Amphiphilic polymers and their use for improved production of nanoparticles for targeted delivery of antigens

Abstract

The present invention provides a nanoparticle 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 also provides a pharmaceutical composition comprising the nanoparticles and the use of the composition for inhibiting a specific immune response.

Description

Technical Field

[0001] The present invention provides nanoparticles for preventing and treating autoimmune diseases, allergies or other chronic inflammatory conditions and for producing regulatory T cells. In particular, the present invention relates to a nanoparticle comprising a micelle comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, such that the nanoparticle is soluble in water, and at least one peptide comprising at least one T cell epitope. The present invention also relates to a pharmaceutical composition comprising the nanoparticle. The pharmaceutical composition can be used to produce regulatory T cells specific for at least one T cell epitope in a subject for the treatment or prevention of a disease, wherein suppression of a specific immune response is advantageous.

[0002] Furthermore, the present invention provides a method for preparing nanoparticles. Background Art

[0003] Autoimmune diseases represent a significant burden for patients and healthcare systems. Current treatments rely primarily on immunosuppressive drugs that have considerable side effects. Autoantigen-specific immunotherapies that specifically target disease-specific immunopathology have no effect on the general immune status, thus representing an unmet medical need.

[0004] Immune tolerance to self-antigens is maintained by a variety of mechanisms that control potentially pathogenic autoreactive lymphocytes, including deletion, clonal inactivation, or suppression of regulatory T cells. Therefore, autoimmune diseases may result from inadequate 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 restore self-tolerance appears to be the manipulation of self-antigen-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 that prevent blood-borne antigens, such as food antigens, from entering the circulation. This fundamental mechanism of the liver can be used to specifically downregulate harmful immune responses against external protein antigens or self-antigens. Antigenic peptides derived from these proteins, when combined with nanoscale carriers and administered intravenously, mimic food antigens triggering uptake by specific liver cells, liver sinusoidal endothelial cells (LSECs), which then generate a tolerogenic immune response. Thus, peptide-specific immune tolerance can be induced against specific immunopathogenic antigens, thereby ameliorating or even eliminating harmful immune responses. This approach can therefore be used to treat existing diseases or to prevent the corresponding diseases by prophylactic means.

[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 ability of the liver to generate neural antigen-specific regulatory T cells (Tregs), which have a powerful ability to control and suppress autoimmune responses. LSECs play a crucial role in achieving this effect. LSECs express MHC / HLA class I and class II molecules on their surface and are therefore able to present peptides to CD8+ (via cross-presentation) and CD4+ T cells, respectively. Peptide antigen presentation by LSECs converts naive and T effector cells into Tregs in an antigen-specific manner in vitro. This is apparently a physiological mechanism by which LSECs establish tolerance to blood-borne antigens.

[0007] Nanoparticles, with disease-specific antigenic peptides bound to their surface, like blood-borne antigens, are targeted to the liver after intravenous injection. After uptake by LSECs, probably by pinocytosis, the nanoparticles accumulate in endosomal compartments and the peptide antigens are released from the particle surface. This leads to the presentation of those antigenic peptides on the surface of LSECs mediated by MHC / HLA molecules. There is evidence that the next generation of Tregs confer specific immune tolerance to the respective self-antigens based on their antigenic peptide epitopes.

[0008] WO2009 / 067349 discloses a pharmaceutical composition comprising biocompatible nanoparticles linked to an aryl hydrocarbon receptor (AHR) transcription factor ligand for treating autoimmune diseases by increasing the number and / or activity of regulatory T cells.

[0009] WO 2013 / 072051 discloses a pharmaceutical composition for producing regulatory T cells specific for at least one T cell epitope in a subject for treating or preventing a disease in which suppression of a specific immune response is beneficial. The nanoparticle comprises a micelle and a peptide, the micelle comprising an amphiphilic polymer that imparts water solubility to the nanoparticle, the peptide comprising at least one T cell epitope associated with the exterior of the micelle. It is generally recommended to use commercially available poly(maleic anhydride-alt-1-octadecene), which has an amphiphilic polymer of 30,000 to 50,000 g / mol molecular weight and about 90% purity.

[0010] However, in certain medical applications, it is important that nanoparticles can be produced in very high purity using efficient purification methods. There remains a need in the art for improved nanoparticles for the treatment and prevention of diseases where suppression of a specific immune response is beneficial, such as in autoimmune diseases, allergies, transplantation, suppression of anti-drug antibodies (ADA) to therapeutic agents or gene vectors, or diseases where inflammation is excessive, chronic or adverse, and where the pharmaceutical composition is suitable for administration to human subjects. Summary of the invention

[0011] According to the present invention, the above problem is solved by the following nanoparticles, the nanoparticles comprising:

[0012] a) a micelle comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, and

[0013] b) at least one peptide comprising at least one T-cell epitope.

[0014] The inventors surprisingly found that nanoparticles comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less can be prepared more easily and with higher purity.

[0015] Without being bound by the underlying theory of the effect of the nanoparticles of the present invention, it is currently understood that at least one peptide associated with the exterior of the micelle is processed like a blood-borne antigen by hydrolysis, proteolytic release or other activities in the endosome when the LSEC ingests the nanoparticles and is presented to T cells in a tolerogenic environment. Low molecular weight amphiphilic polymers are capable of excreting individual polymer molecules upon release in vivo. This provides hepatobiliary excretion, which appears to represent a preferred excretion pathway for nanoparticles.

[0016] It has surprisingly been found that low molecular weight amphiphilic polymers having a number average molecular weight (Mn) of 20,000 g / mol or less can be readily excreted. It is also expected that low molecular weight amphiphilic polymers and their metabolites will be rapidly eliminated from the body after treatment.

[0017] The inventors were surprised to find that low molecular weight amphiphilic polymers have superior performance during the preparation of nanoparticles of the present invention. Compared with high molecular weight amphiphilic polymers, the aggregation formed by low molecular weight amphiphilic polymers during solid core coating is less. In addition, compared with high molecular weight amphiphilic polymers, low molecular weight amphiphilic polymers can be purified more effectively. Especially, unbound polymers, when using low molecular weight amphiphilic polymers in nanoparticles of the present invention, can be separated more effectively.

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

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

[0020] Finally, the present invention provides a method for preparing nanoparticles, comprising:

[0021] i) obtaining an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less,

[0022] ii) optionally purifying said amphiphilic polymer,

[0023] iii) forming micelles of said amphiphilic polymer, and

[0024] iv) adding at least one peptide to form said nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a flow chart showing the synthesis of the iron oleate complex; wherein light blue arrows indicate synthesis steps and dark blue arrows indicate purification steps.

[0026] Figure 2 is a flow chart showing the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs).

[0027] Figure 3 is a flow chart showing the synthesis of low molecular weight poly(maleic anhydride-alt-1-octadecene) (LM-PMAOD).

[0028] Figure 4 is a flow chart showing the synthesis of low molecular weight poly(maleic acid-alt-1-octadecene) (LM-PMAcOD).

[0029] Figure 5 is a flow chart showing polymer coating of SPIONs.

[0030] Figure 6 is a flow chart showing the coupling of peptides to the nanoparticles of the present invention (TPC: Topas particle conjugate).

[0031] Figure 7 A transmission electron microscope (TEM) image of the nanoparticles of the present invention is shown (100 times magnification).

[0032] Figure 8 Graph showing the molecular weight distribution of LM-PMAcOD measured using gel permeation chromatography with polystyrene as the calibration standard.

