ-benzyl ester) (PBLG); phosphatidylserine (PS); poly (isohexyl cyanoacrylate) (PIHCA); poly (allylamine hydrochloride) (PAH); poly (γ-propargyl) (PP); or a combination thereof.

4. according to the pharmaceutical composition described in any one in claim 1 to 3, wherein said polymer comprises poly-oxazoline (POX), and said poly-oxazoline comprises linear part, branched part or its combination, and wherein said poly-oxazoline (POX) comprises poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline) and poly-(isopropyloxazoline), or its combination.

5. The pharmaceutical composition of claim 4, wherein the polyoxazoline is poly(2-ethyloxazoline).

6. according to the pharmaceutical composition described in any one in claim 4 to 5, wherein said polyoxazoline is included in the mol ratio of monomer and initiator in the scope of 50:1 to 80:

1.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein 1% to 100% of the second end groups do not contain primary amines.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein 1% to 100% of the second terminal groups comprise hydroxyl groups.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the size of the nanoaggregates before lyophilization is less than 120 nm.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the nanoaggregates have a weight ratio of the polymer to the bioactive agent in the range of about 2:1 to about 200:

1.

11. The pharmaceutical composition according to any one of claims 1 to 11, wherein the nanoaggregates do not contain human serum albumin, organic solvents, detergents or oils.

12. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition does not contain human serum albumin, organic solvents, detergents or oils.

13. The pharmaceutical composition according to any one of claims 1 to 15, wherein the pharmaceutical composition is a medicament for treating or preventing a disease selected from immune diseases, infectious diseases, and combinations thereof.

14. The pharmaceutical composition according to any one of claims 1 to 16, wherein the bioactive agent comprises at least one compound having Formula (1) to Formula (29), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

15. A pharmaceutical composition according to any one of claims 1 to 17, wherein the bioactive agent comprises a compound having the formula Formula (1) Formula (4) A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

16. The pharmaceutical composition according to any one of claims 1 to 18, wherein the pharmaceutical composition is an adjuvant for a vaccine.

17. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is a prophylactic vaccine, a therapeutic vaccine, or a combination thereof, wherein the pharmaceutical composition comprises the adjuvant and further comprises at least one immunizing agent for stimulating an immune response in a subject in need thereof.

18. The pharmaceutical composition of claim 20, wherein the immunizing agent comprises an inactive microorganism selected from bacteria, viruses, fungi, protozoa, worms, parasites, prions, parts thereof, or combinations thereof; toxins; nucleic acids encoding the toxins; proteins; nucleic acids encoding the proteins; oligonucleic acids; DNA; RNA; mRNA; siRNA; sgRNA; fragments thereof; or combinations thereof.

19. The pharmaceutical composition according to any one of claims 18 to 21, wherein the pharmaceutical composition is formulated for the treatment or prevention of at least one infectious disease.

20. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulated for the treatment or prevention of at least one infectious disease selected from the group consisting of varicella, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika, and combinations thereof.

21. A pharmaceutical composition according to claim 23, wherein the immune agent comprises at least a polypeptide of the spike (S) glycoprotein of a coronavirus, a DNA encoding the spike (S) glycoprotein, an RNA encoding the spike (S) glycoprotein, a receptor binding domain (RBD) of the spike (S) glycoprotein, a DNA encoding the RBD, an RNA encoding the RBD, a portion thereof, or a combination thereof.

22. A pharmaceutical composition according to claim 24, wherein the coronavirus comprises 229E α-coronavirus, NL63 α-coronavirus, OC43 β-coronavirus, HKU1 β-coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, variants thereof, or combinations thereof.

23. A pharmaceutical composition according to any one of claims 24 to 25, wherein the immune agent comprises at least one of the receptor binding domains (RBDs) of the spike (S) glycoprotein of the 229E α-coronavirus, NL63 α-coronavirus, OC43 β-coronavirus, HKU1 β-coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, variants thereof, or combinations thereof.

24. The pharmaceutical composition of claim 26, wherein the immunizing agent comprises at least one polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or a combination thereof.

