Amphiphilic polymers and their use to improve the production of nanoparticles for targeted delivery of antigens - Patent Application 20070122999

Abstract

The present invention provides The present invention provides nanoparticles comprising a) a micelle comprising an amphiphilic polymer having a number-average molecular weight (Mn) of 20,000 g / mol or less, and b) at least one peptide comprising at least one T-cell epitope. The invention further provides pharmaceutical compositions comprising these nanoparticles, and uses of the compositions for suppressing specific immune responses.

Description

[Technical Field]

[0001] The present invention provides nanoparticles for use in the prevention and treatment of autoimmune diseases, allergies, or other chronic inflammatory conditions, and for generating regulatory T cells. In particular, the present invention relates to nanoparticles comprising an amphiphilic polymer having a number-average molecular weight (Mn) of 20,000 g / mol or less, a micelle that renders the nanoparticle water-soluble, and at least one peptide comprising at least one T cell epitope. The present invention also relates to pharmaceutical compositions comprising the nanoparticles. The pharmaceutical compositions can be used to generate regulatory T cells specific for at least one T cell epitope in a subject to treat or prevent diseases in which suppression of a specific immune response is beneficial.

[0002] The present invention further provides a method for producing nanoparticles. [Background technology]

[0003] Autoimmune diseases pose a considerable burden to patients and healthcare systems. Current treatments rely primarily on immunosuppressive drugs, which are associated with significant side effects. Autoantigen-specific immunotherapies that target only disease-specific immunopathology, leaving the general immune status intact, represent an unmet medical need.

[0004] Immune tolerance to self-antigens is maintained by multiple mechanisms that control potentially pathogenic autoreactive lymphocytes, including deletion, clonal paralysis, or suppression by regulatory T cells. Therefore, autoimmune diseases may result from insufficient control of autoreactive lymphocytes, and a major goal of immunotherapy for autoimmune diseases is to induce tolerance to self-antigens by restoring regulation. A particularly promising approach to restoring self-tolerance is believed to be the manipulation of autoantigen-specific CD4+CD25+FOXP3+ regulatory T cells. Adoptive transfer of these cells can prevent autoimmune or inflammatory conditions.

[0005] The liver plays a central role in suppressing unwanted immune responses to blood-borne antigens that enter the circulation, such as food antigens. This fundamental mechanism of the liver can be used to specifically downregulate harmful immune responses to foreign protein antigens or self-antigens. Antigenic peptides derived from such proteins, when coupled to nanosized carriers and administered intravenously, mimic food antigens, eliciting uptake by specific liver cells, liver sinusoidal endothelial cells (LSECs), and subsequently eliciting tolerogenic immune responses. Thus, peptide-specific immune tolerance can be induced against antigens that cause defined immune disorders, potentially leading to the amelioration or even eradication of harmful immune responses. This method can therefore be used to treat ongoing diseases and also to prevent the respective diseases in a prophylactic setting.

[0006] For example, ectopic expression of neural antigens in the liver can prevent autoimmune neuroinflammation in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). This finding can be explained by the liver's ability to generate neural antigen-specific regulatory T cells (Tregs), which have a great capacity to control and suppress autoimmune responses. LSECs play a key role in achieving this effect. LSECs express MHC / HLA class I and class II molecules on their surface and therefore have the ability to present peptides to both CD8+ T cells (via cross-presentation) and CD4+ T cells, respectively. Peptide antigen presentation by LSECs converts naive and T effector cells into antigen-specific Tregs in vitro. This is clearly a physiological mechanism by which LSECs can establish tolerance to blood-borne antigens.

[0007] Like blood-borne antigens, nanoparticles conjugated with disease-specific antigenic peptides on their surface target the liver after intravenous injection. Upon uptake by LSECs, likely via pinocytosis, the nanoparticles accumulate in endosomal compartments, where the peptide antigens are released from the particle surface. This results in presentation of the antigenic peptide on the LSEC surface mediated by MHC / HLA molecules. There is evidence that the subsequent generation of Tregs confers immune tolerance specific to each autoantigen based on the antigenic peptide epitope.

[0008] WO 2009 / 067349 discloses pharmaceutical compositions comprising biocompatible nanoparticles linked to aryl hydrocarbon receptor (AHR) transcription factor ligands for use in the treatment of autoimmune disorders by increasing the number and / or activity of regulatory T cells.

[0009] WO 2013 / 072051 discloses a pharmaceutical composition for use in generating regulatory T cells specific for at least one T cell epitope in a subject to treat or prevent a disease in which suppression of a specific immune response is beneficial. The nanoparticles comprise micelles containing an amphiphilic polymer that renders the nanoparticles water-soluble, and a peptide containing at least one T cell epitope associated with the exterior of the micelles. It is generally proposed to use commercially available poly(maleic anhydride-alt-1-octadecene) as the amphiphilic polymer, with a molecular weight of 30,000 to 50,000 g / mol and a purity of approximately 90%.

[0010] However, for certain medical applications it is important to be able to produce nanoparticles with very high purity using efficient purification methods.

[0011] There remains a need in the art for improved nanoparticles for treating and preventing diseases in which suppression of a specific immune response is beneficial, such as autoimmune diseases, allergies, transplants, suppression of anti-drug antibodies (ADA) against therapeutic agents or gene vectors, or diseases in which inflammation is excessive, chronic, or harmful, and wherein the pharmaceutical compositions are suitable for use in human subjects. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2009 / 067349 [Patent Document 2] International Publication No. 2013 / 072051 Summary of the Invention

[0013] According to the present invention, the above problem is solved by: a) a micelle comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, and b) This is solved by nanoparticles comprising at least one peptide containing at least one T-cell epitope.

[0014] The present inventors have surprisingly found that nanoparticles comprising amphiphilic polymers with number average molecular weights (Mn) of up to 20,000 g / mol can be produced more easily and with higher purity.

[0015] Without being bound by theory underlying the efficacy of the nanoparticles of the present invention, it is currently understood that upon uptake of the nanoparticles by LSECs, at least one peptide associated with the exterior of the micelle is released either by hydrolysis, proteolysis, or other activity in the endosome, where it is processed as if it were a blood-borne antigen and presented to T cells in a tolerogenic environment. Low molecular weight amphiphilic polymers can excrete individual polymer molecules upon in vivo release. This provides hepatobiliary excretion, which appears to represent a preferred route of excretion for nanoparticles.

[0016] Surprisingly, we found that low-molecular-weight amphiphilic polymers with number-average molecular weights (Mn) of 20,000 g / mol or less can be readily excreted, and we also predicted that low-molecular-weight amphiphilic polymers and their metabolites would be rapidly eliminated from the body after treatment.

[0017] The present inventors have surprisingly found that low molecular weight amphiphilic polymers have advantageous properties during the production of the nanoparticles of the present invention. Low molecular weight amphiphilic polymers produce fewer aggregates during the coating of the solid core than high molecular weight amphiphilic polymers. Furthermore, low molecular weight amphiphilic polymers can be purified more efficiently compared to high molecular weight amphiphilic polymers. In particular, the use of low molecular weight amphiphilic polymers in the nanoparticles of the present invention allows for more efficient separation of unbound polymers.

[0018] The present invention further provides a pharmaceutical composition comprising the nanoparticles.

[0019] The present invention also provides a pharmaceutical composition comprising nanoparticles for use in generating regulatory T cells specific for at least one T cell epitope in a subject to treat or prevent a disease in which suppression of a specific immune response is beneficial.

