Clearly sized nanoparticle conjugates

Abstract

The present invention is directed to nanoparticles comprising: (a) an inner core comprising oligomeric (-)-epigallocatechin gallate (OEGCG); (b) an outer core comprising polyethylene glycol-epigallocatechin gallate conjugate (PEG-EGCG); and (c) a protein molecule as a drug substance encapsulated in the inner core, wherein at least 70% of the nanoparticles have a diameter of 50 to 300 nm. The present invention also provides methods for producing the nanoparticles. The present invention further provides methods for treating cancer by administering to a subject in need thereof an amount of the nanoparticles of the present invention effective to treat the cancer.

Description

Technical Field

[0001] The present invention relates to nanoparticles comprising (a) an inner core containing oligomeric (-)-epigallocatechin gallate (OEGCG), (b) an outer core containing a PEG-EGCG conjugate, and (c) a drug molecule encapsulated in the inner core, wherein at least 70% of the nanoparticles have a diameter of 50 to 300 nm. The present invention also relates to a method for producing the nanoparticles.

Background Art

[0002] Green tea catechins have health benefits such as the prevention of cardiovascular diseases and cancer. Among tea catechins, (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and plays a major role in the beneficial effects of green tea. EGCG has antioxidant, anti-inflammatory, and immunomodulatory effects. EGCG has also been shown to effectively inhibit tumor growth and metastasis by targeting multiple signaling pathways essential for cancer cell survival.

[0003] Despite these desirable activities, the clinical application of EGCG has been limited by its low stability and low oral bioavailability. For example, EGCG is unstable and easily decomposed under physiological conditions. As a result, after oral administration, it is impossible to achieve the plasma concentration of EGCG required to achieve the desired therapeutic effect.

[0004] Treating cancer, a complex disease involving multiple signaling pathways, presents three major challenges. First, cancer arises from a person's immune dysfunction. Immunomodulation to restore the host's immune function is essential for long-term treatment solutions. Second, single therapeutic agents can only modify one disease pathway, resulting in limited efficacy, drug resistance, and unresponsiveness. Cancer cells can evade monotherapy through alternative signaling pathways. Third, because tumor size is only a fraction of body size, drug toxicity and ineffective delivery to target tissues are common challenges in cancer treatment. Only a small fraction of the administered drug reaches the target tissue, while the majority invades normal tissue, resulting in low efficacy against the target tissue and high toxicity to normal tissue.

[0005] The molecular size of a drug determines how much of it selectively goes to target tissue (such as inflamed or rapidly growing tissue) or to other unintended tissues (Chem Soc Rev 2019 Oct 28;48(21):5381-5407). Vascular openings in normal, unintended healthy tissue are generally less than 10 nm. Vascular walls in tumor tissue have gaps of approximately 300–1000 nm. Other inflammatory tissues, such as autoimmune disease organs, have a variety of gap sizes, often exceeding 300 nm. [Overview of the project] [Problems that the invention aims to solve]

[0006] To overcome the aforementioned challenges, there is a need for pharmaceutical compositions and drug delivery systems that can effectively penetrate target tissues without potential toxicity. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 shows the nanoparticle micelle composition of the present invention, in which drug molecules are encapsulated within micelles, and the micelles contain PEG-EGCG conjugates and oligomeric EGCG (OEGCG). [Figure 2]Figure 2 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing trastuzumab. [Figure 3] Figure 3 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing IL-12. [Figure 4] Figure 4 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing anti-CD3. [Figure 5] Figure 5 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing IFN (interferon)-α. [Figure 6A] Figure 6A shows a comparison of trastuzumab nanoparticle micelle compositions prepared with different molar ratios of trastuzumab to OEGCG. [Figure 6B] Figure 6B shows a comparison of trastuzumab nanoparticle micelle compositions prepared with different molar ratios of trastuzumab to OEGCG. [Figure 7A] Figure 7A shows a trastuzumab nanoparticle micelle composition prepared without ultrafiltration at a 10K molecular weight cutoff (Figure 7A). [Figure 7B] Figure 7B shows the trastuzumab nanoparticle micelle composition prepared with 10K molecular weight cutoff ultrafiltration (Figure 7B). [Figure 8A] Figure 8A shows a comparison of trastuzumab nanoparticle micelle compositions prepared without 0.22 μm filtration (Figure 7A). [Figure 8B] Figure 8B shows a comparison of trastuzumab nanoparticle micelle compositions prepared with 0.22 μm filtration (Figure 8B). [Figure 9] Figure 9 shows a comparison of trastuzumab nanoparticle micelle compositions prepared by stepwise freezing (invention), continuous freezing (comparative), or one-step freezing (comparative). [Figure 10] Figure 10 shows tumor volume versus time in mice treated with control, trastuzumab, and a trastuzumab nanoparticle micelle composition. [Figure 11] Figure 11 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing anti-CD71. [Figure 12]Figure 12 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing anti-epidermal growth factor receptor (EGFR). [Figure 13] Figure 13 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing anti-Tau. [Figure 14] Figure 14 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing anti-vascular endothelial growth factor (VEGF). [Figure 15] Figure 15 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing IFN-γ. [Figure 16] Figure 16 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing interleukin (IL)-2. [Figure 17] Figure 17 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing IL-6. [Figure 18] Figure 18 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing IL-15. [Figure 19] Figure 19 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing IL-21. [Figure 20] Figure 20 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). [Figure 21] Figure 21 shows the nanoparticle size distribution of the nanoparticle micelle composition of the present invention containing bovine serum albumin (BSA).

Mode for Carrying Out the Invention

[0008] Detailed Description of the Invention Definition The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0009] The term "cytokine" refers to small proteins (about 5 - 70 kDa) that are important in cell signaling. Cytokines have been shown to be involved in autocrine, paracrine, and endocrine signaling as immunomodulators. Cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0010] The term "epigallocatechin gallate" refers to an ester of epigallocatechin and gallic acid and is used interchangeably with "epigallocatechin-3-gallate" or "EGCG".

[0011] The term "nanoparticle" refers to particles with a diameter less than 1 μm and between 1 - 999 nm.

[0012] The term "oligomeric EGCG" (OEGCG) refers to 3 - 20 covalently linked EGCG monomers. OEGCG preferably contains 4 - 12 EGCG monomers. The structure of OEGCG is shown in WO2006 / 124000. For example, OEGCG can be synthesized according to WO2006 / 124000.

[0013] The term "polyethylene glycol - epigallocatechin gallate conjugate" or "PEG - EGCG" refers to polyethylene glycol (PEG) bound to one or two EGCG molecules. The term "PEG - EGCG" refers to both PEG - mEGCG conjugates (monomeric EGCG) and PEG - dEGCG (dimeric EGCG) conjugates. For example, PEG - EGCG can be prepared by binding aldehyde - terminated PEG to EGCG via the reaction of the free aldehyde group with the C6 and / or C8 positions of the A - ring of EGCG. See WO2006 / 124000 and WO2009 / 054813.

