Nanoparticle composition

Abstract

The present disclosure relates to nanoparticle compositions as carriers of pharmaceutically acceptable compounds, methods for preparing the compositions, and the use of the compositions for medical purposes, particularly immunoprophylaxis or immunotherapy. The present invention also relates to vaccines containing the compositions. The compositions may include nanoparticles having silicon dioxide and functional groups on their surfaces, loaded with any type of antigen, where the compound represents the nanoparticle payload and may have a particle size of less than 150 nm. The functional groups on the surface of the nanoparticles are suitable for carrying and / or stabilizing the negative and positive charges of such compounds.

Description

[Technical Field]

[0001] Field of Invention This invention relates to compositions of nanoparticles as carriers for pharmaceutically acceptable compounds, the use of compositions for medical purposes, particularly for immunoprevention or immunotherapy, and the nanoparticles themselves. This invention also relates to vaccines containing compositions and / or nanoparticles. [Background technology]

[0002] Background of the Invention Nanomedicine and nanodelivery systems are a relatively new but rapidly developing science that uses materials in the nanoscale range to function as a means of diagnostic or therapeutic tools, or to deliver therapeutic drugs to specific target sites in the body. In particular, nanoparticles are known for the delivery of chemotherapeutic drugs, biological drugs, immunotherapy drugs, and vaccines in immunotherapy.

[0003] Functionalized nanoparticles are well known as drug delivery systems, for example, as carriers for antigens. The literature describes carrier systems in which the antigen is encapsulated or bound to the surface of the nanoparticles.

[0004] International Publication No. 2006 / 037979 describes gold nanoparticles (GNPs) containing adjuvants and antigens, such as tumor and pathogen antigens, and their use in various applications, including the treatment of cancer and infectious diseases. Immunogenic structures based on nanoparticles or antibodies having carbohydrate ligands are also disclosed, as well as their use for therapeutic and prophylactic purposes, and for the isolation and detection of antibodies against carbohydrate structures.

[0005] International Publication Nos. 2013 / 034741 and International Publication Nos. 2013 / 034726 relate to nanoparticles having linker-linked epitope peptides that have been found to be used as vaccines, for example, in the prophylactic or therapeutic treatment of tumors in mammalian subjects.

[0006] International Publication No. 2011 / 154711 describes glycated gold nanoparticles that act as carriers for the delivery of peptides such as insulin.

[0007] International Publication No. 2010 / 006753 discloses monodisperse nanoparticles of silicon dioxide with at least one antigen bound to its surface. The nanoparticles are used for cancer immunoprevention or immunotherapy. While many nanoparticles have been investigated and developed, particularly in relation to cancer treatment, there remains a high demand for materials with improved properties, especially in terms of their adjuvant effects. Furthermore, there is a need to provide nanoparticles as a flexible and convenient system for delivering pharmacologically relevant drugs to the body's immune system. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2006 / 037979 [Patent Document 2] International Publication No. 2013 / 034741 [Patent Document 3] International Publication No. 2013 / 034726 [Patent Document 4] International Publication No. 2011 / 154711 [Patent Document 5] International Publication No. 2010 / 006753 [Overview of the project] [Means for solving the problem]

[0009] Summary of the Invention Therefore, an object of the present invention is to provide improved nanoparticles and compositions containing them, particularly for use in immunoprevention or immunotherapy. Furthermore, an object of the present invention is to provide a platform technology that enables a simple method for generating readily available nanoparticles suitable for carrying a wide range of ligands for medical procedures. These nanoparticles should have effective immunomodulatory activity.

[0010] These objectives are addressed by providing a composition comprising silicon dioxide and nanoparticles having functional groups on their surface, loaded with a pharmaceutically acceptable compound (preferably any kind of antigen). The compound represents the payload of the nanoparticles. The nanoparticles have a particle size of less than 150 nm. The functional groups on the surface of the nanoparticles are suitable for supporting and / or stabilizing the negative and positive charges of such compounds. The composition has a zeta potential of at least ±15 mV.

[0011] Using the composition according to the present invention, one of the fundamental problems of nanoparticles for drug delivery, namely the lack of stability in colloidal suspensions, is overcome.

[0012] In aqueous systems, surface charges essentially guarantee the stability of the colloidal system. These charges can be positive or negative. To substantially reduce or even avoid particle aggregation, it is crucial that there are sufficient functional groups with the same charge, which results in electrostatic repulsion. If the charge of the colloidal support system is compensated for by the adsorption of molecules with the opposite charge, aggregates will form and the colloidal system will collapse.

[0013] The aggregates have significantly larger particle sizes, which jeopardizes the effective transport of these large particles within the body, resulting in less effective immunomodulatory efficacy.

[0014] The composition according to the present invention, comprising nanoparticles having a particle size of less than 150 nm and a zeta potential of at least ±15 mV, ensures that the composition is sufficiently stable. Particularly under physiological conditions, the nanoparticles dispersed therein can be effectively transported to major active sites in the body. Therefore, the composition according to the present invention enables improved transport of nanoparticles to the lymphatic system, particularly from the administration site to lymph nodes where dendritic cells are located.

[0015] When administered by subcutaneous injection, the particles according to the present invention are too large to enter the bloodstream, thus their rapid elimination and severe systemic adverse effects are essentially avoided. At the same time, they are small enough to enter the lymphatic vessels and enter dendritic cells by phagocytosis. The same applies when the particles are administered intradermally, intraperitoneally, or intramuscularly. Therefore, except for intravenous or intra-arterial administration, parenteral administration of the particles is preferred.

[0016] The inventors have found that particles in the composition according to the present invention can support a large number of compounds on their surface. Considering the desired function of the nanoparticles, the compounds can be pharmaceutically acceptable. The total amount of the compounds is nm 2( The surface loading density (SPL) can be up to 5% (by weight) of the surface of the nanoparticles. The suitability of nanoparticles carrying large amounts of compounds is particularly important when high doses of such compounds (e.g., antigens or active pharmaceutical ingredients) are to be delivered.

[0017] The composition according to the present invention comprises silicon dioxide and nanoparticles having functional groups on their surface. The functional groups can be linked directly or indirectly to the surface of the nanoparticles. In a preferred embodiment of the present invention, the functional groups are linked to the surface of the nanoparticles via a linker (hereinafter referred to as linker compound L). The linker compound L can be linked to the surface of the nanoparticles by any method, particularly by covalent or adsorption bonds, most preferably by covalent bonds.

[0018] In a particularly preferred embodiment, the linker compound L is a carboxyl(-COOH) or carboxylate(-COOH) -) at least one functional group selected from the group consisting of groups and / or a guanidino group (-NHC(=NH)NH2 or -NHC(=NH2 + )NH2) or an amino group (-NH2 or -NH3 + ) and contains at least one functional group selected from the group consisting of. Preferably, it is carboxyl (-COOH) or carboxylate (-COO - ) group and a guanidino group (-NHC(=NH)NH2 or -NHC(=NH2 + )NH2) or an amino group (-NH2 or -NH3 + ) contains both the group and at least one group selected from the group consisting of. Most preferably, the linker compound L is carboxyl (-COOH) or carboxylate (-COO - ) group and at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2 + )NH2). Such a linker compound L enables adsorptive binding of anionic or cationic, and also non-polar pharmaceutically acceptable compounds (e.g., hydrophobic peptides). Thereby, the linker provides a platform technology for simple coupling of a wide range of chemically diverse compounds to the surface of nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1-1] Figs. 1a to 1d show TEM images of the following SiO2 particles. Fig. 1a shows a TEM image of SiO2 nanoparticles having a particle size of 68 nm. Fig. 1b shows a TEM image of SiO2 nanoparticles having a particle size of 40 nm. Fig. 1c shows a TEM image of SiO2 nanoparticles having a particle size of 25 nm. Fig. 1d shows a TEM image of SiO2 nanoparticles having a particle size of 15 nm. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 2] Fig. 2 shows an SEM image of SiO2 nanoparticles having a particle size of 150 nm. [Figure 3]Figure 3 shows a schematic cross-section of the nanoparticles according to the present invention. A represents surface functionalization with linker compound L, B represents the amorphous SiO2 shell, and C represents the core material, which can be voids, water or any other material, as well as amorphous SiO2. The diameter d1 is 0 to 149 nm, and d2 is 10 to 150 nm. [Figure 4] Figure 4b shows a preferred three-part bioconjugate according to the present invention. The peptide used in Figure 4b is a highly hydrophobic epitope derived from human NY-ESO-1. For N-terminal extension, the enzymatically (cathepsin B) cleavable peptide linker Val-Cit was used to ensure the release of the native unmodified antigen to MHC I or MHC II. The extension portion "Lys-Lys-Lys" was used to enhance solubility and electrostatic attraction to nanoparticles. Extension with, for example, Lys-Lys-Lys-Asp or Arg6 is also possible. [Figure 5] Figure 5a shows the Z-mean ratio to peptide concentration, and Figure 5b shows the PDI ratio to peptide concentration. [Figure 6] Up to a 6% poly(I:C) LMW load, the measured Z-mean value increases due to the increase in particle size caused by adsorbed poly(I:C) (Figure 6b). Also, a low PDI (<0.1) indicates a smooth particle load (Figure 6a). [Figure 7-1] Figure 7a: Poly(I:C)LMW without nanoparticles. Figure 7b: SiO2-Arg nanoparticles without poly(I:C). Figure 7c: SiO2-Arg nanoparticles supported with 6 wt% poly(I:C). Figure 7d: SiO2-Arg nanoparticles supported with 8 wt% poly(I:C). [Figure 7-2] Same as above. [Figure 8] Figure 8: Cytokine expression (arbitrary units) after 72 hours of stimulation versus various types of cytokines. [Figure 9] Figure 9: Cytokine expression (arbitrary units) after 96 hours of stimulation versus various types of cytokines. [Figure 10]Figures 10a and 10b: Cytokine release at different time points after stimulation of differentiated THP-1 cells with poly(I:C) [12.5 μg / ml], SiO2-Arg [0.5 mg / mL], or a novel adjuvant (poly(I:C) [12.5 μg / mL] bound to SiO2-Arg [0.5 mg / mL]). [Figure 11-1] The TLR3 agonist adsorbed onto silica nanoparticles (Group E) was shown to be significantly superior to the free TLR3 agonist (Group C) applied at the same concentration and under identical test conditions. The immune booster ZelNate® (Group B) also did not show a significant improvement compared to the placebo group (Group A) in this study. This also applies to the unmodified silica nanoparticles in Group D, which did not show a significant effect (Figures 11a and 11b). [Figure 11-2] Same as above. [Figure 12] Figure 12 shows the clinical scores. The data are presented as the mean clinical score (maximum score = 5) ± SEM (n=10) in relation to the number of days after antigen loading. [Figure 13] To evaluate vaccine-induced HPV16 E711-19-specific CD8+ T cells, ex vivo-restimulated splenocytes were evaluated by IFN-γ intracellular cytokine staining (ICS) followed by flow cytometry (Figure 13). [Figure 14] Figure 14 shows the survival rate of mice treated with either the free antigen HPV16 E7 antigen YMLDLQPET + poly(I:C)(HMW, high molecular weight) "free antigen + TLR agonist" or KKKW-Cit-YMLDLQPET + poly(I:C)(HMW), both adsorbed and bound to arginyl silica nanoparticles with a diameter of 23 nm. [Figure 15] Figures 15a and 15b show the individual tumor growth in both groups. [Figure 16]Figure 16: OVA257-264MHC I expression in H2-Kb-positive cells after incubation for 6 hours with a 5 μM solution of the native or extended OVA257-264 epitope or full-length OVA protein, depending on the presence or absence of SiO2-PO3H2. After labeling with 25-D1.16 detection antibody, quantification was performed by flow cytometry. [Figure 17] Not specified. [Figure 18] Not specified. [Modes for carrying out the invention]

[0020] Detailed description of the invention The zeta potential of nanoparticles is a commonly used parameter known to those skilled in the art for characterizing the surface charge properties of nanoparticles. It reflects the potential of the particles. To avoid particle aggregation, it is important that there are enough functional groups with the same charge that repel each other. Aggregates cause the colloidal system to break down. The resulting aggregates have a particle size significantly larger than the individual particles, and therefore the desired transport mechanism through the fenestrated endothelium of lymphatic vessels is no longer guaranteed. It is accepted as a measure of the stability of the colloidal system.

[0021] Current characterization methods are based on ensemble measurements (e.g., phase analysis light scattering, Doppler velocity measurement, streaming potentiometry) that measure the average electrophoretic mobility of particles in a suspension (Sci.Rep.12 Dec.2017;7(1):17479, PMCID:PMC5727177). It is particularly preferable to measure the zeta potential of the composition according to the present invention using dynamic light scattering (DLS) (ZetaSizer Nano ZS, Malvern Instruments, UK).

[0022] The zeta potential of the composition according to the present invention is at least ±15mV, preferably ±30mV, more preferably ±60mV, and most preferably ±25 to ±40mV. This stabilizes the composition, which is preferably a colloidal suspension, meaning that the collapse of the system is essentially avoided.

[0023] The compositions according to the present invention contain nanoparticles having a particle size of less than 150 nm, preferably less than 100 nm. Nanoparticles having a particle size of less than 50 nm are more preferred, and nanoparticles having a particle size of 20 to 30 nm are most preferred. The particle sizes as defined herein do not have a random distribution over the entire range; instead, defined particle sizes within that range are selected, with a standard deviation of up to 15%, preferably up to 10%, and the standard deviation should always be interpreted as relating to the maximum value in the case of a bimodal or multimodal distribution.

[0024] The particle size index of nanoparticles is the Z-mean diameter. The Z-mean diameter, measured by dynamic light scattering, is also known as the cumulant mean. This is the primary and most reliable parameter produced by this technique. The Z-mean diameter is typically used in quality control settings according to ISO 22412:2017. Dynamic light scattering techniques provide an intensity-weighted distribution, and the contribution of each particle to the distribution will be related to the intensity of the light scattered by the particle. The intensity-weighted distribution is preferably measured using a ZetaSizer Nano ZS (Malvern Instruments, UK).

[0025] In a preferred embodiment, the Z-mean diameter of the nanoparticles according to the present invention is measured according to ISO 22412:2017 and is in the range of 5 nm to less than 150 nm, preferably 15 nm to 60 nm, more preferably 20 nm to 40 nm, and even more preferably 20 nm to 30 nm.

[0026] The particle size and composition stability of the nanoparticles according to the present invention are further confirmed by filtration with a sterile filter having a pore size of up to 0.2 μm. However, this is only practical for nanoparticles with a diameter of less than 50 nm.

[0027] The stability of the compositions of the present invention can be demonstrated by their polydispersity index (PDI). PDI is generally an indicator of the uniformity of a system, and in the context of the present invention, the uniformity and stability of a colloidal nanoparticle suspension. PDI reflects the nanoparticle size distribution. Samples with a wider range of particle sizes have a higher PDI, while samples consisting of particles of uniform size have a lower PDI. Those skilled in the art are familiar with methods and instruments for measuring PDI, in particular the ZetaSizer Nano ZS (Malvern Instruments, UK).