[0033] Figures 9 to 11The SEC chromatogram of the HM-PMAcOD-SPION sample purification is shown; wherein: Fig. 9 SEC chromatogram showing crude HM-PMAcOD-SPION - before TFF purification; Fig.10 SEC chromatogram showing permeation of HM-PMAcOD-SPION - TFF purification - 300 kD membrane pores; Fig.11 SEC chromatogram showing retention of HM-PMAcOD-SPION - TFF purification - 100 kD membrane pores.

[0034] Fig.12 Size exclusion chromatography results of purified LM-PMAcOD-SPION samples are shown.

[0035] Fig.13 SEC chromatograms of the product after coating (MX0373A and MX0374A), TFF (100 kDa membrane) purification (MX0373E and MX0374E) and final 0.1 μm filtration (MX0373F and MX0374F) are shown. DETAILED DESCRIPTION

[0036] According to the present invention, nanoparticles are provided, comprising micelles, comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, such that the nanoparticles are soluble in water, and at least one peptide comprising at least one T cell epitope. The nanoparticles may also comprise a solid hydrophobic core coated by the micelles.

[0037] According to the present application, the terms "nanoparticle" and "nanoscale particle" are used interchangeably. 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.

[0038] In the context of the present invention, the nanoparticle has a structure formed of at least micelles and a peptide associated with the micelles. The peptide may be bound to the outside of the micelles or encapsulated inside the micelles.

[0039] In one embodiment of the present invention, the nanoparticles of the present invention comprise a solid hydrophobic core, a micelle coating the core, the micelle comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, making the nanoparticles soluble in water, and at least one peptide comprising at least one T cell epitope.

[0040] Micelles

[0041] 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 molecules is in contact with the surrounding solvent, isolating the hydrophobic "tail" region of the amphiphilic molecules inside the micelle, thereby making the nanoparticles water-soluble. Such micelles are also called normal phase micelles (or oil-in-water micelles).

[0042] The micelle may be formed of one, but also of more than one, for example two, three or four, amphiphilic polymer molecules. The micelle may be formed of the same or different amphiphilic polymer molecules. Generally, in the context of this specification, "a" or "the" is not intended to be limited to "one", unless otherwise specified.

[0043] In a preferred embodiment, the micelle is formed by a monolayer of amphiphilic polymer.

[0044] Such micelles may be structurally different from bilayers or liposomes formed by amphiphilic polymers. In this case, the nanoparticles of the present invention do not contain or do not contain a significant percentage (e.g., no more than 10%, no more than 5%, or preferably, no more than 1%) of the structure.

[0045] In one embodiment of the present invention, the amphiphilic polymer is used to prepare at least 70%, preferably at least 90% of the micelles. In a preferred embodiment, the micelles consist of the amphiphilic polymer.

[0046] In some embodiments of the invention, the nanoparticles do not comprise a solid hydrophobic core. In other embodiments, the nanoparticles comprise micelles and a solid hydrophobic core.

[0047] The method for preparing the nanoparticles of the present invention is described in detail below.

[0048] Amphiphilic polymers

[0049] 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, more preferably from 16 to 18 carbon atoms.

[0050] The hydrophilic regions of amphiphilic polymers can be negatively charged in aqueous solution.

[0051] In a preferred embodiment of the present invention, the amphiphilic polymer spontaneously forms micelles in solution. When a solid hydrophobic core is present, the amphiphilic polymer forms micelles around the solid core, thereby making the nanoparticles water-soluble.

[0052] 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, most preferably 3,000 to 6,000 g / mol.

[0053] The number average molecular weight can be determined using gel permeation chromatography (GPC), preferably using polystyrene as a calibration standard.

[0054] 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 tetrahydrofuran / acetic acid 90 / 10% (v / v), a flow rate of 1.0 ml / min, combined with a refractive index detector, a temperature of 35°C, and polystyrene as a calibration standard.

[0055] In the most preferred embodiment, the number average molecular weight is determined using GPC and the following measurement conditions:

[0056] Reference Standards Polystyrene Standard (MW (nominal Mp); 1000 to 130000 g / mol column Agilent PL-gel mixed-D,300x 7.5mm ID,5μm Column temperature 40℃ Detector Refractive index detector, at 35°C Flow rate 1.0ml / min Injection volume 20μL Autosampler temperature environment Run time 15min Mobile phase Tetrahydrofuran / acetic acid [90 / 10] % (v / v) Mobile phase program Isocratic

[0057] The amphiphilic polymer may be an alternating copolymer. An alternating copolymer is a copolymer in which two monomer units are distributed in an alternating sequence.

[0058] In one embodiment of the present invention, the amphiphilic polymer is a copolymer of maleic anhydride and at least one olefin.

[0059] The olefin used to prepare the amphiphilic polymer can be selected from one or more of the following: 1-decene, 1-undecene, 1-dodecene, 1-triene, 1-tetradecene, 1-pentadecene, 1-hexadecene, 1-heptadecene, 1-octadecene, 1-nonadecene or 1-eicosene, and the olefin is preferably 1-octadecene.

[0060] In a preferred embodiment of the present invention, the amphiphilic polymer is a copolymer of maleic anhydride and an olefin.

[0061] In a preferred embodiment of the invention, the amphiphilic polymer has a hydrophilic polymaleic anhydride backbone comprising hydrophobic alkyl side chains. Typically, the side chains may be from 5 to 23 carbon atoms, particularly 9 to 21 atoms. In a most preferred embodiment, the side chains are linear and have 10 to 18 carbon atoms.

[0062] The amphiphilic polymer may include the following building blocks:

[0063]

[0064] Wherein R is a hydrogen carbonate group or a substituted hydrogen carbonate group. In a preferred embodiment of the present invention, R is C4 to C22 Alkyl, such as C7 to C 19 alkyl.

[0065] In a more preferred embodiment, R is a linear alkyl group, preferably a linear C7 to C 17 Alkyl, most preferably R is a linear pentadecyl or linear nonyl.

[0066] The amphiphilic polymer may consist of the building blocks defined above.

[0067] In other embodiments of the present invention, the amphiphilic polymer comprises more than 50%, preferably more than 70%, more preferably more than 90% of the structural blocks defined above.

[0068] In a preferred embodiment, the amphiphilic polymer is selected from 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). The average molecular weight of the polymer is 6,000 to 1,000 g / mol.

[0069] In a particularly preferred embodiment, the amphiphilic polymer is selected from poly(maleic acid-alt-1-octadecene), poly(maleic acid-alt-1-dodecene) and poly(maleic acid-alt-1-tetradecene). Poly(maleic acid-alt-1-octadecene). The average molecular weight of the polymer is 5000 to 1000 g / mol.

[0070] The method for preparing the amphiphilic polymer of the present invention is described in detail below.

[0071] Peptides

[0072] Nanoparticles of the present invention further include at least one peptide, including at least one T cell epitope. The peptide may be combined with the outside of the micelle or coated inside the micelle (in the embodiment of the nanoparticles of the present invention without a solid hydrophobic core). Therefore, the peptide can be outside or inside the micelle.

[0073] The peptide may be covalently linked to the micelle or not covalently bound, preferably covalently linked to the micelle.

[0074] In a preferred embodiment, the peptide is covalently linked to the micelle using a method known in the art for covalently coupling peptides (e.g., carbodiimide or succinimide coupling). Preferably, the peptide is covalently linked to the micelle using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry.

[0075] In the context of the present invention, the term "peptide" is not limited in size, in particular the 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, which may be linked to each other as fusion polypeptides.

