Non-adsorbing or super stealth vesicles

Abstract

The present disclosure provides non-fouling or super-stealth vesicles usable for drug delivery, which comprise polycations and polyanions, where the polycations and polyanions are charge-neutralized and cross-linked above a certain level, and have a half-life (T1 / 2) of greater than 24 hours in the blood or serum of a subject.

Description

[Technical Field]

[0001] The present disclosure relates to non-adsorbing or super stealth vesicles that can be used for drug delivery. [Background technology]

[0002] Vesicles capable of delivering targeted drugs to specific sites in the body have been developed to enhance drug efficacy or reduce drug side effects. Polyion complex polymersomes encapsulating enzymes have been developed to improve the blood circulation of administered enzymes and maximize their efficacy (US 2021 / 0346472A). US 2021 / 0346472A discloses polyion complex polymersomes encapsulating L-asparaginase. The polyion complex polymersomes are composed of polycation molecules and PEGylated polyanion molecules (i.e., polyanion molecules modified with polyethylene glycol (PEG), which can improve the biocompatibility of the polymersomes). These molecules are crosslinked to enhance the stability of the polymersomes in the circulation. In US 2021 / 0346472A, the polymersomes demonstrated an improved half-life of less than 24 hours in the blood circulation. Summary of the Invention

[0003] The present disclosure provides non-adsorbing or super stealth vesicles that can be used for drug delivery. The non-adsorbing or super stealth vesicles contain polycations and polyanions, which are charge-neutralized and cross-linked to a certain degree, and have a long half-life (T) of, for example, 24 hours or more, preferably 48 hours or more, more preferably 72 hours or more, and even more preferably 96 hours or more in the blood or serum of a subject. 1 / 2 )

[0004] The present disclosure provides a polyion complex comprising a polycation and a polyanion.

[0005] In one embodiment, the polycation is positively charged and comprises (i) a cationic polymer or a block copolymer comprising a cationic polymer block and, optionally, (ii) an uncharged hydrophilic polymer block. In one embodiment, the polyanion is negatively charged and comprises (iii) an anionic polymer or a block copolymer comprising an anionic polymer block and, optionally, (iv) an uncharged hydrophilic polymer block. In one embodiment, the polycation and polyanion interact with each other to form a polyion complex. In one embodiment, the positive and negative charges in the polyion complex are neutralized, and the ratio of positive to negative charges is in the range of 0.95 to 1.05. In one embodiment, the polycation and polyanion are stabilized in the polyion complex by crosslinking, resulting in a high crosslink density. In one embodiment, the polyion complex has a half-life (T1 / 2) of greater than 24 hours in the subject's blood or serum.

[0006] In one embodiment, the polyion complex forms a polyion complex micelle or polyion complex micelle.

[0007] In one embodiment, the polyion complex forms a polyion complex polymersome or a polyion complex polymersome.

[0008] In one embodiment, the crosslink density is greater than 30%.

[0009] In one embodiment, the crosslink density is greater than 40%.

[0010] In one embodiment, the crosslink density is greater than 30%.

[0011] In one embodiment, the crosslink density is greater than 40%.

[0012] In one embodiment, the sodium content in polyion complex micelles having a crosslinking degree of 3.9% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 60% or less.

[0013] In one embodiment, the sodium content in polyion complex micelles having a crosslinking degree of 3.9% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 50% or less.

[0014] In one embodiment, the sodium content in polyion complex micelles having a crosslinking degree of 7.1% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 60% or less.

[0015] In one embodiment, the sodium content in a polyion complex micelle having a crosslinking degree of 7.1% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 50% or less.

[0016] In one embodiment, the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex micelles with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.5.

[0017] In one embodiment, the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex micelles with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.4.

[0018] In one embodiment, after mixing 0.04 mg / mL of polyion complex polymersome with phosphate buffered saline containing 20% ​​fetal bovine serum, the calorific value measured by isothermal titration calorimetry is in the range of -0.1 to -0.5.

[0019] In one embodiment, after mixing 0.04 mg / mL of polyion complex polymersome with phosphate buffered saline containing 20% ​​fetal bovine serum, the calorific value measured by isothermal titration calorimetry is in the range of -0.1 to -0.4. [Brief explanation of the drawings]

[0020] [Figure 1]Figure 1 is a schematic diagram showing the process for obtaining polyion complex vesicles (polymersomes) with and without polyethylene glycol (PEG) coating. The PEG coating was removed from the vesicles under low pH conditions. [Figure 2] Figure 2 shows electron micrographs of vesicles with and without PEG coating, and a graph demonstrating that certain polyion complex vesicles exhibit remarkable blood retention properties even without PEG coating. The larger graph shows the change in relative fluorescence intensity (%) of fluorescently labeled PIC vesicles without PEG coating, and the smaller graph within the larger graph is a logarithmic scale graph. Mxx% indicates micelles with a cross-linking density of xx% between the polycation and polyanion. Vxx% indicates vesicles with a cross-linking density of xx% between the polycation and polyanion. At Mxx% and Vxx%, the polycation and polyanion are completely neutralized. [Figure 3] Figure 3 shows the Z-average (d nm) and Cy5 / Cy3 ratios of micelles and vesicles with various degrees of crosslinking. In these micelles and vesicles, the polycations are labeled with Cy5 and the polyanions are labeled with Cy3. When the distance between Cy5 and Cy3 becomes within 10 nm due to compression of the micelles or vesicles, excitation of Cy3 causes relaxation of Cy5, resulting in an increase in the Cy5 / Cy3 ratio. [Figure 4] Figure 4 shows the time course of changes in the Cy5 / Cy3 ratio of micelles and vesicles with various degrees of crosslinking, measured in the bloodstream in vivo. [Figure 5] Figure 5 shows a microscopic fluorescence image of micelles with a cross-linking density of 39.5%. [Figure 6] FIG. 6 shows the absorption of sodium ions into micelles and vesicles with different degrees of cross-linking. [Figure 7] Figure 7 shows the relaxation times (T2) of specific carbon atoms a, b, and c in micelles and vesicles with various degrees of cross-linking. [Figure 8] Figure 8 shows the water solubility of M 39.5% and V 30.3%. [Figure 9]Figure 9 shows the amount of protein interacting with micelles and vesicles of various cross-linking degrees measured by calorimetry (top panel) and the amount of uptake of micelles and vesicles by macrophages measured by measuring the fluorescence intensity of Cy5 taken up by macrophages (bottom panel). [Figure 10] Figure 10 shows the rapid clearance of short-circulating micelles (M 33.1%) and vesicles (V 20.9%) by the hepatic sinusoidal wall based on intravital microscopy. Quantification of fluorescence and the proportion of CD45+F4 / 80+ and CD45- cell populations was performed by flow cytometry analysis. [Figure 11] Figure 11 shows that, based on intravital microscopy, long-circulating micelles (M 39.5%) appear in the sinusoids after 10 hours of circulation compared to long-circulating vesicles (V 30.3%). [Figure 12] Figure 12 shows that long circulating micelles (M 39.5%) accumulate more slowly in the bile duct compared to long circulating vesicles (V 30.3%). Quantification of fluorescence and the percentage of CD45+F4 / 80+ cell populations was performed by flow cytometry analysis. [Figure 13] FIG. 13 shows an antitumor therapeutic strategy using asparaginase-encapsulated vesicles. [Figure 14] Figure 14 shows the time course of events in an antitumor treatment strategy using asparaginase-encapsulated vesicles. Before the start of treatment, mice were implanted with tumor cell lines 4T1 or 231 / LM2. Mice with tumor volumes of 50–100 mm3 were subjected to an antitumor treatment strategy using asparaginase-encapsulated vesicles (also called "ASNase@V"). Vesicles were administered intravenously (iv) every 5 days at a daily dose of 4 U per mouse. Tumors and lungs were harvested and analyzed 30 days after the start of treatment for 4T1 and 60 days after the start of treatment for 231 / LM2. [Figure 15] FIG. 15 shows the effect of administration of asparaginase-encapsulated vesicles on tumor weight. [Figure 16] FIG. 16 shows the effect of administration of asparaginase-encapsulated vesicles on lung metastasis. [Figure 17]FIG. 17 shows the effect of administration of asparaginase-encapsulated vesicles on the number of tumor nodules. [Figure 18] FIG. 18 shows the size and number of metastatic foci by hematoxylin and eosin (H&E) staining. [Figure 19] FIG. 19 shows the effect of administration of asparaginase-encapsulated vesicles on plasma asparagine concentrations 4 hours or 96 hours after administration. [Figure 20] FIG. 20 shows the effect of administration of asparaginase-encapsulated vesicles on asparagine levels in tumor cells. [Figure 21] FIG. 21 shows the amount of asparaginase delivered to tumor cells in mice administered asparaginase-encapsulated vesicles. [Figure 22] FIG. 22 shows fluorescent images of tumor tissue sections from mice administered asparaginase-encapsulated vesicles. [Figure 23] Figure 23 shows IVIS images of mice administered asparaginase-encapsulated vesicles and / or an immune checkpoint inhibitor (anti-PD-1 antibody, also known as "aPD-1"), in which tumors were labeled with luciferase and visualized by administration of its substrate, luciferin. [Figure 24] Figure 24 shows the intensity of bioluminescence from luciferase-expressing tumors in mice treated with asparaginase-encapsulated vesicles and / or an immune checkpoint inhibitor (anti-PD-1 antibody). The combination of vesicles and anti-PD-1 antibody treatment demonstrated the most effective anti-tumor effect. [Figure 25] Figure 25 shows abdominal images of mice administered asparaginase-encapsulated vesicles and / or an immune checkpoint inhibitor (anti-PD-1 antibody). [Figure 26] Figure 26 shows the effect of administration of vesicles and / or anti-PD-1 antibody. [Figure 27] Figure 27 shows survival curves for mice administered asparaginase-encapsulated vesicles and / or immune checkpoint inhibitors (anti-PD-1 antibodies). [Figure 28]FIG. 28 shows collagen distribution in tumor tissue sections from mice administered asparaginase-encapsulated vesicles. [Figure 29] FIG. 29 shows the distribution of α-smooth muscle actin (α-SMA) in tumor tissue sections from mice administered asparaginase-encapsulated vesicles. [Figure 30] Figure 30 shows the distribution of anti-PD-1 antibodies in tumor tissue sections from mice administered asparaginase vesicles. In mice administered asparaginase vesicles, the asparaginase vesicles increased the permeability of the tumor tissue, allowing the administered anti-PD-1 antibodies to penetrate into the tumor tissue. [Figure 31] FIG. 31 shows the effect of vesicle administration on collagen 1 (Col1) expression and α-SMA expression in tumor tissues. [Figure 32] FIG. 32 is a schematic diagram showing that depletion induced by vesicle administration can reduce tumor tissue desmoplasia in vivo. [Figure 33] FIG. 33 shows the half-life of V40% when the vesicle-forming polycation molecules exceed the vesicle-forming polyanion molecules by 10%. [Figure 34] FIG. 34 shows the half-life of V40% when the vesicle-forming polyanion molecules exceed the vesicle-forming polycation molecules by 10%. Detailed Description of the Invention

[0021] As used herein, the term "subject" refers to mammals, including dogs, cats, cows, pigs, horses, donkeys, sheep, and primates such as monkeys, gorillas, chimpanzees, orangutans, bonobos, and humans.

[0022] The term "nanoparticle" as used herein refers to particles having a size of, for example, 10 to 1000 nm, 10 to 900 nm, 10 to 800 nm, 10 to 700 nm, 10 to 600 nm, 10 to 500 nm, 10 to 400 nm, 10 to 300 nm, 10 to 200 nm, 10 to 100 nm, 10 to 90 nm, 10 to 80 nm, 10 to 70 nm, 10 to 60 nm, 20 to 50 nm, or 30 to 40 nm.

[0023] As used herein, the term "particle" refers to a particle with or without an internal lumen, including, but not limited to, dendrimers, vesicles, micelles, liposomes, polyion complexes, polyion complex micelles (PIC micelles), and polyion complex polymersomes (PICsomes, also called vesicles).

[0024] The term "vesicle" as used herein refers to a micelle or hollow microparticle. The vesicle preferably has a biocompatible shell. Vesicles such as PICsomes and liposomes can encapsulate low molecular weight compounds, proteins such as enzymes, nucleic acids, and active pharmaceutical ingredients such as antibodies or antigen-binding fragments thereof.