[0028] A PDI greater than 0.7 indicates that the samples have a wide size distribution. A PDI less than 0.1 is considered to be simple variance. Various size distribution algorithms handle data that fall between these two extremes. The calculation of these parameters is defined in ISO standard documents 13321:1996 E and ISO 22412:2008.

[0029] The compositions according to the present invention exhibit a PDI of 0 to 0.32, preferably 0.1 to 0.3, more preferably 0.1 to 0.2, and most preferably less than 0.1. The compositions according to the present invention having a PDI of less than 0.1 are preferably monodisperse.

[0030] In a preferred embodiment, the Z-mean diameter of the nanoparticles according to the present invention is in the range of 20 nm to 40 nm, the PDI is 0.1 to 0.30, and the zeta potential is ±20 mV to ±40 mV.

[0031] In a more preferred embodiment, the Z-mean diameter of the nanoparticles according to the present invention is in the range of 20 to 30 nm, the PDI is 0.1 to 0.2, and the zeta potential is ±25 to ±40 mV.

[0032] In the most preferred embodiment, the Z-mean diameter of the nanoparticles according to the present invention is in the range of 20 nm to 30 nm, the PDI is less than 0.1, and the zeta potential is ±25 mV to ±40 mV.

[0033] Furthermore, it is possible to measure the particle size of the loaded and released particles using TEM or SEM.

[0034] As used herein, the term “transmission electron microscopy (TEM)” refers to a microscopy technique in which an electron beam passes through an ultrathin sample and interacts with the sample as it passes through. An image is formed from the electrons that pass through the sample, which is magnified and focused by an objective lens and appears on an image screen, a fluorescence screen in most TEMs, plus a monitor, or a layer of photographic film, or is detected by a sensor such as a CCD camera.

[0035] As used herein, the term “scanning electron microscope (SEM)” refers to a type of electron microscope that produces an image of a sample by scanning its surface with a focused beam of electrons. Electrons interact with atoms in the sample, generating various signals that contain information about the sample's surface topography and composition. The electron beam is scanned in a raster scanning pattern, and the beam's position, combined with the intensity of the detected signals, generates an image.

[0036] Figures 1a to 1d show TEM images of the following SiO2 particles. Figure 1a shows a TEM image of SiO2 nanoparticles with a particle size of 68 nm. Figure 1b shows a TEM image of SiO2 nanoparticles with a particle size of 40 nm. Figure 1c shows a TEM image of SiO2 nanoparticles with a particle size of 25 nm. Figure 1d shows a TEM image of SiO2 nanoparticles with a particle size of 15 nm.

[0037] Figure 2 shows the SEM image of SiO2 nanoparticles with a particle size of 150 nm.

[0038] In relation to the present invention, "nanoparticles" are interpreted as particulate binding matrices having functional groups on their surface that function as recognition sites for antigens to which they are ultimately bound or adsorbed. Here, the surface includes not only the outer surface but also the inner surface, encompassing all areas of the cavities (pores) within the particles. The functional groups may be directly or indirectly bound to the surface.

[0039] According to the present invention, the nanoparticles may contain silicon dioxide (SiO2), which may be a mixture with other materials. Therefore, this material may also be mixed with further components in which silicon dioxide typically has the highest proportion in a multi-component system. The nanoparticles of the present invention may contain at least 80%, preferably at least 90%, of silicon dioxide.

[0040] In a particularly preferred embodiment of the nanoparticles according to the present invention, the material comprises silicon dioxide that is essentially pure, i.e., containing only impurities expected during the preparation process. In a more preferred embodiment of the present invention, the nanoparticle material consists of silicon dioxide.

[0041] Other examples of materials include metals, metal chalcogenides, magnetic materials, magnetic alloys, semiconductor materials, metal oxides, polymers, organosilanes, and other ceramics or glasses. Metals are selected from the group Au, Ag, Cu, Pt, Pd, Fe, Co, Gd, Ru, Rh, and Zn, or any combination thereof.

[0042] In further embodiments of the present invention, the nanoparticles have a coating containing silicon dioxide. The core may contain any other material such as a metal, a polymer, or a ferromagnetic metal such as Fe2O3 or Fe3O4. The core may be devoid of silicon dioxide.

[0043] The silicon dioxide nanoparticles according to the present invention are described, for example, in International Publication No. 2010 / 006753, which is expressly incorporated herein by reference.

[0044] Silicon dioxide nanoparticles can be prepared, in particular, using the classical Stoeber synthesis, in which monodisperse nanoscale silicon dioxide of a defined size can be prepared by hydrolysis of tetraethoxysilane (TEOS) in an aqueous alcohol-ammonia medium (J. Colloid Interface Sci. 1968, 26, 62).

[0045] The process for preparing silicon dioxide nanoparticles is described in detail in European Patent No. 0216 278 and International Publication No. 2005 / 085135, and these documents are therefore incorporated in their entirety into the disclosure of the present invention. At least one amine is preferably used in the medium.

[0046] The silicon dioxide matrix of the nanoparticles according to the present invention may be either porous or non-porous. The porosity is essentially dependent on the manufacturing process. Non-porous particles are obtained in particular by synthesis according to European Patent No. 0216278.

[0047] According to the present invention, nanoparticles contain functional groups on their surface. These functional groups can support and / or stabilize both negative and positive charges. These charges may belong to pharmaceutically acceptable compounds such as proteins, peptides, polynucleotides, oligonucleotides (RNA, DNA), nucleic acids, small molecules, or peptide antigens.

[0048] In preferred embodiments of the present invention, pharmaceutically acceptable compounds are selected from the group consisting of pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), deficient infectious particles (DIPs), or epitopes.

[0049] Functional groups that can support and / or stabilize both negative and positive charges include, for example, -SH, -COOH, -NH2, -guanidino group (-NHC(=NH))NH2), -PO3H2, -PO2CH3H, -SO3H, -OH, and -NR3 + X -The preferred functional groups are -COOH, -guanidino group (-NHC(=NH))NH2), and -NH2. The functional groups may also exist in their salt forms.

[0050] The composition according to the present invention is nm 2 The total number of pharmaceutically acceptable compounds on the surface of a unit nanoparticle [molecule / nm] 2 Regarding ], the nanoparticles include a surface loading density of up to 0.5, preferably 0.01 to 0.5, more preferably 0.03 to 0.4, and most preferably 0.05 to 0.3.

[0051] This surface loading density is calculated by assuming a perfect sphere and using the molar loading per particle. For example, a spherical particle with a diameter of 25 nm would have a surface loading density of 1964 nm. 2 (A = π) * d 2 ) has a surface area and a weight of 1.64E-8ng (assumed density of amorphous silica is 2000 kg / m³). 3 When such particles are loaded with 5 wt% of the peptide KKKV-Cit-YMLDLQPET (M=1,910.31 g / mol), the volume becomes 4.283E-22 mol. Multiplying this value by Avogadro's number gives 1964 nm. 2 This yields 258 single molecules of KKKV-Cit-YMLDLQPET bound to a single particle. This is at a density of 0.13 molecules / nm 2 This corresponds to the surface load density.

[0052] The compositions according to the present invention are formulated to have a pH of 6.0 to 8.0, preferably 6.5 to 7.8, more preferably 6.8 to 7.5, and even more preferably 7.2 to 7.4. The pH of the composition can be maintained by using buffers such as acetates, citrates, phosphates, succinates, TRIS (tris(hydroxymethyl)aminomethane), or histidine, typically used in a range of about 1 mM to 50 mM. Alternatively, the pH of the composition can be adjusted by using a physiologically acceptable acid or base.

[0053] In one embodiment of the present invention, the functional group is a -PO3H group. The phosphorylated nanoparticles according to the present invention can be prepared, for example, by the reaction of (diethylphosphatoethyl)triethoxysilane with silica nanoparticles in the presence of ammonia.

[0054] In a preferred embodiment of the present invention, the functional groups are linked to the nanoparticles via a linker L. The linker can be linked to the nanoparticles by, for example, covalent bonds or adsorption bonds.

[0055] In preferred embodiments of the present invention, the linker compound L has at least one carboxyl (-COOH) or carboxylate (-COOH) as a functional group. - It contains a ) group.

[0056] In a more preferred embodiment, such a linker has at least one guanidino group as a functional group (-NHC(=NH)NH2 or -NHC(=NH2 + )NH2) or amino group (-NH2 or -NH3 + ) further includes.

[0057] In the most preferred embodiment of the present invention, the linker compound L is at least a structural unit of general formula (I). * -(-O)3Si-(CH2) n -CH(COOX)-(CH2) p -C(O)-NH-CH(COOX)-(CH2) q -Y(I) [In the formula, X is either H or a negative charge, independently of each other. Y is independent of each other, -NHC(=NH)NH2, -NHC(=NH2 + )NH2, -NH2 or -NH3 + And, n, p, and q are independent of each other and are numbers from 0 to 25. * - includes the linkage point with the nanoparticle.

[0058] In a preferred embodiment, the linker compound L has n = 3, p = 1, q = 4, and Y is independently -NH2 or -NH3 + It contains at least one structural unit of formula (I).

[0059] In a particularly preferred embodiment, the linker compound L has n = 3, p = 1, q = 3, and Y is independently -NHC(=NH)NH2 or -NHC(=NH2 + It contains at least one structural unit of formula (I), which is an NH2 group.

[0060] Another aspect of the present invention is a method for preparing nanoparticles according to the present invention, - In the first step (i), hydrolysis polycondensation of tetraalkoxysilanes and / or organotrialalkoxysilanes is carried out in a medium containing water, at least one solubilizer and at least one amine or ammonia, where a sol of primary particles is first produced, and then the resulting nanoparticles are reduced to a desired particle size in the range of 5 to 150 nm by the sequential metering of the corresponding silane in a controlled manner corresponding to the degree of reaction, thereby limiting further nucleation. - The second step (ii) is to provide a method for reacting the nanoparticles from step (i) with [(3-triethoxysilyl)propyl]succinic anhydride to simultaneously form an amide with L-arginine or L-lysine.

[0061] It is preferable to use L-arginine in step (ii) of the method.

[0062] In another embodiment, the linker compound L could also be obtained by the reaction of L-arginine with N-(3-triethoxysilylpropyl)maleimide. The reaction is preferably carried out at a pH value greater than 8.

[0063] In one embodiment of the present invention, it is also possible to produce linker compound L by reacting [(3-triethoxysilyl)propyl]succinic anhydride with agmatine, histamine, cadaverine, or spermidine.

[0064] Further embodiments of the present invention include nanoparticles comprising a silicon dioxide-based surface to which a linker compound L is covalently or adsorbed, wherein the linker L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2) as a functional group. + )NH2) or amino group (-NH2 or -NH3 + ) contains. In a preferred embodiment, linker L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2 + )NH2) or amino group (-NH2 or -NH3 + ) and at least one carboxyl (-COOH) or carboxylate (-COO - In a more preferred embodiment, linker L includes at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2+)NH2) and at least one carboxyl (-COOH) or carboxylate (-COO) as functional groups. - ) Includes the group.

[0065] One embodiment of the nanoparticles according to the present invention is shown in Figure 3.

[0066] Figure 3 shows a schematic cross-section of the nanoparticles according to the present invention. A represents surface functionalization with linker compound L, B represents the amorphous SiO2 shell, and C represents the core material, which can be voids, water or any other material, as well as amorphous SiO2. The diameter d1 is 0 to 149 nm, and d2 is 10 to 150 nm.

[0067] In one embodiment of the present invention, a pharmaceutically acceptable compound is conjugated to nanoparticles by adsorption or covalent bonding. Adsorption bonding is preferred.

[0068] A further object of the present invention is a nanoparticle having silicon dioxide and a functional group on its surface, having a particle size of less than 150 nm, comprising a linker L covalently or adsorbed thereto, wherein the linker L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2) + )NH2) or amino group (-NH2 or -NH3 + These are nanoparticles containing the ) group.

[0069] The main advantage of adsorption bonding of pharmaceutically acceptable compounds is that, in contrast to most covalent conjugations, no byproducts are formed or remain in the “reaction” mixture. In the case of covalent bonding, it may be necessary to chemically modify the molecule to be bonded by adding reactive functional groups. This is a fundamental intervention in the structure of peptides / antigens and constitutes complex chemical modifications in the case of RNA or DNA-based antigens or adjuvants. Furthermore, covalent bonding of known compounds already approved by the drug regulatory agency can create new, independent substances (novel chemicals, NCEs) that require new approval for regulatory reasons.

[0070] In a preferred embodiment, the linker compound L according to formula (I) can support and / or stabilize pharmaceutically acceptable compounds having a positive or negative charge by adsorption.

[0071] In a particularly preferred embodiment, the linker compound L according to formula (I) can support and / or stabilize a pharmaceutically acceptable compound having a negative charge by adsorption.

[0072] In a more preferred embodiment, the linker compound L according to formula (I) can support and / or stabilize a pharmaceutically acceptable compound having a phosphate group or a phosphonic acid group by adsorption.

[0073] Surprisingly, the electrostatic interaction between the arginine group of linker compound L and the negative charge of the phosphate group of the pharmaceutically acceptable compound was found to have "covalent bond-like" stability.

[0074] In this specification, the term “binding” refers to any kind of interaction between a surface functional group and an antigen, particularly covalent and non-covalent interactions, such as hydrophobic / hydrophilic interactions, van der Waals forces, ionic bonds, hydrogen bonds, ligand-receptor interactions, nucleotide base pairings, or interactions between an epitope and an antibody binding site.

[0075] When a pharmaceutically acceptable compound is hydrophobic, for example, a hydrophobic peptide, it is advantageous to increase its hydrophilicity. In the case of proteins or peptides, this can be achieved, for example, by N-terminal or C-terminal elongation with polar amino acids. In preferred embodiments of the present invention, the protein or peptide is elongated with one or more polar amino acids selected from the group including aspartic acid, glutamic acid, histidine, lysine, arginine, serine, threonine, or tyrosine, preferably lysine, arginine, glutamic acid, and aspartic acid, more preferably lysine.

[0076] Accordingly, the present invention relates in particular to a bioconjugate comprising three parts, including an N-terminal or C-terminal extension having polar amino acids for improving solubility, and linked by adsorption to a linker compound L, a linker unit U, and an antigen / epitope shown in Figure 4a.

[0077] The linker unit U according to the present invention is used, for example, in antibody-drug conjugates (ADCs) containing various types of linkers. There are three main types of chemically cleavable linkers: those cleavable by acid, reductible disulfides, and those cleavable by exogenous stimuli.

[0078] Examples of ADCs include gemtuzumab ozogamicin (Pfizer's Mylotarg®, with a linker of 4-(4-acetylphenoxy)butanoic acid), inotuzumab ozogamicin (Pfizer's Besponsa®, with a linker that is a condensation product of 4-(4'-acetylphenoxy)-butanoic acid (AcBut) and 3-methyl-3-mercaptobutane hydrazide (known as dimethyl hydrazide)), trastuzumab emtansine (Roche's Kadcyla®, with a linker of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), or brentuximab vedotin (also known as SGN-035; Adcetris® by Seattle Genetics Inc., with a linker of dipeptide valine-citrulline).

