Nanoparticles containing peptides with N-terminal linkers

Abstract

The invention provides nanoparticles comprising: (a) an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less; and (b) a peptide covalently linked to the polymer, the peptide comprising 8-50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope. The invention further includes compositions comprising the respective nanoparticles and a liquid or lyophilized carrier for use in inducing tolerance to therapeutic compounds (proteins, viral vectors, lipid vesicles), allergens or autoantigens, or for use in treating allergy, autoimmune disease, exogenous antigens (transplantation antigens, drugs), or food intolerance, as well as the nanoparticles and compositions of the invention.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides nanoparticles for use in the prevention and treatment of autoimmune diseases, allergies, anti-drug antibodies, or other chronic inflammatory conditions. These nanoparticles induce antigen-specific immune tolerance by exploiting the liver's innate immune capabilities, targeting liver sinusoidal endothelial cells (LSECs), which generate tolerance to blood-borne antigens. These nanoparticles comprise amphiphilic polymers with a number-average molecular weight (Mn) of 20,000 g / mol or less.

[0002] The present invention particularly relates to nanoparticles comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less; and a peptide covalently linked to the polymer, wherein the peptide comprises 8-50 amino acids, including an N-terminal linker sequence comprising at least one arginine amino acid residue, and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope.

[0003] The invention further relates to compositions comprising these nanoparticles.

[0004] The invention further relates to compositions comprising nanoparticles and a liquid or lyophilized carrier for use in inducing tolerance to therapeutic compounds (proteins, viral vectors, lipid vesicles), allergens or to self-antigens, or for use in treating allergies, autoimmune diseases, exogenous antigens (transplantation antigens, drugs) or food intolerance. [Background technology]

[0005] BACKGROUND OF THEINVENTION 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 down-regulate harmful immune responses to foreign protein antigens or self-antigens.

[0006] Antigen presentation within the uniquely tolerogenic environment of the liver is designed to maintain immune tolerance and homeostasis. This is achieved through various mechanisms of tolerance in the liver, including both intrinsic and active regulatory mechanisms, including the maintenance and elimination of activated T cells and the induction of regulatory T cells.

[0007] Ectopic expression of a neuronal cell-derived antigen in the liver can prevent autoimmune neuroinflammation in mouse experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

[0008] To direct antigens to the liver for effective uptake and tolerogenic presentation, the highly effective capture function of liver sinusoidal endothelial cells (LSECs) can be used to remove blood-borne antigens. To this end, antigenic peptides are conjugated to small (<200 nm) nanoparticles designed to mimic blood-borne antigens. These injected nanoparticle conjugates, like blood-borne antigens, target the liver, where they are first taken up by LSECs. Proof-of-concept studies in several mechanistic and disease animal models for several indications have demonstrated effective immune modulation for both MHC class I-restricted and class II-restricted peptides mediated by tolerogenic nanoparticle-peptide conjugates.

[0009] Nanoparticles with disease-specific antigenic peptides bound to their particle surface are targeted to the liver after intravenous injection and are primarily internalized by LSECs, similar to blood-borne antigens. Once internalized and processed, these peptides bind to MHC / HLA molecules and are presented on the cell surface, where these peptide / MHC complexes are recognized by specific T cells. Within the tolerogenic environment of the liver, this T cell antigen recognition leads to T cell tolerance. More extensive immune tolerance can be induced by applying a mixture of nanoparticles bound to different antigenic peptides. Of note, these can be derived from a single immunogenic protein (such as desmoglein-3) or from different proteins (e.g., the gluten proteins gliadin, glutenin, or hordein).

[0010] Using nanoparticles conjugated with disease-specific antigenic peptides, it has been shown in several different animal models that both MHC / HLA-class I and class II restricted peptides can induce tolerance in an antigen-specific manner, thereby preventing or ameliorating disease or unwanted immune responses.

[0011] For these purposes, nanoparticles can be used that contain an amphiphilic polymer shell, which forms a micellar structure with a surface structure that allows the covalent attachment of autoantigenic peptides.

[0012] 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 for treating or preventing a disease in which suppression of a particular immune response is beneficial. The nanoparticles comprise micelles comprising an amphiphilic polymer and a peptide comprising at least one T cell epitope bound to the exterior of the micelle.

[0013] EP 20157797.0 relates to nanoparticles comprising micelles comprising an amphiphilic polymer with a number average molecular weight (Mn) of less than or equal to 20,000 g / mol and at least one peptide comprising at least one T-cell epitope.

[0014] However, it was found that it was not possible to produce all combinations of peptide nanoparticles with high density of peptides coupled to the surface of the nanoparticles.For certain medical applications, it is important to be able to produce nanoparticles with very high density of peptide loading as well as high purity using efficient purification methods.

[0015] Thus, 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, e.g., in autoimmune diseases, in allergies, in transplantation, in suppressing anti-drug antibodies (ADA) against therapeutic agents or gene vectors, or in diseases in which inflammation is excessive, chronic, or deleterious, and in which the pharmaceutical composition is suitable for use in human subjects. Summary of the Invention

[0016] (Summary of the invention) According to the present invention, the above-mentioned problems are solved as follows: (a) an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less; (b) a peptide covalently linked to the polymer, the peptide comprising 8 to 50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope; This is solved by nanoparticles comprising:

[0017] In a related embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a) an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less; (b) a peptide covalently linked to the polymer, the peptide comprising 8 to 50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope, wherein the sequence of the peptide comprising the N-terminal linker sequence is [ka] (not) The present invention provides a nanoparticle comprising:

[0018] The nanoparticles can further comprise a solid hydrophobic core (eg, a superparamagnetic ionic core (SPION)) coated by the polymeric micelles, or they do not comprise a solid hydrophobic core.

[0019] The peptides can be covalently attached to carboxyl groups on the surface of the nanoparticle micelles via an N-terminal linker.

[0020] The inventors have surprisingly discovered that using an N-terminal linker sequence comprising at least one arginine amino acid residue to a peptide sequence comprising a T cell receptor epitope is beneficial in increasing the coupling of the peptide on the surface of the nanoparticle.

[0021] It is now known that as a result of nanoparticle uptake by LSECs, at least one peptide bound to the outside of the nanoparticle, containing an MHC-binding sequence that includes a T cell receptor epitope, is released, presumably proteolytically, in the endosome, processed as if it were a blood-borne antigen, and presented to T cells in a tolerogenic environment.

[0022] Surprisingly, it has been found that the yield of peptide coupling onto the surface of nanoparticles improves when its isoelectric point (IEP) is higher than 6. Thus, in one aspect, the invention provides nanoparticles in which a peptide without an N-terminal linker sequence has an IEP lower than 6. In a related embodiment, the addition of an N-terminal linker sequence to a peptide results in an increase in the IEP of the peptide, preferably to an isoelectric point of greater than 6, greater than 7, greater than 8, or greater than 9.

[0023] Moreover, the inventors have surprisingly discovered that the addition of an N-terminal linker sequence to a peptide having an IEP lower than 6 results in an increase in the IEP of the peptide. In this way, the peptide becomes more positively charged, which facilitates coupling to a nanoparticle. Thus, in one aspect, the invention provides a nanoparticle comprising a peptide having an IEP lower than 6 without an N-terminal linker sequence. In a related embodiment, the nanoparticle comprises a peptide comprising a linker of at least one Arg amino acid residue and has an IEP greater than 6, greater than 7, greater than 8, or greater than 9.

[0024] Computational analysis further reveals that this N-terminal linker sequence can increase the binding affinity of certain peptides to MHC / HLA molecules.

[0025] Thus, the present invention further provides methods for optimizing the production and improving the functionality of tolerogenic nanoparticle peptide conjugates. [Brief description of the drawings]

[0026] [Figure 1] 1 is a schematic structure of an exemplary nanoparticle. [Diagram 2] FIG. 2 is a flow chart of the synthesis of iron oleate complex; note that in FIG. 2 (as well as in FIGS. 3-5, 7, and 8), the light blue arrows on the left side of the diagram indicate synthesis steps, whereas the dark blue arrows on the right side of the diagram indicate purification steps. [Diagram 3]1 is a flowchart of SPION synthesis. [Figure 4] 1 is a flow chart of LM-PMAOD synthesis. [Diagram 5] 1 is a flow chart of LM-PMAcOD synthesis. [Figure 6] This is a polymer coating of SPIONs. [Figure 7] 1 is a flow chart of PMAcOD-SPION particle synthesis. [Figure 8] 1 is a flow chart of peptide coupling and nanoparticle synthesis. [Figure 9] SDS PAGE gel of peptide coupling (Example 2) using Bolt 12% Bis-Tris Plus gel (Invitrogen). [Figure 10] SDS PAGE gel of peptide coupling (Example 3, 2,5 hours) using Bolt 12% Bis-Tris Plus gel (Invitrogen). [Figure 11] SDS PAGE gel of peptide coupling (Example 3, 16 hours) using Bolt 12% Bis-Tris Plus gel (Invitrogen). [Figure 12] N-Terminal Modification of an Agonistic CD8 T Cell Epitope with One or Two Arginines Retains Peptide Agonist Properties [Figure 13] N-Terminal Modification of an Agonistic CD4 T Cell Epitope with One or Two Arginines Retains Peptide Agonist Properties [Figure 14] N-Terminal Modification of Autoantigen-Derived CD4 T Cell Epitopes with One or Two Arginines Retains Peptide Agonist Properties DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention 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; and a peptide covalently linked to the polymer, the peptide comprising 8 to 50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue, and an MHC binding sequence comprising a T cell receptor epitope.