[0025] As used herein, a "polyion complex" (hereinafter also referred to as "PIC") is an ionic layer formed between two block copolymers, cationic and anionic, by mixing a PEG-anionic block copolymer and a PEG-cationic block copolymer in an aqueous solution and neutralizing the charges. The polyion complex can encapsulate a therapeutic agent that can induce a therapeutic effect in a subject treated with the therapeutic agent. Examples of therapeutic agents include small, medium, and large molecules. The binding of PEG to each of these charged chains is intended to prevent the polyion complex from aggregating and precipitating, thereby enabling the polyion complex to form monodisperse core-shell nanoparticles with particle sizes of several tens of nanometers. Furthermore, the PEG shell prevents protein adsorption to the nanoparticles in vivo, thereby improving biocompatibility and blood retention. It has been shown that one type of charge-block copolymer does not require a PEG moiety for polyion complex formation; the PEG moiety can be replaced with a homopolymer, surfactant, nucleic acid, and / or enzyme. Furthermore, in the formation of polyion complexes, at least one of the anionic polymer and the cationic polymer may be copolymerized with PEG, or both of these polymers may be copolymerized with PEG. As is well known, increasing the PEG content favors the formation of PIC micelles, while decreasing the PEG content favors the formation of PICsomes. Examples of anionic polymers or blocks commonly used in the preparation of polyion complexes include polyglutamic acid, polyaspartic acid, and nucleic acids (e.g., DNA, mRNA, and siRNA). Examples of cationic polymers or blocks include polylysine and poly(5-aminopentyl aspartic acid). Here, mRNA refers to messenger RNA used in protein synthesis by translation. siRNA refers to double-stranded RNA (nucleic acid) capable of inducing RNA interference (RNAi). siRNA is not particularly limited and is a double-stranded RNA of 20 to 30 bp, preferably 21 to 23 bp, 25 bp, or 27 bp, with a sequence homologous to that of a target gene.

[0026] As used herein, "cationic block" and "cationic polymer" refer to a polymer block and a polymer, respectively, which are cationic as a whole and are obtained by polymerizing monomer units containing cationic monomers. Examples of cationic polymers include homocationic polymers and polymers in which a homocationic polymer is linked to an uncharged hydrophilic chain. When a cationic polymer forms a block copolymer with another polymer, the cationic polymer portion may be referred to as a cationic block. As used herein, a cationic polymer is a pharmaceutically acceptable cationic polymer. As used herein, a "polyvalent cation" refers to a cationic molecule having multiple cationic groups within the molecule. As used herein, a "polyvalent cation" can have cationic properties as a whole molecule in a blood environment. Examples of polyvalent cations include molecules that are cationic in a blood environment, such as cationic polymers and cationic dendrimers. Polyvalent cations are biocompatible. As used herein, the term "dendrimer" refers to a molecule having multiple branches from a single core atom.

[0027] As used herein, the term "hydrophilic block" refers to a polymer chain that is soluble in an aqueous medium, and may be a hydrophilic polymer block. In the present invention, the uncharged hydrophilic chain is a pharmaceutically acceptable uncharged hydrophilic chain. Examples of such hydrophilic chains include polyalkylene glycols such as polyethylene glycol (PEG) and polyoxazolines such as poly(2-ethyl-2-oxazoline). The uncharged hydrophilic chain may contain polar atoms, as long as the charge is neutralized locally and globally. The hydrophilic block may be branched or unbranched. If the hydrophilic block is branched, there may be one or more branching points.

[0028] As used herein, the term "polyion complex polymersome" (hereinafter also referred to as "PICsome") refers to hollow microparticles formed from polyion complexes. It is known that, from the viewpoint of blood retention time, it is preferable to modify the outer surface of a PICsome with polyethylene glycol.

[0029] As used herein, the term "micelle" refers to a molecular assembly formed from a monolayer molecular membrane. Examples of micelles include micelles formed from amphiphilic molecules such as surfactants and micelles formed from polyion complexes (PIC micelles). It is known that it is preferable to modify the outer surface of micelles with polyethylene glycol from the viewpoint of blood retention time. An example of a polyion complex polymersome used in the present invention is a PICsome for drug delivery. A known example of such a PICsome for drug delivery is a PICsome formed from a block copolymer. Examples of block copolymers that constitute a PICsome include a block copolymer of a PEG block, a polycation block, and a homopolyanion, and a block copolymer of a PEG block, a polyanion block, and a homopolycation. Preferably, a biodegradable block copolymer is used. Various copolymers are known as such copolymers, and in principle, any copolymer can be used. For example, poly(aspartic acid-tetraethylenepentamine (Asp-TEP)) block copolymer and polyethylene glycol-poly((5-aminopentyl)-aspartic acid) block copolymer can be used as block copolymers with excellent biocompatibility and biodegradability.

[0030] As used herein, the term "nucleic acid" refers to natural nucleic acids such as natural DNA and natural RNA, antisense oligonucleotides (ASOs), modified nucleic acids such as modified DNA and modified RNA, artificial nucleic acids, and combinations thereof. Examples of modified nucleic acids include fluorescent dye-modified nucleic acids, biotinylated nucleic acids, and nucleic acids containing cholesteryl groups. To enhance stability, RNA bases may be modified with 2'-O-methyl, 2'-fluoro, or 2'-methoxyethyl (MOE), or the phosphodiester bonds in the nucleic acid backbone may be replaced with phosphorothioate bonds. Examples of artificial nucleic acids include nucleic acids in which the 2'-oxygen atom and the 4'-carbon atom are crosslinked. Examples of such artificial DNA include locked nucleic acids (LNAs), which are crosslinked DNAs in which the 2'-oxygen atom and the 4'-carbon atom are crosslinked via a methylene, and peptide nucleic acids (PNAs), which use a polymer backbone composed of N-(2-aminoethyl)glycine with amide bonds instead of deoxyribose or ribose. Examples of RNA include artificial RNAs for gene silencing, such as siRNA and shRNA, and non-coding RNAs, such as natural RNAs, such as microRNA (miRNA) and mRNA. These RNAs may be modified to be stabilized in vivo.

[0031] The term "anti-tumor agent" or "anti-cancer agent" refers to an agent administered to treat cancer in a subject. Cancer includes, for example, solid cancers and blood cancers. Cancer includes, for example, metastatic cancers and non-metastatic cancers.

[0032] As used herein, the term "effective amount" refers to an amount of an antibody that is capable of eliciting a desired effect of the antibody. The desired effect is not limited to a therapeutic effect, but also includes a non-therapeutic effect.

[0033] As used herein, the term "therapeutically effective amount" refers to an amount of a drug effective for treating (preventing and curing) a disease or condition. A therapeutically effective amount of a drug can slow the rate of worsening of symptoms, prevent worsening of symptoms, ameliorate symptoms, cure symptoms, or inhibit the onset or progression of symptoms of a disease or condition.

[0034] In this specification, the term "comprise" is used to include the terms "consist of" and "essentially consist of." "Comprise" means that components other than the target component may be included, and "consist of" means that components other than the target component are not included. In this specification, "consist essentially of" means that components other than the target component are not included in a form that exhibits a specific function (for example, a form in which the effect of the invention is completely lost).

[0035] The present disclosure provides polyion complexes and compositions comprising polyion complexes. The polyion complexes are preferably non-adsorbing or super stealth polyion complexes. The polyion complexes comprise a polycation and a polyanion. The polycation has a positive charge and comprises (i) a cationic polymer or a block copolymer comprising a cationic polymer block, and optionally (ii) an uncharged hydrophilic polymer block. The polyanion has a negative charge and comprises (iii) an anionic polymer or a block copolymer comprising an anionic polymer block, and optionally (iv) an uncharged hydrophilic polymer block. In the polyion complex, the polycation and polyanion interact with each other to form the polyion complex. In one embodiment, the positive and negative charges in the polyion complex are neutralized. In preferred embodiments, the ratio of positive to negative charges is in the range of 0.95 to 1.05, 0.96 to 1.04, 0.97 to 1.03, 0.98 to 1.02, or 0.99 to 1.01, or approximately 1.00. In one embodiment, the polycation and polyanion are stabilized by crosslinking within the polyion complex. Charge neutralization from the polycation and polyanion is important for the non-adsorbing properties of the non-adsorbing or super stealth polyion complex. Stabilization is also important for the non-adsorbing properties of the non-adsorbing or super stealth polyion complex. Those skilled in the art will adjust the ratio of polycation to polyanion and the degree of stabilization to render the polyion complex non-adsorbing. The following examples demonstrate that charge neutralization and high crosslink density are sufficient to render the polyion complex non-adsorbing without attaching uncharged hydrophilic polymer blocks to either or both the polycation and polyanion. The following examples further demonstrate that the lyophilized polyion complex can be reconstituted with water (e.g., water for injection) to form a composition containing the polyion complex. Thus, the composition can be provided in a lyophilized form.

[0036] Thus, the present disclosure provides, for example, a polyion complex comprising a polycation and a polyanion, The polycation has a positive charge and comprises (i) a cationic polymer or a block copolymer comprising a cationic polymer block, and optionally (ii) an uncharged hydrophilic polymer block; the polyanion has a negative charge and comprises (iii) an anionic polymer or a block copolymer comprising an anionic polymer block, and optionally (iv) an uncharged hydrophilic polymer block; the polycation and the polyanion interact with each other to form a polyion complex; The positive and negative charges are neutralized within the polyion complex, and the ratio of the positive and negative charges is in the range of 0.95 to 1.05; The polycation and the polyanion are stabilized within the polyion complex by cross-linking.

[0037] In one embodiment, the polyion complex has a crosslink density. When the ratio of positive to negative charges is about 1.00, a high crosslink density provides the polyion complex with non-adsorbing properties. Therefore, in a preferred embodiment, the crosslink density exceeds a predetermined crosslink density. In a preferred embodiment, the predetermined crosslink density is 25% or more, more preferably 30% or more, even more preferably 35% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or about 100%. The higher the crosslink density, the more stabilized the ion pair network within the polyion complex, making the polyion complex non-adsorbing. In a preferred embodiment of a polyion complex micelle, the predetermined crosslink density is 25% or more, more preferably 30% or more, even more preferably 35% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or about 100%. In an embodiment of a polyion complex micelle, the predetermined crosslink density is in the range of 35% to 65%. In a preferred embodiment of a polyion complex polymersome, the predetermined crosslink density is 25% or more, more preferably 30% or more, even more preferably 35% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or about 100%. In one embodiment of a polyion complex polymersome, the predetermined crosslink density is in the range of 35% to 93%. Crosslink density can be determined by one skilled in the art.

[0038] In one embodiment, when the polyion complex is or forms a polyion complex polymersome, the diameter (e.g., hydrodynamic diameter) of the polyion complex in 300 mM NaCl solution is 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, or 20% or less of the diameter of a polyion complex polymersome having a crosslink density of 7.1% in 300 mM NaCl solution.

[0039] In one embodiment, when the polyion complex is or forms a polyion complex micelle, the diameter (e.g., hydrodynamic diameter) of the polyion complex in 300 mM NaCl solution is 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 11% or less, 12% or less, 13% or less, 14% or less, 15% or less, 16% or less, 17% or less, 18% or less, 19% or less, or 20% or less of the diameter of a polyion complex micelle having a crosslink density of 3.9% in 300 mM NaCl solution.

[0040] In one embodiment, when the polyion complex is a polyion complex polymersome or forms a polyion complex polymersome, the polyion complex is crosslinked so that after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (e.g., ultracentrifugation at 120,000 rpm using a desktop ultracentrifuge Optima MAX-XP (Beckman Coulter)), the sodium content in the polyion complex is 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 15% or less of the sodium content in the polyion complex polymersome having a crosslink density of 7.1%. Reducing the counterion in the polyion complex also reduces the sodium content after centrifugation.

[0041] In one embodiment, when the polyion complex is or forms a polyion complex micelle, the polyion complex is crosslinked such that after 4 hours of centrifugation in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl, the sodium content in the polyion complex is 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less of the sodium content in a polyion complex micelle having a crosslinking density of 3.9%.

[0042] In one embodiment, the relaxation time T2 (seconds) of the crosslinked micelles is 0.03 or less, 0.025 or less, 0.024 or less, 0.023 or less, 0.022 or less, 0.021 or less, or 0.020 or less. In one embodiment, the relaxation time T2 (seconds) of the crosslinked PICsomes is 0.03 or less, 0.025 or less, 0.024 or less, 0.023 or less, 0.022 or less, 0.021 or less, or 0.020 or less. The decrease in relaxation time T2 reflects decreased chain flexibility and increased chain compaction in the polyion complex, which may contribute to a reduction in counterions that can interact with other substances and impart non-adsorbing properties to the micelles.

[0043] In one embodiment, the polyion complex is non-adsorbing. In a preferred embodiment, the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.5, more preferably -0.1 to -0.4. In a preferred embodiment, the polyion complex is a polyion complex micelle, and the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex micelle with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.5, more preferably -0.1 to -0.4. In a preferred embodiment, the polyion complex is a polyion complex polymersome, and the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex polymersome with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.5, more preferably -0.1 to -0.4, and even more preferably -0.1 to -0.3. Measurement can be performed 3 to 5 minutes (e.g., 3, 4, or 5 minutes) after mixing.