[0079] In preferred embodiments of the present invention, the peptide comprises a linker unit U having an N-terminal extension having a linker (catB cleavage linker) that can be cleaved by the enzyme cathepsin B to ensure the release of a native unmodified antigen to MHC I or MHC II. The enzymatic catB cleavage linker comprises one of the cathepsin B-sensitive dipeptides Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Lys, Ala-Lys, or Val-Lys, with the preferred dipeptide being Val-Cit or Trp-Cit, and more preferably Val-Cit.

[0080] In one embodiment, the composition according to the present invention comprises phosphorylated nanoparticles and one or more peptides conjugated to the nanoparticles by adsorption bonding, wherein the peptides include Val-Cit or Trp-Cit as cathepsin B-sensitive dipeptides.

[0081] Figure 4b shows a preferred three-part bioconjugate according to the present invention. The peptide used in Figure 4b is a highly hydrophobic epitope derived from human NY-ESO-1. For N-terminal extension, the enzymatically (cathepsin B) cleavable peptide linker Val-Cit was used to ensure the release of the native unmodified antigen to MHC I or MHC II. The extension portion "Lys-Lys-Lys" was used to enhance solubility and electrostatic attraction to nanoparticles. Extension with, for example, Lys-Lys-Lys-Asp or Arg6 is also possible.

[0082] In a preferred embodiment of the present invention, HPV16 E7 82-90 The epitope LLMGTLGGIV is expanded at its N-terminal site by the enzymatically cleavable linker Val-Cit and the cationic solubilization sequence Lys-Lys-Lys, resulting in KKKV-Cit-LLMGTLGIV.

[0083] In another preferred embodiment of the present invention, HPV16 E7 11-19 The epitope YMLDLQPET is expanded at its N-terminal site by the enzymatically cleavable linker Trp-Cit and the cationic solubilization sequence Lys-Lys-Lys, resulting in KKKW-Cit-YMLDLQPET.

[0084] A further embodiment is a composition according to the present invention comprising SiO2 nanoparticles having a linker compound L on its surface, wherein the linker compound L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2) as a functional group + )NH2) and at least one carboxyl (-COOH) or carboxylate (-COO - The compound comprises a group and one or more peptides or antigens conjugated to linker compound L by adsorption bonding, wherein the peptide or antigen includes a hydrophilic extension portion comprising linker unit U and one or more amino acids.

[0085] A preferred embodiment is a composition according to the present invention comprising SiO2 nanoparticles having a linker compound L on its surface, wherein the linker compound L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2) as a functional group + )NH2) and at least one carboxyl (-COOH) or carboxylate (-COO - The compound comprises a group and one or more peptides or antigens conjugated to linker compound L by adsorption bonding, wherein the peptide or antigen includes a cathepsin B-sensitive dipeptide and a hydrophilic extension portion due to Lys-Lys-Lys.

[0086] A more preferred embodiment is a composition according to the present invention comprising SiO2 nanoparticles having a linker compound L on its surface, wherein the linker compound L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2) as a functional group + )NH2) and at least one carboxyl (-COOH) or carboxylate (-COO - The compound comprises a group and one or more peptides or antigens conjugated to linker compound L by adsorption bonding, wherein the peptide or antigen includes Val-Cit or Trp-Cit as a cathepsin B-sensitive dipeptide and a hydrophilic extension portion by Lys-Lys-Lys.

[0087] Another embodiment is a composition according to the present invention comprising SiO2 nanoparticles having a linker compound L on its surface, wherein the linker compound L has at least one guanidino group -HC(=NH)NH2 or -NHC(=NH2) as a functional group. + )NH2) and at least one carboxyl (-COOH) or carboxylate (-COO - The peptide comprises a ) group and one or more model peptides SIINFEKL conjugated to linker compound L by adsorption bonding, the peptide comprising Val-Cit or Trp-Cit as a cathepsin B-sensitive dipeptide and a hydrophilic extension portion by Lys-Lys-Lys.

[0088] However, this hydrophilic modification of the peptide leads to stronger binding to MHCI (i.e., immunodominance), which can displace the target epitope and trigger an immune response against an epitope not present on tumor cells. To avoid this effect, a spacer may be needed between the hydrophilic submolecule and the epitope.

[0089] An optional embodiment of the present invention is the use of so-called self-sacrificing spacers (PABC, p-aminobenzylcarbamates). Examples of such self-sacrificing spacers are compounds comprising structural units of 4-aminobenzyl alcohol, 2-aminobenzyl alcohol, or 4-hydroxybenzyl alcohol, 2-hydroxybenzyl alcohol. (European Patent Application Publication No. 0648503, International Publication No. 2007 / 031734, International Publication No. 2015 / 162291, U.S. Patent No. 6,180,095, U.S. Patent No. 6,214,345).

[0090] A self-sacrificing spacer is defined as a molecular part that is chemically bonded to at least two further molecular parts such that when one of its bonds to the molecular part is released, the remaining bond is broken and the previously bonded molecule is released.

[0091] When a self-sacrificing spacer is used, it is preferable that the spacer be positioned between the enzymatic catB cleavage linker and the peptide. As an example, the following bioconjugate can be generated: (Arg6)-(Val-Cit)-(PABC)-(SIINFEKL), where SIINFEKL is a model antigen.

[0092] Surprisingly, it was found that the enzymatic catB cleavage linker system could be used without the need for a self-sacrificing spacer.

[0093] The epitopes used in therapeutic vaccines typically consist of 8–11 amino acids (for MHC I) and up to 25 amino acids (for MHC II). Because these epitopes are small in size compared to much larger drugs, self-sacrificing spacers are not required.

[0094] pharmaceutically acceptable compounds are preferably selected from the group consisting of proteins (e.g., antibodies or enzymes), peptides, nucleic acids (RNA or DNA), polynucleotides, lipids, polysaccharides, or organic compounds ("small molecules," preferably, for example, chemotherapeutic agents having a molecular weight of 50 to 1000 Da) or mixtures thereof.

[0095] Preferably, the pharmaceutically acceptable compound is selected from the group consisting of pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), deficient infectious particles (DIPs), or antigens.

[0096] The "antigen" according to the present invention is a structure capable of inducing a cellular or humoral immune response. The antigens are preferably proteogenic; that is, they are proteins, polypeptides, peptides, or fragments thereof. In principle, they can have any size, origin, and molecular weight. They contain at least one antigenic determinant or antigenic epitope.

[0097] In alternative embodiments, the antigen is either a nucleic acid itself or encoded by a nucleic acid that, after transport into the nucleus of an antigen-presenting cell, is translated into a proteogenic antigen presented to an MHC molecule.

[0098] Nucleic acids can be single-stranded or double-stranded DNA or RNA or oligonucleotides. Nucleic acids can also be components of complexes with lipids, carbohydrates, proteins, or peptides.

[0099] The "peptides" according to the present invention are preferably composed of any number of any kind of amino acids linked by peptide bonds, preferably naturally occurring amino acids. In particular, the peptide contains at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12, or at least 15 amino acids. In preferred embodiments, the peptide does not exceed 100 amino acid lengths, more preferably 50 amino acid lengths. Even more preferably, it is 25 amino acids or less. In most preferred embodiments, the peptide has a length of 8 to 20 amino acids. The peptide can also undergo post-translational modifications, such as phosphorylation, glycosylation, lipidation (myristoylation or palmitoylation, etc.), citrullination, acetylation (of lysine), or hydroxylation (of proline or lysine).

[0100] The "epitope" according to the present invention, also known as an antigenic determinant, is a part of an antigen recognized by the immune system, specifically by antibodies, B cells, or T cells. T cell epitopes are presented on the surface of antigen-presenting cells, where they bind to MHC molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, while most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides with a length of 8-11 amino acids, while MHC class II molecules present longer peptides, with a length of 13-17 amino acids, and non-classical MHC molecules also present non-peptide epitopes such as glycolipids.

[0101] In a more preferred embodiment of the present invention, a pharmaceutically acceptable compound is a compound comprising at least one pathogen-associated molecular pattern (PAMP).

[0102] PAMPs are recognized by so-called pattern recognition receptors (PRRs) and function as major sensors for microbial pathogens, initiating immune responses against them. PRRs are secretory forms present in the cytosol and extracellularly, in the bloodstream, lymph, and interstitial fluid, and can be found at different locations associated with intracellular compartments, such as membrane-bound contexts on the cell membrane and endosomal membranes.

[0103] There are four major subfamilies of PRRs: Toll-like receptors (TLRs), nucleotide-binding oligomeric domain (NOD)-leucine-rich repeat (LRR) receptors (NRLs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) or RIG-I-like helicases (RLHs), and type C lectin receptors (CLRs). Further PRRs exist within the complement system.

[0104] According to the present invention, PAMPs recognized by TLRs are preferred. TLRs can be classified into membrane-bound TLRs, which are expressed on the cell membrane, on the membranes of endocytic vesicles or other intracellular organelles, and on cytosolic TLRs. TLR4 has a unique role among TLRs because it can trigger pathways at different cellular locations, such as the plasma membrane and intracellular compartments. TLRs recognize distinctive structures on microorganisms, often called "PAMPs" (pathogen-associated molecular patterns). Ligand binding to TLRs triggers a cascade of intracellular signaling pathways that induce the production of factors involved in inflammation and immunity.

[0105] Table 1 shows TLR agonists suitable as PAMPs according to the present invention. Table 1: TLR agonists and their PAMPs [Table 1]

[0106] A more preferred PAMP according to the present invention is selected from the group of TLR3, TLR7, TLR8, and TLR9 agonists, and most preferably selected from the group of TLR3 and TLR9.

[0107] A particularly preferred embodiment is a composition according to the present invention comprising SiO2 nanoparticles having a linker compound L on its surface, wherein the linker compound L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2) as a functional group + )NH2) and at least one carboxyl (-COOH) or carboxylate (-COO - The compound comprises a ) group and one or more PAMPs conjugated to the linker compound L by adsorption bonding, the PAMPs being selected from poly(U) or CpG ODN.

[0108] Preferred TLR3 agonists are selected from the group of poly(I:C) (dsRNA, TLR3 agonist and RIG-I agonist), polyI:polyC12U (dsRNA, TLR3 agonist, trade name Ampligen®, INN:lintatrimod), and NAB2 (nucleic acid band 2, a dsRNA isolated from yeast and identified as an agonist for pattern recognition receptors TLR3 and MDA-5).

[0109] Preferred TLR4 agonists are selected from MPL-A (monophosphoryl lipid A, e.g., MPL-A adsorbed onto a specific form of aluminum salt, derived from Salmonella minnesota R595 or AS04) or LPS (lipopolysaccharide, e.g., derived from Escherichia coli O55:B5). Another preferred TLR4 agonist is an LPS mimetic, which is a synthetic TLR-4 agonist adjuvant. Examples of peptide sequences that mimic TLR4 ligand function include, for example, APPHALS and QEINSSY (A. Shanmugam et al., PLoS ONE, February 2012, Vol. 7, Issue 2; e30839). By introducing such peptide sequences into the sequence of an active ingredient polypeptide, the polypeptide can acquire adjuvant function.

[0110] Other preferred PRR agonists include polyU(ssRNA), TLR8 agonists (human), and TLR7 agonists in mice.

[0111] According to preferred embodiments of the present invention, pharmaceutically acceptable compounds include nucleotide compounds, particularly oligonucleotide agonists including TLR7 agonists containing dsRNA structures (International Publication No. 2010 / 105819, International Publication No. 2006 / 063252), short ssRNA TLR7 and TLR8 agonists including those described in International Publication No. 2007 / 031319, and TLR9 agonists such as immunostimulant CpG dinucleotides as previously described (International Publication No. 2001 / 022990), particularly CpG dinucleotide ODN 2006 (Hartmann and Krieg, 2000, The Journal of Immunology, 2000, 164 944-952).

[0112] Preferred TLR7 agonists are characterized by a central G:U base pair in a double-stranded structure formed by 4 to 8 G:C base pairs in addition to a G:U base pair. Examples of suitable TLR7 agonists are oligonucleotide agents essentially consisting of the nucleotide sequence 5'-GUCCUUCAA-S' (SEQ ID NO: 1), preferably taken from the 5' end of this sequence, e.g., 5'-GUCC-3' (SEQ ID NO: 2), 5'-GUCCU-3' (SEQ ID NO: 3), 5'-GUCCUU-S' (SEQ ID NO: 4), 5'-GUCCUUC-3' (SEQ ID NO: 5), or 5'-GUCCUUCA-S' (SEQ ID NO: 6) (International Publication No. 2006 / 063252).

[0113] A more preferred TLR7 agonist is described in International Publication No. 2010 / 105819, and the RNA poly / oligonucleotide is characterized by a G:U fluctuation base pair at the center of the double-stranded structure formed by 4 to 8 G:C base pairs, in addition to G:U base pairs, preferably G:U and G:C base pairs and A:U base pairs. The preferred poly / oligonucleotide has a structure defined by the following general formulas (II) and (III), which define two separate RNA strands containing a cassette: 5'X n G / UV m 3'(II), 5' o W m U / GY n 3'(III), In the formula, G / U and U / G are selected so that fluctuating base pairs are formed. 2 <n≦12、2<m≦12、2≦o<12であり、 X defines any base that forms a Watson-Crick base pair with the corresponding base of Y, V defines any base that forms a Watson-Crick base pair with the corresponding base of W in the RNA stem structure, and N is any base in the loop.

[0114] The total length of these separate RNA strands in formulas (II) and (III) is preferably 5 to 45 bases.

[0115] The most preferred poly / oligonucleotides having at least one stem-and-loop structure have a structure defined by the following general formulas (I) and (III), which define two distinct RNA strands containing a cassette: 5'X n G / UV m 3'(II), 5' o W m U / GY n 3'(III), In the formula, G / U and U / G are selected so that fluctuating base pairs are formed. 2≦n≦12, 2≦m≦12, 2≦o≦12, In the formula, X represents a Gs or Cs that forms a Watson-Crick base pair with the corresponding G and C bases in Y, and V represents a Gs or Cs that forms a Watson-Crick base pair with the corresponding base in W.

[0116] Repeats of base pair cassettes (i.e., multiple G·U base pairs in a double-stranded structure) will likely be active.

[0117] The preferred total length of these separate RNA strands is between 5 and 45 base pairs.

[0118] In certain embodiments, the single-chain poly / oligonucleotide comprises, preferably, two parts, a 5' part and a 3'' part, one of the two parts comprising, preferably, n U atoms, and the other part comprising, preferably, p G atoms and q A atoms, where n, p, and q are integers greater than 0, p+q=m, m is an integer up to 25, preferably an integer equal to or greater than 11 and less than or equal to 21, and n is greater than m. In one embodiment, n is less than or equal to 100. In another embodiment, n is greater than 100, preferably greater than 1000, more preferably greater than 3000, and most preferably about 3000-5000. In certain embodiments, n may differ in different molecules in the poly / oligonucleotide preparation of the present invention. In a preferred embodiment, p is equal to 1, and G is central to a part comprising or consisting of G and A.