[0076] In a preferred embodiment of the invention, the peptide comprises 10 to 20, such as 13 to 17 amino acids.

[0077] In a particularly preferred embodiment, the peptide comprises 15 amino acids.

[0078] The peptide includes at least one T cell epitope. The method of identifying T cell epitopes and selecting T cell epitopes is well known in the art, for example, described in documents Rammensee et al., 1999 (Immunogenetics 50 213-219) and Sanchez-Trincado et al. 2017 (J Immunol Res. 2017: 2680160.doi: 10.1155 / 2017 / 2680160.Epub 2017Dec 28). In the case of the present invention, T cell epitopes are peptide sequences that induce regulatory T cells. At least one epitope is required to be able to be expressed by the cells of the subject to whom nanoparticles are to be administered. Preferably, the peptide includes several epitopes so that it can be represented by multiple major histocompatibility complex (Major Histocompatibility Complex) types.

[0079] Since regulatory T cells are primarily CD4+, the presentation of MHC class II is of primary interest. The HLA type of a subject (e.g., a human subject) can be easily tested as part of selecting epitopes. The epitopes of a particular peptide that can be presented on a particular MHC molecule are known and / or routinely selected, e.g., by appropriate software.

[0080] The peptides are designed based on published data to ensure that they (in combination with specific HLA restriction elements) bind MHC / HLA with high affinity and significantly stimulate and activate T cells. Ideally, the selected peptides are extrapolated from naturally occurring peptides and are immunodominant.

[0081] The peptides may be synthesized, recombinantly expressed or isolated or modified from natural sources. The peptides or at least the antigenic epitopes produced by regulatory T cells are preferably extracted from peptides / proteins that inhibit inflammatory immune responses, for example, in the treatment or prevention of autoimmune diseases or allergies. The peptides may be, for example, allergens, known autoimmune antigens or fragments or derivatives thereof. The peptides may bind to various epitopes from various antigens.

[0082] 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 than one Desmoglein 3 peptides characterized by SEQ ID NOs: 1-3.

[0083] According to one embodiment of the present invention, the nanoparticles only comprise one type of peptide containing at least one T cell epitope. According to another embodiment, the present invention provides a composition comprising different nanoparticles, wherein each nanoparticle comprises many peptides with the same amino acid sequence, but the composition comprises a mixture of nanoparticles having peptide sequences different from each other. The composition can, for example, comprise different nanoparticles comprising 2 to 6 different peptides. On the one hand, the composition of different nanoparticles can comprise three different types of nanoparticles, each of which is characterized by one of SEQ ID NOs: 1-3.

[0084] Solid hydrophobic core

[0085] In one embodiment of the invention, the nanoparticles comprise a solid hydrophobic core at least partially encapsulated by micelles.

[0086] The core may be an inorganic core, preferably comprising iron oxide, CdSe, silver or gold.

[0087] The diameter of the core may be 2 to 500 nm, preferably 3 to 25 nm, more preferably 5 to 15 nm. The diameter of the core may be determined using transmission electron microscopy (TEM) or small angle X-ray scattering (SAXS).

[0088] An exemplary inorganic core is composed of oleic acid or another carboxylic acid (C 14 -C 22 , preferably C 16 -C 18) stabilized iron oxide nanoparticles, quantum dots (CdSe / CdS / ZnS, stabilized by, for example, trioctylphosphine oxide), gold nanoparticles, stabilized by, for example, sulfonic acid compounds. The inorganic cores themselves are generally unstable in aqueous solvents such as water, but they are embedded in polymer micelles to make them water-soluble. The hydrophobic portion of the amphiphilic polymer interacts with the hydrophobic core of the nanoparticles, resulting in the formation of a single polymer coating around the core. During the coating process, the amphiphilic polymer can replace the hydrophobic portion of the core by ligand exchange, thereby forming a double-layer micelle around the core. In one embodiment of the invention, the polymer at least partially replaces the oleic acid on the surface of the core particle and 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 a double-layer micelle around the iron oxide core, thereby producing iron oxide coated with a polymer.

[0089] According to a preferred embodiment of the present invention, the core is superparamagnetic.

[0090] In a particularly preferred embodiment of the present invention, the core is superparamagnetic iron oxide nanoparticles (SPIONs), which may be stabilized by oleic acid.

[0091] The core preferably renders the nanoparticles of the invention traceable, for example, by their properties in fluorescence, electron microscopy or other detection methods.

[0092] Nanoparticles

[0093] The inventors have found that the nanoparticles used in the present invention are suitable for transferring peptides to liver sinusoidal endothelial cells of a subject in vivo.

[0094] The nanoparticles may additionally 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 may, for example, enhance or accelerate uptake from the circulation by receptor-mediated endocytosis. Examples of suitable modifications are carbohydrates such as mannose.

[0095] The nanoparticles may be negatively charged or uncharged due to the micelle-forming polymer; preferably, the nanoparticles are negatively charged at a pH of 6 to 7. The polymer coating may contain acid groups such as carboxylic acids, resulting in a negative charge on the nanoparticles.

[0096] 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 pH 6 to 7 (pH during measurement). The zeta potential may be measured using a Malvern Zetasizer NanoZS instrument.

[0097] The hydrodynamic diameter (z-average) of the nanoparticles of the invention may be from 10 to 100 nm or from 10 to 70 nm, preferably from 10 to 50 nm, more preferably from 20 to 40 nm, most preferably from 22 to 32 nm, as measured by dynamic light scattering (DLS).

[0098] The polydispersity index of the nanoparticles of the present invention may be lower than 0.50, preferably between 0.05 and 0.45, more preferably between 0.10 and 0.40, as measured by dynamic light scattering (DLS).

[0099] The determination of the hydrodynamic diameter and polydispersity index is performed using an electrophoretic light scattering analysis method, preferably using a Malvern Zetasizer. In one embodiment, the method for determining the hydrodynamic diameter and polydispersity index uses electrophoretic light scattering, disposable polystyrene cuvettes, Zetasizer Software 7.12, milli-Q water. 20nm and 100nm nanosphere size standards (NIST certified or equivalent) are diluted in 0.9% sodium chloride aqueous solution, and the test samples are diluted in water. All aqueous reagents are filtered through a 0.22μm membrane before use. In the most preferred embodiment of the present invention, the method for determining the hydrodynamic diameter and polydispersity index is performed using electrophoretic light scattering in combination with the following analytical conditions:

[0100] Overview of analytical conditions:

[0101]

[0102]

[0103] Evaluation of the data was based on the mean diameter (Z-Average, intensity nm), a parameter also known in DLS as the cumulant mean, and the polydispersity index (PDI), used as a measure of the size distribution.

[0104] In addition, 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, more preferably 1 to 3 mg / mL. The total polymer content is measured by GPC. In order to measure the total polymer content, the peptide is hydrolyzed and the particles are destroyed (e.g., using a 6M HCl solution). The polymer is extracted after adding EDTA. After evaporating the solvent, the residue is redissolved and the polymer content is measured by GPC.

[0105] The determination of total polymer content preferably uses the following reagents and reference standards: water (HPLC grade), acetonitrile (HPLC grade), tetrahydrofuran with BHT (THF-HPLC grade), 100% acetic acid (analytical grade), hydrochloric acid 37% (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 a reference substance.