[0020] Finally, the present invention provides i) obtaining an amphiphilic polymer with a number average molecular weight (Mn) of 20,000 g / mol or less; ii) optionally purifying the amphiphilic polymer; iii) forming micelles of amphiphilic polymers; iv) providing a method for producing nanoparticles, the method comprising adding at least one peptide to form nanoparticles. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a flow chart showing the synthesis of iron oleate complex. [Figure 2] 1 is a flow chart showing the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs). [Figure 3] 1 is a flow chart showing the synthesis of low molecular weight poly(maleic anhydride-alt-1-octadecene) (LM-PMAOD). [Figure 4]FIG. 1 is a flow chart showing the synthesis of low molecular weight poly(maleic acid-alt-1-octadecene) (LM-PMAcOD). [Figure 5] 1 is a flowchart showing polymer coating of SPIONs. [Figure 6] 1 is a flow chart illustrating coupling of peptides to nanoparticles of the present invention. [Figure 7] FIG. 1 shows a transmission electron microscope (TEM) image (100x magnification) of nanoparticles of the present invention. [Figure 8] 1 is a graph showing the molecular mass distribution of LM-PMAcOD determined using gel permeation chromatography and polystyrene as a calibration standard. [Figure 9] FIG. 1 shows SEC chromatograms of purified HM-PMAcOD-SPION samples. [Figure 10] FIG. 1 shows SEC chromatograms of purified HM-PMAcOD-SPION samples. [Figure 11] FIG. 1 shows SEC chromatograms of purified HM-PMAcOD-SPION samples. [Figure 12] FIG. 1 shows the results of size exclusion chromatography of purified LM-PMAcOD-SPION samples. [Figure 13] FIG. 1 shows the SEC chromatogram of the product after coating. DETAILED DESCRIPTION OF THE INVENTION

[0022] According to the present invention, there is provided a nanoparticle comprising an amphiphilic polymer having a number-average molecular weight (Mn) of 20,000 g / mol or less, a micelle that renders the nanoparticle water-soluble, and at least one peptide that contains at least one T-cell epitope. The nanoparticle may further comprise a solid hydrophobic core coated with the micelle.

[0023] According to the present application, the term "nanoparticles" is used interchangeably with "nanoscale particles". Such particles have a diameter of 1 to 999 nm, preferably 2 to 600 nm, 5 to 500 nm, 10 to 300 nm, 30 to 100 nm or 40 to 50 nm.

[0024] In the context of the present invention, a nanoparticle is a structure formed by at least a micelle and a peptide associated with the micelle. The peptide may be associated with the outside of the micelle or encapsulated inside the micelle.

[0025] In one embodiment of the present invention, the nanoparticles of the present invention comprise a solid hydrophobic core, a micelle coating the core comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, rendering the nanoparticle water soluble, and at least one peptide comprising at least one T-cell epitope.

[0026] Micelle In the context of the present invention, the term "micelle" refers to an aggregate of amphiphilic molecules dispersed in an aqueous solution. The hydrophilic portion of the amphiphilic molecule is in contact with the surrounding solvent, sequestering the hydrophobic "tail" region of the amphiphilic molecule inside the micelle, thus making the nanoparticle water-soluble. This type of micelle is also known as a normal-phase micelle (or oil-in-water micelle).

[0027] Micelles can be formed by not only one but also two or more, for example, two, three, or four, amphiphilic polymer molecules. Micelles can be formed by the same or different amphiphilic polymer molecules. In general, in the context of this specification, "a" or "the" is not intended to be limiting to "one" unless otherwise specified.

[0028] In a preferred embodiment, the micelles are formed by a monolayer of amphiphilic polymers.

[0029] Such micelles may be structurally different from bilayers or liposomes formed by amphiphilic polymers, where the structure is not included in the nanoparticles of the present invention or is not included to a significant extent (e.g., 10% or less, more than 5%, or preferably more than 1%).

[0030] In one embodiment of the present invention, the amphiphilic polymer is used to generate at least 70%, preferably at least 90% of the micelles. In a preferred embodiment, the micelles consist of the amphiphilic polymer.

[0031] In some embodiments of the present invention, the nanoparticles do not comprise a solid hydrophobic core, hi other embodiments, the nanoparticles comprise micelles and a solid hydrophobic core.

[0032] Methods for producing the nanoparticles of the present invention are described in detail below.

[0033] amphiphilic polymers The amphiphilic polymers of the present invention generally comprise a hydrophobic region comprising a hydrophobic aliphatic chain having a length of from 8 to 23, preferably from 8 to 21, and most preferably from 16 to 18 carbon atoms.

[0034] The hydrophilic regions of the amphiphilic polymer may be negatively charged in aqueous solution.

[0035] In a preferred embodiment of the present invention, the amphiphilic polymer spontaneously forms micelles in solution: if a solid hydrophobic core is present, the amphiphilic polymer forms micelles around the solid core, making the nanoparticles water soluble.

[0036] The number average molecular weight (Mn) of the amphiphilic polymer is 20,000 g / mol or less, preferably 10,000 g / mol or less, or 6,000 g / mol or less, more preferably 6,000 to 1,000 g / mol, and most preferably 3,000 to 6,000 g / mol.

[0037] Number average molecular weight can be determined using gel permeation chromatography (GPC), preferably using polystyrene as a calibration standard.

[0038] In a preferred embodiment, the number average molecular weight is determined using a PL gel mixed D column at a temperature of 40°C, a mobile phase consisting of 90 / 10% (v / v) tetrahydrofuran / acetic acid, a flow rate of 1.0 ml / min, a refractive index detector at a temperature of 35°C, and polystyrene as a calibration standard.

[0039] In the most preferred embodiment, the number average molecular weight is determined using GPC and the following measurement conditions: TIFF0007756096000001.tif64133

[0040] The amphiphilic polymer may be an alternating copolymer, which is a copolymer containing two types of monomer units distributed in an alternating sequence.

[0041] In one embodiment of the present invention, the amphiphilic polymer is a copolymer of maleic anhydride and at least one alkene.

[0042] The alkene used to form the amphiphilic polymer may be selected from one or more of 1-decene, 1-undecene, 1-dodecene, 1-tridecene, 1-tetradecene, 1-pentadecene, 1-hexadecene, 1-heptadecene, 1-octadecene, 1-nonadecene or 1-eicosene, preferably the alkene is 1-octadecene.

[0043] In a preferred embodiment of the present invention, the amphiphilic polymer is a copolymer of maleic anhydride and an alkene.

[0044] In a preferred embodiment of the present invention, the amphiphilic polymer has a main hydrophilic polymaleic anhydride backbone with hydrophobic alkyl side chains. Typically, the side chains can have 5 to 23 carbon atoms, especially 9 to 21 carbon atoms. In the most preferred embodiment, the side chains are linear and have 10 to 18 carbon atoms.

[0045] The amphiphilic polymer may comprise the following building blocks: TIFF0007756096000002.tif21159In the formula, R is a hydrocarbyl group or a substituted hydrocarbyl group. In a preferred embodiment of the present invention, R is a C4-C 22 Alkyl groups, such as C7-C 19 It is an alkyl group.

[0046] In an even more preferred embodiment, R is a straight chain alkyl group, preferably a straight chain C-C 17 Preferably, R is an alkyl group, most preferably R is a straight chain pentadecyl group or a straight chain nonyl group.

[0047] The amphiphilic polymer may consist of the building blocks defined above.

[0048] In another embodiment according to the present invention, the amphiphilic polymer comprises at least 50%, preferably at least 70%, most preferably more than 90% of building blocks as defined above.

[0049] In a preferred embodiment, the amphiphilic polymer is selected from the group comprising poly(maleic acid-1-octadecene), poly(maleic acid-1-tetradecene) or poly(maleic acid-1-dodecene), preferably the polymer is poly(maleic acid-1-octadecene) and the number average molecular weight of the polymer is 6,000 to 1,000 g / mol.

[0050] In a particularly preferred embodiment, the amphiphilic polymer is selected from the group comprising poly(maleic acid-alt-1-octadecene), poly(maleic acid-alt-1-dodecene) and poly(maleic acid-alt-1-tetradecene), preferably the polymer is poly(maleic acid-alt-1-octadecene) and the number average molecular weight of the polymer is between 5000 and 1000 g / mol.

[0051] Methods for producing the amphiphilic polymers of the present invention are also described in detail below.