[0119] Examples of single-stranded poly / oligonucleotides having at least one stem-and-loop structure include: C2UC2Gn(wherein n is an integer greater than 4, Preferably an integer greater than 8 or 9). C3UC2Gn(wherein n is an integer greater than 6); C2UC3Gn (wherein n is an integer greater than 6); C3UC3Gn (wherein n is an integer greater than 7); C4UC3Gn (wherein n is an integer greater than 8); C3UC4Gn (wherein n is an integer greater than 8); C4UC4Gn(wherein n is an integer greater than 9); GnC2UC2(wherein n is an integer greater than 5); GnC3UC2(wherein n is an integer greater than 6); GnC2UC3(wherein n is an integer greater than 6); GnC3UC3(wherein n is an integer greater than 7); GnC4UC3(wherein n is an integer greater than 8); GnC3UC4(wherein n is an integer greater than 8); GnC4UC4(wherein n is an integer greater than 9); A5GAgUn7(wherein n is an integer greater than 11); A6GAUn(wherein n is an integer greater than 12); A5GA6Un(wherein n is an integer greater than 12); A6GA6Un(wherein n is an integer greater than 13); A7GA6Un(wherein n is an integer greater than 14); A6GA7Un(wherein n is an integer greater than 14); A7GA7Un(wherein n is an integer greater than 15); A8GA7Un(wherein n is an integer greater than 16); A7GA8Un(wherein n is an integer greater than 16); A8GA8Un(wherein n is an integer greater than 17); A9GA8Un(wherein n is an integer greater than 18); A8GA9Un(wherein n is an integer greater than 18); A9GA9Un(wherein n is an integer greater than 19); A 10 GA9Un(wherein n is an integer greater than 20); A9GA 10Un(wherein n is an integer greater than 20); A 10 GA 10 Un(wherein n is an integer greater than 21); A5GA 10 GA4Un(wherein n is an integer greater than 21); UnA5GA5(wherein n is an integer greater than 11); UnA6GA5(wherein n is an integer greater than 12); UnA5GA6(wherein n is an integer greater than 12); UnA6GA6(wherein n is an integer greater than 13); UnA7GA6(wherein n is an integer greater than 14); UnA6GA7(wherein n is an integer greater than 14); UnA7GA7(wherein n is an integer greater than 15); UnA8GA7(wherein n is an integer greater than 16); UnA7GA8(wherein n is an integer greater than 16); UnA8GA8(wherein n is an integer greater than 17); UnAgGA8(wherein n is an integer greater than 18); UnA8GA9(wherein n is an integer greater than 18); UnA9GA9(wherein n is an integer greater than 19); UnA 10 GA9 (wherein n is an integer greater than 20); UnA9GA 10 (In the formula, n is an integer greater than 20); UnA 10 GA 10 (In the formula, n is an integer greater than 21); UNA5GA 10 GA4 (where n is an integer greater than 21).

[0120] Preferably, in the above example, n is greater than 1000, more preferably 3000, and even more preferably around 3000 to 5000.

[0121] TLR7-specific ligands include poly / oligonucleotides in which one chain or a portion of the chain forming at least one fully double-stranded portion contains, preferably, n G molecules, and the other chain or a portion of the chain forming the same at least one fully double-stranded portion contains, preferably, p U molecules and q C molecules, where n is an integer equal to or greater than 5 and less than or equal to 9, p is an integer less than or equal to n, q is an integer, and p+q=n.

[0122] Further embodiments of the present invention provide RNA poly / oligonucleotides exhibiting selective TLR8 activity, where the U nucleoside in the G:U fluctuation base pair is adjacent to the G base on each side. The RNA poly / oligonucleotides exhibiting selective TLR8 activity may be single-stranded or partially double-stranded. Preferably, RNA poly / oligonucleotides having G nucleosides in the GU fluctuation base pair adjacent to the C nucleosides on each side of the G:U fluctuation base pair form a hairpin structure as defined herein, where n is 5 to 100, preferably 20 to 100. The aforementioned RNA poly / oligonucleotides exhibit highly selective TLR8 activity. The aforementioned RNA poly / oligonucleotides exhibiting selective TLR8 activity preferably have at least two adjacent base pairs on each side of the G:U fluctuation base pair. As used herein, the term “selective TLR8 activity” means that the RNA poly / oligonucleotides defined above exhibit TLR8 activity but not TLR7 activity.

[0123] Preferred TLR9 agonists are selected from CpG ODNs (unmethylated DNA having a CpG-rich motif, TLR9 agonists), as described in U.S. Patent No. 6,429,199, U.S. Patent Application Publication No. 2007 / 0065467, International Publication No. 01 / 22990, and U.S. Patent Application Publication No. 2003 / 0100527.

[0124] RNA helicases such as RIG-I, MDA-5, and LGP-2 are involved in the detection of viral RNA. In contrast to TLRs, RNA helicases are cytosolic and expressed in a wide range of cell types, including immune and non-immune cells, such as fibroblasts and epithelial cells. Therefore, not only immune cells, but also non-immune cells expressing one or more RNA helicases can detect and respond to viral RNA. Both the TLR helicase system and the RNA helicase system work together to optimally detect viral RNA.

[0125] According to the present invention, pharmaceutically acceptable compounds are RIG-I agonists, for example, oligonucleotides having free, uncapped 5' phosphate as described in European Patent Application Publication No. 1920755 or European Patent Application Publication No. 3581656. RIG-I agonists according to the present invention are selected from the group of 5' ppp-dsRNA, 3 p-hpRNA, poly(I:C) / LyoVec complexes recognized by RIG-I and / or MDA-5 depending on the size of poly(I:C); poly(dA:dT) / LyoVec complexes indirectly recognized by RIG-I. Other RIG-I agonists are RIG-I agonists based on blunt-ended 5' triphosphate oligonucleotides as described in International Publication No. 09 / 141146, or 5' phosphate oligonucleotides as described in U.S. Patent Application Publication No. 2012 / 0288476.

[0126] Another preferred RIG-I agonist is poly(dA:dT) (a dsDNA, a synthetic analogue of B-DNA). Poly(dA:dT) is recognized by several PRRs: sensing by cytosolic DNA sensors (CDS), including cGAS, AIM2, DAI, DDX41, IFI16, and LRRFIP1, triggers the production of type I interferon. Sensing by the cytosolic DNA sensor AIM2 triggers inflammasome formation and subsequent secretion of IL-1β and IL-18. Indirect sensing by the cytosolic RNA sensor RIG-I can occur during the transcription of poly(dA:dT) into dsRNA with a 5' triphosphate moiety (5'ppp-dsRNA) by RNA polymerase III. This indirect sensing by RIG-I leads to the production of type I interferon.

[0127] The PRR agonists according to the present invention are CDS ligands and STING ligands. Cytosolic DNA sensors (CDSs) detect damaged, mislocalized, or pathogenic DNA and typically induce type I IFN production via the TBK 1-IRF3 pathway. Several CDSs have been identified, including cyclic GMP-AMP synthase (cGAS), IFN-inducible IFI16 protein, and helicase DDX41. Induction of type I IFN by cytosolic DNA is mediated by the endoplasmic reticulum membrane protein STING (an IFN gene stimulator).

[0128] Suitable CDS ligands according to the present invention include dsDNA-EC, G3-YSD, HSV-60 CDS agonist, ISD, ODN TTAGGG(A 151), poly(dA:dT), poly(dG:dC), or VACV-70.

[0129] Preferred CDS ligands are 70 bp oligonucleotides containing viral DNA motifs derived from pathogen-derived dsDNA (e.g., E. coli K12), HIV-1-derived dsDNA moieties, HSV-60-derived dsDNA moieties (herpes simplex virus), ISD interferon-stimulated DNA (e.g., Listeria monocytogenes), VACV-70, and vaccinia virus DNA.

[0130] Another preferred CDS ligand is poly(dG:dC), poly(deoxyguany-deoxycytidylate) sodium salt, which is a repeating synthetic double-stranded DNA sequence of poly(dG-dC)·poly(dC-dG). Intracellular poly(dG:dC) is detected by several cytosolic DNA sensors (CDS) such as cGAS, DAI, DDX41, IFI16, and LRRFIP1, and induces the production of type I interferon (IFN). This induction of IFN appears to be mediated by the endoplasmic reticulum protein STING. Furthermore, poly(dG:dC) activates the cytosolic DNA sensor AIM2 (absent in melanoma 2), triggering inflammasome formation, which leads to the secretion of pro-inflammatory cytokines IL-1β and IL-18.

[0131] To achieve stimulation of the cytosolic DNA sensor, poly(dG:dC) must be delivered to the cytoplasm, for example, by using a transfection agent. Therefore, the nanoparticles according to the present invention can serve as transfection agents.

[0132] Antisense RNA oligonucleotides for immunostimulation, as described in U.S. Patent No. 8,815,503 or MDA-5 (Melanoma Differentiation Antigen 5) or NAB2 (Nucleic Acid Band 2), and dsRNAs isolated from yeast and identified as agonists of pattern recognition receptors TLR3 and MDA-5 are also suitable.

[0133] STING is also a direct sensor of cyclic dinucleotides (CDNs) in both bacteria and mammals, activating the TBK-1 / IRF-3 and NF-κB signaling pathways to induce robust type I IFNs and pro-inflammatory cytokines.

[0134] The STING ligand according to the present invention is 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP, or c-di-GMP.

[0135] Preferred STING agonists are selected from the group consisting of MK-1454 (a synthetic cyclic dinucleotide (CDN) and agonist of interferon gene protein stimulants), poly(dG:dC), ADU-S100 (a synthetic cyclic dinucleotide (CDN) and agonist of interferon gene protein stimulants), transmembrane protein 173 (TMEM173) (possessing potential immunomodulatory and antineoplastic activity), and MIW815 (when administered intratumorally, the STING agonist MIW815 binds to STING and stimulates the STING-mediated pathway).

[0136] The compositions according to the present invention can be used as immunostimulants. The immunostimulants of the present invention preferably comprise a TLR3 agonist as a pharmaceutically acceptable compound. A further object of the present invention is an immunostimulant comprising the compositions according to the present invention. The immunostimulants of the present invention can be administered to humans or animals.

[0137] In one embodiment of the present invention, the composition according to the present invention is preferably administered before an immunosuppressive event. In the case of administration to animals, preferably cattle, such an immunosuppressive event may be the result of increased stress or fatigue in the animals caused, for example, after transport or when the stabilizer is changed. Unstable climate, suboptimal supply of feed or water, and insufficient supply of colostrum can also affect immunosuppression. In the case of administration to humans, such an immunosuppressive event may be, for example, surgery.

[0138] Therefore, the compositions according to the present invention are used for the prevention and / or treatment of bovine infectious rhinotracheitis (IBR) or bovine respiratory disease (BRD), which are often caused by a weakened immune system, preferably BRD.

[0139] The composition according to the present invention is used for perioperative immunostimulation in humans.

[0140] In one embodiment of the present invention, the composition according to the present invention is preferably administered to the patient before surgery.

[0141] Immunostimulants, comprising one or more adjuvants, are used in adjuvant immunotherapy. As is well known in the art, adjuvants are drugs that enhance the immune response. Adjuvants are well known in the art (see, for example, "Vaccine Design: The Subunit and Adjuvant Approach," Pharmaceutical Biotechnology, Volume 6, Eds. Powell and Newman, Plenum Press, New York and London, 1995).

[0142] Exemplary adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), squalene, squalane, and alum (aluminum hydroxide), which are well-known materials in the art and commercially available from several suppliers. In certain embodiments, aluminum salts or calcium salts (e.g., hydroxide salts or phosphates) can be used as adjuvants. Alum (aluminum hydroxide) is used in many existing vaccines.

[0143] In a particularly preferred embodiment of the present invention, the nanoparticles comprise a compound selected from the group consisting of both, namely pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), deficient infectious particles (DIPs), and at least one antigen.

[0144] Most preferably, the nanoparticles according to the present invention contain a compound comprising PAMP and an antigen.

[0145] A very preferred embodiment is a composition according to the present invention comprising SiO2 nanoparticles having a linker compound L on its surface, wherein the linker compound L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2) as a functional group + )NH2) and at least one carboxyl (-COOH) or carboxylate (-COO - The compound comprises a group, one or more PAMPs, and one or more antigens or epitopes conjugated to linker compound L by adsorption, wherein the antigen or epitope comprises a hydrophilic extension of linker unit U and one or more amino acids. The PAMP is preferably selected from oligonucleotide agonists, including TLR3 agonists or TLR7 agonists containing a dsRNA structure.

[0146] The disclosed compositions and methods are applicable to a wide variety of antigens. In certain embodiments, the antigen is a protein (including recombinant proteins), polypeptide, or peptide (including synthetic peptides). In certain embodiments, the antigen is a lipid or carbohydrate (polysaccharide). In certain embodiments, the antigen is a protein extract, cell (including tumor cells), or tissue.

[0147] In certain embodiments, the antigen can be selected from the following group: (a) polypeptides suitable for inducing an immune response against cancer cells; (b) polypeptides suitable for inducing an immune response to infectious diseases; (c) polypeptides suitable for inducing an immune response to allergens; and (d) Polypeptides suitable for inducing an immune response in livestock or pets.

[0148] In some embodiments, the antigen or antigenic determinant is useful for the prevention of infectious diseases. Such treatments would be useful for treating a wide variety of infectious diseases affecting a broad range of hosts, including cattle, sheep, pigs, dogs, cats, and other mammalian and non-mammalian species, preferably humans. Therefore, the antigen or antigenic determinant selected for the composition would be one that is well known to medical technologists.

[0149] Suitable antigens for use with the compositions according to the present invention may be derived from, but are not limited to, pathogenic bacteria, fungi or viruses, coronaviruses (SARS-CoV-2), bovine respiratory syncytial virus (BRSV), bovine respiratory coronavirus (BRCV), human papillomavirus (HPV), bovine herpesvirus-1 (BHV-1), parainfluenza type 3 virus (PI-3 V), Pasteurella multocida, Mannheimia haemolytica, istophilus somni, and ycoplasma bovis.

[0150] Examples of infectious diseases include, but are not limited to, viral infections such as COVID-19, bovine infectious rhinotracheitis (IBR), bovine respiratory disease (BRD), SARS, and cervical cancer caused by HPV.

[0151] Any antigen associated with any disease or symptom provided herein can be used in the compositions and methods described herein. These include antigens associated with cancer, infectious diseases or infectious diseases or degenerative or non-autoimmune diseases. Preferred epitopes include epitopes associated with human papillomavirus infection (e.g., YMLDLQPET(HPV16 E7)). 11-19 ), FAFRDLCIVY(HPV16 E6 52-61 ), TIHDIILECV(HPV16 E6 29-38 ), RRFHNIAGHYR(HPV18 E6 125-135 ), KATLQDIVLHLE(HPV18 E7 5-16), HVDIRTLEDLLM (HPV 16 E7 73-84 ), LEDLLMGTL(HPV16 E7 79-87 ), LLMGTLGIV(HPV16 E7 82-90 ), is a cancer antigen of New York esophageal squamous cell carcinoma-1 (NY-ESO-1), and is a neoantigen as a class of tumor-specific antigens.

[0152] In another embodiment, the antigen associated with an infectious disease or infectious illness is associated with one of the infectious agents provided herein.

[0153] In one embodiment, the infectious agent is a virus of the papillomaviridae family, specifically a virus of the SARS family. In yet another embodiment, the infectious agent is SARS-CoV-2 or human papillomavirus.