[0106] The chromatographic conditions for determining the total polymer content were:

[0107] Chromatographic columns Agilent PL-gel Mixed-D,300+75mm ID,5μm Column temperature 40℃ Flow rate 1.0mL / min Detector Refractive index detector, at 35°C Sampling rate 2.31Hz or equivalent Injection volume 20μL Autosampler temperature environment Run time 15min Mobile phase THF / acetic acid [90 / 10] % (v / v) Mobile phase program Isocratic

[0108] In a particularly preferred embodiment of the present invention, the nanoparticles comprise an iron oxide core coated with 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 comprising at least one T cell epitope preferably covalently linked to the micelle.

[0109] In one aspect, the present invention thus provides a nanoparticle comprising:

[0110] a) Micelles, including amphiphilic polymers, containing the following building blocks

[0111]

[0112] Wherein R is a hydrocarbon group or a substituted hydrocarbon group, preferably R is a straight chain alkyl group, preferably a straight chain C 11 To C 17 alkyl, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and

[0113] b) at least one peptide comprising at least one T cell epitope; and

[0114] c) a solid hydrophobic core at least partially encapsulated by said micelles, wherein said core comprises a traceable inorganic material selected from the group consisting of iron oxide, CdSe / CdS / ZnS, silver and gold.

[0115] The number average molecular weight of the polymer is preferably 6,000 to 1,000 g / mol.

[0116] The nanoparticles preferably have a hydrodynamic diameter of 50 to 10 nm, as measured by dynamic light scattering.

[0117] Pharmaceutical composition

[0118] The present invention further provides a pharmaceutical composition comprising the nanoparticles of the present invention.

[0119] The peptide used may be present in a concentration of 0.01 to 2 mM, preferably 0.1 to 1 mM, more preferably 0.45 to 1 mM in the pharmaceutical composition. 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.

[0120] The pharmaceutical composition of the present invention may further comprise at least one suitable adjuvant and / or diluent. The diluent is preferably water or water-based, for example a buffer such as phosphate buffered saline (PBS), Ringer's solution, TRIS buffer or sodium chloride solution. Suitable preservatives may or may not be included.

[0121] Obviously, particularly for administration to human subjects, the compositions are preferably sterile and biocompatible.

[0122] In a preferred embodiment of the present invention, the pharmaceutical composition comprises the nanoparticles of the present invention dispersed in D-mannitol, TRIS and / or L-lactic acid.

[0123] In one embodiment of the present invention, the pharmaceutical composition comprises nanoparticles of the present invention without a solid hydrophobic core, and the nanoparticles are dispersed in D-mannitol, TRIS and / or L-lactic acid. The advantage of using such a buffer is that the particles are very stable in such a buffer and can be subsequently lyophilized.

[0124] In addition, the pharmaceutical composition may contain more than one type of nanoparticles of the invention, wherein different types of nanoparticles have different peptides associated with the exterior of the micelle. By using a mixture of nanoparticles, multiple autoantigenic peptides can simultaneously induce a broader immune tolerance. These peptides may be from a single immunogenic protein, or from different proteins.

[0125] In a preferred embodiment of the present invention, the pharmaceutical composition comprises 2 to 6 different types of nanoparticles, wherein preferably, all relevant peptides of different types of nanoparticles comprise at least one T cell epitope.

[0126] In a particularly preferred embodiment of the present invention, the pharmaceutical composition comprises 3 to 4 different types of nanoparticles, wherein all relevant peptides of different types of nanoparticles comprise at least one T cell epitope. In particular, each nanoparticle can be bound to a different antigenic peptide derived from Dsg3.

[0127] The pharmaceutical composition comprises nanoparticles at a concentration of less than 100 μM, preferably 0.5 to 80 μM, more preferably 1 to 50 μM. If more than one nanoparticle is present in the pharmaceutical composition, the concentration of each nanoparticle may be less than 100 μM, preferably 0.5 to 80 μM, more preferably 1 to 50 μM.

[0128] The pharmaceutical composition of the present invention may comprise different types of nanoparticles in equimolar concentrations.

[0129] In one aspect, the present invention provides a pharmaceutical composition comprising nanoparticles, comprising:

[0130] a) micelles comprising an amphiphilic polymer comprising the following building blocks

[0131]

[0132] Wherein R is a hydrocarbon group or a substituted hydrocarbon group, preferably R is a straight chain alkyl group, preferably a straight chain C 11 To C 17 alkyl, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and

[0133] b) at least one peptide comprising at least one T cell epitope; and

[0134] c) a solid hydrophobic core, at least partially coated by the micelles, wherein the core comprises a traceable inorganic material selected from the group consisting of iron oxide, CdSe / CdS / ZnS, silver and gold.

[0135] The number average molecular weight of the polymer in the pharmaceutical composition is preferably 6,000 to 1,000 g / mol.

[0136] The hydrodynamic diameter of the nanoparticles in the pharmaceutical composition of the present invention is preferably 50-10 nm as measured by dynamic light scattering.

[0137] In a preferred aspect, the present invention provides a pharmaceutical composition comprising at least three different types of nanoparticles, each of which comprises: a) micelles comprising an amphiphilic polymer comprising the following building blocks:

[0138]

[0139] Wherein R is a hydrocarbon group or a substituted hydrocarbon group, preferably R is a straight chain alkyl group, preferably a straight chain C 11 To C 17 alkyl, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and

[0140] b) a peptide comprising a T cell epitope; and

[0141] c) a solid hydrophobic core, at least partially encapsulated by micelles, wherein the core comprises a traceable inorganic material selected from the group consisting of iron oxide, CdSe / CdS / ZnS, silver and gold; and

[0142] The three different types of nanoparticles differ from each other in peptide sequence, wherein the first type of nanoparticles comprises a peptide having SEQ ID NO:1, the second type of nanoparticles comprises a peptide having SEQ ID NO:2 and the third type of nanoparticles comprises a peptide having SEQ ID NO:3.

[0143] Medical Uses

[0144] The pharmaceutical compositions of the present invention are intended for and formulated for administration to a subject suffering from a disease in which suppression of a particular immune response is beneficial.

[0145] The pharmaceutical composition can be administered to a subject in need thereof.

[0146] The dosage and concentration required for administration to a subject can be determined by the responsible medical personnel according to the facts and circumstances of the case. Exemplary dosages may include 0.03 μmol to 0.90 μmol per patient body weight, for example, for human subjects.

[0147] Administration can be repeated, for example, two, three or four times, for example, with 1, 2, 3, 4, 5, 6, 7, 10 or 14 days between administrations. Preferably, the pharmaceutical composition is used to suppress a specific immune response, for example, to treat or prevent a disease in which suppression of a specific immune response is beneficial. More preferably, the pharmaceutical composition is used to generate regulatory T cells specific for at least one T cell epitope in a subject, for treating or preventing a disease in which suppression of a specific immune response is beneficial.

[0148] 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 in 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, bullous pemphigoid, cicatricial pemphigoid, Goodpasture's syndrome, microscopic polyangiitis, granulomatosis with polyangiitis (Granulom.Wegener), 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, autoimmune Addison's disease, autoimmune hemolytic anemia, chronic inflammatory demyelination, polyneuropathy, Guillain-Barré syndrome, autoimmune neutropenia, linear morphea, Batten disease, acquired hemophilia A, relapsing polychondritis, Isaac syndrome (acquired neuromyotonia), Rasmussen encephalitis, Morvan syndrome, stiff-man 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, Sjögren's syndrome, scleroderma, vitiligo, and alopecia areata.

[0149] More preferably, the autoimmune disease is selected from 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.

[0150] According to the present invention, the term "treating" is used to refer to the alleviation of the symptoms of a particular disease and / or the improvement of an ascertainable measure associated with a particular condition in a subject.