[0044] When the polycation has a primary amino group and the polyanion has a carboxyl group, the primary amino group and the carboxyl group can be crosslinked, for example, with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The crosslink density is calculated from the ratio of the crosslinked amino groups to the total amino groups in the polycation before crosslinking. The crosslink density can also be calculated from the ratio of the crosslinked carboxyl groups to the total carboxyl groups in the polycation before crosslinking.

[0045] In one embodiment, the uncharged hydrophilic block (e.g., PEG or polyoxazoline) can have an average molecular weight of, for example, 10 kD or more, 15 kD or more, 20 kD or more, 30 kD or more, or 40 kD or more (and 80 kD or less, 70 kD or less, 60 kD or less, or 50 kD or less), preferably 20 kD or more, more preferably 30 kD or more. In one embodiment, the uncharged hydrophilic block (e.g., PEG or polyoxazoline) can have an average degree of polymerization of 15 or more, 20 or more, 30 or more, or 40 or more (and, for example, 80 or less, 70 or less, 60 or less, or 50 or less). In one embodiment, the uncharged hydrophilic block is unbranched. In one embodiment, the uncharged hydrophilic block is branched.

[0046] In one embodiment, exemplary cationic polymers or cationic polymer segments include cationic natural amino acids and cationic unnatural amino acids, such as cationic natural amino acids such as histidine, tryptophan, ornithine, arginine, and lysine, and / or cationic polymers having, as a side chain, -(NH-(CH)) p -NH2 (wherein p represents an integer of 1 to 5) or -NH-(CH2) q Examples of suitable polycationic blocks include polymer blocks having a group represented by -NH2 (where q is an integer of 1 to 10 or 1 to 5), such as polymer blocks of cationic unnatural amino acids having the aforementioned cationic side chains, for example, polymer blocks of cationic unnatural amino acids such as aspartic acid or glutamic acid having the aforementioned cationic side chains. In one embodiment, the polycationic block has a side chain represented by -(NH-(CH2)2) p-NH2 (wherein p represents an integer of 1 to 5) or -NH-(CH2) q It is a polymer block having a group represented by -NH2 (where q represents an integer of 1 to 10 or 1 to 5). Preferred examples of cationic natural amino acids include histidine, tryptophan, ornithine, arginine, and lysine, more preferably arginine, ornithine, and lysine, even more preferably ornithine and lysine, and even more preferably lysine. In one embodiment, the cationic polymer or cationic polymer portion may be polylysine or polyornithine.

[0047] A polycation or polycation block may contain a mixture of cationic amino acids and amino acids with cationic side chains. In one embodiment, the polycation block is a polymer of monomer units comprising cationic natural amino acids, cationic unnatural amino acids, or cationic natural amino acids and cationic unnatural amino acids. In one embodiment, the monomer units in the polycation block are linked via peptide bonds. In a preferred embodiment, the cationic unnatural amino acids have -(NH-(CH)) as a side chain. p -NH2 (p represents an integer of 1 to 5) or -NH-(CH2) q In another embodiment, the polycation block is an amino acid having a group represented by -NH2 (q represents an integer of 1 to 10 or 1 to 5). In another embodiment, the polycation block is a polycation block having cationic natural amino acids, glutamic acid and aspartic acid, in any order, represented by -(NH-(CH2)2) p -NH2 (p represents an integer of 1 to 5) or --NH-(CH2) q The polycation block may be formed by modifying the monomer units in the polymer with a group represented by -NH2 (q represents an integer of 1 to 10 or 1 to 5) and polymerizing the modified monomer units. In one embodiment, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the monomer units in the polymer have a side chain represented by -(NH-(CH2)2). p -NH2 (p represents an integer of 1 to 5) or -NH-(CH2) qIt has a group represented by -NH2 (q represents an integer of 1 to 10 or 1 to 5).

[0048] In one embodiment, the anionic or anionic polymer block blocks natural amino acids and cationic non-natural amino acids, such as cationic natural amino acids such as glutamic acid and aspartic acid. In one embodiment, the anionic or anionic polymer block comprises a polynucleotide having an anionic charge. Polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and modified nucleic acids.

[0049] In one embodiment, the polyion complex has a half-life (T) of 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 96 hours or more, or 120 hours or more. In one embodiment, the polyion complex has a half-life (T) of 24 to 168 hours, 36 to 144 hours, or 48 to 120 hours.

[0050] In one embodiment, the polyion complex can be incorporated into macrophages (e.g., CD45+F4 / 80+ macrophages) in an amount that is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the amount of a reference polyion complex having a crosslinking density of 3% to 10%.

[0051] Isothermal titration calorimetry (ITC) can measure the amount of material bound to a polyion complex. The ITC is equipped with a sample cell and a reference cell. A few microliters of phosphate-buffered saline (PBS) containing 20% ​​fetal bovine serum (FBS) in a titration syringe is continuously titrated against a polyion complex solution in a sample cell maintained at a constant temperature. As the FBS solution is titrated into the sample cell and the two materials interact, heat is generated in direct proportion to the amount of binding. As the titration progresses, the binding sites of the polyion complex in the sample cell become saturated, the heat signal decreases, and only the heat of dilution of the FBS is observed. In one embodiment, the heat generated in the first titration is 200%, 190%, 180%, 170%, 160%, 150%, 140%, 130%, 120%, or 110% of the heat of dilution of the FBS in the ITC. Preferably, 15 or more titrations are required to achieve saturation of binding.

[0052] In one embodiment, the polyion complex as a vesicle can include pharmacologically active components such as small molecules, amino acids, peptides, proteins (e.g., enzymes, metabolic enzymes involved in metabolic pathways), oligonucleotides, and polynucleotides.

[0053] In one embodiment, the protein that can be included in the polyion complex is an enzyme that can degrade nutrients required for tumor or fibrosis.

[0054] When an enzyme is encapsulated in a PICsome and the enzyme's substrate is brought into contact with the outer surface of the PICsome, the enzyme is retained within the PICsome, but the substrate present outside the PICsome can pass through the PICsome membrane and react successfully with the enzyme within the PICsome, and the resulting reaction product can be released outside the PICsome.

[0055] Therefore, the present invention provides a polyion complex polymersome encapsulating an enzyme. In the present invention, the enzyme may act on a substance that passes through the membrane of the polyion complex polymersome as a substrate.

[0056] According to the present invention, the membrane permeability of PICsomes containing PEG with a molecular weight of 6 kDa was low. Furthermore, the membrane permeability of PICSOMEs containing non-fibrous proteins is expected to be even lower. Therefore, in one embodiment of the present invention, enzymes with molecular weights of 5 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, 100 kDa or more, 110 kDa or more, 120 kDa or more, 130 kDa or more, or 140 kDa or more can be used. Enzymes with a molecular weight of 5 kDa or more can be retained within PICsomes. The larger the molecular weight of the enzyme, the more difficult it is for the enzyme to pass through the PICsome membrane, making it easier for the enzyme to be retained within the PICsome.

[0057] According to the present invention, the enzyme substrate is water-soluble and has a molecular weight of less than 5 kDa, 4 kDa or less, 3 kDa or less, 2 kDa or less, 1 kDa or less, 750 Da or less, 500 Da or less, 400 Da or less, 300 Da or less, or 200 Da or less. If the molecular weight of the substrate is less than 5 kDa, the solubilized substrate can pass through the PICsome membrane. The smaller the molecular weight of the substrate, the easier it is for the substrate to pass through the PICsome membrane, resulting in easier contact of the substrate with the enzyme and improved reaction efficiency.

[0058] The present invention has the advantage that fibrous polymers are retained within PICsomes. Branched polymers are easily retained within PICsomes due to the low membrane permeability of PICsomes. Therefore, although fibrous proteins can be used as enzymes in the present invention, globular proteins are preferably used.

[0059] According to the present invention, enzymes encapsulated in PICsomes are more stable under physiological conditions and in blood than unencapsulated enzymes. According to the present invention, enzymes encapsulated in PICsomes are particularly stable in blood than unencapsulated enzymes. Thus, in one embodiment of the present invention, the enzyme may be an enzyme that acts on a plasma component as a substrate. This configuration allows the enzyme and its activity to be stably maintained in blood, thereby enabling it to effectively process its substrate in blood.

[0060] In one embodiment of the present invention, L-asparaginase can be used as the enzyme. L-asparaginase (hereinafter also referred to as "L-ASP") is a protein with a molecular weight of approximately 141 kDa that hydrolyzes asparagine to produce aspartic acid and NH3. L-ASP is commercially available as a therapeutic agent for acute lymphoblastic leukemia, for example, from Kyowa Hakko Kirin Co., Ltd. under the trade name Leunase™. L-ASP is also used in the treatment of mast cell tumors. L-ASP can be administered by intravenous injection.

[0061] In one embodiment, the enzyme is asparaginase, which decomposes asparagine to produce aspartic acid and NH3, and exerts a therapeutic effect on tumor cells, including tumor cells with desmoplasia or tumor cells that require asparagine. When asparaginase is contained in a polyion complex of the present disclosure, the concentration of asparaginase in the blood is maintained for a longer period of time than the concentration of naked asparaginase, and the enzyme exerts a stronger antitumor effect on tumor cells, including tumor cells with desmoplasia (which may not be tumors that require asparagine) or tumors that require asparagine.

[0062] In one embodiment, exemplary tumors include, but are not limited to, solid tumors and hematopoietic cancers, such as cancers selected from the group consisting of malignant melanoma, lung cancer, lung adenocarcinoma, lung cancer, small cell lung cancer, lung squamous cell carcinoma, kidney cancer, bladder cancer, head and neck cancer, breast cancer (including triple-negative breast cancer), esophageal cancer, glioblastoma, neuroblastoma, myeloma, ovarian cancer, colorectal cancer, pancreatic cancer, prostate cancer, hepatocellular carcinoma, mesothelioma, cervical cancer, and gastric cancer. In a preferred embodiment, the tumor is pancreatic cancer. In one embodiment, exemplary tumors include, but are not limited to, metastatic cancers (e.g., metastatic breast cancer and metastatic triple-negative breast cancer) and non-metastatic cancers.

[0063] In one embodiment, polyion complexes having anti-tumor effects (e.g., polyion complexes encapsulating anti-tumor drugs) can be administered in combination with other anti-tumor drugs, such as immune checkpoint inhibitors.

[0064] Treatment with the polyion complex of the present invention, which encapsulates an enzyme capable of degrading nutrients necessary for tumors and fibrogenesis, such as asparaginase, may lead to a reduction in fibrogenesis in tumor tissues in vivo. Fibrogenesis, due to its barrier function, may hinder the delivery of antitumor drugs to tumors. Therefore, the polyion complex of the present invention can enhance the permeability of antitumor drugs (or anticancer drugs), particularly small molecule drugs such as chemotherapeutic agents and targeted therapeutic agents, medium molecule drugs such as peptides, antibodies (e.g., antibodies with ADCC activity or CDC activity, or antibody-derived bispecific molecules), immune checkpoint inhibitors (e.g., small molecules and antibodies), antibody-drug conjugates, and other macromolecular drugs, to tumor tissues. Therefore, the polyion complex of the present invention can be administered in combination with other antitumor drugs. Therefore, a subject being treated with an anticancer drug may be one in which fibrogenesis in tumor tissues is reduced by administering the polyion complex of the present invention, which encapsulates an enzyme capable of degrading nutrients necessary for tumors or fibrogenesis, such as asparaginase.

[0065] Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium), Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), Kline), lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylsulfonates such as busulfan, improsulfan, piposulfan; aziridines such as benzodopa, carboquone, metuledopa, uredopa; ethyleneimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase inhibitors including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatins;aldesleukin, talc, duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma II and calicheamicin omega II (Angew Chem. Intl. Ed. Engl. 1994 33:183-186); dynemicins (including dynemicin A); bisphosphonates (such as clodronate); esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, da Unorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pomycin Antimetabolites such as rufilomycin, puromycin, chelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; ancitabine, Pyrimidine analogues such as azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calstalon, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid;Eniluracil; amsacrine; Bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elfomitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2′,2″-trichlorotriethylamine trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel, Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (Cremophor-free), albumin-processed paclitaxel nanoparticle formulations (American Pharmaceutical Partners, Schumberg, Ill.), and TAXOTERE® (docetaxel, Sanofi-Aventis); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine) (XELODA®; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid;and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0066] In some embodiments, chemotherapeutic agents may include alkylating agents (including monofunctional and bifunctional alkylating agents) such as thiotepa, CYTOXAN® cyclophosphamide, chlorambucil, chromafadine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, temozolomide; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0067] In certain embodiments, chemotherapeutic agents include anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0068] In some embodiments, chemotherapeutic agents include antihormonal agents such as antiestrogens and selective estrogen receptor modulators (SERMs), such as, for example, tamoxifen (tamoxifen citrate, including NOLVADEX®), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0069] In some embodiments, chemotherapeutic agents include aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane, Pfizer), formestany, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole, Novartis), and ARIMIDEX® (anastrozole, AstraZeneca); and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0070] In some embodiments, chemotherapeutic agents include antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0071] In some embodiments, chemotherapeutic agents may include protein kinase inhibitors, lipid kinase inhibitors, or antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras.