[0052] peptide The nanoparticles of the present invention further comprise at least one peptide comprising at least one T-cell epitope. The peptide may be associated with the exterior of the micelle (in embodiments in which the nanoparticles of the present invention do not have a solid hydrophobic core) or may be encapsulated within the micelle. Thus, the peptide may be located either on the exterior or the interior of the micelle.

[0053] The peptide may be covalently or non-covalently bound to the micelle, preferably covalently bound to the micelle.

[0054] In a preferred embodiment, the peptides are covalently attached to the micelles using methods known in the art for covalently coupling peptides, such as carbodiimide or succinimide coupling. Preferably, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry is used to covalently attach the peptides to the micelles.

[0055] In the context of the present invention, the term "peptide" is not intended to be limited in size, and in particular, a peptide may comprise a whole protein or 8 to 2,000 amino acids, preferably 8 to 200 amino acids, 8 to 100 amino acids, 9 to 60 amino acids, or 10 to 20 amino acids. The term also includes combinations of different peptides that can be linked together as a fusion polypeptide.

[0056] In a preferred embodiment of the invention, the peptide comprises 10 to 20, for example 13 to 17, amino acids.

[0057] In a particularly preferred embodiment, the peptide comprises 15 amino acids.

[0058] The peptide contains at least one T cell epitope. Methods for identifying T cell epitopes and selected T cell epitopes are known in the art and are described, for example, in the publications of Rammense et al., 1999 (Immunogenetics 50 213-219) and Sanchez-Trincado et al., 2017 (J Immunol Res. 2017:2680160. doi:10.1155 / 2017 / 2680160. Epub 2017 Dec 28). In the context of the present invention, a T cell epitope is a peptide sequence that induces regulatory T cells. At least one epitope must be capable of being presented by cells of the subject to which the nanoparticles are administered. Preferably, the peptide contains several epitopes that allow it to be presented by multiple major histocompatibility complex types.

[0059] Since regulatory T cells are primarily CD4+, presentation on MHC class II is of primary interest. The HLA type of the subject, e.g., a human subject, can be readily tested as part of epitope selection. Specific peptide epitopes that can be presented on specific MHC molecules are known and / or can be routinely selected, e.g., by appropriate software.

[0060] Peptides are designed based on published data to associate with specific HLA restriction elements to ensure high affinity binding to MHC / HLA and high T cell stimulation and activation. Ideally, preferred peptides are predicted from naturally processed peptides and characterized as immunodominant.

[0061] The peptides may be synthesized, recombinantly expressed, or isolated or modified from natural sources. The peptides, or at least the epitopes, from which regulatory T cells are generated are preferably derived from peptides / proteins that suppress inflammatory immune responses, for example in the context of treating or preventing autoimmune diseases or allergies. The peptides may be, for example, allergens, known autoimmune antigens, or fragments or derivatives thereof. The peptides may combine different epitopes from different antigens.

[0062] In a preferred embodiment of the present invention, the peptide is an antigenic peptide derived from desmoglein-3 (Dsg3), for example, the peptide may be one or more desmoglein-3 peptides characterized by SEQ ID NOs: 1 to 3.

[0063] According to one embodiment of the present invention, the nanoparticles contain only one type of peptide comprising at least one T-cell epitope.

[0064] According to further embodiments, the present invention provides a composition comprising different nanoparticles, each nanoparticle comprising multiple peptides having the same amino acid sequence, but the composition comprising a mixture of nanoparticles in which the peptide sequences differ from one another. The composition can comprise, for example, different nanoparticles comprising 2 to 6 different peptides. In one aspect, the composition of different nanoparticles can comprise three different types of nanoparticles, each characterized by one of SEQ ID NOS: 1 to 3.

[0065] solid hydrophobic core In one embodiment of the present invention, the nanoparticles comprise a solid hydrophobic core at least partially coated with micelles.

[0066] The core may be an inorganic core, preferably comprising iron oxide, CdSe, silver or gold.

[0067] The core diameter may be 2 nm to 500 nm, preferably 3 nm to 25 nm, and more preferably 5 nm to 15 nm. The core diameter can be determined using a transmission electron microscope (TEM) or small angle X-ray scattering (SAXS).

[0068] An exemplary inorganic core is oleic acid or another carboxylic acid (C 14 ~C 22 , preferably C 16 ~C 18 ), quantum dots (e.g., CdSe / CdS / ZnS stabilized by trioctyloxine phosphinoxide), and gold nanoparticles stabilized by, for example, sulfone compounds.

[0069] Although such inorganic cores themselves are typically not stable in aqueous solvents such as water, embedding them in polymer micelles makes them water-soluble. The hydrophobic portion of the amphiphilic polymer interacts with the hydrophobic core of the nanoparticle, resulting in the formation of a single coating layer of polymer surrounding the core. During the coating process, the amphiphilic polymer can replace the hydrophobic portion of the core through ligand exchange, thus forming bilayer micelles around the core. In one embodiment of the present invention, the polymer at least partially replaces the oleic acid on the surface of the core particle, the hydrophilic portion of the polymer interacts with the surface of the iron oxide core, and the hydrophobic portions of the polymer interact with each other to form bilayer micelles around the iron oxide core, resulting in polymer-coated iron oxide.

[0070] According to a preferred embodiment of the present invention, the core is superparamagnetic.

[0071] In a particularly preferred embodiment of the present invention, the core is a superparamagnetic iron oxide nanoparticle (SPION), which can be stabilized by oleic acid.

[0072] The core preferably makes the nanoparticles of the invention traceable, for example by their characteristics in fluorescence, electron microscopy or other detection methods.

[0073] nanoparticles The inventors have found that the nanoparticles for use in the present invention are suitable for transferring peptides into liver sinusoidal endothelial cells of a subject in vivo.

[0074] The nanoparticles can further comprise moieties, such as carbohydrates or proteins, that target them or enhance targeting to specific cells, such as liver sinusoidal endothelial cells and / or Kupffer cells. Such moieties can enhance or accelerate uptake from the circulation, for example, via receptor-mediated endocytosis. An example of a suitable modification is a carbohydrate, such as mannose.

[0075] The nanoparticles may be negatively charged or uncharged depending on the polymer that forms the micelle, and preferably the nanoparticles are negatively charged at a pH of 6 to 7. The polymer coating may contain an acid, e.g., a carboxylic acid group, that results in a negative charge on the nanoparticles.

[0076] The nanoparticles of the present invention may have a zeta potential of -20 to -50 mV, preferably -25 to -45 mV, more preferably -28 to -42 mV at a pH of 6 to 7 (pH during measurement). The zeta potential may be measured using a Malvern Zetasizer Nano ZS instrument.

[0077] The nanoparticles of the present invention can have a hydrodynamic diameter (z average) of 10 to 100 nm or 10 to 70, preferably 10 to 50, more preferably 20 to 40 nm, and most preferably 22 to 32 nm, as measured by dynamic light scattering (DLS).

[0078] The nanoparticles of the present invention may have a polydispersity index, as measured by dynamic light scattering (DLS), of less than 0.50, preferably between 0.05 and 0.45, more preferably between 0.10 and 0.40.

[0079] The determination of hydrodynamic diameter and polydispersity index is performed using electrophoretic light scattering analysis, preferably a Malvern Zetasizer. In one embodiment, the method for determining hydrodynamic diameter and polydispersity index is performed using electrophoretic light scattering, a disposable polystyrene cuvette, Zetasizer Software 7.12, and Milli-Q water. 20 nm and 100 nm nanosphere diameter standards (NIST certified or equivalent) are diluted with 0.9% aqueous sodium chloride solution, and test samples are diluted with water. All aqueous reagents are filtered through a 0.22 μm membrane before use. In the most preferred embodiment of the present invention, the method for determining hydrodynamic diameter and polydispersity index is performed using electrophoretic light scattering in combination with the following analytical conditions: TIFF0007756096000003.tif67133

[0080] The evaluation of the data is based on the mean diameter (Z-average, nm by intensity), a parameter also known in DLS as the cumulant average, and the polydispersity index (PDI), used as a measure of the size distribution.