[0014] Nanoparticle composition The present invention provides a nanoparticle micelle composition in which a drug molecule is encapsulated within a micelle, and the micelle contains a PEG-EGCG conjugate in the outer core and oligomeric EGCG (OEGCG) in the inner core (see Figure 1). The nanoparticle micelle composition has a clear and narrow size distribution in which at least 70% of the nanoparticles have a diameter of 50 to 300 nm, and the nanoparticle size distribution has only one major peak containing more than 90% of all nanoparticles.

[0015] The nanoparticle micelle composition of the present invention comprises three active ingredients that have complementary functions to create a "multi-target combination therapy," addressing both the immune response and signaling pathways by its main chain component (OEGCG / PEG-EGCG) and additional signaling pathways by selected protein drug molecules effective in treating complex diseases. Each nanoparticle is a fixed-dose mixed drug containing the three active ingredients in fixed molar ratios.

[0016] The compositions of the present invention treat diseases through multiple signaling pathways. This provides enhanced efficacy (tumor reduction rate) and enhanced patient response rate (number of patients who respond to treatment) in cancer treatment.

[0017] The nanoparticle micelle composition of the present invention has a particle size of 50-300 nm for the majority of particles. This distinct nanoparticle size of 50-300 nm allows for the preferential distribution of the three active ingredients to tumors, thereby reducing penetration into other non-tumor tissues. If the particle size is less than 50 nm, there is a higher risk of the particles distributing into normal tissues and causing cytotoxicity. If the particle size exceeds 300 nm, it can cause excessive uptake by the reticuloendothelial (RE) system, potentially leading to side effects. In the composition of the present invention, more than 70% of the particles are in the 50-300 nm size range, which ensures that the three active ingredients preferentially penetrate tumors over normal tissues and the RE system.

[0018] The nanoparticle micelle composition of the present invention has a narrow particle size distribution, having only one major peak containing more than 90% of all nanoparticles. It is important that the therapeutic composition has a particle size distribution with only one peak, rather than several or more peaks. If the therapeutic composition has multiple molecular sizes, this can cause significant variability in therapeutic effect, patient response, and side effects (toxicity).

[0019] The nanocomplex of the present invention contains, in the main chain of the micelle composition, two active ingredients, OEGCG and PEG-EGCG, which are immunomodulators and signaling modulators. These are potent immunomodulators and derivatives of EGCG that modulate a wide range of disease signaling pathways. For example, EGCG targets tumor-targeting CD8 + It activates T cells and suppresses anti-PD-L1 expression in cancer. EGCG controls both innate and adaptive immunity in autoimmune diseases. However, EGCG has low bioavailability and is unstable. The nanocomplex composition of the present invention overcomes the bioavailability problem of EGCG by forming nanocarriers for delivering EGCG to tumors, and overcomes the stability problem of EGCG by forming OEGCG and PEG-EGCG, thereby effectively enabling EGCG to become a highly effective therapeutic agent.

[0020] The nanocomplex of the present invention further comprises a third active ingredient, which is a drug molecule encapsulated within the nanoparticles. This drug molecule is preferably a protein drug, but is not limited to, cytokines and antibodies. Cytokines include, but are not limited to, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, TARIL, IGF1, GLP-1, IFN-α, IFN-β, IFN-γ, CCL5, CXCL9, CXCL10, CXCL11, CX3CL1, and recombinant cytokine products. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, antibody-drug conjugates, and bispecific antibodies. Monoclonal antibodies are preferred for the present invention. Antibodies suitable for the present invention include anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG3 antibody, anti-TIGIT antibody, anti-TIM3 antibody, anti-HER2 antibody, anti-HER3 antibody, anti-HGFR antibody, anti-EGFR antibody, anti-EpCAM, anti-FOLR1 antibody, anti-c-Met antibody, anti-GD2 ganglioside antibody, anti-GD3 ganglioside, anti-VEGFR1 antibody, anti-VEGF antibody. body, anti-TGF-β antibody, anti-TNF-α antibody, anti-IGF-1R antibody, anti-IL-4 antibody, anti-IL-10 antibody, anti-IL-13 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD40 antibody, anti-CD4 0L antibody, anti-CD43 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD28 antibody, anti-CD38 antibody, anti-CD20 antibody, anti-B7-H3 antibody, anti-B7- H4 antibody, anti-DR5 antibody, anti-MUC1 antibody, anti-Tau antibody, anti-β-amyloid antibody, avagovomab, abituzumab, adalimumab, aducanumab, alemtuzumab, amatuximab, amivantamab, aniflorumab, atezolizumab, avelumab, bapineuzumab, basiliximab, belimumab, benralizumab, becylesomab, bevacizumab, bezlotoxumab , blinatumomab, brazikumab, bronchicutuzumab, kabilizumab, camrelizumab, carrumab, carotuximab, catumakisomab, sedelizumab, cetrerimab, cetuximab, sibisatamab, crenezumab, kusatuzumab, daclizumab, daclizumab, darotuzumab, daratumumab, detumomab, dinutuximab, dorodizumab, durigotuzumab,Dupilumab, durvalumab, eclomeximab, emibetuzumab, epcolitamab, epratuzumab, eptinezumab, erenumab, erzmakisomab, etalasizumab, etesevimab, faretuzumab, fezakinumab, ficratuzumab, figitumumab, freticumab, foralumab, fresolimumab, futuximab, ganitumab, gantenerumab, gatipotuzumab, gevokizumab, golimumab, guselkumab, iculcumab, igobomab, imarumab, imugatuzumab, inebilizumab, infliximab, intetumumab, ipilimumab Istilazumab, ixekizumab, letrizumab, lexatumumab, lintuzumab, mapatumumab, matuzumab, maprilimumab, mepolizumab, mogamulizumab, monalizumab, mosnetuzumab, natalizumab, naxitamab, nesitumumab, nimotuzumab, nivolumab, okalatuzumab, ocrelizumab, ofatumumab, oraratuzumab, oraratumabopicinumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ramucirumab, ranibizumab, rituximab, samarizumab, sarilumab, secukinumab, cintirimab, solan Zumab, teprotumumab, tigatuzumab, tildrakizumab, timigutuzumab, tocilizumab, tomzotuximab, trastuzumab, ustekinumab, vanucizumab, valisakumab, valirumab, vedolizumab, bepalimomab, besenkumab, bicilizumab, bonrelorizumab, zanolimumab, zatuximab, xenoctuzumab, zolbetuximab, adtrastuzumab emtansine, anetumablavutancine, brentuximab vedotin, cantuzumab meltansine, certolizumab pegol, coltuximablavutancine, depatuxizumab mahodotin N, Enapotamab Vedotin, Gemtuzumab Ozogamicin, Glenbatuzumab Vedotin, Iradatuzumab Vedotin, Inatuzumab Vedotin, Indatuximab Labutansine, Indusatuzumab Vedotin, Rifastuzumab Vedotin, Rilotomab Satetraxetan, Rorobotuzumab Meltansine, Rosatuxizumab Vedotin, Rulizumab Pegol, Milbetuximab Sorabtansine, Naratuximab Emtansine, Notuzumab Ozogamicin, Polatuzumab Vedotin-Piiq, Robalpituzumab Tecillin, Sacituzumab Govitecan, Samlotamab Vedotin,These include terisotuzumab vedotin, trastuzumab deruxtecan, and tucozouzumab cermoloykin. The antibodies also include antibody fragments capable of binding to the corresponding antigen, such as Fab, (Fab)2, or single-chain antibodies.