[0151] Methods for preparing amphiphilic polymers and nanoparticles

[0152] Methods for preparing amphiphilic polymers and nanoparticles comprising the same are described in Examples 1-4.

[0153] One method of obtaining an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less is to synthesize the amphiphilic polymer using a two-step process, comprising a step of preparing the polymer of an anhydride and a step of hydrolyzing the anhydride to obtain the acid. The step of hydrolyzing the anhydride form of the polymer to obtain the acid form can be illustrated as follows:

[0154]

[0155] The present invention also provides a method for preparing nanoparticles, comprising:

[0156] i) obtaining an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less,

[0157] ii) optionally purifying the amphiphilic polymer,

[0158] iii) forming micelles of said amphiphilic polymer, and

[0159] iv) adding at least one peptide to form said nanoparticles.

[0160] In one embodiment of the invention, the peptide is encapsulated by micelles and step iv) is performed before step iii). In these embodiments, the peptide is added to the amphiphilic polymer before micelle formation.

[0161] The amphiphilic polymer used in the nanoparticles of the present invention can be prepared by free radical copolymerization using a free radical initiator (step i). The molecular weight of the polymer can be controlled by changing the concentration of the reactants or the amount of the free radical initiator. The molecular weight of the polymer can be analyzed by gel permeation chromatography.

[0162] The copolymerization process can be carried out in an organic solvent such as 1,4-dioxane, xylene or chlorobenzene.

[0163] Many free radical initiators are known in the art; they include various peroxides and azo compounds. Examples of suitable peroxides are benzoyl peroxide, lauryl peroxide, di-tert-butyl peroxide, 2,4-dichlorobenzyl peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, diacetyl peroxide, diethyl peroxycarbonate, tert-butyl perbenzoate and perborate. Suitable azo compounds include 2,2'-azobis(2-methylpropionitrile), p-bromobenzenediazoniumfluoroborate, p-tolyldiazoaminobenzene, p-bromobenzenediazonium hydroxide, azomethane and phenyldiazonium halides. Preferably, the free radical initiator is 2,2'-azobis(2-methylpropionitrile).

[0164] The copolymerization may be carried out at elevated temperature, such as 70 to 120 °C, preferably 90 to 110 °C.

[0165] Preferably, the copolymerization is initiated by heating the mixture to 70 to 120°C, preferably 90 to 110°C.

[0166] In a preferred embodiment of the process of the invention, step i) comprises the steps of mixing the reactants, deoxygenating the mixture, heating the mixture and then cooling the mixture. Afterwards, the polymer can be dissolved and stirred overnight. The solid formed can be recovered, preferably by centrifugation.

[0167] In a preferred embodiment of the method of the present invention, step ii) comprises adding a base (e.g. NaOH) to the polymer. Preferably, the base reacts with the polymer at an elevated temperature, preferably between 50°C and 70°C, e.g. 60°C, until almost all of the solid is dissolved. The resulting suspension can be acidified (e.g. pH <2). Thereafter, the reaction mixture can be extracted with an organic solvent such as ethyl acetate. The organic layer can be extracted with a sodium hydroxide solution. The aqueous solution can be extracted again with an organic solvent such as ethyl acetate and then dried to obtain a purified amphiphilic polymer.

[0168] The polymer may be further purified (step iii). Preferably, the polymer is further purified by extracting the polymer with n-hexane or n-heptane. The extraction may be performed at a concentration greater than 10 g / L, preferably 100 g / L.

[0169] In addition, an additional amphiphilic polymer purification step can be added between steps i) and ii). In this additional purification step, the polymerized crude reaction product is dissolved and precipitated. In a preferred embodiment, the solvent is dichloromethane and a mixture of methanol / heptane or acetonitrile / isopropanol is used to precipitate the polymer. 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.

[0170] The purity of the amphiphilic polymer after the hydrolysis and post-treatment process can be determined by 1 H NMR measurements.

[0171] In the method of the present invention, the micelles are formed by forming a solution containing an amphiphilic polymer. Preferably, the micelles are formed in an aqueous solution. A co-stabilizer may be added to the amphiphilic polymer to improve micelle formation.

[0172] The peptide used in step iv) can be synthesized using state of the art solid phase chemistry.

[0173] The peptides may be covalently linked or non-covalently associated with the micelles.

[0174] In a preferred embodiment of the method of the present invention, the peptide is coupled to the surface of the nanoparticle using peptide coupling techniques known in the art, such as carbodiimide or succinimide coupling.

[0175] In a particularly preferred embodiment of the method according to the invention, the peptide is bound to the surface of the nanoparticles by means of EDC chemistry (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in aqueous phase.

[0176] The resulting nanoparticles can be purified using intensive washing and filtration steps to remove the coupling agent and any low molecular weight components. In a preferred embodiment, the method for preparing the nanoparticles comprises:

[0177] a) obtaining hydrophobic core nanoparticles,

[0178] b) obtaining an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, preferably using free radical copolymerization,

[0179] c) optionally purifying the amphiphilic polymer,

[0180] d) mixing the hydrophobic core nanoparticles and the amphiphilic polymer to form micelles,

[0181] e) adding at least one peptide to form said nanoparticles.

[0182] The discussion above for steps i) to iii) and iv) also applies to steps b) to d) and e) respectively.

[0183] The hydrophobic core of step a) can be synthesized by using suitable reactants in solution. In a preferred embodiment of the method of the present invention, the hydrophobic core can be synthesized using metal salts and carboxylates as reactants in the presence of an organic solvent. Preferably, the reaction is carried out at an elevated temperature under oxygen restriction.

[0184] The micelles can be formed by arranging the amphiphilic copolymer around the core in step e). Preferably, step d) comprises the following sub-steps: dissolving the amphiphilic polymer and the core particles, removing the solvent until a film is formed, adding an alkaline aqueous solution at elevated temperature and ambient pressure to form an aqueous colloidal dispersion, diluting the solution and optionally filtering it. Afterwards, several washing steps can be performed.

[0185] Example

[0186] The invention is illustrated by the following examples, which describe in detail the synthesis of the nanoparticles according to the invention.

[0187] These examples should not be considered as limiting the scope of the invention but rather as illustrating it.

[0188] Example 1: Preparation of superparamagnetic iron oxide nuclei (SPION)

[0189] The synthesis of iron oleate complex is schematically shown as follows Figure 1 shown.

[0190] In the first step, an iron oleate complex was synthesized by mixing oleic acid, sodium hydroxide and ferric 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 in a rotary evaporator. This formed iron oxide crystal nuclei ( Figure 1 ).

[0191] 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, deoxygenated and heated at 300°C for 3 hours to form iron oxide nanocrystals.

[0192] After cooling, the product was purified by using magnetic separation and several washing steps with acetone / tetrahydrofuran (THF).The purified SPIONS were diluted in chloroform and concentrated in a rotary evaporator to obtain SPIONs with a narrow size distribution and good crystallinity.

[0193] Example 2: Preparation of low molecular weight poly(maleic acid-alt-1-octadecene) (LM-PMAcOD)

[0194] The synthesis of low molecular weight poly(maleic acid-alt-1-octadecene) (LM-PMAcOD) was achieved by a two-step process, as Figure 3 and 4 As shown. 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 of 1,4-dioxane was previously removed by filtration on 3 g of alumina). After that, 5.79 mmol AIBN (2,2'-azobis(2-methylpropionitrile)) was added. The flask was equipped with a cooler, which was then flushed with nitrogen and maintained at a nitrogen overpressure. The mixture was heated to 100°C while stirring. One hour after the start of heating, the heating plate was removed together with the stopper; the reaction was exposed to air. The mixture was cooled to room temperature for two days while stirring. The product was purified by co-evaporation with dichloromethane and precipitated with isopropanol and acetonitrile. A low molecular weight poly (maleic anhydride-alt-1-octadecene) (LM-PMAOD) with a number average molecular weight (Mn) of 2,500 to 4,000 g / mol was obtained.