25. The pharmaceutical composition of claim 27, wherein the immunizing agent comprises at least one polypeptide of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

26. A pharmaceutical composition according to any one of claims 20 to 28, wherein the weight ratio of the immunizing agent to the adjuvant is in the range of 1:50 to 50:1, wherein the weight ratio is based on the weight of the immunizing agent and the bioactive agent as the adjuvant.

27. A pharmaceutical composition according to any one of claims 1 to 29, wherein the pharmaceutical composition further comprises one or more subsequent bioactive agents selected from proteins, peptides, antibodies, antibody fragments, compounds, small molecule drugs, one or more chemotherapeutic drugs, and combinations thereof.

28. A method for treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective dose of a pharmaceutical composition, the pharmaceutical composition include: A nanoaggregate comprising a polymer and at least one bioactive agent, wherein the bioactive agent comprises at least one STING polypeptide or a portion thereof, a nucleic acid encoding the STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof; and Optionally, a pharmaceutically suitable carrier; wherein the pharmaceutical composition is soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution; wherein the polymer is water soluble; and The polymer comprises: A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% of the hydrophobic moiety, the hydrophobic moiety comprises a saturated or unsaturated aliphatic hydrocarbon, an aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, an amide, an imide, an imide, a carboxyl group, a hydroxyl group, an ester, an ether, an acetate, a phosphate, a ketone, an aldehyde, a sulfonate, or a combination thereof; or A second polymer comprising one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); polycaprolactone (PCL); (PPO-PEO); poly(γ-L-glutamic acid) (PGA); poly(L-phenylalanine ethyl ester) (PAE); poly(L-lysine) (PLL); methyl PEG (mPEG); poly(aspartic acid) (PasP); poly(L-histidine) (PLH); poly(vinylamine) (PEI); poly(N-vinylpyrrolidone) (PVP); poly(L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly(hydroxybutyrate) (PHB); polyethylene oxide (PEO); poly(γ-benzyl L-glutamate) (PBLG); phosphatidylserine (PS); poly(isohexyl cyanoacrylate) (PIHCA); poly(allylamine hydrochloride) (PAH); poly(γ-propargyl) (PP); or Its combination.

29. The method of claim 31, wherein the polymer comprises the first polymer.

30. The method of claim 31, wherein the polymer comprises the one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); polycaprolactone (PCL); (PPO-PEO); poly (γ-L-glutamic acid) (PGA); poly (L-phenylalanine ethyl ester) (PAE); poly (L-lysine) (PLL); methyl PEG (mPEG); poly (aspartic acid) (PasP); poly (L-histidine) (PLH); poly (vinylamine) (PEI); poly (N-vinyl pyrrolidone) (PVP); poly (L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly (hydroxybutyrate) (PHB); poly (ethylene oxide) (PEO); poly (L-glutamic acid-γ-benzyl ester) (PBLG); phosphatidylserine (PS); poly (isohexyl cyanoacrylate) (PIHCA); poly (allylamine hydrochloride) (PAH); poly (γ-propargyl) (PP); or a combination thereof.

31. according to the method described in any one in claim 31 to 33, wherein said polymkeric substance comprises poly-oxazoline (POX), and said poly-oxazoline comprises linear portion, branched portion or its combination, and wherein said poly-oxazoline (POX) comprises poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline) and poly-(isopropyloxazoline), or its combination.

32. The method of claim 34, wherein the polyoxazoline is poly(2-ethyloxazoline).

33. according to the method described in any one in claim 34 to 35, wherein said poly-oxazoline is included in the mol ratio of the monomer in the scope of 50:1 to 80:1 and initiator.

34. The method of any one of claims 31 to 36, wherein the size of the nanoaggregates prior to lyophilization is less than 120 nm.

35. The method of any one of claims 31 to 37, wherein the nanoaggregates have a weight ratio of the polymer to the bioactive agent in the range of about 2:1 to about 200:

1.

36. The method of any one of claims 31 to 38, wherein the nanoaggregates are free of human serum albumin, organic solvents, detergents or oils.