[0028] In one alternative, the sequence of the peptide including the N-terminal linker sequence is [ka] isn't it.

[0029] According to the present application, the term "nanoparticles" is used interchangeably with "nanoscale particles". Such particles have a diameter of 1-999 nm, preferably 2-600 nm, 5-500 nm, 10-300 nm, 30-100 nm, or 40-50 nm.

[0030] In the context of the present invention, a nanoparticle is a structure formed by at least one micelle and a peptide bound to the micelle, the peptide being either bound to the outside of the micelle or encapsulated inside the micelle.

[0031] According to one embodiment of the invention, the nanoparticles comprise micelles comprising: an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less; and a peptide covalently linked to the polymer, the peptide comprising 8-50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue, and an MHC binding sequence comprising a T cell receptor epitope.

[0032] In one embodiment of the invention, the nanoparticles of the 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, and a peptide covalently linked to the polymer, the peptide comprising 8 to 50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and an MHC binding sequence comprising a T cell receptor epitope.

[0033] The nanoparticles of the present invention, which comprise a solid hydrophobic core and micelles coating the core, the micelles comprising an amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less, exhibit dissolution-like distribution behavior in aqueous liquids.

[0034] The peptide can be covalently attached to the polymer via an N-terminal linker.

[0035] In a preferred embodiment of the invention, the nanoparticles are a) The following building blocks: [ka] wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a straight chain alkyl group, preferably a straight chain C 11 ~C 17 is an alkyl group) 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 covalently linked to the polymer, the peptide comprising 8 to 50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope; c) a solid hydrophobic core at least partially coated by the micelles, said core comprising a traceable inorganic material selected from the group including iron oxide, CdSe / CdS / ZnS, silver, and gold; Includes.

[0036] In a particularly preferred embodiment, all peptides covalently linked to the polymer of one nanoparticle b) have the same amino acid sequence and are covalently attached to the outside of a micellar structure comprising an amphiphilic polymer shell a) composed of low molecular weight poly(maleic acid-alt-1-octadecene) and a superparamagnetic iron oxide nanoparticle (SPION) core c) (schematic structure shown in Figure 1).

[0037] The different components of the nanoparticles are described in more detail in the following sections.

[0038] (Micell) In the context of the present invention, the term "micelle" refers to an aggregate of amphiphilic molecules dispersed in an aqueous solution. The hydrophilic parts of the amphiphilic molecules are in contact with the surrounding solvent, while the hydrophobic "tail" regions of the amphiphilic molecules are sequestered inside the micelle, thus resulting in a dissolution-like distribution behavior of the nanoparticles in aqueous liquids, i.e., the nanoparticles are water-soluble. This type of micelle is also known as normal phase micelles (or oil-in-water micelles).

[0039] Micelles can be formed by one, but also by more than one, e.g., two, three, or four, amphiphilic polymers. Micelles can be formed by the same or different amphiphilic polymers. In general, in the context of this specification, "a" or "the" is not intended to be limited to "one" unless specifically stated.

[0040] In a preferred embodiment, the micelles are formed by a single layer of amphiphilic polymers.

[0041] Such micelles may be structurally distinct from bilayers or liposomes formed by amphiphilic polymers, in which case these structures are not included in the nanoparticles of the invention, or are not included to a significant extent (e.g., not more than 10%, not more than 5%, or preferably not more than 1%).

[0042] In one embodiment of the invention, at least 70%, preferably at least 90%, of the micelles are formed using amphiphilic polymers, hi a preferred embodiment, the micelles are composed of amphiphilic polymers.

[0043] In some embodiments of the invention, the nanoparticles do not include a solid hydrophobic core, hi other embodiments, the nanoparticles include micelles and a solid hydrophobic core.

[0044] In one embodiment of the invention, the micelles can be co-stabilized with further components such as fatty acids or phosphatidylcholine. In this regard, preferred fatty acids are stearic acid or oleic acid and a preferred phosphatidylcholine is Lipoid S100. Cholesterol can also be used as a co-stabilizer.

[0045] (Amphiphilic polymer) 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.

[0046] The hydrophilic regions of the amphiphilic polymer can be negatively charged in aqueous solution.

[0047] In a preferred embodiment of the 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, which results in a dissolution-like distribution behavior of the nanoparticles in aqueous liquids, i.e., the nanoparticles become water-soluble.

[0048] 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.

[0049] Number average molecular weight can be determined using gel permeation chromatography (GPC), preferably using polystyrene as a calibration standard.

[0050] 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 composed of tetrahydrofuran / acetic acid 90 / 10% (v / v), and a flow rate of 1.0 ml / min, in combination with a refractive index detector at a temperature of 35° C. and polystyrene as a calibration standard.

[0051] In a most preferred embodiment, the determination of the number average molecular weight uses GPC and the following measurement conditions:

[0052] [Table 1]

[0053] The amphiphilic polymer may be an alternating copolymer, which is a copolymer that contains two types of monomer units distributed side by side.

[0054] In one embodiment of the invention, the amphiphilic polymer is a copolymer of maleic anhydride and at least one alkene.

[0055] 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.

[0056] In a preferred embodiment of the invention, the amphiphilic polymer is a copolymer of maleic anhydride and an alkene.

[0057] In a preferred embodiment of the invention, the amphiphilic polymer has a hydrophilic polymaleic anhydride backbone with hydrophobic alkyl side chains. Typically, the side chains can have from 5 to 23 carbon atoms, especially from 9 to 21 atoms. In a most preferred embodiment, the side chains are linear and have from 10 to 18 carbon atoms.

[0058] Amphiphilic polymers are made up of the following building blocks: [ka] where 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, e.g. C7-C 19 It is an alkyl group.

[0059] 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.

[0060] The amphiphilic polymer may be composed of building blocks as defined above.

[0061] In another embodiment according to the invention, the amphiphilic polymer comprises at least 50%, preferably at least 70%, most preferably more than 90% of constitutional units as defined above.

[0062] 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 between 6,000 and 1,000 g / mol.

[0063] 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.

[0064] (peptide) The nanoparticles of the invention further comprise a peptide covalently linked to the polymer, said peptide comprising 8 to 50 amino acids, said peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope.

[0065] The peptide can be covalently linked to the polymer via an N-terminal linker.

[0066] In a preferred embodiment, the peptide is covalently linked to the polymer using methods known in the art for covalently coupling peptides, such as carbodiimide or succinimide coupling. Preferably, the peptide is covalently linked to the polymer using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry.

[0067] In a preferred embodiment of the invention, the peptide comprises 10 to 30, such as 11 to 25 or 12 to 24 amino acids.

[0068] In a preferred embodiment of the present invention, the sequence comprising an MHC-binding sequence comprising a T cell receptor epitope comprises 11 to 22 amino acids. In a particularly preferred embodiment, the sequence comprising an MHC-binding sequence comprising a T cell receptor epitope comprises 13 to 21, preferably 15, amino acids.

[0069] In a preferred embodiment of the invention, the N-terminal linker sequence comprises at least two Arg amino acid residues. In a particularly preferred embodiment, the N-terminal linker sequence comprises two Arg amino acid residues.

[0070] In a preferred embodiment of the invention, peptides comprising a linker of at least one Arg amino acid residue have an isoelectric point greater than 6, greater than 7, greater than 8, or greater than 9. The isoelectric point is the pH at which the net charge of the peptide is zero.

[0071] The net charge Z of a peptide at a given pH is given by the formula:

number

[0072] The isoelectric point can be calculated using online tools such as Innovagen's Peptide Calculator.

[0073] The peptide comprises a sequence that comprises an MHC binding sequence that comprises a T cell receptor epitope.Methods for identifying MHC binding sequences that comprise T cell receptor epitopes are known in the art and are described, for example, in Sidney, Peters, Sette et al., Semin Immunol 2020, Aug;50:101418. doi: 10.1016 / j.smim.2020.101418.

[0074] In the context of the present invention, an MHC binding sequence is a peptide sequence which binds to an MHC molecule.

[0075] In the context of the present invention, T cell epitope is an agonistic peptide sequence that can activate T cells.At least one epitope must be able to be presented by the cells of the subject to which the nanoparticles are administered.Preferably, the peptide comprises several epitopes that can be presented in multiple major histocompatibility complex types.

[0076] Peptide presentation by MHC class II molecules is of particular interest for the induction of CD4+ regulatory T cells.In addition, MHC-class I restricted CD8 T cells are also regulated in the liver by peptides presented by MHC class I.The HLA type of a subject, for example a human subject, can be easily examined as part of epitope selection.The epitopes of a particular peptide that may be presented on a particular MHC molecule are known and / or can be selected by computer, for example by suitable algorithms.

[0077] The peptide is designed based on published data to ensure that it binds with high affinity to MHC / HLA - with respect to the designated HLA restriction element - and highly stimulates and activates T cells. Ideally, the optimal peptide is deduced from a naturally processed peptide and characterized as immunodominant.

[0078] The peptide can be synthetic or recombinantly expressed, or isolated or modified from natural sources. The peptide or at least its epitope against which T cell tolerance is induced is preferably derived from a peptide / protein against which an inflammatory immune response is suppressed, for example in the context of treating or preventing an autoimmune disease or allergy. The peptide can be, for example, an allergen, a known autoimmune antigen, or a fragment or derivative thereof. The peptide can combine different epitopes from different antigens.