[0154] In one embodiment, the composition according to the present invention is used as a vaccine for personalized cancer treatment.

[0155] The compositions according to the present invention are used as vaccines for the prevention of HPV, the treatment of HPV-positive humans, or the treatment of HPV-positive tumors.

[0156] A further object of the present invention is to provide a vaccine comprising a composition according to the present invention.

[0157] The composition according to the present invention is preferably a stable dispersion.

[0158] Nanoparticles can be dispersed in any desired solvent, provided that the nanoparticles are not chemically attacked or physically modified by the solvent, and vice versa. As a result, the resulting nanodispersion is stable, particularly pharmaceutically and physically stable. The dispersion is particularly characterized by the fact that the nanoparticles are monodisperse and non-aggregating, have no tendency to settle, and are suitable for sterile filtration.

[0159] Another object of the present invention is a lyophilized product containing nanoparticles according to the present invention. The lyophilized product may contain additives, such as polymers (e.g., polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, dextran, and ficol), as well as sugars (e.g., trehalose, lactose, sucrose, glucose, galactose, maltose, mannose, and fructose), polyhydroxy alcohols (e.g., mannitol, sorbitol, and inositol), amino acids (e.g., glycine, alanine, proline, and lysine), and methylamines (e.g., trimethylamine-N-oxide, betaine, and sarcosine). The lyophilized product according to the present invention can be resuspended in sterile water before vaccination.

[0160] The pharmaceutical compositions according to the present invention are any compositions that can be used for the prevention, treatment, control, or post-treatment of patients who exhibit at least temporary changes in the overall state or condition of individual parts of the patient's body, particularly as a result of infectious diseases, septic shock, tumors, cancer, autoimmune diseases, allergies, and chronic or acute inflammatory processes. Therefore, in particular, the pharmaceutical compositions in the sense of the present invention can be vaccines and / or immunotherapeutic agents.

[0161] The compositions according to the present invention can be formulated as pharmaceutical compositions, which may be in the form of solid or liquid compositions. Such compositions generally include some kind of carrier, such as a solid carrier such as gelatin, an adjuvant, or an inert diluent, or a liquid carrier such as water, petroleum, animal oil, vegetable oil, mineral oil, or synthetic oil. They may also include physiological saline such as propylene glycol or polyethylene glycol, or glycerol or glycol.

[0162] The compositions according to the present invention may optionally contain other active ingredients, or may contain one or more pharmaceutically acceptable pharmaceutical excipients, carriers, buffers, stabilizers, isotonic agents, preservatives or antioxidants, or other materials well known to those skilled in the art. Such materials should be nontoxic and should not interfere with the efficacy of the pharmaceutically acceptable compounds. The exact properties of the carriers or other materials may depend on the route of administration, e.g., oral or parenteral.

[0163] For intravenous, cutaneous or subcutaneous injection, or injection at the site of the disease, the composition according to the present invention is pyrogen-free and in the form of a parenterally acceptable aqueous solution having appropriate pH, isotonicity, and stability.

[0164] Preservatives are generally included in the composition according to the present invention to slow the growth of microorganisms, extend the shelf life of the composition, and enable multi-use packaging. Examples of preservatives include phenol, meta-cresol, benzyl alcohol, parahydroxybenzoic acid and its esters, methylparaben, propylparaben, benzalkonium chloride, 1-thioglycerol, and benzethonium chloride.

[0165] The nanoparticle-containing composition of the present invention may be administered to a patient by any number of different routes, including enteral or parenteral routes. Parenteral administration of the pharmaceutical composition is preferred. Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, vaginal, rectal, intramuscular, intraocular, transepithelial, intraperitoneal, intracardiac, intraosseous, intradermal, intramedullary, intraperitoneal, transdermal, transmucosal, as well as inhalation and topical (including skin, eye, rectum, nose, inhalation and aerosol), and systemic rectal routes. In preferred embodiments, the pharmaceutical composition is for topical (e.g., mucous membrane, skin) application.

[0166] The nanoparticles are administered, for example, by injection or ballistically using a delivery gun, facilitating transdermal passage through the outer layer of the epidermis. The nanoparticles are then taken up by dendritic cells that mature as they travel, for example, through the lymphatic system, resulting in the modulation of immune responses and vaccination against the epitope peptide and / or antigens from which the epitope peptide originates or which form part of the epitope peptide. The nanoparticles can also be delivered by aerosol, which is possible due to their small size.

[0167] Particularly preferred are intradermal, subcutaneous, intramuscular, or intravenous injection. Administration can be carried out, for example, using a so-called vaccination gun or a syringe. Mucosal administration, especially mucosal vaccination, may be beneficial if the pathogen enters the body via mucosal pathways. It is also possible to prepare the substance as an aerosol that is inhaled by the living organism, preferably a human patient, and taken up by the nasal and / or bronchial mucosa. Other possible forms of mucosal administration are vaginal and rectal suppositories. These forms of administration are cost-effective (no consumables such as syringes and needles), safe (very low risk of infection and operative error), easy to apply, and have high patient compliance (no needle phobia). They also directly address the mucosal immune system (MALT).

[0168] In one embodiment of the present invention, a vaccine comprising the composition according to the present invention is used for mucosal administration.

[0169] The exceptionally small size of the nanoparticles of the present invention is a significant advantage for delivery to cells and tissues, as they can be taken up by cells even when bound to target molecules or therapeutic molecules. Therefore, the nanoparticles can be internalized by APCs, and the epitope peptides are processed and presented via class I and class II MHC.

[0170] Particularly Preferred Embodiments of the Invention 1. A composition comprising nanoparticles loaded with silicon dioxide and a pharmaceutically acceptable compound having a functional group on its surface, - Functional groups can support and / or stabilize both negative and positive charges of pharmaceutically acceptable compounds. - The zeta potential of the composition has a value of at least ±15mV. - The composition comprises nanoparticles having a particle size of less than 150 nm, preferably 100 nm or less.

[0171] 2. Nanoparticles, nm 2 The total number of pharmaceutically acceptable compounds on the surface of a unit nanoparticle [molecule / nm] 2 The composition according to Embodiment 1, having a surface load density of a maximum of 0.5, preferably 0.01 to 0.5, more preferably 0.03 to 0.4, and most preferably 0.05 to 0.3.

[0172] 3. The composition according to Embodiment 1 or 2, wherein the polydispersity index (PDI) of the composition is 0 to 0.32, preferably 0.1 to 0.3, more preferably 0.1 to 0.2, and most preferably less than 0.1.

[0173] 4. The composition according to any one of Embodiments 1 to 3, wherein the pH value of the composition is 6.0 to 8.0.

[0174] 5. A composition according to any of the prior embodiments, wherein the zeta potential of the composition is at least ±30 mV.

[0175] 6. The composition according to any of the preceding embodiments, wherein the Z-mean diameter of the nanoparticles is in the range of 5 nm or more and less than 150 nm, preferably 15 nm or more and 60 nm or less, more preferably 20 nm or more and 50 nm or less, and even more preferably 20 nm to 30 nm.

[0176] 7. A composition according to any of the preceding embodiments, wherein the net charge of the loaded nanoparticles is different from zero in total.

[0177] 8. The composition according to any one of the preceding embodiments, wherein the functional group is linked to a linker L bound to the surface of the nanoparticle via a covalent bond or an adsorption bond.

[0178] 9. The composition according to embodiment 8, wherein the linker compound L comprises at least one carboxyl (-COOH) or carboxylate (-COO - ) group as a functional group.

[0179] 10. The composition according to any one of embodiments 8 or 9, wherein the linker compound L comprises at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2 + )NH2) or amino group (-NH2 or -NH3 + ) group as a functional group.

[0180] 11. The composition according to any one of embodiments 8 to 10, wherein the linker L comprises at least one further functional group L' selected from the group consisting of -SH, -COOH, -NH2, -guanidino group (-NHC(=NH))NH2), -PO3H2, -PO2CH3H, -SO3H, -OH, -NR3 + X - .

[0181] 12. The composition according to any one of embodiments 8 to 11, wherein the linker compound L comprises at least the structural unit of formula (I) * -(-O)3Si-(CH2)n-CH(COOX)-(CH2)p-C(O)-NH-CH(COOX)-(CH2)q-Y(I) [wherein, X is independently H or a negative charge, Y is independently -NHC(=NH)NH2, -NHC(=NH2 + )NH2, -NH2 or -NH3 + , n, p and q are each independently 0 or a number from 1 to 25, * - is the point of attachment to the nanoparticle] .

[0182] 13. A composition according to any of the prior embodiments, wherein a pharmaceutically acceptable compound is conjugated to nanoparticles by adsorption bonding.

[0183] 14. A composition according to any of the preceding embodiments, wherein the pharmaceutically acceptable compound is selected from the group comprising proteins, peptides, polynucleotides, oligonucleotides (RNA, DNA, single-stranded or double-stranded), nucleic acids, or peptide antigens.

[0184] 15. A composition according to any of the preceding embodiments, wherein the pharmaceutically acceptable compound is selected from the group comprising pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), deficient infectious particles (DIPs), or epitopes.

[0185] 16. The composition according to Embodiment 15, wherein PAMP contains at least one phosphate group.

[0186] 17. The composition according to embodiment 15 or 16, wherein PAMP is a pattern recognition receptor (PRR) agonist.

[0187] 18. A composition according to any one of Embodiments 15 to 17, wherein PAMP is selected from the group consisting of a TLR agonist, a retinoic acid-inducible gene I (RIG-I) ligand, a melanoma differentiation-related gene 5 (MDA-5) ligand, a cytosolic DNA sensor (CDS) ligand, and a STING ligand (cyclic dinucleotide (CDN)).

[0188] 19. A composition according to any one of Embodiments 15 to 18, wherein PAMP is selected from the group consisting of 5'-triphosphate-RNA, cytosolic DNA sensor (CDS), 2'3'-cGAMP, 3'3'-cGAMP, c-di-AMP(bis-(3'-5')-cyclic dimer adenosine monophosphate), c-di-GMP(bis-(3'-5')-cyclic dimer guanosine monophosphate, double-stranded (ds)RNA).

[0189] 20. The composition according to embodiment 18, wherein the TLR agonist is selected from the group consisting of TLR1 / 2, TLR3, TLR4, TLR7, TLR8 and TLR9 agonists.

[0190] 21. The composition according to embodiment 20, wherein the TLR agonist is lipopolysaccharide (LPS), monophosphoryl lipid A (MPL-A), poly(U), CpG oligodeoxynucleotide (ODN) (unmethylated DNA), or Pam3CSK4 (synthetic triacylated lipopeptide).

[0191] 22. The composition according to embodiment 14, wherein the peptide comprises 8 to 100 amino acids.

[0192] 23. The composition according to embodiment 22, wherein the peptide is an MHC class I epitope or an MHC class II epitope.

[0193] 24. The composition according to embodiment 22 or 23, wherein the peptide is extended at the N-terminus or C-terminus with a polar amino acid.

[0194] 25. The composition according to embodiment 24, wherein the polar amino acid is selected from the group consisting of aspartic acid, glutamic acid, histidine, lysine, arginine, serine, threonine and tyrosine.

[0195] 26. The composition according to any one of embodiments 22 to 25, wherein the peptide comprises an N-terminal extension having a linker cleavable by the enzyme cathepsin B.

[0196] 27. The composition according to embodiment 26, wherein the enzyme-cleavable linker comprises one of cathepsin B-sensitive dipeptides Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Lys, Ala-Lys or Val-Lys.

[0197] 28. The composition according to embodiment 26 or 27, wherein a spacer is disposed between the enzyme-cleavable linker and the peptide.

[0198] 29. The composition according to Embodiment 28, wherein the spacer is a self-sacrificing spacer.

[0199] 30. The composition according to Embodiment 28 or 29, wherein the spacer comprises a structural unit of 4-aminobenzyl alcohol, 2-aminobenzyl alcohol, or 4-hydroxybenzyl alcohol, or 2-hydroxybenzyl alcohol.

[0200] 31. A composition according to any of the preceding embodiments, comprising at least silicon dioxide (SiO2), wherein the nanoparticles may be a mixture with another material.

[0201] 32. A composition according to any of the prior embodiments, wherein the nanoparticles consist of SiO2.

[0202] 33. The composition according to any of the preceding embodiments, wherein the composition is a stable dispersion.

[0203] 34. A composition according to any of the prior embodiments, wherein the pH value of the composition is 7.2 to 7.4.

[0204] 35. A composition according to any of the preceding embodiments, wherein the composition is isotonic.

[0205] 36. A composition according to any of the prior embodiments, wherein the composition contains further pharmaceutically acceptable additives.

[0206] 37. The composition according to Embodiment 1 for use as a vaccine.

[0207] 38. The composition according to Embodiment 1 for use as an immunostimulant.

[0208] 39. The composition according to Embodiment 38 for use in activating the immune system and as an adjuvant.

[0209] 40. The composition according to embodiment 38, for use in the prevention and / or treatment of bovine respiratory disease (BRD).

[0210] 41. The composition according to embodiment 38, for perioperative immunostimulation in humans.

[0211] 42. The composition according to embodiment 1, for personalized cancer treatment.

[0212] 43. The composition according to embodiment 37, for use as a vaccine for preventing HPV or treating HPV-positive humans or treating HPV-positive tumors.

[0213] 44. A nanoparticle comprising silicon dioxide and a functional group on the surface, having a particle size of less than 150 nm, comprising a linker L covalently or adsorptively bound to the nanoparticle, wherein the linker L has at least one guanidino group (-NHC(=NH)NH2 or -NHC(=NH2 + )NH2) or an amino group (-NH2 or -NH3 + ) group as the functional group.

[0214] 45. A lyophilizate comprising the nanoparticle according to embodiment 44.

[0215] 46. A vaccine comprising the composition according to embodiment 1.

[0216] 47. An immunostimulant comprising the composition according to embodiment 1.

Examples

[0217] Example 1 Preparation of monodisperse silicon dioxide nanoparticles with a diameter of 25 nm 500 mL of anhydrous ethanol was collected from a 500 mL volumetric cylinder into a 1000 mL glass bottle with a screw cap. 358 mL of sterile DI water was added, and the mixture was shaken thoroughly.

[0218] 69.6 mL of tetraethyl orthosilicate (TEOS) was added to the bottle. The bottle was tightly closed and shaken thoroughly. After a waiting time of 1 hour, the colorless, transparent mixture reached room temperature (22°C). Then, 9.5 mL of aqueous ammonia (25%) was quickly added.

[0219] The bottle was shaken very vigorously by hand for 10 seconds and then stored at room temperature.

[0220] After 24 hours at room temperature, an additional 34.8 mL of TEOS was added to the mixture to obtain a narrower size distribution (seed growth process). After another 24 hours at room temperature, another 34.8 mL was added to the mixture (continued seed growth process). After 24 hours at room temperature, 50 μL of the reaction mixture was placed in a unidirectional PMMA cuvette (10 mM width) and diluted with 1.5 mL of DI water (0.2 μm filtration). The cuvette was placed in a ZetaSizer Nano (ZetaSizer Nano ZS, Malvern Instruments, UK) for particle size measurement. Applicable parameters: Measurement angle: 173° backscattering (NIBS default) Measurement cell: DTS1070 (used for particle size and zeta potential) Refractive index SiO2: 1.460 Absorption: 0.010 Dispersant: Water Refractive index: 1.330 Viscosity: 0.8872 cP (= sample viscosity) Temperature: 25℃ Performing more than 10 particle size measurements result: Z-average (volume basis): 23.86 nm PDI: 0.108 Intensity peak: 26.77nm pH: 11.0 (Newly calibrated composite electrode)

[0221] The nanoparticle suspension was transferred to a 2-liter round-bottom flask, and ethanol and ammonia gases were removed using a rotary evaporator equipped with a heated water bath. 400 mL of sterile DI water was added in small increments. The volume was reduced to 198.15 g.