[0021] Nanoparticles with a size of 10-50 nm tend to penetrate both normal and target tissues from the bloodstream.

[0022] Nanoparticles with a size of 50-300 nm preferentially reach tumor or inflamed tissue. Larger nanoparticles (500-999 nm) or micron-sized particles (1000-5000 nm) due to aggregation of smaller nanoparticles can cause toxicity because larger nanoparticles are efficiently taken up by the reticuloendothelial system (RES), also known as mononuclear phagocyte systems (MPS), which are often found in the liver, lungs, and bone marrow. This can reduce the effectiveness of nanoparticle drugs in the desired disease lesion and lead to potential toxicity.

[0023] The inventors have discovered a nanoparticle micelle composition comprising EGCG and a drug for targeted delivery to a target tissue, wherein at least 70% of the nanoparticles have a diameter of 50 to 300 nm, and the nanoparticle size distribution has only one major peak containing more than 90% of all particles. The inventors have also discovered a method for preparing such a nanoparticle composition.

[0024] The present invention relates to a nanoparticle composition comprising nanoparticles having (a) an inner core containing oligomer (-)-epigallocatechin gallate (OEGCG), (b) an outer core containing a PEG-EGCG conjugate, and (c) a drug molecule encapsulated in the inner core, wherein at least 70% of the nanoparticles have a diameter of 50 to 300 nm, and the nanoparticle size distribution has only one major peak containing more than 90% of all particles.

[0025] The structure of the nanoparticles of the present invention is shown in Figure 1.

[0026] In one embodiment, at least 80%, or at least 85%, or at least 90%, or at least 95% of the nanoparticles have a diameter of 50 to 300 nm.

[0027] In one embodiment, the median nanoparticle diameter in the nanoparticle composition is 50-250 nm, 50-200 nm, 80-200 nm, 100-200 nm, or 50-150 nm.

[0028] In one embodiment, the nanoparticle size distribution shows only one major narrow peak containing over 80%, over 85%, over 90%, over 95%, or over 98% of all particles.

[0029] In one embodiment, the protein drug is trastuzumab, and at least 80% or at least 90% of the nanoparticles in the nanoparticle composition have a diameter of 50 to 300 nm. The median nanoparticle diameter in the nanoparticle composition is 60 to 200 nm.

[0030] In one embodiment, the protein drug is IL-12, and at least 75% of the nanoparticles have a diameter of 50–300 nm. The median diameter of the nanoparticles is 60–200 nm.

[0031] In one embodiment, the protein drug is an anti-CD3, and at least 80% or at least 90% of the nanoparticles have a diameter of 50–300 nm. The median nanoparticle diameter is 60–200 nm.

[0032] In one embodiment, the protein drug is IFN-α, and at least 80% or at least 90% of the nanoparticles have a diameter of 50–300 nm. The median nanoparticle diameter is 60–200 nm.

[0033] The nanoparticle composition of the present invention has a particle size of mostly 50-300 nm in diameter, with OEGCG, PEG-EGCG, and drug molecules bound together by hydrophobic interactions. This is stable in hydrophilic environments such as blood circulation, but dissociates in hydrophobic environments such as tumor tissue. This can selectively diffuse from leaky blood vessels into surrounding tissues due to inflammation or other excessive activity (e.g., rapid and uncontrolled tumor growth). Due to its size, its penetration into normal tissue with less leaky blood vessels is limited. When the nanoparticle complex enters hydrophobic tissue, it dissociates, releasing its active components, OEGCG, PEG-EGCG, and drug molecules within the nanocomplex. The released active components regain their bioactivity in slowing cancer progression. The active components within the nanoparticles have a long circulating half-life and act as a sustained-release mechanism, further reducing the required dose of the drug. As a result, adverse effects on normal tissue are further reduced.

[0034] Preparation process for nanoparticle compositions The present invention also relates to a method for preparing nanoparticle compositions of fixed-dose combination drugs. This process is optimized so that at least 70% of the particles are nanometer-sized particles having a diameter of 50 to 300 nm.

[0035] This process includes (a) mixing drug molecules with OEGCG and PEG-EGCG in an aqueous solution, (b) filtering the mixture through a membrane with a molecular weight cutoff of 8,000 to 300,000 daltons to remove low molecular weight molecules and retain high molecular weight molecules, and (c) filtering the high molecular weight molecules through a 0.2 to 0.3 μm membrane and collecting the filtrate.

[0036] The method of the present invention optionally further comprises a freeze-drying step (d) after step (c). Step (d): The filtrate is freeze-dried by stepwise freezing at (i) about 0 to 5°C, (ii) about -20 to -30°C, and (iii) about -60 to 100°C, and then dried.

[0037] In step (a), the drug molecule is dissolved in an aqueous solvent, such as phosphate-buffered saline, saline, water, bicarbonate buffer, oxyhemoglobin buffer, bis-trisalkane, tris-HCl, HEPES, histidine buffer, NP-40, RIPA (radioimmunoprecipitation assay buffer), tricine, TES, TAPS, TAPSO, bicine, MOPS, PIPES, cacodylate, or MES. Preferred solvents are phosphate-buffered saline, saline, or water. The concentration of the protein drug is generally 0.01 to 50 mg / ml, preferably 0.05 to 10 mg / ml, and more preferably 0.1 to 5 mg / ml.

[0038] OEGCG, PEG-EGCG, and optionally EGCG are soluble in ketones, acetonitrile, alcohols, aldehydes, ethers, acetates, sulfoxides, benzenes, organic acids, amides, aqueous buffers, and any combination thereof. Preferred solvents are alcohols, acetonitrile, sulfoxides, amides, and any combination thereof. The concentrations of OEGCG / EGCG and PEG-EGCG are generally independently 0.001 to 10 mg / ml, preferably 0.005 to 1 mg / ml, or 0.1 to 5 mg / ml.

[0039] It is important that OEGCG is in molar excess compared to the drug. Generally, the molar ratio of EGCG to drug molecules in OEGCG is 1-500:1, 2-500:1, 3-500:1, or 5-500:1, preferably 3-100:1, 5-100:1, or 10-50:1. The molar ratio is calculated by the number of moles of monomer EGCG and the number of moles of drug molecules in the OEGCG. With molar excess EGCG, almost all or all of the drug is encapsulated by the OEGCG molecules. Unencapsulated drug may not be selectively distributed to the target tissue, potentially leading to lower efficacy and safety issues, which is avoided in this process by controlling the molar ratio of OEGCG to protein.

[0040] The drug, OEGCG, and PEG-EGCG are mixed at a temperature of approximately 0°C to 60°C, preferably 0°C to 45°C, or 0°C to 37°C, for 1 minute to 2 days, preferably 1 minute to 12 hours.