[0072] In certain embodiments, chemotherapeutic agents may include VEGF expression inhibitors (eg, ANGIOZYME®) and ribozymes such as HER2 expression inhibitors.

[0073] In certain embodiments, chemotherapeutic agents may include cytotoxic or antitumor antibiotics such as mitomycins, including dactinomycin, actinomycin, bleomycin, plicamycin, mitomycin C, and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0074] In certain embodiments, chemotherapeutic agents include proteasome inhibitors such as bortezomib (VELCADE®, Millennium Pharm.), carfilzomib (KYPROLIS®, Onyx Pharm.), marizomib (NPI-0052), MLN2238, CEP-18770, epoxomicins such as oprozomib, and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0075] In certain embodiments, chemotherapeutic agents include anti-microtubule agents such as vinca alkaloids, such as vincristine, vinblastine, vindesine, and vinorelbine; taxanes, such as paclitaxel and docetaxel; podophyllotoxin; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0076] In certain embodiments, the chemotherapeutic agent includes an "EGFR antagonist," which refers to a compound that binds to or directly interacts with EGFR and inhibits or reduces its signaling activity, also referred to as an "EGFRi." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579 (ATCC CRL HB 8506), MAb455 (ATCC CRL HB8507), MAb225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.), and variants thereof, such as chimeric 225 (C225 or cetuximab, ERBUTIX®) and reshaped human 225 (H225) (WO 96 / 40210, Imclone Systems, Inc.). Inc.); IMC-11F8 (Imclone), a fully human EGFR-targeting antibody; antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR, as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) (EMD / Merck), a humanized EGFR antibody against EGFR that competes with both EGF and TGF-α for EGFR binding; HuMax-EGFR (GenMab), a human EGFR antibody; fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3, and described in U.S. Patent No. 6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29):30375-30384(2004)). Anti-EGFR antibodies may be conjugated with cytotoxic agents to form immunoconjugates (see, for example, EP659439A2, Merck Patent GmbH).EGFR antagonists include small molecules, such compounds are described in U.S. Patents 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, and 6,140,332. , 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO98 / 14451, WO98 / 50038, WO99 / 09016, and WO99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals), PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (Iressa®) 4-(3′-chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca; ZM105180 ((6-amino-4-(3-methylphenylamino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluorophenyl)-N2-(1-methylpiperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); Dual EGFR / HER2 tyrosine kinase inhibitors such as EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0077] In certain embodiments, the chemotherapeutic agent may be a tyrosine kinase inhibitor, including an EGFR-targeted agent as described in the previous paragraph; a small molecule HER2 tyrosine kinase inhibitor, such as TAK165, available from Takeda Pharmaceutical; CP-724,714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); a dual HER inhibitor, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; the oral HER2 and EGFR tyrosine kinase inhibitor lapatinib (GSK572016, available from Glaxo-SmithKline); PKI-166 (available from Novartis); a Pan-HER inhibitor, such as canertinib (CI-1033, Pharmacia); a Raf-1 inhibitor, such as ISIS-5132, an antisense agent from ISIS Pharmaceuticals that inhibits Raf-1 signaling; imatinib mesylate (GLEEVEC®, Glaxo) non-HER-targeted TK inhibitors such as SmithKline; multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, Novartis / Schering AG); MAPK extracellular-regulated kinase I inhibitor CI-1040 (Pharmacia); quinazolines such as PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP59326, CGP60261 and CGP62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to nucleic acids encoding HER); quinoxalines (U.S. Patent No. 5,804,396); tryphostin (U.S. Patent No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG);pan-HER inhibitors such as CI-1033 (Pfizer), Afinitac (ISIS3521, Isis / Lilly), imatinib mesylate (Gleevec®); PKI166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth), semaxinib (Pfizer), ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (imclone); rapamycin (sirolimus, RAPAMUNE®), or those described in any of the following patent publications: U.S. Pat. No. 5,804,396; WO1999 / 09016 (American Cyanamid); WO1998 / 43960 (American Cyanamid); WO1997 / 38983 (Warner WO1999 / 06378 (Warner Lambert); WO1999 / 06396 (Warner Lambert); WO1996 / 30347 (Pfizer, Inc); WO1996 / 33978 (Zeneca); WO1996 / 3397 (Zeneca) and WO1996 / 33980 (Zeneca).

[0078] In certain embodiments, the chemotherapeutic agent includes a retinoid, such as retinoic acid, and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0079] In some embodiments, the chemotherapeutic agent includes an antimetabolite. Examples of antimetabolites include folic acid analogs and antifolates such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as 5-fluorouracil (5-FU), ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; nucleoside analogs, and nucleotide analogs.

[0080] In some embodiments, the chemotherapeutic agent comprises a topoisomerase inhibitor. Examples of topoisomerase inhibitors include topoisomerase 1 inhibitors such as LURTOTECAN® and ABARELIX® rmRH, topoisomerase II inhibitors such as doxorubicin, epirubicin, etoposide, and bleomycin, and the topoisomerase inhibitor RFS2000.

[0081] In some embodiments, chemotherapeutic agents include histone deacetylase inhibitors such as vorinostat, romidepsin, belinostat, mocetinostat, valproic acid, panobinostat, and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0082] Chemotherapeutic agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17- butyric acid, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate; phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN) immunoselective anti-inflammatory peptides (ImSAIDs) such as BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), d-penicillamine, gold salts, hydroxychloroquine, leflunomide minocycline, and sulfasalazine; tumor necrosis factor alpha (TNFα) inhibitors such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi); interleukin-1 (IL-1) inhibitors such as anakinra (Kineret); T-cell costimulation inhibitors such as abatacept (Orencia); interleukin-6 (IL-6) inhibitors such as tocilizumab (Actemera®); interferon alpha (IFN) blockers such as rontalizumab; rhuMAb beta7 integrin blockers such as Beta7; IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin alpha (LTa); radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb212 and radioactive isotopes of Lu); various investigational drugs such as thioplatin, PS-341, phenylbutyric acid, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta -lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin), podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®), clodronate (e.g., BONEFOS® or OSTAC®), etidronic acid (DIDROCAL®), NE-58095, zoledronic acid / zoledronic acid (ZOMETA®), alendronate (FOSAMAX®), pamidronate bisphosphonates such as thiazolinone (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; oblimersen sodium (GENASENSE®) pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is an abbreviation for a treatment regimen with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0083] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs), which have analgesic, antipyretic, and anti-inflammatory effects. NSAIDs include nonselective inhibitors of the cyclooxygenase enzyme. Specific examples of NSAIDs include propionic acid derivatives such as aspirin, ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen; acetic acid derivatives such as indomethacin, sulindac, etodolac, and diclofenac; enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam; fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid; and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib. NSAIDs are indicated for the relief of symptoms such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathy, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, mild to moderate pain due to inflammation and tissue injury, fever, intestinal obstruction, and renal colic.

[0084] For example, targeted therapeutic agents include B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, dual phosphatidylinositol 3-kinase / mTOR inhibitors, and combinations thereof. As used herein, the term "inhibitor" is used in the broadest sense to encompass any small molecule, protein, or other macromolecule that interferes with the biological activity of its target.

[0085] In certain embodiments, the targeted therapeutic agents include B-Raf inhibitors such as vemurafenib (also known as Zelboraf®), dabrafenib (also known as Tafinlar®), and erlotinib (also known as Tarceva®); MEK inhibitors such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2), cobimetinib (also known as GDC-0973 or XL-518), and trametinib (also known as Mekinist®); K-Ras inhibitors, c-Met inhibitors such as onartuzumab (also known as MetMAb); Alk inhibitors such as AF802 (also known as CH5424802 or alectinib); phosphatidylinositol inhibitors such as idelalisib (also known as GS-1101 or CAL-101), BKM120, and perifosine (also known as KRX-0401). 3-kinase (PI3K) inhibitors; Akt inhibitors such as GSK690693, MK2206, and GDC-0941, sirolimus (also known as rapamycin), temsirolimus (also known as CCI-779 or Torisel®), everolimus (also known as RAD001), ridaforolimus (also known as AP-23573, MK-8669, or deforolimus), OS mTOR inhibitors such as I-027, AZD8055, and INK128; and dual phosphatidylinositol 3-kinase (PI3K) / mTOR inhibitors such as XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502, and PF-05212384 (also known as PKI-587).

[0086] In some embodiments, the anti-cancer agent is a T cell expressing a chimeric antigen receptor. Chimeric antigen receptor (or CAR), as used herein, can refer to any engineered receptor specific to an antigen of interest that, when expressed in a T cell, confers CAR specificity to the T cell. Chimeric antigen receptor-expressing T cells, created using standard molecular techniques, can be introduced into patients using techniques such as adaptive cell transplantation. For example, chimeric antigen receptor-expressing T cells may express a dominant-negative TGF-beta receptor, e.g., a dominant-negative TGF-beta type II receptor. An example of a treatment using T cells expressing a chimeric antigen receptor and a dominant-negative TGF-beta receptor includes the HERCREEM protocol (see, e.g., ClinicalTrials.gov identifier NCT00889954).

[0087] In some embodiments, the anti-cancer agent is an antibody or antigen-binding fragment thereof. For example, antibodies or antigen-binding fragments thereof include alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth), and combinations thereof.Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidutuzumab, clivatuzumab, daclizumab, mab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, natalizumab, and the like. tuzumab, nimotuzumab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, btetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab-celmoleukin, tuxituuzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, anti-IL-12 (e.g., ABT-874 / J695, Wyeth Research and Abbott Laboratories)—that is, recombinant, full-length, human-sequence IgG1λ antibodies engineered to recognize the IL-12 p40 protein, anti-IL-17 (e.g., MCAF5352A or RG7624), and combinations thereof.

[0088] In certain embodiments, the anti-cancer agent is an antibody or antigen-binding fragment thereof that specifically binds to a target selected from CD52, VEGF-A, EGFR, CD20, HER2, HLA-DRB, CD62L, IL-6R, amyloid beta, CD44, CanAg, CD4, TNF-alpha, IL-2, CD25, complement C5, CD11a, CD22, CD18, respiratory syncytial virus F, interferon gamma, CD33, CEACAM5, IL-5, integrin alpha 4, IgE, IL-4, IL-5, CD154, FAP, CD2, MUC-1, AFP, integrin alpha IIb beta 3, ClfA, IL6R, CD40L, EpCAM, Shiga-like toxin II, IL-12, IL-23, IL-17, and CD3. In certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to IL-17 (such as anti-IL-17 antibodies described above) include antibodies or antigen-binding fragments thereof that specifically bind to IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, and combinations thereof.

[0089] In some embodiments, the anti-cancer drug is an antibody-drug conjugate. For example, the antibody-drug conjugate may include mertansine or monomethyl auristatin E (MMAE), such as an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599), trastuzumab emtansine (also known as T-DM1, adotrastuzumab emtansine, or KADCYLA®, Genentech), DMUC5754A, bivatuzumab mertansine or cantuzumab mertansine, and an antibody-drug conjugate targeting endothelin B receptor (EDNBR) (e.g., an antibody against EDNBR conjugated with MMAE). For example, the antibody-drug conjugate may also include calicheamicin or esperamicin (e.g., calicheamicin k or esperamicin A1), such as gemtuzumab ozogamicin (MYLOTARG®, Wyeth) or inotuzumab ozogamicin. For example, an antibody-drug conjugate may also include a radioisotope chelator, e.g., a tetraxetan, such as tacatuzumab tetraxetan or clivatuzumab tetraxetan, or a tiuxetan, such as ibritumomab tiuxetan (ZEVALIN®, Spectrum Pharma.). The term "antibody," with respect to the antibody-drug conjugates of the present disclosure, is used in the broadest sense and specifically covers monoclonal antibodies formed from at least two intact antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab fragments, scFv, minibodies, diabodies, scFv multimers, or bispecific antibody fragments), so long as they exhibit the desired biological activity, i.e., specific binding to an antigen and the ability to be conjugated to a drug.

[0090] In some embodiments, the anti-cancer drug is an angiogenesis inhibitor. For example, angiogenesis inhibitors include VEGF antagonists, for example, VEGF-A antagonists such as bevacizumab (also known as AVASTIN®, Genentech), and angiopoietin 2 antagonists (also known as Ang2) such as MEDI3617. In some embodiments, angiogenesis inhibitors include antibodies.