37. The method of any one of claims 31 to 39, wherein the pharmaceutical composition does not contain human serum albumin, organic solvents, detergents or oils.

38. The method according to any one of claims 31 to 40, wherein the pharmaceutical composition is a medicament for treating or preventing a disease selected from an immune disease, an infectious disease, and a combination thereof.

39. The method according to any one of claims 31 to 41, wherein the bioactive agent comprises at least one compound having Formula (1) to Formula (29), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

40. The method of any one of claims 31 to 42, wherein the bioactive agent comprises a compound having the formula Formula (1): Formula (4) A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

41. The method of any one of claims 31 to 43, wherein the pharmaceutical composition is an adjuvant for a vaccine.

42. The method of claim 44, wherein the pharmaceutical composition is a prophylactic vaccine, a therapeutic vaccine, or a combination thereof, wherein the pharmaceutical composition comprises the adjuvant and further comprises at least one immunizing agent for stimulating an immune response in a subject in need thereof.

43. The method of any one of claims 43 to 45, wherein the pharmaceutical composition is selected for the treatment or prevention of at least one infectious disease selected from the group consisting of varicella, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika, and combinations thereof.

44. A method according to claim 46, wherein the immune agent comprises at least a polypeptide of the spike (S) glycoprotein of a coronavirus, a DNA encoding the spike (S) glycoprotein, an RNA encoding the spike (S) glycoprotein, a receptor binding domain (RBD) of the spike (S) glycoprotein, a DNA encoding the RBD, an RNA encoding the RBD, a portion thereof, or a combination thereof.

45. The method of claim 47, wherein the immune agent comprises the receptor binding domain (RBD) of the spike (S) glycoprotein of at least one coronavirus, the coronavirus being 229Eα coronavirus, NL63α coronavirus, OC43β coronavirus, HKU1β coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2, variants thereof, or combinations thereof.

46. ​​The method of claim 48, wherein the immunizing agent comprises at least one polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or a combination thereof.

47. The method of claim 49, wherein the immunizing agent comprises at least one polypeptide of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

48. The method of any one of claims 45 to 50, wherein the weight ratio of the immunizing agent to the adjuvant is in the range of 1:50 to 50:1, wherein the weight ratio is based on the weight of the immunizing agent and the bioactive agent as the adjuvant.

49. The method of any one of claims 31 to 51, wherein the pharmaceutical composition is administered to the subject by intravenous (IV) injection, subcutaneous (SC) injection, intramuscular (IM) injection, intradermal (ID) injection, nasal spray, inhalation, oral tablets, eye drops, rectal immunization, vaginal immunization, or a combination thereof.

50. The method of any one of claims 31 to 52, wherein from 1% to 100% of the second end groups are free of primary amines.

51. The method of any one of claims 31 to 53, wherein from 1% to 100% of the second end groups comprise hydroxyl groups.

52. The method of any one of claims 31 to 54, further comprising the step of administering to the subject one or more subsequent bioactive agents selected from the group consisting of proteins, peptides, antibodies, antibody fragments, compounds, small molecule drugs, one or more chemotherapeutic drugs, vaccines, and combinations thereof, wherein each of the one or more subsequent bioactive agents is administered to the subject before, simultaneously with, or after administration of the pharmaceutical composition.

53. The method of any one of claims 31 to 55, further comprising the step of administering to the subject one or more booster injections of the pharmaceutical composition.