[0041] In step (b), the mixture is filtered using a membrane with a molecular weight cutoff of 8,000 to 300,000 daltons, preferably 8,000 to 200,000 daltons, 8,000 to 150,000 daltons, or 8,000 to 12,000 daltons, to remove low molecular weight molecules and retain large molecular weight molecules. The material for the ultrafiltration membrane is selected from the group consisting of cellulose (and its derivatives), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, polyvinylidene fluoride, or polyvinylidene difluoride (PVDF), and polypropylene (PP), preferably from the group consisting of cellulose (and its derivatives), PTFE, and PVDF.

[0042] The mixture is optionally diluted with an aqueous solvent, such as that described in step (a) above, before ultrafiltration.

[0043] Ultrafiltration step (b) removes unwanted small molecular weight impurities such as unreacted OEGCG or EGCG or reaction by-products. These impurities may reduce the efficacy and safety of the drug. Excess unreacted OEGCG or EGCG can cause individual nanoparticles to aggregate into particles approximately 1000 nm in size, which can reduce efficacy and potentially lead to toxicity.

[0044] In step (c), the retained high molecular weight molecules are filtered through a membrane with a pore size of approximately 0.2–0.3 μm, for example, 0.22 μm, and the filtrate is collected. This is to remove unwanted impurities of large molecular size, such as giant aggregates. Due to their large size, these aggregates may be excreted once they enter tissue. These aggregates reduce overall efficacy / safety and are more likely to induce immunogenicity in patients. Large nanoparticles are more easily taken up by RES in the liver, lungs, and other undesirable organs.

[0045] The membrane material for step (c) is selected from the group consisting of cellulose (and its derivatives), PES, PTFE, nylon, PVDF, and PP, preferably from the group consisting of cellulose (and its derivatives), PES, and PP.

[0046] In one embodiment, steps (b) and (c) are repeated at least once, for example 1, 2, 3, or 4 times, before step (d) to effectively remove unwanted small molecular impurities and large aggregates.

[0047] After step (c), the filtrate is stored at 2-8°C and remains stable for at least 100 days.

[0048] This process optionally further includes a freeze-drying step (d) after step (c) to provide long-term stability of the nanoparticle composition.

[0049] In step (d), the filtrate collected after filtration through a 0.2-0.3 μm membrane is freeze-dried by first (i) freezing the filtrate in stages at approximately 0-5°C for, for example, approximately 1-3 hours, and (ii) freezing it at approximately -25°C to -30°C for, for example, approximately 1-3 hours, and then (iii) freezing it at, for example, -60°C to -100°C or -70°C to -100°C for, for example, at least 8 hours.

[0050] After freezing, the materials are freeze-dried for 1 to 7 days.

[0051] Freezing and freeze-drying often cause nanoparticles to form aggregates or clusters. These larger particles may be too large to pass through blood vessels and penetrate the tissue environment. As a result, efficacy and safety may be reduced, and immunogenicity may increase. To avoid these changes caused by freeze-drying, this process uses a stepwise freezing process instead of a continuous freezing process (gradually and continuously lowering the temperature during freezing) to maintain nanoparticle size during freeze-drying.

[0052] Pharmaceutical composition The present invention provides a pharmaceutical composition comprising the nanoparticle composition of the present invention and one or more pharmaceutically acceptable carriers optionally. The nanoparticle composition in the pharmaceutical composition is generally about 1 to 90%, preferably 20 to 90%, or 30 to 80%, in the case of tablets, powders, or parenteral formulations. The nanoparticle composition in the pharmaceutical composition is generally 1 to 100%, preferably 20 to 100%, 50 to 100%, or 70 to 100%, in the case of capsule formulations. The nanoparticle composition in the pharmaceutical composition is generally 1 to 50%, 5 to 50%, or 10 to 40%, in the case of liquid suspension formulations.

[0053] In one embodiment, the pharmaceutical composition may be in dosage forms such as tablets, capsules, granules, fine granules, powders, suspensions, patches, parenteral preparations, or injections. The pharmaceutical composition can be manufactured by conventional methods.

[0054] Medicinally acceptable carriers that are non-active ingredients can be selected by those skilled in the art using conventional criteria. Medicinally acceptable carriers include, but are not limited to, physiological saline and electrolyte solutions; ionic and non-ionic osmotic agents, e.g., sodium chloride, potassium chloride, glycerol, glucose; pH adjusters and buffers, e.g., hydroxides, phosphates, citrates, acetates, borates, and trolamine; antioxidants, e.g., bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and salts of ascorbyl palmitate, acids, and / or bases; surfactants such as lecithin and phospholipids, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; Poloxamers and poloxamines; polysorbates, e.g., polysorbate 80, polysorbate 60, and polysorbate 20; polyethers, e.g., polyethylene glycol and polypropylene glycol; polyvinyls, e.g., polyvinyl alcohol and polyvinylpyrrolidone (PVP, povidone); cellulose derivatives, e.g., methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, and salts thereof; petroleum derivatives, e.g., mineral oil, white petrolatum; fats, e.g., lanolin, peanut oil, palm oil, soybean oil; monoglycerides, diglycerides, and triglycerides; polysaccharides, e.g., dextran; glycosaminoglycans, e.g., sodium hyaluronate. Such pharmaceutically acceptable carriers may be used, but are not limited to, known preservatives including benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol to preserve against bacterial contamination, or they may be formulated as non-preservative formulations for single or multiple uses.

[0055] For example, tablets, capsules, or parenteral formulations of active compounds may contain other excipients that are not biologically active and do not react with the active compound. Excipients for tablets or capsules may include fillers, binders, lubricants, and flow enhancers, disintegrants, wetting agents, and release rate regulators. Examples of excipients for tablets or capsules, but not limited to, include carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone.

[0056] For example, tablet formulations may contain non-active ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and titanium dioxide. Capsule formulations may contain non-active ingredients such as gelatin, magnesium stearate, and titanium dioxide. Powdered oral formulations may contain non-active ingredients such as silica gel, sodium benzoate, sodium citrate, sucrose, and xanthan gum.

[0057] How to use The present invention relates to a method for producing combination drugs for the treatment of cancer and other diseases. The method comprises administering an effective amount of the nanoparticle composition of the present invention to a subject in need of treatment. As used herein, “effective amount” means an amount effective in treating a disease by improving a pathological condition or reducing the symptoms of the disease.

[0058] The pharmaceutical compositions of the present invention can be administered by topical and systemic administration. Local administration includes topical administration. Systemic administration includes oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, or rectal), and other systemic administration routes. In systemic administration, the active compound first reaches the plasma and then distributes to the target tissue. Parenteral administration, such as intravenous bolus injection or intravenous infusion, and oral administration are preferred administration routes for the nanoparticle compositions.