[0091] In some embodiments, the anti-cancer agent is an anti-tumor agent. For example, anti-tumor agents include agents that target CSF-1R (also known as M-CSFR or CD115), such as anti-CSF-1R (also known as IMC-CS4); interferons, such as interferon alpha or interferon gamma, such as roferon-A (also known as recombinant interferon alpha-2a); GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor, rhu GM-CSF, also known as sargramostim or Leukine®); IL-2 (also known as aldesleukin or Proleukin®); IL-12, and antibodies that target CD20, such as obinutuzumab (also known as GA101 or Gazyva®) or rituximab.

[0092] In some embodiments, the anticancer agent is a cancer vaccine. For example, the cancer vaccine includes a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long-chain peptide vaccine, a multi-peptide vaccine, a peptide cocktail vaccine, a hybrid peptide vaccine, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013).

[0093] In certain embodiments, the anti-cancer agent is an adjuvant. Substances that enhance, for example, the anti-cancer immune response to cancer-associated antigens or that aid in the presentation of cancer antigens to components of the immune system may be considered anti-cancer adjuvants of the present disclosure.

[0094] In certain embodiments, the anti-cancer agent is an agent selected from a TLR agonist, e.g., poly-ICLC (also known as Hiltonall®), LPS, MPL, or CpG ODN, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonist, IL-4 antagonist, IL-13 antagonist, CX3CL1-targeted therapy, CXCL9-targeted therapy, CXCL10-targeted therapy, CCL5-targeted therapy, LFA-1 or ICAM1 agonist, and a selectin agonist.

[0095] It is known that tumors express PD-L1 to suppress the activity of immune cells that express PD-1, and that blocking the interaction between PD-1 and PD-L1 reactivates tumor immunity, making it effective in tumor treatment. PD-1 and PD-L1 are classified as immune checkpoint molecules, and inhibitors of immune checkpoint molecules are called immune checkpoint inhibitors.

[0096] Examples of immune checkpoint inhibitors include, but are not limited to, checkpoint inhibitors that block PD-1-mediated signaling or checkpoint inhibitors that block CTLA-4-mediated signaling. In a preferred embodiment, a checkpoint inhibitor that blocks PD-1-mediated signaling may be an antibody that can neutralize the binding of PD-1 to PD-L1 or PD-L2. In a preferred embodiment, a checkpoint inhibitor that blocks CTLA-4-mediated signaling may be an antibody that can neutralize the binding of CTLA-4 to CD80 or CD86. Antibodies that neutralize the binding of PD-1 to PD-L1 include anti-PD-1 antibodies and anti-PD-L1 antibodies that can neutralize the binding of PD-1 to PD-L1. Antibodies that neutralize the binding of PD-1 to PD-L2 include anti-PD-1 antibodies and anti-PD-L2 antibodies that can neutralize the binding of PD-1 to PD-L2. Antibodies that can neutralize the binding of CTLA-4 to CD80 or CD86 include anti-CTLA-4 antibodies that neutralize the binding of CTLA-4 to CD80 or CD86. Immune checkpoint inhibitors that can be used in the present invention include, but are not limited to, anti-PD-1 antibodies such as nivolumab, pembrolizumab, and pidilizumab, anti-PD-L1 antibodies such as atezolizumab, durvalumab, and avelumab, and anti-CTLA-4 antibodies such as ipilimumab.

[0097] The present disclosure provides a composition comprising the polyion complex described above. The composition is suitable for delivering a substance of interest to a target tissue. The composition may be a pharmaceutical composition. The pharmaceutical composition is used to treat a subject in need thereof to obtain clinical benefit from the composition. The pharmaceutical composition may be formulated as a parenteral preparation for parenteral administration. The pharmaceutical composition may be administered intravenously, intraperitoneally, intratumorally, intramuscularly, intraarticularly, subcutaneously, or intradermally. The pharmaceutical composition may comprise the polyion complex of the present disclosure and pharmaceutically acceptable additives such as carriers, excipients, and diluents. The pharmaceutical composition may be in lyophilized form. The lyophilized pharmaceutical composition may be provided with water for reconstitution or water for injection.

[0098] The present disclosure provides a method of treating a subject comprising administering to the subject a polyion complex of the present disclosure.The present disclosure provides a method of administering a molecule to a subject comprising administering to the subject a polyion complex of the present disclosure comprising the molecule.

[0099] The present disclosure provides a method for producing the polyion complexes of the present disclosure. The method may include: A polyion complex is formed by mixing a positively charged polycation and a negatively charged polyanion, and the amounts of the polycation and polyanion are adjusted so that the positive and negative charges are neutralized. Crosslinking the polycation and polyanion with a crosslinking reagent in the polyion complex to reduce the counter ions in the polyion complex; A crosslinked polyion complex is obtained. The method may comprise the steps of: A polyion complex is formed by mixing a positively charged polycation and a negatively charged polyanion, and the amounts of the polycation and polyanion are adjusted so that the positive and negative charges are neutralized; crosslinking the polycation and the polyanion with the crosslinker in the polyion complex to reduce the counter ions in the polyion complex; A crosslinked polyion complex is obtained.

[0100] The present disclosure provides, for example, the following inventions:

[0101] Item 1 A polyion complex comprising a polycation and a polyanion, The polycation has a positive charge and comprises (i) a cationic polymer or a block copolymer comprising a cationic polymer block, and optionally (ii) an uncharged hydrophilic polymer block; the polyanion has a negative charge and comprises (iii) an anionic polymer or a block copolymer comprising an anionic polymer block, and optionally (iv) an uncharged hydrophilic polymer block; the polycation and the polyanion interact with each other to form the polyion complex; the positive and negative charges are neutralized within the polyion complex, and the ratio of the positive and negative charges is in the range of 0.95 to 1.05; the polycation and the polyanion are stabilized by crosslinks within the polyion complex and have a crosslink density; The polyion complex has a half-life (T1 / 2) of greater than 24 hours in the blood or serum of a subject.

[0102] Item 2 2. The polyion complex according to item 1, which forms a polyion complex micelle.

[0103] Item 3 2. The polyion complex according to item 1, which forms a polyion complex polymersome.

[0104] Item 4 3. The polyion complex of claim 2, wherein the crosslinking density is greater than 30%.

[0105] Item 5 3. The polyion complex of item 2, wherein the crosslinking density is greater than 40%.

[0106] Item 6 4. The polyion complex of claim 3, wherein the crosslinking density is greater than 30%.

[0107] Item 7 4. The polyion complex of claim 3, wherein the crosslinking density is greater than 40%.

[0108] Item 8 3. The polyion complex according to item 2, wherein the sodium content in the polyion complex micelles is 60% or less after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and having a crosslinking density of 3.9%.

[0109] Item 9 3. The polyion complex according to item 2, wherein the sodium content in the polyion complex micelles is 50% or less after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and having a crosslinking density of 3.9%.

[0110] Item 10 4. The polyion complex according to item 3, wherein the sodium content in the polyion complex polymersome is 60% or less and has a crosslinking density of 7.1% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl.

[0111] Item 11 4. The polyion complex according to item 3, wherein the sodium content in the polyion complex polymersome is 50% or less and the crosslinking density is 7.1% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl.

[0112] Item 12 3. The polyion complex according to item 2, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex micelles with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.5.

[0113] Item 13 3. The polyion complex according to item 2, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex micelles with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.4.

[0114] Item 13 Item 4. The polyion complex according to Item 3, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex polymersome with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.5.

[0115] Item 14 Item 4. The polyion complex according to Item 3, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex polymersome with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.4. [Example]

[0116] Example 1

[0117] material and method

[0118] Materials. α-Methoxy-ω-aminopoly(ethylene glycol) (Mn = 2,200, 5,500, or 12,000) was purchased from Nippon Oil & Fats Co., Ltd. (Tokyo, Japan). β-Benzyl-L-aspartic acid N-carboxyanhydride (BLA-NCA) was purchased from Chuo Kasei Co., Ltd. (Tokyo, Japan). 1,5-Pentanediamine, n-butylamine, 2,2-bis(aminoethoxy)propane, acetic anhydride, fluorescamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 5-carboxyfluorescein diacetate were purchased from Sigma-Aldrich. α-Methoxy-ω-4-nitrophenoxycarbonylpolyoxyethylene (Mw = 2,000) and methoxy-PEG succinimidyl carboxymethyl ester (PEG-NHS, Mw = 5,000) were obtained from Yuka Sangyo Co., Ltd. (Tokyo, Japan). The solvents used for NCA polymerization (dichloromethane and dimethylformamide) were purified using an Ultimate Solvent System 4-2S-TKII (Osaka, Japan). Asparaginase (10,000 U) was provided by Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Sulfo-Cy3 NHS ester (Cy3-NHS) and Sulfo-Cy5 NHS ester (Cy5-NHS) were purchased from Lumiprobe Corporation (Hallandale Beach, Florida, USA). Bouin's solution and 4% paraformaldehyde were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cell lysis buffer was purchased from Promega Corporation (Madison, USA). ACK lysis buffer was purchased from Gibco, Life Technologies (New York, USA). DAPI was obtained from Thermo Scientific (Waltham, USA). The deproteinized sample preparation kit-TCA (ab204708) and asparagine assay kit (ab273333) were purchased from Abcam (Chambridge, UK). FITC anti-mouse CD45 antibody (#103108) and PE anti-mouse F4 / 80 antibody (#123110) were provided by Biolegend (San Diego, USA).Anti-mouse F4 / 80 antibody (eFluor450, #48480182) was purchased from eBioscience. Alexa Fluor® 488 anti-alpha smooth muscle actin antibody [1A4] (ab184675), anti-collagen I antibody (ab21286), and anti-rabbit IgG H&L (Alexa Fluor® 488, ab150077) were obtained from Abcam. InVivoMAb anti-mouse PD-1 (CD279, clone: ​​RMP1-14) was purchased from Bio X Cell (Lebanon, USA).

[0119] Cell lines. The primary KPC cell line was derived from a genetically engineered mouse model (LSL-Kras G12D / +, LSL-Trp53 R172H / +, and Ptf1a-Cre) and was provided by Dr. Ryo Tsumura (National Cancer Center, EPOC, Development and Therapeutics Division, Kashiwa, Japan). The MDA-MB-231-LM2-4175 (referred to as 231 / LM2) human triple-negative breast cancer (TNBC) cell line, which developed spontaneous lung metastasis after orthotopic transplantation, was kindly provided by Professor Joan Massague (Memorial Sloan Kettering Cancer Center). The 4T1 murine TNBC cell line and RAW264.7 cell line were purchased from the Japan Bioresource Research Center (JCRB) Cell Bank (Osaka, Japan). KPC, 231 / LM2, and 4T1 cells were stably transfected with a lentiviral vector carrying firefly luciferase (Luc). 231 / LM2 cells were also transfected with GFP. Cells were routinely cultured at 37°C in DMEM medium (Gibco) supplemented with 10% FBS (Invitrogen), 100 U / ml penicillin (Invitrogen), and 100 U / ml streptomycin (Invitrogen) in a humidified atmosphere containing 5% CO .

[0120] Animals. Five-week-old female C57BL / 6J mice, BALB / c mice, and BALB / c nude mice were purchased from Charles River (Tokyo, Japan). Animal experiments were performed in accordance with the ethical guidelines of the NanoMedicine Innovation Center (Kawasaki, Japan).

[0121] Polymer synthesis and characterization. PEG-b-poly(5-aminopentyl-aspartamide) (PEG-bP(Asp-AP)), homo-poly(5-aminopentyl aspartamide) (P(Asp-AP)), and PEG-b-poly(aspartic acid) (PEG-b-PAsp) were prepared according to a previous report. 16 Typically, PEG-b-poly(β-benzyl-L-aspartate) (PEG-b-PBLA) and homo-poly(β-benzyl-L-aspartate) (homo-PBLA) were prepared using PEG-NH2 (M) as an initiator, respectively. n = 2,200, 5,500, or 12,000 g mol -1 ) and n-butylamine by ring-opening polymerization (ROP) of β-benzyl-L-aspartate N-carboxyanhydride (PBLA-NCA). Subsequently, further hydrolysis (under alkaline conditions) and aminolysis (using 1,5-pentanediamine) of the benzyl ester groups of the PBLA segments afforded carboxyl-based polyanions (PEG-b-PAsp) and amino-based polycations (PEG-bP(Asp-AP) and homo-P(Asp-AP)), respectively, as white powders after lyophilization. The degree of polymerization (DP) was recorded on an NMR JNM-ECS400 (JEOL) spectrometer (Akishima, Japan) using DMSO-d6 or DO as the solvent. 1 The molecular weight (MW) and molecular weight distribution (MW / Mn) were determined by H NMR spectroscopy. The molecular weight (MW) and molecular weight distribution (MW / Mn) were determined by gel permeation chromatography (GPC) using a Superdex 200 10 / 300 column (GE Healthcare, Little Chalfont, UK). The eluents were 10 mM phosphate buffer (pH 7.4) containing 500 mM NaCl for polyanions and 10 mM acetic acid solution containing 500 mM NaCl for polycations, with a flow rate of 0.5 mL min. -1In fluorescence studies, the ω-amino group of PBLA was used to label sulfo-Cy3 or sulfo-Cy5. Similarly, further hydrolysis or aminolysis yielded homo-P(Asp-AP)-Cy5 and PEG-bP(Asp-AP)-Cy5 as blue powders, and PEG-b-PAsp-Cy3 as purple powder after lyophilization.