[0195] In the second step, LM-PMAOD was hydrolyzed to poly(maleic acid-alt-1-octadecene) (LM-PMAcOD) in sodium hydroxide solution. Acid-base extraction was performed using H2SO4, ethyl acetate and NaOH to purify the product and remove impurities, such as residual 1-octadecene. The product was dried over magnesium sulfate, co-evaporated with chloroform, and finally purified by solid-liquid extraction in n-heptane ( Figure 4 ).

[0196] The number average molar mass (Mn) and the mass average molar mass (Mw) of the obtained polymer were determined by gel permeation chromatography.

[0197] 1.5 mg of LM-PMAcOD sample was dissolved in 1.0 mL of THF (without stabilizer) and submitted to GPC. Polystyrene was used as calibration standard. Tetrahydrofuran was used as eluent with a flow rate of 1 ml / min. The temperature was set at 30°C.

[0198] The number average molar mass (Mn) of the generated LM-PMAcOD was 1540 g / mol and the mass average molar mass (Mw) was 2410 g / mol. Therefore, the PDI value of the sample was calculated to be 1.56, which is characteristic of polymers produced by uncontrolled free radical polymerization (see Figure 8 ).

[0199] Example 3: Polymer coating of SPIONs

[0200] The polymer coating of SPIONs is schematically shown in Figure 5 .

[0201] Example 3a: 100mg M-PMAcOD obtained in Example 2 was dissolved in 4mL chloroform in a 100mL round bottom bottle. The mixture was heated until the polymer was completely dissolved. The 3.3mL Oleate-SPION solution obtained in Example 1 was added to the mixture and then evaporated for 15 minutes at 40°C with <10mbar by a 280rpm rotary evaporator. Then, 10mL of 5mM NaOH was added to the mixture and stirred at 50°C for 15 minutes in a rotary evaporator until all black solids were dissolved. 70mL of 25mM NaOH was used to dilute the solution to dissolve the entire polymer. The obtained solution was stirred for 15 minutes in a rotary evaporator to produce a brown solution.

[0202] The product was filtered on 0.45 μm and 0.2 μm PES filters. Afterwards, the probe was purified by tangential flow filtration (TFF).

[0203] Example 3b: Commercially available 30-50 kDa polymer (#419117 Merck) was subjected to the same process after hydrolysis as in Example 2. As TFF was insufficient to remove unbound polymer from polymer-coated SPIONs, an additional magnetic separation using a Miltenyi column was performed to purify PMAcOD-SPION batch MX0194.

[0204] Example 4: Peptides and Peptide Coupling

[0205] Peptide coupling and nanoparticle synthesis are schematically shown in Figure 6 .

[0206] The synthesis of peptides was accomplished using solid phase peptide synthesis (SPPS) via Fmoc chemistry from C to N. The α-amino group of each amino acid was protected by fluoro-9-methoxycarbonyl (Fmoc), while the side chain functional groups were also capped with various appropriate protecting groups.

[0207] Typically, SPPS consists of repeated cycles of N-terminal deprotection and subsequent coupling reactions. The first Fmoc-protected amino acid is coupled to the resin. The amine group is then deprotected using a mixture of piperidine in dimethylformamide (DMF) and then coupled to the free acid of the second Fmoc-protected amino acid. The cycle is repeated until the desired sequence is obtained. The resin is washed between each step. The completion of each coupling reaction is monitored by a qualitative Ninghai standard test. In the last step of the synthesis, the crude peptide-resin is washed with DMF and methanol in sequence and dried. The protecting group is then removed from the peptide and the peptide is separated from the resin using trifluoroacetic acid (TFA). The crude peptide is separated from the separated mixture by ether precipitation.

[0208] In addition, the peptide was purified by preparative HPLC to achieve the required purity, and the counter ion TFA was replaced by chloride using an appropriate solvent buffer system. Finally, the purified peptide was lyophilized.

[0209] The peptide used had one amino acid at the N-terminus, one free acid (HCl salt) at the C-terminus and was 15 amino acids in length. The identification of the free peptide (starting material) was performed by LC-MS.

[0210] The peptide sequences and calculated monoisotopic masses used in the examples of this application are shown in Table 1.

[0211] Table 1: Peptide sequences and monoisotopic masses

[0212] Serial Number sequence Molecular formula Monoisotopic mass (theoretical) (Da) 1 LNSKIAFKIVSQEPA <![CDATA[C 75 H 125 N 19 O 22 ]]> 1643.9 2 TPMFLLSRNTGEVRT <![CDATA[C 74 H 124 N 22 O 23 S]]> 1720.9 3 REGIAFRPASKTFTV <![CDATA[C 76 H 122 N 22 O 21 ]]> 1678.9

[0213] The molecular weight of the peptides was measured by multimode electrospray atmospheric pressure chemical ionization mass spectrometry.

[0214] The peptide was conjugated to the surface of the micelles obtained in Example 3a using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry in boric acid / tetrasodium disodium dehydrogenase (SBB) buffer.

[0215] EDC in SBB buffer was added to the micelles obtained in Example 3. After 15 min at RT, the peptide was added and stirred for 2 h 15 min at RT.

[0216] The resulting nanoparticle solution was filtered and purified by a tangential flow filtration (TFF) purification process.

[0217] Example 5: Identification of Nanoparticles

[0218] The nanoparticles obtained in Example 4 were further characterized by using various analytical methods.

[0219] The iron oxide core was identified using TEM and SAXS. TEM examination was performed on nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM Tris and 6 mM L-lactic acid.

[0220] Table 2 summarizes the particle size results calculated based on TEM analysis of the nanoparticles produced in Example 4. Representative TEM images are shown in Figure 7 .

[0221] Table 2: Average iron oxide core size results based on TEM analysis

[0222]

[0223] SAXS measurements of nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM Tris and 6 mM L-lactic acid.

[0224] The calculated size results based on SAXS analysis of the nanoparticles formed in Example 4 are summarized in Table 3.

[0225] Table 3: Average iron oxide core size results based on SAXS analysis

[0226]

[0227] Characterization of size and distribution is performed by dynamic light scattering (DLS). The hydrodynamic diameter (Z-average) and polydispersity index are determined using a Malvern Zetasizer NnoZS or equivalent instrument in unimodal mode.

[0228] These measurements were performed on nanoparticles produced in Example 4, dispersed in 5% (w / v) D-mannitol, 5 mM Tris and 6 mM L-lactic acid.

[0229] The results are summarized in Table 4. From these results, it can be observed that the formulation can stabilize the particles and can reduce the percentage of large particles.

[0230] Table 4: Size distribution results based on DLS analysis

[0231]

[0232] Analysis of the surface charge of the particles was performed by measuring the Zeta potential at pH 6 to 7 (pH during measurement) using a Malvern Zetasizer nano ZS analyzer. These measurements were performed on nanoparticles produced in Example 4 dispersed in 5% (w / v) D-mannitol, 5 mM Tris and 6 mM L-lactic acid.