[0059] In one embodiment, the protein drug in the nanoparticle composition is an anti-HER2 antibody such as trastuzumab, which is approved for the treatment of breast cancer caused by the HER2 receptor pathway. Only 20% of breast cancer patients respond to trastuzumab therapy. The remaining 80% of breast cancers are caused by mutations in alternative signaling pathways. OEGCG and PEG-EGCG in the nanoparticle composition of the present invention can modulate alternative signaling pathways in breast cancer to treat 80% of breast cancer patients who are trastuzumab nonresponders. Multitarget immune nanocarrier mixture (MINC)-trastuzumab is also suitable for the treatment of bladder cancer, brain cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, and uterine cancer.

[0060] In one embodiment, the protein drug in the nanoparticle composition is IL-12, and includes clinically tested drugs such as NHS-IL-12 and rHU-IL-12. IL-12 is CD4 + T, CD8 +It is known to broadly affect major immune cells such as T, B, and NK cells. However, IL-12 is highly toxic because it significantly impacts the human immune system. MINC-IL-12 greatly reduces its toxicity while increasing its efficacy, making it possible to use IL-12 as a broad-spectrum immunotherapy for many cancers. MINC-IL-12 is suitable for the treatment of bladder cancer, brain cancer, breast cancer, cervical cancer, childhood cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, gastric cancer, and uterine cancer. MINC-IL-12 can be combined with other cancer immunotherapies, such as anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-TIGIT, anti-LAG3, or anti-TIM3, to enhance therapeutic effects.

[0061] In one embodiment, the protein drug in the nanoparticle composition is an anti-CD3 antibody comprising teprizumab, muromonab, otelixizumab, and bicilizumab. Type 1 diabetes is an autoimmune disease caused by the attack of T cells against pancreatic β cells. Anti-CD3 is highly toxic, and severe adverse reactions have been reported in clinical trials. MINC-anti-CD3 improves efficacy and safety by preferentially delivering anti-CD3 to pancreatic β cells and reducing exposure to normal cells. Furthermore, EGCG has the function of promoting the proliferation of pancreatic β cells, helping to restore their function in insulin secretion. MINC-anti-CD3 is suitable for the treatment of rheumatoid arthritis, inflammatory bowel disease, psoriasis, and several other autoimmune diseases.

[0062] In one embodiment, the protein drug in the nanoparticle composition is α-interferon. OEGCG and PEG-EGCG activate T cells and suppress anti-PD-L1, thereby enhancing the response to cancer immunotherapy in cancer patients. IFN-α can also induce the production of IFN-γ, further inhibiting tumor growth. The nanoparticles deliver the active ingredients, OEGCG, PEG-EGCG, and IFN-α, to tumors rather than normal tissue, enhancing the efficacy of IFN-α and reducing its toxicity. MINC-IFN-α can be applied to most cancer types that require an enhanced response to immunotherapy (including bladder cancer, brain cancer, breast cancer, cervical cancer, childhood cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer, gastric cancer, and uterine cancer).

[0063] In one embodiment, the protein drug in the nanoparticle composition is anti-CD71. Anti-CD71 targets CD71, also known as transferrin receptor protein 1, which is a target in human cases of leukemia and lymphoma.

[0064] In one embodiment, the protein drug in the nanoparticle composition is an anti-EGFR. Anti-EGFR is an epidermal growth factor receptor (EGFR) inhibitor used to treat metastatic colorectal cancer, head and neck cancer, and more EGFR-positive cancers.

[0065] In one embodiment, the protein drug in the nanoparticle composition is anti-Tau. The Tau protein causes neurological disorders and dementia, such as Alzheimer's disease and Parkinson's disease. Anti-Tau can be used to treat neurological disorders by targeting the Tau protein.

[0066] In one embodiment, the protein drug in the nanoparticle composition is anti-VEGF. Anti-VEGF is used to block vascular endothelial growth factor in order to treat certain cancers and age-related macular degeneration.

[0067] In one embodiment, the protein drug in the nanoparticle composition is IFN-γ. IFN-γ is a cytokine important for innate and adaptive immunity against viral, certain bacterial, and protozoan infections. IFN-γ is also useful in the treatment of cancer.

[0068] In one embodiment, the protein drug in the nanoparticle composition is IL-2. IL-2 enhances the cytotoxic activity of both natural killer cells and cytotoxic T cells. IL-2 can be used to treat cancers, including malignant melanoma and renal cell carcinoma.

[0069] In one embodiment, the protein drug in the nanoparticle composition is IL-6. IL-6 is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine.

[0070] In one embodiment, the protein drug in the nanoparticle composition is IL-15. IL-15 is a cytokine structurally similar to interleukin-2 (IL-2). IL-15 is CD8 + It has been shown to enhance T-cell anti-tumor immunity.

[0071] In one embodiment, the protein drug in the nanoparticle composition is IL-21. IL-21 has a regulatory effect on immune system cells, including natural killer (NK) cells and cytotoxic T cells, which can destroy virus-infected cells and cancer cells.

[0072] In one embodiment, the protein drug in the nanoparticle composition is TRAIL. TRAIL is a protein that functions as a ligand to induce a process of cell death called apoptosis.

[0073] In one embodiment, the protein drug in the nanoparticle composition is IL-21. IL-21 is associated with sustained and elevated CD8 + It has an antitumor effect mediated by T cell responses, achieving sustained antitumor immunity.

[0074] In one embodiment, a protein such as BSA is used in the nanoparticle composition. BSA is bovine serum albumin protein derived from cattle. It is commonly used as a standard substance for protein concentration in laboratory experiments.

[0075] The administration method of the nanoparticle composition is based on known administration methods for protein drugs to treat specific diseases and subject conditions. For example, when treating adult breast cancer, trastuzumab is administered via IV infusion at a dose of 4-8 mg / kg once a week for 52 weeks. The effective dose of MINC-trastuzumab is within the same dose range, but the administration frequency is lower, once every 12-16 weeks for 52 weeks.

[0076] For the treatment of type 1 diabetes, a 14-day course of anti-CD3 is administered only once in a person's lifetime by IV injection at a dose of 1-20 μg / kg, and repeated administration is not performed due to drug toxicity. The effective dose of MINC-anti-CD3 within the same dose range is administered in courses of 3-5 days per year, and can be repeated once a year.

[0077] In the treatment of melanoma, interferon-alpha is introduced as an IV infusion of 20 million IU / m². 2 This is administered five days a week for four consecutive weeks. The effective dose of MINC-interferon-α is within the same dose range, and administering it once a week for two weeks yields the same efficacy and reduced toxicity.

[0078] For the treatment of kidney cancer, IL-12 at a dose of 600,000 international units / kg (0.037 mg / kg) is administered three times a day for up to 14 doses. After a 9-day rest period, if tolerated, this schedule is repeated for another 14 doses. An effective dose of MINC-IL-12 within the same dose range is administered once a day for 3 days for a total of 9 doses.

[0079] This invention is useful in human medicine and veterinary medicine. This invention is useful in the treatment of humans and non-human animals. For example, this invention is useful in the treatment of mammalian subjects such as humans, horses, pigs, cats, and dogs.