[0122] A typical synthetic procedure for the preparation of PEG-ketal-PAsp, a PEG-removable polyanion with a pH-responsive ketal linker, was as follows. Briefly, 2,2-bis(aminoethoxy)propane (0.81 g, 5 mmol) was dissolved in dichloromethane (30 mL), followed by the slow dropwise addition of α-methoxy-ω-4-nitrophenoxycarbonyl polyoxyethylene (PEG-pNP, Mw 2,000, 1 g, 0.5 mmol) in dichloromethane (30 mL). The reaction was continued at 25 °C for 6 h, and the resulting solution was concentrated and precipitated in diethyl ether to obtain PEG-ketal-NH2. Next, PEG-ketal-NH2 (0.1 g, 0.05 mmol) lyophilized from benzene was used to initiate the ROP of BLA-NCA (1.18 g, 4.75 mmol) in anhydrous dimethylformamide / dichloromethane (5 mL / 20 mL) at 25 °C for 96 h under an argon atmosphere. The reaction mixture was precipitated into diethyl ether to obtain PEG-ketal-PBLA. Next, PEG-ketal-PBLA (185 mg, 0.01 mmol) was dissolved in chloroform (2 mL) and sodium hydroxide (32 mg) in water / methanol / 2-propanol (0.2 mL / 0.4 mL / 0.4 mL) was added. After vigorously stirring at 4 °C for 15 min, 270 μL of acetic acid was added to the reaction mixture. The chloroform was removed by evaporation, and the remaining solution was dialyzed against 10 mM PB (pH 7.4) and then against deionized water at 4 °C. After lyophilization, a white powder of PEG-ketal-PAsp was obtained. The properties of all polymers used are shown in Table 1.

[0123] [Table 1] a The degree of polymerization (DP) per polymer is 1 Determined by 1 H NMR. b Mw / Mn was determined by GPC.

[0124] Construction of PIC nanoparticle continuum. All polycations and polyanions were at a final concentration of 1 mg mL. -1 Stock solutions were prepared by dissolving PEG-b-PAsp and PEG-bP(Asp-AP) separately in 10 mM PB (pH 7.4). Stoichiometric charge ratios of PEG-b-PAsp and PEG-bP(Asp-AP) were physically mixed and vortexed for 2 minutes. Various amounts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 20, and 50 equivalents relative to carboxyl / amino groups) were then added. The crosslinking reaction continued for 6 hours at 25°C. The resulting continuum of PIC micelles with different crosslinking densities was purified by ultrafiltration using a polyethersulfone membrane (MWCO 50 kDa). PEG-b-PAsp and homo-P(Asp-AP) were used to construct continuum PIC vesicles using the same procedure as described above. Similarly, PEG-ketal-PAsp and homo-P(Asp-AP) were used to prepare PEG-removable PIC vesicles. For fluorescence studies, 25 mol% Cy5-labeled polycations (PEG-bP(Asp-AP)-Cy5 or P(Asp-AP)-Cy5) were added. To formulate PIC nanoparticles conjugated with FRET pairs, 25 mol% Cy5-labeled polycations (PEG-bP(Asp-AP)-Cy5 or P(Asp-AP)-Cy5) and 25 mol% Cy3-labeled polyanions (PEG-b-PAsp-Cy3) were added. Note that for each independent assembly of the continuum, all PIC nanoparticles were prepared in large quantities (e.g., 100 mL) and subsequently dispensed for further crosslinking.

[0125] Quantification of crosslink density. The crosslink density of the continuum was quantified independently in two ways. First, the fluorescamine assay was used to detect the content of primary amines. The concentrations were 0.01, 0.005, 0.0025, and 0.00125 mg mL in 10 mM PB (pH 7.4) containing 500 mM NaCl (75 μL). -1 of PIC nanoparticles, 0.3 mg mL -1 The nanoparticles were mixed with fluorescamine / acetone solution (25 μL) and incubated at 25°C for 15 minutes. Fluorescence signals were monitored using a microplate reader (TECAN, Infinite M1000 PRO) equipped with a filter set of Ex / Em = 390 / 475 nm. Linear regression analysis of the fluorescence density plots was used to obtain the slope. The crosslinking density was calculated as follows: Crosslinking density (%) = (1-slope / slope 100%) × 100%, where 100% represents the slope of the fluorescence density plot for uncrosslinked polyion complex nanoparticles. The crosslinking density was further independently confirmed through quantification of carboxyl groups using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy.

[0126] Construction of PIC vesicles without PEG coating. PEG-removable PIC vesicles with 32.6% cross-linking were treated overnight with 10 mM hydrochloric acid solution, followed by ultrafiltration using a polyethersulfone membrane (MWCO 50 kDa). The complete removal of PEG was confirmed. 1 Confirmed by 1 H NMR.

[0127] Construction of therapeutic nanoreactors. Asparaginase (ASNase) was loaded into PIC vesicles to construct therapeutic nanoreactors (ASNase@V). ASNase in 10 mM PB (pH 7.4) was simply added to the solution during the mixing of homo-P(Asp-AP) and PEG-b-PAsp, followed by vortexing for 2 minutes. Nanoreactors were obtained by further cross-linking with 2 equivalents of EDC and purification using a polyethersulfone membrane (MWCO 300 kDa). The loading amount was determined by the ASNase feed concentration (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, and 5 mg mL).-1 The loading of ASNase@V was optimized by varying the concentration of ASNase@V and quantified by fluorescence of Cy5-labeled ASNase. ASNase@V with a loading of 2.91% was used for further studies.

[0128] Dynamic light scattering (DLS), Förster resonance energy transfer (FRET), and (cryo)transmission electron microscopy (TEM / cryo-TEM) measurements were performed. PIC nanoparticles were prepared at 0.5 mg mL in 10 mM PB (pH 7.4) with or without 150 mM or 300 mM NaCl. -1 The particle size, particle size distribution, scattering intensity, and zeta potential were then measured using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK) equipped with a He-Ne laser (633 nm) and 173° focusing optics. FRET spectra were recorded using a microplate reader (TECAN, Infinite M1000 PRO) with an excitation wavelength of 540 nm and an emission wavelength range of 540–780 nm. The emission intensity ratio (Cy5 / Cy3) at 670 nm and 566 nm was quantified. Meanwhile, 3 mg mL of Cy5 in deionized water was used. -1 PIC nanoparticles were used to prepare TEM and cryo-TEM samples and imaged with a JEOL JEM-1400 transmission electron microscope (120 kV) and a Tecnai G2 Spirit BioTWIN electron microscope (120 kV), respectively.

[0129] Spin-spin relaxation time (T2) measurements. PIC nanoparticles were prepared in 10 mM phosphate buffer / D2O. 150 mM NaCl was added to the PIC nanoparticles at 20 mg mL. -1The purified solution containing the compound was used for NMR relaxation experiments on an NMR JNM-ECS400 (JEOL) spectrometer (Akishima, Japan). The echo peak 180° intensity was recorded at 24 different pulse values ​​at 0.005-second intervals, from 0.005 to 0.12 seconds. The data from the T2 relaxation measurements were fitted to a single exponential curve: signal = B × exp(-TE / T2), where TE is the echo time. The hydrogen atoms of three methylene groups in the side chains of the polycation were selected for analysis of the PIC nanoparticles.

[0130] Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Analysis. PIC nanoparticles in 10 mM pH 7.4 PB containing 150 mM NaCl were separated by ultracentrifugation at 120,000 RPM for 4 hours using a desktop ultracentrifuge, Optima MAX-XP (Beckman Coulter). Separation efficiency was monitored by Cy5 fluorescence of the solution. The separated samples were digested in concentrated HNO3 at 150 °C in an acid- and alkali-resistant fume hood, then redissolved in 1% HNO3 and analyzed for sodium content using an ICP-MS 7700x (Agilent). Standard calibration curves were obtained from 0.1, 1, 10, and 100 ppm NaCl solutions.

[0131] Protein binding and macrophage uptake. In vitro protein binding was analyzed by fluorescence correlation spectroscopy (FCS) using a Zeiss LSM 880 confocal laser scanning microscope equipped with a 40x C-apochromat water immersion objective (Zeiss, NA 1.2) and an isothermal titration calorimeter (MicroCal PEAQ-ITC, Malvern). For FCS, briefly, Cy5-labeled PIC nanoparticles (1 mg mL) were incubated with 100 μg of PIC nanoparticles. -1) was added to 200 μL of 10 mM PB (pH 7.4) or 200 μL of 100% FBS in a μ-slide 8-well (Ibidi GmbH, Germany). After 10 min, FCS measurements were performed with a 647 nm He-Ne laser excitation. Ten independent repeats of the autocorrelation function were collected and analyzed on a Zeiss ConfoCor system. For ITC, 0.04 mg mL of PIC nanoparticles was added to phosphate-buffered saline containing 20% ​​FBS at a stirring speed of 750 rpm. -1 The titration was performed by titrating FBS to a volume of 0.4 μL, followed by 18 injections of 2 μL FBS with an injection time of 4.0 seconds and an interval of 150 seconds between injections. For simplicity, the molar concentration of FBS was calculated based on the albumin molecular weight of 66 KDa (since albumin is the major protein in FBS) and an average protein concentration of 38 g L−1. -1 The total polymer concentration was used as the molar concentration of the sample. The ITC data were fitted to a one-site binding model to obtain thermodynamic parameters (enthalpy (ΔH), entropy (ΔS), free energy (ΔG), binding affinity (KD), and stoichiometry (N)). To assess macrophage uptake, RAW 264.7 cells were plated at 3 × 10 in a 96-well plate. 4 After seeding at a density of 100 cells / well and incubating overnight, Cy5-labeled polyion complex nanoparticles were added to the wells at a final concentration of 0.1 mg mL. -1 After 2 h of incubation, the medium was removed, and 100 μL of passive lysis buffer was added and incubated for an additional 1 h. Fluorescence was recorded using a microplate reader (TECAN, Infinite M1000 PRO) equipped with an Ex / Em = 649 / 670 nm filter set.

[0132] Intravital Real-Time Confocal Laser Scanning Microscopy (IVRTCLSM) Observation and Analysis. For intravital observation, an A1R confocal laser scanning microscope system mounted on an upright ECLIPSE FN1 with a CFI Plan Apo λ 20x objective (Nikon, Tokyo, Japan) was used. The pinhole diameter was adjusted to obtain 10 μm optical slices. To determine the lifetime and FRET signal in blood, earlobe dermis was fixed under a coverslip with immersion oil. 10 seconds after the start of fluorescence recording, Cy5-labeled or Cy3 / Cy5-colabeled PIC nanoparticles (200 μL, 0.5 mg / mL) were intravenously administered to BALB / c mice. Cy5 was excited with a 640 nm diode laser equipped with a 700 / 75 nm bandpass emission filter. The relative fluorescence intensity (%) of a region of interest (ROI) defined on a vein was calculated as follows: Fluorescence Intensity (%) = I / I max × 100%, where I and I max The values ​​represent the background-subtracted intensity of the ROI at the specified time point and the maximum background-subtracted intensity of the ROI, respectively. FRET measurements were performed with a spectral emission range of 569–712 nm and an excitation of 561 nm at a spectral resolution of 5 nm. The spectrally unmixed images (Cy3 and Cy5) were then analyzed to identify the FRET signal of the ROI as follows: FRET signal = (area under the curve of the Cy5 spectrum) / (area under the curve of the Cy3 spectrum). All image analyses were performed using NIS Elements software (Nikon).

[0133] To observe interactions with the liver, a midline abdominal skin incision was carefully made to expose the liver lobe. The exposed liver lobe was secured under a coverslip, and 10 seconds after the start of Cy5 fluorescence recording, 200 μL of Cy5-labeled PIC nanoparticles (0.5 mg / mL) were intravenously administered to BALB / c mice. For bile duct imaging, 5-carboxyfluorescein diacetate (CFDA) was intravenously injected at a dose of 0.2 mg / kg at the indicated time points (9, 24, 48, and 96 hours) after intravenous administration. Five minutes later, CFDA and Cy5 fluorescence were recorded. CFDA was excited with a 488 nm laser equipped with a 520 / 50 nm bandpass emission filter. To track macrophages, 10 μg of F4 / 80 antibody (eFluor 450) was intravenously injected. Five minutes later, F4 / 80 and Cy5 fluorescence were recorded. The F4 / 80 antibody (eFluor 450) was excited with a 403 nm laser with a 520 / 50 nm bandpass emission filter. All image analysis was performed using NIS Elements software (Nikon).