[0233] The results are summarized in Table 4.

[0234] Table 4: Zeta Point Results

[0235]

[0236] The total polymer content was determined by using GPC. The peptide was hydrolyzed and destroyed in 6M 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.

[0237] The results are summarized in Table 5.

[0238] Table 5: Total polymer content

[0239]

[0240] The spectral properties of the nanoparticles were determined by Fourier transform infrared spectroscopy (FTIR).

[0241] The assignments of the main absorption bands are summarized in Table 6.

[0242] Table 6: Suggested descriptions of IR vibrational modes of nanoparticles

[0243]

[0244] Example 6: Effect of different sizes of amphiphilic polymers

[0245] The effect of different sizes of the amphiphilic polymers in the nanoparticles of the present invention was investigated. SPIONs were coated with PMAcOD polymers having average molecular weights of 3100, 4800 and 5900 g / mol.

[0246] After these polymers were synthesized, purified and characterized, they were used for coating of oleate-SPIONs. After coating, the excess polymer was removed by TFF purification process.

[0247] a) Synthesis of poly(maleic acid-alt-1-octadecene) (PMAcOD) with different molecular weights

[0248] Three different lengths of poly(maleic acid-alt-1-octadecene) were synthesized via 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 an initiator. The polymerization was started by heating the mixture to 100 °C. Next, after purification, the polymer was hydrolyzed using sodium hydroxide solution to obtain PMAcOD.

[0249] By conducting the polymerization under more concentrated conditions and / or with lower amounts of initiator, polymers with higher molecular weights can be synthesized.

[0250] The amounts of reactants used in the synthesis of the three polymers are summarized in Table 7.

[0251] Table 7: Amounts of reactants

[0252]

[0253] The purity of the polymer after hydrolysis and post-treatment of maleic anhydride was determined by 1 H-NMR (400 Hz, 30 mg sample, 10 mg benzoic acid standard, 700 μL D-chloroform) was measured, and the length of the polymer was analyzed by gel optical layer (Agilent PL-gel mixed-D, 300 x 7.5 mm ID, 5 μm, 2 mg / mL in THF / acetic acid (90 / 10), 15 min run).

[0254] b) Effect of different lengths of amphiphilic polymers

[0255] PMAcOD with different lengths was used to coat Oleate-SPIONs.

[0256] First, the same polymer to SPION weight ratio was used for coating. In order to find the best coating conditions, the polymer to SPION molar ratio was kept constant. The amount of aggregates was compared. The method of minimum aggregates was repeated and then purified using TFF. Size exclusion chromatography (SEC) was used to monitor the removal of aggregates and the amount of aggregates during TFF.

[0257] Oleate-SPIONs were coated with PMAcOD3100, PMAcOD4800 and PMAcOD5900 to determine the optimal polymer / SPION ratio.

[0258] To coat Oleate-SPIONs, the polymer and nanoparticles were dissolved in chloroform. Subsequently, the chloroform was evaporated. In the last step of the coating process, an aqueous sodium hydroxide solution was added to the flask and mixed at 50°C. For PMAcOD3100, 100 mg of polymer was used. For PMAcOD4800, coatings with 100 mg of polymer and 145 mg of polymer were compared. For PMAcOD5900, coatings with 100 mg of polymer and 177 mg of polymer were compared. In this way, an equal amount of monomer was compared to an equal amount of polymer chains relative to PMAcOD3100.

[0259] By using size exclusion chromatography, determine which conditions result in the least amount of aggregation.

[0260] The results are summarized in Table 8.

[0261] Table 8: Summary of aggregates formed by SPIONs coated with different polymers

[0262]

[0263] c) Effect of different polymer lengths after purification

[0264] Oleate-SPIONs are coated with the same flow used before. For all polymers, the same amount of polymer chain is used. After coating, PMAcOD-SPION is purified by TFF. Due to the difficulty in removing the polymer PMAcOD5900 of final amount, 0.15g polymer (compared with the standard experiment of PMAcOD3100, equivalent to 0.79 equivalent) is therefore decided to also attempt to use.

[0265] SEC was used to determine how much aggregates were left in the samples and how much polymer. After coating, PMAcOD3100 contained 5.9% aggregates, PMAcOD4800 contained 6.2% aggregates, and PMAcOD5900 contained 10.7% aggregates. This matches the data described in the previous section.

[0266] Next, by TFF purification PMAcod-SPIONs.Particle is loaded on the film, then rinsed with 5mM NaOH / 45mMNaCl.Every 10 DVs (for PMAcOD3100) or 20 DVs (for PMACO4800 and PMAcOD5900) (diafiltration volume in the DV=TFF purification), by SEC (afterwards) extraction sample and measurement.In the TFF purification process, also extracted process sample, to follow the minimization of polymer and the increase of aggregation.

[0267] The results are summarized in Table 9.

[0268] Table 9: TFF Experiment Summary

[0269]

[0270] The TFF products of passes 1, 2 and 4 were filtered on a 0.2 μm filter. Pass 1 was also filtered on a 0.1 μm filter to reduce the amount of aggregates.

[0271] Dynamic light scattering (DLS) measurements were performed before and after sample filtration to determine the Z-average diameter and polydispersity index (PDI). All batches of PMAcOD-coated SPIONs showed a Z-average diameter of 26 to 28 nm and a PDI of 0.25 to 0.33 (see Table 11).

[0272] The amount of iron was determined using atomic absorption spectroscopy (AAS) to be between 38 and 50 mg, which corresponds to an iron recovery of 83 to 110%.

[0273] The results are summarized in Table 10.

[0274] Table 10: DLS data; before and after filtering.

[0275]

[0276]

[0277] Example 7: Comparison of purification of PMAcOD-SPION with polymers having different molecular weights

[0278] As described in Examples 1-4, SPIONs coated with commercial high molecular weight PMAcOD (Mn is 30,000 to 50,000 g / mol) and low molecular weight PMAcOD (Mn is 3,000 to 5,000 g / mol) were prepared. Afterwards, tangential flow filtration (TFF) purification was used to remove unbound materials. TFF is a preferred method for purifying coated SPIONs because it is easy to adjust the scale. Size exclusion chromatography (SEC) analysis was performed on the retentate and permeate samples to monitor the TFF purification process. The purification efficiency of SPIONs coated with high molecular weight and low molecular weight polymers was compared.

[0279] a) Purification of high molecular weight PMAcOD-SPIONs

[0280] In the first step, a sample of crude HM-PMAcOD-SPIONs was analyzed by SEC before purification by TFF (crude sample, see Fig. 9 The batch of crude HM-PMAcOD-SPIONs showed a very small shoulder to the left of the main HM-PMAcOD-SPION peak (RT: 15.541), indicating that the batch contained a small amount of large aggregates, with 21.2% (a / a) free unbound polymer in the dispersion (RT: 16.698).

[0281] Subsequently, the high molecular weight PMAcOD-SPION sample was filtered by TFF using a 300 kDa TFF membrane to separate the HM-PMAcOD-SPION after polymer coating from the unbound polymer molecules. Fig. 9 and 10 As shown, no separation was achieved, but the permeate after filtration contained both HM-PMAcOD-SPION and HM-PMAcOD polymers.

[0282] 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 between SPIONs and polymer without losing too much product. Lowering the pore size of the membrane to 100 kDa allowed retention of the polymer-coated SPIONs while simultaneously obtaining retention and concentration of the polymer (e.g. Fig.11 shown).