[0080] The following embodiments further illustrate the present invention. These embodiments are intended merely to illustrate the present invention and should not be construed as limiting the invention. [Examples]

[0081] Table 1 shows the suppliers of the raw materials used in the examples. [Table 1]

[0082] OEGCG and PEG-dEGCG were synthesized according to WO2006 / 12400,

[0099] and

[0102] .

[0083] Example 1. Method for preparing MINC-trastuzumab nanoparticles MINC (Multi-Target Immuno-Nanocarrier Mixture)-Trastuzumab nanoparticles were prepared according to the following protocol. Incubate a 5 mg trastuzumab (34.4 nmol) solution in 1.10 ml of PBS at 37°C for 1 hour. 2.16.7 μl of OEGCG (30 mM, 501 nmol in DMSO) is added. Add 3.65 μl of PEG-EGCG (16 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid volume to 1 ml. Add 7.9 ml of 0.9% NaCl and mix. 8. Repeat steps 6 and 7 three more times. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then 80°C overnight. Freeze-dry for 11.3 days.

[0084] Nanoparticle sizes were measured by DLS (Malvern Zetasizer Nano ZS). The final product is shown in Figure 2. The median nanoparticle size was 102.1 nm. The standard deviation was 41.1 nm. The molar ratio of trastuzumab to OEGCG was 1:15. 100% of the nanoparticles were distributed within the range of 50–300 nm.

[0085] Example 2. Method for preparing MINC-IL-12 nanoparticles MINC-IL-12 nanoparticles were prepared according to the following protocol. Incubate a solution of 0.5 mg IL-12 (8.7 nmol) in 1.1 ml of PBS at 37°C for 1 hour. Add 2.5 μl of OEGCG (30 mM, 150 nmol in DMSO). Add 3.10 μl of PEG-EGCG (16 mM in DMSO). 4. Incubate the mixture at 25°C for 15 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid volume to 0.1 ml. Add 7.0.9 ml of 0.9% NaCl and mix. 8. Repeat steps 6 and 7 three more times. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0086] Nanoparticle sizes were measured by DLS (Malvern Zetasizer Nano ZS). The final product is shown in Figure 3. The median nanoparticle size was 74.8 nm. The molar ratio of IL-12 to OEGCG was 1:17. 90% of the particles had a desirable molecular size of 50–300 nm.

[0087] Example 3. Method for preparing MINC-anti-CD3 nanoparticles MINC-anti-CD3 nanoparticles were prepared according to the following protocol. Incubate 0.5 mg of anti-CD3 (3.43 nmol) in 1.1 ml of PBS at 37°C for 1.0 hour. 2. Add 6.7 μl of OEGCG (30 mM, 201 nmol in DMSO). 3. Add 6.6 μl of PEG-EGCG (16 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 three more times. 9.0.Filter three times through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then 80°C overnight. Freeze-dry for 11.3 days.

[0088] Nanoparticle sizes were measured by DLS (Malvern Zetasizer Nano ZS). The final product is shown in Figure 4. The median nanoparticle size was 90.24 nm. The standard deviation was 31.7 nm. The molar ratio of anti-CD3 to OEGCG was 1:59. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0089] Example 4. Method for preparing MINC-INFα nanoparticles MINC-INFα nanoparticles were prepared according to the following protocol. Incubate 0.067 mg (3.48 nmol) IFN-α in 1.1 ml of 1x PBS at 37°C for 80 minutes. 2.1. Add 67 μl of OEGCG (30 mM, 50.1 nmol in DMSO). Add 3.6.5 μl of PEG-EGCG (16 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 three more times. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then 80°C overnight. Freeze-dry for 11.3 days.

[0090] Nanoparticle sizes were measured by DLS (Malvern Zetasizer Nano ZS). The final product is shown in Figure 5. The median nanoparticle size was 105.3 nm. The standard deviation was 50.43 nm. The molar ratio of INFα to OEGCG was 1:14. Over 80% of the nanoparticles were distributed within the range of 50–300 nm.

[0091] Example 5. Comparison of MINC-trastuzumab nanoparticles prepared in different molar ratios of trastuzumab versus OEGCG. MINC-trastuzumab nanoparticles (Figure 6B) were prepared according to Example 1. MINC-trastuzumab nanoparticles (Figure 6A) were prepared according to Example 1, but in step 2, 3.34 μl of OEGCG (30 mM in DMSO, 100.2 nmol) was added. In Figure 6B, 16.7 μl of OEGCG (30 mM in DMSO, 501 nmol) was added. The nanoparticle size distributions in Figures 6A and 6B are significantly different. The results show that when the protein-to-OEGCG (EGCG units) molar ratio changes from 1:15 (Figure 6B) to 1:3 (Figure 6A), the nanoparticle size distribution changes to multiple peaks. Furthermore, the median size of the main peak (peak 1) in Figure 6B is 164.2 nm (intensity 94.8%), while the median size of the main peak (peak 1) in Figure 6A is 267.7 nm (intensity 68.0%).

[0092] Example 6. Comparison of MINC-trastuzumab nanoparticles with and without ultrafiltration, each with a molecular weight of 10K. MINC-trastuzumab nanoparticles (Figure 7B) were prepared according to Example 1. MINC-trastuzumab nanoparticles (Figure 7A) were prepared according to Example 1, but 10 kDa MWCO centrifugation (steps 5-8) was omitted. The results show that in the absence of 10 kDa MWCO ultrafiltration, the nanoparticle size distribution changed to multiple peaks (Figure 7A). Furthermore, the median size of the main peak (peak 1) in Figure 7B is 119.7 nm (intensity 98.3%), while the median size of the main peak (peak 1) in Figure 7A is 130.4 nm (intensity 70.8%).

[0093] Example 7.0.2 Comparison of MINC-trastuzumab nanoparticles prepared with and without 2 μm filtration. MINC-trastuzumab nanoparticles (Figure 8B) were prepared according to Example 1. Nanoparticles (Figure 8A) were prepared according to Example 1, but without 0.22 μm filtration (step 9). The size of MINC-trastuzumab nanoparticles was measured using DLS (Malvern Zetasizer Nano ZS) with and without 0.22 μm membrane filtration (Figure 8A). With 0.22 μm filtration (Figure 8B), undesirable nanoparticles (larger than 300 nm) were removed, and purity increased. The nanoparticle size distribution in the 50-300 nm range improved from 75.7% (Figure 8A) to 100% (Figure 8B).

[0094] Example 8. Comparison of trastuzumab nanoparticles prepared by stepwise freezing for lyophilization with those prepared by other freezing methods. In Figure 9, MINC-trastuzumab nanoparticles were prepared according to Example 1 or in a different procedure, Step 10. The size of the MINC-trastuzumab nanoparticles was measured using DLS (Malvern Zetasizer Nano ZS) under (i) stepwise freezing, (ii) continuous freezing at -1°C / min down to -80°C, and (iii) a single step (immediate freezing) at -80°C in Step 10 (see Figure 9). In the continuous freezing and single-step freezing procedures, the samples were placed in a container at -80°C with or without a freezing container (-1°C / min, Thermo Scientific). In stepwise freezing, the samples were placed at 4°C for 1 hour, at -30°C for 1 hour, and then transferred to -80°C overnight. The results showed that stepwise freezing procedures (4°C, 30°C, -80°C) preserved the size of MINC-trastuzumab, while continuous or immediate freezing significantly increased the size of MINC-trastuzumab after lyophilization (Figure 9).