[0079] In a preferred embodiment of the invention, the peptide comprises an N-terminal linker comprising at least one Arg amino acid residue; [ka] and a peptide sequence selected from the group consisting of:

[0080] In a related embodiment of the invention, the nanoparticle comprises a peptide sequence linked to a polymer via an N-terminal linker. [ka] The peptides include those comprising N-terminal linkers each composed of two Arg amino acid residues, such as selected from the group consisting of:

[0081] In one embodiment, the nanoparticles of the invention comprise a peptide, wherein the peptide is: (a) an N-terminal linker comprising at least one Arg amino acid residue; [ka] or (b) [ka] is selected from the group consisting of:

[0082] In another embodiment, the nanoparticles of the invention comprise a peptide, wherein the peptide is: (a) an N-terminal linker comprising at least one Arg amino acid residue; [ka] or (b) [ka] is selected from the group consisting of:

[0083] In a further aspect, the nanoparticle of the invention comprises a peptide, wherein the peptide is: (a) an N-terminal linker comprising at least one Arg amino acid residue; [ka] or (b) [ka] is selected from the group consisting of:

[0084] (Solid hydrophobic core) In one embodiment of the invention, the nanoparticles comprise a solid hydrophobic core at least partially coated with a polymer.

[0085] The core may be an inorganic core, preferably comprising iron oxide, CdSe, silver, or gold.

[0086] The core diameter can be 2 to 500 nm, preferably 3 to 25 nm, and more preferably 5 to 15 nm. The core diameter can be determined using transmission electron microscopy (TEM) or small angle X-ray scattering (SAXS).

[0087] 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 trioctyloxinphosphinoxide), and gold nanoparticles stabilized by, for example, sulfone compounds.

[0088] By themselves, such inorganic cores are typically not stable in aqueous solvents such as water, but by embedding the inorganic cores in polymeric micelles, they become 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 around the core. In the coating process, the amphiphilic polymer can displace the hydrophobic portion of the core by ligand exchange, thus forming bilayer micelles around the core. In one embodiment of the invention, the polymer at least partially displaces 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, forming bilayer micelles around the iron oxide core, resulting in polymer-coated iron oxide.

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

[0090] In a particularly preferred embodiment of the invention, the core is a superparamagnetic iron oxide nanoparticle (SPION), which can be stabilized by oleic acid.

[0091] The core preferably makes the nanoparticle of the invention traceable, for example by its characteristics in fluorescence, electron microscopy, or other detection methods.

[0092] (Nanoparticles) The inventors have discovered that the nanoparticles for use in the present invention are suitable for transferring the peptide to liver sinusoidal wall endothelial cells of a subject in vivo.

[0093] The nanoparticles may further comprise a moiety, for example a carbohydrate or a protein, that targets or facilitates targeting to specific cells, such as liver sinusoidal endothelial cells and / or Kupffer cells. Such a moiety may 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.

[0094] The nanoparticles of the present invention may have a hydrodynamic diameter (z-average) of 10-100 nm or 10-70, preferably 10-50, more preferably 20-40 nm, and most preferably 26-36 nm, as measured by dynamic light scattering (DLS).

[0095] The nanoparticles of the present invention may have a polydispersity index of less than 0.50, preferably between 0.05 and 0.45, and more preferably between 0.10 and 0.40, as measured by dynamic light scattering (DLS).

[0096] The determination of hydrodynamic diameter and polydispersity index is carried out using electrophoretic light scattering analytical method, preferably Zetasizer by Malvern. In one embodiment, the method for determining hydrodynamic diameter and polydispersity index is carried out using electrophoretic light scattering method, disposable polystyrene cuvettes, Zetasizer software 7.12, Milli-Q water. 20 nm and 100 nm nanosphere size standards (NIST certified or equivalent) are diluted in 0.9% aqueous sodium chloride solution, and test samples are diluted in water. All aqueous reagents are filtered through 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 carried out using electrophoretic light scattering method in combination with the following analytical conditions:

[0097] List of analytical conditions: [Table 2]

[0098] The evaluation of the data is based on the mean diameter (z-average, nm, by intensity), a parameter also known in DLS as the cumulants mean and polydispersity index (PDI), used as a measure of size distribution.

[0099] The nanoparticles of the present invention contain a large amount of peptides covalently linked to a polymer. Specifically, the nanoparticles of the present invention can have a total peptide content, as determined by GC / MS, of greater than 0.1 mg / mL, preferably greater than 0.5-4 mg / mL, more preferably greater than 1 mg / mL.

[0100] Capillary gas chromatography with chiral stationary phase is used for the separation of all proteinogenic amino acids with their enantiomers and most non-proteinogenic amino acids. Quantitative amino acid analysis is carried out by enantiomeric labeling. In this case, the optical antipode of the amino acid is added to the sample before the analysis. Thus, the enantiomeric purity of the sample and the standard is taken into account.

[0101] A mixture of all amino acids of the peptide is added to the sample at equal concentrations. A second sample is prepared without amino acid standards. The dried sample is hydrolyzed in concentrated HCl containing thioglycolic acid at 110° C. in vacuum for 48 hours. After 48 hours, the HCl is removed in a Speed-Vac. After hydrolysis, the sample is separated from the matrix using solid phase extraction. The amino acids are then purified, esterified with HCl (in ethanol), purified again, dried, and the residue is dissolved in dichloromethane and injected into the GC / MS.

[0102] List of analytical conditions: [Table 3]

[0103] In a preferred embodiment, the peptide has a peptide content as determined by a BCA assay of greater than 0.8 mg / mL, preferably greater than 1 mg / ml, more preferably greater than 1.5 mg / ml.

[0104] The BCA assay is a commercially available kit from Sigma Aldrich that determines the amount of peptide in a sample. Peptide standards are prepared from the same peptide batch used for nanoparticle coupling using the following peptide concentrations (0, 0.020, 0.040, 0.060, 0.080, and 0.100 mg / mL). Samples (peptide-coupled nanoparticles) and standards are mixed with the BCA reagent. All samples and standards are then incubated at 60° C. for 15 minutes, followed by centrifugation at 12000 rpm for 10 minutes. All samples and standards are then measured on a plate reader using absorbance at 562 nm. The absorbance of the standards is used to plot a calibration curve (absorbance vs. concentration). A linear regression is calculated to calculate the concentration of the samples.

[0105] (composition) The present invention further provides a composition comprising the nanoparticles of the present invention and a liquid or lyophilized carrier.

[0106] In a preferred embodiment, the composition comprises the nanoparticles of the present invention in a liquid carrier. The liquid carrier is preferably water or a water-based solution, such as a buffer solution, such as phosphate buffered saline (PBS), Ringer's solution, TRIS buffer, or sodium chloride solution. It may or may not contain a suitable preservative.

[0107] The peptides used may be present in the composition at a concentration of 0.01 to 2 mM, preferably 0.1 to 1 mM, most preferably 0.45 mM to 1 mM.

[0108] In particular, for administration to human subjects, it will be appreciated that the compositions are preferably sterile and biologically compatible.

[0109] In a preferred embodiment of the invention, the composition comprises the nanoparticles of the invention dispersed in D-mannitol, TRIS, and / or L-lactic acid.

[0110] Moreover, the composition can contain two or more types of nanoparticles of the present invention, where different types of nanoparticles have different peptides covalently linked to the polymer.By using a mixture of nanoparticles, it is possible to induce broader immune tolerance by several autoantigenic peptides at the same time.These peptides can be derived from a single immunogenic protein or from different proteins.

[0111] In a preferred embodiment of the invention, the composition comprises at least two different types of nanoparticles, each type comprising at least one peptide sequence that differs from the peptide sequence(s) of the other type of nanoparticle.

[0112] In a preferred embodiment of the invention, the composition comprises at least two different types of nanoparticles, each type comprising a peptide sequence having a specific peptide sequence that differs from the peptide sequence of the other type of nanoparticle.

[0113] In a particularly preferred embodiment, the composition comprises five different types of nanoparticles, where: (a) each type of nanoparticle comprises an N-terminal linker of at least one Arg amino acid residue; [ka] and a specific peptide sequence selected from the group consisting of: (b) Each type of nanoparticle is [ka] The peptides include identical peptides having a sequence selected from the group consisting of:

[0114] In another particularly preferred embodiment, the composition comprises three different types of nanoparticles, where: (a) each type of nanoparticle comprises an N-terminal linker of at least one Arg amino acid residue; [ka] and a specific peptide sequence selected from the group consisting of: (b) Each type of nanoparticle is [ka] The peptides include identical peptides having a sequence selected from the group consisting of:

[0115] In a particularly preferred embodiment, the composition comprises four different types of nanoparticles, where: (a) each type of nanoparticle comprises an N-terminal linker of at least one Arg amino acid residue; [ka] and a specific peptide sequence selected from the group consisting of: (b) Each type of nanoparticle is [ka] The peptides include identical peptides having a sequence selected from the group consisting of:

[0116] The composition may contain nanoparticles at a concentration of less than 100 μM, preferably 0.5 to 80 μM, and most preferably 1 to 50 μM. If more than one type of nanoparticle is present in the composition, each may be present at a concentration of less than 100 μM, preferably 0.5 to 80 μM, and more preferably 1 to 50 μM.

[0117] The compositions of the invention can contain equimolar concentrations of different types of nanoparticles.