[0222] The solid content of the suspension (pure nano-dispersed silicon dioxide) was measured in a triple-scale system by evaporation of 250 μL, and the residue was weighed. result: Solid content: 163.3mg / mL pH: 8.0 (Newly calibrated composite electrode)

[0223] To obtain a solid content of 70.0 mg / mL, the distillation residue was diluted with 264.5 mL of sterile DI water. Example 2: Functionalization of nanoparticles with L-arginine to obtain alginated silica nanoparticles (SiO2-Arg)

[0224] 462.65 g of the 462.65 g nanoparticle suspension obtained in Example 1 was placed in a 500 mL glass bottle. A magnetic stirrer was added, and the bottle was placed in the magnetic stirrer. 17.35 g of L of (+)-arginine (CAS No. 74-79-3, PanReac AppliChem, European Pharmacopoeia / United States Pharmacopoeia, product code A1345.0500) was added to the nanoparticle suspension. Once the arginine was completely dissolved, a pH of 10.8 was obtained, and then 5.34 mL of 3-(triethoxysilyl)propyl succinic anhydride (CAS No. 93642-68-3, Gelest Corp., product code: SIT8192.6) was slowly added at room temperature. The reaction mixture was very turbid but cleared within about 30 minutes. During this time, two reactions occurred simultaneously:

[0225] The ethoxy group of the silane was hydrolyzed due to the high pH value. The resulting silanol group precipitated on the nanoparticles, forming a surface coating on the nanoparticles. In parallel, the excess amino group of arginine reacted with the succinic anhydride group to form an amide bond.

[0226] By opening the ring of the cyclic anhydride, the pH value decreased to 9.8 due to the consumption of arginine and the formation of mainly carboxylic acid groups.

[0227] After stirring for 24 hours, the suspension was transferred to a 20 Falcon tube fitted with a 100 kDa membrane (Pall Corp. Macrosp® Advance, product code MAP 100C38). The nanoparticle suspension was centrifuged at 4,000 rpm (2,737 g on the centrifuge used) for 10 minutes to a volume at least 5 less than the starting volume. After centrifugation, the volume in the Falcon tube was restored to its original level with sterile DI water, and centrifugation was repeated 5 times. The centrifugation step is necessary to remove excess arginine and any unbound reaction products.

[0228] After centrifugation, the solid content of the recovered supernatant was measured using a triplicate analyzer. The method was the same as in Example 1. result: Yield: 178.3g Solids content: 97.16 mg / mL

[0229] The nanoparticle suspension was diluted with 168.17 mL of sterile DI water to obtain a final concentration of 50.0 mg / mL. Example 3 Synthesis of 25 nm diameter phosphorylated silica nanoparticles

[0230] 200 mL of anhydrous ethanol was transferred from a 250 mL graduated cylinder to a 500 mL pressure-resistant glass bottle with a screw cap. 143.5 mL of sterile DI water was added, and the mixture was shaken thoroughly. 27.85 mL of tetraethyl orthosilicate (TEOS) was added to the bottle. The bottle was tightly closed and shaken thoroughly.

[0231] After a 1-hour waiting period, the colorless, transparent mixture reached room temperature (22°C). Next, 3.95 mL of 25% aqueous ammonia was quickly added. The bottle was shaken vigorously by hand for 10 seconds and stored at room temperature. After 24 hours at room temperature, an additional 1.5 mL of (diethylphosphatoethyl)triethoxysilane (CAS number 757-44-8, Gelest Corp., product code SID3412.0) was added to the mixture at room temperature. 10 mL of 25% aqueous ammonia was also added, and the reaction mixture was placed in an 85°C water bath for 24 hours.

[0232] At this temperature, two reactions occurred in parallel: due to the high pH value, the ethoxy group of the silane was hydrolyzed. The silanol group precipitated on the silica nanoparticles, forming a surface coating on the nanoparticles. In parallel, but much slower, the phosphate ester was hydrolyzed to obtain the corresponding acid with ammonia as the counterion. 50 μL of the reaction mixture was placed in a unidirectional PMMA cuvette (10 mM width) and diluted with 1.5 mL of DI water (0.2 μm filtration). The cuvette was placed in a ZetaSizer Nano for particle size measurement. result: Z average (volume basis): 21.3nm PDI: 0.144 Intensity peak: 27.7nm

[0233] The nanoparticle suspension was transferred to a 500 mL round-bottom flask, and ethanol and ammonia gases were removed using a rotary evaporator equipped with a heated water bath. 200 mL of sterile DI water was added in small increments. The volume was reduced to 120 mL. This volume was placed in six Falcon tubes fitted with a 100 kDa membrane (Pall Corp. Macrosp® Advance, product code MAP100C38). The nanoparticle suspension was centrifuged at 4,000 rpm (2,737 g on the centrifuge used) for 10 minutes to a volume at least 80 less than the starting volume. After centrifugation, the volume in the Falcon tubes was returned to its original level with sterile DI water, and centrifugation was repeated five times. The centrifugation step is necessary to remove excess potentially unbound reaction products. After centrifugation, the solid content of the recovered supernatant was measured in triplicate. The method was the same as in Example 1. result: Yield: 83.3g Solids content: 104.0 mg / mL

[0234] The nanoparticle suspension was diluted with 86.6 mL of sterile DI water to obtain a final concentration of 50.0 mg / mL. Reaction scheme 1: Example 1=a) and Example 2=b) [ka] Reaction scheme 2: Example 3 [ka] Example 4 Preparation of poly(I:C)@SiO2-Arg by adsorption bonding of poly(I:C)

[0235] A mixture of 200 mL of ethanol, 143.5 mL of sterile deionized water, and 27.85 mL of tetraethyl orthosilicate (TEOS) was prepared. 3.70 mL of ammonia, 25% (NH3 in water), was added at room temperature. The reaction mixture was vigorously mixed by shaking for 10 seconds and left at room temperature for 24 hours without stirring. The following day, another portion, 27.85 mL of tetraethyl orthosilicate (TEOS), was added to the mixture. 50 μL of the resulting mixture was taken and measured by dynamic light scattering (DLS) using a ZetaSizer Nano ZS (Malvern). The following results were obtained: Z average: 25.53nm Average: 20.36nm Polydispersity index (PDI): 0.136

[0236] Next, a portion of the previously generated 100 mL of silicon dioxide particles was concentrated to approximately 30 mL using a rotary evaporator and then refilled to 100 mL. This process was repeated three times to remove ethanol and ammonia from the reaction solution. The resulting suspension was washed five times with sterile deionized water on a 100 kDa membrane. After the final washing step, the solid content of the suspension was measured by gravimetric analysis using 7.9% SiO2, and the suspension was adjusted to a solid content of 5.0% by adding the calculated amount of water.

[0237] To 15 mL of silicon dioxide particles (750 mg of SiO2) produced in Example 1, 500 mg of L-arginine (CAS No. 74-79-3; abcr GmbH, Germany) (=2.87 mmol) was added, and the mixture was stirred while completely dissolving the arginine. A clear particle suspension was obtained, and to this suspension, 127 μL of (3-triethoxysilylpropyl) succinic anhydride group (CAS No. 93642-68-3) (=0.45 mmol) was slowly added while stirring at room temperature.

[0238] The 2.00 mL of "arginylated" silica nanoparticles obtained above were mixed with 3.00 mL of a low molecular weight solution of poly(I:C) (Poly(I:C)-LMW, size 0.2-1 kb, CAS number 31852-29-6, manufactured by InvivoGen (Toulouse, France), with a concentration of 0.833 mg of poly(I:C) per mL). After thoroughly mixing in a vortex mixer for 15 seconds, the clear suspension was mixed with 250 mg of glucose after 1 hour. The formulation included the following: 100mg "Arginylated" silica nanoparticles, SiO2-Arg(c=20mg / mL) 2.5mg poly(I:C)-LMW (c=0,5mg / mL) 250 mg glucose (c=50 mg / mL) (for isotonicity of the formulation)

[0239] Next, the mixture was packed into three sterile 2.0 mL HDPE vials using a 0.2 μm sterile filter. The resulting suspension was clear and perfectly transparent.

[0240] Poly(I:C) loaded particles easily pass through sterile filters. Two types of filters were used: Pall Life Sciences, Acrodisc Supor® membrane (low protein binding) 0.2 μm, catalog number PN4602 and VWR 0.2 μm cellulose acetate membrane 0.2 μm, catalog number 514-0061.

[0241] Even after adding a larger amount of poly(I:C), the suspension remains clear and completely transparent. Example 5 Peptide loading capacity of SiO2-Arg nanoparticles

[0242] In the peptide loading experiment, different amounts of the model peptide KKKW-Cit-SIINFEKL were loaded onto 100 μL of silica nanoparticles with a diameter (Z-average) of 25 nm, a solid content of 20 mg / mL and an arginylated surface. To simulate physiological conditions, sodium chloride (NaCl) was added to obtain an isotonic suspension (0.9% NaCl). Up to 10% peptide was added to the nanoparticles. KKKW-Cit-SIINFEKL has an isoelectric point of pH 10.24 and exhibits cationic properties determined by four basic amino acids (lysine) and one acidic amino acid (glutamic acid).

[0243] 50 μL of the corresponding particle-peptide-NaCl mixture was diluted with 1.5 mL of sterile filtered deionized water, and measured by dynamic light scattering (DLS) in a ZetaSizer Nano (Malvern Instruments, UK). For each sample No. 1 to 10, 100 μL of SiO₂-Arg stock solution was used. The obtained results are shown in Table 2. Table 2: Results of dynamic light scattering [Table 2] 1 Stock solution SiO₂-Arg: 20 mg / mL SiO₂-Arg 2 Stock solution peptide: 4 mg / mL peptide (KKKW-Cit-SIINFEKL) 3 Stock solution NaCl: 100 mg / mL NaCl

[0244] Figure 5a shows Z-average versus peptide concentration, and Figure 5b shows PDI versus peptide concentration.

[0245] As shown in Figures 5a and 5b, up to 5 wt% of this peptide can be added to the nanoparticles, and a stable suspension can be maintained without aggregation or precipitation. Compared with bare particles, the diameter increases slightly (+5 nm), and the PDI still indicates a very narrow particle size distribution. This optically transparent suspension still passes through a sterile filter.

[0246] Particles loaded with peptides easily pass through sterile filters. Two types of filters were used: Pall Life Sciences, Acrodisc Supor® membrane (low protein binding) 0.2 μm, catalog number PN4602 and VWR 0.2 μm cellulose acetate membrane 0.2 μm, catalog number 514-0061.

[0247] As the peptide load increases, the particle size increases, indicating that the adsorbed peptide binds to the surface of the silica nanoparticles. At higher peptide concentrations (6% in the example above), the nano-suspensions disintegrate, which is indicated by a measurable increase in particle size due to turbidity and aggregation. These suspensions do not pass through sterile filters and are highly unsuitable for parenteral administration. Example 6 Poly(I:C) loading capacity of SiO2-Arg nanoparticles

[0248] In the loading experiment, 50 μL of silica nanoparticles with a diameter of 25 nm (Z-average), a solid content of 20 mg / mL, and an arginylated surface were loaded with different amounts of poly(I:C) solution in RNase / DNase-free water prepared by Gibco®. Poly(I:C) (LMW) was obtained from Invivogen Europe (catalog number tlrl-picw). To simulate physiological conditions, sodium chloride (NaCl) was added to obtain an isotonic suspension (0.9% NaCl). Up to 8% poly(I:C) was added to the nanoparticles. The colloid remained stable: no precipitation or turbidity was observed. Particle size and distribution, as well as zeta potential, supported the visual observations.

[0249] The zeta potential was measured under the following conditions, according to the Smoluchowski calculation model: Measurement temperature: 25℃ Equilibration time: 30 seconds 10 to 100 measurements per measurement, and 5 measurements per sample (zeta potential = average of 5 measurements). Table 3: Results of dynamic light scattering [Table 3] * ) Two peaks: 27.38nm (67.8%) and 362.6nm (32.2%) ** ) Two peaks: 7.691nm (22.9%) and 18.4nm (77.1%)

[0250] Up to a 6% poly(I:C) LMW load, the measured Z-mean value increases due to the increase in particle size caused by adsorbed poly(I:C) (Figure 6b). Also, a low PDI (<0.1) indicates a smooth particle load (Figure 6a). At an 8% load, two peaks (7.691 nm and 18.4 nm) appear, and there is also a strong peak broadening towards lower diameter values, and the PDI value deteriorates (Figure 7d). These data indicate particle "overloading": unbound poly(I:C) generates additional peaks, each broadening the measured peak. Figure 7a: Poly(I:C)LMW without nanoparticles Figure 7b: SiO2-Arg nanoparticles that do not contain poly(I:C) Figure 7c: SiO2-Arg nanoparticles supported with 6 wt% poly(I:C) Figure 7d: SiO2-Arg nanoparticles supported with 8 wt% poly(I:C) Example 7 Loading of SiO2-Arg nanoparticles with peptides and poly(I:C)

[0251] In peptide and poly(I:C) loading experiments, 100 μL of silica nanoparticles with a diameter of 25 nm (Z-average), a solid content of 20 mg / mL, and an arginylated surface were loaded with different amounts of the model peptide KKKW-Cit-SIINFEKL and 50 μg each of polyIC(LMW) (in a 20 μL poly(I:C) stock solution with a concentration of 2.5 mg / mL).

[0252] To simulate physiological conditions, sodium chloride (NaCl) was added to obtain an isotonic suspension (0.9% NaCl). Up to 4% of the peptide was added to the nanoparticles. The colloid remained stable: no precipitation or turbidity was observed. Particle size and distribution, as well as zeta potential, supported the visual observations. Table 4: Results of dynamic light scattering [Table 4] * (pept. = peptide) Example 8 Loading of SiO2-Arg nanoparticles by ssRNA (poly(U))

[0253] In the ssRNA loading experiment, 400 μL of silica nanoparticles with a diameter of 25 nm (Z-average), a solid content of 50 mg / mL, and an arginylated surface (total 20 mg of silicon dioxide) were combined with 500 μL of poly(U) solution at a concentration of 1.0 mg / mL (total 0.5 mg of poly(U)) and 100 μL of sodium chloride (NaCl) at a concentration of 90 mg / mL to obtain an isotonic suspension (0.9% NaCl). In this case, the poly(U) load on the silica nanoparticles was 2.44 wt%. 50 μL of this sample was measured by DLS. Poly(U) was obtained from InvivoGen (Toulouse, France) (catalog code: tlrl-sspu). Table 5: Results of dynamic light scattering [Table 5] Example 9 Loading of SiO2-Arg nanoparticles by unmethylated DNA (CpG ODN)

[0254] In the DNA loading experiment, 40 μL of silica nanoparticles with a diameter of 25 nm (Z-average), a solid content of 50 mg / mL, and an arginylated surface (total 2 mg of silicon dioxide) were combined with 50 μL of ODN2395 solution at a concentration of 1.0 mg / mL (total 0.5 mg of ODN2395) and 10 μL of sodium chloride (NaCl) at a concentration of 90 mg / mL to obtain an isotonic suspension (0.9% NaCl). The loading amount of silica nanoparticles in this case was 2.44 wt%. This 50 μL sample was measured by DLS.