54. A nanoaggregate comprising a polymer and at least one bioactive agent, wherein the bioactive agent comprises at least one STING polypeptide or a portion thereof, a nucleic acid encoding the STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof; wherein the nanoaggregates are soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution; wherein the polymer is water soluble; and The polymer comprises: A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% of the hydrophobic moiety, the hydrophobic moiety comprises a saturated or unsaturated aliphatic hydrocarbon, an aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, an amide, an imide, an imide, a carboxyl group, a hydroxyl group, an ester, an ether, an acetate, a phosphate, a ketone, an aldehyde, a sulfonate, or a combination thereof; or A second polymer comprising one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); polycaprolactone (PCL); (PPO-PEO); poly(γ-L-glutamic acid) (PGA); poly(L-phenylalanine ethyl ester) (PAE); poly(L-lysine) (PLL); methyl PEG (mPEG); poly(aspartic acid) (PasP); poly(L-histidine) (PLH); poly(vinylamine) (PEI); poly(N-vinylpyrrolidone) (PVP); poly(L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly(hydroxybutyrate) (PHB); poly(ethylene oxide) (PEO); poly(γ-benzyl L-glutamate) (PBLG); phosphatidylserine (PS); poly(isohexyl cyanoacrylate) (PIHCA); poly(allylamine hydrochloride) (PAH); poly(γ-propargyl) (PP); or Its combination.

55. according to the nano-aggregate described in claim 57, wherein said polymer comprises poly-oxazoline (POX), and said poly-oxazoline comprises linear portion, branched portion or its combination, and wherein said poly-oxazoline (POX) comprises poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline) and poly-(isopropyloxazoline), or its combination.

56. The nanoaggregate of claim 58, wherein the polyoxazoline is poly(2-ethyloxazoline).

57. according to each nano-aggregate in claim 57 to 59, wherein said polyoxazoline comprises the mol ratio of monomer and initiator in the scope of 50:1 to 80:

1.

58. The nanoaggregate according to any one of claims 57 to 60, wherein 1% to 100% of the second end groups are free of primary amines.

59. The nanoaggregate according to any one of claims 57 to 61, wherein 1% to 100% of the second end groups comprise hydroxyl groups.

60. The nanoaggregate according to any one of claims 57 to 62, wherein the size of the nanoaggregate before lyophilization is less than 120 nm.

61. The nanoaggregate of any one of claims 57 to 63, wherein the nanoaggregate has a weight ratio of the polymer to the bioactive agent in the range of about 2:1 to about 200:

1.

62. The nanoaggregate of any one of claims 57 to 64, wherein the nanoaggregate has a weight ratio of the polymer to the bioactive agent in the range of about 5:1 to about 8:

1.

63. The nanoaggregate according to any one of claims 57 to 65, wherein the nanoaggregate does not contain human serum albumin, organic solvents, detergents or oils.

64. The nanoaggregate according to any one of claims 57 to 66, wherein the pharmaceutical composition is a medicament for treating or preventing a disease selected from immune diseases, infectious diseases, and combinations thereof.

65. The nanoaggregate according to any one of claims 57 to 67, wherein the bioactive agent comprises at least one compound of Formula (1) to Formula (29), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

66. The nanoaggregate of claim 68, wherein the bioactive agent comprises a compound having the formula Formula (1) Formula (4) A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

67. Use of a nanoaggregate for preparing a medicament for treating a disease, the nanoaggregate comprising a polymer and at least one bioactive agent, the bioactive agent comprising at least one STING polypeptide or a portion thereof, a nucleic acid encoding the STING polypeptide or a portion thereof, a STING inhibitor, a STING activator, a STING agonist, a STING antagonist, a STING modulatory molecule, or a combination thereof; and optionally a pharmaceutically suitable carrier; wherein the disease is selected from immune diseases, infectious diseases, and combinations thereof; wherein the nanoaggregates are soluble in an aqueous solution to produce at least 1 mg / mL of the bioactive agent dissolved in the aqueous solution; wherein the polymer is water soluble; and The polymer comprises: A first polymer comprising at least one first end group modified with H or a hydrophobic moiety and a second end group modified with a hydrophilic moiety, wherein the first end group comprises 1% to 100% H and 0% to 99% of the hydrophobic moiety, the hydrophobic moiety comprises a saturated or unsaturated aliphatic hydrocarbon, an aromatic hydrocarbon, or a combination thereof having 1 to about 22 carbons, and the second end group comprises a group modified with an amine, an amide, an imide, an imide, a carboxyl group, a hydroxyl group, an ester, an ether, an acetate, a phosphate, a ketone, an aldehyde, a sulfonate, or a combination thereof; or A second polymer comprising one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); poly(caprolactone) (PCL); (PPO-PEO); poly(γ-L-glutamic acid) (PGA); poly(L-phenylalanine ethyl ester) (PAE); poly(L-lysine) (PLL); methyl PEG (mPEG); poly(aspartic acid) (PasP); poly(L-histidine) (PLH); poly(vinylamine) (PEI); poly(N-vinylpyrrolidone) (PVP); poly(L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly(hydroxybutyrate) (PHB); poly(ethylene oxide) (PEO); poly(γ-benzyl L-glutamate) (PBLG); phosphatidylserine (PS); poly(isohexyl cyanoacrylate) (PIHCA); poly(allylamine hydrochloride) (PAH); poly(γ-propargyl) (PP); or Its combination.