[0081] Furthermore, the nanoparticles of the present invention can have a total polymer content of 0.1 to 5 mg / mL, preferably 0.5 to 4 mg / mL, and more preferably 1 to 3 mg / mL. The total polymer content is determined by GPC. To measure the total polymer content, the peptide is hydrolyzed and the particles are disrupted (e.g., using a 6 M HCl solution). The polymer is extracted after the addition of EDTA. After evaporation of the solvent, the residue is redissolved, and the polymer content is determined by GPC.

[0082] The determination of the total polymer content is preferably carried out using the following reagents and reference standards: water (HPLC grade), acetonitrile (HPLC grade), tetrahydrofuran with BHT (THF-HPLC grade), 100% acetic acid (analytical grade), 37% hydrochloric acid (analytical grade), ethylenediaminetetraacetic acid disodium salt dihydrate (analytical grade), ethyl acetate (analytical grade), sodium hydroxide (analytical grade) and poly(maleic acid-alt-1-octadecene) as reference material.

[0083] The chromatographic conditions for determining the total polymer content are as follows: TIFF0007756096000004.tif61133

[0084] In a particularly preferred embodiment of the present invention, the nanoparticles comprise an iron oxide core encapsulated by a coating of poly(maleic acid-alt-1-octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol or less, and a peptide, preferably comprising at least one T-cell epitope peptide, covalently attached to the micelle.

[0085] Thus, in one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) A micelle comprising an amphiphilic polymer comprising the following building blocks: TIFF0007756096000005.tif20159 wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear C 11 ~C 17 A micelle in which the alkyl group is an alkyl group and the number average molecular weight (Mn) of the polymer is 6,000 to 1,000 g / mol. b) at least one peptide comprising at least one T cell epitope, and c) providing nanoparticles comprising a solid hydrophobic core at least partially coated by micelles, the solid hydrophobic core comprising a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0086] The number average molecular weight of the polymer is preferably 6,000 to 1,000 g / mol.

[0087] The nanoparticles preferably have a hydrodynamic diameter of between 50 nm and 10 nm as measured by dynamic light scattering.

[0088] Pharmaceutical Composition The present invention further provides a pharmaceutical composition comprising the nanoparticles of the present invention.

[0089] The peptides used may be present in the pharmaceutical composition at a concentration of 0.01 to 2 mM, preferably 0.1 to 1 mM, and most preferably 0.45 to 1 mM.

[0090] In a preferred embodiment, the amount of free (unbound) polymer in the composition is less than 10%, preferably less than 5%, and most preferably less than 2% of the total amount of polymer.

[0091] The pharmaceutical composition of the present invention may further comprise at least one suitable excipient and / or diluent. The diluent is preferably water or an aqueous solution, such as a buffer solution such as phosphate-buffered saline (PBS), Ringer's solution, TRIS buffer, or sodium chloride solution. Suitable preservatives may or may not be present.

[0092] It will be appreciated that, particularly for administration to human subjects, the compositions are preferably sterile and biologically compatible.

[0093] In a preferred embodiment of the invention, the pharmaceutical composition comprises nanoparticles of the invention dispersed in D-mannitol, TRIS and / or L-lactic acid.

[0094] In one embodiment of the present invention, the pharmaceutical composition comprises nanoparticles of the present invention that do not contain a solid hydrophobic core, the nanoparticles being dispersed in D-mannitol, TRIS and / or L-lactic acid. The use of this buffer has the advantage that the particles are very stable in this buffer and can be subsequently lyophilized.

[0095] Furthermore, pharmaceutical compositions can contain two or more types of nanoparticles of the present invention, with different types of nanoparticles having different peptides associated with the outside of the micelles. By using a mixture of nanoparticles, broader immune tolerance can be induced by several autoantigenic peptides simultaneously. These peptides may be derived from a single immunogenic protein or from different proteins.

[0096] In a preferred embodiment of the present invention, the pharmaceutical composition comprises 2 to 6 different types of nanoparticles, and preferably the associated peptides of all of the different types of nanoparticles comprise at least one T-cell epitope.

[0097] In a particularly preferred embodiment of the present invention, the pharmaceutical composition comprises three to four different types of nanoparticles, and the associated peptides of all the different types of nanoparticles contain at least one T cell epitope. In particular, each nanoparticle can be associated with a different antigenic peptide derived from Dsg3.

[0098] The pharmaceutical composition can contain nanoparticles at a concentration of less than 100 μM, preferably 0.5 to 80 μM, and most preferably 1 to 50 μM. When two or more types of nanoparticles are present in the pharmaceutical composition, each can be present at a concentration of less than 100 μM, preferably 0.5 to 80 μM, and more preferably 1 to 50 μM.

[0099] The pharmaceutical composition of the present invention may contain different types of nanoparticles in equimolar concentrations.

[0100] Thus, in one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) A micelle comprising an amphiphilic polymer comprising the following building blocks: TIFF0007756096000006.tif20159 wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear C 11 ~C 17A micelle in which the alkyl group is an alkyl group and the number average molecular weight (Mn) of the polymer is 6,000 to 1,000 g / mol. b) at least one peptide comprising at least one T cell epitope, and c) providing nanoparticles comprising a solid hydrophobic core at least partially coated by micelles, the solid hydrophobic core comprising a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0101] The number average molecular weight of the polymer in the pharmaceutical composition is preferably in the range of 6,000 to 1,000 g / mol.

[0102] The nanoparticles in the pharmaceutical composition of the present invention preferably have a hydrodynamic diameter of between 50 nm and 10 nm, as measured by dynamic light scattering.

[0103] In a preferred embodiment, the present invention provides a pharmaceutical composition comprising at least three different types of nanoparticles, each nanoparticle comprising: a) A micelle comprising an amphiphilic polymer comprising the following building blocks: TIFF0007756096000007.tif21159 wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear C 11 ~C 17 A micelle in which the alkyl group is an alkyl group and the number average molecular weight (Mn) of the polymer is 6,000 to 1,000 g / mol. b) one peptide containing one T cell epitope, and c) a solid hydrophobic core at least partially coated by micelles, the solid hydrophobic core comprising a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold; The three different types of nanoparticles differ from each other in their peptide sequences: the first type of nanoparticles contains a peptide having SEQ ID NO: 1, the second type of nanoparticles contains a peptide having SEQ ID NO: 2, and the third type of nanoparticles contains a peptide having SEQ ID NO: 3.

[0104] Medical Use The pharmaceutical compositions of the present invention are intended and formulated for use in administration to a subject having a disease in which suppression of a specific immune response would be beneficial.

[0105] The pharmaceutical composition can be administered to a subject in need thereof.

[0106] The dosage and concentration required for administration to a subject can be determined by the attending medical professional depending on the facts and circumstances of the case. Exemplary dosages, for example, for a human subject, can include 0.03 μmol to 0.90 μmol per patient body weight.

[0107] The administration may be repeated, for example, 2, 3 or 4 times, for example, with an interval of 1, 2, 3, 4, 5, 6, 7, 10 or 14 days between administrations.

[0108] Preferably, the pharmaceutical composition is for use in suppressing a specific immune response, such as treating or preventing a disease in which suppression of a specific immune response is beneficial. More preferably, the pharmaceutical composition is for use in generating regulatory T cells specific for at least one T cell epitope in a subject to treat or prevent a disease in which suppression of a specific immune response is beneficial.