[0095] Example 9. Bioactivity of MINC-trastuzumab nanoparticles MINC-trastuzumab nanoparticles were prepared according to Example 1. In Figure 10, lyophilized MINC-trastuzumab was reconstituted with phosphate-buffered saline (PBS) and used in an in vivo tumor suppression assay in a BT474 xenograft mouse model (n=7-10 in each group). In the BT474 xenograft mouse model, Balb / nude mice were subcutaneously injected with a 17β-estradiol pellet (0.72 mg, released for 60 days). The following day, 8 × 10 6 Each mouse was subcutaneously injected with a suspension of BT474 cells (in 100 μL of Matrigel). Tumors were allowed to grow for 2 weeks prior to treatment. Two weeks after tumor injection, the mice were intravenously injected with the drug twice a week for 4 weeks. The mice were divided into three treatment groups: trastuzumab (2.5 mg / kg), MINC-trastuzumab (equivalent to 2.5 mg / kg of trastuzumab at the start of formulation), and PBS as a vehicle control. Tumor size was measured by length (l) and width (w) using calipers. Tumor volume (V) was measured twice a week, V = lw 2 The values ​​were calculated as / 2 and normalized to the tumor size at the time of the first measurement as described (WPMcGuire et al., N.Engl.J.Med.1996,344(1),1268). The results showed that the anticancer activity was well maintained throughout the complete preparation process, and the activity of MINC-trastuzumab nanoparticles was superior to that of trastuzumab alone.

[0096] Example 10. Method for preparing MINC-anti-CD71 nanoparticles MINC-anti-CD71 nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg of anti-CD71 (0.42 pmol) in 1.0.5 ml of PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (1.25 mM, 6.25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (1.1 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0097] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 11. The median nanoparticle size was 106.33 nm. The standard deviation was 17.34 nm. The molar ratio of anti-CD71 to OEGCG was 1:15. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0098] Example 11. Method for preparing MINC-anti-EGFR nanoparticles MINC-anti-EGFR nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg of anti-EGFR (0.42 pmol) in 1.05 ml of PBS at 37°C for 1 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0099] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 12. The median nanoparticle size was 80.57 nm. The standard deviation was 19.52 nm. The molar ratio of anti-EGFR to OEGCG was 1:60. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0100] Example 12. Method for preparing MINC-anti-Tau nanoparticles MINC-anti-Tau nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg of anti-Tau (0.42 pmol) in 1.0.5 ml of PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0101] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 13. The median nanoparticle size was 119.31 nm. The standard deviation was 35.47 nm. The molar ratio of anti-Tau to OEGCG was 1:60. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0102] Example 13. Method for preparing MINC-anti-VEGF nanoparticles MINC-anti-VEGF nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg of anti-VEGF (0.42 pmol) in 1.05 ml of PBS at 37°C for 1 hour. Add 2.5 μl of OEGCG (1.25 mM, 6.25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (1.1 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0103] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 14. The median nanoparticle size was 140.07 nm. The standard deviation was 27.55 nm. The molar ratio of anti-VEGF to OEGCG was 1:15. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0104] Example 14. Method for preparing MINC-IFN-γ nanoparticles MINC-IFN-γ nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg IFN-γ (3.98 pmol) in 1.0.5 ml PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0105] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 15. The median nanoparticle size was 55.61 nm. The standard deviation was 9.47 nm. The molar ratio of IFN-γ to OEGCG was 1:6. Over 85% of the nanoparticles were distributed within the range of 50–300 nm.

[0106] Example 15. Method for preparing MINC-IL-2 nanoparticles MINC-IL-2 nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg IL-2 (4.06 pmol) in 1.0.5 ml PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0107] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 16. The median nanoparticle size was 114.2 nm. The standard deviation was 24.37 nm. The molar ratio of IL-2 to OEGCG was 1:6. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0108] Example 16. Method for preparing MINC-IL-6 nanoparticles MINC-IL-6 nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg IL-6 (2.85 pmol) in 1.0.5 ml PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0109] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 17. The median nanoparticle size was 134.98 nm. The standard deviation was 26 nm. The molar ratio of IL-6 to OEGCG was 1:9. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0110] Example 17. Method for preparing MINC-IL-15 nanoparticles MINC-IL-15 nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg of IL-15 (4.66 pmol) in 1.0.5 ml of PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0111] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 18. The median nanoparticle size was 77.6 nm. The standard deviation was 13.18 nm. The molar ratio of IL-15 to OEGCG was 1:5. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0112] Example 18. Method for preparing MINC-IL-21 nanoparticles MINC-IL-21 nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg of IL-21 (4.40 pmol) in 1.0.5 ml of PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0113] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 19. The median nanoparticle size was 126.59 nm. The standard deviation was 42.65 nm. The molar ratio of IL-21 to OEGCG was 1:6. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0114] Example 19. Method for preparing MINC-TRAIL nanoparticles MINC-TRAIL nanoparticles were prepared according to the following protocol. Incubate 0.0625 mg TRAIL (2.75 pmol) in 1.0.5 ml PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0115] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 20. The median nanoparticle size was 123.89 nm. The standard deviation was 18.34 nm. The molar ratio of TRAIL to OEGCG was 1:9. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0116] Example 20. Method for preparing MINC-BSA nanoparticles MINC-BSA nanoparticles were prepared according to the following protocol. Incubate 0.5 mg BSA (0.42 pmol) in 1.1 ml PBS at 37°C for 1.0 hour. Add 2.5 μl of OEGCG (5 mM, 25 pmol in DMSO). Add 3.10 μl of PEG-EGCG (4.4 mM in DMSO). 4. Incubate the mixture at 25°C for 3 hours. 5. Transfer the liquid to the 10K MWCO centrifugal filter unit. 6. Centrifuge to reduce the liquid to 0.1 ml. Dissolve in 7.0.9 ml of 0.9% NaCl. 8. Repeat steps 6 and 7 one more time. 9.0.Filter through a 22 μm membrane. 10. Transfer to a cryotube and freeze in stages: 4°C for 1 hour, -30°C for 1 hour, then -80°C overnight. Freeze-dry for 11.3 days.

[0117] Nanoparticle sizes were measured using DLS (Anton Paar, Litesizer 500). The final product is shown in Figure 21. The median nanoparticle size was 100.57 nm. The standard deviation was 27.12 nm. The molar ratio of BSA to OEGCG was 1:19. Over 95% of the nanoparticles were distributed within the range of 50–300 nm.