[0118] Thus, in one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) The following building blocks: [ka] wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a straight chain alkyl group, preferably a straight chain C 11 ~C 17 is an alkyl group) a micelle comprising an amphiphilic polymer having a number average molecular weight (Mn) of 6,000 to 1,000 g / mol; b) a peptide covalently linked to the polymer, the peptide comprising 8 to 50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope; c) a solid hydrophobic core at least partially coated by the micelles, said core comprising a traceable inorganic material selected from the group including iron oxide, CdSe / CdS / ZnS, silver, and gold; The present invention provides a composition comprising nanoparticles comprising:

[0119] In a preferred embodiment, the present invention provides a nanoparticle comprising: a) The following building blocks: [ka] wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a straight chain alkyl group, preferably a straight chain C 11 ~C 17 is an alkyl group) a micelle comprising an amphiphilic polymer having a number average molecular weight (Mn) of 6,000 to 1,000 g / mol; b) a peptide covalently linked to the polymer, the peptide comprising 8 to 50 amino acids, the peptide comprising an N-terminal linker sequence comprising at least one Arg amino acid residue and a sequence comprising an MHC binding sequence comprising a T cell receptor epitope; c) a solid hydrophobic core at least partially coated by the micelles, said core comprising a traceable inorganic material selected from the group including iron oxide, CdSe / CdS / ZnS, silver, and gold; wherein the peptide sequences of the at least two different types of nanoparticles differ from one another, and the different peptides comprise an N-terminal linker comprising at least one Arg amino acid residue; [ka] and a peptide sequence selected from the group consisting of:

[0120] (Use of the composition) The compositions of the invention, comprising the nanoparticles of the invention in a liquid carrier, can be used in inducing tolerance to therapeutic compounds (proteins, viral vectors, lipid vesicles), allergens or to self-antigens, or to treat allergies, autoimmune diseases, exogenous antigens (transplantation antigens, drugs), or food intolerance.

[0121] It can be formulated for administration to a subject having a disease in which suppression of a particular immune response is beneficial.

[0122] This pharmaceutical composition can be administered to a subject in need thereof. The dosage and concentration required for administration to a subject can be determined by a responsible medical attendant according to the facts and circumstances of the case. An exemplary dosage would include, for example, for a human subject, 0.03 μmol to 0.90 μmol per patient weight.

[0123] The administration can be repeated, for example, 2, 3, or 4 times, with administration intervals of, for example, 1, 2, 3, 4, 5, 6, 7, 10, or 14 days.

[0124] In a preferred embodiment, the composition comprises 2 to 8, preferably 2 to 6, different types of nanoparticles, each type comprising at least one peptide sequence that differs from the peptide sequence(s) of the other types of nanoparticles.

[0125] Preferably, the composition comprises at least two different types of nanoparticles, e.g., from 2 to 8 different types of nanoparticles, each type comprising at least one peptide sequence that differs from the peptide sequence(s) of the other type of nanoparticle.

[0126] More preferably, the composition comprises 3 to 6 different types of nanoparticles, each type comprising at least one peptide sequence that differs from the peptide sequence(s) of the other types of nanoparticles.

[0127] In a preferred embodiment, the composition comprises at least two different types of nanoparticles, e.g., from two to eight different types of nanoparticles, each type comprising a peptide sequence that differs from the peptide sequence of the other type of nanoparticle.

[0128] More preferably, the composition comprises 3 to 6 different types of nanoparticles, each type comprising a peptide sequence that differs from the peptide sequence(s) of the other types of nanoparticles.

[0129] In one embodiment, the present invention provides a composition for use in inducing tolerance to a celiac disease antigen, comprising: (a) a nanoparticle comprising different types of nanoparticles, each type of nanoparticle comprising at least one N-terminal linker of Arg amino acid residue; [ka] and a specific peptide sequence selected from the group consisting of: (b) comprising different types of nanoparticles, each type of nanoparticle comprising: [ka] The peptides each having a sequence selected from the group consisting of The composition is provided.

[0130] In an alternative embodiment, the present invention provides a composition for use in inducing tolerance to a celiac disease antigen, comprising: (a) a nanoparticle comprising different types of nanoparticles, each type of nanoparticle comprising at least one N-terminal linker of Arg amino acid residue; [ka] and a specific peptide sequence selected from the group consisting of: (b) comprising different types of nanoparticles, each type of nanoparticle comprising: [ka] The peptides each having a sequence selected from the group consisting of The composition is provided.

[0131] In a further embodiment, the present invention provides a composition for use in inducing tolerance to a pemphigus vulgaris antigen, comprising: (a) a nanoparticle comprising different types of nanoparticles, each type of nanoparticle comprising at least one N-terminal linker of Arg amino acid residue; [ka] and a specific peptide sequence selected from the group consisting of: (b) comprising different types of nanoparticles, each type of nanoparticle comprising: [ka] The peptides each having a sequence selected from the group consisting of The composition is provided.

[0132] 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 to be as defined by Hayter et al. (Autoimmunity Reviews 11 (2012) 754-765).

[0133] 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 (Wegener's granulomatosis), thrombotic thrombocytopenic purpura, immune thrombocytopenic purpura, uveitis, HLA-B27-associated acute anterior uveitis, multiple sclerosis, neuromyelitis optica, type I diabetes mellitus, 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, autoimmune Addison's disease, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre 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.

[0134] In a preferred embodiment, the allergy is selected from a peanut allergy, a pollen allergy, or a cat allergy.

[0135] In addition to tolerance induction to self-antigens in autoimmune diseases, Topas nanoparticles can be bound to alloantigen peptides to promote transplant tolerance, and to allergen-derived T cell epitopes for therapeutic intervention in food allergies, e.g. peanut allergy, and airborne allergies to pollen and animal hair components, e.g. cat allergy.

[0136] Additionally, Topas nanoparticles can bind T cell epitopes derived from biological drugs. The therapeutic compound can be a therapeutic protein, a therapeutic antibody, a viral vector, or a lipid vesicle.

[0137] In accordance with 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 identifiable measurements associated with a particular disorder.

[0138] (Method of producing nanoparticles) The nanoparticles of the present invention are a) obtaining hydrophobic core nanoparticles; b) obtaining an amphiphilic polymer having 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 the hydrophobic core nanoparticles with the amphiphilic polymer to form micelles; e) adding at least one peptide to form nanoparticles; The composition can be produced by a method comprising:

[0139] In embodiments of the invention where the peptide is encapsulated by a micelle, step e) is carried out before step d), in these embodiments the peptide is added to the amphiphilic polymer prior to micelle formation.

[0140] The hydrophobic core of step a) can be synthesized using suitable reactants in solution. Preferably, the hydrophobic core is synthesized using a metal salt and a salt of a carboxylic acid as reactants in the presence of an organic solvent. Preferably, the reaction is carried out at elevated temperature under oxygen limitation.

[0141] One method of obtaining amphiphilic polymers with number average molecular weights (Mn) of 20,000 g / mol or less results in their synthesis using a two-step method involving the formation of the polymer as an anhydride and the hydrolysis of the anhydride to obtain the acid.

[0142] The amphiphilic polymers used in the nanoparticles of the present invention can be prepared by radical copolymerization using a radical initiator.

[0143] 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.

[0144] The copolymerization can be carried out in an organic solvent such as 1,4 dioxane, xylene, or chlorobenzene.

[0145] Many radical initiators are known in the art; these include various peroxides and azo compounds. Examples of suitable peroxides are benzoyl peroxide, lauryl peroxide, di-t-butyl peroxide, 2,4-dichlorobenzyl peroxide, t-butyl hydroperoxide, cumene hydroperoxide, diacetyl peroxide, diethyl peroxycarbonate, perbenzoic acid, and t-butyl perborate. Suitable azo compounds include 2,2'-azobis(2-methylpropionitrile), p-bromobenzenediazonium borate fluoride, p-tolyldiazoaminobenzene, p-bromobenzenediazonium hydroxide, azomethane, and phenyldiazonium halides. Preferably, the radical initiator is 2,2'-azobis(2-methylpropionitrile).

[0146] The copolymerization can be carried out at a high temperature of, for example, 70 to 120°C, preferably 90 to 110°C.

[0147] Preferably, the copolymerization is initiated by heating the mixture to a temperature of from 70 to 120°C, preferably from 90 to 110°C.

[0148] Step b) can include mixing the reactants, deoxygenating the mixture, heating the mixture, and then cooling the mixture. The polymer can then be dissolved and stirred overnight. The solids formed can be collected, preferably using centrifugation.

[0149] Step b) may involve the addition of a base to the polymer (e.g. NaOH). The base is preferably reacted with the polymer at elevated temperature, preferably 50°C-70°C, e.g. 60°C, until almost all solids have dissolved. The resulting suspension may be acidified (e.g. pH<2). The reaction mixture may then be extracted with an organic solvent, such as ethyl acetate. The organic layer may be extracted with sodium hydroxide solution. The aqueous solution may be extracted again with an organic solvent, such as ethyl acetate, and then dried to obtain the purified amphiphilic polymer.

[0150] The polymer can be further purified (step c). Preferably, the polymer is further purified by extracting the polymer 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. Furthermore, an additional purification step of the amphiphilic polymer can be added. 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.

[0151] The purity of the amphiphilic polymer after hydrolysis and post-treatment was determined by: 1 It can be measured by H NMR.