[0109] The disease may be an autoimmune disease associated with a defined autoantigen. In the context of the present invention, the term "autoimmune disease" is understood as defined by Hayter et al. (Autoimmunity Reviews 11 (2012) 754-765). In a preferred embodiment, the autoimmune disease is selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, epidermolysis bullosa acquisita, bullous pemphigoid, cicatricial pemphigoid, Goodpasture's syndrome, microscopic polyangiitis, granulomatosis with polyangiitis (granulomatous, Wegener's), thrombotic thrombocytopenic purpura, immune thrombocytopenic purpura, uveitis, HLA-B27-associated acute anterior uveitis, multiple sclerosis, neuromyelitis optica, type 1 diabetes, narcolepsy with or without cataplexy, celiac disease, dermatitis herpetiformis, allergic airway disease / asthma, myasthenia gravis, Hashimoto's thyroiditis, autoimmune thyroid disease, Graves' disease, autoimmune thyroid disease, autoimmune hypoparathyroidism, autoimmune thyroiditis, and autoimmune vasculitis. The autoimmune disease is selected from the group consisting of immune-mediated thyroid disease, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, autoimmune neutropenia, linear localized scleroderma, Batten disease, acquired hemophilia A, relapsing polychondritis, Isaacs syndrome (acquired neuromyotonia), Rasmussen encephalitis, Morvan syndrome, stiff-person syndrome, pernicious anemia, Vogt-Koyanagi-Harada syndrome, primary biliary cirrhosis, autoimmune hepatitis type I, autoimmune hepatitis type II, systemic lupus erythematosus, rheumatoid arthritis, polymyositis / dermatomyositis, Sjogren's syndrome, scleroderma, vitiligo and alopecia areata.

[0110] More preferably, the autoimmune disease is selected from the group comprising pemphigus vulgaris, Goodpasture's syndrome, microscopic polyangiitis, thrombotic thrombocytopenic purpura, multiple sclerosis, neuromyelitis optica, type I diabetes, narcolepsy with or without cataplexy, celiac disease, autoimmune Addison's disease, autoimmune hemolytic anemia and acquired hemophilia A.

[0111] According to the present invention, the term "treating" is used to refer to the alleviation of symptoms of a particular disease in a subject and / or the improvement of an identifiable measurement associated with a particular disorder.

[0112] Amphiphilic polymers and methods for producing nanoparticles Methods for producing amphiphilic polymers and nanoparticles containing same are provided in Examples 1-4.

[0113] One method for obtaining amphiphilic polymers with number average molecular weights (Mn) of 20,000 g / mol or less is to synthesize them using a two-step process that involves forming the anhydride form of the polymer and hydrolyzing the anhydride to obtain the acid form. The process for hydrolyzing the anhydride form of the polymer to obtain the acid form can be exemplified as follows: TIFF0007756096000008.tif18159

[0114] The present invention also provides i) obtaining an amphiphilic polymer with a number average molecular weight (Mn) of 20,000 g / mol or less; ii) optionally purifying the amphiphilic polymer; iii) forming micelles of amphiphilic polymers; and iv) providing a method for producing nanoparticles, the method comprising adding at least one peptide to form nanoparticles.

[0115] In embodiments of the invention in which the peptide is encapsulated by a micelle, step iv) is performed before step iii). In these embodiments, the peptide is added to the amphiphilic polymer prior to micelle formation.

[0116] The amphiphilic polymers used in the nanoparticles of the present invention can be prepared by radical copolymerization using a radical initiator (step i).

[0117] The molecular weight of the polymer can be controlled by varying the concentration of reactants or the amount of radical initiator. The molecular weight of the polymer can be analyzed by gel permeation chromatography.

[0118] The copolymerization can be carried out in an organic solvent such as 1,4-dioxane, xylene or chlorobenzene.

[0119] Many radical initiators are known in the art, including various peroxides and azo compounds. Examples of suitable peroxides include benzoyl peroxide, lauryl peroxide, di-t-butyl peroxide, 2,4-dichlorobenzyl peroxide, t-butyl hydroperoxide, cumene hydroperoxide, diacetyl peroxide, diethyl peroxycarbonate, t-butyl perbenzoate, and perborate. Suitable azo compounds include 2,2'-azobis(2-methylpropionitrile), p-bromobenzenediazonium fluoroborate, p-tolyldiazoaminobenzene, p-bromobenzenediazonium hydroxide, azomethane, and phenyldiazonium halide. Preferably, the radical initiator is 2,2'-azobis(2-methylpropionitrile).

[0120] The copolymerization can be carried out at a high temperature of 70 to 120°C, preferably 90 to 110°C.

[0121] Preferably, the copolymerization is initiated by heating the mixture to 70 to 120°C, preferably 90 to 110°C.

[0122] In a preferred embodiment of the method of the present invention, step i) comprises mixing the reactants, deoxygenating the mixture, heating the mixture, and then cooling the mixture. The polymer is then dissolved and allowed to stir overnight. The solid formed can be recovered, preferably using centrifugation.

[0123] In a preferred embodiment of the method of the present invention, step ii) comprises the addition of a base (e.g., NaOH) to the polymer. Preferably, the base is reacted with the polymer at elevated temperature, preferably 50-70°C, such as 60°C, until almost all solids are dissolved. The resulting suspension can be acidified (e.g., pH <2). The reaction mixture can then be extracted with an organic solvent, such as ethyl acetate. The organic layer can be extracted with sodium hydroxide solution. After further extraction with an organic solvent, such as ethyl acetate, the purified amphiphilic polymer can be obtained by drying.

[0124] The polymer can be further purified (step iii). Preferably, the polymer is further purified by extracting it with n-hexane or n-heptane. The extraction can be carried out at a concentration of more than 10 g / L, preferably 100 g / L.

[0125] Furthermore, an additional purification step of the amphiphilic polymer can be added between steps i) and ii). In this additional purification step, the crude reaction product of the polymerization is dissolved and precipitated. In a preferred embodiment, the solvent is dichloromethane, and the polymer is precipitated using a mixture of methanol / heptane or acetonitrile / isopropanol. The mixture used can contain, for example, 95 / 5% (v / v%) methanol / heptane, 10 / 90 (v / v%) acetonitrile / isopropanol, or 5 / 95 (v / v%) acetonitrile / isopropanol. In a preferred embodiment, the precipitation mixture is added at a temperature of -10 to 10°C, preferably -5 to 5°C.

[0126] The purity of the amphiphilic polymer after hydrolysis and post-treatment is 1 It can be measured by 1 H NMR.

[0127] In the method of the present invention, micelles are formed by forming a solution containing an amphiphilic polymer. Preferably, the micelles are formed in an aqueous solution. A co-stabilizer can be added to the amphiphilic polymer to improve micelle formation.

[0128] The peptides used in step iv) can be synthesized using state-of-the-art solid phase chemistry.

[0129] The peptide may be covalently or non-covalently attached to the micelle.

[0130] In a preferred embodiment of the method of the present invention, the peptides are coupled to the surface of the nanoparticles using peptide coupling techniques known in the art, such as carbodiimide or succinimide coupling.

[0131] In a particularly preferred embodiment of the method of the present invention, peptides are coupled to the surface of nanoparticles via EDC chemistry (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in aqueous phase.

[0132] The resulting nanoparticles can be purified using extensive washing and filtration steps to remove the coupling reagent and any low molecular weight components.

[0133] In a preferred embodiment, the method for producing nanoparticles comprises: a) obtaining hydrophobic core nanoparticles; b) obtaining amphiphilic polymers with a number average molecular weight (Mn) of less than or equal to 20,000 g / mol, preferably using radical copolymerization; c) optionally purifying the amphiphilic polymer; d) mixing hydrophobic core nanoparticles with amphiphilic polymers to form micelles; e) adding at least one peptide to form nanoparticles.

[0134] The above discussion of steps i) to iii) and iv) also applies to steps b) to d) and e), respectively.

[0135] The hydrophobic core of step a) can be synthesized using appropriate reactants in solution. In a preferred embodiment of the method of the present invention, the hydrophobic core can be synthesized using metal salts and salts of carboxylic acids as reactants in the presence of an organic solvent. Preferably, the reaction is carried out at elevated temperatures under oxygen limitation.

[0136] Micelles can be formed by disposing the amphiphilic copolymer around the core in step e). Preferably, step d) comprises the substeps of solubilizing the amphiphilic polymer and core particles, removing the solvent until a thin film is formed, adding an aqueous basic solution at elevated temperature and ambient pressure to form an aqueous colloidal dispersion, diluting the solution, and optionally filtering the solution. Several washing steps can then be applied. [Example]

[0137] The invention is illustrated by the following examples which describe in detail the synthesis of nanoparticles according to the invention.