[0283] In summary, it was found that neither filtration with a 300 kDa TFF membrane nor filtration with a 100 kDa TFF membrane resulted in the purification of HM-PMAcOD-SPION from the unbound HM-PMAcOD material.

[0284] b) Purification of low molecular weight PMAcOD-coated SPIONs

[0285] In the second method, low molecular weight PMAcOD coated SPIONs were purified by TFF (100 kDa filter membrane). Again, size exclusion chromatography was performed on the retentate and permeate samples to monitor the TFF purification process. The results of the two batches (Batch MX0373A and MX0374A) are shown in Fig.12 shown.

[0286] The use of LM-PMAcOD (MN of 3,000 to 5,000 g / mol) showed that unbound polymer diffused efficiently across the 100 kDa TFF membrane and removed more than 90% of unbound polymer from LM-PMAcOD-SPION.

[0287] 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.13 .

[0288] Additionally, SEC data for samples after TFF purification (100 kDa filter; batches MX0373E and MX0374E) and SEC data for samples after TFF purification followed by 0.1 μM filtration (batches MX0373F and MX0374F) are shown in Table 12 below.

[0289] Table 12:

[0290]

[0291]

[0292] From the data presented, it can be seen that LM-PMAcOD-SPIONs can be efficiently purified by TFF, however, the purification efficiency of HM-PMAcOD-SPIONs is low and no pure product can be obtained.

[0293] Example 8: In vivo safety of intravenous injection of low molecular weight polymer micelles containing iron oxide cores compared with high molecular weight polymer micelles containing iron oxide cores in mice

[0294] a) Batch MX0194 produced in Example 3b was injected intravenously in female CD1 mice at doses of 1 mmol Fe / kg bw and 2 mmol Fe / kg bw. One mouse died at 2 mmol Fe / kg, defined as related to the test item. Mice were euthanized 14 days after injection. Absolute organ weights increased significantly compared to matched controls (see Table 13), which was defined as an adverse effect of the test item injection.

[0295] b) PMAcOD-SPION batches produced according to Example 3a were injected 3 times in female CD1 mice at a dose of 1 mmol Fe / kg BW (3 mmol Fe / kg bw in total), 14 days between injections 1 and 2 and injections 2 and 3. Animals were euthanized within 24 hours after the last injection. Only a slight but non-significant increase in liver weight was found, with no increase in lung weight (see Table 13).

[0296] The effect of high molecular weight polymer based nanoparticles on organ weights is related to the long term adverse effects of high molecular weight polymers since the iron cores were similar in the batches produced in Examples 8a and 8b.

[0297] The results of the comparative study are summarized in Table 13.

[0298] Table 13:

[0299]

[0300] This suggests that low molecular weight polymers are less toxic than high molecular weight polymers.

Claims

1. A nanoparticle comprising: a) micelles comprising an amphiphilic polymer having a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, wherein the amphiphilic polymer is an alternating copolymer of maleic anhydride and an olefin, and b) a peptide comprising a T cell epitope, and The nanoparticles have a hydrodynamic diameter of 20 to 40 nm as measured by dynamic light scattering (DLS).

2. Nanoparticles according to claim 1, wherein the nanoparticles comprise a solid hydrophobic core partially or fully coated by the micelles, wherein the core comprises a traceable inorganic material selected from 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. The nanoparticle according to claim 1 or 2, wherein the amphiphilic polymer has a hydrophilic polymaleic anhydride backbone containing hydrophobic alkyl side chains.

5. The nanoparticle of claim 1 or 2, wherein the amphiphilic polymer comprises the following building blocks: Wherein R is a hydrocarbon group or a substituted hydrocarbon group.

6. The nanoparticle according to claim 5, wherein R is C4 to C 22 alkyl.

7. The nanoparticle according to claim 5, wherein R is C8 to C 20 alkyl. The nanoparticle according to claim 5 , wherein R is a linear alkyl group.

9. The nanoparticle according to claim 8, wherein R is a linear C 11 To C 17 alkyl.

10. The nanoparticle according to claim 8, wherein R is a linear pentadecyl group.

11. The nanoparticle of claim 1 or 2, wherein the amphiphilic polymer is selected from poly(maleic acid-alt-1-octadecene), poly(maleic acid-alt-1-dodecene) and poly(maleic acid-alt-1-tetradecene).

12. The nanoparticle of claim 1 or 2, wherein the polymer is poly(maleic acid-alt-1-octadecene), and the number average molecular weight of the polymer is 6,000 to 1,000 g / mol.

13. The nanoparticle of claim 1 or 2, wherein the peptide is covalently linked to the micelle.

14. The nanoparticle of claim 13, wherein the peptide is covalently linked to the micelle via carbodiimide or succinimide coupling.

15. The nanoparticle of claim 1 or 2, wherein the peptide is non-covalently bound.

16. The nanoparticle of claim 1 or 2, wherein the peptide comprises 9 to 60 amino acids.

17. The nanoparticle of claim 16, wherein the peptide comprises 10 to 20 amino acids.

18. The nanoparticle according to claim 1 or 2, wherein the nanoparticle is negatively charged at a pH of 6 to 7.

19. A pharmaceutical composition comprising the nanoparticles according to any one of claims 1 to 18.

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

21. A pharmaceutical composition for suppressing a specific immune response according to claim 20, wherein the response is associated with an autoimmune disease.

22. The pharmaceutical composition for suppressing a specific immune response according to claim 1, wherein the immune disease is selected from the group consisting of: pemphigus vulgaris, pemphigus foliaceus, epidermolysis bullosa, bullous pemphigoid, cicatricial pemphigoid, Goodpasture's syndrome, microscopic polyangiitis, granulomatosis with polyangiitis (Granulom. Wegener), 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, autoimmune Addison's disease, autoimmune hemolytic anemia, chronic inflammatory demyelination, polyneuropathy, Guillain-Barré syndrome, autoimmune neutropenia, linear morphea, Batten disease, acquired hemophilia A, relapsing polychondritis, Isaac syndrome (acquired neuromyotonia), Rasmussen encephalitis, Morvan syndrome, stiff-man 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, Sjögren's syndrome, scleroderma, vitiligo, and alopecia areata.

23. A method for preparing nanoparticles, comprising: i) obtaining an amphiphilic polymer having a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, wherein the amphiphilic polymer is an alternating copolymer of maleic anhydride and an olefin, ii) forming micelles of said amphiphilic polymer, and iii) adding peptides to form said nanoparticles; The method described herein generates nanoparticles having a hydrodynamic diameter of 20 to 40 nm as measured by dynamic light scattering (DLS).

24. The method of claim 23, comprising purifying the amphiphilic polymer prior to step (ii).

25. The process according to claim 23 or 24, wherein step i) is a free radical copolymerization step.

26. The process according to claim 25, wherein 2,2'-azobis(2-methylpropionitrile) is used as free radical initiator.

27. A composition of nanoparticles, wherein each of the nanoparticles independently comprises: a) micelles comprising an amphiphilic polymer having a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, wherein the amphiphilic polymer is an alternating copolymer of maleic anhydride and an olefin, and b) a peptide comprising a T cell epitope, and The nanoparticles of the composition have a z-average hydrodynamic diameter of 20 to 40 nm as measured by dynamic light scattering (DLS).

28. The method of claim 27, wherein the nanoparticles have a polydispersity index of 0.10 to 0.40 as measured by dynamic light scattering (DLS).

29. The method of claim 27, wherein the polymer is poly(maleic acid-alt-1-octadecene), and The polymer has a number average molecular weight of 6,000 to 1,000 g / mol.

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