[0152] The micelles can be formed by forming a solution containing the amphiphilic polymer (step d)). Preferably, the micelles are formed in an aqueous solution. A co-stabilizer can be added to the amphiphilic polymer to improve micelle formation. Preferably, step d) includes the substeps of solubilizing the amphiphilic polymer and the core particles, removing the solvent until a thin film is formed, adding a basic aqueous solution at elevated temperature and ambient pressure to form an aqueous colloidal dispersion, diluting the solution and optionally filtering it. Several washing steps can then be applied.

[0153] The peptides to be used in step e) can be synthesised using state-of-the-art solid phase chemistry.

[0154] In a preferred embodiment, the synthesis of peptides is accomplished in the C to N direction using solid phase peptide synthesis (SPPS) via Fmoc chemistry. The alpha amino group of each amino acid is protected with a fluoren-9-ylmethoxycarbonyl (Fmoc) group, while the side chain functional groups are also blocked with various suitable protecting groups. In general, SPPS consists of repeated cycles of N-terminal deprotection followed by coupling reactions. A first Fmoc-protected amino acid is coupled to the resin. The amine group is then deprotected with a mixture of piperidine in dimethylformamide (DMF) and then coupled with the free acid of the second Fmoc-protected amino acid. This cycle is repeated until the desired sequence is obtained. Between each step, the resin is washed. Completion of each coupling reaction is monitored by a qualitative ninhydrin test. In the last step of the synthesis, the crude peptide resin is washed successively with DMF and methanol and dried. The peptide is then cleaved from the resin using trifluoroacetic acid (TFA). The resulting crude peptide is isolated from the cleavage mixture by ether precipitation. The peptide is further purified through preparative HPLC to reach the purity requirements, and the counterion TFA is replaced with chloride ion by using an appropriate solvent-buffer system. Finally, the purified peptide is lyophilized.

[0155] In a preferred embodiment of the method of the invention, the peptide is coupled to the surface of the nanoparticles using peptide coupling techniques known in the art, such as carbodiimide or succinimide coupling.

[0156] In a particularly preferred embodiment of the method of the invention, the peptides are coupled to the surface of the nanoparticles via EDC chemistry (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in aqueous phase.

[0157] The resulting nanoparticles can be purified using extensive washing and filtration steps to remove the coupling reagent(s) and any low molecular weight components. EXAMPLES

[0158] (Example) The invention is illustrated by the following examples which detail the effectiveness of peptide coupling according to the invention.

[0159] These examples should not be considered as limiting the scope of the invention but as being illustrative.

[0160] Example 1: Preparation of nanoparticles (a) Preparation of superparamagnetic iron oxide crystalline cores (SPIONs) The synthesis of iron oleate complexes is shown diagrammatically in Figure 2. The synthesis of SPIONs is shown diagrammatically in Figure 3.

[0161] The iron oleate complex was dissolved in 1-octadecene with oleic acid at room temperature and stirred until complete dissolution. The solution was deoxygenated and dehydrated at 110°C, then heated at 300°C for the formation of iron oxide nanocrystals. After cooling, the product was purified by several washing steps with acetone and tetrahydrofuran using magnetic separation. The purified SPIONs were then diluted with chloroform, concentrated in a rotary evaporator, and finally diluted with chloroform for use in further manufacturing steps as described below.

[0162] In the second step (Figure 3), the oleate iron complex was dissolved in 1-octadecene with oleic acid at room temperature and stirred until complete dissolution. The solution was deoxygenated and dehydrated at 110°C, then heated at 300°C for the formation of iron oxide nanocrystals. After cooling, the product was purified by several washing steps with acetone and tetrahydrofuran using magnetic separation. The purified SPIONs were then diluted with chloroform, concentrated in a rotary evaporator, and finally diluted with chloroform for use in further manufacturing steps as described below.

[0163] (b) 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 diagrammatically in Figures 4 and 5 .

[0164] In the first step (Figure 4), copolymerization of 1-octadecene with maleic anhydride initiated by AIBN (2,2'-azobis(2-methylpropionitrile)) in 1,4-dioxane was achieved. The product was purified by coevaporation with dichloromethane and precipitation with isopropanol and acetonitrile to yield low molecular weight poly(maleic anhydride-alt-1-octadecene), i.e., LM-PMAOD, with number average molecular weight (Mn) of 2500-4000 g / mol.

[0165] In the second step (Figure 5), LM-PMAOD was hydrolyzed to poly(maleic acid-alt-1-octadecene) (LM-PMAcOD) in sodium hydroxide solution. Further acid-base extraction with H2SO4, ethyl acetate, and NaOH was performed for purification of the product and to remove impurities such as residual 1-octadecene (Figure 6). The product was dried over magnesium sulfate, coevaporated with chloroform, and finally purified by solid-liquid extraction in n-heptane.

[0166] (c) Polymer coating of SPIONs The polymer coating of SPIONs is shown diagrammatically in Figure 6. A flow chart of PMAcOD-SPION particle synthesis is shown in Figure 7.

[0167] The amphiphilic polymer PMAcOD was arranged around the SPION core to form a micellar structure. Most of the oleic acid molecules present on the surface of the SPION were replaced by ligand exchange with PMAcOD units. The hydrophobic polymer side chains then formed a hydrophobic bilayer, resulting in a negatively charged micellar structure. The charged carboxyl groups on the surface of the micelles served as anchors for the peptides.

[0168] For the coating procedure, the polymer and SPIONs were dissolved in chloroform. The solvent was removed via rotary evaporation until a thin film was formed. Sodium hydroxide solution was added and the flask was rotated at elevated temperature and ambient pressure until a clear dark brown aqueous colloidal dispersion was formed. This dispersion was diluted with sodium hydroxide solution and filtered through a 0.2 μm filter. Several washing steps with water and NaOH / NaCl (aq) were then performed using tangential flow filtration (TFF) for separation of low molecular weight components, followed by filtration through a 0.1 μm filter to remove larger particles and aggregates and for sterilization of the PMAcOD-SPION dispersion. The final nanoparticles were dispersed in water (Figure 7).

[0169] (d) Peptides and peptide coupling The peptide coupling and nanoparticle synthesis is shown diagrammatically in FIG.

[0170] Synthesis of the peptides was achieved in the C to N direction using solid phase peptide synthesis (SPPS) via Fmoc chemistry. The α-amino group of each amino acid was protected with a fluoren-9-ylmethoxycarbonyl (Fmoc) group, while the side chain functional groups were also blocked with various appropriate protecting groups.

[0171] The peptides were purified through preparative HPLC to reach the purity requirements, and the counterion TFA was replaced with chloride ion by using an appropriate solvent-buffer system. Finally, the purified peptides were lyophilized.

[0172] Characterization of the free peptides (starting material) was performed by LC-MS. The molecular weights of the peptides were determined by multimode electrospray-atmospheric pressure chemical ionization mass spectrometry.

[0173] The peptide was coupled to the polymer surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry in boric acid / sodium tetraborate decahydrate (SBB) buffer. The resulting TPC preparation was then filtered and purified by TFF purification. The final nanoparticles were dispersed in water, filtered through a 0.2 μm filter, and collected in a sterile container.

[0174] Example 2: Peptide coupling of tolerogenic antigenic peptides 0051, 0078, and 0080 (without N-terminal linker) In this experiment, three antigenic peptides (see Table 1) representing antigenic epitopes of gluten proteins found in celiac disease (CeD) patients with the HLA-DQ8 genotype were used in SPION-containing nanoparticles as described in Example 1. In this case, no N-terminal linker sequence was added to the native peptide epitope sequence.

[0175] Table 1: Peptide sequences of antigenic epitopes of gluten proteins (HLA-DQ8 genotype) [Table 4] *Isoelectric point **Molecular weight

[0176] Peptides were coupled to the polymer surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry in boric acid / sodium tetraborate decahydrate (SBB) buffer.

[0177] For this, 30 mL of 2x SBB (pH 9.0, 100 mM (185.4 mg) boric acid, 100 mM (1.14 g) sodium tetraborate decahydrate) was freshly prepared. EDC solution (aqueous solution containing 28.76 mg / mL (150 mM) EDC HCl) was freshly prepared.

[0178] Peptide solutions were freshly prepared as described in Table 2.

[0179] Table 2: Peptide solutions (0051, 0078, 0080, and 0088) [Table 5]

[0180] Additionally, 154 mM NaCl solution was purchased from BBrau and further diluted to 50 mM NaCl. A 100 kDa Amicon 50 mL spin filter and a 0.2 μm PES filter were used for nanoparticle purification and filtration.

[0181] The following reactions were set up in Nalgene vials, with the reagents added in the following order (see Table 3):

[0182] Table 3: Peptide coupling conditions (TPC0051, TPC0078, TPC0080, and TPC0088) [Table 6]

[0183] While the reaction was incubating, the 100 kDa spin filter was pre-washed with 50 mM NaCl by centrifugation at 4200 rpm for 5 minutes, after which the collection tube was emptied completely.

[0184] The reaction mixture was transferred to a pretreated centrifugal spin filtration tube and diluted to 15 mL with 50 mM NaCl solution. The tube was centrifuged at 4200 rpm for 5 minutes. Filters that contained less than 2 mL after the first centrifugation cycle were considered complete. Filters that contained more than 2 mL were centrifuged for an additional 2 minutes at 4200 rpm.

[0185] The filtrate was removed and the residue was diluted to 15 mL with 50 mM NaCl and centrifuged as in the previous step.