[0138] These examples should not be considered as limiting the scope of the invention, but as being illustrative of the invention.

[0139] Example 1: Preparation of Superparamagnetic Iron Oxide Crystalline Cores (SPIONs) The synthesis of iron oleate complex is shown schematically in Figure 1.

[0140] In the first step, iron oleate complex was synthesized by mixing oleic acid, sodium hydroxide, and iron chloride under reflux at 70 °C. The product was purified by several washing steps in a separatory funnel, then dried over sodium sulfate and concentrated on a rotary evaporator, yielding the iron oxide crystalline core (Figure 1).

[0141] In the second step (Figure 2), the iron oleate complex was dissolved in 1-octadecene at room temperature and stirred until completely dissolved. Then, oleic acid was added, and the mixture was deoxygenated and heated at 300 °C for 3 h to form iron oxide nanocrystals.

[0142] After cooling, the product was purified by magnetic separation and several washing steps using acetone / tetrahydrofuran (THF). The purified SPIONs were diluted with chloroform and concentrated on a rotary evaporator to obtain SPIONs with a narrow size distribution and good crystallinity.

[0143] Example 2: Preparation of low molecular weight poly(maleic acid-alt-1-octadecene) (LM-PMAcOD) The synthesis of low molecular weight poly(maleic acid-alt-1-octadecene) (LM-PMAcOD) was achieved in a two-step process, as shown schematically in Figures 3 and 4.

[0144] In the first step, maleic anhydride (48.9 mmol) and 1-octadecene (48.2 mmol) were dissolved in 10 ml of 1,4-dioxane (the inhibitor in 1,4-dioxane was previously removed by filtration through 3 g of aluminum oxide). Then, 5.79 mmol of AIBN (2,2'-azobis(2-methylpropionitrile)) was added. The flask was equipped with a condenser, followed by a nitrogen flush, maintaining a nitrogen overpressure. The mixture was heated to 100 °C with stirring. One hour after heating began, the heating plate was removed along with the stopper, exposing the reaction to air. The mixture was allowed to cool to room temperature with stirring for 2 days. The product was purified by coevaporation with dichloromethane and precipitation with isopropanol and acetonitrile. Low molecular weight poly(maleic anhydride-alt-1-octadecene) (LM-PMAOD) with number average molecular weight (Mn) of 2,500-4,000 g / mol was obtained.

[0145] In the second step, LM-PMAOD was hydrolyzed to poly(maleic acid-alt-1-octadecene) (LM-PMAcOD) in sodium hydroxide solution. To purify the product and remove impurities such as residual 1-octadecene, acid-base extraction with H2SO4, ethyl acetate, and NaOH was performed. The product was dried over magnesium sulfate, coevaporated with chloroform, and finally purified by solid-liquid extraction in n-heptane (Figure 4).

[0146] The number-average molar mass (Mn) and weight-average molar mass (Mw) of the resulting polymer were determined by gel permeation chromatography.

[0147] A 1.5 mg sample of LM-PMAcOD was dissolved in 1.0 mL of THF (without stabilizer) and subjected to GPC. Polystyrene was used as a calibration standard. Tetrahydrofuran was used as the eluent, and the flow rate was 1 mL / min. The temperature was set at 30°C.

[0148] The number-average molar mass (Mn) of the resulting LM-PMAcOD was 1540 g / mol, and the mass-average molar mass (Mw) was 2410 g / mol. A PDI value of 1.56 was therefore calculated for the sample, which is characteristic of a polymer produced by uncontrolled free radical polymerization (see Figure 8).

[0149] Example 3: Polymer coating of SPIONs The polymer coating of SPIONs is shown schematically in Figure 5.

[0150] Example 3a: 100 mg of LM-PMAcOD obtained in Example 2 was dissolved in 4 mL of chloroform in a 100 mL round-bottom flask. The mixture was heated until the polymer was completely dissolved. 3.3 mL of the oleate-SPION solution obtained in Example 1 was added to the mixture, followed by evaporation on a rotary evaporator at 280 RPM for 15 minutes at 40°C and above 10 mbar. 10 mL of 5 mM NaOH was then added to the mixture, followed by stirring on a rotary evaporator at 50°C for 15 minutes until all the black solid was dissolved. The solution was diluted 8 times with 70 mL of 25 mM NaOH to completely dissolve the polymer. The resulting solution was stirred on a rotary evaporator for 15 minutes, resulting in a brown solution.

[0151] The product was filtered through 0.45 μm and 0.2 μm PES filters, and the probe was then purified by tangential flow filtration (TFF).

[0152] Example 3b: After hydrolysis as per Example 2, the same procedure was performed using a commercially available 30-50 kDa polymer (#419117 Merck). Because TFF was insufficient to remove unbound polymer from the polymer-coated SPIONs, an additional magnetic separation was performed using a Miltenyi column to purify PMAcOD-SPION batch MX0194.

[0153] Example 4: Peptides and peptide coupling The peptide coupling and nanoparticle synthesis are shown schematically in FIG.

[0154] Synthesis of the peptides was achieved via C to N Fmoc chemistry using solid-phase peptide synthesis (SPPS). The α-amino group of each amino acid was protected with a fluoren-9-ylmethoxycarbonyl (Fmoc) group, and side chain functional groups were also blocked with various appropriate protecting groups.

[0155] Generally, SPPS consists of repeated cycles of N-terminal deprotection followed by coupling. The first Fmoc-protected amino acid was coupled to the resin. The amine group was then deprotected with a mixture of piperidine in dimethylformamide (DMF), followed by coupling with the free acid of the second Fmoc-protected amino acid. The cycle was repeated until the desired sequence was obtained. The resin was washed between each step. Completion of each coupling reaction was monitored by a qualitative ninhydrin test. In the final step of the synthesis, the crude peptide-resin was washed successively with DMF and methanol and dried. The protecting groups were then removed from the peptide, and the peptide was cleaved from the resin using trifluoroacetic acid (TFA). The resulting crude peptide was isolated from the cleavage mixture by ether precipitation.

[0156] The peptides were further purified by preparative HPLC to reach the purity requirements, and the counterion TFA was exchanged for chloride by using an appropriate solvent-buffer system. Finally, the purified peptides were lyophilized.

[0157] The peptides used have an amino acid at the N-terminus, a free acid (HCl salt) at the C-terminus, and are 15 amino acids in length.

[0158] The free peptide (starting material) was characterized by LC-MS.

[0159] The peptide sequences and calculated monoisotopic masses used in the examples of this application are shown in Table 1.

[0160] [Table 1]

[0161] The molecular weights of the peptides were determined by multimode electrospray atmospheric pressure chemical ionization mass spectrometry.

[0162] Peptides were coupled to the surface of the micelles obtained in Example 3a using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry in boric acid / sodium tetraborate decahydrate (SBB) buffer.

[0163] EDC in SBB buffer was added to the micelles obtained in Example 3. After 15 minutes at room temperature, the peptide was added and the reaction mixture was stirred at room temperature for 2 hours and 15 minutes.

[0164] The resulting nanoparticle solution was filtered and purified by tangential flow filtration (TFF) purification.

[0165] Example 5: Characterization of nanoparticles The nanoparticles obtained in Example 4 were further characterized using various analytical methods.

[0166] Characterization of the iron oxide core was performed using TEM and SAXS. TEM measurements were performed on nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS, and 6 mM L-lactic acid.

[0167] Calculated particle size results based on TEM analysis of the nanoparticles produced in Example 4 are summarized in Table 2. Representative TEM images are shown in FIG.

[0168] [Table 2]

[0169] SAXS measurements were carried out on nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS, and 6 mM L-lactic acid.

[0170] The calculated particle size results based on SAXS analysis of the nanoparticles produced in Example 4 are summarized in Table 3.