[0134] T of ultra-long period circulating PIC nanoparticles 1 / 2 After intravenous administration (n = 4), blood was collected by venipuncture at various time points (24, 48, 72, 96, 120, and 144 hours), and Cy5 fluorescence in plasma was quantified using a microplate reader. A semi-logarithmic plot of relative fluorescence intensity (%) versus time was generated. Linear regression of the plot was applied to obtain the slope -K. Monophasic T 1 / 2 was calculated as follows: T 1 / 2 =ln2 / K.

[0135] Flow cytometry and immunofluorescence analysis of frozen sections. Cy5-labeled PIC nanoparticles (200 μL, 0.5 mg mL) -1) were administered intravenously to BALB / c mice (n = 3 for short-circulating nanoparticles and n = 5 for long-circulating nanoparticles). At the indicated time points, harvested liver tissue was passed through a 100 μm cell strainer to generate a single-cell suspension, followed by removal of red blood cells using ACK lysis buffer. The single-cell suspension was further incubated with antibodies against CD45 and F4 / 80 according to the manufacturer's instructions. Cells were resuspended in PBS containing 1% BSA and DAPI for flow cytometry (Cell Analyzer LSRFortessa X-20, BD Bioscience). Data were analyzed using FlowJo software. For immunofluorescence analysis of liver tissue sections, livers were cut into 10 μm-thick slices using a Leica cryostat CM1950, subsequently stained with F4 / 80 antibody, and imaged using a Zeiss LSM 880 confocal laser scanning microscope. Pearson's correlation coefficients were quantified using Imaris software.

[0136] Biodistribution. Cy5-labeled PIC nanoparticles (200 μL, 0.5 mg mL -1 ) was intravenously administered to BALB / c mice (n = 5). At the designated time points, the mice were perfused with saline and sacrificed. Organs were removed and analyzed for fluorescence using an in vivo imaging system (IVIS Spectrum SP-BFM-T1, Perkin Elmer). Tissues were further homogenized in passive lysis buffer, followed by analysis of Cy5 fluorescence using a microplate reader (TECAN, Infinite M1000 PRO).

[0137] Antitumor and anti-metastatic effects in orthotopic metastatic triple-negative breast cancer. Orthotopic luciferase-tagged 4T1 or 231 / LM2 tumors (50-100 mm) were transfected into the mammary gland. 3Female BALB / c mice bearing the 4T1 gene or BALB / c nude mice were randomly assigned to the study (n = 5). Mice received intravenous administration of ASNase or ASNase@V (4 U of ASNase equivalent) every 5 days (q5d). Whole-body bioluminescence was monitored via IVIS after intraperitoneal injection of 150 mg kg-1 luciferin. Mice were injected intraperitoneally with luciferin on day 30 for 4T1 and day 60 for 231 / LM2. Ten minutes later, excised lungs were subjected to bioluminescence imaging, and tumor weights were measured. Lungs were further fixed in Bouin's solution for 24 hours, and metastatic nodules were counted. In another independent study (n = 6), mice were perfused with saline followed by 4% PFA at the end of treatment. Lungs were excised and stained with H&E. Images were acquired using an all-in-one fluorescence microscope (BZ-X810, KEYENCE).

[0138] Quantification of asparagine concentration. Mammary glands were cultured with orthotopic luciferase-tagged 4T1 or 231 / LM2 tumors (50–100 mm). 3 Female BALB / c mice bearing HIV-1 or BALB / c nude mice were intravenously administered ASNase or ASNase@V (4 U of ASNase equivalent dose) (n = 5). At designated time points, blood was collected from the abdominal aorta, and tumors were excised. Single-cell suspensions of tumors were counted and lysed in 0.4% trypan blue. Blood samples were centrifuged at 4°C. The resulting tumor lysates and plasma were deproteinized using the Deproteinizing Sample Preparation Kit-TCA (Abcam, ab204708) and a 10 kDa spin column, respectively. Asparagine was quantified using the Asparagine Assay Kit (Abcam, ab273333) according to the kit's protocol.

[0139] Quantification of tumor delivery efficiency. Inject orthotopic luciferase-tagged 4T1 or 231 / LM2 tumors (50–100 mm) into the mammary gland, respectively. 3Female BALB / c mice bearing BALB / c or BALB / c nude mice were intravenously administered Cy5-ASNase, Cy5-ASNase@V (4 U of ASNase equivalent), or empty Cy5-labeled vesicles V 30.3% (n = 5). At the designated time points, mice were perfused with saline and sacrificed. Tumors were harvested and further homogenized in passive lysis buffer, followed by analysis of Cy5 fluorescence using a microplate reader (TECAN, Infinite M1000 PRO). For fluorescence analysis of frozen sections, tumors were cut into 10 μm-thick slices using a Leica Cryostat CM1950 and then imaged using a Zeiss LSM 880 confocal laser scanning microscope.

[0140] Antitumor effect of orthotopic KPC pancreatic cancer. KPC pancreatic cancer was established by orthotopically injecting 5 × 10 5KPC-Luc cells into the pancreatic tail of female C57BL / 6J mice. Six days later, KPC tumor-bearing mice were randomly assigned (n = 5) to receive intravenous injections of ASNase, ASNase@V, PD-1 antibody (aPD-1), or aPD-1 and ASNase@V (ASNase: 4 U per mouse, aPD-1: 5 mg per kg body weight) every 5 days (q5d × 3) for a total of three doses (150 mg kg). -1 Luciferin was injected intraperitoneally, and whole-body bioluminescence was monitored by IVIS, and survival time was recorded. In another independent study (n = 5), mice were sacrificed 22 days after treatment, and the excised tumors were weighed.

[0141] Quantification of desmoplasia and anti-PD-1 antibody delivery in KPC tumors. C57BL / 6J mice bearing KPC tumors were intravenously injected with saline, ASNase, or ASNase@V (4 U per mouse). Five days later, tumors were excised. Immunofluorescence analysis was performed on tumor sections stained with Alexa Fluor® 488 anti-α-smooth muscle actin antibody or anti-collagen I antibody (secondary antibody: Alexa Fluor® 488 anti-rabbit IgG H&L). Meanwhile, mice were injected with Cy5 anti-PD-1 antibody. 12 hours later, to visualize Cy5 anti-PD-1 antibody extravasation, 0.25 mg of fluorescein isothiocyanate dextran was injected 10 minutes before tumor excision to label blood vessels. Collagen I-positive area, α-SMA-positive area, Cy5-αPD-1-positive extravascular area, and mean Cy5-αPD-1 fluorescence were quantified using Image J software.

[0142] result

[0143] The polycations and polyanions synthesized above were mixed to neutralize the charge, yielding polyion complex vesicles such as polymersomes, which were then crosslinked. Vesicles with different crosslinking densities were administered to mice, and the half-life (T) of the vesicles in the blood circulation was measured using fluorescence intensity as an indicator of blood vesicle levels. 1 / 2 The results showed that vesicles with higher cross-linking density have a longer half-life (T 1 / 2 ) was shown. The half-life (T 1 / 2 ) was less than 10 minutes, while the half-lives (T 1 / 2 ) were 97.2 ± 9.7 hours and 43.1 ± 3.4 hours, respectively (see Figure 1). Of note, the half-life (T 1 / 2 ) was 111.1 minutes (see Figure 33), and the half-life (T 1 / 2 ) was 2.2 min (see Figure 34). Thus, neutralization of the positive and negative charges increases the half-life of cross-linked vesicles and micelles.

[0144] Next, the surface polyethylene glycol (PEG) was completely removed under low pH conditions to evaluate the effect of PEG surface coating. Despite the removal of surface PEG, the polyion complex vesicles exhibited a very long blood circulation ability, and the half-life (T) of vesicles without PEG coating was significantly longer. 1 / 2 ) was 104.9 ± 6.4 hours (see Figure 2). This result indicates that although PEG alone is thought to confer stealth effects on substances in the body, PEG is not required to make the vesicles highly stealthy.

[0145] The hydrodynamic diameters of crosslinked micelles and vesicles were observed. The higher the crosslinking density, the smaller the hydrodynamic diameter, indicating that the polyion complex forms an extensive intermolecular ion-pair network, inducing a compact structure. Polyion complex micelles or vesicles consisting of Cy5-labeled polycations and Cy3-labeled polyanions were prepared, crosslinked, and subjected to FRET experiments. When Cy5 and Cy3 are located within 10 nm of each other within the polyion complex, excitation of Cy3 results in emission from Cy5. In these FRET experiments, the crosslinking reaction increased the Cy5 fluorescence / Cy3 fluorescence ratio (Cy5 / Cy3) (see Figure 3). This indicates that the polycation and polyanion become compact within the crosslinked polyion complex depending on the crosslinking density. To confirm whether this compaction is maintained in the blood circulation, mice were administered polyion complex micelles or vesicles consisting of Cy5-labeled polycations and Cy3-labeled polyanions with varying crosslinking densities. Figure 4 shows that micelles with a crosslinking density of 33.1% or higher exhibited Cy5 emission for at least 60 minutes (see also Figure 5 for Cy5 and Cy3 emission from M39.5%). On the other hand, micelles with a crosslinking density of less than 25.7% exhibited low Cy5 emission. Figure 4 also shows that vesicles with a crosslinking density of 30.3% or higher exhibited Cy5 emission for at least 60 minutes. On the other hand, micelles with a crosslinking density of less than 20.9% exhibited low Cy5 emission. These results indicate that the aggregation of polyion complexes due to crosslinking can be maintained even during blood circulation.

[0146] In crosslinked polyion complexes, almost all ion pairs formed an ion-pair network, minimizing the number of counterions. To confirm this, we investigated the degree of interaction between the crosslinked polyion complexes and sodium ions. Crosslinked polyion complexes in phosphate buffer (pH 7.4) containing 150 mM NaCl were subjected to ultracentrifugation. After washing the centrifuged crosslinked polyion complexes, the amount of sodium ions contained in the centrifuged crosslinked polyion complexes was measured. Figure 6 shows that both highly crosslinked polyion complex micelles and polymersomes avoid interactions with sodium ions. This result indicates that neutralized crosslinked polyion complexes have a reduced number of counterions that can interact with ionic substances such as sodium ions.

[0147] Furthermore, we measured the spin-spin relaxation time (T2) of the crosslinked polyion complexes. As shown in Figure 7, the more crosslinked polyion complexes exhibited shorter T2 (seconds), indicating that the crosslinking process reduced chain flexibility and increased the compression of the polyion complexes. We conclude that the ion-pair network formed in the polyion complexes with the minimized number of counterions is stabilized by the crosslinking process.

[0148] The solubility of the polyion complex stabilized by the ion-pair network was investigated. 39.5% and V 30.3% ) were freeze-dried to prepare powder compositions. The freeze-dried polyion complexes were then reconstituted in pure water to examine the solubility of each polyion complex. Figure 8 shows that the polyion complexes exhibited high water solubility and superhydrophilicity. The superhydrophilicity of the ion-pair network is achieved by balancing van der Waals attraction and hydrophobic interactions via hydration repulsion.

[0149] The non-adsorption properties of the cross-linked polyion complexes were examined by isothermal titration calorimetry (ITC). The uptake of the cross-linked polyion complexes by macrophages, which are involved in innate immunity, was also examined. As shown in Figure 9, the highly cross-linked polyion complexes escaped binding to serum proteins, indicating the non-adsorption properties of the polyion complexes. Furthermore, as shown in Figure 9, the highly cross-linked polyion complexes escaped uptake by macrophages, suggesting that the highly cross-linked polyion complexes can escape immune clearance. For dePEGing the vesicles, V 32.6% Even after low pH treatment, dePEGed vesicles were able to escape uptake by macrophages, further suggesting that PEG coating is not necessary if the degree of cross-linking is sufficiently high.

[0150] In vivo nanobio interactions and clearance were examined by intravital microscopy and flow cytometry. As shown in Figure 10, M33.1% and V20.9% adsorbed to the sinus wall immediately upon entering the bloodstream, compared with M39.5% and V30.3%. Flow cytometry analysis indicated that not only macrophages (CD45+F4 / 80+) but also CD45- populations were involved in the uptake of M33.1% and V20.9%. The clearance of the long-circulating M39.5% and V30.3% was also examined. As shown in Figure 11, fluorescence began to appear in the sinus after 10 hours of circulation for M39.5% compared with V30.3%. Furthermore, time-dependent intravital imaging clearly demonstrated that the fluorescence colocalized with bile ducts (Figure 12). Flow cytometry analysis reaffirmed the distinct metabolic pathways of M39.5% and V30.3% (Figure 12). V (30.3%) was found to be slowly captured by CD45+F4 / 80+ macrophages, whereas M (39.5%) did not clearly accumulate in CD45+F4 / 80+ macrophages.