[0186] The filtration was repeated four more times, and in the last three repetitions, washing was performed with ultrapure water (centrifugation 4 min).

[0187] The residue was filtered through a sterile 0.2 μm PES filter followed by 0.1 μm filtration, transferred to a sterile vial and the peptide-coupled PMAcOD-SPION particles (Topas Particle Conjugate (TPC)) were stored at 4° C. for further characterization.

[0188] The TPC is then analyzed by size exclusion chromatography (SEC), dynamic light scattering (DLS) to confirm the percentage of the main peak (see Table 4), SDS-PAGE to determine the particle size distribution (see Table 5), and the peptide coupling efficiency (see Figure 9) and BCA assay to qualitatively determine the peptide coupling efficiency (see Table 6).

[0189] Size exclusion chromatography was performed using a Sepax SRT SEC-2000 column at 50° C. with an eluent of 0.1% SDS (in LiChrosolv water) at 0.5 mL / min. Sample injection of 10 μL at a wavelength of 215.8 nm with a run time of 30 minutes. The results are shown in the table below.

[0190] Table 4: SEC results [Table 7]

[0191] Determination of hydrodynamic diameter and polydispersity index (PDI) is carried out using dynamic light scattering (DLS) analysis with a Malvern Zetasizer. Samples are diluted with Milli-Q water in disposable polystyrene cuvettes to reach an average count rate of 200-500 kcps (1:50 v / v). All aqueous reagents are filtered through a 0.22 μm membrane before use. Samples are measured by DLS according to the analytical conditions described below:

[0192] List of analytical conditions: [Table 8]

[0193] 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 particle size distribution.

[0194] Table 5: Particle size distribution by DLS (intensity) [Table 9]

[0195] A BCA assay (BCA kit from Sigma Aldrich) was performed to determine the amount of peptides in each sample. For preparation of the BCA reagent (4.59 mL), 4.5 mL of Reagent A (a solution containing bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1 N NaOH) from the BCA kit was mixed with 0.09 mL of Reagent B (CuSO4.5HO (4% (w / v)). Peptide standards were then prepared from the same peptide batch used for nanoparticle coupling. A peptide stock solution in water (1.0 mg / mL) was prepared and diluted in water with the following peptide concentrations (0, 0.020, 0.040, 0.060, 0.080, and 0.100 mg / mL). Samples (peptide-coupled nanoparticles) were dispersed in water and 25 μL of samples and standards were mixed with 500 μL of previously prepared BCA reagent. All samples and standards were then incubated at 60° C. for 15 min, followed by centrifugation at 12000 rpm for 10 min. All samples and standards were then transferred in triplicate (150 μl) to a 96-well plate and then measured on a plate reader using absorbance at 562 nm. The absorbance of the standards was measured and a calibration curve was plotted (absorbance vs. concentration). A linear regression was calculated to calculate the concentration of the samples.

[0196] Table 6: BCA assay results [Table 10]

[0197] Samples were analyzed by SDS PAGE using Bolt 12% Bis-Tris Plus gels (Invitrogen). Gels were run at 200V for 23 minutes and then imaged. The gels were rinsed with water and stained in InstantBlue gel staining solution (Expedeon) for approximately 1 hour. The staining solution was then discarded. The gels were rinsed twice with water and then placed in water overnight for destaining. The gels were then imaged again. Samples were prepared as described below:

[0198] [Table 11]

[0199] (SDS PAGE analysis) Peptide 0051, 0078, and 0080 conjugates were coupled to PMAcOD-SPION particles (Topas particles (TP)). Size exclusion analysis was performed and the amount of aggregation was measured to be approximately 16%-25% (main peak 84%-75%). The observed hydrodynamic diameters were 28-34 nm with PDI values ​​of 0.210-0.232.

[0200] The results of the SDS PAGE gel using Bolt 12% Bis-Tris Plus gel (Invitrogen) are presented in FIG. 9. This SDS-PAGE confirms that all three peptides were coupled to the TP. Samples no. 2, 4, and 6 are pure peptides (0051, 0078, and 0080, respectively), samples no. 1 and 8 are protein standards, and samples no. 3, 5, and 7 are TPC0051, TPC0078, and TPC0080, respectively. The SDS PAGE gel is used to qualitatively confirm the coupling of the peptides to the TP. Before staining, it is possible to clearly see the brown color of the TPC, thanks to the iron core. The smearing of the TPC samples (3, 5, and 7) is due to the covalent binding of the peptides to the TP surface. The pure peptides (samples 2, 4, and 6) are shown at 3 kDa in the gel. The peptide content was quantified via BCA assay and the results obtained were 0.12 mg / mL (TPC0051), 0.17 mg / mL (TPC0078) and 0.23 mg / mL (TPC0080).

[0201] SDS PAGE gels qualitatively demonstrated the coupling of peptides 0051, 0078, and 0080 to the TP, but the BCA assay quantitatively demonstrated that these peptides coupled poorly to the surface of the TP.

[0202] Example 3: Peptide coupling of tolerogenic antigenic peptides 0051 and 0087 (without N-terminal linker) using different coupling conditions Two antigenic peptides (0051 and 0087) (see Table 7) representing antigenic epitopes of gluten proteins found in celiac disease (CeD) patients with the HLA-DQ8 genotype were coupled to PMAcOD-SPION particles. As before, in this case no N-terminal linker sequence was added to the native peptide epitope sequence. Different coupling conditions were used in this experiment in an attempt to optimize the amount of peptide coupling.

[0203] Table 7: Peptide sequences of antigenic epitopes of gluten proteins (HLA-DQ8 genotype) [Table 12] *Isoelectric point **Molecular weight

[0204] The first amino acid residue of peptide 0087, a glycine, is not part of the gliadin γ1a sequence and is merely added to facilitate coupling.

[0205] Peptides were coupled to the polymer surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry in boric acid / sodium tetraborate decahydrate (SBB) buffer. In this experiment, different coupling conditions were used in the presence of EDC to improve peptide loading onto the surface of Topas particles (TP).

[0206] For this, 30 mL of 2x SBB (pH 9.0, 100 mM (185.4 mg) boric acid, 100 mM (1.14 g) sodium tetraborate decahydrate) was freshly prepared. EDC solution (aqueous solution containing 28.76 mg / mL (150 mM) EDC·HCl) was freshly prepared. Peptide solutions were freshly prepared as described in Table 8.

[0207] Table 8: Peptide solutions (0051 and 0087) [Table 13]

[0208] Additionally, 154 mM NaCl solution was purchased from BBraunn and further diluted to 50 mM NaCl. A 100 kDa Amicon 50 mL spin filter and a 0.2 μm PES filter were used for nanoparticle purification and filtration.

[0209] The following reactions were set up in glass vials. The reagents were added in the following order (see Table 9):

[0210] Table 9: Peptide coupling conditions (TPC0051 and TPC0087) [Table 14]

[0211] While the reaction was incubating, the 100 kDa spin filter was pre-washed with 50 mM NaCl by centrifugation at 4200 rpm for 5 minutes, after which the collection tube was emptied completely.

[0212] The reaction mixture was transferred to a pretreated spin filter centrifuge tube and diluted to 15 mL with 50 mM NaCl solution. The tube was centrifuged at 4200 rpm for 5 minutes. Filters that contained less than 2 mL after the first centrifugation cycle were considered complete. Filters that contained more than 2 mL were centrifuged for an additional 2 minutes at 4200 rpm.

[0213] The filtrate was removed and the residue was diluted to 15 mL with 50 mM NaCl and centrifuged as in the previous step.

[0214] The filtration was repeated four more times, and in the last three repetitions, washing was performed with ultrapure water (centrifugation 4 min).

[0215] The residue was filtered through a sterile 0.2 μm PES filter followed by 0.1 μm filtration, transferred to a sterile vial and the Topas Particle Conjugates (TPC) were stored at 4° C. for further characterization.

[0216] The nanoparticles are then analyzed by size exclusion chromatography (SEC) after two reaction times, i.e., 2.5 h and 16 h, to confirm the main peak percentage (see Table 10) by dynamic light scattering (DLS), to determine the particle size distribution (see Table 11) by SDS-PAGE, to qualitatively confirm the peptide coupling efficiency (see Figures 10 and 11) and the BCA assay, and to qualitatively determine the peptide coupling efficiency (see Table 12).

[0217] Size exclusion chromatography was performed using a Sepax SRT SEC-2000 column at 50° C. with an eluent of 0.1% SDS (in LiChrosolv water) at 0.5 mL / min. Sample injection of 10 μL at a wavelength of 215.8 nm with a run time of 30 minutes. The results are shown in the table below.

[0218] Table 10: SEC results [Table 15]

[0219] Determination of hydrodynamic diameter and polydispersity index (PDI) was performed using dynamic light scattering (DLS) analysis with a Malvern Zetasizer. Samples are diluted with Milli-Q water to reach an average count rate of 200-500 kcps (1:50 v / v) in disposable polystyrene cuvettes. All aqueous reagents are filtered through a 0.22 μm membrane before use. Samples are measured by DLS according to the analytical conditions described below:

[0220] List of analytical conditions: [Table 16]

[0221] 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 particle size distribution.