[0171] [Table 3]

[0172] Particle size and distribution characterization was performed by dynamic light scattering (DLS): hydrodynamic diameter (z-average) and polydispersity index were determined using a Malvern Zetasizer Nano ZS or equivalent instrument in unimodal mode.

[0173] These measurements were carried out on the nanoparticles produced in Example 4 dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L-lactic acid.

[0174] The results are summarized in Table 4. From these results it can be observed that the formulation is able to stabilize the particles and reduce the proportion of large particles.

[0175] [Table 4]

[0176] The surface charge of the particles was analyzed by measuring the zeta potential at pH 6-7 (pH during measurement) using a Malvern Zetasizer Nano ZS instrument. These measurements were performed on the nanoparticles produced in Example 4 dispersed in 5% (w / v) D-mannitol, 5 mM TRIS, and 6 mM L-lactic acid.

[0177] The results are summarized in Table 4.

[0178] [Table 5]

[0179] The total polymer content was determined using GPC. The peptide was hydrolyzed and the particles were disrupted in 6 M HCl. After adding EDTA, PMAcOD was extracted with ethyl acetate. After evaporation of the solvent, the residue was redissolved in a THF / acetic acid mixture before analysis.

[0180] The results are summarized in Table 5.

[0181] [Table 6]

[0182] The spectroscopic properties of the nanoparticles were determined by Fourier transform infrared spectroscopy (FTIR).

[0183] The assignment of the main absorption bands is summarized in Table 6.

[0184] [Table 7]

[0185] Example 6: Effect of different sized amphiphilic polymers The effect of amphiphilic polymers of different sizes on the nanoparticles of the present invention was studied: SPIONs were coated with PMAcOD polymers with number average molecular weights of 3100, 4800, and 5900 g / mol.

[0186] These polymers were synthesized, purified, characterized, and then used in the coating reaction of oleate-SPIONs. After coating, excess polymer was removed using TFF purification.

[0187] a) Synthesis of poly(maleic acid-alt-1-octadecene) (PMAcOD) with different molecular weights Three types of poly(maleic acid-alt-1-octadecene) with different lengths were synthesized by free radical polymerization of maleic anhydride and 1-octadecene. The reaction was carried out in 1,4-dioxane using 2,2'-azobis(2-methylpropionitrile) as the initiator. The polymerization was initiated by heating the mixture to 100°C. After purification, the polymer was hydrolyzed with sodium hydroxide solution to obtain PMAcOD.

[0188] Higher molecular weight polymers were synthesized by conducting the polymerization under more concentrated conditions and / or by conducting the polymerization with a lower amount of initiator.

[0189] The amounts of reactants used in the synthesis of the three polymers are summarized in Table 7.

[0190] [Table 8]

[0191] The purity of the polymer after hydrolysis of maleic anhydride and post-treatment was 1 The polymer length was measured by H-NMR (400 Hz, 30 mg sample, 10 mg benzoic acid standard, 700 μL D-chloroform), and the polymer length was analyzed by gel permeation chromatography (Agilent PL-gel mixed-D, 300 × 7.5 mm ID, 5 μm, 2 mg / mL in THF / acetic acid (90 / 10), 15 min run).

[0192] b) Effect of amphiphilic polymers of different lengths PMAcODs of various lengths were used to coat oleate-SPIONs.

[0193] Initially, coating was performed using the same polymer to SPION weight ratio. To find the best coating conditions, the polymer to SPION molar ratio was kept constant. The amount of aggregates was compared. The method with the least aggregates was repeated and subsequently purified using TFF. Polymer removal and aggregate amount were monitored during TFF using size exclusion chromatography (SEC).

[0194] Oleate-SPIONs were coated with PMAcOD3100, PMAcOD4800, and PMAcOD5900 to determine the optimal polymer / SPION ratio.

[0195] To coat the oleate-SPIONs, the polymer and nanoparticles were dissolved in chloroform. The chloroform was then evaporated. In the final step of the coating procedure, aqueous sodium hydroxide was added to the flask and mixed at 50°C. 100 mg of polymer was used for PMAcOD3100. PMAcOD4800 coatings with 100 mg and 145 mg of polymer were compared. PMAcOD5900 coatings with 100 mg and 177 mg of polymer were compared. In this way, an equal amount of monomer was compared to an equal amount of polymer chain for PMAcOD3100.

[0196] Size exclusion chromatography was used to determine which conditions resulted in the least amount of aggregates.

[0197] The results are summarized in Table 8.

[0198] [Table 9]

[0199] c) Effect of different polymer lengths after purification The same protocol as previously used was used to coat the oleate-SPIONs. The same amount of polymer chain was used for all polymers. After coating, the PMAcOD-SPIONs were purified by TFF. Because it was difficult to remove the final amount of polymer PMAcOD5900, we decided to also attempt this coating using 0.15 g of polymer (0.79 equivalents compared to the standard experiment with PMAcOD3100).

[0200] SEC was used to determine the amount of aggregates and polymer remaining in the samples. After coating, PMAcOD3100 contained 5.9% aggregates, PMAcOD4800 contained 6.2% aggregates, and PMAcOD5900 contained 10.7% aggregates. This is consistent with the data described in the previous section.

[0201] PMAcOD-SPIONs were then purified by TFF. The particles were loaded onto a membrane and then rinsed with 5 mM NaOH / 45 mM NaCl. Samples were taken every 10 DV (for PMAcOD3100) or 20 DV (for PMAcO4800 and PMAcOD5900) (DV = diafiltration volume in TFF purification) and subsequently measured by SEC. In-process samples were also taken during TFF purification to track the loss of polymer and the increase of aggregates.

[0202] The results are summarized in Table 9.

[0203] [Table 10]

[0204] The TFF products of Runs 1, 2, and 4 were filtered through a 0.2 μm filter. Run 1 was also filtered through a 0.1 μm filter to reduce the amount of aggregates.

[0205] Dynamic light scattering (DLS) measurements were performed to determine the Z-average diameter and polydispersity index (PDI) of the samples before and after filtration. All batches of PMAcOD-coated SPIONs exhibited Z-average diameters of 26–28 nm and PDIs of 0.25–0.33 (see Table 11).

[0206] The amount of iron was determined using atomic absorption spectroscopy (AAS) to be 38-50 mg after filtration, which corresponds to an iron recovery of 83-110%.

[0207] The results are summarized in Table 10.

[0208] [Table 11]

[0209] Example 7: Comparison of purification of PMAcOD-SPIONs with polymers of different molecular weights SPIONs coated with commercially available high-molecular-weight PMAcOD (Mn 30,000-50,000 g / mol) and low-molecular-weight PMAcOD (Mn 3,000-5,000 g / mol) were produced as described in Examples 1 to 4. Tangential flow filtration (TFF) purification was then used to remove unbound material. Because it can be easily scaled, TFF is the method of choice for purifying coated SPIONs. Size-exclusion chromatography (SEC) analysis of retentate and permeate samples was performed to monitor the TFF purification process. The purification efficiency using TFF of SPIONs coated with high- and low-molecular-weight polymers was compared.

[0210] a) Purification of high molecular weight PMAcOD-SPIONs In a first step, a sample of crude HM-PMAcOD-SPION was analyzed by SEC before purification by TFF (crude sample, see Figure 9). The crude HM-PMAcOD-SPION batch showed a slight shoulder (RT: 15.541) on the main HM-PMAcOD-SPION peak, indicating that this batch contained minimal large aggregates and 21.2% (a / a) of unbound polymer was free in the dispersion (RT: 16.698).

[0211] To separate the polymer-coated HM-PMAcOD-SPIONs from unbound polymer molecules, the high-molecular-weight PMAcOD-SPION sample was subsequently filtered by TFF using a 300 kDa TFF membrane. As shown in Figures 9 and 10, separation was not achieved, but both the HM-PMAcOD-SPIONs and the HM-PMAcOD polymer were present in the permeate after filtration.