68. The use of claim 70, wherein the polymer comprises the first polymer.

69. according to the purposes described in any one in claim 70 to 71, wherein said polymer comprises poly-oxazoline (POX), and said poly-oxazoline comprises linear portion, branched portion or its combination, and wherein said poly-oxazoline (POX) comprises poly-(2-methyloxazoline), poly-(2-ethyloxazoline), poly-(2-propyloxazoline) and poly-(isopropyloxazoline), or its combination.

70. The use of claim 70, wherein the polymer comprises the second polymer, the second polymer comprising the one or more hydroxy dendrimers (HD); ethylenediamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated-4, 5, 6, 7, 8, 9, 10 generation dendrimers or combinations thereof; poly(ethylene glycol) (PEG); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(propylene oxide) (PPO); poly(caprolactone) (PCL); (PPO-PEO); poly (γ-L-glutamic acid) (PGA); poly (L-phenylalanine ethyl ester) (PAE); poly (L-lysine) (PLL); methyl PEG (mPEG); poly (aspartic acid) (PasP); poly (L-histidine) (PLH); poly (vinylamine) (PEI); poly (N-vinyl pyrrolidone) (PVP); poly (L-leucine) (PLLeu); deoxycholic acid (DOCA); hydroxypropyl methylcellulose (HPMC); poly (hydroxybutyrate) (PHB); poly (ethylene oxide) (PEO); poly (L-glutamic acid-γ-benzyl ester) (PBLG); phosphatidylserine (PS); poly (isohexyl cyanoacrylate) (PIHCA); poly (allylamine hydrochloride) (PAH); poly (γ-propargyl) (PP); or a combination thereof.

71. The use according to any one of claims 70 to 73, wherein the pharmaceutical composition is a medicament for treating or preventing a disease selected from an immune disease, an infectious disease, and a combination thereof.

72. The use according to any one of claims 70 to 74, wherein the bioactive agent comprises at least one compound of Formula (1) to Formula (29), a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

73. The use according to claim 75, wherein the bioactive agent comprises a compound having the formula Formula (1) Formula (4) A pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, an isomer thereof, or a combination thereof.

74. The use according to any one of claims 70 to 76, wherein the medicament is an adjuvant for a vaccine.

75. The use according to any one of claims 70 to 77, wherein the medicament is a prophylactic vaccine, a therapeutic vaccine, or a combination thereof, wherein the medicament comprises the adjuvant and further comprises at least one immunizing agent for stimulating an immune response in a subject in need thereof.

76. The use of any one of claims 70 to 78, wherein the medicament is formulated for the treatment or prevention of at least one infectious disease selected from the group consisting of varicella, coronavirus, dengue fever, diphtheria, Ebola, influenza, hepatitis, Hib disease, HIV / AIDS, HPV (human papillomavirus), Japanese encephalitis, measles, meningococcal disease, monkeypox, mumps, norovirus, pneumococcal disease, poliomyelitis, rabies, respiratory syncytial virus (RSV), rotavirus, rubella (German measles), herpes zoster, tetanus, pertussis, Zika, and combinations thereof.

77. The use according to any one of claims 70 to 79, wherein from 1% to 100% of the second end groups are free of primary amines.

78. The use according to any one of claims 70 to 80, wherein 1% to 100% of the second end groups comprise hydroxyl groups.

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