[0118] The present invention, as well as the processes and methods of its manufacture and use, are described in complete, clear, concise, and precise terms so that those skilled in the art can manufacture and use it. The foregoing describes preferred embodiments of the present invention, and it should be understood that modifications can be made without departing from the scope of the invention as defined in the claims. This specification is concluded by the following claims, which specifically point out and clearly claim the subject matter considered to be the present invention. Examples of embodiments of the present invention are listed in the following sections [Aspect 1] to [Aspect 18]. [Aspect 1] A nanoparticle composition comprising nanoparticles having (a) an inner core containing oligomer(-)-epigallocatechin gallate (OEGCG), (b) an outer core containing polyethylene glycol-epigallocatechin gallate conjugate (PEG-EGCG), and (c) a drug molecule encapsulated in the inner core, wherein the drug molecule is an antibody or cytokine, at least 70% of the nanoparticles have a diameter of 50 to 300 nm, and the size distribution of the nanoparticles has only one major peak containing more than 90% of all the particles. [Aspect 2] The nanoparticle composition according to embodiment 1, wherein at least 80% of the nanoparticles have a diameter of 50 to 300 nm. [Aspect 3] The nanoparticle composition according to embodiment 1, wherein at least 90% of the nanoparticles have a diameter of 50 to 300 nm. [Aspect 4] The nanoparticle composition according to embodiment 1, wherein the median diameter of the nanoparticles is approximately 50 to 250 nm. [Aspect 5] The nanoparticle composition according to embodiment 1, wherein the median diameter of the nanoparticles is approximately 50 to 200 nm. [Aspect 6] The nanoparticle composition according to embodiment 1, wherein the size distribution of the nanoparticles has only one major peak containing more than 95% of all the particles. [Aspect 7] The nanoparticle composition according to embodiment 1, wherein the antibody is a monoclonal antibody. [Aspect 8] The nanoparticle composition according to embodiment 1, wherein the antibody is anti-HER2, anti-CD71, anti-EGFR, anti-VEGF, or anti-Tau. [Aspect 9] The nanoparticle composition according to embodiment 1, wherein the cytokine is interferon, interleukin, lymphokine, or tumor necrosis factor. [Aspect 10] The nanoparticle composition according to embodiment 1, wherein the cytokine is IL-12, IL-2, IL-6, IL-15, IL-21, IFN-α, IFN-γ, or TRAIL. [Aspect 11] (a) A step of mixing drug protein molecules with OEGCG and PEG-EGCG in an aqueous solution. (b) A step of filtering the mixture through a membrane with a molecular weight cutoff of 8,000 to 300,000 daltons to remove low molecular weight molecules and retain high molecular weight molecules, and (c) A step of filtering the high molecular weight molecules through a 0.2-0.3 μm membrane and collecting the filtrate. A method for preparing the nanoparticle composition according to embodiment 1, which includes the following. [Aspect 12] After step (c), Step (d): A freeze-drying process in which the filtrate is frozen in stages at (i) approximately 0 to 5°C, (ii) approximately -20 to -30°C, and (iii) approximately -60 to -100°C, and then dried. The method according to embodiment 11, further comprising: [Aspect 13] The method according to embodiment 11 or 12, wherein the molar ratio of EGCG to the drug molecule in OEGCG is 5 to 100 to 1. [Aspect 14] The method according to embodiment 13, wherein the molar ratio of EGCG to the drug molecule in OEGCG is 10 to 50 to 1. [Aspect 15] The method according to embodiment 11 or 12, wherein the molecular weight cutoff in step (b) is 8,000 to 12,000 daltons. [Aspect 16] The method according to embodiment 11 or 12, wherein steps (b) and (c) are repeated one, two, three, or four times before step (d). [Aspect 17] The method according to embodiment 12, wherein the stepwise freezing is carried out for (i) at about 0 to 5°C for at least 1 hour, (ii) at about -20°C to -30°C for at least 1 hour, and (iii) at -60°C to -100°C for at least 2 hours. [Aspect 18] A method for treating cancer, comprising administering an effective amount of the nanoparticle composition described in Embodiment 1 to a subject in need of treatment.

Claims

1. A nanoparticle composition comprising nanoparticles having (a) an internal core containing oligomer(-)-epigallocatechin gallate (OEGCG), (b) an external core containing polyethylene glycol-epigallocatechin gallate conjugate (PEG-EGCG), and (c) a drug molecule encapsulated in the internal core, wherein the drug molecule is an antibody or cytokine, at least 70% of the nanoparticles have a diameter of 50 to 300 nm, and the size distribution of the nanoparticles has only one major peak containing more than 90% of all the particles.

2. The nanoparticle composition according to claim 1, wherein at least 80% of the nanoparticles have a diameter of 50 to 300 nm.

3. The nanoparticle composition according to claim 1, wherein at least 90% of the nanoparticles have a diameter of 50 to 300 nm.

4. The nanoparticle composition according to claim 1, wherein the median diameter of the nanoparticles is 50 to 250 nm.

5. The nanoparticle composition according to claim 1, wherein the median diameter of the nanoparticles is 50 to 200 nm.

6. The nanoparticle composition according to claim 1, wherein the size distribution of the nanoparticles has only one major peak containing more than 95% of all the particles.

7. The nanoparticle composition according to claim 1, wherein the antibody is a monoclonal antibody.

8. The nanoparticle composition according to claim 1, wherein the antibody is anti-HER2, anti-CD71, anti-EGFR, anti-VEGF, or anti-Tau.

9. The nanoparticle composition according to claim 1, wherein the cytokine is interferon, interleukin, lymphokine, or tumor necrosis factor.

10. The nanoparticle composition according to claim 1, wherein the cytokine is IL-12, IL-2, IL-6, IL-15, IL-21, IFN-α, IFN-γ, or TRAIL.

11. (a) A step of mixing drug protein molecules with OEGCG and PEG-EGCG in an aqueous solution. (b) A step of filtering the mixture through a membrane with a molecular weight cutoff of 8,000 to 300,000 daltons to remove low molecular weight molecules and retain high molecular weight molecules, and (c) A step of filtering the high molecular weight molecules through a 0.2-0.3 μm membrane and collecting the filtrate. A method for preparing the nanoparticle composition according to claim 1, comprising:

12. After step (c), Step (d): Freeze-dry the filtrate by freezing it in stages at (i) 0 to 5°C, (ii) -20 to -30°C, and (iii) -60 to -100°C, and then drying it. The method according to claim 11, further comprising:

13. The method according to claim 11 or 12, wherein the molar ratio of EGCG to the drug molecule in OEGCG is 5 to 100 to 1.

14. The method according to claim 13, wherein the molar ratio of EGCG to the drug molecule in OEGCG is 10 to 50 to 1.

15. The method according to claim 11 or 12, wherein the molecular weight cutoff in step (b) is 8,000 to 12,000 daltons.

16. The method according to claim 12, wherein steps (b) and (c) are performed two, three, four, or five times before step (d).

17. The method according to claim 12, wherein the stepwise freezing is carried out for (i) 0 to 5°C for at least 1 hour, (ii) -20°C to -30°C for at least 1 hour, and (iii) -60°C to -100°C for at least 2 hours.

18. A pharmaceutical composition for treating cancer, comprising the nanoparticle composition described in claim 1 and a pharmaceutically acceptable carrier.

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