[0212] Therefore, several options were tested to see if this could be reduced. However, none were successful. The retention of HM-PMAcOD-SPIONs was too low to obtain a good separation of SPIONs and polymer without significant loss of product. A membrane with a smaller pore size (100 kDa) allowed for the retention of polymer-coated SPIONs, but also resulted in the retention and concentration of the polymer (shown in Figure 11).

[0213] In conclusion, it was found that neither filtration through a 300 kDa nor 100 kDa TFF membrane allowed for the purification of HM-PMAcOD-SPIONs from unbound HM-PMAcOD material.

[0214] b) Purification of low molecular weight PMAcOD-coated SPIONs In the second method, low molecular weight PMAcOD-coated SPIONs were purified using TFF (100 kDa membrane filtration). Again, size exclusion chromatography analysis of retentate and permeate samples was performed to monitor the TFF purification process. Results are shown in Figure 12 for two batches (Batch MX0373A and MX0374A).

[0215] The use of LM-PMAcOD (Mn of 3,000–5,000 g / mol) demonstrated efficient diffusion of unbound polymer across a 100 kDa TFF membrane and removal of over 90% of unbound polymer from LM-PMAcOD-SPIONs.

[0216] SEC chromatograms of the products after coating (MX0373A and MX0374A), TFF (100 kDa membrane) purification (MX0373E and MX0374E) and final 0.1 μm filtration (MX0373F and MX0374F) are shown in FIG.

[0217] Additionally, SEC data for samples after TFF purification (100 kDa membrane filtration, batches MX0373E and MX0374E) and after TFF purification followed by 0.1 μm filtration (batch MX0373F and MX0374F) are shown in Table 12 below.

[0218] [Table 12]

[0219] As can be seen from the data provided, LM-PMAcOD-SPIONs could be efficiently purified by TFF, whereas purification of HM-PMAcOD-SPIONs was inefficient and did not result in a pure product.

[0220] Example 8: In vivo safety of low molecular weight polymer-based micelles containing an iron oxide core compared to high molecular weight polymer-based micelles containing an iron oxide core after intravenous injection in mice a) Batch MX0194 produced in Example 3b was injected intravenously into female CD1 mice at doses of 1 mmol Fe / kg body weight and 2 mmol Fe / kg body weight. One mouse died at 2 mmol Fe / kg, which was defined as test article related. Mice were euthanized 14 days after injection. A significant increase in absolute organ weights was observed compared to matched controls (see Table 13), which was defined as an adverse effect of test article injection.

[0221] b) PMAcOD-SPION batches produced according to Example 3a were intravenously injected three times into female CD1 mice at a dose of 1 mmol Fe / kg body weight (total of 3 mmol Fe / kg body weight), with 14 days between injections 1 and 2 and between injections 2 and 3. The animals were euthanized 24 hours after the last injection. A slight but non-significant increase in liver weight was observed, and no increase in lung weight was observed (see Table 13).

[0222] The impact on organ weights from high molecular weight polymer-based nanoparticles is related to the long-term deleterious effects of high molecular weight polymers due to the fact that the iron cores were similar for the batches produced in Examples 8a and 8b.

[0223] The results of the comparative study are summarized in Table 13.

[0224] [Table 13]

[0225] Thus, low molecular weight polymers were shown to be less toxic than high molecular weight polymers.

Claims

1. A nanoparticle, a) a micelle comprising an amphiphilic polymer having a number average molecular weight (Mn) of 6,000 to 1,000 g / mol; b) at least one peptide comprising at least one T-cell epitope; The amphiphilic polymer comprises the following building blocks: wherein R is a hydrocarbyl group or a substituted hydrocarbyl group.

2. 10. The nanoparticle of claim 1, wherein the nanoparticle further comprises a solid hydrophobic core at least partially coated by the micelles, the core comprising a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver, and gold.

3. The nanoparticle of claim 1 or 2, wherein the peptide is associated with the exterior of the micelle.

4. R is C 4 ~C 22 The nanoparticles according to any one of claims 1 to 3, which are alkyl groups.

5. R is C 8 ~C 20 The nanoparticles according to any one of claims 1 to 3, which are alkyl groups.

6. 4. The nanoparticles according to claim 1, wherein R is a linear alkyl group.

7. R is linear C 11 ~C 17 The nanoparticles according to any one of claims 1 to 3, which are alkyl groups.

8. 4. The nanoparticles according to claim 1, wherein R is a linear pentadecyl group.

9. 9. The nanoparticles of claim 1, wherein the amphiphilic polymer is selected from the group consisting of poly(maleic acid-alt-1-octadecene), poly(maleic acid-alt-1-dodecene), and poly(maleic acid-alt-1-tetradecene).

10. 10. The nanoparticle of claim 9, wherein the amphiphilic polymer is poly(maleic acid-alt-1-octadecene).

11. The nanoparticle of any one of claims 1 to 10, wherein the peptide is covalently bound to the micelle.

12. The nanoparticle of any one of claims 1 to 10, wherein the peptide is non-covalently bound to the micelle.

13. The nanoparticles of any one of claims 1 to 12, wherein the nanoparticles are negatively charged at a pH of 6 to 7.

14. 14. The nanoparticles of any one of claims 1 to 13, wherein the nanoparticles have a hydrodynamic diameter of 100 to 10 nm as measured by dynamic light scattering.

15. 15. The nanoparticles of claim 14, having a hydrodynamic diameter of 50 to 10 nm.

16. 15. The nanoparticles of claim 14, having a hydrodynamic diameter of 20 to 40 nm.

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

18. A pharmaceutical composition for use in suppressing a specific immune response, comprising nanoparticles according to any one of claims 1 to 16.

19. 20. The pharmaceutical composition for use in suppressing a specific immune response according to claim 18, wherein the response is associated with an autoimmune disease.

20. The autoimmune disease is selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, epidermolysis bullosa acquisita, bullous pemphigoid, cicatricial pemphigoid, Goodpasture's syndrome, microscopic polyangiitis, granulomatosis with polyangiitis (granulomatous, Wegener's), thrombotic thrombocytopenic purpura, immune thrombocytopenic purpura, uveitis, HLA-B27-associated acute anterior uveitis, multiple sclerosis, neuromyelitis optica, type I diabetes, narcolepsy with or without cataplexy, celiac disease, dermatitis herpetiformis, allergic airway disease / asthma, myasthenia gravis, Hashimoto's thyroiditis, autoimmune thyroid disease, Graves' disease, autoimmune thyroid disease, autoimmune hypoparathyroidism, autoimmune thyroid disease, antiphospholipid syndrome, and autoimmune thyroid disease.

20. The pharmaceutical composition for use in suppressing a specific immune response according to claim 19, wherein the specific immune response is selected from the group consisting of immune Addison's disease, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, autoimmune neutropenia, linear localized scleroderma, Batten disease, acquired hemophilia A, relapsing polychondritis, Isaacs syndrome (acquired neuromyotonia), Rasmussen encephalitis, Morvan syndrome, stiff-person syndrome, pernicious anemia, Vogt-Koyanagi-Harada syndrome, primary biliary cirrhosis, autoimmune type I hepatitis, autoimmune type II hepatitis, systemic lupus erythematosus, rheumatoid arthritis, polymyositis / dermatomyositis, Sjogren's syndrome, scleroderma, vitiligo, and alopecia areata.

21. 17. A method for producing nanoparticles according to any one of claims 1 to 16, comprising the steps of: i) obtaining an amphiphilic polymer having a number average molecular weight (Mn) of 6,000 to 1,000 g / mol; ii) forming micelles of said amphiphilic polymer; iii) adding at least one peptide to form said nanoparticles; The amphiphilic polymer comprises the following building blocks: wherein R is a hydrocarbyl group or a substituted hydrocarbyl group.

22. 22. The method of claim 21, wherein step i) is a radical copolymerization synthesis step.

23. 23. The method of claim 22, wherein the radical copolymerization synthesis step uses 2,2'-azobis(2-methylpropionitrile) as a radical initiator.

24. The method according to any one of claims 21 to 23, further comprising purifying the amphiphilic polymer obtained in step i).

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