[0255] ODN2395 was obtained from InvivoGen (France) (catalog code: tlrl-2395). Its structure is: 5'-tcgtcgtttt cggcgc:gcgccg It is a 22-mer with a -3' base (the base is a phosphorothioate (nuclease-resistant), and the palindromic sequence is underlined). Table 6: Results of dynamic light scattering [Table 6] Example 10 Examples of improving the solubility of epitopes

[0256] The solubility of the entire peptide can be increased by adding polar amino acids to the N-terminal region of the linker cleaved by enzymes. For example, the HLA-A:02 immunogenic HPV 16 E782-90 epitope LLMGTLGIV is extremely nonpolar and has very poor solubility in water. Its use in human vaccines is difficult. In animal studies, researchers dissolve it in DMSO. Solubility after subcutaneous injection is questionable, for example: dilution can lead to precipitation of large peptide particles, which is very undesirable for transport to lymph nodes. According to the present invention, the epitope LLMGTLGIV is expanded at the N-terminal region by an enzymatically cleavable linker Val-Cit and a cationic solubilization sequence Lys-Lys-Lys. KKKV-Cit-LLMGTLGIV This occurs.

[0257] The complete synthesis was performed in a single run using automated microwave-assisted solid-phase peptide synthesis (SPPS).

[0258] This peptide exhibits excellent water solubility. Following cleavage of endosomes and / or cytosols by cathepsin B, native HPV 16 E782-90 is released. Furthermore, the remaining KKKV-Cit is not immunogenic because it is too short to present to either MHC I or MHC II. Example 11 Human TLR-induced cytokines in the original human macrophage model (THP-1)

[0259] Cytokine production after stimulation of differentiated THP-1 cells with poly(I:C), SiO2-Arg, and poly(I:C)@SiO2-Arg was performed using a Multi-Analyte ELISArray from Qiagen. The nanoparticle diameter was 22.1 nm (Z-mean), and the PDI was 0.08.

[0260] All experiments were performed using low molecular weight poly(I:C)-LMW manufactured by InvivoGen Europe (Toulouse, France).

[0261] Cytokine screening revealed significant cytokine production. Figure 8. Cytokine expression (arbitrary units) after 72 hours of stimulation versus various types of cytokines.

[0262] TNF-α, IL8 (CXCL8), MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), MIG (CXCL9). After 72 hours of stimulation with poly(I:C)@SiO2-Arg. High cytokine release of IL8 (CXCL8), MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), and MIG (CXCL9) was also observed with SiO2-Arg nanoparticles. Figure 9. Cytokine expression (arbitrary units) after 96 hours of stimulation versus various types of cytokines.

[0263] After 96 hours of stimulation, the expression rates of cytokines IL1-β, IL12, IL17A, TARC, and IFN-α increased, and high cytokine release of IL8 (CXCL8), MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), and MIG (CXCL9) was also observed with SiO2-Arg nanoparticles.

[0264] To investigate whether the combination of the TLR3 agonist poly(I:C)-LMW and SiO2-Arg can stimulate cytokine release, differentiated human macrophage-like THP-1 cells were incubated with poly(I:C)-LMW (poly(I:C)@SiO2-Arg) adsorbed to SiO2-Arg or with the individual compounds, and then subjected to cytokine-specific enzyme-linked immunosorbent assay (ELISA). The supernatant of THP-1 cells was analyzed for interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) at ​​several time points (Figure 9). IL-8 and TNF-α are important mediators of the innate immune response and regulate the activity of various immune cells. The release of these two cytokines is evidence of successful stimulation of the immune system.

[0265] Figures 10a and 10b: Cytokine release at different time points after stimulation of differentiated THP-1 cells with poly(I:C) [12.5 μg / ml], SiO2-Arg [0.5 mg / mL], or a novel adjuvant (poly(I:C) [12.5 μg / mL] bound to SiO2-Arg [0.5 mg / mL]).

[0266] Figure 10a: IL-8 release was quantified using ELISA MAX™ Deluxe Set Human IL-8 (BioLegend, USA).

[0267] Figure 10b: TNF-α release was quantified using ELISA MAX™ Deluxe Set Human TNF-α (BioLegend, USA). Example 12 Experiments in a mouse influenza A model

[0268] To determine the immunostimulatory efficacy of the TLR3 agonist, the suspension prepared according to Example 6 was tested in vivo with other active compounds as immunostimulants for its prophylactic effect against a 5-fold LD50 dose of influenza A virus PR8 / 34. The following active compounds were tested: - Placebo: Glucose solution (5%) - Free poly(I:C)LMW: Low molecular weight poly(I:C) contains short chains of inosinate poly(I) homopolymer annealed to cytidic acid poly(C) homopolymer chains. The average size of poly(I:C)LMW in a glucose solution (5%) is 0.2kb to 1kb (InvivoGen, France). - ZelNate®: An FDA-approved immunostimulant that helps prevent bovine respiratory disease (BRD) caused by Mannheimia haemolytica (Bayer AG, Germany). - Poly(I:C)@SiO2-arginylated preparation prepared according to Example 6 - Unmodified silicon nanoparticles with a diameter of 25 nm in a 5% glucose solution Table 7: Overview of a preventive study in mice, with each group consisting of 10 mice. [Table 7]

[0269] In all cases, the injection volume was 100 μL.

[0270] Each animal group (A-E), consisting of 10 C57BL / 6 mice, was treated with either the respective active compound or placebo 24 hours before subcutaneous administration of influenza A virus. The virus was then administered intranasally.

[0271] This study used body weight as a reliable and easily measurable marker for animal health. Sick animals eat less and lose weight very rapidly. For ethical reasons, the study defined a 25% weight loss based on body weight on the day of viral administration as the termination criterion. In contrast, in many publications from older studies, the “termination criterion” is death of the animal due to viral disease.

[0272] In this study, a formulation of poly(I:C) prepared according to Example 6, adsorbed and bound to silica nanoparticles having an "arginylated" surface, showed a remarkably strong immunostimulatory effect, resulting in statistically significantly longer survival rates and significantly less weight loss in animals in group E.

[0273] Free poly(I:C) not bound to nanoparticles with the same concentration of active ingredient (Group C) showed no statistically significant improvement compared to the placebo group (Group A). ​​Unmodified silica nanoparticles (Group D) also did not show a statistically significant improvement.

[0274] The comparative experiments described above demonstrated that the TLR3 agonist adsorbed onto silica nanoparticles (Group E) was clearly superior to the free TLR3 agonist (Group C) applied at the same concentration and under identical test conditions. The immune booster ZelNate® (Group B) also did not show significant improvement compared to the placebo group (Group A) in this study. This also applies to the unmodified silica nanoparticles in Group D, which showed no significant effect (Figures 11a and 11b).

[0275] Figure 12 shows the clinical scores. The data are presented as the mean clinical score (maximum score = 5) ± SEM (n=10) in relation to the number of days after antigen loading. Example 13 HPV16 E7 in tumor-free A2.DR1 mice 11-19 Generation of a specific CD8 T cell immune response

[0276] The immunogenicity of HLA-A2 binding epitopes derived from human HPV16 E6 / E7 can only be studied in genetically modified mice. Therefore, in vivo studies were conducted using HLA-humanized A2.DR1 BL6 mice. A2.DR1 mice underwent numerous genetic modifications to exhibit the HLA-A2+ / HLA-DR1+, H-2 phenotype and functional CD4 binding to multiple epitopes constrained by HLA-A2 and HLA-DR1. + and CD8 + This is a highly sophisticated mouse model, as it has been shown to assemble a T cell response (Kruse, S. Therapeutic vaccination against HPV-positive tumors in a MHC-humanized mouse model, Ruperto Carola University Heidelberg, 2019, Dissertation; Kruse et al. Therapeutic vaccination using minimal HPV16 epitopes in a novel MHC-humanized, Oncoimmunology, 2019, Vol.8, 1, p.e1524694).

[0277] The efficacy of various formulations was evaluated by high-frequency epitope-specific CD8 after three weekly subcutaneous immunizations (Day 0: Prime, Day 7: Boost, Day 14: Boost, Day 21: Spleen sampling). + They were investigated for their ability to induce T cells. Substances used: - Poly(I:C)-HMW: The average size of Poly(I:C)-HMW is 1.5-8kb, InvivoGen (France) - SiO2-PO3H2 of Example 3 ●KKKW-Cit-E7 11-19 Preparation of poly(I:C)-HMW@SiO2-Arg:

[0278] Dissolve 2.72 mg (1.48 μmol) of KKKW-Cit-E 711-19 in 592 μL of DNase / RNase-free distilled water under sterile conditions. Add 2.37 mL of 50 mg / mL SiO2-Arg stock solution to the solution under vortexing. Add 296 mg of solid glucose and mix the solution until the solid is completely dissolved. Finally, add 2.96 mL of poly(I:C)-HMW as a 1.0 mg / mL stock solution while vortexing the sample. Filter the vaccine through a 0.45 μm filter. Fill each of the 1.8 mL solution into three separate vials (one vial per immunization) and store at 4°C.

[0279] HPV16 E7 11-19 The effects of N-terminal peptide elongation (= single-letter amino acid notation YMLDLQPET) and nanoparticle surface modification were investigated. Vaccine-induced HPV16 E7 11-19 specific CD8 + To evaluate T cells, ex vivo-restimulated splenocytes were assessed by IFN-γ intracellular cytokine staining (ICS) followed by flow cytometry (Figure 13). Figure 13: Y-axis: HPV-001:RW-Cit-E7 11-19 [50nmol]+Poly(I:C)-HMW[50μg] HPV-002:RW-Cit-E7 11-19 [50nmol]+Poly(I:C)-HMW[50μg]@SiO2-Arg[2.25mg] HPV-003:RW-Cit-E7 11-19 [50nmol]+Poly(I:C)-HMW[50μg]@SiO2-PO3H2[2.25mg] HPV-004:KKKW-Cit-E7 11-19 [50nmol]+Poly(I:C)-HMW[50μg]@SiO2-Arg[2.25mg] X-axis: Spleen IFN-y + / CD8 + T cells (%)

[0280] Golgi apparatus - E7 in the presence of transport inhibitors 11-19 IFN-γ positive E7 after ex vivo stimulation of spleen cells 11-19 Specific CD 8 + T cell frequency after subsequent IFN-γICS: IFN-γ positive E7 11-19 Specific T cells were identified by flow cytometry. Data are expressed as mean + / - SEM. Each point represents one mouse.

[0281] As shown in Figure 13, the three immunization methods using nanoparticles conjugated to epitopes showed a higher frequency of antigen-specific spleen IFN-γ compared to epitope injection. + CD8 + We were able to induce T cells. Furthermore, direct comparison of the two surface modifications of the nanoparticles (HPV-002 vs. HPV-003) demonstrated better performance when the epitope was conjugated to SiO2-Arg (HPV-002).

[0282] Furthermore, analysis of the effects of N-terminal peptide elongation (HPV-003 vs. HPV-004) revealed that the "KKKW-Cit-" modification (HPV-004) was associated with epitope-specific CD8 + This results in a higher frequency of T cells.

[0283] This experiment shows that the HPV16 vaccine according to the present invention exhibits high frequency epitope-specific CD8 + The ability to induce T cells was successfully demonstrated. In summary, SiO2-Arg and the "KKKW-Cit-" peptide elongation exhibited more beneficial properties than SiO2-PO3H2 nanoparticles and the "RW-Cit-" peptide elongation. Therefore, both combinations were used for therapeutic efficacy in tumor research. Example 14 Therapeutic efficacy in slow-growing PAP-A2-HPV16 tumors

[0284] The ultimate research goal is the development of a therapeutic anti-HPV16 vaccine to be administered to patients diagnosed with either a prodromal lesion or established cancer. Therefore, we tested the ability of a novel vaccine to induce control of tumor growth in therapeutic vaccination experiments. Immunotherapy schedules for early treatment studies in slow-growing PAP-A2-HPV16 tumor models:

[0285] To evaluate the therapeutic efficacy of SiO2-Arg conjugated with an epitope derived from HPV16 E7, we used HPV16 E6 in HLA-humanized A2.DR1 BL6 mice. + / E7 + A PAP-A2 tumor model was used. 1.5·10 6 Individual PAP-A2 cells were subcutaneously injected, which should result in a large tumor within 2-3 weeks. Treatment began 4 days after tumor inoculation, with an ethical endpoint (tumor volume of 1000 mm²). 3 Tumor-bearing mice were treated weekly with either a complete vaccine (HPV-008) or individual compounds as controls (a total of three immunizations, prime-boost-boost) until they reached [a certain stage].

[0286] Figure 14 shows the survival rate of mice treated with either the free antigen HPV16 E7 antigen YMLDLQPET + poly(I:C)(HMW, high molecular weight) "free antigen + TLR agonist" or KKKW-Cit-YMLDLQPET + poly(I:C)(HMW), both adsorbed and bound to arginyl silica nanoparticles with a diameter of 23 nm.

[0287] As shown in Figure 14, treatment of tumor-carrying mice with KKKW-Cit-YMLDLQPET + poly(I:C)(HMW), both adsorbed onto arginyl silica nanoparticles, resulted in complete tumor regression in 5 out of 9 mice, with an overall survival rate of 55%. In contrast, 90% of carrier control (free antigen + TLR3 agonist) mice had to be eliminated due to excessive tumor growth. Figures 15a and 15b show individual tumor growth in both groups.

[0288] Combining the results from therapeutic vaccination using single compounds and SiO2-Arg conjugated with epitopes, we can conclude that the best therapeutic antitumor outcomes were achieved using KKKW-Cit-YMLDLQPET vaccine conjugated with triple-surface arginyl nanoparticles. Example 15 Cross-presentation experiment OVA after intracellular processing in DC2.4 cells 257-264 MHC I cross-presentation of

[0289] To demonstrate the functionality of the enzymatic cleavage (W-Cit) site bound to the immunogenic epitope, we used a model antigen OVA in mouse dendritic cells (DC2.4 cells, immortalized mouse dendritic cells). 257-264 Experiments were conducted on the cross-presentation of SIINFEKL (represented by the single letter of the amino acid). Therefore, the expression of SIINFEKL on major histocompatibility complex I (MHCI) after incubation with various OVA-derived constructs, with or without nanoparticles, was determined by antibody-specific detection of the native epitope presented on MHCI (25-D1.16, PE / Cy7 anti-mouse H-2Kb conjugated to SIINFEKL antibody, Biolegend, Inc., USA). Free MHC I-bound epitopes or elongated epitopes on MHC I that are not bound to MHC I are not recognized by antibodies that are highly selective for SIINFEKL presented on MHC I.