[0222] Table 11: Particle size distribution by DLS (intensity) [Table 17]

[0223] The samples were analyzed by SDS PAGE using Bolt 12% Bis-Tris Plus gels (Invitrogen) (see Figures 10 and 11). The gel was run at 200V for 23 minutes and then imaged. The gel was rinsed with water and stained in InstantBlue gel staining solution (Expedeon) for approximately 1 hour. The staining solution was then discarded. The gel was rinsed twice with water and then placed in water overnight for destaining. The gel was then imaged again. Before staining, it is possible to clearly see the brown color of the TPC due to the iron core. To qualitatively confirm the coupling of the peptides to the TPs, the gel is run with pure peptides (0051 and 0087) and samples DM008, DM009, DM010, DM011, DM012, MS005, MS006, MS007, and MS008 as well as protein standards.

[0224] Unfortunately, in this experiment the staining was very poor, but it was still possible to see the coupling of the peptide to the TP (light blue color, "smearing" in the sample). Unfortunately, no staining of the pure peptide was discernible.

[0225] BCA assays were performed with these probes using the same method as described above.

[0226] Table 12: BCA assay results [Table 18]

[0227] Coupling of peptides 0051 and 0087 under different coupling conditions results in colloidally stable nanoparticles. However, the coupling efficiency in terms of peptide loading in all conditions remained very low. Therefore, it was not possible to optimize the coupling using peptides without an N-terminal linker sequence.

[0228] Example 4: Peptide coupling of tolerogenic antigenic peptides 0149, 0151, 0153, 0155, and 0159 (with N-terminal linker) In this experiment, five antigenic peptides (see Table 13) representing antigenic epitopes of gluten proteins found in CeD patients with the HLA-DQ8 genotype were coupled to PMAcOD-SPION particles (Topas particles (TP)), in this case by adding an N-terminal linker sequence containing two Arg amino acid residues to the native peptide epitope sequence.

[0229] In this experiment, the sequence of peptide 0149 matches that of previously used peptide 0051, with the addition of two Arg residues at the N-terminus. The sequence of peptide 0151 matches that of previously used peptide 0087, with the addition of two Arg residues at the N-terminus. However, the first glycine added to peptide 0087 to facilitate coupling has been omitted in peptide 0151. The sequence of peptide 0155 matches that of previously used peptide 0078, with the addition of two Arg residues at the N-terminus. Finally, the sequence of peptide 0159 matches that of previously used peptide 0080, with the addition of two Arg residues at the N-terminus.

[0230] Table 13: Peptide sequences of antigenic epitopes of gluten proteins (HLA-DQ8 genotype) [Table 19] *Isoelectric point **Molecular weight

[0231] Peptides were coupled to the polymer surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry in boric acid / sodium tetraborate decahydrate (SBB) buffer.

[0232] For this, 30 mL of 2x SBB (pH 9.0, 100 mM (185.4 mg) boric acid, 100 mM (1.14 g) sodium tetraborate decahydrate) was freshly prepared. EDC solution (aqueous solution containing 28.76 mg / mL (150 mM) EDC HCl) was freshly prepared.

[0233] Peptide solutions were freshly prepared as described in Table 14.

[0234] Table 14: Peptide solutions (0149, 0151, 0153, 0155, and 0159) [Table 20]

[0235] Additionally, 154 mM NaCl solution was purchased from BBraunn and further diluted to 50 mM NaCl. A 100 kDa Amicon 50 mL spin filter and a 0.2 μm PES filter were used for nanoparticle purification and filtration.

[0236] The following reactions were set up in Nalgene vials. Reagents were added in the following order (see Table 15):

[0237] In this example, the same reaction conditions and the same molar equivalent ratio of EDC / peptide (750:150) were used as described in Table 3 in Example 2 (Topas coupling standard procedure). The difference is in the reaction scale, which is larger in this example than in Example 2. The reaction scale does not affect the coupling efficiency.

[0238] Table 15: Peptide coupling conditions (TPC0149, TPC0151, TPC0153, TPC0155, and TPC0159) [Table 21]

[0239] While the reaction was incubating, the 100 kDa spin filter was pre-washed with 50 mM NaCl by centrifugation at 4200 rpm for 5 minutes, after which the collection tube was emptied completely.

[0240] The reaction mixture was transferred to a pretreated spin filter centrifuge tube and diluted to 15 mL with 50 mM NaCl solution. The tube was centrifuged at 4200 rpm for 5 minutes. Filters that contained less than 2 mL after the first centrifugation cycle were considered complete. Filters that contained more than 2 mL were centrifuged for an additional 2 minutes at 4200 rpm.

[0241] The filtrate was removed and the residue was diluted to 15 mL with 50 mM NaCl and centrifuged as in the previous step.

[0242] The filtration was repeated four more times, and in the last three repetitions, washing was performed with ultrapure water (centrifugation 4 min).

[0243] The residue was filtered through a sterile 0.2 μm PES filter followed by 0.1 μm filtration, transferred to a sterile vial and the TPC was stored at 4° C. for further characterization.

[0244] The nanoparticles are then analyzed by size exclusion chromatography (SEC) to confirm the percentage of the main peak (see Table 16) by dynamic light scattering (DLS), to determine the particle size distribution (see Table 17), BCA assay, and to qualitatively determine the peptide coupling efficiency (see Table 18) and peptide content by GC / MS, and to qualitatively determine the peptide coupling efficiency (see Table 19).

[0245] Table 16: SEC results [Table 22]

[0246] Table 17: Particle size distribution by DLS (intensity) [Table 23]

[0247] Table 18: BCA Assay Results [Table 24]

[0248] Table 19: Peptide content by GC / MS [Table 25]

[0249] Peptides 0149, 0151, 0153, 0155, and 0159 were successfully coupled to PMAcOD-SPION particles. Size exclusion analysis was performed and the amount of aggregation was determined to be approximately less than 12% (main peak approximately 89%). DLS measurements were used to observe the particle size distribution and polydispersity index. The observed hydrodynamic diameters were 30-34 nm with PDI values ​​of 0.222-0.282. The peptide content of all five batches ranged from 1.20 mg / mL (0.667 mM) to 2.39 mg / mL (0.879 mM).

[0250] Thus, the coupling efficiency was greatly improved by the addition of an N-terminal linker sequence containing an Arg amino acid residue.

[0251] Example 5: Peptide coupling of antigen peptides 0151 and 0152 (with N-terminal linker) In this experiment, two antigenic peptides (see Table 14) representing antigenic epitopes of gluten proteins found in CeD patients with the HLA-DQ8 genotype were coupled to PMAcOD-SPION particles (Topas particles (TP)), in this case by adding an N-terminal linker sequence containing one and two Arg amino acid residues to the native peptide epitope sequence.

[0252] Table 14: Peptide sequences of antigenic epitopes of gluten proteins (HLA-DQ8 genotype) [Table 26] *Isoelectric point **Molecular weight

[0253] Peptides were coupled to the polymer surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry in boric acid / sodium tetraborate decahydrate (SBB) buffer.

[0254] For this, 30 mL of 2x SBB (pH 9.0, 100 mM (185.4 mg) boric acid, 100 mM (1.14 g) sodium tetraborate decahydrate) was freshly prepared. EDC solution (aqueous solution containing 28.76 mg / mL (150 mM) EDC HCl) was freshly prepared.

[0255] Peptide solutions were freshly prepared as described in Table 15.

[0256] Table 15: Peptide solutions (0151 and 0152) [Table 27]

[0257] Additionally, 154 mM NaCl solution was purchased from BBraunn and further diluted to 50 mM NaCl. A 100 kDa Amicon 50 mL spin filter and a 0.2 μm PES filter were used for nanoparticle purification and filtration.

[0258] The following reactions were set up in Nalgene vials. Reagents were added in the following order (see Table 16):

[0259] In this example, the same reaction conditions and the same molar equivalent ratio of EDC / peptide (750:150) were used as described in Table 3 in Example 2 (Topas coupling standard procedure). The difference is in the reaction scale. The scale of the reaction does not affect the coupling efficiency.

[0260] Table 16: Peptide coupling conditions (TPC0151 and TPC0152) [Table 28]

[0261] While the reaction was incubating, the 100 kDa spin filter was pre-washed with 50 mM NaCl by centrifugation at 4200 rpm for 5 minutes, after which the collection tube was emptied completely.

[0262] The reaction mixture was transferred to a pretreated spin filter centrifuge tube and diluted to 15 mL with 50 mM NaCl solution. The tube was centrifuged at 4200 rpm for 5 minutes. Filters that contained less than 2 mL after the first centrifugation cycle were considered complete. Filters that contained more than 2 mL were centrifuged for an additional 2 minutes at 4200 rpm.

[0263] The filtrate was removed and the residue was diluted to 15 mL with 50 mM NaCl and centrifuged as in the previous step.

[0264] The filtration was repeated four more times, and in the last three repetitions, washing was performed with ultrapure water (centrifugation 4 min).

[0265] The residue was filtered through a sterile 0.2 μm PES filter followed by 0.1 μm filtration, transferred to a sterile vial and the TPC was stored at 4° C. for further characterization.

[0266] The nanoparticles are then analyzed by size exclusion chromatography (SEC) to confirm the percentage of the main peak (see Table 17) by dynamic light scattering (DLS), to determine the particle size distribution (see Table 18) and the BCA assay to qualitatively determine the peptide coupling efficiency.