[0151] Asparaginase, which can convert asparagine into aspartic acid and NH3, can be used to inhibit the growth of tumors that require asparagine. In this study, asparaginase was incorporated into the vesicles of the present disclosure to obtain asparaginase-encapsulated vesicles (see Figure 13). Because water-soluble molecules can penetrate the hydrophilic polyion complex layer within the vesicles, asparagine enters the vesicles from the outside, and aspartic acid and NH3 are released from the vesicles. As shown in Figure 13, in Figure 14, vesicles containing asparaginase (crosslinking density approximately 31.2%) were administered to tumor-bearing mice to examine their antitumor effects. Tumor-bearing mice were obtained by implanting tumors (4T1 or 231 / LM2) into the mammary glands of each mouse. The tumor-bearing mice were 50-100 mm in volume. 3 Vesicles were administered intravenously (q5d) at a dose of 4 U per mouse to mice bearing tumors of 4T1 and 231 / LM2 tumors. Mice were analyzed 30 days after treatment. Tumor weights were measured 30 days after treatment. Vesicles containing asparaginase (ASNase@V) inhibited tumor weight in 4T1 and 231 / LM2 tumor-bearing mice (Figure 15). The lungs of each mouse were observed under a fluorescent microscope. Figures 16 and 17 show that asparaginase-encapsulated vesicles (ASNase@V) prevent metastasis of transplanted tumors. Figure 18 shows the reduction in the size and number of metastatic foci after treatment with ASNase@V, as determined by hematoxylin and eosin (H&E) staining.

[0152] Plasma and intratumor asparagine concentrations were measured. Asparaginase-encapsulated vesicles (ASNase@V) effectively reduced plasma and intratumor asparagine concentrations (Figures 19 and 20). Furthermore, orthotopic transfection of luciferase-tagged 4T1 or 231 / LM2 tumors (50-100 mm) into the mammary gland, respectively, significantly reduced plasma and intratumor asparagine concentrations. 3 Delivery of Cy5-labeled asparaginase to tumors was observed in BALB / c nude mice bearing asparaginase. Asparaginase-encapsulated vesicles (ASNase@V) successfully delivered asparaginase to tumors (see Figures 21 and 22). Tumor cells were stably transfected with GFP.

[0153] Asparaginase-encapsulated vesicles (ASNase@V) were administered in combination with an immune checkpoint inhibitor (anti-PD-1 antibody) to mice bearing pancreatic tumors. While asparaginase-encapsulated vesicles (ASNase@V) successfully inhibited tumor growth to the same extent as anti-PD-1 antibody, the combination of ASNase@V and anti-PD-1 antibody synergistically and dramatically reduced tumor weight (Figures 23-26). The survival of mice bearing pancreatic tumors was monitored. Mice administered asparaginase died approximately 30 days after tumor inoculation, whereas ASNase@V significantly improved survival (Figure 27). The combination of ASNase@V and anti-PD-1 antibody synergistically and dramatically improved survival (Figure 27).

[0154] Depletion inhibits fibrogenic responses, such as the proliferation of α-smooth muscle actin-positive fibroblasts and the deposition of extracellular matrix components, in the tumor environment. When ASNase@V successfully induces depletion in tumors, the desmoplastic response of the tumor is reduced (see Figure 32). Therefore, the effect of ASNase@V on the fibrogenic response was observed. As shown in Figures 28, 29, and 31, ASNase@V successfully inhibited the deposition of collagen I and the proliferation of α-smooth muscle actin-positive fibroblasts. As a result, it was observed that anti-PD-1 antibody (aPD-1) penetrated into the tumor tissue (see Figure 30).

[0155] The high surface energy of synthetic nanomaterials significantly limits their biomedical applications. To date, constructing stealth nanomaterials without steric repulsion remains challenging. Here, we report an unprecedented biostealth technology based on cooperative supramolecular interactions (ion-pair networks) that does not rely on steric repulsion. Starting with model polyion complex nanoparticles, we reveal that the cooperative ion-pair network sheath is characterized by both superhydrophilicity and minimal imbalanced binding sites. Remarkably, fine-tuning the stability of the ion-pair network sheath improves circulation half-life by 5500-fold (>100 hours). With minimal interference from nonspecific interactions, these model super-stealth nanoparticles for the first time reliably define a size threshold for targeting the liver parenchyma, revealing limited impact of individually tailored size. Finally, the super-stealth vesicular nanoreactors activate asparaginase for therapeutic efficacy against metastatic breast and pancreatic cancers by significantly improving bioavailability. The alleviation of fibrillation by severe asparagine deficiency further enhances the delivery of anti-PD-1 antibodies, leading to synergistic deficiency immunotherapy. Our innovative observations highlight the importance of integrating natural hierarchical biostructures and may shift the paradigm for the design of non-adsorbing (stealth) materials from molecular to structural holism.

[0156] The high surface energy of nanomaterials, which notoriously leads to undesirable interactions, severely limits their biomedical applications. 1,2 Surface hydration plays an important role in lowering the interfacial energy and resisting interactions at nano-bio interfaces. 1,3However, hydration alone cannot achieve stealth properties. For example, liposomes and silica nanoparticles have highly hydrophilic surfaces due to zwitterions and hydroxyl groups, yet they still tend to absorb proteins and be rapidly removed by the reticuloendothelial system (RES). In other words, fully hydrated surfaces still have non-negligible interfacial energy (or, more precisely, disproportionate binding sites) that trigger nanobio interactions. To solve this problem, the addition of flexible hydrophilic polymers (e.g., PEG, polyzwitterions) has become widespread as a leading conceptual approach to protect disproportionate binding sites by forming an entropic barrier (i.e., steric repulsion). 4-8 In particular, steric repulsion is susceptible to multiple factors. First of all, sufficient polymer coverage is a prerequisite for effective protection. Despite several attempts to understand the influence of polymer density / thickness and chain topological structure / conformation on steric repulsion, improvements remain limited. 9-15 For example, bilayer PEG (i.e., a dense inner layer to adequately protect the core and a more dynamic outer layer to balance the entropic cost) extended the circulation half-life of PLGA nanoparticles up to approximately 10 hours. 15 Moreover, in most cases, polymer modifications are applied semi-empirically or purely theoretically without quantification. Most of the reported hydrophilic polymer-modified nanomaterials still lack a distribution half-life (T 1 / 2 α) is short. 7,8 This indicates that nano-bio interactions are rapid. Therefore, it is essential and rational to go beyond the unshakable belief in steric repulsion and develop conceptually different stealth technologies.

[0157] Thinking boldly outside the box, another, more straightforward way to minimize interfacial energy is to fundamentally eliminate unbalanced binding sites rather than shielding them by entropic barriers. To date, this has been an insurmountable challenge due to the inherent high surface energy of nanomaterials. Here, we report this unprecedented approach to construct low-energy surfaces on nanomaterials through cooperative supramolecular interactions.

[0158] The present invention provides polyion complex (PIC) nanoparticles composed of polycations and polyanions that naturally integrate an extensive network of intermolecular interactions primarily through ion-pairing. Surprisingly, we discovered that PEG steric repulsion is not necessary for the stealth effect. Instead, the ultralong circulation life of PIC nanoparticles is solely due to the stabilization of the ion-pair network sheath by crosslinking. While this represents a conceptually different stealth technology by producing a stable surface at the expense of entropy, entropic stabilization through molecular flexibility is at the very heart of steric repulsion (Supplementary Video 1 and Supplementary Figure 1). This ion-pair network sheath is characterized not only by ultrahydrophilicity due to the formation of a hydration barrier through ion solvation, but also by stable intermolecular interactions that minimize unbalanced binding sites. First, the ionic hydration barrier is stronger than the barrier formed by hydrogen bonding (e.g., PEG hydration). Second, crosslinking reduces chain flexibility and increases chain compression, allowing positive and negative charges to seamlessly interact to form the ion-pair network. This cooperative ion-pair network minimizes unbalanced charges (counterions) and dipole moments, promoting electrostatic binding. Results showed that fine-tuning the stability of the ion-pair network of PIC micelles (30 nm) and PIC vesicles (100 nm) improved the monophasic T1 / 2 by 5,500-fold (from 1 min to 97.2–121.5 h), verifying the super-stealth effect. Based on these model super-stealth nanoparticles, we reliably elucidated the size threshold for targeting the liver parenchyma while minimizing the interference of nonspecific interactions. Furthermore, the extremely slow distribution of 30 nm micelles revealed that individually tailored size has limited impact on targeting the liver parenchyma. Finally, asparaginase-loaded PIC vesicles as super-stealth therapeutic nanoreactors improved the efficacy of deficiency therapy in metastatic breast and pancreatic cancers and demonstrated excellent bioavailability. These results raise the possibility of sensitizing solid tumors to asparagine depletion, a possibility previously limited to the treatment of asparagine-auxotrophic acute lymphoblastic leukemia (ALL).More intriguingly, severe asparagine deficiency, which limits protein synthesis, may alleviate the fibrogenic response known to impede therapeutic drug delivery in pancreatic cancer. Consequently, combining it with an anti-PD-1 antibody resulted in a synergistic deficiency immunotherapy and extended survival in KPC mice. Our disruptive observations highlight the importance of integrating hierarchical bioarchitectures where the majority of the surface is surrounded by zwitterions or opposite charges, potentially shifting the paradigm for the design of non-adsorbing (stealth) materials from molecular to structural holism.

[0159] The publications and patents are incorporated herein by reference in their entirety.

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Claims

1. A polyion complex comprising a polycation and a polyanion, The polycation has a positive charge and comprises (i) a cationic polymer or a block copolymer comprising a cationic polymer block, and optionally (ii) an uncharged hydrophilic polymer block; the polyanion has a negative charge and comprises (iii) an anionic polymer or a block copolymer comprising an anionic polymer block, and optionally (iv) an uncharged hydrophilic polymer block; the polycation and the polyanion interact with each other to form the polyion complex; Within the polyion complex, the positive and negative charges are neutralized, and the ratio of the positive and negative charges is in the range of 0.95 to 1.05; the polycation and the polyanion are stabilized by crosslinks within the polyion complex and have a crosslink density; the polyion complex has a half-life (T1 / 2) of greater than 24 hours in the blood or serum of the subject; Polyion complexes.

2. The polyion complex of claim 1 , which forms a polyion complex micelle.

3. The polyion complex of claim 1 , which forms a polyion complex polymersome.

4. 3. The polyion complex of claim 2, wherein the crosslink density is greater than 30%.

5. The polyion complex of claim 2 , wherein the crosslinking density is greater than 40%.

6. The polyion complex of claim 3 , wherein the crosslinking density is greater than 30%.

7. The polyion complex of claim 3, wherein the crosslinking density is greater than 40%.

8. 3. The polyion complex of claim 2, wherein the sodium content in the polyion complex micelles having a crosslinking density of 3.9% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 60% or less.

9. 3. The polyion complex of claim 2, wherein the sodium content in the polyion complex micelles having a crosslinking density of 3.9% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 50% or less.

10. 4. The polyion complex of claim 3, wherein the sodium content in the polyion complex polymersome having a crosslinking density of 7.1% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 60% or less.

11. 4. The polyion complex of claim 3, wherein the sodium content in the polyion complex polymersome having a crosslinking density of 7.1% after centrifugation for 4 hours in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl is 50% or less.

12. 3. The polyion complex of claim 2, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex micelles with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.

5.

13. The polyion complex of claim 2, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / ml of the polyion complex micelles with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.

4.

14. 4. The polyion complex of claim 3, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex polymersome with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.

5.

15. 4. The polyion complex of claim 3, wherein the calorific value measured by isothermal titration calorimetry after mixing 0.04 mg / mL of the polyion complex polymersome with phosphate buffered saline containing 20% ​​fetal bovine serum is in the range of -0.1 to -0.

4.

16. 16. The polyion complex according to claim 1, wherein the heat generated during the first titration is 200% or less of the heat of dilution upon addition of FBS in isothermal titration calorimetry (ITC), and preferably (i) the polyion complex is a polyion complex micelle having a crosslinking density of more than 40%, or (ii) the polyion complex is a polyion complex polymersome having a crosslinking density of more than 30%.

17. A pharmaceutical composition comprising the polyion complex of any one of claims 1 to 16.

18. 18. The pharmaceutical composition of claim 17, wherein the therapeutic agent is encapsulated.

19. 19. The pharmaceutical composition of claim 18, wherein the therapeutic agent is asparaginase.

20. 20. The pharmaceutical composition of claim 19, administered in combination with other anti-tumor agents.

21. The pharmaceutical composition of claim 20, wherein the other anti-tumor agent is an immune checkpoint inhibitor.

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