[0290] In the experiment, the presence or absence of nanoparticles affected the full-length protein (OVA = ovalbumin), the N-terminal and C-terminal elongation epitopes (OVA). 247-264 A5K, a so-called synthetic long peptide (SLP), has an N-terminal elongation epitope with a cathepsin B cleavable sequence (exemplified by RW-Cit-OVA). 257-264 ) or natural epitopes (OVA) 257-264 ) 5 μM solution and 5·10 4 OVA after incubation of individual DC2.4 cells 257-264Presentation effectiveness was compared. Surface phosphorylated silica nanoparticles (SiO2-PO3H2) with an average diameter of 25 nm (comparable in size to SiO2-Arg used in other experiments) were used as a support. After 6 hours of incubation, the test material was removed by washing with phosphate-buffered saline (PBS). In the next step, cells were incubated with CD16 / CD32 antibody to block nonspecific binding of the detection antibody to the Fc (fragment, crystalline) receptor in the cells. After incubation with 25-D1.16 antibody, cross-presentation OVA was performed. 257-264 The amount was quantified by flow cytometry.

[0291] Figure 16: H2-Kb positive cells are classified based on the presence or absence of SiO2-PO3H2, and are either natural or elongated OVA cells. 257-264 OVA after incubation with a 5 μM solution of epitope or full-length OVA protein for 6 hours. 257-264 MHC I expression was observed. After labeling with a 25-D1.16 detection antibody, quantification was performed by flow cytometry.

[0292] As shown in Figure 16, only the native epitope and the elongated epitope with an enzymatic cleavage site are presented in significant amounts on the MHC molecule. Incubation with ionically bound peptides on nanoparticles can slightly increase the uptake and thus the amount of the presented epitopes. Native epitopes do not necessarily need to be internalized within the cell, but can be exogenously loaded onto MHC molecules; therefore, the N-terminal elongated epitope must be internalized to release its native sequence. Cells are then subjected to RW-Cit-OVA. 257-264 The detection of native epitopes on MHC I after incubation supports evidence of the functionality of the enzymatic cleavage site. Example 16 Stability measurement in human serum

[0293] HEK-Blue® hTLR3 cells are designed to measure human TLR3 stimulation by human TLR3 by monitoring NF-κB activation. HEK-Blue® hTLR3 cells were obtained by co-transfecting HEK293 cells with the hTLR3 gene and an optimized secretory embryonic alkaline phosphatase (SEAP) reporter gene under the control of an NF-κB and AP-1 inducible promoter. Stimulation with TLR3 ligands activates NF-κB and AP-1, which induce SEAP production. SEAP levels can be easily determined using HEK-Blue® detection, a cell culture medium that enables real-time detection of SEAP. Hydrolysis of the substrate by SEAP produces a purple / blue color that can be easily detected with the naked eye or measured with a 96-well plate reader (see Invivogen, https: / / www.invivogen.com / hek-blue-htlr3).

[0294] Poly(I:C) and poly(I:C)@SiO2-Arg were exposed to human serum (HS) at 37°C for 60 minutes. Serum was used at concentrations of 5%, 10%, and 20%. The 20% serum concentration corresponds very well to the composition of peripheral lymphoid tissue.

[0295] Poly(I:C) and poly(I:C)@SiO2-Arg were added separately to human serum to obtain a concentration of poly(I:C) of 1 μg per mL. For poly(I:C)@SiO2-Arg, the SiO2 concentration was 40 μg / mL. In this case, the poly(I:C) payload in SiO2-Arg was approximately 2.5%. After exposure to HS, 20 μL of culture medium was taken and added to 180 μL of HEK-Blue® hTLR3 cells in HEK-Blue® detection medium. This mixture was incubated at 37°C for 13 hours, and the plates were analyzed using a 96-well plate reader (Tecan Reader, model: Infinite M200 Pro).

[0296] The results are shown in Figure 17.

[0297] The calculated half-life of free poly(I:C) in 20% human serum is 24.2 minutes (18% of the initial concentration after 60 minutes) under selected conditions. Half-life calculation: t 1 / 2 = ln 2 / (ln 100 / ln 18) * 60 [minutes]

[0298] Under the same conditions, the half-life of poly(I:C)@SiO2-Arg is calculated to be 395 minutes (90% of the initial concentration after 60 minutes). t 1 / 2 = ln 2 / (ln 100 / ln 90) * 60 [minutes]

[0299] Therefore, the binding of poly(I:C) to arginyl silica nanoparticles increases the half-life by 16 times (=395 / 24.2). Example 17 Stability measurement in bovine serum

[0300] HEK-Blue® hTLR3 cells are designed to measure human TLR3 stimulation by human TLR3 by monitoring NF-κB activation. HEK-Blue® hTLR3 cells were obtained by co-transfecting HEK293 cells with the hTLR3 gene and an optimized secretory embryonic alkaline phosphatase (SEAP) reporter gene under the control of an NF-κB and AP-1 inducible promoter. Stimulation with TLR3 ligands activates NF-κB and AP-1, which induce SEAP production. SEAP levels can be easily determined using HEK-Blue® detection, a cell culture medium that enables real-time detection of SEAP. Hydrolysis of the substrate by SEAP produces a purple / blue color that is easily detectable with the naked eye or can be measured with a 96-well plate reader (Invivogen).

[0301] Poly(I:C) and poly(I:C)@SiO2-Arg were exposed to fetal bovine serum (FBS) at 37°C for 60 minutes. Serum was used at concentrations of 5%, 10%, and 20%. The 20% serum concentration corresponds very well to the composition of peripheral lymphoid tissue.

[0302] Poly(I:C) and poly(I:C)@SiO2-Arg were added separately to bovine serum to obtain a concentration of 1 μg of poly(I:C) per 1 mL. For poly(I:C)@SiO2-Arg, the SiO2 concentration was 40 μg / mL. In this case, the poly(I:C) payload in SiO2-Arg was approximately 2.5%. After exposure to FBS, 20 μL of culture medium was taken and added to 180 μL of HEK-Blue® hTLR3 cells in HEK-Blue® detection medium. This mixture was incubated at 37°C for 13 hours, and the plates were analyzed using a 96-well plate reader (Tecan Reader, model: Infinite M200 Pro). The results could be confirmed from Figure 18.

[0303] Free poly(I:C) shows clear signs of degradation at higher serum concentrations, while poly(I:C)@SiO2-Arg exhibits significantly higher stability. Half-life calculations for poly(I:C)@SiO2-Arg are not useful due to its extremely low decay rate. The present invention provides, for example, the following items: (Item 1) A composition comprising nanoparticles loaded with silicon dioxide and a pharmaceutically acceptable compound having a functional group on its surface, - The functional group can support and / or stabilize both the negative and positive charges of the pharmaceutically acceptable compound. - The zeta potential of the composition has a value of at least ±15mV, - The nanoparticles have a particle size of less than 150 nm. composition. (Item 2) The aforementioned nanoparticles are nm 2 The total number of pharmaceutically acceptable compounds on the surface of the nanoparticles [molecules / nm] 2 The composition according to item 1, having a maximum surface load density of 0.5. (Item 3) The composition according to item 1 or 2, wherein the polydispersity index (PDI) of the composition is 0 to 0.32. (Item 4) The composition according to any of the preceding items, wherein the zeta potential of the composition is at least ±30mV. (Item 5) The composition according to any of the preceding items, wherein the total net charge of the loaded nanoparticles is different from 0. (Item 6) The composition according to any of the preceding items, wherein the functional group is linked to a linker L bonded to the surface of the nanoparticle by covalent or adsorption bonds. (Item 7) The linker compound L has at least one carboxyl (-COOH) or carboxylate (-COOH) as a functional group. - A composition according to item 6, comprising the group ) (Item 8) The linker compound L has at least one guanidino group (-NHC(=NH)NH) as a functional group 2 Or -NHC(=NH 2 + )NH 2 ) or amino group (-NH 2 Or -NH 3 + A composition according to item 6 or 7, comprising the group ) (Item 9) The linker L is -SH, -COOH, -NH 2 , -Guanidino group (-NHC(=NH))NH 2 ), -PO 3 H 2 ,-PO 2 CH 3 H, -SO 3 H, -OH, -NR 3 + X - A composition according to any one of items 6 to 8, comprising at least one further functional group L' selected from the group. (Item 10) The linker compound L is at least one structural unit of formula (I) * -(-O)3Si-(CH2)n-CH(COOX)-(CH2)pC(O)-NH-CH(COOX)-(CH2)qY(I) [In the formula, X is either H or a negative charge, independently of each other. Y is independent of each other, -NHC(=NH)NH 2 , -NHC(=NH 2 + )NH 2 , -NH 2 or -NH 3 + And, n, p, and q are independent of each other and are numbers from 0 to 25. * - is the linkage point with the aforementioned nanoparticles. A composition containing any of the items 6 to 9. (Item 11) The composition according to any of the preceding items, wherein the pharmaceutically acceptable compound is conjugated to the nanoparticles by adsorption bonding. (Item 12) The composition according to any of the preceding items, wherein the pharmaceutically acceptable compound is selected from the group comprising proteins, peptides, polynucleotides, oligonucleotides (RNA, DNA, single-stranded or double-stranded), nucleic acids, or peptide antigens. (Item 13) The composition according to any of the preceding items, wherein the pharmaceutically acceptable compound is selected from the group comprising pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), deficient infectious particles (DIPs), or epitopes. (Item 14) The composition according to item 13, wherein PAMP is selected from the group consisting of TLR agonists, retinoic acid-inducible gene I (RIG-I) ligands, melanoma differentiation-associated gene 5 (MDA-5) ligands, cytosolic DNA sensor (CDS) ligands, and STING ligands (cyclic dinucleotides (CDNs)). (Item 15) The composition according to item 12, wherein the peptide comprises a C-terminal extension having a linker that can be cleaved by the enzyme cathepsin B. (Item 16) The composition described in item 1 for use as a vaccine or immunostimulant. (Item 17) Nanoparticles having silicon dioxide and functional groups on their surface, with a particle size of less than 150 nm, comprising a linker L covalently or adsorbed to the nanoparticles, wherein the linker L has at least one guanidino group (-NHC(=NH)NH) as a functional group. 2 Or -NHC(=NH 2 + )NH 2 ) or amino group (-NH 2 Or -NH 3 + Nanoparticles containing the ) group.

Claims

1. A composition comprising nanoparticles loaded with silicon dioxide and a pharmaceutically acceptable compound having a functional group on its surface, - The functional group can support and / or stabilize both the negative and positive charges of the pharmaceutically acceptable compound, and the functional group is -COOH, -COO － , -NH ３ ＋ , -NH ２ , and the -guanidino group (-NHC(=NH)NH ２ or -NHC (=NH ２ ＋ ) NH ２ Selected from the group consisting of, - The zeta potential of the composition is 15 mV or greater, or -15 mV or lower. - The nanoparticles have a particle size of less than 150 nm. The nanoparticles further comprise a linker compound L, wherein the linker compound L is bonded to the surface of the nanoparticles at linking points. The linker compound L contains the functional group, The linker compound L is at least one structural unit of formula (I) ＊ - (-) 3Si - (CH2) n - CH (COO) - (CH2) p - C (O) - N - CH (COO) - (CH2) q - Y (I) [In the formula, X is either H or a negative charge, independently of each other. Y is independent of each other, -NHC (=NH)NH ２ , -NHC (=NH ２ ＋ ) NH ２ , -NH ２ or -NH ３ ＋ And, n, p, and q are independent of each other and are numbers from 0 to 25. ＊ — represents the connection point with the nanoparticle. A composition containing the following:

2. The composition according to claim 1, wherein the linker compound L is bonded to the surface of the nanoparticles at the linking point by covalent bonds.

3. The aforementioned nanoparticles are nm ２ The total number of pharmaceutically acceptable compounds on the surface of the nanoparticles per unit [molecules / nm] ２ The composition according to claim 2, having a maximum surface load density of 0.

5.

4. The composition according to any one of claims 1 to 3, wherein the polydispersity index (PDI) of the composition is 0 to 0.

32.

5. The composition according to any one of claims 1 to 4, wherein the zeta potential of the composition is 30 mV or greater, or -30 mV or lower.

6. The composition according to any one of claims 1 to 5, wherein the zeta potential of the composition is at least 15 mV.

7. The composition according to any one of claims 1 to 6, wherein the zeta potential of the composition is at least 30 mV.

8. The composition according to any one of claims 1 to 7, wherein the total net charge of the loaded nanoparticles is different from zero.

9. In equation (I), n is 3, p is 1, q is 4, and Y is -NH independently of each other. ２ or -NH ３ ＋ A composition according to any one of claims 1 to 8, which is the base.

10. In equation (I), n is 3, p is 1, q is 3, and Y is -NHC(=NH)NH independently of each other. ２ or -NHC (=NH ２ ＋ ) NH ２ A composition according to any one of claims 1 to 9, which is a base.

11. The composition according to any one of claims 1 to 10, wherein the pharmaceutically acceptable compound is conjugated to the nanoparticles by adsorption bonding.

12. The composition according to any one of claims 1 to 11, wherein the pharmaceutically acceptable compound is selected from the group comprising proteins, peptides, polynucleotides, oligonucleotides (RNA, DNA, single-stranded or double-stranded), nucleic acids, or peptide antigens.

13. The composition according to any one of claims 1 to 12, wherein the pharmaceutically acceptable compound is selected from the group comprising pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), deficient infectious particles (DIPs), or epitopes.

14. The composition according to claim 13, wherein PAMP is selected from the group consisting of a TLR agonist, a retinoic acid-inducible gene I (RIG-I) ligand, a melanoma differentiation-related gene 5 (MDA-5) ligand, a cytosolic DNA sensor (CDS) ligand, and a STING ligand (cyclic dinucleotide (CDN)).

15. The composition according to claim 12, wherein the peptide comprises a C-terminal extension having a linker that can be cleaved by the enzyme cathepsin B.

16. A composition according to any one of claims 1 to 15 for use as a vaccine or immunostimulant.

17. Nanoparticles for use as an immunostimulant, having silicon dioxide and functional groups on the surface and having a particle size of less than 150 nm, comprising a linker compound L covalently or adsorbed to the nanoparticles, wherein the linker compound L comprises at least one guanidino group (-NHC(=NH)NH ２ Or -NHC (=NH ２ ＋ ) NH ２ ) and at least one carboxyl (COOH) or carboxylate (COOH) － The linker compound L contains at least one structural unit of formula (I). * -(-O)3Si-(CH2)n-CH(COOX)-(CH2)p-C(O)-NH-CH(COOX)-(CH2)q-Y (I) [In the formula, X is either H or a negative charge, independently of each other. Y is mutually independent of -NHC(=NH)NH2, -NHC(=NH2+)NH2, -NH2, or -NH3+, n, p, and q are independent of each other and are numbers from 0 to 25. * - indicates a connection point with the nanoparticle. Nanoparticles containing these nanoparticles.

18. The nanoparticles according to claim 17, wherein the use is perioperative immunostimulation in humans.

19. The nanoparticles according to claim 17, wherein the use comprises administering the nanoparticles to an animal or human prior to an immunosuppressive event.

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