[0267] Table 17: SEC results [Table 29]

[0268] Table 18: Particle size distribution by DLS (intensity) [Table 30]

[0269] Table 19: BCA Assay Results [Table 31]

[0270] Peptides 0151 and 0152 were successfully coupled to PMAcOD-SPION particles (TP). Briefly, size exclusion analysis was performed and the main peak was measured to be approximately less than 16%. DLS measurements were used to observe the particle size distribution and polydispersity index. The observed hydrodynamic diameter was approximately 30 nm with PDI values ​​of 0.222-0.234. The peptide content was quantified via BCA assay. The results obtained were 1.20 mg / mL for the probe with two Arg amino acid residues (TPC0151) and 1.06 mg / mL for the probe with one Arg amino acid residue (TPC0152).

[0271] Thus, the coupling efficiency was greatly improved by the addition of N-terminal linker sequences containing one and two Arg amino acid residues, whereas the addition of two Arg amino acid residues showed even higher coupling efficiency compared to the probe with only one amino acid residue.

[0272] Example 6: Functional validation of CD4 and CD8 T cell epitopes modified with one or two N-terminal arginines Experiments were performed to demonstrate that CD4 and CD8 T cell epitopes retain their agonistic properties when extended at the N-terminus with one or two arginines.

[0273] The functional properties of TPCs coupled with N-terminally modified or unmodified peptides were examined in an in vitro T cell activation assay. Using the example of an exogenous peptide derived from ovalbumin and a peptide derived from the autoantigen myelin oligodendrocyte glycoprotein (MOG), it was shown that T cells expressing receptors with specificity for these peptides were able to respond similarly to R- and RR-modified peptides and the respective unmodified peptides.

[0274] These peptides were coupled to nanoparticles and characterized according to the procedure described in Example 2. Tables 20, 21, 22, and 23 summarize the coupling parameters and analytical data.

[0275] Table 20: Peptide sequences [Table 32] *Isoelectric point **Molecular weight

[0276] Table 21: Peptide solutions [Table 33]

[0277] Table 22: Peptide coupling conditions [Table 34]

[0278] Table 23: Analysis results [Table 35]

[0279] The functional properties of TPCs conjugated with N-terminally modified or unmodified peptides were verified in an in vitro T cell activation assay. All data presented in Figures 12-14 below show the concentration of interferon gamma (IFNγ) as a readout for proliferative T cell activation.

[0280] (CD8 T cell epitope) OVA 257-264 Single cell suspensions were prepared from pooled spleen and lymph node cells of OT-1 mice expressing a transgenic T cell receptor specific for "SIINFEKL". 5 × 10 cells were cultured per well of a flat-bottom 96-well plate. 5 Cells were incubated with adjusted amounts of the indicated peptides in solution or as TPC conjugates. After 3 days of culture, supernatants were harvested and stored at -80°C until use. Supernatants were measured for IFNγ using a standard enzyme-linked immunosorbent assay (ELISA, R&D Systems). IFNγ concentrations were determined from a standard curve according to the manufacturer's instructions.

[0281] FIG. 12 demonstrates that peptides representing CD8 T cell epitopes, with one or two N-terminal arginines, retain their agonistic properties.

[0282] (CD4 T cell epitope) OVA 323-339 Single cell suspensions were prepared from pooled spleen and lymph node cells of OT-2 mice expressing a transgenic T cell receptor specific for IgG1. 5 × 10 cells were placed per well of a flat-bottom 96-well plate. 5 Cells were incubated with adjusted amounts of the indicated peptides in solution or as TPC conjugates. After 3 days of culture, supernatants were harvested and stored at -80°C until use. Supernatants were measured for IFNγ using a standard enzyme-linked immunosorbent assay (ELISA, R&D Systems). IFNγ concentrations were determined from a standard curve according to the manufacturer's instructions.

[0283] FIG. 13 demonstrates that peptides representing CD4 T cell epitopes, with one or two N-terminal arginines, retain their agonistic properties.

[0284] (CD4 T cell autoantigen epitope) MOG 35-55 Single-cell suspensions were prepared from pooled spleen and lymph node cells of 2D2 mice expressing a transgenic T cell receptor specific for this peptide. These T cells and this peptide serve to modulate the autoimmune response in a mouse model of experimental autoimmune encephalomyelitis (EAE), a model of the human autoimmune disease multiple sclerosis.

[0285] 5 x 10 per well of a flat-bottom 96-well plate 5 Cells were incubated with adjusted amounts of the indicated peptides in solution or as TPC conjugates. After 3 days of culture, supernatants were harvested and stored at -80°C until use. Supernatants were measured for IFNγ using a standard enzyme-linked immunosorbent assay (ELISA, R&D Systems). IFNγ concentrations were determined from a standard curve according to the manufacturer's instructions.

[0286] FIG. 14 demonstrates that peptides representing CD4 T cell autoantigen epitopes, with one or two N-terminal arginines, retain their agonistic properties.

Claims

1. (a) An amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less; and (b) A peptide covalently linked to the polymer, the peptide comprising an amino acid sequence of 8 to 50 amino acids, the amino acid sequence comprising (i) an N-terminal linker sequence containing at least one Arg amino acid residue, and (ii) an MHC-binding sequence containing a T cell receptor epitope, said peptide, A nanoparticle comprising.

2. (a) An amphiphilic polymer having a number average molecular weight (Mn) of 20,000 g / mol or less; and (b) A peptide covalently linked to the polymer, the peptide comprising a sequence containing an N-terminal linker sequence containing at least one Arg amino acid residue and an MHC-binding sequence containing a T cell receptor epitope, and comprising 8 to 50 amino acids (wherein the amino acid sequence of the peptide containing the N-terminal linker sequence is 【Chemical 1】 not), said peptide, A nanoparticle comprising.

3. The amphiphilic polymer has the following structural units 【Chemical 2】 (wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, and optionally, R is C 4 to C 22 alkyl group, preferably R is C 7 to C 19 alkyl group, preferably R is linear C 11 to C 17 alkyl group) The nanoparticles according to claim 1.

4. The amphiphilic polymer is selected from the group consisting of poly(maleic acid-1-octadecene), poly(maleic acid-1-tetradecene), or poly(maleic acid-1-dodecene), preferably, the amphiphilic polymer is poly(maleic acid-1-octadecene), and the number average molecular weight of the amphiphilic polymer is 6,000 to 1,000 g / mol. The nanoparticle according to Claim 1.

5. The nanoparticle according to Claim 1, wherein the peptide having no N-terminal linker sequence has an IEP lower than 6.

6. The nanoparticle according to Claim 1, wherein the peptide having the N-terminal linker sequence has an IEP greater than 6, greater than 7, greater than 8, or greater than 9.

7. The nanoparticle according to Claim 1, wherein the peptide is covalently linked to the amphiphilic polymer via the N-terminal linker.

8. The amino acid sequence of the peptide comprises (i) the N-terminal linker containing at least one Arg amino acid residue, and (ii) 【Chemical Formula 3】 A nanoparticle according to Claim 1, comprising a peptide sequence selected from the group consisting of.

9. The nanoparticle according to Claim 1, wherein the N-terminal linker sequence contains at least two Arg amino acid residues.

10. The nanoparticle according to Claim 1, wherein (a) Comprising a superparamagnetic iron oxide core (SPION); or (b) Not comprising a solid hydrophobic core (e.g., a superparamagnetic iron oxide core (SPION)), said nanoparticles.

11. The nanoparticles according to claim 1, having a size of 0.1 to 200 nm, optionally having a hydrodynamic diameter of 10 to 100 nm, 10 to 70 nm, 10 to 50 nm, 20 to 40 nm, or 26 to 36 nm when measured by dynamic light scattering.

12. A composition comprising the nanoparticles according to any one of claims 1 to 11 and a liquid or lyophilized carrier.

13. The composition according to claim 12, comprising at least two different types of nanoparticles, each type comprising at least one peptide sequence different from the peptide sequence(s) of the other type(s) of nanoparticles.

14. The composition according to claim 13, comprising 2 to 8 different types of nanoparticles, each type comprising one peptide sequence different from the peptide sequences of all other types of nanoparticles.

15. The composition according to claim 14, comprising 5 different types of nanoparticles, wherein: (a1) Each type of nanoparticle comprises the same peptide comprising an N-terminal linker of at least one Arg amino acid residue and a specific peptide sequence selected from the group consisting of [Chemical Formula 4] ; or (a2) Each type of nanoparticle comprises the same peptide having a sequence selected from the group consisting of 【Chemical Formula 5】 .

16. The composition according to claim 14, comprising 3 different types of nanoparticles, wherein: (b1) Each type of nanoparticle comprises the same peptide comprising an N-terminal linker of at least one Arg amino acid residue and a specific peptide sequence selected from the group consisting of 【Chemical Formula 6】 ; or (b2) Each type of nanoparticle comprises the same peptide having a sequence selected from the group consisting of 【Chemical Formula 7】 .

17. The composition according to claim 14, comprising 4 different types of nanoparticles, wherein: (c1) Each type of nanoparticle comprises the same peptide comprising an N-terminal linker of at least one Arg amino acid residue and a specific peptide sequence selected from the group consisting of 【Chemical 8】 ; or (c2) Each type of nanoparticle comprises 【Chemical Formula 9】 Said composition, comprising the same peptide having a sequence selected from the group consisting of [

18. ] The composition according to claim 12, for use in inducing tolerance to a therapeutic compound (e.g., a protein, a viral vector, or a lipid vesicle), an allergen, or an autoantigen, or for use in treating an allergy, an autoimmune disease, an exogenous antigen (e.g., a transplantation antigen, or a drug), or a food intolerance.

Images